IntroductionThyroid autoimmune (TAI) disease with a prevalence varying between 5 and 15%, represents the most common endocrine disorder in women with reproductive age. Not only is TAI disease five to 10 folds more common in women than men but also TAI diseases is often undiagnosed because it may be present without overt thyroid dysfunction for several years. Studies found an increased prevalence of TAI in women referred to fertility clinics compared with normal population. In this analysis we aimed to study the prevalence of TAI among women seeking fertility care in Damascus, Syria in order to understand its clinical and public health importance in population.MethodsThis study is a retrospective cross-sectional study on women patients seeking fertility care at Orient Hospital, Damascus city, Syria from April 2011 to March 2018. A total of 2526 women, with available biochemical data of anti-thyroid antibodies (anti-TPO) and anti-thyroglobulin antibodies (anti-TG) were included in our study. Thyroid stimulating hormone (TSH) titers data were also included in the statistical analysis.ResultsTAI was found positive in 559 patients (22.1%) of our studies population. TAI was more prevalent in patients with abnormal TSH levels.ConclusionThyroid autoimmunity prevalence in women seeking fertility care in Damascus, Syria was 22.1% which is significantly higher than normal population. Further studies are needed to assess the relation of these antibodies in thyroid, gynecological and other factors for the Syrian population. Chronic morbidity and impairment are caused by autoimmune diseases, and the thyroid gland is the most commonly affected organ by these diseases . ThyroidHyperthyroidism is caused by Graves' disease which has an approximated prevalence of 80/100,000/year in women and 8/100,000/year in men in Western countries, while Hashimoto's disease is considered the main cause of hypothyroidism in the West . IntolerAnti-thyroid peroxidase (anti-TPO) antibodies are distinct for the autoantigen TPO, a 105-kDa glycoprotein that is responsible for catalyzation of iodine oxidation and thyroglobulin tyrosyl iodination reactions in the thyroid .Anti-TPO antibodies present in almost 90% of Hashimoto's thyroiditis which makes them the most common anti-thyroid autoantibodies, in addition around 75% of Graves' disease and 10-20% of nodular goiter or thyroid carcinoma have positive anti-TPO antibodies. Also, 10-15% of normal individuals can have high-level anti-TPO antibody titers .Thyroglobulin antibodies are specific for thyroglobulin, a 660-kDa matrix protein that contributes to the production of thyroid hormone. Seventy percent of Hashimoto's thyroiditis, 60% of idiopathic hypothyroidism, 30% of Graves' disease, a small proportion of thyroid carcinoma and 3% of normal individuals have positive anti-TG antibodies .Recently the prevalence of other autoimmune endocrine disorders, particularly type 1 diabetes mellitus, has considerably increased . While tTAI has also been accused of increased unfavorable pregnancy outcomes, including implantation failure, recurrent pregnancy loss, placental abruption, preterm birth and perinatal mortality . 5-20% oTAI was also recently accused of negative outcomes on reproductive biology, including spermatogenesis, folliculogenesis, fertilization rates (FRs), embryo quality and pregnancy rates . SubsequSo far, in this analysis we aimed to study the prevalence of TAI among women seeking fertility care in Damascus, Syria in order to understand its clinical and public health importance in population. To the best of our knowledge, this study is the first to investigate thyroid autoimmunity burden in Syria.DesignThis study is a retrospective cross-sectional study on women patients seeking fertility care at Orient Hospital, Damascus city, Syria from April 2011 to March 2018. Participants were referred by their physician for the screening of their thyroid issues relating to fertility and/or gynecological issues.Study populationA total of 2526 women, with available biochemical data of anti-thyroid antibodies (anti-TPO) and anti-thyroglobulin antibodies (anti-TG), were included in our study. Thyroid stimulating hormone (TSH) titers data was also included in the statistical analysis.Immune assaysAll tests were done in the same lab . All tests were done using the same immune assay. These were primarily measured on the instrument Cobas 6000 (Roche Diagnostics). The instrument is a closed system, random access auto-analyzer. Performance characteristics of measurement previously explained . The stuElectrochemiluminescence (ECL) is Roche’s technology for immunoassay detection. Based on this technology and combined with well-designed, specific and sensitive immunoassays, Elecsys delivers reliable results. The development of ECL immunoassays is based on the use of a ruthenium-complex and tripropylamine (TPA). The chemiluminescence reaction for the detection of the reaction complex is initiated by applying a voltage to the sample solution resulting in a precisely controlled reaction. ECL technology can accommodate many immunoassay principles while providing superior performance.Lab tests cutoff values were considered as follows: TPO antibodies ≥ 35 IU/ml, TG antibodies ≥ 40 IU/ml, TSH was defined as ‘abnormally low’ (<0.45), ‘low normal’ (0.45-2.5), ‘high normal’ (2.5-4.5) and ‘abnormally high’ (>4.5) uIU/ml.Statistical analysisThyroid autoimmunity status was considered positive in the presence of anti-TPO and/or anti-TG higher than the upper limit of the reference range. Subgroup analysis for TSH levels between 0.45 and 2.5 was defined as low normal, and between 2.5 and 4.5 was defined as high normal . StatistEthical considerationThis study was approved by Syrian Private University Research Committee with the approval of the Orient Hospital Board of Directors.A total of 2526 women who referred to our fertility center were studied for the presence of TAI. TAI was considered positive in 559 patients 22.1%) of our studies population  while it was 30.10 ± 0.10 (range: 18-43) in TAI negative group   and in abnormally high TSH patients  than patients with TSH levels within normal ranges   and high normal (2.5-4.5 uIU/ml) TSH levels groups revealed that TAI positivity was higher in high normal TSH levels group  than in low normal TSH levels group   , for low normal TSH levels group (30.08 ± 1.17 years), for high normal TSH levels group (30.17 ± 1.26 years) and for abnormally high TSH levels group (30.28 ± 1.79 years) . While, when comparing the mean age of low normal TSH levels group with the mean age of high normal TSH levels group, no difference was found   Table . Among tFinally, when detecting the prevalence of TAI positivity according to the years of lab tests we established that it was 28.18% (n = 122) in 2011, 31.83% (n = 92) in 2012 but 15.10% n = 61) in 2015  , this is consistent with the study done by Poppe et al. . A studyTSH levels in our study showed a significantly higher prevalence of TAI in abnormally low and abnormally high TSH levels. Similarly, TSH abnormally high were frequently more reported in the positive group in comparison to negative TAI women than abnormal low TSH. This finding was previously established .TSH level in correlation with TAI in the pathophysiology of infertility is still controversial. Small case-control prospective studies and a recent systematic review, which included retrospective studies, resulted in no association of TAI and assisted reproductive techniques outcomes, while others found the contrary -30.One limitation that prevented to provide a definitive comparison of our results with other studies is using different immune assays, different lab methods in TAI measurements as suggested in previous studies , 29. We Thyroid autoimmunity prevalence in women seeking fertility care in Damascus, Syria was 22.1% which is significantly higher than the normal population. Further studies are needed to assess the relation of these antibodies in the thyroid, gynecological and other factors for the Syrian population.


Preventive chemotherapy with donated anthelminthic drugs is the cornerstone for the control of helminthiases. However, reinfection can occur rapidly in the absence of clean water and sanitation coupled with unhygienic behaviour. The purpose of this study was to assess the effect of an integrated package of interventions, consisting of preventive chemotherapy, community-led total sanitation (CLTS) and health education, on the prevalence of helminth and intestinal protozoa infections and on participants’ knowledge, attitude, practice and beliefs (KAPB) towards these diseases including water, sanitation and hygiene (WASH).A cross-sectional survey was carried out in nine communities of south-central Côte d’Ivoire to assess people’s infection with helminths and intestinal protozoa and KAPB. Subsequently, interventions were targeted to five communities, while the remaining communities served as control. The intervention encouraged latrine construction and an evaluation was done 6–7 months later to determine open defecation status of the respective communities. Anthelminthic treatment was provided to all community members. A follow-up cross-sectional survey was conducted approximately one year later, using the same procedures.Schistosoma haematobium, Trichuris trichiura, Schistosoma mansoni and Ascaris lumbricoides was 31.1%, 7.0%, 2.0%, 1.0% and 0.3%, respectively. Four of the five intervention communities were classified open-defecation free. For hookworm infection, we observed higher negative changes in terms of proportion of decrease : - 0.16, -0.04) and higher egg reduction rate (64.9 vs 15.2%) when comparing intervention with control communities. For intestinal protozoa, prevalence reduction was higher in intervention compared to control communities (8.2 vs 2.6%) and WASH indicators and intervention outcomes associated with lower odds for infection at follow-up. The intervention significantly impacted on reported latrine use , open defecation in the community surroundings  and awareness for environmental contamination through open defecation .Overall, 810 people had complete baseline and follow-up data and were given anthelminthic treatment. The baseline prevalence of hookworm, An integrated package of interventions consisting of preventive chemotherapy, health education and CLTS reduces the prevalence of helminth and intestinal protozoa infection. Additional studies in other social-ecological settings are warranted to confirm our findings.The online version of this article (10.1186/s13071-018-2642-x) contains supplementary material, which is available to authorized users. More than half of the human population is at risk of soil-transmitted helminthiasis and schistosomiasis, over 1 billion people are infected and the global burden of these neglected tropical diseases in 2015 was 6.3 million disability-adjusted life years (DALYs) –6.vs open defecation) were associated with significantly lower odds of diarrhoea  multiplied by a factor of 100. Significant changes between baseline and follow-up frequencies for infection status, behaviour and awareness with regard to defecation and hygiene within the respective group were analysed using the McNemar’s test. To assess differences over time in infection status between control and intervention communities, taking into account for different baseline prevalence levels, we compared (i) total proportion of change; (ii) proportion of decrease; and (iii) proportion of increase , 34. DifSE is the standard error, pg1 is the proportion of change in group 1, pg2 is the proportion of change in group 2, Ng1 is the number of subjects in group 1, and Ng2 is the number of subjects in group 2.where Univariate and multivariable relationship analysis to assess guiding risk factors for the follow-up infection status was done using logistic regression modelling, including a random effect coefficient on community level. Results of this analysis were presented as crude and adjusted odds ratios (ORs). The latter were adjusted for sociodemographic indicators, namely sex, age group, socioeconomic status and ethnic origin.There were 3420 people living in the nine selected communities according to the Taabo HDSS census database and 3289 were present and participated during the baseline survey in 2011. A total of 2413 provided stool samples, 2294 submitted urine samples and questionnaire data were available from 3152 people. Overall, 1894 people had complete parasitological and questionnaire data. Results from the baseline survey have been presented elsewhere . For theA total of 810 individuals had complete parasitological and questionnaire data from both the baseline and 1-year follow-up surveys and had received anthelminthic treatment after the second ODF evaluation in March 2012. Age and sex characteristics of this final cohort are presented in Additional file Table S. haematobium infection, the predominant Schistosoma species, was mainly found in Sahoua, belonging to the control group. This is reflected in a significantly reduced OR for schistosomiasis in intervention communities . Intervention communities were more affected by soil-transmitted helminth infection (mainly hookworm) than control communities at baseline . However, at follow-up helminth infection prevalence was low among both control and intervention communities with 7.0% and 11.8% for soil-transmitted helminths and 5.7% and 1.4%, respectively, for Schistosoma spp. infections. For intestinal protozoa infections that were equally frequent in both groups at baseline, a significant decline from 72.0% to 63.8% was observed in intervention communities whereas control communities still showed similar frequencies as at baseline (73.0% and 70.4%).The prevalence of helminth and intestinal protozoa infection at baseline and follow-up is shown in Table vs 15.2%) due to higher baseline egg counts in the intervention communities. There was no significant difference in hookworm egg counts between intervention and control group after treatment.Helminth infection intensities at baseline and follow-up are shown in Additional file E. histolytica/E. dispar infection, while control communities showed significantly higher changes in proportion of increase for E. nana and Blastocystis sp. infections.Table P <  0.001). In the former, almost half of all new infections , were diagnosed in school-aged children. Similar to age, reinfection patterns in control communities showed continued sex differences, as already observed at baseline with significantly higher rates of males infected in 2012 than females , while no significant sex differences in infection status in 2012 was observed among intervention communities .Figure P <  0.001) and age groups 6–15 years  or 16–29 years , were positively associated with reinfection with soil-transmitted helminths, whereas individuals from households with higher socioeconomic status showed significantly lower adjusted ORs . Stratification into control and intervention communities revealed that socioeconomic factors associated with soil-transmitted helminth infection in 2012 diverged. In intervention communities, socioeconomic status  and age group 6–15 years  were still significantly related to reinfection. In control communities, in contrast, male gender  and age groups 16–29 years  and 30–45 years  still had higher odds for soil-transmitted helminth infection in 2012.Multivariable regression modelling adjusted for age, sex, socioeconomic status and ethnic origin showed no significant relationship between specific WASH indicators (e.g. toilet ownership and use) and intervention indicators (i.e. ODF status and group) with the 1-year follow-up soil-transmitted helminth infection status Table . SociodeP = 0.015) and ODF community  had lower odds for intestinal protozoa infection at the 1-year follow-up. Among the WASH indicators, households with newly constructed latrines  showed the highest effect on reduction of intestinal protozoa infection.In contrast to soil-transmitted helminth infection, WASH indicators and intervention group . This, however, dramatically changed into the opposite direction after implementation of CLTS . Nearby bushes as the most often stated place for open defecation in 2011  was significantly less often mentioned among households of intervention communities  in 2012, while it remained constantly high in control communities (64.8%). With regard to children’s defecation behaviour, a similar trend was observed. In 2012, children from households of intervention communities were reported to mainly use latrines/toilets (81.4%), while nearby bushes (64.1%) was still the major place for defecation of children from control communities.Table P <  0.001).Not only reported practice with regard to defecation and hygiene changed among households from intervention communities, but also awareness for problems associated with open defecation. Table vs continued open defecation).Our study presents new evidence on potential gains of an integrated control approach consisting of preventive chemotherapy, CLTS and health education against helminthiases and intestinal protozoa infection in a rural setting of south-central Côte d’Ivoire. Over the past decade, CLTS as a means to prevent diarrhoeal diseases has gained traction and is being applied at large scale in various low- and middle-income countries , 35. YetOur package of interventions resulted in a strong response in the five selected communities. Indeed, four out of the five communities were declared ODF within six months, whilst in the fifth community more than 80% of the households had latrines Fig. . ParticuInterestingly, we did not observe significant differences in soil-transmitted helminth reinfection rates when comparing intervention and control communities. This may be partly explained by still relatively low reinfection rates in both groups , perhaps indicating a too short follow-up period (in our case five months). Our assumption was that infection levels in the control communities after a single round of preventive chemotherapy would have approached baseline levels (here > 25%) within about a year . Impact E. nana or Blastocystis sp. if they belonged to the intervention group as highlighted by a statistically significantly lower proportion of increase , which might, at least partly, be explained by children attending the nearest school that might be located in another community not yet declared ODF. Such reinfections might be considered as imported infections, dampening the impact for soil-transmitted helminthiasis control in intervention communities. Another potential source for reinfection could be seen in plantation sites, where open defecation is continued, as latrines were only built in close proximity to the households. In intervention communities, reported open defecation while people pursue agricultural activities remained relatively high (41.1%). Logistic regression modelling indicated potential cultural difference in hygiene practices, as expressed by a higher risk for intestinal protozoa (re)infections among the autochthonous population and reduced risk among Muslim community members. In previous research, males have been found to be more prone for reinfection, as they are less adhering to good defecation practices and hygiene behaviour , 46, 47.Apart from access to sanitation facilities, attitudes towards defecation and hygiene practices, acceptance and (appropriate) use of these infrastructures are key determinants for discontinuation of open defecation and thus successful interruption of faecal contamination of the environment , 16. OurNevertheless, it is difficult to say to what extent CLTS alone or the combination of health education and preventive chemotherapy improved people’s behaviour of defecation and general hygiene and cleanliness. Lawrence et al.  identifiA strength of our study is the rigorous sanitation approach, namely CLTS, which implies a very high private latrine ownership coverage and, ideally, the certification of ODF status in an intervention community. Furthermore, adherence may be enhanced due to non-subsidized latrine provision , 50, 51.Certain considerations with regard to study design should be taken into account in future studies to generate stronger evidence. Specifically, inclusion of more localities is pivotal, so that bias from local heterogeneity of helminth and intestinal protozoa infection can be mitigated. Furthermore, a cluster randomised controlled trial design generates stronger evidence, since potential baseline differences in disease prevalence, population size, level of existing water and sanitation infrastructure are addressed through randomisation . SustainCLTS in combination with health education and preventive chemotherapy holds promise as an effective participatory approach that can be applied at community level to improve people’s health and wellbeing. In our view, the results obtained here from a primarily rural setting of Côte d’Ivoire with high levels of open-defecation and low access to sanitation programmes at the start of the interventions are relevant for other settings in low- and middle-income countries . CLTS imAdditional file 1: Table S1. Characteristics of the final cohort. (DOCX 14 kb)Additional file 2: Table S2. Mean infection intensity in control and intervention communities during baseline and follow-up surveys. (DOCX 17 kb)


Intake of large neutral amino acids (LNAA) may inhibit phenylalanine (PHE) transport across the blood brain barrier and assist with blood PHE control in patients with phenylketonuria (PKU). We evaluated the interrelationship between LNAA in plasma and diet on Phe:Tyr (P:T) ratio in patients with PKU and the influence of dietary factors on plasma LNAA markers.Plasma amino acid values and 3‐day food record analysis from two studies  were examined. For pediatrics (<18 years) and adults (≥18 years) the relationship between P:T ratio, plasma LNAA, and dietary intake patterns were investigated.P < .05). Associations were found between plasma valine and other dietary and plasma LNAA in pediatrics (P < .05) and plasma LNAA with dietary LNAA intake in adults (P = .019). Plasma P:T ratio was inversely associated with plasma LNAA concentrations in both age groups (P < .05). Aside from histidine in pediatrics (P = .024), plasma LNAA did not differ by having plasma PHE levels within or above the therapeutic range (120‐360 μmol/L). Plasma LNAA in both age groups was similar to reported healthy control values.Dietary factors influencing P:T ratio included intake of total protein (g/kg), medical food (MF) protein , and LNAA (g) in the full cohort (P:T ratio is significantly tied to dietary LNAA, adherence to MF Rx, and plasma LNAA concentrations. Additionally, P:T ratio and valine may be effective clinical proxies for determining LNAA metabolic balance and LNAA quality of the diet in patients with PKU. Dietary factors play a role in LNAA plasma concentrations for the potential benefit of PHE control in the blood and across the blood brain barrier, while plasma valine and P:T ratio may serve as effective biomarkers of overall LNAA status.1Phenylketonuria (PKU) is an autosomal recessive genetic disorder in which the enzyme, phenylalanine hydroxylase (PAH), produced in the liver, is deficient. The reduced activity of PAH causes phenylalanine (PHE) to build up in the blood and body tissues which is toxic to the central nervous system, most notably the brain.Plasma concentration of large neutral amino acids (LNAA) may also play an important role in the treatment of PKU, particularly with the presence of LNAA in prescription medical foods and with prescribed LNAA supplements becoming more prevalent as a treatment option.Mechanism of action regarding LNAA influence on metabolic control in PKU and brain function most likely involves competitive uptake inhibition in the gut and across the blood brain barrier (BBB).Therefore, the purpose of this study is to examine the effect LNAA intake from medical food and intact protein have on both P:T ratio and plasma LNAA in children and adults with PKU. These data are useful for determining intervention for optimizing LNAA status and P:T ratio in patients with PKU so as to improve clinical outcomes.22.1The study sample  approved Emory Metabolic Camp and sapropterin study protocols were used as described in the prior section. Each patient was assigned a random study identification number. Variables included demographic characteristics , anthropometrics , nutrient intake from dietary food records, plasma amino acids, and determinants of dietary compliance. Ethics approval for this secondary retrospective study was received from the Georgia State University IRB and met Emory University IRB standards.2.32]. Subjects under 18 years have BMI reported as a percentile. All measures were conducted by trained research staff. Participants at the Emory Metabolic Camp and at the baseline visit for the sapropterin study completed a 3‐day food record prior to their visit. A registered dietitian (RD) from Emory University analyzed collected food records for nutrient intake using Nutrition Data System for Research . Participants were determined to be adherent with their PKU diet if blood PHE levels were within recommended range (120‐360 μmol/L).Weight in kilograms (kg) was measured with a standing scale. Height in centimeters (cm) was determined using a stadiometer. Body mass index (BMI) was calculated [weight (kg) / height (meters)2.4Green top sodium heparin vacutainers were used for blood sampling. Plasma amino acids were analyzed using quantitative ion exchange chromatography at the Emory Genetics Laboratory. Plasma LNAA reported, aside from PHE and Tyr, included valine, leucine, isoleucine, methionine, threonine, and histidine. For a subcohort in the study (n = 50) tryptophan was also reported. Sum plasma LNAA was calculated by adding the individual plasma LNAA concentrations, a method previously utilized in the literature.2.5t test comparisons were used to detect potential group differences between sum (plasma and intake) LNAA values with and without tryptophan. Compliance status of the study sample was determined by plasma PHE concentrations. Those with plasma PHE exceeding 360 μmol/L were categorized as “noncompliant” with their PKU treatment plan independent of reported intakes on their diet record Differences based on PKU diet compliance were investigated with Mann‐Whitney U test or t test. Margins of error are reported as 25th and 75th percentiles or SD dependent on normality of the data. Multiple comparisons due to separation of sample by age group were Bonferroni adjusted. All statistical analyses were performed with SPSS . IBM SPSS Sample Power (version 3) was used to assess needed sample size for a regression model with up to three variables at a power of 0.80. Alpha was set at 0.05.Frequency analysis was used to describe demographic, anthropometric, nutrient intake, and clinical characteristics of the combined sample. Normality testing was conducted on all continuous variables to determine whether parametric or nonparametric statistics should be used. Correlations were used to identify potential cofactors such as age, gender, and dietary compliance. To control for possible age related differences relevant to dietary and metabolic parameters, the sample was divided into pediatrics (<18 years) and adults (≥18 years). Kendall's tau and multivariate regression were used to assess relationships between plasma LNAA, plasma P:T ratio, LNAA intake from diet sources and in association with adherence to MF Rx. The 33.1The final cohort included 64 participants  with an age range of 4.6‐47 years. The majority of participants were in the sapropterin study (n = 55) while the remaining (n = 9) attended the Emory Metabolic Camp in the summer of 2016 , validating outcomes in the subcohort in which plasma tryptophan concentrations were included.Comparisons of dietary sum LNAA (g) with and without tryptophan, and plasma sum LNAA (μmol/L) with and without tryptophan yielded no significant differences (3.2P = .001) , emphasizing the importance of dietary adherence on metabolic control. Moreover, the percentage of MF protein the patient consumed below their prescription had a direct correlation with P:T ratio , though not after Bonferroni correction.In the separate age groups, dietary LNAA (g) were not associated with P:T ratio. For pediatrics, intake of protein , energy , and ratio of MF to intact protein were also not associated with plasma P:T ratio. In adults, g/kg intake of total protein and MF protein were each inversely correlated with P:T ratio  other than threonine , tryptophan , and PHE . The full cohort, as with the adult subcohort, also demonstrated strong association of P:T ratio with MF intake   and total protein intake  .Within the full cohort, when controlled for linear age, P:T ratio was inversely associated with dietary intake (g) of the LNAA sum Figure A, and wi3.3P = .029) and valine . In adults, positive associations were found with valine  and with sum LNAA, . Dietary tyr correlated with plasma valine for both pediatrics  and adults  along with sum plasma LNAA (P ≤ .044). Dietary tyr for pediatrics also correlated with plasma isoleucine  and tryp . Once Bonferroni corrected, the only significant correlations were with valine in the pediatric group, and between dietary sum LNAA and plasma sum LNAA (with tryptophan) in the adults.Evaluating correlations between individual dietary LNAA intake (g) and plasma LNAA levels, significant positive associations were found in the full cohort with the sum LNAA values Figure B and in P ≤ .015) excepting PHE and tryptophan, and with plasma LNAA (P ≤ .019), excepting PHE. In adults, valine likewise directly associated with the plasma BCAA (P ≤ .002), threonine , and the sum plasma LNAA . Only dietary threonine (g)  and tryptophan (g)  intakes correlated with plasma valine in adults once Bonferroni corrected.Of note, in the pediatric cohort, plasma valine had strong positive correlation with intake of all dietary LNAA (P = .004) and total protein intake (g/kg) in adults . Histidine associations held after Bonferroni correction. No significant correlations were found between plasma LNAA and intake measures of dietary PHE  in dietary compliant vs noncompliant pediatrics (mean ± SD: 80.7 ± 13.4 vs 69.0 ± 12.6 μmol/L). Even so, histidine intake (g/day) was similar across both pediatric compliance groups .Plasma histidine correlated with MF protein (g) intake in pediatrics  except for valine, isoleucine, and leucine which were not statistically significant. Plasma tyr had significant positive associations with LNAA outcomes (P ≤ .028) other than plasma methionine  and histidine . Median plasma P:T values for adults was 16.2  and 9.8  for pediatrics. For both pediatric and adult groups with PKU, plasma P:T ratio had significant inverse associations with plasma threonine, histidine, and sum plasma LNAA (tyr removed) (Table P ≤ .028) except for isoleucine . When, controlling for linear age, isoleucine was also inversely correlated with P:T ratio .In the full cohort controlled for age, plasma PHE associated inversely with the sum plasma LNAA Figure D, and wi3.5There is no general population standard, or matched non‐PKU cohort, to compare against the mean plasma LNAA concentrations in our study group. Nevertheless, a study conducted by Scriver et al4This study adds to the research literature on LNAA and their role in managing the metabolic needs of those with PKU. As nutritional and pharmacologic therapies for PKU change, dietary patterns do as well, which can affect health outcomes in diverse ways. The general literature consensus is that LNAA supplementation or specific MF higher in LNAA could benefit patients with PKU, even more so combined with metabolic PHE control. Particularly worth noting are the LNAA supplements available now for PKU patients to be taken in addition to or as a replacement for traditional MF.Our study focused on plasma LNAA and LNAA intake within the limits of PKU dietary adherence. Interestingly, we found in our study an inverse association of P:T ratio with multiple LNAA and the sum of LNAA in plasma Table  even thoAlthough human research into the effects of LNAA intake on brain PHE is limited, one study observed a lowering of brain PHE levels with increased LNAA intake, as measured by Magnetic Resonance Spectroscopy. This occurred despite no observed increase in plasma LNAA concentrations.Rohde et alWithin our study, dietary influence regarding protein source, MF or intact protein and dietary compliance status as measured by plasma PHE, had little bearing on plasma LNAA concentrations, although extent of noncompliance as determined by plasma PHE was a factor in lower plasma histidine concentrations among pediatrics. Even so, most still fell within the clinical reference range of 50‐104 μmol/L (LabCorp) for histidine.There are limitations within the current study. The majority of participants (75%) were noncompliant as evidenced by plasma PHE levels >360 μmol/L, which reduced the size of the compliant subgroup for our analysis. Unfortunately, high numbers of noncompliance with plasma PHE and diet requirements, particularly among adolescents and adults, are common in the PKU medical population.5Our research has demonstrated a clear inverse relationship between LNAA concentrations in the plasma and P:T ratio, with dietary and plasma tyrosine of greater weight than PHE. We also found that P:T ratio correlated with dietary intake of total protein, MF protein, and LNAA  and the Sapropterin (Kuvan) Study (IRB #7828). Ethical approval was also received from the Georgia State University Institutional Review Board (H17510).All patients who participated in the study and are represented in the data provided written informed consent and/or assent as age appropriate.N/A.T.D.D., A.M.N., A.M.B., S.T.H., and R.H.S. have studied the concept and design, original drafting of manuscript and follow up revisions, and critical review of the manuscript. A.M.B., A.M.N., T.D.D., and R.H.S. have prepared the interpretation of data. T.D.D., A.M.N., and A.M.B. prepared the statistical analysis.


Lactobacillus spp. predominantly shows its presence as a normal mucosal flora of the mouth and intestine. Therefore, the objective of our research is to investigate the in-vitro conditions for the prospective of medically valuable biosurfactants (BSs) derived from Lactobacillus spp. Biosurfactant (BS) obtained from Lactobacillus spp. exhibit antibiofilm and antiadhesive activity against broad range of microbes. In the present study we investigated the production, purification and properties of key components of the cell-associated-biosurfactant (CABS) from Lactobacillus acidophilus NCIM 2903.Extracted, purified, freeze-dried CABS shows reduction in surface tension (SFT) of phosphate buffer saline (PBS @pH 7.0) from 71 to 26 mN/m and had a critical micelle concentration (CMC) of 23.6 mg/mL. The CABS showed reduction in interfacial tension (IFT) against various hydrocarbons and had effective spreading capability as reflected through the decrease in contact angle (CA) on different surfaces . The anionic nature of CABS displayed stability at different pH and temperatures and formed stable emulsions. Thin layer chromatography (TLC) and Fourier transform infrared spectroscopy (FTIR) revealed CABS as glycolipoprotein type. The Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) showed presence of multiple bands in a molecular range of 14.4 to 60 kDa, with prominent bands of 45 kDa. The CABS has significant antiadhesion and antibiofilm activity against tested bacterial strains.The current challenging situation is to develop methods or search for the molecules that will prevent the formations of biofilm on medical bioimplants of PDMS based materials. These findings are supportive for the use of Lactobacilli derived BS as potential antiadhesive agent on various surfaces of biomedical devices. Biosurfactants (BSs) are amphiphilic molecules produced by diverse microorganisms such as bacteria, fungi and yeasts at the microbial cell surface or excreted out with pronounced surface and emulsifying activities –3. ThesePseudomonas, Bacillus, Acinetobacter, Arthrobacter, Rhodococcus, Clostridium, Halomonas, Lactobacillus, Leuconostoc, Myroides, and Serratia, also in some genera of yeast viz., Debrayomyces and Rhodotorula [Lactobacillus gained importance in biomedical and therapeutic field for the exploration of BS synthesis due to two reasons, (i) they constitute an important part of natural microflora [Lactobacillus in the microflora of healthy human play a key role in maintaining stability and diversity of the gut microbiome [Lactobacillus prevent the enteropathogen-mediated infection by competing for nutrients and binding sites , by secreting antimicrobial substances such as (i) organic acids, (ii) H2O2, (iii) bacteriocins and reducing gut pH and producing BSs, eventually by counteracting the spread within the colonized body [Lactobacillus, BS derived from other microorganisms may raise the issue of health risk. For example, Serratia marcescens produce serrawettin which plays some role in the virulence of this species [Pseudomonas strains producing rhamnolipids exhibit pathogenic potential, suggesting that these biomolecules may contribute to its opportunistic pathogen characteristics [Lactobacillus is a subject of research interest. Different species of Lactobacillus viz., L. plantarum [L. agilis [L. paracasei [L. pentosus [L. casei [L. helveticus [L. fermenti [L. rhamnosus [L. acidophilus [Lactobacillus derived BS are increasingly applied as antiadhesive agents in therapeutic field, which play an important role in the prevention and control of infections caused by biofilm forming pathogens from various groups of microbes [L. acidophilus NCIM 2903 and structurally characterized for antibiofilm properties using various model organisms.The BS synthesis has been reported by various genera of bacteria, viz., dotorula . Lactobacroflora  and (ii)croflora . Whereascrobiome , the antzed body . Unlike  species . Likewiseristics . Therefolantarum , L. agil. agilis , L. paraaracasei , L. pentpentosus , L. caseL. casei , L. helvlveticus , L. fermfermenti , L. rhamhamnosus , L. aciddophilus  have beemicrobes –25. Biofmicrobes . Biofilmmicrobes . FormatiL. acidophilus NCIM 2903 was procured from the National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratory (NCL), Pune, Maharashtra, India. For antibacterial and antiadhesive assays, Escherichia coli NCIM 2065, Staphylococcus aureus NCIM 2079, Proteus vulgaris NCIM 2027 cultures were all obtained from NCIM, NCL, Pune, India. Other cultures viz., Bacillus subtilis MTCC 2423, Pseudomonas putida MTCC 2467 and P. aeruginosa MTCC 2297 were obtained from Microbial Type Culture Collection - MTCC, Chandigarh, India. NCIM 2903 was grown and maintained in the laboratory on DeMan, Rogosa and Sharpe (MRS) (Himedia) agar [The BS production was assayed by different screening methods, since no single method is sufficiently accurate to assess the BS biosynthesis. The methods used in the study are as follows: Drop collapse test (DCT), Hemolytic Activity (HA), Emulsification Index, (EI), Oil Spread Method (OSM), Blue Agar Plate method (BAP), Tilted Glass Slide Test (TGST). All methods were carried out as described from our previous work . Apart fL. acidophilus into 10 ml of fermentation medium (FM) and incubated it overnight at 30 °C under shaking conditions (170 rpm). For BS production, a 5% (V/V) seed culture was transferred into sterile 150 ml of FM in 1 L Erlenmeyer flask and incubated at 170 rpm and 30 °C up to 120 h. The BS biosynthesis was monitored after every 12 h. The biomass during the BS biosynthesis was determined as dry cell weight. For this purpose, a volume of 10 ml of FM was filtered through sterile filter paper  and dried at 105 °C for 24 h and reweighed. Dry bacterial biomass was repeatedly weighed till constant weight was achieved.For production of BS three different media were used, viz., (i) Fermentation medium (FM)  MRS with Tween 80 (MRS-T) (0.1%), (iii) MRS without Tween 80. The media were examined for better production of BS and the examination for the same was done by measuring the SFT by pendant drop method at different time intervals. Further, FM medium was selected for the BS production throughout the research. A seed culture was prepared by transferring a single colony of g, 20 min, 4 °C), washed in demineralized water and resuspended in 50 mL of phosphate buffered saline . Cells were gently resuspended at room temperature (RT) of 30 °C for 2 h to release BS associated with bacterial cell followed by centrifugation . Remaining supernatant was filtered through filter . The supernatant was dialyzed against demineralized water with dialysis membranes having molecular mass cut-off 12,000 Da  and further freeze-dried and stored at − 20 °C until further use.For the extraction of cell CABS, cells were harvested by centrifugation , relative emulsion volume (REV), temperature and pH stability. Importantly, other physical properties, SFT, critical micelle concentration (CMC) and also interfacial tension (IFT) was determined by a pendant drop technique using OCA Goniometer. The spreading capacity of BS was determined by measuring the contact angle (CA) of CMC solution of BS in a sessile drop technique. Synthetic surfactants (their CMC concentrations) viz., cetyl trimethyl ammonium bromide (CTAB), Aerosol-OT/sodium bis (2-ethylhexyl) sulfosuccinate (AOT), Sodium dodecyl sulphate (SDS), Tween 80 were included as reference surfactants for testing all physical properties of BS.The CMC value of BS was determined by measuring the SFT of BS in PBS solution (pH 7.0). The CMC is the point where an increase in the concentration of BS does not have any further reduction in SFT. In this technique, drop of liquid of different concentrations of BS, ranging from 1 to 60 mg/ml prepared in PBS was allowed to hang from the end of a capillary and the asymmetric shape of a pendant drop was determined by using Laplace-Young equation , 30 for l = 35 mm, b = 22 mm and, h = 25 mm) was cleaned in ultrasonic bath using aqueous detergent solution, followed by washing with distilled water, acetone, ethanol and finally isopropyl alcohol. After cleaning and drying procedures, the glass cubical was filled with 15 ml of kerosene  and placed on the platform of Goniometer. Further, a syringe was filled with reference and BS solutions (at CMC concentration) and the tip of the syringe was dipped completely in kerosene filled in the glass cubical. Subsequently a pendent drop of BS solution in kerosene was formed, and measurements for IFT were taken between two immiscible liquids as discussed earlier.The IFT of the BS sample was also determined. A glass cuvette (The spreading ability or wettability of BS was determined by measuring the contact angle (CA) on various surfaces by using sessile drop techniques A drop of 25 μl of sample was placed and measurements were taken on five different surfaces viz., Teflon polymer (Polytetrafluoroethelene-PTFE) tape (highly hydrophobic), Polydimethyl siloxane (PDMS) (highly hydrophobic), polystyrene, overhead projector (OHP) transparency sheet (Intermediate hydrophilic-hydrophobic) and glass slide (highly hydrophilic) surfaces.l = 2.0 cm, b = 2.0 cm, h = 0.1 cm) for overnight and dried then further in dust free airflow. After complete drying procedures, the CA of a drop of 25 μl water, PBS (@pH 7) and various synthetic surfactants (as mentioned earlier) were also measured to analyse any change in the hydrophobicity of PDMS surface before and after coating of CABS.The CA measurements were taken on both PDMS surface (Control) and PDMS coated with BS (Test). A thin layer CABS (CMC solution 23.6 mg/ml) was coated on PDMS surface by immersing the PDMS sections  were used to examine the stability of BS. Similarly, to evaluate stability at different temperatures, the freeze-dried CABS was dissolved in PBS (pH 7.0) and incubated at 28 °C, 37 °C and 60 °C for 48 h and SFT for each sample was determined before and after incubation procedures. In addition, the activity of BS was also checked before and after autoclaving.The emulsion-stabilizing capacity of the BS was tested on five different hydrocarbons . In this technique, 2 ml of BS (CMC solution) and 2 ml each of hydrocarbons were taken in a graduating test tube, shaken vigorously for 2 min, and left stand still for 24 h. The relative emulsion volume (EV in %) and emulsion stability (ES in %) were calculated as per the method described by Das et al. .Briefly, 10 μl aliquot of BS sample was concentrated on a pre-coated silica gel plate –TLC , and run in following system: chloroform: methanol: acetic acid (65: 25: 2); butanol: ethanol: water (5: 3: 2); chloroform: methanol: water (65: 15: 2); butanol: acetic acid: water (12: 3: 5) and developed respectively with anisaldehyde, diphenylamine, iodine and ninhydrin reagents for the respective determination of glycolipid, sugar, lipids and amino acid moieties of BS.− 1 at a resolution of 4 cm− 1 was obtained and analysed.The FTIR facilitates identification of types of chemical bonds i.e. functional groups present in BS. One milligram of BS powder was grounded with 100 mg of potassium bromide (KBr) and translucent pellet was analyzed using FTIR device . The spectrum in the range of 400–4000 cmw/v) resolving gel and 4% stacking gel and run at a constant current (25 mA) till the dye front reaches the bottom. The gels were developed by staining with Coomassie blue R250. The molecular weight of protein was determined by comparing it with the protein ladder. The protein ladder  used in the study is as follows: (weights in kDa) β-Galactosidase (116.2); Phosphocyclase –B (97.4); Serum albumin (66.2); Ovalbovine (45); Carbonic unhydrase (31); Trypsin inhibitor (21.5); Lysozyme (14.4).Gel electrophoresis of BS (50 μg) was carried out using 12% (w/v). The lower row of wells was filled with the BS solution (at CMC concentration) and the upper rows of well were filled with reference surfactants  of different ionic charges, and monitored for the presence of precipitation line over a period of 48 h at RT.Agar double diffusion technique was used to determine the ionic charge of BS . For thiE. coli, S. aureus, P. vulgaris, B. subtilis, P. putida, and P. aeruginosa. Micro-dilution technique in 96-well flat-bottom plastic tissue culture plates  was carried out to determine the antibacterial and antiadhesive activity of BS against selected bacterial cultures [Antibacterial and antiadhesion assays of BS were performed by using six bacterial cultures viz., cultures .− 3 to 1 Torr). Circular PDMS discs (diameter 0.8 cm and thickness of 0.1 cm) were cut and used for evaluating adhesion inhibition activity of BS against pathogenic bacteria.PDMS based contact lenses were prepared by mixing 184 silicone elastomeric base with a curing agent (10:1)  followed by gentle stirring to obtain a uniform and clear solution. The clear solution of PDMS was poured in sterile disposable petri dish. It was allowed to solidify at temperature of 80–100 °C for 6 h in vacuum oven at a pressure of 100 Pa (10Two sets of sterile PDMS discs were used to demonstrate the antiadhesion and antibiofilm effect of formulation. One set without any BS coating was considered as control. The other set coated with BS (CMC solution in buffer) was considered as test. For coating, all discs were immersed in sterile nutrient culture medium and then inoculated with test culture and incubated at 37 °C. Further discs were removed and rinsed with sterile distilled water to remove un-adhered microorganisms. Further whole discs were observed under light microscope (10 X), and scanning electron microscopic  images were also taken.P values < 0.05 were considered significant. Error bars are represented as as ± SD.All experiments were performed in triplicates. The standard deviation of the data was analyzed using the software ‘GraphPad Prism version 7.00 ’ and is presented in the form of Figs. The results of the screening assays used to confirm BS production including SFT, CA measurements were positive.L. acidophilus NCIM 2903 was determined by measuring the SFT of cell free broth and in case of cell bound BS extracted in PBS. In this paper, our work is solely focused on CABS. L. acidophilus did not synthesized BS in MRS and MRS-T evident from SFT values. Both MRS and MRS-T supported the growth of L. acidophilus culture; however, no drastic change in pH was observed . The synthesis of BS  occurred in FM. At 72 h, the value of SFT reduced from an initial 55 to 28 mN/m. However, after 72 h, the synthesis of BS was decreased   and have not changed over a broad range of pH and temperature. The SFT remained relatively stable over a pH range from 6 to 10, and temperature range of 4 °C to 60 °C for 120 h. The SFT values at pH and temperature outside these range increased slightly (31 to 35 mN/m).The EC and ES activity of BS against different water immiscible hydrocarbons revealed a relative EV between 25 and 65% and a relative ES between 45 and 87%. The highest EV (65%) and ES (87%) were detected against n-decane, followed by xylene, (46%) and (87%).The comparison of the BS with reference compounds on TLC showed the presence of sugar Fig. a, lipid − 1 confirming the presence of –OH groups while the small peak at 2851 cm− 1 corresponds to the existence of C-H bond. Peak at 1666 cm− 1 determines C=O stretching while peak at 1550 cm− 1 determines presence of -NH stretching, both confirming the presence of proteins. The peak at 1069 indicates the presence of C–O stretching in sugars. Peak at 978 cm− 1 reveals the presence of mono-substituted alkenes. The FTIR spectrum obtained in present study suggests the glycolipoprotein type of BS.The molecular composition of BS was further studied by FTIR spectroscopy Fig. . The FTIThe SDS-PAGE of the BS illustrated multiple bands in a molecular range of 14.4 to 60 kDa, with prominent bands of 45 kDa. It might be because of contamination of peptides which could not be removed completely during the purification process. Please note that the BS reported in this study is cell associated (proteinaceous) and not cell free (not released in the medium), therefore higher is the possibility of cell associated BS got contaminated with other proteins.Ionic characteristics as revealed by agar double diffusion tests (which are based on the passive diffusion of two compounds possessing charges of the similar or opposite types in a weakly-concentrated gel) displayed precipitation lines between the BS and cationic compounds (CTAB); such precipitation line was not observed between the BS and anionic compounds (SDS) Fig. .Fig. 8DoE. coli and P. vulgaris by 34 and 33% respectively, followed by B. subtilis (26%) and P. putida (14%)  Fig. . Similarely Fig. .Fig. 9AnP. vulgaris and S. aureus on PDMS based contact lenses surface illustrates the antiadhesion property of CABS. SEM studies also demonstrated the antibiofilm and antiadhesive potentials of BS against P. vulgaris and B. subtilis on PDMS discs  disrupt membranes leading to cell lysis and metabolite leakage (ii) disrupt protein conformations of important membrane functions such as transport and energy generation  (iii) fon et al.  reviewedn et al.  showed aLactobacillus spp. Therefore, we designed a new medium (FM) to support the synthesis of BSs. The decrease in SFT values of FM medium inoculated with Lactobacillus culture (test) was compared with control medium (without inoculation). Interestingly, the synthesis of BSs was accompanied by change in the pH of the FM medium from neutral to alkaline condition, which is rather contrary with available reports [2 gas to replace dissolved oxygen. Gudiña et al. [Lactobacillus genus which might be due to production of lactic acid and other metabolite. However, in present study, an increase in the pH   and a peak at 1′480 cm − 1 (AmII band: NOH bending in proteins). The BS showed comparatively higher amounts of protein and less polysaccharides in the structure. Determination of accurate molecular structure is extremely challenging [L. acidophilus. The FTIR spectrum obtained in our study is comparable with FTIR spectrum of BSs obtained from L. pentosus [L. agilis CCUG31450 produces 84 mg/L CABS in MRS medium. Whereas usage of cheese whey results increase in CABS up to 960 mg/L [It is often important to know the molecular composition of BSs to explore their potential applications in various fields. Therefore, the molecular compositions of the BSs synthesized by ycolipid , 20 or gycolipid , 16, 38.ycolipid , Vecino ycolipid  and Moraycolipid  which shH groups  whereas searches , 19. In r et al. , showed llenging  and is bpentosus , 39, sugpentosus . L. agilLactobacillus. Most of the literature on SFT values for BS produced by Lactobacilli species are in the range of 41 to 46 mN/m with varied CMC values [Lactobacillus spp. have not been frequently reported in literature. The ability of BS to reduce the CA on hydrophobic surfaces has been successfully utilized to interfere the initial adhesion of uropathogenic bacteria and yeasts on silicone based surfaces [Lactobacilli showed some good emulsification abilities as well as stabilities which is in an agreement with Portilla-Rivera et al. [L. pentosus. The agar double diffusion tests performed to know the ionic characteristics of BS revealed an anionic nature of BS derived from Lactobacillus. The test is actually an indication of the passive diffusion of two compounds having same or opposite types of charges in a weakly-concentrated gel. Anionic nature of BS (in present study) has also been reported by Sharma et al. [L. helveticus strain MRTL91.Knowledge of various physical properties of BSs such as SFT, IFT, CMC, CA, EA and ES presents number of opportunities to explore them for undeniable applications proving the efficiency of BS. Unlike the most explored method (DuNouy Ring Method- to determine the efficiency of BS), we used a pendant drop technique to determine the profile of the drop of BS solution suspended in another liquid at equilibrium . A measure of a decrease in the IFT value between liquid and kerosene interphase by BS is an indication of the effectiveness of BS, which can be explored further to remediate oil spills in oceans and petroleum contaminations in soils. The CA determination between BS and surfaces is an important to determine the wettability. The CMC determination is important because it represent the concentration of BSs at which no further reduction in the SFT value of BSs solution occurs despite an increase in the concentration of BSs. Therefore, lower the CMC value, more efficient the BSs. In the present study, the CMC value of BS was quite close to the CMC of SDS, indicating an efficient SFT reducing ability of BS derived from C values , 16, 21.surfaces . This haa et al.  for emula et al.  for BS dLactobacillus could be due the molecular and physiological characteristics of biofilm. Hydrophobicity and surface charge of a bacterial cell are important factors that govern the non-specific attachment of the bacteria to a surface. It is well known that most of microorganisms possess negative charges; therefore, the antiadhesion property shown by BS in present study could be due to an anionic nature of BS, which could have repelled the microbial adherence to the surfaces. The physical forces responsible for bacterial adhesion to surfaces include steric interactions, van der Waals forces and electrostatic (double layer) interaction. Also alteration/ fluctuation in the microenvironment make it difficult for bacterial cells to adapt easily. Gram negative bacteria tend to adhere to moderately hydrophobic surfaces owing to their hydrophobic LPS (lipopolysaccharide) layer [Lactobacillus derived BS are in agreement with Gudiña et al. [Lactobacillus spp. like L. jensenii and L. rhamnosus do exhibit antimicrobial, antibiofilm as well as antiadhesive properties against multidrug resistant pathogens [S. marcescens exhibits antimicrobial activity against C. albicans, P. aeruginosa and B. pumilus. Similar studies carried out by Velraeds et al. [Lactobacillus spp. on E. faecalis biofilms on glass surfaces. We investigated antibiofilm effect of BS on PDMS based surfaces where biofilms P. vulgaris, S. aureus and B. subtilis were nearly inhibited. Previous studies demonstrated by Mireles et al. [Lactobacillus spp. derived BS exhibiting antioxidant and antiproliferative properties against biofilm producers indicating the promiscuous applications in prevention of oral diseases. BS produced by Lactobacillus spp. have offered its applications in cosmetic formulations and medicine [Lactobacillus is proving to be exceptionally promising candidate for the designing of newer BS based products. The proposed fact of our studies can be supported by Brzozowski, et al. [L. acidophilus BS can be utilized against used against one of the nosocomial infectious pathogen like S. marcescens. Infections from various microorganisms at vaginal, urinary and gastrointestinal tracts infections can be protected with the help of BS derived L. acidophilus [L. acidophilus RC14 as antiadhesive against various uropathogenic bacteria to silicone rubber surfaces. One of the pathogens namely S. mutans associated with dental caries and biofilm formations are effectively inhibited by the CABS produced by L. acidophilus [Lactobacillus having huge potential as antiadhesive agent which can be used for various surfaces of biomedical devices.After studying the physical, molecular, properties of BS, we decided to investigate the antibacterial, antiadhesive and antibiofilm properties. The study of antiadhesive and antibiofilm activities of BS become essential due the reports of the failure of bio-implants due to colonization of biofilm forming organisms, and an increased resistance of such microorganisms (due to biofilm) to the treatment of antibiotics. The improved antiadhesive and antibiofilm activities of BS from e) layer . In addia et al. , 17, 45.athogens , 47. Ourathogens  who reves et al.  showed is et al.  indicates et al. . Recent s et al.  demonstrmedicine , 53. Thu, et al.  where BSdophilus . We havedophilus  who havedophilus . FinallyLactobacillus spp. and we are reporting the lowest surface tension value. We are also proposing a simple fermentation medium for BS production; where MRS medium was the routine practice. Our work demonstrated that BS from L. acidophilus exhibited antibiofilm and antiadhesive activities against biofilm producers on PDMS based medical implant surfaces.To the best of our knowledge, this is the first report extensively discussing the characterization of various physical properties of BS derived from


Factor VIII (FVIII) activity ranged from <1.0% to 3.0%, and anti-FVIII inhibitor titers ranged from 8.8 to 240 BU/mL. Treatment was administered at the discretion of the responsible physician. Hemostatic agents applied in the six patients comprised rFVIIa (NovoSeven®) (n=4), APCC (Feiba®) (n=2), and fresh frozen plasma/plasma exchange (n=1). Agents employed for inhibitor eradication comprised prednisolone only (n=3), prednisolone with cyclophosphamide (n=1), prednisolone with cyclosporine (n=1), and prednisolone with rituximab (n=1). In five patients, management was successful, with complete response. Treatment failed in the patient with the highest inhibitor level (240 BU/mL) in whom treatment with APCC  and prednisolone (0.5 mg/kg/day) was followed by several episodes of relapse. The present data demonstrate that AHA severity shows wide variation in elderly subjects, indicating the necessity of individualized management.This report describes six elderly patients with acquired hemophilia A (AHA), including four individuals aged ≥90 years. Bleeding symptoms were subcutaneous or intramuscular hemorrhage ( Acquired hemophilia A (AHA) is a potentially life-threatening bleeding disorder caused by antibodies against factor VIII (FVIII) . PreviouThe summarized case histories of the six patients are presented below, who were treated at two institutions where board certified hematologists were available . All casµg/kg/dose) and prednisolone (1.2 mg/kg/day and then tapered). On day 33, combined cyclophosphamide  and prednisolone therapy was initiated, which was administered for 5 weeks. Within 7 weeks, a CR was obtained.A 91-year-old female presented with a 4-day history of subcutaneous as well as intramuscular hemorrhages of the upper and lower extremities. In her past medical history, she had thoracic and lumber compression fractures. Laboratory investigations revealed a severe anemia (Hb 4.6 g/dL) with a prolonged APTT . Initially, she was taken care of with red blood cell  and fresh frozen plasma  transfusions. On day 5, interventional radiology was carried out to examine femoral vasculatures because of severe extravasation in the right thigh; however, no abnormalities were noted. On day 10 of admission, she was found to have an FVIII activity level <1.0% and an anti-FVIII inhibitor level of 110.6 BU/mL, in addition to positive lupus anticoagulant (LAC) . A diagnosis of AHA was therefore assigned with positive LAC. Following consultation with a hematologist, the patient received 36 doses (4.5 doses/day) of rFVIIa  over 2 days in combination with prednisolone (0.6 mg/kg/day and then tapered). Within 5 days, the hematuria and subcutaneous hemorrhages improved. AHA was controlled within 2 weeks, and a CR was obtained.A 93-year-old male patient presented with hematuria, epistaxis, and a subcutaneous hemorrhage in the region of the right shoulder two weeks after admission for influenza B pneumonia. Laboratory investigations revealed anemia (Hb 7.5 g/dL), a prolonged APTT , and microscopic hematuria (>100/HPF). Five days later, the patient was diagnosed as having AHA when laboratory investigations revealed an FVIII activity level <1.0% and an anti-FVIII inhibitor level of 8.8 BU/mL. After consultation with a hematologist, the patient was administered six doses of rFVIIa , and prednisolone (0.5 mg/kg/day and then tapered) was continued as maintenance therapy. AHA was controlled within 7 weeks, and a CR was obtained. The patient remained stable for 2 years. However, she then developed further subcutaneous hemorrhages. Laboratory investigations revealed an FVIII activity level of 6.2% and an anti-FVIII inhibitor level of 2.1 BU/mL. With a diagnosis of AHA relapse, reintroduction of prednisone (0.6  mg/kg/day) was performed.A 92-year-old female presented with generalized subcutaneous hemorrhages for a few days. Three weeks before, she had undergone surgery for a left femoral trochanteric fracture. Because of anemia (Hb 7.3 g/dL) and a prolonged APTT , she was administered RBC (10 units) and FFP (2 units) transfusions, and oral prednisolone (0.5 mg/kg/day and then tapered) was started. On day 7 of admission, laboratory investigations revealed an FVIII activity level of 3.0% and an anti-FVIII inhibitor level of 10.0 BU/mL. A diagnosis of AHA was therefore assigned. The patient received two doses of rFVII  were administered to control the bleeding with a limited effect. Due to the persistence of high anti-FVIII inhibitor levels, the patient was transferred to a tertiary center for further management.A 90-year-old male presented with a left gluteal intramuscular hemorrhage. The patient was in a state of chronic heart failure and was anemic (Hb 6.4 g/dL) with a prolonged APTT . Based on the information that he had previously been diagnosed as having AHA, a relapse of AHA was suspected, and the patient was immediately managed with RBC infusion (2 units), APCC , and prednisolone (0.6 mg/kg/day and then tapered). Later, it was confirmed that the patient had an FVIII activity level <1.0% and an anti-FVIII inhibitor level of 240 BU/mL. With the above measures, the patient remained in a state of no bleeding but FVIII activity <50% over the subsequent several months. Thereafter, he developed repeat intermittent cutaneous hemorrhages, which required three further periods of hospitalization. During each admission, minimal doses of rFVIIa , and hemostasis was achieved. Inhibitor eradication was achieved using prednisolone (0.6 mg/kg/day and then tapered) and two doses of rituximab (375 mg/m2/dose). During the treatment for AHA, reactivation was noted around the 7th week of treatment, but administration of two more doses of rituximab was successful in eradicating the inhibitor. Together with the treatment of AHA, the underlying CNL was also treated with hydroxycarbamide  and controlled well. A CR of AHA was attained within 26 weeks.An 80-year-old male presented with soft tissue hemorrhages in the left forearm and right lower extremity for 3 weeks, which progressed compartment-like symptoms . He was A 62-year-old female presented with hemorrhagic shock a few hours after a tooth extraction and was referred to the emergency department . The patIn this report, four of the present patients were ≥90 years of age. All cases except for one were admitted through the emergency department with bleeding episodes, and one had undergone a recent surgical procedure (tooth extraction). Hemorrhages ranged from subcutaneous, soft tissue, and intramuscular bleeding episodes to epistaxis and hematuria. Severe hematuria in AHA has been described by Shander et al. . AHA is Review of the present cases was performed retrospectively, and treatment of each case was determined at the discretion of the responsible physician rather than in accordance with a specific AHA protocol. The management of hemostasis and inhibitor eradication therefore differed on a case-by-case basis and was based on FVIII activity, anti-FVIII inhibitor levels, and comorbidities. Of the three patients with high inhibition, two required high doses of rFVIIa (NovoSeven®) with or without APCC (Feiba®) and were appropriately managed. In the remaining patient (Case 4), only three doses of APCC (Feiba®) were administered initially, but complete bleeding control was not achieved. Among the three low inhibitor cases, two attained a control with 2–6 doses of rFVIIa (NovoSeven®), while the patient with liver cirrhosis was successfully managed with FFP, plasma exchange, and prednisolone. To determine the efficacy of prednisolone in the hemostatic management of AHA, Vautier et al.  evaluateAlthough individualized, treatment in the present series did not deviate substantially from that proposed in the AHA Guidelines of the Japanese Society on Thrombosis and Hemostasis . These gIn terms of inhibitor eradication, since usage of immunosuppressive agents for AHA may develop severe side effects , treatment depended on the inhibitor levels with use of low-dose prednisolone . Among cases in the present high-inhibitor group, Case 1 was treated with prednisolone + CPA, while Case 5 (who had a comorbid disease of CNL) required both rituximab and prednisolone. AHA in Case 4 was refractory and persisted in a state of NR probably because he was treated with prednisolone only for inhibitor eradication. Expert review of the case suggests that more intensive inhibitor eradication was indicated. Cases in the low-inhibitor group were treated with prednisolone only (Cases 2 and 3) or prednisolone + CSA (Case 6). Prednisolone dosages and rate of tapering varied according to the degree of impaired glucose tolerance and other age-related factors. Given the potential adverse effects of high-dose prednisolone in elderly patients, rituximab appears to be an ideal agent for anti-FVIII inhibitor eradication in this population , 16. HowTo facilitate the evaluation of treatment effect in AHA, Tiede et al. previously defined PR as the absence of active bleeding and an FVIII level >50 IU/dL in cases in which hemostatic treatment had been discontinued for >24 hr . In theiIn summary, elderly patients frequently present to emergency services with various bleeding episodes. Given the aging demographic in Japan, this is likely to increase. In view of its life-threatening potential, emergency department physicians must consider AHA when formulating a differential diagnosis in such cases. Also, since the severity of AHA is variable, individualized therapy is needed to provide the patients with the best care.


EVD-negative cases. Key-performance indicators for EVD response were also measured. Overall, 454 EVD-positive cases were reported. At multivariable analysis, the odds of EVD was higher among those reporting contacts with an EVD-positive/suspected case (odds ratio (OR) 2.47; 95% confidence interval (CI) 2.44–2.50; P < 0.01) and those attending funeral . EVD cases from Kunike chiefdom had a lower odds of death  and were also more likely to be hospitalised . Only 25.1% of alerts were generated within 1 day from symptom onset. EVD preparedness and response plans for Tonkolili should include social-mobilisation activities targeting Ebola/knowledge-attitudes-practice during funeral attendance, to avoid contact with suspected cases and to increase awareness on EVD symptoms, in order to reduce delays between symptom onset to alert generation and consequently improve the outbreak-response promptness.In Sierra Leone, the Ebola virus disease (EVD) outbreak occurred with substantial differences between districts with someone even not affected. To monitor the epidemic, a community event-based surveillance system was set up, collecting data into the Viral Haemorrhagic Fever (VHF) database. We analysed the VHF database of Tonkolili district to describe the epidemiology of the EVD outbreak during July 2014–June 2015 (data availability). Multivariable analysis was used to identify risk factors for EVD, fatal EVD and barriers to healthcare access, by comparing EVD-positive  Ebola virus disease (EVD) is a life-threatening illness due to Ebola virus (EBOV) . Human EThe 2013–2016, West-African EVD epidemic was the largest, deadliest and most complex outbreak since EBOV was discovered in 1976 . DiffereFirst EVD cases in Sierra Leone were reported from Kailahun district in May 2014  and the Located in the centre of Sierra Leone, Tonkolili district borders with seven districts, is divided in 11 chiefdoms with a population of 455 383 people  .Although Tonkolili accounted for 5.2% of EVD cases and 4.5% of related deaths reported in the country , during http://epiinfovhf.codeplex.com/) [To monitor the EVD epidemic, the Ministries of Health (MoH) of Sierra Leone, Guinea and Liberia, the Centers for Disease Control and Prevention (CDC) and the WHO, set up a community event-based surveillance (CEBS) system using the Epi Info Viral Haemorrhagic Fever (VHF) application (ex.com/) . The sysex.com/) . The sysex.com/) .This study presents EVD surveillance data from the VHF database of Tonkolili district, Sierra Leone, with the principal objectives to describe the epidemiology of the EVD outbreak into the district during July 2014–June 2015 (data availability), to identify risk factors for EVD, fatal EVD and to assess barriers to healthcare access among EVD cases. Secondary objectives were to measure the MoH/WHO key-performance indicators for EVD response, and to present strategic analyses that can be performed from the VHF database, in order to guide decision making for national and international partners planning and implementing the response, also in terms of future preparedness.The EVD CEBS works by detecting alerts generated in the community and/or in healthcare facilities (HCF)/Ebola Treatment Centre/Unit (ETC/ETU) . AccordiOR at least one of the following signs: bleeding, bloody diarrhoea, bleeding into urineOR any sudden death.Alerts are collected at the DERC through the Ebola-hotline (117) and screened by trained surveillance officers. Only if the alert meets the EVD definition of suspected/probable case, trained case investigators and/or contact tracers perform the case investigation using a standardised form. In parallel, a clinical sample is collected  and sent to the district's reference laboratory for confirmation through real-time reverse-transcriptase polymerase chain reaction (RT-PCR) test specific for EBOV .Investigated alerts are daily reported to the WHO/CDC Epidemiologist (surveillance pillar) that also receives and matches the laboratory results in order to assign the final case classification. There are four final case classifications according to the WHO EVD case definition :SUSPECTED CASE: any person, alive or dead, suffering or having suffered from a sudden onset of high fever and having had contact with:–a suspected, probable or confirmed Ebola case;–a dead or sick animal (for Ebola)OR: any person with sudden onset of high fever and at least three of the following symptoms:headaches, vomiting, anorexia/loss of appetite, diarrhoea, lethargy, stomach pain, aching muscles or joints, difficulty swallowing, breathing difficulties, hiccupOR: any person with inexplicable bleedingOR: any sudden, inexplicable death.PROBABLE CASE: any suspected case evaluated by a clinicianOR: any deceased suspected case (where it has not been possible to collect specimens for laboratory confirmation) having an epidemiological link with a confirmed case.LABORATORY-CONFIRMED CASE (EVD+): any suspected or probably cases with a positive laboratory result. Laboratory-confirmed cases must test positive for the virus antigen, either by detection of virus RNA by RT-PCR, or by detection of IgM antibodies directed against Marburg or Ebola.NON-CASE (EVD–): any suspected or probable case with a negative laboratory result. ‘Non-case’ showed no specific antibodies, RNA or specific detectable antigens.Epidemiological, clinical and laboratory data are finally entered into the VHF database by a trained data-clerk and double checked with the WHO/CDC epidemiologist.Alert's-related variables entered into the VHF database include demographic , epidemiological and clinical information, as the date of symptom onset, the clinical presentations, the outcome , laboratory results and laboratory turnaround time, the number of contacts traced and whether the patient was hospitalised  and was in a contacts tracing list. Finally, the following information on risk factors is collected: (1) direct contact with known or suspected/probable EVD case, including the date of exposure, (2) funeral attendance touching/carrying the body.We retrospectively analysed alerts reported into the VHF database of Tonkolili district during the period 1 July 2014–30 June 2015 . Incidence, mortality and hospitalisation rates per 100 000 inhabitants were calculated using the 2015 population data (only available by chiefdom) provided by the DHMT of Tonkolili.χ2) and multivariable analysis. Variables with a P-value <0.2 at the univariate analysis were included in a multivariable stepwise regression model, with a P-value >0.10 for backward elimination [vs. EVD− cases to assess risk factors for EVD+; EVD+ dead vs. EVD+ alive cases to assess death's risk factors among EVD+ cases; hospitalised EVD+ vs. non-hospitalised EVD+ cases to evaluate barriers to healthcare access among EVD+ cases. For the hospitalisation and death analyses, we also assessed the symptoms  associated with these two outcomes, adjusting by age groups and gender.Risk factors for EVD+, fatal EVD+ and barriers to healthcare access among EVD+ cases were assessed through univariate , chiefdom of residence , occupation (HCW/other occupation), hospitalisation (yes/no), time interval from symptom onset to hospitalisation , clinical presentations (symptoms), and whether in the 21 days before symptom onset the patient attended a funeral touching the body and/or had contact with a confirmed/suspected/probable EVD case.For EVD+ cases with a known exposition timing, we also calculated the incubation period.Percentage of alert generated within 1 day from symptoms onsetNumber and percentage of HCWs infectedPercentage of samples tested within 1 day of collectionPercentage of deaths buried within 1 dayPercentage of lives alert tested for EBOVPercentage of deaths alert tested for EBOVPercentage of reported community deaths that were tested for EBOVPercentage of new confirmed cases from registered contactsNumber of hospitalised within 3 days from symptom onsetReporting the case fatality rate among hospitalised EVD+ casesReporting the number of Ebola survivors by gender, age group, chiefdomReporting the number of contacts traced per EVD+ caseFinally, the following MoH and WHO key-performance indicators for EVD response were measured:To evaluate failures of the surveillance system over the time and by place, the achievement of indicators was assessed by trimesters and chiefdom using a logistic regression model, adjusting by age group and gender. Indicators included in this analysis were numbers: 1, 3, 5, 6, 7, 9.P-value <0.05. R-software (version 3.3.1) was used for data analysis.The others were excluded because close to 100% or not suitable for logistic regression analysis. Results were expressed in terms of odds ratios (ORs), with 95% confidence interval (95% CI). Statistical significance was set at Ethical clearance was obtained from: Sierra Leone Ethics and Scientific Review Committee; MoH; Directorate of Policy, Planning and Information (DPPI). Informed consent was not requested because data were collected in the framework of a national surveillance system.During the 1-year study period, 4550 alerts were reported and classified as follows: 454 (10%) EVD+, 3344 (73.5%) EVD–, 53 (1.2%) probable and 699 (15.4%) suspected cases .Table 1n = 53) of the study population; of them, 11 (20.8%) were EVD+. The distribution of EVD+ cases was proportioned between male (51.4%) and female (48.6%), whilst 35.5% of EVD+ alert was reported among those aged <6 years.Overall, 40.2% of alerts were from two chiefdoms , concerned people aged >30 years (40.5%) and dead persons (63.7%). HCWs represented the 1.2% . The first EVD+ alert was a 14-year-old female from Kholifa Rowalla chiefdom (reported on 19 August 2014). In September 2014, 56 EVD+ additional cases were reported, with a district incidence rate (IR) of 147.6/100 000 inhabitants; then the EVD+ case count increased, with the higher IR during October  and November 2014 . After that, the EVD+ case count decreased continuously: 63 EVD+ cases (IR = 168.6/100 000) in December 2014, 24 (IR = 63.2/100 000) in January 2015 and six (IR = 15.8/100 000) in February 2015. The last recorded EVD+ case was imported from Western Area Rural district in Yoni chiefdom on 15 March 2015 (epi-week 12).Alerts increased gradually with time: from six (0.1%) in July 2014, to 332 (7.3%) in October 2014, 531 (11.7%) in March 2015, reaching the peak in June 2015 .The average EVD+ R during the study period  was 99.7n = 0/3) to 40% in Kafe Simiria (n = 2/5)).In two chiefdoms , no EVD+ cases were reported. Globally, 9 (2.0%) EVD+ cases were imported from other Sierra-Leonean districts . Out of the 454 EVD+ cases, 93 died, determining an EVD+ case fatality ratio (CFR) of 24.5% ; a median interval form symptom onset to hospitalisation of 3.0 days (range: 0–21) and a median incubation period of 11 days (range: 4–17).P < 0.05). In comparison to other occupations, HCWs were 10% less likely to become EVD+ . Having had a contact with an EVD+ or suspected/probable case increased the odds of being EVD+ , as well as attending a funeral and touching/carrying the body .At the multivariable analysis, no difference was found by gender and chiefdom of residence; whereas, those aged 16–30 years had a higher odd of being EVD+ ; whilst the odd of death was inversely proportional to the duration of the time interval from symptom onset to hospitalisation: the longer was this interval, the lower was the risk of death . Among symptoms, only headache  and abdominal/stomach pain  were statistically significantly correlated, with a protective effect vs. the odds of death.At the multivariable analysis, only two risk factors remained statistically significant: EVD+ cases from Kunike chiefdom had a lower odds of death , aged 6–15  and >30 years , were more likely to be hospitalised, as well as EVD+ cases from Kunike  and Yoni chiefdoms  and EVD+ cases reporting a contact with an EVD+ or suspected/probable case .Among factors, at the multivariable analysis, EVD+ male patients  and unexplained bleeding  were more likely to be hospitalised.Among symptoms, EVD+ cases that presented at the HCF/ETC/ETU admission with difficult breathing  occurred within 3 days from symptom onset, and the CFR among EVD+ hospitalised cases was 17.8%. Among the 454 EVD+ cases, 424 (93.4%) were already recorded in a contacts-tracing list.The median interval from symptom onset to alert generation was 4 days (range: 0–28), with 25.1% of alerts generated within 1 day from symptom onset. Among the 53 alerts related to HCWs, 11 (20.7%) resulted EVD+. Out of 4550 alerts, 3798 (83.5%) were laboratory investigated . The median interval from alert generation to sample collection was 0 days (range: 0–20), with 79.4% of samples collected within 1 day from the alert; while the median interval from sample collection to sample testing was 1 day (range 0–20), with 75.2% of samples tested within 1 day from collection. The median interval from death-to-burial was 0 days (range: 0–9), with 97.9% buried within 1 day from death. Furthermore, 62.8% of hospitalisations  was reported in the Tonkolili VHF surveillance system (P < 0.05); whilst no differences were observed over the time for the other indicators, which were all statistically significant above the OR of 1.00.Logistic regression model assessing the achievement of indicators over the time and by chiefdom  showed gP < 0.05).Concerning the number of sample tested within 1 day of collection, several chiefdoms had a statistically significant lower odd of reaching the target. The same was observed with the number of deaths occurred at the HCF/ETC/ETU and in the community tested for EBOV, with several chiefdoms showing a statistically significant lower odd of reaching the indicator. Finally, only the chiefdom of Kafe Simiria had a statically significant odd that the hospitalisation occurred within 3 days from symptom onset , many suspected/probable cases may have never been reported, investigated and tested for EVD, as suggested in the literature  and by tOverall, during the 1-year study period, 454 EVD+ cases were reported by the VHF surveillance system of Tonkolili district, with the last on 15 March 2015. After the study period, other three EVD+ cases occurred in Tonkolili district: on the 24 July 2015 , 13 Septvs. 383.1/100 000) [vs. 122.2/100 000 inhabitants) [The monthly EVD+ IR in Tonkolili district and Sierra Leone showed a similar temporal pattern, reaching the peak in October 2014, when the Tonkolili IR was higher than the national IR 400.5 100 000) . Overall0 000 . Obitants) , 27.Concerning risk factors for EVD+, similarly to other studies , 29, no n = 11/454; 2.4%), one of the lowest recorded in Sierra Leone [Differently from other studies conducted during the West Africa EVD outbreak , 31, HCWra Leone .This finra Leone  showed tra Leone .n = 424/454) of EVD+ cases, similarly to what observed in smaller studies from other districts of Sierra Leone [History of contact with confirmed or suspected/probable EVD cases increased the odd of being EVD+ in this study, being reported by 93.4% , with all the chiefdoms reporting values below the national morality rate [vs. 41.2%), and with the CFR of other Sierra Leonean districts  [The EVD+ mortality rate in Tonkolili district was lower than that of Sierra Leone (20.4 ity rate . Also thmba 58%) , 27, 36.vs. the odd of death; this could be explained by the fact that patients reporting these two symptoms had not more severe clinical presentation, and on the other hand, patients with severe symptoms might have not been able to report these two mild symptoms.Concerning risk factors for death among EVD+ cases, no difference was found by gender and age group. In a nation-wide retrospective study conducted in Guinea, the age was the only factor independently associated with EVD-related mortality , as in avs. the odd of death and also had more probability to be hospitalised, suggesting a protective effect of the hospitalisation. On this regard, in Tonkolili district, there were only two ETCs: one was at the Magburaka hospital , and one was an Médecins Sans Frontières’ (MSF) ETC opened in December 2014 in Kholifa Rowalla chiefdom [Although in the present study, the hospitalisation was not associated with death, as instead reported in the literature , EVD+ cachiefdom . Kunike Also EVD+ cases with history of contact with EVD+ or suspected/probable cases and with unexplained bleeding were more likely to be hospitalised, suggesting an effective implementation of the contact-tracing process. Findings related to EVD+ hospitalisation cannot be compared with others, due to the lack of these data in the literature.Observed key-time periods were similar with those reported in the literature. The median EVD incubation period was 11 days, in line with what reported from Pujehun district (10 days)  and fromGlobally, the assessment of WHO/MoH key-performance indicators for EVD response found gaps mainly related to the interval from symptom onset to alert generation in particular in the last trimester of the study, and this is probably due to the exponential increase in the number of alerts over the time. Acceptable performances were observed once the alert is generated, even if 25% of samples were not collected <1 day from the alert.A comparison of these indicators with those attended in other districts is not possible due to the lack of data in the literature.n = 2806/4096), but data on cause of death were not available. This high mortality is strictly related to the chronic weakness of health systems of resource-limited countries in Africa that can easily collapse when epidemics, as the EVD outbreak, strikes [Albeit beyond the objectives of the study, it is noteworthy to be underlined that the CFR among EVD or suspected/probable cases was extremely high  during funeral attendance (b) to avoid contact with EVD+ or suspected/probable cases and (c) to increase awareness on EVD symptoms, in order to reduce the delays between symptom onset and treatment seeking and so the delays between symptom onset and alert generation that consequently affect the promptness of the outbreak response.


HindIII single nucleotide polymorphism of OCN gene (BGLAP) promoter region and T2DM development in Ukrainian population was carried out. 153 individuals diagnosed with T2DM and 311 control individuals were enrolled in the study. The genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The lack of association between BGLAP HindIII single nucleotide polymorphism (SNP) and T2DM development among Ukrainians was found. Further studies with extended groups of comparison are needed to confirm the obtained results.Type 2 diabetes mellitus (T2DM) belongs to the diseases with hereditary predisposition, so both environmental and genetic factors contribute to its development. Recent studies have demonstrated that the skeleton realizes systemic regulation of energy metabolism through the secretion of osteocalcin (OCN). Thus, the association analysis between  Recently, it was established that a final cellular processing step of OCN is provided by proprotein convertase furin regardless of carboxylation process. Furin recognizes C-terminal motif RX(R/K)R (RPRR for Homo sapiens) which is conserved for all vertebrates and cleavages 28 aa of propeptide region. Thus, mature OCN of 49 aa with three γ-carboxyglutamate residues is secreted by osteoblasts into the extracellular bone matrix where its final modification takes place determining mineralization or endocrine function of the protein [Osteocalcin (OCN) is the most prevalent noncollagenous protein in bone . It is e protein .α-helical domains (counting from N-terminus) with short unstructured region. The vast area of negative charge in α1 helix due to the presence of three Gla residues was found. These γ-carboxyglutamates and Asp30 residue (located in α2 helix) bind five calcium ions that were detected to be in unexpectedly regular order reminding crystalline lattice [The large amount of studies were dedicated to investigation of OCN role in mineralization process on the analogy of matrix Gla protein (MGP) which is characterized by pronounced anticalcinogenic action , 4. More lattice . Such st lattice .κ-B ligand (RANKL), which activates osteoclasts through the binding with RANK receptors. Activated osteoclasts create acidic surroundings in the resorption lacunae, and cOCN loses its carboxylic groups. Such undercarboxylated OCN (unOCN) cannot be fixed by HA and releases from bone mineral matrix to systemic circulation [Nowadays, it is believed that OCN's crucial role is the regulation of systemic energy metabolism after its release from bone mineral matrix to circulation. It was found that insulin renders metabolic and mitogenic effects on osteoblasts through the activation of insulin receptor and insulin growth factor receptor located on the cellular surface. This leads to the activation of MAPK and PI3K/Akt signaling pathways resulting to enhanced glucose uptake and glycogen synthesis as well as changing in osteoblastic gene profile expression . On the culation , 9.in vivo evidences that bone regulates systemic energy metabolism through the secretion of unOCN. Thus, the increased expression of Insulin and CyclinD1 in β-cells as well as Adiponectin in adipocytes after the unOCN treatment was showed [Gprc6a knockout in mice β-cells leads to reduction of pancreatic size and β-cell amount as well as decreased insulin expression and secretion. Moreover, these mice are characterized by glucose intolerance but at the same time had normal insulin sensitivity [γ-dependent manner [Lee et al. have provided the first s showed . Furthers showed . Pi et asitivity . Otani et manner . Du et at manner . Mera ett manner . Severalt manner , 17.BGLAP HindIII polymorphism and type 2 diabetes mellitus (T2DM) development in Ukrainian population.To date, the role of OCN in diabetes mellitus (DM) and metabolic syndrome (MS) development is actively studied –20. But The present research enrolled 153 Ukrainians  diagnosed with T2DM and 311  control subjects. The final diagnosis of T2DM was established on the basis of typical symptoms , fasting glucose level, and glucose tolerance test result according to the World Health Organization criteria . Healthy subjects without any carbohydrate metabolism disorders  and nonburdened family history of diabetes were included in the control group. All participants of the study were selected from hospital records in 5th Sumy Clinical Hospital and Sumy Regional Diagnostic Center since 2011-2019. The protocol of the study was conformed to the Declaration of Helsinki and approved by the Ethic Committee of Medical Institute of Sumy State University (number 4/02.18.09). All individuals provided the written informed consent.100 DNA_Blood kit . Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was carried out for genotyping. The reaction mixture on the amplification stage consisted of 5 μL FastDigest Green Buffer (10X) , 0.5 μL dNTP Mix (10 mM of each deoxynucleotide) , 0.75 U DreamTaq DNA Polymerase (5 U/μL) , 0.1 μL each primer, 75-100 ng DNA, and bidistilled water to 25 μL. The primer sequences and PCR conditions are indicated in Whole venous blood was used for DNA extraction by NeoPrepHindIII , 0.8 μL of 10X Buffer R (10 mM Tris-HCl (pH 8.5), 10 mM MgCl2, 100 mM KCl, 0.1 mg/mL BSA) , and bidistilled water to 2 μL were added to 6 μL of each sample and then incubated at 37°C for 20 hours. The presence of thymine in -198 BGLAP gene position leads to the cleavage of 253 base pairs (bp) amplicon into 21 bp and 232 bp fragments. The specific for HindIII restriction site is lost in case of thymine to cytosine replacement, and 253 bp amplicon remains undigested. Horizontal electrophoresis (10 V/cm) in 2.5% agarose gel (with 10 mg/mL ethidium bromide) and subsequent ultraviolet visualization were used for the genotype discrimination . The comparison of allele and genotype frequencies and all categorical variables was done by a chi-squared (χ2) test. The association between BGLAP HindIII and T2DM development was explored in dominant, recessive, overdominant, and additive models of inheritance using binary logistic regression. Further adjustment for age, sex, body mass index (BMI), smoking, and the presence of arterial hypertension in multivariable logistic regression contributes to increasing reliability of the obtained results. Then, logistic models with “BMI × genotype” and “sex × genotype” interaction terms were performed in order to estimate whether BMI or sex is an effect modifier, respectively. The Bonferroni correction for multiple comparisons was used to reduce the probability of type I error from occurring; thus, the obtained P values were multiplied by 4 (as four regression models were tested). The value P < 0.05 was accepted as significant.The statistical analysis was done using Statistical Package for the Social Sciences software . Categorical variables are presented as absolute and percentage values, and continuous variables are indicated as mean ± SD with previous checking of distribution normality by the Kolmogorov-Smirnov test. The mean values of two and more groups were compared using two-tailed Student's P < 0.05). In contrast, the T2DM and control groups were comparable by mean age (P = 0.379), diastolic (P = 0.883) and mean (P = 0.187) BPs, and amount of current smokers (P = 0.452).The general characteristics of comparison groups are presented in P = 0.888), but not in the studied group (P = 0.009). No significant differences between alleles and genotypes were found in comparison groups (P > 0.05).The allele and genotype distributions in the T2DM and control groups are indicated in BGLAP HindIII and T2DM development are shown in Pc = 0.04;ORc = 0.638,95%CI = 0.415-0.979) of inheritance as well as after adjustment for age, sex, BMI, smokers, and arterial hypertension presence . However, the significance of association was lost after the Bonferroni correction applying (Pab = 0.12).The results of association analysis between BGLAP HindIII genotypes and T2DM development depending on sex and the presence of obesity (BMI ≥ 30 kg/m2). Similar results were obtained for males, females, and subjects without obesity (Pint > 0.05). In contrast, a significant association between rs1800247 and T2DM emergence was found among individuals with obesity in dominant , overdominant , and additive  models. The strong protective effect of C allele was preserved after the adjustment for age, sex, smoking, and the presence of arterial hypertension in dominant , overdominant , and additive  models of inheritance .Then, we have prepared regression models with interaction terms in order to explore the association between BGLAP HindIII genotypes were found (P > 0.05).Clinical characteristics of patients with T2DM stratified by genotype are demonstrated in BGLAP) gene, is located on 1q22 chromosome with the length of 1134 bp (NC_000001.11). The thymine to cytosine transition that occurred in the -198 position of BGLAP promoter region (rs1800247) was discovered by Dohi et al. [H (22.8%) and h (77.2%) alleles among postmenopausal Japanese women and showed that HH and Hh genotypes had greater risk for osteopenia compared to hh genotype. According to 1000 Genomes Project phase 3 browser [The OCN gene, bone gamma-carboxyglutamate protein , and neuropeptide Y (NPY). unOCN is considered to be an insulin secretagogue, on the one hand, as it induces β-cell proliferation and insulin expression, and an insulin sensitizer, on the other hand, as it promotes adiponectin upregulation and fatty acid and glucose utilization by muscle tissue [Nowadays, it is known that the skeleton acts as the glucose metabolism regulator through the secretion of various osteokines, such as OCN, lipocalin 2, bone morphogenetic protein 7 (BMP7), receptor activator of nuclear factor e tissue . Based oe tissue . Bao et e tissue . Moreovee tissue . Moreovee tissue . The line tissue .BGLAP and T2DM development neither in Caucasians nor in African Americans. The sizes of comparison groups were large enough to exclude HindIII SNP as potential T2DM risk factor. Moreover, no links were found between metabolic values as well as obesity measures and rs1800247. Ling et al. postulated that there is no significant association between rs1800247 and BMI, waist circumference, HOMA-IR, fasting insulin, fasting glucose, oral glucose test, and any lipid fractions. But they have found the protective effect of C allele against hypertension development and the reduced diastolic blood pressure in CC and TC individuals. Moreover, the significant association between arterial hypertension and rs1800247 due to SNP with HOMA-IR interaction was shown. The carriers of C allele have a decreased risk of hypertension in the subgroup with HOMA-IR less than 1.93, and no link was found among subjects with greater values [In the present study, we have established no association between rs1800247 SNP and T2DM emergence in Ukrainian population. These results are in accordance with the previous research of Das et al. . They har values .HindIII SNP of BGLAP promoter region and T2DM development among Ukrainians.In our study, we have also investigated the interaction effects between rs1800247 genotypes and other covariates in T2DM development. Thus, the protective influence of minor C allele (in the dominant model) and TC genotype (in the overdominant and additive models) against T2DM emergence among patients with obesity was shown, and this association remained statistically significant after adjustment for age, sex, smoking, and arterial hypertension presence. However, the link was lost in all models of inheritance after the Bonferroni correction adjustment. Thus, neither obesity nor gender was detected as effect modifier in the association between HindIII SNP and to explore the BGLAP expression rates depending on the T or C presence in promoter region. In addition, it would be interesting to value OCN fractions and to investigate the appropriate correlations between genotypes and OCN concentrations.Several limitations of this study should be noted. First of all, the small size of comparison groups requires increasing the amount of participants to get the results that are more reliable. Then, it is necessary to find out the functional significance of BGLAP HindIII and T2DM development in Ukrainian population. There was no association between rs1800247 SNP and T2DM emergence in Ukrainian population. Further studies are necessary to expand the comparison groups and reveal the functional significance of SNP.In the present study, we have explored the association between