Child under-nutrition is a leading factor underlying child mortality and morbidity in Sub-Saharan Africa. Several studies from Uganda have reported impaired growth, but there have been few if any community-based infant anthropometric studies from Eastern Uganda. The aim of this study was to describe current infant growth patterns using WHO Child Growth Standards and to determine the extent to which these patterns are associated with infant feeding practices, equity dimensions, morbidity and use of primary health care for the infants.A cross-sectional survey of infant feeding practices, socio-economic characteristics and anthropometric measurements was conducted in Mbale District, Eastern Uganda in 2003; 723 mother-infant (0–11 months) pairs were analysed. Infant anthropometric status was assessed using z-scores for weight-for-length (WLZ), length-for-age (LAZ) and weight-for-age (WAZ). Dependent dichotomous variables were constructed using WLZ < -2 (wasting) and LAZ < -2 (stunting) as cut-off values. A conceptual hierarchical framework was used as the basis for controlling for the explanatory factors in multivariate analysis. Household wealth was assessed using principal components analysis.The prevalences of wasting and stunting were 4.2% and 16.7%, respectively. Diarrhoea during the previous 14 days was associated with wasting in the crude analysis, but no factors were significantly associated with wasting in the adjusted analysis. The adjusted analysis for stunting showed associations with age and gender. Stunting was more prevalent among boys than girls, 58.7% versus 41.3%. Having brothers and/or sisters was a protective factor against stunting , but replacement or mixed feeding was not . Lowest household wealth was the most prominent factor associated with stunting with a more than three-fold increase in odds ratio . This pattern was also seen when the mean LAZ was investigated across household wealth categories: the adjusted mean difference between the top and the bottom wealth categories was 0.58 z-scores, p < 0.001. Those who had received pre-lacteal feeds had lower adjusted mean WLZ than those who had not: difference 0.20 z-scores, p = 0.023.Sub-optimal infant feeding practices after birth, poor household wealth, age, gender and family size were associated with growth among Ugandan infants. Child under-nutrition is a leading factor underlying child mortality and morbidity in Sub-Saharan Africa, and contributes to 2.2 million deaths and a fifth of all disability-adjusted-life-years lost worldwide for children under five years old . Early iin utero [), which is a cluster-randomized multi-centre trial of the safety and efficacy of exclusive breastfeeding (EBF) promotion by peer-counsellors among both HIV-1 infected and uninfected mothers in Burkina Faso, Zambia, South Africa and Uganda [[Several anthropometric studies in Uganda have described impaired linear growth among children up to five years old. Stunting (length/height-for-age less than -2 z-scores) occurs in 25% of children under two years ,7 and inin utero . There hd Uganda . The aim Uganda [.. The populations were sampled on the basis of probability proportional to size: the appropriate number of villages was randomly selected in each parish according to parish size and seven households were randomly selected in each village. The sample was not stratified on urban/rural status. The study site, design, questionnaire details and definitions are fully described elsewhere [The study was conducted from September to November 2003 in Mbale District, Eastern Uganda, in one urban area  and the surrounding rural county of Bungokho. Administrative information was retrieved from the Uganda Bureau of Statistics in Entebbe, which gave us parish sizes and the number of villages within each parish lsewhere . A natiolsewhere . The poplsewhere . We exclWe used a structured questionnaire that included 24-hour dietary recall and dietary recall since birth on 35 food and liquid items. It also included questions on breastfeeding in general, pre-lacteal feeding, initiation of breastfeeding, socio-demographic characteristics, water and sanitation, education of mothers and fathers, having brothers and/or sisters, type of work, marital status, immunisation status, primary health care usage for the infants and recent sickness.Weight and recumbent length were taken according to WHO standardized techniques . Undress.Data were entered using EpiData 3.0 and analysed using SPSS 15.0.1 and STATA 9.2. Anthropometric indices were generated using WHO Anthro 2005 software st day; and (3) after the 1st day. Infant feeding mode was categorised on the basis of WHO definitions and recommendations: (1) exclusively breastfed (EBF) infants were fed on breast milk only; (2) mixed-fed (MF) infants were fed on breast milk and other feeds; and (3) replacement-fed (RF) infants did not receive breast milk [Children's health and nutritional status can be assessed by evaluating their anthropometric data using the z-scores for weight-for-length (WLZ), length-for-age (LAZ) and weight-for-age (WAZ). Anthropometric status was assessed using the WHO Child Growth Standards . Wastingast milk ,17. For Household wealth was assessed by constructing an index using principal components analysis. The first component, which explains most of the variance in the observed set of variables, is expected to reflect an unobserved dimension, and in the given model 'wealth.' This method was established in epidemiology during the 1990s and has been used as a proxy for wealth assessment in the literature -20. The Dependent dichotomous variables were constructed using WLZ and LAZ less than -2 as cut-off value. Potential associated determinants were assessed by crude and adjusted analyses, the latter conducted according to a conceptual hierarchical framework. This framework was established in paediatric epidemiology by Mosley and Chen in 1984 for the study of infant mortality, and refined and elaborated upon by Victora et al. in 1997 with the example of diarrhoea; and similar models have been used by Chopra and Wamani, among others, for the study of anthropometry ,22-24. TAnthropometric mean indices were assessed with respect to pre-lacteal feeding, mode of feeding and household wealth. Linear regression analysis was performed for all the explanatory factors before factors were selected for the adjusted means in order to identify potential confounding between feeding practices and anthropometric z-scores, and household wealth and anthropometric z-scores. The following factors were included from Figure p < 0.05. The cluster effect arising from the sample design was accounted for by setting the village number (113) as the sampling unit using the 'svyset' command in Stata [All 723 infants up to one year of age were included in all data analyses. Because exclusive breastfeeding is recommended only for those under six months, we looked at this group in particular (n = 412) and did sub-group analyses of it. Descriptive statistics were used to record the frequencies of those who were wasted, stunted and underweight. Mean WLZ, LAZ and WAZ were assessed for boys and girls. Means with 95% confidence intervals, t-tests and one-way analysis of variance (ANOVA) were used for continuous variables and a chi-square test for categorical data. The significance level was set to in Stata .Approval of the study was granted by Makerere University Faculty of Medicine Ethics and Research Committee, the Uganda National Council for Science and Technology and the Regional Committee for Medical Research Ethics, Western Norway. Informed consent was obtained from all the study participants, and permission was also obtained from the local administrative units.The mean WLZ was 0.04 (95% CI -0.07 to +0.14), the mean LAZ was -0.76 (95% CI -0.88 to -0.65) and the mean WAZ was -0.49 (95% CI -0.59 to -0.39) Table . Among tThe mean ages for introducing feeds were one month for water-based liquids and two months for complementary feeds. More than half the infants (57%) had received pre-lacteal feeding, predominantly water-based feeds, which skewed the median age for introducing water-based liquids to three days. The latest time-point reported for introducing feeds other than breast milk was seven months. Breastfeeding was practised by 98% at the time of the interview. Thirty-eight percent of the mothers perceived colostrum as good and 35% perceived it as bad, and the remaining mothers had less polarised views.The baseline characteristics are presented together with the crude ORs (95% CI) for the dichotomized variables WLZ and LAZ, with < -2 as cut-off value (Table The crude factors associated with stunting were: (1) age; (2) lowest household wealth; (3) coughing during the previous 14 days; and (4) being mixed or replacement fed. The mother's marital status was borderline significant in the crude analysis, with a two-fold increase in odds ratio for being stunted if the mother was single, divorced, separated or widowed. In contrast, having brothers and/or sisters was protective .The conceptual hierarchical framework was the basis for adjusted logistic regression analysis. No determinants were found to be significantly associated with wasting when all infants, n = 723, were included. In a sub-group analysis of infants under six months, n = 412, pre-lacteal feeding was significantly associated with wasting .Stunting was associated with many factors including all infants, n = 723  Table . Mixed fThe aim of this study was to describe growth patterns in Eastern Uganda using the WHO Child Growth Standards, and to investigate factors that might be associated with growth outcomes, including infant feeding practices. From the results it is clear that lower anthropometric status was associated with household wealth; in particular, the lowest household wealth was related to stunting with a threefold increase in OR. This study showed an increased prevalence of stunting with age and a significant difference between boys and girls in length-for-age: boys were more vulnerable. No factors were significantly associated with wasting in the adjusted analysis. This was probably due to the low proportion of wasted children. Adjusted mean differences showed that pre-lacteal feeding was associated with lower WLZ. Mixed feeding or replacement feeding was associated with lower LAZ after adjustment for other explanatory factors.The cross-sectional design of the study entails certain limitations. First, only associations can be described; causal relationships cannot be established. It would for example be tempting to say that mixed feeding or replacement feeding leads to worse length-for-age outcomes. Theoretically, the converse might be true  ,27: thosThose who had received pre-lacteal feeds tended to have poorer weight-for-length outcomes according to the adjusted means in the multivariate general linear model. Various explanations are possible. First, there may be a physiological explanation . The posWe used 'weighing at birth,' 'having a child health card' and 'being adequately immunised' as indicators of 'contact with primary health care units.' No association was seen with wasting and stunting in the crude or adjusted analyses. These variables were included when the mean anthropometric indices were adjusted for feeding practices and household wealth because they were associated with WLZ and LAZ in the linear regression. Improved anthropometric status could be attributable to better contact with respective health units because of improved health care, including nutritional messages with growth-protective value, actual treatment and immunisation. Mothers who delivered at health units or took their new-borns for weighing could also have been more trained and empowered. An association was seen between the ranking of sub-counties and stunting and this could possibly be explained by access to health services within the respective sub-counties. The present study was not designed to address this, but recent Indian studies suggest such relationships .A more meaningful term than 'feeding mode' would have been a constructed feeding index where infant phase-specific preferred feeding gave higher credits and non-recommended feeding lower credits. Two comprehensive nutritional studies from Africa in which such an index was utilised have been published recently ,42. UnfoThis study describes the growth patterns of a randomly-selected community-based infant population in Eastern Uganda and factors explaining these patterns. It utilised a conceptual hierarchal framework and found associations with growth at all levels of the models, supporting the view that child growth is multi-dimensional. Household wealth showed the strongest association with stunting. These findings are in line with earlier anthropometric descriptions from Western and Northern Uganda including older children, and this study indicated no changes or improvements ,7,30,31.The authors declare that they have no competing interests.IE was active during the design, implementation, analysis and writing. HW and CK contributed to the design and analysis. JKT and TT initiated the study and contributed throughout the whole process of design, implementation, analysis and co-writing. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


We assessed the effect of medical staff role models and the number of health-care worker sinks on hand-hygiene compliance before and after construction of a new hospital designed for increased access to handwashing sinks. We observed health-care worker hand hygiene in four nursing units that provided similar patient care in both the old and new hospitals: medical and surgical intensive care, hematology/oncology, and solid organ transplant units. Of 721 hand-hygiene opportunities, 304 (42%) were observed in the old hospital and 417 (58%) in the new hospital. Hand-hygiene compliance was significantly better in the old hospital  compared to the new hospital  (p<0.001). Health-care workers in a room with a senior  medical staff person or peer who did not wash hands were significantly less likely to wash their own hands ; p<0.001). Our results suggest that health-care worker hand-hygiene compliance is influenced significantly by the behavior of other health-care workers. An increased number of hand-washing sinks, as a sole measure, did not increase hand-hygiene compliance. One of the key components for limiting spread of health-care–associated infectious disease is adequate infection control practice. A cornerstone of infection control is ensuring that health-care workers wash their hands at appropriate times. The Association for Professionals in Infection Control and Epidemiology (APIC), the Guidelines for Handwashing and Hospital Environmental Control  from the Centers for Disease Control and Prevention (CDC), and the Hospital Infection Control Practices Advisory Committee each highlight specific indications for handwashing compliance , and the 11-bed medical intensive-care unit (MICU). Sink-to-bed ratios in the units were 8:33 in the hematology/oncology unit, 4:23 in the solid organ transplant unit, and 1:1 in both ICUs.Sinks were located in various sites in the old hospital. The non-ICUs had a limited number of handwashing sinks for health-care worker use located on walls in the middle of each hallway, in clean storage rooms, and in soiled-linens utility rooms. The hematology/oncology unit had a single handwashing sink located in each of three hallways, two handwashing sinks located in each corridor for the bone marrow transplant patient rooms, and a handwashing sink in the anteroom to the bone marrow transplant suite. The solid organ transplant unit had a single handwashing sink located in each of two hallways. ICUs had private rooms with a sink located inside the entrance of every patient room but no hallway sinks.The new hospital opened with 492 individual (private) patient rooms. Observations in the new facility were done in the 30-bed hematology unit, the 30-bed oncology unit, 30-bed solid organ transplant unit, the 12-bed SICU, and the 17-bed MICU. A sink dedicated for hospital personnel use is located inside every patient room. No sinks are available in the hallways.We defined hand hygiene as any duration of washing with soap and water. No waterless alternatives were available for other types of hand hygiene during the study. We recorded hand-hygiene compliance on room entry and after each hand-hygiene opportunity. The definitions of hand-hygiene opportunities, patient contact, and invasive procedures used for this analysis are consistent with APIC or CDC guidelines . InanimaDuring the two study periods, 1-hour observation periods were conducted weekdays between 8:00 a.m. and 5:00 p.m. The first observation period  took place from October 8, 1998, to April 29, 1999, in the old facility. The second observation period  took place in the new building from July 7 to December 23, 1999. A physician, two infection-control professionals, and a microbiologist were trained to individually observe, as follows: 1) protocol guidelines and study definitions were explained in detail from a printed handout that could be carried to the floor for reference during the study, and the data collection form was discussed; 2) the new observer accompanied the physician to a study unit and observed how to perform surveillance and complete the data collection form; 3) after observing several handwashing opportunities, the new observer made and recorded observations along with the physician; 4) the physician and new observer compared observations, and discrepancies were discussed to assess understanding of the protocol guidelines and study definitions; and 5) side-by-side comparisons were performed on a subsequent day, and the training was considered complete.After entering the nursing unit, observers followed the initial worker or group of health-care workers they encountered who went into a patient room. To maintain health-care worker anonymity, individual identities were not recorded; therefore, we could not eliminate or control for repeat observations during analysis. The first persons entering a room were observed until departure from the room. We then went back into the hallway, and the next new persons entering a room were followed for the next observation, thus avoiding repeat observations of an individual health-care worker on any single observation day. Although health-care workers were not informed regarding the purpose of this study, if questioned during hand-hygiene observations, the investigators replied that infection-control measures were being monitored. No immediate feedback was provided to the health-care workers regarding hand-hygiene behavior.Data gathered during observations included time of day, type and number of health-care workers entering the room, patient or equipment contact, compliance with hand-hygiene practices, glove use, invasive procedures, nursing unit and hospital, and whether isolation precautions had been posted. Health-care workers were designated as one of the following categories: physician; registered nurse; patient-care technician; respiratory, physical, or occupational therapist; pharmacist; radiology, electrocardiogram, or ultrasound technician; dietician; food service worker; unit secretarial staff; housekeeping staff; transportation staff; student; chaplain; volunteer staff; or technical sales support. Each physician was further classified as an attending or fellow, resident, or medical student.A hierarchy was defined to assess the effect of other medical staff on hand-hygiene compliance. Health-care workers were ranked in the following order: 1) attending physician or fellow, 2) resident or intern, 3) nurse, 4) technical staff consisting of respiratory therapists, physical, or occupational therapists, radiology, electrocardiogram, ultrasound technicians, pharmacists, or dieticians, 5) patient-care technicians, and 6) housekeeping or transportation staff. Because the number of observations of medical students was small and determining the proper rank for medical students was difficult, we did not include students in the analysis of the influence of other personnel in the room. During analysis of the influence of other health-care workers present in the room, each health-care worker room entry was placed in one of the following categories: single person, highest ranking person in the room, in the room with a higher ranking person who performed hand hygiene, in the room with a higher ranking person who did not perform hand hygiene, in the room with a peer who performed hand hygiene, or in the room with a peer who did not perform hand hygiene. Peer status was defined as two or more health-care workers in the same category listed above . Hierarchy status was related to a purely theoretical belief of clinical expertise and knowledge held by the health-care worker.Changes in accessibility and availability of hand-hygiene products and supplies between the two study periods included product modifications of soap, hand lotion, towels, and gloves. In general, products were more accessible in the new hospital. A para-chloro-meta-xylenol soap,  was the hand-hygiene agent in the ICUs and hematology/oncology unit in the old hospital. A 5-chloro-2-[2,4-dichlorophenoxyl, triclosan product  was used in the solid organ transplant unit in the old hospital and is now used in all units of the new hospital. An aloe vera lotion with triclosan was available in clean utility rooms of the old hospital . In the new hospital, an amino lotion product is mounted at every health-care worker sink next to the soap dispenser . Single-unit paper towel dispensers were available at each handwashing sink in both hospitals . In the old hospital, various glove sizes were kept in clean utility rooms. In the new hospital, three sizes of gloves are located in wall-mounted dispensers next to towels and sinks. Powder-free gloves were available only for staff requiring them in the old hospital. Powder-free gloves are used exclusively in the new hospital by all personnel.Data were collected on standardized forms and entered in Microsoft Excel . To evaluate predictors for hand-hygiene compliance after a hand-hygiene opportunity, we compared categorical variables using chi-square or Fisher’s exact test; odds ratios (OR) and 95% confidence intervals (CI) were calculated by using Epi Info version 6.04c . A total of 560 health-care worker–patient interactions were observed, resulting in 729 hand-hygiene opportunities. A total of 305 (41.8%) hand-hygiene opportunities were observed in the old hospital and 424 (58.2%) in the new hospital. Of the 560 health-care worker–patient interactions observed, 237 (42.3%) of the workers were registered nurses, 190 (33.9%) were physicians, and 133 (23.8%) were other health-care workers. The old and new hospitals were similar in performance of invasive procedures and health-care worker type. In the older facility, health-care workers were more likely to wear gloves or touch a patient during the hand-hygiene opportunity.Hand-hygiene compliance on room entry was significantly greater in the old hospital at 12% (36/304) compared to the new hospital at 6% (26/424) (p=0.006). After all hand-hygiene opportunities were assessed, we found that hand-hygiene compliance was significantly better in the old hospital compared to the new hospital . Hand-hygiene compliance was significantly better after a hand-hygiene opportunity  compared to before a hand-hygiene opportunity . By univariate analysis, characteristics significantly associated with hand-hygiene compliance after a hand-hygiene opportunity included working at the old hospital, having patient contact, performing an invasive procedure, using gloves, and performing hand hygiene on room entry. A key finding was that when a higher ranking person in the room did not perform hand hygiene, other health-care workers were significantly less likely to wash their hands .During multivariate analysis, we identified the following independent predictors of hand-hygiene compliance: using gloves, performing an invasive procedure, working at the old hospital, performing hand hygiene on room entry, and having patient contact. Again, health-care workers present in the room with a higher ranking person or peer who did not perform hand hygiene were significantly less likely to wash their hands .When we further evaluated group behavior, we found that compared to single person room entry, health-care workers in a room with a higher ranking person who did not wash were significantly less likely to wash their own hands. In each of these episodes, the higher ranking person was a physician or nurse. Surprisingly, if either a higher ranking person or peer was in the room and performed hand hygiene, then the frequency of hand hygiene for others in the group was no better than that of a room which only one person entered . This obDespite construction of a new hospital with an increased number of sinks, we found that hand-hygiene compliance in the new facility decreased substantially. We demonstrated that health-care workers were significantly less likely to wash their hands if they were in a room with a peer or higher ranking person who did not perform hand hygiene. Not unexpectedly, hand-hygiene compliance was better after patient contact, performing an invasive procedure, and removing gloves.Health-care workers were much less likely to perform hand hygiene if a peer or a higher ranking person in the room did not perform hand hygiene. Compared to health-care workers who entered a room alone, group behavior did not seem to improve if the higher ranking person or peer did wash their hands. Although these findings suggest that hand-hygiene behaviors can be affected by role model or peer hand-hygiene compliance, learned behaviors or time constraints may negatively influence group compliance with hand-hygiene procedures.As suggested by some studies, physician hand-hygiene compliance has an impact on peer and group behaviors  was similar to the frequency of hand-hygiene compliance reported by other investigators (>0.2).While some studies . To ensure adequacy of training, institutional education activities are systematic. All new employees are required to attend a hospital orientation that includes general information on infection-control issues. On the first day of work in the department, new and transferred employees also participate in individual departmental orientation. The orientation includes a review of policies and procedures, job-specific responsibilities, performance expectations, and unit-specific infection control measures . For agency and float pool nursing staff, a 3-hour orientation is provided, which includes a review of policies, procedures, and paperwork. Float pool nurses have at least 1 year of experience before being hired and are required to complete annual competencies that incorporate infection-control measures. While they are used throughout the hospital, only nurses having cardiac-care certification may work in the ICUs.>0.2). Since we did not perform observations at night or on the weekends, and duration or efficacy of hand hygiene was not evaluated during our study, we cannot comment on hand-hygiene compliance for these shifts or on the effectiveness of health-care workers’ hand-hygiene technique.Our study had several limitations. First, the two study periods were in different seasons and involved different house staff. A greater proportion of observations in the older hospital were conducted in the winter months, which we postulated would decrease hand-hygiene compliance because of increased skin dryness ; however, the decrease was similar to that observed in other monitored units, which suggests any change in soap product was not the major factor. Additionally, a change from powdered to nonpowdered gloves may have negatively influenced hand hygiene. Reviewing our data set for the potential influence of this factor, we found that if gloves were worn and removed, then hand-hygiene frequency in the old hospital was 131/176 (74%), whereas in the new hospital, the frequency was 76/128 (59%) (p=0.005). If gloves were not worn, then hand-hygiene frequency in the old hospital was 30/116 (26%), and in the new hospital 21/260 (8%) (p<0.001). Thus, both the absolute and relative decreases in hand-hygiene frequency were greater for the nongloved health-care workers, which suggests that powder in the gloves was not the reason for diminished hand hygiene.Pittet has posed the problem of hand-hygiene compliance: How can we change the behavior of health-care workers and how can we maintain such a change (


Short abstract: A multi-level cross-species comparative analysis of gene-expression changes accompanying increased longevity in mutant nematodes, fruit flies and mice with reduced insulin/IGF-1 signaling revealed candidate conserved mechanisms. To what extent are the determinants of aging in animal species universal? Insulin/insulin-like growth factor (IGF)-1 signaling (IIS) is an evolutionarily conserved (public) regulator of longevity; yet it remains unclear whether the genes and biochemical processes through which IIS acts on aging are public or private . To address this, we have applied a novel, multi-level cross-species comparative analysis to compare gene expression changes accompanying increased longevity in mutant nematodes, fruitflies and mice with reduced IIS.Surprisingly, there is little evolutionary conservation at the level of individual, orthologous genes or paralogous genes under IIS regulation. However, a number of gene categories are significantly enriched for genes whose expression changes in long-lived animals of all three species. Down-regulated categories include protein biosynthesis-associated genes. Up-regulated categories include sugar catabolism, energy generation, glutathione-S-transferases (GSTs) and several other categories linked to cellular detoxification . Protein biosynthesis and GST activity have recently been linked to aging and longevity assurance, respectively.These processes represent candidate, regulated mechanisms of longevity-control that are conserved across animal species. The longevity assurance mechanisms via which IIS acts appear to be lineage-specific at the gene level (private), but conserved at the process level (or semi-public). In the case of GSTs, and cellular detoxification generally, this suggests that the mechanisms of aging against which longevity assurance mechanisms act are, to some extent, lineage specific. Caenorhabditis elegans and the fruitfly Drosophila melanogaster. This is also true of other functions, such as cellular metabolism and neurobiology. But what about aging? According to evolutionary theory, aging is not a genetically programmed process, but rather a side-effect either of mutation pressure [Saccharomyces cerevisiae) [Podospora anserina [Growth and development in living organisms, from bacteria to higher animals, are genetically programmed processes involving molecular mechanisms, many of which are evolutionarily ancient and shared across a broad range of taxa. Consequently, it is possible to understand genes and processes controlling mammalian growth and development by studying invertebrate model organisms such as the nematode pressure  or of sepressure . From thpressure . Gross pevisiae) , and extanserina ; neitheranserina .daf-2 in C. elegans (GenBank: NM_065249), the insulin/IGF-1 receptor dINR and insulin-receptor substrate (IRS) chico in Drosophila (GenBank: NM_164899), and the IGF-1 and insulin receptors in mice can all increase lifespan [df/df and Ghrhrlit/lit (the Ames and Little dwarf mice), also increase lifespan [However, recent studies have shown that the insulin/insulin-like growth factor (IGF)-1 signaling (IIS) pathway is a public determinant of aging. For example, mutation of the insulin/IGF-1 receptor lifespan -12. Addilifespan ,14.C. elegans that IIS exerts effects on longevity via regulated effector genes [It has been demonstrated in or genes -18. ThatC. elegans, Drosophila and the mouse, surveyed using oligonucleotide microarray analysis (Affymetrix). To do this we used a novel analytical approach to examine conservation of regulation in which conservation was viewed at each of three different levels: that of gene orthologs, that of paralogous gene sets, and that of broader gene classes (defined by InterPro or Gene Ontology (GO) categories). We find that, in contrast to the public role in aging of IIS itself, IIS-regulated genes are not conserved at the level of gene orthology or of paralogous gene groups. However, if IIS-regulated genes are compared across species at the level of gene category , cross-species similarities are visible. Notably, we see down-regulation of categories linked to protein synthesis, consistent with recent findings that lowered protein translation increases lifespan in the yeast S. cerevisiae [C. elegans [C. elegans [To begin to address these questions, we have compared the genes that are transcriptionally regulated during IIS-linked lifespan extension in three animal species: revisiae  and C. e elegans -22. We a elegans ,24. In tTo search for public, IIS-regulated determinants of longevity, we used previously published microarray data from long-lived mutant worms and mice with lowered IIS, and generated new microarray data for a long-lived IIS mutant in flies  [daf-2 mutants and non-long-lived daf-16; daf-2 double mutants, effectively a comparison of DAF-16 ON and DAF-16 OFF [daf-2, q < 0.1, here and below). Other transcript profiles of C. elegans IIS-regulated genes are available [In 1026423) . We reanF-16 OFF . This idvailable ,16, whicvailable ; these lDrosophila, we compared wild-type (Dahomey) and long-lived chico1/+ heterozygotes [chico1/+). Initially, we also examined transcript profiles from homozygous chico1 mutants, which are slightly longer lived than chico1/+. However, the proportion of genes showing differential expression was so high as to make data analysis impracticable (data not shown). This difficulty was likely due to the fact that homozygous chico1 flies are sterile dwarfs, with different quantities of eggs and oocytes, and altered allometry of tissues and organs and, as a result, the mRNAs that they contain. By contrast, chico1/+ flies are fertile and normal sized. Thus, the present analysis was only possible thanks to the semi-dominant effect of chico1 on aging but not on fertility and size.For ozygotes . This iddf/df (Ames dwarf) and Ghrhrlit/lit (Little) mutants to normal-lived controls [Finally, for the mouse, we reanalyzed data comparing gene expression in the liver of long-lived Prop-1controls . Both mucontrols ,28. In oIf IIS controls aging via regulated public mechanisms, we would expect to see similarities between transcriptional changes in long-lived mutants in each species. We initially reasoned that such similarities could occur on either of two levels. Firstly, IIS could regulate a set of orthologous genes in all species. Secondly, IIS could regulate genes contributing to similar biological processes in different species  that result in increased longevity. This might or might not involve orthologous genes in the three species.q < 0.1) changes in expression in each species, and in the same direction (up- or down-regulated given reduced IIS). Surprisingly, very few orthologous genes changed expression co-ordinately in different species, and the number of such genes differed little from that expected by chance alone. For example, only nine ortholog pairs were significantly up-regulated in the worm and fly datasets (approximately 14 would be expected by chance). However, four ortholog sets were up-regulated in the worm, fly and Little mouse, significantly more (p = 0.003) than expected by chance alone (Tables For gene-level (as opposed to process-level) analysis, we first identified orthologous pairs of genes between each species, and orthologous sets of genes between all three species . We then screened for ortholog pairs or sets (triplets) that showed significant  in the long-lived, RNAi-hypersensitive strain rrf-3(pk1426); daf-2(m577) . Of the test genes, RNAi of only one, the pantothenate kinase pnk-1, significantly shortened lifespan. However, pnk-1 RNAi also did this in a normal-lived control strain (data not shown), and it also causes sterility, larval arrest, and embryonic lethality [pnk-1 for overall viability rather than prevention of aging. Pantothenic acid is a component of coenzyme A, the acetylated form of which plays a key role in the citric acid cycle. Pantothenate kinase catalyzes the first step in coenzyme A synthesis. In conclusion, the transcriptional response to reduced IIS shows very little evolutionary conservation at the level of gene orthology.To further test whether the nine worm-fly ortholog gene pairs might be longevity determinants, we reduced expression of each gene in  rrf-3pk126; daf-2ethality . The redn ≤ 2 or n ≤ 3 paralogous genes present in the gene list for each individual species . We counted the number of paralog sets  where at least one gene was differentially expressed in each species, and in the same direction. Secondly, we examined all paralog sets, whatever their size, and counted the number of paralog sets where a substantial number of genes showed differential expression in the same direction (we used the arbitrary cut-off of >50%). In addition, we counted again the number of orthologs with altered expression in more than one species, using the same statistics . For each of these four levels of conservation , we asked whether the number of ortholog or paralog sets identified were more than expected by chance alone. To this end we performed bootstrap analysis on paralogous groups, comparing the observed number of differentially expressed paralogous groups with the numbers obtained by drawing the lists of differentially expressed groups at random .The lack of conservation seen at the level of gene orthology was unexpected. It led us to wonder whether perhaps, in some cases, IIS-regulated functions might be performed in different species by paralogous genes rather than orthologous ones. To this end, we looked at expression of paralogous genes in long-lived worms, flies and mice in two ways. Firstly, we examined all sets of paralogs where there was either p = 0.01) . According to this analysis, for most comparisons the number of shared categories is more than predicted by chance alone, particularly where genes are up-regulated in the long-lived mutants . However, it should be borne in mind that the statistical test used assumes that the various categories are independent of one another, and in some cases this may not be the case. For example, cytochrome P450 (CYP) enzymes and GSTs can be subject to coordinate regulation [Next we tested whether the number of shared gene categories enriched for differentially regulated genes was more than predicted by chance alone. To do this, we performed bootstrap analysis of gene categories, drawing categories at random and computing gulation ; moreovedf/df and Ghrhrlit/lit datasets [df/df mutation is more pleiotropic, blocking production of thyroid stimulating hormone and prolactin in addition to growth hormone. It may also reflect the larger size of the lists of differentially expressed genes from the dwarf mouse studies, which can reduce the sensitivity of the test for overlapping gene categories. More positively, it suggests that comparing datasets from the two mouse strains has acted as a strong filter to exclude numerous gene categories unlinked to the increased lifespan phenotype.An expected outcome of this analysis was that the two microarray datasets from the mouse would share more over-represented gene categories with one another than with the two invertebrate datasets. In terms of the individual genes showing altered expression, there are strong overlaps between the Prop-1datasets . HoweverC. elegans is increased by loss of function of several genes promoting protein translation, including translation initiation factors and ribosomal proteins [The majority of the common up-regulated GO categories are involved in sugar catabolism and energy generation Table , implyinproteins -22. ThusMost of the Interpro domain gene families showing conserved up-regulation in IIS mutants are linked to cellular detoxification  Table . These cC. elegans daf-2 mutants and long-lived dauer larvae [The association of increased expression of gene classes linked to cellular detoxification with longevity in three species, coupled with the lack of gene-level orthology, prompted us to examine the evolutionary relationships of these gene families in more detail. To do this, we constructed phylogenetic trees for each of three families in worms, flies, and mice, and then examined the distribution of IIS-regulated gene expression. Figure r larvae . In eachr larvae . This isr larvae ,34.Cis-eLement OvEr-Representation) [Finally, we explored whether IIS transcriptional responses are regulated by conserved DNA binding factors. Using the program Clover (ntation) , we examntation) . These fThe role of IIS as a regulator of aging shows evolutionary conservation. The effects of IIS on lifespan reflect the action of IIS-regulated genes and biochemistries of aging and longevity. In this study, we have asked the question: are these genes and processes public (evolutionarily conserved) or private (lineage specific)? We have done this by means of a cross-species comparison of transcript changes seen in long-lived nematodes, insects and mammals with lowered IIS when compared to normal-lived controls. To be able to do this we developed a novel, multi-level cross-species comparative method, comparing gene expression at the levels of genetic orthology, paralogy , and gene classes. We detected little evolutionary conservation of IIS regulation at the orthologous or paralogous gene levels. However, at the genes class or process level some evolutionary conservation was observed, including several processes previously associated with aging.C. elegans and Drosophila, focusing on 17 C. elegans-Drosophila ortholog gene pairs with predicted DAF-16 binding sites in their promoter regions [C. elegans orthologs showed IIS regulation, suggesting possible evolutionary conservation of IIS-regulated genes at the level of orthology. However, no data on IIS regulation of Drosophila orthologs were reported in that study. Our findings point to the opposite conclusion: that the set of genes regulated by IIS is largely lineage specific.The absence of detectable regulation by IIS of orthologous genes in the three animal models tested was unexpected, for several reasons. Firstly, even if the same IIS-regulated genes did not regulate aging in worms, flies and mice, one would expect that some of the genes mediating the effects of IIS on growth and sugar metabolism would be conserved at the level of orthology. Secondly, an earlier study examined putative direct transcriptional targets of FOXO in  regions . There, C. elegans or Drosophila. Secondly, if the direction of IIS regulation is different in different tissues in the invertebrate models, this could prevent detection of IIS regulation. Thirdly, it may be that in extra-hepatic tissues, transcript profile changes resulting from Prop-1df/df and Ghrhrlit/lit are more similar to those in C. elegans and Drosophila IIS mutants. The liver consists mainly of dividing cells whereas, in the invertebrate models, adult somatic tissues consist largely of post-mitotic cells. Recent mouse studies suggest that age-related changes in gene expression may differ between mitotic and post-mitotic tissues [If significant numbers of orthologous genes were robustly IIS regulated in similar ways in multiple tissues, then it is likely that the analytical approaches that we have employed would have detected this. However, it remains possible that orthologous genes regulated similarly by IIS eluded our analysis, for several reasons. Firstly, microarray analysis may have failed to detect small but functionally significant changes in transcript levels, for example, genes showing IIS-regulated expression in only a small proportion of cells in  tissues . Fourthl tissues  and to pIn contrast to our studies of orthologous or paralogous genes, our comparative analysis at the gene class level identified a number of candidate gene classes and processes showing an evolutionarily conserved pattern of regulation in long-lived mutants with reduced IIS Table . We perfClearly, the presence of any of the gene categories in Table C. elegans [S. cerevisiae [C. elegans, Drosophila and mice implicates this process as a public, IIS-regulated mechanism controlling aging. However, it should be noted that the individual genes involved in protein biosynthesis whose expression was shown to affect C. elegans aging were not themselves IIS regulated [C. elegans these perturbations increase heat stress resistance, suggesting that lowered protein synthesis leads to induction of somatic maintenance functions [Several recent studies imply that increased protein biosynthesis accelerates aging. Lowered expression of a number of genes involved in mRNA translation, ribosomal proteins, translation initiation factors and ribosomal protein S6 kinase results in reduced rates of protein biosynthesis and increased lifespan in  elegans -22. Simirevisiae . Over-reegulated . How lowunctions .GSTs detoxify a wide range of electrophilic  and often toxic compounds by conjugation with glutathione (GSH) . Such elC. elegans genes gst-5 and gst-10 encode GSTs that detoxify 4-hydroxy-2-nonenal (HNE), which is a major product of peroxidation of membrane lipids and a mediator of the pathophysiological effects of oxidative stress [C. elegans, Drosophila and mice with reduced IIS suggests that GST activity may represent a public, IIS-regulated mechanism of longevity assurance.More importantly, there is some direct experimental evidence for a role of GSTs in longevity assurance. The e stress . RNAi kne stress ,44. Overe stress . The ovesod genes are up-regulated in C. elegans, this is not the case in Drosophila or the mouse ; however, for the purposes of analysis in this study, ctl-3 data were excluded due to predicted promiscuity in probe binding between clt-3 and ctl-1. In Drosophila there is a possible increase in catalase mRNA levels . The absence of increased transcript levels of catalase and Mn SOD genes in Prop-1df/df mouse liver was unexpected, since increased catalase levels have been reported in this tissue [The possible broader implications of the observed association between GST gene expression and extended lifespan Table  may be cse Table . Consist elegans , but notosophila . In termnd ctl-3 . Our mics tissue . OverallDrosophila overexpression of glutamate cysteine ligase (γ-glutamylcysteine synthetase), the major rate-limiting enzyme in GSH biosynthesis, extends lifespan [Drosophila lifespan [A second perspective on possible GST function in aging is within the context of a broader, GSH-associated biochemistry. Besides its role in detoxification by GSTs, GSH itself acts as an antioxidant , and thelifespan . Moreovelifespan .df/df (Ames dwarf) mice appears to be geared up for increased GSH production and usage [Hepatic metabolism in Prop-1nd usage -55. Bothnd usage , and thend usage ,55. PossBeyond the biology of GSH, GSTs may be viewed as part of a wider system of cellular detoxification involving two phases: phase 1 , and phase 2 (conjugative reactions)  Figure . CYPs anC. elegans, Drosophila and mice  and livth cases ), althoulifespan . This delifespan .In principle, toxins contributing to aging that are lineage-specific could contribute to the lineage specificity of aging-related pathologies. According to this view, aging involves stochastic mechanisms that are partially public and partially private. A summary overview of this interpretation is shown in Figure C. elegans, Drosophila and the mouse. Our aim was to identify genes and processes regulated by IIS that might correspond to evolutionarily conserved (public), proximal determinants of aging. While our analysis suggests that IIS regulation of genes shows relatively little evolutionary conservation at the level of individual orthologous or paralogous genes, we identified two processes that are both IIS regulated in all three animal models, and linked to aging. In each long-lived mutant, there is evidence of lowered protein biosynthesis and increased cellular detoxification . This evolutionary conservation suggests that these processes might play a role in the control of other animal lineages, for example, primates. More research is therefore needed on the impact of these two processes on aging.We have compared changes in transcript profiles occurring in long-lived mutants with reduced IIS in C. elegans datasets are available from the Gene Expression Omnibus [D. melanogaster datasets are available from ArrayExpress [M. musculus datasets are available from ArrayExpress, accession number E-MEXP-153. For an overview of microarray datasets, see Table All microarray datasets analyzed in this study are publicly available. The  Omnibus , accessiyExpress , accessiD. melanogaster used for microarray analysis were generated as follows: chico1/+ heterozygotes were selected from the progeny of a Dahomey wild type × Dahomey chico1/CyO cross. Wild-type Dahomey control flies were age-matched as previously described, and all flies were raised under standard culture conditions [chico1 stock [Drosophila Genome 2.0 Genechip. In total, five biological replicates of each genotype (wild type and chico1/+) were performed.nditions . The chio1 stock ,62 has bC. elegans and M. musculus (Prop1df/df and Ghrhrlit/lit) strains, growth conditions, sample preparation and microarray hybridization protocols used to generate the raw Affymetrix data (cel files) analyzed in this study are previously described [C. elegans, hermaphrodite; D. melanogaster, female; M. musculus, male.Information regarding the escribed ,26. For To assign gene orthologs between the three species, the Ensembl Biomart tool (Ensembl version 37) was used to download lists of orthologous genes between each species . These oTo examine the statistical significance of the number of differentially expressed orthologs that were observed when comparing the microarray datasets, we performed a simulation to determine the number of expected differentially expressed orthologs given the total population sizes  and the number of differentially expressed genes in each microarray experiment. Full data from this analysis are available in Tables 6 random draws of genes. For each random draw, we drew random samples of size n1 and n2 (and n3 for the three species comparison) from the populations of N1 and N2, respectively (and N3 when necessary). Now both use subscript numerals but no italics For example, for the fly-worm up-regulation comparison, the pool of genes contained N1 = 10,395 fly and N2 = 12,414 worm genes, while the sample sizes were n1 = 893 and n2 = 558 up-regulated genes in fly and worm, respectively : two groups were called orthologous when any gene from the first groups had any orthologous or paralogous relationships with any other gene from the second group (similar to single linkage clustering). For the three species comparison, three groups were called orthologous if at least one group was orthologous to the groups in both other species. A group was considered differentially expressed when at least one of its members was differentially expressed , or when at least half of the groups present on the chip were differentially expressed . The probabilities reported in additional Table The potential role of conserved orthologous genes in aging was examined using an RNAi feeding protocol . Bacterirrf-3(pk1426); daf-2(m577)) was used to examine the effect of RNAi on the increased lifespan of daf-2. Eggs from gravid adult animals maintained at 20°C were isolated by hypochlorite treatment [For the lifespan assays, the RNAi-hypersensitive strain GA303   and Ensehomology . Phylogehomology , or by pnsai 1.2 . Phylogeq-value) between different genotypes that takes into consideration the variance of probe set intensity across the different normalizations. The Goldenspike methodology has been shown to out-perform most commonly used normalization methods [We used the following statistical and computational tools in the analysis of our microarray datasets: The R computer program (version 2.0.1) , Goldens methods . The GolC. elegans genome release WS140, D. melanogaster genome release version 4.2.1, and M. musculus genome release NCBIm34) [Prior to further analysis, we performed a quality control procedure on all three Affymetrix microarrays used in this study to ensure the specificity of each individual probe set. All individual probes have been mapped against all known and predicted transcripts of the corresponding genome using recent genome releases (NCBIm34) ,76,77. TTo identify significant differential expression of functionally related categories of genes, we used the program Catmap . We first statistic from the Goldenspike analysis was used as input. The Wilcoxon rank sum was used to generate a score based on the sum of the rankings of all genes with a particular functional annotation, and the significance of that score  was calculated analytically based on a random gene-rank distribution. Gene categories were considered significantly differentially regulated at a Catmap p value < 0.05. Full output from Catmap for each of the comparisons  is available in Additional data file 6.For Catmap analysis, a ranked gene list based on the Bayes Nobs common gene categories among the n1  categories significantly differentially expressed in the various gene lists. To do this we performed 10,000 random draws of n1  gene categories from the set of N1  gene categories annotating the genes in the first  list. The probability is defined as the proportion of the 10,000 random draws where the number of common categories is greater or equal to Nobs. It should be pointed out that this procedure will underestimate the true probability of finding a large number of common categories because it neglects the correlations between gene categories (see Results). A further drawback with this methodology is that drawing genes at random and performing the Catmap analysis on random ordering of genes is costly in terms of computer time and resources.We estimated the probability of finding by chance alone q < 0.1) up- or down-regulated genes compared to the upstream sequences of all known genes. The output from Clover for each dataset is available in Additional data file 8. RepeatMasker [The Clover program  was usedatMasker  was usedp value ≤ 0.05, raw score cut-off >5) in the up- or down-regulated genes in each dataset.To identify motifs that occur in the promoters of differentially expressed genes in all three species, we examined the output of Clover for motifs that were significantly over-represented (C. elegans, Drosophila and mouse. Additional data file C. elegans, Drosophila and the Little mouse. Additional data file The following additional files are available with the online version of this paper. Additional data file Legends for Additional data files 2-9Click here for fileC. elegans, Drosophila and mousePhylogenetic trees for the four main families of drug metabolizing enzymes for Click here for fileC. elegans, Drosophila and the Little mouse.Table Click here for fileOrthologs used in this studyClick here for fileResults of RNAi lifespan experimentsClick here for fileOutput of the Catmap analysis of the microarray dataClick here for fileSummary of the results of statistical tests for over-representation of gene categories identified by the Catmap analysisClick here for fileOutput of Clover analysis for gene regulatory motifs for each datasetClick here for fileFinal gene lists from our analysis or reanalysis of microarray dataClick here for file


African-Americans admitted to U.S. hospitals with community-acquired pneumonia (CAP) are more likely than Caucasians to experience prolonged hospital length of stay (LOS), possibly due to either differential treatment decisions or patient characteristics.We assessed associations between race and outcomes  for African-American or Caucasian patients over 65 years hospitalized in the Veterans Health Administration (VHA) with CAP (2002-2007). Patients admitted to the ICU were analyzed separately from those not admitted to the ICU. VHA patients who died within 30 days of discharge were excluded from all LOS analyses. We used chi-square and Fisher's exact statistics to compare dichotomous variables, the Wilcoxon Rank Sum test to compare age by race, and Cox Proportional Hazards Regression to analyze hospital LOS. We used separate generalized linear mixed-effect models, with admitting hospital as a random effect, to examine associations between patient race and the receipt of guideline-concordant antibiotics, ICU admission, use of mechanical ventilation, use of vasopressors, LOS, and 30-day mortality. We defined statistical significance as a two-tailed p ≤ 0.0001.Of 40,878 patients, African-Americans  were less likely to be married and more likely to have a substance use disorder, neoplastic disease, renal disease, or diabetes compared to Caucasians. African-Americans and Caucasians were equally likely to receive guideline-concordant antibiotics  and experienced similar 30-day mortality when treated in medical wards . African-Americans had a shorter adjusted hospital LOS . When admitted to the ICU, African Americans were as likely as Caucasians to receive guideline-concordant antibiotics , but experienced lower 30-day mortality  and shorter hospital LOS .Elderly African-American CAP patients experienced a survival advantage  in the ICU compared to Caucasians and shorter hospital LOS in both medical wards and ICUs, after adjusting for numerous baseline differences in patient characteristics. There were no racial differences in receipt of guideline-concordant antibiotic therapies. Prior studies have documented health disparities between African-Americans and Caucasians who present to U.S. hospitals with common infectious diseases -4. HealtIn the treatment of community-acquired pneumonia (CAP), several initial processes of care have been associated with improved survival, including the timely delivery and appropriate selection of antibiotic therapy, and acquisition of blood cultures prior to initiating antibiotic therapy -11. ThesDespite the observed racial disparities in several evidence-based processes of care for CAP, most Veteran and civilian studies to date have found equal or lower adjusted mortality rates for African-Americans compared to Caucasians -18, withThe Veterans Health Administration (VHA), the largest vertically integrated healthcare system in the United States, has been at the forefront of a national quality movement to provide high quality and equitable healthcare for all VHA patients. Therefore, we sought to determine whether there were racial differences in processes of care, hospital LOS, or 30-day mortality for African-American and Caucasian VHA patients with CAP, after adjusting for baseline differences in patient characteristics.This retrospective, cohort study of hospitalized patients with CAP is a secondary analysis of data collected as part of a project to assess the association of treatment with statins on clinical outcomes  of patients hospitalized with CAP and/or sepsis in the Department of Veterans Affairs Health Care System . We used administrative data from the VHA to examine trends in pneumonia care and mortality. The VHA databases are repositories of clinical data from more than 150 VHA hospitals and 850 VHA clinics . The InsWe included VHA patients 65 years and older with a diagnosis of pneumonia in fiscal years 2002-2007. We used previously validated ICD-9 codes (480.0-483.99 or 485-487) for pneumonia to identify potential cases . We exclWe recorded the patient's age, sex, and marital status (married versus unmarried) at the time of admission. We used VHA Priority Group (PG) as a surrogate for socioeconomic status and disease severity, a previously validated approach -24. PrioWe defined 19 comorbid illnesses using ICD-9 codes in accordance with the Charlson comorbidity scoring system , using IMissing data on VHA measures of health services utilization is rare in the administrative data extracts of the electronic medical record system. However, while VHA race data have been validated especially for Caucasian and African-American patients, ,31 missiWe evaluated antibiotics received within the first 48 hours of hospital admission per joint American Thoracic Society (ATS) and Infectious Diseases Society of America (IDSA) CAP medical practice guidelines . We defiAdditional processes of care included ICU admission, vasopressor use, and mechanical ventilation. We defined mechanical ventilation using ICD-9A code 96.7, recorded during an ICU stay. We defined vasopressor and inotrope use based upon the receipt of any of the following medications during the hospital stay: dobutamine, dopamine, epinephrine, isoproterenol, metaraminol, norepinephrine, phenylephrine, or vasopressin.We abstracted the admission and discharge dates for each stay, then defined hospital LOS as the date of discharge minus the date of admission plus one day for the overall hospitalization. The VHA vital status file provided date of death to assess 30-day mortality. The 30-day mortality metric includes those patients who died in the hospital as well as those who died outside the hospital. We chose this follow-up duration because previous research demonstrated that 30-day mortality is primarily due to underlying illness with pneumonia, whereas 90-day mortality is generally attributable to other comorbid conditions . PreviouWe conducted all analyses using JMP 7.0, SAS 9.2 , and Stata 10 . Due to the large sample size, we defined statistical significance as a two-tailed p ≤ 0.0001. Bivariate statistics (chi-square and Student's t-test) compared patient demographics, comorbid conditions, processes of care, LOS, and 30-day mortality for African-American and Caucasian patients with CAP. When assessing study endpoints, patients admitted to the ICU were analyzed separately from those not admitted to the ICU. VHA patients who died within 30 days of discharge were excluded from all LOS analyses. This was done because patients who died rapidly experienced abbreviated lengths of hospital stay. In other words, the length of stay for those very severe patients is artificially short because the patients expired before they could complete an entire episode of care. This approach  is consistent with numerous other peer reviewed publications -38.We used chi-square and Fisher's exact statistics to compare dichotomous variables, the Wilcoxon Rank Sum test to compare age by race, and Cox Proportional Hazards Regression to analyze hospital LOS. We used separate generalized linear mixed-effect models, with admitting hospital as a random effect, to examine associations between patient race and the receipt of guideline-concordant antibiotics, ICU admission, use of mechanical ventilation, use of vasopressors, hospital LOS, and 30-day mortality. Hazard ratios, odds ratios, and 95% confidence intervals were calculated from the generalized linear mixed-effects models. Covariates included all 18 variables listed in Table a priori to be clinically important, and we included them as covariates in each of the multivariable models. The bulk of these variables were chosen because they were key components of well-validated scoring systems that predict patient mortality among pneumonia patients  [The 18 variables listed in Table disease) ,40. OtheOverall, 4,936 African-American and 35,942 Caucasian VHA patients met all eligibility criteria . The study population had a median (interquartile range) age of 78 (72-83) years; 98% were male and 53% were married. History of tobacco dependence (37%), chronic obstructive pulmonary disease (51%), diabetes (30%), heart failure (22%), neoplastic disease (21%), and cerebrovascular disease (16%) were common. One-quarter (25%) had organ failure while 4% had sepsis, and 12% were admitted to the ICU at some point during hospitalization.As shown in Table Among VHA patients managed in the medical wards, African-American and Caucasian patients were equally likely to receive guideline-concordant antibiotic therapy in bivariate (92% versus 93%) and multivariable  analyses (Tables African-American and Caucasian VHA patients experienced similar rates of admission to the ICU (12% versus 13%). Of those patients admitted to the ICU, African-American and Caucasian patients were equally likely to receive guideline-concordant antibiotic therapy in bivariate  and multivariable  analyses Tables  and 5. AAfrican-American and Caucasian patients managed on medical wards experienced similar adjusted 30-day mortality . African-American patients had a shorter adjusted hospital LOS   and shorter adjusted hospital LOS  compared to Caucasian patients admitted to VHA ICUs  compared to Caucasian patients admitted to VHA ICUs. While this survival advantage has been documented in other studies -18, therThe difference in mortality could also be due to differences in advanced directives between the two cohorts. Caucasian patients admitted to VHA hospitals are more likely to have "do not intubate" or "do not resuscitate" orders on file as compared to African-American patients . If thisWe acknowledge the growing body of literature on frailty -44, and In addition to differences in mortality, Caucasian patients in our study remained hospitalized longer than African-American patients. This was the case for both the ward and ICU subgroups. This finding contradicts previous studies reporting longer stays for African-Americans admitted to U.S. hospitals with CAP ,19. The When differences in hospital LOS are observed between two groups, it is important to consider if the group with the shorter hospital LOS experiences any clinical detriment for having been discharged sooner. This could be evaluated by considering hospital readmission rates or mortality post hospital discharge. We analyzed 30-day mortality and found that African-American and Caucasian patients discharged from VHA hospitals experienced similar 30-day mortality; we can therefore conclude that the African-American patients admitted to VHA ICUs did not experience any additional clinical detriment for having been discharged from the hospital sooner than their Caucasian counterparts.African-American and Caucasian VHA patients in our study were equally likely to receive guideline-concordant antibiotic therapy in ward and ICU settings, a finding consistent with a previous study . This fiWe did not evaluate individual guideline-concordant therapies, nor did we attempt to correlate certain guideline-concordant therapies with patient race. We recognize that selection from among the various guideline-concordant therapies must be somewhat customized on the basis of patient history, risk factors for multi-drug resistant pathogens, prior antibiotic exposure, and patient comorbidities. National pneumonia guidelines encourage prescribers to consider these things when selecting empiric antibiotic therapies . Since AAfrican-American VHA patients were more likely to have a history of substance abuse or dependence, neoplastic disease, renal disease, and diabetes. In contrast, Caucasian VHA patients were more likely to use tobacco, or have a history of heart failure or chronic obstructive pulmonary disease. These observed differences in comorbidities are consistent with other studies in various infectious diseases, which demonstrate that African-Americans often present to hospitals with more comorbidities compared to Caucasians ,7,19,55.Past studies have demonstrated that African-American patients are less likely to receive timely antibiotic therapy and diagnostic bronchoscopy ,3,13. WeOur study has limitations that are inherent to all retrospective cohort studies. The results attributed to one explanatory variable could be the result of some unmeasured variable. Any relationships uncovered cannot be definitively considered causative.The VHA patients in this study are not necessarily representative of all U.S. patients or even all U.S. veterans . The VHAAdditionally, this study was potentially limited by the fact that the investigators were unable to fully exclude VHA patients with healthcare-associated pneumonia (HCAP); however, the stringent exclusion criteria should have minimized these patients. For instance, VHA patients admitted to the hospital or a nursing home within the 90 days prior to the index admission for CAP were excluded. Prior studies have demonstrated that these two exclusions account for a large proportion of HCAP patients ,59. As wElderly African-American CAP patients experienced a survival advantage  in the ICU compared to Caucasians and shorter hospital LOS in both medical wards and ICUs, after adjusting for numerous baseline differences in patient characteristics. There were no racial differences in receipt of guideline-concordant antibiotic therapies.The authors declare that they have no competing interests.CRF participated in the design, data analysis and interpretation, performed statistical analyses, and was the primary person responsible for drafting the manuscript. EMM conceived and designed the primary study; and participated in the design, data analysis and interpretation, performed statistical analyses, and revised the manuscript for important intellectual content. MJP participated in the design, data acquisition, and manuscript revisions. RTA participated in data analysis and interpretation and was the secondary person responsible for drafting the manuscript. MJP and LAC participated in the design, data acquisition, and manuscript revisions. MIR, AA, BN, and MJF participated in the design and manuscript revisions. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/10/143/prepub


A new family of natural products has been described in which cysteine, serine and threonine from ribosomally-produced peptides are converted to thiazoles, oxazoles and methyloxazoles, respectively. These metabolites and their biosynthetic gene clusters are now referred to as thiazole/oxazole-modified microcins (TOMM). As exemplified by microcin B17 and streptolysin S, TOMM precursors contain an N-terminal leader sequence and C-terminal core peptide. The leader sequence contains binding sites for the posttranslational modifying enzymes which subsequently act upon the core peptide. TOMM peptides are small and highly variable, frequently missed by gene-finders and occasionally situated far from the thiazole/oxazole forming genes. Thus, locating a substrate for a particular TOMM pathway can be a challenging endeavor.itrogen-fixing proteins, can serve as natural product precursors (N11P), but not always of the TOMM variety. Indeed, a number of cyanobacterial genomes show extensive N11P paralogous expansion, such as Nostoc, Prochlorococcus and Cyanothece, which replace the TOMM cluster with lanthionine biosynthetic machinery.Examination of candidate TOMM precursors has revealed a subclass with an uncharacteristically long leader sequence closely related to the enzyme nitrile hydratase. Members of this nitrile hydratase leader peptide (NHLP) family lack the metal-binding residues required for catalysis. Instead, NHLP sequences display the classic Gly-Gly cleavage motif and have C-terminal regions rich in heterocyclizable residues. The NHLP family exhibits a correlated species distribution and local clustering with an ABC transport system. This study also provides evidence that a separate family, annotated as Nif11 nThis study has united numerous TOMM gene clusters with their cognate substrates. These results suggest that two large protein families, the nitrile hydratases and Nif11, have been retailored for secondary metabolism. Precursors for TOMMs and lanthionine-containing peptides derived from larger proteins to which other functions are attributed, may be widespread. The functions of these natural products have yet to be elucidated, but it is probable that some will display valuable industrial or medical activities. Bacteriocins are polypeptide-based natural products of ribosomal origin, usually functioning as antibiotics toxic to rival strains or species of bacteria . PeptideA subset of microcins has been recently described in which the amino acid side chains of cysteine, serine and threonine from a ribosomally produced precursor undergo heterocyclization to generate a product with thiazole or (methyl)oxazole moieties. These include trichamide , the patIn a simplified view, the purpose of the TOMM biosynthetic machinery is to recognize substrate and install structural constraints that restrict peptide bond rotation, thus endowing the modified peptide with a rigidified tertiary structure. By restricting conformational flexibility at the correct locations, the altered steric and electronic properties of the molecule, in conjunction with the physiochemical properties of the adjacent amino acids, lead to a specific biological activity. This type of rationale could also be extended to another family of post-translationally modified peptides, the lantibiotics, with the only major differences being the chemical composition (lanthionine containing) and biosynthetic installation of the structural constraints Figure  15,16].,16.15,16Again, similar to the lanthionine-containing peptides (lantipeptides), TOMM precursor peptides are bipartite: they contain an N-terminal leader sequence and a C-terminal 'core' peptide. The leader sequence has been shown in several cases to be critical to substrate recognition by the modifying enzymes, while the core peptide serves as a foundation upon which the active molecule is built ,17-19. ORecent TOMM precursor identification by several groups ,24,26-29Using a combination of informatics tools against a large number of sequenced genomes, we discovered several protein families that appear to represent an entirely new class of post-translationally modified peptide. The precursors have uncharacteristically long leader sequences and large paralogous family counts per genome. Analysis of the local genomic region predicts that these precursors will have variable chemical fates, including thiazole/oxazole and lanthionine formation. These families, surprisingly, include one set of sequences with strong similarity to the alpha subunit of the enzyme nitrile hydratase (NHase) ,32 whileSyntrophomonas wolfei subsp. wolfei str. Goettingen, Pelotomaculum thermopropionicum SI), proteobacteria , NHLP precursors are found adjacent to a cyclodehydratase-docking scaffold fusion protein (TIGR03882), a required component of TOMM biosynthesis [Microscilla marina ATCC 23134 (chlorobi group) and Methylobacterium sp. 4-46 (proteobacteria) represent two examples of this genetic organization. Akin to the NHLP system, previous informatics work has shown that the Bacillus anthracis and B. cereus TOMM precursors are encoded more than one megabase away from the modification cluster [M. marina and Methylobacterium, recognizing an orthologous cluster in B. licheniformis, in which all components were clustered, accelerated the identification of the precursors in B. anthracis and other members of the B. cereus group.One family of the newly discovered precursor peptides is described by TIGRFAMs model TIGR03793 Table  and desihicus SB ) and the cluster . As withAn unmistakable feature of the NHLP family is its close sequence similarity to the alpha subunit of NHase, which is described by TIGR01323 (Table indel) relative to each other. The median E-value for these HMM genome search results is 1e-7, despite the short length (82 amino acids) of the TIGR03793 model. As these sequences are neither repetitive nor low in complexity in the regions covered by the HMM, the consistently low E-values for alignment between the two families predicts substantial sequence similarity between NHases and NHLPs. Furthermore, over three-quarters of the hits from TIGR03793 (fragment model) to NHases found two match segments, straddling a large indel region present in the alpha subunit of NHase, but not in TIGR03793 family sequences. The above described similarity and indel are clearly evident in the alignment [For over 90% of genomes containing a member of the NHase family (TIGR01323), that member occurs as the highest scoring sequence in the genome to a search using the fragment version  Hidden Markov Model (HMM) of TIGR03793. Fragment model searches are preferred when match regions do not span the full length of the seed alignment or the target sequence. This is certainly the case when comparing sequences that have either a large insertion or deletion , Anabaena variabilis ATCC 29413 (cyanobacteria), M. marina ATCC 23134 (chlorobi group), C. luteolum DSM 273* (chlorobi group), Victivallis vadensis ATCC str. BAA-548 (chlamydia group), and P. thermopropionicum SI*  , in whicynthesis ,42  and an unfused docking scaffold protein (AZL_a09740). While these two genes are plasmid-borne, an additional copy of the unfused docking scaffold protein (AZL_022710) can be found on the chromosome, along with a cyclodehydratase encoded 3 ORFs away (AZL_022680). Therefore, it is not possible to determine the chemical fate of the Azospirillum peptide precursors at this time , if this organism is to produce an extensive array of secondary metabolites, it must do so in a highly genome-efficient manner. This is in contrast to the much larger genome sizes of organisms renowned for secondary metabolism, such as Streptomyces coelicolor  [Nostoc punctiforme PCC 73102  . Such exquences) . It appeBurkholderia order, including Delftia acidovorans SPH-1, two subspecies of Acidovorax avenae and multiple members within the genus Burkholderia: B. cenocepacia, B. ambifaria, B. pseudomallei, B. thailandensis, B. oklahomensis and B. mallei [Burkholderia genus, suggesting these may form a two-peptide product, which are well-known  pair contains either Cys-Cys, or a single Cys, as the C-terminus . Members of this family were discovered as the top hits in their respective genomes to TIGR03793, suggesting a sequence relationship to the NHLP substrates described above. An alignment of NHase, NHLP and NHLP-Burk reveals a moderate level of sequence similarity. Relative to NHLP and NHase, NHLP-Burk contains an insert of about 15 amino acids N-terminal to a Pro-Xaa-Xaa-Pro motif conserved amongst the three families. A major difference between NHLP and NHLP-Burk lies in the leader peptide cleavage region  in C. luteolum, Synechococcus sp. WH 7803, C. phaeobacteroides, Desulfitobacterium hafniense and Eggerthella lenta DSM 2243, among others. Additional N11P members occur adjacent to LanM-like lanthionine-forming enzymes in numerous species of cyanobacteria, including N. punctiforme PCC 73102, Nostoc sp. PCC 7120, Prochloroccocus marinus sp. MIT9313, and Cyanothece sp. PCC 7425  when querying all NHases against any N11P family member. Nevertheless, N11P does exhibit regions of local sequence similarity to NHLP  identified by PPP have a close homolog in species with NHLP-Burk family members. This implies that the export mechanism, if any, must differ. The occurrence of NHLP-Burk members in pairs, fused in some genomes, suggests a two-chain structure. If exported, these metabolites will likely require a different transport mechanism. The NHLP and NHLP-Burk families do exhibit extensive sequence similarity  long leader peptide regions, (ii) similarity to proteins and enzymes assigned to other functions and (iii) locations distant to the genomic regions used to encode their modification and export genes. Microcins recognized by TIGR01847 have leader peptides predicted to end at an average length of 24 amino acids. However, the corresponding Gly-Gly motifs in the new discovered families presented here end at an average position of 83 and 70 for NHLP and N11P, respectively. NHLPs demonstrate significant sequence similarity to the alpha subunit of NHase, suggesting strongly that they share a common ancestor. NHase is an enzyme with a function unrelated to microcin production and, thus, a broader implication of our findings is that a small protein cannot be automatically excluded from classification as a precursor to a natural product, even if it is homologous to a protein with a known function.The proposed precursor families described in this report dramatically expand the current repertoire of ribosomally produced natural products. This revision includes hundreds of peptides that exhibit , using composition-based statistics and an E-value of 0.5. This search strategy provides a working definition for the set of lichenicidin-related bacteriocins homologous in the leader peptide, rather than the core peptide. All non-identical sequences scoring above 0 bits to the respective motif HMMs were aligned to the HMM, resulting in gapless alignments. For each of these, a final HMM was built in order to emit a consensus sequence.In order to model regions of local sequence similarity between different protein families, multiple alignments were first generated, trimmed and used to train HMMs for searches to gather additional candidate sequences through an iterated, manual process. HMM construction was performed with the Logical Depth 1.5.4 package software-accelerated emulation of HMMER 2.3. The resulting motif models, of lengths 17 and 13, were searched against the individual families TIGR01323, TIGR03793, TIGR03795, TIGR03798 and the set of 20 proteins that resulted from PSI-BLAST . The PSITrichodesmium erythraeum [Prochloron didemni [Previous work has identified many cyclodehydratase, dehydrogenase and docking scaffold genes ,24,27. I didemni . The cor didemni ,68. Succet al. [An examination of the genes in the vicinity of orphan cyclodehydratase-docking scaffold fusion proteins revealed no examples of short peptides qualitatively similar to those previously presented by Lee and Mitchell et al. . Previou10 of the score is maximized, corresponding to an optimum for the working size of a candidate protein family. Each phylogenetic profile was used to query all genomes assigned as YES in the profile. Top-scoring proteins were identified for further analysis. In essence, PPP makes it possible to detect a protein family that matches a query profile, even if that family has never previously been defined.Selected TIGRFAM models were searched against a collection of 1450 complete or nearly complete bacterial and archaeal genomes. All genomes with at least one protein scoring above the trusted cutoff of the model were assigned the value 1 ('YES') in the phylogenetic profile built to represent that model, while all other genomes were assigned the value 0 ('NO'). By PPP , the phyindel: insertion-deletion; N11P: Nif11-derived peptide; NHase: nitrile hydratase; NHLP: nitrile hydratase leader peptide; NHLP-Burk: Burkholderia type TOMM substrate family; Nif11: nitrogen fixation protein of unknown function; ORF: open reading frame; PPP: partial phylogenetic profiling; TIGR: The Institute for Genomic Research; TOMM: thiazole/oxazole-modified microcin.ABC: ATP-binding cassette; ATP: adenosine triphosphate; HMM: hidden Markov model; DHH conceived the study and constructed the TIGRFAM models. DAM participated in the model validation and expanded the scope of the study. MKB developed improved software tools for detecting protein family relationships. DHH and DAM analysed and interpreted the data. DHH and DAM wrote the paper. All authors read and approved the final manuscript.


The aim of this study was to evaluate the relationship between intakes of subgroups of energy-providing carbohydrate, and markers of cardiometabolic risk factors in high BMI African American (AA) children.th percentile. Fasting hematological and biochemical values for selected markers of cardiometabolic risk factors were related to intakes of carbohydrates and sugars.A cross sectional analysis was performed on data from a sample of 9-11 year old children (n = 95) with BMI greater than the 85After adjusting for gender, pubertal stage and waist circumference, multivariate regression analysis showed that higher intakes of carbohydrate (with fat and protein held constant) were associated with higher plasma concentrations of triglycerides (TG), VLDL-C, IDL-C, and worse insulin resistance . After dividing carbohydrate into non-sugar versus sugar fractions, sugars were significantly related to higher TG, VLDL-C, IDL-C, lower adipocyte fatty acid insulin sensitivity (ISI-FFA), and was closely associated with increased HOMA-IR. Similar trends were observed for sugars classified as added sugars, and for sugars included in beverages. Further dividing sugar according to the food group from which it was consumed showed that consuming more sugar from the candy/soda food group was highly significantly associated with increased TG, VLDL-C, IDL-C and closely associated with increased HOMA-IR. Sugars consumed in all fruit-containing foods were significantly associated with lower ISI-FFA. Sugars consumed as fruit beverages was significantly associated with VLDL-C, IDL-C and ISI-FFA whereas sugars consumed as fresh, dried and preserved fruits did not show significant associations with these markers.Sugars consumed from in all dairy foods were significantly associated with higher TG, VLDL-C and IDL-C, and with significantly lower HDL-C and ISI-FFA. These effects were associated with sugars consumed in sweetened dairy products, but not with sugars consumed in unsweetened dairy products. This analysis suggests that increases in carbohydrate energy, especially in the form of sugar, may be detrimental to cardiometabolic health in high BMI children. Childhood obesity has reached epidemic levels in developed countries . Obesitytype of carbohydrate, rather than total carbohydrate, has begun to clarify the role of carbohydrate subgroups in the obesity epidemic among adults; however studies of this nature have not yet been performed in children. Numerous short-term studies have shown that diets high in carbohydrates, particularly sugars, and even more particularly sucrose and fructose, increase serum TG concentrations and decrease serum HDL cholesterol; and may therefore increase the risk of CVD [The African American (AA) population has been shown to be at higher risk for both obesity and the metabolic syndrome than are Caucasians . As the k of CVD ,7. It isTaking Action Together study, an inner-city YMCA-based intervention trial that aimed to reduce risk factors for type 2 diabetes. A total of 128 African-American (AA) children  were evaluated in this cross-sectional analysis, and a full set of data was available for 99 children.Subjects included in this analysis represented a cross-sectional convenience sample of 9-11 year-old African American children enrolled in the th percentile, fasting glucose <120 mg/dl, were free from any known metabolic diseases, and were not taking medications known to affect the study outcomes. Parental informed consent was obtained for all subjects, and all protocols were approved by institutional review boards at the University of California, Berkeley and San Francisco. Anthropometric characteristics were measured using procedures previously described [All participants had BMIs above the 85Subjects reported to Children's Hospital and Research Center, Oakland, CA after a 12 hr overnight fast when their blood was drawn. Plasma lipids (lipoprotein cholesterol and triglycerides) were measured by a comprehensive lipoprotein analysis (VAP-cholesterol method) performed by a commercial lab (Laboratory Corporation of America) as previously described . PubertaThree-day food diaries were used to assess intakes of nutrients . MacronuStatistical procedures were performed using SPSS for Windows version 16.0 . Statistical significance was defined to be p ≤ 0.05. Results with 0.05 < p < 0.10 are noted to show close associations. Differences in anthropometric indices and lipoprotein profiles in boys versus girls were performed using independent t-tests. Dixon's test for outliers was used to identify unusual values. When identified, all data for that participant were excluded from further analyses. Using Dixon's test, data for 4 of the 99 children were excluded; thus, data are presented for a total of 95 children . Data were not significantly skewed for any of the variables of interest. Multiple linear regression analyses were used to assess the relationship of intake from different carbohydrate subgroups to TG, VLDL-C, IDL-C, HDL-C, HOMA-IR and ISI-FFA, after adjusting for gender, pubertal stage, waist circumference of the participating child, protein intake and fat intake. These markers were selected for further study since they were found previously to be significantly associated with total carbohydrate intake .In comparison to boys, girls in this cohort had significantly higher values for measures of body fatness and were less insulin sensitive Table . CarbohyUsing data from a similar cohort, we previously reported  that higIn further analysis, when sugar was subdivided according to the dietary food group from which it was consumed, beverage sugar was significantly related to elevated TG, VLDL-C and IDL-C, whereas non-beverage sugar intakes were not Table  Model 4.When the total fruit group was further divided, consumption of sugars from fruit-containing beverages was significantly associated with increased VLDL-C and IDL-C, and with decreased ISI-FFA. By contrast, sugar intakes from fresh, dried and preserved fruits did not show any significant associations with these markers Table Model 6.When the total dairy group was sub-divided, sugar intakes from sweetened dairy products showed significant association with increased TG and with decreased HDL-C and ISI-FFA, whereas sugar intake from unsweetened dairy foods did not Table  Model 7.Our main outcomes highlight the association of intakes of carbohydrate from different subgroups with key markers of cardiometabolic risk in high BMI AA children. Importantly, the variance in TG, VLDL-C, IDL-C and ISI-FFA contributed by total carbohydrate appeared to be mainly from sugars, suggesting that sugar fractions contributed to the undesirable effects of increasing total carbohydrate intake. Thus, increases in total carbohydrate intake, due to increased sugar intake, were associated with undesirable increases in several classes of plasma lipids. Additionally, sugar intake was closely associated with decreases in HOMA-IR. Table  Model 2.New evidence on the relationship between intake of sugars and cardiovascular health has emerged since the last American Heart Association (AHA) scientific statement was published in 2002 . In 2006Our results are also consistent with AHA's recent statement that high intake of added sugars in the setting of a worldwide pandemic of obesity and cardiovascular disease have heightened concerns about the adverse effects of excessive consumption of added sugars , suggestResults from the Framingham Heart Study suggest that soft drink consumption is associated with a higher prevalence and incidence of multiple metabolic risk factors in middle-aged adults . Many clIn our study, sugar intakes from the dairy, and to a lesser extent, the fruit food groups were associated with increased cardiometabolic risk factors in these high-BMI children. When these food groups were further divided, this risk was associated with sugars in fruit-containing beverages and with sweetened dairy foods Table . Thus, oth percentile when matched for age and gender. These limitations preclude comparisons among children of different races, ages and socioeconomic backgrounds, and comparisons with lower BMI children. The limitations inherent in collecting dietary data, regardless of population, are also recognized. This is a cross-sectional analysis of data, precluding a cause and effect relationship. Future longitudinal studies, with measurements at several time-points, would be needed to evaluate a causal relationship. Also, replications in longitudinal studies with larger sample sizes, and in multiracial cohorts are warranted.Limitations of this study include restriction to low-income, inner-city, African American children and exclusion of children with BMI's less than the 85Based on our analysis, we conclude that increases in carbohydrate energy in the form of sugars were associated with undesirable increases in several classes of plasma lipids and with decreases in both hepatic glucose and adipocyte fatty acid insulin sensitivity. Higher intakes of sugars from the candy/soda food group, from fruit-containing beverages, and from sweetened dairy foods were associated with increases in several cardiometabolic risk factors. This analysis suggests that increases in many types and sources of sugar may be detrimental to cardiometabolic health in high BMI children.AA: African American; BMI: Body mass index; CVD: Cardio vascular disease; HOMA-IR: Homeostasis model assessment of insulin resistance; IDL-C: Intermediate density lipoprotein cholesterol; ISI-FFA: Fatty acid insulin sensitivity; NEFA: Non-esterified fatty acids; TG: Triglyceride; VLDL-C: Very low density lipoprotein cholesterol; HDL-C: High density lipoprotein cholesterol; WC: Waist circumference.The authors declare that they have no competing interests.Contributor's list: SS contributed in statistical analysis, preparation of the manuscript and submission. LSR participated in the development of the protocol, analytical framework for the study and patient screening. RHL provided expertise as a pediatric endocrinologist and child health specialist. SEF was the principal investigator of the study. She supervised the design and execution of the study and manuscript. All authors have read and approved the final manuscript.