Crack use is prevalent among street drug users in Brazilian cities, yet despite recent help system reforms and investments, treatment utilization is low. Other studies have identified a variety of – often inconsistent – factors associated with treatment status among crack or other drug users. This study compared socio-economic, drug use, health and service use characteristics between samples of young adult crack users in- and out-of-treatment in Rio de Janeiro, Brazil.Street-involved crack users (n = 81) were recruited by community-based methods, and privately assessed by way of an anonymous interviewer-administered questionnaire as well as biological methods, following informed consent. In-treatment users (n = 30) were recruited from a public service in-patient treatment facility and assessed based on the same protocol. Key indicators of interest were statistically cross-compared. Not-in-treatment users were less likely to: be white, educated, stably housed, to be involved in drug dealing, to report lifetime marijuana and current alcohol use, to report low mental health status and general health or addiction/mental health care; they were more likely to: be involved in begging and utilize social services, compared to the in-treatment sample .In-treatment and not-in-treatment crack users differed on several key characteristics. Overall, in-treatment users appeared to be more socio-economically integrated and connected to the health system, yet not acutely needier in terms of health or drug problems. Given overall low treatment utilization but high need, efforts are required to facilitate improved treatment access and use for marginalized crack users in Brazil. Crack use is prevalent and entails extensive health and social problems in Brazil. There may be up to 1 million crack users in Brazil ; most arWhile care (including treatment) services for substance use problems have been expanded in Brazil in recent years, their utilization – specifically by crack users – appears rather limited ,9. HowevStudies examining differences between crack/cocaine as well as opioid/heroin user populations in and out-of-treatment have predominantly found the latter sub-groups to be more commonly characterized by social marginalization  and limited social supports; lengthier, more complex and/or problematic drug use histories or patterns; more intensive  health problems; and higher crime or criminal justice involvement; however, findings are heterogeneous and inconsistent -13. WhilThe purpose of this study was to compare key socio-economic, drug use and health characteristics of two samples of young adult in- and out-of-treatment crack users in RdJ, Brazil; given the acute extent of crack use in Brazil yet low treatment utilization, this comparative analysis aids to identify potential factors differentiating treatment status, and to inform interventions towards improving treatment access and/or utilization.The study conducted a comparative, cross-sectional assessment of two community-recruited convenience samples of young out-of-treatment and in-treatment crack users. Eligibility criteria for both samples were: 1) Crack use on 3+ days/week in last 3 months ; 2) 18–24 years of age; 3) ability to consent to study protocol, facilitating basic comparability of retrospective data on these grounds. Street-involved users were recruited by way of community-based outreach methods in a poor neighborhood (Jazarezinho) of RdJ known for street drug use. Local community contacts distributed key study information among users, and prospective participants underwent a brief in-person eligibility assessment. The treatment sample was recruited from among in-patients in a public drug treatment clinic  in RdJ. Similarly, potentially eligible patient participants recruited in the clinic were informed about the study by clinic staff in the clinic setting, then contacted study staff if they expressed interest and were subsequently screened for eligibility. The locale from which street users were recruited and the clinic are situated within different parts of RdJ. The clinic is the only general and public referral clinic in RdJ for in-patient drug abuse treatment mainly catering to poor and marginalized users. While the clinic’s crack user patients are referred from different neighborhoods across the city, including the one where street users were recruited, the general profile of the clinic’s patient population allows to reasonably assume the inclusion of overall similar and comparable study populations.Upon eligibility confirmation and provision of informed consent, participants were assessed via an interviewer-administered, anonymous questionnaire comprising social, drug use, and health characteristics; furthermore, blood samples for anonymous BBV testing were collected. Assessments took about 45–60 minutes, and were conducted by field research assistants trained and experienced in field research with marginalized populations. Participants received a public transportation pass for their time and efforts. The study protocol was approved by the Ethical Review Committee, Institute of Psychiatry, Federal University of Rio de Janeiro, as well as the Brazilian National Ethics Committee , and statistically compared these indicators between the two groups by way of chi-square and t-tests, respectively, with significance levels set at p < .05.The mean age in both samples was 21 years . Respective majorities in both samples were male; single or separated; unemployed and had been arrested; a minority in both groups engaged in sex work. The street sample: included fewer white people; had lower education; was more likely to be unstably housed and to be involved in begging – but less likely to be involved in drug dealing – than the treatment sample . None except one participant had an IDU history. A larger proportion of non-treatment participants shared crack pipe implements. Approximately half or more participants in both samples reported lifetime and current use of alcohol, tobacco, marijuana and cocaine. Larger proportions in the treatment sample reported current  alcohol use  positive; a small proportion in the street group was HIV-positive. Minorities in each group reported general health, specialized addiction/mental health, and social service utilization; social service utilization was higher among non-treatment users; general and specialized health service utilization was higher among in-treatment users Table .We found notable differences in key characteristics between the non-treatment and treatment samples of crack users, several of which mirror findings from other studies. For example, lower rates of visible minorities  and higher education status have been found among in-treatment samples elsewhere, suggesting that socio-economic status may play a role in treatment seeking or access; this could relate to the access of relevant information, the management of bureaucratic systems or simply monetary resources ,12,13. HWhile the two groups showed similar crack and IDU patterns, the non-treatment group featured a higher prevalence of crack implement sharing – a risk behavior that possibly facilitates BBV  transmission . This miUsers’ physical and mental health status are associated with treatment access and status ,21. Our While extensive treatment needs for crack use appear to exist, treatment service access and utilization have been found to be distinctly low in Brazil e.g., ,9). In-t. In-t9])Our study’s limitations include that it relied on data from relatively small, non-representative convenience samples, including possible selection biases, e.g. as related to sampling, which therefore cannot be generalized. Data collection utilized self-report methods (except for BBV testing) which cannot be objectively validated and social desirability dynamics may have influenced responses however, study and assessment design  and experiences from other studies have shown similar data to be valid . SamplesIn sum, we found important differences between in- and out-of-treatment crack users in the specific context of RdJ, Brazil; these suggest a need for improved treatment service availability, access and delivery – with particular attention to the most marginalized and high-risk users – in the target population.BBV: Blood borne virus; HBV: Hepatitis B virus; HCV: Hepatitis C virus; HIV: Human immunodeficiency virus; IDU: Injection drug use; Rdj: Rio de Janeiro; SPSS: Statistical package for the social sciences.The authors declare that they have no competing interests.MC, FB, BF designed the study protocol and data analysis plan. NB managed the study data and conducted the data analysis. CB and JG conducted relevant literature searches and reviews, and integrated them with study data. BF, MC and FB jointly led the manuscript writing. All authors contributed to data interpretation and manuscript revisions, and have read and approved the final manuscript.


The role of Lipoprotein (a) cholesterol {Lp(a)-C}as an additional and/or independent risk factor for cardiovascular disease (CVD) is not clear. We evaluated the associations between Lp(a)-C and other CVD risk factors including plasma lipoprotein concentrations and body fatness in overweight and obese African American children.A cross-sectional analysis was carried out using data from a sample of 121 African American children aged 9-11 years with Body Mass Index (BMI)'s greater than the 85th percentile. Body height, weight and waist circumference (WC) were measured. Fasting plasma concentrations of Lp(a)-C, Total cholesterol (TC), High density lipoprotein cholesterol (HDL-C), Very low density lipoprotein cholesterol (VLDL-C), Intermediate density lipoprotein cholesterol (IDL-C), Low density lipoprotein cholesterol (LDL-C), and Triacylglycerides (TAG) were analyzed using the vertical auto profile (VAP) cholesterol method.After adjusting for child age, gender, and pubertal status, Lp(a)-C was positively associated with both HDL-C and TC, and negatively associated with VLDL-C and TAG. Including BMIz and WC as additional covariates did not alter the direction of the relationships between Lp(a)-C and the other lipoproteins. Finally, after adjusting for the other plasma lipoproteins, Lp(a)-C remained strongly associated with HDL-C, whereas the associations of Lp(a)-C with the other lipoproteins were not significant when HDL-C was simultaneously included in the regression models.Lp(a)-C was positively associated with HDL-C and this association is not influenced by other lipoprotein subclasses or by the degree of obesity. We conclude that Lp(a) cholesterol is not an independent risk factor for CVD in African American children. Lipoprotein (a) particles, Lp(a), were first described by Berg in 1963 . Since bDespite these reports, there have been conflicting results from prospective studies that evaluated Lp(a) as an independent risk factor for cardiovascular disease (CVD). Although the majority of studies, performed in primarily Caucasian populations, have found Lp(a) concentrations to be an independent risk factor for CVD, studies that have evaluated these relationships in African Americans have been less consistent. For example, in the biracial cohort ARIC (Atherosclerosis Risk in Communities) study of 15, 800 individuals, Lp(a) lipoprotein levels were reported to be positively associated with CVD as well as with preclinical atherosclerosis in both Black and White adults ,5. In a In the U.S., plasma lipoprotein concentrations and blood pressures have been used as predictors for CVD screening, with cutoffs based on American Heart Association guidelines . IndividLp(a) has been found also to be associated with intermediate markers of CVD risk including obesity and other lipoproteins in African Americans. Lp(a) was reported to be positively associated with TC in African American women  and AfriTo the best of our knowledge, associations between Lp(a) and intermediate markers of CVD risk have not previously been reported for young African American children. Because of an epidemic increase in childhood obesity and a subsequent increase in risk for CVD, it has been recommended that children and adolescents should be screened for lipid profiles even at a young age ,19. TherA complete set of data for this cross-sectional analysis were available for a convenience sample of 121 (56 boys and 65 girls) African-American children who were part of a community-based lifestyle modification program that aimed to reduce the risk for type 2 diabetes mellitus as described more fully elsewhere [http://www.cdc.gov/nccdphp/dnpa/growthcharts/resources/sas.htm. Using a plastic, non-elastic measuring tape, waist circumference (WC) was measured just above the iliac crest with the child in the standing position and hip circumference was measured at the widest point above the greater trocanthers. Measurements were taken twice and if agreement between repeats exceeded 0.4 cm, a third measurement was taken and the mean calculated using the closest two values.Body weight and height were measured to the nearest 0.1 kg and 0.1 cm using a digital electronic scale , and a portable stadiometer, respectively. Body mass index (BMI), BMI percentiles and BMI z-scores were generated using an age and gender specific CDC calculator program After a 12-h overnight fast, participants reported to the Children's Hospital and Research Center Oakland where their blood was drawn. Plasma lipids were measured by a comprehensive lipoprotein analysis performed by a commercial lab (LabCorp). Using the vertical auto profile (VAP) cholesterol method, a modified density gradient centrifugation technique, concentrations of the following components were determined on a single sample: TC, HDL-C, VLDL-C, IDL-C, LDL-C and TAG ,23.All participants were asked to report, after an overnight fast of at least 12 h, at the Children's Hospital and Research Center in Oakland, CA for blood sample collection. Pubertal development was assessed by measurements of serum luteinizing hormones (LH) in boys, and estradiol and LH in girls. Children were classified into stages 1 through 5 using literature-derived values and with cutoffs previously reported .p < 0.05 were defined to be statistically significant.Initially, data for 124 participants were included in the analysis. Values for several of the plasma lipoproteins were significantly skewed, and tests for non-normality remained significant even following log transformation. Using Dixon's test for outliers, data for three children were excluded to avoid unintended bias of associations in this analysis. Thus, data for 121 participants were included in the final analysis. Gender differences for anthropometric and lipoprotein characteristics were assessed using independent two-tailed t-tests following Levene's test for equality of variances. Pearson's correlations were used to determine bivariate associations. To evaluate the primary study objectives, hierarchical multiple linear regression analyses were used, with Lp(a)-C as the dependent variable. Other plasma lipoproteins were included as independent variables and models were adjusted as indicated for child age, gender, pubertal stage and body fatness. Socioeconomic status of the family was evaluated as a potential covariate but was not included in the final models since it was found to have r < 0.20 and to not be significantly related to dependent and independent variables. Statistical procedures were performed using SPSS for Windows version 18.0 PASW . Results with Plasma lipoprotein concentrations were considered to be in the normal range for many, but not all, of the children in this sample. Using reference interval cutoffs, less than 5% of the children had values outside the normal range for IDL-C, VLDL-C and TAG; 10-20% fell outside the normal range for TC, HDL-C and LDL-C; and nearly 25% fell outside the normal range for Lp(a)-C Table .Body fatness, assessed using either BMIz scores and WC, was significantly and negatively correlated with HDL-C and Lp(a)-C, and significantly but positively associated with TAG Table . TC was Using multiple regression analysis, and following adjustments for child age, gender and pubertal stage, Lp(a)-C remained significantly and positively associated with TC and HDL-C, and remained significantly and negatively associated with VLDL-C and TAG Table . Since oSince Lp(a)-C appeared to be consistently and more strongly related to HDL-C than to the other lipoprotein subclasses Table , a finalFinally, we evaluated the frequency with which children in our cohort were simultaneously "at risk" of CVD using the cutoffs provided in Table To our knowledge this is one of the first studies to report a strong positive association between Lp(a)-C and HDL-C in overweight and obese African American children. Positive correlations between Lp(a)-C and HDL-C concentrations have been reported previously in premenopausal African American women , and ourThe strong association of Lp(a)-C with HDL-C observed in our study suggests that increasing Lp(a)-C concentrations may not be reliably used as a marker of CAD risk in this population. Instead, our results agree with those of others  who obseLp(a) is known to consist of an LDL-like particle covalently linked via an apo-B100 molecule to apolipoprotein (a), and these particles are variable in its size and cholesterol content. Lp(a) and LDL have been reported to be positively correlated in African American females ,25 and iIn our cohort of overweight African American children, Lp(a) was significantly and negatively associated with TAG concentrations prior to an following adjustment for obesity. Others have reported also a negative association between LP(a) and TAG in American Americans aged 12-75 , AfricanOthers have previously reported that African American children and adolescents have higher levels of Lp(a) and HDL than their non-Hispanic white peers ,25,26. IIn our population, Lp(a)-C concentrations were negatively related to body fatness assessed using either BMIz or WC, and children between the 85th and 95th BMI percentile had significantly higher Lp(a)-C concentrations than children with BMIs above the 95th percentile. Previously, Lp(a)-C concentrations were reported to be negatively correlated with body weight and waist-to-hip ratio in African American women , althougThis pilot analysis includes restriction to low-income, inner-city, African American children and exclusion of children with BMI's less than the 85th percentile when matched for age and gender. These limitations preclude extrapolation to the wider population of children of different races, ages and socioeconomic backgrounds, and comparisons with lower BMI children. This is a cross-sectional analysis of data, precluding a cause and effect relationship. Lipoprotein subclasses were analyzed using the VAP-cholesterol technique, precluding extrapolation to results based on other analytical techniques that quantify numbers or size of particles or associated protein.Lp(a)-C was positively associated with HDL-C in overweight and obese African American children and this association remained following adjustments for other lipoprotein subclasses and degree of obesity. Based on our study, we propose that Lp(a) is not associated with lipoprotein recognized as markers of increased risk of CVD in overweight and obese African American children. Further studies will be needed to explain the basis for the association of Lp(a)-C and HDL-C in these children.Lp(a): Lipoprotein (a); Lp(a)-C: Lp(a) cholesterol; LDL-C: Low density lipoprotein cholesterol; HDL-C: High density lipoprotein cholesterol; IDL-C: Intermediate density lipoprotein cholesterol; VLDL-C: Very low density lipoprotein cholesterol;TAG: Triglycerides; TC: Total cholesterol; BMI: Body mass index; BMIz: Body mass index z-score; WC: Waist circumference; CVD: Cardiovascular disease.The authors declare that they have no competing interests.Contributor's list: Dr.S contributed in statistical analysis, prepared the manuscript and submission. Ms. Merchant provided analytical support and expertise in interpretation of data. Prof.F was the principal investigator of the study. She supervised the design and execution of the study and manuscript. All authors read and approved the final manuscript.


The most part of our genome encodes for RNA transcripts are never translated into proteins. These include families of RNA molecules with a regulatory function, which can be arbitrarily subdivided in short (less than 200 nucleotides) and long non-coding RNAs (ncRNAs). MicroRNAs, which act post-transcriptionally to repress the function of target mRNAs, belong to the first group. Included in the second group are multi-exonic and polyadenylated long ncRNAs (lncRNAs), localized either in the nucleus, where they can associate with chromatin remodeling complexes to regulate transcription, or in the cytoplasm, acting as post-transcriptional regulators. Pluripotent stem cells, such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), represent useful systems for modeling normal development and human diseases, as well as promising tools for regenerative medicine. To fully explore their potential, however, a deep understanding of the molecular basis of stemness is crucial. In recent years, increasing evidence of the importance of regulation by ncRNAs in pluripotent cells is accumulating. In this review, we will discuss recent findings pointing to multiple roles played by regulatory ncRNAs in ESC and iPSCs, where they act in concert with signaling pathways, transcriptional regulatory circuitries and epigenetic factors to modulate the balance between pluripotency and differentiation. In mammals, this property is restricted to embryonic cells until the stage of blastocyst, in which a small number of cells that constitute the Inner Cell Mass (ICM) are still pluripotent. Such pluripotent cells exist very transiently during embryonic development. Upon gastrulation and during subsequent embryonic development, they lose pluripotency and progressively acquire a specialized character. Embryonic stem cells (ESCs) can be derived from the ICM of human and mouse blastocysts; are pluripotent and can self-renew ESC pluripotency is tightly regulated. Amongst several signaling pathways, the TGF-β pathway has been shown to play a central role . InteresIn vitro, they require Leukemia Inhibitory Factor (LIF) and BMP signaling for self-renewal and differentiate in presence of basic Fibroblast Growth Factor (bFGF). They express a typical set of markers and female cells have two active X chromosomes. Functionally, they are able to form chimeric embryos upon injection in a recipient blastocyst. Conversely, mouse Epiblast Stem Cells (EpiSCs) are in a primed state, which corresponds to the post-implantation epiblast. EpiSCs require bFGF and Nodal/Activin signaling for self-renew, present X chromosome inactivation in female cells, and fulfill some criteria for pluripotency, such as the ability to form teratomas, but cannot generate chimeric mice. There is some plasticity in pluripotent cells, as mESCs can be induced to differentiate into EpiSCs by culturing in presence of Activin and bFGF and EpiSCs can be converted to the ground state by transfection with Klf4 or Nanog and culturing in the presence of LIF and BMP [Pluripotent cells exist in two different states, defined as naïve and primed . Mouse E and BMP ,13. Huma and BMP .in vitro into all adult cell types. Moreover, they can be derived from human patients as patient-specific iPSCs (PS-iPSCs) that hold the same disease-causing genetic alteration [The seminal work by Shinya Yamanaka showed that pluripotency could be induced in mouse somatic cells by ectopic expression of a defined set of reprogramming factors (RFs) . Since tteration . The mecteration .The ability to differentiate into multiple tissues makes ESCs and iPSCs promising tools for regenerative medicine and cell-replacement therapy approaches . HoweverDespite lacking an open reading frame (ORF), long (>200 nucleotides) non-coding RNAs (lncRNAs) are transcribed by RNA polymerase II and have a structure that resemble protein-coding mRNAs. They are generally spliced from multi-exonic precursors, have a 5′cap and are polyadenylated ,36. SomeHere we will review recent findings that point to regulatory non-coding RNAs as important players in the maintenance of ESCs pluripotency and cell fate choices during differentiation. We will also discuss the roles played by ncRNAs during somatic cells reprogramming.Among all miRNAs expressed in embryonic stem cells, a single family of miRNAs with the AAGUGC seed sequence is the most highly expressed and has the most functional impact. Members of this family are organized in two major clusters . The conThe miR-302 family miRNAs are abundantly expressed in undifferentiated ESCs and decline upon differentiation –58. AccoThe specific expression of miR-302 family miRNAs is ensured by their regulation, at the transcriptional level, by the core ESC transcriptional regulatory circuitry ,9,62,63.Interestingly, the conversion from a naïve to a primed state in mESCs correlates with a switch between the miR-290-295 and the miR-302/367 clusters in terms of miRNA abundance  Figure . mESCs ein vitro and in vivo, as assessed by defects in lineage markers expression and teratoma formation. Moreover, inactivation of Dicer impairs the proliferation potential of mESCs [Inactivation of genes involved in the processing of miRNAs represents a useful approach to study the global function of miRNAs. Dicer −/− pre-gastrulation embryos showed lack of Oct4-positive epiblast cells and could not undergo gastrulation , indicatof mESCs . As Diceof mESCs . Moreoveof mESCs , indicatof mESCs . This seof mESCs . For theof mESCs . Specifiof mESCs ,73,75. Oof mESCs . Interesof mESCs . The actof mESCs .Similar to mESCs, also hESCs are characterized by a shortened cell cycle, accumulating in the S phase at the expense of the G1 phase . RegulatA genome-wide approach has been recently performed in hESCs to systematically identify bona fide miRNA targets . PAR-CLIDespite holding normal morphology and expression of pluripotency markers, both mESCs and hESCs depleted of miRNAs cannot properly differentiate ,71,79. TInterestingly, a regulatory loop comprising an unrelated miRNA, miR-125, and components of the BMP signaling has recently been described in mouse embryonic stem cells . Such remiR-302 is deeply integrated in the core transcriptional regulatory circuitry of ESCs. As mentioned above, the miR-302 host gene is under the control of Oct4, Nanog and Sox2, which ensure high miRNA levels in undifferentiated ESCs ,62,63. WEpigenetic silencing of the Oct4 locus is necessary to ensure proper differentiation upon exit from pluripotency. This is achieved by de novo DNA methylation by DNMT3 factors. It has been shown that miR-290-295 play a role in this process by inhibiting Rbl2, that in turn is an inhibitor of DNMT3 . In the Among other miRNAs with a role in ESC differentiation, the let-7 family plays a prominent role. This is a highly conserved family with orthologues in all metazoa. In mammals it comprises several let-7 species (let-7a to let-7i) and other miRNAs, such as miR-98 and miR-202 . MembersFor both let-7 and the miR-302 families feedback loops with Myc are in play. The promoter of the miR-290-295 cluster is directly bound and activated by c-Myc and N-Myc , establiESCs and iPSCs express a similar signature group of miRNAs, including the miR-302 family, with small differences between the two cell types –116. WheRecently, several miRNAs have been directly involved in the reprogramming process . KnockdoAs expected, let-7 has an opposite role during reprogramming. MEFs express high levels of let-7 and this anti-pluripotency miRNA family must be shut down during conversion into iPSCs. In the presence of let-7 inhibitors, reprogramming by the OSK cocktail was enhanced . When OSet al. have reported that miR-302 alone could convert skin cancer and hair follicle cells into iPSCs in the absence of other miRNAs or RFs [Given their multiple roles in ESCs, the increase of reprogramming efficiency by miRNAs in combination with reprogramming factors was not unexpected. More surprisingly, recent work has shown that cocktails of miRNAs, without canonical reprogramming factors, can be sufficient to reprogram both mouse and human somatic cells. The Morrisey lab demonstrated that the miR-302-367 cluster induced pluripotency with two orders of magnitude more efficiency than standard methods . Importas or RFs ,130. It The mechanisms underlying miRNA-mediated reprogramming have been only partially clarified. As mentioned before, introduction of miR-302 family members in DGCR8 −/− mESCs increases expression of endogenous c-Myc and N-Myc downstream genes . HoweverOther miRNAs that affect reprogramming are integrated in the p53 pathway. p53 activation elicits apoptosis and cell cycle arrest, representing a roadblock to reprogramming. Consistently, p53 inhibition promotes reprogramming . The antA mesenchymal-to-epithelial transition (MET) is one of the early events occurring during fibroblasts reprogramming to iPSCs ,134. SinmiR-302 overexpression sustains pluripotency markers in differentiating hESCs ,139. SimA number of differentially expressed lncRNAs were detected in undifferentiated mESCs and upon induction of differentiation by microarray analysis . They coMany mESC lincRNA genes are regulated by core transcription factors that bind directly their promoters . In partA biological role for mESC lincRNAs, initially postulated on the basis of their evolutionary conservation, has been recently experimentally demonstrated. RNAi against the lncRNA AK028326  resulted in morphological differentiation, reduced proliferation, decreased levels of Oct4 and other pluripotency markers and increase of trophoblast markers , suggestLarge-scale analysis suggests that the role of lncRNAs in pluripotent stem cells may be greater than expected. Loss-of-function by RNA interference was systematically achieved for 147 mESC lincRNAs . StrikinMechanistically, mESC lncRNAs may exert their function by modulating the activity of chromatin-modifying factors. Both “readers”, “writers” and “erasers” histone modifiers and the chromatin-associated DNA binding protein Yy1 have been found in association with mESCs lncRNAs ,144. ForA subset of human lincRNAs demonstrated expression at higher levels in iPSCs with respect to both somatic cells of origin and hESCs . Among tIn ESCs and iPSCs, pluripotency is the result of: (i) the interplay between multiple signaling pathways; (ii) a core transcriptional regulatory circuitry; (iii) epigenetic control of gene expression by specific chromatin modification patterns; (iv) the maintenance of a peculiar cell cycle. Recent work suggests that regulatory ncRNAs have a role in modulating all these biological pathways. Similar to the core set of transcription factors, ESCs are characterized by a core set of miRNAs, belonging to the miR-302 family. These miRNAs are hierarchically on top of multiple targets, which participate in the maintenance of self-renewal, and regulate exit from pluripotency and cell fate choice. Moreover, miR-302 family members are required for efficient reprogramming of somatic cells and, in combination with other miRNAs, are sufficient for iPSC generation in the absence of canonical reprogramming factors. A similar role has been shown for other miRNAs in different systems. For instance, neural-specific miRNAs can induce trans-differentiation of fibroblasts into induced neurons without other factors . Since mThe functions of lncRNAs in pluripotent cells are less characterized. So far, large datasets generated by RNA-seq projects have been followed only by a limited number of functional studies. Such are hampered by the fact that unlike protein-coding genes and miRNA the function of a lncRNA cannot be normally predicted by the analysis of its sequence . However


High-fat programming, by exposure to a high-saturated-fat diet in utero and/or during lactation, compromises beta-cell development and function in neonatal and weanling offspring. Therefore, high-fat programming effects were investigated on metabolism and islet architecture in young adult rats. Three-month-old male and female Wistar rat offspring were studied: HFG , HFP (high-fat diet maintenance from birth to 3 months), and HFGP . Control rats were maintained on a standard laboratory diet. Pancreata were double immunolabeled for insulin and glucagon to assess islet morphology and with Ki-67 to determine islet and acinar cell proliferation. HFP and HFGP males were heavier, hyperleptinemic, and hyperinsulinemic. Hyperglycemia presented in HFP males, HFP females, and HFGP males. HFGP males and HFP females were insulin resistant. HFP males displayed beta- and alpha-cell hyperplasia with alpha-cell hypertrophy evident in HFP females. Acinar cell proliferation rates were increased in HFP males. Postnatal high-fat programming induced the most diabetogenic phenotype with high-fat maintenance throughout fetal and postnatal life resulting in a severely obese phenotype. Fetal and postnatal nutrition shapes offspring health outcomes. The pancreatic beta-cells secrete insulin in response to fluctuations in circulating glucose concentrations to stimulate glucose uptake into peripheral organs. Beta-cell failure (insufficient insulin secretion) and insulin resistance (impaired action of insulin to stimulate glucose uptake) precede the development of type 2 diabetes. Both beta-cell failure and insulin resistance contribute to hyperglycemia, the hallmark of type 2 diabetes. Alpha-cells secrete glucagon which is antagonistic to insulin and help to regulate glucose homeostasis.Obesity is strongly associated with insulin resistance. Leptin, an anorexic hormone, secreted by adipose tissue, binds to the appropriate leptin receptor triggering intracellular signaling processes in the ventromedial hypothalamus (satiety center) which in turn provides a negative feedback system to inhibit caloric intake and prevent obesity , 2. In oEnvironmental factors during intrauterine development may alter the structure, physiology, and metabolism of the body . Developn = 3-4 per group) were fed either a low-fat-control (10% fat as energy) or high-fat (40% fat as energy) diet; offspring were therefore maintained on either a control or high-fat diet during defined periods of fetal and postnatal life. At 3 weeks of age, the offspring were weaned and directly fed either control or high-fat diets. Male offspring and female offspring were studied separately. The 3-month-old male and female offspring studied were: HFG , maintained on a high-fat diet throughout gestation  and a control diet throughout lactation and postnatal life); HFP , maintained on a control diet throughout gestation and a high-fat diet from birth to 3 months ; and HFGP , maintained on a high-fat diet throughout gestation, lactation, and postnatal life). The control rats were maintained on a standard laboratory diet throughout fetal and postnatal life. The standard laboratory (control) diet comprised 10% fat, 15% protein, and 75% carbohydrate  whereas the high-fat diet contained 40% fat, 14% protein, and 46% carbohydrate . The experiments were approved by the Institutional Ethics Committee and rigorously adhered to the National Institutes of Health Ethical Guidelines for the care and use of laboratory animals.Female Wistar rats were individually caged with free access to food and water at the South African Medical Research Council (MRC) Primate Unit  in a room at a temperature of 24°C with a 12 : 12 h light-dark cycle. Upon mating with pregnancy confirmed with the presence of vaginal plugs, mothers , blood glucose , and serum insulin  were measured. HOMA-insulin resistance ([fasting plasma glucose (mmol/L) × fasting serum insulin (mU/L)]/22.5)) and HOMA-beta-cell function ([20 × fasting insulin (mU/L)]/glucose (mmol/L) − 3.5)) were calculated.μm2 thick, were selected for immunolabeling. Slides were analyzed from at least one animal per litter. Alpha-cells were first immunolabeled with a polyclonal glucagon antibody  followed by immunolabeling of the beta-cells with a monoclonal insulin antibody  as previously described [Whole pancreata were harvested for immunolabeling experiments. Serial sections, 4 escribed , 13.For image analysis, a Canon Powershot S40 digital camera  mounted on an Olympus BX60 light microscope  attached to a personal computer was used to capture images. The camera focus, light intensity, and image resolution were controlled remotely and the acquired images were transferred to the computer using Remote Capture Software from Canon. The final digitized images were all 768 × 1024 pixels. Using the ×10 objective, images were acquired for alternate fields of view until the entire section was covered. For each field of view, the islets were captured using a ×40 objective for nuclei counts. An islet was defined as a cluster of eight or more endocrine cells. Image analysis was performed with the Leica Qwin Plus Software .μm2. The relative beta-cell volume was obtained by calculating the ratio between the area obtained by immunoreactive beta-cells and the area occupied by total islet cells. The same procedures were applied to estimate the alpha-cell number, size, and volume. The ratio of beta-cell area to alpha-cell area  was determined by dividing the total beta-cell area measured by the total alpha-cell area measured. The inverse calculations were applied to determine the alpha-cell : beta-cell ratio.Tissue parameters were measured using either an interactive option or color segmentation. Total tissue area was determined by adding the tissue measured in each field of view, using the interactive measurement option of the Leica software. Total islet area, total beta-cell area, and total alpha-cell area were determined by color segmentation and thresholding. Beta-cell number was assessed by counting the number of beta-cell nuclei. Beta-cell size was calculated by dividing the measured beta-cell area by the number of beta-cell nuclei counted and expressed in Proliferation indices of islet and acinar cells were calculated after immunostaining with the proliferation marker, Ki-67 . A minimum of 500 nuclei of each cell type (islet and acinar cells) were counted. The number of positive proliferative cells was divided by the total cell number of cells to determine the islet and acinar cell proliferation indices. Cell proliferation was not assessed in female offspring.P < 0.05.One-way ANOVA and Bonferroni's posttest were applied to determine differences in male and La Jolla, female offspring . Genders were analyzed separately. Data are reported as means ± SEM with significance established at HFP and HFGP males were heavier, hyperleptinemic, and hyperglycemic compared to control males . FurtherIn HFP males, beta-cell hyperplasia  presenteUnfortunately, the female HFGP phenotype could not be characterized due to low sample numbers, therefore, comparisons were limited to control, HFG, and HFP females. Despite no significant changes in weight or insulinemia, HFP females were hyperleptinemic, hyperglycemic, and insulin resistant compared to control females . FurtherThere were no changes in beta-cell number , size F, or voluHigh-fat programming generated different phenotypes in young adult rats. Firstly, in male offspring, postnatal high-fat programming from birth to adulthood induced the most adverse effects: a diabetogenic phenotype characterized by hyperglycemia and a mild obese state with increased body weight and hyperleptinemia. Further, this diabetogenic state triggered an islet cell compensatory response evident by beta- and alpha-cell hyperplasia and increased acinar cell proliferation. In female offspring, postnatal high-fat programming induced hyperglycemia, hyperleptinemia, and alpha-cell hypertrophy. Secondly, high-fat maintenance throughout gestation and postnatal life resulted in a severe obese phenotype characterized by the highest body weights, leptin and insulin concentrations, and most severe insulin resistance in male offspring. Finally, gestational high-fat programming induced no changes in either metabolism or islet architecture presenting a normal phenotype with the absence of a diabetogenic and/or obese phenotype in both male and female offspring. In HFP male offspring, the beta- and alpha-cell hyperplasia reflects a compensatory increase in islet cell number likely in an attempt to restore normoglycemia. Glucagon, secreted by alpha-cells, is antagonistic to insulin and stimulates glucose release which potentially contributed to the hyperglycemia in HFP males. Beta-cell compensation is inadequate: beta-cell hyperplasia is countered by alpha-cell hyperplasia, and the absence of beta-cell hypertrophy results in no compensatory increase in beta-cell volume. Therefore, hyperglycemia persists in HFP males.In HFP males, beta- and alpha-cell hyperplasia concomitant with an increase in acinar cell proliferation may mark acinar cells as a source for islet cells. The combination of the transcription factors, MafA, Pdx1, and Ngn3 was reported to drive the reprogramming of acinar cells to islet cells in the pancreas of adolescent immune-compromised mice . Acinar Interestingly, HFP males demonstrated beta-cell hyperplasia relative to HFG and HFGP males which provided further evidence of beta-cell compensation. Compared to HFGP males, beta-cell hyperplasia in HFP males was independent of changes in glycemia and insulinemia suggesting that more beta-cells were required to maintain similar states of glucose homeostasis. With no significant differences in glycemia and insulinemia in HFG males, which were similar to the levels in control males, no islet compensatory response was necessary which may account, in part, for the beta-cell hyperplasia in HFP males relative to HFG males. Alpha-cell hypertrophy presented in HFP females in the absence of any changes in beta-cell morphology. It appears that postnatal high-fat programming therefore only minimally affects islet morphology in female offspring, but does increase alpha-cell sizes; this requires further investigation. A severely obese phenotype presented after high-fat maintenance throughout fetal and postnatal life generating the heaviest, most hyperleptinemic, hyperinsulinemic, and insulin-resistant male offspring that clearly demonstrated the adverse metabolic effects of long-term maintenance on a high-fat diet. Insulin and leptin are secreted into circulation in proportion to adipose tissue  indicatiA diabetogenic and/or obese phenotype was absent in HFG male and female offspring, possibly due to the limited exposure to a high-fat diet of only three weeks of fetal life relative to the 3-month period of maintenance on a low-fat diet (control diet). The lack of an effect of gestational high-fat programming was however surprising. Long-term low-fat diet feeding in male Wistar rats closely mimics the changes in normal middle-aged humans with no onset of disease . HFG proGender influences biological outcomes  due to dn = 3) which increased variability and limited statistical power. Low sample sizes in some of the groups may have limited the finding of significant differences and resulted in type 1 errors. To address this caveat in future studies, we will increase dam numbers thus generating more offspring and allowing one pup per gender to be randomly selected from each dam. Higher sample sizes will reduce biological variability and enhance precision. The differences in sample sizes within groups were attributed to leptin and insulin assay sensitivity and to some sample degradation. The main focus of the study was however on islet morphology and can be further expanded by assessing beta-cell function in future experiments and identifying the proliferating islet cell type(s). The present study has several limitations. We lacked data on islet morphology for HFGP females which limited our investigation on gender-specific effects. Another constraint was not investigating the sole effect of lactational high-fat programming. A major shortcoming was the low sample sizes in some groups. Some sample sizes were on the threshold for statistical analysis (Male offspring maintained on a postnatal high-fat diet presented a diabetogenic and mild obese phenotype. Male offspring maintained on a high-fat diet throughout gestation and postnatal life presented a severe obese phenotype. In contrast, with gestational high-fat programming, there was an absence of a diabetogenic and/or obese phenotype in both male and female offspring. These findings emphasize the importance of gestational and postnatal nutrition in shaping offspring health and disease outcomes and confirm the durable effects of high-fat programming.


The two-stage tissue expander/implant (TE/I) reconstruction is currently the gold standard method of implant-based immediate breast reconstruction in North America. Recently, however, there have been numerous case series describing the use of one-stage direct to implant reconstruction with the aid of acellular dermal matrix (ADM). In order to rigorously investigate the novel application of ADM in one-stage implant reconstruction, we are currently conducting a multicentre randomized controlled trial (RCT) designed to evaluate the impact on patient satisfaction and quality of life (QOL) compared to the two-stage TE/I technique.The MCCAT study is a multicenter Canadian ADM trial designed as a two-arm parallel superiority trial that will compare ADM-facilitated one-stage implant reconstruction compared to two-stage TE/I reconstruction following skin-sparing mastectomy (SSM) or nipple-sparing mastectomy (NSM) at 2 weeks, 6 months, and 12 months. The source population will be members of the mastectomy cohort with stage T0 to TII disease, proficient in English, over the age of 18 years, and planning to undergo SSM or NSM with immediate implant breast reconstruction. Stratified randomization will maintain a balanced distribution of important prognostic factors . The primary outcome is patient satisfaction and QOL as measured by the validated and procedure-specific BREAST-Q. Secondary outcomes include short- and long-term complications, long-term aesthetic outcomes using five standardized photographs graded by three independent blinded observers, and a cost effectiveness analysis.There is tremendous interest in using ADM in implant breast reconstruction, particularly in the setting of one-stage direct to implant reconstruction where it was previously not possible without the intermediary use of a temporary tissue expander (TE). This unique advantage has led many patients and surgeons alike to believe that one-stage ADM-assisted implant reconstruction should be the procedure of choice and should be offered to patients as the first-line treatment. We argue that it is crucial that this technique be scientifically evaluated in terms of patient selection, surgical technique, complications, aesthetic outcomes, cost-effectiveness, and most importantly patient-reported outcomes before it is promoted as the new gold standard in implant-based breast reconstruction.NCT00956384ClinicalTrials.gov:  Mastectomy remains a common form of treatment for breast cancer ,2. In adIn 2007, an estimated 57,000 breast reconstructions were performed to restore mastectomy defects, and of those the majority of cases 34,017) were comprised of the two-stage, tissue expander/implant (TE/I) technique in the USA . The two7 were coTo address the inconvenience associated with the two surgeries in the TE/I method, while avoiding the high complication rates associated with the traditional one-stage technique, a number of newer one-stage implant reconstruction methods have been described ,18. One et al. reported the largest series of immediate one-stage breast reconstruction using ADM  in 260 patients (466 breasts) over an 8-year period  until, unfortunately, it’s suddenly too late’ . The useMost of the risks in this study are common minor and major surgical complications shared by both the one- and two-stage implant breast reconstruction procedures Table . The risClinical equipoise exists in this trial since no prior studies have compared patient-reported outcomes or risks/complications following the two types of implant-based breast reconstruction. Institutional Research Ethics Board (REB) approval has been obtained for all the study sites along with the data share agreements with the primary study site. Research staff will also ensure that protocol subjects receive Notice of Privacy Practices. Any serious adverse events will be reported to the REB as soon as possible but no later than 5 calendar days.in situ versus invasive breast cancer), it would have added too many strata relative to our sample size. In addition, although stratification based on whether the patient will require adjuvant chemo- or hormonal therapy would have been desirable, this information is not generally available at the time of randomization, nor is it in ‘real life’, as it only becomes known with the final pathologic evaluation of the mastectomy and sentinel lymph node. Therefore, our approach will be to adjust these confounders in the analysis. The 1-year duration for follow-up does not take into account revision surgeries that occur later than 1 year, therefore interpretation of the results should also be limited to this duration. The greatest strength of this study is the use of the randomized controlled design as the most stringent study design to elucidate a true cause-effect relationship between our intervention (one-stage ADM reconstruction) and effect (improved patient outcomes) by balancing both known and unknown variables in a relatively mixed mastectomy population.Blinding of participants and surgeons is impossible given the intervention. This may bias the primary outcome since QOL may seem a subjective endpoint; however, the BREAST-Q is a validated, reliable assessment of QOL that should provide a robust measure of satisfaction. The assessment of the overall aesthetic outcome, however, uses independent blinded judges and therefore reduces the bias when evaluating this secondary outcome. Secondly, although it would have been preferable to stratify important baseline covariates such as breast cancer stage . To date, 63 patients have completed the trial and are evaluable for the primary outcome.The protocol was amended in October 2011 to include an additional time-point for completing the BREAST-Q instrument in the control group at 6 months following mastectomy and insertion of TE after the first stage. The research team considered that although patients were still in the mid-process of reconstruction during expansion, answering the BREAST-Q would provide a better understanding of the impact of reconstruction on their interim QOL.AATB: American association of tissue banks; ADM: Acellular dermal matrix; BMI: Body mass index; DSMB: Data and safety monitoring board; HRQOL: Health-related quality of life; ICC: Intraclass correlation coefficient; MCCAT: Multi centre Canadian acellular dermal matrix trial; NSM: Nipple-sparing mastectomy; QALY: Quality-adjusted life year; QOL: Quality of life; RCT: Randomized controlled trial; REB: Research ethics board; SSM: Skin-sparing mastectomy; TE: Tissue expander; TE/I: Tissue expander/implant.TZ, MB, SM, and PL have been paid speakers for LifeCell in the past 5 years.TZ is the primary grant holder of this study. As the primary author, TZ designed the protocol, and is currently leading the study, screening, recruiting patients, operating, and conducting follow-up assessments. TZ will oversee data analysis and interpretation, as well as write the manuscript. CTO is screening, recruiting, operating on patients, conducting follow-up assessments, and will contribute to data interpretation and manuscript writing. SH, BB, JS, and MB designed the study, are screening and recruiting eligible patients, operating on patients, conducting follow-up assessments, and will contribute to data interpretation and manuscript writing. SM and PL are screening and recruiting eligible patients, operating on patients, conducting follow-up assessments, and will contribute to data interpretation and manuscript writing. CM helped to design the study. TP provided statistical support. NB is a grant holder, helped design the study, is contributing expertise to study implementation, and will also contribute to data analysis and manuscript writing. All authors read and approved the final manuscript.


Many stillbirths show evidence of fetal growth restriction, and most occur at preterm gestational age. The objective of this study is to compare birth weights at preterm gestational ages between live births and stillbirths, and between those occurring before or during labour.Based on singleton births from the United States (U.S.) 2003–2005  and Sweden 1992–2001 , we compared birth weights between singleton live births and stillbirths at 24–36 completed weeks of gestation from the U.S. and at 28–42 completed weeks from Sweden.In both the U.S. and Sweden, stillbirth weight-for-gestational-age z-scores were at least one standard deviation lower than live birth z-scores at all preterm gestational ages (GA). In Sweden, no birth weight difference was observed between antepartum and intrapartum stillbirths at preterm GAs, whereas birth weights among intrapartum stillbirths were similar to those among live births at 37–42 weeks.Birth weights observed at preterm gestation are abnormal, but preterm stillbirths appear to be more growth-restricted than preterm live birth. Similar birth weights among ante- and intrapartum preterm stillbirths suggest serious fetal compromise before the onset of labor. Depending on jurisdiction, stillbirth is variably defined as a fetal death from 20, 22, 24 or 28 completed weeks of gestation and can also depend on birth weight -4. In thThe actual time of fetal death is usually unknown. It has been reported that stillbirths are generally delivered within 24 hours of diagnosis -14, and We used period linked birth-infant death data and fetal death data files from the National Vital Statistics System of the United States for the years 2003, 2004, and 2005. These publicly available data files, compiled by the U.S. National Center for Health Statistics (NCHS), include information from the birth certificate on maternal socio-demographic characteristics, birth weight, period of gestation, plurality, and live birth order and is linked to information from the infant death certificate. The fetal death files include information from all reports of fetal deaths but do not include the cause of stillbirth . In the In the United States, gestational age (GA) is usually calculated from the first day of the mother’s last menstrual period (LMP). It has been shown that gestational age derived from the LMP estimate is prone to error, especially for postterm dates -25. The We restricted our primary analysis to singleton births 24–36 completed weeks of gestation. We also excluded implausibly high or low birth weights at given gestational ages from our study sample, based on Table To guard against potential misclassification of GA in the US birth cohort ,25, we a2, which predicts a mean birth weight of 3619 g at 280 days. Hadlock’s formula assumes proportional fetal growth throughout pregnancy and models fetal growth trajectory “by dividing each daily value predicted by this formula by the 280-day value and fitting a third-degree polynomial of gestational age”, which yields the following “proportionality equation” for GAs of 24–39 weeks [2 – 0.01055 × GA3.To evaluate fetal growth, we used estimated fetal weight as the reference standard, rather than the observed birth weight, since the latter is known to be suboptimal at preterm GAs ,22. Inte39 weeks : % EFW =The latter equation can then be used for any estimated 280-day birth weight to estimate fetal weight in 24–39 weeks of gestational age. Sex-specific birth weight z-scores for 40 weeks of gestation were first calculated based on the study sample, and the sex-specific weight-for-gestational-age z-scores were then extrapolated backward by the proportionality equation. Further details have been described in previous studies ,30,31. HThe primary outcomes were birth weight and birth weight-for-gestational-age z-score. We compared mean birth weights and birth weight-for-gestational-age z-scores at preterm gestations between singleton stillbirths and live births at 24–36 weeks of gestation from the U.S. and at 28–36 weeks from Sweden. We further compared birth weight-for-gestational-age z-scores between antepartum and intrapartum Swedish stillbirths both at preterm and term gestations. For all these comparisons, we used two-sample t-test. At each completed week of gestation, stillbirth weight was also expressed as a percentage of the mean (POM) of live birth weights at the same gestation; the mean of these percentages, with its 95% confidence interval (CI), was then calculated. We also carried out race/ethnicity-specific analyses of non-Hispanic Whites, non-Hispanic Blacks, and Hispanics, from the U.S. and compared birth weights among antepartum and intrapartum stillbirths from Sweden. Finally, as a sensitivity analysis, we excluded from the U.S. birth cohort all births diagnosed with congenital anomalies at birth and compared birth weight between preterm live births and stillbirths. All analyses were carried out using SAS version 9.2 .Figure We observed similar patterns in birth weight and z-score differences between live births and stillbirths in race/ethnicity-specific analyses of U.S. non-Hispanic Whites, non-Hispanic Blacks, and Hispanics (data not shown) as we did in the overall U.S. study sample. After excluding all births diagnosed with congenital anomalies at birth, we observed a similar patterns of birth weight and z-score differences between preterm live births and stillbirths (data not shown), although the average difference in birth weight decreased from 1,013 to 876 g.Table Average birth weights are known to be lower than intrauterine  weights early in pregnancy ,22, stroAs shown in our study, stillbirth weights were at least one SD below live birth weights at preterm GAs. The fact that stillbirths weigh less at delivery than live births at the same GA has been previously documented ,35. PrevIn our analysis of the Swedish data, we were surprised not to observe significant birth weight differences between antepartum and intrapartum stillbirths at preterm GAs. This result does not agree with previous reports by Chard  and AlbeAssuming little or no weight loss among most antepartum stillbirths from death to delivery, preterm stillbirths appear to be more growth-restricted than live births at the same GA. It has been shown that size at birth is affected by growth velocity early in gestation and that growth restriction diagnosed at delivery is preceded by slower first- and early second-trimester fetal growth ,38. As sAn important limitation of our study is the lack of a good ultrasound-based fetal weight reference at preterm gestations, which would improve estimation of the difference between the birth weights of live births and the fetal weights of their counterparts who remain in utero. Hadlock’s fetal growth curve , based oOur study is further limited by the use of nation-wide vital statistics databases, in which coding errors are known to occur ,40. In pMany preterm births are growth-restricted. Our study suggests that growth restriction is a cumulative process and that those fetuses who subsequently die preterm in utero grow even more slowly then those born alive at the same GA. Reduced placental blood supply and the consequent reduction in delivery of oxygen and nutrients to developing fetuses may both restrict fetal growth early in gestation and increase the risk of subsequent stillbirth. Suboptimal placental function and poor growth early in pregnancy limit fetal growth for the remainder of pregnancy. Early and routine ultrasound scans to monitor fetal size and growth during pregnancy might theoretically reduce the risk of stillbirth. This is perhaps the most important clinical implication of our findings. Before implementing routine clinical monitoring, however, an improved intrauterine fetal weight reference is required. Large-scale NIH-  and Gates Foundation-funded studies of ultrasound-based fetal size assessment currently under way should provide much-needed data on normal fetal growth patterns. Future research should attempt to understand the pathophysiological mechanisms underlying growth restriction, as a basis for developing and testing preventive and therapeutic interventions to reduce stillbirth risk.Birth weights observed at preterm gestation are abnormal, but preterm stillbirths appear to be more growth-restricted than preterm live birth. Similar birth weights among ante- and intrapartum preterm stillbirths suggest serious fetal compromise before the onset of labor.We have declared no conflict of interest.Dr. XZ designed the study, performed the statistical analyses, and wrote the first draft of the manuscript. Drs. KSJ, SC, and MSK all contributed to the study design, interpretation of results, 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/1471-2393/12/119/prepub


Although ultrasound shows great potential for aiding assessment of LS disease activity, its use has been limited because both image acquisition and interpretation are operator dependent. To facilitate evaluating and to standardize interpretation of jLS ultrasound scans, we generated a scoring measure for evaluating echogenicity and vascularity differences . Objective: To assess the reliability of an ultrasound scoring measure (U-DA) for jLS.A meeting held from 2/21 to 2/24/09 brought together 12 radiologists and sonographers from 5 institutions for hands-on training in acquiring and scoring ultrasound scans of jLS patients. The group had initially met in 2007, and had developed a preliminary ultrasound scoring measure. This preliminary measure was reviewed in conjunction with scans showing the range of sonographic differences that had been observed to date in jLS; this review led to an expansion of scoring ranges. Following finalization of the U-DA, the group was led in the scoring of a jLS scan by Dr. Liebling, and then attendees independently scored two other scans. These scores were jointly reviewed to ensure that participants were comfortable with applying the U-DA measure. Participants then independently scored a set of 16 jLS scans; each participant had a different order of the scans. Ten of the 11 scorers scored these scans a second time on a different day, with scans presented in a different order from the original set. Intraclass correlation scores for total echogenicity and vascularity were calculated to determine intra-rater reliability. The median score for each of the 6 U-DA parameters  was determined, and the distribution of each rater’s score relative to the median plotted.All raters showed moderate to high intra-rater reliability for scoring total echogenicity (ICC 0.591 to 0.806). Poorer intra-rater reliability was found for scoring total vascularity, ranging from poor to high agreement. When evaluating the median and range of scores for each U-DA parameter, for most scans, most raters scored at or ± 1 within the median. However, for a few scans, some parameters showed no consensus or a wide range of scores. Deep tissue layer parameters were more likely to be problematic than those for other layers.Good intra-rater agreement was found for scoring total echogenicity. There was variable intra-rater reliability for scoring total vascularity, and inter-rater reliability varied among the study scans, with some U-DA parameters showing good agreement and others widely divergent scores. Scoring was more often problematic for the deep tissue layer. More review of the scans showing poorer inter-rater agreement, and more focus on deep tissue layer evaluation, may lead to improved inter-rater scoring reliability.Suzanne C. Li: Arthritis Foundation, 2; Melissa S. Liebling: None; Molly Dempsey-Robertson: None; Andrea S. Doria: None; Stephanie Edgerton: None; Carsten Hamer: None; Jose Jarrin: None; Tanicka Kornyat: None; Michael Malone: None; Arun Mohanta: None; Sven Opitz: None; Faridali Ramji: None; Shuzhen Zhang: None; Knut Wittkowski: NIH, 2.


Children who participate in regular physical activity obtain health benefits. Preliminary pedometer-based cut-points representing sufficient levels of physical activity among youth have been established; however limited evidence regarding correlates of achieving these cut-points exists. The purpose of this study was to identify correlates of pedometer-based cut-points among elementary school-aged children.A cross-section of children in grades 5-7 (10-12 years of age) were randomly selected from the most (n = 13) and least (n = 12) 'walkable' public elementary schools , stratified by socioeconomic status. Children  and parents  completed a survey, and steps were collected from children using pedometers. Pedometer data were categorized to reflect the sex-specific pedometer-based cut-points of ≥15000 steps/day for boys and ≥12000 steps/day for girls. Associations between socio-demographic characteristics, sedentary and active leisure-time behavior, independent mobility, active transportation and built environmental variables - collected from the child and parent surveys - and meeting pedometer-based cut-points were estimated (odds ratios: OR) using generalized estimating equations.Overall 927 children participated in all components of the study and provided complete data. On average, children took 11407 ± 3136 steps/day  and 25.9%  achieved the pedometer-based cut-points.After adjusting for all other variables and school clustering, meeting the pedometer-based cut-points was negatively associated (p < 0.05) with being male (OR = 0.42), parent self-reported number of different destinations in the neighborhood (OR 0.93), and a friend's (OR 0.62) or relative's  house being at least a 10-minute walk from home. Achieving the pedometer-based cut-points was positively associated with participating in screen-time < 2 hours/day (OR 1.88), not being driven to school (OR 1.48), attending a school located in a high SES neighborhood (OR 1.33), the average number of steps among children within the respondent's grade (for each 500 step/day increase: OR 1.29), and living further than a 10-minute walk from a relative's house .Comprehensive multi-level interventions that reduce screen-time, encourage active travel to/from school and foster a physically active classroom culture might encourage more physical activity among children. For children and adolescents, participation in regular physical activity provides physical and mental health benefits . Specifith percentile [To date, much of the research examining correlates of physical activity among youth have relied on self-report methods for capturing physical activity behavior, including parent proxy. However, self-reported physical activity is prone to memory and recall bias ,14 and crcentile  while lorcentile .Achieving any of the existing pedometer-based cut-points does not guarantee that a child will be healthy weight. Nevertheless, boys achieving ≥15000 steps/day and girls achieving ≥12000 steps/day for instance, are more likely to participate in at least 60-minutes of moderate-intensity physical activity per day  - notewoIdentifying factors that increase or decrease the likelihood of participating in or above these suggested pedometer-based cut-points may lead to more effective interventions designed to promote physical activity among children and adolescents. The limited evidence regarding correlates of achieving pedometer-based cut-points suggests that sex, day of the week, age, participation in outside play, attending sports clubs, weather conditions, SES, participation in active transport and ethnicity may be important -33. NeveTherefore the aim of this exploratory study is to extend the evidence-base by examining associations between socio-demographic characteristics, sedentary and active leisure-time behavior, independent mobility, active transportation and the built environment, and achievement of sex-specific pedometer-based cut-points , among AThis current study forms part of the TRavel, Environment, and Kids project (TREK). The overall aim of this project was to examine the extent to which the urban design of neighborhoods supports or discourages active transportation among children in grades 5-7 attending public elementary schools in Perth, Western Australia. The research methodology for TREK is presented elsewhere , and is Public elementary schools (n = 238) across metropolitan Perth were assigned a school-specific walkability index and socioeconomic status (SES) score. The walData were collected July-December 2007 using a child questionnaire, parent questionnaire and pedometers in addition to other measures not reported here. Items were assessed for test-retest reliability between March and May 2007 and where available these estimates have been presented here as kappa (κ) or intraclass correlation (ICC) coefficients.Accusplit (AH120 M8) pedometers were used to record children's step counts. A similar model of pedometer was recently used to assess recommendations regarding moderate-intensity walking cadence . The pedChildren reported whether they had friends in the neighborhood  while parents reported whether their household had a dog . The child's sex and grade were also collected in the children's questionnaire, and highest education for any parent , marital status , the number of dependents <18 years at home , and home ownership  were assessed in the parent questionnaire.Parents reported the average amount of time per day their child spent using a computer or internet for pleasure, watching television/videos, and playing passive or active electronic games. Minutes of each activity were summed and dichotomized to reflect current screen-based activity recommendations for Australian youth . ChildreParents were asked whether or not their child was allowed to play in the street (κ = 0.56), play at the closest park, playground, or playing field (κ = 0.55), or walk in their neighborhood (κ = 0.59) without an adult. A dichotomous variable was derived from these items - i.e., not allowed vs. allowed to play or walk in the neighborhood unsupervised. Parents also reported whether their child travelled to and from school by motor vehicle during a usual week (κ = 0.72).On a five-point Likert scale  parents reported whether: they had to drive to get to a park with appealing equipment for their child; they often saw adults walking in their neighborhood; they often saw children walking in their neighborhood; their neighborhood was friendly; there was a lot of traffic in their neighborhood; the neighborhood was a nice place to walk around; there were safe crossings to reach a local shop; there were safe crossings to reach the closest local park; and drivers near the school often exceeded the speed limit . Parents also reported the time  required for their child to walk from home to their nearest: primary school; shop; newsagent; library; transit station; park; bushland; sports field; beach, and; river. The number of different types of destinations perceived to be within a 10-minute from home was counted. The presence of a relative and friend's house within a 10-minute walk of home were examined separately. Reported time to walk to these destinations had acceptable reliability (ICC = 0.51-0.84).The school-specific walkability index, as previously described , was dicThe analytical sample included cases who participated in all components of the study and had complete survey and valid pedometer data (n = 927). Pedometer-based physical activity was considered valid if counts were between 1000 and 30000 steps per day --step coDescriptive statistics were computed for all characteristics. Moreover, characteristics of the analytical sample (n = 927) and excluded  respondents were compared using Pearson's Chi-square and independent t-tests. To reduce the number of parent neighborhood environment perception variables  a principal component analysis was performed. Items belonging to the same construct were identified  and summed. Two factors were identified, including: 1) neighborhood friendliness ; and 2) traffic barriers . A single item--drive to get to a park with appealing equipment for their child--did not load on either factor and was examined separately in the analysis.To account for school-level clustering, generalized estimating equations (GEE) were used  to estimate odds ratios (OR) and 95 percent confidence intervals (95%CI). Using GEE, achievement of the pedometer-based cut-points was regressed onto the built environment, social environment, sedentary behavior, active leisure-time behavior, independent mobility, travel to school, and peer pedometer-based physical activity. To assess the moderating effect of sex, interaction terms between sex and the other correlates were estimated and a backward stepwise removal of non-significant interaction terms (p > 0.05) undertaken to derive the final model. All main effects were retained in the model regardless of statistical significance. Based on bivariate correlations (r < 0.35) and variance inflation factors (VIFs < 1.4) among the correlates, the level of multicollinearity within the regression model was not an issue. Statistical analysis was undertaken using Predictive Analytic Software 17 .Compared with the excluded study participants, the analytical sample included a significantly higher proportion of girls, those spending <2 hours on screen-based activity/day, those allowed to play in the neighborhood without adult supervision, and those attending a school in a higher SES neighborhood Table . MoreoveThe estimated exchangeable working correlation matrix suggested clustering of achievement of pedometer determined physical activity recommendations among students within the same school . Seven variables were associated (p < 0.05) with achieving the suggested pedometer-based cut-points  . The presence of more local destinations might also result in the neighborhood being less supportive of physical activity among children of this age because of more destination-related motor vehicle traffic, limited area for play spaces and the presence of more strangers visiting local neighborhoods. An alternative explanation is that fewer steps are needed to travel to destinations that are closer to home. Nevertheless, Frank et al. , for exaChildren whose friend's house was within a 10-minute walk were more likely to achieve pedometer-based cut-points. Children with friends residing nearby might visit more frequently using active modes, may be involved in active play, and may have company to walk to and from school. Together this may result in more walking and physical activity. There is evidence for associations between friend support and physical activity among children . In suppSeveral limitations should be considered when interpreting the findings of this study. Differences between the analytical sample and the excluded participants were observed. In particular the analytical sample was more physically active, thus the results presented might underestimate the magnitude of the associations between the correlates and pedometer-based physical activity. Moreover, the moderate reliability of some variables may have attenuated the estimated associations between these correlates and the outcome. The generalizability of these results are limited to school-aged children in grades 5-7 attending elementary schools in high and low walkable neighborhoods determined based on pedestrian connectivity and traffic exposure. Other objectively-assessed environmental characteristics not measured here could be important for determining pedometer steps among children, although more research is needed to identify these characteristics. We examined the correlates of one set of recently suggested pedometer-based physical activity cut-points , thus thFor children, participation in regular physical activity can provide physical and mental health benefits and physical activity behaviors adopted during childhood often track into adulthood . These r(BMI): body mass index; (ICC): intraclass correlation; (OR): odds ratio; (SES): socioeconomic status; (95%CI): 95 percent confidence interval;The authors declare that they have no competing interests.BGC, AT, and GRM conceived this study. GW and KW contributed to data collection. GRM conducted the analysis. All authors contributed to interpretation and writing of the manuscript. All authors read and approved the final manuscript.


Few studies have examined outcomes for children treated for multidrug-resistant tuberculosis (MDR-TB), including those receiving concomitant treatment for MDR-TB and HIV co-infection. In Lesotho, where the adult HIV seroprevalence is estimated to be 24%, we sought to measure outcomes and adverse events in a cohort of children treated for MDR-TB using a community-based treatment delivery model.We reviewed retrospectively the clinical charts of children ≤15 years of age treated for culture-confirmed or suspected MDR-TB between July 2007 and January 2011.Nineteen children, ages two to 15, received treatment. At baseline, 74% of patients were co-infected with HIV, 63% were malnourished, 84% had severe radiographic findings, and 21% had extrapulmonary disease. Five (26%) children had culture-confirmed MDR-TB, ten (53%) did not have culture results available, and four (21%) subsequently had results indicating drug-susceptible TB. All children with HIV co-infection who were not already on antiretroviral therapy (ART) were initiated on ART a median of two weeks after the start of the MDR-TB regimen. Among the 17 patients with final outcomes, 15 (88%) patients were cured or completed treatment, two (12%) patients died, and none defaulted or were lost to follow-up. The majority of patients (95%) experienced adverse events; only two required permanent discontinuation of the offending agent, and only one required suspension of MDR-TB treatment for more than one week.Pediatric MDR-TB and MDR-TB/HIV co-infection can be successfully treated using a combination of social support, close monitoring by community health workers and clinicians, and inpatient care when needed. In this cohort, adverse events were well tolerated and treatment outcomes were comparable to those reported in children with drug-susceptible TB and no HIV infection. Between 2000 and 2009, there have been an estimated five million new cases of multidrug-resistant tuberculosis (MDR-TB), defined as resistance to isoniazid and rifampicin. 3.5 million patients received no treatment or treatment of unknown quality, and 1.5 million died Pediatric tuberculosis presents several unique diagnostic and treatment challenges. Treatment delay is particularly problematic for children with MDR-TB, and outcomes are poor when treatment is delayed Additional challenges arise when diagnosing and managing MDR-TB in the context of HIV co-infection. Poor response to first-line TB treatment in HIV co-infected patients can occur for reasons other than drug resistance, including poor adherence and the presence of other opportunistic infections with pulmonary manifestations, and HIV co-infected patients are at increased risk of recurrent TB infections A small number of studies and case series have examined outcomes for children treated for MDR-TB We sought to describe the clinical features, treatment course, and final outcomes in a cohort of children treated for MDR-TB in Lesotho using a community-based treatment delivery model.As this was a retrospective study of medical information previously collected in the course of routine clinical care, informed consent was not required. This study was approved by the Partners HealthCare Human Research Committee.Lesotho is a small, mountainous country completely surrounded by South Africa, with a population of approximately 1.8 million. Lesotho has a high burden of both HIV and TB: 24% of the adult population is HIV infected We conducted a retrospective analysis of medical charts of all patients ages 15 years or younger who initiated second-line TB treatment for confirmed or suspected MDR-TB between July 27, 2007 and January 1, 2011. We excluded one patient who began an MDR-TB regimen and was subsequently switched to first-line tuberculosis treatment based on drug-susceptibility testing (DST) results, patient history, and clinical and radiographic response to MDR-TB treatment.Treatment protocols, including pediatric dosing, followed international standards of care The management of patients who were started empirically on MDR-TB treatment and subsequently had DST results indicating susceptibility to isoniazid and rifampicin was considered on an individual case-by-case basis. Based on the patient’s clinical history, duration of MDR-TB treatment, and treatment response, the clinical team decided whether to transfer the patient to the local health center to receive first-line TB treatment or maintain the patient on the second-line regimen with the addition of rifampicin and high-dose isoniazid.Lesotho experienced an uninterrupted supply of second-line antituberculous drugs during the study period. Because none of antituberculous drugs used in the standard regimen were available in pediatric formulations, patients were provided with adult formulations, and the clinicians and community health workers instructed caregivers to divide doses and cut tablets into the appropriate size.Patients with unknown HIV serostatus were offered testing at the first visit. All patients with HIV co-infection who were not yet receiving ART were initiated on an ART regimen as soon as they were tolerating the MDR-TB treatment regimen, regardless of CD4 cell count. The standard ART regimen consisted of two nucleoside reverse transcriptase inhibitors (zidovudine or stavudine plus lamivudine) and one non-nucleoside reverse transcriptase inhibitor . All patients received pyridoxine supplementation to prevent peripheral neuropathy M. tuberculosis at baseline and at each monthly clinic visit.MDR-TB treatment was provided in the community for patients in stable, ambulatory condition. Trained, paid community health workers (CHWs) delivered second-line antituberculous drugs and antiretroviral therapy twice-daily under direct observation. The CHWs also monitored patients daily for adverse events. Routine laboratory monitoring–including full blood count, liver function tests, electrolytes, and creatinine–was performed monthly at clinic visits, and a specialist clinician evaluated patients at these visits. Thyroid-stimulating hormone (TSH) was initially measured within the first six months of MDR-TB treatment initiation and subsequently every four to six months. Patients with at least one TSH result elevated above 10.0 mIU/l were initiated on a weight-based dose of levothyroxine, which was continued until one month after completion of MDR-TB therapy; TSH was monitored throughout the treatment period at regular intervals A community team communicated regularly between the clinics and the community, tracking patients who missed any appointment and assessing the stability of each patient’s household situation. The community team provided patients and their caregivers with counseling about MDR-TB and HIV, food supplements, reimbursement for treatment-related transportation, and other types of psychosocial support as needed. Patients who were clinically unstable or experienced severe adverse events were admitted to Botsabelo Hospital, a specialized MDR-TB inpatient facility in the capital (Maseru).Baseline characteristics, treatment history, and clinical signs and symptoms, including radiographic findings, laboratory values, adverse events, and treatment outcomes, were abstracted from patient records and entered into a Microsoft Access 2003  database. Standard definitions for retrospective MDR-TB analyses were used to determine final treatment outcomes Nineteen children were included in this analysis . At the Patients were treated with a median of six antituberculous drugs . Sixteen patients (84%) were hospitalized at treatment initiation for a median of 1.5 months  . Most ofFinal outcomes were available for 17 patients: 15 patients (88%) had treatment success (seven patients cured and eight patients completed treatment) and two patients (12%) died . Both paEighteen patients (95%) experienced at least one adverse event . Most ad3  weight gain among HIV co-infected patients was 4 (3–6) kg. There were no incidences of immune reconstitution inflammatory syndrome (IRIS) after ART initiation. Three patients were switched from zidovudine to stavudine when they experienced anemia, and one patient was switched from stavudine to zidovudine after the onset of peripheral neuropathy. Another patient who had experienced immunological deterioration prior to MDR-TB treatment was switched from a second-line ART regimen to a first-line regimen when genotypic resistance testing revealed no mutations conferring resistance to first-line antiretroviral drugs.Among the 14 patients with HIV co-infection, the median (interquartile range [IQR]) CD4 cell count at the start of MDR-TB treatment was 486.5 (49.3–714.8) cells/mm3 . Eight pOur results demonstrate that, even in a setting of high HIV prevalence, it is possible to achieve favorable outcomes among children treated for MDR-TB using early empiric treatment delivered through a comprehensive community-based program. The latter included referral to inpatient care as needed. Almost 90% of the children treated experienced cure or treatment completion, despite the geographically challenging setting and the high rate of baseline malnutrition, severe lung damage, and co-infection with HIV. Excluding the four patients who subsequently had DST results suggesting drug-susceptible TB, the mortality rate among confirmed and suspected MDR-TB cases in this study was 13%. Our outcomes were similar to outcomes among children in MDR-TB treatment programs with largely HIV-negative patients, where treatment success rates have ranged from 80 to 100% and crude mortality rates, from 0 to 6% In contrast to the favorable outcomes achieved in low HIV prevalence settings, pediatric case series with a high prevalence of HIV have reported lower rates of treatment success Two recent studies of DR-TB and HIV co-infected children treated concurrently with ART and second-line TB treatment have reported more favorable outcomes In contrast to previous reports on co-infected children, children in the Lesotho program were initiated on empiric second-line TB treatment immediately after evaluation for suspicion of MDR-TB, without waiting for DST confirmation. Treatment delay is particularly problematic among children because of the difficulty in obtaining sputum and the high likelihood of paucibacillary TB. In a study of 39 children with MDR-TB in South Africa, Schaaf et al. found that treatment delay  was a median of two days if the DST of the source case was considered and 246 days if the DST of the source case was not considered MDR-TB should be suspected when a child has been in contact with a TB patient who died while on treatment and who was suspected to have had MDR-TB Efforts to obtain sputum samples for culture and DST confirmation for all pediatric patients were aggressive. Clinicians attempted to collect sputum specimens at monthly clinic visits, and gastric lavage and sputum induction were used for young children. For the diagnosis of extrapulmonary MDR-TB, pleural fluid sample and lymph node aspirates are the only non-pulmonary specimens routinely collected and cultured in Lesotho. Compared with adults, children with TB are more likely to have extrapulmonary TB The five patients with culture-confirmed MDR-TB all experienced cure or treatment completion. Of the other 14 patients, six had household contact with a confirmed MDR-TB patient or high-risk MDR-TB suspect, and the remaining patients had failed to respond to at least one previous course of first-line TB treatment. For several patients who initiated an empiric MDR-TB regimen, subsequent DST results indicated susceptibility to isoniazid and rifampicin. However, all had responded well to MDR-TB treatment, experiencing significant clinical and radiological improvement, after having been unsuccessfully treated with at least one first-line TB regimen, so the clinical team decided to continue them on MDR-TB treatment with the addition of rifampicin and high-dose isoniazid. We did not have data on patients’ adherence to previous courses of first-line TB treatment, and it is possible that non-adherence, despite directly observed therapy, may have contributed to failure of first-line treatment. Additionally, three of the four patients had HIV co-infection, making them susceptible to other potential causes of clinical deterioration during first-line TB treatment Although we were striving to provide rapid and appropriate care to these patients, we note that the majority (58%) of the children in our cohort had already received two or more previous regimens of first-line TB drugs, and all but one of the household contacts had been treated previously with first-line drugs, suggesting that there was a delay in recognizing them as MDR-TB suspects and initiating them on appropriate treatment. Additionally, nearly half of the HIV co-infected patients were not receiving ART at the time of MDR-TB treatment initiation, despite the fact that pulmonary TB is a criteria for initiating ART in the clinical staging of HIV/AIDS (irrespective of CD4 cell count) Children with HIV co-infection who were not already on ART were initiated on ART as soon as they were tolerating MDR-TB treatment, as early as one week post initiation. Optimal time to introduce ART following the start of TB treatment is uncertain, as early introduction of ART after initiation of TB treatment is believed to be associated with higher risk of IRIS In this cohort, children experienced a higher incidence of adverse events compared to other reports Patients in this study also experienced a high incidence of hypokalemia (68%), which occurred a median of 39 days after the start of the MDR-TB regimen. In adults, electrolyte disturbances, including hypokalemia, have been attributed to the use of aminoglycosides and capreomycin Although ART drugs and second-line TB drugs have potential overlapping or additive toxicities In addition to close monitoring of the patients’ clinical status, the CHWs and community team provided close monitoring of the patients’ household situation and psychosocial needs. Many patients had been orphaned by HIV and TB , and somThe favorable outcomes reported in this study are particularly notable because they were achieved in a predominately ambulatory setting. Because of the complexity of managing MDR-TB and HIV co-infection, the traditional model of MDR-TB treatment in this region of the world has been hospital-based care, and many children with MDR-TB continue to be hospitalized unnecessarily. The community-based treatment model minimized the risk of nosocomial transmission of MDR-TB, which is particularly important in high HIV prevalence settings like Lesotho. Our findings demonstrate that good MDR-TB outcomes in the context of HIV are achievable using a community-based model. However, successful implementation of community-based care in this geographically challenging setting where many patients live in rural, mountainous areas required several key elements, including strong links between the community team and clinicians, well-trained and supervised CHWs, and reimbursement for travel expenses related to treatment For children suspected of having MDR-TB disease, particularly those with HIV co-infection, empiric second-line TB therapy should be initiated while awaiting DST results. Patients with HIV co-infection not already receiving ART may benefit from early initiation of ART once the MDR-TB treatment regimen is tolerated. Our community-based treatment delivery model included empiric treatment, as well as close monitoring, frequent routine screening, and timely interventions for adverse events. Treatment outcomes for this strategy are comparable to those reported among children with drug-susceptible TB and those without HIV infection.


Bacillus (Gram-positive) and Pseudomonas (Gram-negative) have received the most attention because they produce a wide range of effective LPBSs that are potentially useful for agricultural, chemical, food, and pharmaceutical industries. The biosynthetic mechanisms and gene regulation systems of LPBSs have been extensively analyzed over the last decade. LPBSs are generally synthesized in a ribosome-independent manner with megaenzymes called nonribosomal peptide synthetases (NRPSs). Production of active-form NRPSs requires not only transcriptional induction and translation but also post-translational modification and assemblage. The accumulated knowledge reveals the versatility and evolutionary lineage of the NRPSs system. This review provides an overview of the structural and functional diversity of LPBSs and their different biosynthetic mechanisms in Bacillus and Pseudomonas, including both typical and unique systems. Finally, successful genetic engineering of NRPSs for creating novel lipopeptides is also discussed.Lipopeptide biosurfactants (LPBSs) consist of a hydrophobic fatty acid portion linked to a hydrophilic peptide chain in the molecule. With their complex and diverse structures, LPBSs exhibit various biological activities including surface activity as well as anti-cellular and anti-enzymatic activities. LPBSs are also involved in multi-cellular behaviors such as swarming motility and biofilm formation. Among the bacterial genera,  Biosurfactants are biological surface-active compounds largely produced by a wide variety of microorganisms. They have environmentally friendly properties, such as low toxicity and high biodegradability. Biosurfactants are capable of lowering surface and interfacial tensions effectively and thus are potential substitutes for widely used chemically synthesized surfactants. Thus, biosurfactants show promise for use in agricultural, food, pharmaceutical, cosmetic, specialty chemical, and oil production industries as well as in bioremediation technology. The hydrophobic portion of these molecules is commonly made up of fatty acids , whereas the hydrophilic portion is usually composed of peptides or mono-, di-, or polysaccharides . Due to Bacillus subtilis IAM1213  in the T-domains were expected to reflect the recognition by external racemase. However, Balibar and coworkers later demonstrated that Arf contains unique dual C/E domains, which contribute to the conversion of l-amino acids to the d-form . An. An98]. oduction . This reter unit . Althouge d-form . This noficiency . Moreove_347945) . Arf repE-domain . This obP. fluorescens SBW25 and P. fluorescens SS101, respectively , was produced by the recombinant B. subtilis at a level similar to that of wild-type surfactin and it also exhibits stronger surface activity than surfactin   . Along trmediate . A reductivities , 162. Am product . C-termiS system . SwappinS system . Using panalysis . Althougoduction . Recentltivities ,169.Bacillus and Gram-negative Pseudomonas strains produce a variety of lipopeptides with remarkable surface and biological activities. In contrast to the structural diversity of these lipopeptides, their biosynthetic mechanism is basically conserved. They are synthesized nonribosomally by a mega-peptide synthetase unit, NRPS, which is composed of several cooperating multifunctional modules, each capable of performing one cycle of peptide elongation. To become an active form, they are post-translationally modified by a PPTase and properly assembled by DnaJ/K and HtpG proteins. However, recent analysis of the lipopeptide synthetases suggests that there are several variants of NRPS architecture. Modification of NRPS by genetic engineering of the encoding genes is a promising method to produce useful variants. Accumulation of genetic information for lipopeptide synthetases should contribute to design biosurfactants with higher surface activity and/or novel features. Moreover, understanding of their biosynthetic pathways and genetic regulation mechanisms will facilitate not only uncovering the evolution of nonribosomal peptide synthesis mechanisms, but also the development of cost-effective methods for large-scale production of useful lipopeptides.Gram-positive