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+{"text": "The C677T MTHFR variant has been associated with the same third trimester pregnancy complications as seen in women who have elevations of maternal serum \u03b1-fetoprotein (MSAFP). We hypothesized that these women with third trimester pregnancy complications and MSAFP elevations would have an increased frequency of the variant compared to an abnormal study control group (women with MSAFP elevations without pregnancy complications) as well as to normal population controls.Women who had unexplained elevations of MSAFP in pregnancy were ascertained retrospectively. The frequency of the C677T MTHFR variant among those women with unexplained elevations of MSAFP who had experienced later pregnancy complications was compared to that of women with unexplained elevations of MSAFP without complications as well as to that of the previously established Manitoba frequency.p = 0.03). The frequency was also higher than that of the population controls . The frequency in women with MSAFP elevations without pregnancy complications was not significantly different from that of the population controls (p = 0.41).Women who had complications of pregnancy and an unexplained MSAFP elevation had a higher allele frequency for the C677T MTHFR variant  compared to women with MSAFP elevations and normal pregnancy outcomes (q = 0.25, OR 1.73 95% CI 1.25\u20132.37, Women with unexplained elevations of MSAFP and who experience complications in later pregnancy are more likely to have one or two alleles of the C677T MTHFR variant. Significant elevations of amniotic fluid and maternal serum alpha-fetoprotein (MSAFP) have been shown to be associated with spina bifida and other neural tube defects (NTD). The province of Manitoba, Canada offers province-wide midtrimester MSAFP screening to all pregnant women. It has been recognized that some pregnant women with midtrimester unexplained elevations of MSAFP ,2.Increased total plasma homocysteine alters placental function and has been associated with the same complications that are associated with unexplained elevated MSAFP -7. The Cp = 0.151, two tailed) indicating the frequency was increased but, this could have been a random result.In a small pilot study of 32 couples, we found that women who had an unexplained elevation of MSAFP and a normal midtrimester fetal ultrasound, and their partners, had a significantly increased C677T MTHFR frequency compared to Manitoba newborns  . The newAll pregnant women in Manitoba are eligible for routine serum screening through the voluntary MMSSP. In Manitoba, an elevation of MSAFP is defined as 2.3 multiples of the median (MOM) or greater. Candidates for inclusion in this study were women with an unexplained MSAFP elevation  with either a complicated or uncomplicated pregnancy outcome. After appropriate approvals had been obtained from The University of Manitoba Health Research Ethics Board, review of the screening records began in 1999 and took three years. For a study using a two step consent to participate methodology administered by mail, the expected response rate (after excluding lost to follow-up) would be 20% . Our goaAll screening records from 1995\u20131999 were reviewed, accounting for 783 invitations. Records for 2000\u20132002 were reviewed systematically as outcome information on each pregnancy became available to MMSSP. Records for 1990\u20131994 were then reviewed systematically in order to bring the total up to 1000. If a woman had more than one pregnancy with an elevation of MSAFP screened by the MMSSP, only the first pregnancy encountered in the retrospective review was used for the study. Previous or subsequent pregnancies were not included. Women with preexisting conditions known to influence pregnancy outcome, such as essential hypertension, and mothers of babies with major congenital anomalies were excluded. Eight women who had relinquished their babies for adoption or whose babies were placed in foster care were also excluded.th percentile), pregnancy induced hypertension, preeclampsia, eclampsia, postpartum hemorrhage, retained placenta requiring manual delivery, abruptio placenta, premature delivery  and unexplained fetal demise. Study controls were women with normal outcomes which were defined as those with delivery at term \u2265 36 weeks gestation), no complications of pregnancy, a normal placenta and a healthy baby. Definition of complications was based on ICDC-9 codes in the MMSSP outcome charts for each patient [Women who met the inclusion criteria were divided into two groups for analysis. Cases were defined as women with pregnancies complicated by one of the complications previously shown to be associated with an unexplained elevation of MSAFP at midtrimester . These i patient  which ar patient .12 and B6, were calculated from the FFQ for intake both during pregnancy and at the time of the study. A correction of an additional 0.1 mg for folic acid fortification that began in Canada in 1998 was included for pregnancies that began after fortification [Women who agreed to participate in the study were mailed the appropriate questionnaires and blood requisitions. The questionnaire included a semi-quantitative food frequency questionnaire (FFQ) based on standard methodology but, modified to suit Manitoba residents and previously validated for this population by biochemical analysis during the pilot study ,16. The fication . FFQ anaTotal plasma homocysteine, red blood cell folate, and serum folate were determined using established methodology ,19. Sampt test for difference between means with Bonferroni correction for multiple comparisons. Data not normally distributed were analyzed using the nonparametric Mann-Whitney Rank Sum Test. Linear regression was used to test the validity of the dietary survey. A multivariate analysis included age, smoking, maternal weight at the time of MSAFP testing, presence of C677T MTHFR, gender and weight of infant, biochemical parameters, and FFQ results for folate, B12 and B6, both at the time of the survey and for during the pregnancy was undertaken. In order to avoid convergence due to the large number of variables, the analysis was completed in subsets of six variables. Variables with the higher association scores from these analyses were then combined for further testing in various combinations using stepwise multiple linear regression. Also linear regression analysis of each continuous variable with genotype results was performed. Corrections for multiple comparisons were included. Software used was NCSS Statistical Systems for Windows [Chi-squared analysis (one tailed unless otherwise noted) was used for allele frequency. Comparisons of potentially confounding factors between the case group and the study control group were undertaken. Parametric data were analyzed with the Student's  Windows .p = 0.030). There was no difference in the proportions of cases and controls that were lost to follow-up (p = 0.157). We had anticipated a 20% response rate and we achieved 24%.Nine hundred and ninety four women were identified as eligible (342 cases and 652 controls). Of the 590 women successfully contacted, 130 (22%) agreed to participate (56 cases and 74 controls). Four hundred and four women were lost to follow-up. Cases were more likely to choose to participate than controls and this difference was significant . The frequency was also higher than in the population controls . The frequency in women without pregnancy complications and MSAFP elevations (study controls) was not significantly different than that seen in population controls (p = 0.41).Genotypes were available for 54 cases and 73 controls for this analysis. Results are summarized in Table ever had a pregnancy with an unexplained elevation of MSAFP followed by complications, the association would still be present when compared to the population control .The case and study control groups included women at various stages of their child bearing years. Only one woman recruited as a control subject had a previous or subsequent pregnancy with an unexplained elevation of MSAFP and complications. She was a heterozygote for C677T MTHFR. No case subjects had a previous or subsequent pregnancy with an unexplained elevation of MSAFP and a normal outcome, but four case subjects had had a previous or subsequent pregnancy with complications after an elevated MSAFP. If the case versus control classification had been based on whether or not a woman had p = 0.019) for serum folate than those who did not have the variant. None of the women were deficient in either serum folate (defined as <7.0 nmol/L) or red blood cell folate (defined as <430 nmol/L RBC). There was no significant difference in mean homocysteine levels  and serum folate . Higher intake of dietary folate (including synthetic folic acid from supplements) as reported by the FFQ for the time of study was associated with higher serum folate  and higher red blood cell folate .Seven cases and one study control declined to fill out their dietary surveys. Mean values were inserted in the multivariate analysis for these eight women. The validity of the dietary survey was demonstrated again for this study by linear regression analysis. Consistent with known homocysteine metabolism , a negatp = 0.0062) consistent with accurately reporting their smoking habits [We did several checks to determine that the women were answering their surveys accurately. Comparisons of specific data items available in the women's MMSSP charts at the time of pregnancy with the data reported in the surveys showed excellent agreement for every item examined indicating women answered questions accurately. Women who reported smoking  showed a negative correlation with serum folate  was 84% Caucasian, 5% Aboriginal. Mixed ethnicity was reported for 11% of the infants with one parent Caucasian and the other parent Aboriginal, or rarely Black or Asian. The ethnic distribution was the same for controls and is typical for the Manitoba population ,26. Ther12, or B6, or in the biochemical parameters of case and control mothers. There was no difference in the percentage of cases and controls who reported taking prenatal vitamin supplements during pregnancy (37/48 cases and 55/72) or taking vitamin and/or folate supplements preconceptionally (17/48 cases and 25/72 study controls).There were no significant differences in dietary and supplemental intake of folate, Bp = 0.013). Homozygosity for the C677T MTHFR variant is associated with IUGR in women who do not take vitamin supplements according to one large study of Canadian women [We attempted to divide our cases into smaller groups by type of pregnancy complication. We also separated isolated IUGR and IUGR associated with hypertensive disorders of pregnancy. Most of the groups lacked power for statistical analysis due to small numbers. However, normotensive women whose fetus had IUGR (N = 12) had a higher frequency of the C677T MTHFR variant compared to the population controls . This was also not unexpected given that some of the complications we were examining are associated with obesity [p = 0.0462). There was no association between weight and MTHFR status .There was a trend towards higher mean weight for mothers who had complications at the time of MSAFP test in the individual comparisons, but this was not significant after correction for multiple comparisons Table  and 3. T obesity . Even afUnexplained elevations in MSAFP are known to be associated with an increased risk for complications of pregnancy . Others The lack of folate deficiency in this population was unexpected, given previous research which showed that 23.6% of Newfoundland and Labrador women are folate deficient at their first prenatal visit . As our As is the situation with NTDs, lack of folate deficiency by current definitions in a non-pregnant woman may not indicate that her folate intake is adequate for pregnancy. This would especially be true for women with the C677T MTHFR variant. Reexamination of the current definition of what constitutes a normal biochemical result for folate intake for women of child bearing age should be undertaken to clarify this.We suggest that the negative effects of the C677T MTHFR variant are more likely to occur in early pregnancy before women began taking prenatal vitamins because the majority of our study participants took prenatal vitamins, but only 35% took preconceptional supplements. We suspect that reduced methylation interfering with cell proliferation in the placenta as originally suggested by Eskes (2000) [In conclusion, using a retrospective case/control study, we have found that women with unexplained MSAFP elevations who have complications in later pregnancy are more likely to have the C677T MTHFR allele. Our resultsdo not suggest that C677T MTHFR predisposes a woman to having an elevation of MSAFP level (as we did not compare the C677T MTHFR frequency in women with and without elevated MSAFP), but having one or more copies of this variant predisposes such screen positive women to having complications in later gestation. It remains to be seen if other risk factors can be identified which can more accurately define this high risk group.All authors participated in original study design except CS. CG, BC and CS all acted as principal investigators for funding. NB assisted with all grant proposals. NB undertook the review of the individual MSAFP files and MMSSP database searches, designed the dietary and family history surveys, classified cases and controls, acted as study coordinator handling all aspects of participant contact, and provided data analysis. All authors also participated actively with NB for various aspects of the study. CG provided MTHFR genotyping. LS provided biochemical analysis. NB drafted the original manuscript with assistance from BC. CG and BC handled ethics approval assisted by NB. All authors read and approved the final manuscript."}
+{"text": "Hypertensive disorders in pregnancy are leading causes of maternal, fetal and neonatal morbidity and mortality worldwide. However, studies attempting to quantify the effect of hypertension on adverse perinatal outcomes have been mostly conducted in tertiary centres. This population-based study explored the frequency of hypertensive disorders in pregnancy and the associated increase in small for gestational age (SGA) and stillbirth.We used information on all pregnant women and births, in the Canadian province of Nova Scotia, between 1988 and 2000. Pregnancies were excluded if delivery occurred < 20 weeks, if birthweight was < 500 grams, if there was a high-order multiple pregnancy (greater than twin gestation), or a major fetal anomaly.The study population included 135,466 pregnancies. Of these, 7.7% had mild pregnancy-induced hypertension (PIH), 1.3% had severe PIH, 0.2% had HELLP , 0.02% had eclampsia, 0.6% had chronic hypertension, and 0.4% had chronic hypertension with superimposed PIH. Women with any hypertension in pregnancy were 1.6 (95% CI 1.5\u20131.6) times more likely to have a live birth with SGA and 1.4 (95% CI 1.1\u20131.8) times more likely to have a stillbirth as compared with normotensive women. Adjusted analyses showed that women with gestational hypertension without proteinuria (mild PIH) and with proteinuria  were more likely to have infants with SGA . Women with pre-existing hypertension were also more likely to give birth to an infant with SGA  or to have a stillbirth .This large, population-based study confirms and quantifies the magnitude of the excess risk of small for gestational age and stillbirth among births to women with hypertensive disease in pregnancy. Hypertensive disorders in pregnancy complicate approximately 10\u201316% of pregnancies and are leading causes of maternal, fetal and neonatal morbidity and mortality worldwide . DefinitWe, therefore, carried out a population-based study to quantify the frequency of hypertensive disorders in pregnancy and also the excess risk of SGA and stillbirth that is associated with this pregnancy complication. We investigated the way in which SGA and stillbirth were modified by other factors that also have a serious influence on SGA and stillbirth, for example, whether a twin gestation modifies the effect of hypertension on SGA, or whether smoking modifies the effect of hypertension on stillbirth.The study population included all births to residents of the province of Nova Scotia, Canada between 1988 and 2000. Information on these births was obtained from the Nova Scotia Atlee Perinatal Database. The Nova Scotia Atlee Perinatal Database includes several hundred variables containing maternal and newborn information, such as demographic variables, procedures, interventions, maternal and newborn diagnoses and morbidity and mortality information for every pregnancy and birth occurring in Nova Scotia hospitals and to Nova Scotia residents since 1988.Pregnancies were excluded if delivery occurred < 20 weeks, if birthweight was < 500 g, if there was a higher order pregnancy (greater than twin gestation), or a known major fetal anomaly. Information in the database on the type of hypertensive disorder is directly coded from medical charts. The diagnosis of hypertensive disorders in pregnancy was made if it occurred in the antepartum or postpartum period. Mild pregnancy induced hypertension (PIH) included physician-diagnosed mild pregnancy induced hypertension if in the chart, transient hypertension or a diastolic blood pressure exceeding 90 mmHg on two or more occasions in a 24-hour period. Severe pregnancy induced hypertension included physician-diagnosed severe pregnancy induced hypertension, diastolic blood pressure \u2265 110 mm Hg on at least two occasions within a 6-hour period, if magnesium sulfate was administered for seizure prophylaxis, if there was \u2265 2+ proteinuria, low platelets , and/or elevated liver enzymes . HELLP syndrome  included physician-diagnosed HELLP. Eclampsia included physician-diagnosed eclampsia or one or more convulsions not attributable to other cerebral conditions such as epilepsy or cerebral hemorrhage in a patient with hypertension. Chronic hypertensive disease included a history of hypertensive disease when not pregnant, prior to current pregnancy or prior to 20 weeks of the current pregnancy. Pregnancy induced hypertension superimposed on chronic hypertension included physician-diagnosed pregnancy induced hypertension superimposed on chronic hypertension or if there was hemolysis, elevated liver enzymes or low platelets.We first examined the maternal characteristics of study subjects with hypertension in pregnancy. For this analysis, hypertensive disorders in pregnancy were defined according to Nova Scotia Atlee Perinatal Database definitions: mild PIH, severe PIH, HELLP, eclampsia, chronic hypertension and chronic hypertension with superimposed PIH. Maternal characteristics which were considered included age, marital status, parity, prepregnancy weight, pregnancy weight gain, administration of antenatal steroids, smoking, drug abuse, the presence of anemia (Hgb < 10 gm%), gestational diabetes , pre-existing diabetes, and twin gestation.For the multivariate analyses, these database definitions were grouped to more closely approximate commonly used definitions such as those proposed by the Canadian Hypertensive Society and other international organizations . These gth percentile cut-off of a recently published Canadian fetal growth reference [Only live births were considered in the analysis of small for gestational age, while all births were considered for the stillbirth analyses. Small for gestational age was defined as birthweight for gestational age that was less than the sex-specific 10eference . StillbiEthical approval for the study was obtained from the Research Ethics Boards at Dalhousie University in Halifax, Nova Scotia, the Reproductive Care Program of Nova Scotia and the IWK Health Centre in Halifax, Nova Scotia.Exact binomial 95% confidence intervals were calculated for rates of hypertensive disorders in pregnancy. Descriptive analyses were carried out on maternal data to ascertain the association between maternal characteristics and hypertensive disorders in pregnancy. Categorical data between hypertensive and normotensive pregnancies were compared using chi-square and Fisher's exact tests, where appropriate.P < .05 and all tests were two-tailed. Statistical analyses were performed using the SAS programming package for Windows (Version 8.0).Logistic regression analyses were carried out to determine the adjusted odds ratios (OR) and 95% confidence intervals (CI) expressing the relationship between any hypertensive disorder in pregnancy and groups of hypertension  and the two dichotomous primary outcomes (SGA and stillbirth). Backward stepwise elimination of variables was carried out to identify all significant determinants of the outcome. Modification of the effect of hypertensive disorders on SGA or stillbirth was investigated, based on clinical understanding. Variables considered to be in the causal pathway between the determinant and the outcome  were not adjusted for in the model -24. WhenThe study population included 135,466 pregnancies. Of these, 7.7%  had mild PIH, 1.3%  had severe PIH, 0.2%  had HELLP, 0.02%  had eclampsia, 0.6%  had chronic hypertension and 0.4%  had chronic hypertension with superimposed PIH. The overall rate of hypertensive disease in pregnancy was 10.1% .Table Table P < .001) times more likely to have a live birth with SGA as compared with normotensive women. Women with gestational hypertension without proteinuria were 1.5  times more likely to have a live birth with SGA as compared with normotensive women. Similarly, women with gestational hypertension with proteinuria were 3.3  times more likely and women with pre-existing hypertension were 2.5  times more likely to have a live birth with SGA as compared with normotensive women.After controlling for potential confounders, women with any hypertensive disorder were 1.8  times more likely to have a stillbirth as compared with normotensive women (Table P < .001) times more likely to have a stillbirth as compared with normotensive women.After controlling for potential confounders, women with any hypertensive disorder were 1.4  was similar to that reported in the literature (10\u201316%). The rate of mild PIH in this population was 7.7%, compared with 6\u20137% in the literature. The rate of severe PIH (1.3%), HELLP (0.2%) and eclampsia (0.02%) were lower than expected (5\u20136%). The rate of pre-existing hypertension (0.6%) and pre-existing hypertension with superimposed PIH (0.4%) were lower than expected  ,26. ThesSmall for gestational age is a more appropriate measure of fetal growth than birthweight alone. Our study evaluated birthweight for gestational age and gender among live births to normotensive and hypertensive pregnancies using a recent population-based Canadian reference . Women wP values associated with the results be interpreted with caution.Chance and confounding are unlikely explanations for the results of our study, because of the large study size, and adjustment for potential confounders using logistic regression. This study was not able to correct for the degree of blood pressure control, which may have an effect on fetal growth. While retrospective studies in general are limited by the reliability of data, information in the Nova Scotia Atlee Perinatal Database is of high quality. Routine data checks and edits are made at the time of data collection, and validation  and reabth percentile) and stillbirth in comparison with women with normotensive pregnancies. Twin gestation and smoking were important modifiers of the effect of hypertension on SGA and stillbirth. This study allowed the quantification of risks of adverse outcomes in women with pregnancies complicated by hypertension, confirming associations in the published literature and allowing appropriate counseling and monitoring in the management of these women, as well as providing baseline risks which may be used in future intervention studies.This population-based cohort study demonstrates that women with hypertensive disease in pregnancy are at significantly higher risk of having pregnancies complicated by small for gestational age (<10None declared.VMA proposed the study, carried out the preliminary analyses and wrote the paper. All authors discussed the analyses, contributed to the intellectual content of the paper and approved the final version. VMA and KEM provided the maternal-fetal medicine perspective, KSJ provided the general medical and epidemiologic input, LAM provided the general and obstetrical medicine perspective, and AO provided the neonatal-perinatal perspective and epidemiologic input.The pre-publication history for this paper can be accessed here:"}
+{"text": "Documentation of superfoetation is extremely rare in humans., The younger foetus has invariably been small for gestational age  in all the cases reported in the literature. We report a case where the younger twin was of appropriate size for gestation.The first of twins was of 32 weeks gestation and the baby was of appropriate size and development for the gestational age. The second twin was of 36 weeks gestation. Gestational age was estimated with the New Ballard score, x-ray of the lower limbs, dental age on x-ray, and ophthalmic examination.Bleeding on implantation of the first foetus probably helped demarcate the two pregnancies. Dental age and the New Ballard score can be used to diagnose superfoetation in discordant twins, when detailed first trimester ultra-sound data is not available. Superfoetation implies fertilization and subsequent development of an ovum when a foetus is already present in the uterus. Growth discordance in multiple pregnancies due to placental insufficiency, twin to twin transfusion or aneuploidy need to be differentiated from superfoetation. In most instances the larger twin is nearer appropriate size for gestational age calculated from the last menstrual period (LMP). We report a case where superfoetation was likely superfoetationbecause the smaller of the twin was of appropriate maturity, weight and length for gestational age. These circumstances argued against intrauterine growth retardation in the smaller twin.A 21-year-old mother of two had an ante-natal ultrasound examination done 26 weeks after her last menstrual period (LMP). This showed twins, one was of appropriate size for duration of amenorrhoea while the other was approximately four weeks too large. The ultrasound findings are described in Table Six weeks later, after 32 weeks of amenorrhoea, live twins were delivered. They had not received ante-natal steroids. The first of the twins (Twin A) weighed 980 grams and the next baby (Twin B) weighed 2160 grams. Detailed neurological assessment using the New Ballard Scoring [X-ray chest with mandible showed absence of calcified crowns of the first and second deciduous molar in Twin A and both crowns calcified in Twin B Figure , 4. The Retinal vessels normally reach nasal ora serrata by 36 weeks and periphery on the temporal side by 40 weeks.Ophthalmological examination of Twin Ashowed a hazy cornea and the underlying papillary membrane was not visualised. The retinal vessels had not reached the nasal ora serrata. In Twin B the cornea was clear, there was no papillary membrane and the retinal vessels migration was complete on the nasal side and near complete on the temporal side.Intrauterine growth retardation (IUGR) is the usual cause of discordance in multiple pregnancies. We did not find anyreport in the literature of discordance due to one baby being large-for-date. In this case the smaller twin was of appropriate size and maturity for gestation assessed from LMP. The second twin was approximately a month too large and mature. Superfoetation was considered as a possible explanation for the observation. Bleeding one month after conception occurs in about 8% pregnancies and represents a physiological response to implantation or slight bleed from the endometrium in early pregnancy . We therDetailed neurological and physical assessment is considered the most reliable method of estimation of gestational age, in circumstances where IUGR is suspected and there is uncertainty in using LMP . Using tAmong the evidence listed above, anthropometric measurements and bone maturation are delayed in first-trimester-malnutrition and results in symmetric growth retardation ,5. HowevThe author(s) declare that they have no competing interests.NB, SM, NV and AK investigated the case, searched the literature and helped with the write up, NP helped with the radiological input, and JMP conceived of the study and helped with its write up. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Linked population health data are increasingly used in epidemiological studies. If data items are reported on more than one dataset, data linkage can reduce the under-ascertainment associated with many population health datasets. However, this raises the possibility of discrepant case reports from different datasets.We examined the effect of four methods of classifying discrepant reports from different population health datasets on the estimated prevalence of hypertensive disorders of pregnancy and on the adjusted odds ratios (aOR) for known risk factors. Data were obtained from linked, validated, birth and hospital data for women who gave birth in a New South Wales hospital  2000\u20132002.Among 250173 women with linked data, 238412 (95.3%) women had perfect agreement on the occurrence of hypertension, 1577 (0.6%) had imperfect agreement; 9369 (3.7%) had hypertension reported in only one dataset (under-reporting) and 815 (0.3%) had conflicting types of hypertension. Using only perfect agreement between birth and discharge data resulted in the lowest prevalence rates , while including all reports resulted in the highest prevalence rates . The higher prevalence rates were generally consistent with international reports. In contrast, perfect agreement gave the highest aOR  for known risk factors: risk of chronic hypertension for maternal age \u226540 years was 4.0  and the risk of pregnancy hypertension for multiple birth was 2.8 .The method chosen for classifying discrepant case reports should vary depending on the study question; all reports should be used as part of calculating the range of prevalence estimates, but perfect matches may be best suited to risk factor analyses. These findings are likely to be applicable to the linkage of any specialised health services datasets to population data that include information on diagnoses or procedures. Population health datasets (PHDS) provide a valuable tool for epidemiological and health services research with the capacity to address health care, policy and planning issues,2. PHDS 'Hypertensive disorders of pregnancy' encompass two different, but related, conditions,12. ChroPrevious PHDS studies examining outcomes reported on more than one dataset have accepted any report of the condition of interest as a \"case\" -15. Howeth revision of the International Classification of Diseases (ICD10)[The study population included all women, discharged from hospital following birth in New South Wales (NSW) Australia, 1 January 2000 to 31 December 2002. Only 1% of women have homebirths. Data fos (ICD10). Hospitas (ICD10),23. In ts (ICD10),23. The s (ICD10). This stPregnancy hypertension and chronic hypertension are reported on both the MDC and the ISC. The conditions are not mutually exclusive, as some women have chronic hypertension with superimposed pre-eclampsia. The MDC uses a check-box format for hypertension reporting. In the ISC, an ICD10 code for a hypertensive disorder of pregnancy (O10\u2013O16) in any oThe true positive rate for case identification of the type of hypertension is dependent on both the sensitivity and the specificity of the reporting on the datasets and the prevalence of the condition. The validation studies of the MDC and ISC found both datasets suffer from under-reporting of hypertension (sensitivities 50\u201386%) but that specificities were very high (99.1\u201399.8%),23. BecaAlternative methods of classifying inconsistent and discrepant reports from the two datasets were examined. The occurrence and type of hypertension reporting on the two datasets were classified in the following ways Table :1. Totally consistent reports on hypertension occurrence and type (perfect agreement)2. Partially consistent reports, such that if one dataset recorded one type of hypertension and the other recorded both only the common type was accepted, and where the ISC reported unspecified hypertension and the MDC specified a type then the MDC report was accepted (imperfect agreement).3. One dataset reported hypertension and the other did not report any hypertension, then the hypertension report was accepted (under-reporting), or4. Conflicting reports on the type of hypertension, where chronic hypertension was reported on one dataset and pregnancy hypertension on the other (conflicting). The hypertension of interest in any analysis was accepted to be true. So in the analysis of chronic hypertension, the conflicting cases were accepted as chronic hypertension and similarly for the pregnancy hypertension analysis.These categories were then sequentially combined to make four alternative methods for classifying a woman as a \"case\" (having the hypertension type of interest): 1) only perfect agreement counted 2) perfect agreement + imperfect agreement counted 3) perfect agreement + imperfect agreement + under-reporting agreement counted 4) any report . These four alternative methods were assessed to determine the effect on both the prevalence of hypertension and on the strength of association of known risk factors for hypertension.The resulting prevalence rates of chronic hypertension and pregnancy hypertension, using the above four classification methods as well as the rates from the individual datasets, were compared. We then modeled hypertensive status as a dependent variable, with adjustment for risk factors, focusing on a type-specific risk factor: maternal age for chronic hypertension and multiple pregnancy for pregnancy hypertension. The risk factor information was obtained from the MDC and is accurately reported: maternal age has 97% agreement with the medical record  and multiple pregnancy has 99.5% agreement (kappa 0.89). Because misclassification of an outcome or exposure usually  biases measures of association towards the null, we hypothesized that the magnitude of risk  for known risk factors would move further from unity with less misclassification. The goodness-of-fit of the logistic regression models was assessed with the Hosmer Lemeshow Test .Data from previous validation studies were used to calculate \"corrected\" prevalence rates for the MDC and ISC, for comparison purposes. The corrected prevalence was calculated by multiplying the crude rate from the MDC or ISC by the positive predictive values (PPV) from the validation studies and dividing by the sensitivity,23.Among the 250173 women with linked data available, 238412 (95.3 percent) women had perfect agreement on the occurrence and type of hypertension, 1577 (0.6 percent) had imperfect agreement; 9369 (3.7 percent) were classified as under-reported and 815 (0.3 percent) had conflicting reports on the type of hypertension ; \u226540 years: aOR = 1.22 .Pregnancy hypertension showed less sensitivity to how the condition was classified when examining the risk associated with multiple pregnancy Table . Using tThe aOR's for pregnancy hypertension should not have been shifted further from unity by misclassification of chronic hypertension, as the prevalence of this condition is low and in the ISC, multiple pregnancy was not significantly associated with chronic hypertension .This study demonstrates the importance of choosing a method of classifying outcomes of interest that is appropriate to the purpose of the analysis. For many population cross-sectional studies, low sensitivities and under-enumeration of cases may be a real concern. Using data from more than one dataset offers the opportunity to identify more cases, albeit with uncertain effects on specificity and PPV. Examination of possible causal factors may be a secondary consideration and potential under-estimation of risks due to misclassification of the outcome not a prime concern. But if examination of potential risk factors is the main reason for a study, care needs to be taken to minimize the possibilities for misclassification of outcomes and the resultant under-estimation of risks. This is particularly true when the outcome of interest is rare, such that the number of true positives and false positives may be similar. When the incidence or prevalence of a condition is less than one percent, false positives may outnumber true positives even if specificity is greater than 99 percent. In this study, the odds of having chronic hypertension for a woman aged 40 years or more would have been underestimated by 40 percent if perfect and imperfect matches plus under-reporting were used to classify cases as opposed to using only perfect matches (aOR = 2.78 vs 3.95).Using two separate datasets to try to capture cases is known to increase sensitivity,7,9 and Capture-recapture methods have been used elsewhere to evaluate the completeness of case ascertainment and estimate prevalences corrected for under-ascertainment -18,29. HWithout estimates of the PPV and sensitivity of each method of classifying outcomes, it is not certain which of the classification method results in the most accurate estimates for prevalence. However, for a variable which is already known to be a risk factor for the outcome of interest, movements in the aOR away from unity should reflect a higher rate of true positives and increased PPV. Comparing the aOR's for different methods of classifying reports makes it possible to assess which method, and which dataset, is likely to have relatively better PPV's. For both of the conditions in this study, the ISC appeared to more accurately identify cases than the MDC. This is consistent with other findings that hospital discharge data are more accurately reported than birth data, which may be because hospital reporting is tied to compensation,7,9.An important aspect of the design of this study was to select risk factors which were specific to either pregnancy hypertension or chronic hypertension in pregnant women. The aOR's for these risk factors would only move further away from unity if more of the relevant type of hypertension was identified.It is possible that cases of hypertension reported on both databases had a higher proportion at the more severe end of the hypertension spectrum. However, we could not identify any studies that support this conjecture. In 1992, Iezzoni suggested that among elderly hospitalised patients, those who were severely ill and in the process of dying have more severe acute conditions and complications that take precedence over the coding of chronic diseases . HoweverAlthough this study is based on perinatal data, the findings are likely to be applicable to the linkage of any specialised health services datasets that include information on diagnoses or procedures. Increasing linkage of such datasets and additional linkages with population health registries, such as cancer, stroke, coronary heart disease and pharmaceuticals -34, willIn conclusion, we have demonstrated that how cases are classified, when more than one dataset is used to identify cases, can impact on estimates of risk for study factors. A more restrictive method for classifying cases which minimizes misclassification may be warranted when risk assessment is a primary concern for a study. One approach to comparing the relative strength of the PPVs of different datasets and of different methods of classifying reported cases of the condition of interest may be to examine how the adjusted OR (for a previously established risk factor) is affected when different methods are used.PHDS Population health datasetsNSW New South WalesMDC Midwives Data CollectionISC Inpatient Statistics Collectionth revisionICS10 International Classification of Diseases, 10PPV Positive Predictive ValueaOR adjusted Odds Ratio95 percent CI 95 percent confidence intervalThe author(s) declare that they have no competing interests.CLR conceived the study, undertook the data analysis and drafted the manuscript. CSA participated in the design of the study, advised on the statistical analysis and helped to draft the manuscript. JBF participated in the study design and interpretation of the data. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "The A prospective cohort study was carried-out in Alexandria University Maternity Hospital. We planned to examine the records of twin deliveries over 2 years. The inclusion criteria were twin deliveries with gestational age at delivery at least 36 completed weeks. Twins of mothers with chronic illness and those with congenital anomalies were excluded. Perinatal outcome parameters  were defined and evaluated.Out of 273 twin sets, 197 (72.2%) met the inclusion criteria. They were classified into 3 groups according to the gestational age at delivery. Neonatal morbidity and maternal complications were higher in those delivered earlier. Twins electively delivered had worse outcome than those delivered spontaneously. In the elective group, there was no difference in the outcome between those delivered earlier or later.Twins, when the pregnancy is uncomplicated, continue to grow and mature with advancement of the gestational age. In the absence of significant maternal complications, it is advisable to deliver twins only at 38 completed weeks' gestation or later to avoid neonatal complications. Lots of controversies are raised about the optimal gestational age for twin delivery. Whether multiple pregnancy has an adverse effect or not on fetal lung maturity remains very debatable. Luke et al [1] suggested that twins attain more rapid pulmonary maturation than singletons and therefore complications of postmaturity may arise sooner in twins than in singletons. This has been also ascertained by the study of Leveno et al [2], and referred to in the recommendations of the American College of Obstetricians and Gynecologists since 1989 [3]. This has resulted in a tendency to electively deliver twins earlier than singletons of comparable gestational age. Hack et al have found an association between elective delivery and adverse perinatal outcome, in particular respiratory morbidity [4]. This was also reported by Wax et al in 2002 [5]. Friedman et al[6] recently reported that twins do not have accelerated maturation compared with singletons for the same gestational age.Complications related to twin pregnancy and delivery are enormous. Our local statistics point to an incidence of 1.4% for twin gestations. With the recent introduction of assisted reproduction techniques, the number seems to be increasing. Perinatal mortality is known to be approximately seven times higher in twins than singletons [8]. Many of these complications are related to preterm delivery, low birth weight, and respiratory problems [9]. This issue is furthermore complicated in developing countries, where the level of neonatal intensive care is not optimal. The fact that, complications of prematurity and low birth weights are more common in these set-ups,[10] further complicates the decision.The question about outcome in twins delivered early versus late remains unanswered [11]. However, a better understanding of the relationship between timing of delivery and perinatal outcome in local set-ups could be used to decrease the morbidity, mortality associated with twin deliveries [12]. The purpose of this study was to evaluate the association of the timing of delivering twins and the outcome. We planned a study to test the perinatal outcome of twin pregnancies delivered at 36\u201337 weeks' gestation and twin pregnancies delivered at \u2265 38 weeks' gestation.This observational cohort study was carried-out at El-Chatby Maternity Hospital, University of Alexandria, Egypt. El-Chatby hospital is a busy maternity hospital with a large number of deliveries approaching 10,000 per year. Twin deliveries between January 1, 2000 and December 31, 2002, with gestational age at delivery at least 36 completed weeks, were included. Exclusion criteria were significant maternal chronic ill health not related to pregnancy, twin-twin transfusion syndrome, congenital anomalies and the death of one fetus. The protocol was approved by the University board.Patient information was abstracted from the maternal and infants' charts. Maternal data  and newborn data  were collected. Respiratory distress syndrome was diagnosed if respiratory distress was associated with the classic X-ray findings. Hypoglycemia was considered if random blood glucose level was < 40 mg/dl. The diagnosis of neonatal sepsis necessitated a positive blood culture in the presence of suggestive clinical signs. Oligohydramnios was determined by sonographic examination (amniotic fluid index of \u2264 5) [13]. Maternal hypertension with proteinuria or edema were used to diagnose preeclampsia. Fetal distress, antepartum or intrapartum, was defined according to the international standards [14]. Truly elective deliveries were defined as those that were not spontaneous or complicated with preeclampsia, oligohydramnios, fetal growth restriction (FGR) or abruption. Gestational age at delivery was defined as the number of completed weeks from the first day of the last normal menstrual period to the date of delivery. Early ultrasound examination (less than 20 weeks) results were used if the dates were unreliable. FGR was considered if the fetal weight is below the 10th percentile of gestational age [15].t-test and were used for the analysis. P values < 0.05 were considered significant.Perinatal outcomes were compared between twin gestation delivered at < 38 weeks' gestation and those delivered at \u2265 38 weeks' gestation. Chi-square test student's During the 2-year study period, there were 18794 deliveries and 273 twin sets (1.45%). Out of the 273 sets, 197 (72.2%) met the inclusion criteria. Classified according to the gestational age at delivery, 115 sets (58.4%) were born at 36\u2013<37 weeks[Group I], 66 (33.5%) at 37\u2013<39 weeks [Group II] and 16 (8.1%) at > 39 weeks [Group III].Table Eighty-eight sets have undergone elective deliveries for various causes. Table The confusion, created by the belief that fetal lung maturity occurs several weeks earlier in twins compared with uncomplicated matched singletons [2], led to the widespread impression that twins mature sooner than the singletons counterpart. Moreover, the demonstration of advanced placental maturation in twins supported that impression [16]. All that encouraged the practice of delivering twins earlier. In our study, the incidence of neonatal respiratory distress syndrome was significantly higher at 36\u201337 weeks' gestation. It decreased and disappeared at higher gestational ages. Our findings go along with the findings of Friedman et al; who reported that twins < 36 weeks' gestation did not show accelerated maturation or improved neonatal outcome compared with their singletons counterpart [6]. The studies of Luke et al and Kiely et al suggested that Contrary FGR increases in twins after 38 weeks' gestation . In our series, the incidence of FGR significantly decreased as the gestational age at delivery advanced. This might be explained by the fact that complicated pregnancies with FGR might need to be terminated earlier. Moreover, the mean birth weights increased steadily with advancement of the gestational age. Twins do continue to grow in utero till 40 weeks. The presence of higher maternal complications in group I (deliveries before 37 weeks) may, in part, explain the earlier termination of these pregnancies.Comparing the electively delivered group with the spontaneously delivered group showed that the overall neonatal morbidity was worse in those delivered electively. This is in agreement with the findings that electively delivered newborns, even at term, have a higher incidence of severe respiratory distress and other complications . The same was found for the maternal complications. Again this may explain the reasons for their elective delivery. When the elective deliveries were analyzed according to the gestational age at labor, no significant difference was found in the perinatal outcome parameters. Early and late elective twin deliveries had similar outcomes.One of the limitations of our study was the design. A well-designed prospective randomized study involving all twin pregnancies and outcomes, at all the gestational ages will be more informative. Another limitation is the nature of our hospital. It is a tertiary referral centre which receives all complicated deliveries. This could make the sample not totally representative of the general population. Excluding twins of chronically ill mother is another issue. These may really experience advanced maturation.Twins, when the pregnancy is uncomplicated, continue to grow and mature with advancement of the gestational age. In the absence of significant maternal complications, it is advisable to deliver twins only at 38 completed weeks' gestation or later to avoid neonatal complications.The author(s) declare that they have no competing interests.AB carried out the study design, data collection, analysis and writing of the manuscript. TK shared participated in the design of the study and shared in the data collection. All authors read and approved the final manuscript."}
+{"text": "Cleary-Goldman and d'Alton discuss the implications of a new study in PLoS Medicine examining the risk of fetal death in uncomplicated monochorionic diamnotic twin pregnancies. In the past 20 years, advancing maternal age and greater reliance on assisted-conception techniques have dramatically increased the incidence of multiple births, including monochorionic twins see  1,2]. C,2. C1,2]Diamniotic: Twins with two amniotic sacs.Monochorionic: Twins that share one placenta.Monochorionic diamniotic: Twins that share one placenta with two amniotic sacs.Monoamnionicity: Twins that share one placenta and one amniotic sac.Twin-to-twin transfusion syndrome (TTTS): This occurs in monochorionic twins. Twins with TTTS have vascular connections that result in unequal sharing of blood. The smaller twin (donor) does not get enough blood, while the larger twin (recipient) becomes volume overloaded.Intrauterine fetal demise: This occurs when a fetus dies in utero (before birth). This is also known as stillbirth.Multicystic encephalomalacia: This refers to the formation of multiple cystic lesions in the cerebral cortex of fetuses, neonates, and infants following injury usually secondary to a hypoxic event.Twin reversed arterial perfusion sequence: This occurs in monochorionic twins. In this disease, there is a normally developing twin (pump twin) and a twin that does not develop a normal heart (acardiac twin). The pump twin pumps blood for the acardiac twin and for itself by reversing the blood through the umbilical artery. The pump twin is at risk for heart failure due to the increased demands on its heart.Nuchal translucency: This describes the area in the back of the fetal neck during ultrasound between ten and 14 weeks' gestation. Nuchal translucency screening can be used as a method of first-trimester screening for fetal aneuploidy.Nonstress test: A test assessing fetal well-being. It involves attaching fetal heart rate and uterine contraction monitors around the patient's abdomen for approximately 20 to 30 minutes and recording the fetal heart-rate patterns. A reactive nonstress test suggests fetal well-being.The offspring of multiple pregnancies are at greater risk for adverse perinatal outcomes compared to their singleton counterparts, predominantly due to increased risks for preterm delivery and due to monochorionicity . The linDespite these advances in knowledge about risks, there is very little consensus about the management of these high-risk pregnancies. In addition, there is the temptation to be reassured by increasing gestational age as the potential complications of prematurity recede. Recent studies suggest, however, that the offspring of a multiple gestation may benefit from delivering prior to their expected date of delivery ,8.Several studies have focused on the \u201cprospective risk of fetal death\u201d to help determine by which gestational age a multiple pregnancy should be delivered ,8. For tPLoS Medicine, Barigye et al. report the prospective risk of fetal death in uncomplicated monochorionic diamniotic twin pregnancies derived from a cohort of pregnancies that were managed at a single tertiary care referral center and that delivered after 24 weeks' gestation [In this issue of estation . PatientThere were ten unexpected deaths (three double IUFDs and four single IUFDs) in a total of seven (4.6%) of the 151 seemingly uncomplicated monochorionic diamniotic pregnancies. These IUFDs occurred at a median gestational age of 34 weeks 1 day (range 28 weeks 0 days to 36 weeks 3 days). Between 24 and 34 weeks' gestation, the prospective risk of fetal death varied between 1/22 and 1/30 pregnancies. After 32 weeks' gestation, the prospective risk of unexpected antepartum stillbirth was 1/23. In six out of the seven pregnancies, the fetal death was incidental and found on routine ultrasound.  There were no significant differences between the IUFD-affected pregnancies and the unaffected pregnancies with regards to antenatal indicators of intrauterine growth restriction and TTTS. In three of the five pregnancies (autopsy was declined in two cases), deaths remained unexplained after autopsy. Two cases, both double IUFDs, exhibited signs of TTTS. The authors concluded that despite intensive fetal surveillance, uncomplicated monochorionic diamniotic twin pregnancies are at risk for unexpected intrauterine death. The deaths occurred in the third trimester and predominantly after 32 weeks' gestation. As a result, the authors suggested that after 32 weeks' gestation, the prospective risk for fetal death in these pregnancies might be eliminated by elective preterm delivery.when is the ideal gestational age to deliver apparently uncomplicated monochorionic twins? The authors suggest that 32 weeks' gestation may be reasonable. At that gestational age, many of the risks associated with prematurity, such as intraventricular hemorrhage, necrotizing enterocolitis, and respiratory distress syndrome, have abated.Despite the limitations of the study, which Barigye et al. elucidate well , this study highlights an important question that many practicing obstetricians are confronting increasingly often: apparently uncomplicated monochorionic twin pregnancy with the risk of double IUFD or single IUFD with the concomitant risk of multicystic encephalomalacia for the surviving co-twin is challenging. In our practice, we have been conducting antenatal surveillance more frequently than once every two weeks, and we have been using the nonstress test in addition to ultrasound and Doppler studies. Although in our preliminary experience we have not had any unexplained IUFDs, we are uncertain if the frequency of testing could account for the prospective risk of fetal death found in the study by Barigye et al. In addition, we have been offering delivery of these apparently uncomplicated monochorionic twins at approximately 34 to 35 weeks' gestation following antenatal corticosteroid administration and thorough counseling regarding the risks of expectant management versus elective preterm delivery. While we acknowledge that our practice pattern is by no means a standard-of-care requirement, we feel it is a reasonable approach to this dilemma until larger, prospective observational studies have been conducted to better elucidate the natural history of these high-risk pregnancies and to better answer the question of when the ideal gestational age is to deliver apparently uncomplicated monochorionic twins.Nonetheless, the risks of prematurity are not negligible at 32 weeks' gestation. Balancing the risk of iatrogenic preterm birth in an"}
+{"text": "Monochorionic (MC) twins are at increased risk for perinatal mortality and serious morbidity due to the presence of placental vascular anastomoses. Cerebral injury can be secondary to haemodynamic and hematological disorders during pregnancy  or intrauterine co-twin death) or from postnatal injury associated with prematurity and low birth weight, common complications in twin pregnancies. We investigated neurodevelopmental outcome in MC and dichorionic (DC) twins at the age of two years.This was a prospective cohort study. Cerebral palsy (CP) was studied in 182 MC infants and 189 DC infants matched for weight and age at delivery, gender, ethnicity of the mother and study center. After losses to follow-up, 282 of the 366 infants without CP were available to be tested with the Griffiths Mental Developmental Scales at 22 months corrected age, all born between January 2005 and January 2006 in nine perinatal centers in The Netherlands. Due to phenotypic (un)alikeness in mono-or dizygosity, the principal investigator was not blinded to chorionic status; perinatal outcome, with exception of co-twin death, was not known to the examiner.Four out of 182 MC infants had CP (2.2%) - two of the four CP-cases were due to complications specific to MC twin pregnancies (TTTS and co-twin death) and the other two cases of CP were the result of cystic PVL after preterm birth - compared to one sibling of a DC twin  of unknown origin. Follow-up rate of neurodevelopmental outcome by Griffith's test was 76%. The majority of 2-year-old twins had normal developmental status. There were no significant differences between MC and DC twins. One MC infant (0.7%) had a developmental delay compared to 6 DC infants . Birth weight discordancy did not influence long-term outcome, though the smaller twin had slightly lower developmental scores than its larger co-twin.There were no significant differences in occurrence of cerebral palsy as well as neurodevelopmental outcome between MC and DC twins. Outcome of MC twins seems favourable in the absence of TTTS or co-twin death. Monochorionic (MC) twins are at increased risk for perinatal mortality and serious morbidity compared to dichorionic (DC) twins Although long-term outcome has been studied extensively in TTTS All monochorionic (MC) twin pregnancies delivered between January 2005 and January 2006 in nine perinatal centers in The Netherlands, all tertiary referral centers with neonatal intensive care units (NICUs), were recorded. During the study period, 111 MC twin pairs were born in these hospitals. Eleven twin pregnancies were complicated by death of both infants. Four of the MC twins were monoamniotic; these pregnancies were excluded. Therefore, 96 MC twin pairs with at least one survivor were eligible for this prospective follow-up study. Perinatal mortality data of these twins will be reported separately in a large cohort, collected over a longer time period . A group of 98 dichorionic (DC) twins was matched for gestational age at delivery, gender, birth weight, ethnicity of the mother and study center.The study was approved by the institutional review board of the University Medical Center Utrecht, and local permission was obtained in the other centers. Study participation by written consent was obtained from the parent(s). All twins with identical gender (i.e. boy-boy and girl-girl twins) were identified from the electronical databases of each hospital. We checked the medical charts to assess chorionicity. Chorionicity was determined on the basis of first-trimester ultrasound assessment and/or postpartum pathological examination of placentas and intertwin membranes. The diagnosis of TTTS was made antenatally by standard ultrasound criteria Information on the occurrence of cerebral palsy (CP) was obtained from all children. The diagnosis of cerebral palsy (CP) was made according to standard criteria The participants were tested with the Griffiths Mental Developmental Scales Statistical analysis was performed with the SPSS 12.0 statistical package. Continuous variables were compared by independent Student t-testing if normally distributed or otherwise by means of Mann Whitney U testing. Categorical variables were compared by Fisher's exact test. Linear regression was used to estimate mean differences of the DQ-scores according to chorionicity and donor or recipient of a TTTS-twin. These mean differences were adjusted for possible confounders. Statistical significance was based on two-sided tests with a cut-off level of 0.05.During the study period 182 MC and 189 DC long-term survivors were approached for this prospective follow-up study. In forty-six twin pairs the developmental test could not be performed, either due to being lost-to-follow-up (15 MC and 20 DC infants) or parents did not approve participation  and one out of 189 long-term DC survivors had CP . Two MC infants had CP due to complications specific to MC twin pregnancies. In one case fetal death of the smaller co-twin occurred at 26 weeks of gestation most likely due to placental insufficiency, with subsequent very preterm delivery of the surviving twin. This boy had a severe intraventricular hemorrhage (grade III) with posthemorrhagic ventricular dilatation and atrophy of brain tissue and developed spastic diplegia. In the other case, the pregnancy was complicated by TTTS (Quintero stage III) with laser coagulation of the connecting placental vascular anastomoses. Both infants were delivered at 37 weeks of gestation. The recipient showed normal development in contrast to the donor twin. The antenatal ultrasound of the brain showed echogenicity. The MRI after birth showed generalized cerebral atrophy and signs of an old hemorrhage in the periventricular area and in the basal ganglia. This child had a quadriplegia with severe mental retardation. The other two MC infants with CP were born very preterm  and both developed spastic quadriplegia as a result of cystic periventricular leukomalacia (PVL). The cause of CP in the infant of the DC group (born at 31 weeks of gestation) is currently unknown, but may be genetically determined as this child also suffers from albinism. This child has diplegia with psychomotor retardation.After losses to follow-up, 140 MC infants and 142 DC infants without CP participated in this prospective follow-up study and were tested with the Griffiths Mental Developmental Scales. Baseline characteristics of the study population are shown in The majority of the tested infants showed normal development (98.2%). Five out of 282 long-term survivors had a mild developmental delay and two infants had a severe developmental delay. There was a significant difference in development between boys and girls. Boys had lower DQ scores  and the proportion of mildly delayed development was higher among male infants. Infants born before 32 weeks of gestation and/or with a birth weight <1500 gram had significantly lower DQ-scores than infants born after 32 weeks and/or \u22651500 gram . There was no significant difference between infants who were small-for-gestational age and infants with a birth weight above the tenth percentile.p\u200a=\u200a0.423), which was mainly due to the higher proportion of mildly delayed hearing and speech development in MC twins. These results were in agreement with the parental impression of the development of their infant(s), as assessed by the HSCS-PS questionnaire (ionnaire .p\u200a=\u200a0.109).Developmental outcome of the twins after an uneventful course of pregnancy  is shown in p\u200a=\u200a0.338). This trend was found in both MC and DC discordant twins.Eighteen MC pregnancies were complicated by antenatal signs of TTTS (17.6%), of which six had been treated by laser coagulation of vascular anastomoses. Five pregnancies had been treated by serial amnioreduction. The other pregnancies did not receive any treatment, either due to low stage TTTS or immediate delivery after diagnosis. Three TTTS pregnancies were complicated by death of one of the twins. There were 33 long-term survivors, of which one had CP (3.0%). Twenty-seven long-term survivors were tested. All of them had a normal development . However+0 and 25+5 weeks, respectively), one IUD at 26+1 weeks was most likely due to placental insufficiency, and the other IUD remained unexplained (at 32+2 weeks' gestation). One DC fetal death at 31+1 weeks was caused by placental insufficiency and the other two deaths remained unexplained . Only one (MC) survivor developed CP, all other survivors showed normal development.In this cohort of 282 infants, four MC and three DC twin pregnancies were complicated by single intra-uterine death. In two MC cases IUD occurred after laser ablation for TTTS ; two CP-cases were due to complications specific to MC twin pregnancies  and two cases of CP as a result of cystic PVL after preterm birth. One DC twin had CP (0.5%) without clear cause. The majority of 2-year-old twins had a normal developmental status. There were no significant differences between MC and DC twins, apart from a slight delay in hearing and language development in MC twins. Birth weight discordancy did not influence long-term outcome, although there was a trend towards lower DQ-scores in the smaller twin than in its larger co-twin.The response rate of this follow-up study was 76%. This raises concern about the possibility of a non-response bias. In order to reduce this possibility to a minimum, we obtained information on neurological status and development from the attending pediatrician of the non-participants. In this way, we were able to report the study population based CP incidence. From five twin pairs no follow-up data were retrieved. We made an assumption that these children did not develop CP, since they did not have any major  complications during their neonatal period.A limitation of this study is that it was not population-based. The data in this study should thus be interpreted with care since a selection bias may have been introduced due to the specific nature of tertiary referral centres. However, long-term outcome of the twins was even better than reported in previous publications.With regard to the analyses based on the Griffiths test, it may be that our results are somewhat biased in favor of the DC group. That is, non-responders in the DC group were found to be born earlier than the responders in this group, a difference that was not found in the MC group, which may have led to an underestimation of developmental delay especially in the tested DC group. However, since we expected a worse outcome of MC twins due to possible cerebral damage in the presence of haemodynamic imbalance, this bias does not influence our conclusion that long-term outcome of MC twins was not shown to be worse than that of DC twins.However, it should be noted that neurological handicaps and mental retardation may become evident only several years after birth, so a new evaluation of CP and developmental delay at the age of five years is preferable.It is well known that the incidence of abnormal long-term neurodevelopmental outcome in MC pregnancies complicated by TTTS is high. After treatment with serial amniodrainage, most studies on long-term outcome report a 22\u201326% incidence of neurological impairment It is also well known, that the risk of neurological abnormality in the surviving twin after co-twin intra-uterine death is higher in MC than in DC twins. The mechanism leading to damage of the surviving sibling is an acute blood loss through the placental vascular anastomoses into the dying fetus, leading to hypovolemia in the survivor, which in turn might cause death from hypovolemic shock or cerebral ischemia due to hypoperfusion Intrauterine growth restriction (IUGR) is another important risk factor for cerebral palsy and impaired neurological development. Gratacos and co-workers p<0.05). TTTS was considered to be responsible for most cases of adverse outcome in the MC group. In the same period, Adegbite and collegues The increased risk of neurological disability in MC survivors of TTTS or co-twin fetal death suggests that cerebral injury in MC twins is related to the vascular anatomy of the placenta. Placental vascular anastomoses, which are nearly always present, can cause haemodynamic imbalance and subsequent ischemic injury to the brain. Cranial ultrasound abnormalities in the neonatal period also suggest that monochorionity itself is a risk factor for cerebral damage In summary, the majority of 2-year-old twins had a normal neurodevelopmental status. There were no significant differences between MC and DC twins. CP may occur more frequently in MC twins and was mainly due to complications specific to MC twin pregnancies (TTTS and co-twin death). Outcome of MC twins was favourable in the absence of TTTS or co-twin death. More studies are needed to evaluate the effect of improved obstetric management on the long-term neurodevelopmental status of \u2018uncomplicated\u2019 MC twins. Meanwhile we suggest a routine cranial ultrasound examination after birth and neurodevelopmental follow-up."}
+{"text": "To investigate the influence of inhomogeneity corrections on stereotactic treatment plans for non-small cell lung cancer and determine the dose delivered to the PTV and OARs.For 26 patients with stage-I NSCLC treatment plans were optimized with unit density (UD), an equivalent pathlength algorithm (EPL), and a collapsed-cone (CC) algorithm, prescribing 60 Gy to the PTV. After optimization the first two plans were recalculated with the more accurate CC algorithm. Dose parameters were compared for the three different optimized plans. Dose to the target and OARs was evaluated for the recalculated plans and compared with the planned values.20 Gy are harder to fulfill. After recalculation of the UD and EPL plans large variations in the dose to the PTV were observed. For the unit density plans, the dose to the PTV varied from 42.1 to 63.4 Gy for individual patients. The EPL plans all overestimated the PTV dose (average 48.0 Gy). For the lungs, the recalculated V20 Gy was highly correlated to the planned value, and was 12% higher for the UD plans (R2 = 0.99), and 15% lower for the EPL plans (R2 = 0.96).For the CC algorithm dose constraints for the ratio of the 50% isodose volume and the PTV, and the VInhomogeneity corrections have a large influence on the dose delivered to the PTV and OARs for SBRT of lung tumors. A simple rescaling of the dose to the PTV is not possible, implicating that accurate dose calculations are necessary for these treatment plans in order to prevent large discrepancies between planned and actually delivered doses to individual patients. Treatment outcome of conventional radiotherapy for early-stage lung cancer has been rather poor, while possibilities for dose escalation are limited. In recent years several studies have shown promising results using stereotactic body radiotherapy (SBRT) for lung tumors, with local control rates at 3 years up to 90% -3.A wide variety of treatment planning algorithms is used for SBRT. As a result, large differences exist in the way that inhomogeneities in the target volume are handled in the planning phase. In two important SBRT of lung cancer trials, on which many current clinical implementations of SBRT are based, different algorithms were used; the RTOG 0236 phase-II trial planning was performed without using inhomogeneity corrections assuming the patient has unit density , while iPlanning algorithms can roughly be separated in (a) ones which do not take into account changes in lateral electron transport  and (b) ones that do take into account these changes  . In the Several authors have studied the influence of inhomogeneity corrections on dose distributions specifically for stereotactic treatments of lung cancer ,11-14. CIn this study, the influence of inhomogeneity corrections on the dose distributions was investigated for a large group of patients with small stage I lung cancer tumors. These results are e.g., important for the correct interpretation of previous clinical trials and for the definition of planning criteria for new clinical trials of this treatment, and are being used for designing a Dutch multicenter randomized phase-III trial comparing SBRT with surgery for stage-I NSCLC .3 treatment planning system . Using the MIP dataset, an experienced radiation oncologist delineated the internal target volume (ITV). The GTV was delineated on the CT dataset of the maximum inhale phase; tumor mobility was determined by translating the delineated GTV from this phase to the maximum exhale phase. Organs at risk were delineated on an average-density CT reconstruction. As dictated by the RTOG 0236 protocol, no ITV to CTV margin was applied [et al [All patients in this study received a respiration-correlated 4D-CT using a Philips Brilliance Big Bore CT prior to treatment. The 4D-CT was reconstructed in ten equally spaced time bins using phase binning. From these phases, a maximum intensity projection (MIP) was reconstructed. The datasets were then imported in the Pinnacle3, respectively.Twentysix consecutive patients with non-small cell lung cancer (NSCLC) were included. All patients had solitary stage-I tumors, were medically inoperable and were treated at our institute with SBRT. A summary of the volumetric and motion characteristics of these tumors is shown in Table 3 treatment planning system. Dose calculations were performed on an average-density CT using a 3 \u00d7 3 \u00d7 3 cm dose calculation grid size.The treatment plans consisted of 9 equally spaced coplanar 6 MV beams. Beams consisting of 2 segments were not allowed to enter through the esophagus, heart, spinal cord or contralateral lung. The plans were inversely optimized using the direct aperture optimization module of the Pinnacle20 Gy) below 10%. For the V20 Gy, both lungs minus the ITV were delineated, in accordance with the RTOG protocol. Maximum dose to the spinal cord was limited to 18 Gy, to the esophagus to 27 Gy, and to the heart to 30 Gy. To prevent the generation of very small segments the minimum beam segment area was set to 4 cm2, but generally the segment area was significantly larger. The minimum number of monitor units per segment was limited to 50 MU to ensure that the delivery time at least covers one breathing cycle. All plans consisted of 9 beams with in total generally 18 segments and in a few cases 17 segments.Plans were optimized until 95% of the PTV received the prescription dose of 60 Gy in 3 fractions (according to RTOG 0236), and more than 99% of the PTV received 90% of the prescribed dose (54 Gy). No limitations to the maximum dose were applied within the PTV as highly inhomogeneous dose distributions are commonly accepted in stereotactic treatments. Objectives were added to ensure that the prescription isodose closely conforms to the PTV and the dose to healthy lung tissue was minimized. The goal was to keep the fraction of healthy lung receiving more than 20 Gy , in accordance with the RTOG 0236 protocol. The third plan was optimized while only accounting for the decreased attenuation of the primary photons, thus resembling an equivalent pathlength (EPL) correction that is incorporated in less advanced dose calculation algorithms. These plans will be referred to as the CC, UD and EPL plans respectively. For all patients, these three plans were optimized until all planning criteria were met. A small renormalization was applied to all plans to ensure that they had exactly identical PTV coverage (60 Gy to 95% of the PTV). Next, the UD and EPL plans were copied and recalculated without re-optimization using the collapsed-cone algorithm.95) and 99% (D99) of the PTV, and the isocenter dose. Conformality of the PTV coverage was evaluated by the ratio of the volume of the prescription isodose (60 Gy) and the PTV (V100%/VPTV). For evaluation of the low dose spillage, the ratio of the 50% isodose volume (30 Gy) and the PTV was calculated (V50%/VPTV). The influence on lung dose was studied by scoring the lung volume receiving 20 Gy, 10 Gy (V10 Gy) and the mean lung dose (MLD). For all the other organs at risk, no further analysis was done as these received doses far below the tolerance dose by choosing appropriate beam arrangements. All differences in dosimetric parameters were tested using a paired-sample t-test.For each plan the maximum, minimum and mean dose to the PTV was determined, as well as the dose received by 95% (D100%/VPTV < 1.2) could however be met by all treatment plans except for 3 patients with very small tumors having a minor violation (V100%/VPTV < 1.4).For all three calculation algorithms, clinically acceptable treatment plans could be obtained for all patients Table . The dos50%/VPTV) which is plotted as a function of the PTV in Figure The maximum dose for the CC plans is considerably higher than for the other two Figure . Dose ho20 Gy for the CC plans was on average 15% and 21% higher as the planned UD and EPL values, respectively  similar results are found as for D95 , and large variations per patient existed. For the EPL plans the mean V20 Gy was significantly lower, with a mean value of 4.1%. The recalculated V20 Gy is plotted against the planned V20 Gy for the UD and EPL plans in Figure 2 = 0.99 and 0.96 for UD and EPL plans, respectively) although a reasonable amount of scatter is visible. For the V10 Gy and mean lung dose even stronger correlations were found between planned and recalculated values  as mentioned in the introduction is of course a simplification of the differences that exist between the various clinically implemented algorithms. The comparison between the EPL and CC algorithms presented here can be seen as a good quantitative analysis of the differences that can be found between type a and b algorithms. However, slightly different results are expected if two other (implementations of) type a and b algorithms would have been used.The dose criteria as prescribed in the RTOG 0236 trial  have beeDose coverage of the recalculated plans varied widely among different patients. The dose to 95% of the PTV for the plans optimized with unit density ranged from 30% lower to slightly higher than planned for individual patients. A simple rescaling of the planned dose to the actual dose given to the patient is thus not possible, making a recalculation of the plan with accurate dose calculations necessary.et al. who recently presented clinical results of more than 200 patients with NSCLC [The overestimation of the dose using the EPL algorithm seen in all patients varies with increases tumorsize, lung density and location. This type of algorithm is still widely used in clinical practice, and has also been used by Lagerwaard th NSCLC . The poset al.[In a study by Haedinger et al. the ster20 Gy, V10 Gy) in the lungs. On the other hand the algorithms often underestimate the required number of MUs due to lateral electron disequilibrium. As a result, the plans optimized with unit density calculations tend to underestimate the dose to the healthy lung , and the EPL plans overestimate it. In accordance with De Jaeger et al. [et al did not find a difference between V20 Gy values calculated for heterogeneity corrected and UD plans [et al. With yet another beam set-up, namely for breast irradiation, Brink et al. also found significant differences between algorithms in deriving optimal radiation pneumonitis NTCP values [As the UD and EPL calculations do not account for the increased lateral electron range, recalculation of the plans results in an increase of the low-dose region (Vr et al.  a correlUD plans . HoweverP values . Thus, iAlthough Monte Carlo simulations are considered to be the gold standard in the presence of inhomogeneities, the collapsed cone algorithm has proven to be reasonably accurate. Krieger and Sauer did find up to 10% difference between CC and Monte-carlo calculations. However, these deviations were found using a slab geometry phantom and single beam set-up which does not resemble a clinical set-up very well. Furthermore, the authors indicate that part of these errors might be explained by an incorrect choice of the CC parameters . In mostMaybe even more important, the collapsed cone algorithms have now become widely available in clinical practice, while the use of Monte Carlo treatment planning is still very limited. Thus, the CC results presented here can be used to generate optimization criteria in clinical practice, while this would be less straightforward for results based on Monte-Carlo calculations.The implications of the results in this study are twofold. In the first place, planning dose criteria are often easier achieved with plans created using simple dose calculation algorithms, which should be considered in study designs involving multiple institutions using different planning systems. Secondly, the actually delivered dose to the tumor can significantly deviate from the planned value when not using appropriate inhomogeneity corrections. As large variations exist in the actual dose per individual patient, clinical studies evaluating the effectiveness of this treatment should rely on the most accurate dose calculation which is clinically available at the time, or at least retrospectively re-evaluate the actual dose given to the target. Fortunately, the dose to the healthy lung tissue calculated with a simple algorithm can retrospectively easily be recalculated using the correlation parameters derived in this study. Before clinical introduction, the fractionation scheme and dose optimization procedure should be very well tailored to the calculation algorithm and TPS one uses clinically.DS was responsible for study design, carried out treatment planning, analysis of data and results, and writing and editing the manuscript, CWH worked on study design, analysis of data and results, and writing and editing the manuscript. All authors have read and approved the final manuscript."}
+{"text": "The Victorian Government Department of Health funded a diphtheria, tetanus and acellular pertussis vaccine for parents of infants from June 2009 to June 2012 as part of a cocooning strategy for the control of pertussis. The aim of this study was to assess parents\u2019 attitudes and awareness of the vaccination program, and to estimate vaccine uptake.A cross-sectional survey of 253 families with a child born in the first quarter of 2010 residing within five metropolitan and four rural local government areas in Victoria was conducted. Univariate analyses were performed to describe the relationship between demographic variables, knowledge and awareness of the disease, the vaccine program and vaccine uptake. Multivariate analyses examining predictors for awareness of the vaccine program and for the uptake of vaccination were also conducted.One hundred and five families were surveyed (response rate 43%). Of these, 93% indicated that they had heard of \u2018pertussis\u2019 or \u2018whooping cough\u2019 and 75% of mothers and 69% of fathers were aware the pertussis vaccine was available and funded for new parents. Overall, 70% of mothers and 53% of fathers were vaccinated following their child\u2019s birth, with metropolitan fathers less likely to be vaccinated as rural fathers . Being a younger mother (p\u2009=\u20090.02) or father (p\u2009=\u20090.047), and being an Australian-born father  were found to predict uptake of the vaccine in parents.Parents indicated a reasonable level of knowledge of pertussis and a willingness to be vaccinated to protect their child. However, vaccine uptake estimates indicated further opportunity for program improvement. Future cocooning strategies would benefit from specifically targeting fathers and metropolitan maternity hospitals to increase vaccine uptake. Wider promotion of the availability of vaccine providers may increase uptake to maximise the success of cocooning programs. Further investigation of the effectiveness of the cocooning strategy in decreasing infant morbidity and mortality is required. Victoria is a south-eastern state of Australia, which has a population of 5.6 million and comprises approximately 25% of the Australian population . BetweenPeri-natal vaccination of close contacts of infants is known as cocooning. Close contacts may include mothers, fathers, grandparents and other household members. The primary objective of cocooning is to reduce disease transmission to infants from their closest contacts, whilst secondarily reducing morbidity from pertussis in adults . A time-This study aimed to assess parents\u2019 knowledge of pertussis infection, their attitudes towards the free vaccination program, and to estimate uptake of the vaccine among eligible parents following the birth of their child. It also sought to examine factors predicting awareness of the vaccine program and uptake of the vaccine.A cross-sectional survey of parents of infants was designed to assess the attitudes, awareness and uptake of the pertussis vaccine for parents in Victoria. Victorian Local Government Areas (LGAs) were stratified into metropolitan and rural localities, and five LGAs were randomly selected from each stratum. Rural LGAs were oversampled due to a perception that vaccination would be less accessible outside the metropolitan areas. Five metropolitan LGAs and four rural LGAs participated in the study Figure\u00a0. A samplA four page questionnaire was designed to assess parents\u2019 awareness of pertussis and the cocooning program, their attitudes towards a cocooning strategy and uptake of the vaccination. The disease was referred to as both pertussis and whooping cough in the introductory letter; however the term whooping cough was used throughout the questionnaire. The questionnaire consisted of Likert scales to assess knowledge and attitudes, and closed questions to assess program awareness and vaccine uptake. Parents were also given the opportunity to comment on the program. Demographic details were collected on parents and the self-reported vaccination status for their child. Parents were asked about their knowledge of pertussis illness in adults and infants, the adult pertussis vaccine, whether parents had heard about the vaccination program, if they were vaccinated, and if not vaccinated their reasons for not being vaccinated.A study database was created in EpiInfo\u2122 and data analyses were completed using Intercooled Stata\u2122 Version 9.0. Percentages were calculated for most responses including questions utilising Likert scales. Maternal demographic data was compared to the most recent available data on births in Victoria . The proEthics approval to conduct this research was gained from the Australian National University Human Research Ethics Committee. The research was also approved by the Victorian Government Department of Education and Early Childhood Development\u2019s Early Childhood Research Committee.Of the 253 questionnaires mailed, 108 were completed and returned (response rate\u2009=\u200943%). Six were returned without being received by their intended recipients. Three families did not meet the inclusion criteria for the study, leaving 105 eligible families. Responses from these families represented 4-5% of registered births in participating metropolitan LGAs and 4-40% of the registered births in rural LGAs (Table\u00a0The median age of mothers was 33 years (range 20 to 41 years) and fathers was 34 years (range 22 to 59 years). Most families (92%) were either married or in a defacto heterosexual relationship and 8% were single mothers. Eighty-four percent of mothers and 78% of fathers were born in Australia; none identified as Aboriginal and/or Torres Strait Islander. A bachelor degree or higher was the most common highest level of educational attainment reported by mothers and fathers, with 46% and 36%, respectively, reporting having completed a tertiary degree. Twenty-seven percent of mothers and 34% of fathers reported having completed Year 11 or 12, with 22% and 20% having completed a diploma or certificate level education, and 6% and 10% having completed Year 10 or below.Combined household income was slightly skewed to higher income households with 29% of respondents with a household income greater than (AUD) $95,000 and 22% between $65,000 and $95,000. One quarter of those surveyed reported a household income of $45,000 to $65,000, 11% reported an income of $25,000 to $45,000 and 13% of households earned less than $25,000. Almost all children from the families surveyed (98%) were born in hospital and 97% of families indicated that their child had either completed, or intended for their child to complete, the primary course of pertussis-containing vaccines as per the National Immunisation Program schedule. A comparison between mothers in this sample and the most recently available births data for Victoria (2008) showed that the sample roughly approximated the population for maternal age, marital status and place of birth, however there was a statistically significant difference between the proportions of mothers born in Australia between the survey sample and the Victorian population  pertussis vaccine was available for new parents. Mothers commonly heard about the vaccine from the Maternal and Child Health Nurse (43%) or their GP (13%). Friends, family, professional colleagues, immunisation staff, childcare, or a poster at a health centre were cited as other sources of information about the vaccine (13%). Fathers commonly heard about the program from their partner (35%), a Maternal and Child Health nurse (23%) or their GP (13%). Several factors were examined as predictors for awareness of the vaccine program, including parent\u2019s age, country of birth, relationship status, household income, child\u2019s place of birth , and whether their child was vaccinated for pertussis, however no statistically significant predictors were found.Seventy percent of mothers and 53% of fathers had received the pertussis vaccine following the birth of their most recent child. This equated to both parents being vaccinated in 56% of families (including single parents if they were vaccinated), one parent in 17% of families and none in 27% of families. Maternity hospitals were the most common place for mothers to receive vaccine (37%) followed by the local council (35%) and general practice (28%). Fathers were more likely to have the vaccine administered in general practice 46%) followed by maternity hospital (30%) and local council (23%). Not being aware of the availability of the free vaccine was the main reason reported for not being vaccinated (Table\u00a06% followThere was a considerable difference in vaccination of mothers between metropolitan (64%) and rural residence (80%), however this difference was not statistically significant (p\u2009=\u20090.08). In contrast, the difference in vaccination levels of fathers between metropolitan (40%) and rural (73%) residence was statistically significant (p\u2009=\u20090.002). Fathers residing in metropolitan LGAs were less likely to be vaccinated than fathers in rural LGAs . After adjustment for vaccine awareness, age and whether born in Australia or overseas, metropolitan fathers had 5.5 fold lower odds compared with rural fathers of being vaccinated . Most parents residing in rural LGAs were vaccinated at their maternity hospital . In the metropolitan LGAs, most mothers were vaccinated at local councils (62%) and the majority of fathers were vaccinated either at their general practitioner (48%) or at local councils (43%) (p\u2009<\u20090.001). The overall proportion of mothers vaccinated was 68%, and for fathers it was 49% when adjusting for population sizes of the contributing Victorian LGAs.Nearly all surveyed mothers and fathers (96%) indicated they would be prepared to be vaccinated to prevent transmitting an infectious disease to their child, with a general practice clinic the preferred location to receive the vaccine indicated by most mothers 65%) and fathers (70%). Parents were asked about their preferred place for vaccination: local council was preferred by 19% of mothers and 10% of fathers and maternity hospital by 15% of mothers and 19% of fathers. Parents indicated that knowledge about the disease, the level of risk to themselves or their child from the disease, how well the vaccine works, and potential side effects were important when making decisions regarding adult vaccination. The majority of parents gave low or no importance to the cost of the vaccine, the cost of going to the doctor to be vaccinated, the time and effort to be vaccinated and religious beliefs . It needs to be acknowledged that this finding may be indicative of the presence of selection bias in this study, with vaccinated parents possibly overrepresented in our sample. Of the parents who were not vaccinated, the most common reason given was a lack of awareness that a free vaccine was available. This suggests that additional funding to promote or advertise the vaccine to new parents may have resulted in greater uptake under the program implemented in 2009. In conducting these types of programs, funding allocated towards the development of a communications campaign aimed at promoting the program to parents, and considering incentives for hospitals, local councils, and general practitioners to inform and vaccinate their patients, may also assist with the uptake of vaccine.In general, there was a high level of knowledge and awareness of pertussis as a childhood disease among parents in this study; 93% had heard of whooping cough or pertussis, 96% agreed or strongly agreed that pertussis could cause serious illness in infants, and 82% thought that infants were at risk of contracting pertussis. Although household contacts have been shown to be the most important source of infection for infants both in Australia  and worlThe contrast between the numbers of parents residing in metropolitan areas who were vaccinated in hospitals compared with rural parents was striking. Only 6% of metropolitan mothers and 10% of metropolitan fathers were vaccinated in hospital, compared with 70% of rural mothers and 42% of rural fathers. The higher vaccine provision through rural hospitals could potentially be explained by differences in hospital policies between metropolitan and their  rural counterparts, particularly regarding the vaccination of fathers who would not be considered patients of the hospital. Maternity hospitals have been shown to be effective and timely providers of the pertussis vaccine to new parents, particularly amongst families of high risk infants such as neonates, with standing orders having been shown to successfully raise the vaccination rate to more than 80% of eligible women in the United States . FurtherWhilst only 21% of mothers and 23% of fathers agreed that the time and effort to be vaccinated was important in their consideration of vaccination, the results indicated that this, in fact, was a common barrier for parents, particularly for fathers. Interestingly, some parents who indicated that they were not vaccinated due to time pressures responded that, in general, it was of little or no importance in their decision-making process. This suggests that, despite good intentions, the reality of life once a child is born can mean that time and effort are indeed important barriers to vaccination. This disconnection between intentions to be vaccinated versus actually obtaining a vaccination suggests that positive attitudes towards vaccination do not necessarily predict behaviour. Lack of time was found to be a major reason for not being vaccinated in a previous study where, despite an education program that demonstrated a significant increase in knowledge and willingness to be vaccinated for pertussis, only 8% of participants were vaccinated .The major limitation in this study was the response rate of 43% which reduced the power of the study, and may have introduced some bias. A post-hoc power calculation estimated study power at 41.2%. One likely reason for the low response rate was due to the fact that parents of young children have multiple competing demands on their time and thus have limited time to respond to surveys. It is unclear as to whether those that responded to the survey were more likely to be vaccinated or had greater knowledge of pertussis but this possibility has to be considered. Unfortunately due to time and resource constraints, only one reminder letter was sent to parents requesting their participation in this study. Additional contact from the researchers may have increased participation, which may have minimised responder bias. Despite this, the comparison with the most recent report available on births in Victoria in 2008 [A further limitation in this study was that participants were not asked when they were vaccinated relative to the birth of their child. As the questionnaire was administered approximately six months after the birth of their child, parents may not have been vaccinated in the initial two month period at which infants are most vulnerable prior to receiving their first dose of pertussis-containing vaccine at two months of age. Anecdotal information provided by parents in the comments section of the questionnaire indicated that some parents were vaccinated at the time of their child\u2019s two month vaccinations. It is therefore possible that, among parents who were vaccinated at local councils or general practitioners, vaccination was received some time after their child\u2019s birth: leaving their new baby vulnerable to infection, and thereby defeating the purpose of the program. Furthermore, the infant\u2019s age at the time parents were surveyed may have impacted upon their responses to questions relating to their attitudes towards vaccination. It is plausible that a younger infant may be perceived by parents as more vulnerable to infection and hence a parent may be more supportive of vaccination. However, it is also plausible that the converse is true \u2013 an older infant might be considered more \u2018robust\u2019 to cope with a vaccination. The impact that the infant\u2019s age had on parental attitudes towards vaccination was not able to be assessed.It is also important to note that minor methodological differences between participating LGAs may have introduced some biases to the findings reported. Specifically, the study period was extended by one month in two rural LGAs in order to recruit sufficient study participants, although there was no change in the way the program was advertised or delivered during this time. Two potential participants in one rural shire were excluded due to incomplete names being provided by the LGA. One metropolitan LGA chose to post the questionnaires to participants directly, but unlike other LGAs, the introductory letters were not personalised and may account for a response rate of 30% for that LGA, which was the lowest response rate of participating metropolitan LGAs.It is important to consider that the Victorian Government Department of Health initiated the free parental pertussis booster program in mid-2009 in response to the rising incidence of pertussis. The program ceased in Victoria on 30 June 2012 due to declining numbers of notified pertussis cases as well as limited evidence of the effectiveness of cocooning and doubts as to the cost effectiveness of the program. Similar cocooning programs introduced by other Australian jurisdictions, including South Australia, the Australian Capital Territory, Western Australia, and Queensland also ceased around the same time as Victoria, citing lack of evidence of effectiveness. A narrowed program targeting mothers in maternity hospitals continues in New South Wales . ResultsAlthough many countries around the world promote the cocooning strategy, the World Health Organization statement on pertussis vaccines states that \u201cthere is insufficient evidence to include this strategy in national immunisation programs\u201d . SeveralThis study found 70% of mothers and 53% of fathers were vaccinated for pertussis following their child\u2019s birth . Whilst there is continued uncertainty regarding the effectiveness of cocooning as a strategy to reduce transmission of pertussis and subsequent morbidity and mortality to vulnerable infants, several factors were identified in this study that may encourage vaccine uptake should similar programs be implemented in future. These include stronger, specifically targeted communication messages, particularly relating to the susceptibility of adults to pertussis and the potential of pertussis to cause severe disease in infants. Further promotion of the widespread options and availability of vaccine providers may increase uptake for those who indicated time as a barrier to vaccination.AUD: Australian dollar; CI: Confidence interval; dTpa: Diphtheria, tetanus and acellular pertussis vaccine; GP: General practitioner; LGA: Local government area; MCH: Maternal and child health; OR: Odds ratio; RR: Relative risk.The authors declare that they have no competing interests.ED designed the study protocol, prepared the research and ethics applications, coordinated questionnaire design, administered the survey  and coordinated the writing of the manuscript. JF contributed to questionnaire design, interpretation of data and significant drafting of the manuscript. SR assisted in research concept and study protocol development, garnered support for the research and contributed to the manuscript. LF identified important background material for the study protocol and manuscript, provided coordination with Maternal and Child Health Programs to identify participants and assisted in the drafting of the manuscript. HV contributed to the study design, questionnaire design, data analysis and drafting of the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/676/prepub"}
+{"text": "Maternal mortality and morbidity are among the top public health priorities in Brazil, being quite high, especially among the most disadvantage women. A case control study was developed to identify risk factors for severe maternal morbidity in Sao Luis, one of the poorest Brazilian State Capitals.The case\u2013control study was carried out between 01/03/2009 and 28/02/2010 in two public high-risk maternities facilities and in two intensive care units (ICUs) for referral of obstetric cases. All cases hospitalized due to complications during gestation period, childbirth or up to 42 days of puerperium and who fulfilled any of Mantel's and/or Waterstone's criteria were identified. Two controls per case were randomly selected among patients of the same clinics discharged for other reasons. Data were obtained through a structured interview as well as from medical charts and prenatal cards and included sociodemographic variables, clinical and obstetric histories, behavioral factors and exposure to stress factors during pregnancy, pre-natal assistance and obstetric complication and childbirth care.In the final model of the unconditional logistic regression analysis, being older than 35 years , previous hypertension , history of abortion , 4\u20135 pre-natal consultations  and 1\u20133 pre-natal consultations  were independently associated with severe maternal morbidity.The results corroborate the importance of reproductive healthcare, of identifying a high-risk pregnancy and of a qualified and complete prenatal care to prevent severe morbid events.A mortalidade e morbidade maternas est\u00e3o entre os t\u00f3picos priorit\u00e1rios da Sa\u00fade P\u00fablica brasileira, especialmente na popula\u00e7\u00e3o de menor n\u00edvel socioecon\u00f4mico. Um estudo caso-controle foi desenvolvido para identificar os fatores de risco para morbidade materna grave em S\u00e3o Lu\u00eds, capital de um dos estados mais pobres do Brasil.priori.Estudo caso-controle realizado em duas maternidades p\u00fablicas de alto risco e duas UTIs de refer\u00eancia aos casos obst\u00e9tricos entre 01/03/2009 e 28/02/2010. Foram inclu\u00eddas todas as pacientes internadas por complica\u00e7\u00e3o do per\u00edodo gr\u00e1vido-puerperal e que preenchiam os crit\u00e9rios de Waterstone e/ou Mantel para morbidade materna grave. Foram selecionados para cada caso, dois controles por sorteio aleat\u00f3rio dentre as pacientes internadas no mesmo per\u00edodo e mesma maternidade que o caso. As informa\u00e7\u00f5es de dom\u00ednio sociodemogr\u00e1fico, cl\u00ednico, obst\u00e9trico, comportamental, exposi\u00e7\u00e3o a eventos estressores na gesta\u00e7\u00e3o, assist\u00eancia ao pr\u00e9-natal, intercorr\u00eancias obst\u00e9tricas e aten\u00e7\u00e3o ao parto, foram obtidas por meio de entrevista estruturada. As vari\u00e1veis foram analisadas por modelo de regress\u00e3o log\u00edstica m\u00faltipla n\u00e3o condicional, baseado em modelo hierarquizado a Foram identificados como fatores de risco para morbidade materna grave: idade >35 anos , hipertens\u00e3o pr\u00e9via \u00e0 gesta\u00e7\u00e3o , antecedente de aborto , ter realizado 4\u20135 consultas pr\u00e9-natais  ou 1\u20133 consultas .Os resultados do estudo corroboram a import\u00e2ncia da assist\u00eancia \u00e0 sa\u00fade reprodutiva e o pr\u00e9-natal completo e qualificado na preven\u00e7\u00e3o de eventos m\u00f3rbidos graves durante o ciclo gr\u00e1vido-puerperal. The reduction of maternal mortality is one of the United Nations\u2019 Millennium Development Goals . Maternanear miss refers to any severe complication during gestation, childbirth or puerperium [near miss situation have a profile probably similar to those who progress towards death, representing, thus, a proxy model for maternal mortality [Severe maternal morbidity or erperium -6. Pregnortality ,8. To clortality .In Brazil, maternal mortality is still elevated  , maternaA case\u2013control study was carried out to identify risk factors for severe maternal morbidity in public maternities in Sao Luis, Capital of Maranh\u00e3o, with a population estimated at one million people, 85% of whom depend exclusively on the health care provided by the public national system .Data were collected between March 1, 2009, and February 28, 2010. In order to seize the greatest number of severe maternity morbidity cases, we assessed all inpatients at the two public maternity clinics for high risk patients and the two general ICUs which are references of obstetric cases. There are no obstetric ICUs in the city. The two selected maternities are responsible for approximately 8,000 births per year, corresponding to half of the total number of deliveries performed in all seven existing public clinics.All patients admitted with complications during gestation period, childbirth or up to 42 days of puerperium and who fulfilled at least one of the requirements proposed by Mantel\u2019s  and/or WUsing simple random sampling, two controls were selected for each case. All the patients who did not fulfil the criteria for severe maternal morbidity and who were hospitalized in the same maternity clinic where the cases occurred or had been discharged during the same period were eligible as controls. Patients who did not reside in the city were excluded.The sample size calculated as 115 cases and 230 controls allowed estimating an Odds Ratio equal or greater than 2.5, with 90% power and 95% confidence interval, considering a 16% prevalence of exposure  among controls. The expected prevalence of inadequate prenatal care \u2013 the main variable of interest in this study \u2013 was estimated using the Ministry of Health data system .The risk factors were classified as 1) sociodemographic, 2) clinical and obstetric history, 3) behavioural and stress events during the current pregnancy, 4) prenatal care, 5) current obstetric complications and 6) childbirth assistance. The data was obtained through a structured interview applied after obtaining a signed free and informed consent and before hospital discharge. The sociodemographic and health assistance data was also obtained from medical charts and prenatal cards.a priori .In the multivariable analysis, all variables in each group statistically associated with the outcome at the level of p<0.20 were considered following the hierarchical structure indicated above. From the second group of variables on, after adjusting for the previous groups, the variables associated at the level of p<0.10 were kept in the model. After the inclusion of the last group of variables, only those factors which remained statistically significant  were kept. The magnitude of the association between the exposure variables and the outcome was estimated by the The study was approved by the Research Ethics Committee of the Federal University of Minas Gerais (ETIC no: 589/08).ResultsWe identified 127 cases of severe maternal morbidity during the study period. Five cases (3.9%) of severe maternal morbidity were lost, three of whom refused to participate. Therefore, 122 women diagnosed with severe maternal morbidity participated in the study: 66.4% severe pre-eclampsia; 11.5% eclampsia; 11.4% obstetric haemorrhage; 5.7% HELLP syndrome; 2.5% infected abortion, 1.6% pre-eclampsia superimposed upon chronic hypertension; and 1.6% non-obstetric complications. Three of the 244 selected controls were substituted by random sampling; two of them declined to participate and one was excluded for presenting a clinical picture similar to seizure.In the study period, the maternal mortality in the city was 84.5/100,000 live births . Table\u20092With regard to the obstetric variables, the proximal factors, the great majority (94%) of the participating women reported prenatal care. Cases had a higher risk of having had less than six consultations during the gestation period, but the statistical significance was borderline at the level of p<0.10. Previous hypertension was five times more frequent among cases than controls. Cases and controls did not differ regarding to history of gestational haemorrhage at any period or excessive weight gain. The chance of being referred to a high risk care was four times greater among cases than controls Table\u2009.Table\u2009In general, cases and controls were young, from very low income families, married and had at least 8 years of schooling. Among the great number of variables investigated, we identified four risk factors for severe maternal morbidity: age equal or over 35 years, previous history of hypertension, history of abortion and having had less than six prenatal consultations, minimum number of prenatal visits recommended by the Brazilian Ministry of Health.Higher risk of severe maternal morbidity for older women had already been identified in prior studies ,15 Age iLower monthly income per-capita was not associated with being a case in the final model, after considering the effect of prenatal and hospital assistance. Waterstone and cols (2001) , on the A previous history of hypertension has also been reported as a risk factor for severe maternal morbidity ,20. In BAbout one third of cases and one fourth of controls reported having had a previous abortion. These prevalences were much higher than the rate reported for the whole country (16.2%)  .A reseaThe risk of severe maternal morbidity is almost two times higher among women who had less than six prenatal consultations, as recommended by the Ministry of Health. Other studies in Brazil and Argentina, involving severe maternal morbidity cases also found a high prevalence of insufficient number of prenatal consultations ,26,27. AThe most important message of this study, for both clinicians and policymakers, is that an adequate prenatal assistance may contribute to prevent severe maternal morbidity among poor woman, even in the presence of other poor social conditions.near miss experience answered differently from those who did not. Nevertheless, as the cases were interviewed in the same period of the puerperal cycle and close to being discharged, when they were well recovered, we believe that such influence was minimized. The sample used did not have the statistical power to identify as significant risk factors that were infrequent among the women included in the study.However, the present study has some limitations that must be considered. We have not collected data on intra partum care problems because we think that such problems are too closed to the case definition used in this study. Regarding the data gathered, there may be some divergence between the information obtained from medical charts and those present in prenatal cards, considering that the quality and completeness of these sources can vary. But, if such problem occurred it is unlikely to have been differential, thus biasing the odds ratios found towards the null. As to the information collected in the interviews, we cannot rule out that the women who had a The results of this study reinforce the importance of providing prenatal care in adequate number and quality in order to identify high risk women and prevent severe morbid events.The authors declare that they have no competing interests.APPM participated in the design of the study, collection of data, analysis, interpretation of data and wrote the first manuscript draft. SMB participated in the design of the study, statistical analysis, interpretation of data, coordination, helped to draft the manuscript and reviewed of manuscript. VMAP participated in the design of the study, statistical analysis, interpretation of data, coordination, helped to draft the manuscript and reviewed of manuscript. PSG, JEC and MXV participated in data collection and reviewing of the manuscript. All authors read and approved the final manuscript.APPM. MD, Doctor of Public Health, Universidade Federal do Maranh\u00e3o (UFMA), Sao luis, Maranhao, BrazilSMB. MD, PhD. Full professor, Faculty of Medicine, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil.VMAP. MD, PhD. Associate professor, Faculty of Medicine, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil.PSG. MD, MSc. Lecturer of Obstetrics, Ceuma Universidade, S\u00e3o Luis, Maranh\u00e3o, Brazil.JEC. MD. Gynecologist, University Hospital, Universidade Federal do Maranh\u00e3o (UFMA), Sao luis, Maranhao, BrazilMXV. MD. Gynecologist, University Hospital, Universidade Federal do Maranh\u00e3o (UFMA), Sao luis, Maranh\u00e3o, BrazilSMB and VMAP are research fellows of the National Research Council ."}
+{"text": "CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs. CARs in vitro , but hade clinic \u20139. Theree clinic \u201313, incre clinic , promotee clinic  and impro models . CARs cosistence \u201319. 2G Csistence . Howeversistence \u201326. The esponses . CARs coing CD28 . The pering CD28 , 30. DesIn the present study, we compare 2G CAR T cells containing CD28 to a 3G CAR containing both CD28 and 4-1BB to generate a rationale for the use of the latter in clinical trials. We investigated the functional capacity of 3G compared to 2G CARs and have initiated a mapping of the intracellular signaling capacity post antigen stimulation in both 2G and 3G CARs.PBMCs were isolated from blood of patients with CLL (n = 4) or healthy donors (n = 2) using Ficoll paque gradient centrifugation . Written consent was obtained from all patients in concordance with the Helsinki Declaration and the study was approved by the Uppsala Regional Ethical Review Board, Uppsala, Sweden (DNr: 2006:145). Peripheral blood from healthy donors was obtained from the blood bank at Uppsala University Hospital. Deidentified cord blood (CB) units were obtained through the MD Anderson Cord Blood Bank  on a Baylor College of Medicine (BCM) IRB-approved protocol.CD19+ Daudi   and the following day (day 3) transduced on RetroNectin-coated  plates pre-incubated with retroviral supernatant and kept in 100IU IL-2/ml.CAR T cells were generated from peripheral mononuclear blood cells (PBMCs) and cultured in RPMI medium supplemented with 10% fetal bovine serum and 1% Penicillin-Streptavidin, all purchased from Life Technologies. B cells were isolated using \u03b1CD20 magnetic beads  and T cells were subsequently purified from the CD20 negative fraction using pan T magnetic beads , both from Miltenyi Biotec GmbH . T cells were activated with 1\u03bcg OKT-3/mL  at day 0. On day 2, T cells were stimulated with 100IU IL-2/mL  once a week  and 3 days after the last B cell stimulation (endpoint of co-culture) and used for Western blot and cell signaling multiplex assay. Cell culture supernatants were collected 24h after B cell stimulation (once/week), and analyzed for IFN\u03b3  and Granzyme B  using ELISA.B cells were isolated from PBMCs through \u03b1CD20 magnetic bead isolation. CD20 negative cells were subsequently purified using pan T magnetic beads to generate CAR T cells (Miltenyi Biotec GmbH). Due to the slow proliferative capacity of T cells generated from leukemic patients, the expansion was ongoing for 14 days. On day 14, 1x10e a week  similarl2 Fragment Goat Anti-Human IgG (H+L) purchased from Jackson ImmunoResearch Europe LTD (cat no 109-496-088), or negative isotype control antibodies (Biolegend). Cells were washed with PBS. Analyzed on BD Canto II  and evaluated with FlowJo . The transduction efficiency of 3G CAR was lower than that of 2G CAR. Therefore, a correction factor was calculated based on the CAR expression of the 2G and 3G T cells from the same donor at the same time point. This correction factor was then used to normalize the values of the functional assays.Cells were incubated with 1% BSA in PBS for 10 min, centrifuged and stained for 15 min with the following antibodies: CD3-FITC (cat no 300406), CD4-APC/Cy7 (cat no 300518), CD8-PE/Cy7 (cat no 301012), CD19-PerCP (cat no 302228), CCR7-BV421 (cat no 353208), CD45RA-APC/Cy7 (cat no 304128), CD27-PE (cat no 302808), CD28-PE/Cy7 (cat no 302926), Tim-3-BV421 (cat no 345007), PD-1-PE (cat no 329906) all purchased from Biolegend, \u03b1-CAR-DyLight649 AffiniPure F(ab')B cells were isolated from PBMCs through \u03b1CD20 magnetic bead (Miltenyi Biotec GmbH) isolation and the CD20 negative fraction was subsequently purified using pan T magnetic beads all according to manufacturer\u2019s protocol. Pan T purified T cells were used to generate Mock, 2G and 3G CAR T cells. The T cells were stimulated with \u03b1CD20-labelled B cells (4:1 ratio) for 1 and 3h. At these time-points the co-culture was run through a LS column (Miltenyi Biotec GmbH) and the T cell fractions (99.9% CD3+) were washed with cold PBS and resuspended in lysis buffer containing M-PER mammalian protein extraction reagent (cat no 78503) supplemented with 1% Halt phosphatase inhibitor cocktail (cat no 78415) and 1% protease inhibitor cocktail (cat no 1861277) all from Thermo Scientific. Protein lysates were assayed for kinase tyrosine phosphorylation analyzed on PamStation12 .The MILLIPLEX\u00ae MAP T-Cell Receptor Signaling Magnetic Bead Kit 7-Plex from Millipore Merck (cat no 48-690MAG) was used to detect changes in phosphorylated CREB (Ser133), Erk/MAP kinase 1/2 (Thr185/Tyr187), and phosphorylated tyrosine residues on CD3 epsilon chain, Lck, ZAP-70, LAT, and Syk in T cell lysates from day 14, hence before and 3 days after the last B cell stimulation (endpoint of the co-culture). The assay was set up according to manufacturer\u2019s instructions loading 15\u03bcg protein/well and analyzed on the Luminex\u00ae system.51Cr, PerkinElmer, cat no NEZ030S005MC) for 2 hours and subsequently washed prior to co-culture with CAR T cells at different ratios for 4 hours. Co-culture supernatants were mixed with Optiphase supermix  and analyzed on the beta counter Wallac Trilux, 1450 microbeta . Cytotoxicity (% lysis) was calculated as follows: (sample-spontaneous release)/(maximum release\u2013spontaneous release) x 100.Target cells (Daudi and K562) were labeled with radioactive chromium  and treated upon measurable tumor (2 weeks post tumor cell injection) with 5x106 2G, 3G or mock transduced human T cells via i.p. injection (6 mice per group). Survival and tumor size was monitored throughout the experiment using a caliper. Mice were euthanized if tumor \u22651cm2. Animals were housed at the Rudbeck Animal Facility and cared for by the staff according to local regulations. The in vivo experiments were approved by Uppsala Animal Ethics Committee (ref no: C319/9).C57BL/6J nu/nu mice  were injected subcutaneously with 5x105 CD34+ cells selected from umbilical cord blood (UCB) units . Engraftment was confirmed within 6 weeks by measuring human CD45+ and CD19+ cells in the peripheral blood by flow cytometry. Engrafted mice received via tail injection T cells derived from the corresponding UCB negative fraction, transduced with 2G or 3G vector construct and further labeled with FFluc (3x106/mouse). In vivo expansion of T cells was monitored over time by IVIS imaging , while the elimination of CD19+ cell was measured by flow cytometry of peripheral blood samples. By day 60 or 100 mice were euthanized and spleen, bone marrow and blood collected for flow cytometry analysis (human CD45+ and CD3+). CAR expression by T cells was detected using the Cy-5-conjugated goat anti human IgG (H+L) antibody (Jackson ImmunoResearch) that recognizes the human IgG1-CH2-CH3 component incorporated as a spacer region within the CARs. Samples were analyzed in a FACSCalibur (BD Pharmingen) and were analyzed by CellQuest pro software (BD Biosciences). At least 10000 positive events were acquired in each sample. The NSG mice were maintained at Baylor College of Medicine Animal Facility on a BCM IACUC approved protocol.NSG mice 6 weeks of age (Jackson laboratories) were sublethally irradiated (280Gy) and engrafted with 2x10Statistical evaluations were performed using GraphPad Prism . Differences between groups generating p-values <0.05 were considered significantly different.Activated T cells from healthy donors and CLL patients were efficiently transduced with Mock, 2G or 3G virus (MLV vector), resulting in a median 2G CAR expression of 33% ranging from 5 to 90% and a 3G median CAR expression of 27.1% ranging from 3 to 87% at day 14,prior to stimulation. There was a difference in CAR expression when comparing 2G and 3G CAR and also between donors. Therefore, to enable comparison of 3G CAR T cells to 2G, CAR expression was normalized for all functional assays. T cells were expanded with autologous B cells, IL-2 or a combination of the two. After one month\u2019s expansion, CAR T cells stimulated with both B cells and IL-2 had a significantly larger fraction of CAR positive cells  as well 3G CAR T cells were harvested and phenotyped using multicolor flow cytometry before and after one month\u2019s stimulation with IL-2 +/- B cells. As controls, 2G CAR T cells and Mock transduced T cells were used. The majority of cells were of CD8 lineage, yet there were CD4+ T cells present in all cultures  . CAR posAn extended T cell phenotype was determined in the different groups before and after expansion. The phenotypes were defined as follows; na\u00efve: CD45RA+ CCR7+, effector: CD45RA+ CCR7-, central memory: CD45RA- CCR7+ and effector memory: CD45RA- CCR7-. CAR or Mock transduced T cells had a similar phenotype prior to co-culture independently of CAR expression. The majority of T cells had a memory-like phenotype of either central or effector type . After eTo evaluate the activation status of intracellular signaling pathways downstream CAR and signaling pathways activated secondary to CAR stimulation, CAR T cells were stimulated with autologous B cells and analyzed by the PamGene tyrosine kinase array. The Tyrosine Kinase PamChip\u00ae Array by PamGene consists of 144 peptides with known phosphorylation sites derived from 100 different proteins. Intact cells or cell lysates can be stimulated and analyzed for the presence of active kinases that will phosphorylate the Array peptides. Hence, an increased phosphorylation of a certain peptide do not mean that the cells contained that certain peptide, but has activated the kinases that can act on those peptides. CAR T cells showed an overall increased activation status compared to Mock transduced cells and 3G T cells were generally more activated than 2G T cells. The activation status in 3G CARs was also maintained over time compared to 2G CARs. Nevertheless, the pattern of what kinases that were activated was similar between 2G and 3G. As expected, signaling molecules downstream of the TCR were phosphorylated also by stimulating CAR receptors such as CD3 zeta, LAT, LCK and ZAP-70. Interestingly, also other proteins involved in regulation of the cell cycle (CDK2), cell adhesion  and exocytosis (Annexins) were phosphorylated. Stimulating cell adhesion may be of importance especially for treatment of solid tumors, and increased exocytosis may result in enhanced serial killing capacity. The most interesting phosphorylation targets are listed in Since the PamGene array only demonstrate the capacity of cells to phosphorylate a library of peptides, we confirmed some of the data using the MILLIPLEX\u00ae MAP T-Cell Receptor Signaling Magnetic Bead Kit 7-Plex and Western blots. LAT, ZAP-70, SYK and Erk all showed a similar pattern of higher levels of phosphorylated proteins in 3G CAR T cells compared to 2G T cells . Hence, Cytotoxic capacity was evaluated with a chromium release assay before and after one month\u2019s expansion. 3G CARs were equally efficient as 2G CAR T cells in specific killing of CD19+ target cells . The celProliferation was analyzed weekly during expansion, prior to the addition of autologous B cells and IL-2. After one week\u2019s stimulation, 3G CAR T cells proliferated better than Mock transduced cells (p = 0.0302) . The proCell supernatants were collected 24 hours post stimulation and analyzed for IFN\u03b3 and Granzyme B using ELISA. IFN\u03b3 was mainly seen in supernatants from the T cell expansion cultures where the CAR T cells had been stimulated with antigen, while Mock and untransduced cells showed low or undetectable levels . Granzym+ cells  were injected subcutaneously with Daudi cells and mice with established tumors were treated with one 3G or 2G CAR T cell infusion. Both 3G and 2G CAR T cells efficiently reduced tumor growth . Additio+ cells  were use T cells . In addiefficacy . Mice euefficacy  or day 1efficacy  also shoEx vivo engineering of the patients\u2019 own T cells with CAR receptors to generate an army of trained killers is now under evaluation for both leukemia, lymphoma as well as for other malignancies [The recent success stories of immunotherapy have demonstrated the potency of the immune system to specifically target and kill tumor cells. The induction of Th1 immunity with T cells, M1 macrophages and NK cells as effectors has proven important for potent and sustained responses. gnancies . There agnancies . 4-1BB ignancies . 2G and gnancies . Howevergnancies , 39, 40 gnancies .In this study we aimed to evaluate the functional capacity of CD19-targeting 3G CAR T cells with CD28, 4-1BB and CD3 zeta signaling domains and compare their capacity to 2G CARs lacking 4-1BB as well as to Mock transduced T cells. CAR T cells could be generated from both healthy donors and CLL patients but the CAR expression of 2G CARs was always slightly higher than 3G CAR expression both related to number of positive cells and number of CAR molecules per cell as judged by a higher mean fluorescent intensity upon analysis of CAR expression. Hence, for some of the functional assays we correlated the data to the number of CAR+ cells to be able to more appropriately compare the ability of the 2G and 3G cells. Further, the T cells were analyzed before and after repeated stimulation with autologous B cells to determine the persistence of CAR T cells.in vivo response. For example, in a primate model genetically modified T cells derived of central memory phenotype was shown to persist longer than effector cells derived from effector memory cells [in vivo expansion of CD19-targeting CAR T cells in lymphoma patients [Co-culture of CAR T cells with autologous B cells stimulated an accumulation of both 3G and 2G CAR positive cells in the cultures, which was not seen in cultures with IL-2 alone. Further, antigen-driven proliferation generated a memory-like phenotype of the cultured CAR T cells with low expression of the exhaustion markers PD-1 and Tim-3. There is emerging evidence that such a phenotype will generate a better ry cells . Furtherry cells . Furtherry cells . Xu et apatients . In our patients  and can ex vivo expansion phase of manufacturing, suggesting in the light of recent studies that proliferation capacity might be more important than cytotoxic capacity [in vivo since the complex network of stimulatory and inhibitory pathways are difficult to mimic in preclinical settings. Tammana et al showed that CARs with costimulatory molecule 4-1BB alone or combined with CD28 provided a more robust antitumor response in vivo compared to CARs with CD28 alone or without costimulatory molecules [The proliferative capacity of 3G CAR T cells upon IL-2 stimulation was higher than that of 2G CAR T cells. 2G CARs were not significantly different from Mock or untransduced cells. Upon repeated stimulation with autologous B cells, both 2G and 3G CAR T cells showed increased proliferation capacity compared to Mock and untransduced cells. With the exception of one donor, 3G CARs had still a higher proliferative capacity than 2G CARs. Barrett et al emphasizes the importance of maintaining proliferation capacity during the capacity . Yet, diolecules . Zhong eolecules . 2G and olecules , yet CARThe intracellular signaling events downstream different CARs affect the efficiency and longevity of the T cells. Considering the use of the CD3 zeta domain in the CAR, TCR-associated proteins will likely participate in CAR signaling but it is not known to what extent other signaling pathways are affected in different types of CAR T cells. In this study, the PamGene station was utilized to investigate phosphorylation status of multiple targets. Upon antigen stimulation both 3G and 2G T cells could rapidly activate kinases that have the capacity to phosphorylate a number of proteins. 3G T cells showed a higher activation status than 2G but both were increased compared to Mock T cells. After 3 hours the activation status declined but more slowly in 3G T cells. As expected, all the common proteins downstream the TCR signaling pathway were activated such as CD3 zeta, LAT, LCK and ZAP-70. These data were confirmed also by analyzing the T cells using the MILLIPLEX\u00ae MAP T-Cell Receptor Signaling kit. 3G CAR T cells had a significantly higher phosphorylation status on antigen-mediated signaling molecules compared to 2G CAR T cells. The PamGene array also revealed other possibly phosphorylated molecules that will need further confirmation. For example, Calmodulin phosphorylation was increased post antigen stimulation. Calmodulin is a calcium-binding messenger protein important for many Ca2+ regulated pathways in lymphocytes and other cells. For example, upon Ca2+ release from the endoplasmatic reticulum (ER), calcium-bound Calmodulin activates the phosphatase calcineurin, which in turn promotes NFAT-mediated IL-2 transcription . FurtherCDK2 is a protein involved in the progression of the cell cycle as CDK binding to cyclin E during G1 phase promotes the transition from G1 to S phase. Antigen stimulation together with costimulation activates a CDK cascade including CDK2. However, in the absence of costimulation the T cells are entering a state of anergy, which is enforced by the CDK inhibitory protein p27kip1. The major target of p27kip1 is CDK2. In a study of allograft rejection it was shown that CDK2 was highly active in T cells that infiltrated the allograft . InteresThere were also enhanced phosphorylation of molecules that can potentially be involved in self-regulation of the antigen-induced activation such as Crk, Protein 4.1R, FAK and DYRK1A. Crk adaptor molecules interact with a number of signaling molecules in the cell. Their function is not fully understood but there is one report stating that T cells lacking both Crk and CrkL have a defective chemotactic response to multiple chemokines and may show defective diapedesis. In a cutaneous DTH model, Crk and CrkL were required for efficient T cell migration to sites of inflammation. Crk proteins also controlled T cell activation . NeverthDespite of CAR T cell expansion, there was a remaining CAR+ na\u00efve T cell population in the cultures. Ras is activated by TCR stimulation and has a role in activating the MAP kinases leading towards cell growth. Ras is inactivated by Ras GTPase-activating proteins (RasGAPs) such as RASA1. In a paper by Lapinski PE et al it was shown that RASA1 is an important regulator of thymocyte survival during positive selection as well as promoting the survival of na\u00efve T cells in the periphery . In RAFAOther proteins that can be phosphorylated in CAR T cells post activation are Paxillin, PECAM-1 and EGFR. Paxillin is a cytoskeletal adaptor protein that localizes to the microtubule-organizing center in T cells and is part of the immunological synapse of cytotoxic T cells . Paxilliin vivo. 3G CAR T cells had an increased activation of intracellular signaling pathways concerning both TCR signaling and adhesion molecules compared to 2G CARs. However, the tyrosine kinase array indicates potentially interesting pathways that can be activated or blocked to generate even more efficient CAR designs.Taken together, these data demonstrates that antigen-driven expansion of CAR T cells generates memory-like T cells with maintained cytotoxic capacity. Further, while the phenotype and cytotoxic capacity post repeated antigen stimulation were similar between 3G and its predecessor (2G). The 2G and 3G CAR T cells also showed efficient killing S1 FigA. Experimental outline of the co-culture experiment. 3G, 2G CAR T, mock or untransduced T cells were cultured with autologous B cells, IL-2 or the combination of the two. Proliferation was assessed every week. CAR T cell phenotype and cytotoxic capacity was analyzed before and after co-culture using flow cytometry and chromium release assay, respectively. B. Overview of cell preparation. Autologous T and B cells were isolated from healthy donors and CLL patients using MACS beads. The purity of the B cell population (\u03b1CD20-isolation) was confirmed with \u03b1CD19 FACS staining (mean expression was 96.4% ranging from 90.5 to 99.5). T cells were isolated from the CD20- fraction using pan T beads, rendering an unlabeled CD3+ population with a mean CD3 expression of 99.3% (range 99\u201399.7).(TIF)Click here for additional data file.S2 FigCAR- T cells were gated as viable singlet CD3+ CAR- cells. Memory phenotype defined as; na\u00efve: CD45RA+, CCR7+, effector: CD45RA+, CCR7-, central memory (CM): CD45RA-, CCR7+, effector memory (EM): CD45RA-, CCR7. CAR negative T cells show similar expression pattern of memory markers as CAR positive cells before co-culture. However, after co-culture with antigen, the increase in memory phenotype and decrease in effector cells was only seen in the CAR positive population.(TIF)Click here for additional data file.S3 FigComparable expression levels of FF-luciferase was seen in 2G and 3G CAR+ T cells as measured by GFP expression (A). NSG Mice were euthanized on day 60 (B) or day 100 (C). CD19+ B cells were completely eliminated and longterm persistence of CAR+ T cells was seen. T and B cells are gated from the CD45+ population. Panels show phenotype from a representative mouse for each construct.(TIF)Click here for additional data file.S1 TableThe table demonstrates mean values from the tyrosine kinase array by PamGene. CAR T cells (2G and 3G) as well as Mock T cells were stimulated with autologous B cells for 1 and 3 hours. The cells were purified using MACS beads and lysed prior to analysis. Data was normalized for CAR expression.(DOCX)Click here for additional data file."}
+{"text": "Host-microbe interactions are influenced by complex host genetics and environment. Studies across animal taxa have aided our understanding of how intestinal microbiota influence vertebrate development, disease, and physiology. However, traditional mammalian studies can be limited by the use of isogenic strains, husbandry constraints that result in small sample sizes and limited statistical power, reliance on indirect characterization of gut microbial communities from fecal samples, and concerns of whether observations in artificial conditions are actually reflective of what occurs in the wild. Fish models are able to overcome many of these limitations. The extensive variation in the physiology, ecology, and natural history of fish enriches studies of the evolution and ecology of host-microbe interactions. They share physiological and immunological features common among vertebrates, including humans, and harbor complex gut microbiota, which allows identification of the mechanisms driving microbial community assembly. Their accelerated life cycles and large clutch sizes and the ease of sampling both internal and external microbial communities make them particularly well suited for robust statistical studies of microbial diversity. Gnotobiotic techniques, genetic manipulation of the microbiota and host, and transparent juveniles enable novel insights into mechanisms underlying development of the digestive tract and disease states. Many diseases involve a complex combination of genes which are difficult to manipulate in homogeneous model organisms. By taking advantage of the natural genetic variation found in wild fish populations, as well as of the availability of powerful genetic tools, future studies should be able to identify conserved genes and pathways that contribute to human genetic diseases characterized by dysbiosis. In vertebrates, the gut microbiome promotes the normal development of host physiology , skeleta12\u2013Inbred mouse models have traditionally been used to study host-microbe interactions. More than 450 strains have been described since the first inbred mice were created nearly 100 years ago. These strains are valuable not only because of their isogenicity, which allows the isolation of a particular genetic variant of interest, but also because phenotypic differences among strains have been described in great detail . The ricHowever, studies using mouse models have been restricted in several ways. Use of inbred lines limits understanding of how complex genetic variation influences microbial community composition . For exaIn addition to genetic constraints, the inability to observe microbe interactions in live mice can prevent in-depth studies of host-microbe interactions. Most mouse studies rely upon indirect characterization of gut microbial communities from fecal samples, which are not consistently reliable indicators of gut microbial communities \u201334 and c\u2013The 28,000 characterized fish species comprise nearly half of all vertebrate diversity and possess extensive variation in physiology, ecology, and natural history  that canTeleosts possess physiological and immunological features common to all vertebrates as well as a complex gut microbiota. Both teleosts and mammals have a digestive tract consisting of a liver, gallbladder, pancreas, and intestine that develop in a similar trajectory, from the rostral gut to the hindgut and midgut. Guts are separated along the rostral-caudal axis and have an intestinal epithelium made up of absorptive enterocytes, secretory goblet cells, and enteroendocrine cells . IntestiProteobacteria  and breast milk , cannot Danio rerio). However, threespine stickleback (Gasterosteus aculeatus), which is a widely used model organism in evolution, genetics, and ecology, has recently also been adapted for host-microbe interaction research. Advantages of these two systems lie in the powerful genetic tools that have been developed and their rich history of study, dating back to the 1800s Danio rerhe 1800s  and permhe 1800s , 50, 56,The transparency of zebrafish eggs and juveniles allowed the first successful examination of the colonization dynamics of bacteria within live, developing hosts . The abiA primary advantage of using threespine stickleback as a model organism is the ability to study how natural genetic variation, which is of a magnitude similar to that found in the human population , influenex utero and eggs can be surface sterilized shortly after fertilization (Xiphophorus maculatus), followed by tilapia , salmon , sheepshead minnow (Cyprinidon vairegatus), Atlantic halibut (Hippoglossus hippoglossus), and turbot   \u201377. Howeaximus) \u2013, 79 and aximus) \u2013. Gnotobiaximus) \u2013, but zebaximus) \u2013, 56. Howaximus) \u2013, this isGnotobiotic studies in zebrafish have revealed that the gut microbiota stimulates intestinal epithelial cell proliferation , 14 throGnotobiotic studies of laboratory-reared oceanic and resident freshwater stickleback have demonstrated that these two ecotypes have common gut microbial communities and similarities in intestinal development, despite their separation in the wild for at least 10,000 years . HoweverWild-caught and laboratory-reared zebrafish populations have similar gut microbial communities, suggesting the existence of a core gut microbiota , which m82\u2013The colonization of thousands of lakes throughout the Northern Hemisphere by oceanic ancestral stickleback resulted in an adaptive radiation of freshwater populations that are locally adapted to their environments. This \u201cnatural experiment\u201d allows researchers to study the influences of environmental factors, such as water chemistry and predation regimes, on the evolution of a vertebrate host , 135. HoSalmo salar) revealed differences between environmental and gut microbial communities that were driven largely by ontogeny rather than geography (Oncorhynchus mykiss) has been shown to be highly variable temporally, spatially, and interindividually (Fundulus grandis), with decreased bacterial counts in winter and increases in spring that were associated with rising temperature   . HoweverProteobacteria, Firmicutes, and Bacteroidetes , will allow further insight into the relative influences of phylogeny and environment in shaping microbial communities. Fishes living in extreme environments, such as Death Valley pupfish  and Antarctic icefish (Notothenioidei), can help us understand how microbes may enable vertebrates to adapt to extreme environments. Finally, live-bearing fishes have advantages in understanding colonization dynamics early in development. Now that so much is known about how microbial communities influence many aspects of a host's life, including its physiology, immune response, and behavior, fish models can help us better understand the effects of microbial community diversity and disruption on host development and adaptation to its environment.Since fishes exhibit dramatic variations in physiology, natural history, and ecology, they can be used as model organisms to address a wide range of factors relevant to host-microbe interactions. For example, studies of fishes that are of economic and cultural significance, such as salmonids, have potential to improve aquaculture  and safe"}
+{"text": "We have developed a modified FlowCAM procedure for efficiently quantifying the size distribution of zooplankton. The modified method offers the following new features: 1) prevents animals from settling and clogging with constant bubbling in the sample container; 2) prevents damage to sample animals and facilitates recycling by replacing the built-in peristaltic pump with an external syringe pump, in order to generate negative pressure, creates a steady flow by drawing air from the receiving conical flask (i.e. vacuum pump), and transfers plankton from the sample container toward the main flowcell of the imaging system and finally into the receiving flask; 3) aligns samples in advance of imaging and prevents clogging with an additional flowcell placed ahead of the main flowcell. These modifications were designed to overcome the difficulties applying the standard FlowCAM procedure to studies where the number of individuals per sample is small, and since the FlowCAM can only image a subset of a sample. Our effective recycling procedure allows users to pass the same sample through the FlowCAM many times (i.e. bootstrapping the sample) in order to generate a good size distribution. Although more advanced FlowCAM models are equipped with syringe pump and Field of View (FOV) flowcells which can image all particles passing through the flow field; we note that these advanced setups are very expensive, offer limited syringe and flowcell sizes, and do not guarantee recycling. In contrast, our modifications are inexpensive and flexible. Finally, we compared the biovolumes estimated by automated FlowCAM image analysis versus conventional manual measurements, and found that the size of an individual zooplankter can be estimated by the FlowCAM image system after ground truthing. Body size has been recognized as the most critical trait determining metabolic rates of organisms; as a consequence, population traits, such as abundance, production, and turnover rate, all scale with size . In addiThere is active interest in developing efficient methods for obtaining size distributions of plankton \u201313, espeAt present, the two most frequently used systems for automated plankton analysis are the ZooScan and FlowCAM. The ZooScan is suitable for imaging particles ranging in size from 200 \u03bcm to several centimetres  and the While recognizing the utility of the FlowCAM to count and size mesozooplankton, several challenges remain. First, the FlowCAM only takes images of a subset of a sample. Second, processed samples are destroyed by the built-in peristaltic pump using the standard FlowCAM procedure. Third, the accuracy of zooplankton biovolume estimation based on the FlowCAM image analysis has not been evaluated. These issues are especially problematic if only a limited number of plankton specimens are available and so almost every particle needs to be measured, or if the samples are precious and need to be conserved. A practical case example is the need to estimate the growth rate of zooplankton using the artificial cohort method, in which the size distribution of zooplankton before and after incubation needs to be quantified . In typiIn this study, we developed a modified FlowCAM setup and procedure to achieve a non-destructive recycling capacity. Our modification is low cost and is applicable to any existing FlowCAM model and flowcell size. We also test the reliability of zooplankton biovolume estimation by automated FlowCAM image analysis. Here, we demonstrate the efficacy of our modified FlowCAM procedure using samples from artificial cohort incubations of copepods in the East China Sea.As a novel method for measuring copepod biovolume, we designed a new FlowCAM operational setup and procedure . Before Note, a fixed, or universal, flow rate is not recommended; rather, the optimal flow rate should depend on the actual size range and shapes of the animals in the particular sample. We recommend adjusting the flow rate to a speed at which complete body images of individual copepods can be captured. To achieve this optimal speed, we suggest starting the FlowCAM analysis with a slow flow rate and then gradually increasing the rate to the point at which complete copepod body images can be captured without severe image duplication.th to 7th, 2013. The sampling and experiments of animals in this study requires no permit. For each sampling station, two separate Norpac zooplankton nets (with a ring diameter of 45 cm) with mesh size 50 and 100 \u03bcm were deployed to collect copepod nauplii and copepodites, respectively. The nets were set to 10 m depth and allowed to drift with the ship for 10 minutes. The contents of each net were then used for three replicate incubations, following the standard protocol of the artificial cohort method [We demonstrated the modified FlowCAM procedure using samples from artificial cohort incubation experiments of copepods in the East China Sea. Sampling was carried out on board the R/V Ocean Researcher II at three stations in the East China Sea  from Mayt method . The samThe size distribution of copepods in each sample was measured using the modified FlowCAM procedure, as described above. In this case study, images of copepods were captured using the FlowCAM autoimage mode, with an imaging rate of 20 frames per second. We used the flowcell with a 300 \u03bcm chamber depth and the 40x magnification microscope lens, for an optimum, factory defined particle size range of 30\u2013300 \u03bcm. The flow rate was controlled by the pulling rate of the syringe pump, which was adjusted to an optimal speed such that complete body images of whole individual copepods were captured. As a reference, the size of the syringe used in this study is 100 ml with diameter 3.4 cm, and the general withdrawal rate of the syringe is 1.5\u20131.8 ml/min by the syringe pump.The images captured by the FlowCAM were semi-automatically classified into 7 target morphotypes, assisted by our existing image libraries  is based on the diameter of the circle obtained by arranging all the pixels deemed part of the particle into a solid circle. We then compared the VABD with biovolume calculated from manual prosome length and width measurements using the FlowCAM ruler tool on copepod images. For simplicity, we term this manual measurement of biovolume using the ruler tool as \u201cmicroscopic measurement\u201d, because this manual procedure is analogous to conventional procedures for size measurements under the microscope.As a workaround, we used the Area-Based-Diameter (ABD) volume as the proxy for biovolume; this measure is only mildly affected by antennae and appendages since they occupy only a small relative area. ABD-based volume  as: W0 is the mean carbon biomass of copepods at the beginning of incubation, WT is the mean carbon biomass at the end of incubation, and T represents the incubation time. Here, carbon biomass can be estimated from the biovolume of plankton [g. The same procedures have been applied successfully for artificial cohort experiments from other stations. As the objective of this paper is to demonstrate the procedure, we do not present those results here. Importantly, however, our procedure is reproducible, and zooplankton samples were successfully recycled without damage.As an empirical demonstration of the capacity of our modified FlowCAM procedure, we provide examples of the size distribution of copepods before and after incubation in our artificial cohort experiments from station 5 . Compariplankton . In thisWe introduced a modified FlowCAM procedure that facilitates sample recycling and thus overcomes the limitation of small sample size in various practical situations. The advantage of automatic measurements based on the FlowCAM is that the tool is efficient and free from human errors associated with subjectivity. Based on our assessment, the modified FlowCAM procedure is 3 times more efficient than classical microscopic measurements in terms of the time-savings and manpower . HoweverS1 Table(XLSX)Click here for additional data file."}
+{"text": "Malnutrition is an underlying cause of mortality in about half of the cases that occur among children less than five years in developing countries. In Africa including Kenya, this problem may be exacerbated by socio-demographic and socio-economic factors. This study aimed at determining nutritional status and association of demographic characteristics with malnutrition among children aged 1 day to 24 months in Kwale County, Kenya.A cross-sectional study was done in Mwaluphamba Location, Kwale County, Kenya. Data was collected using a semi-structured questionnaire and administered to 380 randomly selected mothers who had children under the age of two years. Nutrition status was determined using anthropometric measurements. Data was analyzed using descriptive statistics and associations were determined by univariate logistic regression.Malnutrition prevalence for children in Kwale was high with 29.2% of the children being stunted and 13.4% being severely stunted. Underweight prevalence was at 20.8% of whom 9.5% were severely underweight. The global acute malnutrition rate was 18.9%. Stunting differed significantly between sex . Significant differences were also observed in stunting and underweight due to age (p < 0.005).The prevalence of stunting, underweight and global acute malnutrition rates was high among the children. Male children were associated with a significantly higher prevalence of stunting than the females. The prevalence of underweight and stunting significantly increased with increasing age. Malnutrition in children is among the leading health problems that contribute to the high child morbidity and mortality in the developing world including Kenya. Globally, child malnutrition declined in the 1990s', but in Africa the number of malnourished children increased from 26 million to 32 million from 1990 to 2000. Additionally, 25% of all children under five years old were underweight by 2000, indicating little change . MalnutrStunting affects women adult height, which in turn affects their reproductive health, survival of their children. In men, economic productivity is negatively affected as a result of stunting in childhood. Secondly, stunting reflects damage that affects, in some cases, irreparably health and development over the long term . Stuntin2 as of July 1, 20The study was conducted in Mwaluphamba Location, using a descriptive cross-sectional design that applied quantitative methods to elucidate nutritional status and association of demographic characteristics with malnutrition among children aged 1 day to 24 months in Kwale County, Kenya. The area is within Kwale Health and Demographic Surveillance System which was established in July 2010 and covered 7,617 households and 51,000 inhabitants, in an area of 384.9 KmChildren 0-6 months were weighed using Slovakia Aid  scale and the weight was recorded to the nearest 0.1 kg. Older children who could not stand were measured using baby trousers and a portable electronic scale to measure the weight. They were first hung from suitable secure points such as trees and doorframes and the scale adjusted to zero with an empty weighing pant suspended from the lower hook. The minimally clothed child was then placed in the weighing pant and suspended on the weighing scale to hang freely and weight recorded to the nearest 0.1 kg. For children who could stand, weight was taken using an electronic Tanita THD-650 scale . The scales were placed on a flat surface and set at zero. The mothers were lightly dressed with no shoes and heavy clothing. Weight was recorded to the nearest 0.1 kg. Height measurements were done using height boards. Height for children below Bilateral oedema was determined by applying thumb pressure on the top part of both feet for 3 seconds. If pitting occurred on both feet upon release of the fingers, the child was recorded as having nutritional oedema, denoting severe malnutrition and referred to the nearest health facility for management. Calibration of weighing scales with known weights was done every day for quality assurance purposes. In addition, pretesting of the questionnaire was done on a sub sample of 30 mother-child pairs. Permission to conduct the study was given by the Kenya Medical Research Institute KEMRI Scientific Steering Committee (SSC) and the Ethical Review Committee (ERC) SSC NO. 2534. Informed consent was obtained from the participants and those who agreed to participate were requested to sign the informed consent forms. For the illiterate, consent was given in form of finger print before commencing the study. Confidentiality was assured and data was anonymized. Data was entered in Microsoft Access 2013  and then imported to Stata Statistical software  for cleaning and data analysis. Comparisons were done for the anthropometric measurements with Z scores established by WHO. Moderate stunting (low height-for-age) in children, was categorized to children that had a score of between negative two and negative three standard deviation below (-2SD/Z to-3SD/Z) of the median height-for-age established by the WHO. Severe stunting was classified to children with a Z-score of -3SD three standard deviation and below from the mean established standard value. Moderate low weight-for-age (underweight) in children was obtained as a measure for weight-for-age who were between negative two and negative three standard deviation score (-2SD/Z to -3SD/Z) from the median weight-for-age. Severe underweight was classified to children with a Z-score of negative three standard deviation (-3Z) and below from the mean standards while children with a Z-score of between negative two and negative three standard deviation (-2SD/Z to -3SD/Z) were categorized as moderately underweight.Wasting, weight-for-height (WHZ) was described as low weight-for-height a measure below (-2Z) two standard deviation from the median weight-for-height standard. Severe wasting was classified to children with a Z-score of three standard deviation and below from the mean standards of WHZ). MUAC was recorded for children aged between 6 months and 24 months. Classification of MUAC was on the basis of the WHO established cutoffs of less than 110mm (11.0cm), for Severe Acute Malnutrition (SAM). Children with a MUAC between 110mm (11.0cm) and 125mm (12.5cm), were grouped to Moderate Acute Malnutrition (MAM). MUAC of between 125mm (12.5cm) and 135mm (13.5cm), indicated that the children were at risk of acute malnutrition while MUAC over 135mm (13.5cm), was showing good nourishment. Calculations of z-scores was done using the formula: /Standard deviation of the reference population. Body Mass Index (BMI) for non-pregnant women was computed using the formula: BMI (Body Mass Index) = weight in kilograms/height in meters squared. The BMI values for mothers/caregivers were categorized as underweight if their BMI was below 18.5, normal or healthy if their BMI ranged between 18.5-24.9, overweight if BMI ranged 25.0-29.9 and obese if the BMI was 30.0 and above. Descriptive statistics of the data were determined by frequencies, percentages, range, mean \u00b1 standard deviation, Association between stunting and explanatory variables were determined by univariate logistic regression analyses. Multiple logistic regression analysis was used to determine association of numerous factors on the outcome variables-stunting, under-weight and wasting.Five mother-child pairs were excluded from the research study because of the following reasons: two mother- child pairs migrated outside from the HDSS area; two mothers refused to give consent to participate in the study and one child was severely sick. The final number of mother child pairs that participated in the study were 380. The final number of children who met the set inclusion criteria were 380 in total of whom 214 (56.3%) were boys and 166 (43.7%) were girls. The median age (months) for the children was 9 months with IQR of 10 (4-14). Median age (months) for boys was 9 and Interquartile Range (IQR) of 11(4-15) while for the girls it was median of 8 and IQR of 11 (3-14). Children in the study were categorized into 3 age-groups; \u2264 5 months who were 125 (32.9%), 6-11 months were 114(30%) and 12-24 months were 141(37.1%). There were 380 mothers/caretakers for the 380 children with a mean age of 28.6 years and a range of 15-61 years. More than half of the mothers (63%) were below 30 years of age, with the highest proportion (28.2%) being between 25-29 years. The mothers/caregivers were classified into eight age categories: 15-19 years 47(12.4%), 20-24 years 85(22.4%), 25-29 years 107(28.2%), 30-34 years 67(17.6%), 35-39 years 48(12.6%), 40-44 years 18(4.7%), 45-49 years 3(0.8%) and 50+ years 5(1.3%). Majority of mothers/caregivers (62.4%) had achieved primary education while 29.5% had no formal education. Overall summary of social demographic characteristics is shown in Differences observed with regard to underweight  were not significant between the sexes while underweight between age-groups was statistically significant  as shown in In this study, the prevalence of malnutrition among the children was relatively high, with stunting at 29.2%, underweight 20.8% and wasting 19.0%. The stunting rate is above the national mean of 26% for children under five . This hiThe study showed a relatively high prevalence of malnutrition among the children. The stunting, underweight and wasting rates were above the national means. Male children were associated with a significantly higher prevalence of stunting than the female children. Male children were more likely to be stunted than female children. The prevalence of underweight and stunting significantly increased with increasing age. Based on these results, more efforts and study programs are needed to address the problem of malnutrition among children. There should also be focus on practices that lead to malnutrition differences between gender and age among children.Childhood undernutrition is declining in Kenya generally, but remains significantly higher is some parts of the country;Prevalence of malnutrition is higher in male than in female children.Malnutrition differences between genders in children is depended on childcare and practices;Social demographic characteristics are determinants of malnutrition differences between gender and age among children in the marginalized regions.The authors declare no competing interests."}
+{"text": "Variant Interpretation Community Of Special Interest Group  provides an international forum for researchers to share progress in their work to address the need for strategies to detect, annotate and interpret variants in the context of health and disease. The 8th edition of the VarI-COSI meeting [Technological advances and decreasing costs of DNA sequencing have created a deluge of genomic data \u20133. These meeting \u20139 was heThis year\u2019s meeting of the VarI-COSI featured three keynote talks, 11 research presentations and two industry presentations describing new datasets, bioinformatic methods, and scientific studies aimed at advancing our understanding of genetic variation. In addition, we heard presentations from our sponsors, Variantyx and Qiagen describing technological developments for variant detection and prioritization.This year, the VarI-COSI was divided into a morning and afternoon session. The morning session began with a keynote by Bonnie Berger describing the potential of genomic crowdsourcing with privacy to significantly boost data availability for genomic studies. Talks in this session described approaches to mine position-specific information from the genome to better annotate and study genetic variation. Speakers described using information about protein structure to study variants at protein interaction interfaces, identifying positions involved in the functional tuning of proteins, using signatures of purifying selection to implicate non-coding Mendelian variants, and annotating variants with an expanded set of conservation categories using machine learning.The afternoon included two additional keynotes. Mona Singh described work studying variation that affects protein interactions with target molecules to gain insights into the pathogenesis of cancer. Olga Troyanskaya described the application of deep learning to predict the effect of single nucleotide variants on the expression of nearby genes. Talks in the afternoon session included statistical developments to improve meta-analysis in GWAS studies in the presence of heterogeneity, new methods for detecting splicing and structural variants, and different approaches using molecular measurements or diverse phenotypes to gain improved insight into the role of genetic variation in disease processes.We welcome Antonio Rausell from the Institut Imagine, Paris (France) to the VarICOSI organizing committee.The VarI-COSI executive committee invites the community to provide feedback regarding meeting content and format, as well as to participate in future sessions of the meeting.Acknowledgements). After 2 rounds of review 11 manuscripts were accepted for publication. These presented novel strategies for implicating biological functions underlying diverse human phenotypes [For this year\u2019s VarI-COSI special issue received 12 manuscript submissions. All manuscripts were evaluated by at least two reviewers, selected by a panel of three editors and 25 experts in the field .We are working to organize the next VarI-COSI meeting . Further information about this coming meeting is available on our website ("}
+{"text": "In common with local government organisations across the world, local authorities in the UK have responsibility for promoting health. A key part of this function is the frontline enforcement activities of officers responsible for compliance with health and nutrition claims. This study identifies attitudes, values and practices of enforcers: namely trading standards and environmental health officers, when faced with the problem of non-compliance with the Regulation. Semi-structured interviews with frontline enforcers from local authority regulatory services to investigate challenges with the enforcement of Regulation (EC) 1924/2006. Twenty participants were interviewed; sixteen were based in North West England and two in the North and two in the South of England. The participants were selected for their specialist knowledge and experience of enforcement of nutrition and health claims. Regulation (EC) No. 1924/2006 on nutrition and health claims presents particular challenges for enforcers seeking to apply an optimal strategy to flawed regulatory design. As with other regulations, when faced with non-compliance, enforcers, specifically trading standards and environmental health officers have a wide discretion to determine their response: ranging from the deterrent or accommodative styles of enforcement. The participants reported using advice rather than action and by doing so confronting their bifurcating identity of prosecutor and advisor. Enforcers used advice as a regulatory tool in enforcing the law relating to nutrition and health claims. Along with other market driven consumer protection in the EU and internationally, the aimAn \u2018information asymmetry\u2019 as defined by Stigler  exists bThe rationale of the Regulation  is to prThe purpose of the study was to identify attitudes, values and practices of enforcers: namely trading standards and environmental health officers, when faced with the problem of non-compliance with the Regulation. There were similarities between the enforcement of the Regulation and the broad range of other legislation enforced by officers.The study was a phenomenological investigation into the experiences and beliefs of enforcers, where the themes were confined by a reductive approach. A non-probability purposive sample  was chosEthical approval was obtained by Manchester Metropolitan University prior to data collection. In this respect, the key concerns for participants were preserving the integrity of their investigations and commercial sensitivity of the businesses who they were investigating. Assurances of confidentiality were provided and the data were anonymised at transcription to prevent identification of participants or food suppliers. The participants provided their consent to be interviewed by email.In the course of this study, eighteen semi-structured interviews were conducted with twenty respondents. Pseudonyms have been adopted to refer to the participants to conceal their identities see .The interviews were conducted in meeting rooms at the offices of the respondent\u2019s employer: the regulatory services department of a local authority. The interviews were of a duration of between 45 and 90 minutes. The interviews were recorded on a digital recording device after obtaining the respondent\u2019s consent.All of the above officers were employed in authorities in the North West of England except for Dexter and Theo who are based in the North and William and Julie who were attached to authorities in the South. Each of the interviews were conducted, transcribed and coded by one investigator, AP. The transcripts were uploaded into NVivo  and a thorough process of data coding and identification of themes completed. The coding process involved identification of a significant statement and encoding it .While the general direction of the work was influenced by the literature and the broader themes might be said to exist a priori, a number of more detailed and particular themes emerged as a result of the analysis of the interview data. There was broad agreement among the interviewees in the major areas of contention and this lead to the emergence of themes. Some of the themes are explored in detail and form the basis of the discussion here and this is based on their relevance to the research question of how enforcers enforce the Regulation and their use of discretionary powers in taking enforcement action.A notable theme which emerged from the data is the enforcement officers\u2019 identification of themselves as prosecutors with sole and direct responsibility to the public: a view consistent with the deterrence model presented by Reiss  and deveEthan captures the duality of his position as an enforcer and how he reconciles the functions:We always have to play two roles. Supporting businesses and the enforcement but they do tend to blend together quite well I find. And most businesses want to be compliant, and they know that, so if you can help them be compliant then they\u2019ll you know, go with you.Traditionally enforcers have had an arm\u2019s length relationship with business. However, Ethan, like his contemporaries, is not uncomfortable with modern enforcement practice where enforcers play a role in supporting businesses in their efforts to ensure compliance with the law. This would support the notion that \u2018an advisory approach to regulatory enforcement may be a necessary component of an effective enforcement strategy\u2019 .Similarly, at the start of the interview where the enforcement officer is invited to describe their role and main duties, Chloe describes her view of her role primarily in terms of enforcement as her main purpose:Well, enforcement is our main, we are regulators, enforcement is our main business advice and primary authority advice is probably secondary to that.integral part of the compliance strategy as described by Law . Environmental health stories would often be front-page news. And if we didn\u2019t take the action they wanted, they would report us to the paperwe\u2019d give assistance with small businesses because we\u2019ve got obviously priorities to promote prosperity and business growth in xxxx.\u2019In her view, there is a public perception that she should be taking a deterrence-based approach as described by Reiss. It is not clear why Chloe feels that public expectations would require that she does not provide advice to business. This may be due to a failure by the public to appreciate the nature of the enforcer\u2019s role and in turn, this may be attributed to the lack of communication by enforcers about their duties. Certainly, Chloe\u2019s remark regarding consumers indicates that she faces some resistance to the idea of helping business and that she ought to police and prosecute non-compliant business. However, in her practice Chloe actually adopts an advisory role. In fact Chloe goes much further when she actually speaks of promoting economic growth as an objective in itself and part of her work rather than as a means of securing compliance: \u2018The role of economic regulators to deliver a competitive marketplace in order to provide an environment for business growth is well established ,20. The Charlotte revealed that the provision of advice can provide the initial engagement between enforcer and a firm on the subject of the application of nutrition and health claims. When asked what experience, if any, she had of nutrition and health claims, she responded:Some of the food manufacturers based in this area or other food businesses will ring up saying I want to make a claim on my label and I\u2019ll give advice.Amelia went on to provide specific instances of the cases of such advice:We have provided advice to a sports supplements importer\u2026We have also been involved in giving advice to an internet trader for vitamins.Really Responsive Regulation Baldwin and Black stress the case for regulators to be responsive to not only the attitude of the firm but also the \u2018operating and cognitive frameworks of firms\u2019  were seen to act inconsistently, both within the individual business and between businesses. SMEs complained that different food safety requirements were made each time the premises was inspected, despite conditions remaining the same and the same EHP visiting. SMEs also believed that EHPs would forget or fail to enforce requirements made previously and therefore failed to take action\u2026Regulating Business: Law and Consumer Agencies  inspection, I\u2019d have to go and look at their inspection plan, any advice that had been given, to know where to direct my resources when I go into my local store.There\u2019s a section just for regulators on the BRDO [Better Regulation Delivery Office] website so I can go on that register and look for like a national supermarket and see if they\u2019ve been advised on health claims. If I was to go and visit that supermarket, I have to look at the primary authority register to see if they\u2019ve got a primary authority and I have to have regard to advice that\u2019s already been given. Chloe seems concerned that the intricate nature of the regulation is not optimal rather than that there is any at all. The research evidence points to inefficiencies in command and control systems of regulation and a preference for market based regulation. Therefore, the questions are around when and how to regulate and how to avoid problems of regulatory capture . Chloe\u2019sThe primary authority relationship where business receives advice in return for payment, may provide a level of indemnity which commercial advisors cannot provide, as Chloe explains:And as much as we wanted to be able to say, yes, we couldn\u2019t so we\u2019ve not heard from them since. And the primary authority has gone, because that was, to be fair, I did say to them, if we can\u2019t approve and give primary authority advice to say that you can use that, there\u2019s very little point in us actually being the primary authority.Where enforcers can provide an indemnity against action from either their own authority or from another authority, this provides a valuable benefit and assurance to business. This goes beyond the benefit that might be provided by an independent professional advisor such as a lawyer while raising the issue of liability for advice provided and the provision of a benefit which cannot be matched by any other provider. It also raises questions of the conflict of interest between the provision of paid for advice and assistance on the one hand and the duty to enforce against that same party (who turns from client to defendant), on the other.Enforcers can demonstrate a surprising level of involvement with the commercial aims of the business as Scarlett shows:I\u2019m trying for them not to change their trademark, I know they want to keep their trademark. How can we go round\u2013not circumvent the legislation obviously, but get to a point where they can continue to use itThere are specific provisions in the Regulation which make it clear that a claim contained within a trademark is subject to the same control as if it were made without the trademark. In other words, that the registration of a trademark does not provide immunity to a claim made within it. Local authority enforcers do have experience and knowledge of law for business. However, it would be unusual for that to extend to providing advice relating to intellectual property issues arising from the protection of trademarks and to provide advice about how the trademark might be protected.At times, there was an expectation from a business that enforcers would be on hand to provide advice whenever it was required. James describes that expectation:certain companies would be phoning you all the time so you felt like you were doing their work for them which is ok but when we are stretched it\u2019s not possible to do that as much so it\u2019s something that\u2019s in formation at the moment with primary authority, so we\u2019ll see how it works out.Enforcers would need to adopt strategies to manage the expectations of businesses because they were unable to deliver the service levels which business would have liked and possibly had become accustomed to from other providers. The problem of managing business expectations was most acutely felt following the severe cuts to local authority budgets imposed from 2009 onwards. James described the circumstances:We\u2019re short staffed now but we used to have a chap who was office based and people would send their labels for approval and raise issues they have and he could spend quite a lot of time doing it. We don\u2019t provide that service any more.It will be instructive to see whether the withdrawal of a label copy clearance service will lead to more ex post liability breach and with enforcers playing a lesser role in the provision of ex ante advice. The effect of withdrawing \u2018free\u2019 advice may implement a cost structure and more efficient distribution of resources and avoid the waste associated with services provided free at the point of use. Alterna\u2018Initially advise them, yes. I mean if they don\u2019t listen to advice then you can take more formal sanctions but most of them will comply.\u2019It was clear from all of the data that advice represents the first step in the \u2018enforcement ladder\u2019 with further steps becoming more formalised as Ethan says, most businesses are reasonable. We advise them \u2018 you must get rid of this\u2019 and they do or are in the process of doing so.\u2019In the majority of cases, matters will not proceed any further than the advice, \u2018Ethan explains the broader position:All enforcement tends to be a hierarchy of actions, we decide the proportion as best we can. And it ranges from just, you know, simple verbal advice up to a full prosecution. It sort of increases in more formality if you will, as you need to, if somebody doesn\u2019t listen to written advice you give them, sorry verbal advice, then you tend to give written advice, then formal written advice\u2026prosecution is the final resort.Attempts to capture and illuminate the negotiation between enforcers and regulated firms have found this hierarchy of actions ,30,31. TIn the pyramid of responses, as one regulatory intervention fails, the regulator moves upward to the next more serious level and as the risk subsides, the regulator should move back down to a lower level. In this way, the pyramid provides an inherently proportionate and reasonable exercise of power the justification of which is based on the failure of the less serious previous action.The commercial response to the advice demonstrates the level of reliance placed on the officer\u2019s judgement as shown by Charlotte:The first step would be advisory. Then you would ask them to change the label. And most of the time they do. Sometimes they stop making the product altogether.It is unclear whether the advice to change a label resulting in the withdrawal of the claim, and finally to stop making the product altogether, is wholly dependent on the opinion of the enforcement officer or whether the opinions of others have been sought. It is clear however, that business can place great reliance on the opinion of the officer and it plays a significant role in influencing the decision.In relation to the enforcement of the Regulation, Hawkins and Hutter capture the central difficulty thus; \u2018the task of a regulatory bureaucracy is, by various means, to induce a potentially unwilling business organisation to bear costs which it would in many circumstances not wish to assume\u2019 . The cosWhile the emphasis of this study was on the enforcement of the Regulation, the study found evidence for both the deterrence and accommodative approaches of enforcement across the range of legislation. When interviewed, enforcers identified themselves primarily as enforcers whose first duty was to the public. A further related theme, which is not found in the literature, but which has more recently emerged is the view of enforcers as advisors to business. Such an approach is rooted firmly in the accommodative model of Braithwaite et al where officers seek to \u2018educate, persuade and cajole\u2019 . In thisMarc Law raises the notion that \u2018an advisory approach to regulatory enforcement may be a necessary component of an effective enforcement strategy\u2019 . The datIn examining this advisory function, the communication between the firm and the regulator is found to demonstrate an awareness of the responses required to not only the needs and attitude of the firm but also the \u2018operating and cognitive frameworks of firms\u2019 . ExampleThe data from the sample interviews of enforcers in this study shows how the roles of advisor and enforcer are taken on and adapted to suit the circumstances. This supports the notion of escalation up the regulatory pyramid of Ayres and Braithwaite et al . SometimThe study found the relationship of local authority advisor and firm differed from that of the retained advisor and firm in some important respects. There was a willingness to challenge the advisor based on observations of what appeared to be inconsistent practice and the tolerance of non-compliant claims in circulation. This raises questions of fairness to which there appears to be no wholly satisfactory answer from enforcers. The conflict between the role of advisor and prosecutor is felt most keenly when faced with the prospect of action from another authority which challenges or undermines the advice provided to the firm. The overriding concern from firms however is for consistency and certainty for the advice which they receive. This places great pressure on enforcers to provide independent, bespoke, complex and commercially aware advice that will face up to scrutiny and possible challenge by experienced advisors. Not surprisingly, the data showed some faltering of confidence among officers (see Dexter above) particularly when faced with the absence of the coordination previously provided by Local Authorities Coordinators of Regulatory Services (LACORS).The data were collected soon after the implementation of the Health and Social Care Act 2012 which imposes significant obligations on local authorities for improving the health of their populations. The responsibility lies primarily with a specialist public health team led by the director of public health supported by existing expertise in areas such as environmental health and trading standards. As such the study highlights the scope for a joined up and strategic approach within local authorities. Many of the officers interviewed reported being subjected to increasing demands from business for advice with expectations of service levels resembling those within commercial practice. Against a background of cuts in local authority services, this represents a further strain on resources. The difficulties to which it gives rise are underscored by the fact that, in the absence of a primary authority agreement, such advice is provided without charge.The study sought to identify attitudes, values and practices of enforcers: namely trading standards and environmental health officers, when faced with the problem of non-compliance with the Regulation. The interviews with enforcers revealed several themes; including that of the application of bureaucratic discretion, deterrence, accommodation and the enforcer as advisor, which were considered to be most relevant in influencing the type of action to take in applying their discretionary powers. The recommendations from this work would suggest the need to promote an awareness of the costs of the very broad nature of the work of enforcers and the extent to which their duties extend to an advisory function. Such insights can contribute to the understanding of the enforcement action for enforcers and health professionals and may be applied to other jurisdictions where enforcers enjoy similar discretion. A fertile direction for future research would be a comparative study to explore how enforcers in other jurisdictions approach similar problems.S1 Checklist(DOCX)Click here for additional data file."}
+{"text": "Transition metal multi-principal element alloys (MPEAs) are novel alloys that may offer enhanced surface and mechanical properties compared with commercial metallic alloys. However, their biocompatibility has not been investigated. In this study, three CoCrFeNi-based MPEAs were fabricated, and the in vitro cytotoxicity was evaluated in direct contact with fibroblasts for 168 h. The cell viability and cell number were assessed at 24, 96, and 168 h using LIVE/DEAD assay and alamarBlue assay, respectively. All MPEA sample wells had a high percentage of viable cells at each time point. The two quaternary MPEAs demonstrated a similar cell response to stainless steel control with the alamarBlue assay, while the quinary MPEA with Mn had a lower cell number after 168 h. Fibroblasts cultured with the MPEA samples demonstrated a consistent elongated morphology, while those cultured with the Ni control samples demonstrated changes in cell morphology after 24 h. No significant surface corrosion was observed on the MPEAs or stainless steel samples following the cell culture, while the Ni control samples had extensive corrosion. The cell growth and viability results demonstrate the cytocompatibility of the MPEAs. The biocompatibility of MPEAs should be investigated further to determine if MPEAs may be utilized in orthopedic implants and other biomedical applications. Metallic biomaterials are used in a variety of applications, including fixtures like screws and plates; electrical connections; and load-bearing applications, including total hip arthroplasty (THA), which is the standard treatment for degenerative hip pain. Over 60,000 THA surgeries were performed in the United States in 2015, with metallic implants paired with polymeric articulating surfaces as the most common implant configuration [Despite the prominent use of metallic implants in THAs, complications including wear ,4,5, looNovel materials could be used to reduce the risk of complications in THA patients and other orthopedic implant patients. However, any novel material should improve on the material properties of existing THA materials. A new class of materials called multi-principal element alloys (MPEAs) may provide such an opportunity. MPEAs contain several different elements in near-equiatomic concentrations, unlike conventional alloys, which are comprised of a principal solvent matrix alloyed with other constituents in minor and trace amounts. MPEAs can exhibit a single-phase microstructure with all elements in substitutional solid solution, and are also referred to as high entropy alloys, baseless alloys, or complex concentrated alloys . MPEAs m2SO4 aqueous solutions [The mechanical properties of TM MPEAs may be advantageous for use in orthopedic applications. The relevant properties of common implant materials and TM MPEAs are shown in olutions ,22,23. Tolutions , althougolutions . TM MPEAolutions ,28,29,30olutions . The intolutions ,33. Desp20Cr20Fe30Ni30, Co30Cr30Fe20Ni20, and Co20Cr20FeeMn20Ni20 . The MPEA cytotoxicity was assessed by analyzing fibroblast cell number and viability following 24, 96, and 168 h of cell culture in direct contact with the alloys, as well as positive and negative controls.This study investigated the cytotoxicity of CoCrFeNi-based MPEAs in direct contact with fibroblasts during in vitro culture, using an adaptation of the ASTM F813 standard. Evaluating the cytotoxicity of MPEAs is a critical first step in assessing MPEA biocompatibility. This is particularly important, as Co, Cr, Fe, and Ni are constituents in the majority of TM MPEAs  and may 25Cr25Fe25Ni25 alloy, Co20Cr20Fe30Ni30 and Co30Cr30Fe20Ni20; and an equiatomic quinary alloy, Co20Cr20Fe20Mn20Ni20. The quinary alloy was cast to analyze the effect of adding a fifth transition metal, Mn, to the CoCrFeNi-base alloy.Three TM MPEAs were fabricated for this study to evaluate the effects of alloy compositions; two off-equiatomic variations of the Co20Cr20Fe30Ni30 sample, indicated by the lack of features in the micrograph; the microstructures of the other MPEA samples were similar. The XRD patterns in Following casting and homogenization, the alloys were characterized using scanning electron microscopy (SEM) and X-ray diffraction (XRD). The backscatter electron micrograph in p = 0.67) in cell number between groups, but after 96 and 168 h, significant differences between groups were observed (p < 0.05), as shown in 20Cr20Fe30Ni30 and Co30Cr30Fe20Ni20 alloy samples had similar responses to the stainless steel and untreated control samples at each time point, indicating negligible cytotoxic effect on cell growth. The cell number for the Co20Cr20FeeMn20Ni20 samples was reduced after 168 h, indicating potential cytotoxicity. The Ni samples (positive control) elicited a cytotoxic response, resulting in significantly reduced cell numbers at 96 and 168 h.The cell number in each well was quantified using the alamarBlue assay at three time points . After 220Cr20Fe20Mn20Ni20- and Ni-containing wells to contrast the responses. The Co20Cr20Fe20Mn20Ni20 samples shown in 20Cr20Fe20Mn20Ni20 samples, with no discernable change in cell morphology and good cell viability, indicates that the cell metabolism may have been affected by leaching of Mn ions, resulting in lower alamarBlue readings.The fibroblasts were evaluated with a LIVE/DEAD assay at 24, 96, and 168 h. The cultures were imaged using fluorescence microscopy to visualize the cell viability in response to the samples. The MPEAs, stainless steel, and untreated samples showed a high percentage of viable cells at each time point. The cell viability is clearly demonstrated after 168 h for the MPEAs, stainless steel, and untreated samples . Few dea20Cr20Fe30Ni30, stainless steel, and Ni after 168 h of cell culture . The surface of the Ni metal appeared dull following the cell culture, and OM revealed surface degradation suggestive of extensive corrosion . The target compositions were obtained by converting the nominal alloy concentrations to weight percents, and the corresponding amounts of the elemental constituents were combined. The alloys were melted in an Ar-filled chamber, flipped, and re-melted 5\u20136 times to promote homogeneous compositional distribution. Following melting, the alloys were sealed in Ar-backfilled quartz tubes and homogenized at 1100 \u00b0C for 48 h in a high temperature furnace . The MPEAs were sectioned using a low-speed diamond saw, followed by grinding and polishing using sequentially finer SiC polishing pads from 400 to 1200 grit, and finally polished using 1 \u00b5m diamond paste. The final thickness of each sample ranged between 1 and 1.5 mm. The sample shapes were irregular due to the casting and cutting process, but the sample masses differed by \u22640.3 g. Stainless steel 304 (SS) and commercially pure Ni rods were used as negative and positive controls, respectively. The SS and Ni samples were cut and polished using the same methods as the MPEAs. The sample areas  ranged between 0.201 to 0.630 cm2.Three TM MPEAs\u2014CoThe chemical composition, microstructure, and crystal structure of the MPEA samples was analyzed. The microstructure and composition were analyzed using a Zeiss Ultra-55 field-emission scanning electron microscope (FE-SEM) operating at 20 keV accelerating voltage. An X-ray energy dispersive spectroscope (XEDS) detector was used to examine the compositional homogeneity. The MPEA crystal structures were analyzed via X-ray diffraction (XRD) using a Cu K-\u03b1 source (\u03bb = 1.54 \u00c5) with 45 keV accelerating voltage and 40 mA current over a range of 2\u03b8 angles from 30\u2013100\u00b0. After the cell culture, bright-field and dark-field optical microscopy (OM) were used to examine the sample surfaces for evidence of corrosion.2 in a fully humidified incubator. The cells were detached with trypsin-EDTA , centrifuged at 225\u00d7 g for 10 min, then re-suspended in media and seeded in 12-well culture plates at 100,000 cells per well. Cells were cultured in the plates with media changes every 48 h until the monolayers became nearly 100% confluent.BJ fibroblasts  were cultured and expanded to 80% confluency in T-150 flasks, according to the manufacturer\u2019s instruction, in \u03b1-MEM with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 \u00b0C and 5% COn = 3 samples per group. Untreated fibroblast seeded wells were also used as controls. The media was changed every 48 h during the evaluation.The cytotoxicity of the MPEA samples was evaluated using an adaptation of ASTM F813, where confluent fibroblast cell monolayers are in direct contact with the alloy sample. ASTM F813 indicated 24 h of direct contact in vitro culture; however, metallic implants typically remain in the patient for several years, so the samples were cultured for 168 h to observe the cell response as function of time. The three experimental MPEAs, stainless steel (negative control), and Ni (positive control) were placed on confluent monolayers of BJ fibroblast cells in 12-well plates with n = 3 per group) were incubated at 37 \u00b0C for 2 h. Following incubation, 300 \u00b5L samples in duplicate from each well were placed in a black 96-well plate. Fluorescence measurements were performed using a Cytation5 multimode imaging microplate reader  with an excitation wavelength of 560 nm and emission wavelength of 590 nm. The emission intensities were converted to cell numbers using a standard curve generated from a series of known cell numbers.The alamarBlue assay was performed at 24, 96, and 168 h time points to evaluate cell number. The media was aspirated and the cells were washed with Dulbecco\u2019s phosphate buffered saline (D-PBS). A 10 vol % alamarBlue solution was made by adding the alamarBlue reagent into fully supplemented \u03b1-MEM media, and 1.5 mL of the 10% alamarBlue solution was added to each well. The samples  was used to assess the viability of BJ fibroblasts in contact with MPEA samples at 24, 96, and 168 h time points. The cell viability was evaluated using calcein AM and ethidium homodimer-1 (EthD-1). The calcein binds to live cells to produce green fluorescence, while EthD-1 binds to dead cells and produces red fluorescence. The staining solution was prepared in D-PBS at a concentration of 2 \u00b5M calcein and 4 \u00b5M EthD-1. The media in the sample wells was aspirated and replaced with 500 \u00b5L of the staining solution and incubated at room temperature for 20 min. The samples were washed with D-PBS, then imaged with the Cytation5 using GFP and RFP filters for live and dead cells, respectively. The entire well was inspected and representative images were obtained.A LIVE/DEADn = 3 per group) was measured following 168 h of cell culture and compared with the mass prior to cell culture. After removal from the wells, the metal samples were washed with D-PBS, then placed in an ethanol bath and ultrasonically cleaned using a Fisher CPXH 1900 ultrasonic cleaner. The samples were dried with warm air then weighed using a Mettler AE166 balance.The mass of the metal samples . The data are presented as the mean \u00b1 standard deviation of the mean. Statistical significance was set at"}
+{"text": "Autism is an intellectual developmental disorder characterized by insidious disability in communication, social interaction, and using language and abstract concepts. This organic disorder is known to have deformities in brain, i.e., cerebellum and limbic system, showing wide spectrum of systemic and behavioral symptoms. The oral health care of such patients can be complicated as they cannot verbalize complaints about any dental problems they may be experiencing and can display a variety of behaviors and reactions to small changes also.This study summarizes etiology and diagnosis of this disorder with the special emphasis on the issues encountered while coping with children with autistic spectrum.How to cite this article: Chandrashekhar S, Bommangoudar JS. Management of Autistic Patients in Dental Office: A Clinical Update. Int J Clin Pediatr Dent 2018;11(3):219-227. Autistic disorder is categorized into the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) under the section, Pervasive Developmental Disorders (PDD), which is also referred to as autism spectrum disorder (ASD).Kanner,4According to DSM-IV, PDD is an umbrella term used under which specific diagnoses are defined, which include autism disorder (AD), Asperger syndrome, rare disorders: Rett syndrome and childhood disintegrative disorder, and PDD not otherwise specified . Individuals with ASDs will have difficulty in three domains: Social interaction, communication and repetitive behaviors, or restricted interests.The cause of ASD is not clear yet. Based on the available research, one can say that the disorder may be multi-factorial, involving genetics, environmental poisons, neuropsychopathy.de novo mutations, mitochondrial defects,5 increased level of inflammatory cytokines, maternal bleeding during pregnancy, metabolic syndromes, and advancing maternal age6 are all persuasively linked to autism.Recent research work has elucidated that parameters, such as CNTNAP2 gene, 7 and hypoplasia of cerebellar lobules VI and VII.8Neurological autism is an organic disorder associated with abnormalities in brain structure and function. Characteristic findings are a reduced number of Purkinje cells in the posterior-inferior regions of the cerebellar hemispheres, truncation in the dendritic tree development of neurons in the limbic system,9 Prenatal factors like intrauterine viral infections, or metabolic disorders and intrauterine exposures to the teratogenic drugs, thalidomide and valproate,10 may all play important role in the pathogenesis of AD.Some patients with AD showed abnormal levels of serotonin or other neurotransmitters that can affect the brain development.Coexisting medical conditions of AD may be linked with seizure disorder, fragile-X syndrome, tuberous sclerosis, allergies, immune system problems, gastrointestinal disturbances, developmental delay, dysmorphic features, obstetric complications, and phenylketonuria.11 Most recent study suggests the current prevalence rate as 1 in 110 children.12 The male:female ratio is 3.7:1,13 but females exhibit severe form of mental retardation. Higher prevalence rate in males suggests X-linked mode of inheritance.14Incidence of autism is about 0.2% in the US.1 The mean age noted for these deviations is 17 to 44 months.15 Early detection is necessary for early introduction of learning and behavioral guidance, which will give abiding benefits for these children and their families.16 Early signs and symptoms include baby does not seek parent attention, fails to cuddle, making direct eye contact, imitation games, no reaction to known faces, or other warm joyful expressions by 6 months of age,17 afraid of new things, try to make repeated actions to gain attention by 12 months of age, oversensitivity to textures, not able to understand pretend play by 16 months of age; 50% of patients do not develop speech. Abnormal and immature speech develops in virtue of intonation, pitch, rate, rhythm, grammar and word integration, understanding, and nonliteral speech.1The onset of AD usually occurs before 3 years. There are some specific criteria referred by DSM for a patient to be diagnosed as AD, which include impairments in social functioning, deficits in communication, and restricted interests.18 They don\u2019t enjoy group activities and want to be in their own world and unable to share another child\u2019s interest in an activity.19 Temper tantrums, hyperactivity, short attention span, impetuous, anxiety, anger, and a tendency for aggressive and self-injurious behaviors (SIBs) are common behavioral features in these patients. An automatic stereotyped behavior of psychogenic origin-like arm flapping and toe walking are common.About 75% autistic patients suffer from moderate range of mental retardation.20 The SIB can occur in 4 to 5% of individuals of autistic patients. It may be simple one, like self-pinching, or severe reactions, like self-biting or head banging, can even involve oral structure, such as lip biting and deep gingival cleft on canine caused by scrapping with finger nail.21 The cause is not clear. It might be to gain attention of caregivers or avoid unwanted events.Echolalia and delayed echolalia are also present. Even small change in daily routine may increase or initiate it. This repetitive behavior is more frequent in females. The head and neck region is usually affected.Understanding interpersonal dynamics will help in giving therapeutic approaches. Suggested therapeutic approaches consist of rewarding good conduct after completion of every step in a procedure, distracting patient from an undesired action, and inserting a prefabricated oral screen as a temporary physical distraction also helps diagnosis. Diagnosis of autism is completely based on observation of child\u2019s behavior, psychosomatic testing, and history from parents, as there is no specific test or biomarker for autism.Diagnosing AD involves developmental screening, followed by a comprehensive diagnostic evaluation. General developmental screening is performed for children to evaluate basic learning skills, such as behavior, speech, and movement are at appropriate time. Age intervals for this screening are 9, 18, 24, and 30 months. This screening specifically designed for autistic child are at the age of 18 and 24 months: Modified checklist for autism in toddlers (M-CHAT) and screening tool for autism in toddlers and young children (STAT).After developmental screening, if any child is showing delay in attaining the developmental milestones, then comprehensive diagnostic evaluation is done to diagnose the AD. The evaluation is done by specialists, such as developmental pediatrician, psychologists, and child neurologists.22This evaluation involves thorough review of child behavior and development, interview with the parents, genetic and neurological tests of patients. The diagnostic criteria for autism  is given23 Autism disorder is a lifelong disorder, with no remission. The symptoms may change or lessen or disappear. The patients with low intelligence quotient (IQ) and low functioning need a protected environment throughout their life, whereas those patients with higher IQs will be able to live and can work with only minor supervision.Development of speech is the best favorable outcome by 5 years.24 Larger doses of pyridoxine are the most widely used alternative medication.25 Many of these drugs cause systemic as well as oral side effects. It is essential for the dentists to know the properties of these drugs , and tactile cues.During the dental treatment, the main challenge is reduced ability of autistic kids to communicate and relate to others.29 Kopel32 reported that there will be high level of peripheral vision in autistic children. So lateral movements of any toy just before the patient are potential distractions should be avoided. These movements can change the behavior of child.30 Peripheral vision can be used by the autistic children to get more reliable information.31The dental team should be organized for changeable and atypical responses to sensory stimuli, as these patients dislike even minute changes in their surroundings and require resemblance in continuity.Children with autism ought to have a patient-centered medical home that provides a constant, complete, family-centered, harmonized, empathetic, and culturally effective care, which can be easily reachable.33The objectives of management of autistic patients are to enhance independent performance, improve community engagement, and provide support to their parents and caregiver. A successful enduring management plan requires coordinating the efforts of educators, therapists, physicians, and mental health professionals.The role of family parental counseling: Previsit meeting is to prepare the family for first dental visit, which will be beneficial for both parents as well as dental team. Previsit meeting will help the parents in preparing the child for dental treatment and any hesitant problems related to child behavior should be discussed and must conquer.21 Dentist should know the medical conditions of the child and should be ready for co-occurring medical and physical issues.The parents should understand that though their child has difficult time, the dental team support remains the same. Carefully listening to the parents/caretakers is a key element in gaining their trust, which in turn will help tremendously in gathering data.22 Dentists should explain the parent and endeavor to give quality care without harming the child.Potential areas of concern during dental treatment for an ASD may worsen the symptoms, like use of fluoride, amalgam, and exposure to dental products with gluten and casein.22 The amalgam is durable restoration. When it comes to composites, it raises the health concern as it contains Bisphenol A, which is endocrine disrupter.The major controversy about the fluoride application is possible neurotoxin, and two other possible effects are gastrointestinal irritation and dental fluorosis. So fluoride application in autistic patients is controversial, until unless the patient is under high risk for caries.22Generally, gluten-free, casein-free diet is recommended in autistic patients as these patients have a \u201cleaky gut\u201d where intestine is permeable to larger molecules like gluten from wheat, rye, and barley and casein from dairy products. These large molecules enter into bloodstream and tend to have negative effects on brain function and immune system.The front desk receptionist is a key in helping patient\u2019s future visits. For a successful dental visit of autistic child, the whole staff should be caring, empathetic, and aware of how to communicate with these patients.34 The experimental introduction of relight conditions, rhythmic music, and deep pressure in the dental setting diminished adverse patient reactions and enhanced active involvement in oral prophylactic procedures.34Patients are likely to be disturbed emotionally by surrounding distracting stimuli like sound, light, and taste. Such discomfort may be reduced by adjusting the dental clinic environment sensitively.21 A single operating room may be also reserved to accommodate the treatment of the autistic child.It may be feasible to treat the patient in a calm, secured sole operatory with reduced decoration and dimmed lights.35 It is important to learn what the patient is capable of doing vs learning what the patient is not able to do.36 Short,37 well-organized appointments should be planned and the waiting time should not exceed 10 to 15 minutes to avoid upset, as these patients have very limited attention span.28 To address the autistic individual\u2019s preference for sameness and aversion to change, a routine should be established by maintaining days, times, and personnel for each dental visit.38 Discussions of any procedure of actual work should be avoided during its course.The goal of the initial appointment is to establish trust and develop a relationship.39 Pictures of dental clinic or a story can be given to parents to make acquaint the patient about clinic and reassure the comfort.35Anyone who is involved in the management should reduce the movements because the autistic child is easily distracted.40Typically, it is the dental assistant or hygienist who will first contact with patient. Assistants should be able to identify triggering points of deviant reactions without fail.41 However, higher rate of pliability is needed to meet the needs of quickly changing patient.21 Inappropriate behavior should be ignored. Hand over mouth is not considered an appropriate technique for these patients.32Goals of behavior guidance are to develop rapport, lessen anxiety, and provide quality dental treatments while building a trusting and positive relationship for a lifetime between the professional and patient.Communication guidance helps to establish trust and builds needed cooperation. Oral commands should be short, clear, and simple sentences. It is important to maintain good, ongoing communication throughout the visits and even after that. The ability to follow directions, learn new things, and articulate wants and needs may be difficult for some patients with autism. Some require assistive communicative devices, such as a Smart/Scan 32 pro, an augmentative communication device , or a Pi36The PECS consists of a book of pictures to express desires, observations, and feelings. The book grows as the patient grows, with more words and pictures and is very helpful for those who are nonverbal.42 For individuals with limited language, use pictures or objects to explain what will occur. Example: Pictures of radiographic film, disposable plastic mouth mirrors, mouth props or rests, saliva ejectors/ suction tips. Some individuals will benefit from practicing certain aspects of a procedure before experiencing them in a dental office.\u201cTell-Show-Do\u201d is a basic and effective exposure therapy and a way to introduce dental instruments, equipment, or procedures to a patient.43 But some disapprove. McDonald and Avery31 reported that restraints were used in challenged patients to obtain safer working conditions and more predictable working conditions. Various studies reported that applying a more or less firm wrap, pressure, and/or touch on emotionally disturbed or oversensitive persons can have a positive calming and comforting effect.21 Deep touch had a soothing effect, whereas light pat can be a tonic to the nervous system.44 One method that being wrapped in blankets or applying tight pressure to the entire body  lets them feel the limits of their body, and this has a soothing effect.45Concerning the use of restraints for autistic children, there is a controversy. Some authors advocate restraint board for calming down these patients in dental setting.32 suggested familiarizing the child with basic dental procedures in home. This technique involves dividing dental procedures into smaller steps. Each procedure should be successfully completed by a slow and step-wise approach and achievement of specific behavior. Then only next step is introduced.Desensitization techniques based on classical conditioning theory are required to diminish the apprehension present in autistic patients, which is very severe. Such techniques are time-consuming. Kopel46Before the use of this technique, the parents/caregiver should be well versed about the voice control, to avoid any misunderstandings during treatment. It can be used in any patients; however, autistic patients with hearing deficits would not be good candidates. Phrases, such as \u201ceyes to me,\u201d \u201clook at me,\u201d \u201chands on tummy,\u201d or \u201cfeet straight out\u201d can be used to elicit appropriate behaviors. If the patient is able to understand nonverbal communication, the use of nonverbal cues is a good way of eliciting appropriate behavior.46Positive reinforcement rewards preferred behaviors and thus strengthens the recurrence of the behavior. Oral admire and the pat of warmth, along with tokens of appreciation, can be used as positive reinforcers. The presence of the parent during the procedure is a good positive reinforcer. The parent presence is used to get the patient\u2019s attention and increase compliance, decrease negative behaviors, establish appropriate roles during treatment, provide effective communication between dental provider and patient, and provide a positive dental experience.46Distracting techniques like watching a favorite cartoon, listening to music, or holding onto special toys might help autistic patient to getting distracted while undergoing some procedures. Some techniques can engage the patient like holding balloon filled with water, an accordion tube. Autistic patients with high intellectual can be distracted by soothing and relaxed enough to undergo a procedure.37 pertaining to oral hygiene, objectionable taste of toothpaste, and the sensation of toothbrush may hinder the effect of brushing.47 A gentle introduction to toothbrushing using alternatives, such as a washcloth, toothbrushes of different texture and design, or an electric toothbrush may enhance the acceptance of toothbrush by the child with ASD. Dentist or parent can be helpful by selecting the toothpaste with tolerable taste.It is essential to decrease the exposure of auditory and taste stimuli for autistic patients. During the dental visit, any drastic exposure to senses should be least,Social stories help an individual to understand the events and what to look forward during the dental visit. During the previsit consultation with the family, functional behavioral evaluation can be done by the dentist.48 Visual pedagogy  helps to change the behaviors by teaching specific skills. It functionally analyses the antecedents of behavior and consequences that follow.48 By increasing the likelihood of patients who accept simple and routine dental procedures, dentists can decrease the need for more intrusive procedures, such as restraints and sedation.33 Each component of this skill would be divided into specific steps, each step would be taught separately, and a child would be rewarded as they learned each component skill. In shaping, the child is reinforced to adopt the behavior eventually on his own initiative.In dentistry, the use of ABA practices has a high chance of improving the outcome of conventional behavior management techniques.48 Reinforcement represents one of the elemental concepts. It is considered to occur when there is an increase in certain behavior, as a consequence of a stimulus. The positive and negative reinforcements are linked to initiation and termination of the stimulus respectively.With this method, the child can be trained to sit on the dental chair by themselves.39For example, positive reinforce like rewarding with a toy or praising may lead to enhanced compliance in the dental chair. Conversely, negative reinforce like drilling can be handled by doing the procedure for predetermined period like counting from 1 to 10. Immediately after that, the procedure is interrupted for a while. The sequence of events is repeated as long as necessary for the procedure to be completed.49 Although using physical restrainers in treating autistic child is also controversial, it may be applied effectively in some situations as a protectively supporting device for the patient, when used properly under informed consent.50 The use of restraints needs to determine according to each child.45The practice of hand-over-mouth exercise is rarely used these days because its use may be misconstrued as an assault.Conscious sedation had variable effect on autistic child. Usually, the physician is aware of an underlying health problem that would be a contraindication for sedation. If the patient has minimal dental treatment needs that can be accomplished in two operative appointments or less, then conscious sedation can be selected as a treatment plan. The sedation drugs most commonly used alone or in combination are: Versed, Vistaril, Demerol, Chloral Hydrate, and Nitrous Oxide.42 Management of autistic patients under general anesthesia is effective and it will help the patients to tolerate conventional treatment.During sedation, a patient must be monitored with a blood pressure and heart monitor, pulse oximeter, and a precordial stethoscope. There must be a second assistant employed to document these vital signs every 5 minutes during a sedation appointment. Some authors noted that administration of long duration and higher concentration of nitrous oxide than usual is required to get the preferred level of sedation in patients with AD.As each patient is an individual, a thorough understanding about each patient is necessary for dentist and assistant. Simultaneously, parent should also have knowledge about the treatment given to their offspring is suitable and what is comfortable for him. Emotional skills will be more useful than intellectual and clinical skills.The ability to handle the patients should be guided by instinct and creativity, rather than by strict reasoning. This article has projected on slight modifications of each behavior management techniques that are helpful for treating autistic patients. Over that, it is the flexibility and creativity of dentist and staff to give optimal care to the patients."}
+{"text": "In breast cancer patients, the diversity of the microbiome decreases, coinciding with decreased production of cytostatic bacterial metabolites like lithocholic acid (LCA). We hypothesized that LCA can modulate oxidative stress to exert cytostatic effects in breast cancer cells. Treatment of breast cancer cells with LCA decreased nuclear factor-2 (NRF2) expression and increased Kelch-like ECH associating protein 1 (KEAP1) expression via activation of Takeda G-protein coupled receptor (TGR5) and constitutive androstane receptor (CAR). Altered NRF2 and KEAP1 expression subsequently led to decreased expression of glutathione peroxidase 3 (GPX3), an antioxidant enzyme, and increased expression of inducible nitric oxide synthase (iNOS). The imbalance between the pro- and antioxidant enzymes increased cytostatic effects via increased levels of lipid and protein oxidation. These effects were reversed by the pharmacological induction of NRF2 with RA839, tBHQ, or by thiol antioxidants. The expression of key components of the LCA-elicited cytostatic pathway (iNOS and 4HNE) gradually decreased as the breast cancer stage advanced. The level of lipid peroxidation in tumors negatively correlated with the mitotic index. The overexpression of iNOS, nNOS, CAR, KEAP1, NOX4, and TGR5 or the downregulation of NRF2 correlated with better survival in breast cancer patients, except for triple negative cases. Taken together, LCA, a metabolite of the gut microbiome, elicits oxidative stress that slows down the proliferation of breast cancer cells. The LCA\u2013oxidative stress protective pathway is lost as breast cancer progresses, and the loss correlates with poor prognosis. Evidence is accumulating for the role of bacterial dysbiosis in the pathogenesis of different cancers ,14,15,16LCA, in particular, inhibits the proliferation of breast cancer cells ,24,25,26low, PgRhigh), Luminal B , Her2+ , and triple negative cases  [Breast cancer is a heterogeneous disease and there are well-established systems for the classification of breast cancer cases. The AJCC TNM classification ,28 is ba\u2212, PgR\u2212) .Oxidative stress evasion is critical in cancers ,33. ThusAll chemicals were from Sigma-Aldrich unless otherwise stated. Lithocholic acid (LCA), cholic acid (CA), chenodeoxycholic acid (CDCA), glutathione (GSH), N-acetyl-cysteine (NAC), and tert-butylhydrquinone (tBHQ) were from Sigma-Aldrich . LCA was used at concentrations of 0.1 \u00b5m, 0.3 \u00b5m, and 1 \u00b5m, which corresponded to LCA concentrations in the breast . GSH and2. The MCF7 cells were maintained in MEM (Sigma-Aldrich) medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 2 mm L-glutamine at 37 \u00b0C with 5% CO2. The SKBR3 cells were maintained in DMEM  medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 2 mm L-glutamine at 37 \u00b0C with 5% CO2. The human primary fibroblast cells were maintained in DMEM  containing 20% FBS, 1% penicillin/streptomycin, 2 mm L-glutamine, and 10 mm HEPES at 37 \u00b0C with 5% CO2.The 4T1 cells were maintained in RPMI-1640 (Sigma-Aldrich) medium containing 10% FBS and 1% penicillin/streptomycin, 2 mm L-glutamine, and 1% pyruvate at 37 \u00b0C with 5% COMCF7, SKBR-3, and 4T1 cells were purchased from the American Type Culture Collection (ATCC). Cells were regularly checked for mycoplasma contamination. In the cellular experiments, control cells received vehicle (0.001% DMSO in medium) but no LCA. All cellular experiments were performed in the presence of 10% FBS unless stated otherwise.w/v) sulphorhodamine B solution in 1% acetic acid. Unbound dye was removed by washing with 1% acetic acid. Bound stain was solubilized with 10 mm Tris base and the absorbance was measured at 540 nm.Cellular proliferation was assessed using a sulforhodamine assay, as described in Reference . Cells wRNA isolation and RT-qPCR reactions were performed similarly to in Reference . Total R3VO4, 1 mm NaF, 1 mm PMSF, protease inhibitor cocktail). Protein samples (30\u201350 \u00b5g) were separated on 10% SDS polyacrylamide gels and electrotransferred onto nitrocellulose membranes. After blocking for 1 h with TBST containing 5% BSA, the membranes were incubated with primary antibodies overnight at 4 \u00b0C. After washing with 1\u00d7 TBST solution, the membranes were probed with IgG HRP-conjugated secondary antibodies . Bands were visualized by enhanced chemiluminescence reaction . Densitometry was performed using the Image J software [Protein isolation, SDS-PAGE, and western blotting were performed similarly to in Reference . Cells wsoftware . AntibodWe used the Abcam (ab31163) antibody in our studies, which we validated, due to the ambiguity in the molecular weight of NRF2. The calculated molecular weight of NRF2 is 68 kDa, however, recent studies have claimed that NRF2 has a molecular weight of ~130 kDa ,47. In tLipid peroxidation was assessed using the thiobarbituric acid-reactive substances (TBARS) assay as described in . The 4T1Cells were seeded in 24-well plates (MCF7\u201450.000 cell/well). On the following day, cells were transiently transfected with TGR5, CAR, VDR, or PXR siRNA or the negative control at a final concentration of 30 nM using Lipofectamine RNAiMAX transfection reagent (Invitrogen). Cells were incubated with transfection complexes in medium containing LCA (0.3 \u03bcm) for 48 h. CTL stands for vehicle-treated (0.001% DMSO in medium), non-transfected cells, while NEG stands for negative control siRNA-transfected, LCA-treated cells.Preparation of 2,2\u2032-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was performed as described . The absThe kmplot.com database  was usedLeica Bond Max\u2122 was used. The antibodies and the conditions used are summarized in This study was authorized by the institutional ethical body. Tissue microarray (TMA) and immunohistochemistry were performed as described in Reference . The TMAAnimal experiments were authorized by the local and national ethical board (reg. 1/2015/DEM\u00c1B) and were performed to conform to the relevant EU and US guidelines. We re-analyzed samples from a previous experiment in line with the 3R principles.We assessed the effects of the supplementation of LCA on tumor growth and behavior by grafting 4T1 cells to Balb/c female mice, as described in Reference . LCA was6/mL) in ice-cold PBS\u2013matrigel  at a 1:1 ratio. From this suspension, female BALB/c mice received 50 \u00b5L injections to their second inguinal fat pads on both sides (105 cells/injection). Tumor growth and animal wellbeing were monitored daily.The 4T1 cells were suspended  for storage at \u221220 \u00b0C. LCA stock was diluted each day to a working concentration of 75 \u00b5m in sterile PBS immediately before the treatment. Ethanol vehicle (1% in PBS) was prepared and diluted similarly. Animals received a daily oral dose of 200 \u00b5L/30 g bodyweight from the LCA solution or the vehicle. Researchers administered LCA and vehicle solutions blind. Treatment was administered every day at the same time between 8 a.m. and 10 a.m.Experimental animals were female BALB/c animals between 8\u201310 weeks of age (20\u201325 g). Mice were randomized for all experiments. Animals were bred in the \u201cspecific pathogen free\u201d zone of the Animal Facility at the University of Debrecen, and kept in the \u201cminimal disease\u201d zone during the experiment. Animal studies have been reported in compliance with the ARRIVE guidelines ,54.2; 1284 L Eurostandard Type II. L from Techniplast) with Lignocel Select Fine  as bedding. Mice had paper tubes to enrich their environment. The dark/light cycle was 12 h, and temperature 22 \u00b1 1 \u00b0C. Cages were changed once a week, on the same day. Mice had ad libitum access to food and water (sterilized tap water). The animal facility was overseen by a veterinarian. A total of 28 mice were used in this study and group sizes are indicated in the figure captions.Mice were purchased from Jackson Laboratories  and were subsequently bred at the animal facility of the University of Debrecen. No more than six mice were housed in each cage  was used followed by Tukey\u2019s or Dunnett\u2019s honestly significant (HSD) post hoc test. Data are presented as mean \u00b1 SEM unless stated otherwise. Statistical analysis was done using GraphPad Prism VI software. Correlation studies were done using Pearson correlation test and linear regression. Mitotic index was log2 transformed before the analysis. Values of p < 0.05 were considered statistically significant. Calculations were performed by R project [We used a two-tailed Student\u2019s  project  version NQO1), glutamate\u2013cysteine ligase catalytic subunit (GCLC), catalase (CAT), and heme oxygenase 1 (HMOX1),  . LCA tre(HMOX1), . Taken tNOX4), a major ROS producing enzyme , a key antioxidant protein, decreased upon LCA treatment A. Oxidatg enzyme B, and ing enzyme C.\u2212 [An imbalance between pro-oxidant and antioxidant systems leads to oxidative or nitrosative stress. We detected increased lipid and protein oxidation after LCA treatment, as shown by increases in thiobarbituric acid reactive species (TBARS) D and 4-h\u2212 ,58 and t\u2212 . LCA hadTo assess whether the phenomena described above were restricted to 4T1 cells, the effects of LCA were assayed in MCF7 and SKBR3 cancer cell lines. LCA treatment decreased NRF2 expression A,D and is\u03b1-selective antagonist that can inhibit the downstream signaling of the TGR5 receptor. LCA-mediated reduction in NRF2 protein expression was blocked by NF449 and CINPA1, while the other inhibitors (GSK2033 and DY268) were ineffective  can bind and respond to LCA . First, ffective A.The pharmacological experiments above were complemented by siRNA depletion experiments. Other possible LCA receptors, including the vitamin D receptor (VDR) and the pregnane X receptor (PXR), were also assessed. To provide a comprehensive view, we silenced TGR5, CAR, VDR, and PXR in MCF7 cells. Silencing of TGR5 and CAR efficiently blocked the LCA-induced decreases in NRF2 protein B,C, simiAs a next step, we assessed whether the supplementation of LCA could hamper the redox status of tumors in an in vivo setting. We re-analyzed samples from a previous study we published . In thatIn the tumors of control and LCA-treated mice, we assessed the expression of anti- and pro-oxidant genes. LCA supplementation reduced the expression of NRF2 and a set of antioxidant genes: catalase (CAT), glutamate\u2013cysteine ligase catalytic subunit (GCLC), glutathione peroxidase 2 (GPX2), glutathione peroxidase 3 (GPX3), heme oxygenase 1 (HMOX1), inducible NO synthase (iNOS), NADPH oxidase 4 (NOX4), NAD(P)H quinone dehydrogenase 1 (NQO1), nuclear factor, erythroid 2-like 2 (NRF2), superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), and superoxide dismutase 3 (SOD3) A. Furthehttps://www.ncbi.nlm.nih.gov/geoprofiles/) and kmplot.com [We assessed the expression of the LCA-elicited oxidative/nitrosative stress markers  using a tissue microarray (TMA) made up of tumor samples from 88 breast cancer patients. In parallel, we assessed the available public expression databases, GEO Profiles . In ourNext, we stratified patients based on the pathological grade (Nottingham grade) of the disease. 4HNE expression significantly decreased in grade II and grade III patients compared to grade I patients B. In linCAR, KEAP1, iNOS, nNOS, and NOX4 or lower expression of NRF2 was associated with better survival when we assessed all patients or ER+ positive cases, but not TNBC cases  or ER+ cases. The expression of TGR5, iNOS, and 4HNE decreased in TNBC cases as compared to ER+ cases C. In linBC cases .Finally, we grouped patients as a function of the mitosis score. Staining for 4HNE, the most direct indicator of tissue oxidative stress, decreased as mitosis score increased D. FurtheTaken together, LCA-elicited oxidative stress correlated well with the mitotic rate in breast cancer. Furthermore, the LCA-elicited cytostatic system was hampered at higher breast cancer stages, higher grade carcinomas, or in TNBC cases that had a poor prognosis. In line with that, we identified cases in the TMA study where the staining for iNOS was well-maintained in the surrounding healthy breast tissue, but was lost in the cancerous tissues, or when NRF2 expression was upregulated in the cancerous tissue as compared to the neighboring healthy tissue. Further supporting our observations, we found a dataset in the GEO database  in whichIn breast cancer, the diversity of the gut microbiome is reduced . The gutWe widened the scope of LCA-mediated effects by showing that, when LCA was applied in concentrations corresponding to tissue LCA concentrations , oxidatiThe role of oxidative stress in breast cancer is controversial , as incrThe NRF2\u2013KEAP1 system and reactive species were shown to modulate the clinical behavior of breast cancer. NRF2 overexpression is an independent adverse prognostic factor for cancer recurrence and disease-free survival for breast cancer patients . Moreove+ HER+ or HER+ cases. Similar trends were true for trace amino acid receptors (TAAR) 1, 2, 3, 5, 8, and 9, which are receptors of another bacterial cytostatic metabolite, cadaverine [+/HER+ cancers has not been elucidated, other studies have substantiated the importance of the HER2 signaling pathway, such as in the finding that HER2 signaling induces iNOS and reduces cell proliferation [Our dataset revealed that estrogen and HER2 signaling affect the activity of the LCA-elicited pathways. The expression of the pro-oxidative iNOS and the oxidative stress marker, 4HNE, was lower in TNBC cases than in ERdaverine . Althougferation .The gut microbiome loses its diversity in breast cancer , and bacPreviously, we showed that LCA exerts its effects through the TGR5 receptor . TGR5 ovWe have shown that LCA, a metabolite of the microbiome, induces oxidative and nitrosative stress by creating an imbalance in pro- and antioxidant systems in breast cancer cells. LCA and other similar bacterial metabolites  have properties that are similar to human hormones, as they are produced at a site (gut microbiome) different from the one(s) where they elicit their effects (breast). We have also provided evidence that the lower expression and activity of the LCA\u2013TGR5 signaling system that characterizes triple negative breast cancers correlates with worse clinical outcomes in breast cancer. These findings have translational applicability by targeting TGR5/CAR signaling and oxidative stress for the treatment of breast cancer.In this study, we showed that the cytostatic effects of the bacterial metabolite LCA depend on oxidative stress brought about by the downregulation of the NRF2/Keap1 system and the induction of iNOS, and, hence, nitrosative stress. LCA elicits these effects by activating TGR5 and CAR receptors. The LCA-induced oxidative stress pathway provides better survival in human breast cancer, and the downregulation of the expression of its components characterize the triple negative cases."}
+{"text": "Recently, increasing study have found that DNA methylation plays an important role in tumor, including clear cell renal cell carcinoma (ccRCC).We used the DNA methylation dataset of The Cancer Genome Atlas (TCGA) database to construct a 31-CpG-based signature which could accurately predict the overall survival of ccRCC. Meanwhile, we constructed a nomogram to predict the prognosis of patients with ccRCC.P\u2009<\u20090.0001; testing set: HR\u2009=\u20094.1, CI 2.2\u20137.7, P\u2009<\u20090.0001; entire set: HR\u2009=\u20097.2, CI 4.9\u201310.6, P\u2009<\u20090.0001, Moreover, combined with clinical indicators, the prediction of 5-year survival of ccRCC reached an AUC of 0.871.Through LASSO Cox regression analysis, we obtained the 31-CpG-based epigenetic signature which were significantly related to the prognosis of ccRCC. According to the epigenetic signature, patients were divided into two groups with high and low risk, and the predictive value of the epigenetic signature was verified by other two sets. In the training set, hazard ratio (HR)\u2009=\u200913.0, 95% confidence interval (CI) 8.0\u201321.2, Our study constructed a 31-CpG-based epigenetic signature that could accurately predicted overall survival of ccRCC and staging progression of ccRCC. At the same time, we constructed a nomogram, which may facilitate the prediction of prognosis for patients with ccRCC. Renal cell carcinoma (RCC) is a cancer that originates in the renal epithelial cells and accounts for more than 90% of renal cancer, of which clear cell RCC (ccRCC) is most common subtype and causes the most deaths . AccordiThe aberrant methylation status of CpG islands located in the promoter region of tumor genes is becoming increasingly important in the search for new potential biomarkers for cancer \u20136, becauIn this study, we used the methylation data of the ccRCC of TCGA database to construct a 31-CpG-based epigenetic signature, which can accurately predict the overall survival rate of ccRCC through the most recently used The Least Absolute Shrinkage And Selection Operator method (LASSO) algorithm. We then verified this epigenetic marker using two validation sets (testing set and entire set). Furthermore, we constructed a nomogram to facilitate clinicians to accurately predict overall survival in ccRCC patients. Our results identified a new 31-CpG-based epigenetic marker that may be a new target for predicting overall survival of ccRCC.http://cancergenome.nih.gov/), The details were listed in Table\u00a0A total of 319 cases of ccRCC were randomly assigned to two groups, which had a 2:1 ratio. The former was defined as the training set (n\u2009=\u2009213) and the latter as testing set (n\u2009=\u2009106). The corresponding clinical follow-up information of all cases included the survival time, survival outcome, tumor staging, grading and other information of the cases. The level three of RNA-seq (Illumina RNASeqV2) and the Infinium HumanMethylation450 BeadChip array (Illumina) data were downloaded from The Cancer Genome Atlas (TCGA) database . Frist, the function \u201cglmnet\u201d returned a sequence of models for us to choose from. Second, we used the function \u201ccv.glmnet\u201d to perform Cross-validation, we followed the protocol, did 100 times cross-validations, and finally got an \u03bb average. Based on this \u03bb value, we constructed an epigenetic signature based on 31 CpGs from training set. The accuracy of the epigenetic signature was then verified in the testing set and the entire set by using the time-dependent ROC curve and Kaplan\u2013Meier survival curve analysis. Time-dependent ROC curve analysis was performed by R package \u201csurvivalROC\u201d [LASSO cox regression analysis was conducted for the DMPs obtained in the previous step, based on R packet \u201cglmnet\u201d. The degree of LASSO regression complexity adjustment was controlled by the parameter ariables . We consivalROC\u201d  while thTo investigate whether the epigenetic signature we constructed could be independent of other factors affecting the prognosis of ccRCC, we conducted univariate and multivariate analyses by R package \u201csurvival\u201d. First of all, the epigenetic signature score, age, gender, neoadjuvant treatment, lymph node count, histologic stage and pathologic grade were performed the univariate analysis. Then, we put the univariate analysis has significant difference factor to carry on the multivariate analysis. Finally, we used the forest map to visualize our results by R package \u201cforestplot\u201d.Based on the results of the previous step, we used the factors with significant differences (epigenetic signature score and histologic stage) in multivariate regression analysis to construct a nomogram by using the R package \u201crms\u201d. In order to verify the accuracy of the nomogram, we used the calibration curve to evaluate the nomogram. In the calibration curve, if the observed value and the actual value are more coincident, it indicates that the prediction accuracy of the nomogram is higher.https://david.ncifcrf.gov/) for GO analysis and KEGG pathway analysis. The following, the STRING database (https://string-db.org/) was used to performed protein\u2013protein interaction (PPI) network [http://software.broadinstitute.org/gsea/msigdb/index.jsp). And it was chosen as the signature to perform ssGSEA. The R package \u201climma\u201d was used to identify the differentially expressed gene sets. The threshold for screening differentially expressed gene sets were the p-value\u2009<\u20090.05.We calculated the top 500 genes with the highest correlation with epigenetic signature score, and then enriched the functions of these 500 genes, thus indirectly predicting the functional annotation of epigenetic marker score. The 500 genes were uploaded to the DAVID website , under the threshold of FDR\u2009<\u20090.05 and | \u0394\u03bb value (\u03bb\u2009=\u20090.054), and finally we constructed an epigenetic signature based on 31 CpGs. The formula of epigenetic signature score were obtained by calculating the LASSO regression coefficient: epigenetic signature score\u2009=\u2009n is the number of the CpG site; \u03b2 is the LASSO regression coefficient of the CpG site; X is the methylation value of CpG k and patient i; k is the CpG site. Details of the 31 CpGs were listed in Table\u00a0We used the R package of \u201cglmnet\u201d to construct LASSO cox regression model. After 100 cross - validation, we got an optimal entclass1pt{minimaentclass1pt{minimaentclass1pt{minimaP\u2009<\u20090.0001, Fig.\u00a0P\u2009<\u20090.0001, Fig.\u00a0P\u2009<\u20090.0001, Fig.\u00a0We divided the patients in the training set into high and low risk groups based on the median epigenetic signature scores , age , histologic grade  and pathologic stage  have significant prognostic value  and pathologic stage  could accurately predict the overall survival rate of ccRCC independently of other factors  were screened under the threshold FDR\u2009<\u20090.05 and |LogFC|\u2009>\u20090.015. The result of ssGSEA revealed that patients in high risk group mainly enriched in p53, nod like receptor, cytosolic DNA sensing and other signaling pathways. The patients in low risk group were mainly enriched in the fatty acid metabolism pathway, PPAR signaling pathway, renin angiotensin system and so on \u00a0and 2 kidney cancer cell lines ACHN and 769-P), and sequenced them with targeted methylation . The results showed that CpG island involved in the construction of prognostic model had different expression patterns between carcinoma and paracancer , which will provide a lot of data to verify our epigenetic markers in the future.We performed methylation sequencing on ccRCC tissue samples and kidney cancer cell lines, and the results showed that CpG islands involved in the construction of the prognosis model had significantly different expression patterns. However, our study has some limitations, we lack more data to verify it, but we have built a new biological sample database called Biological Repositories Zhongnan Hospital of Wuhan University . At the same time, we also constructed a nomogram for the convenience of clinicians to predict the prognosis of ccRCC.We developed a 31-CpG-based signature using bioinformatics methods . We found that this epigenetic signature could accurately predict the overall survival rate of ccRCC, contributing to the prediction of the prognosis of ccRCC. And we constructed a nomogram based on this epigenetic signature, which could accurately and conveniently predict the prognosis of ccRCC patients for clinicians.Additional file 1. Supplementary tables 1\u20132, Supplementary Information: Related file 1. Ethics Committee Approval (number: 2020102).Additional file 2. Supplementary figure S1: Functional annotation of the epigenetic signature. Additional file 3. Supplementary figure S2: The PPI network diagram."}
+{"text": "Listeria monocytogenes may persist in food production environments and cause listeriosis. In Norway, a product of concern is the traditional and popular fermented fish product \u201crakfisk\u201d, which is made from freshwater salmonid fish by mild-salting and brine maturation at low temperatures for several months. It is eaten without any heat treatment, and L. monocytogenes, therefore, poses a potential hazard. We investigated the effect of salt and temperature on the growth of L. monocytogenes in rakfisk during the 91 days of maturation. The amounts of organic acids produced during fermentation were too low to inhibit growth of L. monocytogenes. Temperature was clearly the most important parameter for controlling L. monocytogenes. At 7 \u00b0C, approximately 2 log growth was observed during the first 14 days of fermentation, and the level of L. monocytogenes thereafter remained constant. At 4 \u00b0C, only a little growth potential of the pathogen was recorded. We also investigated the effect of the anti-Listeria bacteriophage P100 on rakfisk with added L. monocytogenes. The phage was introduced to the L. monocytogenes-inoculated fish before fermentation, and an average of 0.9 log reduction was observed throughout the fermentation period. This is the first study of L. monocytogenes behavior in rakfisk and points to possible measures for increasing the product safety. Listeria monocytogenes is a facultatively anaerobic, Gram-positive bacterium, resistant to various harsh conditions, and found ubiquitously in the environment. L. monocytogenes causes listeriosis, a relatively rare foodborne infection affecting mainly elderly and immunocompromised persons [L. monocytogenes of primary concern for public health. The bacterium is found in a range of foods including dairy products, meat and egg products, seafood, freshwater fish, vegetables, and other ready-to-eat (RTE) foods [L. monocytogenes in RTE food products in different countries was summarized by Jami et al. [L. monocytogenes in infant foods and RTE foods used for medical purposes, while standard RTE foods that contain less than 100 colony-forming units (CFU)/g at the end of shelf-life are accepted [ persons . The incE) foods ,2, and tE) foods  or at thE) foods ,5. Legisi et al. . The Fooi et al. . The Euraccepted .rakfisk\u201d is regarded as a national specialty food, which is manufactured in a traditional, artisanal, and localized manner. Rakfisk is a seasonal product, mainly consumed from late fall and through the Christmas celebration period, and it grew in popularity in Norway as a delicatessen, currently traded at approximately 400 tons annually [w/w). During the storage, a fermentation and maturation/ripening of the fish occurs, resulting in a characteristic taste, odor, and somewhat spreadable texture of the product, attributes that increase with the ripening time. In most cases, especially in productions where low salt (4\u20135%) and high temperature (6\u20137 \u00b0C) are used, psychrotrophic and relatively salt-tolerant lactic acid bacteria (LAB), such as Lactobacillus sakei, dominate the fermentation [Fermentation of food is an ancient technique that preserves, alters the flavor, odor, and texture, and may enrich the nutritional value ,10,11. Fannually . The proentation .rakfisk is produced and consumed without any heat treatment step, the product is the subject of food safety concerns. Historically, botulism was of major concern due to outbreaks originating from homemade productions with inadequate control of proper salt amounts and storage temperatures [L. monocytogenes since, in theory, growth of this pathogen is not entirely inhibited by the conditions prevailing during production and storage of rakfisk, i.e., salt concentrations around 5% and temperatures around 5 \u00b0C [rakfisk. As a result of a relatively extensive control program, L. monocytogenes is from time to time detected in rakfisk and occasionally causes product recalls. However, outbreaks of listeriosis, where specific batches of rakfisk were implicated, were not recorded until fairly recently. In the last six years, two documented outbreaks  and a very recent suspected outbreak occurred [Since eratures . Presentund 5 \u00b0C ,2. The Noccurred ,15,16.rakfisk that guarantee the complete elimination of L. monocytogenes in the final product. Given the ubiquitous nature of L. monocytogenes, the lack of listericidal steps in the production procedure, and the ability of the organism to become established in the processing environment, it is challenging to produce rakfisk free of L. monocytogenes. Adhering strictly to good manufacturing practices (GMPs) and good hygienic practices (GHPs) to prevent recontamination events and ensuring good quality of the raw materials are measures taken by the producers to limit its prevalence. However, this does not preclude the need for additional hurdles. The lytic bacteriophage P100 specifically infects the majority of L. monocytogenes strains [L. monocytogenes on a variety of RTE foods such as sliced turkey meat, cabbage, mixed seafood [L. monocytogenes on cold smoked salmon [9 plaque-forming units/g food [rakfisk brine of six producers during the course of normal production [L. monocytogenes in the production of rakfisk is described. The purpose of the present work was to determine the effect of different ripening temperatures and salt concentrations on the growth and viability of a mix of L. monocytogenes strains during rakfisk production. The chosen salt concentrations and temperatures reflect the prevailing types of commercial rakfisk production [L. monocytogenes in brine during the rakfisk ripening process and on vacuum-packed rakfisk fillets. Our findings are of particular significance to rakfisk producers and food safety authorities.There are no critical control points during the production process of  strains . It may  seafood , cooked  seafood  fresh cu seafood , and che seafood . The phad salmon  and raw d salmon ,24. Seved salmon ,26,27. Td salmon , and thes/g food . We receoduction . Howeveroduction . We alsoL. monocytogenes strains used in this work are listed in rakfisk, marinated salmon, and herring, one strain originated from RTE meat (2230/92) and was responsible for a food outbreak in Norway in 1992 [v/v). Rifampicin -resistant (RifR) derivatives were prepared by growing strains in liquid media containing 200 \u00b5g/mL Rif as described by Heir et al. [g for 10 min and washed twice with sterile phosphate-buffered saline , before the cell pellets were resuspended in PBS. A five-strain suspension of L. monocytogenes was prepared by mixing equal amounts of washed cells (109 CFU/mL) of each strain. Appropriate dilutions were prepared in PBS.E meat 22/92 and w in 1992 . The strr et al. . For eacrakfisk producers. The fish was gutted, cleaned, and transported on ice to the laboratory facilities within 24 h after slaughtering. Fish heads, gills, and remnants of kidneys were removed before the fish was rinsed under running water and air-dried on paper. Rakfisk was prepared in a traditional dry salting manner [rakfisk was produced with (C1\u2013C8) and without  the addition of L. monocytogenes to the trout or char used. Production conditions were 4.8% or 6.3% NaCl (w/w) with storage at 4 or 7 \u00b0C. Three containers were prepared for each condition. In addition, three containers with char spiked with L. monocytogenes and phage P100 were prepared. The workflow and experimental set-up are schematically depicted in Trout and char of approximately 600 g were obtained from Norwegian fish farmers that deliver fish to g manner  where fiL. monocytogenes viability and growth in rakfisk during product ripening, the fish were spiked with the L. monocytogenes multi-strain mix to obtain an inoculation level of 104\u2013105 CFU/mL brine at day zero for easy detection of both growth and possible inactivation. The spiking procedure was as follows: 100 \u03bcL of the multi-strain mix (5 \u00d7 107 CFU/mL) was added to the fish (2 on each side of the fish belly (muscle side). After 30 min in room temperature, the dry salting procedure was initiated by adding the appropriate amount of NaCl; finally, spiked fish were placed in the containers.For investigating the fish , C1\u2013C8 b2 holes were placed along the container side. Lids with small holes  aligned with the perforated tubes were used to close the containers. Brine samples from the mid layers of the containers were collected aseptically and without opening the container by using syringes with needles inserted through the septa.For logistic reasons explained previously and not to disturb the fermentation , brine sL. monocytogenes as described above. After 30 min, 100 \u00b5L of P100 phage solution , diluted to 1010 plaque-forming units (PFU)/mL in PBS, was spread over the same area, resulting in approximately 108 PFU/cm2 on the fish surface. After another 30 min, the dry salting procedure was initiated by adding NaCl to a final concentration of 4.8% (w/w), before the fish were placed in the containers and stored at 7 \u00b0C for 91 days  with a bent sterile glass rod, resulting in phage levels of approximately 108 PFU/piece and 109 PFU/piece, respectively. The pieces were then air-dried for 30 min before being vacuum-packed and stored at 8 \u00b0C for five days. Controls were fish pieces treated in the same manner, but using PBS instead of phage solution.g period , C6, werRakfisk from char produced under the same conditions, but without added L. monocytogenes (L. monocytogenes. The mix (100 \u00b5L per fish piece of 10 g at 107 CFU/mL) was distributed dropwise on all sides and spread with a bent glass rod. The pieces were air-dried for 30 min. Subsequently, phage P100 was spread onto the surface as described above. After another 30 min of air-drying, the pieces were vacuum-packed and stored at 8 \u00b0C for five days. Controls were fish pieces spiked with L. monocytogenes in the same manner, but using PBS instead of phage solution. Both sets of vacuum-packed rakfisk pieces were analyzed on days one and five.ytogenes , C14, weL. monocytogenes from fresh brine samples was carried out by plating appropriate dilutions on BHI agar  supplemented with rifampicin (200 \u00b5g/mL), incubating for four days at 30 \u00b0C. For determination of L. monocytogenes on fish samples, pieces of 10 g were added to 90 mL of peptone water (0.1% peptone and 0.02% Tween-80) and homogenized for 2 min in a stomacher . Ten milliliters of the homogenate was concentrated by centrifugation at 12,000\u00d7 g for 5 min. The supernatant containing phage P100 was removed, and the pellets containing L. monocytogenes cells were resuspended in 1 mL of peptone water before plating on BHI agar. Total anaerobic bacterial counts were determined by plating brine samples from one of the biological replicates collected on days zero, seven, 14, 28, 42, and 91 on plate count agar . For logistic reasons, this analysis used frozen brine samples, and all samples were processed at the same time. This procedure was previously shown not to affect the number of bacteria in rakfisk brine [Enumeration of sk brine . The pla2SO4 (1:1), and perchloric acid (PCA) precipitation was performed to remove interfering proteins. To each diluted sample (1 mL), 100 \u00b5L of PCA 35% was added. After 10 min incubation on ice, 55 \u00b5L of KOH 7 M was added to neutralize the sample and precipitate excess PCA. The samples were centrifuged at 13,000 rpm for 10 min, and supernatants were transferred to a new tube and centrifuged again for 5 min. Resulting sample supernatants were filtered through a 0.45-\u00b5m Millex-HV Durapore PVDF filter  and used for metabolite analysis. Concentrations of glucose, pyruvic acid, lactic acid, formic acid, acetic acid, acetoin, butanediol, and ethanol in the samples were determined using an Agilent 1100 series high-pressure liquid chromatography (HPLC) system  as previously described [The salinity was determined in brine samples collected on day 91 by following AOAC International official volumetric method 937.09 for measuring salt (chlorine as sodium chloride) in seafood . The pH escribed . ChemStarakfisk production experiment  using three-way ANOVA with fish species, temperature, and NaCl concentration as factors. The growth curves for L. monocytogenes in the production with the addition of P100 phage were analyzed with treatment (P100 or control) and day as factors. All analyses were performed in R [Three containers  were prepared for each condition used in the periment , and eaclmerTest  was appllmerTest  was applmed in R .rakfisk batches were controlled at day 91 (end-point) and confirmed to be at the two intended levels of 4.8%  and 6.3%  (w/w). The pH remained essentially stable at around 6\u20136.5 for all conditions and time-points (not shown). Metabolites were measured in end-point (day 91) control samples (w/v) in all samples with no significant differences between different temperature and salt conditions when analyzed for the same fish species. However, there was a small, but statistically significant difference in overall lactic acid concentration between the fish species (higher in char) when analyzed collectively for all conditions  than at 4.8% NaCl (LS) after day 28. Results from ANOVA of the responses at specific days  gave a slightly different picture, but also determined temperature as the main effect at all days. However, in the early ripening phase (day 14), the effect of salt concentration was also significant. This effect disappeared after day 28, and, at day 91, only the effect of temperature was significant. As displayed in L. monocytogenes showed rapid growth to the maximum level, which was 106\u2013107 CFU/mL at day 14 for the LS batches. Growth was slightly delayed in HS batches with maximum levels reached at day 28. After this time-point, the numbers of L. monocytogenes remained at approximately 106 CFU/mL for the rest of the ripening period, and there were no differences between LS and HS batches. For trout at 4 \u00b0C, L. monocytogenes numbers were essentially unchanged throughout the ripening period. For char at 4 \u00b0C, some initial growth of L. monocytogenes was observed early in the process. The number of L. monocytogenes thereafter remained essentially constant for the LS/LT samples, while a decrease was observed in the HS/LT batches, leading to the abovementioned difference in growth/survival curve appearance for this fish species.The intended inoculation level of the five-strain mix of nditions  are dispnditions . There wL. monocytogenes was added), and growth resulted in total numbers ranging from approximately 5 \u00d7 105 CFU/mL  to 5 \u00d7 107 CFU/mL  at day 91  was only estimated in selected samples and not in all biological replicates, and a complete dataset was, therefore, not obtained. The results nevertheless indicated that bacteria were present at low levels (<100 CFU/mL) at day zero  compared with fish not treated with phage P100. The reduction was maintained during the entire ripening period.Thirty minutes after the addition of turation , C17. Gr91 days, . The pharakfisk LS/HT .In the samples where the  process , C6, theL. monocytogenes during production of the Norwegian traditional fermented fish product, rakfisk, is described. The temperature and salt conditions chosen in this study essentially represent the ranges found in commercial production. The process of rakfisk production is generally described as a bacterial fermentation in combination with so-called \u201cautolysis\u201d, i.e., the activity of endogenous fish enzymes that degrade and transform proteins and fat of the fish [L. monocytogenes at pH above 5 [L. monocytogenes occurred in the LS/HT batches where the total concentration of acids was highest , reaching levels of 106 CFU/mL well before the end of the maturation period. The inoculation level was unrealistically high in this study, but even very low contamination levels would result in numbers of food safety concern (>100 CFU/g or mL) assuming a similar growth rate. High NaCl concentration (6.3%) delayed growth somewhat, but L. monocytogenes eventually reached essentially the same levels also in these batches. Although even higher salt levels could inhibit the pathogen more and have a general preservative effect, this is not a realistic option, as the product will not achieve the desired rakfisk attributes. Low temperature (4 \u00b0C) restricted growth of L. monocytogenes at both salt levels. There were some variations of the numbers during the maturation period, with some initial growth in the char batches, but the level of L. monocytogenes at end-point (day 91) was essentially the same as at inoculation level, or somewhat lower. The variations seen in the early phase could be an effect of the salt equilibration in the dry salting and spontaneous brining process. Earlier, preliminary studies indicated that complete salt equilibration  may take 10\u201312 days [This work represents the first study where the behavior of the fish ,13. Chemthe fish , althoug above 5 . This wa highest . Low-tem\u201312 days .L. monocytogenes growth. However, this low temperature poses the disadvantage of prolonged rakfisk maturation time, and producers using this temperature generally employ a maturation time of minimum five months. In accordance, a preliminary sensory analysis of the products in our study  contained 3 \u00d7 105 CFU/g fish and 6 \u00d7 105 CFU/mL brine. Some samples of rakfisk were stored for eight and nine months. In these samples, the levels of L. monocytogenes remained essentially equal to those after three months of storage (not shown).Using a temperature of 4 \u00b0C during the maturation period, thus, seems like a feasible option for achieving some control of  in 2018 . In addiL. monocytogenes during production, additional strategies to reduce Listeria should improve the product safety. One such strategy is treatment with the bacteriophage Listex P100. In our study, the low-salt and high-temperature (LS/HT) condition was chosen as representing a \u201cworst-case scenario\u201d, where growth of L. monocytogenes was anticipated to be most prominent. The present recorded effects of phage P100 against L. monocytogenes in brine during the rakfisk ripening process and on vacuum-packed rakfisk fillets demonstrate their anti-listerial potential when introduced at certain steps in a production line. The contamination experiments with L. monocytogenes mimic likely scenarios in commercial production where fish are contaminated from the production environment or production equipment. From the salmon industry, it was documented that this route of contamination is very common [L. monocytogenes ceased to grow around day 14. The ceased growth is probably due to inhibition from the competitive background flora, which reached high levels by this day. The inhibition agrees well with that obtained using the FSSP mathematical model [L. monocytogenes at 4 \u00b0C and about 2 log growth at 7 \u00b0C after 14 days, with no further growth thereafter when competition from lactic acid bacteria is taken into consideration (not shown). Inhibition of L. monocytogenes by a competitive flora was shown for other products like cooked ham [L. monocytogenes at the start of the rakfisk ripening presented an additional product safety measure. Furthermore, in the contamination experiments with L. monocytogenes after fermentation during packaging of the product, which also mimic a likely scenario in the production line, a weak, short-term P100 phage effect was also demonstrated. It should be noted that the number of phage per cm2 was relatively low in this experiment (4 \u00d7 107 PFU/cm2 at the highest dose). Further experiments using higher doses are, therefore, warranted.As low temperature and high NaCl concentration only lead to a limited extent of reduction of y common ,41. The al model . Under toked ham ,43. Repooked ham , althougL. monocytogenes added before fermentation, which indicates that these bacterial cells resided on the fish for an extended time and were not readily available for phage binding. The cells may be shielded in inaccessible crevices on the surface or in the complex food matrices of the fish [2 (see above) could have affected the outcome of this experiment.No effect of phage addition on ripened fish could be seen against the fish  or proteL. monocytogenes, including their self-perpetuating nature and their ability to target their host bacterium with high specificity [L. monocytogenes isolates of serovar groups 1/2 and 4. The phage genome was sequenced and no virulence genes were found [Bacteriophages have a number of attributes that make them candidates for control of foodborne pathogens such as cificity . The lytre found . From a Application of phages can be carried out by simple spraying or dipping of the gutted fish before fermentation. In addition, phages may also be added after fermentation before packaging of the final product. Phages may, however, to some extent be inactivated after being sprayed on products, which varies with different foods. More importantly, intact phage particles appear to be immobilized soon after addition to solid foods and become inactive due to limited diffusion. The concentration of phages, therefore, needs to be high enough to ensure contact between phages and the target cells. Although the use of P100 phages is considered safe by EFSA, there is little legislation in Europe and Asia to regulate the use of phages in food and food production. More information is still required on immediate and long-term efficacy of phages and application methods. Potential problems are the possible emergence of phage-resistant mutants, phage spread, and inhibition in microbiological monitoring ,46,47.L. monocytogenes, could be relevant in rakfisk production.In addition to phage treatment, other measures, such as protective cultures or bacteriocins for inhibiting L. monocytogenes growth during rakfisk production. Low ripening temperatures result in essentially no growth, although the conditions are not entirely inhibitory. Introducing listericidal steps such as addition of suitable bacteriophages may reduce L. monocytogenes numbers during the ripening process and on packed products, and this presents a possible measure for increasing the product safety.The ripening temperature had the largest impact on"}
+{"text": "The mats were able to remove 1 mg l\u20131 of Cr(VI) in 7 days under aerobic conditions. MiSeq sequencing of the original mats yielded 46\u201399% of the sequences affiliated to Proteobacteria, Firmicutes and Actinobacteria. When the mats were incubated with Cr(VI), the bacterial community shifted in the favor of Alphaproteobacteria and Verrucomicrobiae. We conclude that microbial mats in the quarry sumps harbor diverse microorganisms with the ability to remove toxic Cr(VI), hence these mats can be potentially used to remove chromium from polluted waters.Microbial mats are rarely reported for chromium-polluted ecosystems, hence information on their bacterial diversity and role in chromium removal are very scarce. We investigated the role of nine microbial mats, collected from three quarry sumps of chromium mining sites, in the removal of hexavalent chromium [Cr(VI)]. Bacterial diversity in these mats and community shifts after incubation with Cr(VI) have been investigated using MiSeq sequencing. In nature, a chromium content of 1,911 \u00b1 100 mg kg The use of heavy metals such as chromium has remarkably increased over the last decades because of rapid industrialization and urbanization. Chromium is used in metal processing, leather tanning, cement dyeing, wood preservation, paints, textile and canning industries . High vo\u20131) compared with the continental crust (ca. 20 mg kg\u20131)  . Such romg kg\u20131) . The weamg kg\u20131) . The chrmg kg\u20131) . Howevermg kg\u20131) .In the past decades, a rise in the need for chromite export has increased the number of chromite mining operations in several locations around Oman. The mining practices include solely open pit excavations (quarry sumps) and the bulk rocks rich in chromite are exported without prior enrichment or other beneficiation processes. In these quarry sumps, different types of microbial mats, either attached to the sediment or floating at the water surface, have been observed. Such mats have been rarely reported for chromium-contaminated sites and so far, few studies have been performed to investigate their microbial community composition and their possible role in chromium removal . In prevThis study was undertaken to investigate the ability of chromium-contaminated microbial mats from excavation quarry sumps to remove Cr(VI) and to reveal the composition of their bacterial communities. This basic research will promote our understanding of what kind of microorganisms survive in chromium-impacted microbial mats and what mechanisms they employ to remove Cr(VI) from the environment. Such knowledge can eventually help in designing strategies to decontaminate Cr(VI) polluted environments.Microbial mats and water samples were collected from quarry sumps in three abandoned chromite mining sites  while another fraction (450 ml) was preserved in a cooler for nutrient analysis with a portable LaMotte photometer.Water samples were collected from the three chromite mines. Physical and chemical parameters such as pH, temperature, electrical conductivity (EC), oxidation and removal potential (ORP) and dissolved oxygen (DO) were measured with Aquaprobe AP-800 from AquaRead. The water samples were filtered with Whatman paper of 0.45 \u03bcm pore size. A fraction of the water sample (50 ml) was acidified with 3% HNOIn order to cover all the possible sources of leachable  chromium, samples were collected only from Nakhl-1 quarry sump. The mineralogical analysis was restricted to Nakhl-1 quarry sump because the three sumps are considered of the same rock unit with the same lithology of harzburgite with dunite in concordant lenses . Harzbur\u20131 of Cr(VI). Pre-prepared packets provided by TRACE\u2013HT22 chromium hexavalent batch code R09A, were used to prepare the Cr(VI) stock solution. The removal of Cr(VI) was assessed with a colorimetric analysis at 540 nm of the complex developed by binding of Cr(VI) with diphenylcarbizide under acidic conditions according to EPA 7196A  and to identify the optimum conditions at which maximum removal occurred. These experiments were performed in 160 ml glass bottles. Each bottle received 30 ml of filter sterile site water, 0.5 g of the mat and 1 mg lPA 7196A . The mat1 for illumina MiSeq sequencing of the bacterial 16S rRNA genes using the primers 341F (5\u2032-CCTACGGGNGGCWGCAG-3\u2032) and 805R (5\u2032-GACTACHVGGGTATCTAATCC-3\u2032) with barcode on the forward primer  were calculated using the Mothur software . ClusterThe water temperature at the sampled quarry sumps was between 21.0 \u00b1 0.1 to 25.1 \u00b1 0.1\u00b0C while pH was alkaline, in the range of 9.05 to 9.44 . The ele\u20131), as expected, followed by the mud sample from the bottom of the quarry sump  (\u20131) was close to that of the harzburgite  and the weathered harzburgite  . The bul\u20131 d\u20131. Incubation of the five selected mats in 1 mg l\u20131 Cr(VI) under 12h light and 12 h dark showed the inability of these mats to decrease Cr(VI), except in the case of Nakhl-2A mat  after 7 days of incubation in the dark under aerobic conditions revealed a complete removal by mat samples from Nakhl-1 but lesser in the mats from Nakhl-2 and Samail . In the l-2A mat . Cr(VI) \u20131 which corresponded to 96\u201398% of Cr(VI) removal (2O4), which was not associated with other minerals and originated from the quarry sump by being bound to the mat prior to sampling  and the lowest in Nakhl-2B mat  in mats B and C  . All theClostridium, Acetivibrio and Bacillus in both sites (Azospirillum and Brevundimonas (\u20131 Cr(VI), the growth of Alphaproteobacteria and Verrucomicrobiae was most favored in Nakhl-1 mats (53\u201368%) with most sequences belonging to the genera Rhizobium, Brevundimonas and Verrucomicrobium (Verrucomicrobium and Luteolibacter in Nakhl-2 mats (The most noticeable community shift in mats without Cr(VI) was the increase in the relative abundance of Clostridia and Bacilli from <1% to 44% and 66% in Nakhl-1 and Nakhl-2, respectively . Majoritth sites . In Nakhth sites . In Nakhndimonas . After iicrobium , 7. Howeicrobium . Verrucol-2 mats .+ and also contribute to the release of Cr(III) (\u20131) was as high as in alluvial fans (30\u201375 \u03bcg l\u20131)  . Moreove Cr(III) . However Cr(III) . These m Cr(III) . Cr(VI)  \u03bcg l\u20131) . Interes2  in our study. This suggests that the process of Cr removal is not physical rather facilitated by microbes actively secreting EPS. Previous studies have shown that when microorganisms like cyanobacteria and bacteria (Ochrobactrum sp.) are exposed to heavy metals, they secrete large amounts of EPS mainly constituting macromolecules with charged functional groups exhibiting adsorptive properties. This results in the EPS serving as a binding site for heavy metal ions like Cr(III) or Cr(VI) leading to complex formation . A clear decline in Cr(VI) levels in the site water based on spectrophotometry and the detection of Cr(III) residues based on our ICP-OES analysis indicates that most of the Cr(VI) added into the solution was not converted to Cr(III) rather incorporated into the microbial mat. SEM and EDX analyses confirmed this by the detection of low levels of chromium within the EPS of the mat. Furthermore, the inability of the autoclaved mats to remove Cr(VI) can be attributed to the fact that the EPS was disintegrated due to autoclaving  and prevormation . Howeverormation . TherefoCyanothece spp., Nostoc PCC7936 and Nostoc linckia) and algal  strains were capable of directly removing Cr(VI) from wastewaters  removal was mostly performed by aerobic heterotrophic bacteria. Light incubations suggest that phototrophic organisms in these mats were unable to remove Cr(VI) and also lowered the removal rate of Cr(VI) by the associated heterotrophic bacteria. This is in contradiction with previous reports, which showed that cyanobacterial  in 24 h  under aerobic conditions with the involvement of a putative chromate reductase enzyme (Bacillus strains were capable of reducing Cr(VI) at concentrations as high as 1500 mg l\u20131  contaminated microbial mats. To the best of our knowledge, next generation sequencing has been used to reveal the diversity of bacteria that can resist or tolerate heavy metals such as iron and copper , 2018, b in 24 h . The stre enzyme . Many Ba0 mg l\u20131 . In facts strain . AlthougStreptomyces have been shown to possess the ability to bioaccumulate and/or reduce Cr(VI) (Streptomyces sp. MC1 showed an ability to produce a bioemulsifier and to reduce 90% of Cr (VI) in soil samples amended with 50 mg kg\u20131 after 1 week of incubation without any pretreatment (Zea Mays for the bioremediation of soils contaminated with Cr(VI)  . The strreatment . This sth Cr(VI) . IsolatiRhizobium and Brevundimonas exhibited higher relative abundance in the presence of Cr(VI). Indeed, several Rhizobium and Brevundimonas spp. with the ability to resist heavy toxic metals including chromium have been isolated (Brevundimonas spp. were shown to be the main group of Cr(VI) tolerant bacteria in the magnetite mine drainage water from Hebei China and could reduce up to 350 mg l\u20131 of Cr(VI)  . While tf Cr(VI) , there aWe conclude that the microbial mats in Nakhl and Samail quarry sumps were able to decrease Cr(VI) concentration under aerobic conditions and, thus, playing a vital role in chromium removal. These mats harbored diverse microbial communities, with the dominance of the bacterial classes Proteobacteria, Firmicutes, Actinobacteria and Verrucomicrobiae and these communities shifted in the favor of Alphaproteobacteria and Verrucomicrobiae when incubated in the presence of Cr(VI). Further research should attempt isolating aerobic Cr(VI)-reducing bacteria from these mats.https://www.ebi.ac.uk/ena/data/view/PRJEB33895.The 16S datasets generated for this study can be found in the European Nucleotide Archive (ENA), RA designed the project, analyzed the MiSeq data, and wrote the manuscript. MS, RA, and DM collected the samples. MS performed the chromium removal experiments and together with TM did the molecular work. ZA-R, BP, and DM performed the geochemical analysis. All authors read and corrected the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "SO4), a naturally occurring mixed-valent iron layered double hydroxide has shown to be highly effective in the reduction of Cr(VI) to poorly soluble Cr(III), giving promise for its use as reactant for in situ remedial applications. However, little is known about its immobilization efficiency inside porous geological media, such as soils and sediments, where this reactant would ultimately be applied. In this study, we tested the removal of Cr(VI) by GRSO4 in quartz sand fixed-bed column systems (diameter\u2009\u00d7\u2009length\u2009=\u20091.4\u00a0cm\u2009\u00d7\u200911\u00a0cm), under anoxic conditions. Cr(VI) removal efficiency  was determined by evaluating breakthrough curves performed at different inlet Cr(VI) concentrations (0.125\u20131\u00a0mM) which are representative of Cr(VI) concentrations found at contaminated sites, different flow rates (0.25\u20133\u00a0ml/min) and solution pH . Results showed that (i) increasing Cr(VI) inlet concentration substantially decreased Cr(VI) removal efficiency of GRSO4, (ii) flow rates had a lower impact on removal efficiencies, although values tended to be lower at higher flow rates, and (iii) Cr(VI) removal was enhanced at acidic pH conditions compared to neutral and alkaline conditions. For comparison, Cr(VI) removal by sulphidized nanoscale zerovalent iron (S-nZVI) in identical column experiments was substantially lower, indicating that S-nZVI reactivity with Cr(VI) is much slower compared to GRSO4. Overall, GRSO4 performed reasonably well, even at the highest tested flow rate, showing its versatility and suitability for Cr(VI) remediation applications in high flow environments.Chromate, Cr(VI), contamination in soil and groundwater poses serious threat to living organisms and environmental health worldwide. Sulphate green rust (GR Faster and less disruptive clean-up is obtained by in situ treatment via emplacement of reactants in the subsurface to create permeable reactive barriers (PRBs) or zones, where contaminants are degraded and/or immobilised, often under reducing conditions.2+-rich environments , assuming a Fe(II):Fe(III) ratio of 2 for GRSO4  is very low at circum-neutral pH ) and S-nZVI (=\u20093\u2009\u00d7\u2009[Fe0]s):The RE factor reflects the number of electrons needed to reduce Cr(VI) to Cr(III), which is equal to 1 in the case of GRSO4 (from Fe(II) to Fe(III)) and equal to 3 in the case of S-nZVI (from Fe0 to Fe(III)).SO4 in anoxic batch sand experiments is shown in Fig.\u00a0tot), followed by the formation of Fe(III)-hydroxides as previously observed , complete Cr(VI) removal was achieved within less than an hour of reaction. However, at the highest Cr(VI) concentration, where Cr(VI) was in excess , 97% of the added Cr(VI) was immobilized. While incomplete removal was indeed expected for that experiment, because of insufficient reducing equivalents, it was actually 15% higher than the expected immobilization capacity based on reduction only. This may be due to additional Cr(VI) removal by Cr(VI) adsorption onto the newly formed goethite particles, which is favoured at the neutral pH studied here , as observed previously \u2009~\u200946.7\u00a0\u00b5mol).Lastly, the aqueous Fe(II) that was initially present in the added GR slurry (~\u200925\u00a0\u00b5mol) was mostly flushed out during the MilliQ water rinse (5 pore volumes) performed before each experiment  concentrations, [Cr(VI)0], are shown in Fig.\u00a0SO4 indicating that Cr(VI) was successfully immobilised by GRSO4.Normalised experimental breakthrough curves obtained for GR0\u2009=\u20090.05) and exhaustion (C/C0\u2009=\u20090.9) points, which increased from 1.5 to 11 PVs and from 2.5 to 19 PVs, respectively, for [Cr(VI)0] decreasing from 1 to 0.125\u00a0mM  removed, this value substantially decreased with increasing [Cr(VI)0], from about 76.6\u00a0mg to 29.3\u00a0mg Cr(VI) per g GR 0].Moreover, this delay in breakthrough became more pronounced with decreasing inlet Cr(VI) concentration, as shown by the breakthrough 0] (Table\u00a00] from 0.125 to 1\u00a0mM. Such early GRSO4 exhaustion was not observed in batch sand experiments performed at the 3 different Cr(VI) concentrations. The occurrence of early GRSO4 exhaustion in columns can be explained by the constant Cr(VI) influx, which will keep reaction rates high on GRSO4 particle surfaces, which in turn is more likely to induce the formation of passivating reaction rims. Such passivating rims were shown by Skovbjerg et al. [SO4 exhaustion occurs even faster if [Cr(VI)0] is higher. Moreover, some GRSO4 may not actually come in contact with the Cr(VI) solution, because of where the GRSO4 particles are situated in the columns (for example near to dead-ends and/or in static flow areas); however, that effect should have been similar amongst the different [Cr(VI)0] as the added GRSO4 mass and the flow rate were constant.In terms of Cr(VI) removal efficiency relative to the added GR0] Table\u00a0. The remg et al.  and Willg et al.  in GR baSO4 amended sand columns as a function of flow rate, i.e., 0.25, 1 and 3\u00a0ml/min  for the different [Cr(VI)0] are shown in Fig.\u00a00] do not greatly change with a change in flow rate: at all three tested flow rates, Cr(VI) removal (mg/g) and removal efficiencies (%) steadily decrease with increasing [Cr(VI)0]  solution with increasing flow rate. Furthermore, with increasing flow rate, advection  becomes more dominant, while flow close to pore surfaces decreases, and hence contact with immobilised GRSO4 is further reduced. At the lower [Cr(VI)0] , an increase in flow rate has less of an impact  solution.Normalised experimental breakthrough curves obtained for GRSO4 amended sand columns as a function of inlet solution pH , where [Cr(VI)0] and the flow rate were kept constant at 0.5\u00a0mM and 1\u00a0ml/min, respectively, are shown in Fig.\u00a0Normalised experimental breakthrough curves obtained for GRs, can readily react with the Cr(VI). The increased solubility of green rust and the dominance of aqueous Fe2+ over green rust under acidic conditions have been demonstrated in previous studies \u2009=\u20090.25\u00a0mM. For comparison, breakthrough curves obtained in GRSO4 amended sand columns at the same flow rates and [Cr(VI)0] conditions are also shown. Similar to the GRSO4 column results, flow rate had little effect on absolute Cr(VI) removal and removal efficiencies in S-nZVI columns, yielding 26.2, 20.7 and 22.1\u00a0mg/g and 3.47%, 2.73% and 2.91% at 0.25, 1 and 3\u00a0ml/min, respectively, while lower pH conditions enhanced Cr(VI) removal (53.4\u00a0mg/g and\u00a07.05%)  and exhaustion (C/C0\u2009=\u20090.9) points under those conditions are listed in Additional file SO4 observations, Cr(VI) removal in S-nZVI amended columns is significantly lower than in batch sand experiments  inside porous sand media. Batch sand studies confirmed similar fast reduction of Cr(VI) by GRSO4 as observed for batch studies in aqueous media , with >\u200995% Cr(V) removed within only 10\u00a0min. In comparison, Cr(VI) removal efficiencies in sand columns under dynamic flow conditions were substantially lower than in batch studies, particularly at higher inlet Cr(VI) concentrations. This is likely explained by the constant influx of Cr(VI) solution which keeps reaction rates high on GR particle surfaces likely promoting the formation of passivating rims on GR surfaces, as observed before in batch experiments with high initial Cr(VI) concentrations. Furthermore, lower Cr(VI) removal efficiencies were observed at higher flow rates and alkaline pH conditions (compared to acidic). For comparison, similar batch and column studies were also performed with S-nZVI, an alternative reductant material. These results clearly showed that Cr(VI) reduction and immobilisation by GRSO4 is substantially faster and yields 2.5 times higher efficiencies compared to S-nZVI, meaning GRSO4 performs substantially better under the tested flow conditions.This study demonstrated the high effectiveness of GRSO4 in Cr(VI) remediation applications, given the presence of sand matrices, dynamic flow conditions and changing pH conditions, we still observe high immobilisation yields that are considerable higher than what is observed for alternative reductants such as S-nZVI. Moreover, the observed trends suggest that at much lower flow rates, Cr(VI) removal efficiencies by GRSO4 would likely be much higher. Thus, at flow rates closer to average groundwater flow , GRSO4 might achieve 100% removal efficiency because of higher contact time with Cr(VI).Overall, these results demonstrate the high potential for use of GRSO4 as well as S-nZVI may not necessarily suit all sites. Overall, with this study we show that, under specific conditions, both GRSO4 and S-nZVI can successfully reduce and immobilise Cr(VI) in porous media. Therefore, these materials certainly warrant further study on how to apply them at larger scale.Obviously, any Cr(VI) contaminated site has its own characteristics depending on the hydrogeological and geochemical properties of the contaminated subsurface , and remediation with GRSO4 and S-nZVI synthesis, mineral characterization of oxidation products, manual and on-line measurements of effluent aqueous samples, as well as breakthrough analysis procedures, can be found in the attached Additional file Detailed description of the GRAdditional file 1: Text S1. Synthesis and characterization of GRSO4 and S-nZVI. Figure S1. XRD patterns (Cu-K\u03b1) of freshly synthesized and glycerol-coated a) GRSO4 (PDF# 13-0092) and (b) S-nZVI (Fe0 PDF# 06-0696 and FeS PDF# 89-6268). Figure S2. XRD patterns (Cu-K\u03b1) of oxidation products after reaction with Cr(VI) solution (pH 7) of a) GRSO4: goethite  and GRSO4, and (b) S-nZVI: Fe0, FeS, lepidocrocite (PDF# 44-1415) and magnetite (PDF# 19-0629). Text S2. Calculation of the iron content, mass and volume of GRSO4 and S-nZVI. Figure S3. Control batch experiment with sand and Cr(VI) only (no added GRSO4 or S-nZVI) showed that Cr(VI) sorption to grain surfaces was negligible over 48\u00a0h (note that x-axis is not linear). Text S3. NaNO3, Cr(VI) and total Cr measurements. Figure S4. (a) Effect of Cr(VI) solution pH  on UV\u2013Vis absorption spectra; (b) UV\u2013Vis spectra of Cr(VI) solutions of varying concentration  used to make the calibration curves shown in (c) and (d) where adsorption readings were taken at 274.3 (R2\u2009=\u20090.999) and 371.3\u00a0nm (R2\u2009=\u20090.998), respectively. Figure S5. (a) BTCs (as a function of time) obtained by injecting 0.4\u00a0M NaNO3 at 0.25, 1 and 3\u00a0ml/min. (b) Comparison of BTCs (as a function of pore volume) obtained by injecting 0.5\u00a0mM Cr(VI) solution (control) at 0.25, 1 and 3\u00a0ml/min and 0.4\u00a0M NaNO3 solution (tracer) at 1\u00a0ml/min. All BTCs show no delay in breakthrough, i.e., are typical of non-reactive solutes. Figure S6. Comparison of BTCs obtained by measuring effluent Cr(VI) concentrations using the on-line UV\u2013Vis set-up (black symbols) and by determining total Cr concentrations via ICP-OES in manually collected samples (red symbols). The test conditions were identical with [Cr(VI)0]\u2009=\u20090.5\u00a0mM, pH\u2009=\u20097 and flow rate\u2009=\u20091\u00a0ml/min. The fact that the two BTCs overlap demonstrates that Cr(VI) is the only Cr species detected in the effluent. Thus, any Cr(III) forming due to reduction is immobilised within the sand column. Figure S7. Eh\u2013pH diagram for chromium based on experimental chromium concentration (10\u22123 M). Dashed line is based on lower concentrations (10\u22126 M). Calculations were made using PHREEQC (USGS). Figure S8. Prior to each Cr(VI) injection, columns were pre-flushed with 5 PVs of MilliQ water to remove aqueous Fe2+ present in the GR slurry that was mixed with the sand. Manually collected samples were analysed via ICP-OES to determine loss in total Fe as a function of flushed MilliQ pore volumes, which is depicted here. The plotted data show average values of 6 columns (3 columns run at 1\u00a0ml/min and 3 run at 3\u00a0ml/min). Figure S9. Cr(VI) breakthrough curves in S-nZVI amended packed sand columns as a function of inlet solution pH: 4.5 and 7.0 ([(Cr(VI)0]\u2009=\u20090.25\u00a0mM, flow rate\u2009=\u20090.25\u00a0ml/min). The dashed lines help to identify the breakthrough (C/C0\u2009=\u20090.05) and exhaustion (C/C0\u2009=\u20090.9) points on each curve. Table S1. Breakthrough (C/C0\u2009=\u20090.05) and exhaustion (C/C0\u2009=\u20090.9) points expressed in terms of pore volumes (PV) for GRSO4 and S-nZVI sand column experiments performed at different [Cr(VI)0], flow rates and solution pHs (data taken from Fig."}
+{"text": "The influence of initial pH, humic acid (HA), and catalyst dosage was investigated for Cr(VI) containing solutions. Then, a real galvanizing industry effluent (Cr(VI) = 77 mg L-1mg.L\u22121, Zn = 1789 mg L\u22121) was treated. It was observed that Cr(VI) adsorption and photoreduction are greatly favored at low pH values. HA can decrease Cr(VI) adsorption but also acts as holes scavenger, reducing the electron\u2013hole recombination, favoring then the photoreduction. With the immobilized Ce\u2013ZrO2, more than 97% of Cr(VI) was removed from the diluted effluent. These results indicate the feasibility to treat Cr(VI) effluents even in the presence of other metals and natural organic matter. The developed material has great chemical and mechanical resistances and avoids the use of nanoparticles, dangerous for the environment and hard to recover. Moreover, solar light can be used to drive the process, which contributes to the development of more sustainable, cleaner, and cost-effective wastewater treatments.Cr(VI) has several industrial applications but it is one of the most dangerous pollutants because of its carcinogenicity and high toxicity. Thus, the removal of Cr(VI) by photocatalytic reduction was investigated. The catalyst applied, Ce\u2013ZrO Hexavalent chromium is widely used in several industrial processes, such as metal plating, leather tanning, pigment, and refractory production ,2,3. TheSeveral methods can be applied to treat Cr(VI) effluents, such as chemical precipitation, ion-exchange, filtration, solvent extraction, electrochemical, and biological processes ,9. Adsor2  = 77 mg L\u22121, [Zn] = 1789 mg L\u22121).In this context, the objective of this work was to study the photocatalytic reduction of Cr(VI) using Ce-doped ZrO2 nanopowder was synthesized by a sol\u2013gel route developed in previous work total = 89 mg L\u22121, [Cr(VI)] = 77 mg L\u22121, [Zn] = 1789 mg L\u22121.Stock Cr(VI) and humic acid solutions were prepared by dissolving the desired amounts of K2, a specific amount of the catalyst was added to the Cr(VI) or Cr(VI) + HA solutions in order to achieve the desired dosage. As a reactor, a 100 mL borosilicate glass was used. The pH was then adjusted to the desired value. Prior to irradiation, the mixture was kept at room temperature (25 \u00b1 3 \u00b0C), under stirring, in the dark, for 3 h in order to achieve the adsorption equilibrium. Then, the mixture was irradiated with visible light for 3 h under stirring, without any injection of gas. Samples were collected in specific intervals of time and filtered with 0.27 \u00b5m siring filters prior to Cr(VI) analysis. All experiments were performed in duplicate.For the first part of the photocatalytic reduction experiments, with powdery Ce\u2013ZrO\u22122.As the light source, an 18 W white LED lamp  was used. This lamp has a color temperature of 6500 K and, as indicated by the manufacturer, no UV/IR emission. In order to confirm the lamp emission, the UV irradiance (\u03bb < 400 nm) was measured with the light meter HD 2302.0 . No UV emission was detected; thus, no filter was applied in the photocatalysis experiments. When the lamp was positioned just above the beaker, the measured VIS-light irradiance (400 < \u03bb < 700 nm) was 1000 W m2 immobilized on the support (SiC/ZrO2) was applied to the Cr(VI) photoreduction. A piece of 4 \u00d7 6 cm was added to a petri dish containing 30 mL of a Cr(VI) model solution or the galvanizing industry effluent (diluted 7\u00d7 in order to achieve a Cr concentration of 11 mg L\u22121) and positioned under the above-mentioned lamp  concentration was determined by applying the 1,5-diphenylcarbazide (DPC) method , in whic2O was generated using the HSC Chemistry 6.0  software. The stability areas of the Cr species were calculated considering the reduction potentials and equilibrium constants provided by the software at the temperature of 25 \u00b0C and for a total Cr(VI) concentration of 10 mg L\u22121.The Eh\u2013pH (Pourbaix) diagram for the system Cr\u2013H2 nanoparticles was studied. An extensive characterization of the material was performed, including XRD, SEM, TEM, N2 adsorption/desorption, diffusive reflectance, and photo-electronic characterization. In summary, these analyses demonstrated that Ce\u2013ZrO2, prepared by the same sol\u2013gel procedure applied in the present work, presents both tetragonal and monoclinic phases with crystallite size around 15 nm [2 g\u22121, the total pore volume is 0.33 cm3.g\u22121, and pore sizes from 3 to 10 nm [3+ species and holes as O\u2212 species, when irradiated by visible light (>420 nm) [\u25cfOH radicals under visible light irradiation using the spin trap DMPO  [In previous works ,24,25,26>420 nm) . The forN-Oxide)  was alsoN-Oxide) , methyleN-Oxide) , and humIn the following items, we will discuss the activity of Ce\u2013ZrO2 nanopowder on photocatalytic reduction of Cr(VI) and the effects of the catalyst dosage and the initial pH on the Cr(VI) removal from model solutions containing only Cr(VI) or Cr(VI) + humic acid. It can be observed in 2 exposed more active sites available for adsorption [Considering first the system without HA a, for pHsorption . The incsorption ,59.42\u2013 to the monovalent anion HCrO4-. At the same time, in a lower pH range, the surface charge of Ce\u2013ZrO2 becomes less negative, as shown by \u03b6-potential measurements  amounts were adsorbed in more acidic environments. This can be explained by the reduction of the electrical repulsion between chromium species and the zirconia surface. In fact, analyzing the Cr(VI) species distribution b, it canurements a, with t systems ,16.2, this material exhibits a negative charge in a wide range of pH (>3.5) because of the presence of hydroxyl groups (ZrO2\u2013OH) on zirconia surface [2 as the pH increased.Concerning the \u03b6-potential of Ce\u2013ZrO surface . Another surface  also obsConsidering now the effect of humic acid, it is known that both Cr(VI) and HA compete for the adsorption at the zirconia surface  and that\u22121, the presence of humic acid decreased the Cr(VI) adsorption, it may be concluded that HA is preferably adsorbed by zirconia, occupying the active sites of the catalyst. Nevertheless, for the catalyst dosage of 0.5 g L\u20131, the presence of HA has slightly increased the Cr(VI) adsorption. Since humic acid can form complexes with Cr(VI) species [Since, for the catalyst dosage of 1.0 g L species , these CIn 2 amount. However, the comparison with In the systems without humic acid a, as exp2 surface. At the same time, also the photocatalytic reduction of Cr(VI) is favored at lower pH values, as shown in The initial pH value has two effects on Cr(VI) removal. As discussed previously, acid media favor the Cr(VI) adsorption by reducing the electrical repulsion between Cr(VI) species and the Ce\u2013ZrOThere are many reasons explaining this phenomenon: 1. The Cr(VI) photocatalytic reduction is controlled by a surface-reaction step ,65; thus4\u2013 (E\u00b0 = 1.35 V) is higher than that of CrO42\u2013 (E\u00b0 = \u22120.13 V) [4\u2013 is the predominant species, the Cr(VI) reduction is favored;2. As shown in \u22120.13 V) . Since a3. The photocatalytic reduction of Cr(VI) is driven by the difference between the potential of the photo-induced electron at the catalyst CB and the reduction potential of Cr(VI) ,15. From\u22121, higher Cr(VI) total removal percentages were obtained in comparison with the systems without HA  total removal. The important effect of HA was to increase the percentages of the Cr(VI) removed by photocatalytic reduction  adsorption and reduction, decreasing Cr(VI) removal efficiency; 2 valence band, which reduces the electron\u2013hole recombination rate [2 in the presence of organic compounds, such as humic acid [2. The adsorbed HA, being an electron-rich molecule, acts as a scavenger for positive holes at the ZrOion rate  and consmic acid ,16, phenmic acid , ethanolmic acid ,69, and mic acid . \u22121, the HA increases both Cr(VI) adsorption and photocatalytic reduction, which leads to higher Cr(VI) total removals. For a loading of 1.0 g L\u22121, the Cr(VI) adsorption is hindered because HA is preferably adsorbed and occupies the active sites of the catalyst, but still, HA increases the Cr(VI) photocatalytic reduction by scavenging the holes on the VB. In agreement with these statements, the observed rate constants, considering a first-order reaction, were 0.11 and 0.16 h\u22121 for Cr(VI) and Cr(VI) + HA systems, respectively, using 1.0 g L\u22121 of the catalyst. It is worth noting that in all the tests the removal of HA after the end of the irradiation was higher than 90% (data not shown). Therefore, the final effect of HA on Cr(VI) removal is a balance between these two effects, which depends on the solid loading, pH, and HA concentration. For the zirconia loading of 0.5 g L2 works [In view of the experimental results and previous works with zirconia ,25,56 an2 works ,4,68, a 2, the catalysts used for the experiments shown in \u22121 of Ce\u2013ZrO2 and initial pH of four) were recovered and washed with strong basic solutions to promote the complete desorption. Then, the washing solutions were analyzed in order to determine the amount of Cr(VI) released. Once knowing the initial and final Cr(VI) concentrations, and the amount that was adsorbed, it was calculated, through a mass balance, how much Cr(VI) was reduced to Cr(III). As a result, it was observed that ~60% of the adsorbed chromium was reduced to Cr(III) and the rest remained as Cr(VI). For the non-adsorbed Cr(VI), which remained in the starting solution, 36% was reduced to Cr(III) in the presence of HA, whereas this value dropped to 25% without HA. This result corroborates with the proposed mechanism, in which the photocatalytic reduction depends on the adsorption of the Cr(VI) species at the catalyst surface and that the HA favors the photoreduction by acting as a hole scavenger. In addition, the presence of HA can be considered beneficial since, in its presence, Cr(III)\u2013HA complexes are formed when Cr(VI) is reduced to Cr(III). These complexes are less toxic than Cr(III) species [In order to evaluate the fate of the adsorbed Cr species on Ce\u2013ZrO species . 2, only with Cr(VI) and humic acid, were performed, very low Cr(VI) removals were observed, which confirms the actual photocatalytic activity of Ce\u2013ZrO2.Another positive effect of HA reported in the literature is the direct electron transfer from the light-excited HA to Cr(VI), promoting the metal reduction, which would require lower energies than the semiconductor bandgap . However2 was immobilized on a silicon carbide support coated with ZrO2 as the intermediate layer (2 immobilized on the support (2 nanoparticles are a mix of nanometric grains of about 10 nm with twinned grains of a relatively larger dimension (from 15 to 60 nm) and some bigger crystals of about 100 nm [In order to increase the photocatalytic reduction efficiency and avoid the use of suspended nanoparticles, hard to recover, Ce\u2013ZrOte layer a. The FE support b indicatt 100 nm . 2  and tetragonal  phases form during the synthesis. No evidence related to a preferred phase for the photocatalytic activity were reported; however, the presence of both phases could enhance the charge separation at the interface between the two polymorphs [2 P25 (Evonik) [In the X-ray diffractogram of the immobilized Ce\u2013ZrO2 c, it is zirconia , both molymorphs  reducing(Evonik) . 2 intermediate layer and the immobilized Ce\u2013ZrO2. Since the silicon carbide support is black, it absorbs light in the whole UV\u2013Vis spectra, whereas when coated with the white ZrO2 intermediate layer, only UV light is absorbed. The spectrum obtained for ZrO2 layer matches that reported for pristine ZrO2 [2 immobilized on the SiC support with the ZrO2 intermediate layer, an absorption shoulder centered at ca. 330 nm with a tail in the visible region is observed. Analyzing the bandgap transitions, two Egap values are reported, 4.9 and 2.6 eV. The first value is associated with the fundamental VB \u2192 CB transition of ZrO2, which was practically unaffected by the Ce doping, the second value is due to the absorption band associated to the VB \u2192 Ce 4f charge transfer transition [ine ZrO2 ,23,24, iine ZrO2 . In the \u22121 of Cr(VI), no humic acid, and at an initial pH of four. It can be observed that a strong adsorption of Cr(VI) occurred in the dark period, as reported for the suspended catalyst. However, a direct comparison with the powdery catalyst is not possible since it is difficult to determine the amount of Ce\u2013ZrO\u2082 that was immobilized on the support and the related active fraction. Nevertheless, when the visible light irradiation started, it was clearly observed that the photocatalytic reduction of Cr(VI) occurred. Considering a first-order reaction, the observed rate constant (Kobs) was 0.13 h\u22121, value compatible with the ones obtained in the presence of a suspended catalyst.In 2 at pH 4 is shown in \u22121, high removal of chromium was achieved, whereas, for the concentrations of 50 and 100 mg L\u22121, the Cr(IV) removal did not overcome the values around 11%. Thus, it can be concluded that the removal of Cr(VI) is significantly dependent on the initial Cr(VI) concentration in the feed and follows an inverse relationship with the concentration, as reported by other authors [The effect of the initial Cr(VI) concentration on the Cr(VI) removal with immobilized Ce\u2013ZrO authors ,73. The 2 for the model Cr(VI) solution, the next step was to evaluate the efficiency of this material in the treatment of a real Cr(VI) effluent, since complex matrixes can affect significantly the photocatalytic mechanism [In view of the satisfactory results obtained with the supported Ce\u2013ZrOechanism ,75.\u22121, respectively. As reported in the previous item, the Cr(VI) removal efficiency decreases considerably at higher Cr(VI) concentrations. Therefore, prior to the experiments, the effluent was diluted seven times in order to achieve a Cr(VI) concentration of 11 mg L\u22121. It is worth noting that the total Cr concentration in the effluent is 89 mg L\u22121, which means that a small amount of dissolved Cr(III) is also present. Since the effluent does not contain dissolved organic matter, one test was made with the diluted effluent spiked with HA. The results of the photocatalytic reduction of Cr(VI) using immobilized Ce\u2013ZrO2 are shown in The galvanizing industry effluent studied here contains a high concentration of Zn and Cr(VI), 1789 and 77 mg L2+ cations present in the solution, which also competes for adsorption on the negatively charged Ce\u2013ZrO2 surface. As shown in 2+ (\u22120.76 V) is much lower than the one of Cr(VI) [From f Cr(VI) .The Cr(VI) removal efficiencies for the model Cr(VI) solution and the diluted galvanizing effluent were ~76% and >97%, respectively. The first reason for the higher removal of Cr(VI) with the galvanizing effluent is that the effluent pH value is lower (~3.5) than the one tested for the model solution (4.0). As discussed previously, the Cr(VI) photoreduction is strongly favored in more acidic media. Another reason for the higher removal of Cr(VI) is the presence of Cr(III) and Zn(II) in the galvanizing effluent, which help to maintain pH stability by forming hydroxo-complexes acting as pH buffer . 2 and also by the mesoporous silicon carbide support. Therefore, there was no humic acid available anymore to participate in the reaction and affect the Cr(VI) removal. On the contrary, the presence of HA does not seem to have a significant effect on Cr(VI) removal, even if the effect was clearly visible when the suspended catalyst powder was used. Actually, in the case of the diluted effluent spiked with HA, the humic acid was no longer detectable by the UV\u2013Vis analyses carried out after a 1 h experiment in the dark, as it was completely adsorbed by the immobilized Ce\u2013ZrO\u22121 [The final concentrations of Cr(VI) in the samples  indicate\u22121 ,77. Ther2 under visible light irradiation. For the model Cr(VI) solution, it was observed that higher catalyst dosages and lower initial pH values favor Cr(VI) adsorption and photoreduction. The reason is that in more acidic media, the electrostatic repulsion between the zirconia surface and Cr species is limited. At the same time, the driving force for the reaction is increased because CrO42\u2013 converts to HCrO4-, which has a higher reduction potential and less repulsion by the zirconia surface.In the present work, we studied the removal of Cr(VI) by photocatalytic reduction promoted by suspended and immobilized Ce\u2013ZrO2, it reduces Cr(VI) adsorption by occupying the active sites. On the other hand, HA acts as a hole scavenger, reducing the electron\u2013hole recombination, favoring then the photocatalytic reduction of Cr(VI). Therefore, the combination of these effects on the total Cr(VI) removal was dependent on the catalyst dosage, but in all cases, the amounts of Cr(VI) reduced with the irradiation were higher in the presence of HA, even at higher pH values. In the best condition, it was possible to remove 70% of Cr(VI) from the model solution containing only Cr(VI) at an initial pH of four, using 1.0 g L\u22121 of Ce\u2013ZrO2. Regarding the effects of humic acid, there is a balance between beneficial and detrimental effects. Since HA is preferably adsorbed by Ce\u2013ZrO2 demonstrated the feasibility of immobilizing this catalyst without changing its photocatalytic properties. The immobilized Ce\u2013ZrO2 was able to treat a diluted galvanizing industry effluent with a high concentration of zinc, achieving Cr(VI) removals greater than 97% after visible light irradiation, which would allow its discharge into surface waters. The presence of HA did not affect the processes.The experiments with the supported Ce\u2013ZrOThese results indicate that immobilized Ce-doped zirconia can be applied to treat Cr(VI) effluents even in the presence of other metals and naturally occurring organic matter, such as humic-like substances. The advantages of this material are its great chemical and mechanical resistances and the chance to avoid nanoparticle recovery. In addition, there is the possibility to use visible light sources, such as solar light, which contributes to the development of more sustainable, cleaner, and cost-effective wastewater treatments."}
+{"text": "C44, bulk modulus (B), shear modulus (G), and Young\u2019s modulus (E), with high concentrations of Mo + W. Element W is brittle and has high density. Thus, low-density Mo can substitute part of W. Vanadium (V) has low density and plays an important role in decreasing the brittleness of the V\u2013Mo\u2013Nb\u2013Ta\u2013W system. Niobium (Nb) and tantalum (Ta) have relatively small influence on elastic properties. Furthermore, the calculated results can be used as a general guidance for the selection of a V\u2013Mo\u2013Nb\u2013Ta\u2013W system.The elastic properties of seventy different compositions were calculated to optimize the composition of a V\u2013Mo\u2013Nb\u2013Ta\u2013W system. A new model called maximum entropy approach (MaxEnt) was adopted. The influence of each element was discussed. Molybdenum (Mo) and tungsten (W) are key elements for the maintenance of elastic properties. The V\u2013Mo\u2013Nb\u2013Ta\u2013W system has relatively high values of  HEAs haONEL@718 . In a VMONEL@718 .Recently, many reports have shown that the best properties of RHEAs may generally be displaced from equilibrium compositions; thus, the studied compositions become complicated ,8. Some The present study reports a first-principles design of a VMoNbTaW alloy. The aims are to decrease the brittleness and density of a V\u2013Mo\u2013Nb\u2013Ta\u2013W system. The elastic properties of seventy different compositions were calculated. The influence of each element was discussed.\u22127 Hartree. A Broyden\u2013Fletcher\u2013Goldfarb\u2013Shanno (BFGS) minimization algorithm was introduced to deal with the geometry optimization problems. The convergence criteria for the maximum geometry change and force were 1 \u00d7 10\u22123 Bohr and 1 \u00d7 10\u22123 Hartree/Bohr, respectively.CP2K was introduced for first-principles calculation and it is efficient for larger systems. CP2K is a quantum chemistry and solid-state physics software package . QUICKSTB and C44 were also calculated. All bulk moduli fluctuated around 143.3 (\u00b12) GPa and all C44 fluctuated around 36.2 (\u00b13) GPa. The scattered diagram is shown in MaxEnt structures were generated by a Monte Carlo simulation code in python. A repeat loop was written in the code to make sure all the elements were distributed homogeneously in the model . In orde0.1Mo0.2Nb0.1Ta0.4W0.2 is shown as an example in All components of the VMoNbTaW alloy have a BCC lattice and, thus, the formation of BCC substitution solutions was the most probable. This was confirmed by diffraction analysis of these alloys . The 4 \u00d7B and equilibrium lattice configuration were determined from the minima of the curves according to the Birch\u2013Murnaghan equation of state (B\u2013M EOS), as presented in Equation (1). V, V0, B, E, and E0 are volume, equilibrium volume, bulk modulus, total energy, and equilibrium energy, respectively. In order not to exceed the elastic limit, the changes in V should be kept within 3%.The calculated bulk modulus C11, C12, and C44. They can be calculated by applying small strains to the equilibrium lattice configuration, which transforms the lattice vector a according to the rule [The cubic crystal has three independent elastic constants: the rule  shown ine =  is the strain vector. The different values of e were applied to the equilibrium lattice configuration according to G, Young\u2019s modulus E, and Poisson\u2019s ratio \u03c5.The following equations were used to calculate Shear modulus Due to the lack of experimental data of VMoNbTaW, the elastic properties of pure V, Mo, Nb, Ta, and W were calculated to prove the accuracy of the calculated data. Seventy different compositions of the V\u2013Mo\u2013Nb\u2013Ta\u2013W system were calculated. The results are shown in C11 + 2C12 > 0, C11 > C12, C44 > 0. C44, B, G, E, B/G and \u03bd are presented in scatter-plots in All the structures were found to fulfill the mechanical stability criteria. The mechanical stability criterion of the cubic structure is C44. In C44 are bigger than the area below. There is also a data blank area between them. C44 increases with the increase of the W + Mo concentration. Thus, W and Mo show significant influence on C44. This may be due to the fact that the C44 of Mo (125 Gpa) and W (163 Gpa) are higher than the C44 of V (46 Gpa), Nb (31 Gpa), and Ta (82 Gpa). The densities of W and Mo are 19.350 g/cm3 and 10.390 g/cm3. In order to decrease the density and keep the high hardness of the V\u2013Mo\u2013Nb\u2013Ta\u2013W system, increasing Mo concentration and decreasing W concentration may be a feasible method.According to Reference , there iB can be used to describe the average atomic bond strength. The overall trend of the influence of alloying elements on B is shown in B decreases, while B increases with the increase of Mo concentration. Additionally, B decreases slightly with the increase of Nb concentration. B. B increases with the increase of W concentration, as shown in B decreases in the Nb = 0.1 area. This can be attributed to the increase in the concentration of element V. Arrow a in B decreases with the increase of Nb. Arrow c in B decreases with the decrease of W. A sharp variation in some points  can be seen in B increases sharply. In summary, Mo and W can help to increase B. Elements V and Nb have a negative effect on B. B has a high value in each period with W + Mo \u2265 0.4.According to Reference , bulk moE and the shear modulus G [B/G < 2 are associated with brittleness; otherwise, the materials may behave as ductile. Materials with \u03c5 > 0.31 have good ductility. Otherwise, the materials are considered brittle.The hardness of materials can be related to Young\u2019s modulus odulus G . The genodulus G , materiaG and E have the same trend, while B/G and \u03bd also have the same trend. It can also be seen that there is an inverse relationship between them. Element V has a negative effect on G and E, and a positive effect on B/G and \u03bd. Thus, the trend of E, B/G, and \u03bd can be predicted from the trend of G. G and E and exhibits a negative effect on B/G and \u03bd, while elements Nb and Ta have no obvious effect. In summary, element V can help to increase the ductility of the V\u2013Mo\u2013Nb\u2013Ta\u2013W system. G and E have a relatively high value with W + Mo \u2265 0.4.C44, B, E, and G, with high concentrations of Mo + W. The concentration of Mo + W shows the most prominent effect on C44. Due to the high density of W, element Mo can be used to substitute part of W. In this case, the concentration of W should be reduced.Mo and W are key elements in the V\u2013Mo\u2013Nb\u2013Ta\u2013W system. The V\u2013Mo\u2013Nb\u2013Ta\u2013W system has relatively high values of 3) and plays an important in decreasing the brittleness of the V\u2013Mo\u2013Nb\u2013Ta\u2013W system.V has low density (6.110 g/cmIn comparison, Nb and Ta have relatively small influence on elastic properties.In order to improve the ductility and decrease the density of the V\u2013Mo\u2013Nb\u2013Ta\u2013W system, the elastic properties of seventy different compositions were studied. This work concludes as follows:"}
+{"text": "Over a range of stimulation intensities, we found that the monosynaptic current varied significantly between the cell types, in the order of eTC > sTC > MC. The prolonged component was smaller in sTCs vs. both MCs and eTCs. sTCs also had much higher whole-cell input resistances than MCs, reflecting their smaller size and greater membrane resistivity. To evaluate how these different electrophysiological aspects contributed to spiking of the output MCs and sTCs, we used computational modeling. By exchanging the different cell properties in our modeled MCs and sTCs, we could evaluate each property's contribution to spiking differences between these cell types. This analysis suggested that the higher sensitivity of spiking in sTCs vs. MCs reflected both their larger monosynaptic OSN signal as well as their higher input resistance, while their smaller prolonged currents had a modest opposing effect. Taken together, our results indicate that both synaptic and intrinsic cellular features contribute to the production of parallel output channels in the olfactory bulb.A common feature of the primary processing structures of sensory systems is the presence of parallel output \u201cchannels\u201d that convey different information about a stimulus. In the mammalian olfactory bulb, this is reflected in the mitral cells (MCs) and tufted cells (TCs) that have differing sensitivities to odors, with TCs being more sensitive than MCs. In this study, we examined potential mechanisms underlying the different responses of MCs vs. TCs. For TCs, we focused on superficial TCs (sTCs), which are a population of output TCs that reside in the superficial-most portion of the external plexiform layer, along with external tufted cells (eTCs), which are glutamatergic interneurons in the glomerular layer. Using whole-cell patch-clamp recordings in mouse bulb slices, we first measured excitatory currents in MCs, sTCs, and eTCs following olfactory sensory neuron (OSN) stimulation, separating the responses into a fast, monosynaptic component reflecting direct inputs from OSNs and a prolonged component partially reflecting eTC-mediated feedforward excitation. Responses were measured to a wide range of OSN stimulation intensities, simulating the different levels of OSN activity that would be expected to be produced by varying odor concentrations  In many sensory systems, the brain structure that is involved in the initial processing of information is endowed with multiple types of output cells that carry different types of information about the stimulus. One example is in vision, where the many types of retinal ganglion cells (RGCs) create parallel pathways from the retina to the Lateral Geniculate Nucleus (LGN). The diversity of RGCs is theorized to increase the sensitivity of the retina to a wider range of inputs  at postnatal age 13\u201326 days, of both sexes. While housed in the UCAMC facility, mice had full, and continuous access to food and water.Horizontal slices (300\u2013400 \u03bcm) were prepared from OBs of mice following general isoflurane anesthesia and decapitation, as described previously . Patch pipettes for whole-cell recordings (4\u20138 M\u03a9) contained 125 K-gluconate, 2MgCl2, 0.025 CaCl2, 1 EGTA, 2 NaATP, 0.5 NaGTP, 10 HEPES, pH 7.3 with KOH. The sodium channel blocker QX-314 (10 mM) was included to block action potential firing. Current and voltage signals were recorded with a Multiclamp 700A amplifier (Molecular Devices), low-pass filtered at 1\u20132 kHz, and digitized at 10 kHz. The reported value for the holding potential for our excitatory current measurements (\u221277 mV) has been corrected for a liquid junction potential. OSN stimulation was performed by placing a broken-tip patch pipette (10 \u03bcm diameter) in the ON layer, ~50 \u03bcm superficial to the target glomerulus of the test cell. Brief pulses (100 \u03bcs) triggered by a stimulus isolation unit were applied, with an interstimulus interval of 20 s. Data were acquired using AxographX. Morphological analysis of the cells, including determination of target glomeruli, was done for whole-cell recordings by including Alexa 488 (100 \u03bcM) in the patch pipette. Selected cells had apical dendrites targeted to glomeruli at the slice surface, which facilitated stimulation of OSNs at target glomeruli.The base extracellular recording solution contained the following (in mM): 125 NaCl, 25 NaHCOCell types were defined based on several criteria, as described previously  that was near the reversal potential for chloride for our experiments (\u221289 mV). This should have minimized the contribution of potentially contaminating GABAIn the analysis of EPSCs evoked by OSN stimulation, the peak of the OSN-EPSC was defined as the maximum current response within 6 ms of stimulus onset. Peak OSN values that were plotted as a function of OSN stimulation intensity were fitted to a sigmoidal function:a2 was defined as the maximum response, x was the OSN stimulation intensity, x0 was the stimulus%max50 or the stimulus necessary to elicit half of the maximum response, dx was the steepness of the sigmoidal curve, and a1 was the minimum response.with In the estimates of unitary EPSCs (uEPSCs), a minimum stimulus intensity was found that produced a response in <50% of trials, for at least 10 trials. We then measured the amplitude of the unitary EPSC by taking the average amplitude over 1.5 ms of each uEPSC event, centered on the time frame with the largest current response. Spontaneous EPSCs (sEPSCs) were found using an event detection search in AxographX. The amplitudes were measured with the same methodology as the uEPSC amplitudes.In the analysis of the prolonged current components, estimates of its magnitude were obtained by integrating the current measured 6\u2013300 ms after stimulation. In most cells, the decay of the OSN-EPSC appeared to be complete by 6 ms after the stimulus. We also measured the amplitude of the current at 300 ms after stimulus by taking the average current over 1 ms.n = 11) was ~29% that of MCs . The values for the relative size of the sTC soma and lateral dendrites were based on values reported for sTC reconstructions .The MC and sTC models were first used to estimate the spiking properties of MC/sTCs at different OSN stimulation intensities. The spiking properties (number of spikes and spike probability) were determined for each cell and at each OSN stimulation intensity based on three trials of experimentally recorded current at each stimulation intensity that were input into the model cell. This procedure applied over our population of test MCs and sTCs enabled us to accumulate statistics about the spike responses of each cell type. An estimate of the stimulus-response relationship for each MC and sTC was obtained by fitting a sigmoidal function (see Eqtn 1 above) to the relationship between stimulation intensity and the number of spikes for each cell (see nMC = 8) with a Fisher Combined Test to determine the statistical significance of the difference in MC responses caused by substituting the OSN-EPSC.An exchange procedure in NEURON was used to estimate the contribution of each of three cellular properties to the sTC/MC spike response. The properties exchanged included the OSN-EPSC, the prolonged current, and the intrinsic cell properties. When the prolonged current was exchanged, we grouped the slow transient and step current together as one component. For the intrinsic property substitution, we grouped cell morphology, cell resting potential, and resistance per unit area to be a single component to be exchanged at once. Whether the exchange of a particular cellular property induced a statistically significant change in spike number at a given OSN stimulation intensity was determined through the following procedure, as described here for substituting the OSN-EPSC of the sTC into the MCs. We first determined a Mann-Whitney U statistic for each MC by comparing the number of spikes across three experimental trials in the intact version of that MC (with its \u201cnative\u201d OSN-EPSC) with the number of spikes in that MC with an OSN-EPSC substituted in that reflected the corresponding trials in all sTCs. Because there were seven test sTCs and three trials were recorded per sTC, this per-MC statistical analysis involved comparing 21 spike number values representing substituted OSN-EPSCs from the sTCs to three spike number values in the native MC . We then combined the Mann-Whitney U results for each MC  differed modestly from the mean values reported for sTCs by Tavakoli et al. . For exaFigure 4F). The sensitivity of spiking to changes in OSN stimulation intensity for each cell was then defined by the squared derivative of the fitted sigmoidal function at every stimulus intensity.This analysis compared the sensitivity of a population of bulbar output cells under two conditions, Condition A, when the population included MCs and sTCs with distinct relationships between OSN stimulation intensity and spike number , and Condition B in which the cells had properties that were the average (\u201chybrid\u201d) between that of MCs and sTCs. The stimulus-response behavior of any one cell was defined by the sigmoidal curve fitted to the simulated OSN stimulation vs. spike number data for that cell . The overall sensitivity measure for the cell population in Condition A  was the average of the squared derivatives for MCs and sTCs, weighted by the number of cells in each subgroup . For Condition B, the overall sensitivity measure  was the squared derivative of the stimulus response relationship of the average hybrid MC/sTC .For Condition A, we wished to remove the contribution to the sensitivity measurement of the variability of spiking behavior amongst the cells within a subgroup (MCs or sTCs). Hence, the stimulus response curve for each cell within a subgroup was the average of the fitted sigmoidal curves for that group . Files to be included in ModelDB will be the hoc files with the morphologies of the model MC and sTCs, mod files that specify the intrinsic properties and excitatory conductances (OSN and prolonged), example current recordings, and binary search python files that can reproduce the example traces in Figures 4Aii,v,B,C. These files can also be used to fit other current recordings with excitatory conductances and convert simulated conductances into voltage traces and spiking activity. We will also include hoc and mod files with pre-fitted conductances to reproduce the traces in Figures 5C,D. All other raw experimental data supporting the conclusions of this article will be made available upon request by the authors, without undue reservation.Our modeling results will be made available at the time of publication in ModelDB (Vhold = \u221277 mV) in response to electrical stimulation of OSN fibers in mouse OB slices. Cell types were determined based on their location in the bulb  included a component that occurred with a short onset delay (\u2264 2 ms) and fast rise time, consistent with it being the monosynaptic EPSC reflecting direct transmission from OSNs  and the stimulus intensity needed to reach 50% of this maximum (stimulus%max50). In terms of OSN-EPSCmax, eTCs had much larger values  than sTCs  or MCs . OSN-EPSCmax at high intensities should reflect the current arising from the activation of all OSN axons that targeted the test cell. Hence, differences in OSN-EPSCmax between cell types should reflect differences in the current produced when all such OSN axons were activated. Importantly, the amplitudes of each cell's OSN-EPSC appeared to saturate or nearly saturate at high stimulation intensities  and sTCs (84 \u00b1 4 \u03bcA) had similar values , consistent with the cells receiving input from populations of OSN axons with similar spatial distributions and levels of excitability. We found somewhat smaller values for stimulus%max50 for eTCs . We were careful not to over-interpret this difference however since the leftward shift in the stimulus-response relationship for eTCs could have reflected series resistance errors generated by the very large OSN-EPSCs in eTCs (since the errors would have most impacted the largest currents).In the analysis of OSN-EPSCmaxproduced by stimulation of all  OSN axons at a given glomerulus. The different values for OSN-EPSCmax in MCs, sTCs, and eTCs could be explained by differences in the number of OSN axons that target the cells or differences in the current produced by each single OSN axon. To disambiguate between these possibilities, we used two approaches, the first of which was to record currents at \u201cminimal\u201d stimulus intensities, when current responses were produced in <50% of the trials (n = 6) than either MCs  or sTCs , suggesting that larger currents generated by single OSN axons are a major factor underlying the larger OSN-EPSCmax in eTCs vs. MCs/sTCs. That the single OSN axon current is much larger in eTCs than MCs/sTCs was also supported by recordings of spontaneous EPSCs (sEPSCs) that were conducted in parallel (n = 11) than sTCs  or MCs . This was distinct from the OSN-EPSCmaxvalues, which were ~2-fold larger in sTCs than in MCs (see above). One interpretation of these data is that sTCs and MCs have similar sized currents arising from stimulation of single OSN axons but sTCs have more convergent axons than MCs. However, we caution against such an interpretation because the small size of the uEPSCs/sEPSCs in MCs/sTCs meant that we were generally operating at the limits of our experimental detection capabilities. That the average sEPSC in MCs was likely to be much smaller than our reported values was also supported by the extremely low frequency of sEPSCs in MCs  is not the rapid monosynaptic EPSC but rather a prolonged current that in part reflects feedforward excitation mediated by eTCs  and smaller than that of MCs at 100 \u03bcA . The prolonged currents were also somewhat larger in eTCs vs. MCs at 100 \u03bcA stimulation (p = 0.0378).We first compared the size of the prolonged currents by integrating the charge in a window (6\u2013300 ms) after OSN stimulation that should have mainly excluded the OSN-EPSC. Across cell-types and stimulation intensities, the most consistent observation was that the prolonged current was smaller in sTCs than in MCs or eTCs . The curnMC= 10; sTCs: 27 \u00b1 10 pA, nsTC = 13; p = 0.028, Mann-Whitney U test) and 200 \u03bcA . eTCs also had much a larger sustained current than sTCs at 200 \u03bcA . These results indicate that sTCs are unique in not having a large sustained current at high OSN stimulation intensities.Direct visualization of the data traces in in vitro current response to OSN stimulation relates to the spike responses of the output neurons of the bulb. For this comparison, we focused our analysis on MCs and sTCs, since eTCs are not output neurons, and there have been no in vivo characterizations of eTCs . Our strategy, as outlined in n = 8; sTC: 447 \u00b1 29 M\u03a9, n = 7; p = 0.0001, Mann-Whitney U test).We next set out to understand how the n = 8) and sTCs (n = 7), the number of spikes and probability of spiking in a given trial, at each of four OSN stimulation levels. At all stimulus levels except 30 \u03bcA, sTCs had a significantly higher average number of spikes than MCs . sTCs also had a significantly higher spike probability at 50 \u03bcA  and 100 \u03bcA , but the spike probabilities converged to near 100% for both cells at 200 \u03bcA. These spiking characteristics are consistent with MCs having a reduced sensitivity vs. sTCs to similar levels of OSN activity.Within this framework, we quantified two aspects of the spiking behavior of MCs  to the stimulus intensity vs. spike number data for that cell . This yiin vivo in studies examining the odor concentration dependence of spiking  into MCs (n = 8 cells) increased the number of spikes . This can be seen in the example traces in MCSTCmono in p = 0.0003; Fisher Combined tests of Mann-Whitney U tests). This can be seen in the example traces in sTCMCmono in Using this approach, we found that the monosynaptic OSN conductance had a modest but significant impact on the number of spikes . At everincreased the number of spikes at every stimulus intensity  led to a significant decrease in the number of spikes at every stimulus intensity , the cellular morphology, and resting potential  in which we assumed that the MC and sTC populations each individually carried information about the level of OSN activity  and a second (Condition B) in which we grouped all MCs and sTCs into one population with a stimulus-response relationship that was average between that of MCs and sTCs. The stimulus-response curves were generated from the sigmoidal curves that were fitted to the OSN stimulation intensity vs. spike number data for individual MCs/sTCs , either It should be noted that our analysis, which was based on the average behavior of MCs and sTCs for both Conditions A and B, ignored cell-to-cell variabilities within the subpopulation of MCs or sTCs. Such variance in the stimulus-response relationships for individual MCs or sTCs can be observed in the different positions along the x-axes for the fitted sigmoidal curves in In this study, we combined experimental and computational methods to examine mechanisms that could contribute to differences in the spiking properties of different populations of excitatory neurons in the olfactory bulb. The principal focus was on MCs and a subpopulation of output TCs, the sTCs. Our main findings in our comparison between MCs and sTCs were that: (1) sTCs have a number of differences in their excitatory currents from MCs, including larger direct OSN input currents but smaller prolonged currents; (2) sTCs are more sensitive than MCs, producing more spikes at lower levels of OSN activity; (3) the greater spiking in sTCs reflects both the greater OSN input signals as well as differences in their intrinsic properties; and (4) differences in sensitivity of sTCs vs. MCs enhances the ability of the bulb to encode changes in stimulus intensity. We also characterized experimentally a number of novel properties of the excitatory currents of another class of TCs, the eTCs. These points are discussed below.in vivo have indicated that MCs and TCs have markedly different responses to odors. MCs require higher concentrations of an odorant to be activated, displaying rightward-shifted odor-concentration vs. spike response curves (Igarashi et al., in vivo (Griff et al., in vivo differences between MCs and TCs.Recent physiological studies in vivo analysis (Griff et al., in vivo for MCs vs. TCs. For example, we found in our analysis that many of the largest changes in spike number and probability upon exchanging the MC/sTC components occurred at low OSN stimulation intensities, when sTCs but not MCs were significantly active. This argued that the specific components in question contributed to shifts in the activation curves.Our study adds to a growing literature that has examined the mechanistic basis for differing odor-evoked spike responses in MCs vs. TCs. In one such study, conducted by Burton and Urban , the autin vitro rather than in vivo. Perhaps chief amongst them was the nature of the stimulus. While an odor activates OSNs in a distributed fashion over the duration of a sniff, we examined responses to a single electrical stimulus pulse applied to OSN axons. A priori, it is not clear whether this difference in stimuli should impact any of our fundamental conclusions about what underlies the spike differences in MCs and sTCs, but it is nevertheless a notable limitation of our study. At the same time, our in vitro approach using a discrete stimulus applied locally to OSNs offered the advantage of a much-simplified system in which we could largely ignore the contribution of interglomerular interactions and centrifugal feedback mechanisms on spike differences between MCs and sTCs. This enabled us to focus on the impact of local excitatory mechanisms and cell intrinsic properties on the spike differences. There are certainly a number of cellular and synaptic mechanisms that we did not examine that could contribute to spiking differences between MCs and sTCs in addition to the ones we identified. For example, differences in GABAergic inhibition are already known to contribute to spike timing differences between MCs and some TCs (Fukunaga et al., There are naturally caveats associated with the fact that our analysis of MCs and sTCs was conducted Besides providing insight into mechanisms underlying spiking differences between MCs and sTCs, our study also provided a number of novel insights into the basic cellular and synaptic properties of various excitatory bulbar neurons. First, we extended the available information about what is known about differences in OSN-EPSCs between MCs and eTCs, which are a class of glutamatergic interneurons that reside in the glomerular layer. Prior reports have shown that, at least at some OSN stimulation intensities, the OSN-EPSC in eTCs is much larger than in MCs (Najac et al., We also characterized more prolonged excitatory currents that are evoked by OSN stimulation across MCs, sTCs, and eTCs. Interestingly, we found that sTCs stood out from MCs and eTCs in the small magnitude of the prolonged currents, especially in a sustained component that persisted for hundreds of milliseconds after OSN stimulation. The sustained evoked current, at least in MCs, reflects activation of Group I mGluRs (Schoppa and Westbrook, It should be noted that there were some sources of uncertainty in the analysis of the prolonged excitatory current components. For example, the extracellular solution in which the slow currents were recorded did not include added glycine, which is a co-agonist for NMDA receptors. Because slow excitation of at least MCs partially depends on NMDA receptors (Carlson et al., A final issue that we addressed in our study was what broader function might be served by having two classes of output cells in the bulb with differing sensitivities to OSN activity (Igarashi et al., Amongst sensory systems, the olfactory bulb is hardly unique in having multiple types of output cells that provide parallel pathways for information flow. Such pathways are perhaps best characterized in the retina where there are at least 30 types of ganglion cells each with distinct tuning to visual stimuli Masland, . Some ofThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Institutional Animal Use and Care Committee, University of Colorado, Anschutz Medical Campus.SJ, JZ, and NS: design of experiments, interpretation of results, and writing of manuscript. SJ: acquisition and analysis of data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Objective: The elective subject \u201ccareer management for medical students\u201d is presented as an example of teaching gender sensitivity issues among medical studies at Leipzig University. The project report shows the interim results of promoting gender-sensitive teaching at the Medical Faculty of Leipzig University, as well as the elective\u2019s contribution to the development of gender sensitivity at the entire university.Method: Project Description and Results show the organization/procedure, participants and detailed contents of the elective since it began in Winter Term 2010/11. The research examines the elective\u2019s mandate at the Medical Faculty and beyond, i.e. by comparing with the efforts of other universities. Results: The elective is the first subject for credit within the clinical curriculum of medical studies at Leipzig University that connects the topics of gender sensitivity and career management. It creates a view of the specialties of medicine and research as they relate to gender, and also on the options of a medical career and touches the topic of gender equality. A faculty survey in the winter semester of 2011/12 reveals that nearly one third of the medical students want an extension of the curriculum around the topic of gender or even an independent subject \u201cGender Medicine\u201d. The elective is part of a cycle promoting gender equality at Leipzig University.Conclusion: The elective initiates and continues the implementation of gender-sensitive teaching at the Medical Faculty of Leipzig University. The management of the elective aims at the permanent establishment of the subject in the curriculum in order to encourage career ambitions early \u2013 especially for women. In their 2013 equality report, Leipzig University (LU) conjectured \u201ca particular problem of compatibility within the university medicine\u201d . The MedWhat are the interim results for the promotion of gender-sensitive teaching at the MF of LU \u2013 as they relate to the establishment of the elective \u201ccareer management for medical students\u201d?Is it legitimate to interpret the establishment of the elective \u201ccareer management for medical students\u201d at the MF of LU as a contribution to improving gender sensitivity for the entire university?https://www.gesetze-im-internet.de/_appro_2002/BJNR240500002.html]. The regulations require that at most six medical students are entrusted to a single instructor or mentor. The elective is designed as a one-week course, consisting of 27 lessons of 45 minutes each. The students\u2019 work and contributions throughout the course are graded by the supervising instructor at the end of the week. An evaluation of the class can be provided by faculty if the instructor requests it. Several times during the elective, two groups of six students and at least two instructors congress to discuss in plenum. The subject is matter of the educational branch of the Institute for Social Medicine, Occupational Health and Public Health of the LU. Some lessons take place in the mentor\u2019s occupational areas. The elective subject has been offered to medical students since Winter Term 2010/11 by the MF of the LU. It was approved by the local study committee and faculty council. It is one of many electives for the official course distribution requirements, which need to be fulfilled by every student of advanced medical studies to get admitted for second state examination . RegardiThe elective is presumed to be a short-term mentorship. Medical students are mentees. They can register for the course in their ninth semester. The mentors are recruited by the managing instructor from the diverse field of medicine. In this way the mentees meet with several professors, chief physicians, general practitioners and other office-working physicians, researchers, interns, and also with service providers like coaching experts or management consultants. These experts play a key role since they teach the mentees by personal interactions.https://www.insala.com/Articles/what-is-flash-mentoring.asp]. Supportive questions are: Do you want to start out as an intern in a hospital? Would you prefer a general public hospital to a university clinic? Do you care for any additional conditions? Career management based on economic maximum principle brings focus to the benefit of synergy and claims calculability. The mentorship inspires the students to shape their own career. Individual expectations of the students from the subject are welcome with willingness to respond and fulfill. While the mentors keep up their main assignment as role models, they bear in mind the opportunities for bidirectional learning through first-hand interaction  or any other valid reason obtains accommodation for the disadvantages.\u201d The elective deals proactively with compatibility of study or rather occupation with family, among other things. In Winter Term 2012/13 the Ulm University was the first nationwide to implement an independent course called \u201cGender Medicine\u201d as part of its Medical Faculty\u2019s curriculum so that each of the medical students acquires the key qualification \u201cgender competence\u201d. Applying this knowledge to patients and defining intrapersonal and interpersonal roles through self-reflection are the explicit goals . The eleYes. The small-group-based short-term mentoring program asks about all the individuals at the LU and the LUM, respectively. What are their experiences regarding their right to equal opportunities and equality? Political correctness should be evident in all actions. As a constructive tool in the elective the principle of error-friendliness by Urmila Goel is lived out: The irreversibility of the consequences of discrimination is set aside within the learning environment . A simplWhat are the implications? In its guiding principles, the LU proclaims: \u201cThe university promotes actual equality of men and women.\u201d . In ordeThe authors thank the Medical Faculty of Leipzig University (deanship and teaching department) for the continuous support of the elective \u201ccareer management for medical students\u201d and in the writing of this article.The authors declare that they have no competing interests."}
+{"text": "Staphylococcus epidermidis, found in all but one of the HM samples; the presence of several species of coagulase-negative Staphylococci was also noted. Next, we sought to develop a platform towards antibacterial treatment using Zn2+ ions that have recently been found to be potent against contaminants isolated from bovine milk. Zn2+ efficiently inhibited the growth of viable aerobic population and S. epidermidis in HM. Growth was also inhibited in other Gram-positive bacteria such as Bacillus cereus, a well-known food-borne pathogen. S. epidermidis and B. cereus cells grown in the presence of zinc were taken for microscopic evaluation, aiming to demonstrate zinc\u2019s antimicrobial mode of action morphologically. Images obtained using scanning electron microscopy indicated leakage of cellular content and cell lysis in S. epidermidis. Besides, B. cereus cells showed abnormalities in their cell surface and complete loss of flagella upon treatment with zinc. Along with the above findings, it should be noted that this was a pilot study that tested how high doses of Zn2+ affect breast milk as a product. Further research is likely needed on the safety of consumption of Zn2+-treated HM in infants and older children. Donor human milk (HM) obtained at HM banks is exceptionally crucial for the feeding and treatment of preterm infants. Bacterial contaminations of HM in various stages of its handling are very common and can lead to disqualification of donations or severe infections in worse cases. Hence, HM donations are subject to strict bacteriological evaluations pre- and post-pasteurization. The main contaminating species vary between countries, banks and donors and even exhibit inter-individual variation. We initiated an assessment of the bacteriological composition of HM donated by women hospitalized in a neonatal intensive care unit in Israel. The most common bacterium identified was  Exclusive human milk (HM) feeding for the first six months of life, with continued breastfeeding for one to two years or longer, is considered as the recommended standard for infant feeding . HM offeStaphylococcus aureus and Enterobacteriaceae before heat treatment , filamentous fungi and listeria. The results were then transferred to a Krona visualization program. Strains identified by the instrument as \u201chighly probable species identification\u201d  were included and listed for their presence in the original sample. Isolates were taken for further identification using 16S rRNA sequencing in order to assure that the identification was accurate.HM samples were collected from women hospitalized at the Beilinson Women Hospital in Petach Tikva, Israel. Fourteen randomly selected raw HM samples were defrosted after being stored at temperature of \u221280 \u00b0C. Aliquots of 100 \u00b5L were plated on four types of agar plates: Lysogeny broth , PCA , brain heart infusion (BHI) and De Man, Rogosa and Sharpe , then incubated at 37 \u00b0C for 48 h. Based on differentiating morphologies, colonies were selected from each agar plate. The selected colonies were then streaked and grown separately on agar plates to assure purity. Following separate growing, colonies were identified using matrix-assisted laser desorption/ionization MALDI TOF mass spectrometry . Isolates were prepared for the analysis as follows: the selected colonies were suspended in 300 \u00b5L pure - polymerase chain reaction (PCR)-grade water in 2 mL sterile plastic tubes, along with 900 \u00b5L of ethanol. Tubes were then centrifuged at 13,000\u00d7 2 as the source for Zn2+ ions. Addition of Zn2+ ions was performed by diluting a 1 M solution of ZnCl2  directly into the growth medium to achieve a desirable experimental concentration. For evaluation of bacterial growth in isolates in HM, milk was first heat-treated in order to eliminate any previous bacterial presence. Pasteurization of HM was done using \u201cHolder\u201d pasteurization, i.e., heat treatment at 62.5 \u00b0C for 30 min.Bacterial strains were propagated in LB consisting of 10 g of tryptone, 5 g of yeast extract and 5 g of NaCl per liter, or on solid LB medium supplemented with 1.5% agar. Each bacterial strain was plated on the solidified LB plates at 37 \u00b0C until visible growth of bacterial colonies occurred. Afterwards, a fresh starter culture was generated from a single bacterial colony, by incubating it in LB broth overnight at 23 \u00b0C with shaking at 150 rpm. Being a highly soluble compound and an FDA generally recognized as safe (GRAS) approved for food, we chose to use ZnCl2+ was added to separate flasks at concentrations of 1, 3 and 5 mM from 1 M stock of ZnCl2; an additional unsupplemented HM flask was used as control. Concentrations were selected based on previous work [Three different HM samples were pooled into a sterile container, then divided into three Erlenmeyer flasks, each flask containing 5 mL of HM. Znous work , adjustious work .B. cereus and Staphylococcus epidermidis, isolated from HM and identified using a MALDI TOF biotyper, were grown in a LB medium. For starter cultures, strains were grown as described above, then introduced into the LB medium in 1:100 ratio, with or without supplementation of zinc. ZnCl2 was added at concentrations of 1, 3 and 5 mM; flasks were then incubated at 37 \u00b0C with shaking at 150 rpm. 100 \u00b5L of each sample was collected every 2 h for the duration of 8 h. Viable cell count was determined using the CFU method as previously described. For evaluation of bacterial growth of the isolates in HM, a similar assay was conducted using pasteurized HM, with pasteurization being done as described above. Two major bacterial species, The bacterial cells were prepared and visualized using the SEM observation similar to that as reported recently . t-tests using Microsoft Excel 2010 and GraphPad Prism 6 software. p-Values < 0.05 were considered significant. The results are based on three biological repeats performed in duplicates.The experimental data were analyzed through ANOVA following post hoc S. epidermidis was the bacterial strain with the highest prevalence as it was present in 13 out of the 14 milk donations sampled. 16S rRNA sequencing results matched those received from the MALDI TOF assay. The prevalence of bacterial species among our samples was similar to those previously reported; the majority of bacterial strains identified belonged to the Staphylococcus genus and seemingly originated from commensal skin flora [S. epidermidis is a non-motile, Gram-positive, commensal microorganism that often colonizes the skin and mucous membranes of mammals and is the most prevalent staphylococcal species found in humans [S. epidermidis has been identified as the most common cause for primary bacteremia and infection of indwelling medical devices, particularly in immunocompromised individuals and neonates [S. epidermidis isolates from HM originating from healthy women have shown resistance to various types of antibiotics [B. cereus, in pasteurized HM. Therefore, isolation and identification by sequencing was also done for B. cereus isolates, intending to use them to test zinc\u2019s effect against this bacterium. Hence, the ability of Zn2+ to mitigate microbial contaminations in HM was further tested against S. epidermidis and B. cereus.As previously mentioned, HM stored in HM banks is highly prone to contaminations arising from its biologically rich composition. At the same time, the core contaminating bacterial strains may vary between different banks and countries of origin ,26,27. Tin flora ,29,30. Sn humans . Althougneonates . Moreoveibiotics . Another2+ ions on the growth of general viable bacterial populations in pooled unpasteurized HM. Following that, we tested zinc\u2019s activity against S. epidermidis and B. cereus previously isolated from HM, first in growth medium, then in pasteurized HM. As can be seen in B. cereus and S. epidermidis was done by growing the isolates in a growth medium for 8 h, with or without the presence of zinc. An examination of zinc\u2019s effect on their growth in HM medium was done next. As can be seen in B. cereus were able to grow in the presence of 1 mM of zinc, although to a lesser extent compared to the control, and it seemed that growth was somewhat delayed in the overall period of incubation. On the other hand, growth was completely inhibited when B. cereus cells were exposed to 3 and 5 mM of zinc. S. epidermidis cells were grown in the presence of zinc, with a concentration-dependent reduction in bacterial levels . One previous study showed comparable results, as the second-most frequent bacteria isolated from the expressed HM was S. epidermidis [Klebsiella spp., which was not found in any of the HM we sampled. This further stresses the diversity in bacterial populations that can be found in bacteriological screenings in expressed HM, which can be credited to various factors. S. epidermidis and other coagulase-negative Staphylococcus are considered commensal among most milk banks\u2019 criteria, yet they may cause nosocomial infection in preterm infants. However, since they are most likely to be eliminated by pasteurization, the main concern is that their presence will lead to disqualification of donations. For the most part, samples of donor HM prior to pasteurization are colonized with normal skin flora, including coagulase-negative  to find . Normalllla spp. ,34,35,36lla spp. . The firdermidis . On the Bacillus spp. Furthermore, in some cases, HM samples that showed no bacterial presence before pasteurization will test positive for Bacillus spp. after the heat treatment [B. cereus. This bacterium is highly pathogenic in preterm infants; in 2013, two infants born at very low birth weight presented with severe intestinal infection after consuming donated HM contaminated with B. cereus in an NICU in France [B. cereus at levels below the detection threshold of the reference method. Post-pasteurization bacterial presence is not as common since most bacteria populating raw HM do not survive Holder pasteurization ,8. Stillreatment . This careatment . In thisn France . RetrospS. epidermidis and B. cereus as solid candidates to exemplify zinc\u2019s effect against the major and common HM contaminants, both before and after pasteurization. As we expected, both strains were highly sensitive to zinc, as the presence of Zn2+ ions in various concentrations inhibited their ability to grow in a growth medium and in pasteurized HM. After observing the bactericidal effect caused by exposure to zinc, we wished to further elaborate its antimicrobial effect. A closer inspection of zinc\u2019s influence on bacterial cells was achieved using scanning electron microscopy, which demonstrated a diverse strain-dependent antimicrobial effect. The antimicrobial effect of zinc over S. epidermidis has been previously described [2+ concentrations above optimal levels disturbs this homeostasis and allows entry of Zn2+ ions to inside the cells, resulting in a cytotoxic effect. The antimicrobial mode of action for zinc against various bacterial and fungal species has been addressed before. It has been suggested that zinc\u2019s antimicrobial activity can be a result of the multiple inhibitory effects this ion has on crucial metabolic processes in bacterial cells, such as glycolysis, glucosyltransferase production, polysaccharide synthesis, transmembrane proton translocation and acid tolerance [2+ ions can also enhance proton permeabilities of bacterial cells\u2019 membranes and reduce adenosine triphosphate (ATP) synthesis in glycolyzing cells. Disturbance to energy-producing and respiratory processes in bacterial cells can be a result of zinc\u2019s ability to inhibit the activity of key enzymes, such as glycolytic enzymes [S. aureus and E. coli cells, upon treatment, bacteria displayed strong evidence of membrane disorganization and increased roughness, leading to a leak-out of the intracellular components and subsequent cell lysis in both strains [2+ ions and the negatively charged cell membrane of the bacterial cells or via production of intracellular reactive oxygen species (ROS). Similar mechanisms of action can be advanced to explain the cell lysis of S. epidermidis observed in our study. Crossing results of the HM bacteriological analysis with previous reports marked escribed , while iescribed ,41. Zincescribed ,43. Zincolerance . Further enzymes . Another enzymes ,45. In aS. epidermidis cells, B. cereus cells did not show a phenotype of cell lysis when grown in the presence of zinc, despite showing a bactericidal effect when treated with the same concentrations. Interestingly, when exposed to 5 mM of ZnCl2, cells showed abnormalities in the cell surface, as well as complete loss of flagella. Not much is known about the relation between ions\u2019 homeostasis and flagella formation in bacterial cells. Zn2+ ions have been reported to be crucial for the activity of anti-sigma factors in various species [B. cereus, flagella formation is linked to the activity of Sigma 54, a sigma factor which is somewhat characterized as a modulator of bacterial cell exterior [B. cereus ATCC 14579, was obtained after deletion of the rpoN gene encoding for sigma 54 [2+ ions can possibly promote the expression of anti-sigma factors and subsequently, suppress the activity of sigma 54, causing for a reduction in flagella biosynthesis. However, a direct effect of Zn2+ ions on the levels of anti-sigma factors or on the expression of the rpoN gene in B. cereus has not been tested. The detailed mechanism by which Zn2+ ions affect physiological processes such as flagella formation in B. cereus needs further research as literature on this subject is lacking. Furthermore, the microscopically visible changes in bacterial cell morphology obtained after exposure to zinc does not necessarily account for the observed bactericidal effect. Hence, this promotes the notion that, in B. cereus cells, the antimicrobial effect of Zn2+ ions, resulting in cell death, could likely be related to some intracellular changes in physiological state of the cell.Unlike  species ,47. In Bexterior . A phenosigma 54 . AlteratBacillus spp. [2+ ions before heat treatment should lead to a significant reduction in post-pasteurization survival rates of spoiling Bacillus species. Enrichment of HM with zinc could be fundamental for not only the bacterial safety of the milk, but also for the prevention of zinc deficiencies, especially considering the fragility of the target recipients. Zinc is a critical micronutrient for preterm infants, having a significant role in immune function and growth [2+ supplementation, reaching up to 5 mM . Such a dose is substantially higher than the physiologic requirement for growth, hence concerns about toxicity and other complications need to be evaluated. For instance, in adults, high doses of zinc caused inhibition of absorption of other micronutrients (such as Cu2+ and Fe3+), which could also be the case in preterm infants [2+ supplementation should be lowered upon optimization of possible treatment, along with ruling out any negative effects of this practice on infants\u2019 health. Optimization and further research should also include use of other zinc compounds, namely those used as dietary supplements, which might exert similar antimicrobial ability with lower risks and costs. It should be noted that the current investigation was designed as a proof-of-concept study, aiming to evaluate the feasibility of a zinc-based treatment in targeting bacterial species populating pumped HM. Thus, the results of this study open a possibility for developing a mineral-based antimicrobial approach that can possibly offer a future solution to microbiological problems in the food industry, including HM handling. Incorporation of minerals can be done in various stages of food preparation and treatment, as different stages can be the source for bacterial contaminations. Previously, it was reported that incubation with magnesium or zinc before heat treatment led to a reduction in post-pasteurization survival rates in lus spp. ,22. Hencd growth . Moreoved growth ,52. Evidd growth . Howeverd growth . Our stu infants ,56. Ther"}
+{"text": "Antenatal care service satisfaction is a measure of the degree to which a woman seeking care is happy with the antenatal care service provided to her. Thus, this systematic review and meta-analysis aims to identify factors that determine antenatal care service satisfaction among women in Ethiopia.  PubMed, Hinari, and Google Scholar were systematically searched for eligible studies. In addition, national university digital libraries were also searched. The Joanna Briggs Institute's (JBI) critical appraisal tools were used to assess the quality of the included articles. The Cochrane Q-statistics and I2 tests were used to assess heterogeneity among the included studies. Publication bias was assessed using Egger's test. Joanna Briggs Institute Critical Appraisal Checklist for Analytical Cross-Sectional Studies. The extracted data were analyzed using STATA version 14 software and the results were presented using the forest plot.  Of the 274 articles identified through the systematic search of the literature, 13 studies fulfilling the inclusion criteria were included in this meta-analysis. First antenatal care visit , women waited <60\u2009min (AOR: 1.87 and 95% CI: 1.40\u20132.50), women whose privacy was maintained (AOR: 3.91 and 95% CI: 1.97\u20137.77), women treated respectfully (AOR: 5.07 and 95% CI: 2.34\u201310.96), and unplanned pregnancies (AOR\u2009=\u20090.28 and 95% CI: 0.10\u20130.77) were significantly associated with antenatal care service satisfaction.  The study assessed the determinants of antenatal care service satisfaction in Ethiopia. First antenatal care visit, waiting time (<60\u2009min) to see the care provider, maintenance of privacy, respectful treatment, and pregnancy unplanned were found to be determinants of antenatal care service satisfaction. Counseling a woman to comply with a minimum required antenatal care visits and compassionate and respectful maternity care will increase maternal satisfaction with the antenatal care services. Patient satisfaction is a measure of the degree to which a health care seeking client or patient is happy with the care given by health care providers. It is important to define the success of health care facility . DissatiPatient satisfaction is an indirect indicator of the quality of health care services . It is aAntenatal care is care provided to pregnant women by skilled health care professionals to ensure the best health conditions for both the baby and mothers during pregnancy. Risk prevention, identification, and management of pregnancy-related or coexisting diseases, and health education and promotion are the components of antenatal care . The WorAlthough a number of studies have identified determinants of antenatal care service satisfaction in Ethiopia, these tend to be limited to certain areas and present findings which are highly variable, inconsistent, and nonrepresentative. In this regard, the absence of a nation-wide study was identified as a significant gap. Thus, this systematic review and meta-analysis are meant to summarize the determinants of antenatal care service satisfaction among women in Ethiopia. The findings from this study will help antenatal care providers, policymakers, and concerned bodies to know antenatal care dissatisfying factors, and modifying or intervening them.The study protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO), the University of York Centre for Reviews and Dissemination (ID number: (CRD42019137013). This review and meta-analysis were conducted according to the guideline of Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) . Protoco(1) Setting. Only quantitative studies carried out in health institutions in Ethiopia were included in this systematic review and meta-analysis.(2) Language. Only articles published in English were retrieved for review. No articles published in another language were found.(3) Publication Condition. Both published and unpublished articles were considered for this review.Intervention(s)/exposure(s): All pregnant women attending antenatal care.\u2009Outcome: The primary outcome is the determinants of ANC service satisfaction among woman in Ethiopia. It is defined as the degree to which an antenatal care seeking woman is happy with the antenatal care given by health care providers, which was measured by different sociodemographic and care provider factors.\u2009Studies for which we are unable to get the necessary details after contacting the authors were excluded. Studies on intranatal care, case series, and case studies were also excluded.The following databases were searched to find potentially relevant articles: PubMed, Hinari, and Google Scholar. No date limit was applied. National university digital libraries, such as the electronic library of Addis Ababa University, were searched to include gray literature. Hand searches of the reference lists of all included studies were also conducted. Identified articles were directly transferred to endnote citation manager for inclusion.Search terms such as \u201cmagnitude\u201d, \u201cprevalence\u201d, \u201cdeterminants,\u201d \u201cassociated factors,\u201d \u201cantenatal care,\u201d \u201cprenatal care,\u201d and \u201csatisfaction\u201d were used. The Boolean operators (OR and AND) were used to combine search terms. Examples of a searching strategy fit for all the databases searched are available in supporting information  and all duplicates were removed. In the second step, all articles were screened by their title, abstract, and full text for eligibility against the predefined inclusion and exclusion criteria. Third, a full document manuscript review was conducted and studies were removed through the predefined exclusion criteria. Finally, the included articles were evaluated based on the Joanna Brigg's Institute (JBI) quality assessment tool , 16.The articles for this study were selected guided by a PRISMA flow diagram. Data charting and screening process was performed independently (KS) based on eligibility criteria by using Microsoft Excel\u2122. This was conducted after extracting the titles and abstracts of the eligible studies and after removing duplicates. The retrieved articles were uploaded to endnote software citation manager and duplicates were removed. The data extraction form included the name of the author, year of publication, regions where the study was conducted, sample size, response rate, setting, and type of study design. The log odds ratios with 95% confidence intervals for the included variables were extracted in a binary format. The extracted data were imported to STATA version 14 for data analysis. The data collection and checking was conducted by a single author (KS) and two corresponding authors of the included studies were contacted twice through e-mail provided on their published papers.Risk of bias was assessed using JBI Critical Appraisal Checklist for Analytical Cross-Sectional Studies . The cheI2statics. I2 test statistics result of 25%, 50%, and 75% was declared as low, moderate, and high heterogeneity, respectively [The odds ratio for determinant data were computed by pooling odds ratio reported in the original studies and the standard error (SE) for the natural logarithm of odds ratios (ln OR) were calculated using formula SE (ln OR)\u2009=\u2009 ectively . STATA vP-value of less than 0.05 was used to declare the publication bias.Egger's test was used to assess publication bias. A A leave-one-out Sensitivity analysis using a random-effects model was performed to assess the influence of a single study on the overall pooled estimate. The result of sensitivity analysis is given in an additional Unplanned pregnancies occur when no children or extra children are not needed or when the pregnancy occurs earlier than desired.\u2009Respect refers to what pregnant women think about their treatment with dignity and compassion.\u2009Satisfaction shows how happy an antenatal care seeking woman is with the care provided by the antenatal care provider.\u2009Privacy in this study refers to absence of another person in the antenatal care room during providing antenatal care and consultation service to a woman.\u2009A total of 274 articles were retrieved during our primary search. Of these, 163 articles were excluded due to duplication. The titles and abstracts of the remaining 111 articles have been screened and 41 articles that do not meet the inclusion criteria were removed. The remaining 70 articles were screened in full, and 30 articles that were found to be outside the study area were removed. The full text of the remaining 40 articles were also assessed in detail against the eligibility criteria, and 27 articles were excluded as they did not have any important information . Finally, 13 articles that meet the inclusion criteria are left for final analysis .Thirteen studies are included in this review. All of them are cross-sectional by design. Six of the included studies were from Southern Nation Nationalities And Peoples Region (SNNPR) \u201323, fourI2\u2009=\u200949.2% and P\u2009=\u20090.140)  . Hence, P\u2009\u2264\u20090.01. Egger's test revealed the absence of publication bias (P\u2009=\u20090.41)  .2 test showed the existence of high heterogeneity . Egger's test revealed the absence of publication bias with a P-value of 0.85   .I2\u2009=\u200990.4% and P\u2009\u2264\u20090.01). Hence, a random effect meta-analysis was employed . Hence, a random effect meta-analysis was employed . Hence, a random effect meta-analysis was employed . Hence, a random effect meta-analysis was employed  may think the service/care is good and that may make her to continue to seek health care in the future also. This is supported by a study conducted in Malawi which stated that women will not seek service if she is disrespected .This study showed a negative association between antenatal care service satisfaction and unplanned pregnancy. The possible explanation is women who had an unplanned pregnancy may be too sensitive in terms of confidentiality and privacy due to possible stigma if the pregnancy is out of marriage. Women who had an unplanned pregnancy may also experience greater relationship instability than women whose pregnancies were intended . AnotherThe presence of high heterogeneity among the included study was sought as a limitation for this study. Absence of data upon requesting the corresponding authors of few articles might affect the results of this study. Due to the absence of primary studies, other regions were not covered in this study. Hence, primary studies aimed to reach those regions are very important.Antenatal care service satisfaction is an indicator of the quality of antenatal care. Low satisfaction or dissatisfaction with the antenatal care services hinder women from going to a health facilities to receive prenatal care. Factors such as decreased frequency of visits, long waiting time to see care, provider, privacy, unmaintained, disrespectful treatment, and pregnancy unplanned cause low satisfaction with antenatal care services. In the future, I hope the findings of this research will lead to improvement of the quality of antenatal care services. If we cannot avoid those factors, antenatal care dissatisfaction will make the women flee the health institutions and we will continue to have low antenatal care utilization.Hence, antenatal care service providers should counsel the women to finish the recommended visits, should treat the mother friendly and respectfully whenever they come for the service. In addition, reproductive health counselling shall be provided to all women of reproductive age to prevent unplanned pregnancy."}
+{"text": "Logistic regression was used to analyze the covariates of having initiated antenatal care up to the 16thgestational week, 49.1% attended four or more visits, and 34.1% reported both events. Having concluded high school  or college  were aspects associated with reporting both situations. The reasons for the late initiation of antenatal care and attending less than four visits were as follows: not finding it important to attend several visits, not having easy access to the health facility, not being aware about pregnancy, and not having a companion for the visits.all postpartum women underwent antenatal care, but only 39.9% started it until the 16the gestational age at the beginning of antenatal care and the number of antenatal visits are lower than the current recommendations in the country. Currently, antenatal care is recognized as an important strategy to prevent or reduce the risk of maternal and newborn morbidity and mortality. The literature around this issue show that adequate antenatal care is closely linked to better perinatal outcomes and to a reduction in maternal and neonatal morbidity and mortality-4.Antenatal care consists of a set of procedures and measures aimed at diagnosing, treating and preventing situations that are undesirable for women\u2019s health - during pregnancy, delivery, postpartum - and also for the infant,5.Antenatal care encompasses various guidelines, recommendations, measures and procedures that, in some way, differ across countries. Even so, there are two fundamental elements that are the basis for good quality antenatal care: its early initiation and a minimum number of visitsthand 12thgestational week-7. However, some low-income countries have different recommendations regarding the number of visits, which at least four; and endorse the gestational age at which antenatal care is initiated with greater flexibility, up to the 16thweek-10. Although there are differences in the recommendations in various contexts, the World Health Organization(WHO) recommends at least eight antenatal visits, with gestational age up to the 12thweek at its initiation,5. It is noteworthy that these recommendations can be adapted to the socioeconomic context and to the population and health system of each country.The vast majority of high- and middle-income countries recommend at least six antenatal visits; they also recommend initiating antenatal care between the 8thweek at its initiation-12. In that country, few studies have assessed gestational age at the beginning of antenatal care and the number of visits-14. A national survey showed that approximately 90% of the pregnant women in Mozambique undergo antenatal care, but only 55% attend the minimum number of four visits. Apparently, among other reasons, late initiation is due to the guidelines provided by the health professionals themselves, who inform women that antenatal care should be started when fetal movements are felt or when the baby can be palpated; another reason is the experience of multiparous women who start antenatal care late as they already know the guidelines of the health facilities.This is what the Ministry of Health(MISAU) of Mozambique did, a country located in Sub-Saharan Africa and scenario of this study, which recommends a minimum number of four antenatal care visits and gestational age up to the 16per 100,000 live births, which reveals that there is a need to improve the quality of maternal health care, and antenatal care does not seem to be an exception. Given the importance of antenatal care for the promotion of maternal health, especially in a country with high rates of maternal deaths, this study has the following objectives: 1)to assess the gestational age at the beginning of antenatal care and its covariates; 2)to assess the number of antenatal visits and its covariates; and 3)to identify the reasons for the late initiation of antenatal care and for reporting less than four antenatal care visits among postpartum women living in Nampula, Mozambique.Therefore, it seems that access to antenatal care in Mozambique is not necessarily the main problem in the field of reproductive health, but quality of care. The country has a high maternal mortality rate, 452.This is a quantitative, cross-sectional and analytical study, guided by the STROBE tool, developed to assess the quality of observational studiesthplace out of a total of 188. Data from the population census conducted in 2017 indicate that there are almost 28 million inhabitants, with nearly 67% of the population living in rural areas. The mean life expectancy is 56.5 years old and the total fertility rate is 5.3 children per woman.The study was conducted in the city of Nampula, located in the inland of the province with the same name, in northern Mozambique, Africa. According to the Human Development Report of the United Nations Development Program(UNDP), in 2015 Mozambique was among the nine countries in the world with the lowest Human Development Index(HDI), ranking in the 180The research was conducted from August to December 2019.The study population consisted of postpartum women aged between 18 and 49 years old.The inclusion criteria were all women whose delivery had taken place within the previous 24 hours, in maternity hospitals, and women whose delivery had taken place within the previous 15 days, at their own homes, as long as they reported being physically and emotionally willing to answer the questionnaire. The exclusion criterion was having antenatal care classified as high risk., female population of Nampula of 337,839 women at the time of the study, significance level of 95% and 5% margin of error. Sample calculation indicated the need to interview 381 women.The sample size considered the proportion of Mozambican women with four or more antenatal visits:p=54.6thweek and after the 16thweek), number of antenatal care visits(up to three and four or more) and access to basic antenatal care, herein defined as the one in which women initiated antenatal care up to the 16thgestational week and reported four or more antenatal care visits(dichotomous).The dependent variables were gestational age at the beginning of antenatal care; the reasons for late initiation of antenatal care; and the reasons for attending less than four visits.The independent variables referring to the sociodemographic characteristics were age group; schooling in years: elementary education(\u22647), high school education(8-12) or higher education(>12); religion; place of residence: peripheral region or urban region; paid jobs(no or yes); use of private healthcare services(no or yes); and living with a partner(no or yes). Regarding reproductive history, the number of pregnancies, including the current one(1-3 or \u22654) was assessed, as well as antenatal care in previous pregnancies. As for the planning of the last pregnancy, the instrument called London Measure of Unplanned Pregnancy(LMUP) was used, validated for the Portuguese languageThe instrument used for data collection consisted of a specific form, prepared by the main researcher, which contained questions about the dependent and independent variables, in addition to the LMUP. LMUP is an instrument that measures pregnancy planning retrospectively, regardless of the outcome of pregnancy, birth or abortion. The classification regarding the planning of the last pregnancy is obtained by the sum of points for each question; the total score can vary from zero to 12. Women who scored between 0 and 3 points are classified as having an unplanned pregnancy; between 4 and 9 points, as ambivalent, that is, simultaneous prevalence of apparently contradictory feelings and actions about pregnancy planning and non-planning; and between 10 and 12 points as having a planned pregnancy.Data collection took place in a maternity hospital and at the women\u2019s residence. Selection of the women took place through information from the puerperium registry book available at the Health Sciences College/UniL\u00fario and from the maternity hospital, where information on all births that took place in the city is available. Women were interviewed using a structured instrument created in GoogleForms, pre-tested and applied using a tablet, respecting their privacy. The questionnaire included information regarding sociodemographic characteristics, reproductive history, planning of the last pregnancy and antenatal care.Analyses were performed using the Stata 15.0 software. To describe the sociodemographic characteristics, reproductive history and antenatal care, absolute and relative numbers, mean, median and standard deviation were used. The test of difference between two proportions(Chi-Square or Fisher\u2019s Exact) was applied to verify differences between gestational age at the beginning of antenatal care, number of visits and access to basic antenatal care, and the sociodemographic characteristics, women\u2019s reproductive history, and planning of the last pregnancy. The analysis of the covariates was performed using univariate and multiple logistic regression. In the multiple logistic regression analysis, the variables were simultaneously inserted into the model. A5% significance level was considered statistically significant.Comit\u00ea Institucional de Bio\u00e9tica para Sa\u00fade da Universidade L\u00fario,CIBSUL) of Nampula, Mozambique, under No.26/August/CIBSUL/19.The project was approved by the Institutional Bioethics Committee for Health of the L\u00fario University, most had elementary education(52.4%), lived in the peripheral region of the city(75.3%), used public health system(91.1%) and lived with a partner(88.8%). Nearly one out of 10 women had a paid job(10.9%). Almost half reported professing the Islamic religion(43.4%), number of pregnancies greater than or equal to four(44.5%) and had their pregnancies classified as planned(41.0%) .thgestational week; 49.1%(n=193) reported at least four antenatal visits, and 34.1%(n=134) were categorized as having undergone basic antenatal care, that is, they started antenatal care up to the 16thgestational week and had at least four antenatal visits(data not shown in the table).All women reported having undergone antenatal care in their last pregnancy(which is being considered in this study), and 39.9%(n=157) started antenatal care up to the 16thgestational week, the main reasons were thinking that the belly was small(42.6%), not having a spouse as a companion for the visits(24.1%) and not knowing that they were pregnant(19.8%), among others. Among women who reported less than four antenatal visits, the main reasons were not finding it important to have several antenatal visits(36.0%), not having a spouse as a companion for the visits(25.5%) and not having easy access to the health facility or living far from the health service(24.5%), among others were more likely to start antenatal care up to the 16thweek, when compared to those who used the public healthcare system, as was also the case of the women with paid job. In the multiple model, adjusted for all the variables entered simultaneously, only education remained associated with the initiation of antenatal care up to the 16thweek, considering both women with high school education and with higher education, when compared to those with elementary education.The analysis of the aspects associated with the initiation of antenatal care up to the 16The association between women\u2019s characteristics and reporting at least four antenatal care visits is shown in The analysis of the aspects associated with having accessed basic antenatal care is shown in . Antenatal care coverage was found to be universal in the municipality of Nampula, Mozambique. However, the quality of prenatal care was still low, as less than half of women started antenatal care early and attended the minimum number of recommended visits. When these two indicators are analyzed together, the condition of access to basic antenatal care seemed even more fragile.This study aimed at assessing both the gestational age at the beginning of antenatal care and the number of antenatal care visits and their associated aspects; as well as identifying the reasons for the late initiation of antenatal care and less than four visits, according to the MISAU recommendations. Similar findings were identified in another study conducted in five Mozambican provinces, in which 55% of pregnant women started antenatal care between 20 and 28 weeks of gestational age. Therefore, it seems that little progress has been made on Mozambican context regarding women`s ability of making full use of antenatal care, starting it early and accessing a greater number of contacts with health professionals.Our findings are similar to the few studies already conducted in Mozambique on the topic. In a qualitative study conducted in the provinces of Cabo Delgado, Tete and Maputo, which represented the three regions of the country, it was observed that, in general, women started antenatal care at the end of the second trimester, and that the reasons for this late initiation were lack of a pregnancy test in the health services and waiting for the presence of visible signs of pregnancy,8,19. It is noteworthy that, despite the different political, economic and social contexts across these countries, the results are similar. Although our findings cannot be directly compared with other studies that assess the quality of antenatal care, as we analyzed only two indicators and some other studies often consider other indicators, the access to adequate antenatal carein Mozambique was observed in 45% of the pregnancies elsewhere. That said, our results confirm that are still limitations in the health services regarding the early recruitment of pregnant women in the country.Such results were also observed in other countries that adopted similar antenatal quality parameters: 9%to 48%variation in the adequacy of gestational age at the beginning of antenatal care and 27% to 45% in the number of visits, considering other African countries, as well as countries in the American and Asian continents,5, the reasons for its late initiation and lower number of recommended visits were also investigated in this study. Considering the women\u2019s reports, the reasons that stood out the most were the fact that they did not think it was important to attend several visits; did not have easy access to the health facility; found their belly small; did not know that they were pregnant, and did not have a companion for the visits. Similar data were found in studies carried out in Mozambique-14 and in other regions of the world,19. This is a very worrying finding, as it shows the influence of beliefs and values on access to and continuity of antenatal care in the city of Nampula. In addition to promoting access to rapid pregnancy diagnosis methods, which allow for earlier recruitment of the pregnant woman, to encouraging the partner\u2019s participation in antenatal visits, to supporting women who do not want their pregnancy, and to reframing strategies that facilitate access to the healthcare facilities, it is fundamental to demystify certain cultural aspects related to pregnancy.Considering that early initiation of antenatal care and more visits allow for more qualified care and better maternal and fetal results, showed that women perceive pregnancy as a normal life event, that is, as a physiological event in which antenatal care is only necessary in situations of disease. Thus, little understanding of the benefits of antenatal care and a preference for popular care methods are observed. In addition to that, antenatal care visits demand long wait at the healthcare services, and this time could be used for household chores and for taking care of other children; in addition to the fact that the inadequate transportation structure in some regions of Mozambique represents exposure to risks for the pregnancy. Our results confirm the findings of the aforementioned review.Furthermore, a systematic review study on the perception of the importance of antenatal care and barriers to access in 15 low-income countries, including Mozambique-9,21-26. Education enables women to develop the confidence needed to make decisions about their own body and health and to more easily understand the importance of the antenatal care services; in addition to the fact that a higher schooling level is generally related to greater autonomy in the decision to seek antenatal care. Other studies have also shown that women with more years of schooling are the ones who most perform preconception preparation and who experience less unplanned pregnancies-28, events that are markedly related to better levels of maternal health. Therefore, investment in women\u2019s education will benefit women and their fetuses.Women with more years of schooling were more likely to start antenatal care early, report the appropriate number of visits and report both situations simultaneously, which is widely documented in several other studies,24. It is understood that women with paid job are often those with greater autonomy in the decision to seek antenatal care and better schooling. In addition to that, paid job enables economic independence so that woman can pay for the costs of transportation to the healthcare service, which enables access to healthcare.Having a paid job was associated with attending four or more antenatal visits which is similar to other studies-30. Thus, the absence of a partner can constitute a potential weakness for accessing antenatal care. This may be because the presence of a partner encourages the performance of high quality antenatal care, since the presence of men in antenatal visits offers well-being to women and, somehow, encourages adherence to the treatment and to preventive measures,5.Additionally, women who lived with their partners were more likely to attend four or more visits when compared to those who did not live with a partner, a fact that is similar to results obtained in studies carried out in Brazil. Social support from family members and/or friends can be strengthening for pregnant women who are not in a stable relationship.On the other hand, the lack of support from an intimate partner or from family members leaves women more fragile and vulnerable and, in turn, negatively affects the search for adequate antenatal care. Culturally, pregnancies outside marriage can be considered disapproved and considered a shameful act in many African societies, and this may discourage single or unaccompanied women from seeking antenatal visitsthgestational week and reporting a minimum of four antenatal care visits, it is necessary to highlight that the responsibility for improving the access and offering high-quality antenatal care lies in the MISAU, as well as in the healthcare services themselves, which need to respond to the health needs of pregnant women or those planning to become pregnant.Although we have only considered variables related to the women\u2019s individual characteristics in our analysis of the covariates of the initiation of antenatal care up to the 16To our knowledge, this is the first study in Nampula, Mozambique, which assessed the covariates of gestational age at the beginning of antenatal care, the number of visits, and the simultaneity of these two events. Another potential of our study lies in the fact that we used a validated instrument to assess pregnancy planning. Knowing the profile of women with access to so-called basic antenatal care contributes to the proposal of public health and social policies that effectively improve women\u2019s health, as well as that of their future children. Thus, it is necessary to think of strategies so that women with low schooling and without any paid job may, through the public healthcare service, diagnose pregnancy early and access information about the importance of starting antenatal care early and, consequently, attend more antenatal care visits.Health professionals, especially nurses and maternal nurses, common in Mozambique, can also act to demystify beliefs and values, such as finding the belly small and not considering it important to attend antenatal care; they can also contribute to expanding access to reproductive planning, as some women reported not knowing that they were pregnant and not knowing how to take care of a future pregnancy..In addition to that, Mozambique adopts a system of fewer antenatal care visits than the number recommended by the WHO. It is noteworthy that countries that adopt a reduced number of antenatal visits present a reduction in the associated cost without changes in the maternal mortality rates, with the aggravating factor of an increase in perinatal mortality and lower women\u2019s satisfactionThis study has some limitations. In the first place, women living in a single municipality and attending the Mozambican National Healthcare System were evaluated; therefore, the findings cannot be generalized to Mozambican women in general, nor to those who use the private healthcare system. However, considering that antenatal care coverage should be universal, results of research implemented around the quality of public health services may help to improve the quality of antenatal care for every women, irrespective if attending public or private services. In the second place, our data were obtained from the women\u2019s reports and memory bias can have occurred, as some women may have omitted or forgotten some information during the interviews. To minimize this bias, interviews were done with postpartum women.Our study also contributes by identifying the difficulties of the antenatal care services in a country in Sub-Saharan Africa, which may support the implementation of initiatives aimed at increasing the number of visits and anticipating the initiation of antenatal care, such as increasing the schooling of children, adolescents and women; offering support so that they can reach the health facility without major difficulties; and providing information so that pregnant women understand the importance of antenatal care. Such initiatives are not only necessary in African countries, but also in other low- and middle-income countries that still struggle to achieve the best possible levels of maternal health.Our findings showed that, despite the universal coverage of antenatal care, the gestational age at the beginning of antenatal care and the number of visits are lower than the current recommendations in Mozambique. For the social and public health policy-makers, improvements are suggested in the access to health care facilities, in the early recruitment of pregnant women, in the demystification of beliefs for adherence to antenatal care, and in the provision of care on reproductive planning to support women who plan to become pregnant."}
+{"text": "The use of vinylelectrophiles in synthesis has been hampered bythe lack of access to a suitable reagentthat is practical and of appropriate reactivity. In this work we introducea vinyl thianthrenium salt as an effective vinylating reagent. Thebench-stable, crystalline reagent can be readily prepared from ethylenegas at atmospheric pressure in one step and is broadly useful in theannulation chemistry of (hetero)cycles, N-vinylation of heterocycliccompounds, and palladium-catalyzed cross-coupling reactions. The structuralfeatures of the thianthrene core enable a distinct synthesis and reactivityprofile, unprecedented for other vinyl sulfonium derivatives. Here we report thereagent vinyl thianthrenium tetrafluoroborate  that functions as a versatile reagent for differentsynthetic transformations. Reagent 1 is accessible directlyfrom ethylene (1 atm) in a single step from commercially availablematerial on multigram scale and is a bench-stable, nonhygroscopicsolid that can be stored at room temperature in air without signsof decomposition for at least one year. Despite its high stability, 1 displays a rich reactivity profile and has been implementedin several polar and palladium-catalyzed cross-coupling reactions,which differentiates it from all other vinylating reagents reportedto date. The unusual direct conversion of ethylene into a versatilebuilding block for organic synthesis sets the approach apart fromprevious syntheses of alkenylsulfonium salts; in addition, 1 can participate in useful reactions such as a Suzuki cross-couplingthat have not been realized with other alkenylthianthrenium salts.Owing to the rich chemistryof alkenes, the presence of a terminal alkenyl  are uncommon and almost exclusiveto metal-mediated reactions, owing to the ability of metal centersto activate ethylene via coordination.14 Metal-free reactions utilizing ethylene at 1 atm are manily restrictedto photochemical cycloadditions with high-energy UV light.17 Overall, the general requirement for specialized equipment (high-pressurereactors or UV-photoreactors) has traditionally restricted the useof ethylene as a reagent in organic synthesis involving complex smallmolecules.Ethylene is an inexpensive gas ,2 bonds  derivatives, the use of vinyl halides as electrophiles  is challenging owing to the difficulty of handlingthe gaseous compounds that are acutely toxic and carcinogenic, whichhas historically thwarted their utilization in synthesis.20 Alternatively, numerous nucleophilic vinyl-[M]reagents 2, etc.) have been developed over the years,21 but most of them are prepared in several steps from vinyl bromideitself, display high toxicity and low stability, or are poorly reactivecycles.34 However, neither the reagent nor its precursors have ever been reportedas suitable electrophiles in cross-coupling reactions owing to theirfundamental reactivity profile (vide infra). In fact, only a few substitutedalkenyl sulfonium salts have been successfully engaged in cross-couplings,37 but no examples of vinylations have been reported. Our group recentlyreported the synthesis of alkenyl thianthrenium salts,38 but a general reactivity profile in polar andcross-couling reactions has not been explored yet. Moreover, we wereunsuccessful in engaging these salts in efficient couplings with arylboronic acids via Suzuki-type reactions.The development of palladium-catalyzed cross-couplingreactionshas allowed researchers to reliably construct C\u2013Csp2 bonds 1A.18,19 reactive1B. Moreo+) could provide a valuablesolution to this task. The perchlorate salt of vinyl-TT+ was published three decades ago while exploring the reactivity ofthianthene radical cation (TT\u2022+) with (vinyl)4Sn,39 but only milligram quantitieswere accessed owing to the involvement of potentially explosive perchloratesalts,40 and its synthetic use has neverbeen reported. While, in comparison with other olefins, ethylene gastypically requires high pressure and an autoclave for cycloadditionreactions,41 we sought to capitalize on the high reactivityof the highly electrophilic thianthrenium dication species generatedby treatment of thianthrene-S-oxide (2) with activating reagents such as Tf2O (1) can now beprepared on multigram scale (50 mmol) with a simple balloon of ethylene(1 atm) in 86% yield. The isolation of 1 as a crystallinesolid can be carried out by simple precipitation, to afford an analyticallypure compound without the need for further purification. An alternativelab-scale synthetic route from vinyl-SiMe3 (2 equiv) wassimilarly effective . The salt 1 is a nonhygroscopic solidthat can be stored in the presence of air and moisture without signsof decomposition for at least one year, which makes it practical andeasy-to-handle. DSC-TGA reveals that 1 does not decomposeat temperatures lower than 280 \u00b0C, which underscores a desirablesafety profile . In contrast,attempts of implementing this protocol using other sulfoxides suchas dibenzothiophene-S-oxide or diphenylsulfoxidewere unsuccessful .The structural features of thianthrene that allow the formation ofa [4 + 2] adduct with ethylene seem crucial for a productive reaction,and indeed has enabled the first report on the formation of sulfoniumsalts directly from ethylene gas. To further confirm the key roleof the [4 + 2] cycloadduct under the reaction conditions, we isolatedand characterized intermediate 3, the crystal structureof which shows the \u201csnapshot\u201d of ethylene activationby the formal thianthrenium dication  while avoiding certain limitations associatedwith other reagents.We recently aimed todesign a strategy to trap ethylene efficientlyand convert it into a practical and crystalline reagent 1C. Ideal as Tf2O 2A. Follo profile S6. In co1 we startedbenchmarking the reagent in annulation reactions reported for vinyl-SPh2(OTf) or its precursor, which proceed via sulfonium ylideintermediates.25 As depicted in 4 \u2192 5),28 the assemblyof morpholine (6 \u2192 7)24 and azetidine (8 \u2192 9)31 scaffolds, and a tandem N-nucleophilicaddition/Corey\u2013Chaykovsky epoxidation (10 \u2192 11).32 In all cases, the isolatedyields were comparable or superior to those obtained with vinyl-SPh2(OTf) under the same conditions.To evaluate the reactivity profile of 1 in new reactions toeffectively transfer the vinyl moiety to nucleophilic nitrogen. Duringhis studies on annulation reactions, Aggarwal reported an annulation\u2013vinylationsequence on 1,2-aminoalcohols42 only whenCbz is the N-protecting group, but, beyond these examples, a generalplatform for N-vinylation of heterocycles using sulfonium salts isnot yet established43 and current methodsrequire harsh conditions47 or metal-mediated reactions (85\u2013100 \u00b0C).51 We developed a simple protocol that uses 1 in the presenceof a base at room temperature (12), indole (13 and 14), imidazole(19), pyrazole (15 and 16),triazole (17), and pyridone (18). A broadtolerance to an array of polar groups was displayed as demonstratedby the compatibility of nitro (13) and aldehyde groups(14), which are not tolerated using calcium carbide,47 or aryl halides  that are reactive in SNAr and cross-coupling reactions.Other scaffolds of relevance such as deazapurine (20)or theophylline (21) were also vinylated, as well asthe amino acids tryptophan and histidine (22 and 23). Finally, we explored the use of 1 for late-stageN-vinylation. The mild conditions and fast reaction times enabledmodification of the drugs metaxalone (24), carvedilol(25), lansoprazole (27), and the laser dyecoumarin 7 (26), further showcasing the compatibilitywith groups such as alcohols, alkylamines, and sulfoxides.Next, we aimed to implement perature 1. A dive53 radical chemistry,55 and electrophilic reactions.57 In contrast to alkenylation,the assembly of styrenes using vinylating reagents in metal-catalyzedcross-couplings often face several additional challenges,21 such as undesired Heck-type reactivity on thevinyl\u2013[M] reagent or polymerization styrene-type products.Vinyl sulfonium salts are ideally positioned to undergo metal-catalyzedvinylations, but no examples have been reported. One of the main reasonsis the unselective cleavage of the different C\u2013S bonds in sulfoniumsalts,37 which can result in mixtures ofproducts. We conceived 1 as a suitable coupling partnerthat could overcome the above-mentioned challenges and enable fastoxidative addition in view of its electropositive character (Ered = \u22121.13 V vs SCE). Moreover, in linewith what has been observed in palladium-catalyzed reactions of arylthianthrenium salts,63 cleavage of the Cvinyl\u2013S bond selectively overthe two Caryl\u2013S bonds may be explained by irreversibleoxidative addition into the vinyl bond but reversible oxidative additioninto the aryl bond65 of the annulated structure ofthe thianthrene core (see Supporting Information for a discussion). To demonstrate the performance of 1 in cross-coupling reactions, we developed a Suzuki-type vinylationof aryl boronic acids (28\u201336), including electrophilicgroups that are not tolerated by Wittig olefination-based synthesis66 , with proto-deborylationobserved as the main side reaction in those examples with lower yields.The fast rate of oxidative addition of the C\u2013S bond allowedthe vinylation of substrates containing C\u2013Br bonds (33) that are otherwise reactive in Suzuki reactions.19 Likewise, a competition experiment between vinyl thianthrenium 1-d3 and vinyl bromide establishedthat the thianthrenium compound reacts substantially faster than vinylbromide; less than 1% of reaction product based on vinyl bromide couldbe detected by either NMR spectroscopy or mass spectrometry analysis(38) and trifluoroborates (39), ispossible. Alkenyl boronic acids were also suitable substrates, yieldingvaluable dienes (40) that can be employed for furtherelaboration .Vinylatedarenes (styrenes) are activated alkenes with widespreaduse in transition-metal catalysis,ic acids 2A. The sanalysis2B. Exten2(OTf) under the samereaction conditions did not afford the desired products or resultedin <15% yield in all the cases studied , which may be the result of a faster reagent decompositionor catalyst poisoning. Moreover, analysis of the reaction mixturerevealed the presence of equimolar amounts of side-product 43, arising from aryl\u2013Ph instead of aryl\u2013vinyl bond formation,while no related product resulting from aryl\u2013aryl couplingcould be detected in the reaction with 1. A similar outcomewas observed with substrate 44. These results underlinethe key benefits of the structural design of thianthrene electrophiles,effectively channelling the oxidative addition process toward thedesired C\u2013S bond and allowing, for the first time, a vinylationreaction based on cross-coupling with vinyl sulfonium salts.In contrast, the use of vinyl-SPh studied 2C. For eIn summary, we have developed a convenient vinyl electrophile reagentthat is prepared directly from ethylene and can be stored in the presenceof air and moisture. The salt has proven to be an effective vinylatingreagent and C-2 synthon for the synthesis of carbo- and heterocycles,N-vinylated products, styrenes, and dienes. The distinct structuralfeatures of thianthrenium salts in comparison with other sulfoniumsalts enable both its unique synthesis from ethylene and its superiorperformance in cross-coupling reactions. Its one-step synthesis, easy-to-handlefeatures, and robust reactivity make it a valuable and versatile reagentthat will find synthetic utility in further organic and transition-metalcatalyzed transformations."}
+{"text": "There is an astonishing diversity of ways in which people benefit from coral reefs. They provide recreation, resource extraction, inspirational, and educational opportunities, among many others as well as being valued just for their existence. As the condition of coral reef ecosystems decline, so do their ability to provide these benefits. Prudent management of coral reefs and the benefits they provide are important as some predict most coral reefs globally will be lost by the mid-21st century. Meanwhile, coral reef managers have limited tools and relevant data to design and implement effective environmental management practices that will enable coral reefs to provide benefits demanded by society. We demonstrate an approach to identify and measure environmental components of coral reefs that directly benefit human well-being. The approach views ecosystems through the lens of a specific set of beneficiaries and the biophysical features directly relevant to each. We call these biophysical features Final Ecosystem Goods and Services (FEGS). In our demonstration, we (1) identify a range of beneficiaries of coral reefs; (2) identify metrics of FEGS for those beneficiaries; and (3) describe how data quantifying those biophysical metrics might be used to facilitate greater economic and social understanding. Human well-being is reliant upon ecosystems goods and services (EGS) that sustain our society, human health, and economy, and they are often assumed to be available for free . The chaIncreasingly, greater emphasis is placed on integrating the full set of benefits when considering decisions that can impact EGS with a growing awareness about the complexity and diversity of connections between natural and human systems. Ecosystems provide goods or tangible biophysical components of nature that provide services to humans . These EEcological products and processes directly experienced by human beneficiaries are final ecosystem goods and services (FEGS) . BeneficAlthough the Millennium Ecosystem Assessment  was pivoThe FEGS framework is distinguished from other ecosystem service definitions  by takinFinal ecosystem goods and services serve as the linking metrics to clarify the benefits experienced by people in the specific ways in which they directly interact with ecosystems. Final ecosystem goods and services are contrasted with the broader set of essential intermediate ecosystem goods and services (IEGS) that are required to support or regulate FEGS ; Fig. 1.Coral reefs were chosen as one of seven ecosystems examined as part of a larger U.S. national effort developing FEGS metrics using a similar structured process and shared expertise on metric development across these ecosystems . Coral rProtection of ecosystem benefits is important for coral reef managers; a priority heightened by the presence of rapidly increasing coastal human populations increased sea temperatures and ocean acidification  increasePrevious studies addressing coral reef EGS have primarily focused on identifying ecological characteristics that contribute to the resistance, recovery, and conservation of ecosystem services based on key ecological traits, life history strategies, and functional ecology of coral reefs . This woOur objective was to demonstrate how to apply the FEGS conceptual framework to link people\u2019s well-being to coral reef ecosystems by adopting a user-centric perspective. Coral reefs were chosen as one of seven ecosystems examined as part of a larger U.S. national effort developing FEGS metrics using a similar structured process and shared expertise on metric development across these ecosystems . The metAcknowledgments) who were biophysical scientists familiar with the principles of biophysical metric development and selection  and a social scientist with a specialty in decision science and EGS (L. Sharpe). The metrics team worked to select the beneficiaries, attributes, and FEGS biophysical metrics that related to elements of human well-being . MetricsStep 1: Delineate ecosystem boundaries;Step 2: Specify beneficiaries and begin to define the final good or service for each beneficiary by asking \u201cWhat directly matters to that beneficiary?\u201d;Step 3: Select attributes guided by the questions from a standardized list of ecosystem attributes directly used, appreciated, or enjoyed by each beneficiary. Refine ecosystem attributes at the level necessary to support the specification of metrics of the FEGS for each beneficiary; andStep 4: Specify metrics for each beneficiary to develop the FEGS and FEGS metrics using these steps:Define the ideal metric;Define the available biophysical measures closely related to that ideal metric;Use the metrics team expertise to evaluate the ideal metric to determine if the metric(s) proposed sufficiently translate the FEGS into the desired information most easily understood by the beneficiary; andMetrics team validate metrics and metrics vetted by FEGS team, review, revise, and repeat until consensus among both groups.Key to the FEGS approach is designating the beneficiaries, then identifying relevant biophysical attributes and how to measure them . The folCoral reef ecosystems were categorized employing the Final Ecosystem Goods and Services Classification System (FEGS-CS) environment classification . FEGS-CSThe metrics teams attempted to identify all likely beneficiary groups to evaluate the utility of this approach across a diverse spectrum of uses. We included beneficiaries from direct use, indirect use, optional use, and the least tangible non-use value necessary for a total economic benefits analysis . We did https://www.census.gov/naics/ last accessed May 2021; We chose beneficiaries using the National Ecosystem Services Classification System (NESCS) Plus  that conThe metrics team identified which ecosystem attributes provide a final good or service for each beneficiary and their defined use by answering \u201cWhat matters to this beneficiary?\u201d as a heuristic question . GeneralIn NESCS Plus, Tier 1 attributes for FEGS are basic components of all ecosystems classified as Water, Air, Weather, Soil and Substrate, Natural Materials, Flora, Fungi, Fauna, and Extreme Events , Composite , all Tier 1 attributes are mutually exclusive except for the last one. Final ecosystem goods and services Tier 2 attributes divided each Tier 1 attribute into multiple and more specific attributes . For exaEach attribute was considered by the metrics team and appraised how well and to what degree the FEGS attribute was appreciated by the beneficiary. We defined how each beneficiary directly interacted with the coral reef by considering all the ways that the beneficiary  used, apFirst, the metrics defined the desired information that lead to a biophysical measurement for each sub-attribute of the ecosystem to identify an ideal metric that was most meaningful to that beneficiary. The metrics team identified metrics that reflected the sub-attributes to embody biophysical aspects of nature that ecologists could measure and monitor directly, often those used for environmental assessment programs. Many potential metrics were scrutinized to select subsets of metrics that were most meaningful to the beneficiaries\u2019 interests. The metrics team considered whether the biophysical metric chosen represented the most apparent, tangible, and intuitive features that resonated with the specific beneficiary. The metrics team described this information so that it would be easily understood by each beneficiary group and ensured the metric was not too technical. Best professional judgment and review of the ecological literature guided the development and identification of the best biophysical measures of reef condition contributing to FEGS .Frequently more than one metric was suggested for most beneficiaries as they might directly and simultaneously experience or perceive multiple metrics of an ecosystem at the same time. For example, coral reef viewers enjoy the seascape that encompass the sub-attributes of reef type, color, shape, rarity, diversity, richness, and abundance that directly contribute to their appreciation, enjoyment, and usage of the coral reef. The technical metrics and units for each one of these sub-attributes would not have much meaning to a lay beneficiary, rather an indicator of overall pleasure for viewing a coral reef seascape might integrate all or a combination of those sub-attribute metrics to develop a categorical metric or indicator. A rating of excellent, good, fair, or poor seascape viewing experience of coral reefs for beneficiaries might be more meaningful and easier to communicate to a nontechnical user . Final eThe proposed FEGS metrics were evaluated by assessing face validity, common sense, and qualitative research . Drafts The results for a single beneficiary were incorporated into a table designed to guide selection of appropriate metrics for additional FEGS-based assessments that could be conducted for specific decision contexts or locations beyond those identified as exemplified in the U.S. Environmental Protection Agency (U.S. EPA) national effort for FEGS development for seven different ecosystems . The tabA diverse spectrum of ecosystems services provided by coral reefs was identified and translated using the FEGS structured framework into related metrics to better facilitate economic and social evaluations for environmental management and policy decisions. The FEGS analysis results for 10 coral reef beneficiaries are in separate tables , cultural, spiritual, and ceremonial perspectives. Finally, Non-use beneficiaries cared about the existence of coral reef ecosystems in the present and future. Other beneficiaries analyzed were Ornamental Extractors using live reef organisms for display in aquariums or dead for jewelry or decorative products; Commercial or Industrial users focusing on Pharmaceutical Extractors using organisms for medical, cosmetic, and beauty products; and Aquaculturists rearing juvenile or adult corals for multiple purposes such as aquaria trade and reef restoration.Tier 1 attributes assigned for coral reefs were Water, Soil and Substrate, Flora, Fauna, and Extreme Events, Composite; Tier 2 attributes and sub-attributes developed for coral reef beneficiaries are bolded text in For this step, we detail the development FEGS metrics for two beneficiary classes to illustrate the application of the FEGS approach: recreational SCUBA divers and snorkelers, and anglers who catch fish on coral reefs. Results for the other eight beneficiaries are found in The metric team limited analysis of SCUBA divers and snorkelers (now referred to as divers) to those who were primarily interested in recreational diving or snorkeling . Their interests posited as questions were \u201cWill my dive be enjoyable and safe?\u201d and \u201cIs the environment appealing?\u201d , Step 2 Fourteen FEGS and metrics were developed for divers , after bThe presence of chemicals and contaminants in the seawater was identified as a critical sub-attribute to determine \u201cIs the water quality high enough to be safe for diving?\u201d . RecreatFish and coral community metrics reflecting what was most desired by divers were overall abundance; presence of rare species; biodiversity; species richness; size; color and unique behaviors and morphologies, most of which are surrogate metrics. A multimetric index expressed as a simple categorical indicator would be easier for divers to decide whether their dive would be enjoyable. Literature identifies the amount of live coral cover as the second most common coral community metric associated with making dives pleasurable  with corThe angler beneficiary merged three angler types: recreational catch and release and catch and eat, and subsistence anglers. We assumed anglers fished from boats and had minimal contact with the seawater, posing no health concerns from contaminated seawater exposure. Anglers were interested whether \u201cIs this a good place to go fishing?\u201d and \u201cWill the boat be enjoyable and safe to fish from?\u201d .The angler subclasses overlapped in their interest in Fauna , Step 3,There were many FEGS metrics which crosscut the spectrum of 12 coral reef beneficiaries as each angler subclass was considered individually for this exercise. Both wave intensity and current strength metrics were identified for 10 of the 12 (83%) beneficiaries for coral reefs . The mosThe suite of FEGS metrics identified for beneficiaries exemplify the wide diversity of ways stakeholders use and benefit from coral reefs \u2013S8. We dWe developed our FEGS metrics using the hierarchical NESCS Plus classification to exemplify selection of the beneficiaries, attributes, FEGS, and biophysical metrics after defining the context using general and specific questions. It is likely that our selected elements may differ considerably, be less familiar, or of less importance for others with different management responsibilities. In those cases, there might not be consistent criteria to propose or select FEGS metrics on a comprehensive basis, so surrogate metrics can be substituted. In other cases, direct measurement of some attributes might be difficult or expensive, justifying the use of surrogate metrics that best approximate valued attributes while acknowledging inherent limitations of using surrogates.Coral reef managers desire tools and approaches to assist them in problem definition and finding solutions, because many have very limited resources, time, and expertise to make important decisions. Ecosystem-based management aims to guide local and regional experts to organize and streamline the level of information required to formulate the desired results and identify trade-offs and uncertainty in predicting ecosystem outcomes while weighing socioeconomic concerns against ecosystem condition . Final eThose identifying our current time as the Anthropocene Era have proposed that the trajectory of change imparted by humans is irreversible, and scientists must acknowledge that the forces of human impacts and intervention are rapidly changing the structure and function of reefs . ReportsThe FEGS framework can be adapted to many different applications, additional beneficiaries, and scaled up or down both spatially and temporally as required by decision needs of the environmental manager or communities. The identification of beneficiaries and FEGS linking metrics can be tailored to the local scale for a specific ecosystem and period of time. Examples include streams  or seasoA primary advantage of the FEGS framework is the ability to be very flexible for operationalizing EGS from a beneficiary perspective, particularly when paired with standardized and hierarchal classification systems  that canThe conceptual framework for ecosystem goods and services continues to be expanded and incorporated into decision-making by governmental, national, and international organizations as these entities better define their values. Supplement1"}
+{"text": "Prunus sibirica is detrimental to fruit production. The late flowering (LF) type, which is delayed by 7\u201315 days compared with the normal flowering (NF) type, avoids damages at low temperature, but the molecular mechanism of LF remains unclear. Therefore, this study was conducted to comprehensively characterize floral bud differentiation. A histological analysis showed that initial floral bud differentiation was delayed in the LF type compared to the NF type. Genome-wide associated studies (GWAS) showed that a candidate gene (PaF106G0600023738.01) was significantly associated with LF type. It was identified as trehalose-6-phosphate phosphatase (PsTPPF), which is involved in trehalose-6-phosphate (Tre6P) signaling pathway and acts on floral transition. A whole-transcriptome RNA sequencing analysis was conducted, and a total of 6,110 differential expression (DE) mRNAs, 1,351 DE lncRNAs, and 148 DE miRNAs were identified. In addition, 24 DE mRNAs related with floral transition were predicted, and these involved the following: three interactions between DE lncRNAs and DE mRNAs of photoperiod pathway with two mRNAs  and three lncRNAs ; one interaction between DE miRNAs and DE mRNAs with one mRNA, encoding trehalose-6-phosphate synthase , and one miRNA (miRNA167h). Combined with the expression profiles and Tre6P levels, functions of PsTPPF and PsTPS1 in Tre6P regulation were considered to be associated with flowering time. A new network of ceRNAs correlated with LF was constructed, and it consisted of one mRNA (PsTPS1), one lncRNA , and one miRNA (miR167h). This study provided insight into the molecular regulatory mechanism of LF in Prunus sibirica.Freezing during the flowering of  Prunus sibirica is a stone fruit and woody oil plant that has excellent resistance to extreme environment conditions and is both economically and ecologically valuable  in photoperiod pathway  in age pathway  in trehalose-6-phosphate (Tre6P) signaling pathway  have been conducted to identify candidate genes associated with important traits in perennial fruit trees. For example, studies have investigated l traits , Prunus sistance , Prunus l traits , and Maly traits . Recentl persica , Heliants annuus , and Braca napus  have beeCOLD-INDUCED LONG ANTISENSE INTRAGENIC RNA (COOLAIR) and COLD-ASSISTED INTRONIC NON-CODING RNA (COLDAIR) acted on vernalization process associated with controlling flowering time by silencing the transcription of FLC  involved in floral transition have also been studied, such as miR159 , which participate in multiple biological and abiotic functions. NcRNAs are divided into two categories by length, small RNAs and long non-coding RNAs (lncRNAs). Notably, ncRNAs played important roles in the control of flowering; for example, two well-known lncRNAs, n of FLC . The stus miR159 , miR168 s miR159 , milR169s miR159 , and miRs miR159 . Competis miR159 . Furthers miR159 . ceRNAs s miR159 . Howeverpersicum  and Brasmpestris .P. sibirica, we conducted a GWAS to identify the loci and candidate genes associated with LF. We also conducted an integrated analysis of mRNAs, lncRNAs, and miRNAs to construct ceRNAs networks by whole transcriptome RNA-sequencing. This study provides new insight into the genetic regulation of LF mechanisms in P. sibirica.To elucidate the molecular mechanism of late flowering (LF) in P. sibirica accessions were used in this study, which grown in the Inner Mongol Forest Seed Breeding Center in Horinger County within the Inner Mongolia Autonomous Region. Full bloom date (FBD) was defined as when up to 50% of the flowers opened  of floral buds collected from LF and normal flowering (NF) accessions were respectively used to conduct histological analyses and whole-transcriptome RNA sequencing.A total of 66 s opened , and thes opened . Fresh yFloral buds  from the LF and NF types were sampled every 10 days from June 20 to July 10, 2017, every 3 days from July 10 to October 11, and every 7 days from October 11, 2017 to April 3, 2018. After fixation in an FAA fixative solution for 24 h, the buds were kept in a 70% alcohol solution under 4\u00b0C. The materials were then subjected to alcohol dehydration, xylene treatment, waxed treatment, embedding, and slicing at a thickness of 6\u201310 \u03bcm. Samples were subsequently dyed with Fast Green and counterstained with safranin. Tissues were observed, and images were obtained under an optical microscope, Olympus BX-51 .P. sibirica accessions were used in this study, of which 41 have been used in our previous study  and 25 were newly sequenced. The leaf DNA of 25 P. sibirica accessions was extracted with CTAB methods, and sequence libraries were constructed with fragment sizes of up to 300 bp. The libraries were then sequenced on an Illumina HiSeq X Ten platform. After sequencing, clean reads were obtained using fastp  by removing low-quality reads with adapters at both or either end, and with the number of N bases accounting for more than 5% (P. sibirica accessions were submitted to Genome Sequence Archive (PRJCA006925).A total of 66  than 5% . The genP. sibirica \u201cF106\u201d reference genome  using BWA-MEM  (Whole-genome sequences for each accession were mapped to the -v 3 -Y) . Duplica-v 3 -Y) . A GATK -v 3 -Y) .To improve the statistical power of the analysis, a set of bi-allelic SNPs with missing rates of less than0.2 and minor allele frequency (MAF) of >0.05 were obtained to conduct subsequent analyses. Principal component analysis was performed using Smartpca . A distaThe total RNA was extracted from floral buds of the LF and NF types obtained on July 10, 2017 using a ethanol precipitation protocol and a CTAB-PBIOZOL reagent. The results were qualified and then quantified using a NanoDrop and an Agilent 2100  bioanalyzer. Two biological replicates were analyzed for each RNA sample.P. sibirica genome using the HISAT2 software (version 2.0.4)  (epicenter) to deplete rRNA. The transcription was reversed, and the adapters were ligated. The qualified libraries were sequenced pair end on the Hiseq X-Ten  platform. After sequencing, clean reads were obtained by removing low-quality reads and reads, with the number of N bases accounting for more than 10%. The clean reads generated by high-throughput sequencing were mapped on the n 2.0.4) , and then 2.0.4) . The rawSmall RNA library was prepared with 1 \u03bcg total RNA for each sample. Total RNA was purified by electrophoretic separation on a 15% urea denaturing polyacrylamide gel electrophoresis (PAGE) gel, and small RNA regions corresponding to the 18\u201330 nt bands in the marker lane  were excised and recovered. The adaptors were then ligated, transcribed into cDNA, and purified. Final ligation PCR products were sequenced using the BGISEQ-500 platform . The small RNA sequencing raw data were submitted to the Genome Sequence Archive (PRJCA006925).The CPC , txCdsPrp-value \u2264 0.001 by DEG-seq   .The function of lncRNAs is mainly realized by their action on target genes in a cis or trans relationship, and cis regulation of lncRNAs and their target mRNAs are based on a location relationship. Trans-regulation was predicted here by calculating the binding energy. In addition, the two correlation coefficients of lncRNA and mRNA were calculated using Spearman and Pearson (Spearman_COR \u2265 0.6 and Pearson_COR \u2265 0.6). A cis relationship was defined as lncRNA within 10 K upstream of mRNA, or within 20 kb downstream of mRNA. If the lncRNA and mRNA binding energy was beyond this range, RNAPlex  was usedpsRobot , TAPIR , and TarA hypergeometric distribution model was used to test whether the DE Tre6P signaling pathway genes shared a significant number of miRNA binding sites with DE lncRNAs. The LF-related ceRNAs were selected according to the following criteria: (1) the DE floral transition pathway coding-genes, lncRNAs, and miRNAs were significantly differentially expressed; (2) the DE floral transition pathway coding-genes shared the same DE miRNA with ceRNA (mRNA/lncRNA) and with the same MRE; and (3) the expression levels of the floral transition pathway coding-genes and predicted ceRNAs were opposite to those of the shared miRNA. The ceRNA network was presented using Cytoscape (version 3.8.0) .g and 4\u00b0C. Supernatants were then used for detection of Tre6P content using Plant Trehalose-6-Phosphate (T6P) ELISA Kit . The optical density (OD) of the samples and six standard products was measured at the wavelength of 450 nm with BioTek\u2019s Gen5\u2122 Microplate Readers  and calculated based on the standard curve (with a correlation coefficient R2 \u2265 0.99), which was established using the concentrations and ODs of six standard products . The Non-linear Curve Fit was established to build the standard curve using the Origin Pro 2021 software  of three independent biological replicates.Trehalose-6-phosphate (Tre6P) was extracted from the frozen pulverized floral bud tissues of eight randomly selected accessions , and three biological replicates were performed. Tissue homogenates were extracted for 30 min of ice-cold PBS (pH 7.4) and centrifuged for 15 min at 3,000 \u00d7 software . Student1 The actin gene was used as an internal reference to normalize the qRT-PCR data. All the primers are listed in \u2013\u0394\u0394Ct method. Student\u2019s t-test was performed in the analysis and conducted using the Origin Pro 2021 software. The data are presented as the means \u00b1 standard deviation (SD) of three independent biological replicates.Total RNA was extracted using GenePure Plus Plantpoly RNA Kit (rich in polysaccharides and polyphenols) . One microgram of DNA-free RNA was transcribed into first-strand cDNA using All-in-One First-Strand Synthesis MasterMix (with dsDNase) . qRT-PCR was conducted with a Roche LightCyler 480 instrument using 2 \u00d7 SYBR Green qPCR Premix  . Primers for target genes were designed using Primer 3.0.Both NF and LF type accessions were selected to explore the process of floral bud differentiation. The FBD of LF types was found to be delay by 7\u201315 days compared to that of NF types . HistoloP. sibirica. The phenotypes of 66 accessions were classified into NF (43) and LF (23) types base on the FBD from the 3 years in succession. A total of 2,598,398 high-quality SNPs were identified by mapping against the P. sibirica reference genome, and these were used in the subsequent analyses. Principal component analysis (PCA) showed two differentiated clusters, which were consistent with the classifications from the FBD (ubiquitin-conjugating enzyme E2 (UBC) and trehalose-6-phosphate phosphatase (TPP), respectively, whereas the predicted function of PaF106G0500018905.01 was unknown. In particular, an SNP mutation (Chr. 6: 18107219 bp) was found to be located on the promoter of PsTPPF (PaF106G0600023738.01), which resulted in a point mutation in the third nucleotide of a G-box core sequence (TACGTG), suggesting that the mutated site might hinder the binding of G-box interaction proteins, thus affecting the transcriptional expression PsTPPF. These results will be valuable for the development of molecular markers for late flower breeding in P. sibirica.To better identify the candidate genes associated with flowering time, we conducted a GWAS for this trait in  the FBD . The GWA the FBD , which w the FBD . Among t the FBD , includi the FBD . The preP. sibirica floral buds was conducted, and 515,976,068 raw reads were obtained. After filtering of adaptor sequences and low-quality reads, over 97% clean reads remained  mRNAs and lncRNAs. A total of 6,110 DEmRNAs and 1,351 DE lncRNAs were identified. Of these, 3,017 known mRNAs, 586 novel mRNAs, and 731 lncRNAs were upregulated. In contrast, 2,038 known mRNAs, 469 novel mRNAs, and 620 lncRNAs were downregulated . Notably by GWAS .A DE lncRNA-DE mRNA network was constructed according to location relationship and binding sequence. A total of 9,345 pairs of cis-regulatory  and 1,87A total of 116,046,350 raw reads were generated from the small RNA libraries . After fWe identified 148 DE miRNAs in LF vs. NF types, including 80 upregulated miRNAs and 68 downregulated miRNAs . Among tPsTPS1 (PaF106G0100001132.01), encoding trehalose-6-phosphate synthase, were predicted, and these have been previously identified as being involved in the Tre6P signaling pathway of floral transition , one miRNA , and one lncRNA (TCLR)  . In the P. sibirica is reduced when temperatures are low . The expression patterns of PIF4 and ARP6 in regulating LF are in accordance with those of earlier studies conducted on A. thaliana, which found PIF4 and ARP6 controlling flowering time by activating the expression of FT and FLC, respectively, in response to warm temperatures  involved in Tre6P signaling pathway were found by GWAS and the whole-transcriptome RNA sequencing analysis in our study, and their expression levels showed inhibitory effects to the content level of Tre6P. However, as floral transition is controlled by an intricate genetic network, it is considered that the other 22 floral transition genes may also regulate the LF trait together with PsTPPF and PsTPS1 in the LF types of P. sibirica.Previous studies have shown a correlation between sugar content and floral transition in thaliana , M. domeomestica , and R. hinensis . Tre6P ihinensis . Moreovethaliana . Two canpho2-induced phosphate starvation1 (IPS1)-miR399 was reported in A. thaliana , and one mRNA (PsTPS1), which shared with one miRNA (miR167h). We hypothesize that the low expression of PsTPS1 is negatively regulated by TCLR competitively binding to miR167h, and that the high expression of PsTPPF is caused by a single SNP located in its promoter, which might result in the LF trait in P. sibirica. Studies have shown that miR167a-d played important roles in floral organ development in A. thaliana  is related to LF in P. sibirica, and we constructed a new ceRNA network  for the LF types of P. sibirica. However, it is also considered that the other 22 differentially expressed floral transition genes with their interactional lncRNAs and miRNAs may also be involved in the regulation of LF of P. sibirica, and further studies are, thus, required. This study identified the molecular regulation mechanism associated with LF in P. sibirica, and the results can be used as a valuable genetic resource for breeding LF varieties of P. sibirica.We proposed that a new regulatory flowering gene (The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/TW and LW designed and supervised the experiments. WX conducted the experiments, analyzed the data, and wrote the manuscript. WB, HL, CC, and HB helped analyzed the sequencing data and experiments. WB, MH, GZ, HZ, NG, and YC performed the sample collection. TW, LW, and WX revised the manuscript. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Bifidobacterium genus, and also, higher alpha-diversity compared to the rest of the women. Our results confirm the importance of controlling weight during pregnancy and breastfeeding practices in terms of milk microbiota. Further studies to clarify the potential impact of these maternal factors on milk and infant development and health will be necessary.Breastfeeding is critical for adequate neonatal microbial and immune system development affecting neonate health outcomes in the short and long term. There is a great interest in ascertaining which are the maternal factors contributing to the milk microbiota and the potential relevance for the developing infant. Thus, our study aimed to characterize the effect of mixed and exclusive breastfeeding practices on the milk microbiota and to determine the impact of pre-pregnancy body mass index (BMI) and weight gain over pregnancy on its composition. Breast milk samples from 136 healthy women were collected within the first month post-partum and milk microbiota profiling was analyzed by 16S rRNA gene sequencing. Information on breastfeeding habits and maternal-infant clinical data were recorded. Breastfeeding practices (exclusive vs. mixed), maternal pre-gestational BMI, and weight gain over pregnancy contributed to the milk microbiota variation. Pre-gestational normal-weight women with exclusive breastfeeding habits harbored a significantly higher abundance of  Breast milk is the best and the primary food for infants since it covers the infant\u2019s needs during a critical period of human development and health programming. Beyond nutritional aspects, it contains a diverse array of biologically active components which guide the adequate infant\u2019s growth and development . The WorGrowing evidence suggests that breast milk microbiota would be influenced by different perinatal factors such as delivery mode, antibiotic treatment, diet, and maternal psychosocial status ,7,8,9. Hn = 136) were included in the study  were collected. Regarding maternal age, participants were categorized into three age groups as following: <30\u2009years, 30\u201335\u2009years, and\u2009>35\u2009years, corresponding approximately to the quartiles (Q1 = 32 and Q3 = 37 years).2) and OW (\u226525.0 kg/m2). Additionally, mothers were also classified according to their weight gain over pregnancy according to their pre-gestational BMI following the recommendations of the Institute of Medicine [Mothers were classified according to their pre-gestational BMI  as normaMedicine  in norman = 136). Breast milk collection was performed following a standardized protocol. Briefly, milk was collected using a sterile pumper in sterile bottles upon the cleaning of breast skin with 0.5% chlorhexidine solution. Milk samples were kept at \u221220 \u00b0C until its transport to the laboratory, where they were stored at \u221280 \u00b0C until further analysis.Breast milk samples were compiled within 30 days post-partum  as previously described . DNA pur\u00ae Green PCR Master Mix (Roche), 0.25 \u03bcL of each of the specific primers in a LightCycler\u00ae 480 real-Time PCR System , at a concentration of 10 \u03bcM, and 1 \u03bcL of template DNA. All amplifications were performed in duplicates. Standard curves for the targeted bacterial-amplicon were generated using Ct values and the calculated gene copies numbers were determined based on the fragment amplification length.Total bacterial load was determined by qPCR amplification as described elsewhere . PrimersBreast milk microbiota composition was assessed by the sequencing of the V3\u2013V4 variable region of the 16S rRNA gene following Illumina protocols as described by Garc\u00eda-Mantrana et al.  on a MiSn = 48 ASV). Sequence data have been deposited in the National Center for Biotechnology Information (NCBI) under the project accession number BioProject ID PRJNA614975.Trimmomatic software  was usedwww.graphpad.com accessed on 1 April 2021). Redundancy analysis (RDA) was applied to study the statistical effect of breastfeeding practices on breast milk microbiota. The differences between the groups were visualized by the discriminant of principal components analysis (DAPC) at the amplicon sequence variant (ASV) level and the Adonis test was achieved based on the Bray-Curtis distance.The following software was used for analysis: Calypso online platform (V8.84) , SPSS V.T-test and Mann-Whitney analysis were used depending on data normality assessed by Kolmogorov-Smirnov and Shapiro-Wilk test (Graphpad Prism V5.04). Spearman correlations between relative abundances of bacterial and maternal age were using RStudio [ RStudio . Multiva2 and 12.0 kg, respectively. In the study cohort, 62.5% of deliveries were vaginal and 81.6% of the mothers followed the EBF practices during the first month of life.Maternal-infant characteristics are shown in p < 0.001) shorter in women with MF at 1 month . In our cohort, 81.6% followed the EBF practices during the first month of life.We found significant differences in the total breastfeeding duration being significantly (Streptococcus (29.2%) and Staphylococcus (27.8%), followed by Ralstonia (10.1%) and Acinetobacter (9.6%)  and Proteobacteria (28.8%) phyla, followed by Actinobacteria (3.7%) and Bacteroidetes (1.18%) A. At ther (9.6%) B.p = 0.034) significantly affected the breast milk microbiota.A permutational multivariate analysis of variance Adonis, with Bray-Curtis distance metric, showed the main contributors to the breast milk microbiota variation C. BreastR2 = 0.0174, and p < 0.01) at the ASV level. Indeed, breast milk microbiota was significantly different between those mothers that had or not an EBF in the multivariate analysis (RDA) , Ralstonia (p = 0.05) and Pseudomonas (p < 0.001) compared with MF mothers, was observed.Breast milk microbiota was significantly different according to breastfeeding practices (Adonis test p < 0.001) and diversity (measured by Shannon index) (p < 0.001) were observed in milk from EBF mothers compared to those observed in MF  ) and Lactobacillus (6.43 (3.42\u201312.10)) and lower incidence of Staphylococcus (0.69 (0.64\u20130.74)), and Escherichia/Shigella (0.42 (0.31\u20130.58)) in milk compared to MF mothers : 3.72 (95% CI: 2.94\u20134.72)) in their milk compared to MF mothers (p = 0.440).In the Poisson regression models based on breastfeeding practices (EBF or MF) adjusted by covariates, including mode of delivery and antibiotic treatment for one month and pre-gestational BMI, breastfeeding practices were found to be consistently associated with some genera relative abundance . EBF mot mothers . EBF mot mothers . FurtherBifidobacterium (p = 0.029) genus was observed in NW mothers compared to OW mothers (p = 0.031).We tested the impact of pre-gestational BMI and weight gain over pregnancy in the whole population. We observed that breast milk microbiota profile and also, alpha-diversity were influenced by pre-gestational BMI and weight gain. However, a higher relative abundance of  mothers A. RegardStreptococcus (incidence rate ratio (IRR): 1.21 (95% CI: 1.12\u20131.30)) in their milk compared to OW mothers (Bifidobacterium (4.67 (2.53\u20138.64)) and Ralstonia (1.16 (1.03\u20131.32)) and a lower incidence of Staphylococcus (0.89 (0.83\u20130.96)) in milk compared to OW mothers  while this observation was not reported in MF women . Furthermore, significant differences were observed in the whole microbial community between breastfeeding practices (EBF vs. MF) in NW mothers , whereas no differences were detected in OW mothers  (Bifidobacterium (p = 0.033) genus were found in EBF_NW mothers compared to the other groups and significantly lower abundances of Pseudomonas (p < 0.01) was observed in MF_OW mothers compared to the other groups.Due to the impact of breastfeeding practices on breast milk microbiota, we explored the possible potential effect of pre-gestational BMI and weight gain over the pregnancy in the milk microbiota . Pre-ges = 0.78) . Discrim = 0.78) B. Indeed = 0.78) . Higher p = 0.014; and Adonis Bray\u2013Curtis R2 = 0.015, p = 0.111), whereas no differences were detected in EWG mothers according to breastfeeding practices  (Staphylococcus (p = 0.049) genus and lower relative abundances of Pseudomonas (p = 0.019) genus were found in MF_NWG mothers compared to the other groups  . Higher r groups .Streptococcus in their milk compared to EWG mothers (incidence rate ratio (IRR): 1.38 (95% CI: 1.27\u20131.51)) (Bifidobacterium (3.20 (1.71\u20135.98)) and lower incidence of Ralstonia genus(0.53 (0.46\u20130.61)) in breast milk samples as the incidences observed in the EWG mothers which showed a higher relative abundance of Escherichia/Shigella genus (0.19 (0.14\u20130.26)) (p = 0.926).In the Poisson regression modeling, NWG mothers displayed a greater incidence of 7\u20131.51)) . Contrar4\u20130.26)) . FurtherAn association between the diversity and richness of breast milk microbiota and breastfeeding practices modulated by pre-gestational BMI and weight gain over pregnancy was found. Higher microbial diversity and richness were observed in women from EBF and NW mothers compared to the other groups A,B. Regap < 0.001) and diversity (p < 0.001) were observed in NW compared to OW mothers (p = 0.026) was observed in NWG mothers to EWG mothers, but this difference was not statistically significant in the case of bacterial richness  and lower weight gain over pregnancy , and also, lower microbial richness was associated to higher pre-gestational BMI  and weight gain over pregnancy .A relationship between the diversity and richness of breast milk microbiota, pre-gestational BMI, and weight gain over the pregnancy is presented . Signifi mothers . Furtherrichness . BesidesBreastfeeding practices, EBF vs. MF, have a key impact on the composition and diversity of the breast milk microbiota. Likewise, we found that pre-gestational BMI and weight gain over pregnancy are related to the composition and, especially, the diversity of breast milk microbiota. Our results confirm the importance of breastfeeding practices and also, maternal factors that would shape the milk microbial composition.Breastfeeding, maternal diet, pre-gestational BMI, weight gain over pregnancy, maternal age, genetics, and geographical location have been reported to influence breast milk microbial composition ,9,15,17.Bifidobacteriaceae and Enterobacteriaceae in infants who were EBF [Nevertheless, the impact of these factors and others, such as breastfeeding type, pollution exposure, maternal age, parity, and others, on breast milk microbial communities is still unclear. EBF during the first months of life is important for the growth and development of the child . Breastfwere EBF , though,were EBF ,39. Regawere EBF . It has were EBF ,43,44, awere EBF .The differences in breast milk microbiota composition and diversity according to breastfeeding practices would be partially explained by the impact of the infant oral microbiota which is influenced by mixed/exclusive breastfeeding practices as described previously ,46. OtheStaphylococcus genus has been observed in breast milk microbiota from women who exhibited an obese pre-pregnancy BMI [Staphylococcus genus was also associated with OW mothers while Bifidobacterium was associated with NW mothers compared to OW. Similarly, another study reported lower Bifidobacterium relative abundance on the gut microbiota of OW mothers even though they reported increased levels of Staphylococcus, and Escherichia coli in OW women compared to those NW women [5\u2013106 bacterial gene copies per mL in agreement with other studies performed using culture-independent techniques [3\u2013104 per mL of breast milk [Streptococcus and Bifidobacterium genera. Similarly, previous studies have been reported that OW mothers had lower levels of Bifidobacterium in their milk microbiota [Streptococcus and Bifidobacterium in their milk compared to EWG mothers. Colonization with the Bifidobacterium genus has been associated with several positive health outcomes in observational studies [Regarding maternal nutritional status, in our study, pre-gestational BMI was associated with the relative abundance of some genera as well as with microbial diversity and richness. Previous studies have reported associations between pre-gestational BMI and breast milk microbial composition . In accoancy BMI . In thisNW women . In our chniques . Howeverast milk . These oast milk . Our fincrobiota . Howevercrobiota . In thiscrobiota . Besides studies  and it h studies . EWG has studies . DespiteBifidobacterium genus in the breast milk of mothers from the EBF_NW group. Indeed, we found that mothers with EBF and NW were characterized by higher diversity and richness. These data are in agreement with previous studies [Considering the impact of breastfeeding practices on milk microbiota depending on pre-gestational maternal BMI and weight gain over pregnancy, we found an increase in the abundance of  studies . Other s studies . These rStaphylococcus genera and maternal age in mothers from the <30 group and 30\u201335 years group. However, a positive correlation between maternal age and Streptococcus genus was observed in older mothers (>35 years) (p < 0.001) in their breast milk microbiota compared to those from younger mothers  . A signi mothers . HoweverThis study has some limitations, including the limited sample size which reduces the possibility of detecting other significant associations as well as the lack of obese mothers. Our study is a part of an observational birth cohort and we identified that 27.9% of our population was overweight. In general, the maternal pre-gestational BMI was normal weight, thus further studies aimed to analyze the impact of breastfeeding practices and microbiota according to different BMI and weight gain are needed. Furthermore, we also evaluated these relationships in the first postpartum month period, and the results obtained need to be studied at other time points as well as during lactation. However, even if associations could be modified over lactation, these relations would be established in an extremely important period of infant maturation and therefore, they may impact the child\u2019s development with long-lasting consequences. Moreover, most of the studies focused on breast milk microbiota have relied on small cohorts and thus their findings require replication in larger studies. Our study reported data from 136 women but longitudinal data, as well as a bigger cohort, are needed in parallel to the identification of the potential impact on infant development and health outcomes. Further, differences in sample handling protocols, collection procedures and processing data pipelines could also be the source of variability and act as confounding factors.Despite all those potential limitations, our results shed light on the effect of some maternal factors on the composition and diversity of the breast milk microbiota, mainly the effect of breastfeeding practices, as limited evidence is available. Despite the importance of breastfeeding for infant growth, factors that modulate the microbial composition of breast milk have been underexplored. Therefore, efforts should be taken to ascertain the exact mechanisms by which maternal health and related factors could influence the breast milk microbiota to open the door to the design of strategies that target the modulation of breast milk microbiota composition and diversity with consequences in infant health, and in this way verify if the difference in the composition of the microbiota of breast milk has clinical relevance."}
+{"text": "Eosinophilic esophagitis (EoE) is a chronic disease, characterized clinically by esophageal disfunction. Topical corticosteroids (tCS), predominantly fluticasone and budesonide, are considered the effective first line treatment, as well as an option of maintenance therapy in EoE. The way that tCS are administered significantly affects their effectiveness. There is still no ready-to-use steroid drug to be applied topically to the esophagus in children\u2014a few experimental viscous slurries (mainly of budesonide) have been shown in trials to be more effective than steroids administered via metered dose inhalers (MDIs) and swallowed. The best examined steroid solvent of all is sucralose, a high-intensity artificial sweetener. Although it has been shown in a critical review that it is non-toxic and safe for all consumers, there are still some concerns among patients about its potential adverse effect on humans. Due to that fact, we developed a new viscous formulation and evaluated its effectiveness in the treatment of children with EoE. In an open, prospective, single-center study, we administered our new formulation of viscous budesonide twice daily for 8 weeks in patients with an active EoE. After treatment, we performed a control gastroscopy with the collection and evaluation of histopathological samples. We have proven our formulation effectiveness at 64%, as far as histological remission is concerned. We have also shown a reduction in the mean endoscopic reference score (EREFS) from 3.1 points at the beginning of the study to 1.6 points at the end of the study. Bearing in mind how important the acceptance of the solvent is for long-time compliance, especially among children, we also decided to assess the taste of the formulation. Therefore, we asked 46 adults and 10 children to swallow a sample of the solvent and fill in a short anonymous questionnaire about its taste, smell, consistency and easiness of swallowing. General acceptance for the proprietary solvent was high, reaching 7.5/10 among adults and 6.5/10 in children. To be able to compare the results of our preliminary experience, we reviewed the studies which evaluated substances that have been used so far as steroid solvents for the treatment of EoE. The overall effectiveness of the oral viscous budesonide (OVB) ranged from 65% to 90%, which is consistent with the results obtained in our study. Unfortunately, the high heterogeneity of the studies did not allow us to draw reliable conclusions. Eosinophilic esophagitis (EoE) is a chronic, multifactorial, immune-mediated disease characterized clinically by esophageal dysfunction resulting from mucosal inflammation. Histologically, it manifests by eosinophilic infiltration with a count of \u226515 eosinophils per high-power field (HPF) .Clinical symptoms of EoE vary with age. Adults and adolescents usually present with dysphagia, heartburn, chest pain, or food impaction, whereas infants and children present more commonly with symptoms of feeding problems, failure to thrive, or vomiting. Some patients may also manifest respiratory symptoms, such as cough or sleep disorders . Treatme\u00ae) were recently designed. However, although Jorveza\u00ae is effective, it is not widely available. Moreover, a liquid budesonide oral suspension is currently still in a phase III trial in the United States (Takeda) [Topical corticosteroids (tCS), predominantly fluticasone and budesonide, are considered the most effective first-line treatment, as well as an option for maintenance therapy in EoE ,6. These(Takeda) .In children, due to the lack of other possibilities, swallowed metered-dose inhalers (MDI) steroids are used. Another possibility for topical budesonide to be used in the esophagus is by combining it with another substance. Until now, only a few experimental viscous slurries (mainly of budesonide) have been shown in trials to have greater mucosal contact, and hence, to be more effective than steroids administered via MDI or swallowed .An ideal diluent for topical corticosteroids used in EoE should meet certain features: it should be safe and hypoallergic, ensure long mucosal contact, and have an acceptable taste. The best-examined steroid solvent of all is sucralose\u2014a product of sucrose, a high-intensity sweetener, which is not metabolized and does not affect blood glucose levels . The bigThe possibility of offering patients alternative solutions would give us a chance of involving the patient in the process of making therapeutic decisions, which increases his sense of responsibility for his treatment, and thus increases the chance of good compliance in the future. To treat our patients with EoE budesonide topically as effectively as possible, we developed a new viscous formulation. In a pilot study, we evaluated its effectiveness in the treatment of children with EoE. To be able to compare the results of our pilot study, we reviewed studies that evaluated substances used to date as a steroid solvent for the treatment of EoE.The new formulation was invented by our team of researchers, which included clinicians and pharmacists in the Faculty of Pharmacy of the Medical University of Warsaw, Poland as previously described . We aimeWe evaluated the taste of the formulation among several groups of people. We asked doctors, nurses, medical students, and patients with EoE to swallow a sample of our formulation and complete a short anonymous questionnaire about its taste, smell consistency, and easiness of swallowing. Additionally, we asked about general acceptance of the formulation and if they would agree to swallow the formulation for 8 weeks daily. All ratings were on a scale from 1 to 10 points, with 1 point being unacceptable and 10 points being excellent. The evaluation was conducted in the Department of Pediatric Gastroenterology and Nutrition, Medical University of Warsaw, Poland.In total, 56 people participated in the study (46 adults and 10 children). Participants rated the taste of the solution the worst, while they felt its smell and texture were more tolerable. The median score for the taste was 4/10 among both adults and children. General acceptance for the proprietary solvent was high, reaching a median score of 7.5/10 among adults and 6.5/10 among children. Notably, only three children refused to use our slurry for 8 weeks and four for 12 weeks, regardless of whether it was taken once or twice a day. The acceptance in the adult group was a bit higher and exceeded 90% when taken once a day. The detailed results concerning the acceptance of our formulation in all groups are shown in We conducted an open-label (without a control group), prospective, single-center study at the Department of Paediatric Gastroenterology and Nutrition, Medical University of Warsaw, Poland between 2019 and 2020. Research participants were recruited among children diagnosed with EoE based on standard, clinical, endoscopic, and histologic findings . Eleven participants were divided into two groups based on height: group A \u2265150 cm and group B < 150 cm. The two groups received 2 mg of budesonide/6 mL formulation or 1 mg of budesonide/3 mL formulation, respectively. The budesonide formulation was administered twice daily for 8 weeks. Participants were asked to prepare the viscous slurry by themselves, just before administration, by mixing one or two ampules of 2 mL of budesonide solution for nebulization (0.5 mg/mL) with the appropriate volume of proprietary solvent, depending on the group (6 mL for group <150 cm and 10 mL for patients \u2265150 cm). After swallowing the slurry, patients were asked not to eat or drink for at least 30 min. At the time of enrollment and in the 8th week of the study, patients underwent a gastroscopy. EoE severity was determined using the eosinophilic esophagitis reference score (EREFS) that assesses the following findings: edema (0\u20132 points), exudates (0\u20132 points), furrows (0\u20132 points), rings (0\u20133 points), and strictures (0\u20131 points) . At leasIn total, we enrolled 13 patients who met the inclusion criteria. All agreed to participate in the study, but two did not complete the trial: one did not report for a follow-up visit and the second did not take medications regularly. Finally, 11 male children aged 5 to 17 years (median age = 11 years) were included in the analysis . Six chiWe performed a search of the MEDLINE database for studies investigating the efficacy of oral viscous budesonide (OVB) slurries in EoE. In the search, we used the following keywords: EoE, oral budesonide, viscous slurry, and topical steroids. We collected data on the type of slurry, number of patients, dose and duration of therapy, histological response, clinical improvement, and, if possible, taste assessment.In total, we found 14 studies (733 participants) that assessed the effectiveness of various budesonide slurries in the treatment of EoE. Of them, nine were performed exclusively or partially in a child population (323 participants). In most trials (9/14), the OVB slurry was made using 5 to 10 g of sucralose mixed with 4 to 8 mL of budesonide solution intended for nebulized administration (0.5 mg/2 mL). The final volume of a single dosage of the solution was between 8 and 10 mL and the concentration was about 0.2 mg/mL. Some studies analyzed different solvents, such as xanthan gum, xylitol, honey, infant formulas, or apple sauce; for the sake of clarity, we have presented them in separate lines. The effectiveness of the OVB ranged from 65% to 90%; however, the high heterogeneity of the studies did not allow us to draw reliable conclusions. Detailed data are presented in p < 0.001) [p = 0.002 and MD 2.45, 95% CI: 0.76\u20134.15, p = 0.005) [The effectiveness of OVB in inducing and maintaining the remission of EoE has been proven in many clinical trials. Our study found histopathological remission (<5 eos/HPF) in 64% of patients (7/11) and response to treatment in 81% (9/11). This is slightly lower than in the study by Oliva et al. , which, < 0.001)  as well p < 0.001) and the reduction in eosinophilic infiltration  [The effect of OVB treatment appears to be quite fast. Straumann et al., after only 15 days of treatment with a medium dose of OVB (1.0 mg 2\u00d7 daily), found statistically significant differences from a placebo in terms of both the severity of dysphagia  .The effectiveness of OVB treatment is partially dose-dependent. Most studies used a cumulative daily dose of 4 mg in older children and adults. Gupta et al. studied whether dose reduction affected the effectiveness of the treatment . Daily dp = 0.30). OVB was also compared in head-to-head trials with fluticasone. Despite numerous theoretical premises favoring OVB, no statistically significant differences were found in the effectiveness of both steroids [OVB was contrasted not only with a placebo. In an interesting study, Dohill et al. compared OVB and a placebo while using lansoprazole in both groups . After 1steroids ,19 or thsteroids .p = 0.131) [p = 0.647). Similar conclusions were drawn from another study [OVB has also been shown to be effective in maintaining EoE remission. When such treatment was continued after successful induction of remission, the relapse rate after 36 weeks was significantly lower compared to a placebo  . Importa= 0.131) . Once ager study . An obseAn important aspect of the analyzed treatment is patients\u2019 compliance. Since better taste can potentially improve compliance, especially among children, we decided to analyze the taste, smell, and consistency of our slurry. The median general acceptance of the formulation in the group of patients with EoE was 6.5/10 points. Surprisingly, better acceptance was found in the group of younger children (8/10 points). Importantly, they maintained their acceptance of the formulation for the 8 or 12 weeks of treatment. To the date of publication, only one trial had assessed the taste of viscous slurries. In the Hefner et al. study, adult volunteers assessed the tolerability of a single dose of three slurries mixed with budesonide: sucralose, honey, and xanthan gum . ValidatAbnormal, only transient, cortisol levels were observed in only one patient. This is in line with the results of previous studies ,28 and tMuch research is still ongoing to develop an ideal budesonide carrier for the topical treatment of EoE. Most studies are based on the search for an ideal, viscous solvent ensuring the longest possible contact of the steroid with the esophageal mucosa. At the same time, new steroid-eluting esophageal-targeted drug delivery devices are also being tested, such as the fluticasone-eluting strings or fluticasone-loaded rings described by Prasher.Our pilot study covered a comparatively small group of participants; however, the formulation was assessed not only based on its effectiveness but also on the opinions of patients regarding its taste and tolerance. The effectiveness of the slurry was measured in accordance with international standards described in the guidelines. The open-label character of the study as well as the lack of control group comparison makes our conclusions precarious. It means the necessity to confirm the results in further studies. The study was prospective, and the formulation studied is composed of hypoallergenic ingredients and is widely available, safe, and inexpensive.Our proprietary formulation of budesonide solvent seems to have similar effectiveness to most popular sucralose-based viscous slurries used in trials to date. However, the small size of the study group and the heterogeneity of the population require a cautious approach to this statement. Due to the limitations of the study, such as the lack of a control group and the mentioned small size of the study group, we plan to confirm it in further research. The smell and consistency of the solvent were judged to be very good, and the overall tolerance to be good enough. This may suggest good compliance, which is of particular importance for the long-term treatment that EoE requires."}
+{"text": "The determination of acylcarnitines (AC) in dried blood spots (DBS) by tandem mass spectrometry in newborn screening (NBS) programs has enabled medium-chain acyl-coA dehydrogenase deficiency (MCADD) to be identified in presymptomatic newborns. Nevertheless, different confirmatory tests must be performed to confirm the diagnosis. In this work, we have collected and analyzed the NBS results and confirmatory test results  of forty individuals, correlating them with clinical outcomes and treatment, with the aim of obtaining useful diagnostic information that could be applied in the follow-up of the patients. Our results led us to classify patients into two groups. The first group (14 cases) had high increased octanoylcarnitine (C8) levels, biallelic pathogenic variants, and severe impaired enzyme activity (<10% of the intra-assay control (IAC)); all of these cases received nutritional therapy and required carnitine supplementation during follow-up, representing the most severe form of the disease. The second group (16 patients) was a heterogeneous group presenting moderate increases in C8, biallelic likely pathogenic/pathogenic variants, and intermediate activity (<41% IAC). All of them are currently asymptomatic and could be considered as having a milder form of the disease. Finally, eight cases presented a normal\u2013mild increase in plasma C8, with only one pathogenic variant detected, and high\u2013intermediate residual activity (15\u2013100%). Based on our results, we confirm that combined evaluation of acylcarnitine profiles, genetic findings, and residual enzyme activities proves useful in predicting the risk of future metabolic decompensation, in making decisions regarding future treatment or follow-up, and also in confirming the clinical effects of unknown clinical variants. Medium-chain acyl-CoA dehydrogenase MCAD, EC1.3.8.7) is a mitochondrial matrix flavoprotein essential for the beta-oxidation of medium-chain fatty acids, and its deficiency is the most commonly known genetic disorder of fatty acid oxidation (FAO) (OMIM 201450). In Iberia, and influenced by Portuguese population data, MCADD has a birth prevalence of 1:11,945, which is one of the highest reported in European countries, alongside the U.K., Denmark, and the Netherlands  (c.683C > A), reported as a conflicting variant in ClinVar, and three patients were detected in combination with [p.Glu43Lys](c.127G > A). Four patients combined c.985A > G with other nucleotide variants: [p.Pro209Leu](626C > T), [p.Cys116Gly] (c.346T > G), [p.Leu84Phe] (c.250C > T), or [p.?] (c.599 + 3A > G) (The most common pathogenic variant described in  3A > G) . Only th 3A > G) .All variants but one (c.626C > T) were previously described ,22, althp < 0.001).Regarding our biochemical confirmatory analysis, statistically significant differences in the C8 levels were found via both DBS and plasma confirmatory testing between cases with two pathological variants and patients with only one variant found (mean)  value of 2.5 \u00b1 1.7 nmol/min/mg protein. Enzyme activity was also determined in the two obligate carriers to validate the method. P39 and P40 presented residual activity levels of 24% and 32% of the control value, respectively.n = 9), presented the least MCAD activity (<6% IAC) (The enzyme activity in lymphocytes was clearly impaired (<10% intra-assay control (IAC)) in 14 individuals with abnormal NBS results. All presented biallelic pathogenic defect variants, ranging from 0.7 to 8.2% IAC . Patients homozygous for the most frequent mutation, c.985A > G (<6% IAC) .Patients with combined variants c.985 A > G and c. 683C > A presented residual activity ranging from 11 to 31%. The patient bearing a LoF variant (Gln38Term) and p.Thr228Asn presented 31% activity. Patients bearing c.985G > A and c.127G > A presented residual activity ranging from 16 to 41%. The remaining patients with two pathogenic variants presented intermediate activity (13\u201321% IAC) .Regarding cases with only one exonic variant detected, we can distinguish a group with residual activity of more than 22%, and two cases with residual activity close to 15%.Sixty-five percent of the NBS cases with two variations were asymptomatic with normal clinical parameters and growth when first referred to clinical units. Four presented minor clinical symptoms: P13, hypotonia (HP:0001252) and food intolerance (HP:0012537); P16, small gestational age (HP:0001518) with persistent creatine kinase elevation (HP:0003236) and 8% weight loss at birth (HP:0001824); P17, jaundice (HP:0000952); and P23, low birth weight (HP:0001518).During childhood, P4 developed autistic behavior (HP:0000729), language impairment (HP:0002463), social interaction problems (HP:0000735), and macrosomia (HP:0001520). P1 at 10 years exhibits abnormal behavior (HP:0012433).P5, P9, and P12 have been admitted to hospital several times for food intolerance (HP:0012537), vomiting (HP:0002013) or diarrhea (HP:0002014); bronchiolitis (HP:0011950); or leishmaniasis, respectively. In addition, P9, who has poor compliance with treatment, presented metabolic decompensation at 2 years of age and now presents autistic behavior (HP:0000729).P8 was not studied by expanded NBS and attended hospital at 16 months owing to his clinical manifestations of convulsive status epilepticus (HP:0032660) and hypoglycemia (HP:0001943).Two cases (P33 and P38) with only one variant identified presented some symptomatology. P33 presented with severe interventricular communication (HP:0010438), which was intervened, liver malfunction (HP:0001410), neurofibromatosis, coagulopathy (HP:0003256), and metabolic decompensations; P38 has had cyclical vomiting (HP:0002572) since 5 years of age, abdominal pain (HP:0002027), tiredness (HP:0012378), pallor with muscular effort (HP:0000980), and temperature episodes (HP:0001954), MCADD was suspected at 10 years of age as this case was not studied by expanded NBS.After confirmatory studies, a dietary intervention based on avoiding prolonged fasting and medium-chain triglycerides (MCT) was recommended in those cases with two mutations or clinical symptoms. In cases with hypocarnitinemia, a maintenance dose of carnitine was supplemented, individualized according to carnitine levels . The decThe plasma AC levels were monitored periodically in the 30 cases with two variants and also in P33 and P38 due to their clinical manifestations. Under treatment, plasma C8 levels remained high (1\u201310.8 \u00b5mol/L) in cases P1\u2013P12, P25\u2013P27, and P30, all with residual activity (<18%). Only patients with moderate C8 levels in NBS or in their confirmatory test presented normalized levels in some of the routine AC controls.Cases with a single variant that did not receive dietary interventions, and did not avoid long-period fasting, did not always manage to maintain C8 levels in the control range (<0.25 \u00b5mol/L).In this cohort of patients, even though there were no major events or deaths after diagnosis by NBS, there were cases with significant adverse health outcomes.The detection of MCADD has been mandatory in Spanish expanded NBS programs since 2013 because early detection of this disease may improve the outcome if prophylactic measures are implemented , along wp < 0.001), highlight the informative value of C8 plasma levels in a first stratification. As described in the literature, there is a correlation between the levels of C8 at diagnosis and clinical severity  has only a modest effect in raising plasma C8 levels, as also occurred in . Most ofConcerning c.127G > A, all three patients also exhibited intermediate MCAD activity and C8 levels. Most of them remain asymptomatic, except P23, who also presented the lowest activity of the group and hypocarnitinemia. Thus, this variant could be also considered a mild one.The remaining patients (P21\u2013P30) are compound heterozygotes of different nucleotide variations and, as could be expected, are heterogeneous. Most of them presented significant elevations of C8 in DBS and plasma and intermediate MCAD activity, except for P25 and P26, who had increased acylglycines and also showed less than 10% IAC activity, suggesting a severe presentation of the illness. In both cases, their mutations c.250C > T and c.346T > G have not been the subject of functional studies described in the literature and are classified as VUS in ClinVar; these patients showed similar MCAD activity to homozygote patients for the most frequent mutation, highlighting the importance of knowing the residual activity to emphasize the importance of treatment and follow-up in these patients. Similarly, P21 and P22 presented the highest activity of all the patients with two biallelic mutations, which could provide important information about the prognosis of the illness.p < 0.001) have been calculated.The results presented here allowed us to stratify the true cases into two categories: (i) The first comprises 14 cases, namely, those homozygous for c.985A > G  with high increased C8 levels at diagnosis and follow-up analysis, and severely impaired enzyme activity (<10% of IAC). These constitute the most severe presentation of the deficiency and require treatment and follow-up. (ii) The second comprises 16 patients (P13\u2013P25 and P28\u2013P30), forming a genetically heterogeneous group, with moderate increases in C8 levels at diagnosis and follow-up, and intermediate activity in lymphocytes (<41% IAC). This group can be classified as presenting milder forms of the disease; most of them remain asymptomatic or mildly symptomatic. Strong statistically significant differences in MCAD activity between severe cases and mild form patients (The patients\u2019 stratification presented in this work has been based mainly on biochemical/genetic confirmation data and NBS C8 levels as we do not own C8/C2 and C8/C10 ratios of all the national neonatal screening units. This information could be very informative as it is supposed to be predictive of severe cases and could help to achieve more conclusions.In summary, the results presented here indicate that the addition of the non-invasive enzyme activity to the diagnostic algorithm improves the diagnosis capacity, especially in cases with VUS variants or even in unsolved cases where only one variant is detected, adding information to determine whether further genetic analysis beyond exome sequencing, such as transcriptomic, methylomic, or genomic sequencing using long reads, might be performed to conclude diagnosis. In addition, analysis of the enzymatic activity can provide clues to determine clinical outcomes and to apply future tailored treatments."}
+{"text": "Clinical decision aids may support shared decision-making for screening mammography. To inform shared decision-making between patients and their providers, this study examines how patterns of using an EHR-integrated decision aid and accompanying verbal patient-provider communication predict decision-making satisfaction.For 51 patient visits during which a mammography decision aid was used, linguistic characteristics of patient-provider verbal communication were extracted from transcribed audio recordings and system logs automatically captured uses of the decision aid. Surveys assessed patients\u2019 post-visit decisional satisfaction and its subcomponents. Linear mixed effects models assessed how patients\u2019 satisfaction with decision making was related to patterns of verbal communication and navigation of the decision aid.The results indicate that providers\u2019 use of quantitative language during the encounter was positively associated with patients\u2019 overall satisfaction, feeling informed, and values clarity. Patients\u2019 question-asking was negatively associated with overall satisfaction, values clarity, and certainty perception. Where system use data indicated the dyad had cycled through the decision-making process more than once (\u201clooping\u201d back through pages of the decision aid), patients reported improved satisfaction with shared decision making and all subcomponents. Overall satisfaction, perceived support, certainty, and perceived effectiveness of decision-making were lowest when a high number of navigating clicks occurred absent \u201clooping.\u201dLinguistic features of patient-provider communication and system use data of a decision aid predict patients\u2019 satisfaction with shared decision making. Our findings have implications for the design of decision aid tools and clinician training to support more effective shared decision-making for screening mammography.The online version contains supplementary material available at 10.1186/s12911-022-02058-3. Each year in the U.S., approximately 237,000 women are diagnosed with breast cancer, and about 41,000 women die from the disease , 6. The Though SDM may bring many benefits , it presPrior work has categorized decision support aids into several types, including patient decision aids that can be used independently , those that digitally mediate interactions between a provider and patient, and those used in face-to-face clinical encounters , 22. WhiThe inconsistent effectiveness of CDSTs may reflect, in part, how providers use these tools within encounters. While limited work has assessed how use of CDSTs affects the encounter, related work has examined use of electronic health records (EHR) during clinical encounters. These studies suggest that increased navigation of the EHR via mouse clicks can lead to provider fatigue and cause medical errors , and mayEffective verbal communication between provider and patient is also a central component of clinical encounters. Several studies examine the linguistic features of provider communication, finding associations with patient satisfaction and outcomes \u201334. HoweThis study seeks to provide an initial understanding of how patterns of interaction, considering how both patient-provider verbal communication and navigation of the CDST are associated with women\u2019s satisfaction with SDM. We examine these questions in the context of a CDST supporting mammography SDM that has been embedded in the EHR within a healthcare system and available to all providers as a resource to support mammography decision making since 2016. Our approach may inform future research examining heterogeneity in CDST use and its association with SDM outcomes. Findings may also suggest considerations related to the design of decision aids, and help identify areas where providers may need more training and support in enacting an effective technology-supported SDM process to help women make complex cancer screening decisions.Our analyses are guided by a series of research questions and hypotheses about the ways SDM outcomes may relate to provider-patient verbal communication and CDST navigation.As far as the effective elements of provider-patient verbal communication, prior work suggests that patient satisfaction is positively associated with both the volume of communication between provider and patient, and with the frequency of questions from patients , 35\u201337. Next, we examine the impact of CDST navigation, measured by clicks (selecting or navigating between elements of the tool\u2019s interface) and loops (backtracking in the tool to revisit a prior page). The implications of clicks are unclear in the context of a CDST for SDM, where clicks may also serve to uncover personalized information but might also be associated with fatigue, errors, and distraction. Loops may reflect a more directed type of use, allowing providers to work with patients to understand the implications of varying decisions and their consequences, helping patients to clarify their values and preferences, and ultimately supporting SDM satisfaction. Finally, the relationship between clicks and loops is unclear. Thus, we examine whether the potential association between clicks and SDM satisfaction may change as a function of whether those clicks are devoted to looping, or to other activities within the tool.We propose the following hypotheses and research questions:RQ1 & 2: What is the relationship of providers\u2019 use of affect words [RQ3: What is the relationship between clicks in a CDST and SDM satisfaction?RQ4: What is the relationship between looping in a CDST and SDM satisfaction?RQ5: Is there an interaction between clicks and loops on SDM satisfaction? H1a & b: Overall word count (a) and more patient questions (b) will be positively associated with SDM satisfaction.This study examined these issues in the context of a CDST called the Breast Cancer Risk Estimator-Decision Aid (BCaRE-DA), a software platform designed for collaborative use by patients and providers to support informed breast cancer screening decisions, and emphasizing average risk women aged 40\u201349, for whom these decisions can be particularly challenging , 40. BCaWhile verbally communicating with a patient, a provider clicks through a series of pages in the BCaRE-DA, first entering individual data and the patient family history, then viewing personalized risk information, and ultimately making a collaborative decision. BCaRE-DA prompts a chronological sequence of activities. The provider moves sequentially through several pages: data entry, baseline assessment, and screening decision options. First, on the \u201cData\u201d page  and who had a patient panel meeting the inclusion criteria (women 40\u201349) were invited to participate. Eleven providers agreed to participate and provided informed consent. Their female patients aged 40\u201349 who had an average risk of developing breast cancer were invited to the study if they scheduled a primary care appointment with these providers in which a discussion of mammography screening would be appropriate. During the visit, providers used the tool to guide a discussion leading to a decision regarding screening mammography. These discussions were audiotaped and transcribed, and all page views within the BCaRE-DA tool were automatically recorded in a system use log. Within a week after the visit, patients were mailed a survey to gauge satisfaction with various elements of SDM. The study was approved by the institution\u2019s Institutional Review Board.Eligible participants had no history of dementia or breast cancer, spoke English as their primary language, and were willing to use the BCaRE-DA tool to support their decision-making regarding screening mammography and to have their interactions audio recorded and logged by the BCaRE-DA tool. While women were eligible whether or not they had past mammography experience, those who had a mammogram in the nine months preceding their scheduled visit were excluded as they would not yet be eligible for their next mammographic screening. Sixty-three eligible patient participants provided informed consent. We had complete data for 51 of these patients , comprising the audio transcription, completed post-visit patient survey, and system use logs for the BCaRE-DA tool. Figure\u00a0Audio recordings of the study visits were transcribed by a professional transcriptionist. Transcripts were then automatically coded for linguistic dimensions of providers\u2019 and patients\u2019 verbal contributions during the encounter using the Linguistic Inquiry and Word Count (LIWC) software , a dictiOverall Decisional Satisfaction was measured using the Decisional Conflict Scale (DCS) developed by O\u2019Connor [Strongly Agree) to 4 (Strongly Disagree). The formula defined by O\u2019Connor calls for items to be summed, divided by 15, and multiplied by 25, such that total scores range from 0 to 100. For ease of interpretation, decisional conflict scores were reverse coded such that higher scores indicate higher satisfaction with SDM, whereas lower scores represent lower satisfaction with SDM [O\u2019Connor .The measwith SDM .Our study also examined five subscales of the DCS,Covariates/control variables were years of education and prior mammography experience. The level of education may affect SDM by influencing patients\u2019 confidence to participate in SDM or ability to process complex or technical information. We recoded patients\u2019 highest completed education level into a continuous variable by estimating the total years of education corresponding to each categorical response . Prior mammography was a binary variable capturing whether patients had ever had a mammogram. As participants\u2019 eligibility for the study was based on their age and being at average risk of breast cancer, we did not control for age or for aspects of patients\u2019 medical history.The CDST automatically recorded system navigation in usage logs. Two variables were extracted from the logs: 1) the number of mouse clicks, and 2) the number of loops. We define a loop as returning to any previous page in the sequence. For example, one patient\u2019s record might show two loops, one where the clinician navigated from the Assessment page back to the Data page, and then later from the Decision page back to the Assessment page. Such loops allow clinicians to re-enter or edit patient data and to re-populate personalized recommendations or adjust how they are visualized, thus allowing for testing alternative scenarios, further processing information, or simply confirming and reinforcing the decision.Providers may each have a unique approach to facilitating SDM. To take account of the effects of providers\u2019 differing styles, we conducted a series of linear mixed-effect models wherein patients were nested within clinicians. The use of linear mixed-effect models can provide standard errors corrected for non-independence in the data and information about effects within and between groups . We contWe ran separate models predicting overall decisional satisfaction and each of its five subscales in relation to language use and system use data. The independent variables in the fixed effects were: provider word count, patient word count, patient question asking, provider affect words, provider quantifier words, clicks, loops, and the click by loop interaction (only entered in model 2). Covariates were patients\u2019 years of education and prior mammography. Provider identifier was entered as a random effect.The mean age of participants was 44.1\u00a0years (SD\u2009=\u20092.7). As far as the highest completed level of education, 7.8% had some high school or a high school diploma, 20.4% had some college or 2-year degree, 38.9% had a 4-year college degree, and 33.3% had completed more than a 4-year college degree. Forty-five of the 51 participants (88.2%) reported having mammography before. All had health insurance.Ten female clinicians and one male clinician working at the study University participated in this study. Their ages ranged from 37 to 63\u00a0years old, and they had 9\u201336\u00a0years in practice in primary care. Three were physicians from Internal Medicine, six were physicians from Family Medicine, and two were nurse practitioners from Ob/Gyn. Providers conducted between 2 and 9 visits each as part of this study. Table p\u2009=\u20090.01) (Table p\u2009=\u20090.001)   (Table p\u2009=\u20090.002)   (Table p\u2009=\u20090.04)    (Table p\u2009=\u20090.04)      Table , feelingp\u2009=\u20090.003, see Table p\u2009=\u20090.0001, see Additional file p\u2009=\u20090.003, see Additional file p\u2009=\u20090.001; see Additional file We finally examined the interaction effects between clicks and looping on SDM satisfaction (RQ5). We found significant interactions between clicks and looping such that, when click count was higher in the presence of more loops, patients felt more satisfied overall  and provider affect words showed no association with satisfaction, patient satisfaction was associated with the rate at which providers used quantitative language. Patient question asking was negatively associated with overall decision-making satisfaction. Regarding CDST use, our findings suggest that more clicks within the tool were negatively associated with SDM satisfaction, whereas looping back through pages in the tool was positively associated with SDM satisfaction. Moreover, patients felt more satisfied when high click counts occurred with looping, whereas patients felt less satisfied when high click counts occurred without looping.These findings are consistent with prior studies in highlighting the importance of quantitative information in facilitating decision-making , 16. ManOther findings are not consistent with the prior literature. Past studies suggest positive associations between word count, as a proxy of depth of the interaction, and patient satisfaction, whereas we did not find any such association. However, past studies have not been conducted in the context of using CDSTs, and it is possible that word count might work differently in this context. For example, gestures may play a role in communication when using a CDST , relatively brief utterances could prompt important actions in the tool , or too much verbal communication concurrent with tool use could disrupt or distract processing information conveyed in the tool. This interplay of communication volume and tool use warrants further research.Likewise, regarding affect words, we did not find a significant relationship to SDM satisfaction, which could perhaps reflect the sample in this study, with most patients having completed mammography before. Most patients therefore have prior experience weighing potential affective consequences of outcomes like false positives or cancer diagnoses. For such patients, perhaps discussing up-to-date quantitative information that clarifies their personal risks and benefits from mammography is of greater value.Surprisingly, whereas we had hypothesized that question asking would indicate patients\u2019 active involvement in SDM and associate with higher satisfaction, we found that patients who asked more questions tended to feel less satisfied overall, less clear about their values, and less certain about their decision at follow-up. These negative associations need to be further explored but could suggest that information was not clearly conveyed, or that clinicians did not adequately address patients\u2019 questions. Prior studies showed that the extent to which patients ask questions is contingent on the clinicians\u2019 communication style . MoreoveOur findings also suggest that looping in a CDST may play an important role in SDM. Specifically, we found that the relationship between clicks within the CDST and SDM satisfaction varied based upon looping in the system. With fewer clicks, SDM outcomes were not related to the extent of provider looping in the CDST. However, with more clicks, patients reported better SDM outcomes when clinicians looped through more scenarios and worse outcomes without looping. This finding may relate to prior work showing that more clicks in the EHR are associated with lower patient satisfaction , 30. OurTo improve the process of navigating CDSTs, it may be important to identify where and when clicks occur in the tool. Higher clicks may be important markers of a challenging decision process or a provider\u2019s lack of experience with the tool , perhapsThese findings can inform SDM provider training to optimize CDST use in clinical encounters. There may be value in providing proper training before large scale implementation of SDM supported by CDSTs, as it is possible that inexperience with a CDST could lead to inefficient use that detracts from SDM. Moreover, it may be beneficial to educate clinicians on verbal communication skills relevant to SDM, such as how to verbalize information in quantitative terms  to accompany the visual displays in the CDST, provide adequate answers to questions, and verbally engage patients while simultaneously managing data in the CDST . MoreoveThese findings also have implications for the design of CDSTs. To improve patient satisfaction, the digital interface must be user-friendly for the patient as well as the clinician , 48. OurOur study has several limitations. First, we cannot draw conclusions about the causal relationships among the measured variables. We assessed relationships between linguistic patterns and tool use and SDM satisfaction while controlling for potential confounding variables, but there may be additional unmeasured factors that contribute to the observed relationships. For example, patients\u2019 health literacy should likely be a covariate .Second, more work may be needed to distinguish activities in the CDST that predict SDM in different ways, including examining when and why clinicians clicked on different parts of the interface, and whether certain patterns drove the negative association between click count and patients\u2019 SDM satisfaction. There may also be particular types of loops that are helpful or unhelpful, based on the specific scenarios explored.Additional limitations relate to generalizability. As the participants are all from one geographic region, it would be beneficial to replicate this study in other locations. Our study also has a large proportion of patients who had a college education or higher, which may limit the ability to generalize to others. Our patient sample was also entirely women, and all but one of the providers in our study were women. Past work suggests that gender can shape clinical interactions, and it would likely also have implications for how decision aid tools can be effectively used. For example, some work suggests that women are more motivated than men to participate actively in SDM , and thaAs far as future directions, time is an important element to consider in relation to any CDST. Time in clinical settings can be understood as the available length of consultation time and the time constraints perceived by the provider and the patients . LimitedVery limited work has addressed heterogeneity in uses of CDSTs, and how this predicts satisfaction with decision-making. As such, this study provides an important but preliminary step toward understanding these issues. Future work should seek to confirm these findings with a larger sample size that provides increased power.As CDSTs are increasingly deployed to support SDM, it is critical to understand how specific uses of these tools, and the accompanying patient-provider communication, affect SDM satisfaction. This study suggests that elements of both system navigation and verbal communication are associated with women\u2019s post-visit satisfaction with SDM, suggesting areas for future study, as well as implications for the design of CDSTs and for training clinicians in effective use of these tools.Additional file 1. Figure S1a. \u2018Data\u2019 page from the Breast Cancer Risk Estimator Decision Aid. Figure S1b. \u2018Assessment\u2019 page from the Breast Cancer Risk Estimator Decision Aid.Figure S1c. \u2018Decision\u2019 page from the Breast Cancer Risk Estimator Decision Ai. Table S3. Linear Mixed-effect Model of Linguistic Features and System Use on Patient\u2019s Feeling Informed. Table S4. Linear Mixed-effect Model of Linguistic Features and System Use on Patient\u2019s Benefit/Risk Clarity. Table S5. Linear Mixed-effect Model of Linguistic Features and System Use on Patient\u2019s Support/Advice Perception. Table S6. Linear Mixed-effect Model of Linguistic Features and System Use on Patient\u2019s Value Clarity. Table S7. Linear Mixed-effect Model of Linguistic Features and System Use on Patient\u2019s Confidence/Certainty about Decision Making. Figure S2a. Interaction effect of clicks and loops on patient support/advice perception. Figure S2b. Interaction effect of clicks and loops on patient value clarity. Figure S2c. Interaction effect of clicks and loops on patient confidence/certainty about decision making."}
+{"text": "A sample size justification is required for all studies and should give the minimum number of subjects to be recruited for the study to achieve its primary objective. The aim of this review is to describe sample sizes from agreement studies with continuous or categorical endpoints and different methods of assessing agreement, and to determine whether sample size justification was provided.th September 2018 to 28th September 2020. The search returned 5257 studies of which 82 studies were eligible for final assessment after duplicates and ineligible studies were excluded.Data were gathered from the PubMed repository with a time interval of 28We observed a wide range of sample sizes. Forty-six studies (56%) used a continuous outcome measure, 28 (34%) used categorical and eight (10%) used both. Median sample sizes were 50 (IQR 25 to 100) for continuous endpoints and 119 (IQR 50 to 271) for categorical endpoints. Bland\u2013Altman limits of agreement  were the most common method of statistical analysis for continuous variables and Kappa coefficients for categorical variables . Of the 82 studies assessed, only 27 (33%) gave justification for their sample size.Despite the importance of a sample size justification, we found that two-thirds of agreement studies did not provide one. We recommend that all agreement studies provide rationale for their sample size even if they do not include a formal sample size calculation.The online version contains supplementary material available at 10.1186/s12874-022-01723-5. Agreement is defined as the extent to which measurements or ratings are the same as one another. Inter-rater agreement is the similarity of measurements from different instruments or raters on the same subjects, and intra-rater agreement is the consistency of repeat measurements by the same instrument or rater on the same subjects . AgreemeAgreement studies are important to facilitate the development of new clinical methods of evaluation, ensuring they are consistent with the current \u2018gold standard\u2019 approach, or to ensure diagnostic consistency between and within assessors. Agreement is commonly tested using statistical methods such as Bland\u2013Altman limits of agreement (LoA), the intraclass correlation (ICC) and Kappa coefficients. However, methods inappropriate for the assessment of agreement are also often used .Quantifying an appropriate sample size for research studies is important to prevent the recruited sample from being overly small or large. A small sample size can lead to inconclusive results with wide confidence limits, whereas a too large a sample could be expensive and time-consuming, study participants could be exposed to unnecessary burden, and it could be considered unethical as patients continue to be enrolled after a time when the research questions can be answered .Determining the target sample size is an important step in any study design and should be considered and justified a priori. However, in the design of agreement studies, sample size determination often does not receive the same level of attention as the choice of method for assessing agreement , 5.In this study we reviewed sample sizes used in agreement studies in the medical literature, and assessed whether the authors justified the sample size and conducted formal sample size estimation.To describe the sample sizes used or reported in clinical agreement studies with a categorical  or continuous endpoint;To describe the sample sizes used in agreement studies when using different statistical methods to assess agreement;To describe the use of formal sample size estimation and calculations in agreement studies.The research aims were:https://pubmed.ncbi.nlm.nih.gov, accessed 29th September 2020) was used to identify medical research studies that investigated intra-rater or inter-rater agreement or method comparison between different clinical instruments using the same units of measurement. The time scope of the search result was two years between 28th September 2018 and 28th September 2020. An online search was conducted on 29th September 2020 using the following search terms: \u2018Agreement Study\u2019 OR \u2018Test Repeatability\u2019 OR \u2018Method Comparison\u2019. Studies reporting agreement of categorical  or continuous variables were considered. The selection was limited to clinical studies relating to only human participants with full text available in the English language.The PubMed repository (Search results were identified and exported to Microsoft Excel where duplicates were removed. We excluded studies that compared techniques that used different units of measurement and studies not involving human subjects. The selection of studies was conducted independently by two researchers (OH and HT). In the event of disagreement, a third researcher was to be called in for evaluation; however, no disagreement was found between the two researchers during the primary selection stage. The initial extraction of data for the analysis was conducted by the same two researchers.After the initial extraction by OH and HT the data for each study was reviewed by two additional researchers  and verified against the original source. If there was any disagreement on the final data extracted SJ and LS adjudicated with OH and HT. The data extracted from the papers were analysed by OH and HT.Bland\u2013Altman LoAICCKappa coefficientsSignificance testsOther methods Studies were categorised into four fields: medicine, surgery, radiology and allied health. Studies were also classified into five groups according to the main statistical method used to assess agreement:Further categorisation was made into types of endpoints: categorical and/or continuous.Data pertaining to planned sample sizes, sample size estimation and actual sample sizes were identified. Where no planned sample size was given the actual sample size was reported. To describe the distribution of sample sizes, the mean, median, interquartile range and range were calculated.We assessed whether sample size justification was provided. The justification could be through a formal sample size calculation or narratively to explain the rationale for the sample size.The PubMed repository search returned 5,257 studies. After removal of duplicates, 4,473 titles were screened. There were 235 titles eligible for further review based on heading relevancy. Three studies did not have full text available; their respective authors were contacted, however no reply was received and the studies were excluded. After exclusion of a further 150 ineligible studies that did not report agreement analyses, 82 studies were included in the present analysis. The study selection process is summarised in Fig.\u00a0A summary of the characteristics of the 82 studies meeting the review inclusion criteria is presented in Table n\u2009=\u20093), fixed by calendar time , or selected based on the sample size of similar studies (n\u2009=\u20091).Each study reported the sample size used. However, only 27 out of 82 studies (33%) provided justification for the sample size for agreement analysis. Of the 27 studies that had a formal justification for the sample size, 22 (82%) showed evidence of sample size calculation having been performed, including parameter estimates and/or reference to formulae or software packages used. All but one of those 27 studies provided at least some parameter estimates, though not all provided sufficient information for precise replication. Of the five studies providing rationale but no formal calculation, sample sizes were determined by the study being nested within another powered on a different endpoint . This was followed by allied health, surgery and lastly radiology with a median sample size of 50 (IQR 27 to 143).Of the 82 research studies assessed, 30 studies (37%) utilised one statistical method to assess agreement whilst 52 studies (63%) utilised two or more statistical methods. Bland\u2013Altman LoA was the most used statistical method by studies measuring continuous endpoints  and Kappa coefficients were most used by studies measuring categorical endpoints .t-test, used in seven studies. The most common \u2018other\u2019 statistical method employed was a correlation coefficient, used in seven studies.Studies in which agreement was assessed using the Kappa method had the largest median sample size of 71 (IQR 50 to 233) and those using the ICC as the primary method had the smallest median sample size of 42 (IQR 27 to 65). For significance tests, the most common approach was a paired Overall, studies measuring primarily categorical endpoints had a larger median sample size of 119 (IQR 50 to 271), compared to those focussing primarily on continuous endpoints, with a median of 50 (IQR 25 to 100). It was noted that all median sample sizes were smaller than mean sample sizes, indicative of positively skewed sample size distributions.Our review of the PubMed repository identified 82 eligible agreement studies published in the medical literature between 2018 and 2020. The studies covered a variety of disease areas. We observed a wide range of sample sizes and variability in typical sample size according to clinical field, statistical method and type of endpoint.Continuous endpoints were the more common, for which Bland\u2013Altman LoA was the most frequent statistical approach used, with a median sample size of 89 (IQR 35 to 124). Finding Bland\u2013Altman LoA the most common approach is consistent with the review of Zaki et al. . HoweverWe found Kappa statistics to be the most common approach used with categorical variables, with a median sample size of 71 (IQR 50 to 233). Kappa is commonly used for the assessment of agreement using binary and ordinal scales  and defiWe found that all included studies reported a sample size, but only one-third provided justification for their sample size, and of those, not all reported use of statistical sample size formulae. Kottner et al.  recommenVariation in the quality of sample size reporting has been examined in the context of clinical trials, with 95% of the trails published in high impact journals reviewed by Charles et al.  reportinSome authors suggest general rules of thumb for sample sizes for agreement studies, for example, Liao  recommenSome agreement studies may be constrained by the sample size available, for example when embedded within studies powered on a different outcome, or the pre-determined target sample may not be achieved for financial, temporal or other reasons. Nevertheless, the target and actual samples used should still be described and justified. The quality of agreement studies could be improved by following the Guidelines for Reporting Reliability and Agreement Studies (GRAAS) recommendations , which rStrengths of this review are that this is the first to investigate how typical sample sizes in recent medical agreement studies differ by field, types of endpoints and statistical method. A team of statisticians was involved in the assessment of studies, allowing for increased accuracy of data review and extraction, and reduction of bias. Limitations include the use of only one electronic repository; research studies not present within the PubMed registry would not have been captured.\u00a0Relatively few search terms were used, meaning some relevant studies may have been missed. Searches were limited to English language, meaning studies in other languages were also not included.We reviewed clinical agreement studies and noted that typical sample sizes varied according to research area, statistical approach and type of endpoint. We found that for continuous and categorical endpoints, the median sample sizes for agreement analyses were 50 (IQR 25 to 100) and 119 (IQR 50 to 271), respectively.A sample size justification should be provided in all research studies even if a formal sample size calculation is not possible. However, despite the importance of a sample size justification, we found that only a third of papers reporting agreement studies provided one. The quality of reporting of agreement studies would be improved by following the guidelines in the GRAAS checklist  as this Additional file 1: Supplementary Table 1. Distribution of sample sizes by field of study and type of endpoint. Supplementary Table 2. Distribution of sample sizes by statistical methods and type of endpoint.Additional file 2."}
+{"text": "Wealthy nations must step up support for Africa and vulnerable countries in addressing past, present and future impacts of climate changeThe 2022 report of the Intergovernmental Panel on Climate Change paints a dark picture of the future of life on earth, characterised by ecosystem collapse, species extinction and climate hazards such as heatwaves and floods.While the Paris Agreement of 2015 outlines a global action framework that incorporates providing climate finance to low-income and middle-income countries, this support has yet to materialise.The climate crisis has had an impact on the environmental and social determinants of health across Africa, leading to devastating health effects.Droughts in sub-Saharan Africa have tripled between 1970\u20131979 and 2010\u20132019.The damage to Africa should be of supreme concern to all nations. This is partly for moral reasons. It is highly unjust that the most impacted nations have contributed the least to global cumulative emissions, which are driving the climate crisis and its increasingly severe effects. North America and Europe have contributed 62% of carbon dioxide emissions since the Industrial Revolution, whereas Africa has contributed only 3%.Yet it is not just for moral reasons that all nations should be concerned for Africa. The acute and chronic impacts of the climate crisis create problems like poverty, infectious disease, forced migration and conflict that spread through globalised systems.The primary focus of climate summits remains to rapidly reduce emissions so that global temperature rises are kept to below 1.5\u00b0C. This will limit the harm. But, for Africa and other vulnerable regions, this harm is already severe. Achieving the promised target of providing US$100 billion of climate finance a year is now globally critical if we are to forestall the systemic risks of leaving societies in crisis. This can be done by ensuring these resources focus on increasing resilience to the existing and inevitable future impacts of the climate crisis, as well as on supporting vulnerable nations to reduce their greenhouse gas emissions: a parity of esteem between adaptation and mitigation. These resources should come through grants not loans, and be urgently scaled up before the current review period of 2025. They must put health system resilience at the forefront, as the compounding crises caused by the climate crisis often manifest in acute health problems. Financing adaptation will be more cost-effective than relying on disaster relief.Some progress has been made on adaptation in Africa and around the world, including early warning systems and infrastructure to defend against extremes. But frontline nations are not compensated for impacts from a crisis they did not cause. This is unfair, and drives the spiral of global destabilisation, as nations pour money into responding to disasters, but can no longer afford to pay for greater resilience or to reduce the root problem through emissions reduction. A financing facility for loss and damage must now be introduced, providing additional resources beyond those given for mitigation and adaptation. This must go beyond the failures of COP26, where the suggestion of such a facility was downgraded to \u2018a dialogue\u2019.The climate crisis is a product of global inaction, and comes at great cost to disproportionately impacted African countries, and to the whole world. Africa is united with other frontline regions in urging wealthy nations to finally step up, if for no other reason than that the crises in Africa will sooner rather than later spread and engulf all corners of the globe, by which time it may be too late to effectively respond. If so far they have failed to be persuaded by moral arguments, then hopefully their self-interest will now prevail.East African Medical Journal; Gregory E. Erhabor, Editor-in-Chief, West African Journal of Medicine; Aiah A. Gbakima, Editor-in-Chief, Sierra Leone Journal of Biomedical Research; Abraham Haileamlak, Editor-in-Chief, Ethiopian Journal of Health Sciences; Jean-Marie Kayembe Ntumba, Chief Editor, Annales Africaines de Medecine; James Kigera, Editor-in-Chief, Annals of African Surgery; Laurie Laybourn-Langton, University of Exeter; Bob Mash, Editor-in-Chief, African Journal of Primary Health Care & Family Medicine; Joy Muhia, London School of Medicine and Tropical Hygiene; Fhumulani Mavis Mulaudzi, Editor-in-Chief, Curationis; David Ofori-Adjei, Editor-in-Chief, Ghana Medical Journal; Friday Okonofua, Editor-in-Chief, African Journal of Reproductive Health; Arash Rashidian, Executive Editor, and Maha El-Adawy, Director of Health Promotion, Eastern Mediterranean Health Journal; Siaka Sidib\u00e9, Director of Publication, Mali M\u00e9dical; Abdelmadjid Snouber, Managing Editor, Journal de la Facult\u00e9 de M\u00e9decine d\u2019Oran; James Tumwine, Editor-in-Chief, African Health Sciences; Mohammad Sahar Yassien, Editor-in-Chief, Evidence-Based Nursing Research; Paul Yonga, Managing Editor, East African Medical Journal; Lilia Zakhama, Editor-in-Chief, La Tunisie M\u00e9dicale; Chris Zielinski, University of Winchester.Lukoye Atwoli, Editor-in-Chief, https://www.bmj.com/content/full-list-authors-and-signatories-climate-emergency-editorial-october-2022This comment is being published simultaneously in multiple journals. For the full list of journals, see:"}
+{"text": "The 2022 report of the Intergovernmental Panel on Climate Change (IPCC) paints a dark picture of the future of life on earth, characterised by ecosystem collapse, species extinction, and climate hazards such as heatwaves and floods.While the Paris Agreement of 2015 outlines a global action framework that incorporates providing climate finance to developing countries, this support has yet to materialise.The climate crisis has had an impact on the environmental and social determinants of health across Africa, leading to devastating health effects.,Droughts in sub-Saharan Africa have tripled between 1970\u20131979 and 2010\u20132019.The damage to Africa should be of supreme concern to all nations. This is partly for moral reasons. It is highly unjust that the most impacted nations have contributed the least to global cumulative emissions, which are driving the climate crisis and its increasingly severe effects. North America and Europe have contributed 62% of carbon dioxide emissions since the Industrial Revolution, whereas Africa has contributed only 3%.,,Yet it is not just for moral reasons that all nations should be concerned for Africa. The acute and chronic impacts of the climate crisis create problems like poverty, infectious disease, forced migration, and conflict that spread through globalised systems.The primary focus of climate summits remains to rapidly reduce emissions so that global temperature rises are kept to below 1.5\u00b0C. This will limit the harm. But, for Africa and other vulnerable regions, this harm is already severe. Achieving the promised target of providing $100bn of climate finance a year is now globally critical if we are to forestall the systemic risks of leaving societies in crisis. This can be done by ensuring these resources focus on increasing resilience to the existing and inevitable future impacts of the climate crisis, as well as on supporting vulnerable nations to reduce their greenhouse gas emissions: a parity of esteem between adaptation and mitigation. These resources should come through grants not loans, and be urgently scaled up before the current review period of 2025. They must put health system resilience at the forefront, as the compounding crises caused by the climate crisis often manifest in acute health problems. Financing adaptation will be more cost effective than relying on disaster relief.Some progress has been made on adaptation in Africa and around the world, including early warning systems and infrastructure to defend against extremes. But frontline nations are not compensated for impacts from a crisis they did not cause. This is not only unfair, but also drives the spiral of global destabilisation, as nations pour money into responding to disasters, but can no longer afford to pay for greater resilience or to reduce the root problem through emissions reductions. A financing facility for loss and damage must now be introduced, providing additional resources beyond those given for mitigation and adaptation. This must go beyond the failures of COP26 where the suggestion of such a facility was downgraded to \u2018a dialogue\u2019.The climate crisis is a product of global inaction and comes at great cost, not only to disproportionately impacted African countries, but to the whole world. Africa is united with other frontline regions in urging wealthy nations to finally step up, if for no other reason than that the crises in Africa will sooner rather than later spread and engulf all corners of the globe, by which time it may be too late to effectively respond. If so far, they have failed to be persuaded by moral arguments, then hopefully their self-interest will now prevail.Provenance and peer review: Commissioned; not externally peer reviewed.Conflict of interest: In the interest of transparency the authors wish to declare the following roles and relationships: James Kigera is the Ex-Officio, President and Secretary of the Kenya Orthopedic Association; Paul Yonga been paid to speak or participate at events by Novartis, bioMerieux and Pfizer; Chris Zielinski is a paid consultant for the UK Health Alliance on Climate Change; Joy Muhia is an unpaid board member of the International Working Group for Health systems strengthening; David Ofori-Adjei has a relationship with GLICO Healthcare Ltd. The authors declare no further conflicts of interest beyond those inherent in the editorial roles listed."}
+{"text": "To avoid these catastrophic health effects across all regions of the globe, there is broad agreement\u2014as 231 health journals argued together in 2021\u2014that the rise in global temperature must be limited to less than 1.5\u00b0C compared with pre-industrial levels.The 2022 report of the Intergovernmental Panel on Climate Change (IPCC) paints a dark picture of the future of life on earth, characterised by ecosystem collapse, species extinction, and climate hazards such as heatwaves and floods.2 COP27 is the fifth Conference of the Parties (COP) to be organised in Africa since its inception in 1995. Ahead of this meeting, we\u2014as health journal editors from across the continent\u2014call for urgent action to ensure it is the COP that finally delivers climate justice for Africa and vulnerable countries. This is essential not just for the health of those countries, but for the health of the whole world.While the Paris Agreement of 2015 outlines a global action framework that incorporates providing climate finance to developing countries, this support has yet to materialise.3 Impacts on health can result directly from environmental shocks and indirectly through socially mediated effects..4 Climate change-related risks in Africa include flooding, drought, heatwaves, reduced food production, and reduced labour productivity.5The climate crisis has had an impact on the environmental and social determinants of health across Africa, leading to devastating health effects.6 In 2018, devastating cyclones impacted 2.2 million people in Malawi, Mozambique, and Zimbabwe.6 In west and central Africa, severe flooding resulted in mortality and forced migration from loss of shelter, cultivated land, and livestock.7 Changes in vector ecology brought about by floods and damage to environmental hygiene have led to increases in diseases across sub-Saharan Africa, with rises in malaria, dengue fever, Lassa fever, Rift Valley fever, Lyme disease, Ebola virus, West Nile virus, and other infections.9 Rising sea levels reduce water quality, leading to water-borne diseases, including diarrhoeal diseases, a leading cause of mortality in Africa.8 Extreme weather damages water and food supply, increasing food insecurity and malnutrition, which causes 1.7 million deaths annually in Africa.10 According to the Food and Agriculture Organization of the United Nations, malnutrition has increased by almost 50% since 2012, owing to the central role agriculture plays in African economies.11 Environmental shocks and their knock-on effects also cause severe harm to mental health.12 In all, it is estimated that the climate crisis has destroyed a fifth of the gross domestic product (GDP) of the countries most vulnerable to climate shocks.13Droughts in sub-Saharan Africa have tripled between 1970-1979 and 2010-2019.14The damage to Africa should be of supreme concern to all nations. This is partly for moral reasons. It is highly unjust that the most impacted nations have contributed the least to global cumulative emissions, which are driving the climate crisis and its increasingly severe effects. North America and Europe have contributed 62% of carbon dioxide emissions since the Industrial Revolution, whereas Africa has contributed only 3%.15 These knock-on impacts affect all nations. COVID-19 served as a wake-up call to these global dynamics and it is no coincidence that health professionals have been active in identifying and responding to the consequences of growing systemic risks to health. But the lessons of the COVID-19 pandemic should not be limited to pandemic risk.17 Instead, it is imperative that the suffering of frontline nations, including those in Africa, be the core consideration at COP27: in an interconnected world, leaving countries to the mercy of environmental shocks creates instability that has severe consequences for all nations.Yet it is not just for moral reasons that all nations should be concerned for Africa. The acute and chronic impacts of the climate crisis create problems like poverty, infectious disease, forced migration, and conflict that spread through globalised systems.The primary focus of climate summits remains to rapidly reduce emissions so that global temperature rises are kept to below 1.5\u00b0C. This will limit the harm. But, for Africa and other vulnerable regions, this harm is already severe. Achieving the promised target of providing $100bn of climate finance a year is now globally critical if we are to forestall the systemic risks of leaving societies in crisis. This can be done by ensuring these resources focus on increasing resilience to the existing and inevitable future impacts of the climate crisis, as well as on supporting vulnerable nations to reduce their greenhouse gas emissions: a parity of esteem between adaptation and mitigation. These resources should come through grants not loans, and be urgently scaled up before the current review period of 2025. They must put health system resilience at the forefront, as the compounding crises caused by the climate crisis often manifest in acute health problems. Financing adaptation will be more cost effective than relying on disaster relief.18Some progress has been made on adaptation in Africa and around the world, including early warning systems and infrastructure to defend against extremes. But frontline nations are not compensated for impacts from a crisis they did not cause. This is not only unfair, but also drives the spiral of global destabilisation, as nations pour money into responding to disasters, but can no longer afford to pay for greater resilience or to reduce the root problem through emissions reductions. A financing facility for loss and damage must now be introduced, providing additional resources beyond those given for mitigation and adaptation. This must go beyond the failures of COP26 where the suggestion of such a facility was downgraded to \u201ca dialogue.\u201d\u200aThe climate crisis is a product of global inaction and comes at great cost, not only to disproportionately impacted African countries, but to the whole world. Africa is united with other frontline regions in urging wealthy nations to finally step up, if for no other reason than that the crises in Africa will sooner rather than later spread and engulf all corners of the globe, by which time it may be too late to effectively respond. If so far, they have failed to be persuaded by moral arguments, then hopefully their self-interest will now prevail."}
+{"text": "These aWhile the Paris Agreement of 2015 outlines a global action framework that incorporates providing climate finance to developing countries, this support has yet to materialise . COP27 iThe climate crisis has had an impact on the environmental and social determinants of health across Africa, leading to devastating health effects . ClimateDroughts in sub-Saharan Africa have tripled between 1970\u20131979 and 2010\u20132019 . In 2018The damage to Africa should be of supreme concern to all nations. This is partly for moral reasons. It is highly unjust that the most impacted nations have contributed the least to global cumulative emissions, which are driving the climate crisis and its increasingly severe effects. North America and Europe have contributed 62% of carbon dioxide emissions since the Industrial Revolution, whereas Africa has contributed only 3% .Yet it is not just for moral reasons that all nations should be concerned for Africa. The acute and chronic impacts of the climate crisis create problems like poverty, infectious disease, forced migration, and conflict that spread through globalised systems . These kThe primary focus of climate summits remains to rapidly reduce emissions so that global temperature rises are kept to below 1.5\u00b0C. This will limit the harm. But, for Africa and other vulnerable regions, this harm is already severe. Achieving the promised target of providing $100bn of climate finance a year is now globally critical if we are to forestall the systemic risks of leaving societies in crisis. This can be done by ensuring these resources focus on increasing resilience to the existing and inevitable future impacts of the climate crisis, as well as on supporting vulnerable nations to reduce their greenhouse gas emissions: a parity of esteem between adaptation and mitigation. These resources should come through grants not loans, and be urgently scaled up before the current review period of 2025. They must put health system resilience at the forefront, as the compounding crises caused by the climate crisis often manifest in acute health problems Financing adaptation will be more cost effective than relying on disaster relief.Some progress has been made on adaptation in Africa and around the world, including early warning systems and infrastructure to defend against extremes. But frontline nations are not compensated for impacts from a crisis they did not cause. This is not only unfair, but also drives the spiral of global destabilisation, as nations pour money into responding to disasters, but can no longer afford to pay for greater resilience or to reduce the root problem through emissions reductions. A financing facility for loss and damage must now be introduced, providing additional resources beyond those given for mitigation and adaptation. This must go beyond the failures of COP26 where the suggestion of such a facility was downgraded to \u2018a dialogue\u2019 (The climate crisis is a product of global inaction and comes at great cost, not only to disproportionately impacted African countries, but to the whole world. Africa is united with other frontline regions in urging wealthy nations to finally step up, if for no other reason than that the crises in Africa will sooner rather than later spread and engulf all corners of the globe, by which time it may be too late to effectively respond. If so far, they have failed to be persuaded by moral arguments, then hopefully their self-interest will now prevail."}
+{"text": "These are all linked to physical and mental health problems, with direct and indirect consequences of increased morbidity and mortality. To avoid these catastrophic health effects across all regions of the globe, there is broad agreement\u2014as 231 health journals argued together in 2021\u2014that the rise in global temperature must be limited to less than 1.5\u00b0C compared with pre\u2010industrial levels.The 2022 report of the Intergovernmental Panel on Climate Change (IPCC) paints a dark picture of the future of life on earth, characterised by ecosystem collapse, species extinction, and climate hazards such as heatwaves and floods.1 Thes COP27 is the fifth Conference of the Parties (COP) to be organised in Africa since its inception in 1995. Ahead of this meeting, we\u2014as health journal editors from across the continent\u2014call for urgent action to ensure it is the COP that finally delivers climate justice for Africa and vulnerable countries. This is essential not just for the health of those countries, but for the health of the whole world.While the Paris Agreement of 2015 outlines a global action framework that incorporates providing climate finance to developing countries, this support has yet to materialise.2 COP2 Impacts on health can result directly from environmental shocks and indirectly through socially mediated effects. Climate change\u2010related risks in Africa include flooding, drought, heatwaves, reduced food production, and reduced labour productivity.The climate crisis has had an impact on the environmental and social determinants of health across Africa, leading to devastating health effects.3 Impa In 2018, devastating cyclones impacted 2.2 million people in Malawi, Mozambique and Zimbabwe. In west and central Africa, severe flooding resulted in mortality and forced migration from loss of shelter, cultivated land, and livestock. Changes in vector ecology brought about by floods and damage to environmental hygiene have led to increases in diseases across sub\u2010Saharan Africa, with rises in malaria, dengue fever, Lassa fever, Rift Valley fever, Lyme disease, Ebola virus, West Nile virus and other infections. Rising sea levels reduce water quality, leading to water\u2010borne diseases, including diarrhoeal diseases, a leading cause of mortality in Africa. Extreme weather damages water and food supply, increasing food insecurity and malnutrition, which causes 1.7 million deaths annually in Africa. According to the Food and Agriculture Organization of the United Nations, malnutrition has increased by almost 50% since 2012, owing to the central role agriculture plays in African economies. Environmental shocks and their knock\u2010on effects also cause severe harm to mental health. In all, it is estimated that the climate crisis has destroyed a fifth of the gross domestic product (GDP) of the countries most vulnerable to climate shocks.Droughts in sub\u2010Saharan Africa have tripled between 1970\u201379 and 2010\u20132019.6 In 2The damage to Africa should be of supreme concern to all nations. This is partly for moral reasons. It is highly unjust that the most impacted nations have contributed the least to global cumulative emissions, which are driving the climate crisis and its increasingly severe effects. North America and Europe have contributed 62% of carbon dioxide emissions since the Industrial Revolution, whereas Africa has contributed only 3%.14 These knock\u2010on impacts affect all nations. COVID\u201019 served as a wake\u2010up call to these global dynamics and it is no coincidence that health professionals have been active in identifying and responding to the consequences of growing systemic risks to health. But the lessons of the COVID\u201019 pandemic should not be limited to pandemic risk. Instead, it is imperative that the suffering of frontline nations, including those in Africa, be the core consideration at COP27: in an interconnected world, leaving countries to the mercy of environmental shocks creates instability that has severe consequences for all nations.Yet it is not just for moral reasons that all nations should be concerned for Africa. The acute and chronic impacts of the climate crisis create problems like poverty, infectious disease, forced migration, and conflict that spread through globalised systems.15 TheThe primary focus of climate summits remains to rapidly reduce emissions so that global temperature rises are kept to below 1.5\u00b0C. This will limit the harm. But, for Africa and other vulnerable regions, this harm is already severe. Achieving the promised target of providing $100bn of climate finance a year is now globally critical if we are to forestall the systemic risks of leaving societies in crisis. This can be done by ensuring these resources focus on increasing resilience to the existing and inevitable future impacts of the climate crisis, as well as on supporting vulnerable nations to reduce their greenhouse gas emissions: a parity of esteem between adaptation and mitigation. These resources should come through grants not loans, and be urgently scaled up before the current review period of 2025. They must put health system resilience at the forefront, as the compounding crises caused by the climate crisis often manifest in acute health problems. Financing adaptation will be more cost\u2010effective than relying on disaster relief.Some progress has been made on adaptation in Africa and around the world, including early warning systems and infrastructure to defend against extremes. But frontline nations are not compensated for impacts from a crisis they did not cause. This is not only unfair, but also drives the spiral of global destabilisation, as nations pour money into responding to disasters, but can no longer afford to pay for greater resilience or to reduce the root problem through emissions reductions. A financing facility for loss and damage must now be introduced, providing additional resources beyond those given for mitigation and adaptation. This must go beyond the failures of COP26 where the suggestion of such a facility was downgraded to \u201ca dialogue\u201d.18The climate crisis is a product of global inaction, and comes at great cost not only to disproportionately impacted African countries, but to the whole world. Africa is united with other frontline regions in urging wealthy nations to finally step up, if for no other reason than that the crises in Africa will sooner rather than later spread and engulf all corners of the globe, by which time it may be too late to effectively respond. If so far they have failed to be persuaded by moral arguments, then hopefully their self\u2010interest will now prevail.East African Medical Journal; Gregory E. Erhabor, Editor\u2010in\u2010Chief, West African Journal of Medicine; Aiah A. Gbakima, Editor\u2010in\u2010Chief, Sierra Leone Journal of Biomedical Research; Abraham Haileamlak, Editor\u2010in\u2010Chief, Ethiopian Journal of Health Sciences; Jean\u2010Marie Kayembe Ntumba, Chief Editor, Annales Africaines de Medecine ; James Kigera, Editor\u2010in\u2010Chief, Annals of African Surgery; Laurie Laybourn\u2010Langton, University of Exeter; Bob Mash, Editor\u2010in\u2010Chief, African Journal of Primary Health Care & Family Medicine; Joy Muhia, London School of Medicine and Tropical Hygiene; Fhumulani Mavis Mulaudzi, Editor\u2010in\u2010Chief, Curationis; David Ofori\u2010Adjei, Editor\u2010in\u2010Chief, Ghana Medical Journal; Friday Okonofua, Editor\u2010in\u2010Chief, African Journal of Reproductive Health; Arash Rashidian, Executive Editor, and Maha El\u2010Adawy, Director of Health Promotion, Eastern Mediterranean Health Journal; Siaka Sidib\u00e9, Director of Publication, Mali M\u00e9dical; Abdelmadjid Snouber, Managing Editor, Journal de la Facult\u00e9 de M\u00e9decine d'Oran; James Tumwine, Editor\u2010in\u2010Chief, African Health Sciences; Mohammad Sahar Yassien, Editor\u2010in\u2010Chief, Evidence\u2010Based Nursing Research; Paul Yonga, Managing Editor, East African Medical Journal; Lilia Zakhama, Editor\u2010in\u2010Chief, La Tunisie M\u00e9dicale; Chris Zielinski, University of Winchester.Lukoye Atwoli, Editor\u2010in\u2010Chief, https://www.bmj.com/content/full\u2010list\u2010authors\u2010and\u2010signatories\u2010climate\u2010emergency\u2010editorial\u2010october\u20102022This Guest Editorial is being published simultaneously in multiple journals. For the full list of journals see: In the interest of transparency the authors wish to declare the following roles and relationships: J.K. is the Ex\u2010Officio, President and Secretary of the Kenya Orthopedic Association; J.M. is an unpaid board member of the International Working Group for Health Systems Strengthening; D.O.\u2010A. has a relationship with GLICO Healthcare Ltd.; P.Y. been paid to speak or participate at events by Novartis, bioMerieux and Pfizer; C.Z. is a paid consultant for the UK Health Alliance on Climate Change. The authors declare no further conflicts of interest beyond those inherent in the editorial roles listed above."}
+{"text": "The acute general surgical unit (AGSU) model has become a standard of efficient acute surgical care. Whilst the AGSU has been compared to the traditional surgical model, there is a lack of research auditing referrals and admissions. This study evaluates abdominal pain referrals to AGSU and the necessity of admission.A retrospective cohort study of adult abdominal pain admissions was conducted over a two-year period at a single centre in metropolitan Victoria, Australia. The data were extracted from electronic medical records and key endpoints of data included the diagnosis, length of stay, investigations and subjective pain outcomes.p\u2009<\u20090.001). They were less likely to have an abnormal haemoglobin (p\u2009=\u20090.004), elevated white cell count (p\u2009=\u20090.02) or elevated C-reactive protein\u2009>\u200950\u00a0mg/L (p\u2009<\u20090.001). On multivariable analysis, surgical patients had higher odds of having a CRP\u2009>\u200950\u00a0mg/L (p\u2009=\u20090.024) and a positive imaging result (p\u2009<\u20090.001). The patient\u2019s pain control also correlated with length of stay.A total of 1587 patients met the study criteria of which 1116 (70.3%) had a non-surgical diagnosis with the majority having non-specific abdominal pain. The non-surgical patients had a lower median length of stay (25.3\u00a0h) compared to surgical patients (44.2\u00a0h, A large population of patients with non-specific abdominal pain are admitted to AGSU. These patients do not require surgery and have a short length of stay. Incorporating a negative CRP result and negative imaging result may be utilised in conjunction with optimised analgesia to help avoid these unnecessary admissions, thereby improving AGSU efficiency and workload. The acute general surgical unit (AGSU) is a fast-paced surgical unit dedicated and designed for efficient acute general surgical care. It receives the hospital\u2019s general surgical and trauma admissions, providing a diagnostic and therapeutic plan for these patients. Whilst this model has become the standard of surgical practice, there is limited evidence on improving its efficiency , 2.The AGSU model was developed after observing an increase in key performance indicators including length of stay (LOS), time to surgical review in emergency department (ED), time to operating theatre and outcomes in general surgery, as well as an objection to the conflicting demands of balancing emergency and elective surgery with increased after hours operating that would potentially result in suboptimal care \u20135. InitiWhilst the AGSU has been compared to the traditional model for the improvement of care in confirmed surgical pathologies, there is a paucity of research evaluating the referrals to AGSU and the management of patients without a defined surgical pathology. Non-essential referrals and admissions increase the workload for the surgical team, reducing efficiency and depleting financial resources , 9.This retrospective review of a single centre AGSU assesses non-specific abdominal pain admissions with the aim of identifying factors in these patients that may allow identification of patients that can be treated more efficiently in other departments or as an outpatient. The results of this will provide greater clarity in designing models for efficient AGSU referrals and admissions, potentially resulting in improved patient flow, care and health economics.A retrospective cohort study was conducted of all patients who were admitted under the acute general surgical unit (AGSU) at Northern Health  between March 31st, 2017 and March 31st, 2019 and admitted with the diagnosis of \u2018Abdominal Pain\u2019 as per the ICD10 code.The population group was adults aged 18\u00a0years of age and older who were admitted from the emergency department. Patients less than 18\u00a0years of age and trauma admissions were excluded. The study data were collected from electronic medical records (EMR) and the inpatient administration system (iPM). The primary outcome assessed was the discharge and final diagnosis with the secondary outcome being the length of stay.A non-surgical diagnosis was defined as those patients who did not undergo a procedure or have a surgical diagnosis at discharge or follow-up. The \u2018surgical\u2019 group of patients was defined as those patients who underwent a surgical procedure regardless of outcome, or non-operative patients who had a surgical diagnosis during the admission or follow-up based on clinical information and investigations.6\u00a0mmol/L. The normal haemoglobin values were 115\u2013165 and 130\u2013180\u2009\u00d7\u2009106\u00a0mmol/L in females and males, respectively. Abnormal CRP values were classified as proportion greater than or equal to 10 and 50\u00a0mg/L, respectively.Data that were collected included demographical data, investigations performed, surgical interventions, discharge diagnosis, follow-up disposition and final diagnosis. Time parameters including emergency and total hospital length of stay (LOS) were also recorded. The results of pathology testing including haemoglobin (Hb), white cell count (WCC) and C-reactive protein (CRP) were classified and analysed as the proportion abnormal. This was based on the values in relation to the standardised laboratory ranges in which normal WCC ranged from 4 to 11\u2009\u00d7\u200910There was no standardised pain evaluation data available; however, medical and nursing documentations were reviewed for patient\u2019s level of pain prior to discharge. The subjective descriptions were classified as resolved, improved with analgesia, controlled, ongoing or not recorded.T-test for parametric data and the Mann\u2013Whitney U-Test for nonparametric data with significance set at a 5% alpha level. Discrete data were statistically analysed with the Chi-square test. Multivariable analysis was performed through binomial logistic regression.Statistical analysis was performed with SPSS . Median and interquartile range values were selected for descriptive analysis of nonparametric continuous data. Statistical analysis of continuous data was performed with the independent samples Ethical approval was obtained from the Northern Health Research and Governance Office (ALR 53.2019).In the study period, there were 1784 admission episodes for abdominal pain. Figure\u00a0The primary outcome was the proportion of patients who had a non-surgical cause for abdominal pain, and these were compared with the surgical patients Table . Of the p\u2009=\u20090.004), a WCC above normal , and median CRP above 10\u00a0mg/L  and above 50\u00a0mg/L . Imaging investigations were conducted in 90% of the patients with CT scanning used more prevalently in surgical patients , but conversely, ultrasound scanning was used less frequently . LOS was a key outcome assessed and demonstrated a significantly longer LOS for surgical patients .Blood test investigations were performed on all but one patient in the study. In the surgical group, there was a significantly higher number of patients with a haemoglobin below normal  and shortest IQR [16.5\u201342.7] seen in the group with resolved pain  and the majority were able to have outpatient follow-up. As the admissions are short, it prompts the question as to whether these patients even require admission in the first place. In the literature, admission of non-specific abdominal pain patients has been shown to decrease acute surgical theatre efficiency [This study demonstrates that many patients are admitted to an AGSU with a diagnosis of non-specific abdominal pain, consuming considerable resources. The non-surgical group of patients was more likely to have normal results for haemoglobin, WCC, CRP as well as negative imaging to investigate their pain and exclude surgical diagnoses. The admitted non-surgical patients had a shorter length of stay than surgical patients  and six-hour rule  , 13. WhiPain control can be a significant component to early discharge for patients with non-specific abdominal pain. As emphasised by the findings in Fig.\u00a0Between 40 and 75% of GP abdominal pain referrals have been found to be appropriate to discharge from ED or treat in the general practice setting, and suggest the possibility of defensive medicine with patients referred based on an unclear diagnosis as opposed to a clear surgical indication, 15. ThiAlternatives to a surgical admission include utilising a clinical decision unit or follow-up in an ambulatory clinic. The UK has adopted an emergency surgery ambulatory care pathway for appropriate diagnoses such as NSAP with these \u2018hot clinics\u2019 offloading up to 30% of presentations , 17. UtiSelecting appropriate patients for and utilising the outpatient services can reduce the burden on AGSU and provide enhanced efficiency as well as avoiding unnecessary admissions for patients. Whilst studies assessing combined radiology and pathology investigations are lacking, early routine CT for abdominal pain has been found to increase surgical diagnostic accuracy and confidence in addition to reduced 6-month mortality , 22. TheA vast majority of non-specific abdominal pain patients admitted to the AGSU do not require an operation and have a short length of stay. Avoiding an admission in these patients may help improve AGSU efficiency, hospital patient flow and health economics.Utilising an algorithm incorporating inflammatory markers and negative imaging results in addition to pain optimization may enable safe discharge of these patients without compromising care whilst enhancing the efficiency of the AGSU."}
+{"text": "Clear cell renal cell carcinoma (ccRCC) is one of the most common malignancies. Recently, immunotherapy has been considered a promising treatment for metastatic ccRCC. NUF2 is a crucial component of the Ndc80 complex. NUF2 can stabilize microtubule attachment and is closely related to cell apoptosis and proliferation. This research is dedicated to investigating the role of NUF2 in ccRCC and the possible mechanisms.First, analysis of NUF2 mRNA expression levels in ccRCC and normal tissues by The Cancer Genome Atlas (TCGA) database and further verified by analysis of independent multiple microarray data sets in the Gene Expression Omnibus (GEO) database. Moreover, we evaluated and identified correlations between NUF2 expression, clinicopathologic variable, and overall survival (OS) in ccRCC by various methods. We investigated the relationship between NUF2 and tumor immune infiltration and the expression of corresponding immune cell markers via the Gene Expression Profiling Interactive Analysis (GEPIA) and Tumor Immune Estimation Resource (TIMER) databases. Then, we performed functional enrichment analysis of NUF2 co-expressed genes using R software and protein-protein interactions (PPIs) using the search tool used to retrieve interacting genes/proteins (STRING) databases.We discovered that NUF2 mRNA expression was upregulated in ccRCC tissues and was associated with sex, grade, pathological stage, lymph node metastasis, and worse prognosis. In addition, NUF2 was positively linked to tumor immune cells in ccRCC. Moreover, NUF2 was closely related to genetic markers of different immune cells. Finally, functional enrichment and protein\u2013protein interaction (PPI) analysis suggested that NUF2 and its closely related genes may be involved in the regulation of the cell cycle and mitosis. Our results suggested that NUF2 is correlated with a poor prognosis and immune infiltration in ccRCC. Renal cell carcinoma (RCC) is one of the most common malignancies worldwide, accounting for 90% of renal tumors, of which approximately 80\u201390% are clear cell renal cell carcinoma (ccRCC) , 2. AlthNUF2 is a crucial component of the Ndc80/NUF2 complex. As part of a linker between the kinetochore and tubulin subunits of the spindle, it can stabilize microtubule attachment and is closely related to cell apoptosis and proliferation. Disruption of NUF2 could induce defects in kinetochore attachment defects and spindle checkpoints and ultimately induce mitotic cells to undergo cell death \u20138. NUF2 In this study, we first performed differential expression analysis using the Gene Expression Omnibus (GEO) database, The Cancer Genome Atlas (TCGA) database and its derived online tools. Secondly, we investigated the relationship between NUF2 and clinicopathological features. Then,we analyzed the relationship between NUF2 expression and prognosis of ccRCC patients. In addition, we identified specific associations between NUF2 expression and infiltrating immune cells, immune cell markers or immune checkpoints in ccRCC. Finally, we performed functional annotation and protein interaction analysis of NUF2 and its related genes. Collectively, our study shows that NUF2 is likely to be a meaningful gene in the occurrence and development of ccRCC and an effective biomarker for the diagnosis and prognostic assessment of ccRCC.https://portal.gdc.cancer.gov/cart) is a free data portal that contains information derived from a large cancer genome project, providing scholars and researchers withclinical and pathological information of 33 cancers. In our study, the main information, including NUF2 mRNA expression, clinicopathologic features and survival data of ccRCC patients, was derived from the TCGA database. Subsequent analyses were based on RNA-seq data from the TCGA database.The TCGA database(https://www.ncbi.nlm.nih.gov/geo/) is a comprehensive gene expression library in the National Center of Biotechnology Information. In this study, all of the NUF2 data representation validation sets in ccRCC were downloaded from this database, including four independent data sets .The GEO database is an efficient online analysis and mining cancer data tool, mainly used for the analysis of multiple cancer types in the TCGA database. It helps medical researchers with gene identification, differential expression analysis and survival analysis of genes [The UALCAN database(of genes . In our http://gepia.cancer-pku.cn/index.html) is an online tool for expression analysis and interactive analysis of cancer-related and normal genes on the basis of TCGA and Genotype-Tissue Expression (GTEX) data [The GEPIA database (EX) data . In our http://timer.comp-genomics.org/). According to the information of more than 10,000 samples of different cancer types in the TCGA database, the immune infiltration level was analyzed by specific algorithms in the TIMER database. This database can be used to determine the level of tumor invasion according to differential gene expression [The Tumor Immune Estimation Resource (TIMER) is a web server for analyzing the level of immune infiltration in different tumor types (pression . This sthttp://www.linkedomics.org/) is a third-party online analysis website of TCGA data [http://kmplot.com/ananysis/) was used for prognostic analysis. It contains more than 12,000 samples with survival data for 21 cancer types [The LinkedOmics database(CGA data . The Kaper types . This stWe screened genes closely related to NUF2 by the R package \u201cstat\u201d in R (v3.6.3)  and functionally annotated these significantly related genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis .https://string-db.org/) [In addition, The STRING database (db.org/)  is a webWe performed immunohistochemical validation using tissue from patients with cCRCC in our hospital. HE staining was performed on paraffin sections. The details of these experiments have been previously described. All of the above methods are carried out in accordance with the relevant guidelines and regulations.Evaluation of NUF2 transcript levels differentially expressed in multiple cancer samples via the TIMER database. NUF2 transcription levels were found to be high in a variety of human tumor samples, including ccRCC and 15 other kinds of cancers, such as hepatocellular carcinoma, Esophageal squamous cell carcinoma, gastric adenocarcinoma, urothelial carcinoma, lung cancer, glioma, ovarian cancer, breast cancer, cervical squamous cell carcinoma, colorectal cancer, pancreatic cancer and prostate adenocarcinoma  and different T helper cells were deeply investigated.To further analyze the role of NUF2 in tumor immunity, the GEPIA and TIMER databases were used to assess the association between NUF2 expression and immune cell markers in ccRCC tissues. We discovered that NUF2 expression had a significant positive correlation with most specific immune cell markers (Table\u00a0As shown in Fig.\u00a0It is well known that a more comprehensive understanding of protein functions can be achieved by studying protein interactions. We constructed an 11-point, 42-edge interactions network map of NUF2-related proteins via the STRING database Fig.\u00a0D, and thSPC25 and SPC24 are all part of the NDC80 complex, mainly involved in cellular chromosome segregation and spindle checkpoint activity. The 10 most correlated proteins are mainly involved in the cell cycle and essential for normal chromosome alignment, segregation and kinetochore formation during mitosis. In addition, up-regulated BUB1 is associated with a poor prognosis in ccRCC patients.ccRCC, whose pathogenesis mechanism has been unclear thus far, is extremely resistant to chemoradiotherapy . An in-dIt has been reported that NUF2 expression is significantly upregulated in many other cancers. To further clarify the function of NUF2 in ccRCC, multiple databases were analyzed and NUF2 was upregulated in ccRCC tissue compared with normal kidney tissue was found, suggesting that NUF2 is likely to be independently involved in the occurrence and development of ccRCC.NUF2 has been previously reported to play an essential role in tumorigenesis and is upregulated in a variety of human cancers , 7. The Immunotherapy for cancer has made tremendous progress in recent years. Attention is being paid to the role of the immune response in the tumor microenvironment \u201325. ccRCIt has been reported that the targeted regulation of NUF2 can inhibit cell proliferation and invasion \u201312. In oIn conclusion, by mining public databases, we preliminarily elucidated the relationships between the high expression of NUF2 and a poor prognosis and clinicopathologic features in ccRCC. Moreover, our current findings suggest that NUF2 may act as an oncogene by regulating the level of tumor-infiltrating immune cells and the expression of immune checkpoints. In addition, genes closely related to NUF2 are particularly abundant in the cell cycle as well as in the P53 signaling pathway. However, there are some limitations to our research. Firstly, we mined a variety of public databases, but our results should be verified with basic experiments and clinical studies. Secondly, we only focused on the transcription level, and we should analyze the mechanism of NUF2 and the upstream and downstream regulatory mechanisms at many different levels."}
+{"text": "Over 200 health journals call on the United Nations, political leaders, and health professionals to recognise that climate change and biodiversity loss are one indivisible crisis and must be tackled together to preserve health and avoid catastrophe. This overall environmental crisis is now so severe as to be a global health emergency. The research communities that provide the evidence for the two COPs are unfortunately largely separate, but they were brought together for a workshop in 2020 when they concluded that: \u201cOnly by considering climate and biodiversity as parts of the same complex problem\u2026can solutions be developed that avoid maladaptation and maximize the beneficial outcomes.\u201dThe world is currently responding to the climate crisis and the nature crisis as if they were separate challenges. This is a dangerous mistake. The 28. Climate change is set to overtake deforestation and other land-use change as the primary driver of nature loss.As the health world has recognised with the development of the concept of planetary health, the natural world is made up of one overall interdependent system. Damage to one subsystem can create feedback that damages another\u2014for example, drought, wildfires, floods and the other effects of rising global temperatures destroy plant life, and lead to soil erosion and so inhibit carbon storage, which means more global warming. Indigenous land and sea management has a particularly important role to play in regeneration and continuing care.Nature has a remarkable power to restore. For example, deforested land can revert to forest through natural regeneration, and marine phytoplankton, which act as natural carbon stores, turn over one billion tonnes of photosynthesising biomass every eight days. But actions that may benefit one subsystem can harm another\u2014for example, planting forests with one type of tree can remove carbon dioxide from the air but can damage the biodiversity that is fundamental to healthy ecosystems.Restoring one subsystem can help another\u2014for example, replenishing soil could help remove greenhouse gases from the atmosphere on a vast scale, and by the nature crisis. This indivisible planetary crisis will have major effects on health as a result of the disruption of social and economic systems\u2014shortages of land, shelter, food, and water, exacerbating poverty, which in turn will lead to mass migration and conflict. Rising temperatures, extreme weather events, air pollution, and the spread of infectious diseases are some of the major health threats exacerbated by climate change. \u201cWithout nature, we have nothing,\u201d was UN Secretary-General Ant\u00f3nio Guterres\u2019s blunt summary at the biodiversity COP in Montreal last year. Even if we could keep global warming below an increase of 1.5\u00b0C over pre-industrial levels, we could still cause catastrophic harm to health by destroying nature.Human health is damaged directly by both the climate crisis, as the journals have described in previous editorials. Contamination of water on land can also have far-reaching effects on distant ecosystems when that water runs off into the ocean. Good nutrition is underpinned by diversity in the variety of foods, but there has been a striking loss of genetic diversity in the food system. Globally, about a fifth of people rely on wild species for food and their livelihoods. Declines in wildlife are a major challenge for these populations, particularly in low- and middle-income countries. Fish provide more than half of dietary protein in many African, South Asian and small island nations, but ocean acidification has reduced the quality and quantity of seafood.Access to clean water is fundamental to human health, and yet pollution has damaged water quality, causing a rise in water-borne diseases. People losing contact with the natural environment and the declining biodiversity have both been linked to increases in noncommunicable, autoimmune, and inflammatory diseases and metabolic, allergic and neuropsychiatric disorders. For Indigenous people, caring for and connecting with nature is especially important for their health. Nature has also been an important source of medicines, and thus reduced diversity also constrains the discovery of new medicines.Changes in land use have forced tens of thousands of species into closer contact, increasing the exchange of pathogens and the emergence of new diseases and pandemics. Connection with nature reduces stress, loneliness and depression while promoting social interaction. These benefits are threatened by the continuing rise in urbanisation.Communities are healthier if they have access to high-quality green spaces that help filter air pollution, reduce air and ground temperatures, and provide opportunities for physical activity. Linked to this, inequality is also arguably fuelling these environmental crises. Environmental challenges and social/health inequities are challenges that share drivers and there are potential co-benefits of addressing them.Finally, the health impacts of climate change and biodiversity loss will be experienced unequally between and within countries, with the most vulnerable communities often bearing the highest burden. Industrialised countries agreed to mobilise $30 billion per year to support developing nations to do so. These agreements echo promises made at climate COPs.In December 2022 the biodiversity COP agreed on the effective conservation and management of at least 30% percent of the world\u2019s land, coastal areas, and oceans by 2030. If these events were to occur, the impacts on health would be globally catastrophic. Yet many commitments made at COPs have not been met. This has allowed ecosystems to be pushed further to the brink, greatly increasing the risk of arriving at \u2018tipping points\u2019, abrupt breakdowns in the functioning of nature are that it: 1) is serious, sudden, unusual or unexpected; 2) carries implications for public health beyond the affected State's national border; and 3) may require immediate international action. Climate change would appear to fulfil all of those conditions. While the accelerating climate change and loss of biodiversity are not sudden or unexpected, they are certainly serious and unusual. Hence we call for WHO to make this declaration before or at the Seventy-seventh World Health Assembly in May 2024. This risk, combined with the severe impacts on health already occurring, means that the World Health Organization should declare the indivisible climate and nature crisis as a global health emergency. The three pre-conditions for WHO to declare a situation to be a Public Health Emergency of International Concern. As the 2020 workshop that brought climate and nature scientists together concluded, \u201cCritical leverage points include exploring alternative visions of good quality of life, rethinking consumption and waste, shifting values related to the human-nature relationship, reducing inequalities, and promoting education and learning.\u201d All of these would benefit health. Tackling this emergency requires the COP processes to be harmonised. As a first step, the respective conventions must push for better integration of national climate plans with biodiversity equivalents. But first, we must recognise this crisis for what it is: a global health emergency. Health professionals must be powerful advocates for both restoring biodiversity and tackling climate change for the good of health. Political leaders must recognise both the severe threats to health from the planetary crisis as well as the benefits that can flow to health from tackling the crisishttps://www.bmj.com/content/full-list-authors-and-signatories-climate-nature-emergency-editorial-october-2023This Comment is being published simultaneously in multiple journals. For the full list of journals see:"}
+{"text": "A weak volume Raman\u2013Nath grating of dimension 0.5 by 3\u00a0mm and 3\u00a0\u03bcm pitch was placed in front of a Samsung Galaxy S21 FE cellphone to record the spectrum using the 2nd diffraction order. This spectrometer covers the visible band from 401 to 700\u00a0nm with a 0.4\u00a0nm/pixel detector resolution and 3\u00a0nm optical resolution. It was used to determine the concentration detection limit of Rhodamine 6G in water which was found to be 0.5\u00a0mg/L. This proof of concept paves the way to in-the-field absorption spectroscopy for quick information gathering.In this work we demonstrate the integration of a spectrometer directly into smartphone screen by femtosecond laser inscription of a weak Raman\u2013Nath volume grating either into the Corning Gorilla glass screen layer or in the tempered aluminosilicate glass protector screen placed in front of the phone camera. Outside the thermal accumulation regime, a new writing regime yielding positive refractive index change was found for both glasses which is fluence dependent. The upper-bound threshold for this thermal-accumulation-less writing regime was found for both glasses and were, respectively at a repetition rate less than 150\u00a0kHz and 101\u00a0kHz for fluence of 8.7\u2009\u00d7\u200910 This integrated platform has evolved over the years by the increase of its computing power capabilities and the addition of new sensors and features. It has already replaced common commodity items such as the video or photographic cameras, alarm clocks, watches, Global Positioning Systems (GPS), calendars, the calculator, flash lamps, just to name a few becoming as powerful as a small computer with access to the web. The recent Covid pandemic has highlight the potential of this tool to rapidly implement and distribute applications to a vast population in record time.Since their introduction in 19932 for blood sample analysis as well as detection of parasites3, bacteria5 and viruses6. Blood sugar level can be detected by evaluating the ratio of the blue and green spectral components at the RGB camera7. Turbidity level of water using Mie diffusion can be also measured as shown in8. An optical breathalyzer based on the difference in evaporation rate with alcohol content of the fog generated in breath, was as well demonstrated9. Spectroscopy systems capable of measuring the water pH level have been also demonstrated with a resolution of 0.305\u00a0nm/pixel10. Detection of water contaminant like copper, chrome, fluor, lead, mercury or pesticides11 was also investigated. Plasmonic resonance systems can be coupled with spectroscopy to detect agents that are transparent to the optical bandwidth of the camera and offer low concentration detection level of analyte in water (100 picogram/mL) of staphylococcal enterotoxin B as reported in12.Photonics can be an interesting avenue to increase the capabilities and thus the potential of these devices. Manufacturers already have integrated new photonics sensors such as Lidars for augmented reality applications or pulse oximeter to acquire on field blood oxygen level and cardiac frequency to some recent model of smartphones. Simultaneously, many research groups are actively working to create new functionalities on this device using the already on-board sensors or developing new ones. Microscopy systems using smartphone cameras coupled with an algorithm have been demonstrated to count white or red blood cell13. With the help of femtosecond (fs) laser writing at 1030\u00a0nm, they demonstrated low loss single mode waveguide of 0.053\u00a0dB/cm at 1550\u00a0nm. They also demonstrated a refractive index (RI) measurement device based on the evanescent field interaction losses at the surface of the glass14.However, these new functionalities often need additions of component as an add-on which consumes space. The space limitation problem is of concern in conditions when optimized devices are required. To tackle this issue, the idea of using the 750\u00a0\u03bcm thick protecting layers made of Corning Gorilla glass in front of the screen to inscribe photonics devices was proposed by Lapointe et al. in15. Such process uses non-linear effects such as multiphoton absorption or tunnel ionization16 to cause permanent changes in the RI. The non-linear dependence on the electric field strength localizes the RI change only at the focal volume which permits the 3D modification into the bulk of the material. The nature of the RI change is highly dependent on the material and writing condition and arises from the sum of many contributions such as color center formation17, structural changes in the glass matrix18, or thermal effects leading to a change of density19, to name a few. There is also a particular regime of thermal accumulation at high repetition rate which gives rise to large out-of-focal RI modification20.Fs laser functionalization of glassy material was introduce in 1996 by Davis et al.21. In the same study, it was shown that the RI change can be increased by one order of magnitude by reducing the number of photons needed (reducing the wavelength) involved in the writing.Following the research of Lapointe et al., the integrity of the mechanical properties of the protective glass layer by such fs laser modification was studied and it was found that fs writing had negligible impact on the strength of the glass22 (VRNG) in front of the smartphone camera to generate an integrated smartphone spectrometer. The key is to produce a weak VRNG that does not significantly alter the traditional function of the camera but generates a spectrum when exposed to bright light illumination. Therefore, we propose to evaluate the possibility of using fs-written volume gratings in those glasses to realize an integrated spectrum analyzer using the smartphone RGB camera.In this paper, we first demonstrate a new writing regime without heat accumulation, which leads to highly resolved fine writing points with a positive index change. A positive index change is of particular interest for waveguide writing, while small index change region is critical to write grating with fine periods. This regime is not limited to a single glass, as we demonstrate it in two different glasses. Using this novel writing technique, we demonstrate a volume phase grating operating in the Raman\u2013Nath regime23.\u0394\u03c6 is the measured phase change, \u03bb the wavelength of measurement, which is 633\u00a0nm for that system and h is the height of the structure. The cross-section was measured with a classical microscopy system with a 60\u00d7 (0.8 NA) PLAN Olympus objective in the visible.The fs laser writing was performed using an 8 W Pharos laser system from Light Conversion featuring a 250\u00a0fs pulse length. This laser is coupled to an Orpheus OPA to double the frequency from the original 1030 to 515\u00a0nm. The fs laser pulse was focused with a 50\u00d7, Olympus PLAN 0.65 numerical aperture (NA) microscope objective and the sample was put on a 3-axis writing system controlled by an AEROTECH 3200 controller. The repetition rate of the laser was controlled using a pulse picker to conserve the pulse energy. The polarization of the laser was parallel to the writing direction. The writing speed used was varied from 0.1 to 100\u00a0mm/s and the pulse energy from 82 to 825\u00a0nJ. Two types of glass were used for the writing, Corning Gorilla glass which is an alkali-aluminosilicate glass used for protection of multimedia screen device and tempered aluminosilicate glass which is a generic add-on removable protective layer from Bodyguardz easily available commercially. To measure the RI change induced, the Ripper system on loan from Photonovainc.com was used. This microscopy interferometric systems measure the relative phase change from the surrounding and, providing knowledge about 2D cross section profile of the inscription, could determine the RI change profile by using the following Eq.\u00a0. The results are shown in Fig.\u00a0In our previous study on fs laser writing into Corning Gorilla Glassp\u2009=\u20093300\u00a0kW, which is above the self-focusing power threshold (Pth\u2009=\u2009550\u00a0kW) defined by Eq.  definition express at Eq.\u00a0 and different number of passes (1 to 8 passes). A 3\u00a0\u03bcm pitch was selected to ensure the highest line density without overlap between inscription lines. These results are displayed in Fig.\u00a0th order and thus maximises power into diffraction orders.To inscribe VRNGs, a high RI change in a very thin elongated region is required. The writing regime proposed in the last section is ideal for this. To optimize the writing of VRNG structures, we compare two writing regimes: one at low pulse energy with high fluence and the other at high pulse energy with low fluence. To this end, a grid of small VRNGs composed of 30 lines with a 3\u00a0\u03bcm pitch have been written with different fluences . In an effort to produce optimal RI change by fs writing for VRNGs, a new writing regime without thermal accumulation was characterized. The thermal accumulation upper-bound thresholds for both glasses were measured at a repetition rate of 150\u00a0kHz and 101\u00a0kHz for Corning Gorilla glass and aluminosilicate tempered glass, respectively. Below such frequencies, a transition from negative (low fluence) to positive (high fluence) RI change was observed. The fluence required to reach positive index change regime is 8.7\u2009\u00d7\u200910Supplementary Figures."}
+{"text": "Despite recent advancements in knowledge and molecular profiling, recurrence of GBM is nearly inevitable. This recurrence has been attributed to the presence of glioma stem cells (GSCs), a small fraction of cells resistant to standard-of-care treatments and capable of self-renewal and tumor initiation. Therefore, targeting these cancer stem cells will allow for the development of more effective therapeutic strategies against GBM. We have previously identified several 7-amino acid length peptides which specifically target GSCs through in vitro and target intracranial GBM in mouse models. A peptide pull-down performed with peptide EV followed by mass spectrometry determined N-cadherin as the binding partner of the peptide, which was validated by enzyme-linked immunosorbent assay and surface plasmon resonance. To develop cytotoxic cellular products aimed at specifically targeting GSCs, chimeric antigen receptors (CARs) were engineered containing the peptide EV in place of the single-chain variable fragment (scFv) as the antigen-binding domain. EV CAR-transduced T cells demonstrated specific reactivity towards GSCs by production of interferon-gamma when exposed to GSCs, in addition to the induction of GSC-specific apoptosis as illustrated by Annexin-V staining.We have combined two of these peptides to create a dual peptide construct (EV), and demonstrated its ability to bind GSCs These results exemplify the use of phage display biopanning for the isolation of GSC-targeting peptides, and their potential application in the development of novel cytotoxic therapies for GBM. Glioblastoma (GBM) is the most common and malignant form of primary brain tumor and is associated with a poor prognosis with a median survival time of 16 months . RecurreEx-vivo expansion of T cells followed by genetic modification with a chimeric antigen receptor (CAR) is used to redirect the immune cells to target various types of tumor cells. A CAR is a synthetic protein that typically consists of an extracellular antigen-recognition domain that contains the heavy and light chain variable fragments of a monoclonal antibody or B cell receptor, joined to a hinge and transmembrane domain, and an intracellular CD3\u03b6 signaling domain from the T cell receptor, often in addition to a costimulatory domain such as CD28  comprise a subpopulation of tumor cells that possess the unique abilities of self-renewal and tumor recapitulation, in addition to resistance to conventional radiotherapy and chemotherapy \u201312. AddiPhage display biopanning is a discovery tool that employs bacteriophages in which genes are inserted, and then displayed on their surface. Phage display allows for the isolation of peptides that can bind a specific cell type or protein through a series of positive and negative selection steps screening a library of random peptide sequences . The useExperiments performed were approved by the Institutional Reviewed Board at the H. Lee Moffitt Cancer Center and Research Institute in accordance with the ethical guidelines set forth at each institution. GSCs, tumor tissue, and non-malignant normal brain were harvested from patient tumor samples or epileptic brain at Duke University Medical Center, Cleveland Clinic, and University Hospital Cleveland Medical Center in accordance with an approved protocol by the respective Institutional Review Boards. All animal studies were performed according to guidelines under Institutional Animal Care and Use Committee protocol approved at H. Lee Moffitt Cancer Center and Research Institute (IACUC# RIS00010727). Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice  were acquired from Jackson Laboratories , housed with ad libitum access to standard laboratory chow and water and with a 12-hour light/dark cycle.\u00ae CRL-1620\u2122, Manassas, VA) and non-malignant brain cells (NM263) obtained from epileptic brain tissue were also maintained in DMEM supplemented by 10% FBS and 1% PS. Neural stem cells (NSCs) were obtained from EMD  and cultured as described by the manufacturer. Human primary peripheral blood mononuclear cells (PBMCs) were obtained from de-identified buffy coats , cultured in X-VIVO medium  supplemented with 5% human serum , 1% PS, 1% 200 mM L-glutamine, and 300 IU/mL of IL-2 , and activated with the anti-CD3\u03f5 monoclonal antibody OKT3  for two days prior to transduction.T387 and T4121 glioblastoma stem cells were obtained from tissue dissociation of patient derived glioblastoma samples and maintained as previously described , 23. BotIn vitro and in vivo phage display biopanning was performed using a Ph.D.-7 Phage Display Peptide Library Kit . The first step of the in vitro biopanning was a negative selection of the library against extracellular matrix and DGCs to remove non-GSC binding peptides. The peptide library was then screened against GSCs for four rounds of biopanning. Phage clones were then isolated through bacterial infection and peptide sequences were isolated by sequencing. In vivo biopanning was performed by intravenously injecting the phage library in NSG mice bearing intracranial GBM. Following circulation of the peptide library for 24 hours, tumors were harvested, tumor cells were lysed, then bound phage peptides were isolated, purified, and transduced into E. coli for phage clone isolation and sequenced. The in vivo phage display performed against subcutaneous GBM was performed as previously described (escribed .The peptide AWEFYFPGGGGSGGGGSGGGGSSSQPFWS was conjugated with cyanine 3 (EV-Cy3) or with biotin (EV-b), and the scrambled non-targeting peptides FAYPEWFGGGGSGGGGSGGGGSPSWSFSQ (NT-Cy3 and NT-b) were synthesized by LifeTein, LCC . They were reconstituted in DMSO at a concentration of 10 mM and stored at -80\u00b0C.After coating glass coverslips with Geltrex  for 24 hours, cells were seeded and incubated overnight on the coated coverslips. The peptide cell staining was performed by removing the media and incubating the cells with PBS-1%-BSA for 15 minutes at 4\u00b0C. Biotinylated peptides  were incubated for 20 minutes at 4\u00b0C, washed with PBS-1%-BSA for 5 minutes, and fluorescent-labeled streptavidin  was added and incubated for 20 minutes at 4\u00b0C. The cells were then washed , fixed with 4% paraformaldehyde , incubated with a 4\u2019,6-diamidino-2-phenylindole (DAPI) solution  and mounted with Anti-Fade Fluorescence Mounting Medium . Z-stack images were acquired with a Leica SP8 confocal microscope at 63 magnification 1X and 3X. All images were post-processed identically with Image J software .6 cells/mL) were saturated with PBS-1%-BSA for 15 minutes on ice, centrifuged at 150 x g for 3 minutes, then incubated at 4\u00b0C for 30 minutes with 10 \u03bcM or increased concentration of biotinylated peptide (EV-b or NT-b). Cells were washed two times and incubated with an AlexaFluor 647-streptavidin  at 4\u00b0C for 30 minutes in the dark. Cells were washed two times with PBS-1%-BSA and resuspended in 50 \u03bcL of PBS-1%-BSA with DAPI . An Amnis ImageStream flow cytometer  was used for recording and data was analyzed with Image Data Exploration and Analysis Software . Gating strategy shown in For the peptide-binding quantification and binding affinity assays, single cell suspensions of GSCs or DGCs  T cells were washed, stained with CD34-PE , then incubated for 15 minutes at 4\u00b0C in the dark. Cells were then washed and resuspended in FACS buffer with DAPI and incubated for 15 minutes at 4\u00b0C in the dark. Data acquisition was performed with a BD FACSCanto II flow cytometer .5 and 8\u00d7105 cells, respectively) were surface-stained with CSPG4-APC , and CD3-BV711 , following a 4-6 hour co-culture, then incubated for 20 minutes at 4\u00b0C in the dark. Cells were then washed, secondarily stained with Annexin-V-FITC  and DAPI, incubated for 15 minutes at room temperature in the dark, and then analyzed. Data acquisition was performed with BD FACSCanto II or LSR II cytometers  and analyzed with FlowJo software . Gating strategy shown in For apoptosis assay experiments, single cell suspensions of effector and target cells  was injected intravenously in the tail vein of the mice and intraperitoneal injection of 100 \u03bcL Luciferin  was performed to confirm the presence of GBM. Images were acquired on an IVIS Lumina  with excitation and emission filters of 660 and 710 nm, respectively, and a luminescence filter for luciferase cells. Measurements were performed by subtracting the fluorescence before injection from the fluorescence after injection on the lower back, and on the head.For Cells were lysed using the IP Lysis Buffer Protocol  as described by the manufacturer. Biotinylated peptides  were incubated with 100 \u03bcL of streptavidin agarose resin at 4\u00b0C for 1 hour on a rotator to generate the peptide-coupled streptavidin agarose resin. The protein samples were pre-cleared by incubation of 50 \u03bcL of NT-streptavidin agarose resins (4\u00b0C on rotator for 1 hour). After centrifugation, the pre-cleared samples were incubated with 150 \u03bcL of EV-coupled streptavidin agarose resin at 4\u00b0C overnight on rotator. After PBS wash, the resin was pelleted and sent for mass spectrometry analysis  for the profiling assay of the samples. Candidates were identified using the following criteria: (1) The mean signal intensity of the sample group (EV-Cy3) is greater than 1.25-fold of the NT-Cy3 group; (2) p value < 0.05 (t-test); (3) the signal intensity of the candidates is at least three standard deviations above the mean signal intensity of the sample group (EV-Cy3).2 and 150 mM NaCl at a flow rate of 10 \u00b5L/min. The carboxymethyl surface of the CM5 chip was manually activated for 7 minutes using a 1:1 ratio of 0.4 M 1-ethyl-3-(3-diaminopropyl) carbodiimide hydrochloride (EDC) and 0.1 M N-hydroxysuccinimide (NHS) to achieve a density of about 10,000 RU per flow cell. N-cadherin protein stock (1.12 \u00b5M) was diluted to 100 nM in 10 mM sodium acetate, pH 4.5, and manually injected over the surface at the same flow rate. Excess activated functional groups on the surface were blocked using a 7-minute injection of 1M ethanolamine, pH 8.5, at a flow rate of 10 \u00b5L/min. Using this manual injection protocol, approximately 3500 RU of N-cadherin was immobilized on the surface of the CM5 chip.The direct binding measurements between N-cadherin and EV peptide by surface plasmon resonance was performed by immobilizing N-cadherin protein  to a CM5 sensor chip surface docked in Biacore T200  at 25\u00b0C using an amine-coupling method. Prior to immobilization, the chip was primed and equilibrated with a running buffer containing 20 mM HEPES, pH 7.4, 1 mM TCEP, 2 mM CaClFor kinetic titration experiments, the same running buffer was used except with the inclusion of 5% DMSO. SPR single cycle kinetic experiments with EV peptide were carried out in duplicate on two different sensor chips at 25\u00b0C. A threefold concentration series of EV peptide serially diluted in the running buffer ranging from 2 to 167 \u00b5M was injected over the sensor surface at 30 \u00b5L/min flow rate with 80-second contact time and 120-second dissociating time. For control experiments, a non-targeting (NT) peptide (GGGSGGG) was applied in place of EV peptide under identical condition. Sensorgrams were solvent corrected, buffer referenced, and the on-rate, off-rate, and equilibrium binding constants were calculated from the sensorgrams by global fitting of a 1:1 binding model, using analysis software (version 3.0) provided with Biacore T200 instrument (Cytiva).6 per effector condition) were lysed with Laemmli buffer, and protein concentrations were quantified utilizing the Pierce BCA Protein Assay Kit . Membranes were incubated with 0.2 \u03bcg/mL mouse anti-human CD3\u03b6  and 0.04 \u03bcg/mL rabbit anti-human glyceraldehyde-3-phosphate dehydrogenase . Membranes were then washed and subsequently incubated with 0.05 \u03bcg/mL of corresponding secondary antibodies, goat anti-rabbit 680LT , and goat anti-mouse 800CW  in 5% blocking solution for 1 hour. After washing and drying, the membrane blots were developed utilizing the LI-COR Odyssey imaging system with both the 700 and 800 channels (T cells (5\u00d7103 overnight at 4\u00b0C. The wells were washed with PBS-0.025%-Tween 20 and blocked with PBS/BSA-3%-Milk at RT for 2 hours. Biotinylated peptides (EV-b or NT-b), diluted in PBS-2%-BSA at different concentrations, were incubated for 1 hour at RT and washed five times. The streptavidin-HRP (0.1 \u03bcg/mL) was incubated for 1 hour at RT followed by five washes and incubation with 3,3\u2032,5,5\u2032-tetramethylbenzidine substrate  for 10 minutes at RT. 50 \u03bcL of 2 M sulfuric acid (H2SO4) were added to each well to stop the coloration and the absorbance was immediately read at 450 nm. Five replicates were used for each concentration, and wells without N-cadherin coating served as control.For evaluation of the peptide target, Nunc MaxiSorp, 96-well plates  were coated with 2.5 \u03bcg/mL of recombinant N-cadherin  diluted in 0.1 M NaHCO2SO4, and the absorbance was immediately read at 450 nm and 550 nm. Experimental conditions were run in duplicate.For evaluation of IFN-gamma secretion in co-culture experiments, Nunc MaxiSorp 96-well plates were coated at RT overnight with 0.63 \u03bcg/mL of recombinant human IFN-gamma M700A monoclonal antibody  diluted with 1X PBS. Wells were washed with PBS-0.2%-Tween 20 and blocked with PBS-4%-BSA at RT for 1 hour. IFN-gamma recombinant protein was prepared in a 1:1000 ratio with PBS-4%-BSA for use as a serially diluted standard for later interpolating concentrations of IFN-gamma in pg/mL. Co-culture supernatants were prepared with PBS-4%-BSA in a 1:10 ratio, then added to the ELISA plates and incubated at RT for 1 hour. 0.16 \u03bcg/mL biotinylated recombinant IFN-gamma M701B monoclonal antibody  was prepared with PBS-4%-BSA, then added to the ELISA plates and incubated at RT for 1 hour. Precision Protein StrepTactin-HRP Conjugate  was prepared with PBS in a 1:5500 ratio, then added to the ELISA plates and incubated at RT for 30 minutes. 1-Step Ultra TMB Substrate  was added to the ELISA plates, then incubated for 10-15 minutes in the dark. Reactions were stopped by the addition of HE. coli  were transformed for amplification of the CAR plasmids. Retroviruses encoding for our CAR constructs were generated utilizing 293GP cells. These packaging cells were co-transfected for 7 hours on poly-D-lysine-coated plates with RD-114 envelope protein in addition to the CAR plasmid and lipofectamine 2000 . For the T cell transductions, RetroNectin-coated plates  were coated with retrovirus and centrifuged for 2 hours at 4000 x g at 32\u00b0C, after which the virus was aspirated and 2\u00d7106 activated \u03b1\u03b2 T cells were seeded per well. The cells were centrifuged for 10 minutes at 1000 x g at 32\u00b0C, and then incubated for 24 hours before undergoing a second transduction.For CAR cloning, gBlock gene fragments were designed to contain the codon-optimized coding sequence for the indicated target-binding domains, and synthesized by Integrated DNA Technologies. These were flanked by NcoI/NotI restriction sites, which were used for cloning by restriction enzyme digestion followed by ligation. Using this approach, we replaced the scFv-encoding sequence of PSCA-28t28z and PSCA8t28z plasmids, described previously . Chemicain vivo fluorescence of the body and the head of the mice after EV injection was evaluated with a Wilcoxon paired test. IFN-gamma ELISA data was analyzed by two-way ANOVA and Tukey\u2019s multiple comparisons test to determine significance between CAR and GFP conditions within the same target cell group, and to compare CAR conditions across the different targets. The Annexin-V apoptosis data was analyzed by two-way ANOVA and Sidak\u2019s multiple comparisons test to determine significance between CAR and UT conditions against the target cell conditions. Proteome and mass spectrometry data was analyzed by T tests at Moffitt\u2019s core facilities.All statistics were performed with GraphPad Prism 8.2 . ImageStream flow cytometry data was analyzed with a one-way ANOVA and a Dunnett\u2019s multiple comparisons test. The binding affinities (Kd) were calculated with the One site-specific binding equation. A Kruskal-Wallis uncorrected test was used to compare the binding of EV and N-cadherin by ELISA. The difference between the in vitro and in vivo biopanning strategies , was isolated from the subcutaneous flank biopanning. These two peptides were previously demonstrated to have specificity for targeting GSCs in vitro and therefore used to design new CAR constructs.We have previously isolated glioblastoma stem cell targeting peptides through a combination of rategies , 23. In To combine the binding affinities, peptides AWEFYFP (E) and SSQPFWS (V) were joined by a flexible linker (GGGGSGGGGSGGGGS) resulting in a final length of 29 amino acids (EV). A scrambled sequence of both peptides joined by the same linker was used as a control peptide (NT) (A peptide pull-down was performed with EV and NT peptides following incubation with GSC lysates to identify cellular binding partner(s) of EV  of 2615 M-1 s-1 and an off-rate (kd) of 0.01957 s-1, resulting in an equilibrium binding constant KD of 7.50 \u00b5M , separaWe next analyzed the ability of these CAR-T cells to recognize GSCs. To that end, we co-cultured (CAR-)T cells with GSCs or media alone overnight, and quantified the presence of IFN-gamma in the supernatants. All CAR-transduced T cells (except for E-8t28z-tCD34) showed a significant increase in IFN-gamma produced in response to co-culture with T387 GSCs when compared with mock-transduced (GFP) cells and with each respective CAR in presence of cell-free media (p<0.0001) . In addition, due to the expression of CDH2 in healthy neural tissue, we tested if the E-28t28z selectively recognizes GSCs over non-malignant neural stem cells. To that end, E-28t28z-tCD34 engineered CAR-T cells were co-cultured with the abovementioned targets. Untransduced/mock-transduced T cells were used as negative controls, as well as CAR-T cells in media alone. CAR-transduced T cells secreted significantly more IFN-gamma in response to co-culture with either T387 and T4121 GSCs when compared with DGCs, NSCs, and cell-free media (p<0.0001). Additionally, CAR-transduced T cells demonstrated a significant increase in IFN-gamma produced in response to co-culture with either T387 and T4121 GSCs when compared with GFP-transduced T cells co-cultured with the same target cell type (p<0.0001) . N-cadherin is an adhesion molecule with over 96% homology between human and mouse, which involved in the development of neural tissue and in various neurodegenerative processes . By usin results , 40. The results , 8, 41. Our study has illustrated the utility and feasibility of designing novel antigen-binding domains for CAR-T cells by using two short peptides of just seven amino acids each to induce functional recognition of GSCs. We evaluated the expression and reactivity of several different EV-based CAR constructs in alpha-beta T cells, narrowing it down to use of CD28 or CD8 as the hinge/transmembrane domains and CD28 as the superior costimulatory domain, with our final CAR constructs exhibiting specific reactivity and cytotoxicity against GSCs with the dual peptide used in the place of the single-chain variable fragment (scFv) as the antigen-binding domain. By using mass spectrometry and human proteome analysis, we were able to isolate N-cadherin as the likely target of the peptide-based recognition of GSCs. N-cadherin is a transmembrane protein that plays a key role in cell-cell adhesion in the brain, and is involved in the epithelial-mesenchymal transition, with increase of its expression while E-cadherin expression is decreased \u201344. N-caDespite the specificity of the dual peptide-based CAR-T cells for targeting GSCs over DGCs or NSCs, the targeting of N-cadherin can have some limitations. This protein is widely expressed in normal tissues, particularly in the brain, endocrine tissues, and muscle tissues . Though we have demonstrated a lack of EV peptide targeting of non-neoplastic brain tissue, further studies are necessary to understand the role of N-cadherin in EV peptide specificity for the targeting of GSCs. However, the knowledge that we have advanced in producing a functional antigen-recognition domain with tandemly arranged short peptides derived from phage-display biopanning could prove useful for the generation of new tethering or costimulatory receptors. Such receptors can serve as a physical bridge linking tumor cell and T cell, increasing functional avidity, and stabilizing or enhancing the immunological synapse without resulting in an activation cascade on their own . On-targin vivo setting. Given the potential ubiquity of N-cadherin receptors, different delivery methods may need to be tested, such as intrathecal or intratumoral delivery, to avoid non-specific binding outside of the central nervous system. As GSC-targeting CAR-T cells will be directed against a subset of the GBM tumor population, detailed studies will need to be constructed to safely and effectively deliver the CAR-T cells and to test their effectiveness on limiting on tumor recapitulation and invasion.Further studies utilizing GSC-targeting CAR-T cells will need to be tested to demonstrate the effectiveness of these cells in the in vitro and in vivo phage display biopanning approaches to obtain peptides that can serve as the antigen-binding domain of CAR constructs. Our CAR-T cells retained the same specificity toward GSCs as our initial peptides and led to functional reactivity and cytotoxicity. This study demonstrates the potential of developing novel strategies for the treatment and diagnosis of different tumors currently incurable.Our results exhibit the potential for utilizing The datasets presented in this article are not readily available because sharing of materials and data are subject to review and approval by Moffitt\u2019s Office of General Counsel and Office of Innovation. Some materials are bound to MTA. Requests to access the datasets should be directed to james.liu@moffitt.org.The animal study was reviewed and approved by USF IACUC (Protocol RIS00010727).DA-D and JL jointly contributed to conception and design of the study. MP, SS, CS, JK, KT, TT, and MR contributed to experimental design and acquisition/analysis/interpretation of data. MP and SS wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version."
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