Physical functioning (PF) is an essential domain of older persons’ health and quality of life. Health behaviors are the main modifiable determinants of PF. Cross-sectionally, alcohol consumption appears to be linked to better PF, but longitudinal evidence is mixed and very little is known about alcohol consumption and longitudinal PF trajectories.We conducted longitudinal analyses of 28,783 men and women aged 45–69 years from Novosibirsk (Russia), Krakow (Poland), and seven towns of the Czech Republic. At baseline, alcohol consumption was measured by a graduated frequency questionnaire and problem drinking was evaluated using the CAGE questionnaire. PF was assessed using the Physical Functioning Subscale of the SF-36 instrument at baseline and three subsequent occasions. Growth curve modeling was used to estimate the associations between alcohol consumption and PF trajectories over 10-year follow-up.PF scores declined during follow-up in all three cohorts. Faster decline in PF over time was found in Russian female frequent drinkers, Polish female moderate drinkers, and Polish male regular heavy drinkers, in comparison with regular and/or light-to-moderate drinkers. Nondrinking was associated with a faster decline compared with light drinking only in Russian men. Problem drinking and past drinking were not related to the decline rate of PF.This large longitudinal study in Central and Eastern European populations with relatively high alcohol intake does not strongly support the existence of a protective effect of alcohol on PF trajectories; if anything, it suggests that alcohol consumption is associated with greater deterioration in PF over time. Physical functioning (PF) is a key indicator of older persons’ health status and is strongly related to their quality of life. Decline in PF is a consequence of age-dependent physiological changes and onset of diseases and can be modified by medical care, socioeconomic, environmental, psychosocial, and behavioral factors . ChangesCross-sectional studies have shown a protective effect of light-to-moderate drinking on PF , yet theThe rapid population aging in CEE brings major public health challenges, given the often inadequate provision of health services and long-term social care and low private savings . CompareIn this study, we investigated individual PF trajectories in three aging cohorts in CEE over approximately 10 years and how the trajectories were influenced by alcohol consumption, problem drinking and past drinking behavior.We used data from the Health, Alcohol and Psychosocial factors In Eastern Europe (HAPIEE) study in Novosibirsk (Russia), Krakow (Poland), and seven middle-sized towns in the Czech Republic  . In 2002PF was measured at all four occasions using the Physical Functioning Subscale (PF-10) of the Short-Form-36 instrument . The PF-Alcohol consumption in the 12 months before baseline was assessed using a graduated frequency (GF) questionnaire . Six driAverage drinking frequency, annual drinking volume, and average drinking quantity per drinking day were calculated from the GF using midpoints of drinking quantities and corresponding drinking frequencies. Average drinking quantity per drinking day was categorized into non, light, moderate, and heavy drinking . DrinkiAmong Russians, past drinking was determined by a question asking whether participants had cut down their drinking frequencies before baseline. Nondrinkers identified by the GF were then separated into former drinkers and lifetime abstainers. Current drinkers were split into those who had reduced drinking and those who had maintained drinking. According to self-reported reasons for reducing drinking, former drinkers and reduced drinkers were further categorized into due to health versus nonhealth reasons.Several baseline characteristics were included as covariates. Age was classified into 5-year groups. Marital status was dichotomized into married/cohabiting versus others. Educational attainment , sum of ownership of 12 household amenities , and economic activity were selected to reflect participants’ SES. Economic activity included four categories: working, pensioners but still employed, pensioners without employment, and unemployed. Spine/joint problems were based on self-reported diagnosis/hospitalization for a disease of spine or joints in the past year. Objectively measured height (m) and weight (kg) were used to calculate body mass index. Smoking status was coded as never, former, and current smoking.Supplementary Table 1). Missing data were imputed using multiple imputation by chained equations, and 70 imputed data sets were generated in Stata 12   . Most paplus 6.0 . Pooled estimates based on these imputed data sets are presented. Two growth parameters describe the trajectories: initial status (PF-10 score at baseline) and slope (rate of change in PF-10 score per year of follow-up). Maximum likelihood estimation with robust standard errors was used due to the non-normality of the PF-10 scores. Two models were fitted by cohort and sex separately for each drinking index, adjusting for (i) age (Model 1) and (ii) age, marital status, education, household amenities, economic activity, spine/joint problems, body mass index, and smoking status (Model 2). In additional models, we also examined the interaction between alcohol consumption indices and socioeconomic factors.Intra-individual PF-10 changes (trajectories) over the 10-year follow-up and interindividual variations in the trajectories were estimated using growth curve modeling . The shaSupplementary Table 1. Baseline PF-10 scores were higher among men than among women and declined during follow-up in both sexes. More men were drinkers than women, and they drank more frequently and heavily. Very few women were identified as problem drinkers; thus, the relationship between problem drinking and PF-10 trajectories was not examined among women.p < .001). Compared with the age-adjusted models (Supplementary Table 2), some associations between alcohol consumption and the slope became statistically insignificant after multivariable adjustment. In most country-sex groups, the slope did not significantly differ across drinking categories. The PF-10 scores in Polish male regular heavy drinkers declined more steeply than in regular light-to-moderate drinkers . Polish female moderate drinkers were also found experiencing a faster decline than light drinkers . In Russian men, compared with light drinking (average drinking quantity/drinking day), nondrinking was related to a faster decline , but this was not replicated in other drinking indices. Multivariable-adjusted results on drinking frequency and annual drinking volume are provided in Supplementary Table 3. Among Russian women, a more rapid PF decline was observed in those who drank ≥1/week versus 1–3/month . We found no significant associations of the slope of PF decline with problem drinking among male drinkers or with past drinking behavior among Russians. This pattern was replicated after further categorizing past drinking by drinking pattern (Supplementary Table 4).Czech participants’ PF-10 scores declined more slowly than among their Russian and Polish counterparts  (Supplementary Table 5). There were no statistically significant interactions between SES (education and ownership of household amenities) and alcohol consumption on the slope of PF decline (not shown in table).Further adjustment for baseline health conditions reduced the differences in the baseline PF-10 scores between drinking categories, but the associations of alcohol with the slope changed very little .Several limitations in this study need to be acknowledged. First, measurement of alcohol intake in epidemiological studies is subject to measurement error ,28. The Supplementary Table 7).Second, participants might overreport their PF, particularly when being interviewed, because of the stigma attached to being unhealthy . In earlThe third methodological concern is residual confounding. In western societies, both abstainers and heavy drinkers tend to have lower SES, poorer health, poorer social network, and less favorable behavior than moderate drinkers, and it is difficult to entirely control for all these factors . In our Finally, middle-class individuals with relatively favorable drinking patterns and health profile are likely to be overrepresented in studies ; whereasTo our knowledge, this is one of the first investigations of the relationship between alcohol consumption and PF trajectories in aging populations. The multicenter design optimizes the cross-country comparability in this study. Unlike most existing studies, we examined several aspects of alcohol consumption, including drinking patterns, which may have a major influence on health in CEE, particularly in Russia . Data onOur findings are generally consistent with a recent study that found no associations of alcohol consumption with either the onset or rate of change in the severity of functional limitations in middle-aged Americans over 10 years . By contThe discrepancies in PF decline between the three cohorts may reflect a better population health in the Czech Republic than in Russia and Poland. This is exemplified by the highest life expectancy reported in the Czech Republic, followed by Poland, and lastly Russia . In addiAn important issue to consider is the potential bidirectionality of the association between alcohol and PF. Heavy drinkers at baseline might have been able to maintain high consumption because of their good health. During follow-up, once their health deteriorated, they might reduce or stop drinking, and the process of PF decline may slow down or even reverse. This bidirectional relationship may contribute to heavy drinkers’ good PF at baseline but no consistently faster decline during follow-up. We could not explore such bidirectionality because alcohol consumption was not measured repeatedly in this study. Future studies with repeated measurements of alcohol consumption therefore are needed.PF declined during 10-year follow-up in all three aging cohorts, but alcohol consumption was not consistently associated with the decline rate. These longitudinal analyses did not provide strong support for the existence of protective effect of alcohol shown in cross-sectional studies; if anything, they suggested that alcohol consumption was associated with faster PF decline over time. Considering the detrimental effect of alcohol consumption on numerous diseases , it seemhttp://gerontologist.oxfordjournals.org/ to view supplementary material.Please visit the article online at Wellcome Trust “Determinants of Cardiovascular Diseases in Eastern Europe: Longitudinal follow-up of a multi-centre cohort study” (The HAPIEE Project) (Reference number 081081/Z/06/Z); MacArthur Foundation “Health and Social Upheaval (a research network)”; and the U.S. National Institute on Aging “Health disparities and aging in societies in transition (the HAPIEE study)” (Grant number 1R01 AG23522). S.B. is supported by the European Research Council (ERC-StG-2012-309337) and UK Medical Research Council/Alcohol Research UK (MR/M006638/1).The HAPIEE study was supported by


Previous studies revealed that thymus is a targeted immune organ in malnutrition, and high-boron stress is harmful for immune organs. African ostrich is the living fossil of ancient birds and the food animals in modern life. There is no report about the effect of boron intake on thymus of ostrich. The purpose of present study was to evaluate the effect of excessive boron stress on ostrich thymus and the potential role of TLR3/4 signals in this process. Histological analysis demonstrated that long-term boron stress (640 mg/L for 90 days) did not disrupt ostrich thymic structure during postnatal development. However, the numbers of apoptotic cells showed an increased tendency, and the expression of autophagy and proliferation markers increased significantly in ostrich thymus after boron treatment. Next, we examined the expression of TLR3 and TLR4 with their downstream molecular in thymus under boron stress. Since ostrich genome was not available when we started the research, we first cloned ostrich TLR3 TLR4 cDNA from thymus. Ostrich TLR4 was close to white-throated Tinamou. Whole avian TLR4 codons were under purify selection during evolution, whereas 80 codons were under positive selection. TLR3 and TLR4 were expressed in ostrich thymus and bursa of fabricius as was revealed by quantitative real-time PCR (qRT-PCR). TLR4 expression increased with age but significantly decreased after boron treatment, whereas TLR3 expression showed the similar tendency. Their downstream molecular factors  did not change significantly in thymus, except that p100 was significantly increased under boron stress when analyzed by qRT-PCR or western blot. Taken together, these results suggest that ostrich thymus developed resistance against long-term excessive boron stress, possibly by accelerating intrathymic cell death and proliferation, which may bypass the TLR3/4 pathway. In addition, attenuated TLRs activity may explain the reduced inflammatory response to pathogens under boron stress. Thymus is the primary immune organ for T cell development in jawed vertebrates . Normal Struthio camelus) is a terrestrial flightless giant bird and regarded as the living fossil of primitive birds, as it could be a good model for evolutionary research of ancient avians [The African ostrich  are major components of innate immune system in that they can recognize multiple endogenous and exogenous stress signals and induce an immune response . TLRs arThough there is no direct evidence that boron derived compound can be recognized by TLRs, boron stress can increase intracellular stress signals, such as the subunit of heat shock proteins , which aThis study was approved by the ethics committee of Huazhong Agricultural University . Protocols for animal were performed according to pertinent guidelines . Exsanguination and decollation were used for humane form of euthanasia. All efforts were made to minimize animal suffering.Struthio camelus) were obtained from Jinlu Ostrich Co., Ltd. . Animals were randomly divided into 3 groups , and housed under conventional conditions without any vaccinations. Group 45d and 90d received distilled water while Group 90dB received natrium biboricum through drinking water at 640 mg/L dose daily which is known to be excessive and toxic for immune related organs [Healthy 1-day-old African ostriches  ratio as described , and theTotal RNA was isolated from thymus using TRIzol reagent (TaKaRa). First strand cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) and DNase I (Thermo Scientific). Degenerative PCR primers for ostrich TLR4 and TLR3 were designed based on the multiple alignments of previously reported sequences in birds . The firThe qRT-PCR primers for TLR3, TLR4 and actin were designed from our cloning results , since the ostrich genome was not available when we started the research. The qRT-PCR primers for p100, IRF1, IL6 and IFN were designed from subsequent ostrich genomic information .Amino acid sequences were deduced using the NCBI ORF finder program. The protein domains were predicted using the SignalP 4.1, TMPRED and SMART programs. The genomic structure of TLR4 gene was deduced from mRNA and DNA sequence using NCBI SPIDEY program. For adaptive evolution analysis, the 36 known TLR4 sequences of bird were retrieved from GenBank  Tables, Expression patterns of TLRs and related genes in ostrich tissues were analyzed based on quantitative real time PCR (qRT-PCR) using β-actin as the reference gene. All the primer sequences, TLR3, TLR4, P100, IRF1, IL-6, IFN and β-actin were described in Proteins were homogenized in lysis buffer containing a protease inhibitor. Protein concentration was measured by the Bradford method using BSA as standard. Samples of 40 μg of total protein were separated by SDS–PAGE and electrotransferred to a PVDF membrane . Immunodetection was performed using antibodies against TLR4 (1:500) from Boster , JNK (1:500) and Actin (1:1000) from Santa Cruz Biotechnology , ERK (1:1000) from Bioworld , P38 (1:1000) from Epitomics , and the reliability of these antibodies were provided in t-test. All analyses and graphic representations were performed with Prism software 5.01. Data trends are marked with one asterisks (*) for P < 0.1 and two asterisks (**) for P < 0.05 respectively. P < 0.05 was considered significant.Data were presented as mean ± SEM,with samples derived from 3 individuals in each group. Significant differences between groups were studied using a Student’s In previous studies a toxic effect of boron disrupting the immune organs’ structures was shown in other animals when the concentration was more than 400 mg/L , 13. HerP < 0.1) (P < 0.1)  . In boroP < 0.1)  comparedP < 0.05) in normal ostrich developing thymus (P < 0.05)   in boronP < 0.05) (P < 0.05)  . Under b < 0.05) .Considering the possibility of TLR signals in participating ostrich thymic boron stress, we analyzed the expression of TLR3 and TLR4, the two classic ones of TLRs. TLR3 can recognize both host mRNA and viral double stranded RNA, whereas TLR4 can recognize bacterial lipopolysaccharide and endogenous heat shock proteins . At the The partial TLR3 (KM651767) and TLR4 sequence are 712 bp and 546 bp, encoding 236 and 182 amino acids (aa) respectively . These tThe proteins ID from GenBank are as follows: duck TLR3 XP_005009038.1; chicken TLR3 NP_001011691.3; turtle TLR3 XP_006128521.1; human TLR3 NP_003256.1; zebrafish TLR3 NP_001013287.2; ostrich TLR3 XP_009674996.1; duck TLR4 AER24529.1; chicken TLR4 NP_001025864.1; turtle TLR4 NP_001273862.1; human TLR4 NP_612564.1; zebrafish TLR4 NP_997978.1; ostrich TLR4 XP_009666473.1. The proteins for ostrich TLR 3 (KM651767) and TLR4 (KM408431) deduced from our cloning result were indicated in Since the ostrich genome was available after we cloned ostrich TLRs, we adopted ostrich genomic scaffold (KL205999.1) from GenBank and our cloning result to deduce the genomic sequence of ostrich TLR4. The ostrich genomic TLR4 is 5164 bp, consists 2 introns (813 and 1370 bp) and 3 exons , the same as that of duck, chicken, turtle, human and zebrafish . The codStruthio camelus) was more closely related to that of white-throated Tinamou (Tinamus guttatus), both of which belong to the superorder Palaeognathae. This result was in accordance with conventional taxonomy of birds.Phylogenetic trees revealed that TLR4 sequences from birds were more closely related to reptiles than those of mammals and fish . In addiPrevious studies revealed an episodic positive selection of TLR4 (only 270 codons in ECD) in 18 avian species  Fig 6B)Fig 6B). P <0.05)  .The tissue distribution of TLR4 in ostrich thymus and bursa of fabricius was studied subsequently, since there are available antibodies against human TLR4 which has been used to identify avian TLR4 . In ostrFurthermore, we adopted the reliable avian antibodies against PCK  and S100P < 0.05) (P < 0.1) (P < 0.05) (P < 0.1)  , whereasP < 0.1) . Unexpec < 0.05) , and TLRP < 0.1)  in boronP < 0.05) after boron treatment  , ostrichApoptosis, autophagy and proliferation are the classic process to determine the fate of thymic cells. Increasing apoptosis and defective autophagy are associated with the abnormal development of thymus , 30, andThe phylogenetic tree suggested that theTLR4 sequence was conserved among species and ostrich TLR4 was closely related with ancient species, which indicated that ostrich could be a good model for evolutionary analysis in birds. The evolutionary analysis of full-length TLR4 in 37 birds showed that purifying selection occurred across all TLR4 protein, and positive selection on some codons, which is the same as the previous study . MoreoveBoth the transcripts of TLR3 and TLR4 were detected with different levels in ostrich thymus and bursa of fabricius, the central immune organ for the development of T cells and B cells respectively. TLR3 was preferentially expressed in bursa of fabricius, whereas TLR4 expressed in ostrich thymus. Their differential expression pattern in primary immune organs may reflect the diverse demands of TLRs in these organs. Using tested antibody against avian TLR4, we found that TLR4 was distributed in specific regions of both thymus and bursa of fabricius. In thymus, TLR4 was mainly observed in medulla and cortico-medullary junction, the later developing region for thymocytes . In bursFurthermore, in normal thymus, TLR4 expression increased significantly, and TLR3 expression showed an increased tendency with age, similar to what had been described in bird and mammals , 17, 37.In boron-stress thymus of ostriches, TLR4 expression decreased significantly and TLR3 expression decreased modestly compared to those in normal developing thymus at day 90. There is no change in the proinflammatory transcriptional regulators  and proinflammatory cytokines (IFN and IL6) following boron stress. However, the transcriptional regulator (p100) increased. Impaired expression TLRs are associated with inhibition of TLRs function , 40, andIn conclusion, long-term excessive boron intake did not change ostrich thymic organization. The resistance of ostrich thymus against long-term boron stress may rely on the accelerating intrathymic cell renew and death, which may bypass TLR3/4 pathway, since TLR3 and TLR4 expression decreased after boron treatment. The decreased TLRs activity may explain the immune suppression under boron stress in other studies. Yet, the precise mechanisms for regulating thymic cell fate and immune response under boron stress deserve further investigation.S1 FileFigure A) and PCK-positive signals  were found in the similar regions in adjacent sections of ostrich thymus at day 90. TLR4-positive signals  and S100-positive signals  were also found in the similar regions in adjacent sections of ostrich thymus at day 45. However, TLR4-positvie signals were not observed in eosinophils in ostrich thymus . The regions marked by black arrows in Figure E are enlarged in right corner of this panel. Bars, 100 μm.The TLR4-positive signals ((TIF)Click here for additional data file.S1 TableThe identities between the targeted antigen sequences of those antibodies and those found in ostrich were compared using NCBI BLAST program.(DOC)Click here for additional data file.S2 TableMeleagris gallopavo were updated in December 2014.Sequences XM_003211211 (TLR4) and XM_003205774 (TLR3) for (DOC)Click here for additional data file.S3 TableFor adaptive evolution analysis, we adopted 37 TLR4 mRNA sequences from birds, including the 30 known ones, except that from ostrich, listed in this table, the 6 ones listed in (DOC)Click here for additional data file.S4 TableFor compared genomic structure of TLR4 genes, we used 6 genomic sequences of TLR4, including 5 ones from zebrafish, human, chinese soft-shell turtle, chicken and mallard listed in this table, and one from ostrich, which was deduced from our cloning TLR4 mRNA (KM408431) and ostrich genomic scaffold (KL205999.1) provided by BGI. For phylogenetic analysis, we adopted 49 TLR4 protein sequences from different species, including the 13 ones listed in this table, the 35 ones except that of ostrich listed in (DOC)Click here for additional data file.


Despite the fact that UNICEF declared freedom from hunger and malnutrition a basic human right in 1948, more than 20 million children were severely malnourished in 2010 and a further 170 million were stunted. Malnutrition attributes to >50% of child deaths by potentiating infectious diseases.The aim of this study was to determine the extent of malnutrition in Grade 1 pupils in public sector schools in Bloemfontein. An objective of the study was to identify relationships between socio-economic parameters and malnutrition.Grade 1 pupils from ten public schools in Bloemfontein, selected from a random table, were included in the study. Their parents/caregivers gave informed consent and completed a questionnaire regarding baseline characteristics and feeding practices at home. The children were then weighed and measured, and the 2007 WHO Growth Reference for school-aged children and adolescents used as reference.A total of 187 children were included in the study. The combination of underweight, wasting and stunting gave an 18% prevalence of malnutrition in this study. A BMI of less than the fifth percentile occurred in 27% of the pupils. Factors positively associated with malnutrition included: Absence of a fridge and/or running water in the house and low education and/or unemployment of parents. Illness in the previous month was reported by 41% of the malnourished children.As socio-economic factors that contribute to malnutrition are now known, teachers will be able to identify and refer children with or at risk of malnutrition and indirectly decrease child mortality. Most of these deaths (83%) occurred in mildly and moderately malnourished children and not in the severely malnourished group.The World Health Organization (WHO) defines malnutrition as ‘the cellular imbalance between supply of nutrients and energy and the body's demand for them to ensure growth, maintenance, and specific functions’.5 as well as a study in Sri Lanka6 demonstrated a relationship between socio-economic status and malnutrition in pre-school children in developing countries. Several studies8 linked malnutrition with low income, unemployment, low educational status of parents, poor sanitation, type of housing and number of people per household.A recent study by Armstrong et alIf teachers are aware of the specific risk factors for malnutrition they can identify these children and also focus their education programmes on practical issues regarding diet and nutrition. In this way they can indirectly assist in reducing child mortality.Malnutrition in children attributes to 54% of child deaths by potentiating infectious diseases. This study identified risk factors for malnutrition in Grade 1 pupils and will therefore enable teachers to refer and support children at risk and thereby indirectly contribute to a decrease in child mortality.The primary aim of this study was to determine the extent of malnutrition and particularly under-nutrition in Grade 1 pupils in public sector schools in Bloemfontein. An objective of the study was to identify relationships between socio-economic parameters and malnutrition.Malnutrition is a preventable and treatable condition, but it can have serious consequences if it is not managed adequately. If the risk factors for malnutrition are known, teachers will be able to identify children at risk and refer them for proper treatment. Schools will also be able to implement feeding programmes for the children with malnutrition as well as those at risk.Grade 1 pupils from six to seven years of age, attending public schools in Bloemfontein were our study population. From the 46 public schools in Bloemfontein (5893 Grade 1 pupils), 10 were selected using a random number table. One Grade 1 class per school was then chosen for the study according to a random number table. The exclusion criterion was Grade 1 pupils who had not obtained consent from the school and/or a parent/guardian.Bloemfontein is situated in the centre of South Africa and is the capital city of the Free State Province. Together with Botshabelo and Thaba Nchu it forms the metro of Mangaung.This was a cross-sectional study where Grade 1 pupils were weighed and measured to obtain their anthropometric data and parents or caregivers supplied demographic information by means of filling in a questionnaire.A pilot study was conducted at one school to test the questionnaires. Twenty-five questionnaires were filled in and returned. The questionnaire was then changed slightly and therefore the initial results were not included in the actual study. Validity was accepted at face value.Thereafter the principals of each of the selected schools were contacted and the study was explained to them. All gave their written consent for the study. The consent forms and questionnaires developed by the researchers were distributed to the selected Grade 1 pupils. Consent forms as well as questionnaires were supplied in the learner's language of choice – Afrikaans, English or Sotho – to be completed by parents or guardians at home. The questionnaires were returned to the school in sealed envelopes to protect confidentially. In this way demographic data, as well as information on the number of people and children per household, type of housing, sanitation and availability of water, electricity and fridges were collected. Information regarding the child's food consumption and the parents’ education level was also obtained. On the pre-arranged day the researchers then weighed and measured all the children who had obtained consent for the procedure and noted their measurements on the questionnaires. Standardised methods were used for the weight and height of each child, with a calibrated scale and a non-stretchable measuring tape attached to a wall. Data were collected during 2006 and analysed only after the 2007 guideline became available.The study was approved by the Ethics Committee of the Faculty of Health Sciences of the University of the Free State as well as the Department of Education in the Free State (ETOVS 23/04).9 which is regarded as the standard to measure the nutritional status of preschool children. The WHO Growth Reference for school-aged children and adolescents10 was published in 2007 and it was used as reference in this study, together with body mass index (BMI) charts specific for age and gender. The following definitions, quoted from the above guidelines, were used in this study:Underweight: Weight-for-age less than two standard deviations below the median reference value. Underweight reflects a combination of acute and chronic malnutrition.Wasting: Weight-for-height less than two standard deviations below the median reference value. Wasting is considered an indication of acute or recent malnutrition.Stunting: Height-for-age less than two standard deviations below the median reference value. Stunting is considered an indication of chronic malnutrition.Overweight: A BMI above the 85th percentile and lower than the 95th percentile for children of the same age and sex.Obesity: A BMI at or above the 95th percentile for children of the same age and sex.In 2006 WHO published the WHO Child Growth Standards,2). A BMI of less than the fifth percentile is considered as underweight according to WHO as well as the Centers for Disease Control and Prevention (CDC).The body mass index (BMI) for age and sex is calculated as weight (kg)/height  males and 45% (84) females. No child in this study was overweight-for-age or obese.p-values >0.05. Nine per cent of the pupils were underweight.The results of the categories of malnutrition, namely weight-for-age, height-for-age, weight-for-height and gender are displayed in Stunting, which indicated chronic malnutrition, occurred in four per cent of the children. A weight-for-height calculation of minus two to minus three of the SD for median reference value, indicative of acute weight loss, occurred in 11% of the children. A total of 34 pupils (18%) presented with at least one of the three forms of malnutrition. A BMI of less than the fifth percentile occurred in 27% of the pupils.p-value of <0.05 was indicative of a statistically significant difference between two measurements. This information was not available for all the children; it was only available for 153 fathers and 168 mothers. The information was, however, available for all the malnourished children.The presence of running water to prepare hygienic food and a fridge to store the food safely was associated with less malnutrition as displayed in p-value of 0.007.The majority of the children ate breakfast (82%), lunch (92%) and dinner (96%) on a daily basis and these factors were not associated with malnutrition. Twenty-four children did not take food to school and did not eat snacks between meals; 50% of these children were malnourished. This factor had a significant association with malnutrition with a The positive association between malnutrition and unemployment and poor education is demonstrated in p = 0.02), wasting (p = 0.04) and being underweight (p = 0.02).Illness during the previous month was reported by 41% of the malnourished children. This factor was significantly associated with stunting  in Grade 1 pupils in Bloemfontein is comparable to the 14% for the Free State which was recorded by the National Food Consumption Survey (NFCS-FB_I) of 2005, if the fact that this study was performed in an urban area is taken in consideration,The association of malnutrition with the absence of water and fridges was statistically significant in this study population. The presence of water in the house provides better opportunities for the hygienic preparation and consumption of food and a fridge keeps food fresh for longer periods. This may also be an indirect measurement of improved socio-economic circumstances.14 The 50% prevalence of malnutrition in children who did not take food to school or eat snacks between meals is something that needs to be addressed as nutritional snacks may improve the children's nutritional status. The type and amount of food that the children ate and took to school were not identified in this study.It was encouraging to see that most children ate at least three times a day. A concern is the 34 children (18%) who did not eat breakfast before they went to school, although this factor did not contribute to malnutrition. However, the recommendation for children of this age is three meals plus two to three snacks a day.17 Employment and education of parents were identified as protective factors against malnutrition in this study. ‘Educate the girl child to save the next generation from malnutrition’.6Parents’ low education and unemployment levels were significantly associated with malnutrition. It needs to be kept in mind that education and employment provide opportunities to earn money and to support the family. Unemployment and/or low education levels in mothers were identified in the study as important factors contributing to malnutrition. Mothers’ low education levels as a cause of malnutrition was demonstrated in numerous studies.Illness during the previous month was identified as a major contributing factor in malnutrition. Illness during the previous month occurred in 41% of the malnourished children. It is, however, difficult to determine causes and results here, as malnutrition can lead to suppressed immunity, which can lead to disease and as disease can cause malnutrition because of poor food intake during illness. Although BMI is used more often to diagnose and monitor overweight, it can also indicate rapid weight loss as weight changes occur more rapidly than changes in length over a short period of time, e.g. during a time of acute illness. A BMI of less than the fifth percentile, which occurred in 27% of the pupils, may be an indication of acute weight loss because of an illness during the previous month, rather than of chronic malnutrition, as the latter occurred only in five per cent of the children.6 and Bangladesh.7 A study in China18 did not find a relationship between the number of children in the household and malnutrition, either.The ownership of a house, type of house, number of people in the house, number of children in the household and sanitation method could not be linked to malnutrition in this study. It has, however, been linked to malnutrition in studies performed in Sri LankaThe response rate of 67% gave us a confidence interval of 7.1% and a standard error of 3.6% which is a little low, but still acceptable for this study at a confidence level of 95%.Standardised methods and equipment were used to ensure accurate measurements, but measurement errors were still possible.The questionnaires did not include information on the type or amount of food that was given for meals or snacks, but only on how often food was eaten.Although the questionnaires were anonymous the accuracy of the information supplied could not be guaranteed.It was difficult to compare the results of different studies as different parameters for malnutrition were used in different studies and some studies did not indicate which measurement/s they used.Grade 1 teachers should know the risk factors for malnutrition and identify and appropriately refer these children.Feeding programmes at schools should focus on children at risk and also on snacks between meals.The Department of Education should adjust the school curriculum so that the subject of a healthy diet is introduced from the beginning of the school years. The Department should also keep children in school to improve their education and to prevent malnutrition in future.Unemployment and the lack of water and electricity in households should be priorities for local, provincial and national government.6It bears repeating: ‘Educate the girl child to save the next generation from malnutrition’.Although the 18% prevalence of malnutrition in our study population was within the global norms, it means that one in every five Grade 1 pupils suffers from malnutrition. This makes them more prone to infections, which, in turn, can lead to early morbidity.Different factors influence malnutrition in different parts of the world. In the Bloemfontein region the absence of water and fridges in houses, parents’ unemployment and low education levels and the fact that children did not take food to school were the major factors associated with malnutrition. Illness during the previous month was also strongly associated with malnutrition.


Excessive alcohol use is a prevalent and worldwide problem. Excessive drinking causes a significant burden of disease and is associated with both morbidity and excess mortality. Prototype alteration and provision of a cue reminder could be useful strategies to enhance the effectiveness of online tailored interventions for excessive drinking.Through a Web-based randomized controlled trial, 2 strategies  within an existing online personalized feedback intervention (Drinktest) aimed to reduce adults’ excessive drinking. It was expected that both strategies would add to Drinktest and would result in reductions in alcohol consumption by intrinsic motivation and the seizure of opportunities to act.Participants were recruited online and through printed materials. Excessive drinking adults (N=2634) were randomly assigned to 4 conditions: original Drinktest, Drinktest plus prototype alteration, Drinktest plus cue reminder, and Drinktest plus prototype alteration and cue reminder. Evaluation took place at 1-month posttest and 6-month follow-up. Differences in drinking behavior, intentions, and behavioral willingness  were assessed by means of longitudinal multilevel analyses using a last observation carried forward method. Measures were based on self-reports.P<.05) and cue reminder usage  were both more effective in reducing alcohol consumption than when these strategies were not provided. Combining the strategies did not produce a synergistic effect. No differences across conditions were found regarding intentions or willingness.All conditions showed reductions in drinking behavior and willingness to drink, and increased intentions to reduce drinking. Prototype alteration : 4169; http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=4169 (Archived by WebCite at http://www.webcitation.org/6VD2jnxmB). Excessive alcohol use is a prevalent and worldwide problem . In the It is important to further our understanding of how to reduce excessive drinking. A large number of interventions have targeted drinking behavior assuming that behavior is intentional. However, medium-to-large changes in intentions only lead to small-to-medium changes in behavior . Effect Two main reasons may account for the small-to-medium (or lack of long-term) effects. First, individuals may not be fully aware of the opportunities of how to act on their intentions. For example, in the case of drinking behavior, the individual may intend to limit his alcohol consumption. The person needs to be aware of, for example, opportunities and resources to accomplish this limitation, such as responses to others to resist drinks when offered. As a result, many studies and interventions have focused on helping people act on their intentions , acknowThis study addresses these issues by examining the effect of 2 intervention strategies that could potentially help enhance the effect of an existing online  tailored intervention, Drinktest.nl: prototype alteration and cue reminders. Drinktest is based on the TPB , I-ChangDrinktest was developed by the Netherlands Institute for Health Promotion and Disease Prevention (NIGZ). Drinktest offers brief personalized feedback regarding an individual’s personal alcohol consumption patterns. The intervention consists of various components: overview of mean weekly alcohol intake, associated health risks, self-help guidelines to reduce alcohol intake, and normative feedback to compare one’s own alcohol consumption to the level of one’s own cohort.The first strategy that could potentially enhance the effect of Drinktest is prototype alteration. Prototypes refer to the mental image of a typical person engaging in a certain behavior ,18, suchPrototypes have been shown to explain behavior through their effect on willingness and intentions and have also been shown to directly explain drinking behavior -31. The A second strategy is the use of cue reminders. The limited number of studies focusing on cue reminders has shown that cue reminders can help in changing (and maintaining) behavior -45 becauThis study examined whether prototype alteration and provision of a cue reminder can be useful strategies to enhance the effectiveness of an existing online  tailored intervention, Drinktest. Drinking behavior, intentions to reduce drinking, and willingness to drink were targeted as primary outcomes. It was expected that (1) prototype alteration may intrinsically motivate people to drink less, (2) cue reminders may strengthen the salience of alcohol reduction goals, and (3) the combination of prototype alteration and a cue reminder may produce a synergistic effect and thus increase the salience and intrinsic motivation to drink less. As such, we tested whether the strategies of prototype alteration and a cue reminder in addition to the Drinktest intervention would be more effective in addressing excessive drinking behavior than the original Drinktest without those additional strategies. Other outcomes are also addressed, as will be described subsequently.A randomized controlled trial was conducted in the Netherlands in which participants were randomly assigned by computer to 1 of 4 conditions: (1) original Drinktest, (2) Drinktest plus prototype alteration, (3) Drinktest plus cue reminder, and (4) Drinktest plus prototype alteration and cue reminder (further referred to as the “combined condition”). The online tailored intervention consisted of baseline measurements and tailored feedback. Follow-up measurements were conducted at 1 and 6 months (postintervention: T2 and T3). Eligible participants were individuals aged 18 or older engaging in excessive drinking: exceeding ≥14 and ≥21 glasses of alcohol per week or drinking ≥4 and ≥6 glasses per occasion for women and men, respectively . This noParticipants were recruited online and by printed materials (posters and newspaper advertisements) from September 2012 to June 2013. The website of Drinktest was also easily accessible by online search engines. Before entering the intervention (T1), participants read the study information and were told that the existing Drinktest website was being evaluated. It was explicitly stated that participants did not have to commit themselves to reducing their alcohol consumption. Participants were then asked to sign the online informed consent form. In case participants declined to participate, they could close the browser or receive the original Drinktest without taking part in the study. After the informed consent form had been signed, participants were randomized into the conditions. Nonexcessive drinkers (of which the status was known only after drinking behavior was measured) were excluded from the study sample and routed to the original Drinktest.All questions were self-administered and data were collected online. Participants were invited by email to participate in the 2 follow-up measurements and received reminders if necessary (maximum of 3). Participants were invited for the 6-month follow-up irrespective of their participation in the 1-month follow-up. A total of 50 vouchers worth €50 were distributed (by means of a raffle) as incentive. Ethical approval was granted by the independent ethics committee of the Erasmus MC, Rotterdam, the Netherlands (ref no: MEC-2010-112).Power analyses using G*Power3  were peParticipants in the original Drinktest condition only received the standard version, in which they received feedback tailored to demographic background (gender), alcohol consumption, and intentions to reduce drinking. These messages reflected on personal drinking levels in comparison to the Dutch norm and peers’ drinking behavior, the correctness of their absolute and relative perceived drinking risks regarding health risks due to their alcohol consumption, intentions, temptations , correctness of positive effects of alcohol , and correctness of negative effects of alcohol . To improve self-efficacy participants were encouraged to make a plan (without guidance) or to balance the advantages and disadvantages of reducing alcohol consumption. This part took approximately 10 minutes . MultimeAfter completing the original Drinktest, participants in the prototype condition received feedback regarding prototype alteration  to aParticipants selected action plans rather than forming their own because (1) forming plans of good quality has proven to be difficult for participants  and (2) After finishing the original Drinktest modules, cue condition participants followed the same procedure in forming action plans as in the prototype condition . FeeParticipants in the combined condition completed the original Drinktest modules, the prototype alteration module, and the cue module see . These pAll measurements included the same questions and followed the same guidelines for drinking norms unless otherwise specified. Measures from the original Drinktest were left unchanged and items regarding demography, willingness, prototypes, cue reminder, and process evaluation were added.Participants reported on their appreciation of the intervention at the 1-month posttest by answering the statement: “The information and advice of Drinktest.nl were...” Answers ranged from 1=I disagree to 7=I agree regarding reliability, novelty, being informative, ease of understanding, personal relevance, persuasiveness, enjoyability, and usefulness (α=.86).At 1-month posttest, all participants were asked, regarding the past 4 weeks (1) how aware they had been of their alcohol use, (2) how often they had contemplated on the intervention’s feedback, and (3) their perception of having tried to reduce their alcohol consumption. Finally, we checked whether participants had correctly remembered their choice of cue, how aware they were of their cue, and how often they had worn or used the cue reminder. Answers to the Likert scales ranged from 1=not at all to 7=a lot.These items were assessed only at baseline among the prototype and combined conditions because it was part of their manipulation and feedback. Participants were asked to characterize themselves by prototypical characteristics. That is, prototypes are usually assessed by a list of characteristics describing them eg, ,52). In . In 52])Drinking behavior was assessed by the Dutch version of the Quantity-Frequency-Variability (QFV) index of alcohol intake , which aTo assess intentions, the item was framed by Drinktest in behavioral stages in which participants chose from the following options: (1) I do not plan to reduce my alcohol consumption, (2) I plan to reduce my alcohol consumption within half a year, (3) I plan to reduce my alcohol consumption within a month, (4) I already started reducing my alcohol consumption, and (5) I have reduced my alcohol consumption more than half a year ago. This single item was treated as a continuous variable.r=.76-.85). Answers to the second statement were reversed.Willingness was assessed by describing a scenario with 2 possible actions : “ImAbsolute perceived drinking risks was assessed with the item “With regard to my health, I consume too much alcohol” rated from 1=I disagree to 3=I agree. Relative perceived drinking was assessed with the item “Compared to [women/men] of my age, I drink...” rated from 1=a little to 3=a lot.Attitude was examined by the original Drinktest using 12 items measuring advantages and disadvantages of drinking alcohol regarding health, sociability, and coping. For instance, “My alcohol use is healthy for my heart and veins” rated from 1=yes, healthy to 3=no, unhealthy and “My alcohol use is a bad example to others” and “My alcohol use is bad for my liver” both rated from 1=yes, bad to 3=no, good. If needed, items were reversed so that a higher score represented a more positive attitude toward drinking. Because reliability over the 12 items was low, principle component analysis was performed revealing 2 factors. Only the first factor  was used in analyses (T1-T3: α=.73-.78) because the second factor still had low reliability (T1-T3: α=.35-.43).A single item assessed self-efficacy: “I find reducing my alcohol use” rated from 1=very hard to 5=very easy.Twelve items examined temptations, which regarded emotions, coping, habit, and social situations, such as “How tempting do you find it to drink alcohol when you are at a party or in a restaurant?” with answers ranging from 1=not tempting at all to 5=very tempting (T1-T3: α=.86-.87).All analyses were performed in SPSS 21.0 . First, we determined whether dropout between baseline and follow-up measurements was different for condition, gender, age, ethnicity, level of education, intentions, willingness, and drinking behavior. Second, potential differences between conditions at baseline were assessed regarding these measures. Third, the process evaluations were assessed. Fourth, longitudinal multilevel analyses (mixed models) were performed using the last observation carried forward (LOCF) method (1) to account for dropout and (2) because of the nested design . Using the LOFC method implies that if data for a follow-up measurement were missing, then data from the previous known data were used for analyses. For example, if data were available for the first and third measurements and the second was missing, then the data from the first measurement were also used as the second measurement instead of treating this data as missing. It should be noted that reported descriptives are based on LOCF.Following previous research , a multiThe following independent variables were included in the multilevel longitudinal analyses: having received prototype alteration feedback (yes/no), having received a cue reminder (yes/no), and the interaction of prototype alteration (yes/no) and cue reminder (yes/no) to assess the added value of their combination , North America, Oceania, Japan, and Indonesia (including former Netherlands East Indies). Non-Western includes Turkey and all countries of Africa, Latin America, and Asia, except Japan and Indonesia [ndonesia . Also, mIntervention analyses were corrected for age and educational level because these were significantly different between conditions at baseline. P<.001), cue condition , and combined condition ; the 3 added conditions did not differ from one another. Dropout was also higher among men , lower educated participants , and non-Western participants . Additionally, those who dropped out were also slightly younger  and reported a slightly higher alcohol consumption . We used LOCF in the longitudinal multilevel analyses to account for dropout and corrected the analyses for age and education.A total of 1260 participants completed 1 or both of the follow-up measurements . A total of 599 participants participated in all 3 measurements . Dropout analyses were performed for those who did not participate in either of the 2 follow-up measurements. Dropout was highest among the original Drinktest condition  and was significantly higher than the prototype condition . Both Drinktest versions were rated as equally interesting, new, informative, understandable, personally relevant, persuasive, enjoyable, and useful. The results were similar across all 4 conditions.Second, the appreciation of the intervention was assessed. The original  and extended Drinktest  did not differ in their intervention evaluations  received a bracelet and 43.1% (243/564) chose to use their own cue, whereas only 22.7% (128/564) did not wish to be reminded. At follow-up, the vast majority were found to remember their chosen cue reminder correctly  and reported using or wearing their cue reminder frequently . The awareness of the cue was reasonable .Based on the means, participants that received a prototype alteration and/or cue reminder strategy generally had higher awareness of their alcohol consumption, contemplation of the intervention, and perception of having reduced alcohol consumption than those who only received the original Drinktest . For thoP=.03) and a cue reminder  had larger reductions than those who did not receive these strategies in addition to the original Drinktest  than for those who did not (see P=.04) and prototype feedback  resulted in a lower relative drinking perception than when these strategies were not received in addition to Drinktest . The effect of prototype alteration (in addition to Drinktest) on drinking behavior became nonsignificant.Finally, the analyses were repeated including full cases only . Similar patterns of results were found as when the LOCF method was applied, albeit the effect of the cue reminder on relative perceived drinking became nonsignificant  in reducing alcohol consumption. Although all conditions showed reductions in alcohol consumption and willingness, and increased intention to reduce drinking over a period of 6 months, reductions in alcohol consumption were higher among people who had received the prototype alteration or a cue reminder in addition to the original Drinktest compared to those who did not. The combination of the cue reminder and prototype alteration did not enhance the effect of either of the independent strategies. Importantly, participants in all conditions equally appreciated the intervention, but dropout was lower for participants who received the prototype alteration and/or cue reminder in addition to Drinktest than for participants who received the original Drinktest only.Regarding the effect of the prototype alteration strategy, the reduced drinking levels that were found were expected. It is plausible that distancing from heavier drinking prototypes  ,53 was aThe results show that cue reminders may be an effective strategy in addition to an existing intervention such as Drinktest, and that the type of cue that we provided is feasible . Our study adds to the knowledge of testing the effect of cue reminders on drinking behavior -45 by apThe interaction of prototype alteration and cue reminders did not produce an extra effect beyond the separate effects of the 2 strategies. It suggests that both strategies have an independent effect on drinking behavior, but that there is no synergistic effect by combining them. Thus, for those effects that were significant for both strategies, both may be effective but by separate means. Perhaps the link between the characteristics to be achieved and the cue reminder should have been stronger. It could be that the characteristics were already salient in the prototype alteration and hence no additional benefit of cue reminders may have occurred. Or it may be that a cue reminder does not support remembering an abstract construct such as “achievable personal characteristics” but does support the remembrance of concrete implementation intentions and action plans. To our knowledge, a bracelet as a cue reminder has not been used as a means to help decrease drinking behavior. It is conceivable that another type of cue  may have a different but additional effect on the prototype alteration. Future research could shed light on this possibility.The following study limitations must be addressed before discussing the implications. First, dropout was large and the sample largely consisted of Western participants. However, it is unlikely that selection based on ethnicity would have changed the results because non-Western and Western samples have been found to show similar drinking behavior in the Netherlands  and the The findings suggest the following implications and future directions. First, our findings support earlier suggestions that future interventions may benefit from providing relevant prototypes to be achieved and avoided  and to tSecond, the bracelet had the advantage of being self-regulated by participants and that it can be effective even when alcohol is already consumed ,44. HoweThird, it may be important for future interventions to complement the strategies with messages that make people aware of their drinking behavior and that especially informs excessive drinkers about the consequences of their behavior as was done by the original Drinktest . However


Paenibacillus are important plant growth-promoting rhizobacteria that can serve as bio-reactors. Paenibacillus polymyxa promotes the growth of a variety of economically important crops. Our lab recently completed the genome sequence of Paenibacillus polymyxa CR1. As of January 2014, four P. polymyxa genomes have been completely sequenced but no comparative genomic analyses have been reported.Members of the genus P. polymyxa genomes, which revealed a significantly conserved core genome. Complex metabolic pathways and regulatory networks were highly conserved and allow P. polymyxa to rapidly respond to dynamic environmental cues. Genes responsible for phytohormone synthesis, phosphate solubilization, iron acquisition, transcriptional regulation, σ-factors, stress responses, transporters and biomass degradation were well conserved, indicating an intimate association with plant hosts and the rhizosphere niche. In addition, genes responsible for antimicrobial resistance and non-ribosomal peptide/polyketide synthesis are present in both the core and accessory genome of each strain. Comparative analyses also reveal variations in the accessory genome, including large plasmids present in strains M1 and SC2. Furthermore, a considerable number of strain-specific genes and genomic islands are irregularly distributed throughout each genome. Although a variety of plant-growth promoting traits are encoded by all strains, only P. polymyxa CR1 encodes the unique nitrogen fixation cluster found in other Paenibacillus sp.Here we report the comparative and genetic analyses of four sequenced P. polymyxa genomes analysed, despite variations in the accessory genome. This work suggets that plant-growth promotion by P. polymyxa is mediated largely through phytohormone production, increased nutrient availability and bio-control mechanisms. This study provides an in-depth understanding of the genome architecture of this species, thus facilitating future genetic engineering and applications in agriculture, industry and medicine. Furthermore, this study highlights the current gap in our understanding of complex plant biomass metabolism in Gram-positive bacteria.Our study revealed that genomic loci relevant to host interaction and ecological fitness are highly conserved within the The online version of this article (doi:10.1186/1471-2164-15-851) contains supplementary material, which is available to authorized users. Paenibacillus genus is comprised of over 150 species of which many act as important PGPRs in agriculture and horticulture [Paenibacillus polymyxa is a well-studied PGPR and strains have been isolated from diverse geographic regions and ecological niches [P. polymyxa was originally identified and characterized as Bacillus polymyxa as a result of its similarity to Bacillus sp. [P. polymyxa strains are best known for their production of various potent anti-microbial and volatile compounds that improve plant fitness through nutrient cycling, pathogen antagonism and induction of plant defenses [Plant-growth promoting rhizobacteria (PGPR) increase the fitness and growth of host plants through various mechanisms, including biological nitrogen fixation, synthesis of plant hormones, solubilization of inorganic mineral phosphates, and siderophore production –5. Many iculture . Paenibal niches –18. P. pllus sp. . P. polydefenses , 20–25. defenses .P. polymyxa CR1 (hence forth CR1) from the rhizosphere of degrading corn stalks in southern Ontario, Canada. CR1 produces large amounts of indole-3-acetic acid (IAA) and excretes antagonistic compounds active against several plant pathogenic bacteria and fungi (unpublished data). Additionally, CR1 was able to fix-nitrogen and promote the growth of a variety of crops. Interestingly, CR1 metabolized lignin, cellulose and hemi-cellulose as a sole carbon source, and fermented these plant-derived compounds directly into alcoholic compounds . To better understand the relevant P. polymyxa CR1 metabolic pathways and regulatory mechanisms, we sequenced, assembled and annotated the CR1 genome [P. polymyxa genomes have been completely sequenced and annotated [P. polymyxa E681, P. polymyxa M1, P. polymyxa SC2, and P. polymyxa CR1 .Recently, our lab isolated 1 genome . As of Jnnotated –30: P. pP. polymyxa strain to be completely sequenced. It promotes plant growth through production of indole-3-acetic acid (IAA); and produces volatile compounds that elicit induced-systemic resistance in plants, priming the plant host defenses for a rapid response against pathogenic bacteria [P. polymyxa strains possess various non-ribosomal peptide synthetase gene clusters encoding polymyxins, fusaricidins, paenicidins, gramicidins, bacitracins, polyketides, a bacilorin and a lantibiotic [P. polymyxa are capable of strong growth antagonism of various bacterial and fungal plant pathogens and parasitic nematodes [P. polymyxa increase plant-growth in iron-limited conditions by increasing iron availability for the associated plant host and decreasing iron availability for pathogenic organisms [E681, isolated in Korea from the rhizosphere of barley, was the first bacteria , 31. E68bacteria , 32. Bottibiotic , 29. Allematodes –25, 32. rganisms .in silico comparisons and analyses of taxonomically related bacterial species allows for rapid identification of functional similarities and unique genetic elements, uncovering complicated metabolic pathways and regulatory networks in the process [P. polymyxa. Limited information is available regarding the specific adaptations and mechanisms that allow for their survival and growth in the soil environment.As a major feature of the post-genomic era in biology, comparative genomics is recognized as an important tool for identifying and dissecting key metabolic pathways and regulatory networks amongst related organisms –37. Thes process , 40–42. P. polymyxa strains. The genomes of each strain were screened for the presence of loci associated with plant growth promotion and disease control. We also compared genomic regions implicated in metabolic versatility and association with plant hosts. These comparative genomic analyses have expanded our understanding of P. polymyxa biology and highlighted the gap in our current understanding of biomass metabolism pathways in Gram-positive bacteria. This work will provide a foundation for follow-up studies of target genes and functions and will facilitate genetic and metabolic engineering of P. polymyxa to improve agricultural and industrial applications.Here we perform detailed comparative genome analyses of the four sequenced Paenibacillus sp. using the Maximum-likelihood method [P. polymyxa is most distantly related to Paenibacillus mucilaginosus, which is currently employed in microbial fertilizers [Paenibacillus terrae, a diazotrophic, free-living soil bacterium.A phylogenetic tree was generated based on the 16S rRNA of completely sequenced d method  in MEGA6d method   to glean global information into the nucleotide level similarity amongst the sequenced P. polymyxa progenitor strain independently from each other. This notion is further supported when considering the high level of similarity between the core chromosomes of M1 and SC2  categories R and S represent general function prediction and unknown function, respectively, and offer minimal evidence of the substrate specificity and/or protein function of the annotated protein. As evidenced by the large proportion of genes assigned to the COG categories R and S, protein function in the Paenibacillus genus is not well characterized. Regardless of the limitations of available data sets and analytical tools, some important inferences into the metabolic capacities of each strain are still possible. The proportion of genes assigned to each COG is presented in Figure P. polymyxa strains and are omitted [Comparative analyses of protein function from genomic sequence data of less heavily studied taxonomic families, such as  omitted –51. BothP. polymyxa strains were conducted to visualize the similarity of encoded proteins. Average amino acid identities were calculated using the pair-wise orthologous sets of CDSs from the four P. polymyxa genomes. The accessory and core genomes were identified by an in silico subtractive hybridization approach using mGenomeSubtractor [P. polymyxa strains and their respective plasmids. H-values <0.41 were considered strain-specific while those >0.81 were considered conserved, representing approximate amino acid level similarity cut-offs of <41% and >81% respectively. When chromosomes and plasmids are considered together all strains contain between 9% and 18% strain-specific CDS. The largest number of strain-specific genes is encoded by CR1 where 955 CDS were identified as strain-specific , surprising when considering its genome size and absence of a plasmid  is commonly associated with insertion sequence (IS) elements, tRNA genes, tmRNA genes and transposons and is identified by an anomalous GC content, codon bias, di/trinucleotide differences and GC skew . To visu Artemis  and plot Artemis   are thought to be genetic elements acquired during evolution from distantly related organisms and such horizontally transferred genes contribute to genome flux and variation , 58. Inie Figure . Since tP. polymyxa strains  into ammonium (NH4) thereby improving plant growth and crop yields by increasing the concentration of biologically available nitrogen [Paenibacillus sp. WLY78 has demonstrated that a minimal nif cluster can confer nitrogen fixation to Escherichia coli[nif cluster was identified only in CR1 but not the other three P. polymyxa genomes. The gene organization and composition of the CR1 nif cluster was identical to the previously identified nif cluster of WLY78 [nif cluster of CR1 is approximately 10.5 kb and encodes 9 genes; nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, and nifV  than the rest of P. polymyxa genome (45%) and the nif cluster was identified as a GI by IslandViewer . The whole genome phylogeny  . Nitrogenitrogen . Recent hia coli. In thisof WLY78 . The entDatabase  and NCBI HPL-003 , the nexGluconacetobacter diazotrophicus[nifA gene in Paenibacillus sp. or other Gram-positive, diazotrophs such as Clostridium sp. The nifA gene encodes a nif-specific regulatory protein that activates the expression of nifHDK genes [2-fixing alfalfa symbiont Sinorhizobium meliloti, the three sigma-54 type NtrA-dependent regulatory proteins NifA, NtrC, and DctD are required for activation of promoters involved in N2-fixation (nif genes), nitrogen assimilation (pglnII), and C4-dicarboxylate transport (pdctA) respectively [nifA gene is dispensable for N2-fixation since a mutation in ntrC283 allele in S. meliloti restores the N2-fixation ability of a nifA deletion mutant to 70-80% of the wild-type level [nif gene cluster. The other three P. polymyxa strains do not contain the NtrC homolog found in CR1, consistent with the absence of the nif gene cluster in these three strains. However, it is unclear how these three P. polymyxa strains  handle nitrogen assimilation.Unlike free-living Gram-negative nitrogen fixing bacteria such as rophicus, 65, theDK genes . In the ectively , 67. Prepe level , 69. TheRhizobia, DctD, a C4-dicarboxylate response regulator, activates the expression of dctA upon detection of C4-dicarboxylates. Previous studies have found that DctD is not only involved in C4 uptake, but also participates in symbiotic nitrogen fixation through complex signalling mechanisms [dctD homolog in the CR1 genome, which matched to the same protein identified as an NtrC homolog (YP_008912290.1). These results suggest that CR1 maintained the nif cluster and a hybrid NtrC/DctD response regulator required for nitrogen fixation. Conversely the absence of ntrC and dctD gene homologs in the other P. polymyxa strains is unsurprising since these strains lack the nif cluster and do not require their associated signalling pathways. In the case of a recent study that demonstrated Escherichia coli nitrogen fixation by acquisition of the nif cluster from Paenibacillus sp. WLY78 [E. coli partially activates expression of the exogenous nif cluster. Nevertheless, the 9 gene nif cluster identified in CR1 is particularly exciting for its potential use as a transferable nitrogen fixation genetic element to facilitate development of genetically modified plant-growth promoting bacteria. The broad host-range and free-living lifestyle of P. polymyxa coupled with the ability to fix nitrogen is in stark contrast to Rhizobia sp. which require nodule organogenesis (mediated by nod genes) prior to induction of diazotrophy [In chanisms , 70. Intp. WLY78 , it is pzotrophy .P. polymyxa genomes for pathways involved in the production of IAA. Indole-3-pyruvate decarboxylase (IPDC) is a key enzyme involved in the IPA pathway and is encoded by the ipdC gene, which has been established as necessary for the production of IAA in E681 [P. polymyxa. Preliminary results in our lab corroborated the capacity to produce IAA by the IPA pathway in CR1 . Furthermore, all genomes examined here encode putative auxin efflux carrier proteins, suggesting that these bacteria are capable of production and export of IAA in a tryptophan-dependent manner  is the immediate precursor of the plant hormone ethylene. Stressed plants accumulate ethylene, which inhibits root elongation and accelerates abscission, aging and senescence. ACC deaminase-producing rhizobacteria lower plant ethylene levels by converting ACC into ammonia and α-ketobutyrate, preventing inhibition of root growth thereby improving tolerance to environmental or pathogen-induced stress , 8. The Pseudomonas sp. have implicated glucose-1-dehydrogenase (gcd) and gluconic acid dehydrogenase (gad) in the production of gluconic acid and its conversion to 2-ketogluconate respectively [P. polymyxa genomes  is responsible for bacterial degradation of phosphonates, which releases biologically available phosphate for nearby plants. Our comparative genomics revealed that P. polymyxa strains do not carry the complete phn cluster, lacking the genes encoding a C-P lyase protein (phnGHIJKLM) responsible for phosphonate degradation into phosphate and an alkane, a system commonly identified in other phosphonate degrading bacteria . In addition, CR1 and E681 also possess the necessary genes  for 2-aminoethylphosphonate and phosphonopyruvate degradation. Furthermore, CR1 and E681 encode phnW, required for the biosynthesis of phosphonates, possibly utilizing produced phosphonates as a sequestered storage form of phosphate  and polyketides (PK) are modular in nature and are comprised of a multitude of possible modification domains including adenylation, condensation, thiolation and esterification domains. The most well-known NRPs produced by bacteria . There ibacteria . Recent nd fungi –25, 79.P. polymyxa strains, which are summarized in Figure P. polymyxa strains do not possess identical clusters of NRP and PK synthesis genes, likely representing adaptations by strains to their specific ecological niche and the organisms it competes with. Notably, each strain has an impressive repertoire of strain specific NRP, PK and NRP-PK hybrid synthases. For example, CR1 encodes a staphyloferrin-like-siderophore synthetase, tyrocidine synthetase, a type II polyketide synthase, a type I polyketide synthase and a novel NRP-PK hybrid synthase not found in other bacteria. Meanwhile, E681 encodes a novel gramicidin-like synthase, and a NRP-PK hybrid synthase also found in SC2. M1 encodes fuscaricidin synthase, two NRPs, lichenysin and a lantibiotic otherwise found only in SC2 as well as a plasmid encoded NRP and pyroverdine siderophore. Finally, SC2 encodes gramicidin, two NRPs and a lantibiotic otherwise found only in M1 and a NRP-PK hybrid also found in E681 . Since highly active hydrolytic enzymes and bacterial strains are a desirable commodity in the developing biofuel sector we compared the quantity and nature of glycoside hydrolases (GH) amongst P. polymyxa strains to glean an overall outlook of the strain-specific biomass degradation capacity , followed by M1 (123 GH family proteins). CR1 encodes an increased quantity of GH families 1, 2, 3 and 42 proteins (xylanase and endo/exo-glucanase enzymes) while showing a decreased number of GH family 5 (cellulase) proteins. As a result of their increased number of cellulase domain containing enzymes, M1, E681 and SC2 are also likely proficient cellulose degraders. Interestingly, E681 encodes a large number of rhamnogalacturonan lyase and pectin lyase proteins, which are responsible for non-hydrolytic cleavage of pectin, possibly underpinning a previously unknown saprophytic niche occupied by E681 in soils. Carbohydrate hydrolase enzymes function as complex, multi-meric proteins assembled from various numbers of encoded domains. This allows the substrate specificity of the complex to vary greatly dependant on the stoichiometry of the subunits [P. polymyxa an untapped reservoir of potentially valuable hydrolytic enzymes and plant biomass degradation complexes. Furthermore, the large quantity of GH family genes present in P. polymyxa genomes is consistent with the intimate association of P. polymyxa with plant hosts in the rhizosphere where there is an increased concentration of plant-derived compounds.Bacteria that form commensal or mutualistic relationships with plants are at a selective advantage if they can metabolize plant-derived carbon sources, which are enriched in the rhizosphere environment , 80. Thepolymers . Our inisubunits . It is tP. polymyxa contain putative DyP-peroxidase and laccase genes (Table P. polymyxa using hhPRED [Rhodococcus jostii RHA1 (accession no. 3QNR_A) that is responsible for non-specifically degrading lignin substrates [Lignin metabolism by many actinomyces bacteria as well as various fungi occurs through non-specific redox and free-radical generating mechanisms. Fungal laccase enzymes are copper-dependent hydrolases expressed by white and brown rot fungi that non-selectively depolymerize lignin through reduction-oxidation reactions –85. Conves Table . Recent g hhPRED  and PHYRg hhPRED  suggestsbstrates , 89. FurP. polymyxa are noted for their ability to sterioselectively ferment valuable -2, 3-butanediol from monosaccharide feed stocks [Firmicutes lignin-degrading pathways. Further studies are necessary for elucidating the responsible enzymes and regulatory networks. Further complicating our analysis, our research suggests CR1 is incapable of degrading protocatechuate  from characterized species. Therefore, P. polymyxa likely utilizes a yet to be characterized lignin and aromatic degradation pathway and to the best of our knowledge no complete characterization of the lignin metabolism network has taken place in any Firmicutes bacteria.Specific strains of d stocks –92. PrelP. polymyxa encodes a large number of transporters and regulators allowing a wide range of substrates to be up taken. Bacterial two-component signal transduction systems play important roles in enabling detection and response to diverse changes/stresses in the environment [B. subtilis DegS/DegU two component system is involved in the regulation of post-exponential phase processes, including activation of genetic competence, motility, poly-γ-glutamic acid production and biofilm formation [B. subtilis produces several proteolytic and hydrolytic enzymes including an intracellular protease and several secreted enzymes , and xylanase), which are controlled at the transcriptional level by the DegS/DegU two-component system. To understand how P. polymyxa co-ordinates the decision-making processes to ensure appropriate physiological responses occur during different environmental conditions, we attempted to identify a P. polymyxa degS/degU two-component system. Our analysis at the genome level indicates that a conserved degS  and degU  are present among the sequenced P. polymyxa genomes . However, without experimental evidence, we can only speculate the identified two-component system DegS/DegU participates in biomass degradation and provides a selective advantage to P. polymyxa.In variable and changing environments bacteria with larger genomes encoding varied metabolic capabilities have a long-term selective advantage , 93. Theironment , 96. Theormation , 98. DurBacilli, including P. polymyxa, also encode anti-sigma-factors (anti-σ-factors) or anti-anti-σ-factors that are secreted either as a mechanism of competition with other bacteria or to control their own gene expression in rapidly changing environments where tight gene regulation is paramount. Strikingly, all strains of P. polymyxa encode a large number of σ-factors and MCPs . As expected, rpoD, sigE, rpoN and fliA are encoded by all strains, representing σ-factors responsible for basal transcription, general stress responses, nitrogen limitation and flagella assembly respectively. In addition to necessary basal transcription factors, each strain encodes a variable amount anti-σ-factors and anti-anti-σ-factors, which may be involved in sensing environmental conditions and modulating gene expression accordingly . The repertoire of chemotactic signal pathways of a microorganism is correlated to the environments it inhabits and the hosts it colonizes . Many BaP. polymyxa lifestyle has not yet been revealed so far, however, other members of the genus are known for intricate signalling pathways and striking patterns formed during migration [B. subtilis is a typical Gram-positive QS system that controls bacterial competence [comQXPA genes in the P. polymyxa genomes, which may explain the low transformation frequency of CR1 compared to B. subtilis (our unpublished data). In addition to ComQXPA, B. subtilis operates a LuxS-dependent QS system that regulates its morphogenesis and social behaviour [luxS gene product synthesizes autoinducer-2 (AI-2) that modulates biofilm formation. Expression of luxS is negatively regulated by two master regulatory proteins, SinR and Spo0A. SinR is a key regulator for proper biofilm development and Spo0A controls sporulation in B. subtilis[luxS gene homolog among the fully sequenced P. polymyxa genomes . Therefore, it is plausible that in these P. polymyxa strains, LuxS is the main QS signal producer, whereas, the hybrid two-component protein LuxQ plays a major role as a sensor kinase and response regulator for the LuxS-dependent QS amongst strains of the species.Quorum sensing (QS) is a specialized cell-to-cell communication mechanism employed by bacteria to control gene expression on a community level through secretion and detection of extracellular signalling molecules, called autoinducers, which accumulate in the environment proportional to cellular density . QS-contigration . The Commpetence , 101. Hoehaviour , 103. Thsubtilis, 101. Our domain . HoweverP. polymyxa is typically found either in association with plant hosts or as free-living in the rhizosphere and is expected to encode diverse metabolic networks and transporters to adapt to the specialized living environments [P. polymyxa genomes . As expected, ABC transporters responsible for export of bacteriocins and other NRPs and PKs are encoded by all strains. In addition, our comparative genomic analysis found all P. polymyxa genomes encode a C4-dicarboxylate transporter (dctA) , reminiscent of Rhizobia sp. where dicarboxylic acids  are major carbon and energy sources for the symbiotic bacteria [Rhizobia, DctD, a C4-dicarboxylate response regulator, activates the expression of dctA upon detection of C4-dicarboxylates. Our comparative genomic analysis revealed a dctD homolog in CR1 genome (YP_008912290.1), which is absent in the other three P. polymyxa genomes.Soil bacteria are known to be rich in ATP-binding cassette (ABC) transporters. ronments . To gainns Table . Notablyem Table , a high-bacteria . In RhizP. polymyxa genomes encode complete Twin-arginine translocation and Sec systems for protein secretion. Interestingly, all P. polymyxa genomes analyzed here appear to encode various genes responsible for type 3 secretion systems (T3SS), however due to the similarity between flagella structures and T3SS structures, it is plausible these genes are actually involved in flagella structure, transport and function  using the progressiveMauve algorithm in Mauve [The genome of each in Mauve . Furtherin Mauve . Dot-ploin Mauve  on defauGenes putatively responsible for plant-growth promotion, bio-mass degradation and solventogenesis were identified by using KO and homology searches using tBLASTx to previously characterized homologs. Metabolic and signalling pathways were constructed using the KEGG database . HomologNon-ribosomal peptide synthesis clusters, polyketide clusters and siderophore synthesis genes of both chromosomes and plasmids were identified using antiSMASH , and the−25 and ≥60%, respectively, were considered homologous.Protein homology was determined by performing a BLASTp search against identified homologs. Proteins that met an E-value and positive amino acid identity cut-off of ≤10All sequences and annotations referenced in the manuscript are publically available on the Genebank Database at the accession numbers provided. Distances used to compute phylogeny are publically available on the Joint Genome Institute’s Integrated Microbial Genomics Database. The data sets supporting the results of this article are included within the article and its additional files.P. polymyxa genomes. Each strain’s genome is compared pairwise against all other strains. Plasmid comparisons are denoted by their accession numbers and are separated from their respective strains chromosome comparison by a red line. Dots that deviate from the horizontal represent chromosome rearrangements, at the gene level, from the reference genome. The strains listed on the left represent the reference genome for the horizontal row; those listed at the top correspond to the query strain in the vertical column. (TIFF 43 KB)Additional file 1: Figure S1: MUMmer dot-plots comparing protein level homology between completely sequenced Identified genomic islands and their encoded genes.(XLSX 53 KB)Additional file 2:Identified insertion sequences and their homology to known insertion sequences.(XLSX 35 KB)Additional file 3:Table S1. CAzY profile of sequenced P. polymyxa genomes. Table S2. σ-factors and methyl-accepting chemotaxis proteins encoded by P. polymyxa strains. Table S3. Encoded ABC transporters and PTS family transporter specificities. (DOC 106 KB)Additional file 4:Identified non-ribosomal, polyketide and hybrid synthase clusters and their locations.(XLS 67 KB)Additional file 5:Transporter classification homology and the number of identified homologs in the fourP. polymyxastrains.(XLSX 15 KB)Additional file 6:


This study evaluated maternal C-reactive protein (CRP) as a predictor of microbial invasion of the amniotic cavity (MIAC) and histological chorioamnionitis (HCA) in women with preterm prelabor rupture of the membranes (PPROM) before and after 32 weeks of gestation.Ureaplasma species, Mycoplasma hominis and Chlamydia trachomatis and/or positive 16S rRNA gene amplification. HCA was defined based on the Salafia classification.This study was a prospective observational cohort study of 386 women. Maternal serum CRP concentrations were evaluated, and amniotic fluid samples were obtained via transabdominal amniocentesis at the time of admission. Placentas underwent histopathological examination after delivery. MIAC was defined based on a positive PCR for p<0.0001). CRP was a weak predictor of the occurrence of MIAC and HCA before and after 32 weeks of gestation. Only the 95th percentile of CRP and PPROM before 32 weeks exhibited a false-positive rate of 1%, a positive predictive value of 90% and a positive likelihood ratio of 13.2 to predict MIAC and HCA. However, the low sensitivity of 15% limits the clinical utility of this detection.Maternal CRP was significantly higher in women with MIAC and HCA (median 9.0 mg/l) than in women with HCA alone (median 6.9 mg/l), MIAC alone (median 7.4 mg/l) and without MIAC or HCA (median 4.5 mg/l) (CRP is a poor predictor of the occurrence of MIAC and HCA, even at early gestational ages. Preterm delivery is a worldwide public health problem, and it occurs in approximately 6–12% of all pregnancies .Microbial invasion of the amniotic cavity (MIAC) is identified in 30–40% of PPROM, particularly at early gestational ages .Signs of histological chorioamnionitis (HCA) are also observed after placental examination in up to 60% of all preterm deliveries . HCA andThe active management of PPROM in the Czech Republic provides a short latency from membrane rupture to delivery in women with a PPROM diagnosis. This management scheme provides unique information on the subgroup of women presenting MIAC and HCA. We previously demonstrated that women with MIAC and HCA exhibited the greatest intra-amniotic inflammation , 12. SimThe only method to diagnose MIAC involves an invasive procedure, and placental information on HCA is not available antenatally. Therefore, non-invasive approaches were widely proposed  to iEvaluation of 27 biomarkers using a multiplex approach revealed differences in the concentration of IL-18, IL-1β and monocyte chemotactic protein-1 (MCP-1) in maternal serum between PPROM women with or without MIAC. Notably, these differences were only observed prior to 32 weeks of gestation . HoweverMaternal serum CRP has been proposed as a marker of infection and inflammation in several diseases –23 and MIt is clinically important to evaluate the role of CRP as a predictor of the worst infectious conditions, such as the occurrence of MIAC and HCA, because the information for maternal CRP associated with infection might induce changes in the management of PPROM in most clinical settings. Whether gestational age influences the predictive value of CRP for infectious conditions should also be investigated.Therefore, the present study evaluated maternal CRP as a predictor of MIAC and HCA in women with PPROM before and after 32 weeks of gestation.This study was conducted as an observational prospective cohort study. The individuals enrolled in this study were pregnant women with PPROM between gestational ages 24+0 and 36+6 weeks who were admitted to the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove, Czech Republic from January 2008 to December 2013. Gestational age was established based on the first trimester ultrasound scan.th percentile, the presence of either congenital or chromosomal fetal abnormalities, gestational or pre-gestational diabetes, gestational hypertension, preeclampsia and signs of fetal hypoxia were excluded from this study.Inclusion criteria were singleton pregnancies and maternal age ≥ 18 years. Women with signs of clinical chorioamnionitis, fetuses with an estimated weight <10PPROM was diagnosed using a sterile speculum examination to verify the pooling of amniotic fluid and confirmed using a positive test for the presence of insulin-like growth factor-binding protein 1  in vaginal fluid when necessary.All women received prophylactic antibiotic treatment, and steroid therapy was administered when PPROM occurred from 24–34 weeks of gestation. Tocolysis was administered for 48 h during lung maturation.The clinical guidelines of the Czech Republic recommends an active management of pregnancy when PPROM occurs later than 28 weeks of gestation. Labor is induced or Cesarean section is performed after lung maturation within 72 h following membrane rupture in all PPROM later than 28 weeks. The induction of labor or the performance of Cesarean section was indicated based on signs of fetal distress and/or severe vaginal bleeding and/or clinical chorioamnionitis or when high levels of maternal serum CRP were observed before 28 weeks. The remaining women were treated expectantly up to completed 28 weeks of gestation.Written informed consent was obtained from all subjects. The University Hospital Hradec Kralove Institutional Review Board approved the collection and use of these samples and information for research purposes .Maternal blood and amniotic fluids were collected prior to treatment with antibiotics, steroids and tocolysis. Maternal blood was obtained via venipuncture of the cubital vein at admission. Maternal serum CRP was measured using a high-sensitivity immunoturbidimetric analysis , with a method sensitivity of 0.3 mg/L.Ureaplasma species, Mycoplasma hominis and Chlamydia trachomatis and non-specific PCR for 16S rRNA. MIAC was determined based on a positive result of the PCR analysis for Ureaplasma species, Mycoplasma hominis and Chlamydia trachomatis and/or positive 16S rRNA gene amplification.Amniotic fluid was obtained via ultrasound-guided transabdominal amniocentesis. Microbial analyses of amniotic fluid were based on specific polymerase chain reaction (PCR) for Placentas were collected after delivery and fixed in formalin, and tissue samples with placental membranes were embedded in paraffin. Tissue sections of placentas were stained using hematoxylin and eosin for standard histological examination. HCA was defined using the Salafia classification based on the presence of neutrophil infiltration in the chorion-decidua (Grades 3–4) and/or the chorionic plate (Grades 3–4) and/or the umbilical cord (Grades 1–4), and/or the amnion (Grades 1–4).® C. trachomatis/Ureaplasma/M. hominis-FRT  to detect the DNA from Ureaplasma species, Mycoplasma hominis, and Chlamydia trachomatis in a common PCR tube. We included a PCR run for the housekeeping gene beta-actin as a control to examine the presence of PCR inhibitors. The amount of Ureaplasma species DNA in copies/mL was determined using an absolute quantification technique that employs an external calibration curve. Plasmid DNA  was used to prepare the calibration curve . Categorical variables were compared using the Chi-square test and presented as numbers [percentage (%)]. Spearman partial correlation was used to adjust the data for gestational age at admission or delivery. Spearman rank correlation test was used to determine the correlations between CRP concentrations and the microbial load of A total of 495 pregnant women with PPROM met the inclusion criteria and were included in the present study. Sixty-eight women were excluded because of pre-gestational diabetes, gestational diabetes, chronic hypertension, gestational hypertension, preeclampsia or fetal growth restriction in the fetus. Amniocentesis was not possible in 22 of the 427 women, and the results of histopathological assessments of the placenta were not available for 19 women. The remaining 386 women were included in the present study . Table 1The presence of MIAC and HCA was observed in 25% (97/386) of women. The presence of HCA or MIAC alone was observed in 36% (141/386) and 8% (32/386) of women, respectively. The remaining 29% (116/386) of women had neither MIAC nor HCA.p < 0.0001) and after adjustment for the gestational age of the sample (p < 0.0001).Women with MIAC and HCA exhibited the highest concentration of CRP [median: 9.0 mg/L (IQR: 3.9–20.1)] vs. women with HCA alone [median: 6.9 mg/L (IQR: 3.6–12.1)], MIAC alone [median: 7.4 mg/L (IQR: 3.6–15.6)] or with neither MIAC nor HCA [median: 4.5 mg/L (IQR: 2.0–8.6)];  in crudePregnancies with MIAC and HCA exhibited higher median CRP concentrations than women with HCA alone, MIAC alone, and neither MIAC nor HCA.th percentile below and above 32 weeks of gestation. Supplemental information of the graphical distribution of CRP in the 90th percentile below and above 32 weeks is provided in Ureaplasma species and/or Mycoplasma hominis) [median: 6.6 mg/L (IQR: 3.5–12.1)] and women infected with other microorganisms [median: 7.1 mg/L (IQR: 2.63–14.2) p = 0.81].No differences in maternal CRP concentrations were observed between women infected with genital mycoplasmas . Women with MIAC and HCA (n = 70) had a higher microbial load of Ureaplasma species in amniotic fluid compared to women with MIAC alone (n = 16) [MIAC and HCA [median: 465000 (IQR): 3700–6667000)] vs. MIAC alone ].The amount of th percentile in PPROM below 32 weeks of gestation accurately predicted the occurrence of MIAC and HCA. Unfortunately, this low sensitivity prevents the application of this finding in a clinical setting.The main finding of the present study was the weak association between maternal serum CRP and the occurrence of MIAC and HCA, even when different gestational ages were considered. Only CRP levels above the 95The worst scenario in PPROM is the presence of MIAC and HCA , 11, 33.Previous studies reported the influence of CRP in pregnancies complicated with HCA 34] [19 [1934] . Howman th and 95th percentile to further explore the predictive value of CRP in a secondary analysis. The results demonstrated that a PPV of 90%, a FP rate of 1% and a +LR ratio of 13.1 predicted MIAC and HCA only when CRP levels were above the 95th percentile and only PPROM below 32 weeks even when the extreme values of CRP were considered. However, the extremely low detection rate of 15% limits the clinical usefulness of this screening technique. These findings reveal that assessment of CRP levels was a poor predictor of MIAC and HCA, which highlights the importance of considering the amniocentesis as the only accurate method to identify this infectious condition. Therefore, these findings are interesting and clinically relevant.Therefore, the data obtained in the present study revealed differences in CRP levels between different infectious subgroups of women with PPROM. CRP concentrations are gestational age-dependent, and difUreaplasma species. No differences were observed between the intensity of intraamniotic inflammatory response triggered by genital mycoplasmas and other microorganisms[Ureaplasma species in the amniotic fluid. We also did not observe differences in the intensity of maternal CRP between women with genital mycoplasma and other microorganisms. This finding is not consistent with Oh et al., who observed a higher inflammatory response in the amniotic cavity and maternal compartment in women with MIAC compared to the presence of genital mycoplasmas and other microorganisms[Our previous study clearly demonstrated that the intraamniotic inflammatory response depended on the microbial load of organisms, 39. Theorganisms. These cOne of the strengths of the present study is the active management of PPROM in our cohort, in which most women delivered within 72 h from membrane rupture. This management provides information on the placenta, which is consistent with the information obtained from amniotic fluid analyses. We also obtained microbial results from the amniotic fluid using modern non-cultivation technologies with a higher detection rate than classical cultivation approaches. We explored a large cohort of women in one tertiary center hospital with a clearly defined PPROM phenotype.However, the present study also has some limitations. Maternal serum CRP analysis was performed only at admission, and we were unable to longitudinally analyze the progression of this protein until delivery. We also did not have information on IL-6 levels in amniotic fluid. Therefore, we could not evaluate the influence of maternal CRP on the intra-amniotic inflammatory response.th percentile, predicted the presence of MIAC and HCA below 32 weeks of gestation. However, the low sensitivity limits the clinical utility of this detection method. Therefore, there is no evidence to support the use of CRP as a tool for the prediction of the presence of MIAC and HCA, and the results suggest that amniocentesis is the most accurate tool for MIAC and HCA prediction in women with PPROM.In conclusion, maternal CRP was a poor predictor for the identification of women with MIAC and HCA. Therefore, only the extreme cutoff levels of CRP, represented by the 95S1 Dataset(XLSX)Click here for additional data file.S1 FigMaternal serum C-reactive protein (CRP) concentrations (> 90 percentile) based on the presence and absence of microbial invasion of the amniotic cavity (MIAC) and/or histologic chorioamnionitis (HCA) in women with PPROM below 32 weeks of gestation.(TIFF)Click here for additional data file.S2 FigMaternal serum C-reactive protein (CRP) concentrations (> 90 percentile) based on the presence and absence of microbial invasion of the amniotic cavity (MIAC) and/or histological chorioamnionitis (HCA) in women with PPROM above 32 weeks of gestation.(TIFF)Click here for additional data file.