A wide range of cellular responses occur when plants are exposed to elevated temperature, including adjustments in the unsaturation level of membrane fatty acids. Although membrane bound desaturase enzymes mediate these adjustments, it is unknown how they are regulated to achieve these specific membrane compositions. Furthermore, the precise roles that different membrane fatty acid compositions play in photosynthesis are only beginning to be understood. To explore the regulation of the membrane composition and photosynthetic function in response to temperature, we examined the effect of temperature in a collection of mutants with altered membrane lipid fatty acid composition.fad5, fad6, act1 and double mutants, fad7 fad8 and act1 fad6. The results revealed key changes that occur in response to elevated temperature regardless of the specific mutations in the glycerolipid pathway, including marked decreases in trienoic fatty acids and consistent increases in unsaturated 16:0 and in dienoic 18:2 levels. Fluorescence measurements of various mutants indicated that photosynthetic stability as well as whole plant growth at elevated temperature is influenced by certain membrane fatty acid compositions.In agreement with previous studies in other species, the level of unsaturation of membrane fatty acids in Arabidopsis was inversely correlated with growth temperature. The time required for the membrane fatty acids to attain the composition observed at elevated temperature was consistent with the timing required for the synthesis of new fatty acids. Comparisons of temperature-induced fatty acid alterations in membranes were made among several Arabidopsis lines including wild-type Columbia, and the compositional mutants, The results of this study support the premise that defined proportions of saturated and unsaturated fatty acids in membrane lipids are required for photosynthetic thermostability and acclimation to elevated temperature. The results also suggest that changes in the membrane fatty acid composition brought about in response to temperature are regulated in such a way so as to achieve highly similar unsaturation levels despite mutations that alter the membrane composition prior to a high-temperature exposure. The results from examination of the mutant lines also suggest that interorganellar transfer of fatty acids are involved in mediating temperature-induced membrane alterations, and reveal steps in the fatty acid unsaturation pathway that appear to have key roles in the acclimatization of membranes to high temperature. One of the most prevalent environmental challenges encountered by plants is the exposure to a broad range of temperatures. Plants use a variety of anatomical, metabolic and cellular strategies to deal with changing environmental temperatures. Acclimation to elevated temperatures is mediated at the cellular level in part by the induction of general stress responses, which include the increased expression and activity of heat shock proteins -6. TheseAtriplex lentiformis, changes its membrane fatty acid composition by decreasing the level of unsaturated fatty acids such as hexadecatrienoic acid (16:3) and increasing the level of saturated lipids at higher growth temperatures [Early studies of the ability of plants to acclimate to higher temperatures were conducted on plants adapted to high temperature growth -12. For eratures . Membraneratures ,15.Of the numerous cellular processes likely to be affected by the membrane composition, photosynthesis is probably the most critical. Adjustments in the level of unsaturation of thylakoid membranes may therefore affect the capacity of plants to adapt to elevated temperature conditions in order to avoid a reduction in photosynthetic efficiency. In this regard, the reaction center of PSII is considered to be the most sensitive component of the thylakoid membrane to thermal breakdown, and the function of the water-splitting D1 protein within PSII has been implicated as the most readily damaged by high temperature  as well fad8 gene [Studies on cyanobacteria have indicated that the mechanism by which temperature mediates compositional alterations in membranes is at the level of transcription. The level of desaturase transcripts in cyanobacterial cells increases in response to growth at low temperature . It is lad8 gene , and othad8 gene , have beIn this study we examined the leaf membrane fatty acid composition of Arabidopsis in response to temperature. After establishing the changes that occur at different elevated temperatures, we examined a number of mutant lines of Arabidopsis that possess well-characterized alterations in their membrane fatty acid content. The lines studied differ primarily in the degree of unsaturation of various lipids in chloroplast membranes. Examination of the fatty acid profiles of these lines grown at high temperature reveals a regulatory system that mediates membrane compositional changes even in the absence of specific desaturase steps. The results also suggest some general control points that might be important in the mechanism by which the membrane fatty acid unsaturation level is adjusted in response to temperature. In the course of carrying out these studies, we also examined the relationship between membrane fatty acid composition and growth at elevated temperatures.Arabidopsis lines were germinated and grown at 17°C for seven days before being shifted to designated temperatures. Total leaf fatty acids were extracted from lines grown at various temperatures for 30 to 35 d, converted to methylesters and analyzed by gas chromatography. Changes in the relative abundance of the major fatty acid species from the Columbia wild-type line grown at 17°C, 20°C, 29°C and 36°C were plotted and are shown in Fig. Based on the substantial changes detected after the shift to elevated temperature, we wished to determine whether these changes occurred on time scales similar to the timing known for new synthesis of fatty acids. To estimate the time required for a temperature increase to induce measurable changes in the membrane fatty acid composition, Arabidopsis seedlings were germinated and grown at 22°C for seven days and then shifted to 29°C. The fatty acid composition was then evaluated in rosette leaves sampled at various times after the shift to elevated temperature by gas chromatographic analysis Fig. . No signThe time course observed for the fatty acid composition to change in response to elevated temperatures correlates with the time determined for the incorporation of precursors to attain steady state levels in radiolabel feeding experiments . The sams lines possessing well-characterized alterations in their membrane fatty acid profiles , decreased to 6 mol% of total fatty acids at 36°C, the lowest leaf 18:3 levels of all the lines tested. Thus, the general trend of increasing 18:2 and decreasing 18:3 in response to high temperature growth was retained in the absence of most FAD7 and FAD8 desaturase activities, which catalyze the formation of the majority of trienoic fatty acids in the leaf membranes.The deficiency in the oroplast . In thisact1 and fad6 was produced during this study and used to evaluate the performance and temperature response of plants containing a membrane fatty acid composition which is distinguished by an elevated level of 18:1 (due to the deficiency in the FAD6 desaturase) but no increase in the relative amounts of 16:1 . The resulting composition in the act1 fad6 double mutant line grown at 17°C indicates the profile expected for mutations at each of these steps. The fatty acid profile altered in response to elevated temperature is similar to each parental mutant line grown at high temperature .A line derived from a cross between ure Fig. . The actfad5 and act1 lines [in planta measurements in side-by-side comparisons to examine differences in thermal tolerance. In addition, prior to the fluorescence stability measurements, the fatty acid compositions of all lines used in this analysis were determined and all were found to be within the standard error of those values determined for the fatty acid profiles of the lines grown at 17°C  temperature while fad7 fad8 shows a slight but insignificant increase. These results are likely to be due to differences stemming predominantly from the distinct membrane fatty acid compositions in leaves from the lines grown at this temperature. The TP values obtained for these intact leaf measurements here, are very similar to various determinations previously conducted on some of the lines using isolated chloroplast preparations or detached whole leaves [Six Arabidopsis lines possessing distinct membrane fatty acid compositions were evaluated by measuring chlorophyll fluorescence parameters to determine the thermal stability of PSII. The use of chlorophyll fluorescence has been used extensively to investigate photosynthesis and previous studies have examined photosynthetic stability using similar methods on isolated chloroplasts or detached leaves from the t1 lines ,36. Howee leaves ,36,37. Tact1 fad6 provided a line with a fatty acid profile that is distinct from that of other lines analyzed in this study   Fig. . To detety Table .act1++ line is a transgenic line that over expresses GPAT . It was included as an additional line here to test whether its fatty acid composition would impact the thermal stability measurements as determined by chlorophyll fluorescence. However, the leaf fatty acid composition of act1++ plants grown at 17°C is similar to the wild-type composition except that the percentage of 16:0 and 16:3 fatty acids is increased to 2.0 and 1.5 mol%, respectively, over the proportions determined in the wild type. No differences in thermostability as indicated by chlorophyll fluorescence measurements were apparent for this line compared to the wild type.The fad5, fad6 and act1 lines, which showed reproducible increases in thermal stability compared to wild type when grown at 17°C, showed essentially the same TP temperatures as the wild type when grown at the elevated temperature. The lack of a detectable increase in thermal stability in mutant plants above the temperature found for the wild-type line grown at high temperature may be a consequence of the altered composition of the membrane fatty acids after growth at high temperature. The similar TP temperatures are also consistent with the overall similar unsaturation levels as indicated by the double bond index calculated for these lines. These results may also reflect the upper tolerance limit attainable that arises from fatty acid adjustments in response to elevated growth temperature, at least with the different compositions in the lines studied here.Although the most equivalent comparisons for stability measurements among the different lines would be obtained from those grown at temperatures considered moderate for Arabidopsis, such as 17°C used here, we also determined the TP temperatures for those lines that exhibited enhanced thermal stability at 17°C after growth at high temperature. Since this study revealed clear differences in the membrane fatty acid composition at various elevated temperatures, an indication of high-temperature acclimation based on fluorescence measurements may be evident. Four plants including wild type were grown at 29°C for three weeks before conducting the fluorescence stability tests. The results indicated an increase in the TP temperature from 42.7°C for wild type grown at 17°C to about 44.8°C in plants grown at 29°C Table . Howeverfad5, fad6, act1 and fad7 fad8 seedlings are at least the same or slightly higher than wild type within several days after the shift to high temperature. In agreement with findings by Murakami et al. [fad7 fad8 mutant line exhibited the most pronounced tolerance to high temperature based on growth rate. These results indicate that a less polyunsaturated membrane fatty acid composition than normally found in wild type favors seedling growth at elevated temperatures.Most of the mutant lines used in this study were chosen on the basis of their altered fatty acid profiles in chloroplast membranes. As shown here, reductions in the total level of triunsaturated fatty acids in many lines grown at 17°C reflects the composition of the wild-type line grown at elevated temperature. To establish whether the observed changes translates into improved performance at the whole plant level, growth rates were determined for the lines at various temperatures. Growth rates were determined only during the first six days after the shift to elevated temperatures in order to maintain equivalent membrane fatty acid compositions of the lines before changes occurred in membranes during acclimation to high temperature Fig. . As showi et al. , the fadSeveral clear alterations are evident in the membrane fatty acid composition in response to high temperature growth. A decrease in trienoic fatty acids, including strongly diminished 16:3, produced exclusively within the prokaryotic pathway in the chloroplast, is consistent with the general decrease of polyunsaturated fatty acids in response to high temperature. Concurrent with the reduced accumulation of trienoic fatty acids at high temperature is an increase in linoleic acid, 18:2, and an increase in 16:0  activity missing in each mutant Table . The temand 30°C . This fiand 30°C .The time required for the fatty acid composition to adjust to high-temperature growth conditions demonstrated that major alterations in leaf membranes do not occur rapidly in response to elevated temperature. The ~60-h period before changes become evident in the fatty acid profile corresponds with the occurrence of new lipid synthesis and turnover and therefore does not suggest a mechanism by which temperature induces modifications of existing membrane fatty acids. These results are also consistent with labeling studies that show fatty acids produced by the prokaryotic pathway accumulate prior to those synthesized via the eukaryotic pathway. The abrupt changes in 16-C fatty acids beginning 60 h after the shift to high temperature compared to the gradual and more continuous alterations observed for 18-C fatty acids Fig.  also fitfad5, fad6, act1, act1 fad6 and wild-type lines suggests that the percentage of 18:2 may be important in membrane acclimatization. These results also suggest that desaturase activities as well as flux from extrachloroplastic membranes might also be controlled to mediate the response to temperature (see below). The accumulation of 18:2 (as opposed to 18:1) in the lines tested implicates it as a preferred species in membranes adapted to high-temperature growth. Although unknown, one speculation for 18:2 for this may be due to the "intermediate" level of disorder represented by diunsaturated acyl chains in the membrane. Fatty acids with one double bond, such as 18:1, impart a greater relative degree of disorder to the membrane than acyl groups containing two double bonds which, in turn, confer only slightly less disorder to the membrane relative to triunsaturated 18:3. The composition of the fad7 fad8 mutant line supports this contention, in which the mol% of 18:2 is almost 2-fold higher than that of wild-type membranes and this line exhibited the best growth at elevated temperature. However, biological membranes are complex, dynamic structures and it is likely that other, unknown factors will be important to maintain optimally functioning membranes.The fatty acid composition resulting from high-temperature growth in the different genetic backgrounds reveals a complex regulatory system. Thus, despite deficiencies in several enzymatic steps in the different mutants, the membrane fatty acid composition undergoes adjustments similar to those observed in the wild type in response to elevated temperature. The similar increase in 18:2 levels after high temperature growth in the fad6 mutant grown at high temperature, the proportions of 18:2 and 18:3 are highly similar to the proportions observed in the wild type, despite that essentially all of the 18:2 must be derived from the eukaryotic pathway. Labeling studies have shown previously that the fad6 line exhibits a decrease in lipid synthesis via the prokaryotic pathway [fad6 profile also reveals that activity of the FAD2 enzyme, operating in the endoplasmic reticulum, might also be subject to temperature regulation.The fatty acid profiles of several mutants grown at high temperature suggests that the control of flux of fatty acids from the eukaryotic pathway is partly responsible for the changes observed in 18:2 and 18:3 due to temperature. In the  pathway , and thiact1 mutant line, in which entry of fatty acids into the prokaryotic pathway is blocked by the step catalyzed by GPAT, the primary difference is a decrease in the level of 16:0 when grown at elevated temperatures. This presumably reflects an increase into the eukaryotic pathway but with a similar temperature-responsive regulation. Flux of 18:2 from the eukaryotic pathway back into the chloroplast also appears to be modulated, as the proportions of 18:1 and 18:2 are slightly elevated in act1 at both temperatures, while the 18:3 level is highly similar to wild type.Similarly, in the fad7 fad8 mutant reveals alterations that occur in response to elevated temperature in the absence of all trienoic fatty acid-forming desaturase activity in the chloroplast  was determined in intact leaves. The fluorescence measurements were conducted on plants grown at 17°C, to minimize variation and other potential responses, such as increases in the synthesis of heat shock proteins that might be induced at higher temperatures. In addition, only at the 17°C growth temperature were differences observed in the total membrane unsaturation level among the different lines tested as estimated by the double bond index, which might suggest that the largest influence of the fatty acid composition occurs at lower and more moderate temperatures.fad6 mutant exhibited the maximum fluorescence yield enhancement of the lines grown at 17°C. This maximum TP is essentially the same as that obtained from several plants grown at 29°C, including the wild-type line  potting soil irrigated with a commercial fertilizer at every third watering. For growth in pots at elevated temperatures, seeds were germinated and grown at 17°C for seven days and then shifted to growth chambers adjusted to the respective temperatures and set to maintain humidity levels to at least 80% or higher, which was found to be essential to maintain growth at temperatures above 32°C. These chamber-grown plants were utilized for determining leaf membrane fatty acid compositions and for analyzing fluorescence yield characteristics. Growth rates of seedlings were determined on lines germinated and grown in Murashige and Skoog salts media (Sigma) containing 1% sucrose and 0.7% agar. Surface-sterilized seeds were plated, treated at 4°C for 2 to 3 days to synchronize germination, and grown for 16 d at 16°C in a growth chamber before shifting to elevated temperatures. Light intensity remained constant at 60 μmol m-2 s-1. Four seedlings were sampled at days 0, 2, 4 and 6 d after the shift to elevated temperatures and were dried for 2 d at 70°C before weighing. The relative growth rates (ω-1) were calculated as the slope of the natural log of the seedling dry weight plotted over time in days. Growth rate determinations for seedlings grown at 22.5°C and 30°C were conducted 3 separate times to obtain SE and growth rates determined for 28 and 34°C were conducted once.Arabidopsis lines were grown in environmentally controlled chambers  adjusted to a given temperature under continuous light at 120–150 μmol mact1 fad6 double mutant line was derived by first crossing act1 to the fad6 line and then crossing progeny from the resulting heterozygotes back to fad6 before selecting for lines possessing a fatty acid composition indicative of both homozygous mutations. The act1++ line, is a transgenic line containing the gene encoding glycerol-3-phosphate acyltransferase (GPAT)  under tFor each determination, at least two plants were used as a source of leaf material for extraction and analysis. Three mid-to-fully expanded (2 to 5-cm long) rosette leaves were harvested from each plant by removing approximately one-third of the leaf and immediately storing at -80°C in Teflon-capped tubes until needed. Preparation of methyl esters from these leaves and gas chromatographic analysis of the resulting extracts was performed using established procedures  with a HChlorophyll fluorescence was measured using an Opti-Sciences OS500-FL pulse modulated fluorometer . A custom designed aluminum heating block was constructed to hold an Arabidopsis rosette leaf, the associated optical cables and the temperature probes to conduct the leaf chlorophyll excitation and fluorescence measurements. An Arabidopsis leaf (~2.4 cm long) was fitted into a recessed (~0.5-mm) portion of the heating block to prevent damage to the leaf section. An ethylene glycol solution was circulated within the block to control temperature. The block and clamp module also provided an adjustable fixture to stably hold the leaf of a potted plant during the measurements.-1 to a final temperature of 48.5°C using a digital temperature control unit . Leaf temperatures indicated were measured with a thermocouple in contact with the leaf surface. Quantum yield was calculated by the equation F'ms-Fs/F'ms according to Genty [s is the steady state fluorescence under environmental conditions, and F'ms is the maximal fluorescence yield obtained after a saturating light pulse. Fm-Fo/Fm, which is a measure of the photochemical efficiency of photosystem II, is based on the variable fluorescence (Fm-Fo), which is the minimum (Fo) and maximum (Fm) fluorescence of a dark adapted leaf before and after, respectively, of a saturating light pulse. All determinations were made on leaf samples that gave an initial Fm-Fo/Fm ratio of at least 0.80, prior to initiating the temperature increase. This measure was taken to ensure that the integrity of the leaf section introduced into the clamp module was sound and to verify that the leaf was properly fitted into the measurement module. The mean value ratio of 0.83 is indicative of a well functioning photosynthetic apparatus in an unstressed leaf as has been shown in a variety of other plant species [The photosynthetic thermostability of the different lines was evaluated by determining the transition point temperature of fluorescence quantum yield (TP). An attached rosette leaf was fitted into the thermally controlled heating block and fluorescence was monitored with a saturating light pulse (0.8 s duration) every 90 s. After stabilization at 29°C (4–5 pulses), the temperature of the heating block was increased at a rate of 0.75°C minto Genty , where F species .act1 overexpressing lines and assisted in the construction of the double mutant line. CS conceived of the study. All authors read and approved the final manuscript.DF executed the fatty acid analysis, photosynthetic measurements, data analysis and drafted the manuscript. JO undertook the production of the


A paper published in 1998 showed that loss of only one copy of the p53 tumor suppressor gene is sometimes enough to initiate carcinogenesis For people who received their introduction to cancer genetics in college in the first half of the 1990s, everything looked simple and straightforward. It was the stuff you could explain to sincerely interested relatives who wanted to know what you were spending your time on. There were oncogenes and there were tumour suppressor genes. Oncogenes were overactive genes and proteins that somehow caused cancer because they were overactive; therefore, they were dominant. Tumour suppressor genes were genes that would normally prevent a tumour from happening and that needed to be inactivated for a tumour to start to form; both copies of a tumour suppressor gene had to be inactivated, and the mutation was recessive. If inactivation of these genes is a random process, it was understandable that people who inherit an inactivated copy of a tumour suppressor gene had a higher risk of developing the associated form(s) of cancer than people born with two normal copies, as postulated in EMBO Journal describing a detailed study of tumours in mice lacking one copy of the p53 tumour suppressor gene (Trp53). This gene is known to be the most mutated gene in human cancer and its function to be central to many processes that are involved in the cellular prevention of cancer. Mice lacking both copies of this gene are for the most part viable, but succumb to cancer (mainly thymic lymphomas) at three to five months of age  and one downregulated gene (Pcna) in p53-positive tumours after irradiation and showed responses indicative of normal p53 function. Furthermore, it was shown that the p53 protein from the tumours was able to bind to a p53-binding DNA sequence in an in vitro setting. Finally, since it is known that p53 absence in tumours is correlated with chromosomal instability, The p53 protein is known to function as a transcriptional regulator by either up- or down regulating target genes in response to different forms of cellular stress, including irradiation-induced DNA damage. The authors studied the expression of two p53-upregulated genes (Trp53+/− tumours the wild-type allele of Trp53 is not only retained, but also appears to function normally. This strongly suggested that a decrease of dosage in p53 is already sufficient for tumourigenesis, a phenomenon referred to as haploinsufficiency. Shortly before, the group of Moshe Oren (Trp53+/− background leads to a greater than 50% reduction in p53 activity using a p53-responsive lacZ reporter gene in transgenic mice. Venkatachalam and colleagues suggested the strong concentration dependence of p53 function could be explained by the fact that p53 functions as a tetramer. A 50% decrease in p53 monomers can easily be imagined to result in a greater than 50% decrease in functional tetramers, which in turn increases the chances of these cells becoming cancerous.In short, this paper clearly showed that, at least in mice, in many she Oren  had showPLoS Biology, Pten tumour suppressor gene influences the occurrence of prostate cancer. Further genes have been described with other unexpected roles in the tumourigenic process. There is a long-standing debate in the literature about the number and role of mutations in a tumour, and without going into the details, it is clear that haploinsufficient mechanisms for tumour suppressor genes will greatly influence the statistics on which these discussions are based. At a time when microarray analysis has become a standard experiment and the many thousands of changes in tumour cells are analysed across the whole genome, it is important to keep in mind that the correct interpretation of this wealth of information might be more complicated than the widely accepted models would have us believe.This paper by


Critical appraisal skills are believed to play a central role in an evidence-based approach to health practice. The aim of this study was to evaluate the effectiveness and costs of a critical appraisal skills educational intervention aimed at health care professionals.This prospective controlled trial randomized 145 self-selected general practitioners, hospital physicians, professions allied to medicine, and healthcare managers/administrators from the South West of England to a half-day critical appraisal skills training workshop  or waiting list control. The following outcomes were assessed at 6-months follow up: knowledge of the principles necessary for appraising evidence; attitudes towards the use of evidence about healthcare; evidence seeking behaviour; perceived confidence in appraising evidence; and ability to critically appraise a systematic review article.At follow up overall knowledge score [mean difference: 2.6 (95% CI: 0.6 to 4.6)] and ability to appraise the results of a systematic review [mean difference: 1.2 (95% CI: 0.01 to 2.4)] were higher in the critical skills training group compared to control. No statistical significant differences in overall attitude towards evidence, evidence seeking behaviour, perceived confidence, and other areas of critical appraisal skills ability  were observed between groups. Taking into account the workshop provision costs and costs of participants time and expenses of participants, the average cost of providing the critical appraisal workshops was approximately £250 per person.The findings of this study challenge the policy of funding 'one-off' educational interventions aimed at enhancing the evidence-based practice of health care professionals. Future evaluations of evidence-based practice interventions need to take in account this trial's negative findings and methodological difficulties. For clinicians to make sense of scientific evidence and follow an evidence-based approach to their practice it has been stated they should be able to: 1) turn problems of their clinical practice into focused questions; (2) comprehensively search for literature to address these questions; (3) critically appraise this literature for its usefulness and scientific validity; and, (4) apply the results of this appraisal to their practice  (see Table systematic reviews play a key role in informing evidence-based decisions'), in the CAS training group compared to control there were no other significant differences between groups in attitude statements. There was no evidence of difference in overall attitude score between groups ]. However, the difference was not significant when assessed using explanatory analysis. No difference between groups was observed in the ability to appraise 'methodology' or 'relevance/generalisability' of evidence , we failed to obtain a substantial proportion of outcomes in the trial participants – 60% and 85% of the knowledge, attitude and behaviour outcomes were obtained for CAS training and control groups respectively, and even less for the critique of the published systematic review. It is plausible that respondents may have differed in some way to non-respondents, such as in their level of motivation, and may therefore responded more positively to this educational intervention. However, this was not supported by the poor outcome response rate. Moreover there was no evidence of a difference in the baseline characteristics of participants who returned their outcomes, and those who did not. A differential response rate across the two study groups possibly reflects a greater reluctance in those individuals who had undertaken the educational intervention to return their outcomes (i.e. 'more to lose') compared to those in the control group. If true, the direction, in terms of over- or underestimating the impact of the intervention, is uncertain. An interview-administered assessment, rather than a mail based one, may have enhanced outcome response rate.Of the 73 participants allocated to receive CAS training only 52 (71%) actually attended. The reasons for this were unclear, and were not formally addressed within this study. In addition to conventional intention-to-treat analyses, secondary explanatory analyses, i.e. based upon the participants who actually did attend the workshop, were undertaken. That there were no differences between groups for most outcomes, irrespective of whether an intention-to-treat or explanatory analysis, was used (see Tables Evidence Based Medicine) and on-line critically appraised topics ('CATs'), the most important role of CAS training may be simply be to sensitise participants to the availability of high quality evidence. Further debate is therefore needed about refocusing critical appraisals skills training towards finding such evidence and the role of healthcare librarians and the new initiatives such as the National Electronic Library for Health. A number of commentators have criticised previous evaluations of CAS training for not using experimental designs • To critically appraised a published review article.• To understand the terms systematic review and meta-analysis.• To be able to explain why critical appraisal skills are important for provision of health care.• To have greater confidence in your ability to make sense of the research evidence.3 hours attendance JAMA appraisal guideline (~60 mins).• Introductory talk: overview of the importance of evidence based health care practice, the theoretical basis of the appraisal of a systematic review, and orientation to the • Small group work: appraisal of a published systematic review (~60 mins).• Plenary session: feedback from the small group, general discussion of the relevance of the appraisal to clinical scenario and ballot of opinions on the clinical scenario. (~60 mins)All workshops were run by 3 to 4 individuals each of whom had a formal training in health services research methods and were experienced in delivering CASP workshops.One to two weeks prior to the workshop, a pre-workshop pack was sent to participants.• Workshop objectives.• Orientation guide.• Clinical scenario and questions• Systematic review paper.• Glossary.One to two weeks post workshop, a post workshop pack was sent to participants:• Introductory talk slides.• Systematic review checklist.JAMA guidelines for systematic review [• c review .The workshop is based on the Critical Appraisal Skills Programme (CASP) developed by Oxford Regional Health Authority and developed from the educational methods of McMaster University in Canada . The 'McThe pre-publication history for this paper can be accessed here:MS-Word format, contains study outcome questionnaireClick here for file


Addition of glucose to starved yeast cells elicits a dramatic restructuring of the transcriptional and metabolic state of the cell. While many components of the signaling network responsible for this response have been identified, a comprehensive view of this network is lacking. We have used global analysis of gene expression to assess the roles of the small GTP-binding proteins, Ras2 and Gpa2, in mediating the transcriptional response to glucose. We find that 90% of the transcriptional changes in the cell attendant on glucose addition are recapitulated by activation of Ras2 or Gpa2. In addition, we find that protein kinase A (PKA) mediates all of the Ras2 and Gpa2 transcriptional effects. However, we also find that most of the transcriptional effects of glucose addition to wild-type cells are retained in strains containing a PKA unresponsive to changes in cAMP levels. Thus, most glucose-responsive genes are regulated redundantly by a Ras/PKA-dependent pathway and by one or more PKA-independent pathways. Computational analysis extracted RRPE/PAC as the major response element for Ras and glucose regulation and revealed additional response elements mediating glucose and Ras regulation. These studies provide a paradigm for extracting the topology of signal transduction pathways from expression data. Inducing expression of most glucose responsive genes in yeast can be mimicked by activation of the small GTP-binding proteins Ras2 or Gpa2 even in the absence of a physiologic signal Complex intracellular networks inform a cell's developmental and growth decisions in response to external nutrients or signaling molecules. Defining the topology of such networks has generally relied on combinations of genetic epistasis and biochemical techniques to establish the linear order of components that convey information on the presence of a particular stimulus. Generally, only one or a few endpoints, such as enhanced transcription of a responsive gene, are monitored in gauging the output of a pathway. More recently, global transcriptional analysis has allowed reseachers to capture the entire transcriptional output of a signaling process and assess the consequence of eliminating individual components of the signaling network on the entire response . This apWe have used global transcriptional analysis to dissect the signaling network activated by glucose addition to yeast cells, with an emphasis on the role of the small GTP-binding proteins, Ras2 and Gpa2, in that signaling process. Addition of glucose to yeast cells growing on a nonfermentable carbon source induces a dramatic restructuring of the metabolic and transcriptional state of the cell . At the The dramatic change in the metabolic activity and protein synthesis capacity attendant on glucose addition to starved cells is accompanied, and driven in part, by a reprogramming of the transcriptional state of the cell . Cells rCYR1, to yield an increase in intracellular cAMP levels  production to the presence of glucose in the medium . As in oP levels . Additio by BCY1 . Active  by BCY1 . Several by BCY1 , 1999.GPA2, a member of the Gα family of heterotrimeric G proteins, also regulates cAMP levels through a pathway parallel to Ras  and compared that to the changes following glucose addition to glycerol-grown cells. To focus on signaling events, rather than the transcriptional consequences of metabolic changes in the cell, we examined the transcriptional response as it changed immediately following addition of glucose. Similarly, to examine the effects of Ras2 or Gpa2 activation, we constructed gal1 strains that carried an activated form of RAS2 or GPA2 under control of the galactose-inducible GAL10 promoter. Since gal1 strains cannot metabolize galactose, addition of galactose resulted in induction of the activated RAS2 or GPA2 allele and a small number of other galactose-inducible genes, but resulted in no changes in the metabolic state of the cell.In order to examine the role of Ras2 and Gpa2 in effecting transcriptional changes in the cell in response to glucose, we measured the global transcriptional response of yeast cells immediately following induction of an activated allele of tpk-w, lacks the regulatory subunit and two of the redundant catalytic subunits, with the third catalytic subunit crippled in its activity  closely resembled that of the strain carrying the inducible activated Ras allele , with only 0.4% of the genes exhibiting greater than 3-fold differences in absolute expression levels (r = 0.96). This reflects the isogenicity of the strains and indicates that the inducible RAS2G19V allele is not expressed under these conditions.The results of comparing Ras2 activation to glucose addition, provided in r = 0.94). Of those genes exhibiting a change in expression levels of at least 3-fold following addition of glucose, greater than 92% of those showed at least a 2-fold change in the same direction following activation of Ras2 . The pattern of expression at 1 h following activation of Gpa2 strongly resembles that at 20 min following addition of glucose to wild-type cells (r = 0.93). However, the response following activation of Gpa2 under these conditions is not as robust as that following activation of Ras2 or addition of glucose. While the overall magnitude of the Ras2-induced response is essentially equivalent to that obtained by glucose addition, the overall magnitude of the Gpa2-induced response is only half that of the glucose-induced changes , indicating the existence of a glucose signaling pathway that relies solely on the Ras signaling pathway. Inversely, a subset of genes is activated (or repressed) by glucose in both the wild-type and tpk-w strains but is unaffected by activation of Ras2 or Gpa2, indicating the existence of a Ras2-independent glucose signaling pathway (class III). However, the vast majority of genes that respond to glucose are affected by Ras2 activation and also respond in the tpk-w background (classes I and V). This suggests that the majority of glucose-responsive genes are regulated by redundant pathways, one of which requires Ras2 and the other one(s) of which is Ras2 independent. Thus, the major transcriptional response of glucose addition diverges prior to activation of Ras2, but converges before gene activation. This is elaborated further in the Discussion.If those genes induced by glucose and by activation of Ras2 are regulated by glucose solely through the Ras2–Gpa2–cAMP pathway, then we would anticipate that glucose-induced transcriptional alteration would be abrogated in a tpk-w cells compared to that in Tpk+ cells. For those genes whose induction or repression by Ras2 is exerted through PKA, the tpk-w mutations would be expected to abrogate that response. In GAL10p-RAS2V19tpk-w strain versus the change in expression of each gene 60 min after galactose addition to the GAL10p-RAS2V19 strain. As evident, almost all genes fail to respond to Ras2 activation in the tpk-w background. Of the 789 genes  in this experiment whose expression increased by more than 2-fold at 60 min following addition of galactose to the GAL10p-RAS2V19 strain, only 16 (2%) also showed increased expression through activation of Ras2 in the tpk-w background. Similarly, of the 1,121 genes whose expression decreased by more than 2-fold following activation of Ras2 in a wild-type background, only five (0.5%) also showed decreased expression in the tpk-w background. Repetition of these experiments using cDNA microarrays and direct Northern blot analysis of candidate genes failed to confirm that expression of any gene was altered by Ras induction in a tpk-w background (data not shown). Thus, we conclude that the entirety of the transcriptional response to Ras2 activation is mediated through PKA.To assess the extent to which the effects on transcription of Ras2 activation are mediated by PKA, we examined the pattern of expression following activation of Ras2 in tpk-w strain is not as obvious as that seen with Ras2. Of the 444 genes in this experiment whose expression increased 2-fold or more in response to Gpa2 activation in a wild-type background, 75 (17%) also showed increased expression in the tpk-w background. Similarly, of the 831 genes whose expression decreased by 2-fold or more, 24 (3%) also showed decreased expression in the tpk-w background. However, multiple replicates of this experiment using cDNA microarrays failed to identify any gene consistently altered in transcription by Gpa2 in a tpk-w background. Thus, as with Ras, the vast majority, if not all, of Gpa2-responsive genes are regulated exclusively through PKA.The results are similar for Gpa2 activation. As noted above, the response to Gpa2 activation is not as robust as that to Ras2 activation, and, as noted in GPR1 encodes a protein structurally related to seven-transmembrane, G-protein–coupled receptors, and both biochemical and genetic evidence suggests it regulates Gpa2 activity in response to glucose  is higher than that between gpr1 TPK and GPR1 TPK (r = 0.65), the patterns of expression of gpr1 tpk-w and GPR1 tpk-w, as highlighted by the cluster analysis, are similar but notably distinct . Thus, these results could suggest that Gpr1 impinges on both PKA-dependent and PKA-indepen-dent signaling pathways. Alternatively, the steady-state differences between gpr1 and GPR1 strains at the onset of the experiment could render the strains differentially responsive to glucose. This issue could be resolved by appropriate conditional alleles in GPR1 and TPK.The time course data from the We have addressed the nature of the genes regulated by glucose and Ras2 in two different but related ways. First, we asked how those genes that have been annotated as performing related functions behave on average over the set of experiments. Second, we have determined whether genes performing a common function are significantly overrepresented in any cluster of coexpressed genes. Both approaches give essentially the same results.tpk-w cells or following activation of Ras2 or Gpa2  synthesis, and all the genes required solely for gluconeogenesis are significantly downregulated both by glucose addition and by activation of Ras or Gpa2. These functional categories of genes are significantly overrepresented in that class of coexpressed genes that are downregulated in all conditions tested (class V). Thus, Ras2-dependent and Ras2-independent repression pathways redundantly regulate the restructuring associated with conversion from respiration to fermentation.tpk-w strain (class VI), and include those involved in carbohydrate storage  and, to a large extent, in ubiquinone synthesis. A number of genes exhibit induction by glucose in an exclusively Ras2-dependent fashion and include genes involved in ribosome biogenesis.Several groups of genes appear to be regulated by glucose exclusively through a PKA-dependent pathway. These are genes repressed by Ras2 or Gpa2 and by glucose in the wild-type strain, but not in the Reciprocally, a number of genes exhibit induction by glucose in a completely Ras-independent fashion. As noted in We have used a number of computational approaches to identify potential regulatory sequences and regulatory factors responsible for changes in gene expression in response to glucose and/or Ras2 activation. All of these approaches are based on the assumption that genes exhibiting a common expression pattern over all the experiments are more likely to share a common regulatory sequence or respond to a common transcription factor (see Supporting Information).Several motifs  and transcription factor-binding sites  are associated with the class of genes induced by glucose through both a Ras-dependent and a Ras-independent pathway. Rap1- and Fhl1-binding sites have previously been associated with ribosomal protein genes , and theRAS2* or GPA2* alleles. As a positive control, we examined expression of the RPS18B promoter/enhancer region when it was fused to the reporter construct. As evident in RAS2* or GPA2* in our genome-wide expression analysis. Having confirmed the ability of this system to detect Ras-responsive promoters, we examined the ability of the Rap1-binding site or the RRPE or PAC element to enhance transcription in response to activation of the Ras pathway. As noted in Tables To evaluate whether the predicted motifs mediate Ras-activated transcription, we inserted each motif upstream of a reporter gene lacking any other upstream activation sequence (UAS) and then introduced the individual constructs into strains containing the inducible CYC1 UAS and the promoter of a CYC1-lacZ reporter construct and examining expression under different growth conditions. Most of the known elements manifested modest repression activity that was not enhanced by growth on glucose or by Ras or Gpa2 activation. However, multiple copies of an Ume6-like element from PDR10 elicited strong glucose-enhanced repression activity. As evident from UME6  did not alleviate the repressive effects of this element, although this deletion eliminated repression effected by a known Ume6-binding site from CAT8. Repression by the PDR10 site was alleviated by deletion of TUP1 or SSN6. Thus, this element likely functions by recruiting the Tup1/Ssn6 repressor complex to the promoter through a specific DNA binding factor intermediate. Given the lack of correspondence between the sequence of the element and known regulatory motifs, the element likely represents a novel glucose repression mechanism.Several motifs were identified as correlated with repression by glucose and by Ras2 or Gpa2. These included binding sites for Rpn4, Ume6, Hap2/3/4, and Msn2/4 as well as several sequences of unknown association. We tested several of these motifs for their ability to mediate glucose- or Ras-induced transcriptional repression by inserting them between the Transcriptional regulation by glucose has been examined extensively by genetic and biochemical analyses of specific glucose-repressible and glucose-inducible genes as well as by global transcriptional analysis . These sIn this study, we have shown that most of the changes in transcription attendant on glucose addition can be recapitulated by activation of Ras2 or Gpa2. Thus, most of the glucose-induced changes in gene expression can be mediated by Ras2 and Gpa2. This is surprising since most transcriptional responses to glucose, particularly glucose repression, have been associated with Ras-independent mechanisms . In facttpk-w strains of yeast, which, as noted above, contain a PKA that is unresponsive to changes in cAMP levels. The authors found that tpk-w strains not only reverse all the phenotypes of bcy1 strains, but also regain the ability to respond to glucose depletion and readdition  in a timely and appropriate manner. Thus, the authors concluded that, while Ras/PKA could affect the cell's growth response to nutrients, one or more cAMP-indepen-dent pathways regulate the cell's response to nutrient availability. Under circumstances in which the cAMP signaling pathway is maintained at a moderate but constant level, this additional pathway(s) is sufficient for normal nutrient regulation. The presence of redundant glucose signaling in yeast could explain these earlier results.A redundant pathway for glucose signaling is consistent with previous observations suggesting that while activation of Ras/PKA elicits substantial changes in growth and carbohydrate metabolism in the cell, most of those changes can be effected even in the absence of an active Ras/PKA pathway. gal1 background or addition of the gratuitous inducer β-estridiol to a strain with Ras2 or Gpa2 activation driven by a lexA-ER-VP16 chimeric transcription factor  was placed under the control of the GAL10 promoter (plasmid B2364), digested with ClaI, and integrated into the LEU2 locus of Y2864 and Y2895 to obtain strains Y2876 and Y2897. Yeast Consortium Deletion Strains created in the BY4742 background (MATα his3Δ leu2Δ lys2Δ ura3Δ) were obtained from Research Genetics . The hoΔ strain was used as a wild-type control.All strains used in this study were derived from W303–1B and are listed in 600 of 0.25 , at which time an aliquot of cells was removed as the time-0 control. Glucose or galactose was then added to 2% in the remaining culture and aliquots (40 ml) of cells were collected at 20, 40, and 60 min following sugar addition. Cells were mixed with 100 ml of prechilled water and quickly spun down by centrifugation at 2,500 rpm for 3 min at 4°C.Cells were streaked on YEPD plates and grown for 2–3 d at 30°C. Fresh colonies were inoculated into synthetic complete (SC) medium supplemented with 3% glycerol as the only carbon source. Cells were grown at 30°C and shaken at 200 rpm to an ODCell pellets were lysed in TRI reagent  by vortexing with glass beads for 3 min. After a 5-min incubation at room temperature, 0.2 ml of chloroform per 1 ml of TRI reagent was added and mixed well with the homogenate. After centrifugation at 14,000 rpm for 15 min at 4°C, the upper aqueous phase was removed and precipitated with equal volume of isopropanol. RNA pellets were washed with 75% ethanol, air-dried, and dissolved in water. mRNA was purified from the total RNA with oligotex .24 primer  and SuperScript II RT (Invitrogen). Second-strand cDNA was synthesized using DNA ligase (10 U), DNA polymerase I (40 U), and RNase H (2U) from Invitrogen. Biotin-labeled cRNA was made with a BioArray HighYield RNA transcript labeling kit  and purified using an RNeasy mini-kit (Qiagen). The cRNA was fragmented, mixed with control cRNA cocktail, and hybridized to yeast genome S98 array  for 16 h in a 45°C oven rotating at 60 rpm. The probe arrays were washed and stained using the GeneChip Fluidics station 400 (Affymetrix) and scanned at 570 nm with the Agilent GeneArray scanner (Affymetrix).First-strand cDNA was synthesized from mRNA using HPLC-purified T7-(dT)gal1 strains by addition of galactose . All experiments were normalized to the same total signal intensity. Data for all experiments can be obtained from Tables http://www.molbio.princeton.edu/labs/broach/microarray.htm.For each experiment, we examined multiple timepoints, and for samples of significant interest we performed the experiment in triplicate. For initial analysis, we used MicroArray Suite 5.0 software (Affymetrix) to determine whether the hybridization signal for a gene was reliable and incorporated in our analysis only those measurements that were judged present, which generally included greater than 90% of the gene measurements in any one sample, with greater than 75% of all genes yielding reliable values over all the experiments. We also eliminated from our initial analysis those genes that were induced more than 3-fold in the Partitional clustering of gene expression data was performed using the Qtclust algorithm , which czed Saccharomyces cerevisiae transcription factors. These matrices were constructed from a mix of experimentally determined binding sites, augmented with extensive expression and chromatin IP-derived data ; tpk-w = Y2872 ; RAS* = Y2866 ; RAS tpk-w = Y2873 ; GPA2* = Y2876  GPA tpk-w = Y2897  .Strains were as follows: Wild-type = Y2864  gene designation, the MIPS functional category, and the function of the gene product, and the Affymetrix probe was set for that gene. For each time sample, the first column provides the normalized intensity values and the second column provides the determination from the MicroArray Suite 5.0 software as to whether the value was significant (P), insignificant (A), or indeterminate (M). The table is in a tab-delimited text format.Data presentation is as follows. The first five columns provide the gene name (if known), the (1.99 MB TXT).Click here for additional data file.Table S2MATα ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1; Y3159 = MATα ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 gpr1::hphMX; Y2857 = V218G tpk3::TRP1 bcy1::LEU2MATα ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1tpk1::HIS3 tpk2; Y3077 = V218G tpk3::TRP1 bcy1::LEU2 gpr1::hphMXMATα ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1tpk1::HIS3 tpk2.Strains were as follows: Y2092 = 600 = 0.3. Glucose was added to 2%, and samples were removed at 0, 20, 40 and 60 min following the addition.Experimental conditions were as follows. Cells were grown in SC medium plus 3% glycerol to AMicroarrays were performed as follows. Reference samples (RNA from 0 timepoint in each experiment) were labeled with Cy3, and each test sample (RNA from subsequent timepoints) was labeled with Cy5, mixed with the corresponding reference sample, and hybridized to cDNA microarrays printed in-house.Data presentation is as follows. Values are ratios of RNA levels for each gene at the indicated timepoint relative to the level for that gene at time 0 in that particular experiment.(477 KB TXT).Click here for additional data file.Table S3The set of genes, specified by their SGD designation, comprising each of the gene expression classes listed in (29 KB TXT).Click here for additional data file.


The WINPEPI (PEPI-for-Windows) computer programs for epidemiologists are designed for use in practice and research in the health field and as learning or teaching aids. They aim to complement other statistics packages. The programs are free, and can be downloaded from the Internet.There are at present four WINPEPI programs: DESCRIBE, for use in descriptive epidemiology, COMPARE2, for use in comparisons of two independent groups or samples, PAIRSetc, for use in comparisons of paired and other matched observations, and WHATIS, a "ready reckoner" utility program. The programs contain 75 modules, each of which provides a number, sometimes a large number, of statistical procedures. The manuals explain the uses, limitations and applicability of specific procedures, and furnish formulae and references.WINPEPI provides a wide variety of statistical routines commonly used by epidemiologists, and is a handy resource for many procedures that are not very commonly used or easily found. The programs are in general user-friendly, although some users may be confused by the large numbers of options and results provided. The main limitations are the inability to read data files and the fact that only one of the programs presents graphic results. WINPEPI has a considerable potential as a learning and teaching aid. This paper describes the WINPEPI (PEPI-for-Windows) programs recently added to the PEPI suite of computer programs for epidemiologists, and discusses some of their uses and limitations. The programs were developed for use in practice and research in the health field and as learning or teaching aids.Programs for EPIdemiologists) grew from a set of programs for programmable pocket calculators that was published in 1983 to "make life easier for investigators, extend the use of appropriate analytic methods, and enable researchers to concentrate on substantive issues rather than on procedural technicalities" [PEPI , examine a sequence of rates or other values , perform direct and indirect standardization, estimate prevalence from a cluster or stratified sample or by the capture-recapture method, determine required sample sizes, and appraise screening or diagnostic tests .COMPARE2 has 28 modules for use in comparisons of two independent groups or samples, and may be used to analyze cross-sectional, cohort and case-control studies, and trials. It can compare proportions or odds, risks, rates, and categorical and numerical data , appraise the effect of misclassification, and determine power and sample size for a variety of tests.The program can deal with stratified data, analyzing the combined strata as well as each stratum; it permits the control of possible confounding by the stratifying variable or variables, and the assessment of heterogeneity as an indication of effect modification. It can be used in meta-analyses, to compare study results and, if warranted, combine them.kappa or intraclass correlation coefficients. Like COMPARE2, PAIRSetc can deal with stratified data.PAIRSetc has 29 modules for use in comparisons of paired and other matched observations, such as matched-control trials and cohort studies, matched case-control studies, before-after studies, and reliability studies that compare replicate observations or methods of measurement. The "etc" in its name indicates the program's ability to deal with matched sets larger than pairs. The program can compare dichotomous, categorical and numerical data , appraise the effect of misclassification, and determine power and sample size for a variety of tests and for measuring z, t, chi-square, or F, or vice versa, and time-spans.WHATIS is a "ready reckoner" utility program with four modules. It provides a calculator  that stores values and formulae, enabling them to be recalled when needed, and it computes confidence intervals for a variety of statistics, P-values corresponding to given values of Some of the modules have very specific purposes; for example, to determine the sample size required to perform a specific test with a given power or precision, or to appraise the effect of misclassification in a given situation by computing the "true" findings that would give rise to the observed findings. Other modules provide many statistical procedures, as is illustrated by the following summaries of what two of the richer modules do. A module may not only provide numerous tests and measures, it may also use alternative methods of estimation.Q, phi, and lambda. Separate results are shown for studies in which inverse sampling was used.After entry of a 2 × 2 table, this module provides exact one-tailed and two-tailed tests , chi-square tests , an optional equivalence test, the ratio of proportions , the difference between proportions , the odds ratio , Yule's H and I-squared, with their 95% confidence intervals), and  estimates of the fail-safe N and two tests for a skewed funnel plot (regression asymmetry and adjusted rank correlation tests).For stratified data, the combined analysis provides Fisher and mid-P exact and Mantel-Haenszel tests, an optional equivalence test, three estimators of the overall ratio of proportions and of the overall difference between proportions , and four estimators of the overall odds ratio , heterogeneity tests and measures (t-test and Wilcoxon's signed-ranks test), and the Poisson dispersion test for heterogeneity is done .This module appraises a frequency distribution, and also appraises a sequence of numbers. It describes the frequency distribution in terms of its central tendency  and dispersion , and it performs the Grubbs test for outliers. The shape of the frequency distribution is appraised in terms of symmetry or skewness  and peakedness or flatness . The shape of the frequency distribution is pictured in box-and-whisker diagrams, for both raw and log-transformed data. Two tests for normality (Lilliefors and D'Agostini-Pearson tests) are applied to the raw and log-transformed data. The median or mean can be compared with a hypothetical value , trend , a change-point, and centrifugality. The module provides Sen's estimator of slope, parametric and nonparametric linear regression analyses, and Spearman, Kendall's, and Pearson's correlation coefficients, and it smooths the curve, using procedures based on running medians and on Fourier transforms. Regression lines, smoothed curves, and the change-point are shown in a graph.There is no special installation procedure; the programs need only be put in a folder of the user's choice.The appropriate program and module must first be selected. As an aid, a Pepi Finder  is provided; it is called up by clicking on its icon, and can be printed for easy reference. The Pepi Finder is an alphabetical index that shows which programs and modules deal with a specified procedure, measure, or kind of study. As seen in the excerpt shown in Figure Each program has an opening screen Figure  that disThe programs do not read data files, but require the entry of data that have already been counted or summarized, either manually or by using statistical software that processes primary data. The data can be entered at the keyboard, or (in multiple-entry boxes for the entry of tables) can be "pasted" from a file in which they are available. Once entered, tabular data can be pasted to a text file for future re-use by pasting. Alternative forms of data are often accepted, e.g. numerators instead of rates or proportions, and either individual or grouped observations. Warning messages are shown if obvious errors are made when entering data or if essential items are omitted.Simple on-screen instructions are provided, using simple language. For example, dichotomous variables are referred to as "yes-no" variables, and metric-scale observations, continuous or discrete, as "numerical". The term "rate" is used both for rates that have person-time denominators (e.g. incidence density) and for measures whose denominators are numbers of individuals ; when the distinction is important, this is indicated. The instructions make use of terms well-known to epidemiologists, such as "case-control study", "exposed" and "not exposed", and "risk factor". To simplify operation, the program generally performs and reports all the prescribed procedures that the data will permit, without requiring choices by the user. But some options may be offered. In Figure Pop-up hints and help screens are provided.Results are shown in an output screen Figure , from whAll results are saved in a disk file, unless the user changes this default. The WINPEPI package contains a utility program (JOINTEXT) that can merge result files.DESCRIBE (but no other WINPEPI program) displays graphs – box-and-whisker plots, survival curves, seasonal peaks, regression lines, smoothed curves, forest plots, scattergrams, summary ROC curves, and graphs showing required sample sizes under different conditions. In most of the graphs, numerical values can be read by mouse-clicking at any location, optionally after magnifying a segment (zooming). Specimen graphs are shown in Figures Comprehensive manuals are provided. These furnish full information about each module, including explanations of the uses, limitations, and applicability of specific procedures, and formulae or references. The Pepi Finder serves as an index to the manuals.Criteria for the appraisal of statistical software for epidemiology  include The WINPEPI programs are easy to install and easy to use (with the reservations discussed below). Their documentation is very detailed and (at the price of repetitiveness) includes a separate self-contained description of each module. A regrettable shortcoming of WINPEPI is that only one of the programs, DESCRIBE, presents graphic results. This is because DESCRIBE is the only 32-bit program, and the graph unit used by WINPEPI  is approBut the WINPEPI programs do not provide data management facilities, and some other software package must be used if the data require processing. An epidemiologist or student whose data have been stored and maybe processed in another package, and who is well versed in the use of that package, may therefore have no need for the WINPEPI programs, despite their ease of operation, except when these do analyses not done by the other package. The WINPEPI programs aim "to complement – not replace – other statistics packages" .Also , the WINPEPI programs do not read data files. Data must be entered each time a program is used. This drawback is partly overcome by the possibility of pasting tabular data into data-entry boxes. But data entry can be tiresome, and users accustomed to programs that use data files may find it particularly vexatious. On the other hand, for some purposes keyboard entry may be seen as a boon: "Although conventional statistical software packages are adequate when you have a data set to work with, they are not always helpful when you need to do keyboard entry of data and rapidly perform simple analyses. For instance, you may want to replicate some analyses from a journal article and compute a Mantel-Haenszel odds ratio, or you may want to compute the sample size for your study while writing a grant proposal. Maybe you want to demonstrate to your students the impact of increasing sample size on the confidence intervals of a proportion. Perhaps you are a student and would like to do your epidemiology or biostatistics homework with some easy-to-use analytical routines... It is in this niche area that PEPI scores!" .A criticism of version 3 of PEPI as being insufficiently user-friendly  led to aUnfortunately the wide variety of statistical procedures that is offered makes the WINPEPI programs less convenient to use; versatility carries a price. Even the provision made for the entry of alternative forms of data, meant as a convenience, necessitates a decision and may hence be an inconvenience – for example, a simple comparison of two proportions (using module A of COMPARE2) requires a choice between entry of four frequencies, of numerators and denominators, or of proportions and denominators.The DOS-based PEPI package elicited the comments "there are so many modules that sometimes it is difficult to remember which one to use"  and, witThe possibility of confusion is of course much reduced by the fact that related modules – for example, those concerning comparisons of two independent samples – are concentrated in the same WINPEPI program. Having opened the appropriate program, the user need only click on the kind of analysis that is required. But even that may tax some users. In COMPARE2, for example, a choice between modules B and D , for example, provides numerous "exact" and chi-square tests, and three measures of association, with confidence limits computed by different methods, as well as other results, including some that are valid only if inverse sampling was used. Similarly, module D1 of PAIRSetc  provides three tests, six intraclass coefficients and a number of other measures of agreement, appropriate for different purposes. For this reason, every WINPEPI manual carries the admonition: "This program offers more options than most users will ever need, and will usually display more results than are needed. Ignore the options and results you don't require". (This of course assumes that the user knows what he or she wants.)kappa, it can do no harm if the output provides extra results that draw attention to the fact that kappa has a ceiling value, or that its value can be adjusted to avoid paradoxical results. The user may be stimulated to use some of the additional procedures, after (if necessary) learning more about them. The manuals carry the warning: "It is unwise to use a statistical procedure whose use one does not understand. This manual cannot supply this knowledge, and it is certainly no substitute for the basic understanding of statistics and epidemiological thinking that is essential for the wise choice of methods and the correct interpretation of their results".But while all the results cannot be of interest to an ordinary user, each of them may be of interest to some users. As pointed out in a review of epidemiological software , "what oThe provision of alternative tests, and estimators based on alternative methods, may of course be confusing, whatever explanatory comments may be offered in the output or the manuals. But it may permit a knowledgeable user to select the method most appropriate in a particular situation, and it serves as a reminder to the less knowledgeable user that different methods exist, based on different assumptions and using different models, most of them yielding approximations, and none of them having absolute validity for all purposes, and as a warning that caution is indicated if different methods lead to very different conclusions. "Exact" results computed in different ways differ, and "exact" probabilities and confidence intervals are not always preferable to probabilities and confidence intervals computed in other ways.The length of the list in the Pepi Finder testifies to the wide variety of statistical routines offered. "The programs cover an amazing array of applications", says one review . PEPI hakappa  it uses epidemiological language and provides results that are meaningful to epidemiologists. They rapidly learn to use the Pepi Finder and the programs themselves. They find the programs easy to use, although they may at first be confused by the multiplicity of modules and results; but they rapidly learn to focus on the specific modules and results that interest them, and to disregard others. At the same time, the rich output may serve to acquaint the student with other measures and tests, and excite interest in them. The weight the programs give to measures of association and their confidence intervals may help to counteract the belief that significance testing is the be-all and end-all of an analysis."PEPI facilitates a ready understanding of important epidemiologic concepts, unfettered by the complexities of statistical programming", says a reviewer . With apA useful feature is that, by clicking on the "Repeat" button and making changes to the data or options, students can easily do "what if?" exercises . For exaA recent epidemiology textbook makes frequent use of PEPI in its exercises "to relieve students from some of the tedium and anxiety of hand calculation, while opening up possibilities of using advanced techniques that might not otherwise be available. It is time to familiarize even introductory students to these essential tools of the trade" .WINPEPI complements other statistics packages. It is versatile, providing a wide variety of statistical routines commonly used by epidemiologists, but is far from being a complete compendium of such routines. It is a handy source of many procedures that are not very commonly or easily found.The programs are in general user-friendly, although some users may be confused by the large numbers of options and results provided. The main limitation is the inability to read data files, but tabular data can be entered by pasting, and for some purposes keyboard entry of data is an advantage. Only one of the programs presents graphic results.WINPEPI has a considerable potential as a learning and teaching aid.The current version (at the time of this writing) of the software is available for free download as an additional file (WINPEPI.ZIP) attached to this article. It includes the programs, their manuals, and the Pepi Finder. Subsequent versions will be available at http://www.brixtonhealth.com for free download. Information about the latest WINPEPI version can be found at http://www.sagebrushpress.com/pepibook.html, where the DOS-based programs are available for free download.The programs and manuals are copyrighted, but may be freely copied and distributed for personal use; they may not be exploited commercially without permission.COMPARE2, PAIRSetc, and WHATIS are 16-bit programs (written in Delphi version 1) that can be run in any version of Windows. DESCRIBE is a 32-bit program (written in Delphi version 5), and can be run in any version of Windows except Windows 3.The manuals for DESCRIBE, COMPARE2, and PAIRSetc are in PDF format, and can be read or printed with Adobe Acrobat. WHATIS is documented in the version 4 manual .The author wrote the WINPEPI programs and manuals and is co-author of the DOS-based programs and manual, and hence may be biased in their favour.WINPEPI package. WINPEPI programs, with manuals and Pepi Finder.Click here for file


Asynchronous myocardial contraction in heart failure is associated with poor prognosis. Resynchronization can be achieved by biventricular pacing (BVP), which leads to clinical improvement and reverse remodeling. However, there is a substantial subset of patients with wide QRS complexes in the electrocardiogram that does not improve despite BVP. QRS width does not predict benefit of BVP and only correlates weakly with echocardiographically determined myocardial asynchrony. Determination of asynchrony by Tissue Doppler echocardiography seems to be the best predictor for improvement after BVP, although no consensus on the optimal method to assess asynchrony has been achieved yet. Our own preliminary results show the usefulness of Tissue Doppler Imaging and Tissue Synchronization Imaging to document acute and sustained improvement after BVP. To date, all studies evaluating Tissue Doppler in BVP were performed retrospectively and no prospective studies with patient selection for BVP according to echocardiographic criteria of asynchrony were published yet. We believe that these new echocardiographic tools will help to prospectively select patients for BVP, help to guide implantation and to optimize device programming. Heart failure is among the most common chronic diseases in modern civilizations. The dilatation of the left ventricle frequently induces intracardiac conduction delays resulting in asynchronous left ventricular motion. This manifests as left bundle branch block in the surface ECG. Both QRS width and intraventricular asynchrony are predictors of hospitalization and severe cardiac events in patients with heart failure -3.The mechanisms of myocardial asynchrony include a delayed left ventricular regional contraction and relaxation. The right ventricle contracts during left ventricular end-diastole, leading to a "bulging" of the septum into the left ventricle. The intra(left)ventricular delay of the systolic velocity induces the "delayed longitudinal contraction (DLC)". Furthermore, the delay of the contraction of the papillary muscles aggravates mitral regurgitation. This, in summary, leads to an increased oxygen demand of the myocardium .Resynchronization of the intraventricular conduction can be achieved by introducing an additional lead through the coronary venous sinus to stimulate the left ventricle . The combination of BVP and a cardioverter-defibrillator (ICD) combines the clinical improvement by BVP and reduction in mortality . Recent In the current guidelines, LBBB in the surface ECG and a reduced LVEF are the main indications for BVP . HoweverFurthermore, asynchrony is common even in heart failure patients with narrow QRS complexes compared with healthy controls. A prospective study assessed left ventricular systolic and diastolic asynchrony in 67 patients with heart failure (LVEF < 50%) with normal QRS width and 45 patients with CHF and wide QRS complexes (>120 ms). 88 healthy control patients were included. Systolic (diastolic) asynchrony occurs in 51% (46%) of the heart failure patients with narrow QRS complexes and in 73% (69%) in the patients with wide QRS complexes. Systolic asynchrony was defined as the max difference in time-to-peak myocardial contraction of 12 myocardial segments. Diastolic asynchrony was defined as maximum difference of time-to-peak early diastolic relaxation. In summary, the authors state that asynchrony is common in patients with heart failure even without a wide QRS complex . This isA recent study demonstrated that successful BVP can be achieved in patients with a normal QRS duration and asynchrony ,17.These controversial data indicate the need for a more careful patient selection for BVP. Newer echocardiographic techniques, such as Tissue Doppler Imaging and Tissue Synchronization Imaging could potentially improve patient selection and guidance of implantation and programming of the devices for BVP. The risks of pacemaker implantation and expenses in non-responders to BVP could be avoided. Furthermore, the cost-effectiveness of BVP would be augmented.Regarding the nomenclature, the term "asynchrony" is used synonymously to "dyssynchrony" in this article. There is a variety of methods to determine asynchrony. In table Echocardiographic tools include 2D, Doppler and Tissue Doppler methods. Up to date, there is no consensus on the definition of echocardiographically measured myocardial asynchrony. The determination of asynchrony by M-mode echocardiography is limited to septal and inferior segments in parasternal long-axis and is not performed routinely in current studies ,21. EarlTissue Doppler imaging (TDI) measures regional wall motion velocities. TDI can accurately quantify regional left ventricular function . Pulsed TDI technology includes tissue tracking and strain rate imaging. Tissue tracking allows the measurement and visualization of longitudinal motion in each myocardial segment during the different phases of the heart cycle.Strain measures compression and distension of myocardial segments ("deformation imaging") and strain rate imaging expresses strain changes per time interval. Post-systolic movement diagnosed with velocity or tissue tracking can be differentiated into passive or active motion . But in ischemic cardiomyopathy PSS was not an useful criterion for response to BVP because this phenomenon is not only a sign of asynchrony but also a marker for ischemia and/or viability of severe hypo/akinetic segments .Tissue synchronization imaging (TSI) is a newer technique that utilizes color-coded time-to-peak tissue Doppler velocities and visualizes segments of dyssynchrony in real-time by superimposing these temporal motion data on 2D echo images. TSI analysis is possible in all myocardial regions except the apex. The color-coding is green , yellow (150–300 ms) and red (300–500 ms) . Online Interventricular asynchrony can be assessed by comparing pw-Doppler signals in the right and left ventricular outflow tracts. A delay of >60 ms is considered to demonstrate interventricular asynchrony. These measurements in the outflow tracts cannot be performed simultaneously and, therefore, there is a high inter-measurement variability and dependence on cardiac workload. In addition, interventricular asynchrony can measured as the difference of the electromechanical delays in the basal LV segments and the lateral RV segments [Myocardial asynchrony includes inter- and intraventricular asynchrony. segments .Intra(left)ventricular asynchrony is considered to be the most important aspect of the electromechanical delay (EMD). It can be measured by a variety of methods. EMD is defined as the delay between the onset of the QRS complex on the surface ECG and the onset of the systolic TDI wave in corresponding myocardial segments. Recently, the systolic synchronicity index has been introduced [troduced . It is dIntraventricular asynchrony can also be demonstrated by tissue tracking with diastolic color-coded areas called DLC. This is the amount of post-systolic contraction after the closure of the aortic valve (i.e. post systolic shortening = PSS) which was confirmed by strain and strain rate in this study .Intra-left ventricular asynchrony is not only of diagnostic value for selecting patients for BVP, but has prognostic value as well. Bader et al.  examinedIn figures Only limited data are published concerning prospective echocardiography based patient selection for BVP. Bordachar et al.  performeSeveral studies were performed to retrospectively correlate markers of asynchrony to benefit from BVP.Lafitte  has inclIn another study , 49 patiThe role of TDI and 3D echo on the long term (1 year) outcome after BVP was evaluated in 25 patients . The extThe myocardial segments with the best resynchronization after BVP were studied in 18 patients with an LVEF <35% and a QRS width of >120 ms (NYHA III-IV). Color tissue Doppler velocity imaging was performed from the apical four chamber view at baseline and one month of follow-up after BVP . Peak veReverse remodeling and improved synchrony after 3 months of BVP was evaluated in 25 patients . AsynchrOne recent study has compared the value of TDI and SRI and post-systolic shortening in the prediction of reverse remodeling after BVP: The previously introduced asynchrony index (=Ts-SD) based on Tissue Doppler velocity data has the highest predictive value of reverse remodeling after BVP. PSS has predictive power only in non-ischemic heart failure. In ischemic heart failure, PSS seems not to be a marker for reverse remodeling but rather reflects viability and is therefore not altered by BVP. SRI imaging techniques did not predict reverse remodeling after three months of BVP . This isKanzaki has introduced the synchrony index, which is defined as the correlation coefficient of linear regression of velocity of septal and lateral mitral annular region. This index showed an increase after 6 months of BVP paralleled by increased LV contractility .One study  has retrTDI could play a role in identifying patients during catheterization procedures that will profit from BVP. Catheterization studies have shown that the beneficial effects of BVP begin almost immediately ,38. But Furthermore, TDI can assist in finding the optimal pacing site for the coronary sinus lead. In 31 patients, it was documented that LV-stimulation on the site of longest EMD had the best benefit of BVP. The regional asynchrony was assessed by pw-TDI and the pacing site was determined fluoroscopically . LateralAn AV time is considered to be optimal when the end of the A wave coincides with the complete closure of the mitral valve . An optiThe optimal delay between the right ventricular and the coronary sinus stimulation is yet unknown. One study compared simultaneous versus sequential BVP in 29 patients. The optimum interventricular delay was found by maximum reduction of DLC as measured by Tissue Doppler and Tissue Tracking. An optimum sequential BVP could significantly reduce the extent of DLC compared to simultaneous pacing .About one third of patients with heart failure have atrial fibrillation. The large trials, however, have only included patients in sinus rhythm. Only small studies with controversial results were performed in patients with atrial fibrillation and LBBB. Leclercq has performed one study in 59 NYHA III patients with chronic atrial fibrillation, a slow ventricular rate and the need for permanent pacing (VVI-paced QRS width of >200 ms). Due to a high drop out rate, the results did not show a significant increase in 6-min-walk distance after BVP . Larger We have performed a double-blind cross-over study in our clinic to assess the use of new echocardiographic techniques in BVP. Patients (n = 40) with a QRS >140 ms and a LVEF <35% received an InSyncICD 7272 . Preliminary results (n = 8) after two years demonstrate a reduction of the septal-posterior delay from 264 (±23) msec to 234 (±34) msec  and a stabilization of clinical (NYHA class improvement) and hemodynamic status (EF and LV volumes). The study is ongoing.The following video loops underline the utility of TSI and Tissue Tracking to document improvement of synchronicity after BVP. In Many controlled and uncontrolled studies have demonstrated that new echocardiographic tools to determine myocardial asynchrony in heart failure patients will help to select patients for BVP help guidance of implantation and optimize device programming. To date, all studies employing tissue Doppler date were performed retrospectively. No prospective studies that have selected patients for BVP according to echocardiographic evaluation of asynchrony were performed yet. The ongoing CARE-HF study incorporates echocardiographic criteria of asynchrony in a subset of patients with a QRS of 120–150 ms ; resultsUnresolved issues include different opinions regarding the various elements of asynchrony and their contribution to the pathophysiology and progression of heart failure. There is a lack of consensus about the best asynchrony marker for patient selection. There is evidence that ischemic and dilated cardiomyopathy might have different selection parameters for BVP. The practical consequences for patient selection and/or implantation site of the lead are currently under investigation. There are only limited echocardiographic data regarding the programming of the optimal interventricular (V-V) delay. There are no data concerning the long-term effect (i.e. years) of BVP on hemodynamics, amelioration of mitral regurgitation, reverse remodeling and mortality. Another area of uncertainty is the selection of patients for BVP without electrical (QRS < 120 ms) but with mechanical asynchrony.BVP Biventricular PacingDCM Dilated CardiomyopathyDLC Delayed longitudinal ContractionEMD Electromechanical DelayIVMD Interventricular Mechanical DelayLBBB Left Bundle Branch BlockPSS Post-Systolic ShorteningSRI Strain Rate ImagingTDI Tissue Doppler ImagingTs Time-to-peak myocardial contractionTSI Tissue Synchronization ImagingTs-SD Standard deviation of time-to-peak myocardial contractionF Knebel and AC Borges have performed the literature review and have prepared the manuscript. RK Reibis, have performed echocardiographic examinations for this article. HJ Bondke and J Witte and G Baumann have selected patients for BVP. HJ Bondke and J Witte have implanted the biventricular pacing devices. All authors have read and approved the final version of the manuscript.TSI in a patient with LBBB before BVP: The lateral segments show a delayed time-to-peak velocity (red colour). Apical four chamber view.Click here for fileTSI post-implantation: There is only green colour coding indicating synchronous contraction of all segments from apical four chamber view.Click here for fileTissue Tracking without BVP : There are reduced displacement values and no basal-apical gradient in the septal segments and DLC in the lateral wall (no colour-coding) indicating asynchrony of the lateral wall. Apical four chamber view.Click here for fileAcute effect with BVP "on" (Tissue Tracking): There are increased displacement values, a basal-apical gradient. Apical four chamber view.Click here for fileTissue Tracking before BVP : There are reduced displacement values and no basal-apical gradient in the septal segments and DLC in the lateral wall. Apical four chamber view.Click here for fileLong-term effect after 6 months of BVP: Reduction of left ventricular dilatation, increased displacement values, a basal-apical gradient. Apical four chamber view.Click here for file


The ACE gene contains a polymorphism consisting of either the presence  or absence  of a 287 bp alu repeat in intron 16. The D allele is associated with increased ACE activity in both tissue and plasma. The DD genotype is associated with risk of developing ARDS and mortality. The frequency of the D allele is higher in patients with pulmonary fibrosis, sarcoidosis and berylliosis. The role of this polymorphism has not been studied in the development of BPD in the premature newborn.ACE I/D genotype was determined in 245  mechanically ventilated infants weighing less than 1250 grams at birth and compared to outcome (death and/or development of BPD).2 on 28 days or 36 weeks PCA). Secondary outcomes  similarly were not influenced by the ACE ID polymorphism.The incidence of the D allele in the study population was 0.58. Eighty-eight (35.9%) infants were homozygous DD, 107 (43.7%) were heterozygous ID and 50 (20.4%) were homozygous II. There were no significant differences between genotype groups with respect to ethnic origin, birth weight, gestation, or gender. There was no effect of the ACE I/D polymorphism on mortality or development of BPD (OThe ACE I/D polymorphism does not significantly influence the development of BPD in ventilated infants less than 1250 grams. Prematurely born infants who require mechanical ventilation (MV) frequently develop chronic lung disease known as bronchopulmonary dysplasia (BPD). Oxidant injury, mechanical disruption of the lung, inflammation and subsequent failure of lung development are considered the major mechanisms in the pathogenesis of BPD. The development of BPD is characterized by an initial acute inflammatory component followed by variable degrees of lung fibrosis and failure of alveolar septation, both of which ultimately impair the development of the immature lung -5. The iThere is increasing evidence to support the role for the activation of the renin-angiotensin system (RAS) system during ALI. In ARDS there is an increase in bronchoalveolar lavage and a concomitant decrease in circulating angiotensin converting enzyme (ACE) activity,7. This There is significant variation in circulating ACE activity among individuals, which may be genetically determined. The human ACE gene is located on chromosome 17 (17q23) and contains a polymorphism consisting of either the presence  or absence  of a 287 bp alu repeat in intron 16. The delBecause of the relationship between inflammation and activation of the RAS and the association with lung fibrosis and adverse outcome in ARDS, the ACE l/D polymorphism may modify risk for the development of chronic lung disease or death in mechanically ventilated VLBW infants. The purpose of this study was to determine if there is a relationship between the ACE I/D polymorphism and respiratory outcomes of death or the development of BPD, in mechanically ventilated very low birth weight (VLBW) infants.Genomic DNA used for this case controlled study was extracted from archival tracheal aspirate (TA) pellets 223 patients) or blood (22 patients) collected prospectively as part of an ongoing study of genetic factors in the development of complications of prematurity. The TAs that were used as a source of genomic DNA were originally collected as part of long term longitudinal studies examining cytokine concentrations and the development of CLD[ patientsCultures for genital mycoplasmas were performed on samples collected in the first few days of life. Clinical and outcome data were abstracted from the clinical record and included information on respiratory outcome, survival and development of complications of prematurity.Isolation of total DNA from blood or TA pellets was performed using the QIAmp DNA Mini kits™ . Briefly, TA pellets were suspended in 200 μl of sterile phosphate buffered saline by vigorous vortexing, then digested in proteinase K and applied to silica gel spin columns. Columns were washed in the manufacturer's supplied buffers and the total DNA was eluted in 200 μl elution buffer. Blood (200 μl) was extracted similarly to the TA pellets.2 1.5 mM, KCl 50 mM, 5%DMSO, Triton X-100 0.1%, 200 micromolar each of dNTPs, 10 pmol each primer, 1–2 U of Taq polymerase, and 1 microliter of DNA. DNA was denatured at 95°C for 5–10 min, followed by 30 cycles of denaturation at 94° for 60 sec, annealing at 67 for 60 sec, and extension at 72° for 2 minutes, with a final extension at 72° for 7 min. PCR products were separated on 2% agarose gel containing 0.5-microgram/ml ethidium bromide. After gel electrophoresis the bands were visualized by UV-transillumination. The PCR product is a 190 bp fragment in the absence of the insertion (D genotype) and a 490 bp fragment in the presence of the insertion (I genotype).ACE I/D polymorphism was performed by microplate PCR method as described previously. . Tracheal aspirate cultures obtained during the first few days of life grew Ureaplasma urealyticum (Uu) on at least one occasion from 74 of 217 infants cultured (34%) and Mycoplasma hominis (Mh) from 29 (13%). One hundred and fifty one (67%) infants were oxygen dependent at 28 days and 55 (25%) were oxygen dependent at 36 weeks PCA. There were 39 (16%) patients who died  during their initial hospitalization (24 before 28 days of age and 15 after 28 days).Two hundred and forty five (245) patients had complete clinical information on respiratory outcome and genomic DNA available for genotyping. Mean gestational age and birth weight of the study population was 26.4 ± 0.1 weeks and 869 ± 12 grams respectively. One hundred and ninety-four (79%) infants were African-American, 47 (19 %) were Caucasian and 4 (2%) were Hispanic. Male: female ratio was 147:98. All patients required MV at birth and 228 (93%) infants were treated with exogenous surfactant therapy  and Caucasian infants (0.59) (p = 0.664). Fifty (20.4%) infants were homozygous II, 107 (43.7 %) were heterozygous ID and 88 (35.9%) were homozygous DD.Baseline clinical characteristics of birth weight, gestational age, race, gender, TA isolation of Mh, and the need for surfactant replacement were not different between genotype groups Table . IsolatiClinical characteristics of surviving infants who were or were not oxygen dependent at days are shown in Table Because the ACE I/D polymorphism may have different functional effects on plasma and tissue ACE activities in different ethnic groups, we analyzed the effects of ACE on the incidence of BPD separately for Caucasian and African-American infants. (The effects of the ACE I/D polymorphism on the incidence of BPD and other outcomes for the combined group can be found in the online data supplement). Table The effects of ACE genotype on outcome in ventilated African-American infants less than 1250 grams are shown in Table Since birth weight and gestation are the primary determinants of adverse outcome, analysis was repeated in both infants greater than 750 grams and infants ≤ 750 grams. There was no significant effect of ACE genotype when this subgroup analysis was performed (data not shown).The RAS is activated during lung injury and plays a role in several pathological processes. In addition to the pulmonary endothelium, respiratory epithelium also possesses significant ACE activity. Fas-induced alveolar epithelial cell apoptosis is dependent on local AT-II production and interaction with it receptor.,11 FurthThe frequency of the D allele in our study population was not different than reported in our local population or for other groups,24,31. TIn our study population, the ACE ID polymorphism was not associated with an altered risk for the development of BPD, oxygen dependency at 28 days, death  or composite outcomes (BPD or death) suggesting that factors other than genetic variation at this locus contribute to these outcomes. This is in contrast to that seen in adults with ARDS or other lung disorders such as pulmonary fibrosis, sarcoidosis and berylliosis where the D allele is associated with disease -21.1 +915 G/C SNPs on the development of BPD[Indeed genetic factors may not have a large influence in a disease (BPD) where maturity at birth has such an overwhelming influence. Our previous study in this population failed to find any association of the tumor necrosis factor-α-308 G/A, MCP-1-2518 A/G or transforming growth factor-βnt of BPD AdditionUreaplasma urealyticum colonization in these infants. Chorioamnionitis, which is highly correlated with isolation of Uu, is a risk factor for PVL.[The finding of increased incidence of PVL in Caucasian infants with the II genotype may be due more to the greatly increased incidence of  for PVL. Because Our study has several limitations. The ACE I/D polymorphism has only been demonstrated to be functional in Caucasians. Data linThe observations of this retrospective case controlled study are also limited somewhat by selection bias. Because only infants who were mechanically ventilated were included, the true impact of this polymorphism on the complications of prematurity may be underestimated. Since this was a retrospective study using stored material no attempt was made to correlate phenotype with genotype (ie TA ACE measurements).Lung injury and the subsequent maladaptive repair process that leads to the development of BPD is complex with a great many factors that interplay to determine outcome. It is very likely that other genetic play a role in determining the risk of poor outcome. Polymorphisms in cytokines, their receptors, bacterial pattern recognition molecules, surfactant proteins, and heme oxygenase-1 are known to alter the course of other pulmonary diseases and should be examined as to their potential role in the development of BPD in the premature infant.The ACE I/D polymorphism does not significantly influence the development of BPD in ventilated infants less than 1250 grams.The author(s) declare that they have no competing interests.RJB conceived and organized the study, prepared the manuscript and performed the statistical analyses. KY performed the genotyping, and assisted with manuscript preparation. JL oversaw collection tracheal aspirates, assisted with recruitment of subjects into the original studies, and assisted with editing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Contains Tables showing baseline clinical characteristics of African American and Caucasian infants separately by Genotype groupClick here for file