In the management of cutaneous melanoma, it is desirable to complete the regional lymphadenectomy during the initial surgical procedure for wide excision of biopsy site and sentinel lymph node (SLN) biopsy. In this study, we optimized and evaluated a rapid 17 minutes immunostaining protocol. The discriminatory immunostaining pattern associated with the 'MCW Melanoma Cocktail' (mixture of Melan- A, MART- 1, and tyrosinase) facilitated the feasibility of intraoperative evaluation of imprint smears of SLNs for melanoma metastases.Imprint smears of 51 lymph nodes from 25 cases (48 SLNs and 3 non-SLNs, 1 to 4 SLNs/case) of cutaneous melanoma were evaluated.Sixteen percent, 8/51 lymph nodes (28%, 7/25 cases) were positive for melanoma metastases in immunostained permanent sections with the 'MCW melanoma cocktail'. All of these melanoma metastases, except 1 SLN from 1 case, were also detected in rapidly immunostained wet-fixed and air-dried smears . The cytomorphology was superior in air-dried smears, which were rehydrated in saline and postfixed in alcoholic formalin. Wet-fixed smears frequently showed air-drying artifacts, which lead to the focal loss of immunostaining. None of the 5 SLNs from 5 cases exhibiting capsular nevi showed a false positive result with immunostained imprint smears.Melanoma metastases can be detected intraoperatively in both air-dried smears and wet-fixed smears immunostained with the MCW Melanoma cocktail. Air-dried smears rehydrated in saline and postfixed in alcoholic formalin provide superior results and many practical benefits. A rapid intraoperative evaluation of sentinel lymph nodes (SLNs) for melanoma metastases during the interval between the SLN biopsy and the wide excision of the melanoma biopsy site may eliminate the need of an additional surgery for completion of regional lymphadenectomy.Air-dried imprint smears which were postfixed in alcoholic formalin following saline rehydration were optimal for immunocytochemical evaluation with the 'MCW melanoma cocktail'. The rapid evaluation of imprint smears immunostained with the 'MCW melanoma cocktail' is reliable for the intraoperative evaluation of cutaneous melanoma SLNs for melanoma metastases.The prevailing trend in the management of cutaneous melanoma supports the sentinel lymph node (SLN) biopsy as a standard of care -16, but Imprint smears of lymph nodes can be prepared rapidly. When compared to frozen sectioning, imprint smears are more desirable due to lower cost, quicker process, avoidance of tissue loss in the cryostat, prevention of freezing artifact in the tissue, and the elimination of problems associated with cryo-sectioning of fatty lymph nodes. These advantages have resulted in a preference for imprint smears over frozen sections by many investigators for the evaluation of SLNs in breast carcinoma ,29.Although relatively specific, the morphological interpretation of imprint smears alone used for the evaluation of melanoma metastases in SLNs is not very sensitive . This isAt the current time, frozen-section examination  and the morphological evaluation of imprint cytology smears are the methods available for intraoperative evaluation of SLN in cutaneous melanoma. However, these studies have demonstrated relatively low sensitivity and specificity, discouraging the practical application -27.Conventional immunomarkers such as the S-100 protein and HMB45 suffer a significant drawback because of interference by non-melanoma cells resulting in high signal to noise ratio . BecauseIn our previous study, the 'MCW Melanoma Cocktail'- a mixture of monoclonal antibodies- MART-1 {1:500}, Melan- A {1:100}, and Tyrosinase {1:50} (Table We prospectively studied 51 lymph nodes (48 SLNs and 3 non-SLNs) from 25 patients (range- 1–4 SLNs per patient, mean- 2 per patient) under an IRB approved protocol at Froedtert Memorial Lutheran Hospital / Medical College of Wisconsin, Milwaukee, WI. A standard surgical protocol was used to identify the SLN . The SLNFor the evaluation of the maximum surface area of the lymph node and most of the capsular area, the lymph nodes were transected perpendicular to the long axis as thin (not thicker than 2 mm) cross sections. Two pairs of imprint smears (one test and one negative control in each pair) were made by gently touching the cut surfaces to glass slides without allowing the imprint to be smeared. One of the pairs (1 test and 1 negative control) was air-dried, rehydrated in saline, and post-fixed in 'alcoholic formalin'  (see vid® was used in place of 'the cocktail'.The first imprint smears from each pair were used as a 'test' and were immunostained with the 'MCW melanoma cocktail' by rapid protocol. The second imprint smear was used as a 'negative control' and processed in the same manner as the test slide except that Dako diluentNumerous smears of positive controls (both air-dried and wet-fixed smears) were prepared previously from a melanoma tumor with a known immunoreactivity for each of the three components of 'the cocktail'. These smears were prepared by scraping the cut surface of the fresh melanoma tumor and spreading the scraped material between two slides as described previously . The airBoth air-dried and wet-fixed positive control smears were used during immunostaining for each batch of test smears by removing the coverslip following immersion of the slide in xylene for about 24 hours. This dissolves the mounting medium and separates the coverslip. After removal of the coverslip, the smears were put through absolute ethanol and then descending grades of ethanol to water to be combined with the respective step in the rapid immunostaining protocol . The wet-fixed test smear from a given SLN was compared with a respective air-dried test smear by evaluating the sharpness of immunostaining, morphological details of the immunostained tumor cells, frequency of staining of non-melanoma structures such as mast cells and erythrocytes, air-drying artifact, deterioration in the immunostaining of the cells with unequivocal features of tumor cells, and nonspecific background staining. The results were interpreted by pathologists as positive, indeterminate, or negative for melanoma metastases. For statistical analysis, indeterminate interpretations of immunostained imprint smears were considered negative. This was based on the clinical significance with reference to the intraoperative decision algorithm for the completion of regional lymphadenectomies in SLN positive cases.After the preparation of imprint smears, the slices of SLNs were fixed in 10% formalin and processed for formalin-fixed paraffin-embedded tissue sectioning. These sections were evaluated according to the melanoma protocol . The cells with such brown stained nuclei were morphologically consistent with histiocytes Figure  &4g. UneThe non-specific staining was relatively frequent in wet-fixed smears  and in manually immunostained smears (versus smears immunostained with Autostainer). These structures were usually interpreted as negative with ease. However, this factor could increase the interpretation time for negative cases due to the distraction effect and could prolong the crucial turn around time for intraoperative consultation.Seventeen percent (8 out of 48) lymph nodes (28%, 7/25 cases) were positive for melanoma metastases in immunostained permanent sections. All melanoma metastases, except 1 SLN from 1 case, were demonstrated in both wet-fixed and air-dried imprint smears immunostained with the rapid protocol (sensitivity 89% and specificity 100%). On a case by case basis, 86% (6/7) of positive cases showed metastases in imprint smears immunostained with the 'MCW melanoma cocktail'; and demonstrated a sensitivity of 86%, a specificity of 100%, a negative predictive value of 95%, and a positive predictive value of 100%.Imprint smears, immunostained with the rapid protocol, showed unequivocal melanoma metastases in 1 SLN which was negative by immunohistochemical evaluation of permanent sections. This was one of the SLN from a case with two other unequivocally positive SLNs in permanent sections and rapid immunostained imprint smears. This unequivocal positivity with immunostained imprint smears alone underscored the sampling benefit with imprint cytology.Two SLNs (from 2 patients) interpreted as negative for melanoma metastases by immunohistochemical evaluation of permanent sections, were interpreted as indeterminate with the rapid protocol in both wet-fixed and air-dried smears. After retrospective evaluation, the rare doubtful cells observed in immunostained imprint smears were consistent with mast cells Figure .In 2 SLNs from 2 other cases, some scattered cells with non-granular immunostaining but with small, inconspicuous nuclei were observed. These cells were not mast cells and were absent in negative controls. They were also present as scattered single cells in permanent sections immunostained with 'the cocktail'. They were interpreted as benign and negative.Metastases of melanoma tumor cells in SLN could be detected in imprint smears immunostained with the 'MCW melanoma cocktail'. The air-dried imprint smears from different SLNs were easier to prepare than wet-fixed smears. As reported previously, air-dried smears have numerous advantages . BecauseImprint smears (both air-dried and wet-fixed) immunostained with the 'MCW melanoma cocktail' showed excellent sensitivity and specificity . As compared to this, the alternative intraoperative approaches such as frozen-section alone , immunosImmunostained imprint smears unequivocally showed melanoma metastases in one SLN, but these melanoma metastases were not detected in permanent sections immunostained with 'the cocktail'. Two other SLNs from this case showed melanoma metastases in both immunostained permanent sections and imprint smears. This unequivocally positive result with immunostained imprint smears highlights the benefit of the enhanced sampling with imprint cytology.Imprint smearing facilitates the sampling of two surfaces from each slice, except for the first and the last slice, as compared to only one surface of all slices by any sectioning method Figure . In contThe possibility of false positive results due to the cells of capsular nevi was disproved by the negativity of all immunostained imprint smears from 5 SLNs (5 cases) with capsular nevi. The cells in capsular melanocytic nevi located in the capsule and fibrous septa of lymph node did not exfoliate and adhere to slides during preparation of imprint smears. This appears to be due to the greater cohesiveness of the cells in capsular melanocytic nevi than the cells of malignant melanoma.However, scattered cells with non-granular cytoplasmic brown staining which masked the small and inconspicuous nucleus were observed in 2 SLN of 2 cases. Such cells exhibiting benign morphology were also observed as scattered single cells in permanent sections immunostained with 'the cocktail'. These cells were interpreted as negative without significant challenge, but their exact nature could not be established. The possibility of singly scattered nevus cells was considered. Contrary to capsular nevus cells, such cells may be detached easily and picked up by the glass slide during the preparation of imprint smears. In some cases, they may cause an interpretation dilemma leading to indeterminate results even with permanent sections.In 2 patients, 2 SLNs were interpreted as indeterminate with immunostained imprint smears. They were interpreted as negative by the immunohistochemical evaluation of permanent sections. Cytomorphologically, the rare doubtful cells present in immunostained imprint smears were consistent with mast cells. They were also present in the respective negative controls Figure .As endogenous peroxidase activity could not be blocked completely during the short endogenous peroxidase blocking step in the rapid protocol, non-melanoma cells such as mast cells may show brown staining in some cases. Familiarity with the morphological spectrum of immunostained tumor cells Figure  and otheThe positive control smears may be prepared from time to time utilizing fresh, unfixed melanoma tumors for long term availability. Alternatively, the smears of melanoma tumor cell lines immunoreactive to individual components of the cocktail may be used after processing and fixing similar to test smears. These smears may be dehydrated and coverslipped Table . CoverslImprint smears are easy and quick to make without incurring significant expense. They are faster than frozen sectioning and help prevent the loss of tissue associated with cryosectioning. Frozen-sectioning of lymph nodes is frequently problematic because of fat, either adjacent to or in the lymph node. These problems are circumvented with imprint smears, which would also prevent problems associated with the interpretation of final permanent sections of frozen tissue.For billing and reimbursement purpose, the rapid intraoperative evaluation of immunostained imprint smears may be coded with existing CPT  codes- 88329 for the intraoperative consultation, 88161 for the preparation and processing of the imprint smears, and 88342 for the immunostaining of the imprint smears with interpretation .As a future prospect, a 'cocktail' of directly conjugated individual antibodies (with a peroxidase or similar indicator system) used for a one step immunostaining method resulting in a significant reduction in immunostaining time (up to 6 minutes) with fewer staining steps would simplify the procedure . As discIn summary, air-dried imprint smears which were postfixed in alcoholic formalin following saline rehydration were optimal for immunocytochemical evaluation with the 'MCW melanoma cocktail'. Wet-fixed smears did not compromise the immunoreactivity of 'the cocktail', but they were difficult to prepare without air drying artifact and non-specific background staining. Capsular melanocytic nevi did not show false positive results. The rapid evaluation of imprint smears immunostained with the 'MCW melanoma cocktail' is reliable for the intraoperative evaluation of cutaneous melanoma SLNs for melanoma metastases.ABC, avidin-biotin-peroxidase complex; DAB, Diaminobenzidine Hydrochloride; H&E, hematoxylin and eosin; MCW, Medical College of Wisconsin; SLNs, Sentinel Lymph Nodes.None.VS conceived, designed, carried out the entire study in addition to the standardization of MCW melanoma cocktail protocol and preparation of manuscript. RK participated in its design and coordination. GD performed the immunohistochemical staining including standardization of the MCW melanoma cocktail with VS and SK. WD organized the clinical participation including recruiting of cases and arranging patients consent. All authors read and approved the final manuscript.Higher resolution - (for high speed connection)Click here for fileLower resolution - (for low speed connection)Click here for fileScreen shotsClick here for file


Congenital heart defects are the leading non-infectious cause of death in children. Genetic studies in the mouse have been crucial to uncover new genes and signaling pathways associated with heart development and congenital heart disease. The identification of murine models of congenital cardiac malformations in high-throughput mutagenesis screens and in gene-targeted models is hindered by the opacity of the mouse embryo.Ptdsr) deficient embryos. These included ventricular septal defects, double-outlet right ventricle, and hypoplasia of the pulmonary artery and thymus. These results indicate that Ptdsr plays a key role in cardiac development.We developed and optimized a novel method for high-throughput multi-embryo magnetic resonance imaging (MRI). Using this approach we identified cardiac malformations in phosphatidylserine receptor (Our novel multi-embryo MRI technique enables high-throughput identification of murine models for human congenital cardiopulmonary malformations at high spatial resolution. The technique can be easily adapted for mouse mutagenesis screens and, thus provides an important new tool for identifying new mouse models for human congenital heart diseases. Congenital malformations are a major cause of death in childhood, and are typically characterized by lesions that do not compromise fetal survival. For instance, congenital heart disease (CHD) typically consists of lesions such as ventricular and atrial septal defects, which are compatible with fetal hemodynamics . AlthougN-ethyl-N-nitrosourea (ENU) mutagenesis [The mouse is a particularly good model for studying mechanisms of cardiac diseases as its anatomy and development resembles that of the human more closely than any other genetically tractable organism. Importantly, the mouse is amenable to genotype-driven approaches such as transgenic knockouts of defined candidate genes , genome agenesis ,9. Howevagenesis . Currentagenesis -13. To magenesis -14. The Ptdsr) in controlling ventricular septal, outflow tract and pulmonary artery development. In addition, we found thymus hypoplasia in Ptdsr-deficient embryos. These findings suggest that a novel Ptdsr-mediated pathway is required for cardiac and thymus development.We previously reported the development of fast gradient-echo MRI of single mouse embryos -17. ThisWe modified our previously described fast gradient echo magnetic resonance imaging technique -17 to imCited2 deficiency [Cited2 (Cited2-/-) have diverse cardiac malformations, including atrial and ventricular septal defects, outflow tract and aortic arch malformations, and adrenal agenesis [Trp53-repressor gene Cdkn2aP19ARF is a target of Cited2 [Cited2 and Trp53 to determine if this would rescue the heart and adrenal defects in Cited2-/- mice. We imaged 50 embryos using the multi-embryo technique in the 16-embryo mode. Embryonal genotypes included 12 wild-type, 13 Cited2+/-, 14 Cited2-/-, three Cited2-/-: Trp53-/-, four Cited2-/-: Trp53+/-, two Trp53+/-, and two Trp53-/-. We analyzed the data from each embryo for cardiac malformations without knowledge of the genotype. This typically took a maximum of 30 minutes per embryo. We scored each embryo for atrial and ventricular septal defects (ASD, VSD), outflow tract , and aortic arch malformations , and for adrenal agenesis. Each embryo was then re-imaged singly at high resolution, and the data re-analyzed as before. In this group we identified 20 embryos with ASD, 19 with VSD, 18 with outflow tract defects, 11 with aortic arch defects, and 21 with bilateral adrenal agenesis, using high-resolution single embryo imaging. In comparison to single embryo imaging, the overall sensitivity and specificity of multi-embryo imaging for cardiac malformations was 88% and 92% respectively. For ASD (18 identified by single embryo imaging) the sensitivity and specificity was 85% and 95%; for VSD 94% and 94%; for outflow tract malformations 94% and 100%; and for aortic arch malformations 91% and 100% respectively  on a C57BL/6J background, by gene targeting in embryonic stem cells [Ptdsr function in knockout mice causes perinatal lethality, growth retardation [Ptdsr-/- embryos develop complex ocular lesions [Ptdsr mutants, none of those detected could explain the observed perinatal lethality of Ptdsr-/- mice. In the process of phenotypical characterization of our Ptdsr-deficient mouse line, we frequently observed subcutaneous edema of varying sizes in Ptdsr-/- embryos by gross inspection , which is identical to the initially analyzed Ptdsr mutant except that the loxP-flanked neomycin selection cassette was removed by breeding the original Ptdsrtm1 Gbf knockout line [CMV-Cre deleter mouse line [Ptdsrtm1.1 Gbf knockout mouse line we analyzed 8 Ptdsr-/- embryos, and as littermate controls, 3 Ptdsr+/+ and 1 Ptdsr+/- embryos. We found ventricular septal defects in five out of the eight Ptdsr-/- embryos (data not shown). Again we found no evidence for cardiac malformations in wild-type or heterozygous littermate control embryos.We next evaluated the role of multi-embryo MRI in analyzing unexplained lethality in embryos generated in collaborating laboratories. Recently, we have generated mice lacking the em cells . Ptdsr iem cells -24. Ablaardation ,22,24 anardation . In addi lesions  as well pection  would require the analysis of ~1200 – 2400 embryos per year. Our results show that multi-embryo MRI is eminently suitable for such throughput. Although, screening of 32 embryos overnight requires typically about 16 hours of data analysis, multi-embryo MRI mutagenesis screens can be easily performed at a reasonable scale if multiple operators analyze the data in parallel. As the data are permanently stored on DVDs, and can be analyzed easily by commercially available software, they can be without difficulty disseminated to specialists for further and more detailed analysis. Another powerful application of multi-embryo MRI will likely be the investigation and screening of potentially teratogenic drugs.MRI screens performed at late gestation would be expected to identify genes that affect later aspects of development, and identify hypomorphic and haploinsufficient alleles of genes that affect earlier steps of development. Published data from genome-wide ENU mutagenesis screens in the mouse indicate that ~30% progeny carry a heritable recessive phenotype, making 3-generation recessive screens the method of choice for identifying developmental malformations . At leasPtdsr-mediated pathway is required for cardiac and thymus development. Recently, we have demonstrated that in contrast to previously reported hypothetical Ptdsr functions, the Ptdsr protein is not required for the clearance of apoptotic cells [Ptdsr+/+ and Ptdsr-/- embryos during heart development did not reveal any difference in the number and location of apoptotic cells between the genotypes (J.B., A.D.G. and A.L. unpublished observations). This further excludes that Ptdsr has any function in apoptotic cell clearance and points to other developmental mechanisms that are affected by Ptdsr ablation. The neural crest plays an important role in the development of the cardiac outflow tract, aortic arches, and the thymus [Ptdsr-deficient embryos lack intestinal ganglia [Ptdsr-/- mice may have an underlying neural crest defect. Importantly, dysfunction of these Ptdsr-mediated pathways during development could also potentially result in heart defects in humans.Our results presented here indicate a new, and hitherto unsuspected role for the phosphatidylserine receptor in controlling ventricular septal, outflow tract, pulmonary artery, and thymus development. This finding suggests that a novel ic cells . Moreovee thymus . As Ptds ganglia  which arPtdsr is essential for normal cardiac development. Further experiments are needed to define exactly in which pathways Ptdsr is involved during heart development. We expect that multi-embryo MRI will be an important technology for future phenotype-driven mouse mutagenesis screens. The technology can be easily implemented at standard MRI imaging centers, thus allowing by collaboration with individual researchers or mouse mutagenesis centers, a high-throughput functional genetic dissection of mechanisms underlying cardiac development and congenital heart diseases.Our results validate the utility of multi-embryo MRI for high-throughput identification of murine models for human congenital cardiac malformations, and using this technique we have shown that Cited2-/- [Trp53-/- [Ptdsr-/- mice [ited2-/- , Trp53-/Trp53-/- , and Ptd-/- mice  have beeEmbryos were fixed in 4% paraformaldehyde at 4°C for ~1 week, and then embedded in 1% agarose (Seakem) containing 2 mM gadolinium-diethylenetriamine pentaacetic anhydride (Gd-DTPA, Magnevist, Schering UK) in 28 mm nuclear magnetic resonance tubes  at a field of view of 26 × 26 × 30 mm achieved an experimental resolution of 51 × 51 × 39 μm when imaging up to 16 specimens. In case of 32 embryos, a matrix size of 608 × 608 × 1408 at field of view of 26 × 26 × 50 mm, yielded an experimental resolution of 43 × 43 × 36 μm. The total experimental time was ~8.75 hours for 16 embryos, and ~12.3 hours for 32 embryos  whereby each phase encoding step was averaged four times.Single embryo imaging was performed as described previously -17. For t-test in Microsoft Excel.The raw MR data were reconstructed into a stack of 1024 for 16 embryos), or 2048 (for 32 embryos) 2D TIFF files  using purpose-written software as described previously [ embryos,Embryos were dehydrated in ethanol, embedded in paraffin wax, and sections were stained with hematoxylin and eosin.Ptdsr was detected by staining the embryos overnight in X-Gal according to standard protocols. The embryos were postfixed in 4% paraformaldehyde and processed for documentation or histology.Embryos were dissected free of extraembryonic membranes and then fixed in 4% paraformaldehyde at 4°C. Expression of Ptdsr knockout lines and harvested embryos for MRI and histopathological analysis, S.D.B developed the sample preparation for embryonic MRI, A.D.G. carried out the histopathological analysis of Ptdsr-/- mutants, C.B. prepared the embryos for MRI, K.C. and S.N. assisted the experimental development, S.B. analyzed the MRI and histopathological data, A.L. and S.B. were responsible for the co-ordination of the study and the drafting of the paper.J.E.S. developed the multi-embryo MRI technique, J.B. generated both


Low birth weight (LBW) is a public health problem linked to lack of equity in populations. Despite efforts to decrease the proportion of newborns with LBW, success has been quite limited. In recent years, studies focused on explaining how social factors influence this problem have shown that populations with greater inequities have a greater proportion of newborns with LBW.The objective was to describe socioeconomic factors related to LBW adjusted by demographic, reproductive and health services variables in Mexico City. A case-control study was carried out in three hospitals with gynaecological and obstetrics services in Mexico City during the first half of 1996. During the recruiting period all children with LBW (cases), defined as newborns weighing <2500 grams, were matched with children born on the same day to control for time of birth. Upon arrival at the hospital for delivery, women were interviewed to determine if they met our inclusion criteria. Women with a history of chronic conditions and those with twin or multiple pregnancies were excluded. Variables with clinical and statistical significance were included in a multivariate model (logistic regression).We found that low socioeconomic level was the most important risk factor for LBW and was independent of other factors, including those related to reproduction and nutrition, smoking, morbidity during pregnancy, accessibility to health services and prenatal care .We found that socioeconomic factors are relevant to LBW. However further research should be done in different population groups as well as developing precise ways of measuring socioeconomic factors and their role in reproductive health. On average, the worldwide incidence of low birthweight (LBW) is 17% per year, making LBW an important infant health problem in many populations . The LBWIn addition to its impact on infant mortality, LBW has been associated with higher probabilities of infection, malnutrition and handicapping conditions during childhood, including cerebral palsy, mental deficiencies and problems related to behavior and learning during childhood -6. Childin utero are related to the mother's physiology, including her nutrition (mother's weight before pregnancy and history of having newborns with LBW), exercise, infections, and consumption of tobacco, alcohol and other drugs [The biological processes that affect the fetus er drugs ,11.During the fetal phase, growth depends on the nutritional condition of the mother, indicating that pregnant women should not only increase their weight but also consume essential nutrients. For many women in the developing world, however, economic, social and cultural factors make it difficult for them to obtain the necessary food and healthcare, which are closely interrelated .Associations between poorer child health and poverty, inequity and social exclusion have been documented worldwide and have been shown to be independent of research methods, local culture, and available health care services . While tAmong the socio-economic factors are income, education, occupation, household leadership and gender differences related to roles within the family ,18. In MSeveral studies have shown different results on whether socioeconomic factors affect pregnancy outcomes and newborn conditions -21. The In this study, we have analyzed the socio-economic factors related to birthweight adjusted by others known factors in the urban population of Mexico in three hospitals located in three different geographic areas of Mexico City.A case-control study was carried out in three hospitals with gynaecological and obstetrics services in Mexico City during the first semester of 1996. We recruited 154 LBW newborns, defined as newborns weighing <2500 grams, from these three hospitals, as well as 474 controls chosen from births on the same day to control for time of birth. All newborns included in this study provided a statistical power of 80% when we assumed a = 0.05 (one-sided test), a case/control ratio 1:3, and a 3% difference among both groups in the low socioeconomic level .Upon arrival at the hospital for delivery, women were interviewed to determine if they met our inclusion criteria. Women with a history of previous chronic conditions or those with twin or multiple pregnancies were not included. Following delivery, and after obtaining informed consent, each woman was administered a questionnaire by a trained interviewer to obtain information about socio-economic, reproductive, and nutritional factors. Socioeconomic factors included age, level of education, civil status, occupation, income and owning certain goods; reproductive factors included parity, history of preterm delivery and LBW, which were classified as positive or negative for all previous pregnancies; and nutritional factors included calcium and iron supplementation, pre-gestational weight, prenatal care, morbidity during pregnancy and tobacco exposure. Clinical records were also reviewed to verify information about each newborn.To create a socioeconomic level index, we used two variables that have been considered as proxies . OwnershStatistical analysis was performed by describing sociodemographic, reproductive and prenatal care characteristics of the mother and newborn. Univariate analysis was used to evaluate the association between the independent variable (socioeconomic level) and covariates with LBW (outcome variable). Stratified Mantel-Haenszel analysis was performed to evaluate confounding and/or interaction (i.e. parity and age). To obtain the association magnitude of the socioeconomic level adjusted by the covariates, multivariate analysis was performed (logistic regression). Variables with clinical and statistical significance were included in multivariate modeling. We used three models to classify factors relevant to LBW into three groups as known, controversial and unknown risk factors for LBW.Model 1 included socioeconomic level index and maternal age, maternal education, marital status and accessibility to public services. In model 2 we included all variables of the model 1 besides, prenatal care, tobacco exposure, and morbidity during pregnancy were considered as adjusted variables. Finally, model 3 included all variables previously mentioned . However we thought that it will be a non differential error. Although we analyzed other proxies of socioeconomic level, we found that each of them was irrelevant. For example, although education has been used as a proxy of social status, we found that this indicator was not important in our population, perhaps because the women in our study were incorporated into the work force, making exposure to intermediate factors, such as occupational stress and load work, an influence on pregnancy outcomes. Family circumstances and biological processes may also be affected by a wider social context, including cultural and historic issues such as educational opportunities, parent's divorce, unemployment, risk of poverty risk, and risks factors for smoking and obesity.Although many socioeconomic factors related to LBW have been identified, the specific role of each of them is not known, limiting the ability to use preventive actions in exposed populations. Interventions aimed at reducing the number of LBW infants have had limited success on conditions of the newborn, although some showed benefits in pregnant women . To decrHeterogeneity among different populations makes findings related to interventions in one population not applicable to others. Thus, it is necessary to design studies that account for the geographic, racial, cultural, social and economic context of each country and specific group. In the case of Mexico, we believe that our results provide information representing a closer approach to the effect of socioeconomic status on LBW. Our results provide a starting point in the search for better indicators for evaluating socioeconomic status in Mexican populations with other social conditions, including suburban, rural, and indigenous populations.Another aspect to consider within the context of each country is the availability of information. In Mexico, there is no way to obtain uniform information about the socioeconomic characteristics of the population. Information about conditions of different populations is important in designing programs aimed at solving existing inequities. It is a challenge to create a socioeconomic index that will reflect the real living conditions of pregnant women in Mexico. This effort will require including other variables, as well as refining proxy variables.The author(s) declare that they have no competing interests.LPTA, PCC, SFH contributed in the conception and design of the study and statistical analysis. JPVB, ERM reviewed for important intellectual content. All authors participated in the interpretation of data and read and approved the final version to be published.The pre-publication history for this paper can be accessed here:


The C677T MTHFR variant has been associated with the same third trimester pregnancy complications as seen in women who have elevations of maternal serum α-fetoprotein (MSAFP). We hypothesized that these women with third trimester pregnancy complications and MSAFP elevations would have an increased frequency of the variant compared to an abnormal study control group (women with MSAFP elevations without pregnancy complications) as well as to normal population controls.Women who had unexplained elevations of MSAFP in pregnancy were ascertained retrospectively. The frequency of the C677T MTHFR variant among those women with unexplained elevations of MSAFP who had experienced later pregnancy complications was compared to that of women with unexplained elevations of MSAFP without complications as well as to that of the previously established Manitoba frequency.p = 0.03). The frequency was also higher than that of the population controls (q= 0.25, OR 1.70 95% CI 1.11–2.60, p = 0.007). The frequency in women with MSAFP elevations without pregnancy complications was not significantly different from that of the population controls (p = 0.41).Women who had complications of pregnancy and an unexplained MSAFP elevation had a higher allele frequency for the C677T MTHFR variant (q = 0.36,) compared to women with MSAFP elevations and normal pregnancy outcomes (q = 0.25, OR 1.73 95% CI 1.25–2.37, Women with unexplained elevations of MSAFP and who experience complications in later pregnancy are more likely to have one or two alleles of the C677T MTHFR variant. Significant elevations of amniotic fluid and maternal serum alpha-fetoprotein (MSAFP) have been shown to be associated with spina bifida and other neural tube defects (NTD). The province of Manitoba, Canada offers province-wide midtrimester MSAFP screening to all pregnant women. It has been recognized that some pregnant women with midtrimester unexplained elevations of MSAFP ,2.Increased total plasma homocysteine alters placental function and has been associated with the same complications that are associated with unexplained elevated MSAFP -7. The Cp = 0.151, two tailed) indicating the frequency was increased but, this could have been a random result.In a small pilot study of 32 couples, we found that women who had an unexplained elevation of MSAFP and a normal midtrimester fetal ultrasound, and their partners, had a significantly increased C677T MTHFR frequency compared to Manitoba newborns (RR 1.42, 95% CI 1.08–1.85, p = 0.012, two tailed) . The newAll pregnant women in Manitoba are eligible for routine serum screening through the voluntary MMSSP. In Manitoba, an elevation of MSAFP is defined as 2.3 multiples of the median (MOM) or greater. Candidates for inclusion in this study were women with an unexplained MSAFP elevation  with either a complicated or uncomplicated pregnancy outcome. After appropriate approvals had been obtained from The University of Manitoba Health Research Ethics Board, review of the screening records began in 1999 and took three years. For a study using a two step consent to participate methodology administered by mail, the expected response rate (after excluding lost to follow-up) would be 20% . Our goaAll screening records from 1995–1999 were reviewed, accounting for 783 invitations. Records for 2000–2002 were reviewed systematically as outcome information on each pregnancy became available to MMSSP. Records for 1990–1994 were then reviewed systematically in order to bring the total up to 1000. If a woman had more than one pregnancy with an elevation of MSAFP screened by the MMSSP, only the first pregnancy encountered in the retrospective review was used for the study. Previous or subsequent pregnancies were not included. Women with preexisting conditions known to influence pregnancy outcome, such as essential hypertension, and mothers of babies with major congenital anomalies were excluded. Eight women who had relinquished their babies for adoption or whose babies were placed in foster care were also excluded.th percentile), pregnancy induced hypertension, preeclampsia, eclampsia, postpartum hemorrhage, retained placenta requiring manual delivery, abruptio placenta, premature delivery  and unexplained fetal demise. Study controls were women with normal outcomes which were defined as those with delivery at term ≥ 36 weeks gestation), no complications of pregnancy, a normal placenta and a healthy baby. Definition of complications was based on ICDC-9 codes in the MMSSP outcome charts for each patient [Women who met the inclusion criteria were divided into two groups for analysis. Cases were defined as women with pregnancies complicated by one of the complications previously shown to be associated with an unexplained elevation of MSAFP at midtrimester . These i patient  which ar patient .12 and B6, were calculated from the FFQ for intake both during pregnancy and at the time of the study. A correction of an additional 0.1 mg for folic acid fortification that began in Canada in 1998 was included for pregnancies that began after fortification [Women who agreed to participate in the study were mailed the appropriate questionnaires and blood requisitions. The questionnaire included a semi-quantitative food frequency questionnaire (FFQ) based on standard methodology but, modified to suit Manitoba residents and previously validated for this population by biochemical analysis during the pilot study ,16. The fication . FFQ anaTotal plasma homocysteine, red blood cell folate, and serum folate were determined using established methodology ,19. Sampt test for difference between means with Bonferroni correction for multiple comparisons. Data not normally distributed were analyzed using the nonparametric Mann-Whitney Rank Sum Test. Linear regression was used to test the validity of the dietary survey. A multivariate analysis included age, smoking, maternal weight at the time of MSAFP testing, presence of C677T MTHFR, gender and weight of infant, biochemical parameters, and FFQ results for folate, B12 and B6, both at the time of the survey and for during the pregnancy was undertaken. In order to avoid convergence due to the large number of variables, the analysis was completed in subsets of six variables. Variables with the higher association scores from these analyses were then combined for further testing in various combinations using stepwise multiple linear regression. Also linear regression analysis of each continuous variable with genotype results was performed. Corrections for multiple comparisons were included. Software used was NCSS Statistical Systems for Windows [Chi-squared analysis (one tailed unless otherwise noted) was used for allele frequency. Comparisons of potentially confounding factors between the case group and the study control group were undertaken. Parametric data were analyzed with the Student's  Windows .p = 0.030). There was no difference in the proportions of cases and controls that were lost to follow-up (p = 0.157). We had anticipated a 20% response rate and we achieved 24%.Nine hundred and ninety four women were identified as eligible (342 cases and 652 controls). Of the 590 women successfully contacted, 130 (22%) agreed to participate (56 cases and 74 controls). Four hundred and four women were lost to follow-up. Cases were more likely to choose to participate than controls and this difference was significant (1.5, p = 0.03). The frequency was also higher than in the population controls (q = 0.25, OR 1.70 95% CI 1.11–2.60, p = 0.007). The frequency in women without pregnancy complications and MSAFP elevations (study controls) was not significantly different than that seen in population controls (p = 0.41).Genotypes were available for 54 cases and 73 controls for this analysis. Results are summarized in Table ever had a pregnancy with an unexplained elevation of MSAFP followed by complications, the association would still be present when compared to the population control (q = 0.3636, OR 1.72, 95%CI 1.27–2.61, p = 0.0055).The case and study control groups included women at various stages of their child bearing years. Only one woman recruited as a control subject had a previous or subsequent pregnancy with an unexplained elevation of MSAFP and complications. She was a heterozygote for C677T MTHFR. No case subjects had a previous or subsequent pregnancy with an unexplained elevation of MSAFP and a normal outcome, but four case subjects had had a previous or subsequent pregnancy with complications after an elevated MSAFP. If the case versus control classification had been based on whether or not a woman had p = 0.019) for serum folate than those who did not have the variant. None of the women were deficient in either serum folate (defined as <7.0 nmol/L) or red blood cell folate (defined as <430 nmol/L RBC). There was no significant difference in mean homocysteine levels  and serum folate (r = 0.932, p = 0.0001). Higher intake of dietary folate (including synthetic folic acid from supplements) as reported by the FFQ for the time of study was associated with higher serum folate (r = 0.941, p = 0.0001) and higher red blood cell folate (r = 0.949, p = 0.0166).Seven cases and one study control declined to fill out their dietary surveys. Mean values were inserted in the multivariate analysis for these eight women. The validity of the dietary survey was demonstrated again for this study by linear regression analysis. Consistent with known homocysteine metabolism , a negatp = 0.0062) consistent with accurately reporting their smoking habits [We did several checks to determine that the women were answering their surveys accurately. Comparisons of specific data items available in the women's MMSSP charts at the time of pregnancy with the data reported in the surveys showed excellent agreement for every item examined indicating women answered questions accurately. Women who reported smoking  showed a negative correlation with serum folate (r = 0.923, g habits . Based oThe ethnicity of the infants born to the case mothers (based on the ethnicity reported for the infants grandparents) was 84% Caucasian, 5% Aboriginal. Mixed ethnicity was reported for 11% of the infants with one parent Caucasian and the other parent Aboriginal, or rarely Black or Asian. The ethnic distribution was the same for controls and is typical for the Manitoba population ,26. Ther12, or B6, or in the biochemical parameters of case and control mothers. There was no difference in the percentage of cases and controls who reported taking prenatal vitamin supplements during pregnancy (37/48 cases and 55/72) or taking vitamin and/or folate supplements preconceptionally (17/48 cases and 25/72 study controls).There were no significant differences in dietary and supplemental intake of folate, Bp = 0.013). Homozygosity for the C677T MTHFR variant is associated with IUGR in women who do not take vitamin supplements according to one large study of Canadian women [We attempted to divide our cases into smaller groups by type of pregnancy complication. We also separated isolated IUGR and IUGR associated with hypertensive disorders of pregnancy. Most of the groups lacked power for statistical analysis due to small numbers. However, normotensive women whose fetus had IUGR (N = 12) had a higher frequency of the C677T MTHFR variant compared to the population controls (q = 0.33, OR 2.58 95% CI, 1.78–3.73, an women ,27. Our p = 0.024). This was also not unexpected given that some of the complications we were examining are associated with obesity [p = 0.0462). There was no association between weight and MTHFR status (r = 0.431, p = 0.679).There was a trend towards higher mean weight for mothers who had complications at the time of MSAFP test in the individual comparisons, but this was not significant after correction for multiple comparisons Table  and 3. T obesity . Even afUnexplained elevations in MSAFP are known to be associated with an increased risk for complications of pregnancy . Others The lack of folate deficiency in this population was unexpected, given previous research which showed that 23.6% of Newfoundland and Labrador women are folate deficient at their first prenatal visit . As our As is the situation with NTDs, lack of folate deficiency by current definitions in a non-pregnant woman may not indicate that her folate intake is adequate for pregnancy. This would especially be true for women with the C677T MTHFR variant. Reexamination of the current definition of what constitutes a normal biochemical result for folate intake for women of child bearing age should be undertaken to clarify this.We suggest that the negative effects of the C677T MTHFR variant are more likely to occur in early pregnancy before women began taking prenatal vitamins because the majority of our study participants took prenatal vitamins, but only 35% took preconceptional supplements. We suspect that reduced methylation interfering with cell proliferation in the placenta as originally suggested by Eskes (2000) [In conclusion, using a retrospective case/control study, we have found that women with unexplained MSAFP elevations who have complications in later pregnancy are more likely to have the C677T MTHFR allele. Our resultsdo not suggest that C677T MTHFR predisposes a woman to having an elevation of MSAFP level (as we did not compare the C677T MTHFR frequency in women with and without elevated MSAFP), but having one or more copies of this variant predisposes such screen positive women to having complications in later gestation. It remains to be seen if other risk factors can be identified which can more accurately define this high risk group.All authors participated in original study design except CS. CG, BC and CS all acted as principal investigators for funding. NB assisted with all grant proposals. NB undertook the review of the individual MSAFP files and MMSSP database searches, designed the dietary and family history surveys, classified cases and controls, acted as study coordinator handling all aspects of participant contact, and provided data analysis. All authors also participated actively with NB for various aspects of the study. CG provided MTHFR genotyping. LS provided biochemical analysis. NB drafted the original manuscript with assistance from BC. CG and BC handled ethics approval assisted by NB. All authors read and approved the final manuscript.


The idea that cancer cells go through a fateful transition that turns them into fast-growing, invasive, metastasizing tumors first surfaced in the early 1970s. During this conversion, blood vessels form around the tumor, providing a dedicated supply of blood to fuel the tumor's aggressive behavior. By the mid-1980s histological analysis revealed a similarity between the tumor “microenvironment” and that of a healing wound, prompting Harvard pathologist Harold Dvorak to describe cancer as a wound that does not heal. When the body sustains a wound, it coordinates an emergency response defined by rapid cell proliferation, invasion and “remodeling” of connective tissues and extracellular matrix (the network of proteins and molecules around cells), cell migration, and blood vessel formation (angiogenesis). These processes, which are restorative in normal wound healing, may promote cancer by supporting tumor formation, invasion, and metastasis. With no systematic method to measure the “wound-like” features in cancer, however, scientists have no way to evaluate the risk that a wound-healing genetic program may pose in cancer progression.A molecular understanding of the wound-healing process and its connection to cancer would provide insight into the nature of these similarities and perhaps provide molecular indicators of tumor progression. In an effort to create a framework for evaluating this relationship, Howard Chang and his colleagues at Stanford University developed a model to predict cancer progression based on the gene expression profile of a cellular response to serum in cell culture.Part of the problem with evaluating the physiological status of a tumor based on its genetic profile is that current techniques indicate only the expression, not the effect, of genes. To develop a strategy for interpreting biological outcomes from a gene expression profile, Brown's team modeled a physiological process by exposing cultured fibroblasts to serum—the soluble fraction of coagulated blood—and tracking gene expression. Serum is encountered in the body where blood leaks out of blood vessels  and is thought to be a major initiator of the wound response. Fibroblasts exist in the connective tissue of epithelial organs (which include the digestive tract, lungs, and mammary glands) and contribute to organ development, wound healing, inflammation, and a condition called fibrosis.  And fibroblasts can promote tumor formation and metastasis when mixed with epithelial cancer cells.Though fibroblasts from different sites in the body differ in their properties and gene expression profiles, Chang et al. found that they share a common expression pattern in response to serum. From this expression profile, the researchers identified a core group of genes—a genetic signature—associated with a serum response. Because many of the genes in the signature were known to be involved in various wound-healing processes—such as matrix remodeling, cell motility, and angiogenesis—Chang et al. used this signature as a surrogate marker to measure how much tumors may be like wounds. When they compared the wound-like genetic signature with the expression profiles of various clinical tumor samples, they found the signature was always present in certain cancers—prostate and liver-cell carcinomas—and occurred variably in others—breast, lung, and gastric carcinomas. In each of these three latter types of tumors, patients with tumors carrying the serum-activated wound-like genetic signature had a significantly increased risk of metastasis and death compared to patients with tumors that lacked the signature. Therefore, Chang et al. conclude that a wound-like phenotype is a general risk factor for metastasis and the aggressive behavior in many of the most common cancers.These results reveal a robust and useful similarity between the molecular programs in normal wound healing and tumor progression and metastasis. Although Chang et al. point out that their results do not indicate whether this fibroblast “fingerprint” is merely a marker for cancer progression or plays a role in orchestrating this pathway, they conclude that the genetic program activated in response to serum also contributes to tumor invasion and metastasis. This serum-response expression profile, the authors propose, provides a valuable new tool for predicting tumor behavior and determining a patient's prognosis.


Escherichia coli is described.A systematic approach for identifying human proteins and protein fragments that can be expressed as soluble proteins in  Escherichia coli. A cDNA expression library of 10,825 clones was screened by small-scale expression and purification and 2,746 clones were identified. Sequence and protein-expression data were entered into a public database. A set of 163 clones was selected for structural analysis and 17 proteins were prepared for crystallization, leading to three new structures.We describe here a systematic approach to the identification of human proteins and protein fragments that can be expressed as soluble proteins in  Escherichia coli, are well suited for preparing such samples at high throughput. Genetic manipulation of E. coli is easy and large amounts of recombinant protein can be expressed in a short time. However, low success rates have been reported for the expression of eukaryotic proteins in E. coli: only a small proportion of proteins can be successfully expressed, partly owing to the specific requirements of eukaryotic proteins in regard to the cellular environment [E. coli cells. Consequently, one possible approach to structural proteomics for eukaryotic proteins is to study those that can be expressed in E. coli first.Structural genomics and structural proteomics involve the systematic structural analysis of large sets of proteins ,2. Strucironment -6. Alterironment . Cell-frironment . At the E. coli K-12 strain SCS1 was used for cloning the library and subsequent protein-expression experiments. A total of 193,536 clones were arrayed on protein-binding membranes and putative expression clones were detected immunologically, resulting in a smaller library of 37,830 putative expression clones [A human cDNA expression library (hEx1) was constructed for parallel screening of protein function on high-density protein arrays ,10. ThisThe hEx1 cDNA expression library was screened for expression clones on protein macroarrays. Using an anti-His-tag antibody, a subset of 37,830 clones was detected, as described before . On the To identify soluble expression products, small-scale protein expression and purification experiments were performed in microplates in 1 ml cultures. Protein expression was routinely performed at 37°C for 7,316 clones. Because lower induction temperatures have been reported to increase the yield of soluble product for certain proteins , we carrCells were lysed and aliquots were removed twice - before and after pelleting of cellular debris by centrifugation. These aliquots were termed 'whole' and 'soluble' protein extracts, respectively. Small-scale purification by metal chelate affinity chromatography was performed in batches of 96 in microplates, either manually or with the help of a pipetting robot . CellulaOnly clones expressing soluble protein with a size of at least 15 kDa were selected. As found previously , the sizClones expressing soluble protein with a size of at least 15 kDa were subjected to DNA tag-sequencing, starting from the 5' ends of the cDNA inserts. For 1,588 clones, sequences of at least 200 base-pairs (bp) of good quality were obtained. Of these sequences, 1,509 (95%) could be matched to transcript sequences from the Ensembl database . These tTranscript sequences from the Ensembl database are annotated with start and end positions of ORFs. Annotation of the ORF start position in Ensembl depends on experimental data from other databases and is not determined automatically. Many transcript sequences in the Ensembl database were generated automatically using cDNA sequences and exon-detection algorithms. If such a transcript is novel and does not correspond to known proteins, the ORF start position cannot be determined reliably by the automated annotation process of Ensembl. The annotation will often assign an ORF starting at position 1 to such transcripts; this is the case for 33% of transcript sequences in the Ensembl release 20.34c.To determine which cDNA clones contain complete ORFs (full-ORF clones), the Ensembl database was used, despite the limitation outlined above. Of 1,509 cDNA clones, 538 (36%) were identified as full-ORF clones, as their 5'-tag sequences align to an Ensembl transcript sequence at a position upstream of the ORF start position on that sequence. These clones, representing 375 distinct transcripts, were annotated as containing a complete ORF, as the cDNA for the hEx1 library was constructed by oligo(dT) priming and is therefore assumed to contain the 3' end of their transcript templates.For expression of cDNA inserts as His-tag fusion proteins, the respective cDNA insert has to be cloned in-frame to the vector-encoded start codon and His-tag. The reading frames of the clones' cDNA inserts were determined from the positions of Ensembl transcript and vector sequences aligned to the clones' sequences . We determined the reading frame of 1,447 of the 1,509 clones and found that 1,014 (70%) of the sequences were cloned in the correct frame with respect to the vector.The complete clone insert sequences are unknown as only partial 5'-tag sequences were generated. However, if a clone is matched to an Ensembl transcript sequence, it is possible to construct a putative predicted insert sequence by combining the experimentally derived DNA sequence and the Ensembl transcript sequence. Such a strategy can lead to wrong results if a different splice variant is represented by the clone and the Ensembl sequence. By comparison of predicted sequences with experimentally derived, complete sequences we found that in most cases the prediction is correct (data not shown). Predicted insert sequences were generated for 1,133 clones and the corresponding putative sequences of expression products were calculated. For the remaining sequences, the quality of the experimental sequence was not sufficient, or the alignment to the Ensembl transcript suggested that the clone represents a different splice form.E. coli cells.The molecular masses derived from the predicted protein sequences were compared to the sizes of proteins expressed in the corresponding clones. Only clones with inserts in the correct reading frame were considered. As shown in Figure The results of our protein expression screening and DNA sequencing of the hEx1 cDNA library are publicly available . The corClones expressing soluble recombinant protein and containing full-ORF inserts were selected for the structural analysis pipeline of the Protein Structure Factory . Clone sFor preparation of proteins without additional residues such as the His-tag, ORFs were subcloned into the vector pQTEV. This vector allows expression of His-tag fusion proteins and subsequent tag removal by specific protease cleavage using tobacco etch virus (TEV) protease. Of the selected cDNAs, 110 were subcloned into pQTEV, of which 48 were selected for large-scale protein production. A total of 17 of the 48 proteins could be expressed and purified in sufficient yield and quality for protein crystallization.The volume of cultures, grown either in shaker flasks or fermenters, varied between 1 and 5 liters. Protein yields varied from 1.5 to 38 mg/liter of culture volume. Following cell lysis, His-tag fusion proteins were captured by metal chelate affinity chromatography. The His-tag was removed proteolytically and proteins were further purified by ion-exchange and size-exclusion chromatography. The proteins were characterized and prepared for crystallization trials using biophysical methods. A summary of a typical preparation for each clone, and the preparation and characterization data is given in Table Tm) were measured by differential scanning calorimetry (DSC). With one exception, all proteins that were free of aggregation showed high Tm values, of 49-60°C, at pH 7.0 (Table The protein preparations were tested to see whether they were free of aggregates. For 10 of the 17 proteins, this was proven by dynamic light scattering (DLS) analysis. To determine the thermal stabilities, denaturation temperatures (So far, the structures of gankyrin (PDB 1QYM), aortic preferentially expressed protein 1 and prolidase (unpublished data) have been solved by the Protein Structure Factory as a result of the approach described here.The expression of soluble recombinant protein is still a bottleneck for functional and structural genomics projects studying human proteins. We demonstrate here a method for generating and characterizing a large set of expression clones for human proteins from a cDNA library, yielding a pre-selection of clones for large-scale expression. By matching clone sequences to the Ensembl database, it was shown that expression clones with soluble products were found for 1,509 human proteins corresponding to 1,105 distinct genes. To cover a larger set of proteins with our approach, additional libraries from different tissues and developmental stages could be used.It was found that 36% of expression clones are full-ORF clones expressing complete human proteins, while the remaining clones express carboxy-terminal fragments. It should be noted that because the Ensembl database is generated automatically and start codon positions are still unknown for many human transcripts, this number is inaccurate and will probably be higher. Future releases of Ensembl will benefit from the ongoing efforts to generate and annotate human full-length cDNA sequences , and theE. coli could be another reason why many clones expressing carboxy-terminal fragments were obtained.Thre are several reasons for the presence of clones expressing carboxy-terminal fragments. A certain proportion of incomplete inserts is a common feature of cDNA libraries constructed by the cloning technique used here. Furthermore, full-ORF clones containing parts of the 5'-untranslated region (UTR) are not detected in our expression screen if the UTR contains stop codons. The fact that smaller proteins or fragments are often expressed better than very large proteins in Full-ORF clones are generally required for determination of protein structures. However, carboxy-terminal fragments can be interesting for other applications, such as structural analysis of the domain by NMR spectroscopy.As an example of the application of the characterized clone library, we show the selection of clones for structure analysis. The high-throughput screening for expression clones took about a year, while the work on the 163 selected proteins is still in progress and additional proteins are being purified. From the 17 protein preparations, three new protein structures were solved.E. coli expression clones of human proteins is described here. Using this approach, a public resource of 2,746 clones was created that allows functional genomics projects to select clones and express human proteins of interest.In conclusion, a systematic screening approach for cDNA sequences have been submitted to the dbEST database and are available under the accession numbers CD579165-CD580594. Clone DNA sequences were matched to transcript sequences of the Ensembl database, release 20.34c, using the program cross_match, version 0.990329, of the swat/cross_match/phrap package , versionA database was created to store the results of the protein expression and purification experiments as well as clone sequence data. The Oracle database management system 8.1.6 was used. A web-based front end including search functionality was developed, using the Java programming language.The reading frame of a cDNA insert was determined using the following formula:cce,start - (ccv,end + l - vcv.end) + o - ece,end| mod 3,|l is the length of the vector pQE30NST (3,494 bp). In an alignment of a vector and clone sequence, ccv,end and νcv,end denote the positions of the end of the matched region on the clone and vector sequence, respectively. Likewise, cce,start and ece,start denote the start positions of the match of clone and Ensembl sequence. o is the start position of the ORF on the Ensembl transcript sequence. For clones that are in-frame to the vector-encoded start codon and His-tag, the formula returns 0.where Predicted clone insert sequences were generated from experimental tag sequences and Ensembl transcript sequences by the Perl program seqjoin. seqjoin uses alignments generated by cross_match to generate combined sequences. It does not generate output for alignments that indicate alternative splicing. The program and documentation are publicly available online .Tm of 60-65°C and sense and antisense primers were equipped with BamHI and NotI sites, respectively. For ORFs containing these sites, alternative enzymes producing compatible overhangs were used (BglII, Eco31I or Esp3I). PCR products were cloned into the vector pQTEV (GenBank AY243506). A pipetting robot and microplates were used for PCR setup, restriction digest and DNA purification steps. The resulting plasmid was introduced into E. coli SCS1 cells carrying the pSE111 helper plasmid. pSE111 provides resistance to 15 μg/ml kanamycin and carries the lacIQ repressor and the argU gene for the arginine tRNA that recognizes the rare codons AGG and AGA. The low abundance of this tRNA is especially critical when expressing eukaryotic genes in E. coli [ORFs were PCR amplified from hEx1 cDNA clones using gene-specific primers. Primers were automatically designed using a Perl script that is available on request. Primer length was adjusted to obtain a uniform  E. coli . The resg for 10 min and frozen at -80°C.Protein expression was performed as described . The hEx2, 0.1 U/μl Benzonase gradeII (Merck) in 50 mM Tris-HCl pH 8.0, brief vortexing and incubation at room temperature for 30 min. An aliquot was collected for SDS-PAGE analysis (whole cellular proteins). Cellular debris was pelleted by centrifugation of the plates at 6,200 rpm for 30 min. Aliquots of the supernatants were collected (soluble cellular protein). Supernatants were transferred to a filter plate (Millipore Multiscreen MADVN6550) and were filtered on a vacuum manifold. Filtrates were collected in a second filter plate. Imidazole was added to 10 mM, and 25 μl of 20% (v/v) Ni-NTA agarose (Qiagen) equilibrated in 50 mM Tris-HCl pH 8.0. Plates were shaken at room temperature for 30 min, followed by removal of cell lysates on the vacuum manifold. The agarose beads were washed three times by shaking in 200 μl wash buffer (50 mM Tris-HCl pH 8.0, 0.3 M NaCl, 20 mM imidazole). Upon complete removal of liquid from the plate, proteins were eluted by addition of 25 μl wash buffer containing 250 mM imidazole. Eluates were collected in a 96-well plate by brief centrifugation. Seven microliters of the eluates and 3.5 μl of the whole and soluble cellular extracts were analyzed by SDS-PAGE (15% polyacrylamide) and Coomassie staining.Proteins were purified via metal chelate affinity chromatography in a 96-well format. We used an automated procedure on a pipetting robot  or a corA600 of 1.5 and protein expression was induced by addition of 1 mM IPTG for 4 h. The optimal expression temperature was determined in small-scale experiments beforehand (28-37°C). Cells were pelleted by centrifugation and resuspended in a threefold volume of 20 mM Tris-HCl pH 7.4, 300 mM NaCl, 10 mM imidazole, 5 mM 2-mercaptoethanol, 1 mM PMSF, a protease inhibitor cocktail tablet (EDTA-free, Roche) and 500 units Benzonase (Merck). Cells were lysed by treatment with lysozyme and sonification, followed by centrifugation (23,000g, 45 min) and filtration through a 0.22-μm syringe filter. Proteins were applied to a metal chelate chromatography using a Ni-POROS20-column (Applied Biosystems) or a TALON column (Clontech). After washing with 20 mM Tris pH 7.4, 150 mM NaCl, 10 mM imidazole, the protein was eluted with 250 mM imidazole in the same buffer and eluates were supplemented with 2 mM dithiothreitol and 1 mM EDTA. The His-tag was removed by incubation with TEV protease (molar ratio 1:40 protease:substrate) at 4°C overnight. Proteins were diluted fivefold and depending on the theoretical pI of the protein, anion or cation exchange chromatography was performed. Proteins were further purified by gel filtration on a Superose 12 16/50 column (Amersham Biosciences).Proteins were expressed, purified, concentrated and analyzed as described . Cells ws) (~ λ-4) with the assumption that no absorption due to protein chromophores occurs above 320 nm [Protein concentrations were determined from the absorbance at 280 nm using the extinction coefficient calculated from the amino acid sequence . Absorbae 320 nm . Purifieg, 3 min, 4°C) and measured in a 1.5 × 1.5 mm cuvette (Hellma, Müllheim, Germany) for 20 sec. The instrument software allows us to judge the autocorrelation function and deduce the dispersity, that is, the distribution N(Rh), of particles according to their hydrodynamic radius. Protein samples were judged 'free of aggregation' when a single peak indicated a monomodal distribution.DLS measurements were carried out at room temperature, using the Spectroscatter 201 (660 nm laserdiode, 30 mW, scattering angle 90°, PMT detector, 400 nsec to 30 sec correlator, quasi-logarithmic arranged channels, RiNA, Berlin, Germany). The samples were centrifuged .DSC measurements were performed at a rate of 1 K/min using an automated capDSC calorimeter . Proteins were diluted at least 20-fold in a buffer of temperature-independent pH (20 mM Na/K phosphate pH 7.0, 150 mM NaCl). The resulting scans were baseline-corrected and cDNA inserts were PCR-amplified using primers pQE65 (TGAGCGGATA ACAATTTCAC ACAG) and pQE276 (GGCAACCGAG CGTTCTGAAC), annealing temperature 65°C. PCR products were tag-sequenced using primer pQE65.Additional data file A a tab-delimited text file listing information on hEx1 clones with inserts in the correct reading frame, giving their clone ID, Ensembl transcript ID, experimental and predicted expression product size, expression strengthClick here for additional data file


Poor diet is thought to be a risk factor for many diseases, including age-related macular disease (ARMD), which is the leading cause of blind registration in those aged over 60 years in the developed world. The aims of this study were 1) to evaluate the dietary food intake of three subject groups: participants under the age of 50 years without ARMD (U50), participants over the age of 50 years without ARMD (O50), and participants with ARMD (AMD), and 2) to obtain information on nutritional supplement usage.A prospective cross-sectional study designed in a clinical practice setting. Seventy-four participants were divided into three groups: U50; 20 participants aged < 50 years, from 21 to 40 (mean ± SD, 37.7 ± 10.1 years), O50; 27 participants aged > 50 years, from 52 to 77 (62.7 ± 6.8 years), and ARMD; 27 participants aged > 50 years with ARMD, from 55 to 79 (66.0 ± 5.8 years). Participants were issued with a three-day food diary, and were also asked to provide details of any daily nutritional supplements. The diaries were analysed using FoodBase 2000 software. Data were input by one investigator and statistically analysed using Microsoft Excel for Microsoft Windows XP software, employing unpaired t-tests.Group O50 consumed significantly more vitamin C (t = 3.049, p = 0.005) and significantly more fibre (t = 2.107, p = 0.041) than group U50. Group ARMD consumed significantly more protein (t = 3.487, p = 0.001) and zinc (t = 2.252, p = 0.029) than group O50. The ARMD group consumed the highest percentage of specific ocular health supplements and the U50 group consumed the most multivitamins.We did not detect a deficiency of any specific nutrient in the diets of those with ARMD compared with age- and gender-matched controls. ARMD patients may be aware of research into use of nutritional supplementation to prevent progression of their condition. Poor diet is thought to be a risk factor for many diseases ,2. One wAge-related macular degeneration (AMD) is the leading cause of blind registration and visual disability in patients over the age of 60 years in the developed World . The conARM is the early stage of ARMD and is most often clinically apparent over the age of 50 years. The main symptom is increasing difficulty with fine detail discrimination. AMD is the later stage of ARMD and is categorised further in to 'dry AMD' , and 'wet AMD'  . GA is tThe prevalence of GA and exudative AMD in the US population over 40 years of age has been estimated at 1.47% [95% confidence interval (CI), 1.38% – 1.55%] . The likIt is thought that people with low systemic antioxidant levels may be more prone to oxidative damage of the retina and therefore, AMD . OxidatiThe hypothesised role of oxidation in the development of AMD has prompted research into the use of nutritional supplementation . The AgeThe primary aim of this study was to evaluate the dietary food intake of three subject groups: participants under the age of 50 years without ARMD (U50), participants over the age of 50 years without ARMD (O50), and participants with ARMD. The secondary aim was to obtain information on nutritional supplement usage.Prospective cross-sectional in a clinical practice setting.Seventy-four participants gave informed consent to take part in this study, which was approved by the Institutional Human Ethics Committee. Recruitment methods included sending information to Birmingham optometrists, ophthalmologists, and a specialist centre for rehabilitation of people with sight loss, an editorial in a local newspaper, recruitment e-mails sent to the Royal National Institute for the Blind (RNIB) and all staff and students at Aston University and Aston Science Park, Birmingham, UK. For analysis the participants were divided into three groups: U50; 20 participants aged < 50 years, from 21 to 40 (mean ± SD, 37.7 ± 10.1 years), O50; 27 participants aged > 50 years, from 52 to 77 (62.7 ± 6.8 years), and ARMD; 27 participants aged > 50 years with age-related macular disease, from 55 to 79 (66.0 ± 5.8 years). All participants were part of a larger study investigating the effects of nutritional supplementation on visual function in normal and diseased eyes .2 (1) = 0.104 p = 0.305] and groups O50 and ARMD [χ2 (1) = 3.814 p = 0.051]. The difference in age is not significant between groups O50 and ARMD (t = -1.842, p = 0.071).Chi squared analysis for gender yielded no significant difference between groups U50 and O50 [χExclusion criteria were the presence of an ocular condition other than ARMD and the presence of medical conditions indicating a diet in which particular foods or food groups were excluded (e.g. coeliac disease).Participants were issued with a three-day food diary with verbal and written instructions explaining that they should add to their diary every time they eat or drink, describing the food as accurately as possible and giving estimates of amounts. They were also asked to provide details of any daily nutritional supplements. The diary consisted of two week days and one weekend day. The diaries were analysed using FoodBase 2000 software (The Institute of Brain Chemistry and Human Nutrition, 166–220 Holloway Road, London N7 8DB, UK), which is a computerised nutrition database containing data on approximately 3750 foods. It can be used for recipe analysis, meal analysis, and daily or weekly analysis of menus or food intakes. Data were input by one investigator and statistically analysed using Microsoft Excel for Microsoft Windows XP software, employing unpaired t-tests.The values for energy and nutrient intakes for all participants are shown in table Group O50 consumed significantly more vitamin C (t = 3.049, p = 0.005) and significantly more fibre (t = 2.107, p = 0.041) than group U50.The mean intakes for men and women in each group are shown in table By tradition, investigators consider a study to be adequately powered if it has an 80% chance of detecting a significant difference when one exists. The number of study participants needed to detect a clinically important difference with acceptable power, can be calculated using the required power, the expected variability of the outcomes, and the chosen probability of masking a type 1 error . Power aGroup ARMD consumed significantly more protein (t = 3.487, p = 0.001) and zinc (t = 2.252, p = 0.029) than group O50  consumes significantly more types of nutritional supplement than group U50 (0.55 ± 1.11) (t = 2.220, p = 0.032). No difference found between groups O50 and ARMD, and the study was powered to have an 80% chance of detecting a difference in means of 1 at the 10% level of significance. The percentage of supplements taken for each group is shown in figure The aim of this study was to evaluate the dietary intakes of three subject groups; U50, O50 and ARMD, as well as to obtain information on nutritional supplement usage. Participants under the age of 50 years consumed significantly less dietary vitamin C than those aged over 50 years. Supplementation data shows that 7.4 % of the O50 group take uncombined vitamin C compared with 0% of the U50 group. However, a higher percentage of the U50 group take multivitamins (33.3%) compared with the O50 group (22.2%).Vitamin C is water-soluble, is involved with several biological processes. As a reducing agent it is thought to be active in protection against heart disease. It protects LDL (low density lipoprotein) cholesterol from oxidative damage and reduces platelet aggregation . By enhaHigh dose supplementation with an antioxidant and zinc formulation, including vitamin C was associated with a 25% reduced risk of progression of AMD in those participants already suffering with the condition . Some stAlthough the antioxidant properties of vitamin C are well known, there is no clinical evidence suggesting that supplementation with vitamin C can reduce the risk of ARMD, or other ocular conditions such as cataract and glaucoma. The RNI for men and women over the age of 18 years is 40 mg. The mean intakes of men and women in the U50 and O50 groups are all above this value table . The higet al (1988) should be treated with caution [The ARMD group consumed significantly more dietary zinc than age- and gender- matched controls. Zinc has been investigated with regard to its potential preventative role in ARMD. The AREDS group found a suggestive reduction in the risk of progression of AMD in participants supplementing with 80 mg zinc daily. Previous randomized controlled trials (RCTs) using 200 mg zinc daily found conflicting results ,62, and  caution . The hig caution . Our resThe ARMD group consumed significantly more protein than O50 participants. We are not aware of any investigation into a link between protein and risk of ARMD, and table 4 shows that the mean intakes are above the RNI for both men (55.5 g) and women (46.5 g).Previous studies have found a relationship between higher dietary fat intake and risk of ARM (RR 1.6) , and higResearch into the role of alcohol consumption in the development of AMD has produced conflicting results. Several studies have found no relation ,66-70, bThe non-significant differences found between groups for alcohol, copper, cholesterol, selenium, vitamin E, vitamin D, and retinol equivalents may have occurred because there truly was no difference, or because the study had insufficient power to detect a difference. Because of the variability of the data, subject numbers required per group for 80 % power at the 5 % significance level are 467 for alcohol, 50 for copper, 44 for cholesterol, 341 for selenium, 59 for vitamin E, 113 for vitamin D, and 71 for retinol equivalents.Multivitamins were the most commonly taken supplement by the U50 group (30.0 %), compared with cod liver oil for the both the ARMD group (33.3 %) and O50 participants (22.2 %). Seventy-five percent of the specific ocular health related supplements were taken by the ARMD group, 25 % by the O50 group, and 0 % by the U50 group.We did not detect a deficiency of any specific nutrient in the diets of those with ARMD compared with age- and gender-matched controls. A higher percentage of ARMD participants consume specific ocular health nutritional supplements (33.3 %) compared with age- and gender-matched controls (11.1 %) and U50 participants (0 %). The U50 group consumed a higher percentage of multivitamins, but significantly less vitamin C and fibre than the O50 group. This suggests that the younger age-group might use supplementation to ensure adequate consumption of vitamins and minerals. The ARMD group consumed more dietary zinc, more supplemental zinc, and the highest percentage of ocular health related supplements. This may suggest that information regarding the results of studies investigating the role of nutritional supplementation in reducing the risk of onset or progression of AMD is reaching patients. These results however, may be confounded by the fact that the ARMD participants used in this study were enrolled in an RCT investigating the use of nutritional supplementation in ARMD. Participants in research projects may be more aware of scientific developments and more likely to investigate their condition and potential therapies.The authors declare that they have no competing interests.HB participated in the design of the study, carried out data collection, input, and analysis, and drafted and developed the manuscript. FE participated in the design of the study and development of the manuscript. Both authors read and approved the final manuscript.


While a definitive genetic test for Hereditary Prostate Cancer (HPC) is not yet available, future HPC risk testing may become available. Past survey data have shown high interest in HPC testing, but without an in-depth analysis of its underlying rationale to those considering it.Telephone computer-assisted interviews of 400 men were conducted in a large metropolitan East-coast city, with subsequent development of psychometric scales and their correlation with intention to receive testing.Approximately 82% of men interviewed expressed that they "probably" or "definitely" would get genetic testing for prostate cancer risk if offered now. Factor analysis revealed four distinct, meaningful factors for intention to receive genetic testing for prostate cancer risk. These factors reflected attitudes toward testing and were labeled "motivation to get testing," "consequences and actions after knowing the test result," "psychological distress," and "beliefs of favorable outcomes if tested" (α = 0.89, 0.73, 0.73, and 0.60, respectively). These factors accounted for 70% of the total variability. The domains of motivation (directly), consequences (inversely), distress (inversely), and positive expectations (directly) all correlated with intention to receive genetic testing (p < 0.001).Men have strong attitudes favoring genetic testing for prostate cancer risk. The factors most associated with testing intention include those noted in past cancer genetics studies, and also highlights the relevance in considering one's motivation and perception of positive outcomes in genetic decision-making. There are several factors to consider in undergoing genetic testing for cancer risk: potential benefits, possible risks, psychological distress, and the uncertainty in subsequent decision-making about prophylactic interventions -9. WhileA definitive genetic test for prostate cancer is not clinically available yet. Current genetic tests are only conducted in research studies. Several potential genetic loci have been identified as linked to hereditary prostate cancer, including HPC1 , MXI1, KInformed consent for genetic testing for cancer risk is particularly controversial in cancers where knowledge of a positive test result does not provide opportunities for interventions for favorable outcomes, and a negative result does not provide reassurance . High stThis study examines men's beliefs and values toward interest in prostate cancer genetic testing. A survey instrument was developed for men between 40 and 70 years of age, exploring their beliefs, attitudes, and concerns in considering a hypothetical blood test. Exploratory factor analysis was applied to identify the underlying factor dimensions. The relative importance of these factors was then compared to testing intention.The Institutional Review Board (I.R.B.) of the University of Pennsylvania and US Department of Defense Human Subjects Review approved the study. Subjects for this study included healthy outpatient males, identified with the assistance of the institution's Office of Health Services Research for demographic characteristics of age, ethnicity, and absence of past or current history of prostate cancer. Subjects were sent a letter-invitation to participate with an opt-out telephone number to call. Inclusion criteria were that subjects must be English-speaking men in a large metropolitan East-coast city, between the ages of 40 and 70, with no current evident mental incapacity and no present or past personal history of prostate cancer. All others were excluded.A 53-item attitude survey instrument was developed. The items were selected by the collaborators from a pool of more than 100 preliminary items from the data resulting from 12 focus groups of 90 lay men regarding their attitude, beliefs, and concerns about prostate cancer genetic screening . The staThe survey was conducted using Computer-Assisted Telephone Interviewing software (MacCATI, Senecio Software). The survey instrument was pilot tested in face-to-face interviews of randomly selected men, age 40–70, in a primary care office prior to data collection, to verify understandability of the survey's content and format. For the telephone survey, a recruitment packet that included an informed consent letter was first mailed to the prospective participants. Instructions explained the goals of the study and gave them an option to opt-out with a toll-free phone call prior to their interview. An oral informed consent was completed prior to the telephone interview.The number of missing observations ranged from 0 to 13, with an average of 2.38. Missing data were imputed based on an imputation model that predicts the missing values of factors as predicted by all of the other responses, including the outcome (desire to be tested). The algorithm uses Markov Chain Monte Carlo methods to select at random a value from the distribution of the possible values predicted by the missing value model. This method differs in several respects from other methods of filling in for missing data, in that with each imputation a different value will be imputed for the missing value, thus ensuring an added dimension of variability in the resulting analyses. The imputation was repeated multiple times. Each imputation generated an imputed data set. The same factor analysis was applied and no statistically reliable differences were found across the imputed data sets. Thus, only the results from the first imputed data are reported here. The imputation was carried out by SAS PROC MI.a priori as a separate factor. The factor analysis involved methodological criteria for data reduction, which included the rules summarized in Tabachnick [A maximum-likelihood factor analysis with oblique rotation was applied to the 49 non-intent questions to classify men's non-intent beliefs and attitudes according to their underlying dimensions. The four questions that directly probed men's expressed intent were considered bachnick . Items wbachnick . Items tbachnick . Factor bachnick .Interviews were completed with 400 respondents with a cooperation rate of 47% (1675 were contacted, 431 refused to participate either by phone prior to the interview, or at the time of the interview, and 844 were excluded due to no answer, disconnected telephones, and death). Table no new information on timing or severity of prostate cancer is to be learned from the prospective test.About 82% of men interviewed expressed that they "probably" or "definitely" would take the test if one were offered now. This high interest increased to 88% if a positive test result indicates elevated risk in the early onset of cancer; 93% if it indicates graver prognosis of cancer; and the stated interest dropped to a still appreciable 68% if Motivation, i.e., those values relating how strongly the respondent wanted the test, and how strongly the opinions of professionals, spouse, family, relatives, and friends could have influenced the respondent's own strength of intent; 2) Consequences, which measured beliefs with respect to follow-up decision-making and management; 3) Distress, which assessed fear of losing health and life insurance, anxiety, and worsening of quality of life if tested positive; and 4) Positive Expectations, which described beliefs in how the test results will confer useful information in family risk and favorable outcomes. The four intent items were added separately as the fifth subscale 5) Intention directly probing the respondent's stated intent. Table Exploratory factor analysis identified four underlying factors that accounted for 76% of the total variability among the 49 items probing men's beliefs and attitudes. The four factors were 1) a priori for their content, showed a good internal consistency alpha coefficient at 0.79. Among the 49 items that probed men's beliefs, values, and attitudes, the 11 items that loaded high on Motivation accounted for most of the variability (37%) with a very high alpha coefficient (0.89). The nine items in Consequences accounted for the next largest amount of variability (23%) with an alpha coefficient of 0.73. The six items in Distress accounted for 10% of the variability with an alpha of 0.73. Finally, the four items in Positive Expectations accounted for 7% of the variability with an alpha of 0.60.Overall, the five subscales showed satisfactory internal consistency. The four items within Intention scale, although grouped together The factor pattern loadings reflect the correlation between an individual item and its subscale. For example, Table r = 0.69, p < 0.001). There was also a positive and statistically significant correlation between one's motivation and one's expectations that genetic screening may lead to favorable outcomes for the gene carrier and his family (r = 0.39, p < 0.001). Concerns about the consequences of a positive result, including the uncertainties of test validity and accuracy, and the availability of subsequent interventions, were positively correlated with distress (r = 0.34, p < 0.001) and diminished intention to test (r = -0.16, p < 0.01). Distress-based values were associated with diminished intention to test (r = -0.17, p < 0.001). Finally, respondents who expected favorable outcomes were associated with increased intention to test (r = 0.48, p < 0.001).The inter-correlations between the subscales are summarized in Table These data demonstrate that men in the general public, aged 40 to 70 years without a personal history of prostate cancer, consider prostate cancer genetic testing related to four value-based factor domains, similar to past literature findings on genetic testing for hereditary cancer risk. The motivation factor, which measures values of influence by others, is the strongest decision factor in guiding their opting for the test. More than 80% of men interviewed would consider getting tested if the test was available now. Their stated intention, as measured by the four intent items, is highly correlated with how strongly they feel they are motivated toward the test and inversely related to family influences. Men with strong motivation to get tested also have significantly lower concerns about psychological distress and higher levels of positive expectations. The recommendations of physicians and geneticists are important to men's expressed motivation, although the professionals did not appear to be more influential than their kin.family risk and may lead to early identification and prevention of cancer (as part of the Positive Expectations domain). The influences of kin, along with beliefs in family risk, highlight the importance of reviewing family-related risk information as part of genetic consultation and informed consent. Men undergoing informed consent for hereditary prostate cancer risk in the future not only should be provided information on what genetic testing can and cannot do for them, but also what the test results could mean for others surrounding them (as evidenced by the influences of family, etc.).A respondent is more likely to want the test if he believes that the test may be informative of Prior hereditary breast cancer (BRCA) and colorectal cancer (CRC) literature has noted anecdotally that perception of benefit to one's family influences genetic test uptake. Eliciting patient perceptions of concerns regarding their family may be beneficial to consider in oncology genetic testing generally. Similar to this literature, intention was found to be influenced by the respondent's concerns about test validity, test accuracy, and by the availability of interventions that may lead to favorable outcomes. Not surprisingly, men who were concerned about potential psychological distress were less likely to want the test. One unanswered question is how men's anticipatory distress and expected adverse consequences may affect how family risk information is interpreted and discussed. Few men in our study anticipated high levels of distress. Although literature data clearly show elevated distress among patients and their family members . More reThe present study has limitations. Given the exploratory nature of factor analysis, these data are aimed at identifying coherent subsets of variables for data reduction, not at identifying specific attitude statements that discriminate skeptics from supporters. Nevertheless, the reduced set of 34 items is the most important among the administered 57 items, and comprises a coherent and reliable assessment tool of eliciting values and intention toward testing. This item set can thereby serve as a foundation for a confirmatory health beliefs model, using Structural Equation Modeling techniques to better elucidate the interactions of these value-based domains . Also, wFuture directions of this research may include exploring the relationship between stated intent in prostate cancer genetic screening and actual testing behavior when testing is available. Studies have shown that expressed intention does not necessarily translate to actual behavior in taking genetic tests for breast and colorectal cancers ,23-29. TMen in this survey voiced strong attitudes favoring future genetic testing for prostate cancer risk. In the past decade and a half, genetic testing for a variety of cancers concentrated on several key concepts: i.e., stigmatization, privacy, anxiety/stress, and the need to know. These notions of stigma and psychological impact were not as relevant in this population regarding prostate cancer risk genetic testing. For examples, the following statements did not show strong enough factor loadings to warrant their inclusion, such as "I will not be able to keep my job, or get a promotion, if I know I have the prostate cancer risk gene," "The government could use my test results in ways I do not want," "I often worry about getting prostate cancer," and "I would want to get tested because I just want to know if I have the gene for prostate cancer."The most relevant aspect of data reported herein is that they begin to shed new light on the relevance of "others." How men were concerned about the impact on and the effects upon one's family were reflected in the factor analysis. As a result, future informed consent may likely include considerations of 1) how the test results will affect their own future lives, and 2) how the test results will affect their family members. The latter consideration is seldom brought into the informed consent process in the genetic counseling but may be relevant to the patient.The author(s) declare that they have no competing interests.DD designed the overall project study, collaborated in the data analysis, and participated in its coordination. YL designed and carried out the factor analysis statistics. Both authors drafted, read, and approved the final manuscript.The pre-publication history for this paper can be accessed here: