Laparoscopic cholecystectomy is one of the commonest surgical procedures carried out in the world today. Occasionally patients present with undiagnosed situs inversus and acute cholecystitis. We discuss one such case and outline how the diagnosis was made and the pitfalls encountered during surgery and how they were overcome.A 32 year old female presented to our department with epigastric pain radiating through to the back. A diagnosis of acute cholecystitis in a patient with situs inversus totalis was made following clinical examination and radiological investigation. Laparoscopic cholecystectomy was subsequently performed and the patient made an uneventful recovery.Situs inversus presenting with acute cholecystitis is very rare. The surgeon must appreciate that care should be taken to set up the operating theatre in the mirror image of the normal set-up for cholecystectomy, and that right handed surgeons must modify their technique to adapt to the mirror image anatomy. In 1600 the first known case of situs inversus in humans was reported by Fabricius . The conSince Mouret first performed it in 1987, laparoscopic cholecystectomy has become the standard operative procedure for gallbladder disease. It is associated with reduced hospital stay, fewer respiratory complications, less pain and a faster return to work.A thirty two year old female was admitted with a three hour history of epigastric pain radiating into her back in keeping with biliary colic. She had vomited a number of times. In the previous week she had two episodes of a similar nature.On examination there was no jaundice or pyrexia. The apex beat was in the right fifth intercostal space, midclavicular line. She had epigastric tenderness but was not tender in the right or left upper quadrants. Her white cell count and amylase level was normal but her C-reactive protein level (CRP) was elevated at 290 mg/L. An electrocardiograph showed right axis deviation and right ventricular hypertrophy, in keeping with dextrocardia.An ultrasound scan of the upper abdomen identified the gallbladder, which contained stones, in the left upper quadrant. The spleen was visualised in the right upper quadrant. There was no evidence of common bile duct or intrahepatic duct dilatation. Chest X-Ray confirmed the clinical and electrocardiograph diagnosis of dextrocardia.The diagnosis of acute cholecystitis and situs inversus was made. The patient settled clinically over two to three days and was discharged home to be admitted electively for laparoscopic cholecystectomy.2 insufflator were positioned in the mirror image of their normal position. The surgical team also changed sides with the primary surgeon and first assistant on the patients right and the second assistant on the left. The ports were inserted in the usual way but on the left side. At laparoscopy the entirety of the abdominal contents were indeed reversed.In order to conduct the laparoscopic cholecystectomy all theatre equipment including diathermy, monitors and COThe main difficulty encountered was that the primary surgeon, who was right handed, would have had to cross hands to retract on Hartmann's pouch while dissecting Calot's triangle. We overcame this difficulty by allowing the first assistant to retract on Hartmann's pouch, while the primary surgeon dissected Calot's triangle using his right hand via the epigastric port without hindrance. The common bile duct and cystic duct were identified, as was the cystic artery, which lay anterior to the cystic duct. The surgery proceeded without incident and the patient recovered and was discharged the next day.In this case the patient presented with epigastric pain only and had no definite left upper quadrant pain. It has been noted in 30% of previous reported cases of acute cholecystitis in patients with situs inversus that the pain was felt in the epigastrium alone and in 10% the pain was localised to the right upper quadrant . The proPrevious reports have confirmed that situs inversus is not a contraindication for laparoscopic cholecystectomy -3,5,6. T1. Detailed clinical examination is important in diagnosing previously unknown situs inversus.2. Patients with gallabladder disease and situs inversus may have pain in the right upper quadrant, epigastrium or left upper quadrant.3. Theatre equipment must be moved to the mirror image of their normal positions before surgery.4. The surgeon must recognise the mirror image anatomy and modify his or her technique appropriately.CRP- C reactive protein.2- Carbon dioxide.COThe author(s) declare that they have no competing interests.The pre-publication history for this paper can be accessed here:


To investigate whether low birth weight increases the risk of myocardial infarction later in life in women.Nationwide population-based case-control study. Patients and controls: 152 patients with a first myocardial infarction before the age of 50 years in the Netherlands. 568 control women who had not had a myocardial infarction stratified for age, calendar year of the index event, and area of residence.th percentile in controls) compared to higher than 3000 gram was 1.7 (95%CI 1.1–2.7), while the odds ratio for myocardial infarction for children with a low birth weight (< 2000 g) compared to a birth weight ≥ 2000 g was 2.4 (95%CI 1.0 – 5.8). Both figures did not change after adjustment for putative confounders .Birth weight in the patient group was significantly lower than in control women . The odds ratio for myocardial infarction, associated with a birth weight lower than 3000 gram (20Low birth weight is associated with an increased risk of myocardial infarction before age of 50 in Dutch women.


Primary care physicians are caring for increasing numbers of persons with comorbid chronic illness. Longitudinal information on health outcomes associated with specific chronic conditions may be particularly relevant in caring for these populations. Our objective was to assess the effect of certain comorbid conditions on physical well being over time in a population of persons with chronic medical conditions; and to compare these effects to that of hypertension alone.® Health Survey physical component summary score (PCS) over 4 years. We used logistic regression analysis to determine significant differences in longitudinal change in PCS between patients with hypertension alone and those with other comorbid conditions and linear regression analysis to assess the contribution of the explanatory variables.We conducted a secondary analysis of 4-year longitudinal data from the Medical Outcomes Study. A heterogeneous population of 1574 patients with either hypertension alone (referent) or one or more of the following conditions: diabetes, coronary artery disease, congestive heart failure, respiratory illness, musculoskeletal conditions and/or depression were recruited from primary and specialty  practices within HMO and fee-for-service settings in three U.S. cities. We measured categorical change (worse vs. same/better) in the SF-36Specific diagnoses of CHF, diabetes and/or chronic respiratory disease; or 4 or more chronic conditions, were predictive of a clinically significant decline in PCS.Clinical recognition of these specific chronic conditions or 4 or more of a list of chronic conditions may provide an opportunity for proactive clinical decision making to maximize physical functioning in these populations. Comorbid chronic diseases are increasingly recognized as a significant factor in declining health. Of the 125 million Americans with chronic diseases, 48% are estimated to have at least one comorbidity, and 62% of persons over the age of 65 have two or more chronic illnesses. As the pPrimary care for persons with chronic medical conditions differs from specialist care for these same conditions in the need to see both the forest and the trees: to address disease-specific issues and outcomes in the context of both coexisting medical conditions and the patient's psychosocial environment. To this end, practice guidelines developed from randomized controlled trials with strict inclusion criteria may not generalize well to the heterogeneity of primary care practice or the complex individual patient.In the environment of competing demands that characterizes medicine in general, and primary care in particular; information on health outcomes that can be inferred from the medical record problem list may be particularly relevant in clinical decision making for persons with multiple chronic conditions. We chose to investigate the health outcome of physical well being for several reasons: It is relevant to both clinical and quality-of-life decision making; and previous findings have demonstrated that different combinations of chronic medical conditions have been shown to be associated with type and/or severity of disability ,6.Longitudinal studies suggest that the cumulative effect of comorbid conditions is not simply additive: certain combinations of diseases may have a greater effect on outcomes than others. Combinations of diabetes plus obesity or heart disease , and artWe hypothesized that certain chronic diseases that often occur as comorbidities may have a greater impact than others on functional status outcomes over time. To explore this question, we analyzed data from the Medical Outcomes Study (MOS), a longitudinal study focusing on the care and medical outcomes of patients with specific common chronic conditions . The MOSOur study used MOS data to investigate the relative effect of six different common chronic conditions , congestive heart failure (CHF), chronic respiratory disease, musculoskeletal disease and depression) on measures of physical well being over the course of four years in patients with comorbidities. This data base is comprised of information from respondents with certain chronic medical conditions. There is no information on 'healthy' respondents without any chronic conditions. Therefore we examined the relative effects of selected conditions relative to hypertension alone. We examined the presence of a specified level of decline in physical well being that had been verified to be clinically significant, rather than a change in the PCS score that might be statistically significant but of limited practical importance to patients. In addition, we analyzed the effect of specific diagnoses in the context of the total disease burden in an effort to identify possible 'sentinel' conditions that may specifically contribute to functional decline for patients with multiple comorbidities.The MOS was a four-year observational study that included assessment of health outcomes of chronically ill patients. Details of the study including design, sampling, site selection and clinician recruitment have been previously published,16-18MOS study sites were selected from three cities , from both primary care  and specialty  practices, and from both managed care and fee-for-service payment plans.The original study sample consisted of patients with one or more of five chronic "tracer" conditions  approached during a visit with an MOS clinician during a two-week period in 1986. Of the 28,257 patients originally approached, 20,232 agreed to participate. From this group, patients were selected for follow up on the basis of diagnosis and completion of baseline data collection, as described elsewhere ,19. Of tThe final sample included patients who had complete baseline and four-year follow-up information (including deaths), had completed a medical history questionnaire, and had definitive diagnostic information on their original tracer condition . Patient® Health Survey (SF-36) physical component summary (PCS) scores over four years. We chose this dichotomous outcome to emphasize the clinical importance of anticipating functional decline in patients with chronic medical conditions. Categorical change was defined as a decrease of 6.5 or more points in PCS. This was based on standards for PCS scores in which a change of 6.5 points is outside the 95% confidence interval for PCS scores. Longitudinal change norms for PCS classify patients with +/- two standard errors of measurement (SEM) as "better" or "worse" and those within two SEM as "staying the same" [We analyzed categorical change (worse versus same/better) in SF-36he same" ,20. Chanhe same" ,20. We aBased on published sample sizes specifically calculated to detect differences in PCS between two groups using repeated measures over time, our sample size was adequate to detect a difference of five points in PCS with 80% power  . As thesWe used logistic regression to analyze the independent effect of each main chronic disease on categorical change (worse vs. same/better) in PCS relative to hypertension alone adjusting for the effect of the other main diseases. Logistic regression was again used to assess change in PCS over four years in patients with one, two, three or four or more of the main chronic conditions relative to those with hypertension alone.Due to the selection criteria for the original MOS study , the MOSother than the condition of interest. Using similar modeling, linear regression analysis was used to assess the relative contributions of the explanatory variables. There were no significant interactions between each of the main conditions and the total number of conditions. As age has been shown to be associated with functional outcomes in persons with comorbidities [We completed separate regressions to determine the effect on PCS due to each of the main chronic conditions of interest. In these models, the study population was divided into those with hypertension alone (referent group), those with the main chronic disease of interest (with and without other conditions), and those with any other of the main chronic conditions bidities , we asseAnalyses were additionally adjusted for age, a count from a list of 16 additional chronic conditions (in addition to adjustments for main diseases as mentioned above), poverty level, gender, race, educational level, employment status, and marital status. Subjects who died during the course of the study were assigned a four-year PCS score of zero and included in the 'worse' category. Assignment of a zero PCS sore to participants who died during the course of the study has been discussed by Diehr et al. as a reasonable approach for analyses in which a decline in health is the outcome of interest ,24. FailOf a total of 1574 subjects, 281 individuals carried a diagnosis of hypertension exclusive of any of the other major comorbid conditions, and were defined as the referent population for this analysis.  The remaining 1293 subjects had either one or more of the six comorbid conditions of diabetes, CAD, CHF, respiratory disease, musculoskeletal disease and depression with or without hypertension. In this heterogeneous population, subjects with the main conditions of interest had, on average, 1.8 of the main conditions and 0.8 from a list of 16 additional conditions. Referent subjects with hypertension and no other main conditions of interest had 0.3 additional conditions. The majority of all respondents had starting PCS scores within 1 standard deviation of age/gender norms, with an additional 10% above and 26% below age gender norms. Table Subjects with CHF, diabetes or chronic respiratory disease had increased odds of a clinically significant decline in PCS over 4 years. These odds ratios  were 2.9 , 2.1  and 1.7  respectively Table . Subjectper subject was used as criteria for a clinically significant decline in PCS over time based on previous analyses of the MOS data and on validation of the SF-36® survey instrument [population of subjects adjusted for characteristics of that population. Therefore the statistically significant changes in PCS scores over time resulting from the linear regression analysis may not necessarily be greater than 6.5 pointsAn absolute decrease in PCS of 6.5 points strument . The linLittle increase in the odds of functional decline was evident in individuals with 1,2, or 3 of the main chronic conditions. However having 4 or more of these conditions predicted a decline in PCS  (Table Physical well being is particularly relevant for persons with chronic conditions and the clinicians who care for them. Declines in physical well being may have significant social, emotional and economic repercussions, as they correlate with job loss, high health care utilization and increased mortality . Based oIn this analysis of change in physical well being over time in persons with a variety of chronic medical conditions, we found that specific diagnoses of CHF, diabetes and/or chronic respiratory disease; or the presence of 4 or more chronic conditions, were predictive of a clinically significant decline in PCS. We hypothesize that our findings reflect the different clinical courses of each of these conditions as well as the varying potential for therapeutic interventions in each case. The natural history of CHF, diabetes and some respiratory disease is progressive. Treatment of these conditions is aimed at optimizing long-term health outcomes. Ongoing management involves self-care that is replete with complex concepts and tasks. In contrast, management of CAD and musculoskeletal disease may include the potential for surgical intervention and protocol-driven rehabilitation programs. While some sub-populations of patients with these latter conditions may develop increasing disability, others may experience significant improvement in physical well being over time. For example, increases in PCS scores associated with hip replacement and therapy for low back pain can be in the range of 9.5 and 7.6 respectively . We haveDisease management programs have been successful in improving health outcomes for persons with these and other chronic conditions ,26. HoweIn considering the issue of functional decline, any 'clinically significant change' over time is not only a function the starting and ending levels of functional status. It also is determined by the individual for whom the change has meaning, the instrument used to assess the change, and population norms that provide the context for the observed change ,33. PopuOur findings on depression deserve special mention. Depression in particular and mental health in general are well known to affect the management and outcomes of several chronic medical conditions -36. FurtThe study of comorbidity is, by definition, the study of inter-relationships: between different diseases and between diseases and age or other health-related sociodemographic variables. Caring for persons with chronic illness is, similarly, the care of heterogeneous populations of individuals with multidimensional medical, psychological and social issues. The heterogeneity of this study population may be relevant to the provider willing to sacrifice some internal validity in hopes of generalizing the findings to his or her patient population.Our sample size precluded stratification to investigate the relative contributions of different medical conditions to physical well being in sub-populations defined by specific combinations of conditions, smaller age groups, socioeconomic status or other demographic criteria that might further clarify the interactive nature of the comorbid chronic disease process. It is likely that the effect of CHF, diabetes, chronic respiratory disease or other conditions on measures of health related quality of life differs within different subpopulations. Specifically, vulnerable populations may be more at risk of poor health outcomes due to socioeconomic status or process of care factors than specific disease states ,38. In aDue to the nature of the MOS data base, we compared the effect of selected chronic conditions on physical well being to a referent group with hypertension alone. While the effect of hypertension alone on physical well being over 4 years may be minimal ; it is possible that there is a synergistic effect between hypertension and certain conditions such as CAD and CHF on physical well being over time. If so, the effect of CAD or CHF on PCS scores for persons with these conditions may have been slightly magnified. It is unlikely that this bias would change the overall significance of the results of our analysis.We were unable to directly account for severity of illness for all main chronic conditions either at baseline or follow-up. However statistical adjustments for starting PCS score relative to age/gender norms and the inclusion of patients who died in the final analysis should indirectly account for some degree of severity of illness throughout the study population.As in all investigations, the conclusions reflect the population studied: primarily Caucasian, with a majority above 200% of the federal poverty level, relatively well educated and specifically selected for inclusion on the basis of certain medical diagnoses. Results from this study may not be generalizable to populations with different demographic characteristics and different constellations of comorbid conditions.This analysis suggests that long term physical well being in persons with multiple chronic diseases is a function of both the number and type of medical conditions. Relative to persons with hypertension alone, those who carry diagnoses of CHF, diabetes and chronic respiratory disease have an increased risk of a decline in physical well being over 4 years. The presence of CAD, musculoskeletal disease or depression does not predict a similar decline.For the primary care physician for whom 'comorbidity' implies an exponentially increasing ratio of items on the problem list to available time during the office visit, the specific diagnoses of CHF, diabetes and chronic respiratory disease may serve as triggers for management decisions. Clinicians who care for patients with these common conditions should be alert to the possibility that a proactive approach incorporating generalizable principles of disease management may either attenuate this loss of function or help the patient and family anticipate future needs.EB participated in the design of the investigation, performed analysis and drafted the manuscript. MB participated in the design of the investigation, performed additional analysis, data management, and manuscript revision. JW supervised the original data collection and MOS investigations and assisted in manuscript revision. JS participated in the design of the investigation, advised on the analysis and assisted in manuscript revision. All authors read and approved the final manuscript.


Children in urban public housing are at high risk for asthma, given elevated environmental and social exposures and suboptimal medical care. For a multifactorial disease like asthma, design of intervention studies can be influenced by the relative prevalence of key risk factors. To better understand risk factors for asthma morbidity in the context of an environmental intervention study, we conducted a detailed baseline evaluation of 78 children (aged 4–17 years) from three public housing developments in Boston.Asthmatic children and their caregivers were recruited between April 2002 and January 2003. We conducted intake interviews that captured a detailed family and medical history, including questions regarding asthma symptom severity, access to health care, medication usage, and psychological stress. Quality of life was evaluated for both the child and caregiver with an asthma-specific scale. Pulmonary function was measured with a portable spirometer, and allergy testing for common indoor and outdoor allergens was conducted with skin testing using the prick puncture method. Exploratory linear and logistic regression models evaluating predictors of respiratory symptoms, quality of life, and pulmonary function were conducted using SAS.We found high rates of obesity (56%) and allergies to indoor contaminants such as cockroaches (59%) and dust mites (59%). Only 36% of children with persistent asthma reported being prescribed any daily controller medication, and most did not have an asthma action plan or a peak flow meter. One-time lung function measures were poorly correlated with respiratory symptoms or quality of life, which were significantly correlated with each other. In multivariate regression models, household size, body mass index, and environmental tobacco smoke exposure were positively associated with respiratory symptom severity (p < 0.10). Symptom severity was negatively associated with asthma-related quality of life for the child and the caregiver, with caregiver (but not child) quality of life significantly influenced by caregiver stress and whether the child was in the intensive care unit at birth.Given the elevated prevalence of multiple risk factors, coordinated improvements in the social environment, the built environment, and in medical management would likely yield the greatest health benefits in this high-risk population. Asthma morbidity and mortality have been increasing in recent years, with a disproportionate impact on urban minority children -4. HospiRegardless of the relative contributions of these and other factors, children in urban public housing are important to consider, because they likely have elevated exposures across numerous domains, some of which could be addressed through development-wide interventions. However, there has been only limited evaluation to date of asthma in this high-risk subpopulation ,11.The Healthy Public Housing Initiative (HPHI) is a collaborative effort that includes the Boston Housing Authority (BHA), West Broadway and Franklin Hill Tenant Task Forces, Committee for Boston Public Housing, Boston Public Health Commission, Boston University and Harvard University Schools of Public Health, and Tufts University School of Medicine. A primary goal of HPHI is to evaluate the effectiveness of interventions in reducing known asthma triggers and improving the health of pediatric asthmatics in public housing in Boston.The effectiveness of environmental interventions in this context will clearly depend on the prevalence of environmentally-linked risk factors within this cohort , as well as the prevalence of other risk factors for asthma morbidity. In addition, when evaluating the efficacy of environmental interventions, numerous health endpoints may be valuable to consider. Health care utilization will inform cost-effectiveness analyses, typically driven by infrequent but severe events such as hospitalizations ,13. On tPast studies have demonstrated varied relationships among these parameters. For example, percent of predicted forced expiratory volume in one second (FEV1%) has been correlated with asthma attacks  and sympMore broadly, past studies have generally not considered the full array of risk factors and health endpoints for inner-city asthmatics. The most comprehensive assessment to date has been the National Cooperative Inner-City Asthma Study (NCICAS), which evaluated many similar endpoints as our study in a longitudinal baseline assessment , althougThus, the objective of our analysis is to characterize the baseline risk factors and health status of a cohort of asthmatic children enrolled in an intervention study based in public housing developments in Boston and to determine concordance between and risk factors for key health endpoints . We hypothesize that risk factors associated with housing quality and psychosocial stress will be elevated in our cohort when compared with reference groups, and that quality of life will be significantly influenced by asthma symptom severity and other caregiver characteristics.We recruited asthmatic children from the Franklin Hill, West Broadway, and Washington Beech public housing developments in Boston . Recruitment was coordinated by Community Health Advocates, residents of the developments or surrounding neighborhoods who were involved in outreach and data collection, following training about asthma, its risk factors, and interviewing techniques. Recruitment methods included advertised enrollment open houses, community meetings, mailbox drops for flyer circulation, and door knocking. Any children between the ages of 4 and 17, who lived in the developments, had self-reported doctor-diagnosed asthma, and who were willing to enroll in a longitudinal intervention study, were eligible. Enrollment occurred between April 2002 and January 2003. Written informed consent was obtained from all caregivers, with assent forms completed by children above the age of 8, and the protocols were approved by the institutional review boards of all three participating universities.In the intake interview, the caregiver  was asked about family demographics, child and family asthma history, access to health care, exposure to smoking, and medication usage, with questions taken from NCICAS when possible to facilitate comparability . BecauseThe Paediatric Asthma Quality of Life Questionnaire (PAQLQ)  was usedth percentile), at risk of overweight (85th to 95th percentile), or not at risk (below 85th percentile).Pulmonary function was assessed using the NDD EasyOne Diagnostic portable spirometer , an instrument which correlates well with office-based spirometry . AlthougD. pteronyssinus, D. farinae, Alternaria, Aspergillus fumigatus, Cladosporium, and Penicillium.Allergy testing was conducted using similar methods as NCICAS , with skFor our exploratory regression models, we evaluated the relationship between our primary outcome measures  and a subset of demographic variables, intrinsic risk factors, health care risk factors, physical risk factors, and social risk factors. Furthermore, we considered FEV1% as a potential predictor of the respiratory symptom score, and both FEV1% and the respiratory symptom score as hypothetical predictors of quality of life. We treated FEV1% in a logistic regression, using 80% of predicted FEV1 as the cutoff for low FEV1%, and evaluated other health outcomes in linear regressions. Given missing data and numerous covariates, we conducted an initial screen using univariate regressions (retaining variables for which p < 0.2), and then constructed a multivariate stepwise regression with p < 0.1 as the entry and exit criteria. Finally, for covariates with extensive missing data, we constructed multivariate regressions both with and without these terms to evaluate the sensitivity of our findings. All statistical analyses were conducted using SAS version 8.02, using PROC REG for linear regressions and PROC LOGISTIC for logistic regressions.In total, 78 children from 61 households were enrolled in the HPHI intervention study. As indicated in Table Considering prominent non-environmental risk factors associated with asthma, 16% of children were in an intensive care unit upon birth and 10% were on a respirator. Seventy percent of children had a parent or grandparent with asthma, while 43% had eczema or hay fever and 34% had a family history of eczema or hay fever. For the 75 children with recorded height and weight, 56% were categorized as overweight and 9% were categorized as at risk of overweight. Forty-two percent of children lived with a smoker, and 45% of children were around smokers at least several times per month.D. pteronyssinus (59%), cockroach (59%), D. farinae (50%), and tree pollen (30%)  who underwent allergy testing, 78% were sensitized to at least one of the tested substances, with the most prevalent allergies including ) Figure . Only 44For the 70 children for whom we received information about medications, 87% were taking short-acting beta-agonists at the time of the survey. Thirty-one percent of these children on beta-agonists also reported the use of any long-term asthma control medication . All children who did not report the use of beta-agonists indicated that they were using long-term control medication. Seventeen percent of children indicated that they were currently taking allergy medication as part of their asthma control. Of note, for the remaining eight children, we could not ascertain whether non-responses indicated lack of any medication or lack of recall/medication availability.There were additional factors and barriers that indicated potentially sub-optimal asthma care Table . AlthougIn addition, 71% of caregivers indicated that at least one of seven barriers impeded asthma management for their children during the last six months. The most frequently cited barrier was that the pharmacy did not have their asthma medication (38%), followed by the asthmatic child either not being home when it was time to take the medicine (29%) or refusing to take the medicine (26%). The cost of the medication was cited as a barrier by only 12% of caregivers, the lowest percentage among the seven questions, an indication that MassHealth/Medicaid coverage was largely viewed as adequate.On the five-point social cohesion scale, there was a significant difference across developments, with a lower mean value at Franklin Hill than at West Broadway or Washington Beech Figure . There wWe also found significant differences across developments in exposure to violence. We asked caregivers whether they were afraid that they or their children would be hurt by violence in their neighborhood, whether they have had violence used against them or other household members in their neighborhood, and whether they fear letting their children play outside in their neighborhood because of community violence Table . In all As indicated in Table For the subset of 49 children age six or older able to perform spirometry, the mean FEV1% was 88% . Twenty-nine percent of children had FEV1 less than 80% of predicted, though no values were less than 60% of predicted. The mean PEF% was 97% . Twelve percent of children had PEF less than 80% of predicted, with none having PEF less than 60% of predicted. As would be anticipated, these two measures were well correlated (Spearman correlation of 0.69), with all but one of the children with PEF below 80% of predicted also having FEV1 below 80% of predicted.Of note, of the children under 6 tested, seven (30%) were able to record acceptable spirometry values. Only a child who had been recently hospitalized for asthma exacerbation had reduced FEV1 and PEF, both below 60% of predicted . Given pBased on the four questions addressing recent asthma symptoms . The range across children was substantial, with overall quality of life scores ranging from 1.4 to 7 Figure . SimilarThe quality of life questionnaires provide insight regarding the perceived burdens of asthma beyond the aggregate quality of life scores. For example, in the EQ5D responses, 31% of children were reported to be moderately or extremely worried or depressed, with 50% of children having problems doing their usual activities and 53% of children in moderate or extreme pain or discomfort. In addition, 39% of caregivers reported that their child's asthma interfered with their job or work around the house at least some of the time, with 62% reporting sleepless nights related to their child's asthma at least some of the time.One key question is whether any of the primary measures of asthma severity  are significantly correlated with one another in a cross-sectional baseline assessment. For lung function, we consider both FEV1% and PEF%. Quality of life outcomes include the aggregate PAQLQ score for the child, aggregate EQ5D and VAS scores for the child, and the aggregate PACQLQ scores for the caregiver. For respiratory symptoms, we develop a symptom score reflecting the responses to the questions in Table Considering the Spearman correlations among these key covariates Table , the lunGiven our sample size, missing data for selected covariates , and the number of factors hypothesized to influence our outcome measures, we consider our regression analyses to be exploratory in nature. The goal is to better understand the above correlations and factors that might influence the relationships among our outcome measures. For each of the outcome measures, we evaluate a subset of demographic variables , intrinsic risk factors , health care risk factors , physical risk factors , and social risk factors . It should be noted that some of these covariates may be direct causative agents, while others represent proxies or outcomes that could be influenced by asthma severity (such as caregiver stress).The results from this analysis are summarized in Table Alternaria allergy prevalence is quite low, indicating that the most effective interventions might differ between HPHI and NCICAS. In addition, exposure to violence and fear of violence is slightly higher than reported in NCICAS. Although the fraction of children with persistent asthma who are adequately medicated is quite low (36%) and indicative of poorly managed asthma, this figure is actually higher than the percentages reported in other studies, indicating that this is a pervasive problem in asthma management.To address our first hypothesis and interpret this baseline characterization of asthmatic children in public housing enrolled in an intervention study, it is instructive to compare the prevalence of selected risk factors with those reported in previous studies ,25,35-39More broadly, our findings regarding medication usage coupled with the prevalence of children with mild, moderate, or severe persistent asthma and the frequency of respiratory symptoms suggest that this population is being treated sub-optimally. NHLBI guidelines  indicateAlthough exploratory in nature, our correlation and regression analyses provide some useful insights regarding the factors associated with various measures of asthma severity in our cohort. FEV1% was not strongly correlated with other health measures, and was only weakly associated with a subset of risk factors in univariate regressions. As this measurement was taken at a single point in time at varying times of day and seasons, a weak relationship is unsurprising, especially when compared with symptoms over a two week period . The lacThe respiratory symptom score was moderately associated with a number of risk factors in univariate regressions, with all coefficients in the anticipated directions .The robust multivariate relationship with household size and presence of smokers could be indicative of the influence of indoor air quality or respiratory infection related to unit crowding, with the association between obesity and asthma symptom frequency and severity in agreement with past studies ,41. The There are some clear limitations in interpreting the results of our investigation. First, given the numerous risk factors for asthma, both the univariate and multivariate regressions must be interpreted with caution. For example, fear of violence in the neighborhood was significantly associated with reductions in caregiver quality of life in univariate regressions (p = 0.01), but given a strong positive association between fear of violence and caregiver stress as well as the child's respiratory symptom score, this term did not enter into the final regression model. Thus, both the significant and insignificant terms from our regression models should be interpreted with caution, as they may not reflect causal relationships.In addition, the participants in our study represented a convenience sample of individuals from three selected public housing developments in Boston, who were interested in enrolling in a longitudinal intervention study. It is unclear whether our participants are representative of asthmatic children across those developments, or more broadly, asthmatic children in public housing. Families who enrolled in our study may have been more desperate for help given current asthma conditions, or may have had greater confidence in the ability of a research project to improve their child's health. A comparison between the demographics of our study population and the demographics of the developments indicates that the age distribution and racial/ethnic composition are similar, but there is no way of knowing if our cohort represents typical characteristics of all asthmatic children across the BHA.In addition, we have evaluated the prevalence of risk factors and correlations among key indicators of respiratory health in a cross-sectional survey without a defined control group. Thus, while we have found an elevated prevalence of obesity and allergies to cockroach and dust mites when compared with other populations, we cannot infer a causal relationship with asthma. However, this comparison illustrates the relative importance of various risk factors internal to our intervention study. In addition, the correlations among respiratory health measures will not necessarily be identical cross-sectionally and longitudinally. For an intervention study, the crucial question is whether observed changes in lung function would occur simultaneously with changes in quality of life or respiratory symptoms, and knowledge of the cross-sectional correlations is not necessarily informative. That being said, the correlations among health endpoints Table  and the In spite of these limitations, our study provides some important and unique information. To our knowledge, this is the most comprehensive evaluation focused on asthmatic children in public housing to date. Although many attributes of public housing are similar to low-income private housing, the viability of large-scale interventions in public housing based on common indoor environmental exposures and centralized management makes it important to characterize similarities and differences from other low-income populations. More broadly, our study demonstrates that a community-based participatory research paradigm, with members of the community conducting most of the primary data collection, is able to gather valid and meaningful data. Given that recruitment and retention of our study population would have been quite difficult without involvement from community members, it is clear that community-based participatory research is essential for detailed evaluations of asthma in public housing.The findings from this cross-sectional baseline evaluation of asthmatic children in public housing in Boston have multiple policy implications. The inadequacy of medical care for a majority of asthmatic children indicates that medical interventions might yield substantial improvements in asthma status for poor children. The fact that significant differences existed in adequacy of medical care for asthma across housing developments indicates likely variability across providers . In addition, the high prevalence of cockroach and dust mite allergies indicates that interventions aimed at reducing or eliminating these triggers are likely to provide health improvements. This reinforces the expectation that interventions in public housing, a setting with high asthma prevalence and high prevalence of allergic responses to indoor contaminants, are likely to be meaningful and effective. Finally, the responses to the psychosocial questions as well as the percentage of caregivers who feared neighborhood violence and modified their children's activities as a result indicates that violence and stress are substantial risk factors for both respiratory health and other outcomes. Our findings point to the need for coordinated improvements in the social environment, the built environment, and in medical management.Future investigations should similarly evaluate asthma risk factors and severity in other public housing settings to determine whether our conclusions provide generalizable and relevant information for regional or national housing authorities in considering intervention strategies. In addition, a longitudinal comparison of correlations among measures of asthma severity would help determine whether conclusions drawn from a cross-sectional evaluation are robust.We conclude that asthmatic children enrolled in a public housing-based intervention study would likely benefit from a coordinated intervention focused on reduction of indoor allergens , improved medical management, and increased social support.AQL – Asthma-related quality of lifeBHA – Boston Housing AuthorityBMI – Body mass indexFEV1 – Forced expiratory volume in one secondFEV1% – Percent of predicted forced expiratory volume in one secondHPHI – Healthy Public Housing InitiativeNCICAS – National Cooperative Inner-City Asthma StudyNHLBI – National Heart, Lung, and Blood InstituteNICU – Neonatal intensive care unitPACQLQ – Paediatric Asthma Caregiver's Quality of Life QuestionnairePAQLQ – Paediatric Asthma Quality of Life QuestionnairePEF – Peak expiratory flowPEF% – Percent of predicted peak expiratory flowVAS – Visual analogue scale on EuroQol EQ5D quality of life questionnaireThe authors declare that they have no competing interests.JIL participated in study design and implementation, conducted statistical analyses, and drafted the manuscript. LKWH designed and administered survey instruments, provided support to participants as a nurse/case manager, and contributed to analyses related to adequacy of health care. JEC contributed to survey design and analyses for psychosocial risk factors. RED completed a literature review, drafted text, and conducted statistical analyses. SS provided asthma severity classifications and related analyses and conducted allergy testing. HPH collaborated in the conception and design of the study, and co-directed data collection and study coordination. All authors read and approved the final manuscript.


Although leg length has been shown to contribute to the Tc, the role of age is controversial. More confusing, none of the previously performed studies have used strict criteria to rule out subclinical neuropathy, so the results could be misleading. This study has been performed to determine the influence of leg length and age on Tc among a carefully selected group of healthy volunteers.central loop of the gastrocnemius-soleus H-reflex latency  and 14 females (35%) with the age range of 19–65 years (Mean ± SD: 37 ± 10.7) and leg length range of 29.5–43 centimeters (36.4 ± 3.4).c was 6.78 ± 0.3. We found a significant correlation between Tc and leg length , no significant correlation was found between age and Tc , also we obtained the regression equation as: Tc = 0.04L + 5.28Mean ± SD for Tc. Future studies are required to delineate other contributing factors to Tc.in contrast to leg length, age was not correlated with T Pease et al [1 loop latency or Tc) and suggested it might be promising in the diagnosis of S1 radiculopathy [The H-Reflex evaluates Sse et al  were theulopathy .c has been the subject of few studies, and as far as we know, only 5 articles [c. Leg length has been shown to have a significant effect on Tc. It is controversial whether age entails a similar effect. Wang et al [Unfortunately, Tarticles -6 have bc.The current study has been performed to determine the influence of leg length and age on Tp). We defined our exclusion criteria as: history of sacral radiculopathy or diabetes mellitus or any other disease with potential to cause neuropathy, any abnormality in neurological or musculoskeletal physical examination, or any of the following findings: PNCV less than 40 m/s, PDML more than 5 ms or prolonged Tp . The ground electrode was posed proximal to E1 and a disc electrode (anode) was placed on the anterior superior iliac spine. Then we inserted a monopolar 70mm needle (cathode) at a point 1cm medial to the posterior superior iliac spine, perpendicular to the frontal plane, and retracted it just a little after reaching the sacrum. Stimulus duration of 1 ms at 0.5 HZ was then applied while increasing current intensity to obtain both H and M waves simultaneously. M wave is the earlier wave and H is the later one. The interpeak latency was measured in milliseconds (ms) and recorded as Tc. This measurement was only performed on the right lower extremity.For obtaining Tc was assessed by multiple regression model. The analyses were performed using SPSS 10.0 software. Kolmogrov-Smirnov test was used for evaluating the normal distribution of the variables.Descriptive statistics were applied to depict Mean ± SD of age, leg length and Tc. The independent effect of leg length and age on TFrom 46 subjects who volunteered to participate in this study; five cases were excluded after history taking and physical examination  and one case after electrophysiologic evaluation; thus we completed the study with 40 subjects. Subjects' characteristics are shown in table The group consisted of 26 males (65%) and 14 females (35%). Kolmogrov-Smirnov test showed normal distribution of the variables.c and leg length .You are provided with the information below: (Mean ± SD) Age: 37.0 ± 10.7 years (range: 19–65); leg length: 36.4 ± 3.4 cm (range: 29.5–43); Tc= 6.78 ± 0.3 There was a significant correlation between Tc and age  We also found this regression equation: Tc = 0.04L + 5.28 There was no correlation between Tc.In this study we found a significant correlation between leg length and Tc, but we were unable to show such a relation between age and Tc [ms which is very close to our results (Tc= 6.78 ± 0.3). They didn't specifically consider the leg length, age or any other potential confounding variables to Tc.Pease et al were the first, studied Tc ,4, and rc: 6.8 ms and its SD: 0.33 ms, again close to our results. They also reported that Tc and person's height were correlated but didn't study any correlation between age and Tc.Zhu et al  evaluateWang et al  evaluatec = 0.02A + 0.003H + 0.92 (H: Height and A: Age), and stated that age is a contributing factor on Tc.Tc ± SD was 6.9 ± 0.4; two regression equations were also suggested: Tc = 0.097Tp + 4.045 and Tc = 0.051L + 4.92 (L=leg length in centimeters); results are close to ours, and age was not found to affect Tc.Another research was performed by Ghavanini et al , in whicA summary of the above data plus detailed demographic data are provided in the table c. we didn't control, randomize or observe other possible confounding (contributing) factors with potential to affect this parameter.In this study we focused on age and leg length as potential contributing factors on the Tc , that is compatible to a previous published work [We observed a significant correlation between leg length and Tc and age, the question might have been raised that subclinical neuropathy of old age could have been contributive; obviously, this is not the case in our study.Had we found any association between TAlthough F-wave has been used to evaluate the possibility of proximal neuropathy; it was not measured in this study. Alternatively, we measured H-reflex latency to exclude proximal neuropathy .It should be emphasized that noninvasive methodologies for the diagnosis of subclinical S1 radiculopathy are now available . It is ac. It may be reasonable to consider leg length for calculating Tc and to "narrow" the normal limits.We found that between age and leg length, only the latter can affect Tc according to leg length.Further studies with larger sample sizes are required for detecting other contributing factors and standardizing TNone declared.c: central loop of the gastrocnemius-soleus H-Reflex latencyTp: gastrocnemius-soleus H-Reflex latencyTPNCV: right peroneal nerve conduction velocityPDML: right peroneal nerve distal motor latencySS: examining the cases, calculation of TC, writing the paper.MRAG: suggesting the research, supervision and helping with calculation of TC.AA: examining the cases, calculation of TCPJ: statistical consultant The pre-publication history for this paper can be accessed here:


To identify differentially expressed genes, it is standard practice to test a two-sample hypothesis for each gene with a proper adjustment for multiple testing. Such tests are essentially univariate and disregard the multidimensional structure of microarray data. A more general two-sample hypothesis is formulated in terms of the joint distribution of any sub-vector of expression signals.By building on an earlier proposed multivariate test statistic, we propose a new algorithm for identifying differentially expressed gene combinations. The algorithm includes an improved random search procedure designed to generate candidate gene combinations of a given size. Cross-validation is used to provide replication stability of the search procedure. A permutation two-sample test is used for significance testing. We design a multiple testing procedure to control the family-wise error rate (FWER) when selecting significant combinations of genes that result from a successive selection procedure. A target set of genes is composed of all significant combinations selected via random search.A new algorithm has been developed to identify differentially expressed gene combinations. The performance of the proposed search-and-testing procedure has been evaluated by computer simulations and analysis of replicated Affymetrix gene array data on age-related changes in gene expression in the inner ear of CBA mice. The dimension of X is typically very high relative to the number of observations (replicates of experiment). The standard practice is to test the hypothesis of no differential expression for each gene. Formulated in terms of the marginal distributions of all components of X, this hypothesis means that the expression levels of a particular gene are identically distributed under two (or more) experimental conditions. It is commonly believed that the only challenging problem here is that of multiple statistical tests, because the corresponding test statistics computed for different genes are stochastically dependent. This problem is discussed in  xxxx or [Executable file] 0 xxxx. The input file "simulation04_ui.txt" is designed for simulation experiments. To conduct simulations, one has to prepare an input file with the name: XXX_simu_ui.txt, where XXX is a string that follows the naming convention of computer files. An input file for data analysis with the name XXX_ui.txt is also needed. To run simulations, type: [executable file] 1 xxxx.Click here for file


We also examined the relationship between chromatin composition and replication timing of artificial chromosomes.Human centromere regions are characterized by the presence of alpha-satellite DNA, replication late in S phase and a heterochromatic appearance. Recent models propose that the centromere is organized into conserved chromatin domains in which chromatin containing CenH3 (centromere-specific H3 variant) at the functional centromere (kinetochore) forms within regions of heterochromatin. To address these models, we assayed formation of heterochromatin and euchromatin on Heterochromatin factors (histone H3 lysine 9 methylation and HP1α) were enriched on artificial chromosomes estimated to be larger than 3 Mb in size but depleted on those smaller than 3 Mb. All artificial chromosomes assembled markers of euchromatin (histone H3 lysine 4 methylation), which may partly reflect marker-gene expression. Replication timing studies revealed that the replication timing of artificial chromosomes was heterogeneous. Heterochromatin-depleted artificial chromosomes replicated in early S phase whereas heterochromatin-enriched artificial chromosomes replicated in mid to late S phase.Centromere regions on human artificial chromosomes and host chromosomes have similar amounts of CenH3 but exhibit highly varying degrees of heterochromatin, suggesting that only a small amount of heterochromatin may be required for centromere function. The formation of euchromatin on all artificial chromosomes demonstrates that they can provide a chromosome context suitable for gene expression. The earlier replication of the heterochromatin-depleted artificial chromosomes suggests that replication late in S phase is not a requirement for centromere function. In the post-sequencing phase of genome characterization, it is important to understand the contribution of non-coding sequences to higher-order genome structure and stability. Maintenance of genome integrity and the faithful transmission of genetic information in mitosis and meiosis are essential to organism survival and are critically dependent on two repetitive chromosomal elements. Telomeres protect against chromosomal truncation or fusion events , while cde novo centromeres on human artificial chromosomes following transfection of some types of alpha-satellite sequences into human cells [Defining the minimal DNA sequences required for centromere function on a normal human chromosome has proved challenging, owing to the complex nature of inter- and intra-chromosomal homology and variability in genomic DNA content near the primary constriction. Common to all normal human centromeres are large amounts of alpha-satellite DNA, which is comprised of a family of diverged 'monomers' of around 171 base-pairs (bp) that have been amplified in multimeric groups (higher-order repeats) on different chromosomes to form chromosome-specific arrays typically megabases in length -17. In aan cells -14.Paradoxically, despite conservation of the functional role of the centromere in every eukaryotic cell, DNA sequences at eukaryotic centromeres are quite divergent in sequence even between closely related species ,25. AlthRecent evidence suggests that a complex system of epigenetic modifications based on histone variants and histone tail modifications is important for centromere activity , in Schizosaccharomyces pombe. Detailed analyses of a 40-kilobase (kb) S. pombe centromere revealed that it encompasses both the kinetochore, as defined by the exclusive association of Cnp1, the fission yeast CenH3, with the central core element [The most completely studied complex eukaryotic centromere at the molecular level is that of the fission yeast  element  and adja element  that are element -40. With element .per se.There is substantial evidence that HP1 is involved in setting up and/or maintaining a repressed chromatin state in several epigenetic systems (reviewed in ). HP1 prtrans-acting chromatin factors in human centromere function is through the construction of detailed genomic maps of human centromeric regions and evaluation of their associated proteins [de novo centromeres on human artificial chromosomes without large amounts of adjacent heterochromatin demonstrate the same chromatin characteristics as either normal human centromeres or human artificial chromosomes with large amounts of heterochromatin.One way to address the interacting and complementary role(s) of DNA sequence and proteins ,19,56,57proteins  as toolsproteins ,13,58. Hde novo centromere formation and function is likely to be generally conducive to gene expression, as will probably be required for either gene-transfer experiments and/or functional genomic applications of the artificial chromosome technology. Further, the data raise the possibility that functional centromeres may adopt a novel chromatin state that is, contrary to what has been long assumed, quite distinctive from that of conventional heterochromatin.In the present study, we have characterized the nature of heterochromatin and euchromatin formed on a series of human artificial chromosomes derived from higher-order repeat alpha satellite from chromosomes X or 17 ,14. Whilde novo centromere assembled from the transfected DNA, as well as at least one copy of a functioning gene used as a selectable marker. Together, this panel of artificial chromosomes provides an opportunity to examine the nature of heterochromatin and euchromatin assembled on the transfected DNA sequences. The high mitotic stability and de novo composition of artificial chromosomes generated from D17Z1  or DXZ1 (X-4 and X-5) have been described [in situ hybridization (FISH), artificial and host chromosome segregation products can be measured and nondisjunction or anaphase lag defects recorded.To examine the chromatin composition of human artificial chromosomes, we used a panel of artificial chromosomes formed after transfection with vectors containing either synthetic chromosome 17 (D17Z1) or cloned X chromosome (DXZ1) alpha-satellite sequences ,14. EachIn X-4 and X-5, artificial chromosomes mis-segregated in 1.8% and 2.4% of cells, respectively  was applied to metaphase spreads. Methylation of lysines at these sites has been associated with formation of repressive chromatin, including pericentric heterochromatin in mouse cells -53,59,60per se. Such a model [In a parallel approach, we examined the distribution of HP1α in four lines containing D17Z1-based artificial chromosomes. Each line was stably transfected with a Myc-epitope tagged form of HP1α  to permit detection of HP1α using an anti-Myc antibody. The smaller artificial chromosomes stained very weakly (at a level similar to that of the staining on the euchromatic chromosome arms), well below the levels of HP1α detected at the centromeric region of the endogenous chromosome 17s Figure . As seen a model ,3 is alsFor their potential use as gene-transfer vectors or as general vehicles suitable for interrogation of genome function, human artificial chromosomes must also be capable of forming euchromatin to support gene expression. Indeed, one would hypothesize that at least small amounts of transcriptionally active chromatin must form during artificial chromosome formation to permit expression of the selectable marker gene(s) contained on the transfected constructs ,12,14. Iper se.As a test for formation of permissive chromatin on artificial chromosomes, we stained metaphase spreads with an antibody that recognizes histone H3 dimethylated at lysine 4 (H3DimK4). All artificial chromosomes tested stained positively for H3DimK4 modifications Figure ; Table 2Previous structural analyses of artificial chromosomes indicate that they consist of input DNA multimers arranged as blocks of alpha-satellite DNA interspersed with vector sequences ,11,12. TWhile the genomic determinants of potential origins of DNA replication in the human genome, as well as of their timing of replication during S phase, are still not well understood, the generally accepted paradigm is that expressed sequences replicate in the first half of S phase, while non-expressed sequences replicate in the second half . ConsistWhile there was overlap between artificial chromosome replication timing patterns and those of the host 17 centromere regions during mid S phase Table , we founcis- and trans-acting factors necessary for chromosome segregation and offer potential for both functional genomics and gene-transfer applications. The artificial chromosomes we used contain defined alpha-satellite DNA sequences [de novo centromere on artificial chromosomes may reveal the critically important components and may help distinguish between those features that are characteristic of the functional centromere itself and those that are markers of the surrounding heterochromatin. Such a distinction is extremely difficult in normal human chromosomes but should be enhanced by the ability to generate a variety of different artificial chromosomes made with different input sequences.Human artificial chromosomes provide a novel system for analyzing equences ,14. StudRecent detailed molecular studies in the fission yeast have revealed that such epigenetic factors are critical for centromere function. The fission yeast CenH3, Cnp1, is deposited only at the central core domain, while heterochromatin  forms on the surrounding inverted repeats ,36,41. TDrosophila and human chromosomes [While the data presented here are largely consistent with these models, they permit two important refinements. First, large amounts of heterochromatin  are not required for effective chromosome segregation during mitosis; indeed, the small artificial chromosomes examined here do not contain detectable amounts of H3TrimK9/K27  replicated in the first 2 hours of S phase  these fCytological data indicate that the amount of CENP-A modified chromatin  is similar on endogenous human chromosomes and on all artificial chromosomes regardless of the amount of total alpha satellite present. This suggests that the amount of CENP-A chromatin and/or the size of the kinetochore is regulated and/or limited in some manner -14,58,61To evaluate their potential for characterization of genome sequences and, eventually, for gene transfer or gene therapy applications, we sought to determine the extent of transcriptionally competent chromatin formation in artificial chromosomes. Epigenetic modification of histone H3 by dimethylation at lysine 4 (H3DimK4), a marker of transcriptionally competent chromatin, was present on all artificial chromosomes tested. This contrasts with the staining pattern associated with the centromere regions on human metaphase spreads, where this modification is largely undetectable, probably reflecting the general absence of genes mapping to centromere regions  detected in the anaphase assay . All artificial chromosomes were formed in human HT1080 cells. Cell lines were grown as described [Characterization of cell lines containing mitotically stable human artificial chromosomes formed after transfection with either synthetic D17Z1 arrays (PAC17HT1.E29 (17-E29), PAC17HT1.D34 (17-D34), BAC17HT4.B12 (17-B12) or cloned DXZ1 sequences  have been described previously . The artAnaphase assays used to directly measure chromosome segregation defects in 17-B12 and 17-C20  and subcloned using standard techniques [bsr marker gene. Clonal lines (17-C20-1.B22 and 17-B12-1.B10) resistant to 4 μg/ml Blasticidin S HCl (ICN) were selected and expanded. Confirmation of Myc-tagged HP1α expression was by immunofluorescence using a mouse monoclonal anti-Myc antibody (Invitrogen).The nucleotide sequence of human HP1α (NCBI Nucleotide database: S62077) was used in BLAST searches against entries in the human expressed sequence tag (EST) database using the NIH BLAST server . A reprechniques  into pcDchniques  that carMetaphase spreads were prepared for immunofluorescence using previously described protocols . Primary2 flasks for 2 h intervals. Following three PBS washes, medium supplemented with 50 μM thymidine (Sigma) was added. Cells were left in thymidine-containing medium until chromosome harvest. At appropriate intervals, colcemid was added to block cells in mitosis. Cells were harvested and fixed in 3:1 methanol/acetic acid. Metaphase spreads on microscope slides were baked for 1 h at 60°C. Primary anti-BrdU antibody (Roche) was added for 1 h at room temperature. Rhodamine-donkey-anti-mouse secondary antibodies (Jackson ImmunoResearch) were used to visualize sites of BrdU incorporation. Typically, 25 metaphase spreads were captured following BrdU detection and their coordinates on the slide recorded. Subsequent analyses with a D17Z1 probe were used to confirm identity of artificial or endogenous chromosomes.Cells were pulsed with 10 μM BrdU (Roche) in T25 cm


TP53 gene. Isolated and multiplexed PCR products, 108 and 92 bp long, from exons 7 and 8, respectively, were obtained from 24 different samples. Single-stranded target DNA was then prepared from isolated or multiplexed PCR products, through cyclic DNA synthesis. Independent ssDNA's were annealed with the corresponding pairs of labeled stacking oligonucleotides to create partially duplex DNA having a 7-nt gap, which contains the sequence that will be interrogated by the capture probes forming double tandem hybridization. In the case of multiplexed ssPCR products, only two stacking oligonucleotides were added per target, therefore the gap for the PCR products having two consecutive codons to be interrogated in exon 7 was 12 nt long, so only single tandem hybridization was produced with these respective probes.We have developed an oligonucleotide microarray (genosensor) utilizing a double tandem hybridization technique to search for 9 point mutations located in the most frequently altered codons of the 18 codon substitutions were found by DNA sequencing. In 13 of them a perfect correlation with the pattern of hybridization was seen . In 3 other cases a mutation was falsely suggested by the combination of the absence of the wild type signal along with a false signal in the other probe. In the other 2 cases the presence of the mutation was not detected due to the production of a false hybridization signal with the wild type probe. In both cases  relatively stable mismatched target/probe duplexes should be formed. These problems could be avoided by the addition of probes to improve the performance of the array.TP53 microarray employing short (7-mer) probes, used in combination with single or double tandem hybridization approach and a simple or multiplex target preparation method, can identify common TP53 missense mutations from a variety of DNA sources with good specificity.Our results demonstrate that a simple  Cancer is a group of diseases characterized by uncontrolled cell growth. According to The World Cancer Report, cancer rates could further increase by 50% to 15 million new cases in the year 2020 ATP was removed by filtration through an Ultrafree spin filter  , and the retained DNA was dissolved in 20 μL of 1X SSC.To prepare labeled partially duplex (gapped) target DNA's, each pair of γIn the case of multiplexed targets, only 4 stacking oligonucleotides  were added, one pair for each exon in the study, forming gap sizes of 12 and 7 nt for exons 7 and 8, respectively. Thus, with exon 7 a single tandem hybridization was conducted with the glass-tethered 7-mer capture probes, whereas with exon 8 double tandem hybridization was conducted.TP53 targets under study, two negative controls consisting of 7-mers targeted to the wild type sequences from codons 157 and 158 of TP53 gene, plus one blank spot without probe. As depicted in Fig. TP53 capture probe was arrayed in triplicate (to assess the reproducibility of the results) in an upward direction starting from position 1 and the printing was divided into three groups. Positions 1–14 were printed first, then the pattern was repeated twice more across the slide. Position 15 was left empty  and served as a negative glass control. After spotting, each slide was washed with water to remove unbound oligonucleotides, air-dried, then stored in a dessicator at room temperature. This attachment procedure results in a surface density of 1010-1011 molecules mm2, which corresponds to intermolecular spacing of about 30–100 Å across the surface [Glass microscope slides were prepared by soaking in hexane 20 min, then 20 min in absolute ethanol followed by drying 5 h at 90°C. Oligonucleotide probes containing 3'-terminal aminopropanol modification were dissolved in sterile water to a final concentration of 20 μM, and 200 μl droplets of each probe were applied to the glass slides using a model 417 Affymetrix  arrayer to place submicroliter droplets onto glass slides. Each array contained 14 probes: three wild type sequences and nine mutant sequences corresponding to the  surface .32P-labeled target molecules bound across the hybridization array was performed with a scanner (HP Scanjet 4400 c), and analysis of densitometric images was performed using ScanAnalyze 2 software (Stanford University).Just prior to hybridization the slides were soaked for 1 h at room temperature with blocking agent (10 mM tripolyphosphate) and then rinsed twice with water and air-dried. The hybridization cocktail contained: 118 μL 5 M tetramethyl-ammonium chloride (TMAC) (Life Technologies), 9 μL 1 M Tris-HCl (pH 8.0) (Life Technologies), 0.72 μL 0.5 M EDTA, 1.8 μL 10% (w/v) sodium dodecyl sulfate, 14.4 μL 40% (w/v) polyethylene glycol, 20 μL labeled target DNA in 1X SSC, and 35.3 μL HPLC-grade water. A 40-μL aliquot of this cocktail was placed onto each array and a cover slip was applied. Slides were incubated in a humid chamber for 3 h at 25°C. After hybridization, the slides were washed by dipping several times in the same hybridization solution without polyethylene glycol and DNA, air-dried, and then wrapped in plastic film and placed against X-ray film (Kodak BioMax) for autoradiography. Detection and quantitation of To provide confidence in the interpretation of the signals produced by human samples, reference hybridization patterns were prepared using a full set of synthetic targets. For this purpose, thirteen synthetic targets (44–50 nt), representing the wild type and mutant sequences, were used independently and in combination, to represent homozygous and heterozygous conditions.The PCR products obtained were purified using the QIAEX II kit (QIAGEN Inc. USA). For each PCR product the presence or absence of a mutation in exons 7 and 8 was demonstrated via sequencing using the Big Dye Terminator kit (Perkin-Elmer) and a model 373 automated DNA sequencer .ARL performed all molecular assays, data collection and analysis, and drafted the manuscript. RMR conceived and designed the study, coordinated and managed the study, performed data analysis and participated in drafting the manuscript. MSV conceived and designed the study, coordinated and managed the study, performed data analysis and participated in drafting the manuscript. JMEL participated in the design and coordination of the study. AMT designed the oligonucleotide probes, performed bioinformatic analysis of targets, and participated in drafting the manuscript. KLB assisted in coordination of the study, data analysis, and drafting the manuscript. All authors read and approved the final manuscript.


Recombination is a nearly ubituitous feature of genomes; where and when it occurs can provide insights about its evolution and can affect our ability to identify genes that cause disease Consider a piece of text, either this one that you are now reading or any other. Surely they are all pretty much alike, in so far as they are all run-on strings of characters. In this same sense, we can envision that all DNA strands are alike because all are monotonous polymers with the same general chemical makeup. Indeed, this is how we think of DNA when considering its basic function of inheritance, in which all parts of all chromosomes must be duplicated and then passed from one cell generation to the next. The capacity for inheritance is fundamentally a consequence of DNA's general molecular structure, and not of its sequence per se, as But sequence does matter when DNA fulfills its other, more directly functional role. When the DNA that makes up a gene is exposed and expressed, when a gene is serving its individual function, then the detailed sequence means all.So where does recombination  fit in? Recombination hotspots are of strong interest to at least two quite different groups of biologists. For geneticists and cell biologists who study meiosis, the existence of recombination hotspots offers a way to learn what other processes are associated with recombination. This is partly how we know that homologous crossovers in yeast and other eukaryotes are initiated by the cleavage of single chromosomes, called “double-strand breaks” . It turnFor population geneticists, much of the interest in recombination hotspots comes from their possible effect on the patterns of DNA sequence variation along human chromosomes and from the possibility that these patterns could be used to map the position of alleles that cause disease. When multiple copies of the DNA sequence of a gene, or of a larger region of a chromosome, are aligned, they reveal the location and distribution of variation at individual nucleotide positions—single nucleotide polymorphisms (SNPs). Each particular sequence, or haplotype, will carry a configuration for the SNPs for that region . Investihttp://www.hapmap.org/), which has the goal of identifying a subset of SNPs that capture most of the relevant linkage information in the human genome  to fluctuate in location and intensity, in ways that would be hard to precisely predict without knowing what genes have been under selection and what patterns of linkage disequilibria there may have been.PLoS Biology is especially interesting. They report that chimpanzees do not have a recombination hotspot in the TAP2 region where humans have a fairly well characterized recombination hotspot  have not shown evidence of recombination hotspots. If we compare these organisms with yeast and mammals, which do show hotspots, we gain some more insight into the factors affecting the evolution of hotspots.As appealing as the recombination modifier theory of recombination hotspots may be, there is circumstantial evidence that argues against it and that suggests that recombination hotspots are not directly the byproduct of selection on alleles in linkage disequilibrium. Particularly important in this regard is that some wellstudied organisms   and thus elegans . Double-Recombination during meiosis seems to be required for proper chromosome segregation; however, in those organisms where recombination and double-strand-break hotspots occur, these phenomena are also required for proper formation of the SC. It is as if the recombination machinery has been partly co-opted for chromosome alignment in some eukaryotes more so than in others. The implication of these findings is that recombination hotspots are byproducts of other functional constraints associated with the recombination process. This does not rule out the evolutionary theory of recombination modifiers, or that the location and intensity of recombination hotspots may evolve rapidly, but it does suggest that we may not need to invoke the evolutionary modifier theory to explain the existence of recombination hotspots.Recombination hotspots co-occur with double-strand-break hotspots in some eukaryotes, and together these phenomena appear to play an important role in the formation of the SC in those organisms. Given the limited phylogenetic occurrence of recombination hotspots , general theories for the evolution of recombination may not be very helpful for understanding the existence of recombination hotspots. However, in those species where they do occur, it is quite possible that recombination hotspots do evolve in location and intensity. Furthermore, the presence of recombination hotspots in humans may have large effects on the length of local patterns of linkage disequilibrium (haplotype blocks) and thus on our ability to map disease alleles by their association with other markers.Double-strand break: A break in both strands of a DNA molecule, as distinguished from a break in just one strand.Linkage disequilibrium: A pattern of association between two SNPs or two loci that each have multiple alleles, such that pairs of particular SNPs or alleles, one from each locus, tend to co-occur within individuals or genomes more often than would be expected if the loci are sorting independently of each other.Recombination: The process of one double-stranded DNA molecule joining with another; specifically in the context of meiosis, the process of two homologous chromosomes exchanging large portions of their DNA .


Several studies have investigated the association between male pattern baldness and disease such as prostate cancer and cardiovascular disease. Limitations in the lack of standardized instruments to measure male pattern baldness have resulted in researchers measuring balding patterns in a variety of ways. This paper examines the accuracy and reliability of assessment of balding patterns by both trained observers and men themselves, using the Hamilton-Norwood classification system.An observational study was carried out in Western Australia with 105 male volunteers aged between 30 and 70 years. Participants completed a short questionnaire and selected a picture that best represented their balding pattern. Two trained data collectors also independently assessed the participant's balding pattern using the same system and the men's self assessment was compared with the trained observer's assessment. In a substudy, observers assessed the balding pattern in a photo of the man aged 35 years while the man independently rated his balding at that age.Observers were very reliable in their assessment of balding pattern . Compared to trained observers, men were moderately accurate in their self-assessment of their balding status . For the substudy the exact agreement between the men and the observers was 67% and the agreement within balding groups was 87%.We recommend that male balding patterns be assessed by trained personnel using the Hamilton-Norwood classification system. Where the use of trained personnel is not feasible, men's self assessment both currently and retrospectively has been shown to be adequate. Male pattern baldness is the most common form of baldness observed in human beings. The twoSeveral studies have been conducted investigating the association between male pattern baldness (as a proxy for testosterone levels) and various health issues such as prostate cancer -7 and caSubjects were male volunteers between the ages of 30–70 years recruited from sporting clubs, shopping centres, universities, the central business district and other public areas throughout the metropolitan area of Perth, the capital city of the state of Western Australia, during March 2003 to August 2003. Of the 105 volunteers recruited, two were less than 30 years of age, and therefore were ineligible for the study.Participants completed a questionnaire providing minimal information regarding demographic characteristics, including age, ethnicity, and level of education. Included in the questionnaire was an unlabelled Hamilton-Norwood classification scale Figure , which pWhilst the subjects completed the questionnaire, two data collectors with some training in recognising different balding patterns independently assessed the participant's balding pattern using the same classification system. On recruitment, participants were told the study was to help with medical research, however the role of the data collectors with regard to their independent assessment of the participant's balding pattern was not revealed until after the questionnaire was complete.Where possible, (sporting clubs and universities) volunteers were asked to bring a photograph of themselves at age 35 on the day on which the study would be done. The photographs were used by the observers to independently assess the men's balding patterns at age 35 while the participants completed the questionnaire. These participants were also asked to select the picture from the Hamilton-Norwood scale which best depicted their balding pattern at age 35, and were not permitted to look at the photographs while completing the questionnaire.For data analysis, we compared the assessments of the two observers with each other, and the assessment of the participants with the assessments of the observers. Initially, each of the 12 reference pictures included in the Hamilton-Norwood classification system[There were 69 participants (67%) between the ages 30–49 years, and 34 (33%) were between the ages 50–70 years. Whilst 70% of subjects considered themselves of "Australian" ethnicity , the remainder was made up of representatives from Europe (20.4%), Asia (4.9%), New Zealand (3.9%) and South America (1%). A third of subjects had trade education or equivalent (30.1%); 22.3% had completed high school but not gone onto further education; 12.6% had completed junior high school only; and, because of recruitment through universities, 19.4% of subjects had an undergraduate degree and a further 15.5% had completed post-graduate study.Overall, trained observers were found to be highly reliable at analysing balding patterns with an exact agreement of 85.4% and an agreement within balding pattern groups of 90.3% Table . CompareIn regards to how different demographic characteristics affect men's ability to predict their balding pattern Table , the chaThere were 15 subjects who provided photos of themselves aged approximately 35 years and both observers examined 13 of these. The inter-observer reliability for exact match was 81.8%  and agreement within balding pattern groups increased to 100% . Observer 2 only examined 13 subject, so for the 15 subjects examined by Observer 1, agreement between the men and the observer was 66.7% for exact match and 86.7% for agreement within the balding pattern groups.In this study we have shown that trained observers are very reliable in assessing men's balding patterns. Our data also show that, when compared to the trained observers, men themselves can assess their balding patterns quite well. In particular, men are accurate in assessing which balding pattern group they have. This result is important due to previous research suggesting that it is the overall pattern of hair loss rather than extent of balding that determines whether men are at an increased risk of developing negative health outcomes including prostate cancer,4,7.et al[There have been several studies investigating the link between male pattern baldness and prostate cancer-5,7,10, et al assessmeOther methods for assessing baldness have been used in clinical situations including reference grids used with standardized photographs of the scalp or in vivo; and vidThe majority of studies,7,10,15 The strengths of our study included the recruitment of volunteers from a broad cross-section of the population thus allowing for extrapolation of the results back to the wider community. As our research was performed as an observational study, care was taken to avoid the data collectors influencing the results. This included not informing participants that the observers would be assessing their balding pattern until after the questionnaire was complete. The data collectors also refrained from giving advice to participants when asked to help assess their balding pattern.Older subjects appeared to be better at assessing their balding group than men less than 50 years. This may be due to greater hair loss resulting in a more straightforward distinction between balding patterns, or possibly just a greater self-awareness of degree of balding amongst those in the older age group. The results with regard to education were confusing with men who had only senior high school education seeming to be best at assessing their balding patterns. This may have just been due to small numbers in the groups. Other demographic characteristics not included in our questionnaire may have been of interest in determining factors that influence accuracy of men's self assessment of their balding pattern. These include factors such as marital status, occupation, and personality sub-types, and we would encourage any further research into the area to investigate the possible relationship between these aspects and the accuracy of men's self-assessment.Any extrapolation of our data needs to take into account the differing methods of data collection between our study and other studies in which the man may obtain advice from partners or friends in the home environment, as well as have access to mirrors and photographs to assist in their assessment of current and past balding patterns. These factors do not negatively affect our results however, as the use of such help would ultimately increase accuracy of balding assessment from the already acceptable level shown in our results, rather than detract from it.The study of the accuracy of previous balding was limited by small sample size. Participants had to be approached prior to the study to provide a photograph of them at age 35 years. This limited numbers of eligible participants, and also meant that the men may have viewed the photograph of themselves before completing the questionnaire, which may have increased their accuracy of retrospective assessment. Difficulties in assessing vertex balding from the photographs by the observer also became apparent, as often photographs did not provide an adequate view of the top and back section of a participant's head. It is unlikely that these limitations can be overcome, as it would be difficult to devise another method for the observer to retrospectively assess the participant's balding pattern at age 35.Previous research has demonstrated an intra-observer rate of consistency in assessment of 98–99% using the Hamilton-Norwood scale,10. HamiFrom this study, we suggest that any further work requiring assessment of male balding patterns consider the use of trained observers as the gold standard of assessment. Where this is unattainable, we have shown that men's self evaluation is accurate enough to ensure reliability and validity of results. In addition, we believe this study demonstrates that if links are found between male balding patterns and health effects, that men can reliably determine their own balding pattern and assess their own risk.The author(s) declare that they have no competing interests.RT and JM planned the study, collected the data, analysed the data under supervision, and wrote the study report for a student project.JL assisted in planning the study and drafted the manuscriptLF assisted in planning the study, supervised data collection and analysis and edited the manuscript.All authors read and approved the final manuscriptThe pre-publication history for this paper can be accessed here:


When gene therapy was introduced nearly fifteen years ago, it was widely hailed as a panacea. Since many diseases have a genetic component, the hope was that gene therapy could replace compromised genes with healthy versions to treat everything from inherited disorders like cystic fibrosis to cancer and HIV. That great promise was quashed when a teenager suffering from a rare hereditary liver disorder died after participating in an experimental gene therapy trial in 1999: four days after being injected with millions of viruses engineered to deliver healthy genes to his liver, Jesse Gelsinger died. It seems the virus, derived from an adenovirus, targeted his immune cells rather than his liver cells, which triggered an immune response against the virus, resulting in massive organ failure.In another case, two young boys who received gene therapy for the severe immunodeficiency disorder known as “bubble boy disease” developed leukemia-like symptoms 30 months after treatment. In this case, the viral vector inserted itself near a promoter region—a site that initiates gene transcription—of a proto-oncogene, a gene that can initiate cancer. Since viral vectors can integrate at various genomic locations, the safety and effectiveness of gene therapy ultimately depends on being able to predict a virus's particular bias. Comparing retroviral vectors derived from three viruses, including two common gene therapy vectors, Rick Mitchell et al. report 3,127 sites where these viruses typically integrate into the human genome. The different vectors, they found, show different target preferences.Retroviruses use viral enzymes to copy their own genome, which is stored in an RNA transcript, into DNA. Now recognizable by the host's genome, the virus can integrate into one of the host's chromosomes. In this study, Mitchell et al. studied vectors derived from the human immunodeficiency virus (HIV), avian sarcoma-leukosis virus (ASLV), and murine leukemia virus (MLV). Introducing the viral vectors into human cells, the authors analyzed the gene expression profiles of the cells to determine where vectors integrate into human chromosomes and which, if any, genes they activate. Mitchell et al. then compared the integration sites with the transcription profiles.Each retrovirus, they discovered, showed distinct preferences for genome integration. HIV vectors tend to integrate into sites of active transcription, favoring chromosomal regions rich in expressed genes. MLV vectors tend to integrate near transcription initiation sites, confirming the results of a previous study, with a weak bias toward active genes. In contrast, the authors report, the ASLV vector “does not favor integration near transcription sites, nor does it strongly favor active genes.”Early efforts to understand how chromosomes may influence where viruses insinuate themselves into a chromosome focused on factors governing accessibility. Viruses are more likely to be integrated into chromosomal regions that are more accessible, which tend to be transcriptionally active sites. But since each of the three viruses studied here routinely targeted different sequences, the authors note, accessibility is probably just one factor. Specific chromosomal proteins, for example, might interact with the viral integration machinery and facilitate integration at nearby sites. Another possibility, the authors propose, is that DNA-binding proteins that bind to specific DNA sequences assist integration of one virus while impeding another.This could explain why ASLV behaved as it did in the human cells studied here. The virus might have more refined integration preferences during normal infection of chicken cells, the authors note, but its integration machinery can't interact properly with human cells. The leukemia-like effects of the bubble boy gene therapy stemmed from integration of a mammalian retrovirus—the MLV vector—near an oncogene promoter region. Since ASLV tends to avoid both transcription initiation sites and active gene sites, it could be a more promising candidate for human gene therapy. With the draft chicken genome sequence now complete, researchers can investigate whether that proves true. But for now, Mitchell et al. make the case that scientists can gain more control over where viral vectors integrate into the human genome by selecting different retroviral integration systems. Only time will tell whether more control translates into safer gene therapy protocols.