The mannose receptor (MR) is an endocytic receptor of Mφ and endothelial cell subsets whose natural ligands include both self glycoproteins and microbial glycans. It is also expressed by immature cultured dendritic cells (DC), where it mediates high efficiency uptake of glycosylated antigens, yet its role in antigen handling in vivo is unknown. Knowledge of which APC subsets express MR will assist the design of experiments to address its immunological functions. Here the expression of MR by MHC class II positive APC in non-lymphoid organs of the mouse is described.MR positive APC were identified in several peripheral organs: skin, liver, cardiac and skeletal muscle and tongue. MR positive cells in salivary gland, thyroid and pancreas coexpressed MHC class II and the myeloid markers macrosialin and sialoadhesin, but not the dendritic cell markers CD11c or DEC-205. MR and MHC class II colocalised in confocal microscope images, implying that antigen capture may be the primary role of MR in these cells. Distinct ligands of MR were found in salivary gland and pancreas tissue lysates that are candidate physiological ligands of MR positive APC in these organs.The tissue and subcellular distribution of MR suggest it is appropriately located to serve as a high efficiency antigen uptake receptor of APC. Dendritic cells (DC), APC specialised for the efficient stimulation of naïve T cells, are of fundamental importance in the control of antigen-specific immune responses (reviewed in ). ImmatuImmature DC capture antigens by receptor-mediated endocytosis, in addition to macropinocytosis and phagocytosis (reviewed in ). DC areNotwithstanding their role in stimulating immune responses, it is becoming increasingly apparent that DC play a role in maintaining T cell tolerance to self antigens in the periphery . DirWe recently found MR positive interstitial cells in the thyroid, pancreas and salivary gland: secretory organs which bear endogenous ligands of the CRDs of MR . ThyroglWe previously characterised the expression pattern of MR in the adult mouse by immunohistochemistry using a polyclonal Ab raised against MR, and by in situ hybridisation . MR posiA488, was verified by observation of the expected expression pattern of MR in the spleen. Expression was restricted to the red pulp and did not overlap in dual-labeled specimens with the CD11c positive immature DC located at the border of the white and red pulps .The specificity of MR6F3To detect the scattered interstitial DC of non-lymphoid organs, sections were labeled for MHC class II figure . AlthougWe previously showed that secretory cells in the salivary gland, thyroid and exocrine pancreas were rich in ligands of the CRDs of MR, and expression of MR by adjacent interstitial cells was particularly intense . In adjaSince the MR positive cells of secretory organs exhibited a strongly myeloid phenotype but lacked expression of traditional DC markers, the possibility that they may be activated Mφ was considered. IFNγ is the most potent activator of Mφ, leading to expression of MHC class II, so the phenotype of interstitial cells in the secretory organs of IFNγ -/- mice was examined. Constitutive expression of MR and MHC class II was retained (not shown). In summary, MR positive interstitial cells displayed a novel APC phenotype, and constitutively expressed MHC class II in the absence of stimulation with IFNγ.The coexpression of MR and MHC class II in secretory organ APC implied that these cells may have immunologic functions. The distribution of these markers were examined by confocal immunofluorescence microscopy, in cells that expressed comparable levels of the two proteins figure . In someWe previously identified thyroglobulin as an abundant ligand of MR present in the thyroid, and showed that CRD4-7Fc, an Fc-fusion protein bearing four of the CRDs of MR, which retains the same ligand specificity as MR, bound specifically to sections of salivary gland and pancreas . Here, fAs a control, equivalent quantities of lung, thymus and spleen tissue lysates were subjected to lectin blotting with CRD4-7Fc as described above, but defined bands were not detected (not shown). We previously showed that Mφ within lung and thymus tissue sections contained ligands of CRD4-7Fc, as did Mφ and other cells within spleen . The absThe MR was the first non-opsonic C-type lectin receptor shown to be expressed and mediate adsorptive endocytosis in DC ,20, propIn contrast to our earlier observation that MR is not expressed by well-characterised subsets of DC in lymphoid organs , many AP+ and CD8+ T cells by DC has recently been shown using Ag fused to mAb specific for MR [The function of MR in relation to antigen handling in vivo is poorly understood, in part because most studies have used glycosylated Ag that are now known to be ligands of other receptors in addition to MR (reviewed in ). Howevec for MR ,30. Surpc for MR ), MR mayc for MR . Therefoc for MR . A link c for MR .K were compared [Antigen uptake by MR on cultured DC promotes presentation to T cells of antigen-derived peptides on MHC class II . The colcompared . These mcompared . Therefocompared .If MR is poised to direct Ag to the MHC class II compartments of secretory organ APC, the question of which Ag are captured in these environments must be raised. We previously showed that thyroglobulin, a major auto-antigen in murine autoimmune thyroid diseases, is a ligand of MR, and suggested that MR expressed by interstitial cells in the thyroid may be involved in maintaining tolerance to this Ag in normal mice . Here, fIn light of the location of MR positive APC in secretory organs, the abundance of endogenous ligands, and the positive identification of one ligand as thyroglobulin, the available data could suggest a tolerogenic role of MR is more likely than an immunostimulatory role in these organs in the steady-state. Such a mechanism may represent a physiological correlate of the experimentally induced immune tolerance achieved by targeting Ag to DEC-205 in naïve mice ,14,15. FMR positive APC are present in non-lymphoid organs of the naïve mouse, but in most organs examined, only a subset of MHC class II positive cells express MR, in contrast to the ubiquitous expression of MR described in cultured DC. This information will inform the design of experiments to test the function of MR in antigen handling in vivo. An immuno-regulatory role for MR in relation to its endogenous ligands in salivary gland, pancreas and thyroid is suggested by the finding that MR positive APC are abundant in these organs, they co-express MHC class II and appear to have an overlapping subcellular distribution of MR and MHC class II.IFNγ -/- mice  were bac488  according to the manufacturer's instructions. Goat anti-amylase was from Santa Cruz Biotechnology Inc . CRD4-7Fc was prepared as described [2 anti-rat IgG, and goat F(ab')2 anti-Armenian hamster IgG, both cy3 conjugated, and horseradish peroxidase conjugated mouse F(ab')2 anti-human IgG Fc . Horseradish peroxidase conjugated donkey F(ab')2 anti-goat IgG was from Chemicon .Rat mAb were prepared in our laboratory and used at optimal concentrations for immunolabelling. They were directed against MHC class II ; macrosialin  and siaescribed . Seconda488 conjugated MR6F3 mAb or isotype matched control. Nuclei were labeled with Hoechst 33342 dye (Molecular Probes). Sections were mounted in Aqua Polymount . Slides were examined by fluorescence microscopy and 12-bit digital images captured using a CCD camera attached to a Zeiss Axioplan photomicroscope. Slides were prepared in the same way for confocal microscopy, except that cryosections were cut at 20 μm. Confocal microscope z-stack images were collected using a Bio-Rad Radiance 2000 MP laser scanning confocal microscope, with lasers exciting at 367, 488 and 543 nm. Images corresponding to each fluorophore were collected using individual lasers sequentially to eliminate bleed-through. Immunofluorescence and confocal images were processed using MetaMorph version 4.5 software.For immunofluorescence microscopy, 6 μm cryosections were prepared from unfixed frozen tissues. They were permeabilised in an incubation buffer consisting of 0.5% BSA and 0.05% saponin in PBS, then blocked with 5% normal rabbit serum in incubation buffer. Sections were subsequently incubated with rat or hamster mAb specific for APC antigens for 1 hr, then the appropriate cy3-conjugated secondary Ab for 30 min. Sections were blocked with 100 μg/ml rat IgG for 30 min, before being probed with Alexa3, 150 mM NaCl, 10 mM EDTA, 5 mM iodoacetamide, 1 mM PMSF, 1 mg/ml pepstatin, 1μM leupeptin). Proteins were quantified in duplicate using Bicinchoninic acid protein assay kit . Samples were electrophoresed by SDS-PAGE, transferred to nitrocellulose membranes and subjected to western or lectin blotting using standard methods [2) and mannose-BSA  was used as positive control. Gels were also stained with Coomassie R-250 to detect protein.Mouse tissue lysates were prepared in lysis buffer (2% v/v Triton X-100, 10 mM Tris pH 8.0, 10 mM NaN methods . CRD4-7FCRD, carbohydrate recognition domain; DC, dendritic cell; MR, mannose receptor


To examine the prevalence of major chronic diseases and their risk factors in different socioeconomic groups in the Australian population, in order to highlight the need for public policy initiatives to reduce socioeconomic inequalities in health.Data were provided by the Australian Bureau of Statistics (ABS) from the 2001 National Health Survey (NHS) for selected chronic diseases and associated risk factors. Conditions selected were those, which form the National Health Priority Area (NHPA) conditions ; plus other 'serious' chronic conditions, in line with the classification developed by Mathers; and for which sufficient cases were available for analysis by socioeconomic status. Indirectly age-standardised prevalence rates were calculated by broad age group for Australia and for five groups of socioeconomic status; rate ratios were calculated to show variations in prevalence between these groups.Significant socioeconomic inequalities were evident for many of the major chronic diseases; the largest was for diabetes mellitus (at ages 25 to 64 years); and for many diseases, there was also a strong, continuous socioeconomic gradient in the rates.Circulatory system diseases  and digestive system diseases also exhibited a strong differential in the 25 to 64 year age group.In the 65 years and over age group, the strongest inequalities were evident for mental and behavioural problems, diabetes (with a continuous socioeconomic gradient in rates) and respiratory system diseases.A number of risk factors for chronic diseases, namely self-reported smoking, alcohol misuse, physical inactivity and excess weight showed a striking association with socioeconomic status, in particular for people who were smokers and those who did not exercise.This analysis shows that the prevalence of chronic disease varies across the socioeconomic gradient for a number of specific diseases, as well as for important disease risk factors. Therefore, any policy interventions to address the impact of chronic disease, at a population level, need to take into account these socioeconomic inequalities. As in other developed countries, chronic diseases in Australia are major contributors to the extent of illness, disability and premature mortality in the population. They are estimated to make up the greatest proportion of the burden of disease, mental problems and injury for the population as a whole (about 80%), and for particular sub-population groups .Chronic diseases are exemplified by having multifactorial aetiologies, including common disease risk factors and determinants; significant latency periods and protracted clinical courses; and are seldom cured completely ,3. CausaThe inequalities in health observed across populations are many – some of them are inevitable and others, unnecessary and unfair. Those inequalities that are potentially avoidable are deemed 'inequitable' . DespiteThere have been a number of studies published in Australia on socioeconomic inequalities in mortality from various chronic diseases and conditions. The earlier ones were analysed using information on occupation recorded on the death certificate -10. An aHowever, to date, there have been fewer studies that have examined socioeconomic inequalities in chronic disease prevalence in those still living  . One of The work of Mathers is significant for its systematic documentation of health inequalities among working aged Australians (25 to 64 years) in the late 1980s. He examined mortality, disability, disease groups, specific diseases, self-perceived health, risk factors, health service use and use of preventive screening services, using data from the 1989–90 National Health Survey (NHS) . MathersCurrent information on inequalities in health other than mortality is limited in Australia because of a dearth of suitable data collections. However, the release of data from the 2001 NHS provides an opportunity to examine the prevalence of self-reported chronic disease in Australia and the way in which this impacts on different socioeconomic groupings within the population.Information for a selection of chronic diseases is shown in Table The main findings are:The largest differential between those in the most well off and those in the most disadvantaged areas was for diabetes mellitus at ages 25 to 64 years, with the prevalence in the most disadvantaged areas being just over two and a quarter times (a rate ratio of 2.28) the prevalence for the least disadvantaged; there is also a strong, continuous gradient in the rates, with the rate ratios in each of the third to fifth quintiles statistically significant.There was a statistically significant differential of 67% at ages 25 to 64 years, with a strong, continuous gradient, in the prevalence of self-reported mental and behavioural problems across the socioeconomic gradient; differentials  in the 0 to 14 year and 65 years and over age groups were 52% and 56%, respectively.Circulatory system diseases  and digestive system diseases also exhibit a strong differential in the 25 to 64 year age group .In the 65 years and over age group, the strongest differentials were evident for mental and behavioural problems , diabetes  and respiratory system disease .Asthma accounted for almost two thirds of the rate of reporting of respiratory system disease in the 0 to 14 year age group, almost half in the 15 to 24 year age group, and for about a third of the rate in the 25 to 64 year age group.The NHS also included data on a number of risk factors for chronic diseases, namely self-reported smoking, alcohol misuse, physical inactivity and excess weight. A number of these risk factors show a striking association with socioeconomic status, in particular for people who are smokers and those who did not exercise, with continuous gradients and highly elevated rates of statistical significance Table . The difIt is important to note that the inequalities reported above relate to the health of those people living in a geographic area and to the overall level of socioeconomic disadvantage of that area. Most areas will contain varying levels of individual socioeconomic disadvantage and, to the extent that the poorer health is associated with individual economic circumstances and living conditions rather than communal environment, the inequalities will understate the true differences in health status according to socioeconomic disadvantage .Furthermore, there are limitations to the use of area-based measures of SES. Due to misclassification error , estimates of difference across the quintiles will be smaller than if data on individual-level measures of SES were used . Thus, cOur analysis indicates that socioeconomic inequalities in the prevalence of chronic diseases and their concomitant risk factors are evident across the Australian population. However, the diseases with substantial disparities across the socioeconomic quintiles are different, for different stages in the life course. Although these results cannot be directly compared with those of previous studies, because of definitional and methodological differences, the recurring finding of inequalities for chronic disease morbidity and risk factor prevalence across the socioeconomic gradient remains a significant concern.The burden in the Australian population attributable to socioeconomic inequality is large, and has far-reaching implications in terms of unnecessary disability and suffering, the loss of potentially economically productive members of society, and increased costs for the health and social care systems . DespiteWhat should we be doing differently? There is a growing body of knowledge that will help to provide direction for developing policies to reduce inequities across the population. The socioeconomic environment is a powerful and potentially modifiable factor, and public policy is a key instrument to improve this environment, particularly in areas such as housing, taxation and social security, work environments, urban design, pollution control, educational achievement, and early childhood development .However, attention must be paid to the nature of any action that is taken, to ensure that social and economic inequalities are not increased. Some programs, by their very success, can increase inequality by widening the gap between groups in the population; for example, such programs may be more attractive to those who are already healthier, or not as effective for certain groups with poorer health, less education or more stressful lives. In one smoking cessation initiative, it was found that the prevalence of smoking decreased predominately in those adults with higher education, thus increasing the existing difference with those who were more disadvantaged . While sIt is also evident that the ways in which systems such as education and health are delivered and structured can increase existing inequality. For example, schooling can be a way of addressing inequality and also a way of reproducing it. It has been suggested that there are two goals for a social justice program in education: to work to eliminate the contribution that the education system makes to the production over time of social inequality in general; and to maximise the positive contributions that the education system makes to reducing social inequality . TherefoPolicy-makers who wish to address socioeconomic inequalities in health may favour one of the following approaches. Some view the impact of socioeconomic disadvantage on those groups with the poorest health in the population, such as Aboriginal people and Torres Strait Islanders, as the priority policy goal. Others identify the gap between the health of those groups at the outer ends of the socioeconomic hierarchy , and see the narrowing of the gap as the goal. Others prefer to focus on the socioeconomic gradient in health that runs across the whole population .Graham has identified that the last approach widens the policy debate in three ways . FirstlyShe also observes that, "improving the health of poor groups and improving their position relative to other groups are necessary elements in a strategy to reduce the socioeconomic gradient. However, neither is sufficient on its own. To reduce the socioeconomic gradient, health in other socioeconomic groups also needs to improve at a faster rate than in the highest socioeconomic group. Thus, policies to ameliorate health disadvantage, to close health gaps and to reduce health gradients need to be pursued together, and not at the expense of each other" .There is also an urgent need to make health inequalities a research priority for each stage of the life course – not just to monitor the size and extent of the disparities but also to undertake research that will find preventive approaches and further policy interventions that will be effective in reducing them, and that are likely to be implemented by governments and communities.Clearly, any moves to address the impact of chronic disease at a population level must take into account socioeconomic inequalities in prevalence. More research is needed to determine which approaches are effective and why others have failed to have the desired impact, particularly for those who are from socioeconomically disadvantaged areas. Finally, although rates are generally highest at the oldest ages, the development of risk factors for many chronic diseases occurs early in life, and thus, it is essential those health inequities are addressed right across the life course.The ABS conducts the National Health Survey (NHS) on a regular basis, most recently in 2001 . The NHSThe survey includes self-reported details of health conditions (both acute and long term) and major risk factors, as well as demographic and socioeconomic information about the survey respondent. Respondents were asked if they had been told by a doctor or nurse that they had asthma, cancer, heart and circulatory conditions, and/or diabetes. These conditions, together with injuries and mental health, form the NHPAs . HoweverThe particular conditions for which data were requested from the ABS for this analysis were:the NHPA conditions ; plus other 'serious' chronic conditions, in line with the classification developed by Mathers ; and forThe risk factors used by the ABS were those identified for the NHPA conditions .The ABS has coded conditions reported by respondents to output disease categories based on ICD-10. Conditions described as 'chronic' in this article include those long-term conditions reported in the NHS, which are commonly recognised by health practitioners as chronic diseases . The risGiven the policy importance of the NHPAs, the 2001 NHS questionnaire underwent significant revisions to more precisely capture information on several of the NHPAs. Consequently, while the quality of the information on NHPAs has been improved from the 1995 NHS, the degree of comparability with previous surveys has been somewhat compromised for many of the major health conditions. Some specific conditions  appear to be comparable between the 1989–90 and 2001 surveys, however, for most groups of conditions based on ICD chapter headings  the ABS advise that the combined effect of major conceptual changes as well as major classification changes between the 1989–90 and 2001 surveys would make direct comparisons very difficult. This analysis is therefore restricted to the 2001 data.The socioeconomic status (SES) of the address of residence of each survey respondent is available at the Census Collection District (CD) level and was added to the NHS file, as was the quintile of socioeconomic disadvantage of area into which that CD fell at the Census. The measure used to allocate CDs to quintiles was the 1996 Census Index of Relative Socio-Economic Disadvantage (IRSD). The IRSD is one of five Socio-Economic Indexes for Areas produced by the ABS using Principal Components Analysis. It summarises information available from variables collected in the 1996 Population Census including those related to education, occupation, and income. The variables are expressed as percentages of the relevant population.The NHS records were aggregated to the quintiles derived from the Census data, where Quintile 1 comprises the CDs with the highest IRSD scores  and Quintile 5 comprises the CDs with the lowest IRSD scores . Each quintile comprises approximately 20% of CDs.The ABS produced the estimates of the number of people with chronic diseases and risk factors by quintile. The method used resulted in the production of quintiles of varying sizes, ranging from 17.4% of the population in Quintile 5 (most disadvantaged areas) to 22.8% in Quintile 2 and 21.1% in Quintile 1 (most advantaged areas). This is a differential of over five percentage points between Quintile 5 and Quintile 2, or 1,027,030 fewer people in the most disadvantaged areas when compared with Quintile 2 . The effect of this lack of precision on the results by quintile is not known. Although, in part, the difference arises as a result of the method used , it may also reflect different response rates to the survey from different socioeconomic groups.The NHS includes other measures that could potentially be used to measure socioeconomic status. For example, income reporting in ABS surveys is known to be incomplete, in particular for low income groups and, in the 2001 NHS, income was only collected for the respondent and their spouse. Information on education is also available, but a single measure has yet to be agreed. Problems in using education include that it is typically completed early in adulthood; it captures neither differential on-the-job training and other career investments made by individuals with similar levels of formal schooling, nor the volatility in economic status during adulthood that has been shown to have adverse implications for health . The ageChronic disease and risk factor rates are expressed as rates per 100,000 population, indirectly age-standardised. The rates were also calculated by age although, given the small sample size of the NHS, only broad age groups  were available. The standard population and quintile populations are the weighted 2001 survey populations from the NHS. The extent of any differential between the quintiles is shown by the rate ratio, which expresses the ratio of the rate in each quintile to the rate in Quintile 1 .The author(s) declare that they have no competing interests.JG conceived the study, and was responsible for its design and coordination. ST participated in the design of the study and performed the statistical analysis. DH participated in the drafting of the manuscript. All authors read and approved the final manuscript.


Survival in colorectal cancer may correlate with the degree of systemic inflammatory response to the tumour. Atrial fibrillation may be regarded as an inflammatory complication. We aimed to determine if atrial fibrillation is a prognostic factor in colorectal cancer.A prospective colorectal cancer patient database was cross-referenced with the hospital clinical-coding database to identify patients who had underwent colorectal cancer surgery and were in atrial fibrillation pre- or postoperatively.A total of 175 patients underwent surgery for colorectal cancer over a two-year period. Of these, 13 patients had atrial fibrillation pre- or postoperatively. Atrial fibrillation correlated with worse two-year survival . However, in a Cox regression analysis, atrial fibrillation was not significantly associated with survival.The presence or development of atrial fibrillation in patients undergoing surgery for colorectal cancer is associated with worse overall survival, however it was not found to be an independent factor in multivariate analysis. In general, colorectal cancer patients are three times more likely to be in atrial fibrillation preoperatively than matched controls undergoing non-colorectal cancer surgery ,2. It mast January 2000 and 31st December 2001 in a 600-bed district general hospital in the United Kingdom National Health Service were identified. The hospital serves a population of approximately 230,000. About 90 elective and emergency laparotomies are performed each year for colorectal cancer. Patients were identified from the prospectively maintained colorectal cancer database maintained by the colorectal surgical department. Patients with radiological, endoscopic or clinical examinations suspicious of colorectal cancer are referred to the weekly colorectal multi-disciplinary team (MDT) meeting. In the case of suspicious radiology, the referral to the MDT is made automatically by the radiology department. This avoids the possibility of the responsible clinical firm failing to refer a patient for consideration. Similarly, the pathology department automatically refers any patient in whom histology shows colorectal malignancy. In addition, patients who undergo surgery where a suspicion of colorectal cancer is raised are referred for consideration. The colorectal meeting is attended by the colorectal surgeons, radiologists, pathologists, palliative care physicians and nursing staff. Patients determined to have colorectal cancer by the MDT are entered into the database. The colorectal department periodically compares the database to clinical coding data for patients with colorectal cancer in order to ensure complete data capture. All patients are followed-up regularly by a team of colorectal nurse specialists in a dedicated clinic.Patients who underwent a resection for colorectal cancer between 1® version 2 .Age, sex, mode of presentation (emergency or elective), Dukes stage, postoperative anastomotic leakage and adjuvant therapy were recorded for all patients. The colorectal cancer database was cross-referenced with the hospital clinical-coding database to identify those patients who were in atrial fibrillation at any time before or after their surgery. Patients with colorectal cancer who did not undergo surgery or who only underwent palliative stoma formation were excluded. All patients were followed up for at least two years postoperatively. Overall survival and recurrence-free survival were recorded. Recurrence-free survival was defined as the time interval between operation and first diagnosis of local or distant recurrence. Patients with no recurrence were censored at the time of death from any cause other than cancer or at the time they were last seen by the colorectal team. Characteristics between those with and without AF were compared using the Student t-test and Fisher Exact test for continuous and categorical data respectively. Potential prognostic factors were compared by the log-rank test. Significant prognostic factors identified from the univariate analysis were entered into a multivariate Cox regression model of survival to test for independence. The 5% level was considered significant in the multivariate analysis. Statistical analysis was performed using StatsdirectOne hundred and seventy-five patients M:F = 111:64) who underwent bowel resection for colorectal cancer were identified from the database. Their median age was 74 years (interquartile range 66 to 80 years). Tumour site, Dukes stage and mode of operation (emergency or elective) are summarised in Table 11:64 whoCross-referencing with the clinical coding database identified thirteen patients with a history of atrial fibrillation. Five patients were in AF preoperatively. The remaining eight patients developed postoperative AF. A comparison of baseline characteristics between patients with and without AF is shown in table There was no correlation between overall survival and the following variables in the univariate survival analysis: gender, postoperative anastomotic leak, site of tumour  or preoperative radiotherapy. Mode of surgery (emergency or elective), age (<72 years or ≥ 72 years) and Dukes' stage had a significant effect on survival. When patients with atrial fibrillation were compared to those without Figure , atrial Tumours stimulate an inflammatory response when they invade or metastasise . This inIn our cohort, atrial fibrillation was not an independent predictor of survival. Dukes' stage was the strongest predictor of survival. Previous data from colorectal cancer patients suggest the degree of inflammatory response may reflect the degree of disease progression . The incThere are several other limitations to our study. The quality of the clinical coding has not been assessed. Thus, the sensitivity and specificity of our clinical coding department in coding AF is unknown. Only 13 patients (7.4%) developed AF. Previous work suggests that 13% of elective colorectal patients develop postoperative AF . Thus, iThe author(s) declare that they have no competing interests.SRW conceived the study, performed the statistical analysis and drafted the paper.KMG and NJW performed the literature search and collected the data.TAJ and NJK co-wrote the paper with SRW.All authors approved the manuscript.None declared


Arabidopsis uncovers distinct temporal classes of gene expression and opens the door to detailed studies of the regulatory pathways involved.A transcriptome analysis of male gametophyte development in  The haploid male gametophyte generation of flowering plants consists of two- or three-celled pollen grains. This functional specialization is thought to be a key factor in the evolutionary success of flowering plants. Moreover, pollen ontogeny is also an attractive model in which to dissect cellular networks that control cell growth, asymmetric cell division and cellular differentiation. Our objective, and an essential step towards the detailed understanding of these processes, was to comprehensively define the male haploid transcriptome throughout development.Arabidopsis and used Affymetrix ATH1 genome arrays to identify a total of 13,977 male gametophyte-expressed mRNAs, 9.7% of which were male-gametophyte-specific. The transition from bicellular to tricellular pollen was accompanied by a decline in the number of diverse mRNA species and an increase in the proportion of male gametophyte-specific transcripts. Expression profiles of regulatory proteins and distinct clusters of coexpressed genes were identified that could correspond to components of gametophytic regulatory networks. Moreover, integration of transcriptome and experimental data revealed the early synthesis of translation factors and their requirement to support pollen tube growth.We have developed staged spore isolation procedures for The progression from proliferating microspores to terminally differentiated pollen is characterized by large-scale repression of early program genes and the activation of a unique late gene-expression program in maturing pollen. These data provide a quantum increase in knowledge concerning gametophytic transcription and lay the foundations for new genomic-led studies of the regulatory networks and cellular functions that operate to specify male gametophyte development. Development of eukaryotic cells towards particular cell fates is regulated by complex and dynamic changes in gene expression. These changes, when monitored on a genome-wide scale, provide a detailed framework for the analysis and modeling of cellular development. To monitor patterns of gene expression it is important to be able to isolate cells at precise stages along a developmental pathway. Well-developed procedures for cell culture and single-cell PCR techniques have allowed genome-wide changes in gene expression to be monitored during animal cell differentiation -4.Arabidopsis - which include the Affymetrix ATH1 gene arrays. Recently, a significant advance in transcriptome analysis of plant cell types has been achieved through fluorescence-activated cell sorting of cell-type marked and protoplasted root cells using Affymetrix ATH1 micorarrays Arabidopsn Server . The chaThe complete transcriptomic datasets usedClick here for additional data fileThe complete set of 39 clusters of genes coexpressed during male gametophyte developmentClick here for additional data fileThe expression profiles of male gametophyte-expressed transcription factors sorted into individual gene familiesClick here for additional data file


FEZ1/LZTS1, FHIT, TP53, P27kip1, and BCL2).Collecting duct carcinoma (CDC) is a rare but very aggressive variant of kidney carcinoma that arises from the epithelium of Bellini's ducts, in the distal portion of the nephron. In order to gain an insight into the biology of this tumor we evaluated the expression of five genes involved in the development of renal cancer  cases. Fhit protein was absent in three cases (27%). The overexpression of p53 protein was predominantly nuclear and detected in 4 of 11 cases (36%). Immunostaining for p27 was absent in 5 of 11 cases (45.5%). Five of the six remaining cases (90%) showed exclusively cytoplasmic protein expression, where, in the last case, p27 protein was detected in both nucleus and cytoplasm. Bcl2 expression with 100% of the tumor cells positive was observed in 4 of 11 (36%) cases. Statistical analysis showed a statistical trend (P = 0.06) between loss and reduction of Fez1 and presence of lymph node metastases.These findings suggest that Fez1 may represent not only a molecular diagnostic marker but also a prognostic marker in CDC. Renal cancer accounts for 3% of adult malignancies. An estimated 35,710 new cases are expected to occur in the U.S. in the current year and 12,480 patients will die of this disease [Recent evidence suggests that solid renal parenchymal tumors arising in the distal portion of the nephron, such as oncocytomas, chromophobe renal cell carcinomas and collecting duct carcinomas represent an heterogeneous group of neoplasm from both clinical and biological perspectives . CollectHistochemically, CDC cells contain intracytoplasmic mucicarminophilic material, where RCCs do not . CDCs arLoss of heterozygosity (LOH) analysis in six CDCs revealed allelic loss at chromosomes 8p and 13q in 50% of the tumors, and rarely at the short arm of chromosome 3 ,13. ThisTo improve our understanding of the biology of CDC and to explore the possibility that different genes may be involved in the etiology and prognosis of this neoplasm, we analyzed by immunohistochemistry eleven cases of CDC for the expression of five genes  often involved in the development of many common cancers. These findings were correlated with conventional clinical-pathological features including clinical outcome.Eleven patients with primary CDC  underwent radical nephrectomy between 1983 and 2000 in the Department of Urology, University of Padua, Italy. Tissue specimens from these tumors were registered in the Department of Pathology at the same institution. All eleven patients had an adequate clinical follow up and were included in our study. Eight of the 11 patients had metastatic disease, five with lymph node metastasis and five with distant metastases at the time of surgery. None of these eleven patients received any systemic therapy before surgery. Samples were fixed in 10% buffered formalin for routine histological processing and were stained with hematoxylin and eosin.Ulex Europaeus Lectin, polyclonal antibody were performed as previously described [Immunohistochemical reactions using anticytokeratin  monoclonal antibodies, and escribed . The tumescribed ,5-7 and escribed .Paraffin sections containing non-neoplastic kidney as well as neoplastic areas were deparaffinized according to standard procedures followed by rehydration through graded ethanol series, and mounted on positively charged slides. Immunostaining was performed as previously described . BrieflyWe evaluated the association between each marker's expression (positive or negative) and each clinical-pathological outcome (metastasis and stage) with Fisher's exact test. For survival, we used the Kaplan-Meier method and log-rank test, as well as Cox proportional hazards regression.All cases showed several common microscopic features. The main tumor consisted of a tubulo-papillary carcinoma showing pleomorphism and a high mitotic rate with several bizarre mitotic figures, and presence of small cystic spaces surrounded by a highly desmoplastic stroma.Two cases showed a predominant papillary pattern accompanied with dilated and atypical changes in the epithelium of the collecting ducts in the adjacent renal medulla, which was the most convincing feature supporting a collecting duct origin of these tumors. Metastases, when available for examination, were histologically similar to the infiltrating part of the primary tumors. Immunocytochemically, tumor cells from all cases examined did not express keratin 10, while strong positivity to anti-keratin 5, 8, and 18 antibodies, and anti-UEL antibody was observed. The positivity to high molecular weight keratin and UEL further substantiate the diagnosis of CDC. The clinical-pathological features and protein expression of the study's 11 cases are shown in Table Our results showed that Fez1 protein expression was undetectable in six of the eleven (55%) cases. One case showed a substantial reduction of expression with 60% of the tumor cells negative, while the remaining four showed diffuse positivity for Fez1 immunostaining Figures  and 1B. Fhit protein was absent in three of eleven cases (27%), and the remaining eight showed diffuse immunoreactivity Figure . Reductip53 protein was found to be overexpressed mainly in the nucleus in four of eleven cases (36%) Figure . One of Immunoreactivity for p27 was observed in the nuclei of most glomerular and tubular cells in the normal kidney. p27 immunostaining in CDC showed high variability. Protein expression was absent in five of eleven cases (45.5%). All the remaining six carcinomas showed a high percentage (> 40%) of p27 positive cells. The expression of p27 protein was detected exclusively in the cytoplasm in five of the six positive cases (90%) while a mixture of nuclear and cytoplasmic protein staining was observed in the last case Figure . OverallBcl2 protein expression in 100% of the CDC tumor cells was observed in four of eleven cases (36%) Figure , while tClinical-pathological features as well as immunohistochemistry results are listed in Table FEZ1, FHIT, P53, P27kip1, and BCL2) in a relatively large series of CDCs (eleven cases), considering the rarity of this tumor.The application of the most recent molecular cytogenetic techniques revealed that renal parenchymal tumors can be classified into distinct subtypes based on the combination of specific genetic alterations . The patFEZ1/LZTS1 (leucine zipper tumor suppressor 1) is a putative tumor suppressor gene located at 8p22 [ at 8p22 . Studies at 8p22 . LOH at  at 8p22 , suggestFEZ1 encodes a 67-kDa leucine-zipper protein with a region of similarity to cAMP-dependent activated protein [FEZ1 gene have been reported in several solid tumors, including prostate, breast, esophageal, and gastric carcinomas [FEZ1 into Fez1 null cancer cells reduced cell growth with the accumulation of cells at late S to G2/M phase of the cell cycle. Conversely, inhibition of Fez1 expression stimulated cells growth [FEZ1 gene lies (8p21-22) has been also associated with the invasive behavior of breast cancer [FEZ1 in several human cancers including CDC. protein . Mutatiorcinomas ,19. In arcinomas . Recent s growth . Furthert cancer  and witht cancer . These dFHIT maps to the short arm of chromosome 3 (3p14.2), encompasses the common FRA3B fragile region, and encodes for a protein of 16.8 kDa, with diadenosine triphosphate hydrolase activity [FHIT gene has been observed by immunohistochemistry in many types of cancers [FHIT gene in tumorigenesis of this rare tumor cannot be excluded.The tumor suppressor gene activity . Reducti cancers ,27. Alth cancers , Hadacze cancers . The sam cancers . While iTP53 tumor suppressor gene maps to chromosome 17p13.1 and plays a major role in DNA transcription, cell growth, proliferation and apoptosis process [The  process . In norm process  our dataP27 is a member of the universal cyclin-dependent kinase inhibitor (CDKI) family. The expression of this important protein is regulated by cell to cell contact inhibition as well as by specific growth factors, such as transforming growth factor (TGF-β). In addition to its role as a CDKI, p27 is considered a putative tumor suppressor gene, a major regulator of drug resistance in solid tumors, and a promoter of apoptosis. p27 acts also as a safeguard against inflammatory injury and has a role in cell differentiation . It has Nevertheless, we did not find statistical correlation to assess its involvement in CDC biology, possibly due to the limited number of tumors studied.BCL2, implicated in the regulation of cell death by inhibiting apoptosis, seems to be vital in normal kidney morphogenesis. In fact, Bcl2 deficient mice develop polycystic kidneys characterized by dilated proximal and distal tubules [The proto-oncogene  tubules . High le tubules . In the Our results suggest a potential association between FEZ1 expression and CDC pathology and prognosis. No similar patterns were seen for any of the other markers studied. Even so, the statistical power of the study was limited and negative findings should not be construed as evidence that these markers are not important. Rather, a larger study would need to be carried out to further investigate their role in CDC.FEZ1 gene alterations may be linked to the high frequency of LOH found at 8p22, where FEZ1 resides. The lack of similar association for the other four genes studied may be due to the low statistical power of the study.Our results suggest that Fez1 expression may be associated to both clinical-pathological features and survival in patients with CDC. None declared.CDC, Collecting Duct Carcinoma; RCC, renal cell carcinoma; LOH, loss of heterozygosityA.V. carried out the immunohistochemical analysis and reviewed the slides, he also contributed to the draft of the manuscript. T.P.G. was responsible for the clinical study. M.G. carried out the original histopathological diagnosis. H.I. participated in the immunohistochemical analysis and statistical analysis. E.G. participated in the immunohistochemical analysis and statistical analysis. F.P. was responsible for the clinical study. L.G.G. participated in designing the study and in drafting the manuscript. C.M.C. participated in designing the study. R.B. participated in the original design and coordination of the study, and in writing the manuscriptThe pre-publication history for this paper can be accessed here:


Pelvic lymph nodes metastasis is an important prognostic factor for patients with cervical carcinoma. However, the relationships between the number of positive nodes, site of metastases nodes, adjuvant therapy and the prognosis is controversial. The purpose of this study was to investigate the influence of positive lymph nodes on the prognosis of Chinese women with stage IB1-IIB cervical carcinoma.Between January 1992 and December 1997, 398 women with International Federation of Gynecology and Obstetrics (FIGO) stage IB1-IIB cervical carcinoma underwent radical surgery in Cancer Hospital, Fudan University. Of these sixty-six patients (16.6%) who were histologically confirmed to have positive pelvic lymph nodes were analyzed retrospectively. The survival was estimated using Kaplan-Meier method. The differences in survival were compared with Log-rank test. Multivariate analyses were performed with the Cox proportional hazard model.P < 0.05). The 5-year survival of patients with one positive node was higher than that of those with two or more positive nodes . The distant metastasis rate in the former group (5.9%) was lower than the latter's (32.7%) (P = 0.05). However, there was no significant difference of pelvic recurrence between the two groups (P > 0.05). The number of positive nodes positively correlated with the level of positive nodes (P < 0.01). The 5-year survival of the patients who had no adjuvant therapy (12.6%) was much lower than that (53.7%) of those with adjuvant therapy (P < 0.05). However, there was no obvious difference between adjuvant radiotherapy, chemotherapy and chemo-radiotherapy (P > 0.05).The 5-year survival of the patients with pelvic lymph nodes metastases was 40.7%. Cox proportional hazard model analysis showed that cellular differentiation, the number of positive nodes and adjuvant therapy to be the independent prognostic factors (The prognosis of patients with stage IB1-IIB node-positive cervical carcinoma who underwent radical surgery alone was very poor. Adjuvant therapy increases the survival rate, decreases the pelvic recurrence and distant metastasis. Although radical radiotherapy (RT) and radical surgery can be the proper choices for patients with early stage cervical cancer, most of the patients in China prefer the radical surgery to RT. Hence, in China the radical surgery has been widely used as first-line therapy for this group of women. Some poor prognostic subgroups have been identified, among these the pelvic lymph node status has been considered as the most important prognostic factor. Radical hysterectomy with bilateral pelvic lymphadenectomy produces an expected 85–90% survival in women with stage IB and IIA cervical carcinoma without lymphatic spread. However, once tumors involve regional lymph nodes, 5-year survival has been reported to be only 30–60% . In mostBetween January 1992 and December 1997, 398 women with International Federation of Gynecology and Obstetrics (FIGO) stage IB1-IIB cervical carcinoma underwent radical surgery at the Department of Gynecologic Oncology, Cancer Hospital of Fudan University. Of these 66 patients who had undergone Wertheims-Meigs' surgery  and were histologically confirmed to harbor positive pelvic lymph node were included in this study. The median age at diagnosis was 49 years (range 21 to71). Out of 66, 8 patients were in stage IB1 (12.1%), 37 patients (56.1%) in stage IIA and 21 patients (31.8%) in stage IIB. Histologically 41 women (62.1%) had squamous carcinoma, 20 (30.3%) had adenocarcinoma, 4 (6.1%) adenosquamous carcinoma and 1 patient (1.5%) had small cell carcinoma. The tumors in 4 patients (6.1%) were well differentiation, 46 cases (69.7%) moderately differentiated and 16 (24.2%) poorly differentiated. The average lymph nodes resected were 14.8 per patient while the average positive lymph nodes resected were 3.7 (1~28) per patient. The average diameter of the cervical tumors was 3.6 cm (1~7 cm). The details of the patients' clinical characteristics are listed in Table Sixty four of these women (97.0%) had brachytherapy in either three or four fractions with a total dose of 15~20 Gy at point A, two weeks prior to radical surgery because of bulky tumor or vaginal vault involvement. Intra-arterial chemotherapy was administered to 11 patients (16.7%) before surgery because of bulky tumor or parametrial extension. The regimen based on cisplatin (CDDP) + 5-Fluorouracil(5-FU) with 2~3 cycles at 3-weeks intervals was used. All patients underwent Wertheims-Meigs' procedure that included radical hysterectomy and bilateral pelvic lymphadenectomy. Three patients had para-aortic lymph node (PALN) sampling because there was suspicion of metastasis.Postoperative external beam irradiation was delivered to 18 patients (27.3%) with 6 MV X-ray routine anterior and posterior portal at a dose of 35~45 Gy at point B with 1.8 Gy daily fraction. Two patients with positive vaginal margin were given extra brachytherapy with 15 Gy 0.5 cm below the vaginal mucosa. 12 cases with positive common iliac node and 3 with PALN metastasis received an additional 40 Gy to the PALN chain area. 10 patients (15.2%) were given postoperative chemotherapy. The regimen consisted CDDP + 5-FU + Cyclophosphamide (CTX) for squamous carcinoma, and CDDP + 5-FU + Mitomycin (MMC) for adenocarcinoma with 2~6 cycles at 3~4-weeks intervals. A total of 19 patients (28.8%) received adjuvant radiotherapy and chemotherapy as mentioned above.Patients were evaluated every two months for the first two years and then six monthly for the additional years by clinical interview, or telephone, or letters. Disease-free survival (DFS) was defined as the time from the date of surgery to local or nodal recurrence or metastasis. Overall survival (OS) was calculated from the date of surgery to the date of death. Recurrences were defined as local if they were detected in the pelvis or vagina and distant metastases as detected in extra-pelvic locations. The median follow-up time was 32 months (range 2~108 months).P < 0.05 was considered significant.Data analysis was performed with Statistical package for social sciences (SPSS) version 10.0 statistical package. The survival was calculated by Kaplan-Meier method. The differences in survival were compared with Log-rank test. Multivariate analyses were performed with the Cox proportional hazard model. The correlation analysis was performed by Kendall's method. Pearson's chi-square or Fisher's exact test was used to compare the difference of proportions. A probability value of P < 0.05). The former's distant metastasis rate (5.9%) was lower than the latter's (32.7%) (P = 0.05). However, there was no significant difference of pelvic recurrence between them (P > 0.05)  was higher than that (36.4%) of those with two or more positive nodes (atter's 3.7% (P = 5) Table The 5-year survival 33.3%) of the patients with positive common iliac node and paraaortic lymph node (PALN) was less than that (43.1%) of   patients with lower than common iliac node involvement. However, the difference was not significant (P > 0.05). The former's pelvic recurrence rate (13.3%) was lower than the latter's (29.4%) (P > 0.05). However, the patients with common iliac or paraaortic nodes had significantly higher distant metastasis (53.3%) than that of the patients with lower nodes (17.6%) (P < 0.01). The number of the positive lymph nodes was closely correlated with the level of lymph node metastasis. The relative coefficient was 0.557 (P < 0.01)  of the   patients who had no adjuvant therapy was much lower than that (53.7%) of   those with adjuvant therapy (P<0.05). The 5-year survival rates of the adjuvant radiotherapy, adjuvant chemotherapy, and adjuvant radio-chemotherapy groups were 53.5%, 49.2% and 56.1% respectively (P > 0.05). The pelvic recurrence (42.1%) and distant metastasis (31.6%) of the surgery alone were higher than those with adjuvant therapeutic groups. However, the differences were not significant (P > 0.05) (Table .6% of thP < 0.05) . The 5-year survival of patients with one positive node (56.5%) was higher than that (36.4%) of those with two or more positive nodes (P < 0.05). There was no significant difference of pelvic recurrence between them. However, the former's distant metastases rate (5.9%) was lower than the latter's (32.7%) (P = 0.05). In the analysis of the site of lymph node metastases, the 5-year survival (33.3%) of the patients with positive common iliac node and paraaortic lymph node (PALN) was lower than that (43.1%) of patients with lower than common iliac node involvement. Unlike the series of Tsai the difference in our series was not statistically significant [P < 0.01). The result suggested that in patients with more positive pelvic nodes there is higher chance of nodal metastasis at higher level node basins, which supported the external irradiation of PALN chain area for the multiple pelvic nodes involvement. The distant metastasis rate was 32.7% in patients with multiple positive lymph nodes, which showed the limitation of adjuvant radiotherapy and theoretically supported the postoperative combined chemotherapy.Radical surgery has been found to be very effective in patients with early stage (IB-IIA) cervical carcinoma -13. Howenificant . This iset al [The prognosis of patients with positive lymph node was poor because of local recurrence and distant metastasis. How to improve the prognosis of these patients has been the focus of gynecological oncology. Stock et al  comparedPeters' study  demonstret al [Several randomized clinical trials -25 perfoet al  did a syet al ,27. Receet al .The therapy of patients with stage IIB cervical carcinoma is still a controversy . AlthougThe author(s) declare that they have no competing interests.XC collected the data, patients' follow-up and drafted the article.SC, ZL, MT, MX collectively designed the study, participated in collection of data and revising the article.RZ participated in collection and analysis of data, revising the articleNone


How trophic factors regulate the expression of FasL is presently unclear, but two regulators of FasL, FOXO3a (FKHRL1) and JNK have been described to play a role in other cell types. Thus, their potential function in motoneurons was investigated in this study.Programmed cell death of motoneurons in the developing spinal cord is thought to be regulated through the availability of target-derived neurotrophic factors. When deprived of trophic support, embryonic spinal motoneurons We show here that as a result of removal of neurotrophic factors and the consequent reduction in signalling through the PI3K/Akt pathway, Foxo3a translocates from the cytoplasm to the nucleus where it triggers cell death. Death is reduced in Fas and FasL mutant motoneurons and in the presence of JNK inhibitors indicating that a significant part of it requires activation of the Fas/FasL pathway through JNK.Therefore, in motoneurons as in other cell types, FOXO transcriptional regulators provide an important link between other signalling pathways and the cell death machinery. During development of higher vertebrates, motoneurons within the spinal cord are generated in excess, and about half the cells initially generated undergo programmed cell death (PCD) during the days following target muscle contact . The mosin vitro is activation of the Fas/CD95 death receptor by its cognate ligand, FasL [FasL expression is strongly upregulated in motoneurons cultured for 3 days without neurotrophic factors [cis between FasL and Fas on individual motoneurons [We have shown that a major driving force for the death of motoneurons deprived of neurotrophic factors nd, FasL . Fas andnd, FasL . While l factors , as in c factors . Moreoveoneurons . UnderstFasL promoter contains three FOXO DNA-binding sites, and Foxo3a-induced apoptosis of cerebellar neurons is decreased when Fas/FasL interaction is blocked by the decoy fusion protein Fas-Fc [FasL gene.The transcription factor Foxo3a  was a clear candidate. In conditions in which the PI3K/Akt survival and growth pathway is activated, Foxo3a is phosphorylated by Akt and exported to the cytoplasm where it is sequestrated by the 14-3-3 protein . Overexpn Fas-Fc . Thus, iFasL expression in some neuronal cells, through its effects on the transcriptional activity of the AP-1 complex. Although this pathway can play different roles, in neurons it is involved in apoptosis in response to several stresses, including withdrawal of survival factors [FasL upregulation in neurons induced to die results from JNK activation and phosphorylation of c-Jun [gld mice, which are defective for FasL, the killing effect induced by trophic deprivation is reduced compared to wt mice.The JNK pathway has also been shown to regulate  factors . In cereof c-Jun . MoreoveFasL activation both directly and through JNK is a major target of its actions.We therefore wished to study the function of Foxo3a in motoneurons and its relation to JNK signalling. We show that, in the absence of survival signalling through the Akt pathway, Foxo3a is translocated to the nucleus where it triggers motoneuron death. Using appropriate mouse mutants, we show that Fas signalling is required for roughly half of the cell death induced by Foxo3a, suggesting that Methods) to strongly activate the PI3K/Akt pathway . In these conditions, Foxo3a is predominantly detected in the cytoplasm  or the absence Fig.  of neuroResults in all conditions were quantified by counting 2 independent experiments Fig. . They clTo investigate the ability of Foxo3a to trigger death of cultured motoneurons, we took advantage of the electroporation technique we recently developed for high-efficacy transduction of primary neurons . We firsWe therefore used electroporation to analyse the effects of Foxo3a on motoneuron survival. Overexpression of HA-wt-Foxo3a with GFP had no significant effect as compared to GFP alone or GFP with the empty vector . To mimic Foxo3a activation and nuclear translocation, we then overexpressed HA-TM-Foxo3a together with GFP. The triple mutant triggered significant death of motoneurons cultured with or without NTFs Fig. . In the FasL, known to be regulated by Foxo3a and shown by us to be able to trigger motoneuron death. Motoneurons were therefore isolated from mice bearing mutations that lead to reduced signalling through the Fas pathway: gld mice (point mutation in FasL) and lpr mice (regulatory defect in Fas). As before, electroporation of HA-TM-Foxo3a in control motoneurons led to loss of 60 to 70 % of them compared to HA-wt-Foxo3a  show greatly exacerbated death responses to activation of this pathway [We overexpressed the constitutively active mutant TM-Foxo3a in the presence of neurotrophic factors to mimic the nuclear translocation of Foxo3a in their absence. We observed a 50% reduction in death induced by TM-Foxo3a when we used motoneurons that were mutant for Fas/FasL signalling. This could in theory result through several indirect mechanisms. However, the literature on direct regulation of y Foxo3a  and our  support  make it  complex ,18. The  pathway . In vitr pathway . It willlpr or gld mice. This may in part reflect the fact that these mutants are strong hypomorphs, rather than complete nulls. For example, in lpr mice, some Fas is still synthesized following splicing-out of the inhibitory transposon from primary transcripts [kip1 [The killing effect of Foxo3a was not totally abolished in motoneurons from nscripts . Howeverts [kip1 , the gluts [kip1 , transfots [kip1 , the DNAts [kip1  and the ts [kip1 . Of partS, BimL and BimEL [L and 89% by overexpression of BimS imS In conclusion, one of the upstream events now known to trigger death of neurons deprived of trophic support is the failure of Akt to phosphorylate Foxo3a and thereby prevent it from entering the nucleus. The fact that this mechanism is found to occur in motoneurons, a classical system for the study of neuronal cell death, opens the door to a better understanding of its role during development and in neurodegenerative pathology.lpr/lpr and gld/gld mice were purchased from the Jackson Laboratory . lpr/lpr mutants present an insertion of an early transposon into the fas gene, resulting in fas transcriptional repression [Normal CD1 and C57BL/6 mice were obtained from Iffacredo . pression . gld/gldpression . All mutLY 294002 was purchased from Calbiochem and used at a final concentration of 100 μM. L-JNKl1 was purchased from Alexis Biochemicals and used at a final concentration of 1 μM. Rabbit polyclonal antibody to HA-tag was from Clontech. Antibodies against Foxo3A were as described previously  and wereWe are grateful to Anne Brunet for donating vectors used as the basis for our expression constructs. The vectors encoding HA-tagged Akt and Akt ca were as described previously . The vecMotoneuron cultures were prepared from E12.5 mouse spinal cords essentially as described , except Cells dissociated from E12.5 mouse ventral spinal cords were centrifuged over a 6.5% (v/v) Metrizamide cushion at 2000 rpm for 15 min. Cells at the interface were collected and washed on a BSA cushion at 1500 rpm for 5 min. Cells were resuspended in electroporation buffer at a density of 50,000 cells per 100 μL. 100 μL aliquots of the suspension were transferred to 4 mm gap cuvettes (Eppendorf) and five μg of the pCAGGS-GFP vector as well as the same molar amount of the vector of interest were added. After 15 min of incubation at room temperature, cells were electroporated using three pulses of 5 ms at 200 V with intervals of 1 s. Immediately after electroporation, 1.5 ml of culture medium was added to dilute the cells which were plated in four four-wells plates .For anti-HA staining, motoneurons electroporated with HA-tagged wt or TM Foxo3A were seeded on polyornithine-laminin-coated 12-mm diameter glass coverslips and cultured for 24 hr at 37°C in complete Neurobasal medium supplemented or not with NTFs. Motoneurons cultured without NTFs were treated with LY294002 for the last 90 min. They were then fixed in 3.6% (v/v) formaldehyde for 30 min, washed in PBS-50 mM L-Lysine, and blocked for 1 hr in 5% donkey serum, 4% BSA, 0.1% Triton X-100 in PBS-50 mM L-Lysine. Cells were incubated with the anti-HA antibody (dilution 1:500) in blocking buffer, followed by fluorochrome-conjugated anti-rabbit secondary antibody. The cells were then observed under a fluorescence microscope. Cells with typical motoneuron morphology that were both GFP- and HA-positive were analyzed. The fraction of motoneurons expressing HA-wt-Foxo3A or HA-TM-Foxo3A exclusively in the nucleus, exclusively in the cytoplasm or in both compartments was evaluated in a total of 20 cells per well.Staining for endogenous Foxo3A (dilution 1:100) was performed using the same method on non-electroporated motoneurons cultured for 24 hr with NTFs.Motoneurons were counted 48 hr after electroporation under a fluorescence microscope. Only green cells with healthy motoneuron morphology, i.e. with large cell bodies and long non-fragmented neurites, were taken into consideration. Two different wells for each condition were counted (between 50 and 350 green motoneurons) in all the experiments. Cross percentages of test versus control (being set at 100%) were calculated yielding 4 values, used to calculate the medium and the SD for each experiment.C.B. conducted most of the experiments, B.P. some. C.H and B.P. conceived the experiments and the experimental design. C.B., B.P. and C.H. wrote the paper.A – The 3 forms of Bim are expressed in motoneurons: RT-PCR was performed on extracts of 80,000 motoneurones cultured for 3 days in the presence of NTFs, in a 35 mm diameter dish. Primers used for Bim were the following: 5'-GTGACAGAGAAGGTGGACAAT-3' and 5'-ATACCAGACGGAAGATAAAGC-3'. The 3 products were:BimS 284 bp, BimSL 374 bp, BimL 542 bp; S or BimL kills a major proportion of motoneurons: B – Overexpression of Bimmotoneurons purified from E12.5 mice embryos have been electroporated with a vector coding GFP alone or coelectroporated with a vector coding GFP and a vector coding either BimL or BimS, obtained by direct cloning of the PCR products described above first cloned into pGEMTeasy, then subcloned into pcDNA3 into EcoRI sites. Conditions of electroporation were as described in Material and Methods. The surviving electroporated motoneurons were counted after 2 days in culture in the presence of NTFs.Click here for file


In quality of care research, limited information is found on the relationship between quality of care and disease outcomes. This case-control study was conducted with the aim to assess the effect of guideline adherence for stroke prevention on the occurrence of stroke in general practice. We report on the problems related to a variant of confounding by indication, that may be common in quality of care studies.Stroke patients (cases) and controls were recruited from the general practitioner's (GP) patient register, and an expert panel assessed the quality of care of cases and controls using guideline-based review criteria.A total of 86 patients was assessed. Compared to patients without shortcomings in preventive care, patients who received sub-optimal care appeared to have a lower risk of experiencing a stroke . This result was partly explained by the presence of risk factors , as reflected by the finding that the OR came much closer to 1.00 after adjustment for the number of risk factors . Patients with more risk factors for stroke had a lower risk of sub-optimal care . This finding represents a variant of 'confounding by indication', which could not be fully adjusted for due to incomplete information on risk factors for stroke.At present, inaccurate recording of patient and risk factor information by GPs seriously limits the potential use of a case-control method to assess the effect of guideline adherence on disease outcome in general practice. We conclude that studies on the effect of quality of care on disease outcomes, like other observational studies of intended treatment effect, should be designed and performed such that confounding by indication is minimized. There is a long tradition of studying at population level the quality of medical care provided to patients who died from conditions amenable to medical intervention. This type of study , aims to identify deficiencies in medical care that may have contributed to death. It was first systematically carried out on maternal death, and later on other causes of avoidable death -4. This An important limitation of this type of study, without control subjects, is its inability to fully establish a causal relationship between identified deficiencies in care and the adverse outcome, and to determine to what extent identified deficiencies are associated to the occurrence of such an event. Identified deficiencies in care are expected to indicate only to a certain extent an increase in risk of an adverse health outcome, while the probability of having an adverse outcome can be calculated only if we compare the care provided to patients who suffered an adverse outcome with that of patients who did not suffer such an event. For this reason it has been proposed to perform a case-control study with patients with an adverse event as 'cases' and a comparable group of patients without an adverse outcome as 'controls' .We performed a case-control study with the aim to assess the effect of guideline adherence for stroke prevention on the occurrence of stroke in general practice. Unfortunately, we encountered various obstacles in the design and conduct of this study, in particular related to the recruitment of cases and controls, in availability of information on the care delivery process in the GP's data registration system, and in controlling for differences other than differences in the quality of care. The aim of this paper is to highlight the problems related to a variant of confounding by indication, that may be common in quality of care studies.Observational studies of intended treatment effects are particularly prone to 'confounding by indication', and can produce misleading estimates on either, or both, the size and direction of treatment effects ,8. ConfoFrom the Dutch national GP register, a random sample was taken of 58 GPs working in Rotterdam and the surrounding region. The study was restricted to patients with a first-ever stroke meeting the following criteria for inclusion: (a) diagnosis of intracerebral haemorrhage or infarction according to the World Health Organization (WHO) definition of stroke , (b) ageIn a pilot study among 32 GPs, the quality of care measurement instruments (audit procedure and questionnaire) were tested. GPs participating in the pilot study did not participate in this study. Data on the process of care, two years prior to the occurrence of stroke (for controls from January 1995 to January 1997), were collected by means of structured face-to-face interviews with the GP, using separate questionnaires for each stroke patient. GPs were interviewed between March and October 1999. At the time of interview, GPs used either hand-written or electronic patient records to retrieve patient information. In case information was not available in the patient's record, information was drawn from the GP's memory. For each question, the type of data source was registered. The questionnaire comprised questions related to patient characteristics and family and medical history of CVD and risk factors, and the detection and treatment of cardiovascular risk factors such as hypertension, diabetes mellitus, transient ischemic attack (TIA) and cardiac failure. Similarly, data were collected on lifestyle-related risk factors such as smoking status, overweight, and excessive alcohol intake.The quality of preventive care and its potential to prevent stroke was assessed and valued by a six-member panel of experts. The panellists (three neurologists and three GPs) were selected on the basis of their clinical expertise with respect to stroke prevention, experience in quality of care evaluation, academic or non-academic background and professional discipline. Six practice guidelines relevant to stroke prevention  were selected by the panel . These gIn a two-round evaluation, with a final plenary round, cases were assessed by the panellists (panellists were divided in sub-panels). Each sub-panel assessed a specific number of cases. Based on identified elements of sub-optimal care and seriousness of shortcoming in terms of 'minor' and 'major', the panellists allocated grades on a scale of 0 to 3 Table .The two-round process was focussed on detecting consensus among the panellists (providing the same grade), and no attempt was made to force the panellists to consensus. The intersubpanel agreement was k = 0.63 . A detailed description of the assessment method is provided elsewhere .Analysis of the data was done by using simple cross-tabulations, and by using logistic regression analysis to model the chance of getting a stroke as a function of the presence of sub-optimal care (as ascertained by the panel), age and sex, and risk factors for stroke.The rate of participation was 62% (36 GPs). The main reason for GPs not to participate in the study was lack of time and interest (68%). Participating and non-participating GPs did not differ significantly in age, practice type, and date of qualification. Ninety-two percent of the GPs used electronic GP information systems. Among cases and controls there was a nonsignificant difference in mean age, however, cases were slightly older than controls (67 versus 65 years). Initially, before we excluded patients 'without' hypertension, GPs identified and selected 50 cases and 58 controls (1.4 case and 1.6 control per GP). Expected number of cases was 2.5 stroke patients per GP per year . After eOverall, data for verification of the initial diagnosis of stroke, assessment of GPs' guideline adherence, and judgement of the causality of the relationship between non-adherence and the occurrence of stroke could be collected from the patient records. However, information on risk factors such as family history of CVD, body weight (overweight), excessive alcohol intake, and smoking was less easily obtained. Depending on the type of risk factor, in 8–56% of all subjects, information on risk factors was unknown to the GP . In 41–58% information was taken from the GP's memory, instead of the patient register.In 43% of the cases and 31% of the controls, no sub-optimal care could be identified (grade 0), whereas in 57% and 69%, respectively, sub-optimal care was identified grade 1, 2 or 3). Thus the Odds Ratio for a case to receive sub-optimal care was 0.60 (95%CI 0.24 – 1.53) compared to a control (Table  or 3. ThThe mean number of risk factors among cases (6.1 per patient) was higher than among controls (4.4 per patient) Figure . If adjuPatients with a higher number of risk factors for stroke, indeed have a lower risk of sub-optimal care . As expected, higher numbers of risk factors per patient also increases the risk of stroke .This study demonstrated confounding by indication in a case-control study analysing the association between guideline adherence and the occurrence of stroke in general practice. It also provided insight into the possibilities for controlling for this confounding bias. We learned that, at present, difficulties in patient recruitment and data retrieval seriously limit the potential use of a case-control method to assess the relationship between guideline adherence for stroke prevention and stroke in general practice.We found that in specific domains data were incomplete and not readily available in the patient records. As a consequence, in many cases GPs were unable to identify stroke patients from their patient register, which most likely introduced under-reporting of stroke patients. As compared to national frequencies (2.5 stroke patients per GP per year) , GPs parStrong indications for the existence of confounding by indication were found, albeit different from how it is usually described in literature. Confounding by indication, which is conceived as a substantial problem in observational studies of treatment efficacy, usually refers to a situation in which patients who are more in need both receive more care have a higher risk of adverse health outcome . In our A more detailed analysis showed, similar to results found in previous studies, that this result partly emanates from a higher prevalence of risk factors for stroke among patients suffering stroke at a later stage in life, which not only increases the risk of stroke but also GPs' compliance to guidelines. We hypothesize that, on average, patients with more risk factors for stroke receive more attention or visit their GP more frequently, which in turn facilitates guideline adherence (e.g. compliance to quarterly follow-up of treated hypertensive patients) and at the same time results in better quality of care. Controlling for (recorded) risk factors reduced the counter-intuitive result by approximately one half, and we hypothesise that incomplete registration of risk factors for stroke explains why the risk of stroke in stroke-prone or high-risk patients associated with sub-optimal care remained below 1.00, even after controlling for risk factors. We hope that our paper draws the attention of quality of care researchers to this variant of confounding by indication, that may lead to biased associations between process measures of quality of care and care outcomes.This study shows that, at present, difficulties in patient recruitment and data retrieval seriously limit the potential use of a case-control method to assess the relationship between guideline adherence for stroke prevention and stroke in general practice. It demonstrates the role of confounding by indication, causing patients with an adverse health outcome to appear to receive better quality of care.The author(s) declare that they have no competing interestsJK, NK, PK, AP and JM conceived and designed the study. Analyses were performed by JK and GB. All authors contributed to this article and earlier drafts of the manuscript.The pre-publication history for this paper can be accessed here:


Tyger! Tyger! burning brightIn the forests of the nightWhat immortal hand or eyeCould frame thy fearful symmetry?Panthera tigris, three have gone extinct since the 1940s.When William Blake wrote these words in the late 1700s, the deforestation and habitat destruction that would decimate wild tiger populations had already begun. In 1900, an estimated 100,000 wild tigers lived throughout much of Asia, from India in the west to Sumatra and Indonesia in the south to Siberia in the east. Today, the ongoing stresses of habitat loss, hunting, and an illegal trade in tiger parts have spared fewer than 7,000 tigers. Of eight traditionally classified subspecies of Conservation strategies to combat this grinding attrition are tailored to each subspecies. But several lines of evidence suggest that subspecies designations—based on geographic range and morphological traits such as body size, skull traits, coat color, and striping patterns—may be flawed. An earlier molecular study of 28 tigers found little evidence of genetically distinct subspecies, while surveys of tiger habitat found few physical barriers sufficient for subspecies isolation.To get a clearer picture of the genetic structure of existing tiger populations, Shu-Jin Luo, Jae-Heup Kim, Stephen O'Brien, and nineteen colleagues performed a comprehensive genetic analysis of mitochondrial and nuclear genes from over 130 tigers. By identifying distinct patterns of variation within these gene families, the authors reconstructed the evolutionary distribution and ancestry of the tiger. Their results support many of the traditional subspecies designations and identify further subdivisions in others.DRB that's involved in pathogen recognition, and short repeating genetic elements called microsatellites—act as unique signposts that flag significant demographic and evolutionary events in the tiger populations.Luo et al. collected “voucher specimens”  of blood, skin, and hair from 134 tigers representing the entire tiger range, and examined them, along with samples of preserved pelts and hair, for three molecular markers. The markers—a stretch of mitochondrial DNA (mtDNA) sequence, a gene with highly variable DNA sequence called P. t. sumatrae) and (P. t. tigris) Bengal tigers—but others suggested that the Indochinese subspecies (P. t. corbetti) should be divided into two groups, representing a northern Indochinese and a peninsular Malaya population . Interestingly, clusters for the captive South China tigers also fell into two distinct lineages—P. t. amoyensis, the traditional grouping, and P. t. corbetti, though the designation is still tentative. These subdivisions were largely supported by the other genetic analyses.mtDNA sequences were extracted from tigers originating in the Russian far east , south China, northern Indochina, the Malaya Peninsula, Sumatra, and the Indian subcontinent. The mtDNA analysis identified 30 haplotypes—characteristic regions on a chromosome—that could be clustered. Some of the clusters supported traditional classifications—e.g., for the Sumatran (The distinct genetic patterns found in the tiger populations suggest six rather than five living subspecies. Reduced gene flow and genetic drift in isolated populations, as well as human activity, likely caused these partitions. The low genetic variability seen in the Siberian tigers, for example, might be explained by severe population declines: the animals were nearly exterminated in the early 1900s, and today only 500 remain. Sumatran tigers, on the other hand, show relatively high genetic variability and uniqueness, possibly reflecting a historically large breeding population that was later isolated.In what distant deeps or skiesBurnt the fire of thine eyes?Whether recent population and habitat declines, as opposed to earlier events, can fully explain these patterns is not clear. But these results offer valuable data for conservation strategies and captive breeding programs that rely on distinctions in subspecies taxonomy and geographic provenance. Evoking both the darker side of creation and humanity, Blake could not have imagined the modern fate of his “Tyger.” Scholars have long debated the multilayered meaning of his poem, including the second stanza, which starts,Will we reduce future generations to a literal reading?


The single-strand binding (Ssb) protein of phage T4  is a mRNA-specific autogenous translational repressor, in addition to being a sequence-independent ssDNA-binding protein that participates in phage DNA replication, repair and recombination. It is not clear how this physiologically essential protein distinguishes between specific RNA and nonspecific nucleic acid targets. Here, we present phylogenetic evidence suggesting that ssDNA and specific RNA bind the same gp32 domain and that plasticity of this domain underlies its ability to configure certain RNA structures for specific binding. We have cloned and characterized gene 32 of phage RB69, a relative of T4 We observed that RB69 gp32 and T4 gp32 have nearly identical ssDNA binding domains, but diverge in their C-terminal domains. In T4 gp32, it is known that the C-terminal domain interacts with the ssDNA-binding domain and with other phage-induced proteins. In translation assays, we show that RB69 gp32 is, like T4 gp32, an autogenous translational repressor. We also show that the natural mRNA targets  for the 2 proteins are diverged in sequence from each other and yet can be repressed by either gp32. Results of chemical and RNase sensitivity assays indicate that the gp32 mRNA targets from the 2 related phages have similar structures, but differ in their patterns of contact with the 2 repressors. These and other observations suggest that a range of gp32-RNA binding specificities may evolve in nature due to plasticity of the protein-nucleic acid interaction and its response to modulation by the C-terminal domain of this translational repressor. T4 gp32, the single-strand binding (Ssb) protein of bacteriophage T4, is a well studied member of the Ssb protein family, and was the first such ssDNA-binding replication protein to be discovered . The proin vivo. The protein binds to a specific target  in the 5' leader segment of the mRNA from gene 32, and represses translation of this RNA [in vitro measurements of the protein's binding affinities to different nucleic acid ligands. It has been observed that ssDNA is favored over translational operator RNA as a ligand for T4 gp32 and that RNA of nonspecific sequence is the least preferred nucleic-acid ligand for this Ssb protein [In vivo, T4 encoded mRNA for gp32 is intrinsically more metabolically stable than the typical prokaryotic mRNA and is thought to have opportunities to undergo many cycles of gp32-mediated repression and depression during the replication and other processing of phage DNA. The potential for translation of this mRNA in the T4 infected E coli host is thought to be determined by availability of ssDNA in the metabolic pool [in vivo observations relating to levels of T4 gp32 biosynthesis under conditions of DNA damage or abnormal accumulation of ssDNA in the phage infected bacterial host [T4 gp32 has the interesting property of being able to control its own biosynthesis at the translational level this RNA . Anotherthis RNA ,9. It is protein -12. In vlic pool ,13,14. Dial host .In vitro studies suggest that the pseudoknot serves as the initial recognition (nucleation) site for the protein and that this gp32-RNA interaction leads to cooperative binding of additional gp32 monomers to the less structured downstream sequence containing the UUAAA/UAAA elements and ribosome-binding site (RBS) [It is not clear how T4 gp32 distinguishes between specific RNA and the non-specific nucleic acid sequence of ssDNA or ssRNA ligands. It appears that single-strandedness of the nucleic acid is not the most important criterion used by the protein to selectively bind its own message in the phage-induced mRNA pool. The translational operator for T4 gp32 has been mapped by RNA footprinting assays and determined to consist of two contiguous components, a 5' terminal ~28-nucleotide component that forms a folded structure (RNA pseudoknot) and an adjacent, less structured, >40-nucleotide component that lies 3' to the pseudoknot ,16. The The 3-dimensional structure of intact T4 gp32 has not been solved, although a number of biochemical and physiological observations have provided clues that the protein is modularly organized into 3 distinct domains . In partE coli K-12 strain K802  was used as host in cloning experiments and the E coli B strain NapIV  was the host for plasmid-mediated gene expression studies that utilized lambda pL control. E coli B strain BL21(DE3), which harbors a T7 RNA polymerase gene under cellular lac promoter control [32 to bacterial cells.The AseI-digested RB69 genomic DNA to identify and retrieve an ~35-kb DNA fragment that hybridized to a T4 gene 32-specific riboprobe under stringent conditions. The riboprobe was prepared by methods described previously [32 clone pYS69 [AseI fragment in AseI-compatible Eco R1-generated ends of plasmid vectors. However, further digestion of the AseI fragment with ApoI (which generates Nde1-compatible ends) yielded a shorter, ~15-kb, fragment that could be cloned in the NdeI-EcoRI interval of vector pNEB193 . The cloned fragment was sequenced and found to be very similar to the T4 genetic segment extending from gene 59 through the 5' terminal ~2/3 of gene 32, except that the RB69-derived DNA appeared to lack a homologue of the T4 ORF 32.1 (see below). Comparisons between the T4 and RB69 gene 59-32 regions are diagrammed in Fig 32 from RB69 genomic DNA, through PCR amplification using Taq DNA polymerase. For this purpose, we utilized two primers, one perfectly matching a sequence in the cloned AseI-ApoI RB69 fragment  and the other (the downstream primer), an 18-mer bearing the sequence 5'CAGCAGCAGTGAAACCTTTA3', was chosen from a PCR screen of an RB69 primer library. DNA amplification was carried out under low-stringency conditions for primer annealing (30 sec at 25°C), which allowed activity from the imperfectly matched downstream primer. We obtained several products that we resolved by agarose gel electrophoresis Only one of these products, an ~35-kb DNA fragment, hybridized, although poorly, to the T4 gene 32-specific riboprobe initially used for the Southern blot analysis of Ase1-digested RB69 genomic DNA. This fragment was sequenced, using the PCR, and found to contain the 3' terminal segment of RB69 gene 32 as well as some of the region distal to RB69 gene 32 (relative to the T4 genetic map). Collectively, sequence analysis of the cloned and amplified RB69 genomic segments yielded sufficient information for designing new primers to amplify, from genomic DNA, the entire wild-type RB69 gene 32, as well as shorter segments of this gene and its putative control region in the untranslated RB69 IC59-32 region  as to maintain pL-mediated expression at undetectable levels. Minimizing plasmid-driven transcription from pL contributed to stable maintenance of the cloned wild-type RB69 gene 32, the product of which is highly toxic to bacterial cells. RB69 gene 32 mutants still emerged when such clones were grown at ≤30°C. Some of these mutants were archived for use as controls in certain studies , probably because of residual (constitutive) lac-promoter activity in this bacterial host. To circumvent potential toxicity, pSP72-based recombinants were propagated in hosts lacking a T7 RNA polymerase gene. The purified plasmid DNA from these hosts was used for in vitro transcription and translation assays. Methods for the radiolabeling of plasmid encoded proteins and their subsequent analysis by SDS-PAGE have been described elsewhere [We used the lambda r pLY965  to clonelsewhere ,25, and E coli NapIV clones of phage genes, was as described previously for T4 RegA protein [RB69 gp32 and T4 gp32 were purified from the overproducing clones pRBg32Δop (RB69 gp32) and pYS69 (T4 gp32), respectively. We used the gp32 purification protocol outlined by Bittner et al,  with min protein . Anionic protein . Under tin vitro studies originated from in vitro transcription of pSP72 clones of the desired gene 32 sequences Methods have been described elsewhere [32 transcripts and positions -96 to +161 in case of the T4 gene 32 transcripts. These products also included a 10-nt sequence from the plasmid's T7 promoter region RNA sequencing was carried out by using the RVT-catalyzed primer-extension (cDNA synthesis) method described elsewhere [in vitro translation assays, the RNA preparations included full length and truncated versions of the gene 32 open-reading frame from each of the 2 phage sources.RNA preparations used for footprinting and other lsewhere . Phage-slsewhere ,29. Sequ32 recombinant DNA (coupled transcription-translation assays) or purified RNA (DNA-free translation assays) to assess repressor activities of purified RB69 gp32 and T4 gp32. With plasmid-directed gene 32 expression, it was possible to use expression of the plasmid borne bla gene (β-lactamase) as an internal control. Each 50 μl in vitro assay reaction mixture  contained 1 μg of plasmid DNA template or 4 μg RNA, 5 μl of a mixture of all amino acids (1 mM each) except L-methionine, 1 μl of an S30-premix cocktail , 15 μl S30 extract and the balance of volume in nuclease-free water Reaction mixtures, including any added gp32, were constituted in an ice bath before transferring to 37°C for incubations (30 or 60 min). Reactions were stopped by rechilling in the ice bath. Proteins from 5 μl samples were precipitated with 20 μl acetone, collected by centrifugation, dried and suspended in SDS extraction buffer for analysis by SDS-PAGE and autoradiography. Analysis of plasmid encoded  gp32 fragments was carried out in SDS-PAGE (10% gels) using Tricine as the electrophoresis buffer. This buffer system allows for effective resolution of small polypeptides [We used E coli S30 cell-free extracts  with purified pSP72-based gene The RNA-modifying chemical reagents Dimethylsulfate  and Diethylpyrocarbonate  and the ribonucleases  were used to probe RB69- and T4-derived operator RNAs for intrinsically structured regions. The RNases were also used for RNA footprinting (protection by gp32) studies.DMS was diluted in absolute ethanol at ratios of 1:2, 1:4, and 1:5 ratio v/v and its effects were analyzed at the three concentrations. The reaction buffer contained 30 mM HEPES pH 7.5, 10 mM MgCl2. Reactions were stopped in 0.5 M β-mercaptoethanol and 0.75 M sodium acetate. The protocol for DEPC treatment was identical to that for DMS, except that we used 1 μl of DEPC per 100 μl of reaction mix and incubated the reactions at room temperature for 10 min.4Cl, 10 mM Mg acetate, 10 mM Tris-HCl pH 7.4, and 6 mM β-Mercaptoethanol. The buffer for digestions with RNase V1 contained 25 mM Tris-HCl pH 7.2, 10 mM MgCl2, and 0.2 M NaCl Incubations were at 37°C in 30 μl buffer in all cases. RNase treatments were halted with an equal volume of buffer containing 0.4 M Na acetate pH 5.2, 20 mM EDTA, and 30 μg E coli tRNA. When used for RNA footprinting, RB69 gp32 or T4 gp32 was added at concentrations in the range between 1 μM and 5 μM.For the RNase-sensitivity assays, including gp32-mediated RNA footprinting, digestions with RNases A1 and T1 were carried out in 30 μl buffer containing 60 mM NHThe amino acid sequence of RB69 gp32 was deduced from the determined nucleotide sequence of the gene. An alignment between the predicted primary structures of this protein and its T4 homologue is shown in Fig IC59-32 region with its counterpart (the IC32.1-32 region) from T4 The T4 region (GenBank NC_00866) has been experimentally documented to harbor the translational operator for gene 32 expression [IC59-32 region that appears to have no T4 counterpart . However, such experiments, which require 10–30 μM purified protein to demonstrate repression  By these criteria, the T4 and RB69 proteins appeared to be similarly functional in each other's physiological systems. Yet, the natural targets for the 2 proteins are clearly different from each other in topography , the RB69 gp32 footprint on the RNA target from RB69 was 5 residues longer than the footprint of this protein on the T4-derived RNA target; however, the positions of the 2 footprints relative to the respective initiator AUG and 5' terminal boundary of the pseudoknot structure appeared to be identical , it has been reported that there are no indications that an RNA pseudoknot structure exists in the putative TIR for gene 32, although the UUAA/UUAAA sequence units are conserved in the RB49 IC59-32 region [In summary, we envisage that as a mediator of gp32's interactions with other phage induced proteins, the C-terminal domain of gp32 may co-diverge with its protein targets to maintain mutual recognition, and that structural plasticity of a conserved ssDNA-binding domain may allow an also diverging RNA target to establish rearranged contacts within a relatively conserved protein pocket. It is unclear if the 2 sets of divergence are interconnected, but together, they could facilitate the evolution of a high degree of diversity in how the synthesis and/or replication activity of this Ssb protein is regulated among phylogenetic relatives of T4. It will be important to find out if this diversity includes RNA ligands for gp32 that control the DNA-binding activity but not synthesis of gp32, or if autogenous translational repression has been replaced by other mechanisms for control of gene 32 in some T4 relatives. There is at least one reported example where evolution resulted in lack of RNA binding function in the Ssb protein of an M13-like phage . Also, a2 region . It remaORF32.1  homing endonuclease (now named SegG) that mediates its own transfer, along with T4 gene 32, to the ORF32.1-less genome of phage T2 in T4 × T2 genetic crosses. We note that the 5' terminal sequence of the RNA pseudoknot for T4 gp32 translational control overlaps the reading frame of the segG gene, in addition to being very similar (~83% identity) to the corresponding segment of the pseudoknot sequence of RB69, which lacks a segG gene  may be a chimera consisting of an extension of the parental segG reading frame into the recipient genome's pseudoknot sequence. Such lateral transfer events and subsequent mutation may have profound influences on evolution of the RNA binding functions of proteins that have relaxed sequence but stringent structural requirements for their RNA target.Finally, we should comment about None declared.Jamilah Borjac-Natour: Conducted most of the experimental work and initial data analysis and prepared summaries; wrote the first draft and participated in subsequent revisions of the manuscript. Vasiliy Petrov: Conducted independent analysis of data and generated summaries and composite figures for presentation in the manuscript. Participated in revision of the manuscript during later stages of preparation. Jim Karam: Directed the study, evaluated results on an ongoing basis, worked closely with the coauthors during preparation of Figures, played a major role during revision of manuscript drafts and communicated the manuscript to the journal.