DMBT1 , a putative tumor suppressor gene, in normal, proliferative, and malignant breast epithelium and its possible relation to cell cycle.We studied the expression of DMBT1 antibody (DMBTh12) and sections from 36 samples, were double-stained also with anti MCM5, one of the 6 pre-replicative complex proteins with cell proliferation-licensing functions. DMBT1 gene expression at mRNA level was assessed by RT-PCR in frozen tissues samples from 39 patients.Sections from 17 benign lesions and 55 carcinomas were immunostained with anti DMBT1 protein expression was down-regulated in the cancerous lesions compared to the normal and/or hyperplastic epithelium adjacent to carcinomas . In 72% of cases RT-PCR confirmed immunohistochemical results. Most of normal and hyperplastic mammary cells positive with DMBTh12 were also MCM5-positive.Normal glands and hyperplastic epithelium in benign lesions displayed a luminal polarized DMBTh12 immunoreactivity. Normal and hyperplastic epithelium adjacent to carcinomas showed a loss of polarization, with immunostaining present in basal and perinuclear cytoplasmic compartments. DMBT1 in normal and hyperplastic tissues flanking malignant tumours and its down-regulation in carcinomas suggests a potential role in breast cancer. Moreover, the concomitant expression of DMTB1 and MCM5 suggests its possible association with the cell-cycle regulation.The redistribution and up-regulation of  DMBT1  at chromosome 10q25.3–26.1, was considered a tumour suppressor gene because frequent deletions and/or lack of expression had been found in malignant tumours of the brain [he brain ,5, and the brain .DMBT1 is not a typical tumour suppressor gene because it is up-regulated in normal or inflamed epithelium adjacent to lung carcinomas as well as in some primary lung cancers and glioblastomas [DMBT1 inactivation in many tumours including breast cancer [However, recent evidence has shown that lastomas . It was t cancer ,10DMBT1 is a multi-functional protein related to the Mac-2 binding protein and mucins that have a protective function and mediate cell-extracellular matrix interactions [GP340(glycoprotein-340) and DMBT1SAG  are DMBT1 variants, present in the respiratory tract and the oral cavity, respectively, that have been linked to innate host defense and epithelial regeneration [GP340is involved in innate host defence by its interaction with the lectins surfactant protein D and A  and by its ability to stimulate alveolar macrophage migration [in vitro and in vivo [DMBT1 expression and localisation observed in other tumor types originating from mono-layered epithelia such as primary esophageal adeno-carcinomas and lung carcinomas [ractions ,11. DMBTneration -16. In tigration ,13,17. A in vivo ,19. Whil in vivo . Remarkarcinomas ,8.DMBT1 [DMBT1 homologue in the mouse, and a novel homologue in the rat , have been linked to initiation of cell proliferation, differentiation and repair [DMBT1 to the ECM triggers cellular differentiation, whereas a loss of expression of DMBT1 favours tumor cell growth [Hensin, the rabbit homologue of DMBT1, triggers epithelial terminal differentiation by mediating cell-extracellular matrix (ECM) interactions, and this may apply also to DMBT1 ,21-24. Cd repair ,26. It hl growth .DMBT1 in the morphologically normal, hyperplastic, and neoplastic breast epithelium. We also examined in morphologically normal epithelium a possible association of DMBT1 expression with that of an early proliferative marker such as MCM5 (Minichromosome Maintenance protein 5). MCM5 is one of the 6 pre-replicative complex proteins (MCM 2–7) [These findings led us to explore whether similar principles apply to breast epithelium. Here we examined the expression of MCM 2–7)  and its in situ (DCIS) and 45 cases of infiltrating carcinoma (IC) .From the files of the Surgical Pathology at S.Paolo University Hospital, we retrieved paraffin blocks of 72 consecutive breast lesions surgically resected. This material included 16 cases of fibrocystic disease (FD), 1 case of gynecomastia (GM), 10 samples of ductal carcinoma RT-PCR (Reverse-Transcriptase Polymerase Chain Reaction) was performed on 39 samples: i.e. 37 samples out of the 55 carcinomas used for the immunohistochemical studies, 1 sample of normal breast tissue that had been removed with a neoplastic lesion and 1 sample of fibrocystic disease. All 39 samples had been quickly frozen in liquid nitrogen after surgical resection and stored at -80°C. As confirmed by histopathologic examination, in all tumor specimens the amount of tumor cells equalled or exceeded 80% of the overall sample. As controls for each test a frozen sample of the same case positive with immunohistochemical method was used. We also analysed by RT-PCR two breast cancer cell lines (Hs578T and T-47D) obtained from the American Type Culture Collection and maintained as recommended.DMBT1 and anti MCM5 monoclonal antibodies  was performed on 17 benign lesions and a subset of 19 in situ and invasive carcinomas where residual normal breast tissue was well represented. On these selected samples a single incubation with anti MCM5 and anti Ki 67 antibody was performed in order to compare the two respective immunoreactivities. Antibodies were diluted as follows: anti-DMBT1h12, 1:500; anti MCM5, 1:40; anti Ki 67 Mib1, 1:100. For anti-DMBT1 and Ki 67 Abs, sections were pre-treated for antigen retrieval in boiling 0.01 M sodium citrate buffer pH 6, in microwave (MW) oven for 12 minutes. DMBT1 and MCM5 double labelling was performed in two steps following a modification of the method of Lan et al. [Sections from blocks containing the predominant lesion were incubated with anti DMBT1h12 monoclonal antibody  as well n et al. , brieflyThree investigators  evaluated separately DMBTh12 immunopositivity of morphologically normal, hyperplastic, and malignant cells. Immunopositivity was scored in 4 distinct categories according to the percentage of positive cells: 0 = negative, 1 = < 10%, 2 = 10%–50% and 3 = > 50%. MCM5 and Ki67 positivity in morphologically normal epithelium was determined with a semi-quantitative method: percentage of positive glandular structure and number of positive cells observed in each positive gland divided number of glands.Total RNA was extracted from breast specimens using Trizol reagent,  according to the manufacturer's instructions. Total RNA was quantified and checked for purity by UV spectrophotometry.2, 20 units of RNase inhibitor, 50 units of MuLV Reverse Transcriptase, 2.5 μM Random hexamers and 1 μg of total RNA. All reagents were purchased by the supplayer . The samples were incubated at 25°C for 10 min, 42°C for 60 min and at 95°C for 5 min.Reverse transcription of RNA was done in a final volume of 20 μl containing 1x buffer , 500 μM each deoxynucleotide triphosphate, 5 mM MgCl2, 1.25 units of Taq Gold DNA polymerase and each primer at 0.3 μM. The PCR amplification of DMBT1 was carried out in two steps, following a nested procedure. In the first step we used 2 μl of cDNA from the reverse transcription and in the second 2 μl of the first PCR product as template. Primers and thermal condition were used according to Mollenhauer et al [DMBT1) were obtained.Polymerase chain reaction (PCR) of the cDNA was performed in a final volume of 50 μl containing all four dNTPs (each at 200 μM), 1X buffer, 1.5 mM MgClStatistical differences between immunopositive cases/total number of cases were calculated by Fisher's exact test. p value less than 0.05 were considered statistically significant.T-test for independent values was used to evaluate statistically significant differences between immunoreactivity of normal and hyperplastic epithelium in benign, pre-invasive and invasive lesions.Different patterns of immunopositivity were observed in the histologically normal and hyperplastic breast tissue, present in the sections of benign and malignant lesions: a) single supra-nuclear, intra-cytoplasmic dot polarized toward luminal surface Figure ; b) a stDMBT1 at protein level in the mammary gland epithelium. In tumour areas, only 3 out of 55 carcinomas showed variable degrees of immunopositivity with anti-DMBTh12 Ab: in one case there was a widespread and strong decoration of numerous tumour cells (immunopositivity score = 3) with irregularly distributed dots [Figure DMBT1 expression in the cancerous lesions compared to the normal and/or hyperplastic epithelium was statistically significant for both, ductal carcinoma in situ (DCIS)  and invasive carcinoma (IC) .Normal and hyperplastic mammary structures displayed a variable DMBTh12 immunoreactivity: some ducts were decorated while others were negative. Moreover a variable percentage of cells was immunopositive. This heterogeneity suggests that there is no constitutive expression of s Figure ; in the s Figure  and in ts Figure . Down-reDMBT1 expression at protein and mRNA level are summarized in table The results of DMBT1 immunoreactive cells between the normal and the hyperplastic epithelium that flanked benign lesions, in situ and invasive carcinomas  RT-PCR confirmed immunohistochemical results, whereas 11 tumours that were negative with anti-DMBTh12 revealed PCR-products after RT-PCR amplification  and malignant (78%) lesions. Among 55 carcinomas we could detect immunopositivity only in 3 tumours (5%); in one case of infiltrating carcinoma the immunopositivity was widespread with a pattern similar to that seen in hyperplastic lesions near carcinoma; in the other two cases , it was limited to few tumour cells. DMBT1 expression was detected by RT-PCR in 13 of 35 (37%) infiltrating carcinomas and in 2 breast cancer cell lines. Eleven tumours, that were negative with anti-DMBTh12, revealed PCR-products after RT-PCR amplification. This discordance between IHC and RT-PCR results may be due to residual normal epithelial cells present in the sample. Furthermore other possible reasons of this discrepancy could be: 1) the greater sensitivity of molecular technique like RT-PCR in detecting very low expression levels of DMBT1 that were below detection by immunohistochemistry, 2) sampling problems i.e. the presence of few positive tumour cells only in the frozen samples or 3) few immunoreactive cells have been missed by microscopic examination and finally 4) DMBT1 protein could have an altered epitope not recognized by its paratope in the antibody. By either immunohistochemistry or molecular technique we found no correlation between DMBT1 expression and tumor histotype, grade, hormonal receptors status, proliferation index, Her2 Neu expression or lymph-nodal metastases.In the present study we investigated DMBT1 in human breast cancer, but an indirect weak correlation between the degree of differentiation of the breast carcinomas and the extent of the immunoreactivity was found [DMBT1 in breast cancer or to other poorly-defined factors such as race, age or hormonal status of the population under study. Mollenhauer et al. point out that DMBT1 expression may be up-regulated in pathophysiological conditions such as inflammation, liver injury or after carcinogen administration and it is down-regulated in tumors to achieve a growth advantage in a dynamic and complex model. Also according to these authors, the possibility that DMBT1 loss may be a consequence rather than a primary event in carcinogenesis is unlikely (10).In a recent paper on breast carcinogenesis, qualitatively similar data were reported for as found . The disDMBT1 expression by RT-PCR [DMBT1 expression patterns observed in the human respiratory tract and in associated inflammation and carcinomas [DMBT1 expression in the normal mammary gland epithelium may result from the necessity of DMBT1 induction. Indeed, in parallel in vivo studies, it was found that DMBT1 is strongly up-regulated in the mammary gland tissue of the mouse shortly after carcinogen-administration and prior to the onset of histo-pathologically visible symptoms [DMBT1 products [DMBT1 in breast tissue may be part of a protective response as suggested by Kang et al [DMBT1 and Sp-D in pulmonary epithelium.Although the normal mammary gland has been reported to have low y RT-PCR , we founy RT-PCR . Moreovercinomas  and suggsymptoms . It has products . We receproducts ; thus, wng et al  for DMBTDMBT1, the depolarisation of the staining patterns indicates that DMBT1 may also be secreted to other destinations, i.e. the ECM, by the mammary gland epithelial cells. In this regard, the second putative function of DMBT1, i.e. triggering of epithelial differentiation, is noteworthy. It may prevent hyperproliferative epithelial cells from out-of control proliferation and progression to the tumor state. This view is supported by two present findings. The correlation between MCM5 and DMBT1 expression may point to a coupling to cell-cycle processes: basolateral relocation and hyper-expression of DMBT1 at the ECM may provide cells that are licensed to proliferate with protection from unregulated proliferation. On the other hand loss of DMBT1 expression in a significant numbers of DCISs and ICs is consistent with the notion that DMBT1 inactivation may offer growth advantages to neoplastic cells [DMBT1 expression appears to take place early, i.e. already at the time of transition from hyperplastic lesions to carcinoma, as it has been reported in oesophageal squamous cell carcinomas [While this interaction may be attributed to lumenally secreted ic cells . The losDMBT1 inactivation remain unclear. An inactivation by mutation appears not to be a major mechanism for DMBT1 inactivation in tumors [DMBT1 or genes that regulate DMBT1 expression takes place in some tumors [DMBT1 promotor, loss of expression or mutation of DMBT1-regulatory genes might represent alternative candidate mechanisms.The exact mechanisms for n tumors ,31 The se tumors . AccordiDMBT1 expression and cellular location in breast epithelium varies in normal, hyperplastic and neoplastic cells. In normal or hyperplastic tissue flanking carcinomas it is up-regulated and polarized toward glandular lumen or the basolateral membrane while it is down-regulated in carcinomas. These findings closely resemble the patterns observed for its interaction partner SP-A in an earlier study. Additionally we have shown that DMBT1 is co-expressed with MCM5 in normal and hyperplastic tissue. We hypothesize that hyper-expression and basolateral relocation of DMBT1 in these epithelia prevents the out of control growth that characterizes neoplastic epithelium.We have shown that DMBT1: Deleted in Malignant Brain Tumor 1,MCM5: Minichromosome Maintenance protein, 5RT-PCR: Reverse Transcriptase Polymerase Chain ReactionGP 340: Glycoprotein 340SAG: salivary AgglutininSp-A, Sp-D: Surfactant Protein A, DMBP: Mannan Binding ProteinECM: Extracellular MatrixFD: Fibrocystic DiseaseGM: gynecomastiaIn Situ DCIS: Ductal Carcinoma IC: Invasive CarcinomaMW: Micro WaveEDTA Ethylenediaminetetraacetic AcidDAB: DiaminobenzidineNone declared.PB designed the study, participated in the evaluation of immunohistochemical results and drafted the manuscript. PGN participated in the evaluation of immunohistochemical results and performed statistical analysis. JM participated in sequence alignment, criticism and produced DMBTh12 antibody. AP participated in sequence alignment and data evaluation. CP Carried out RT-PCR. AM participated in the evaluation of histologic samples and immunohistochemical results. GB participated in data evaluation and sequence alignment GC participated in study design and data evaluation. SB participated in study design, sequence alignment, criticism and coordinated molecular study. GGP participated in study design, criticism and general revision of paper.The pre-publication history for this paper can be accessed here:


In most parts of the world, microscopy is still the gold standard for diagnosing malaria. An online tool could help to improve your diagnostic skills An online tool allows microscopists to test their knowledge Malaria kills over one million people in Africa each year and contributes 10% of the continent's burden of disease . In thisMalaria affects at least 200–300 million people every year and causes from 1–2 million deaths—mostly children under five and pregnant women in sub-Saharan Africa [Malaria is not limited to Africa—40% of the world's population is at risk of acquiring the disease —and it iDespite the efforts of a global campaign to roll back the disease, the number of deaths from malaria is increasing in Africa . This stThere is another important reason why correct diagnosis matters. Because of rising drug resistance in Africa, the conventional drugs for treating malaria—chloroquine and sulfadoxine-pyrimethamine—are now failing in up to 80% of cases . There iPlasmodium falciparum [We still regard microscopy as the gold standard for the diagnosis and characterisation of malaria infection . Modern lciparum —althoughlciparum ,17—but tSome authors have suggested that these new tests should not be considered a complete substitute for direct microscopic examination of blood smears . We agreP. falciparum or three P. vivax parasites per microlitre of blood [Analysis of DNA by the polymerase chain reaction (PCR) may be a useful tool for diagnosis of malaria when the results of conventional techniques are negative, especially since PCR allows accurate species identification . And wheof blood . Howeverof blood . Even inhttp://www.rph.wa.gov.au/labs/haem/malaria/index.html). The materials are now available in English, French, and Spanish. One of the most important features is a “Test and Teach” section to allow microscopists to develop and sharpen their skills in malaria diagnosis  has been sent on request to institutions and centres in 149 countries, a number that does not include unauthorised (pirated) copies of the CD-ROM. The number of hits to the site (the number of pages or images of the site that are accessed) has risen dramatically, from 1,500 to over 100,000 per week.Graham.Icke@health.wa.gov.au.Through a circulated questionnaire with a 63% response rate, we have received feedback from users in 15 countries. Respondents said they valued the content, presentation, and usefulness of the online information. Who is using the material? A wide range of people—from students through to experienced personnel, and from clinicians to scientific, research, and technical staff. Many tropical medicine institutions have requested copies of the CD-ROM to supplement their own training programs. We have discussed the project at scientific conferences around the world and in scientific journals, advertising the fact that this educational material is freely available. Institutions wishing to obtain a free copy of the CD-ROM should contact E-mail: We also became aware that unauthorised copies of the CD-ROM were being produced. Although there is a real need to guarantee the integrity of the information on the CD-ROMs and ensure proper credit for the authors and patrons, our main aim is to spread the “good word”, and such unauthorised copies are a powerful aid to distribution. Given that there is no need to recoup profit from this project, our only concern is its usefulness. Consequently, we have welcomed the production of these pirated copies, on condition that they were of good quality and were made freely available at no cost.The Internet and associated technologies make it possible for educators with a desire to teach to contact those with a need to learn, regardless of the geographical distances involved. By enlisting assistance from international colleagues, language barriers can be overcome. In addition to the English, French, and Spanish versions, we are aware that our project has been translated into German, Thai, and Vietnamese and has been distributed through small regional health group networks.There can be little doubt that these new technologies have the potential to revolutionise information dissemination, with particularly significant implications for healthcare professionals in developing countries. The malaria educational program could be a model on which future health education programs are based. With rapidly increasing access to these new information technologies, health care professionals from anywhere in the world can now join the global health community.


Waterpipe smoking is becoming a global public health problem, especially in the Eastern Mediterranean region (EMR).We try in this study, which is a cross sectional survey among a representative sample of waterpipe smokers in cafes/restaurants in Aleppo-Syria, to assess the time period for the beginning of this new smoking hype. We recruited 268 waterpipe smokers  30.1 ± 10.2, response rate 95.3%). Participants were divided into 4 birth cohorts  and year of initiation of waterpipe smoking and daily cigarette smoking were plotted according to these birth cohorts.Data indicate that unlike initiation of cigarette smoking, which shows a clear age-related pattern, the nineties was the starting point for most of waterpipe smoking implicating this time period for the beginning of the waterpipe epidemic in Syria.Maassel), and the proliferation of satellite and electronic media during the nineties may have helped spread the new hype all over the Arab World.The introduction of new flavored and aromatic waterpipe tobacco ( This cee smoker , Recent e smoker . This tre smoker ,7. Althoe smoker -11.Maassel , the media, and social trends [Developing effective intervention strategies to curb this emerging public health problem requires clear understanding of factors influencing the take up of this habit, as well its time course . Accordil trends . UnderstThe current analysis is drawn from a survey conducted in 2003 among a representative sample of waterpipe smokers visiting cafes/restaurants in Aleppo, Syria. The survey details can be found elsewhere , but briThe protocols and informed consent documents for this study were approved by the SCTS' IRB and the University of Memphis' IRB. The questionnaire was anonymous and informed consent was obtained prior to all interviews.First, year of birth was calculated by subtracting current age of participant from the year of survey (2003), while year of initiation of waterpipe smoking and daily cigarette smoking were calculated by adding the age of initiation of smoking to the year of birth. Year of birth was then divided into four decade-long categories , and year of smoking initiation into three decade-long categories .p level <0.05 considered significant.The Chi-Square test was used to assess differences between the three smoking initiation time-groups for each birth cohort, with th century, and showing an increasing trend. Based on these results and our previous data regarding factors related to the spread of waterpipe [Maassel was introduced [Figure aterpipe , we prestroduced  simplifytroduced ,15, nicoNone declared.SR designed the study, conducted the analysis and wrote the first draft of the manuscript. KW and TE contributed to the design of survey and revision of the manuscript. WM contributed to the design of survey and wrote the final draft of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


To examine the associations between vision-targeted health-related quality of life (VT-HRQ) and ocular surface parameters in patients with Sjögren's syndrome, a systemic autoimmune disease characterized by dry eye and dry mouth.Forty-two patients fulfilling European / American diagnostic criteria for Sjögren's syndrome underwent Schirmer testing without anesthesia, ocular surface vital dye staining; and measurement of tear film breakup time (TBUT). Subjects were administered the Ocular Surface Disease Index (OSDI) and the 25-item National Eye Institute Vision Functioning Questionnaire (NEI-VFQ). Main outcome measures included ocular surface parameters, OSDI subscales describing ocular discomfort (OSDI-symptoms), vision-related function (OSDI-function), and environmental triggers, and NEI-VFQ subscales.Participants  all had moderate to severe dry eye. Associations of OSDI subscales with the ocular parameters were modest (Spearman r (ρ) < 0.22) and not statistically significant. Associations of NEI-VFQ subscales with the ocular parameters reached borderline significance for the near vision subscale with TBUT  and for the distance vision subscale with van Bijsterveld score . The strongest associations of the two questionnaires were for: ocular pain and mental function with OSDI-symptoms ; and general vision, ocular pain, mental function, role function, and driving with OSDI-function .Associations between conventional objective measures of dry eye and VT-HRQ were modest. The generic NEI-VFQ was similar to the disease-specific OSDI in its ability to measure the impact of Sjögren's syndrome-related dry eye on VT-HRQ. Dry eye is a common disorder of the ocular surface and tear film and is estimated to affect from 2% to over 15% of persons in surveyed populations, depending on the definition used . SymptomPatient-reported measurements used to evaluate the specific impact of eye disease and vision on symptoms (discomfort), functioning (the ability to carry out activities in daily life), and perceptions  are referred to as vision-targeted health-related quality of life (VT-HRQ) instruments. Valid and reliable measurements of VT-HRQ have become essential to the assessment of disease status and treatment effectiveness in ocular disease . There a© [The measurement of the impact of dry eye on a patient's daily life, particularly symptoms of discomfort, is a critical aspect of characterizing the disease . Despite©  was deveSjögren's Syndrome is an autoimmune systemic disease characterized by dry mouth and dry eye signs and symptoms ,26. Its Our purpose in this study was to examine VT-HRQ in patients with primary Sjögren's syndrome, using a generic and a dry-eye-disease-specific instrument. We examined the associations of ocular surface parameters with the VT-HRQ scores, hypothesizing that the disease-specific instrument would be more closely related than the generic to the clinical markers of disease. We also examined the association of the generic and disease-specific VT-HRQ scores with each other.The study protocol was approved by the National Eye Institute Internal Review Board. All patients completed an informed consent prior to examination. Consecutive patients with diagnosed primary Sjögren's syndrome were recruited from the NIH Clinical Center, Bethesda, MD. The diagnosis of primary Sjögren's syndrome was based on European-American criteria, which requires at least four of the following six features: signs and symptoms of dry eye and of dry mouth, histopathologic evidence of inflammation on minor salivary gland biopsy, and positive anti-Ro or anti-La antibodies.Before the clinical examination, a trained interviewer administered two questionnaires (described further below) to measure VT-HRQ to each patient. The subsequent clinical examination included a comprehensive anterior segment evaluation, including slit lamp biomicroscopy, evaluation of lid margin thickness and hyperemia, conjunctival erythema, chemosis, tear film debris and mucus, and extent of meibomian gland plugging. Tests of tear function and ocular surface status were performed as described below.The OSDI   ,42 is a Schirmer tests of tear production without and with anesthesia were performed by inserting a Schirmer tear test sterile strip  into the inferior fornix, at the junction of the middle and lateral third of the lower eyelid margin, for 5 minutes with the eyes closed. The extent of wetting was measured by referring to the ruler provided by the manufacturer on the envelope containing the strips. Possible scores range from 0 to 35 mm, with lower scores indicating greater abnormality in tear production. This test was repeated after instillation of topical anesthetic, 0.5% proparacaine . A SchirThe assessment of ocular surface damage was performed by a cornea specialist using vital dye staining with 2% unpreserved sodium fluorescein and then 5% lissamine green dye. The corneal, temporal, and nasal regions of the conjunctiva were scored individually from 0–5 (for fluorescein) and 0–5 (for lissamine green) using the Oxford grading scheme . The OxfTear film stability was assessed using fluorescein tear film breakup time (TBUT). Five microliters of 2% sodium fluorescein was instilled into the inferior fornix and the patient was asked to blink several times. Using the cobalt blue filter and slit lamp biomicroscopy, the duration of time required for the first area of tear film breakup after a complete blink was determined. If the TBUT was less than 10 seconds, the test was repeated for a total of 3 values and the average was calculated.For analysis, for each individual, the maximum (worse) score for the two eyes was used for Oxford score and VB, and the minimum (worse) score for the two eyes was used for Schirmer with and without anesthesia and for TBUT. TBUT values greater than or equal to 10 seconds  were codHypotheses of specific associations were formulated based on the areas and domains assessed by the two VT-HRQ instruments. Scatterplots and Spearman's correlation coefficient (ρ)  were useA total of 42 patients, 40 female and 2 male, were included in this study. The average age was 55 years range, 31–81 y). Most (81%) were of European descent. Visual acuity in the better eye was 20/20 or better for 68% of the patients; the remainder had 20/25 or better in the better eye, except for one patient who was 20/32 in both eyes. Ocular examination  indicated moderate problems with symptoms, functioning, and adverse environmental conditions. Mean OSDI-symptoms score was 62.5, mean OSDI-function score was 78.2, and mean OSDI-triggers score was 60.2. However, some patients had no problems with these areas: 12% reported no problems with irritation symptoms, 21% reported no problems with functioning, and 24% had no problems with environmental triggers. Associations of the OSDI subscale and overall scores with ocular surface parameters  are shown in Overall, scores on the NEI-VFQ subscales tended to be high. Average scores for near and distance vision, social and mental functioning, dependency, driving, and peripheral vision were over 80, and a substantial percentage reported no problems at all with any of the items on the subscale: 26% for near vision, 24% for distance vision, 83% for social functioning, 17% for mental functioning, 74% for dependency, 38% for driving, and 79% for peripheral vision. The subscale indicating the most impairment was the ocular pain subscale, with a mean score of 66.7. Associations of the NEI-VFQ subscale and overall scores with ocular surface parameters  are shown in Table In general, stronger associations were observed between subscales of the OSDI and NEI-VFQ Table  than betTable disease-specific instrument (OSDI) with a generic VT-HRQ instrument (NEI-VFQ-25) in patients with a systemic autoimmune disease associated with moderate to severe dry eye. We found that patients with primary Sjögren's syndrome had OSDI scores  similar to those previously published [We compared subscale scores for an ocular surface ublished  for modeublished -20. IndeOne explanation could be that the nature of the items for each of these instruments is quite different. The OSDI queries the frequency of a symptom or difficulty with an activity, over a one week recall period. The NEI-VFQ incorporates questions both the frequency and intensity of symptoms and their impact on activities, with no specified recall period. Perhaps this added element of capturing both the frequency and intensity of a symptom or impact accounts for some of the differences we found. For subscales that are similar, agreement was higher but still moderate, possibly due to differences in the nature of the questions or response options. The OSDI is targeted to assess how much the symptoms of dry eye affect the patient's current status , whereas the NEI-VFQ may be more suited to capturing the overall impact of a chronic ocular disease on VT-HRQ.In this group of primary Sjögren's syndrome patients, associations between subscales of the NEI-VFQ and OSDI were moderate to strong (< 0.70) and in hypothesized directions. Significant associations were seen between OSDI and NEI-VFQ overall scales; OSDI-symptoms and NEI-VFQ ocular pain; and OSDI-function and NEI-VFQ general vision and driving. This suggests that both instruments are capturing important aspects of VT-HRQ. It is not surprising that the highest correlations were observed between subscales with similar domains, which serves to validate the use of alternate methodologies. On the other hand, it is counter-intuitive that the generic and disease-specific instruments appeared similar (or that the generic seemed to do a little better) with respect to their association with objectively measured clinical signs of dry eye, as the NEI-VFQ was designed to capture broader aspects of VT-HRQ. For the NEI-VFQ, we found moderate correlations (greater than 0.3) of distance vision with VB and near vision with TBUT. This was surprising, as one may have expected that subscales measuring ocular discomfort or pain  would have the strongest correlations with clinical measures of dry eye. Clinical signs of dry eye include measures of tear production, ocular surface staining, and tear film break-up; visual acuity and other aspects of visual function are not generally as widely used. However, some investigators have reported that visual acuity in dry eye patients is correlated with decreased spatial contrast sensitivity  and is fWould we expect the associations to be different in Sjögren's patients? Sjögren's syndrome is an autoimmune exocrinopathy and effects of its systemic nature and chronicity on dry eye may have been more readily captured by the NEI-VFQ's ability to measure both frequency and intensity of problems with VT-HRQ. In contrast, although the OSDI includes items to measure function, responses are limited to the frequency of problems. Because the type of dry eye in Sjögren's syndrome is more likely to be severe, and all patients in our study had Sjögren's-related dry eye, we speculated that somewhat stronger associations between signs and symptoms might be observed. On the other hand, ocular surface inflammation and decreased corneal sensation are features of severe dry eye which might alter a patient's perception of symptoms of ocular irritation and might be the cause of weaker correlations between signs and symptoms ,55. IndeIn addition to clinical signs, it is important to include assessments of VT-HRQ and visual function to fully characterize the impact of dry eye on health status. The correlation between signs and VT-HRQ are modest at best, indicating that VT-HRQ is capturing an additional component of disease that is not captured by the clinical assessment. This does not necessarily mean that the measures of VT-HRQ or the methods of detecting clinical signs are deficient, but rather that VT-HRQ is an additional element of the overall impact of this disease process on affected individuals. Furthermore, in diseases with systemic manifestations, such as Sjögren's syndrome, that may have an influence on quality of life independent of dry eye symptoms, appropriate tests of VT-HRQ are critical to completely characterize quality of life in these patients. It may also be valuable to explore possible differences in associations of clinical signs with VT-HRQ in patient populations with different manifestations or causes of dry eye.VT-HRQ: Vision-targeted health-related quality of life; TBUT: Tearfilm breakup time; OSDI: Ocular Surface Disease Index; NEI-VFQ: National Eye Institute Visual Function Questionnaire; VB: van BijsterveldSV helped to design the study and performed all analyses and took the lead in writing the manuscript. LG performed the patient interviews and assisted with data analyses. GFR provided advice on statistical methods and presentation of the results. JA conceived and helped to design the study and assisted with writing the manuscript.


The visual cortex responds to repeated presentations of the same stimulus with high variability. Because the firing mechanism is remarkably noiseless, the source of this variability is thought to lie in the membrane potential fluctuations that result from summated synaptic input. Here this hypothesis is tested through measurements of membrane potential during visual stimulation. Surprisingly, trial-to-trial variability of membrane potential is found to be low. The ratio of variance to mean is much lower for membrane potential than for firing rate. The high variability of firing rate is explained by the threshold present in the function that converts inputs into firing rates. Given an input with small, constant noise, this function produces a firing rate with a large variance that grows with the mean. This model is validated on responses recorded both intracellularly and extracellularly. In neurons of visual cortex, thus, a simple deterministic mechanism amplifies the low variability of summated synaptic inputs into the large variability of firing rate. The computational advantages provided by this amplification are not known. A simple model accounts for the high variability of firing rates observed in responses of cortical neurons to visual stimuli In the primary visual cortex (V1), different trials of presentation of an identical stimulus yield highly variable firing rates . This trA key property of trial-to-trial variability is that it depends on the strength of the stimulus: Response variance across trials is approximately proportional to response mean . An examBecause the production of firing rates within a neuron introduces remarkably little noise , trial-tFrom traces of membrane potential obtained at high temporal resolution D, I obtamean,V is the “signal” reflecting the stimulus-driven synaptic input to the neuron  whose slope is 0.08 ± 0.01 and whose intercept at restV = −60.4 mV is 3.3 ± 0.1 mV. Similar values were obtained in the rest of the population , intercept at restV was 3.3 ± 1.4 mV, and mean slope was a shallow 0.14 ± 0.09. In occasional cells . When meanV is higher, however, the tail of the Gaussian that lies above threshold becomes substantially larger, and the distribution of firing rates reaches higher rates (e). The large peak at 0 spikes/s corresponds to the area of the Gaussian that lies below thresh.VIn the Gaussian–rectification model, the stimulus determines the mean of the Gaussian B, and th instant B. The re instant H operate instant A: Each ptly zero A, a. Wheer rates A, e. ThemeanV increases, the distribution of firing rate becomes broader  appears remarkably Gaussian , the firing rate is always zero, whereas larger mean potentials yield a distribution of firing rates that spans values from 0 to 300 spikes/s . Deviations from the predictions are largest where they are least significant, i.e., at high firing rates for the high values of meanV . These high values were achieved seldom; for example, only 21 data points were obtained at meanV = −50 mV (e), compared to 273 at meanV = −54 mV (d) and 1,575 at meanV = −64 mV (a).The model predicts the main features of the distributions of firing rate A. It pre= −50 mV A, e, com= −54 mV A, d and = −64 mV A, a.In fact, the model closely predicts both the firing rate's mean and standard deviation C. It preCrucially, the model closely predicts how firing-rate variance depends on firing-rate mean C and 3D.Overall, the Gaussian–rectification model applied to the trace of mean potential performed as well as the rectification model applied to the individual traces of potential. Both models underestimated the intercept of the lines fitted to the relationship between firing-rate variance and mean: the rectification model by 25 ± 42% A, and thThese results illustrate how the key element in producing the steep growth in firing-rate variance observed with growing stimulus strength is the nonlinear transformation between potential and firing rate H. IndeedThe predictions of the Gaussian–rectification model apply to any neuron that meets minimal criteria: a relationship between synaptic input and firing rate that is monotonic and includes a threshold, and noise in the input that has a Gaussian distribution.As an example, let us consider a neuron that is closer to biological reality than the Gaussian–rectification model, one that receives currents  in its input and produces individual spikes (not continuous firing rates). In particular, consider an enhanced integrate-and-fire neuron, where each spike is accompanied by a temporary increase in spike threshold and by the entry of calcium, which in turn determines an after-hyperpolarization potassium current see .To ensure realism, I fitted the model parameters to responses to injected currents of a regular spiking neuron. This neuron was recorded in vitro in the visual cortex of the guinea pig, in the near absence of synaptic inputs . The injJust as predicted, this spiking neuron responds to noisy injected currents with a firing rate whose variance grows with the mean . To simuThe Gaussian–rectification model captures the essence of this behavior. Once it is given the standard deviation of the noise and the relationship between injected current and firing rate F, the GaSimilar results were obtained when the stimulus parameters were changed to simulate synaptic inputs to a complex cell, or when the parameters of the spiking neuron were changed to simulate other cells measured in vitro, or even chosen randomly within reasonable bounds. As predicted, as long as the relationship between synaptic input and firing rate involved a threshold and the input noise was Gaussian, the variance grew with the mean for firing rate but not for injected current.thresh,V (3) the gain k of the relationship between firing rate and potential above threshold, and (4) the exponent n of this relationship. For the purpose of studying the model, we can assume, without loss of generality, that potential is unitless and has standard deviation σ = 1. Then, because threshV can only determine the range of firing rates that is achieved, only k and n control the shape of the variance versus mean curves , variations in potential are compressed into proportionally ever smaller variations in firing rate; the opposite occurs if the curves expand (n > 1).The gain ve shape . Increasightward , rows. Tsaturate , columnse . Accordingly, a mild form of saturation is common in the plots of firing-rate variance and mean , measurements in this dataset yield more precise estimates of firing-rate variance over a wider range of firing rates than are available in the intracellular sample.k and exponent n of the transformation of potential into firing rate. I thus first computed the model predictions for a variety of combinations of k and n  show evident saturation in firing-rate variance as mean firing rate increases. The remaining five show a milder saturation, as expected from their higher exponents (n = 1.1 to 1.2). Saturation was common, as the median n was 1.06, with n < 1 in 13/37 cells. Yet to my knowledge, except for an anecdotal account  = k[V−thresh]n+V, with [.]+ indicating rectification, k a proportionality factor, and n an exponent. Fitted parameters were threshV = −55.3 mV, k = 16.7, and n = 1.2 for the simple cell in threshV = −46.6 ± 10.5 mV, k = 12.4 ± 7.9, and n = 1.1 ± 0.6 for the whole intracellular population (N = 22). The distance between threshV and restV was 5.1 mV for the simple cell in The relation between potential  R e.g., H was fit R e.g., , where RVmean in response to a stimulus was defined as the mean across trials of coarse potential.The mean potential r  is the relation between firing rate and potential V  depends on whether r is zero or positive:where ential V H, and N .Distributions for potential at a given mean potential B were co)] e.g., B, a.z-scores  of potential ; (2) for each interval i, find the set of times (ikt) when the mean potential is in the i-th bin; (3) pooling across trials j, compute i,σ the standard deviation of jV(ikt); (4) transform each jV(ikt) into a z-score: zijk= [jV(ikt) − iv]/i;σ (5) look at the distribution of (zijk)  e.g., A.The enhanced integrate-and-fire model was derived in collaboration with Davide where the currents are:spiket the time of the last spike. Ca(t) is the (unitless) calcium concentration:with spike < t.twhere the sum extends over all spikes with Nag(t) and Kg(t) derived from Hodgkin–Huxley equations and are scaled to approximate the spikes from the recorded neuron. They occur when mV exceeds a threshold, which depends on the time since the last spike:Spikes result from stereotyped conductances kV = −80 mV and NaV = 55 mV. Passive parameters of the membrane  were obtained by fitting the membrane potential responses to sinusoids. The remaining parameters  were obtained by a search algorithm aimed at maximizing the quality of the predictions for firing rate.The reversal potentials for sodium and potassium were set to


Few studies have focused on the association between the sociodemographic characteristics of a patient with the change in health-related quality of life (HRQOL) following invasive coronary procedures, and the results remain inconclusive. The objective of the present study was to measure the temporal changes in HRQOL of patients with coronary heart disease, and assess how these changes are associated with invasive coronary procedures and sociodemographic characteristics.t-tests and multiple regression analyses were used to assess temporal changes in HRQOL and to identify the associated factors.This was a prospective study of 254 patients with angina pectoris and 90 patients with acute coronary syndrome. HRQOL was assessed with the multi-item scales and summary components of the SF-36, both 6 weeks and 2 years after baseline hospitalization in 1998. Paired Physical components of HRQOL had improved most during the 2 years following invasive coronary procedures. Our findings indicated that patients with angina pectoris who were younger, male, and more educated were most likely to increase their HRQOL following invasive coronary procedures. When adjusting for baseline HRQOL scores, invasive coronary procedures and sociodemographic characteristics did not explain temporal changes in patients with acute coronary syndrome, possibly due to higher comorbidity.Sociodemographic characteristics should be taken into account when comparing and interpreting changes in HRQOL scores in patients with and without invasive coronary procedures. In the medical treatment of patients with coronary heart disease (CHD), invasive coronary procedures – such as percutaneous coronary intervention – are effective in reducing mortality and morbidity ,2. An imMost studies investigating the association between sociodemographic characteristics and HRQOL in patients with CHD focus on cross-sectional group comparisons -11. LongOnly few studies have focused on the association of sociodemographic variables with temporal changes in HRQOL following invasive coronary procedures. A recent observational study indicated that higher income is associated with greater improvement in physical HRQOL following invasive coronary procedures . ImproveIn the present study of patients with CHD, we aimed to (i) describe the effect of invasive coronary procedures on different domains of HRQOL at both 6 weeks and 2 years after baseline hospitalization, (ii) assess the association between sociodemographic characteristics and temporal changes in HRQOL.Baseline data in the present study were derived from the Norwegian study on outcomes research and quality improvement (RESQUA), a cross-sectional postal survey of HRQOL and the experiences of patients receiving hospital care . All patParticipants with CHD discharged from internal medicine wards were selected for a follow-up postal survey approximately 2 years later, in October and November 2000. We used information on primary and secondary diagnoses from the patient-administration systems of the hospitals, and included patients with acute coronary syndrome (ICD-9 410.xx and 411.xx) and angina pectoris (ICD-9 413.xx). Patients with chronic heart failure (ICD-9 428.xx) as the primary diagnosis were classified as angina pectoris or acute coronary syndrome dependent on their secondary diagnoses.HRQOL was measured by the Norwegian version 1.2 of the Short-Form 36 (SF-36), a widely used generic health status measure that enables comparison with normative scores ,29. The To estimate the potential impact of CHD on HRQOL, we compared the SF-36 scores from the patients in our study with normative data from the Norwegian general population . Norm scInvasive coronary procedures referred to diagnostic and therapeutic procedures, such as coronary angiography, which contributes to diagnosis of potential coronary artery disease, and when followed by angioplasty, it can contribute to a relief from chest-pain as well as improve the prognosis in high-risk patients. Procedure codes were derived from the patient-administration systems of the hospitals in 1998 . We defined invasive coronary procedures as a dichotomous variable, differentiating between patients with and without invasive coronary procedures during baseline hospitalization in 1998. The age and sex data were also derived from the administration systems in 1998. Information about the highest level of educational attainment was obtained from self-reported data in the 1998 postal survey. This variable was heavily positively skewed, and we therefore created two groups: (1) below and equal to, and (2) above the median value in our cohort. As a crude estimate of the degree of comorbidity for each patient, we used the total number of secondary diagnoses registered in the administration databases during baseline hospitalization in 1998.2-statistics or the t-test for independent samples to analyze the extent of selective attrition, and differences in the use of invasive coronary procedures across characteristics of respondents.Changes in HRQOL were only assessed in patients who had valid scores on all multi-item scales both in 1998 and 2000. We used χt-tests. As a measure of the minimally important difference in intra-individual scores, we calculated the standardized response mean, a distribution-based approach that compares temporal change by the standard deviation of change [Temporal changes in HRQOL were analyzed with paired-samples f change . Standarf change .Additionally, we applied multivariate linear regression analyses to determine the association of invasive coronary procedures and sociodemographic factors with PCS and MCS scores 2 years after baseline hospitalization. By including baseline PCS and MCS scores in the regression model, the regression coefficients of invasive coronary procedures and sociodemographic factors indicate the one unit increase in 2-year PCS and MCS scores, provided that baseline scores are held constant.All analyses were performed separately in patients with angina pectoris and acute coronary syndrome. We chose a 5% statistical significance level. The Regional Medical Research Ethics Committee, the Data Inspectorate, and the Norwegian Board of Health approved the study.A total of 1,534 patients with CHD were sent a questionnaire in 1998, and 1,059 (69%) responded. In 2000, 700 patients with valid HRQOL scores were sent a follow-up questionnaire , and 473 patients responded. After excluding 38 patients who had recently died and 9 patients with an unknown address, the adjusted response rate in the follow-up study was 72% . Among patients with valid HRQOL scores both in 1998 and 2000, women, elderly patients, and patients with higher comorbidity had fewer invasive coronary procedures during the baseline hospitalization  or percutaneous coronary intervention (N = 45). Twenty-one patients underwent coronary bypass surgery and the remaining patients (N = 4) underwent other medical procedures related to the cardiovascular system. Compared to the original cohort of patients, patients with valid HRQOL scores both in 1998 and 2000, more often had undergone invasive coronary procedures, were male, younger, and had lower comorbidity . PatienP = 0.001) and Social Functioning (P = 0.003) improved in angina pectoris patients without invasive coronary procedures, corresponding to a small effect size (Table 3). Physical Functioning (P = 0.03), Physical Role Limitations (P < 0.001), and Bodily Pain (P = 0.03) improved in patients with angina pectoris undergoing invasive coronary procedures. The change in Physical Role Limitations corresponded to a moderate effect size. A significant deterioration was found in General Health Perceptions (P = 0.04).Over the 2 years analyzed, Physical Role Limitations (P = 0.003), Social Functioning (P = 0.005), and Emotional Role Limitations (P = 0.009) significantly improved. Physical Role Limitations (P = 0.001) improved in patients with acute coronary syndrome undergoing invasive coronary procedures.In patients with acute coronary syndrome without invasive coronary procedures, Physical Role Limitations (P = 0.034 for angina pectoris and P = 0.015 for patients with acute coronary syndrome). MCS scores remained stable during the 2 years of follow-up; only patients with angina pectoris without an invasive coronary procedure experienced a small improvement in MCS scores (P = 0.007).Patients with invasive coronary procedures showed a small improvement in PCS scores . For thIn the present study, most improvement was found in the physical components of HRQOL 2 years following invasive coronary procedures. These results support the findings of Krumholz et. al.  that theWhen adjusting for baseline scores, invasive coronary procedures and sociodemographic characteristics did not explain any additional variation in PCS and MCS scores 2 years after hospitalization in patients with acute coronary syndrome. This may be due to the relatively small sample size. An alternative explanation is that patients with acute coronary syndrome exhibited higher comorbidity that could limit the effect of invasive coronary procedures on HRQOL, and accordingly, the sensitivity of the SF-36 in detecting differences .Our results demonstrated that invasive coronary procedures and sociodemographic characteristics were weakly associated with MCS scores and indicated small deviations from the population norm, which corresponds to previous findings in patients with CHD ,38. ThisOther factors may have influenced our results, for example selective attrition. In our study, the respondents to both surveys represent a survivor cohort, and hence attrition may have reduced the temporal changes in SF-36 scores and possibly lead to underestimation of the associations with invasive coronary procedures and sociodemographic factors. Moreover, the use of self-administered and postal questionnaires may have contributed to missing SF-36 items, especially in elderly subjects who are associated with a higher frequency of missing values for items used to score physical and emotional role functioning . The appAnother limitation of our study is the lack of HRQOL data before the baseline hospitalization in 1998, which prevented us from assessing the full impact of invasive coronary procedures on subsequent HRQOL and its association with sociodemographic characteristics. We also did not examine the influence of use of medical services after the baseline hospitalization. Finally, coronary patients and invasive procedures were defined by registry data from the patient-administration systems of hospitals, which might be inaccurate and mask some of the underlying clinical differences that could influence the HRQOL results.Our findings indicated that patients with angina pectoris who were younger, male, and more educated were most likely to increase their HRQOL following invasive coronary procedures. In patients hospitalized for acute coronary syndrome, temporal change in HRQOL was not associated with invasive coronary procedures and sociodemographic characteristics, possibly due to higher comorbidity. In a usual care setting the occurrence of invasive coronary procedures varies with sociodemographic characteristics ,43. The MV carried out the follow-up study, analyzed the data, and drafted the manuscript. KIP performed the baseline survey. AR participated in the design of the study. KS participated in the design and coordination of the study. All authors read and approved the final manuscript.Table 3: SF-36 multi-item and summary scales 6 weeks and 2 years after baseline hospitalization in patients with angina pectoris and acute coronary syndrome according to invasive coronary procedures (ICP). Norwegian norm data and 2-year change scoresClick here for fileB) and 95% Confidence Intervals (CI)Table 4 Predictors of Physical Component Summary (PCS) and Mental Component Summary (MCS) scores 2 years following the baseline hospitalization in patients with angina pectoris and acute coronary syndrome. Multivariate linear regression analysis; unstandardized regression coefficients (Click here for file


Endosomal Sorting Complexes Required for Transport  largely mediate sorting of plasma membrane receptors at endosomal membranes, the formation of multivesicular bodies and their release into the endosomal lumen. In addition, the human ESCRT-II has been shown to form a complex with RNA polymerase II elongation factor ELL in order to exert transcriptional control activity.Down-regulation of plasma membrane receptors via the endocytic pathway involves their monoubiquitylation, transport to endosomal membranes and eventual sorting into multi vesicular bodies (MVB) destined for lysosomal degradation. Successive assemblies of Here we report the crystal structure of Vps25 at 3.1 Å resolution. Vps25 crystallizes in a dimeric form and each monomer is composed of two winged helix domains arranged in tandem. Structural comparisons detect no conformational changes between unliganded Vps25 and Vps25 within the ESCRT-II complex composed of two Vps25 copies and one copy each of Vps22 and Vps36 [Our structural analyses present a framework for studying Vps25 interactions with ESCRT-I and ESCRT-III partners. Winged helix domain containing proteins have been implicated in nucleic acid binding and it remains to be determined whether Vps25 has a similar activity which might play a role in the proposed transcriptional control exerted by Vps25 and/or the whole ESCRT-II complex. Endosomal compartments receive membrane bound cargo from both the biosynthetic and the endocytic pathways. Receptor downregulation by endocytosis includes transport to early endosomes and either recycling or sorting into late endosomes. The latter have the morphological characteristics of multivesicular bodies (MVB)  that canvacuolar protein sorting (VPS) into the class E compartment and are recruited to endosomal membranes from the cytosol in order to assemble into three ESCRT  complexes that function in MVB formation [Gene deletion and inactivation studies in yeast have identified 17 proteins that directly affect MVB formation (yeast class E compartment) by resulting in aberrant endosomal/vacuolar morphology . All proormation -11. Receormation . Initialormation , which iormation . ESCRT-Iormation ,9. Assemormation .Two recent crystal structures of a core of the ESCRT-II complex reveal a trilobal complex, containing two copies of Vps25, one copy of Vps22 and the C-terminal region of Vps36. Each subunit is composed of two winged helix domains and an N-terminal region of Vps25 interacts with Vps22 and Vps36 ,2.ELL Associating Protein; Vps22/EAP30; Vps25/EAP20; Vps36/EAP45), associated with the RNA polymerase II elongation factor ELL in the nucleus [Although ESCRT-II is essential for the MVB pathway, since cells missing ESCRT-II components fail to localize ESCRT-III to late endosomes ,9 the co nucleus ,15. Cons nucleus . To date that are arranged in tandem  [The structure of Vps25 was solved by single wavelength anomalous diffraction (SAD) using selenomethionine-derivatized crystals. Vps25 consists of two homologous winged helix domains as detected by the program GRATH m Figure . Winged ptional) , which fptional) ,2, folloptional) . Strand Two recent crystal structures of the ESCRT-II core reveal trilobal structures with head to tail interactions of one copy of Vps25 with Vps22 and the other copy of Vps25 with Vps36 at the center. In both cases a conserved proline rich N-terminal region of Vps25  and members of the CHMP protein family  are also found to act in the nucleus as well as in the cytosol and at endosomal membranes [Ste2 is downregulated via the endosomal pathway [Clear evidence suggests that ESCRT-II recruitment is involved in MVB formation leading to plasma membrane receptor downregulation . On the embranes -23. Inteembranes  and pher pathway . Similar pathway ,25. Prot pathway . Therefo pathway  a signalE. coli BL21 codon+ cells. For purification, cell pellets from 6 liter cultures were lysed in 150 mls of buffer A ) and 0.1 mg/ml lysozyme for one hour on ice. The cell lysate was cleared by centrifugation and loaded onto a GST-sepharose (Pharmacia) column. The column was extensively washed with buffer B  and Vps25 fusion protein was eluted with buffer B containing 5 mM reduced glutathione. GST was then removed by TEV cleavage  at 4°C overnight. His-tagged GST and TEV were subsequently both removed on a Ni2+ chelating sepharose column. Vps25 was further purified on a superdex75 column (Pharmacia) in buffer C . Selenomethione-labeled Vps25 was produced using standard procedures and purified as described above.Full length yeast Vps25 DNA (gene bank #CAA89632) was cloned into expression vector pETM30  and the Vps25 GST fusion protein was expressed in Crystallization conditions for Vps25 (7 mg/ml) were first determined by screening 600 conditions using a Cartesian crystallization robot. Initial conditions were refined using the hanging drop method, and the final crystallization condition  produced rectangular- and wedge-shaped selenomethionine-labeled Vps25 crystals in the same drop. Native Vps25 crystallized initially only with rectangular morphology and wedge-shaped crystals were produced by microseeding with the original SeMet crystals. For cryogenic data collection, the crystals were equilibrated in 25% glycerol and flash cooled in a gaseous nitrogen stream at 100 K.12121 with unit cell dimensions as indicated  beamline ID14-EH3 and data from SeMet-labeled crystals were collected at the ESRF beam line ID29 at three wavelengths . Accordingly, mol D is poorly defined (43 residues missing out of 204). The final R factor and R free (0.275/0.327) reflect missing residues and the poor model for molecule D. The model exhibits otherwise overall good stereochemistry with no outliers in the Ramachandran plot as defined in PROCHECK (table . Sequences were aligned using Clustalx [Figures were generated using coordinates of molecule C with programs MOLSCRIPT , Raster Clustalx . SecondaClustalx . The burClustalx  and the Clustalx .W.W. conceived of the study, and participated in its design, coordination and writing of the manuscript. W.W. expressed, purified and established initial crystallization conditions and participated in data collection. A.K.W. carried out data collection, structure solution and refinement and participated in writing of the manuscript. All authors read and approved the final manuscript.


In addition, the cranial muscles that normally attach to the affected cartilage region and their associated nerve are secondarily reduced in −integrinα5 animals. Earlier in development, integrinα5 mutants also have specific defects in the formation of the first pouch, an outpocketing of the pharyngeal endoderm. By fate mapping, we show that the cartilage regions that are lost in integrinα5 mutants develop from neural crest cells directly adjacent to the first pouch in wild-type animals. Furthermore, we demonstrate that Integrinα5 functions in the endoderm to control pouch formation and cartilage development. Time-lapse recordings suggest that the first pouch promotes region-specific cartilage development by regulating the local compaction and survival of skeletogenic neural crest cells. Thus, our results reveal a hierarchy of tissue interactions, at the top of which is the first endodermal pouch, which locally coordinates the development of multiple tissues in a specific region of the vertebrate face. Lastly, we discuss the implications of a mosaic assembly of the facial skeleton for the evolution of ray-finned fish.Pharyngeal endoderm is essential for and can reprogram development of the head skeleton. Here we investigate the roles of specific endodermal structures in regulating craniofacial development. We have isolated an  A developmental mutant of the zebrafish is helping to uncover the processes by which the facial skeleton is patterned in vertebrates The skeletal elements that form and support the vertebrate jaw and gills are derived from a specialized population of ectomesenchyme cells, the cranial neural crest . In the casanova mutant embryos make no endoderm and pharyngeal cartilages fail to form, and transplantation experiments show that wild-type endoderm is sufficient to rescue cartilage development (tbx1 (vgo) mutant embryos, most pouches fail to develop and posterior pharyngeal cartilages are reduced and fused together  23 between the markers Z5141 and Z20492, a region containing a zebrafish integrinα5 homolog   phenotype, HS was reduced to a rod that variably fused with CH; rarely, the first arch joint was fused as well , the CH, and the hyoid opercle bone remained unaffected. In addition, integrinα5 mutants had variably reduced numbers of ceratobranchial (CB) cartilages and rare fusions of adjacent CBs  B. In othn to aHM C. In wha as well D. Howeveintegrinα5 mutants  transgenic line  and persists as pharyngeal cartilages and bones develop. Pouches are evident as nonexpressing regions between the GFP-labeled crest-derived cells of the arches. We sorted live −integrinα5 animals for first pouch defects at 36 hpf and then grew these animals to 4 d to analyze pharyngeal cartilages. Strikingly, we found a strong correlation between reductions of the first pouch and later hyoid cartilage defects in integrinα5 mutants. −integrinα5 sides that lacked the first pouch early had hyoid cartilage defects 93% of the time (n = 116), whereas only 7% of −integrinα5 sides with a normal first pouch early developed hyoid cartilage defects (n = 108). The correlation was highly significant (p < 0.0001).As both the pouch and cartilage phenotypes were incompletely penetrant in nic line . fli1-GFintegrinα5 mutants, the dorsal first and second arch muscles, do and ah, were selectively reduced, whereas levator arcus palatini, ao, and lo were present but appeared closer together  cranial muscles. Second arch mesoderm gives rise to interhyal and hyohyal muscles, ventrally, and adductor hyomandibulae (ah), adductor operculi (ao), and levator operculi (lo) muscles, dorsally M. In a ftogether . The musintegrinα5 mutants had specific defects in the nerve that innervates second arch muscles. Facial motor neurons send a ventral-directed nerve (VII) that innervates dorsal second arch muscles ah and ao, passes through the foramen of HM, and subsequently branches to innervate ventral muscles interhyal and hyohyal , expressed integrinα5, whereas integrinα5 expression was no longer seen in the first pouch . In conclusion, integrinα5 expression in the pharyngeal endoderm is in a pattern that both spatially and temporally corresponds to regions of pouch formation, and expression of integrinα5 in the crest begins during premigratory stages and later becomes refined to patches of crest derivatives within the pharyngeal arches.In order to understand where Integrinα5 might be acting to control pharyngeal arch development, we examined the expression of ribution A. Matern in frog . By gasttegrinα5 B. At theecursors C. In addecursors . By 5-s tegrinα5 D, and phtegrinα5 E. From 1 placode F and 3G.sections H–3J. In st pouch H. At latst pouch K. At 38 pressing L. In addintegrinα5 expression was observed in both pharyngeal endoderm and neural crest, we used transplantation experiments to determine in which tissues Integrinα5 was required for first pouch and hyoid cartilage development   J. Thus, −integrinα5 cartilage phenotype requires a fate map of pharyngeal cartilages in wild-type animals. Here we focus on the origins of the SY, aHM, and pHM regions of HS, and a more complete mandibular and hyoid fate map will be published elsewhere. We used in vivo microelectroporation . These results are consistent with the first pouch being required for the survival of hyoid crest that contributes to aHM and SY.We next investigated whether the losses of aHM and SY regions in oid arch . At 25 h embryos B, and 6Eo 35 hpf D and 6E.o 35 hpf B and 6D,st cells B′. Inter develop E. In addintegrinα5 mutant embryos. Hyoid crest expresses Hox class 2 genes, whereas mandibular crest is Hox nonexpressing (hoxa2 in the hyoid arches of integrinα5 mutants at 36 hpf (data not shown). In 36-hpf wild-type animals, goosecoid (gsc) expression marks dorsal and ventral domains within the hyoid arch  that has variably penetrant and expressive reductions of the first pouch and hyoid aHM and SY cartilage regions. By positional mapping, allele segregation, and morpholino phenocopy, we identified the genetic basis of the lesion as a missense mutation in the ligand-binding domain of Integrinα5. As similar pharyngeal phenotypes were observed in mutant and morpholino-treated animals, we conclude that b926 is a loss-of-function allele of integrinα5. However, we do not know if Integrinα5 activity is completely eliminated in b926. In addition, we observed strong maternal expression of integrinα5 that could mitigate the zygotic loss of integrinα5 in b926. Thus, the variable penetrance and expressivity of the integrinα5 phenotype could be due to partial activity of mutant Integrinα5 or the presence of maternally supplied Integrinα5. Additionally, other integrins may act redundantly with Integrinα5 in pharyngeal development. A survey of nearly finished genome sequence (http://www.ensembl.org/Danio_rerio/) has uncovered at least 15 additional Integrin α chains, for which no expression or phenotypic data are known in zebrafish. Lastly, integrinα5 is expressed strongly in many tissues, such as the otic placode, for which no overt phenotypes were observed in b926. Future studies, in particular those using animals in which both maternal and zygotic integrinα5 have been eliminated, may reveal new functions of Integrinα5 in zebrafish development.In this work, we isolated and characterized a zebrafish mutant allele −integrinα5 animals to show that early first pouch defects are strongly predictive of later hyoid cartilage defects. Furthermore, transplantation experiments show that wild-type endoderm, but not crest, rescues first pouch and hyoid cartilage development in integrinα5 mutants. We infer that Integrinα5 functions in the pharyngeal endoderm for the formation of the first pouch, and that the first pouch, in turn, interacts with postmigratory neural crest to promote cartilage development in a region of the hyoid arch. A role for the first endodermal pouch in promoting regional hyoid cartilage development is consistent with work in chicken showing that domains of pharyngeal endoderm specify region-specific cartilage shapes . Although more embryological studies of Polypterus need to be done, the evolution of the HM plate in ray-finned fishes such as teleosts appears to have involved a new induction event that led to a new region, pHM, being added to an older region, aHM. We propose that the de novo addition of regions to the skeletal pattern represents another mechanism, in addition to differential growth, of generating skeletal diversity during evolution.Our data show that the first endodermal pouch is required for the development of aHM, yet in mutants that lack all pharyngeal endoderm, such as are lost . Thus, o(Danio rerio) raised at 28.5 °C were staged as previously described  . A morpholino  was designed against the exon13-intron splice site of integrinα5 (itga5-MO) with the following sequence: ATGCTTTCTCACCTGGGTAGCCATT. Embryos were pressure-injected with 5 nl of 2 mg/ml itga5-MO as previously described  and goat anti-rabbit Alexa Fluor 568 antibodies . GFP fluorescence survived the procedure.Alcian Green staining was performed as described . For flaintegrinα5 RNA probes were made from plasmids pINT4150 and pINT4853, constructed by inserting RT-PCR fragments corresponding to nucleotides 254–2,207 and 1,679–2,960 of the integrinα5 cDNA, respectively, into the TA vector . Plasmids were linearized with BamHI, and T7 RNA polymerase was used for probe synthesis. Both probes gave identical expression patterns, and pINT4853 was used for photographs. pea3  for 1 min to allow passage of the microelectrode through the skin. Agar mounting of embryos on slides was performed as described in The microelectroporation technique was similar to that described by fli1-GFP-expressing hyoid arch was assessed using confocal microscopy. Only embryos with one or two adjacently labeled cells were used in the analysis. Three-dimensional projections were constructed to determine the position of labeled cells in the arch relative to landmarks. All cell distances were made from the mid point of the cell to the landmark. Distance of the labeled cell from the edge of the first pouch was used to determine A-P position. Similarly, distances of labeled cells from the dorsal and lateral edges of the arch were used to determine D-V and mediolateral positions. In electroporations where two adjacent cells were labeled, the positions of each cell were measured and averaged. To control for variation in arch dimensions among individuals, measurements along the three axes were normalized to total axes lengths. At 4 d, embryos were imaged again to determine the fate of labeled cells in hyoid cartilage. The aHM region was defined as the anterior portion of HM that is characteristically lost in integrinα5 mutants. pHM comprises the rest of the HM region. The SY region begins at the point of attachment to pHM. All graphing and statistical analysis were done using JMP .Shortly after electroporation, the location of labeled cells relative to the Embryos were manually dechorionated, anesthetized with tricane solution, transferred to 0.2% agarose in embryo media with 10 mM HEPES and tricane, and then mounted onto a drop of 3% methylcellulose on a rectangular coverslip with three superglued #1 square coverslips on each side. A ring of vacuum grease was added around the embryo to make an airtight seal upon addition of the top coverslip. A heated stage kept the embryos at 28.5 °C. Approximately 80-μm Z-stacks at 2-μm intervals were captured every 10 min using a Zeiss LSM 5 Pascal confocal fluorescence microscope. Movies of individual Z-sections were made by manually following cells and concatenating sections; further processing was done with Adobe Premiere. For single time point confocal sections, embryos were mounted without vacuum grease.fli1-GFP host embryos. For the crest transplants, donor fli1-GFP embryos were injected at the one-cell stage with the Alexa Fluor 568 mixture (“Alexa568”). At shield stage (ca. 6 hpf), donor tissue was taken from the animal cap and moved to a position approximately two germ ring widths from the margin and 90° from dorsal in a fli1-GFP host embryo. Confocal images of host embryos were captured at 38 hpf and 4 d, and embryos were subsequently fixed and Alcian-stained to visualize cartilages. For endoderm transplants, only embryos in which donor tissue contributed to the second and at least one other pouch were included in the analysis. For crest transplants, only embryos in which at least half the hyoid arch was composed of donor tissue were included in the analysis.Transplant techniques were as described . For endVideo S1fli1-GFP animal from 38 hpf to 86 hpf. At the beginning of the video, the mandibular (1) and hyoid (2) arches are numbered and an arrow denotes the normal position of the first pouch. At the end of the video .Click here for additional data file.Video S2fli1-GFP animal from 38 hpf to 86 hpf. This video is a different depth of the same time-lapse recording as Confocal time-lapse recording shows hyoid cartilage development in a wild-type (7.1 mB MOV).Click here for additional data file.Video S3−; fli1integrinα5-GFP animal from 38 hpf to 86 hpf. At the beginning of the video, the mandibular (1) and hyoid (2) arches are numbered and an arrow denotes the normal position of the first pouch. At the end of the video .Click here for additional data file.http://www.ncbi.nlm.nih.gov) accession number for the zebrafish integrinα5 cDNA is AY550244. Accession numbers for the related Integrinα5 proteins described in Homo sapiens (Genbank P08648), Mus musculus (Genbank P11688), Xenopus laevis (Genbank Q06274), and Fugu rubripes .The Genbank (


Alopecia areata (AA) is a common and chronic skin disease with an unknown etiology. It may significantly affect the patient quality of life. This study was designed to evaluate the illness perception in patients with AA.A questionnaire consisting of 25 questions about causes, timeline, consequences and control of disease were given to 80 patients with AA attending a skin clinic in Tehran, Iran. The impact of age, gender, duration of disease, education, extent of disease and family history of AA were also assessed.Eighty patients  with a mean age of 27.5 years (SD = 9.3) and disease duration of 7.8 years (SD = 7.7) completed the questionnaire. 76.9% of the patients believed that the role of stress was the cause of disease. 17.1 % believed genetic background to be the main cause, this found to be more frequent in patients with positive family history of AA. More than half of patients believed that their illness had major consequences on their lives and 40% of patients believed that their illness would be likely to be permanent rather than temporary, more in patients with longer duration of disease. Only 57.5% of patients considered their treatments to be effective.AA may considerably affect various aspects of patients' lives. The patient knowledge about the causes and course of this disease is limited. Alopecia areata (AA) is manifested as a sudden loss of hairs without any inflammation or scarring. The hair loss might be seen in a circumscribed area or the whole scalp  or whole body  . It is aThe onset of a chronic condition brings with it a range of difficulties that may show considerable variation in their nature and severity as perceived by the patient. In order to make sense of and respond to the difficulties that chronic illness may present, patients construct their own common-sense cognitive model of their condition. Such models are based upon information received from a range of sources including their physician, family, friends, and existing social and cultural notions about health and illness. The resulting system of beliefs can of course be flawed or inaccurate; however there is evidence that it is those beliefs that drive attempts to cope with a condition and issues of compliance with treatment. Patient-held beliefs have important implications for the clinical management of their disease. Studies on patients with psoriasis and acne vulgaris have shown that knowledge of patients about their condition, the course of their disease and current treatments is not appropriate ,7.In this study, we examined the system of beliefs held by AA patients and the factors that might influence such beliefs.The Illness Perception Questionnaire (IPQ)  with a fCause subscale (10 items) measures personal ideas about the cause of AA.Time line (3 items) deals with perceptions about how long the disease will last.Consequences (6 items) are concerned with expected effects and outcomes of the illness.Cure/Control (6 items) details beliefs about recovery from or control of the condition.There were four possible answers for each item in the IPQ to be chosen by patients: I strongly agree, I agree, I do not know, I disagree.Furthermore, some demographic information such as age, sex, family history of AA, and duration and extent of disease , and level of education were obtained from the patients to evaluate their influence on patients' beliefs.Statistical analysis was conducted by means of SPSS statistical software, version 11.0. Because the data were not normally distributed, nonparametric statistics were used. Correlations were processed by Spearman's rank correlation, and differences between means were computed by means of the Mann-Whitney U test. For simplicity of analysis and increasing the power of study, the answers of "I strongly agree" and "I agree" were grouped together and compared with the answers of "I don't know" and "I disagree" which were grouped together as "I do not agree". A p value of less than 0.05 was considered as significant.A total of 80 patients with AA , with a mean age of 27.5 years  were recruited to the study. The mean duration of illness was 7.8 years . In 75% of patients, AA was patchy and it was totalis or universalis in 25%. Fifteen percent of patients had a positive family history of AA in their first degree relatives. Physicians were the main source of patients' information about their disease in 66.2% of them.cause item. A total of 76.9% of patients believed that stress was a major factor in onset of their illness and older patients were more likely to believe in this (p < 0.05). Patients who had a belief that their disease was a result of genetic factor were more likely to have a family history of AA and longer duration of the disease (P < 0.05). Younger patients and those with extensive disease (AT/AU) believed that their illness was because of chance or fate (P < 0.05).Table. consequence item.Majority of the patients 58.2%) believed that their illness had a major consequence on their lives, 53.8% of patients also felt that AA had strongly affected their self-esteem, and 50.6% considered AA as a serious condition. These believes were stronger in younger patients, and in patients who had the disease for a long time (p < 0.05). Table % believeHalf of the patients believed whether their disease cleared, it would always come back and forty percent of patients believed that their illness would be likely to be permanent rather than temporary. They were more likely to have a longer duration of disease (p < 0.05). The minority of patients (25.0%) believed that their illness would last a short time.More than 60% of patients believed that their behavior could determine improvement or worsening of their illness table . This beAlopecia areata is a chronic disease which may influence individual or social aspects of patients' lives. The results of our study confirmed this fact as the majority of patients believed that their illness had strongly affected their lives. It also influenced their self-esteem. The results of studies in other chronic diseases with periods of remission and exacerbation have had different results in this respect. For example in a study on acne patients, the disease had affected patients' self-image in nearly all of them, but it had no impact on interpersonal relationships, work, or school activities in majority of patients . On the The present study also investigated cognitive appraisals held by patients about their illness and showed that such beliefs were not associated in any significant manner with the extent of their condition. Fortune et al also did not find an association between the clinical severity of psoriasis and beliefs held by patients about their condition . Thus thThe results of our study also showed that the beliefs about the consequences of having AA were not influenced by the gender of the patients. Thus men are as vulnerable as women in suffering from the consequences of AA.et al. showed that emotional stress is not an important factor in the initiation of AA [et al. did not find a significant role of stress in the onset of AA but stressful life events had an important role in triggering of some episodes of disease [et al. found that AA patients who were depressed, were more likely to mention stress as the cause of their disease [Patients with AA, including 77% of patients in this study, often attribute the onset of their disease to a specific stressful life event. In a study on 178 patients, Van der Steen on of AA . Brajac  disease . On the  disease .In recent studies, psychologic and psychopathologic factors have been analyzed as modulators of neuroendocrinologic, vascular, and immunologic variables; this is far from the initial concept of stress being the causal agent in the illness. In fact, stress may cause its effect by making alterations in immune responses related to neuropeptides, such as the migration of the macrophages, vasodilator or vasoconstrictor responses, phagocytosis, lymphocytic cellular immunity, and expression of some factors of leukocytic adhesion to the microvascular endothelium . In addiThis study also showed that as AA lasts, the patients feel more hopeless about time line and treatment modalities of their disease. The majority of patients had no hope to get rid of their disease. Almost half of patients expected their disease to relapse after it disappeared. Such perpetual stresses are hardly endurable. The psychiatrists' intervention may alleviate patients' stress and improve their quality of life.This study was performed in a private dermatology clinic. The possibility of socioeconomic homogeneity among recruited patients may be biasing the results. So it should be considered that these results may be different in patients with AA in different socioeconomic and cultural backgrounds.There is a need for accessible, accurate, community-based education on the natural history of AA, the effectiveness and expected duration of treatment. The inadequacy of information provided by current sources is evident in ongoing misconceptions on causality and the perceptions of respondents. Incorporating information on this disease may facilitate patient into therapeutic selection, enhance understanding of treatment options and improve patient compliance.The author(s) declare that they have no competing interests.AF participated in the design and conduct of the study and preparation of the manuscript.MRF participated in the conduct of the study and statistical analysis.BG participated in the conduct of the study and statistical analysis.YD participated in the design of the study and preparation of the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Imagination and creativity are essential traits that medicine, and medical insurers, must again learn to recognize and reward Galvanized by rising costs, increased calls for greater, accountability, and an Institute of Medicine  report suggesting that medical errors may kill nearly 100,000 Americans every year The problem is, most of these reform efforts, while critically important, only capture half the picture. Efficiency isn't everything, and unless we learn to cultivate creativity as avidly as we pursue consistency, future generations of patients may find themselves stuck with the same basic treatments they're receiving today. It will be the same medicine, just served quickly.From its earliest days, medical training was based on an apprenticeship model, in which junior acolytes learned the art from senior practitioners. Even with the evolution of modern medical schools, which offered future physicians a rigorous common training, once doctors entered the real world they essentially did as they pleased. Consequently, there were pronounced differences in approaches to common problems from one clinician to another.There was also little to guarantee that once doctors had hung out their shingle, they were actually competent (and remained competent) to practice their craft. While most physicians remained committed to the general professional standard—do the best that you can for each individual patient— many well-meaning doctors ultimately were not delivering their patients the best care available.More recently, and largely due to the contagious spread of the so-called “business model,” there has been an increased emphasis on the consistency and quality of care. The clear goal is ensuring that all patients truly receive the very best care available, as defined by rigorous scientific studies.This discrepancy between what patients should be receiving and what patients are actually receiving is the major focus of quality reform, and reflects the new recognition that there are truly preferred approaches—pathways—to guide disease management. These pathways are not meant to represent a rigid algorithm reflexively applied to each patient, but are intended as a summary of the best available data, a useful template to guide further medical decisions.The renewed emphasis on quality has also resulted in a newfound appreciation for the role of experience and repetition in patient care. Study after study has shown that the best physician to treat a particular problem is the one who has treated it the most The great paradox here is that the same reforms that are improving our current care may also be endangering our future health. As medicine has become more standardized and increasingly regulated, it turns out there is much less room for innovation. The spirited pursuit of the unknown—so long a defining quality of medicine—now seems seriously endangered. The new world of rapid throughput and endless documentation provides little time to reflect upon important clinical problems and consider fresh approaches. If anything, thinking about a patient or a question too much is now implicitly discouraged because it slows doctors down; contemplation is bad for productivity.Academic medical centers like our own have played a particularly important role in the history of medical discovery; the hallmark of these institutions is our commitment to thinking and reflecting about the patients we see, patients who are often extremely sick and whose management is exceptionally complex. Unfortunately, many of the measurements now used by insurance companies to assess quality pay little attention—if any at all—to the complexity of a patient's illness, or to the importance of spending time trying to define the underlying malady. Insurance companies' major concern seems to be how fast a patient is “processed,” ideally with as few tests as possible. These measures provide no mechanism for distinguishing between the addled physician who inappropriately orders every test that springs to mind, and the reflective physician who is trying to get to the bottom of a patient's complaint, rather than simply throw a Band-Aid over the symptoms Situated on the front lines, clinicians have a unique opportunity to provide new medical insights and to identify critical, unanswered questions. Classic examples include Archibald Garrod, a British physician whose desire to understand why a patient produced black urine led to the hypothesis that diseases can result from defective metabolic enzymes, and Fuller Albright, a clinical investigator at Harvard whose thoughtful approach to his patients yielded insights that revolutionized the field of endocrinology. More recently, the astute clinical observations of UCLA immunologist Michael Gottlieb resulted in the original description of the Acquired Immune Deficiency Syndrome (AIDS) in 1981 But where are these types of insights going to come from today? It seems difficult to imagine that a medical care environment characterized by staccato-quick patient visits covering an ever-increasing number of compulsory topics will support or encourage such reflection and innovation. Our failure to nourish and sustain inquisitive physicians seems particularly tragic because medicine has traditionally attracted some of our brightest and most imaginative individuals. Even at the height of the dot-com boom, for example, there were still more medical school applicants than there were spaces to train them. But if current trends continue, many of these creative minds will head elsewhere, while those who stay will risk becoming stultified by repetitious routine.www.pasteur.hms.harvard.edu) Several medical schools and a handful of foundations have recognized this emerging problem, and have initiated programs aimed at sparking curiosity in young doctors (our own school's program is called the PASTEUR initiative—see Even as we strive to improve the consistency of care—and striving is clearly a very good idea—we must continue to cultivate novelty and originality, rather than penalize it. Imagination is perhaps the most essential trait that medicine, and medical insurers, must again learn to recognize and reward. Even with the best algorithms and the brightest computers, the future of health care ultimately depends upon the creativity of the hardy men and women still entrusted with its delivery.