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+{"text": "Conflicts between professional duties and fear of influenza transmission to family members may arise among health care professionals (HCP).We surveyed employees at our university hospital regarding ethical issues arising during the management of an influenza pandemic.Of 644 respondents, 182 (28%) agreed that it would be professionally acceptable for HCP to abandon their workplace during a pandemic in order to protect themselves and their families, 337 (52%) disagreed with this statement and 125 (19%) had no opinion, with a higher rate of disagreement among physicians (65%) and nurses (54%) compared with administrators (32%). Of all respondents, 375 (58%) did not believe that the decision to report to work during a pandemic should be left to the individual HCP and 496 (77%) disagreed with the statement that HCP should be permanently dismissed for not reporting to work during a pandemic. Only 136 (21%) respondents agreed that HCW without children should primarily care for the influenza patients.Our results suggest that a modest majority of HCP, but only a minority of hospital administrators, recognises the obligation to treat patients despite the potential risks. Professional ethical guidelines allowing for balancing the needs of society with personal risks are needed to help HCP fulfil their duties in the case of a pandemic influenza. Medical providers worldwide are gearing up for a likely pandemic of human influenza. Professional duty of healthcare professionals (HCP) may clash with fear of contracting influenza or its transmission to family members. Triggered by the experience responding to the SARS epidemic in 2003, the lack of official ethical guidelines on balancing public needs and personal risk has been pointed out . Prompte2-test to assess differences between groups, using SPSS v12.0G software (SPSS Inc.). Since the study was anonymous, formal institutional review board approval was not required, but approval by the main board of the medical centre representing all professional groups was obtained before conducting the survey.In February 2006, we distributed anonymous self-administered, multiple-choice paper questionnaires to the 637 physicians and final-year medical students (FYMS), 994 nurses, and 267 hospital administrators at the university hospital in Regensburg, Germany. All questions were multiple-choice. Items (4) on knowledge about H5N1 (avian) influenza, in the 'True/False/Don't know' format, assessed awareness about general human-to-human transmissibility; the availability of a commercial vaccine; the projected efficacy of neuraminidase inhibitors; and the resistance against neuraminidase inhibitors. Further four items (Table We received 644 (34%) completed surveys: 233 of 637 (37%) from physicians and FYMS, 264 of 994 (27%) from nurses, and 147 of 267 (55%) from administrators. There were no statistically significant differences among respondents and non-respondents regarding age (51% vs. 49% younger than 35 years) or gender . Absence of general human-to-human transmissibility of avian influenza was correctly indicated by 566 (88%) respondents, absence of commercially available anti-H5N1 influenza vaccine was known to 543 (84%) respondents, while 171 (27%) participants knew about descriptions of resistance to neuraminidase inhibitors of H5N1 strains . Surprisingly, only 65 (10%) respondents believed that prophylactic use of neuraminidase inhibitors could protect them from H5N1 influenza in the event of human-to-human transmission. Overall, 182 (28%) respondents strongly agreed or agreed that it is professionally acceptable for HCP to abandon their workplace during a pandemic in order to protect themselves and their families, 337 (52%) respondents disagreed or strongly disagreed with this, while 125 (19%) selected the 'no opinion' option  declare that they have no competing interests.BPE designed the study, conducted the paper-based survey, analysed the data, and drafted the manuscript. FH participated in the design of the study and helped to draft the manuscript. BS participated in the design of the study, in the statistical analysis and helped to draft the manuscript. All authors read and approved the final version of the manuscript."}
+{"text": "This study aimed to investigate prevalence and correlates of alcohol consumption frequency in a sample of Swiss conscripts  in order to identify factors that predispose for frequent consumption. A self-report of drinking frequencies, as well as socio-demographic and psychosocial variables, was collected at psychiatric baseline screening. Based on univariate analyses, relevant variables were included in a multivariate multinomial logistic regression model. Six percent were abstainers, 15% reported rarely drinking, 53% occasional drinking, 24% regular drinking and 2% daily drinking. Except for substance use, most associations followed a \u201cJ\u201d-shaped curve across the categories of alcohol frequency. Abstinence and frequent drinking can be perceived as deviations from the social norm. Both behaviors are associated with more psychosocial stressors and might be therefore special targets for further studies and new prevention programs. Ezzati and colleagues  estimateIn Switzerland, one of the few countries still practicing conscription and requiring compulsory military service, all Armed Forces conscripts go through an early screening of any potential alcohol problems, including risk and concomitant factors, as well as adverse implications. Detailed knowledge of this behavior is certainly an important matter for the Armed Forces. It is the Armed Forces\u2019 obligation and responsibility to care for the physical and mental health of everyone serving during basic military training (BMT) followed by repetition courses or being sent on real missions. Alcohol misuse already represents a potentially important safety risk factor, especially for all those in responsible and demanding positions, and even more so when handling vehicles or weapons. Furthermore, evidence exists that higher stress levels are often experienced during military training or service, which can lead to more frequent and heavy alcohol consumption . ParticuThe objective of this study was to evaluate drinking frequencies self-reported by a sample of Swiss conscripts, as well as to draw up sample-specific characteristics associated with alcohol consumption. It specifically focused on associated factors that can be easily observed or assessed at pre-military capability examinations. Easily available data such as age, psychosocial problems, etc., might be used by examiners to identify persons with a clearly increased risk for elevated levels of alcohol consumption. Profiles of known risk factors could facilitate the early detection of individuals at greater risk for hazardous drinking behavior, and allow their triage to subsequent clinical examinations.2.2.1.PST \u2265 70) [Due to required conscription of every Swiss male between ages 18 and 22 years, they are obliged to undergo a pre-military examination for physical and mental fitness, independently of whether the individual will eventually serve in the Armed Forces. This procedure also includes a psychiatric examination with a pre-selection screening procedure via an electronic survey as a first step. The data investigated in this study are the psychiatric screening data collected on Swiss Armed Forces conscripts in 2005. A total of 28,125 conscripts and female volunteers entered recruitment, and hence completed the psychiatric screening questionnaire. After excluding 713 damaged or incomplete records, as well as removing the too-small number of female volunteers (n=171), 27,241 conscripts were left in the dataset. Further restrictions were made for subjects with suspected simulation tendencies   - operatST \u2265 70) , so finaThis project of the Medical Department of the Swiss Armed Forces was undertaken in collaboration with the Centre for Disaster and Military Psychiatry at the University of Zurich. The project was cleared by the Zurich State Ethical Committee (KEK) to fulfill all legal and data privacy protection requirements. All screening sessions were introduced and supervised by military test psychologists.2.2.The psychiatric screening survey consisted of 291 questions covering the primary facets of psychopathology, as well as various additional psychosocial and behavioral aspects of mental health. It includes questions about alcohol consumption as an important correlate of mental and social wellbeing. One of these measures asked about drinking frequencies, i.e. \u201chow frequent alcohol will be used?\u201d The conscripts could specify their drinking behavior on five categories ranging from \u201cnever drink\u201d through \u201cdaily drinking\u201d. Basic characteristics such as age, social factors , career factors , as well as whether the individual was receiving a disability pension. For a better interpretability, all factors were negatively poled in the direction of higher risk for elevated alcohol consumption.Psychosocial stressors were related to problems in the past 12 months in relevant areas of life, such as with work , partnership , and school or training . All three problem-related variables were dichotomized into having at least one of these stressors versus none.Family situation was assessed in terms of having parents with a migration background, being adopted, and/or having parents separated or divorced. All these items were dichotomized to \u201ctrue\u201d versus \u201cnot true\u201d.Substance use contained the three items: \u201cDo you currently smoke tobacco?\u201d, \u201cDo you currently smoke cannabis?\u201d and \u2013 herein after referred to as hard drugs \u2013 \u201cHave you ever tried ecstasy, speed or amphetamines, cocaine, heroine or other opiates?\u201d. Response options were \u201cyes\u201d versus \u201cno\u201d.Use of health care service inquired about the average number of medical consultations per year. Four response categories were given: \u201cnever\u201d, \u201conce or twice\u201d, \u201cthree to four\u201d, and \u201cFive or more times\u201d.Own or family history of mental health was characterized by suicidal behavior (dichotomized to having ever thought about or attempted suicide versus never), past or present psychological treatment (\u201cyes\u201d versus \u201cno\u201d), as well as alcohol abuse in family , other addictions in family  and psychiatric illness in at least one family member (\u201cyes\u201d versus \u201cno\u201d).In order to search for possible risk and other associated factors of alcohol use, we investigated the following variables:Moreover, the study included the Symptom-Checklist-90-Revised , a well2.3.Drinking frequencies of the study sample were analyzed in relation to the variables described above. Chi-Square Tests were used to examine differences in proportions, while mean differences in continuous variables were tested using one-way-ANOVAs. Variables that showed significant differences between categories of the outcome variable in univariate analyses were entered jointly into a multivariate logistic regression model. Frequency of alcohol consumption was measured by using an ordinally scaled variable what requires an ordered logistic regression as appropriate modeling strategy. However, testing the assumption of parallel regression revealed a violation of that assumption, which was to be expected due to such a large study population . Alterna3.3.1.Examining the self-reported drinking frequencies, 6.3% conscripts described themselves as abstainers, 14.7% reported rare (max. five times per year) alcohol consumption, about half of the sample (53.1%) reported not exceeding one through five times per month , whereas 23.8% reported drinking regularly , and 2.2% daily.Univariate analyses revealed that all independent variables significantly varied across the five outcome categories of drinking frequency, whereof most associations followed a \u201cJ\u201d-shaped curve (\u201cU\u201d-shaped for age and disability pension). According to this, \u201cnever\u201d, as well as \u201cdaily\u201d (and \u201cregular\u201d) drinking, was more often related to psychosocial stressors  compared to \u201crare\u201d and \u201coccasional\u201d drinking. For instance, lowest proportions of training- or school-related problems were found in occasional drinkers (15.7%), with an increasing tendency towards abstainers (21.9%) as well as daily drinkers (64.6%). These differences towards the extreme categories were almost higher for daily drinkers than for abstainers, except in those whose parents immigrated to Switzerland, which followed a converse \u201cJ\u201d-shaped curve. Only substance use  was linearly related to the outcome .Health care utilization was related to drinking frequencies as follows: never seeing a doctor was negatively linear related to the drinking frequencies (ranged from 37.6% abstainers to 25.8% daily drinkers), bell-shaped in one or two doctor visits per year with a majority of occasional drinkers (54.2%), and linearly increasing in three through four service uses per year (ranged from 11.3% abstainers to 19.2% daily drinkers), up to an \u201cJ\u201d-shaped curve in heavy service users  with a majority of daily drinkers (14.8%) and a minority of occasional drinkers (4.3%). Further evaluation of the data was performed using a multivariate multinomial logistic regression analysis, with all variables included \u2013 comparing elevated drinking frequencies with abstaining from drinking alcohol as the reference category see . The res3.2.This study analyzed alcohol-drinking frequencies in a sample of young Swiss male conscripts, and aimed to further investigate possible factors that may indicate higher consumption rates. Our analyses accessed a sample of 25,611 subjects aged 18\u201322.The prevalence of male abstainers in Switzerland (14.2% in 2002) is high compared to other European countries, but low when compared to worldwide numbers . In the These drinking levels cannot be attributed to conditions within the Swiss Armed Forces, since the screening was carried out at recruitment and during the pre-military health examination. However, changing life circumstances during and following military service with elevated stress levels may result in increased alcohol consumption. Ong and Joseph  reviewedComparing the drinking frequencies between subgroups reveals that frequent drinking was more prevalent in single males and those who were not living with others, even after adjusting for all other variables. Described in the literature as the \u201cmarriage effect\u201d, living in a partnership is protective against alcohol misuse , whereasversus abstinence) were paired with lower probabilities of having immigrant parents. One possible explanation was suggested by Amundsen and colleagues [A similar picture appeared for conscripts whose parents formerly immigrated to Switzerland. Increased drinking frequencies  had significantly decreased odds ratios when compared to abstainers regarding these last three variables. Similar findings were discussed in a study on Swedish women, in that underlying reasons for not drinking often remain unclear .Although not tested in this study, abstainers are a very heterogeneous group, including \u201csick quitters\u201d, as well as former problem drinkers . JohanssAll in all, it should be noted that, although significant, many of the differences between the categories of drinking frequency are rather small. However, no matter how small these differences are, they are important. Studying a complete age cohort of Swiss males represents a clear strength of this study, because of the large database the findings presented in this paper can be regarded as representative.3.3.Unquestionably, this study suffers from several limitations. The first and major limitation has to be the restricted informative value of causality. Alcohol use seems frequently to be associated with several potential confounders. But due to our cross-sectional design, it still remains unclear whether we are dealing with pre-existing, promoting risk factors, or on the contrary, with the consequences of alcohol use. A further limitation is given by the fact that alcohol consumption was summarized as frequency of alcohol use. However, such information provides a large sense of variability and thus a source of bias. Not every use of alcohol is hazardous. So might one person drink one or two drinks at lunch on every day of the week, whereas somebody else might drink the same accumulated amount on one occasion each weekend. Using the present measure to record drinking habits might overestimate the risk of alcohol use in the first mentioned case, while underestimating it in the latter. Our findings therefore provide rather rough estimations of alcohol use. Additional information about drinking quantities as well as frequencies of binge drinking may contribute to a more complex definition of drinking patterns. Another limitation concerns the validity of self-reported data as a potential source of biasing the results. Although it has shown that self-reports on drinking behavior may be affected by bias of underreporting ; most st4.Our study provides further evidence that frequent use and probable misuse of alcohol is highly related to more problematic behaviors and other risk factors. However, it shows that abstinence is actually not linked to better mental health or less psychosocial problems than moderate drinking, but only to less risky behavior. As long as the underlying mechanisms for abstinence remain unclear, conclusions should be drawn with caution. Our findings only suggest that moderate alcohol use is an important part of social life, whereas more frequent as well as never drinking can somehow be considered as deviations from the norm. These results underscore not only the need for additional research examining factors of problematic alcohol use, but also indicate that further studies and prevention programs should be targeted to both ends of drinking continuum - abstinence and high-frequency."}
+{"text": "During early postnatal life, extensive changes in gene expression occur concomitantly in multiple major organs, indicating the existence of a common core developmental genetic program. This program includes hundreds of growth-promoting genes that are downregulated with age in liver, kidney, lung, and heart, and there is evidence that this component of the program drives the widespread decline in cell proliferation that occurs in juvenile life, as organs approach adult sizes. To investigate epigenetic changes that might orchestrate this program, we performed chromatin immunoprecipitation-promoter tiling array to assess temporal changes in histone H3K4 and H3K27 trimethylation (me3) at promoter regions throughout the genome in kidney and lung, comparing 1- to 4-wk-old mice. We found extensive genome-wide shifts in H3K4me3 and H3K27me3 occurring with age in both kidney and lung. The number of genes with concordant changes in the two organs was far greater than expected by chance. Temporal changes in H3K4me3 showed a strong, positive association with changes in gene expression, assessed by microarray, whereas changes in H3K27me3 showed a negative association. Gene ontology analysis indicated that shifts in specific histone methylation marks were associated with specific developmental functions. Of particular interest, genes with decreases in H3K4me3 with age in both organs were strongly implicated in cell cycle and cell proliferation functions. Taken together, the findings suggest that the common core developmental program of gene expression which occurs in multiple organs during juvenile life is associated with a common core developmental program of histone methylation. In particular, declining H3K4me3 is strongly associated with gene downregulation and occurs in the promoter regions of many growth-regulating genes, suggesting that this change in histone methylation may contribute to the component of the genetic program that drives juvenile body growth deceleration. Posttranslational modifications of histone protein are thought to be important epigenetic marks closely associated with transcriptional regulation Widespread temporal changes in gene expression are not restricted to the differentiation of ESCs and embryogenesis; during early postnatal life, extensive changes in gene expression occur in most major organs. Some of these changes are organ-specific, but other shifts in gene expression occur simultaneously in multiple organs. In rodents, hundreds of growth-promoting genes are downregulated from 1- to 4-wks of age simultaneously in liver, kidney, lung, and heart. There is evidence that this multi-organ genetic program helps drive the widespread decline in cell proliferation that occurs in juvenile life, as organs approach adult size in vivo. Gene ontology analyses were then used to explore the biological functions associated with these epigenetic changes. In addition, we combined the histone methylation analysis with our previous expression microarray analysis performed in the same organs and ages We previously showed that, in a number of these growth-promoting genes, mRNA downregulation is associated with declining promoter H3K4me3 All animal procedures were approved by the National Institute of Child Health and Human Development Animal Care and Use Committee. C57BL/6 male mice at 1- and 4-wk of age were purchased from Charles River and sacrificed to obtain kidney and lung tissue.2O. For each corresponding sample, a portion of chromatin was analyzed without chromatin immunoprecipitation (Input) to represent the unselected DNA content.Cell nuclei from animal tissues were isolated as previously described To confirm bivalency of histone marks H3K4me3 and H3K27me3 on the same promoter, we performed sequential ChIP, using essentially the same methods as individual ChIP. Importantly, the elution after the first ChIP was performed by incubating the Protein A agarose in TE buffer with 10 mM DTT at 37\u00b0C for 30 mins, followed by a 20-fold dilution in ChIP dilution buffer  Fig. S1). Primer sequences are provided in Table S1 in File S1. We also performed ChIP-qPCR validation for genes that showed changes (increases or decreases) in H3K4me3 or H3K27me3 with age, genes that showed bivalent histone modifications and genes that showed concordant declines in H3K4me3 with age. Primer sequences for these three different sets of genes are provided in Table S2, S3, and S4 in File S1. The amount of histone modification at a particular position was quantified with a standard curve generated by serially diluted mouse genomic DNA. Chromatin enrichment was expressed as percent input\u200a=\u200a100\u00d7/(volume of DNA solution used for ChIP/volume of DNA solution set aside as input).Before hybridization, the quality of the immunoprecipitated DNA was tested by real-time PCR using custom primers that amplify promoter regions previously known to show presence or absence of histone modifications being studied DNA recovered from chromatin immunoprecipitation was amplified as previously described Microarray signals were analyzed using the default Affymetrix RMA algorithm for tiling array. ANOVA was performed and false discovery rate (FDR) reports were generated using Partek Genome Suite 6.6 . The Model-based Analysis of Tiling-arrays (MAT) algorithm was used to detect promoter regions enriched by immunoprecipitation with antibody against H3K4me3 or H3K27me3 th), middle quartiles (25th\u201375th), and upper quartile (75th\u2013100th). The lower quartile consisted of genes that showed decreasing MAT score, which represents decreasing histone methylation with age. On the contrary, the upper quartile consisted of genes that showed increasing MAT score and therefore increasing histone methylation with age. The middle quartiles comprised genes that showed little change in histone methylation with age. This approach of categorizing genes into three groups  was used because there is currently no clear understanding about what cut-off value for change in MAT score should be used identify biologically meaningful changes in histone methylation We combined the tiling microarray data for the two age groups to compare the histone modifications between 1- and 4-wk of age in the same organ. We considered genes that showed significant H3K4me3 signals at 1-wk (versus Input DNA) but not at 4-wk to have \u201clost\u201d H3K4me3 with age. On the contrary, genes that showed significant H3K4me3 signals only at 4-wk old were considered to have \u201cacquired\u201d the mark with age. For genes with significant histone methylation at both 1- and 4-wk compared to input DNA, the quantitative level of that histone methylation may have changed substantially with age, which might lead to altered gene expression. To detect these quantitative temporal changes in histone methylation, all genes in the microarray that showed histone signal at both 1- and 4-wk were ranked by the change in MAT score \u2013\u0394Ct\u00d7106, where Ct represents the threshold cycle and \u0394Ct\u200a=\u200a(Ct of gene of interest)\u2013(Ct of 18S rRNA). Values were multiplied by 106 for convenience of comparison.Real-time PCR was used to assess specific mRNA levels in mouse kidney and lung. Organs were dissected from 1- and 4-wk old C57BL/6 mice . Total RNA (100\u2013200 ng) was reverse transcribed using SuperScript III Reverse Transcriptase (Invitrogen). Quantitative real-time PCR was performed for 18S, Aurkb, Ccnd2, Sox11, Rrm2, and Zwilch using commercially available FAM or VIC-labeled Taqman assays . Reactions were performed in triplicate on cDNA derived from each animal using the ABI prism 7900 Sequence Detection System instrument (Applied Biosystems). The relative quantity of each mRNA was calculated using the formula: Relative Expression\u200a=\u200a2We performed chromatin immunoprecipitation (ChIP) with genomic DNA isolated from normal mouse kidney and lung at 1- and 4-wk of age using antibodies that recognize specific histone modifications H3K4me3 or H3K27me3. The ChIP-enriched DNA and total genomic DNA (Input) were hybridized to tiling microarrays that interrogate over 25,500 mouse promoters (Affymetrix Gene Chip Promoter 1.0R). Promoter regions with histone binding was identified by significant enrichment in the immunoprecipitated DNA compared to the Input DNA . Since we were primarily interested in the correlation between histone modifications and gene expression, we combined the promoter analysis with our previous expression microarray Fig. S2).Tiling array analysis of 1-wk old kidney and lung suggested that the distribution of H3K4me3 and H3K27me3 marks across the genome is very similar between the two organs Fig. 1A.P<0.05, ANOVA on ranks). H3K27 marked genes showed a modest, yet statistically significant association with lower expression levels compared to unmarked . Genes marked by both H3K4me3 and H3K27me3 showed an elevated level of expression compared to unmarked genes , which agreed with the observation that H3K4me3 has a stronger association with gene expression than H3K27me3. Similarly, when we divide the genes into groups with different expression levels . For example, the three most significant biological functions in genes with bivalent marks in 1-wk old kidney were tube development , urogenital system development , and kidney development . Similarly, in 1-wk old lung, epithelium development , lung development  and respiratory tube development  ranked among the most significant GO terms.We used the DAVID 2008 web-server Table S7\u20138 in File S1). Some of the major categories that showed the strongest enrichment included protein localization/transportation  and catabolic processes . Interestingly, cell division functions contributed to more than 10 of the 50 most significant GO terms, perhaps reflecting the active transcription of cell cycle-related genes during this juvenile period, when the organs are rapidly growing and many cells are still undergoing active proliferation.Gene ontology analysis also indicated that H3K4me3-positive genes at 1-wk of age showed a wide range of biological functions, which were generally similar between kidney and lung (Table S9\u201310 in File S1). The expression of these genes is important for neurodevelopment, and therefore should presumably be actively transcribed in neuronal cells, but remain silenced in other cell types. It is therefore not surprising that in kidney and lung, H3K27me3 mark is present at the promoter regions for silencing of these genes.Similar to H3K4me3, H3K27me3 positive genes are implicated in a variety of biological functions, among which neuron differentiation constitute an overwhelming proportion  was similar to that observed at 1-wk of age. As at 1 week, H3K4me3 at 4 weeks of age was associated with higher levels of expression and H3K27me3 was associated with lower levels of expression . Gene ontology analyses of genes marked with H3K4me3 and/or H3K27me3 at 4-wk of age yielded results (Table S11\u201316 in File S1) similar to those of 1-wk old mice, except that in the 4-wk old kidney, bivalent genes were not strongly implicated in developmental functions, but rather involved in tissue homeostasis (Table S9 in File S1).We next analyzed the tiling promoter microarray for kidney and lung at 4 wk of age. The overall distribution of H3K4me3 and H3K27me3 marks across the genome at 4 wk of age (th), middle quartiles (25th\u201375th), and upper quartile (75th\u2013100th). The lower quartile consisted of genes that showed decreasing MAT score and therefore decreasing histone methylation with age, while the upper quartile consisted of genes with increasing MAT score and increasing histone methylation with age. The middle quartiles comprised genes that showed little change in histone methylation with age . We used ChIP-qPCR in a subset of genes to confirm the validity of our approach for identifying changes in histone modifications from 1- to 4-wk organs .To gain insight into how changes in histone modifications regulate biological functions from 1- to 4-wk, we combined the tiling microarray data for the two age groups and focused on genes that showed changes in histone methylation with age. As described in the materials and methods section and summarized in Fig. S6, Spearman\u2019s p\u200a=\u200a0.137, kidney; p\u200a=\u200a0.111, lung; both P<2e-08) and negatively correlated with changes in H3K27me3 . In a similar analysis, the set of age-downregulated genes  was significantly enriched with genes showing loss of or decrease in H3K4me3, and with genes showing acquisition of or increase in H3K27me3 . For genes that acquired or showed increased H3K4me3 with age, major implicated biological functions included protein localization/transport (both organs) and catabolism and apoptosis (in kidney) (Table S19\u201320 in File S1). Genes that lost or showed decreasing H3K27me3 with age in both kidney and lung were predominantly implicated in morphogenesis and developmental functions (Table S21\u201322 in File S1). Genes that acquired or showed increasing H3K27me3 with age in kidney and lung were also enriched with developmental functions, along with ion transportation and cell motion/migration (Table S23\u201324 in File S1).Gene ontology analysis Fig. 3 sCcna2, Ccnd2), cyclin-dependent kinases  and condensins . The second biggest protein cluster, which consists of multiple ECM proteins  and integrins , involves cell adhesion and cell signaling. In contrast, proteins encoded by genes with concordant increases in H3K4me3 or concordant changes (increases or decreases) in H3K27me3 only showed sporadic interactions, rather than forming an extensive network . Taken together, these analyses suggest that the concordant downregulation of H3K4me3 in cell-cycle genes may be implicated in the decline of cell proliferation with age in multiple organs during early postnatal life.We next analyzed the overlap in histone methylation changes with age between different organs. We found that the number of genes that showed concordant changes in histone methylation in both kidney and lung was much greater than would be expected by chance if regulation in the two organs were independent events Fig. 4C.Broad shifts in gene expression have been identified during differentiation of specific isolated cell types and during embryogenesis in vivo. Similarly, we found that temporal changes in histone methylation in juvenile animals were associated with temporal changes in gene expression. In both mouse kidney and lung, from 1- to 4-wks of age, loss of H3K4me3 and acquisition of H3K27me3 were strongly associated with downregulation of gene expression, and conversely, acquisition of H3K4me3 and loss of H3K27me3 were associated with upregulation of gene expression. Although these strong temporal associations are consistent with the hypothesis that histone methylation helps coordinate the widespread changes in gene expression occurring during early postnatal life, the actual causal relationship between histone methylation marks and gene expression remain unclear, not only in this setting but in general To explore postnatal changes in histone methylation patterns, we assessed the patterns of H3K4me3 and H3K27me3 at promoter regions across the genome in kidney and lung of 1- and 4-wk-old mice and compared these genome-wide patterns of histone methylation to genome-wide patterns of gene expression. We found that, in both organs and at both ages, the histone mark H3K4me3 was associated with high levels of gene expression, and H3K27me3 was associated with low levels of expression. These associations are similar to associations identified in other biological systems, including human embryonic stem cells differentiation Interestingly, the genome-wide pattern of H3K4 and H3K27 trimethylation showed strong similarity in kidney and lung, at both ages studied. Furthermore, the shifts in both K4 and K27 methylation patterns between 1- and 4-wks of age showed marked similarity in the two organs studied, suggesting that there might be a common core developmental program of histone methylation occurring simultaneously in multiple organs, which accompanies the common core developmental program of gene expression that occurs in multiple organs. Studies of additional organs are needed to determine how widespread this histone methylation program is among other tissues.Fig. S2). Other genes that were not evaluated by ChIP-reChIP may not be truly bivalent.Gene ontology analysis was used to explore the possibility that specific changes in histone methylation might be associated with discrete biological functions. An interesting pattern was observed for the genes marked with both H3K4me3 and H3K27me3. At 1-wk of age, this apparent bivalent histone mark was strongly enriched for genes with developmental functions, which is consistent with previous reports showing that many key developmental genes are bivalently marked in embryonic stem cells Our findings also point to the potential involvement of H3K4me3 in the regulation of cell proliferation. Gene ontology analyses of genes marked with H3K4me3 were enriched for cell-cycle-related functions in both 1- and 4-wk-old kidney and lung. Similarly, in both kidney and lung, genes that showed decreasing H3K4me3 marks from 1- to 4-wk were strongly enriched for cell division and cell cycle-related functions. Furthermore, when the analysis was focused on genes that showed concordant changes in histone methylation in both kidney and lung, those genes with declining H3K4me3 in both organs were particularly enriched for cell division and cell cycle-related functions. Thus, the findings suggest that, during this early postnatal period, decreasing H3K4me3 is associated with downregulation of genes involved in cell-cycle functions. Because the downregulation of growth-regulating genes appears to be involved in the physiological postnatal slowing of body growth, the combined evidence suggests declining H3K4me3 is also involved in postnatal growth deceleration.In summary, our combined ChIP-on-chip and expression array analysis in the mouse suggests that extensive genome-wide shifts in H3K4me3 and H3K27me3 are occurring during early postnatal life, and that these changes in histone methylation are strongly associated with genome-wide changes in gene expression occurring concordantly in multiple organs. First, in postnatal tissues, we observed a strong association between histone methylation marks and gene expression and also an association between temporal changes in histone methylation marks and changes in gene expression during juvenile life. Second, we found substantial similarities between histone methylation patterns in two different organs and similarities between temporal changes in histone methylation in these organs, raising the possibility that there may exist a common developmental program of histone methylation occurring simultaneously in multiple organs. Third, using gene ontology analysis, we found that shifts in specific histone methylation marks were associated with specific developmental functions. Of particular interest, genes showing declining H3K4me3 were strongly enriched for functions related to cell proliferation.Taken together, the findings indicate that the common core developmental program of gene expression which occurs in multiple organs during juvenile life is associated with a common core developmental program of histone methylation. The findings also suggest that one component of this program, declining H3K4me3, is associated with the declining expression of multiple growth-regulating genes. Prior evidence suggests that this declining expression contributes to the physiological slowing of proliferation that occurs in multiple tissues during juvenile life. Therefore, the current findings suggest that declining H3K4me3 may contribute to the developmental program that limits somatic growth in mammals.Figure S1Assessment of DNA quality after chromatin immunoprecipitation. Chromatin from kidney of 1-wk and 4-wk old mice was immunoprecipitated with antibodies to H3K4me3 (A) or H3K27me3 (B). Real-time PCR was used to measure content of indicated genomic regions in the immunoprecipitated DNA compared to input DNA. Igf2 and Inmt served as positive controls for H3K4me3 at 1- and 4-wk, respectively. Chr19\u223623.12 Mb was a negative control for H3K4me3. Mdk intronic region was a positive control for H3K27me3, and Chr10\u223679.15 Mb was a negative control for H3K27me3.(TIF)Click here for additional data file.Figure S2Validation of H3K4me3 and H3K27me3 bivalency using ChIP-reChIP. Chromatin from kidney and lung of 1-wk old mice was immunoprecipitated first with antibodies to H3K4me3, followed by DNA purification (green bar), or a second immunoprecipitation using antibodies to H3K27me3 (red bar) or IgG (yellow bar). Real-time PCR was then used to measure content of indicated genomic regions in the immunoprecipitated DNA compared to input DNA. Chr19\u223623.12 Mb and Chr10\u223679.15 Mb was used as negative control for both H3K4me3 and H3K27me3. Ezh2 was a positive control for H3K4me3 but negative control for H3K27me3. Two-way-ANOVA was performed using genomic position and effect of antibody in the re-ChIP (H3K27me3 versus IgG) as the two independent variables. P values represent the effect of antibody in the re-ChIP. Number of animals\u200a=\u200a5 in each data point.(TIF)Click here for additional data file.Figure S3H3K4me3 is associated with high level of gene expression and H3K27me3 is associated with low level of gene expression in kidney and lung of 4-wk-old mice. Pie chart (left) depict the distribution of H3K4me3 and H3K27me3 marks at promoter regions of genes across the genome, in kidney (top) and lung (bottom) of 4-wk old mice. The number of genes within each category are indicated. Box and whisker plots (center) show the expression microarray signals of genes with different types of histone methylation. The line within each box represents the median microarray signal of the genes in the designated gene set, with the median value displayed over the line. The upper and lower boundaries of the box indicate the 75th and 25th percentiles, respectively. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles and outlying dots indicate the 95th and 5th percentiles. The overall median of the microarray signal from the whole genome is displayed on the upper right corner of each graph. Bar graphs (right) show the distribution of H3K4me3 and H3K27me3 marks in gene sets with different levels of expression. H3K4me3 marks occurred more frequently in genes with higher expression levels, and H3K27me3 marks occurred more frequently in genes with lower expression levels. The values above bars indicate number of genes in each group. The color code for all graphs is shown in below pie charts.(TIF)Click here for additional data file.Figure S4Box and whisker plot showing the change in MAT scores of gene sets as categorized in . The line within each box represents the median MAT score of the genes in the designated gene set. The upper and lower boundary of the box indicates the 75th and 25th percentiles, respectively. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles and outlying dots indicate the 95th and 5th percentiles.(TIF)Click here for additional data file.Figure S5Validation of changes of H3K4me3 and H3K27me3 with age using ChIP-qPCR. Chromatin from lung (top panels) or kidney (bottom panels) of 1-wk and 4-wk old mice was immunoprecipitated with antibodies to H3K4me3 (light green and dark green bars) or H3K27me3 (pink and red bars). Real-time PCR was used to amplify promoter regions of of the indicated genes and measure the concentrations in immunoprecipitated DNA compared to input DNA. The results confirmed the tiling array analysis: In lung, Tnc and Inmt showed increased H3K4me3 with age, Peg3 and Bub1 showed decreased H3K4me3 with age; E2f7 and Ptch2 showed increased H3K27me3 with age, Cdk4 and Gpc3 showed decreased H3K27me3 with age; in kidney, Fgf9 and Fndc4 showed increased H3K4me3 with age, Mdk and Mmp14 showed decreased H3K4me3 with age; Fat4 and Lrrc17 showed increased H3K27me3 with age, Pcdh1 and Hoxa1 showed decreased H3K27me3 with age. P values (ANOVA) are for change in histone level with age. Number of animals\u200a=\u200a5 in each data point.(TIF)Click here for additional data file.Figure S6Temporal changes in H3K4me3 and H3K27me3 were associated with temporal changes in gene expression in kidney and lung. Dot plots show that changes in mRNA from 1- to 4-wk was positively correlated (Spearman\u2019s correlation) with changes in H3K4me3 (left panels) and negatively correlated with changes of H3K27me3 (right panels) from 1- to 4-wk in kidney (upper panels) and lung (lower panels). Spearman\u2019s \u03c1 and P-values are indicated at an upper corner of each graph.(TIF)Click here for additional data file.Figure S7Protein interactions of different sets of genes with concordant changes of histone methylation with age between kidney and lung. STRING9.0 was used to analyze protein interactions. (A) Genes with concordant acquisition of or increase in H3K4me3. (B) Genes with concordant loss of or decrease in H3K27me3. (C) Genes with concordant acquisition of or increase in H3K27me3. None of these gene sets form an extensive network of protein-protein interactions as compared to genes with concordant loss of or decrease in H3K4me3 with age (with age .(TIF)Click here for additional data file.File S1Combined file that contains Table S1\u2013S24.(DOCX)Click here for additional data file."}
+{"text": "Cores were scored for membranous \u00df-catenin, a key functional component of adherens junctions, and for nucleocytoplasmic \u00df-catenin, a hallmark of Wnt/\u00df-catenin pathway activation. Only 82% of benign samples exhibited membrane-associated \u00df-catenin, indicating a finite frequency of false-negative staining. The frequency of membrane positivity was similar in DCIS samples, but was significantly reduced in carcinomas , consistent with loss of adherens junctions during acquisition of invasiveness. Negative membrane status in cancers correlated with higher grade (P\u200a=\u200a0.04) and estrogen receptor-negative status (P\u200a=\u200a0.03), both indices of poor prognosis. Unexpectedly, a substantial frequency of nucleocytoplasmic \u00df-catenin was observed in benign breast tissues (36%), similar to that in carcinomas (35%). Positive-staining basal nuclei observed in benign breast may identify putative stem cells. An increased frequency of nucleocytoplasmic \u00df-catenin was observed in DCIS tumors (56%), suggesting that pathway activation may be an early event in human breast neoplasia. A correlation was observed between HER2/neu expression and nucleocytoplasmic \u00df-catenin in node-positive carcinomas (P\u200a=\u200a0.02). Furthermore, cytoplasmic \u00df-catenin was detected in HER2/neu-induced mouse mammary tumors. The NLSlacZAxin2 mouse strain, a previously validated reporter of mammary Wnt/\u00df-catenin signaling, was utilized to define in vivo transcriptional consequences of HER2/neu-induced \u00df-catenin accumulation. Discrete hyperplastic foci observed in mammary glands from bigenic MMTV/neu, NLSlacZAxin2 mice, highlighted by robust \u00df-catenin/TCF signaling, likely represent the earliest stage of mammary intraepithelial neoplasia in MMTV/neu mice. Our study thus provides provocative evidence for Wnt/\u00df-catenin signaling as an early, HER2/neu-inducible event in breast neoplasia.Wnt/\u00df-catenin signaling is strongly implicated in neoplasia, but the role of this pathway in human breast cancer has been controversial. Here, we examined Wnt/\u00df-catenin pathway activation as a function of breast cancer progression, and tested for a relationship with HER2/neu expression, using a human tissue microarray comprising benign breast tissues, ductal carcinoma  The key role of Wnt/\u00df-catenin signaling in stem cell biology provides another mechanism by which this signaling axis may impact tumorigenesis The goal of this study was to investigate the activation status of Wnt/\u00df-catenin signaling in human breast neoplasia, and to test for a potential relationship between Wnt/\u00df-catenin pathway activation and expression of human epidermal growth factor receptor 2 (HER2/neu). \u00df-catenin protein exists in two discrete functional pools in epithelial cells. Membrane-associated \u00df-catenin is an integral component of the adherens junctions linking membrane-localized E-cadherin to the actin cytoskeleton via alpha-catenin. In contrast, \u00df-catenin protein accumulates in the cytoplasm and nucleus in response to canonical Wnt signaling, the best characterized pathway regulated by Wnt proteins Wnt1, was originally identified as a locus activated by retroviral insertion of mouse mammary tumor virus (MMTV), and transgenic Wnt1 overexpression was subsequently shown to drive mammary tumor formation in mice The role of Wnt/\u00df-catenin signaling in human breast cancer has been subject to much debate APC, Axin, and CTNNB1 genes (encoding \u00df-catenin), leading to \u00df-catenin stabilization, and hence activation of the Wnt/\u00df-catenin pathway, is now recognized as a common event in human tumorigenesis Striking frequencies of aberrant nucleocytoplasmic \u00df-catenin accumulation have now been recorded in multiple human neoplastic conditions, most notably in colorectal cancers. Direct elucidation of the likely contribution of Wnt ligand overexpression to pathway activation in human cancer specimens has been hampered by a dearth of immunohistochemistry-compatible anti-Wnt antibodies. However, mutation of pathway components, including the in situ tumors, and invasive carcinomas. An additional goal was to assess the relationship between canonical Wnt pathway activation and HER2 overexpression based on several lines of evidence indicating that Wnt and epidermal growth factor receptor (EGFR) signaling pathways can interact Our goal in the present study was to further investigate the frequency of Wnt/\u00df-catenin signaling pathway activation in breast neoplasia using \u00df-catenin immunohistochemistry (IHC) to analyze a human breast tissue microarray (TMA) and to systematically catalogue the prevalence of both membrane-associated and nucleocytoplasmic \u00df-catenin protein. We sought to characterize the status of Wnt/\u00df-catenin signaling as a function of breast cancer progression by quantifying and comparing the proportion of tissue samples that exhibited nucleocytoplasmic \u00df-catenin in benign breast tissue, ductal carcinoma ad libitum.All mice were housed in pathogen-free rooms in filter-topped cages at the Laboratory Animal Research Center at the New York Blood Center. This facility is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, and operates in accordance with Federal , State and local laws and regulations. All mice were used in accordance with protocols approved by the Institutional Animal Care and Use Committees of both the New York Blood Center (Protocol Number 266) and Weill Cornell Medical College . Mice received food and water nd Generation Breast Cancer Progression Tissue Microarray developed by the National Cancer Institute (NCI) Cancer Diagnosis Program (CDP). The CDP assembled a collection of 339 breast tissue specimens arrayed on three separate slides (http://cdp.nci.nih.gov/breast/progression_cs2.html). The intended tissue representation was: 69 cores of benign breast tissue , 31 ductal carcinoma in situ (DCIS) specimens, and 239 invasive breast cancers with a principal histology of ductal carcinoma. Of the benign samples, 43 were from women without breast cancer and 26 were from individuals with breast cancer represented elsewhere on the TMA. Of the DCIS samples, 15 were from women without breast cancer and 16 were from individuals with breast cancer represented elsewhere on the TMA. Of the invasive carcinomas, there were 80 from patients that were node-negative at diagnosis, 80 from patients that were node-positive but not metastatic at diagnosis, and 79 from patients with metastatic disease at diagnosis. The TMA slides purchased from the NCI CDP contained coded human biological specimens lacking personal identifiers such that they could not be linked to specific individuals by the research team. Therefore this study did not constitute human subjects research, and Institutional Review Board approval was not required.To analyze \u00df-catenin protein immunohistochemically in human breast tissues we used the 2Three freshly-cut serial sections were obtained from each of the three TMA blocks. Two of the three sections from each block were stained in duplicate for \u00df-catenin protein by IHC as described below. The third intermediate section was stained in parallel omitting primary antibody to provide a negative control. Histopathology of each core on slides stained with \u00df-catenin antibody was evaluated by a breast pathologist (D.D.G.).\u00df-catenin IHC was performed using Clone 14 anti-\u00df-catenin antibody (BD Transduction Labs) as previously described HER2 IHC was performed on an additional set of TMA slides by the MSKCC Pathology Dept using Ventana's PATHWAY anti-HER2 antibody (clone 4B5), an FDA-approved monoclonal for immunohistochemical detection of HER2 protein in breast cancer tissue. Slides were scored by a breast pathologist (D.D.G.) in accordance with the American Society of Clinical Oncology/College of American Pathologists guideline recommendations for HER2 testing in breast cancer HER2/neu allele , under the control of the mouse mammary tumor virus (MMTV) long terminal repeat, that induces mammary hyperplasia and tumorigenesis MMTV/NDL mice express a mammary-targeted, mutationally activated NLSlacZAxin2 strain (Axin2LacZ) provides a useful reporter of in vivo \u00df-catenin/TCF signaling. Axin2 is upregulated in response to canonical Wnt/\u00df-catenin signaling, and functions as a negative feedback regulator NLSlacZAxin2 mice have a bacterial \u00df-galactosidase  expression cassette \u201cknocked-in\u201d to the endogenous Axin2 locus, such that \u00df-gal activity provides a surrogate for Wnt/\u00df-catenin signaling in vivo \u00df-catenin/TCF pathway activation in mouse mammary gland by demonstrating increased \u00df-gal activity in response to expression of a Wnt1 transgene The neu strain (FVB/N-Tg(MMTVneu)202Mul/J; The Jackson Laboratory) expresses a wildtype HER2/neu allele. MMTV/neu females develop palpable mammary tumors with a latency of 2\u201318 months, with subsequent lung metastases HER2/neu transgene, suggesting that mutational activation of the transgene may be a prerequisite for tumor formation The MMTV/+/NLSlacZAxin2 mice were interbred with homozygous MMTV/neu animals. Abdominal (#4) mammary glands were harvested post-mortem from both MMTV/neu and bigenic MMTV/neu, +/NLSlacZAxin2 female offspring, stained with X-gal to detect \u00df-gal activity and wholemounted, as previously described NLSlacZPea3 females were harvested and stained in parallel to provide a positive control for LacZ staining. NLSlacZPea3 mice (Pea3LacZ) have a \u00df-gal expression cassette \u201cknocked-in\u201d to the endogenous Pea3 gene, which is expressed predominantly in the myoepithelial compartment in murine mammary gland Axin2LacZ and Pea3LacZ mice was performed as described previously Here, In this study, the proportion of samples with positive membrane and nucleocytoplasmic \u00df-catenin staining in samples of various clinical pathological categories were summarized and compared. Specifically, the associations between \u00df-catenin staining and the type of tissue sample were examined using mixed-effects logistic regression to take into account possible within-subject correlation because some subjects provided samples of more than one tissue type. Pair-wise comparison of \u00df-catenin staining positivity between any two tissue types of interest were performed using simultaneous inference methods for general parametric models nd Generation Breast Cancer Progression TMA generated by the NCI Cancer Diagnosis Program  and nucleocytoplasmic (NC), for each core. Nuclear and cytoplasmic signals were combined into a single entity because accumulation at both these subcellular locations is indicative of canonical Wnt signaling pathway activation. Strikingly in this respect, Kim P\u200a=\u200a0.72) (P<0.001 compared with either benign or DCIS). Additionally, when cases were sub-divided into node-negative, node-positive or metastatic at diagnosis, a trend of lower odds of having positive membrane staining in higher stage cancer was observed  ; Table 3P\u200a=\u200a1). Interestingly however, the proportion of DCIS tumors positive for nucleocytoplasmic \u00df-catenin was quantitatively increased relative to benign tissue . The two parameters were not associated in DCIS cores, but there was a correlation between membrane and nucleocytoplasmic \u00df-catenin positivity in benign breast tissue samples . In order to control for effects of this correlation, we conducted an additional analysis restricted to those samples with positive membrane staining (n\u200a=\u200a166). In this subset, the proportion of samples with positive nucleocytoplasmic staining was increased for both DCIS  and invasive cancer samples  relative to benign breast tissue .A striking positive correlation was identified between membrane and nucleocytoplasmic \u00df-catenin positivity in individual samples . For carP\u200a=\u200a0.04) and estrogen receptor (ER) negative status (P\u200a=\u200a0.03), both indices of poor prognosis. No statistically significant associations were observed between nucleocytoplasmic \u00df-catenin positivity and individual clinicopathological parameters when all invasive tumors were considered together (P\u200a=\u200a0.02). When all invasive cases were considered together, the data were also suggestive of a potential interaction between HER2/neu and \u00df-catenin stabilization: nucleocytoplasmic \u00df-catenin was detected in 43.8% of HER2/neu-expressing breast carcinomas, but only in 34.2% of HER2/neu-negative cases  from HER2/neu transgenic mice. Firstly, \u00df-catenin IHC was performed on MG sections from MMTV/NDL mice, which develop multiple DCIS-like lesions in each MG due to expression of a mammary-targeted, mutationally activated HER2/neu allele To further investigate the potential relationship between HER2/neu and canonical Wnt signaling in vivo functional significance of HER2/neu-induced \u00df-catenin redistribution, we employed a \u00df-catenin/TCF reporter strain, NLSlacZAxin2, which has a \u00df-gal expression cassette \u201cknocked-in\u201d to the Axin2 locus NLSlacZAxin2 MGs in response to expression of Wnt1, which activates canonical Wnt signaling neu, +/NLSlacZAxin2 mice, to test the ability of HER2/neu to drive \u00df-catenin/TCF-dependent transcription in vivo. Discrete regions of robust \u00df-gal activity were detected in the mammary epithelium of bigenic MMTV/neu, +/NLSlacZAxin2 MGs which coincided with focal regions of hyperplasia , consistent with previous reports of reduced membranous \u00df-catenin in human breast cancers P\u200a=\u200a0.04) and ER-negative status (P\u200a=\u200a0.03).In this study we analyzed \u00df-catenin protein levels in membrane and nucleocytoplasmic compartments in human breast tissues  with the goal of better understanding the activation status of Wnt/\u00df-catenin signaling in human breast cancer, given conflicting findings from previous studies P\u200a=\u200a0.77). Thus the basis for \u00df-catenin stabilization in these samples remains unclear. Notably, the vast majority of previous IHC studies of \u00df-catenin in breast cancer focused exclusively on malignant specimens.In light of the fact that nucleocytoplasmic \u00df-catenin is indicative of activated Wnt/\u00df-catenin signaling, our analysis of nucleocytoplasmic \u00df-catenin signal yielded somewhat unexpected data. Specifically, we observed a finite frequency of positive nucleocytoplasmic staining in normal human breast tissues (36%), with 5 of the 56 assessable cores exhibiting substantial signal  & 2B. ThWnt genes are expressed in human and mouse breast tissues in vivo in mouse MG is predominantly restricted to the myoepithelial compartment Intriguingly, some benign breast cores exhibited discrete staining of nuclei in the basal layer of normal-looking epithelium . It is unclear whether this reflects an intrinsic biologic phenomenon specific to the node-positive subset. Nevertheless, the observed association is of particular interest because EGFR family members have the capacity to modulate \u00df-catenin phosphorylation, localization and transcriptional activity No correlations were detected between pathway activation and any clinicopathological parameter, when invasive cancers were analyzed as a single population. However, a positive correlation between HER2/neu expression and nucleocytoplasmic \u00df-catenin was observed in node-positive cases  wildtype neu transgene, which may be an obligate prerequisite to tumor formation neu allele, and potentially the earliest manifestation of mammary intraepithelial neoplasia (MIN) in this strain. Irrespective of the mutational status of the neu allele, these observations provide intriguing evidence of \u00df-catenin/TCF transcriptional activity in HER2/neu-expressing MIN lesions, and suggest that HER2/neu-induced activation of canonical Wnt signaling may be an early event in breast neoplasia. Consistent with this model, the elevated frequency at which nucleocytoplasmic \u00df-catenin was detected in human DCIS specimens compared with invasive carcinomas in our study could reflect the relatively high frequency of HER2/neu overexpression in human DCIS lesions compared with frank cancers To further investigate the relationship between HER2/neu expression and canonical Wnt signaling, we analyzed mammary tissues from ithelium ; Table 3ene NDL; . FunctioacZ mice . NotablyHER2/neu transgenic breast. Our study thus provides provocative evidence for Wnt/\u00df-catenin signaling as an early, HER2/neu-inducible event in breast neoplasia.In summary, in this study we effected a systematic comparison of membrane and nucleocytoplasmic \u00df-catenin during breast cancer progression from benign breast to metastatic carcinoma. A limitation of the study was that outcome data were not available for the breast cancer cases, and thus prognostic significance of membrane and nucleocytoplasmic \u00df-catenin positivity could not be determined. However, data addressing prognostic significance has been provided by other studies Figure S1\u00df-catenin staining patterns of human breast carcinomas. Images of seven invasive cancer cores demonstrating the range of observed staining patterns. Samples were scored as follows: (A) Membrane (MB), 0; Nucleocytoplasmic (NC), 0. (B) MB, +/\u2212; NC, 0. (C) MB, 1+; NC, 0. (D) MB, 1+; NC, 1+. (E) MB, 2+; NC, 2+. (F) MB, 0; NC, 2+. The image in (G) illustrates the positive cytoplasmic \u00df-catenin staining observed in mitotic cells in some cores . Left-hand panels; slides treated with primary (anti-\u00df-catenin) and secondary antibodies; right-hand panels, negative control slides treated with secondary antibody alone.(TIF)Click here for additional data file."}
+{"text": "Reduced access to healthcare during the outbreak substantially increased mortality rates from other diseases. Response to the 2014\u20132015 Ebola outbreak in West Africa overwhelmed the healthcare systems of Guinea, Liberia, and Sierra Leone, reducing access to health services for diagnosis and treatment for the major diseases that are endemic to the region: malaria, HIV/AIDS, and tuberculosis. To estimate the repercussions of the Ebola outbreak on the populations at risk for these diseases, we developed computational models for disease transmission and infection progression. We estimated that a 50% reduction in access to healthcare services during the Ebola outbreak exacerbated malaria, HIV/AIDS, and tuberculosis mortality rates by additional death counts of 6,269  in Guinea; 1,535  in Liberia; and 2,819  in Sierra Leone. The 2014\u20132015 Ebola outbreak was catastrophic in these countries, and its indirect impact of increasing the mortality rates of other diseases was also substantial. The 2014\u20132015 Ebola outbreak in West Africa debilitated the healthcare systems of affected countries, hampering diagnosis and treatment for endemic diseases such as malaria, HIV/AIDS, and tuberculosis (TB) . Halting the spread of these pathogens is a 2015 Millennium Development Goal and the priority of the Global Fund  Demographic Health Surveys (DHS) Table 1 We developed 3 computational simulation models from a population perspective of disease burden: a disease progression model for malaria and 2 decision tree models for HIV/AIDS and active TB cases. In all models, the decision node branches represent temporal intervals before and during the Ebola outbreak. For each disease, we conservatively considered deaths only for the highest risk groups: malaria among children >5 years of age, HIV/AIDS among persons 15\u201349 years of age, and active TB in all age groups.We used these models to estimate the number of deaths from malaria, HIV/AIDS, and TB that would have occurred in Guinea, Liberia, and Sierra Leone from March 2014, the start of the Ebola outbreak, through March 2015. We compared this estimate with a scenario of reduced access to health services from June 2014, when Ebola was reported in the major cities of the 3 countries, through March 2015, when the outbreak was tapering , and probabilities dependent on CD4 counts of dying without ART, ART failure, and dying while receiving ART  on HIV/AIDS-related deaths in Guinea, Liberia, and Sierra Leone , panel Bving ART Table 1.We developed a decision tree model to estimate the impact of reduced treatment coverage on the TB burdens in Guinea, Liberia, and Sierra Leone throughout the Ebola outbreak , panel CWe assumed that persons with drug-susceptible TB who receive treatment can either be successfully treated, be unsuccessfully treated and remain alive, or die. Those who experience treatment failure or discontinue treatment before taking the full course may progress to MDR TB, and then may seek treatment for MDR TB. We assumed that persons who were infected with MDR TB at the start of the Ebola outbreak would remain infected or die during the 9-month time horizon of our analysis if they were not treated. Given that MDR TB requires 24 months of treatment . PRCC measures the strength of monotonic association between the input parameters and output variable  children <5 years of age from malaria in Guinea, 2,573  in Liberia, and 4,860  in Sierra Leone. We estimated that malaria-attributable mortality rates increased by 48.0% (95% CI 4.9%\u201393.8%) in Guinea, 53.6% (95% CI 4.8%\u2013145.5%) in Liberia, and 50.0% (95% CI 5.0%\u2013118.8%) in Sierra Leone. This increase represents 4,275  additional deaths in Guinea, 788  in Liberia, and 1,755  in Sierra Leone .Given a 50% reduction in ART coverage during the Ebola outbreak, we estimated that 5,151  adults 15\u201349 of age would have died in Guinea, 1,198  in Liberia, and 2,621  in Sierra Leone. The increase in HIV/AIDS deaths attributable to this reduction in ART coverage was estimated to be 16.2% (95% CI 1.3%\u201330.2%) in Guinea, 13.0% (95% CI 2.6%\u201325.4%) in Liberia, and 9.1% (95% CI 1.6%\u201319.1%) in Sierra Leone. This increase represents 713  additional deaths in Guinea, 155 (95% CI 23\u2013297) in Liberia, and 223 (95% CI 29\u2013504) in Sierra Leone .Using a 50% reduction in treatment coverage for both drug susceptible and multidrug-resistant TB, we estimated that 3,463  persons would have died from TB in Guinea, 1,553  in Liberia, and 2,164  in Sierra Leone. The increase in TB deaths attributable to this reduction in TB treatment coverage was estimated to be 51.1% (95% CI 44.7%\u201370.5%) in Guinea, 59% (95% CI 47.9%\u201377.4%) in Liberia, and 61.4% (95% CI 49.2%\u201387.6%) in Sierra Leone. This increase represents 1,281  additional deaths in Guinea, 592 (95% CI 394\u2013691) in Liberia, and 841  in Sierra Leone .We conducted a sensitivity analysis by varying the reduction of treatment coverage over a range of 10%\u201390% of the level before the Ebola outbreak for malaria, HIV/AIDS, and TB in Guinea, Liberia, and Sierra Leone. Because treatment coverage was varied, the additional deaths attributable to the Ebola outbreak in Guinea were estimated to vary from 1,425\u20138,336 for malaria, 146\u20131,237 for HIV/AIDS, and 277\u20132,317 for TB. In Liberia, additional deaths attributable to Ebola outbreak varied from 210\u20131,502 for malaria, 50\u2013314 for HIV/AIDS, and 100\u2013987 for TB. Additional death counts in Sierra Leone varied from 630\u20133,172 for malaria, 70\u2013630 for HIV/AIDS, and 143\u20131,723 for TB.>65% resulted in higher numbers of indirect deaths from malaria, HIV/AIDS, and TB than the reported 3,629 direct deaths from Ebola  additional deaths in Guinea, 1,535  in Liberia, and 2,819  in Sierra Leone.The Ebola outbreak likely had the most detrimental effect on children with malaria, with an estimated 4,275 additional deaths among children <5 years of age in Guinea, 788 in Liberia, and 1,755 in Sierra Leone. Malaria is the most prevalent disease in West Africa and the primary cause of death among children. Although Ebola primarily affected young adults in West Africa  compared with that of Guinea (48.0%) and Sierra Leone (50.0%). These differences are likely attributable to the higher pre-Ebola malaria treatment coverage in Liberia (51.1%); Guinea and Sierra Leone had 22.0% and 42.8% treatment coverage, respectively. The percentage increase in HIV/AIDS deaths attributable to Ebola was higher in Guinea (16.2%) than in Liberia (13.0%) and Sierra Leone (9.1%), consistent with higher pre-Ebola ART coverage in Guinea (51.3%), compared with Liberia (42.9%) and Sierra Leone (33.0%). For TB, the percentage increase in deaths attributable to Ebola was highest in Sierra Leone (61.4%) compared with Guinea (51.1%) and Liberia (59.0%), which corresponds with the high treatment coverage before the Ebola outbreak in Sierra Leone (66.5%) compared with Guinea (52.1%) and Liberia (54.8%).3 in place during the timeframe of our study, it is possible that these guidelines were not yet being fully implemented in West Africa. Furthermore, our model considers only a short time horizon, limiting long-term measurements of the impact of the Ebola outbreak on HIV/AIDS, TB, and malaria deaths.Our analysis was conservative in several respects. Our base case estimates are conservative in the sense that some reports have cited much greater than 50% reduction in treatment accessibility due to the Ebola outbreak on healthcare systems in Guinea, Liberia, and Sierra Leone , ART failure, and dying while receiving ART and description of population sizes used to calculate mortality rates from malaria, HIV/AIDS, and tuberculosis."}
+{"text": "Staphylococcus aureus (SA), the predominant site of carriage of SA in the community is less certain.Although the nares represent the most common carriage site for traditional hospital-associated strains of mec, staphylococcal protein A and multilocus sequence typing.We conducted a cross-sectional study in 285 patients attending sexually transmitted diseases and inner-city clinics to evaluate the prevalence, body site colonisation and risk factors associated with carriage of methicillin susceptible SA (MSSA). All isolates were characterized by pulsed field gel electrophoresis, staphylococcal cassette chromosome The prevalence of colonisation with SA was 57.5% (164/285); 162 (56.8%) participants were colonized with MSSA, and 4 (1.4%) with methicillin-resistant SA (MRSA), 2 of them were co-colonised with both MRSA and MSSA. The most common sites of colonisation were the throat (73.1%), nares (65.2%) and interdigital web spaces of the hand (21.3%). Three out of 4 MRSA isolates were USA300-MRSA strains. Twelve MSSA isolates were closely related to the USA300 CA-MRSA. We identified sexual behaviours such as having more than 6 heterosexual sexual partners in the last 6 months and trimming pubic hair to be independently associated with MSSA colonisation, and more specifically practicing oral sex as a risk factor for throat colonisation.mec element, respectively.There is a high prevalence of MSSA carriage in this population, with a low prevalence of MRSA. The throat was the most common site of carriage and sexual behaviours were found to be risk factors for MSSA colonisation. Close strain relatedness of MSSA and USA300-MRSA isolates suggests either gain or loss of the SCC Staphylococcus aureus (CA-MRSA) have been recognized over the last decade with increasing frequency among patients in ambulatory and hospital settings throughout Canada [mec genotype, pulsed field electrophoresis pattern, multilocus sequence typing and the presence of Panton-Valentine leukocidin (PVL) toxin genes. [Community-acquired strains of methicillin-resistant t Canada  and the t Canada  CA-MRSA n genes.  The domin genes.  CA-MRSA n genes. ,6 The prn genes.  This reln genes. , raisingStaphylococcus aureus colonisation have been recognized including the genital area, as described by a recent study from a population of patients attending a sexually transmitted infections (STI) clinic in Baltimore. [Staphylococcus aureus (SA) is high in the community (~ 50%) and nearly one fifth of colonised individuals would be missed if the genital area was not tested. Lee and colleges identified having a sexual partner with a skin infection as a risk factor for MRSA colonisation amongst HIV-positive patients [Other body sites of ltimore.  This stupatients  Similarlpatients , on a popatients  findingsWe undertook a cross-sectional study of carriage of SA, both methicillin-resistant and methicillin-susceptible, in at-risk populations from STI and inner city clinics in Calgary, Alberta, Canada. The objectives of the study were to determine the prevalence of colonisation with SA, rates of carriage at different body sites and to identify risk factors associated with SA carriage.Between February and November 2014, all individuals attending the STI clinic and an inner city medical clinic: Calgary Urban Project Society (CUPS) were recruited to participate. Inner city clinics provide care to underserved and minority groups in a densely populated central core area of the city. Individuals 18 years of age or older, with no previous hospitalization over last 4 weeks and no use of antimicrobials with anti- staphylococcal activity within the last 6 weeks were eligible to participate. The University of Calgary Conjoint Health Research Ethics Board approved this study.\u00ae, College Station, TX).A sample size of 141 individuals was estimated based on an expected colonisation rate with CA-MRSA USA300 of 8% , with a \u00ae, Murrieta, CA) were collected from traditional sites: nares, throat, axilla, inguinal skin folds, and vagina, and from 3 novel sites: upper back, interdigital web spaces (IDWS) of hands and perineal-perianal area (\u201ckey swab\u201d). The key swab was obtained applying the swab with a continuous movement starting at the upper margin of the perineum and moving downwards to the margins of the anal region and then around it, resembling the shape of a skeleton key.After written informed consent was obtained, a 50-item questionnaire  was admi\u00ae catalogue # 240826) and/or Staphylsoside Latex kit . MRSA were further confirmed by PCR assay for nuc, femA and mecA genes as previously described. [mec (SCCmec) typing [(spa) typing [mec type IVa, spa type t008, MLST type ST8 and identical PFGE pattern with standard USA300 control strain CA-MRSA-10.Samples were inoculated in 5 ml tryptic soy broth with 7% sodium chloride for 72 hrs at 37\u00b0C. Following incubation, broths were subcultured to mannitol salt agar plates and incubated 48 hr at 37\u00b0C.  Presumptscribed.  All isolscribed.  The strascribed.  Any MRSA) typing ,19, and ) typing  and mult) typing  Identifi\u00ae, College Station, Texas) was used for the statistical analysis.Participants with missing questionnaire or laboratory data were excluded from analysis. Univariate analysis was performed using Pearson\u2019s chi-square or Fisher exact test for categorical data and independent sample t-test or Wilcoxon rank sum test as appropriate for continuous data to assess for possible independent risk factors associated with MSSA colonisation . SubsequOverall, 301 participants were recruited and 285 included in the analysis , with 68The overall SA colonisation rate was 57.5%, MSSA and MRSA carriage rates were 56.8% and 1.4% respectively. Two individuals had co-carriage of MRSA and MSSA. Three out of 4 MRSA colonised participants carried the USA300 strain, all MRSA carriers were recruited at CUPS. Overall, colonisation by MSSA was greater among STI clinic compared to CUPS participants . No SA infections were identified among participants.The most common site of SA colonisation was the throat, followed by the nares . OverallParticipants were most commonly colonised at multiple sites . Among tOur hypothesis was based on an estimated prevalence of CA-MRSA of 8%, unexpectedly we found a low CA-MRSA colonisation rate of 1% and high MSSA carriage rate of 56.8%; thus, we assessed for risk factors associated with MSSA carriage instead.As shown in We then looked for risk factors with respect to specific body site colonisation. Throat carriage was associated with practice of oral sex , having more than 6 heterosexual sexual partners in the last 6 months  and trimming the pubic hair  . In contmec type IVa and PVL, spa type t008 and MLST type ST8. Of note, 12 MSSA isolates were spa type t008 and MLST type ST8 and showed more than 85% relatedness in the PFGE profile to the MRSA ST8-t008 (USA300-MRSA strain). A total Staphylococcus aureus in this at-risk population is changing. We found a prevalence of SA carriage of 56.8%, with an high rate of MSSA carriage among participants attending a STI clinic (64.6%), compared to a similar study done in a marginalized urban population in Calgary in 2004 [The results of this study indicate that the epidemiology of  in 2004 , where t in 2004 ,25 In co in 2004  Similar  in 2004 \u201328It has been proposed that the rates of MSSA colonisation in the community may reflect the intensity of environmental exposure  or complOur findings support that certain risk behaviours are related to MSSA colonisation. The practice of oral sex has not been described as a risk factor for SA colonisation in the past. Miko and colleagues  did not Anecdotally, removal of pubic hair was found to be common among sexually active individuals in our local population. Trimming of pubic hair instead of shaving, was associated with higher throat and nasal colonisation. This may reflect the intensity of manipulation of the area resulting in colonisation of the pubic area or MSSA hand inoculation and then self-inoculation to other body sites. MSSA carriage in pelvic sites was 18%, similar to 23% reported among patients attending a STI clinic in Baltimore.  A nasal Additionally, among 3 novel sites assessed, the interdigital web spaces of the hands was the third most common site of SA carriage (21%), followed by the perineal-perianal area (18%) and the upper back (14%). Our study also found the practise of yoga vs any sports with mats, as a novel risk factor associated with MSSA colonisation. This association is biologically plausible, when people exercise with exposed upper back and has close contact with the mat while doing yoga. Prior studies suggest that skin contact is important for transmission of SA, including sharing of gym equipment  includinmec element among the epidemic MRSA strain previously documented in this population [attB insertion site confirmed SCCmec excision in vivo. [mec-excised strains can arise in vivo secondary to spontaneous deletion of the methicillin resistant determinant. [mec deletion has also been associated with increased fitness in mixed culture competition studies [Interestingly, we found 12 MSSA strains with high homology >85%) to the ST8 t008 strains of MRSA (USA300 strain), raising the question of possible loss of methicillin resistance by complete or partial excision of the SCC% to the in vivo.  It has brminant.  The SCCm studies \u201342 that Our study has several limitations; we included participants from an urban inner-city clinic that provides dental and medical care and from an urban-core STI clinic. Thus, participants had significant baseline differences; however, they both represent populations at-risk for SA colonisation. The original study design was based on a sample size to assess risk factors of CA-MRSA assuming a prevalence of 8%, but instead we found a 7-fold higher rate of carriage of MSSA and only 1% of CA-MRSA. We therefore, performed a risk analysis for MSSA carriage with the original sample size, which was strong enough to detect associations. Due to the cross-sectional design of the study, colonisation status and risk factors were only assessed at one time point and these associations cannot be temporally determined. This study targeted a limited at-risk population in Calgary attending inner-city clinics and results may not be generalizable to other community populations.Given the study design, volunteerism bias may have limited the participation of all patients at risk. The accuracy of the data may have been subject of underreporting bias due to lack of willingness to report sexual or drug practices, similarly self-reported comorbidities may not be accurate; we believe these limitations are not major concerns since the information collected in the questionnaires is similar to the profile of the population attending these clinics.The current investigation has a number of strengths, including a large number of participants from a community-based setting, testing multiple body sites including 3 novel sites to minimize the risk of missing intermittent carriers, and the use of broth cultures, which have been shown to increase the yield of recovery of SA by 15\u201320%, compared to solid media. ,43 AdditStaphylococcus aureus in this at-risk population attending inner-city clinics in Calgary, where we found a low prevalence of MRSA colonisation and high prevalence of MSSA compared to 22\u201332% prevalence reported in Canada and worldwide. The throat represents an important reservoir of MSSA in the community and may reflect the degree of exposure through sexual behaviour. Nasal carriage of MSSA was strongly associated with practice of yoga, having important epidemiological consequences for transmission. Close strain relatedness of MSSA and USA300-MRSA isolates suggests gain or loss of SCCmec element that requires further study.Our study suggests a changing epidemiology of S1 File(PDF)Click here for additional data file."}
+{"text": "Sonographers\u2019 perceptions of ergonomic and work-related pain problems at work have so far mostly been researched in quantitative studies by questionnaires. There is a need of experience-based research to deepen the knowledge about how sonographers perceive ergonomic problems at work. Therefore, the aim of this qualitative study was to describe sonographers\u2019 perceptions of ergonomic problems at work, and their suggestions for improvement strategies.Twenty-two female sonographers were individually interviewed regarding different aspects of their physical working environment. Content analysis was applied.The sonographers perceived different ergonomic problems in their working environment, but to offer patient comfort and to obtain the best possible images were often prioritized over working posture. Echocardiography was considered demanding as the examination is performed with little variation in posture. Ergonomic improvements included reducing the manual handling of the transducer, optimizing the adjustability of equipment, and taking the patient\u2019s physique and health into account. As some examinations were perceived to be more ergonomically demanding, variation between examinations was suggested, however, this requires broader skills.Sonography, especially echocardiography is ergonomically demanding but the improvement strategies suggested were perceived useful and applicable. Sonographers constitute a professional group with a high reported prevalence of work-related musculoskeletal pain and discomfort , especiaVillage and Trask reported that sonographers had their scanning arm abducted 30\u00b0 66\u00a0% of their scanning time . ResultsA relation has also been found between ultrasonography and WMSD in radiologic technologists , and an Sonography is an important diagnostic tool in daily medical practice  with litThe level of artificial light has to be low to facilitate interpretation of the images on the screen, which may cause visual strain . ExaminaWorking posture varies depending on the type of sonographic examination being performed. In vascular examinations, for example vein mapping, the handling of the transducer involves diverse movements and postures. In echocardiography the postures and movements are less varied, and the transducer is held in a relatively fixed position. Echocardiography also requires higher grip strength than vein mapping . GreaterThe ergonomic challenges of sonography have been known for many years. The Society of Diagnostic Medical Sonography developed industry standards, in 2003, to prevent sonographers from WMSDs . The staSo far, sonographers\u2019 perceptions of ergonomic problems at work has mostly been researched in quantitative studies , 6, 22. The aim of this study was, therefore, to assess sonographers\u2019 perceptions of these issues. The specific research questions addressed were: 1) What are the perceived ergonomic problems in echocardiography and vascular sonography? and 2) How can the working situation be improved ergonomically?A qualitative study was performed to ascertain sonographers\u2019 perceptions and characteristics of their work . The firThe sonographers were recruited from clinical physiology and cardiology departments in the south of Sweden. The participants were selected according to the following inclusion criteria: woman, interest in participating, and variation in age, seniority in sonography, workplace, examinations performed and musculoskeletal disorders. We thus attempted to enrol as varied a study group as possible, with a wide range of perceptions. A total of 22 female sonographers expressed interest in participating, and these formed the study group. The mean age was 45\u00a0years (24\u201359 years) and mean seniority in sonography was 13.5\u00a0years (0.5\u201336 years). All except one performed echocardiography, and twelve performed both vascular and echocardiographic examinations. Thirteen worked full-time and all worked at least 20\u00a0h per week.N\u2009=\u20099) followed by T3 (N\u2009=\u20098). In vascular scanning the technique varies depending on the type of examination.Three main working techniques are employed and taught in echocardiography, depending on local hospital practice. In technique 1 (denoted T1) the patient is facing the examiner, who holds the transducer in the left hand Fig.\u00a0. In techA semi-structured interview guide was developed to address the research questions. The opening question was: \u201cHow do you perceive your sonographic work?\u201d The interview guide contained open questions concerning ergonomic, psychosocial and organisational problems at work, as well as possible solutions to these problems and improvement strategies. Both positive and negative factors were elicited. The interview was performed as an open conversation, and the interviewer used follow-up questions to ensure that the research questions were answered in depth. The data were collected over a period of 9\u00a0months.This paper focuses on ergonomic problems and improvement strategies. Content analysis was used to analyse the transcribed interviews , 26. FirThe result showed that most of the sonographers perceived their work as stimulating, including contact with the patients, but found it physically exhausting. Four categories emerged from the first research question: I) working postures and type of examination, II) equipment including physical factors , III) the patient\u2019s physique and health, and IV) the sonographer\u2019s working experience and knowledge Table\u00a0. The anaI.Working postures and type of examination\u201cI\u2019m tired because I\u2019ve done a lot of echocardiographs this week, and then I feel more tired.\u201d (9)The sonographers perceived that the type of examination influenced their working posture to a high degree, with less variation in posture in echocardiography compared to vascular examinations. Echocardiography was perceived to be extremely physically demanding as several measurements were required.\u201cIt\u2019s really only in heart examinations that the hand is still for so long.\u201d (20)Regardless of the technique used for echocardiography (T1\u2013T3), the handling of the transducer was perceived as static and strenuous.\u201cIf the patient is too far away on the table, I have to stretch further.\u201d (8)If the patient lay on the table facing away Fig.\u00a0, T3, the\u201cYou map out the veins in the patient\u2019s arms, sometimes both, so you don\u2019t have any support for your own arm, that\u2019s the problem.\u201d (7)When mapping veins the examiner occasionally sat on the floor or on a chair with no possibility to rest the hand holding the transducer.The hand managing the control panel was stretched out several times to reach the touch screen, especially in echocardiography.\u201cWe take the equipment to the ward and do the ultrasound exam on patients that can\u2019t be moved. You have to position the equipment to accommodate the patient in bed. We sometimes stand up as there isn\u2019t much room.\u201d (16)In bedside examinations the ultrasound equipment is taken to the ward, implying poor ergonomic postures and insufficient room for the examination.Obtaining high-quality images was perceived as a higher priority than adjusting the equipment to achieve a comfortable working posture.II.Equipment and physical factors\u201cThe transducers are hopeless\u2026 you use this gel and it makes things slippery. They\u2019re quite heavy, and the cable makes them heavier at the end, they aren\u2019t very easy to grip, they\u2019re made of hard smooth plastic.\u201d (7)Some sonographers reported ergonomic problems associated with the control panel, keyboard and screen, for example, the buttons were not within comfortable reach and the screen was not sufficiently physically adjustable. The leg space in front of the ultrasound unit was too narrow, the transducer uncomfortable to handle, and the cable was heavy. The transducer was sometimes difficult to grip, especially if it became slippery.\u201cInstead of holding the transducer with your hand, you\u2019re supposed to manoeuver it with a little joystick. I\u2019ve tried it, and it\u2019s definitely not easy.\u201d (9)A few of the echocardiographers had experience of a robot-assisted transducer attached to an arm, but perceived it difficult to handle.\u201cPatients who are brought in their beds\u2014they\u2019re so big these days\u2014so for there to be enough room for the patient, my chair and my equipment, as well as the desk, I think the rooms are far too small.\u201d (9)The examination room was perceived to be too small, especially when examining an inpatient in bed. Sometimes, the ultrasound unit had to be moved to make room, which meant extra problems.\u201cWhen the equipment has been on all day, it feels like a sauna in here.\u201d (2)Lack of a patient lift also made it difficult to move the patient onto the examination table. Some of the sonographers also perceived that the tables were not sufficiently adjustable for different examinations and patients. The ultrasound unit gave off heat, even when additional cooling units had been fitted.\u201cThe fans make a noise all the time, but you get used to it\u2026 You only think about it when you turn the machine off. Then it\u2019s quiet.\u201d (9)Exposure to noise was also perceived as a problem. The sonographers were used to the fans and were not aware of the noise until they turned the equipment off or left the room.\u201cYou get mentally tired, and your eyes get tired\u2026 I don\u2019t want all the ceiling lights on as then I have to squint\u2026 the lights mustn\u2019t be visible on the screen\u2026 I mean, there mustn\u2019t be reflections.\u201d (8)Some of the sonographers reported eyestrain and headaches due to poor lighting in the examination room. To avoid glare on the screen the ceiling light was only switched on when the patient entered the room. Daylight sometimes came in through a slit in the curtain, which caused irritation. The same problem occurred when examining a patient on the ward, as there was no means of darkening the room.III.The patient\u2019s physique and health\u201cSo fat that you have to almost lie over the patient to get around\u2026 to get close to the heart. There\u2019s a lot of fat in the way.\u201d (18)The participants reported that the patient\u2019s physique and health often had negative effects on their work load, their posture and the ability to obtain good images. Corpulent patients and patients with lung disease often meant longer examinations (transducer time) due to the poor quality of the images, and the need to press the transducer harder to obtain good images.\u201cYes, because the patients are often very ill, and some of them aren\u2019t very mobile either, and that means heavy work.\u201d (7)Slim patients were also a problem as their ribs were an obstacle to the examination of the heart. When inpatients were brought to the examination room the sonographers sometimes perceived a high workload and apprehensive.Patients in intensive care were sometimes in a respirator, which hampered communication. The priority was high to make the patient comfortable and safe. If the patient was in pain the examination was completed as quickly as possible to reduce discomfort.\u201cSometimes we do it (the examination) in a wheelchair. Not so many heart exams, but there are lots of vascular patients who come in a wheelchair, and they\u2019re so heavy you can\u2019t lift them.\u201d (10)If the inpatient arrived in a wheelchair and was too heavy to move, the examination was performed with the patient sitting in the wheelchair, which was perceived as demanding by the sonographer, who had to adjust her working posture, i.e. bend forward.IV.Work experience and knowledge\u201cThat\u2019s probably right\u2026 a common beginner\u2019s problem is that you press really hard.\u201d (9)\u201cIf you\u2019re not an experienced sonographer, you tend to keep the transducer still for a long time until you get a good picture.\u201d (2)Lack of experience led to extended transducer time and too hard pressure in a static position.\u201cYou need a lot of knowledge to do an ultrasound exam. Then the pictures have to be good enough to interpret, to resolve the question on the referral.\u201d (20)Lack of knowledge and experience also implied stress, such as tense shoulders and forgetting the physical risks. On the other hand, a high degree of skill and experience often meant examining more patients per day.Obtaining images of high quality was given higher priority than good ergonomics.I.Working tasks and postures\u201cI use my left hand as much (as my right) to hold the transducer in all examinations except the heart.\u201d (13)The strategy employed to vary posture was to swap hands. Some of the sonographers changed the hand holding the transducer during some vascular examinations, but not in echocardiography.\u201cI make sure the patient moves until I get a good working position.\u201d (4)Another measure employed was to adjust the position of the equipment and the patient before each examination.\u201cSometimes you can stand up to do the exam, then it\u2019s easier to get to, or around the patient, and relieve the strain in another way.\u201d (2)Standing up during scanning also made it possible to change posture and was also perceived as a relaxed position when managing the control panel and handling the transducer. Resting the transducer hand on the table, on the armrest or on the patient, and the other hand on the control panel were other strategies.\u201cTwo sonographers. I think that\u2019s really good\u2014we work in similar ways and it works really well.\u201d (8)A physiotherapist had instructed them on how to adjust the screen in order to reduce the amount they had to turn their head during scanning. Another way of avoiding strenuous postures was to work in a team of two sonographers, i.e. one managed the control panel and screen, while the other handled the transducer.\u201cWe feel much better when we divide the day into four sessions.\u201d (8)Vascular examinations were perceived as less physically tiring than echocardiography, as they were more varied and involved less static positions. To ensure variation in workload, the working day was divided into four sessions, so that echocardiography was performed in one session, after which other tasks were carried out.\u201cI sit turned more towards the patient if he or she is lying down, so that I don\u2019t have to bend my wrist, so I can keep it straight.\u201d (16)When seated and using techniques (T1 and T2)) for echocardiography, the sonographer tried to sit turned towards the patient to avoid having a bent wrist when handling the transducer.\u201cWe have a chair with armrests that you can rest your arm on. What\u2019s better with the first technique\u2026 is that I don\u2019t have to stretch as much to get where I need to go.\u201d (5)Scanning facing the patient was perceived as preferable as it was possible to rest the arm on the armrest. When the patient was lying down facing away from the sonographer (T3), it was necessary for the examiner to stretch their arm more.Standing and using the weight of their body during echocardiographic examination was a strategy used to facilitate the transducer projection, especially in corpulent or overweight patients, regardless of the working technique.II.Equipment and physical factors\u201cIt must be possible to move the screen on the ultrasound equipment, a better arm, so I can raise and lower it and turn it how I want to. Buttons that are very close to me so I don\u2019t need to reach out my arm, with touch buttons or buttons that are easy to press. A keyboard that can be moved up and down and sideways, that I can pull. Lots of space so you can get your legs in under the equipment. An ultrasound unit that\u2019s easy to move.\u201d (7)An easily and highly adjustable control panel with touch buttons that could be positioned to minimize arm extension and finger pressure was considered a desirable improvement to ultrasound equipment. Likewise, a more adjustable screen would facilitate a comfortable neck position. Sufficient space for the legs when seated was another suggested improvement.A light, wireless transducer with a comfortable grip and a cover to avoid gel smears were also suggested. A cable hook attached under the equipment was perceived to facilitate handling of the transducer.\u201cThen I can steer it so that it moves towards the patient\u2019s chest, and I can make most of the small movements that a hand can do with the remotely controlled control panel. I wouldn\u2019t have to sit and press (the transducer) on the patient myself\u2014it would do the heavy work. I can steer it now without thinking, it comes automatically, just like when I\u2019m using my hand.\u201d (18)The use of a robot-assisted transducer in echocardiography was perceived to reduce awkward postures, manual pressure and pain due to strained positions. Continued training was recommended to improve handling.\u201cIt\u2019s better for me\u2014I know some people stay at the ultrasound unit, but then you have to click on every image, and on the computer I can scroll.\u201d (6)An adjustable examination table, electrically controlled with a foot pedal, would facilitate positioning the patient and adjusting the height during the examination. A more adaptable table would facilitate performing different examinations using the same table. Also, a table resembling a dentist\u2019s chair was suggested. Technical developments and improvements of the ultrasound unit have led to more distinct images suitable for computerized image analysis. A computer workstation was also perceived to be a better ergonomic alternative for reviewing the images, where the keyboard, screen and artificial light were adjustable and daylight could sometimes be used.\u201cThe machines have become quieter\u2026 so that\u2019s better.\u201d (7)\u201cWe have textiles, curtains\u2026 so it doesn\u2019t echo so much.\u201d (8)Larger examination rooms with automatic door openers would facilitate bringing a bed into the room, and examining the patient without having to move the table or the equipment. Two screens were perceived to facilitate the examination when two sonographers worked together. Ergonomic aids such as a sliding sheet, a turntable and patient lift were perceived useful if located close at hand. The noise level was perceived to have improved, as the newer ultrasound units were quieter and silencers had been fitted.\u201cThen there\u2019s the temperature, yes, we have air conditioning in the room, so we can raise and lower the temperature, that\u2019s good.\u201d (18)The air-conditioning system was perceived to be sufficiently adjustable in some workplaces. In others, additional cooling units had been installed to improve the ventilation, especially in smaller rooms.\u201cThe lights in the room have dimmers, so that can be adjusted.\u201d (13)\u201cThe computer screens are better these days, there isn\u2019t so much reflection in them, so I decided to turn the lights on, I can still see just as well.\u201d (7)The participants perceived that the possibility of adjusting the artificial light with a dimmer, and low-glare screens had improved the lighting conditions. These made it possible to increase the level of artificial lighting in the room.Spectacles for computer work were reported to facilitate scanning and computer work.III.Professional working strategies\u201cThe more (sonographers) who know how to do everything, the better it is. It\u2019s also better for that person ergonomically.\u201d (21)Most of the sonographers perceived that achieving good images depended on practice to improve skills and increase experience. Cooperation with the doctor when reviewing the images provided an opportunity to improve knowledge and understanding. The broader the skills of the sonographer, the greater the possibility to alternate between different tasks and kinds of examinations.\u201cI try to help out if I see that someone from sonography is late.\u201d (12)In some workplaces a resource person was available to assist if a colleague was delayed or a patient was difficult to examine.\u201cNo, when I\u2019ve got the image I need, I try to relax my hand and find it again. I\u2019ve been doing this so long it\u2019s no problem.\u201d (7)Being well-accustomed with the equipment was perceived as improving the likelihood of a comfortable scanning posture, especially handling the transducer with less pressure.\u201cThe work afterwards takes longer. I try to do as short examinations as possible to spare my body.\u201d (1)Some employed a strategy that involved shortening the scanning period, i.e. the transducer time, while others believed that taking more images would facilitate the analysis.Depending on local practices, some sonographers analysed the images directly on the unit after scanning, while others did it afterwards at a separate workstation. The latter method was perceived as shortening the time spent at the ultrasound unit, and providing the possibility to change posture.Consultation with a more experienced colleague was recommended if the recommended scanning time was exceeded. During prolonged examinations, e.g. mapping of veins, a team of two examiners was stated to be good practice, which also shortened the total examination time.\u201cI always ask the patient if he or she can stand, and if they can move, and if they can\u2019t, I fetch someone who can help me with the patient, so I don\u2019t try to move them by myself.\u201d (2)It was also stated that preparing for the examination by checking the images from previous examinations was a good strategy. Seriously ill or unstable patients were examined in bed and, if necessary, a colleague or a member of staff from the ward was asked to assist. Scanning facing the patient facilitated observation of the patient\u2019s face.The selection of the sonographers included in this study was based on several inclusion criteria to ensure a variety of perceptions . One of The interview questions were developed by the authors together with two researchers well-versed in the research questions and the aim of the study. The credibility of the results may have been increased by the fact that the first author had performed pre- and pilot interviews, and had a prior understanding of the sonographers\u2019 working situation, which facilitated the development of the interview guide . The secThe results were based on the participants\u2019 unique perceptions, which allowed the researchers to gain a deeper understanding of the sonographers\u2019 working situation. This was a suitable method of obtaining information, i.e. giving the participants a voice . The resThe sonographers were aware of the ergonomic problems in their working environment, but these were usually not prioritized which is in line with previous results . Thus, mEchocardiography was considered physically demanding, regardless of the technique used, as the examination was performed with little variation in working posture and required simultaneous handling of the transducer and the control panel to obtain the required images. Carrying out the examination facing the patient  helped keep the transducer arm less abducted, which is in line with recommendations in previous research also put forward in the industry standards , 31. Beith to the 95th percentiles of the population is recommended in the industry standards [The sonographers\u2019 perceptions of how the equipment could be optimized to make it more individually adjustable should be acknowledged and supported. This is in line with the accommodation to user anthropometrics described by Park et al. . Equipmetandards . Inapprotandards . In thistandards , 21.An articulating support arm system for left-hand scanning was developed and tested in echocardiography to reduce the gripping of the transducer in strenuous and static postures,  which isSome of the sonographers in our study suggested a limitation on transducer time, and image analysis at a separate computer workstation, where individual adjustment, avoidance of noise and better visual conditions are possible. These aspects are prerequisites for good work conditions when working with computers and standard in some of the workplaces , 37. TheSeveral aspects of sonographers\u2019 working situation must be improved in the future. These include ergonomic aids, scheduling and optimization of workplaces used for scanning. Scheduling of varied examinations and tasks, requires both access to different work task and broad skills. It is also important to motivate sonographers to take an active part in the ergonomic aspects of their work. Further research is required on ways in which this can be achieved.The sonographers perceived their work to be stimulating but physically exhausting. They were aware of the ergonomic problems associated with the patient\u2019s poor health. However, the patient\u2019s comfort and obtaining good images were often prioritized to the detriment of working posture. Ergonomic improvements were suggested, such as reducing the manual handling of the transducer, optimizing equipment adjustability and taking the patient\u2019s physique and health into account. As some examinations were perceived more difficult than others, variation in examinations was suggested which, however, also requires broader skills."}
+{"text": "Deinococcus radiodurans. The data reveal two motions, a bend and a twist of the PHY domain with respect to the chromophore-binding domains. Infrared spectroscopy shows the refolding of the PHY tongue. We conclude that a monomer of the phytochrome photosensory core is sufficient to perform the light-induced structural changes. This implies that allosteric cooperation with the other monomer is not needed for structural activation. The dimeric arrangement may instead be intrinsic to the biochemical output domains of bacterial phytochromes.Phytochromes sense red light in plants and various microorganism. Light absorption causes structural changes within the protein, which alter its biochemical activity. Bacterial phytochromes are dimeric proteins, but the functional relevance of this arrangement remains unclear. Here, we use time-resolved X-ray scattering to reveal the solution structural change of a monomeric variant of the photosensory core module of the phytochrome from  I.Phytochromes are photoreceptors in plants and certain microorganism that sense red/far-red light and convert the signal to a biological response. Bacterial phytochromes often function as histidine kinases (HKs) in two-component signaling systems.Phytochromes have a modular architecture with specific photosensory and signaling parts Fig. . The ligZ-to-E isomerization of the C15=C16 double bond in the chromophore.Incident red/far-red light causes a Deinococcus radiodurans phytochrome is associated with the change in the relative orientation of the PHY domains. As a result, the sister PHY domains of the dimeric photosensory module (PAS-GAF-PHY) separate by several nanometers in the Pfr state, compared with the Pr state.We have previously shown how refolding of the PHY tongue in the Phytochromes are usually dimers. It is not understood if both monomers have to be photoactivated for output (de)activation. In bacteria, the function of the HK output domains probably requires interdimer interactions.D. radiodurans have been engineered by mutating residues F145, L311, and L314 in the dimer interface. They were used to create red fluorescent proteins.Monomeric variants of a phytochrome from D. radiodurans (PAS-GAF-PHYmon).Here, we study the light-induced conformational changes of the monomeric variant of the photosensory module from II.A.Escerichia coli strain BL21 DE3 was induced with 1\u2009mM IPTG followed by inoculation at 26\u2009\u00b0C for 18 h. The cells were then lysed with EmulsiFlex\u00ae, and the lysate was cleared by ultracentrifugation. An excess of solubilized biliverdin hydrochloride (Frontier Scientific) was added to the cell lysate, and the samples were incubated overnight on ice to allow holoprotein formation. All steps after biliverdin addition were conducted in dark. The His6-tagged phytochrome samples were first purified with NiNTA affinity purification using HisTrap\u2122 columns , followed by size-exclusion chromatography  in running buffer . The final samples were concentrated to 30\u2009mg/ml (0.53\u2009mM in the case of PAS-GAF-PHYmon) and flash-frozen. The phytochrome solutions were thawed and filtered with a 0.22\u2009\u03bcm centrifugal filter (Amicon) just before X-ray scattering measurements.The expression plasmids for monomeric and dimeric fragments are described elsewhere.B.\u22122, spot size 800 \u00d7 250\u2009\u03bcm, Altechna) and a far-red diode laser . While photoswitching the sample, the X-ray scattering (X-ray energy of 11.2\u2009keV) was recorded simultaneously on two detectors, a Pilatus 2M and a Pilatus 300K-W. The detectors were read out at 25\u2009Hz, with 35\u2009ms for image acquisition and 5\u2009ms for readout. The 2M detector covered the small-angle region (q\u2009=\u20090.05\u2009nm\u22121 to q =\u20096.7\u2009nm\u22121) and the 300\u2009K detector recorded scattering at larger angles (q =\u20095.2\u2009nm\u22121 to q =\u200927.5\u2009nm\u22121). The overlap between the two ranges was used for scaling the two detector readouts. The energy of the red excitation laser pulse was set by controlling the opening time of a mechanical shutter to 5 ms. The turnover was estimated by titrating the laser power and measuring the amplitude of the difference scattering signal of the full-length dimeric phytochrome from D. radiodurans with details described in Ref. Millisecond time-resolved X-ray scattering was recorded at the Coherent Small-Angle X-ray Scattering (cSAXS) beamline at the Swiss Light Source as previously described.\u03bcl/min) through a quartz capillary. A red nanosecond laser  was focused to the capillary together with the overlapping far-red recovery laser  and the X-ray pulses. The X-ray energy was 18\u2009keV, and the X-ray pulse length was 2\u2009\u03bcs. 5-fold dilution of the sample did not affect the signal shape or kinetics .Microsecond time-resolved X-ray scattering measurements were conducted at the beamline ID09b of the European Synchrotron Radiation Facility (ESRF) as described in Ref. \u22121\u2009<\u2009q\u2009<\u200916\u2009nm\u22121. The heating signal was measured and subtracted from the data as before.For both experiments and following standard practice, the 2D readout from the detector was integrated in rings and normalized in the region of 14\u2009nmC.Molecular Dynamics (MD) simulations were performed with GROMACS 4.5.5\u22121 nm\u22121). Afterwards, both structures were solvated in a cubic box with periodic boundary conditions and a side length of 12\u2009nm comprising the protein, with 54\u2009000 (Pr simulation) and 66\u2009000 (Pfr simulation) water molecules and Na+/Cl\u2212 ions corresponding to a salt concentration of 0.1\u2009M. The charge of the protein (-26) was neutralized by adding Na+ ions. After a steepest-descent minimization with the parameters listed above, two 100 ps equilibration MD runs were performed: The first one in the constant particle number, volume, temperature ensemble . In both equilibration runs, a position restraint potential with a force constant of 1000\u2009kJ mol\u22121 was imposed on all protein atoms except hydrogens. For the subsequent MD production runs (588 ns for Pr and 550\u2009ns for Pfr), the same temperature and pressure coupling were used. No position restraints were used, and coordinates were saved every 100 ps.Single chains from the starting Pr and Pfr structures from previous PAS-GAF-PHY simulationsAll bonds to hydrogen atoms were constrained using the linear constrained solver (LINCS)D.sastbxq-range between 0 and 5\u2009nm\u22121. The difference scattering (Pfr-Pr) curves for all combinations of Pr and Pfr structures from the MD simulation were scored against the experimental data by using the sum of squares of the error (SSE) in the q-range between 0.5 and 2.5\u2009nm\u22121. The SSE was determined by minimizing the difference between the experimental and calculated difference scattering against the scaling factor kq is the modulus of the scattering vector, \u0394S is the experimental difference scattering, and Spfr and Spr are the calculated scattering for a given Pfr and Pr structure, respectively.For each MD frame of the Pr and Pfr trajectories, the X-ray scattering was calculated with the software E.\u03bcl was placed on a CaF2 window and sandwiched between two window plates using Glisseal grease (Borer Chemie). The resulting sample thickness was approximately 10\u2009\u03bcm. The extent of hydration was assessed from the absorption ratio between Amide I (at 1645\u2009cm\u22121) and Amide II (at 1580\u2009cm\u22121) bands, which was about 0.9/0.35 in each sample. The FTIR difference spectra were recorded with a FTIR spectrometer (Nicolet) by utilizing red (\u03bb\u2009=\u2009655\u2009nm) and far-red (\u03bb\u2009=\u2009785\u2009nm) light-emitting diodes in a consecutive manner. The spot diameters of red (5 mW) and far-red (40 mW) diodes were approximately 1\u2009cm2. The samples were illuminated until the photo equilibrium was reached, which took typically several seconds. The baseline was recorded during each switch before changing the illumination conditions. About 20 scans of each state were measured with 2\u2009cm\u22121 spectral resolution. All measurements were done at room temperature.The samples were concentrated to an approximate concentration of 2.5\u2009mM with Amicon Ultra Centrifugal Filters (Merck-Millipore). A sample volume of 2\u2009III.mon after illumination with short red-light pulses. The data were referenced to resting state patterns, yielding time-dependent difference X-ray scattering  measurements of pre-illuminated Pr and Pfr samples  in supplementary material).q are resolved .The peak at ved Fig. . Comparet\u2009=\u20093\u2009\u03bcs and t\u2009>\u200920\u2009ms. The kinetics of the components are illustrated in Figures supplementary material).The time-evolution of the data is described by two components, represented by the difference scattering at q value at which a difference peak occurs gives information about the length scale of the structural changes. The positive peak at q\u2009=\u20090.8\u2009nm\u22121 in PAS-GAF-PHYmon indicates a change in a tertiary structure . In order to structurally interpret this difference X-ray scattering, we first created 5888 and 5506 candidate structures by running molecular dynamics simulations in Pr and Pfr, respectively. The simulations started from the Pr and Pfr solution structures of the dimeric PAS-GAF-PHY with one monomer removed.t\u2009>\u200920\u2009ms in Eq. q range of 0.5\u20132.5\u2009nm\u22121. This scattering region gives information about the large-scale movements of the protein. The small structural features indicated by the higher scattering angles (q\u2009>\u20092.5\u2009nm\u22121), however, were not sampled by the simulations.The Figure In order to quantify these observations, we chose two representative angles Fig. . The benq\u2009=\u20090. Third, Fig. S1(a) shows that the reported difference scattering signal agrees with the difference of a SAXS measurement recorded for PAS-GAF-PHYmon in Pr and Pfr.q =\u20090, or I(0), is essentially the same in Pr and Pfr, which is a clear indication that the oligomeric state of the protein does not change upon illumination.We note that light-dependent oligomerization is unlikely to be the reason for the observed difference scattering signal. First, analytical gel filtration analyses indicated that the samples are purely monomeric.mon, PAS-GAFmon, and their dimeric counterparts  and amide II (1530\u20131570\u2009cm\u22121) spectral regions  cm\u22121 stem from the chromophore\u22121 is present in PAS-GAF-PHY and PAS-GAF-PHYmon, but not in PAS-GAF fragment. We therefore conclude that the signal stems from the PHY domain. Amide I signals characteristic for changes in the content of \u03b2-sheets and \u03b1-helices were found at 1637(\u2212) cm\u22121 and 1654(+) cm\u22121, respectively  cm\u22121 and 1644(\u2212) cm\u22121 partially mask the 1637(\u2212) cm\u22121 signal in PAS-GAF-PHY  cmHY Figs. . The peaIV.D. radiodurans (PAS-GAF-PHY) was previously found to undergo a large structural rearrangement, which increases the PHY-PHY distance.mon). Compared to the previous study, the new data reveal more structural details. The dominant structural changes are a bend and a twist of the PHY domains with respect to the PAS-GAF domains (Fig. The dimeric photosensory core fragment of the phytochrome from ins Fig. . SimilarRhodopseudomonas palustris,Agrobacterium tumefaciens,Synechocystis PCC 6803.It is intuitive that the bend and twist movements are a consequence of the same structural rearrangement that is caused by the shortening of PHY tongue in the Pr-to-Pfr transition.The FTIR data further demonstrate that the structural and chemical changes of the PAS-GAF domains are very similar between monomeric and dimeric counterparts. Only small differences are observed in the Amide I and Amide II regions. It is interesting to note that the monomeric PAS-GAF gives stronger difference spectra in the visible spectral region than the dimer.Combining these findings implies that one monomeric photosensory core can be activated structurally even when separated from its dimerization partner. The monomer of the photosensory core is therefore the smallest unit to initiate the structural machinery of the entire phytochrome. It is currently unclear how the two monomer units in a phytochrome dimer work together to activate the output domains. Our data show that the build-up of the final state is slower by approximately an order of magnitude in the monomer mutant than in the dimer. Thus, the dimerization partners have an influence on the kinetics of the light-induced structural turnover. Highly similar dimerization interfaces of the Pr- and Pfr-state crystal structuresDrBphP, RpBphP2, PaBphP, AtPhyB crystallize as parallel dimers,RpBphP1, RpBphP3 crystallize as antiparallel dimers.This conclusion also implies that the photosensory core of bacterial phytochrome likely does not require any specific quaternary arrangement for its activation. Different dimer arrangements have been found among phytochromes. For example, fragments of The photosensory module of phytochromes appears to be structurally highly conserved. Conventional phytochromes have a PAS-GAF-PHY domain arrangement with a long scaffolding helix and a PHY tongue extension. Therefore, we propose that the structural changes identified here are likely widely conserved in plants, fungi, and bacteria.supplementary material for supplementary Figures 1 and 2.See"}
+{"text": "Glioblastoma (GBM) has a high rate of local recurrence despite chemoradiotherapy (CRT). Genome-wide expression profiling was performed on patient tumors before and after chemoradiotherapy to identify genes and gene pathways associated with recurrence.Median time to recurrence was 8.9 months with median time to second surgery of 9.6 months. The microRNA (miRNA) analysis identified 9 oncologic and immune-related miRNAs to be differentially expressed, including the hypoxia-related miR-210 and the immune-modulatory miR-146b. More than 1200 differentially-expressed genes were identified with RNA-sequencing (RNA-seq). Gene set enrichment analysis (GSEA) identified p53 signaling, Notch, Wnt, VEGF, and MEK gene sets enriched in recurrent GBM. Consistent with the miRNA profiling data, the miR-146b target gene set from GSEA analysis was also associated with recurrence.Fourteen patients with GBM recurrence after CRT who had available tumor tissue from the initial diagnosis as well as recurrence were selected. Total RNA was isolated from formalin-fixed paraffin-embedded (FFPE) tumor specimens. Genome-wide expression profiling using RT-PCR for miRNA analysis and RNA-seq for messenger RNA (mRNA) analysis were conducted to identify differentially-expressed genes. GSEA was performed on the differential expression data.Genome-wide expression profiling identifies multiple oncologic and immune-related gene sets associated with GBM recurrence. In particular, immune-related miR-146b is upregulated in recurrence and deserves further investigation. Glioblastoma  is the most common malignant primary brain tumor in adults and has an annual incidence of 3.2 in 100,000 in the United States . Tumor rMicroRNAs (miRNAs) are a family of small non-coding RNA molecules (\u223c22 nucleotides) that regulate expression of thousands of protein-coding genes . With thGiven the importance of miRNAs, we hypothesized that miRNA expression changes in GBM after CRT may allow an improved understanding of pathways responsible for treatment resistance. We also employed a genome-wide expression profiling approach of 14 GBM tumors before CRT and their corresponding recurrent tumor after CRT to identify oncologic and immune-related changes within the recurrent tumors after CRT.6-methylguanine-DNA-methyltransferase (MGMT) methylation and isocitrate dehydrogenase (IDH) status were largely not assessed in this patient cohort.Fourteen patients were eligible for the analysis . There wp = 0.003), which has established roles in cell survival, particularly in hypoxic conditions [p = 0.02). Table http://mirdb.org) [Notch2 [TGFBR1 [STAT3 [FGFRL1 [NOVA1 [miRNA profiling was performed for the initial and recurrent tumors for all 14 patients. A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) based miRNA profiling experiment revealed 9 miRNAs differentially expressed with statistical significance in recurrent GBM  , which c [Notch2 , TGFBR1  [TGFBR1 , STAT3 [1 [STAT3 , FGFRL1  [FGFRL1 , and NOV1 [NOVA1 .p < 0.05) (TF), ferritin (FTL), fibroblast growth factor 1 (FGF1), TNF receptor-associated factor 4 (TRAF4), and signal transducer and activator of transcription 1 (STAT1).Given the multiple oncologic and immune-related miRNAs differentially expressed in recurrent GBM, a large-scale genome-wide expression profiling study using RNA-sequencing (RNA-seq) was employed to identify genes and gene pathways associated with recurrence. Due to limited RNA quality from two patients as well as the initial tumor of a third patient, RNA-seq was employed on 11 initial and 12 recurrent RNA samples. More than 1200 genes were identified that were differentially expressed with statistical significance ( < 0.05) . SelecteGiven the significant number of genes differentially expressed, gene set enrichment analysis (GSEA) was employed to identify pathways associated with recurrence. Using this statistical tool, recurrent GBM was noted to be enriched in gene sets associated with the MEK, KRAS, Notch, VEGF, and Wnt pathways, among others . Additiohttp://mirdb.org). We previously demonstrated increased expression levels of the immune-modulatory miRNA, miR-146b, in tumor tissue isolated from recurrent GBM Table . Multiplp = 0.01) and a trend toward improved freedom from local recurrence (FFLR) . However, the local control did not translate to significant difference on OS  as well as time to salvage surgery , but not with time to death .The median fold change of miR-146b between recurrent and initial tumors was 1.5 (range: 0.6\u20135.4). Low miR-146b change (ratio < 1.5) was associated with significantly improved freedom from salvage surgery ( FFSS)  where they identified 43 miRNAs with at least two-fold changes in expression levels in both of the recurrent tumors (miR-146b is not among them) [et al. [et al. [et al. [et al. [et al. [in vitro culture system which inherently lacks the complexity of GBM recurrence in vivo. Our hypothesis presented in this manuscript is that miR-146b upregulation in recurrence plays a role in GBM immune-suppression potentially at a later stage of the tumor progression event, ultimately resulting in tumor recurrence. This manuscript challenges the notion of miR-146b functioning as solely a tumor suppressor. Based on our investigation of human GBM recurrent tumor tissue in relation to the paired initial tumor, the role of miR-146b in tumorigenesis, tumor progression, and tumor maintenance is likely more complex than has been previously reported in the literature.This pilot study compared miRNA and mRNA expression profiles of 14 pairs of GBM tumors before CRT and after recurrence. It identified several candidate oncologic and immune-related miRNAs and gene sets differentially expressed in recurrent GBM. In particular, the immune-modulatory miRNA miR-146b was significantly increased at recurrence and may be a promising target for future studies. Due to the difficulty after repeated biopsy of intracranial tumors, detailed expression profiling studies of miRNA and transcriptome (or RNA) of GBM tumors before and after CRT have been limited in the literature. Park ng them) . However [et al.  analyzed [et al. , 41. The [et al. , Katakow [et al.  and Yang [et al. , who allA vast number of potential targetable pathways in GBM have been identified in preclinical studies, but few have shown efficacy in the clinical setting. Complicating clinical efficacy is the heterogeneity of these tumors stemming from the complexity of their molecular makeup , 46. Thiet al. implicated miR-146b in dendritic cell survival and cytokine production via targeting of TRAF6 and IRAK1 [et al. also recently showed that miRNA-146b are highly expressed in regulatory T cells (Tregs) and play a vital role in their expansion, survival, and suppressor function [This study implicates the immune-modulatory miRNA miR-146b as an important factor to drive GBM recurrence after chemoradiotherapy. Multiple studies have established an immune-modulatory role for miR-146b, including suppression of IL-6 allowing p16-dependent tumorigenesis in breast cancer , as wellnd IRAK1 . Lu et afunction . Since Tfunction , our finfunction . SNX22 hfunction . As immufunction \u201355.in vitro and in vivo will be needed to further confirm the role of miR-146b in promoting GBM recurrence.Since this study is a small pilot study of genome-expression profiling, its findings need to be interpreted with the following limitations. As this study is based on a limited sample size and a highly selected population of GBM patients who underwent salvage surgery for recurrence after CRT, the finding should be considered hypothesis-generating. The small sample size limits the use of stringent FDR-controlling method. External validation using a larger sample size, ideally using a multi-institutional tumor bank, will be necessary to confirm our finding. Recent data have also shown significant regional and cellular heterogeneity within GBM , 57. TheIn conclusion, this study presents multiple deregulated miRNAs and gene pathways after CRT and implicates the immune-modulatory miR-146b as a potential factor in GBM recurrence. Additional investigations are warranted to validate the association of miR-146b with GBM recurrence and to understand its mechanisms.2 after maximal safe resection. These patients were also required to have sufficient pathologic tissue from the initial surgery before CRT and the salvage surgery at the time of recurrence after CRT. Additional salvage chemotherapies were allowed prior to the salvage surgery. This study was reviewed and granted approval by the IRB.Eligible patients with pathologically diagnosed GBM were identified from our institutional review board (IRB)\u2013approved database. They were required to have received standard fractionated RT and concurrent daily TMZ at 75 mg/mFormalin-fixed paraffin-embedded (FFPE) tumor blocks from the initial and salvage surgeries were collected for pathological analysis. FFPE specimens were stained with hematoxylin and eosin (H&E), reviewed by a board-certified neuro-pathologist (CC) to confirm diagnosis and to demarcate tumor regions. This was followed by macrodissection of 10-micron unstained sections in order to remove non-tumor tissues. Total RNA was then extracted using the miRNeasy FFPE Kit (Qiagen) according to the manufacturer\u2019s protocol.miRNA expression profiling of 96 miRNAs implicated in tumorigenesis was performed using our laboratory\u2019s previously described method, which is based on RT-PCR, the details of which have been described previously . The RT RNA-seq was performed to allow identification and quantification of expression of genes in a given tumor sample by a single massively parallel sequencing run . Detailsn = 12) and the initial tumor (n = 11). Utilizing GSEA software available from the Broad Institute [p-value < 0.05 and FDR  < 0.25. ES (enrichment score) reflects the degree to which a gene set is over-represented in our dataset of differentially expressed genes. NES  is the ES normalized for gene set size.Differential gene expression profiles were generated using RNA-seq from recurrent GBM  for differential expression was calculated using the Student\u2019s T-test. Median value of the fold change in miR-146b expression in recurrent vs initial tumors was used as cutoff to identify high fold change (mir-146b ratio \u2265 1.5) vs low fold change (mir-146b ratio < 1.5). Freedom from local recurrence (FFLR), freedom from salvage surgery (FFSS), and overall survival (OS) were evaluated using the Kaplan-Meier method and compared using the log-rank test. Cox regression analysis was utilized to evaluate the association of miR-146b fold change with clinical outcomes. Statistical analyses were performed with the Statistical Package for Social Sciences, version 22 . Significance was defined as a p value \u2264 0.05. All statistical tests were two sided.All time points were determined from the date of initial surgery. Statistical significance ("}
+{"text": "Various classification systems have been proposed to describe furcation lesions and Glickman\u2019s classification for many years seems to have been the most widely utilized in the sole clinical diagnosis with no reference to the prognostic value of the lesion itself. This article reviews the previous classification systems and proposes a new method to classify furcation lesions based on the position of the gingival margin and its relationship with the furcation area  providing significant aid for a better understanding of furcation involvements and increases the prognostic value of treatments in the long term. According to the glossary of terms of the American Academy of Periodontology, a furcation involvement exists when periodontal disease has caused resorption of bone into the bi- or trifurcation area of a multi-rooted tooth .Currently, the proposed classifications are based on the extension of the defect and the degree of horizontal/vertical attachment loss. Glickman in 1953 , developMoreover, other classifications have been proposed in an attempt to describe the anatomy of the furcation more completely, describing the number of remaining bony walls , the morMost of the classifications of furcation lesions are unable to convey all the relevant information related to the marginal tissue position and its relationship with the furcation involvement . This information could be important for diagnosis, prognosis and treatment planning as well as for the communication between clinicians and researchers. Furthermore, with a broad variety of cases with different clinical presentations, it is not always easy to classify all furcation defects according to classification systems in use to date.The aim of the present review is to summarize the previous classifications that have been proposed in the past years and introduce a new system that provides information on a parameter that has not been taken into account; the gingival marginal tissue position and its relationship with the clinical exposure of the furcation involvement. The goal is to promote this new classification as a promising system for a better understanding of furcation involvements and to predict the prognosis by selecting the correct treatment for each case.Several systems have been proposed based either on the extent of horizontal probing depth into the furcation defect or on the vertical extent of the loss of alveolar bone within the defect .All the classifications proposed to date are showed in One of the first proposed classifications was the one by Glickman . This clGrade I involvement: it is the incipient or early lesion. The pocket is supra-bony, involving the soft tissue; there is slight bone loss in the furcation area. Radiographic change is not usual, as bone changes are minimal.Grade II involvement: the bone is destroyed on one or more aspects of the furcation, but a portion of the alveolar bone and periodontal ligament remain intact, thus allowing only partial penetration of the probe into the furcation area. The radiograph may or may not reveal the grade II furcation involvement.Grade III involvement: the inter-radicular bone is completely absent, but the facial and/or lingual orifices of the furcation are occluded by gingival tissue. Therefore, the furcation opening cannot be seen clinically, but it is essentially a through and through tunnel. If the radiograph of the mandibular molars is taken with a proper angle and the roots are divergent, these lesions will appear on the radiograph as a radiolucent area between the roots. The maxillary molars present a diagnostic difficulty owing to roots overlapping each other.Grade IV involvement: the inter-radicular bone underneath the roof of furcation is completely destroyed. The gingival tissue is also receded apically so that the furcation opening is clinically visible. The radiographic image is essentially the same as in grade III lesions.Degree I: horizontal attachment loss < 3 mm of the total width of the furcation area.Degree II: horizontal attachment loss > 3 mm but not encompassing the total width of the furcation area.Degree III: \u201cthrough and through\u201d destruction of the periodontal tissue in the furcation area.In 1975 Hamp, Nyman and Lindhe  proposedRamfjord and Ash  proposedSubclass A: vertical bone loss 3 mm or less.Subclass B: vertical bone loss from 4 to 6 mm.Subclass C: bone loss from the fornix of 7 mm or more.Later on, a sub-classification referring to the vertical bone loss from the furcation fornix  was intrThe reference point for the classification is the horizontal attachment loss. However, the subjectivity in the term \u201cearly or incipient lesion\u201d (grade I) and the absence of the precise numerical values to identify the horizontal attachment loss create difficulties in the classification between grade I and II.In Glickman\u2019s grade III and IV furcation lesion the inter-radicular bone is completely absent, with the difference that in grade IV the furcation entrance is exposed. In this case, grade III and IV would represent a single group considering that the reference point of this classification is the horizontal attachment loss and it is the same for both groups. The difference between them could be represented by creating two subgroups.In grade I and II furcation lesions the relationship with gingival margin  is not considered. In such cases, a furcation lesion with incipient bone loss (Glickman\u2019s grade I) but clinically exposed for the presence of gingival recession cannot be classified as grade I but neither as grade IV. The same problem arises for Glickman\u2019s grade II.Although Glickman\u2019s classification  has beenThe classification systems quantifying the horizontal attachment loss give rise to the same problem: in none of them one can clearly differentiate between grade I and II since both use the same reference point (less or greater than one third\u2014less or greater than two or three millimeters) ,11,19, wWhen the furcation lesion is partially or not clinically exposed it is difficult to measure the vertical bone loss ,7,15,16.The classification systems that quantify the horizontal/vertical attachment loss ,4,11,19 Considering the above limitations and the fact that none of the proposed classification systems to date consider the clinical exposition of the furcation area as a reference point, a more detailed and informative new classification system is proposed. This classification is based on criteria derived from previously mentioned systems ,3,11,19 Furcation lesions are divided into two main groups according to the following criteria .Non-exposed (NE): Clinically non-exposed furcation lesion .Exposed (E): Clinically exposed furcation lesion .-Class I: incipient lesion. There is slightly horizontal attachment loss in the furcation area. The examiner probe penetrates two millimeters or less from the entrance of furcation .-Class II: partial horizontal bone loss. The examiner probe penetrates three millimeters or more from the entrance of furcation, but there is not a total attachment loss with a through and through opening of the furcation .-Class III: total horizontal attachment loss with a through and through opening of the furcation. The inter-radicular bone is completely absent .Considering the main characteristics of the furcation involvement (according to the aforementioned classification systems) ,3,8,9 eaIf a molar presents marginal tissue recession but the furcation is not clinically exposed, it will be considered as non-exposed group (NE) because the criteria for this classification system is the exposure of the lesion more than the presence of gingival recession. For example, if a molar presents a long root trunk, the furcation lesion may not be visible even in the presence of gingival recession.In order to avoid mistakes in the diagnosis of class II and III lesions\u2014mainly when they are NE\u2014it is important to use two periodontal probes, each of which must be introduced from buccal and lingual (lower molars) and buccal/mesio-palatine\u2013disto-buccal (upper molars); and if they get in contact the diagnosis of a Class III furcation involvement can be confirmed, since many times the anatomical characteristics of the furcation area does not allow to go from \u201cside to side\u201d and this could generate an incorrect diagnosis.The furcation is an area of complex anatomic morphology that may be difficult or impossible to debride by means of routine periodontal instrumentation ,23,24. TTherefore, diagnosis and its correct interpretation are essential when establishing an adequate treatment .The aim of this new classification system is to help clinicians to classify those cases, which cannot be categorized into a particular class with any of the current classification. The limitations of the present classification systems can lead to inadequate diagnosis, prognosis and, therefore, treatment planning.As discussed in a previous article ,27, a coZappa et al. , compareConsidering previous studies ,27,28 anLongitudinal prospective ,30 and rMoreover, according to the new classification of periodontal diseases, once both staging and grading of the periodontitis are established based on evidence of progression, the prognosis can be modified based on the presence of risk factors such as diabetes and smoking. Therefore, these factors could also modify the prognosis of furcated teeth .On the other hand, previous studies comparing scaling and root planing in furcation area with and without surgical access ,23, haveIn fact, a recent retrospective study  concludeTaking into account all of the above mentioned aspects, the prognosis of teeth with furcation lesions and, especially, the choice of an appropriate treatment would depend on the characteristics of the lesion, whether it is clinically exposed or not exposed, since according to our understanding and based on previously evaluated literature, in the case of a Grade II\u2013III non-exposed furcation lesion the surgical treatment could be the right approach for a better long term survival rate.It is important to consider that the present new system measures the horizontal attachment loss alone. Considering previous articles and, to the base of our knowledge and experience, vertical attachment loss in furcation lesion is more difficult to be clinically determined. On the other hand, the morphology of inter-radicular osseous defects is further complicated by the fact that supra-bony and/or infra-bony defects of different morphology may also be associated with furcation involvements . A furthNo classification system can be complete but with its continual use both advantages and disadvantages of each system can be evidenced. This classification attempts to refine the existing drawbacks of the current classifications so that the new system can be applied to a wider variety of cases to provide more accurate characterization of the lesions. This would be of significant aid in communication between clinicians and researchers providing a better understanding of furcation involvements and could be important to predict the prognosis and select correct treatment for each case."}
+{"text": "Self-reactive B cells generated through V(D)J recombination in the bone marrow or through accrual of random mutations in secondary lymphoid tissues are mostly purged or edited to prevent autoimmunity. Yet, 10\u201320% of all mature na\u00efve B cells in healthy individuals have self-reactive B cell receptors (BCRs). In patients with serologically active systemic lupus erythematosus (SLE) the percentage increases up to 50%, with significant self-DNA reactivity that correlates with disease severity. Endogenous or self-DNA has emerged as a potent antigen in several autoimmune disorders, particularly in SLE. However, the mechanism(s) regulating or preventing anti-DNA antibody production remain elusive. It is likely that in healthy subjects, DNA-reactive B cells avoid activation due to the unavailability of endogenous DNA, which is efficiently degraded through efferocytosis and various DNA-processing proteins. Genetic defects, physiological, and/or pathological conditions can override these protective checkpoints, leading to autoimmunity. Plausibly, increased availability of immunogenic self-DNA may be the key initiating event in the loss of tolerance of otherwise quiescent DNA-reactive B cells. Indeed, mutations impairing apoptotic cell clearance pathways and nucleic acid metabolism-associated genes like DNases, RNases, and their sensors are known to cause autoimmune disorders including SLE. Here we review the literature supporting the idea that increased availability of DNA as an immunogen or adjuvant, or both, may cause the production of pathogenic anti-DNA antibodies and subsequent manifestations of clinical disease such as SLE. We discuss the main cellular players involved in anti-DNA responses; the physical forms and sources of immunogenic DNA in autoimmunity; the DNA-protein complexes that render DNA immunogenic; the regulation of DNA availability by intracellular and extracellular DNases and the autoimmune pathologies associated with their dysfunction; the cytosolic and endosomal sensors of immunogenic DNA; and the cytokines such as interferons that drive auto-inflammatory and autoimmune pathways leading to clinical disease. We propose that prevention of DNA availability by aiding extracellular DNase activity could be a viable therapeutic modality in controlling SLE. Anti-DNA antibodies (Abs) are not exclusive to systemic lupus erythematosus (SLE or lupus), yet, their persistence in serum is the most reliable serological marker for lupus diagnosis \u20134. High Rheumatic diseases like SLE, RA, Sjogren's syndrome, vasculitis, antiphospholipid syndrome etc., which cause development of anti-DNA Abs in several patients, are driven by B cells , 14. MorIn accordance with the predominantly short-lived nature of DNA-reactive B cells, B cell targeting therapies like Rituximab (anti-CD20) and Belimumab (anti-BAFF) have been partially effective in SLE treatment , 14, 26.+ T cells expressing high CXCR5, ICOS, and PD-1, named follicular helper T cells (Tfh) are particularly implicated in several autoimmune diseases. Tfh promote the generation of germinal center-driven anti-DNA Abs in several lupus mouse models by providing key cytokines like IL-21 and IL-4 to B cells in the germinal centers  with anti-RNP Abs, ox-mtDNA is present, and so are antibodies to it (The stimulatory NET components are a composite of neutrophil genomic DNA (gDNA), mitochondrial DNA (mtDNA) and neutrophil granular proteins, which are interferonogenic , 83, 84.es to it . Increases to it , 84 and Apart from self-DNA and ox-mtDNA, the DNA-associated neutrophil microbial peptides LL37 and human neutrophil proteins (HNPs), human beta-defensin 2 and 3 are strong potentiators of IFN responses. LL-37 cause aggregation of DNA fragments, making them resistant to nucleases and facilitating their endocytosis in pDCs via autoantibody-Fc receptor-mediated uptake and IFN production , 77, 96.In summary, autoimmune responses to NETs studied so far provide evidence for NET-DNA (gDNA/ mtDNA) as a TLR9 ligand and as an adjuvant promoting IFN production and polyclonal proliferation of B cells, including DNA reactive B cells in SLE, RA , psoriaslpr mice caused increased oxidative stress and impaired acidification of lysosomes. This promoted prolonged accumulation of internalized nucleic acids in endolysosomes and leakage into the cytosol, activating TLRs, and cytosolic sensors   . Moreoven = 44) . Overallrd of the SLE patients with sporadic SLE, have DNase-sensitive chromatin on the surface of their MPs  sensors do not discriminate between foreign and self-NAs, hence the processing or metabolism of endogenous NAs is of paramount importance to prevent immune stimulation. Therefore, it is not surprising that ~40% of the genes involved in monogenic or Mendelian-inherited forms of autoimmunity are nucleases. Nucleases can be broadly classified into two main categories depending on their spatial expression: (1) Intracellular nucleases\u2014cleave NAs inside the cells, during apoptosis or after uptake of apoptotic bodies. (2) Extracellular nucleases\u2014cleave NAs exposed extracellularly during apoptosis or generated outside of the cells. The tissue expression profile, structure, enzymatic activity, and functions of the two main classes of DNases in various pathological conditions were recently reviewed , 119.ADAR1, and SAMHD1 cause abnormal induction of type-I IFNs and lead to Aicardi-Gouti\u00e8res syndrome (AGS) and related interferonopathies. In addition to these RNases, an autosomal recessive mutation in the cytoplasmic\u2013ER membrane-resident 3\u2032-DNA repair exonuclease1 (TREX1 or DNASEIII) also causes AGS and SLE  was performed, using an autoantibody array to assess their antigen-specificity. The study revealed their specificity to nuclear antigens like gp210, PCNA, Ro/SSA, Sm/RNP, SS-A/SS-B etc. Even though AGS and SLE share several overlapping disease manifestations, ss/dsDNA specific antibodies were not detected in any of the AGS patient sera in this study  with putative phospholipase activity were shown to have a functional 5\u2032 exonuclease activity preferentially on unstructured ssDNA. PLD3 or PLD4-deficient mice displayed a TLR9-stimulated inflammatory syndrome while PLD3/4 double-deficient mice were unable to survive beyond the age of 21 days due to severe liver inflammation. Interestingly, the observed autoinflammatory syndrome was mediated by IFN\u03b3 instead of IFN\u03b1. Although there was excessive TLR9 activity causing IFN\u03b3 production, no autoantibody responses were reported . PolymorDNase1\u2212/\u2212 mouse model generated on a mixed B6/129 background, in which some mice developed anti-DNA and anti-nucleosome-Abs (predominant), as well as glomerulonephritis in a gender-independent manner. However, in subsequent studies it was shown that the B6/129 mixed background itself caused most of the observed SLE phenotype  caused pediatric-onset familial SLE  Extracellular availability of immunogenic DNA as a direct antigen drives anti-DNA reactive B cell responses in SLE, making a strong case for the regulatory role of extracellular DNases in anti-DNA antibody production.Altogether, as depicted in It is now well-established that microbial NA sensors also recognize self-NAs under autoimmune conditions , 156. FoMice deficient in RNaseH2 complex proteins, SAMHD1 and ADAR1 are either embryonically lethal or do not recapitulate human AGS. Yet, they revealed that RNaseH2 or SAMHD1-dependent NA-accumulation led to the stimulation of the cGAS-STING pathway, while loss of ADAR1 (deaminase) stimulated the RNA sensing MDA5-MAVS pathway . CytosolDNASE2 mutations. The absence of DNase2 in mice causes accumulation of apoptotic cell derived DNA in the lysosomes of liver and bone marrow macrophages causing lethal anemia and cell death. DNase2KO mice can be rescued by the deletion of IFNAR. However, \u201cIFNAR-deficient DNase2KO mice develop chronic polyarthritis, splenomegaly, and ANA. The accumulated DNA stimulates the cGAS-STING-IRF3/7 pathway leading to massive type-I interferon production, because the deficiency of either cGAS  cause excessive type-I IFN production leading to severe disease pathologies (The three main pathways of type-I interferon induction include sensing by (1) cGAS-STING, (2) RIG-I/MDA5-MAVS, and (3) TLR-MyD88/TRIF. Their involvement in the pathogenesis of several rheumatic diseases and the current therapeutic interventions targeting the type-I interferon pathway have been extensively discussed in excellent reviews elsewhere , 193. Tyhologies , 196. HoSle1,2,3, reviewed in Liu et al.  has been implicated in both human and mouse lupus . Accumulstations , 208. Instations . Alternang cells . Moreoveng cells , where ang cells . Indeed,ive IFN\u03b3 . AdditioFN\u03b3 mRNA . Conside limited .Overall, the role of interferons in anti-DNA Ab production is well-established. However, due to the significant overlap between the genes induced by IFN\u03b1 and IFN\u03b3 , 214, itEmerging genetic and functional evidence suggests that the efficient degradation of extracellular DNA is an important checkpoint in preventing the stimulation of DNA-reactive B cells. As depicted in All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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": "Furthermore, HP reduced the surface roughness of AgNWs, which solved the inter-electrode short circuiting problem for devices. We studied the performance of poly-poly(styrene sulfonate) and zinc oxide-based PSC devices. The power conversion efficiency of HP-AgNW-derived polydithiophene-2,6-diyl-alt-(4-(2-ethylhexyl)-3-fluorothienothiophene-)-2-carboxylate-2-6-diyl] (PTB7-Th):-phenyl-C71-butyric acid methyl ester (PC71BM) devices was 7.83%, which was slightly lower than the performance of the device using ITO (8.03%) as a substrate. After a bend test (100 times) at a 2-cm curvature radius, the efficiency of AgNW/PET-derived PSCs was more than 70%. The performance of PSCs made with AgNWs and ITO electrodes is comparable, but the cost of using AgNWs for electrodes is much lower; therefore, HP-derived AgNWs demonstrate great potential for optoelectronic applications.In this study, chemical treatment  and physical treatment  were applied to improve the performance of silver nanowire (AgNW)-derived electrodes on a glass or flexible polyethylene terephthalate (PET) substrate. The four-point probe method, UV-Vis spectroscopy and scanning electron microscopy (SEM) were used to measure the properties of AgNW electrodes and compare them with those of indium tin oxide (ITO) electrodes for exploring the possibility of using CT- and HP-based AgNW electrodes for polymer solar cell (PSC) applications. Using the CT or HP method, the sheet resistance of electrodes decreased to lower than 40 \u03a9 sq However, this brittle characteristic limits the application of ITO on polyethylene terephthalate (PET) or polyethylene-naphthalate substrates. Other TCOs, such as SnO2 fluoride and zinc oxide (ZnO)-mixed aluminum, have similar issues when used on plastic substrates   and  , polydithiophene-2,6-diyl-alt-(4-(2-ethylhexyl)-3-fluorothienothiophene-)-2-carboxylate-2-6-diyl] , [-phenylC3), ethanol, nitric acid (HNO3) and ascorbic acid to form an acidic alcohol-based solution with a pH value of 3 [2 with various temperatures and time periods. The AgNW nodes were melted under the high temperature and high pressure, which caused the welding of AgNWs and the formation of a stable AgNW network electrode . AgNWs were welded using silver nitrate (AgNOlue of 3 . To formlectrode b.3/active layers/ZnO/AgNW/substrates were fabricated. ZnO (self-synthesized) was used as an electron transport layer, which was formed on the AgNW film through spin coating at 2000 rpm for 30 s, followed by annealing at 160 \u00b0C for 20 min. PEDOT:PSS was used as the hole transport layer, which was formed on the AgNW films through spin coating at 2000 rpm for 30 s, followed by annealing at 120 \u00b0C for 20 min. Various blend films were used as active layers for devices, including P3HT:PC61BM, PTB7-Th:PC71BM and PffBT4T-20D:PC71BM, which were all spin-coated at 450 rpm/30 s, followed by a second stage of spin coating at 1200 rpm/2 s for P3HT:PC61BM, 2000 rpm/30 s for PTB7-Th:PC71BM and 800 rpm/30 s for PffBT4T-20D:PC71BM. PffBT4T-20D: PC71BM received a third stage of spin coating at 1200 rpm/2 s. The annealing was performed at 140 \u00b0C for 20 min. The metal electrodes, Ca 30 nm/Ag 100 nm for normal PSCs and MoO3 3 nm/Ag 100 nm for inverted PSCs, were prepared through vapor deposition (Both PSCs with the structure Ag/Ca/active layers/PEDOT:PSS/AgNW/substrates and the inverted structure Ag/MoOposition c.Rs) of 100\u2013180 \u03a9 sq\u22121 and an average transmittance of 85% . Because the values of the Rs and transmittance were trade-offs, we evaluated the effects of the CT and HP treatments on the optoelectronic properties of electrodes and compared them with the normal AgNW electrode. First, we modified the normal AgNW electrode by using CT and used ascorbic acid as a reducing agent to reduce Ag ions into Ag atoms; the nitrate ions were introduced into the solution to prevent the rapid precipitation of Ag ions. The optimized CT process allowed Ag ions mainly to precipitate between the AgNW nodes and form well-defined AgNW network films. We scanned the concentration of AgNO3 and evaluated its effect on the Rs and transparency of the AgNW electrodes. As shown in Rs values of the AgNW films decreased significantly with an increase in the AgNO3 concentration. Using an optimized AgNO3 concentration of 8 \u00d7 10\u22124 M, we fabricated the CT-based AgNW electrode with an Rs lower than 60 \u03a9 sq\u22121, which was 40\u201350% lower than that of the normal AgNW electrode. We further compared the optical transmittance of the ITO and AgNW electrodes before and after CT. We found no significant difference in optical transmittance. The transmittance in the visible region was higher than 80%  for 100 \u00b0C, 120 \u00b0C, 140 \u00b0C, 160 \u00b0C, 180 \u00b0C and 200 \u00b0C (with 5-min treatment) samples by 32%, 32%, 38%, 50%, 39% and 47%, respectively. The decrease in the Rs value for the electrodes treated for 30 min was 77%, 63%, 57%, 59%, 51% and 60% under the temperatures of 100 \u00b0C, 120 \u00b0C, 140 \u00b0C, 160 \u00b0C, 180 \u00b0C and 200 \u00b0C, respectively. The results suggest that the key factor in determining the Rs value of the HP process is the time period. When the HP time period was long enough, AgNWs became well welded, and thus, the contact resistance and Rs decreased under the appropriate HP energy and pressure. Fabricating an AgNW-based transparent electrode that simultaneously has high conductivity and transparency, as well as low surface roughness was challenging. Surface roughness and poor adhesion to the substrate resulted in unsatisfactory stability of derived optoelectronic devices. Here, AgNWs were treated separately using CT(oxidation\u2013reduction method) and HP (hot-pressing method) to overcome these disadvantages. We fabricated the normal AgNW electrode from the IPA-diluted solution. Through optimizing the concentration and the spin-coating rates of the AgNW solution, we obtained a sheet resistance  for normal and CT-AgNW electrodes. Because of the high surface roughness of these AgNW electrodes, fabricating optoelectronic devices with high reproducibility is challenging , which was close to the Rs value (10 \u03a9/sq) of the ITO glass. Compared with CT-AgNWs, the HP-AgNWs treatment showed a stronger welding effect, and the derived AgNW network was rather flatter than that for CT-AgNWs.llenging a,b. To f61BM (approximately 140 nm)/Ca/Ag PSC devices. Because of the high transparency and conductivity of the optimized CT-AgNWs, we observed the highest power conversion efficiency (PCE) of 2.8% , which was close to that of the ITO-based device (3.5%). Nevertheless, because of the large surface roughness of CT-AgNWs, the reproductivity was low, with six of the 10 devices short circuiting. We fabricated the HP-AgNW-based (ca. 50 \u03a9 sq\u22121) P3HT device and obtained a PCE of 2.96%, which was 16.5% higher than the normal AgNW-based (120 \u03a9 sq\u22121) device (PCE = 2.54%), because of the increase in the fill factor (FF) value benefiting from the low Rs of the electrode ) device. As shown in Rs value of the electrode.We first fabricated the glass/CT-AgNWs/PEDOT:PSS/P3HT:PClectrode . Figure Rs values of electrodes before and after various HP conditions. The Rs of the electrode decreased by 40\u201350% under the HP process. To evaluate the HP-AgNWs for flexible PSCs, we optimized the fabrication condition of the AgNW-PET substrate. Because the glass transition temperature of PET is ca. 100 \u00b0C, we applied the temperatures of HP at 80 \u00b0C, 90 \u00b0C and 100 \u00b0C for 10 min and then for 30 min. Rs for the HP-AgNW/PEDOT:PSS and HP-AgNW/ZnO films. The inset shows the schematic of the resistance measurement for the electrodes. We found that the Rs values of the HP-AgNWs/PEDOT:PSS electrodes increased markedly. The Rs value of HP-AgNWs/PEDOT:PSS increased by almost three orders of magnitude after storing in air for 140 h, indicating its high air sensitivity. By contrast, the Rs value of the HP-AgNW/ZnO electrodes can remain constant for a long time. Because the ZnO layer is not sensitive to humidity and is a neutral material, ZnO plays the role well of a protective layer preventing AgNWs from direct exposure to air and improving stability. J-V curves of inverted PSCs made through the active layers of PffBT4T-2OD:PC71BM or PTB7-Th: PC71BM, respectively. The J-V characteristic is listed in ocV, scJ, FF and PCEs values were 0.80 V, 17.17 mA cm\u22122, 0.57, 7.83% for PTB7-Th:PC71BM and 0.76 V, 15.59 mA/cm\u22122, 0.49, 5.81% for PffBT4T-2OD:PC71BM. For the efficiency of PSCs based on the ITO/glass electrode and these three active layers, PTB7-Th:PC71BM and PffBT4T-2OD:PC71BM were 8.03% and 7.16%, respectively. The results demonstrate that the PCEs obtained from HP-AgNW-based PSCs are close to those measured from ITO-based PSCs. We calculated the series (Rs) and shunt (Rsh) resistances from the respective J-V curves. The values of Rsh and Rs of the ITO/ZnO/PTB7-Th, HP-AgNWs/ZnO/PTB7-Th, ITO/ZnO/PffBT4T-2OD:PC71BM and HP-AgNWs/ZnO/PffBT4T-2OD devices are 540/3.3, 574/6.0, 603/2.3 and 298/7.0 \u03a9 cm2, respectively . ectively . The valistances . The val71BM-based PSC devices using HP-AgNW electrodes with a high performance of 5.09%, and the cell maintained more than 70% of its efficiency after a bend test (100 times). We further fabricated an AgNW/ZnO-based inverted PSC device and observed a PCE of 7.83% for the device using PTB7-Th:PC71BM as an active layer. The results obtained from this study demonstrate that the efficiency of PSCs made with AgNWs or ITO electrodes can be similar, but the cost of using AgNWs for electrodes is much lower than that using ITO. Because of the easy nature of HP treatment and the high reproducibility of the derived devices (because of lower surface roughness), the HP-AgNWs have the potential to replace ITO electrodes in the application of PSCs.Because of the trade-off between the conductivity, surface roughness and transparency of AgNW electrodes, it was challenging to fabricate a high-performance AgNW-derived flexible optoelectronics system. In this study, we used CT and HP treatment to improve the conductivity of AgNW electrodes  and simultaneously maintained their high transparency (T% > 80%). The CT-and HP-derived AgNW electrodes were used to replace expensive and brittle ITO electrodes for the application in flexible PSCs. Simultaneously, the surface roughness could also be modified, which solved the problem of short circuiting for the HP-derived AgNW electrodes. We successfully demonstrated the flexible PffBT4T-20D:PC"}
+{"text": "Vector-borne diseases are a major public health concern inflicting high levels of disease morbidity and mortality. Vector control is one of the principal methods available to manage infectious disease burden. One approach, releasing modified vectors (such as sterile or GM mosquitoes) Into the wild population has been suggested as an effective method of vector control. However, the effects of dispersal and the spatial distribution of disease vectors (such as mosquitoes) remain poorly studied. Here, we develop a novel mathematical framework using an integrodifference equation (discrete in time and continuous in space) approach to understand the impact of releasing sterile insects into the wild population in a spatially explicit environment. We prove that an optimal release strategy exists and show how it may be characterized by defining a sensitivity variable and an adjoint system. Using simulations, we show that the optimal strategy depends on the spatially varying carrying capacity of the environment. Vector borne diseases are a major public health concern, causing high levels of morbidity and leading to nearly one million deaths, annually WHO . Often vIn evaluating the efficacy of SIT, mathematical modelling is an effective tool  by focusing more on their biology, ecology and behaviour , which are discrete in time and continuous in space, incorporate a dispersal kernel for the spatial distribution of mosquitoes, model populations in which growth and dispersal do not happen at the same time , which is a probability density function (pdf) and gives the probability that an individual starting at point y, will settle at point x by the next time step. The number of individuals moving to location x is found by integrating the dispersal kernel k over the domain of interest. Hence, we have In order to familiarise the reader with the integrodifference equation (IDE) framework  in a one dimensional domain S is the average dispersal success over the domain For the SIT control problem, we assume an unstructured mosquito population with no overlapping generations. Let Clements . This meentclass1pt{minimand Lewis  and Reimnd Lewis  for a mox and time step t, where the initial distribution In this section, we describe the optimal control framework for minimizing the cost of controlling the wildtype mosquito population. We propose to control a vector population over a time period f is twice differentiable in The kernels are bounded and measurable such that X is the solution ofb and d are defined by: Given an optimal control Proof of this Theorem is given in the \u201cAppendix\u201d. We find the characterization of the optimal control by solving Eq.\u00a0. Using tr is considered to be optimal.We use a forward-backward sweep numerical method  is the same across the whole domain Here, we apply the optimal control theory to homogeneous environment. To replicate such environments we assume that the value of We have chosen an exponential form for the dispersal kernel, as these dispersal conditions have received the most support from the available data . We consider situations (a) when the centre of the domain has more favourable Conditions  and predation. In this section, we analyse how landscape heterogeneity affects the dispersal and the optimal control of mosquitoes. We replicate this behaviour by varying the value of Here, we formulated a novel mathematical model to understand the effects of releasing sterile mosquitoes into wild populations as well as the effect of spatial spread on mosquito population dynamics. The approach described here has not been used before in designing and optimizing sterile insect release strategies.k in the integrodifference equation.The model is described by an integrodifference equation, which are used to model populations where growth and dispersal do not happen at the same time. In our model we consider a homogeneous population consisting of wild and sterile mosquitoes with no overlapping generations. The growth function is based on the Ricker model and we assume releases proportional to wild population equilibrium. The spatial spread of the mosquitoes is described by the dispersal kernel A. aegypti control  that more mosquitoes should be released where densities are higher. Another (expected) result from this model is that the optimal strategy is to release significantly more sterile mosquitoes at the beginning of the vector management control programme until the wild mosquito population is suppressed to a level that the imposed burden is so low.J(r) and the characterization of the optimal control. Importantly, our results emphasize that optimal control does not necessarily lead to population elimination. Varying the parameters  to achieve cost-efficient control strategies needs more thorough investigation (Hackett and Bonsall"}
+{"text": "Bayesian inference and ultrasonic velocity have been used to estimate the self-association concentration of the asphaltenes in toluene using a changepoint regression model. The estimated values agree with the literature information and indicate that a lower abundance of the longer side-chains can cause an earlier onset of asphaltene self-association. Asphaltenes constitute the heaviest and most complicated fraction of crude petroleum and include a surface-active sub-fraction. When present above a critical concentration in pure solvent, asphaltene \u201cmonomers\u201d self-associate and form nanoaggregates. Asphaltene nanoaggregates are thought to play a significant role during the remediation of petroleum spills and seeps. When mixed with water, petroleum becomes expensive to remove from the water column by conventional methods. The main reason of this difficulty is the presence of highly surface-active asphaltenes in petroleum. The nanoaggregates are thought to surround the water droplets, making the water-in-oil emulsions extremely stable. Due to their molecular complexity, modelling the self-association of the asphaltenes can be a very computationally-intensive task and has mostly been approached by molecular dynamic simulations. Our approach allows the use of literature and experimental data to estimate the nanoaggregation and its credible intervals. It has a low computational cost and can also be used for other analytical/experimental methods probing a changepoint in the molecular association behaviour. The long-term impacts to the ecosystem by oil spills are reviewed elsewhere6. Water-in-oil emulsions (WOE) form during petroleum spills as a result of petroleum mixing with sea water, whereby very little energy is required for emulsification to occur7. Such emulsions are very stable and problematic to remove due to their high viscosity and stability9. The efficient removal of WOEs thus requires phase separation into water and oil phases. The WOE stability is a function of factors that include a high water content (30\u201390%)11, water salinity13 and pH15.Petroleum spills occur due to anthropogenic  phenomena, such as petroleum exploration, transportation and refining17, have been extensively reported to be the main cause of the high WOE stability. The importance of waxes and the water droplet size distribution in increasing the viscosity of WOEs has also been reported numerously18. Asphaltenes is a class of compounds that is operationally defined as soluble in toluene and insoluble in n-pentane or n-heptane21. The features of an asphaltene fraction, therefore, are defined by the precipitating solvent and can comprise a vast structural polydispersity22. A subfraction of the asphaltenes24, that is reported to be more polar25, stabilises the WOEs by adsorbing at the water/oil interface forming rigid films resisting droplet coalescence27.Asphaltenes, and specifically the natural interfacially-active emulsifiers within them20 is one of the most widely-accepted. It suggests that in low concentrations, asphaltenes in petroleum exist as monomers. As their concentration increases to the critical nanoaggregate concentration (CNAC) of 50\u2013150 mg/L asphaltenes self-associate into nanoaggregates. Further, as their concentration reaches 2\u20133 g/L asphaltenes start forming clusters20. The surface-active asphaltenes self-associate at the nanoscale28 forming nanoaggregates, the latter were reported to form films that stabilise WOEs31. The nanoaggregates are ca. 3\u201310 nm in size, have an ellipsoidal shape and can entrap solvent within the aggregate interior33. There are two primary forces governing nanoaggregation, attraction between the aromatic cores and repulsion from the aliphatic appendages20. We have previously suggested that the abundance of longer side-chains 16. A discussion about this and further literature review of asphaltene nanoaggregation is presented in Svalova et al.34. In what follows it is assumed that asphaltene nanoaggregates contribute to the WOE stability.In understanding the asphaltene phase properties, the Yen-Mullins model40, corresponding to the critical nanoaggregate concentration (CNAC)39. This concentration can be determined using conductivity41, centrifugation42, nuclear magnetic resonance43 and high-Q ultrasonic measurements39. We have used the latter technique to test the CNAC of four asphaltene samples34. At higher concentrations (g/L) asphaltene nanoaggregates start forming clusters corresponding to the critical cluster concentration.Asphaltene nanoaggregation is assumed to be the first stage of their self-association, which occurs at concentrations of ca. 100 mg/L  with E2 (mildly-moderately biodegraded) and from E3 (highly biodegraded) with E4 (mildly-moderately biodegraded) are from two different source rocks respectively and all are from different reservoirs34. Noteworthy, there was significant noise in the data which we removed before modelling the data by analysing the outliers in the ultrasonic trace versus time. However diversions from the two-line model remain e.g. in the high-concentration tail of E3. These diversions may have been caused by the high molecular heterogeneity of the asphaltenes51, micro air bubbles52 or trace impurities. This is illustrated numerically in our synthetic data study where we emulate one of our specimens and a specimen from Andreatta et al.39 to validate our MCMC scheme. Bayesian inference can serve to remediate this as it combines expert opinion40 and experimental data to estimate the aggregation point. The synthetic data study also illustrates this as the aggregation point is recovered for both data sets.The asphaltenes were precipitated from four petroleum samples: E1 with E2 and E3 with E4 are from two different source rocks respectively and all are from different reservoirs. E1 and E2 are from South America and E3 with E4 are from North America. The asphaltene preparation procedures, including precipitation and purification, geochemical description of the samples and ultrasonic methodology can be found in our earlier studyThis subsection provides an introduction to Bayesian inference and Markov chain Monte Carlo simulation which the familiar reader should feel free to skip.Y generating an outcome y that is governed by a model M and controlled by a collection of parameters (vector) n, and has been used in our previous analysis34. Often, however, experiments and/or events cannot be repeated (a meaningful number of times). The Bayesian approach53 is extremely useful in conditions of low/noisy data availability and will be used here.In statistical modelling, we assume a process/experiment Y follows some probability distribution (e.g. Gaussian) with a probability density function f, and the probability of M controlled by M suppressed in notation). The likelihood function of f over all data values of y, Required in both approaches is the likelihood. To formulate the likelihood, it is assumed that L is maximised to obtain the maximum likelihood estimate (MLE) of M. This implies that the likelihood alone can be used to draw inference about M is suitable to model y. However, when n is small then the uncertainty around the MLE will be high. The Bayesian approach, on the other hand, allows the incorporation of expert/literature knowledge about y. This distribution is also known as the posterior distribution, which is denoted by 53:In the likelihood approach, 54 is the most popular.Formalism  can alsorepresent/approximate the posterior distribution 54. Specifically, the random samples are generated according to a Markov chain, which is a stochastic process whereby the value  aggregation52, Equation\u00a0 only. In particular, the sound velocity u is related to apparent molar solution quantities following the relationv denotes specific volume, c- weight concentration, tilde- apparent quantities and subscripts refer to solvent (0), monomer (1) and aggregated (a) quantities. Also,For multi-phase fluids which are well-dispersed, and ignoring the effects of sound scattering Equation\u00a0 can be aEquation\u00a0 allows tThe model\u00a0 implies y varies with asphaltene concentration x as follows.n denotes the total number of measurements, Formalism\u00a0 can be eIn\u00a0n is the total number of measurements (sample size).In the above, 39 summarised in Table\u00a0The prior distributions mentclass2pt{minim39. Also note that Noteworthy, the values of mentclass2pt{minimN and Ga denote normal and Gamma distributions respectively. The prior mean and standard deviation of 20. The mean of the 34. All the parameters apart from 54, details of which can be found in the Supplementary Information . The39 and our E2 specimen. Noteworthy, the precision value to emulate the E2 specimen was found to be two orders of magnitude lower than that of UG8, Figure 39. The synthetic study included testing the impact of prior mean misspecification on the posterior estimation of We carried out a study (Supplementary Information) on synthetic data designed to emulate the UG8 asphaltene by Andreatta et al.The chain mixing was very efficient for all parameters Figure , and all54 based on Equations  was was54 baentclass1pt{miniman Figure , where mentclass1pt{minimaFigure\u00a0The specimen split between posterior uni-/bimodality of the regression parameters is likely to be related to the accepted values of 61. It is clear that our measurements have a greater noise that those reported by e.g. Andreatta et al.39 and the use of Bayesian inference has allowed us to estimate the 34 we have illustrated using pure surfactant solutions that the Resoscan instrument can detect molecular self-association at a similar scale to the asphaltenes. Thus, we infer that the fluctuations in the ultrasound velocity versus concentration for asphaltenes are mainly caused by the physico-chemical properties of the sample.Bayesian inference and MCMC sampling of the posterior distribution can be very useful in the conditions of sparse and noisy data. Figure\u00a0mentclass2pt{minim20. For all of the specimens, one point or less lie outside the credible intervals which is consistent with a 5% outlier rate.The changepoint Noteworthy, the behaviour of E1 is different to the remaining samples in that the velocity gradient is increasing in the monomeric region as opposed to vice versa in the remaining samples. The gradients in Fig.\u00a034. In the latter study, the architecture of the asphaltenes was linked to their aggregation behaviour by considering the abundance of the long side-chains 64. Asphaltene side-chains are released as n-alkanoic fatty acid methyl esters which are the dominant products, and are also extremely volatile. Also, the reaction produces a significant amount of di-alkanoic fatty acid di-methyl esters and other products which can interfere with the main peaks of interest65. Therefore, the results of RICO should be used as indicative. The specimens E1\u2013E4 were reported to have 14%, 21%, 18% and 11% of long n-alkanoic side-chains respectively.The estimated changepoint values are similar to those we reported using non-Bayesian methodsComparing to the present study, the the relative abundance of the long side-chains is linearly related to 39, herein referred to the changepoint Asphaltene nanoaggregation ultrasonic characterisation data has been studied using Bayesian inference to produce an estimation of the critical nanoaggregation concentration66 and nuclear magnetic resonance67. The computational burden of the proposed sampling scheme is very low and can be run within minutes.Given an appropriate prior distribution specification our model can be applied to any asphaltene characterisation data that is assumed to follow a changepoint regression behaviour and has uncertainty, such as calorimetry22 is too high for the model by Zielinski et al. to be appropriate. Additional geochemical investigations can also complement the current study, for example further understanding the composition and architecture of the aliphatic moieties of the asphaltenes through elemental analysis.Further developments of this work include a hierarchical Bayesian structure of the single-changepoint model that would allow to estimate the regression coefficients by borrowing strength from all of the specimens/pooling the data. A two-changepoint model may also be proposed to estimate the Supplementary material 1"}
+{"text": "Women who were overweight/obese, had excessive GWG or GWGrate had 1.996-, 1676- or 1.673-fold higher risks for delivering large-for-gestational-age (LGA) infant. The effects of GWG and GWGrate on birth weight varied by pre-pregnancy BMI statuses. Dose-response analysis demonstrated L-shaped and S-shaped relationships between pre-pregnancy BMI, GWG, GWGrate and neonatal birth weight.Of the 3585 participants, women who were underweight, had insufficient GWG or GWGrate were associated with neonatal birth weight among Chinese women. Both body weight before and during pregnancy should be maintained within the recommendations to prevent abnormal birth weight.Pre-pregnancy BMI, GWG or GWG Since the 1980s, the overweight and obesity rates have doubled during the past four decades in more than 70 countries worldwide, and the rates are still increasing . For womrate), which is also the period of maximal growth and weight gain for fetuses, is significantly associated with newborn weight while weight gain in the 1st trimester mostly affects overweight or obesity risk in the offspring\u2019s childhood [rate is preferable for exploring the relationship between trimester-specific weight change and infant birth weight even though the data collection is complicated. The overall prevalence of insufficient and excessive GWGrate varies in different regions [rate respectively in 2013 [Although GWG is commonly used to evaluate fetal development, it has some limitations. Weight gain has different effects on fetal development in different trimesters, and total GWG failed to show trimester-specific weight change during pregnancy. A large number of clinical studies have demonstrated that women\u2019s weight gain during the 2nd and 3rd trimester  infants but a lower risk of delivering large for gestational age (LGA) infants . Similarnsistent , 16. Furrate in the 2nd and 3rd trimester, and to explore their associations with abnormal neonatal birth weight (i.e. SGA and LGA) among Chinese women, as well as the dose-response relationship between women\u2019s weight status before or during pregnancy and abnormal birth weight risks.This study aimed to evaluate the prevalence of pre-pregnancy BMI, GWG and GWGThe Chinese Pregnant Women Cohort Study (CPWCS) is a multicenter, prospective cohort study focusing on antenatal women and their neonates. Twenty-four hospitals [see Table S1 in Participants were required to complete an electronic self-designed questionnaire in their first trimester and were followed up three times: in the second trimester, in the third trimester, and 42\u2009days postpartum. Information provided by respondents includes basic sociodemographic characteristics, lifestyle behaviors, complications in each trimester and pregnancy outcomes. The pre-pregnancy weight and height of each respondent were self-reported when women were enrolled in this study, and pre-pregnancy BMI was assessed. Body weight at the beginning of the second trimester and at delivery were also collected, and the weight difference was GWG. Some important variables, such as pre-pregnancy body weight and weight at delivery, height, pregnancy complications, gestational age and pregnancy outcome were also checked with data recorded in the hospital information system (HIS) to maintain data quality.2) was calculated by respondent\u2019s height and pre-pregnancy weight and was categorized into four groups according to standard WHO criteria: underweight (BMI\u2009<\u200918.5\u2009kg/m2), normal-weight (18.5\u2009kg/m2\u00a0\u2264\u2009BMI\u2009\u2264\u200924.9\u2009kg/m2), overweight (25.0\u2009kg/m2\u00a0\u2264\u2009BMI\u2009\u2264\u200929.9\u2009kg/m2) and obese (BMI\u2009\u2265\u200930.0\u2009kg/m2) [Pre-pregnancy BMI (weight(kg)/ height (m)0\u2009kg/m2) .GWG was defined as the difference in weight at delivery and weight before pregnancy. According to the IOM recommendation, for pre-pregnancy underweight, normal weight, overweight and obese women, the appropriate GWGs are 12.5\u201318\u2009kg, 11.5\u201316\u2009kg, 7\u201311.5\u2009kg and 5\u20139\u2009kg, respectively. Women with GWG below the recommended range were defined as having insufficient GWG, and those with GWG above the appropriate range were defined as having excessive GWG [rate was calculated as (the difference of weight at delivery and weight at the beginning of 2nd trimester)/, whereas 13 was the cutoff value of the 1st and 2nd trimesters. According to IOM, for pre-pregnancy underweight, normal weight, overweight and obese women, the appropriate GWGrate ranges are 0.44\u20130.58\u2009kg/w, 0.35\u20130.50\u2009kg/w, 0.23\u20130.33\u2009kg/w and 0.17\u20130.27\u2009kg/w, respectively [rate below the recommended range were defined as having insufficient GWGrate, and those with GWGrate above the appropriate range were defined as having excessive GWGrate.GWGectively . Women wThe main outcomes of this study were SGA and LGA. Birth weights below the 10th or above the 90th percentile, respectively, for the same gestational age by sex were defined as SGA and LGA according to the Chinese neonatal birth weight curve .In this study, covariates included sociodemographic characteristics , lifestyle behaviors , and clinical characteristics  and gestational hypertension). Gestational age was calculated as the difference in date from the last menstrual period to delivery. Alcohol consumption was defined as consuming any alcoholic beverage more than once per month. GDM was defined as meeting one or more of the following criteria: fasting plasma glucose \u22655.1\u2009mmol/L, 1\u2009h plasma glucose levels \u226510.0\u2009mmol/L, 2\u2009h plasma glucose levels \u22658.5\u2009mmol/L after overnight fasting with a 75\u2009g glucose load at 24\u201328 gestational weeks according to the diagnostic criteria amended by WHO in 2013 [rare, and birth weight and the results are shown as odds ratios (ORs) and 95% confidence intervals (CIs). Dose-response relationships were explored by restricted cubic spline (RCS) logistic regression models. Three knots were located at the 25th, 50th and 75th percentiles of the distribution of each continuous dependent variable. RCS analysis was conducted by SAS 9.4 software with the RCS_Reg macro. All P values in this study were two-sided and P\u2009<\u20090.05 was regarded as a significant difference.Qualitative variables were described as the mean values and standard deviations while quantitative variables were described as frequencies and percentages. The chi-square test and Kruskal-Wallis test were used for univariable analyses. Multinomial logistic regression models were conducted to explore the relationship between pre-pregnancy BMI, GWG, GWGP\u2009<\u20090.05). SGA mothers were shown to have fewer gravidities and parities than LGA mothers. In addition, women who gave birth to SGA infants were less likely to have GDM but had higher risk of gestational hypertension compared with women in the LGA group .A total of 3585 women were enrolled and maternal characteristics presented by neonatal birth weight are shown in Table\u00a0rate categories. The prevalence of underweight, overweight and obesity before pregnancy was 14.17, 12.86 and 1.73%, respectively. According to the IOM recommendation, 24.69 and 33.78% of women had insufficient and excessive GWG, respectively. Table S4 [see rate categories. Approximately 26.86% of women showed insufficient GWGrate while 46.95% had excessive GWGrate. The average birth weight of infants was associated with women\u2019s pre-pregnancy BMI, GWG and GWGrate (P\u2009<\u20090.001).Table S2 to Table S4 see  show thee S4 see  illustrarate were 1.9-,1,9- and 1.5-fold more likely to have SGA infants, respectively, compared with reference groups, and women who were overweight or obese before pregnancy, had excessive GWG or had excessive GWGrate were 2.0-,1.7- and 1.7-fold more likely to have LGA infants, respectively. Additionally, protective effects were shown between excessive GWGrate and SGA, as well as pre-pregnancy underweight, insufficient GWG and LGA.The multinomial logistic regression analysis results are shown in Table\u00a0rate [see Table S5 to Table S7 in rate were associated with infant birth weight under different pre-pregnancy BMI levels (P\u2009<\u20090.05).Chi-square analysis demonstrated the relationship between birth weight and jointed pre-pregnancy BMI and GWG/GWGStratified analysis results are shown in Table\u00a0rate under each pre-pregnancy BMI level is shown in Table\u00a0rate was associated with a 1.6 times higher risk for LGA but a 0.35 times lower risk for SGA. Excessive GWGrate also contributed to a 1.7 times higher risk for LGA among pre-pregnancy overweight and obese women, and insufficient GWGrate was associated with a 0.81 times lower LGA risk in this group.The association of neonatal birth weight with GWGrate and LGA (Pnonlinear\u2009<\u20090.001), the other associations were linear (Pnonlinear\u2009>\u20090.05).Figure\u00a0rate was also associated with neonatal birthweight. The association of GWG and GWGrate with birth weight varies with pre-pregnancy BMI status. Dose-response relationships existed between pre-pregnancy BMI, GWG, GWGrate and newborn weight.In this study, we found that pre-pregnancy underweight and insufficient GWG were associated with higher SGA risk and lower LGA risk while pre-pregnancy overweight or obese and excessive GWG were associated with higher LGA risk and lower SGA risk. GWGrate are consistent with the findings of some existing studies [rate, another Chinese cohort study indicated that excessive GWGrate increased LGA risk, but insufficient GWGrate had no impact on SGA, and this result was inconsistent with our finding [The relationships this study found between pre-pregnancy BMI, GWG and GWG studies \u201323, incl studies . A meta- studies . We also studies  found th studies  conducte finding . Differerate and birth weight varies with different pre-pregnancy BMI statuses. Stratified analysis showed that insufficient GWG increased SGA risk only in pre-pregnancy underweight or normal weight women, rather than overweight or obese women. This finding is consistent with a cohort study targeted at US women [rate was associated with a higher risk of LGA except in pre-pregnancy underweight women, and it was found to decrease SGA risk only in pre-pregnancy normal weight. However, some research [rate could decrease LGA risk in pre-pregnancy underweight women, but we did not find the same results. This study also did not show relationship between insufficient GWGrate and the risk of SGA, which are commonly believed to be associated. This difference indicates that more comprehensive and well-designed studies are needed to help determining consistent results.The association of GWG and GWGUS women . This stUS women , which sresearch  found thrate and LGA, which showed an \u2018S-shaped\u2019 carve, others were \u2018L-shaped\u2019 in this study. These results were different from those of the previously mentioned studies, which may be attributed to different research populations or study designs. Nevertheless, dose-response research on this topic is mere, and far more studies are needed.There are relatively few studies focusing on the dose-response relationship between women\u2019s body weight before and during pregnancy and infant birth weight. Consistent results can be found through these studies that a dose-response relationship exists, but the shapes of these curves are different , 32. Excrate on LGA infants among Chinese women and the protective effects of pre-pregnancy overweight and obese/ excessive GWG and GWGrate on SGA infants, which are still inconsistent yet. Second, stratified analysis was adopted to explore the relationships between GWG/GWGrate and birth weight under different pre-pregnancy BMI levels, and this made our study more comprehensive and accurate; Third, this study analyzed GWGrate, which was less commonly researched due to cumbersome measurement, and the results can provide references for further relevant research. Fourth, we explored the dose-relationships between women\u2019s body weight before and during pregnancy and neonatal birth weight, and this was focused by only few studies around the word.The novelty of this study is significant. First, this study explored the protective effects of pre-pregnancy underweight/ insufficient GWG and GWGThis study has some strengths and limitations. This nationwide multicenter prospective cohort study enrolled people living in 15 different provinces in China, and this sample can well represent Chinese women. Core data, including weight before pregnancy and weight at delivery, gestational age and neonatal birth weight, were double checked to maintain data quality, which means data collected by questionnaire were checked with that recorded in the HIS system of each hospital. If there was any inconsistency, a medical record was preferred unless it was illogical. For limitations, respondents were asked to recall their pre-pregnancy body weight and height, which may result in recall bias. In addition, convenience sampling may decrease the representativeness of our results. We did not conduct subgroup analysis stratified by obesity class due to the small sample size of pre-pregnancy obese women. However, this subgroup analysis may provide more detailed references for body weight control before pregnancy and it should be explored in the future.rate increased the risk of SGA while pre-pregnancy overweight or obese, excessive GWG and GWGrate increased the risk of LGA. In addition, these relationships varied according to different pre-pregnancy BMI statuses. Dose-response relationships were observed between independent and dependent variates mentioned above. Our results emphasized the significance of body weight control both before and during pregnancy. Although it may be difficult for women to adhere the IOM recommendation, interventions such as education should be imposed to help achieving suitable pre-pregnancy BMI and GWG [In summary, this study showed that pre-pregnancy underweight, insufficient GWG and GWG and GWG .Additional file 1: Table S1.Table S2. Maternal characteristics presented by pre-pregnancy BMI; Table S3. Maternal characteristics presented by GWG; Table S4. Maternal characteristics presented by GWGrate; Table S5. Birth weight classified by pre-pregnancy BMI, GWG and GWGrate; Table S6. Birth weight classified by jointed pre-pregnancy BMI and GWG; Table S7. Birth weight classified by jointed pre-pregnancy BMI and GWGrate. (DOCX 53 kb)Twenty-four hospitals sites in the CPWCS project;"}
+{"text": "Odds ratios of the complications ) were calculated in the multivariate logistic regression. In the cohort, 10.8% of the women had pre-pregnancy obesity (body mass index (BMI) \u2265 30 kg/m2), and 36.8% had gestational weight gain (GWG) above the range of the Institute of Medicine recommendation. After correction for excessive GWG and other confounders, pre-pregnancy obesity  was associated with a higher odds ratio of GH , PE , GDM-1 , and GDM-2 . The threshold risk of development of GDM-2 occurred at lower BMI values (26.9 kg/m2), compared to GDM-1 (29.1 kg/m2). The threshold point for GH was 24.3 kg/m2, and for PE 23.1 kg/m2. For GWG above the range (vs. GWG in the range), the adjusted odds ratios of GH, PE, GDM-1, and GDM-2 were AOR = 1.71 (p = 0.045), AOR = 1.14 (p = 0.803), AOR = 0.74 (p = 0.245), and AOR = 0.76 (p = 0.672), respectively. The effect of maternal edema on all the results was negligible. In our cohort, hypertension and diabetes were associated with incorrect birth weight and gestational age at delivery. Conclusions: This study highlights the importance and influence of excessive pre-pregnancy maternal weight on the risk of pregnancy complications such as diabetes and hypertension which can impact fetal outcomes.Excessive pre-pregnancy weight is a known risk factor of pregnancy complications. The purpose of this analysis was to assess the relationship between several categories of maternal weight and the risk of developing hypertension and diabetes in pregnancy, and the relationship of these complications with the results of the newborn. It was carried out in a common cohort of pregnant women and taking into account the influence of disturbing factors. Our analysis was conducted in a prospective cohort of 912 Polish pregnant women, recruited during 2015\u20132016. We evaluated the women who subsequently developed diabetes with dietary modification (GDM-1) ( According to global estimates, the incidence of obesity has doubled in the last four decades, and over 30% of women are obese . BecauseMany studies have shown that pre-pregnancy obesity and overweight are independent pregnancy-induced hypertension (PIH) and gestational diabetes mellitus (GDM) risk factors . In mostThe aim of our study was to check the relationship between various maternal weight categories and the risk of developing pregnancy complications such as hypertension or diabetes, which in turn could affect the results of the newborn. We evaluated the main forms of these diseases separately. Our goal was to take into account important disturbing factors including maternal edema. We excluded women with pre-existing hypertension or diabetes.The entire research process carried out to implement this analysis was in line with the Helsinki Declaration. The women recruited for the study were informed about the voluntary character of their participation. All recruited participants read and signed the Conscious Consent Form. The study was approved by the Bioethics Committee of the Medical University of Poznan (Poland) (769/15).We examined a cohort of pregnant women who were enrolled at the Obstetric-gynecological and Neonatological Hospital . This is the university hospital with the highest degree of references for obstetric cases, in which the number of deliveries per year is about 7000. The recruitment process was carried out during 2015\u20132016.Polish white women from the Wielkopolska region were recruited . Other inclusion conditions involved single pregnancy, gestational age between 10th and 14th week, no fetal aneuploidy, gestational age of delivery \u2265 25th week, and delivery of a phenotypically normal child. The inclusion criterion was also the mother\u2032s age during conception of 18\u201345 years.We excluded women with chronic diseases, primarily diabetes mellitus, and/or arterial hypertension. Kidney disease and/or liver disease were excluded as well. The same regarded thromboembolism and diseases related to immune disorders. Excessive body mass index on the other hand was not an exclusion criterion.Information related to the characteristics of the participants was collected during recruitment. For this purpose, we used a questionnaire created specifically for the purposes of our study. The women completed these questionnaires in the presence of a medical worker (midwife), but did it on their own. They were asked about their age, height, and weight before pregnancy. The following questions concerned the course of the current pregnancy as well as the number and history of previous births. We collected information about the multivitamin preparations and other drugs they took. We asked them about addictions such as smoking, drinking alcohol, and other drugs . We also collected the information about other previous illnesses and operations. An important part of the questionnaire was questions about socio-economic indicators and demographic data.The data we collected after the end of pregnancy were taken from medical records. Information on pregnancy outcomes included fetal sex, birth weight, gestational age at birth, intrauterine growth restriction, and APGAR  results. The information on pregnancy complications mainly included the development of gestational diabetes mellitus (GDM) and pregnancy-induced hypertension (PIH). The information on mother\u2032s edema was also recorded.The PIH covered the development of de novo hypertension after 20th gestational week and its resolution within 12 weeks after delivery. Hypertension was defined as a pressure value \u2265 140/90 mmHg . The pressure was measured in a sitting position, and an oscillometric device was used to measure the pressure. Isolated gestational hypertension (GH) (a milder form of hypertension in pregnancy) was diagnosed if no organ disorder was found. A severe form of the PIH, or preeclampsia (PE), was defined as hypertension accompanied by organ disorders involving the kidneys and/or liver and/or brain, as well as pulmonary edema. Proteinuria was not a mandatory criterion . In all The definition of GDM was in accordance with national and international guidelines . This definition was based on the \u201ca 2-h 75 g oral glucose tolerance test (OGTT)\u201d. This is a test performed on pregnant women after an overnight fast. This test is performed between the 24th and 28th gestational week . Two forThe participants of the study were recruited in hospital laboratory. Brief information about the study and recruitment criteria was posted in the laboratory. In total, 1300 women volunteered for the study over a 12-month period during recruitment during 2015\u20132016, of whom 388 participants were excluded after the end of pregnancy . Finally, the studied cohort consisted of 912 participants.n = 137)  cases and 24 preeclampsia (PE) cases). Controls for the cases (n = 775) were the participants who remained normotensive. In the second analysis, the study group included the women who subsequently developed gestational diabetes mellitus (GDM) (n = 146) . Controls for the cases (n = 766) were the participants without diabetes.In the current study, two large analyzes were conducted. In the first analysis, the study group included the women who subsequently developed pregnancy-induced hypertension (PIH) . Body mass index (BMI) was estimated for each participant. The formula for calculating BMI was typical: weight (in kilograms) divided by height (in meters) squared. BMI definitions were in line with the recommendations of the World Health Organization (WHO). The BMI definition within the standard range included values of 18.50\u201324.99 kg/mMaternal weight before delivery  was used to calculate weight gain (GWG). To obtain the GWG value, the weight before pregnancy was subtracted from the weight before delivery. The optimal GWG ranges were different for each BMI category: it was 12.5\u201318 kg for underweight women before pregnancy; 11.5\u201316 kg for the women with BMI in the normal range; 7\u201311.5 kg for women with overweight before pregnancy; and 5\u20139 kg for obesity before pregnancy. The ranges were defined in line with the recommendations of the (US) Institute of Medicine-IOM from 2009.We also included information on swelling in women (most often edema of the lower limbs), which was noted in medical reports.p-Value< 0.05 was assumed as significant.We performed statistical analyses using the Statistica 13 software . The Shapiro\u2013Wilk test was used to assess the normality of the data distribution. We used the Mann\u2013Whitney U test  for comparisons of continuous variables , ranges, and medians are given as description). For categorical ordered categories, the Cochran\u2013Armitage test for trend was applied. For binomial categories, the Pearson chi-square test (or Fisher exact test when Cochran assumption was not met) was applied. p-value (p < 0.05 was assumed to be significant). The whole cohort and the subgroups  were assessed.The diseases risk for pre-pregnancy BMI and gestational weight gain (GWG) categories was assessed in the logistic regression. The Wald test was used to estimate Odds ratios of the diseases ) were determined for each pre-pregnancy BMI category with respect to the normal BMI  as well as for GWG above (or below) the IOM recommendation with respect to the GWG in the range (OR = 1). Crude odds ratios (OR) were established in the univariate logistic regression. Adjusted odds ratios (AOR) were obtained after correcting for confounders in several models. The investigated variable was always excluded from confounding variables.The risk of hypertension in pregnancy was adjusted for hypertension in previous pregnancies, smoking, infertility treatment, age, being primiparous, and for gestational weight gain (GWG) above the range (when BMI was investigated) or for BMI before pregnancy (when GWG was investigated). The risk of gestational diabetes mellitus was adjusted for diabetes in previous pregnancies, maternal age, being primiparous, and for gestational weight gain (GWG) above the range (when BMI was investigated) or for BMI before (when GWG was investigated). The risk results after adjustment for edema are presented in the supplement.Graphs of risk profiles (in the Lowess method) for pre-pregnancy BMI are presented.2 (range 14.6\u201342.9). The median of gestational weight gain (GWG) was 13.8 (25\u201375% ranges 10\u201317). Overall, 65.0% of the women had normal pre-pregnancy BMI (18.50\u201324.9 kg/m2) and 10.8% were obese (BMI \u2265 30 kg/m2). In the cohort, 37.1% of the women had GWG in the range of the IOM recommendations (regardless of the pre-pregnancy BMI category) and 36.8% had GWG above the range. In medical records, edema was reported in 43 (4.7%) women.General maternal characteristics are presented in 2) was associated with a higher risk of birth weight > 90th percentile  and > 4000 g , as well as with a higher risk of preterm birth < 34th week . The results were obtained after correcting for many variables, including GWG above the IOM recommendation, as well as for pre-eclampsia and gestational diabetes mellitus in present pregnancy.Relationships between pre-pregnancy BMI and the newborn outcomes are presented in The effect of maternal edema on the results was negligible .2). The higher pre-pregnancy BMI was associated with the higher risk of studied diseases, especially of the severe forms (PE and GDM-2). The threshold point (of pre-pregnancy BMI) was 24.3 kg/m2, above which the risk of GH was higher, and 23.1 kg/m2, above which the risk of PE was higher (this first point). The threshold risk of development of GDM-2 occurred at lower BMI values (26.9 kg/m2), compared to GDM-1 (29.1 kg/m2).In our study, higher values of maternal BMI before pregnancy increased the risk of pregnancy complications such as hypertension and diabetes. Pre-pregnancy obesity was a strong  risk factor of these diseases, to a greater extent than overweight or excessive gestational weight gain (GWG). Gestational weight gain (GWG) above the range (vs. GWG in the range) was a weak risk factor  for studied diseases, and was associated with higher odds ratios of GH and PE, and lower odds ratios of GDM-1 and GDM-2.Similar connections between obesity before pregnancy and hypertension or diabetes in pregnancy have been demonstrated in the literature ,21,22,23Our study was based on the data from a prospective cohort study of women with a single pregnancy. The body mass index (BMI) was defined in line with the World Health Organization (WHO) guidelines, and the categories of weight gain in pregnancy (GWG) were defined in line with the 2009 IOM guidelines. The risk was corrected by many confounding variables, including GWG above the range (in the study of BMI) and pre-pregnancy BMI (in the study of GWG). The general profile of our cohort, especially for GH 12.4%) and GDM-1 13.7%), was in accordance with results in the literature [% and GDM%, was inSeveral results we obtained need to be highlighted.Firstly, the results for risk of severe forms of the pregnancy complications (PE and GDM-2) for pre-pregnancy obesity were stronger than for isolated gestational hypertension (GH) and diabetes with diet modification GDM-1. However, the number of PE and GDM-2 cases was small (24 (2.6%) and 21 (2.3%), respectively). Our methodology was probably influenced by the low incidence of PE in our study; we excluded some risk factors of preeclampsia, such as multiple pregnancy and pre-existing chronic diseases ,24. Othe2) and within the limit of overweight. The threshold point for the increase in the odds ratios of isolated gestational hypertension (GH) was 24.3 kg/m2, and for the increase in the preeclampsia (PE) odds ratios was 23.1 kg/m2 (this first point) (2), compared to gestational diabetes without insulin (29.1 kg/m2). This requires examination in a larger cohort.Secondly, our results attract attention because the pre-pregnancy BMI threshold points (above which an increase in the risk of the pregnancy complications was recorded) are within the upper limit of the normal BMI (18.5\u201324.99 kg/mt point) . We founp < 0.001) and GDM-2 . The results were statistically significant, but the result for GDM-2 was close to the materiality limit. This requires examination in a larger cohort.Thirdly, we found that gestational weight gain (GWG) above the range (vs. GWG in the range) was associated with higher odds ratios of hypertension forms (GH and PE), and lower odds ratios of diabetes forms (GDM-1 and GDM-2) . The resImportantly, all the results were obtained after correcting for many confounders and in the subgroups . The eff2) was associated with a higher risk of adverse newborn outcomes  and unfavorable pregnancy results are difficult to investigate. Firstly, it is known that the appropriate gestational weight gain differs depending on the pre-pregnancy BMI. It was also found that the relationship between GWG and a specific complication may vary between pregnancy periods . It is nOur research has several advantages. It was based on the data from a prospective cohort. We calculated the minimum sample size before recruitment. We excluded chronic diseases during recruitment (except for overweight and obesity), reducing the number of risk factors having negative influence on the results. We applied multivariate logistic regression and adjusted the risk for existing confounding variables. However, although we accumulated many data, other factors are possible. We also assessed the impact of maternal edema on the risks we examined. In the joint cohort, we evaluated the pre-pregnancy BMI and gestational weight gain simultaneously, and at the same time assessed two important pregnancy complications and its forms. We evaluated the whole cohort and the subgroups.We are also aware of the limitations our research. The participants reported their body weight before pregnancy themselves, but this practice is often used. Both methods, examining the BMI or GWG by means of categorical variables or continuous variables have their limitations. Information about edema came from medical records. Sizes of subgroups were smaller, but the results of risk after adjustment were statistically significant. We did not assess the degree of glucose control, but the number of cases of diabetes treated with insulin was small. Despite the limitations of our study, we believe that the methodology we chose allowed systematically assessing and comparing two important risk factors of adverse pregnancy outcomes.This study highlights the importance and influence of excessive pre-pregnancy maternal weight on the risk of pregnancy complications such as diabetes and hypertension, which can impact fetal outcomes.Continued emphasis should be placed on the normalization of maternal weight before pregnancy, because higher pre-pregnancy BMI values  were strongly associated with a higher risk of pregnancy-induced hypertension and gestational diabetes mellitus.Our results for severe forms of hypertension and diabetes in pregnancy require examination in a larger cohort."}
+{"text": "To investigate the efficacy of middle meatal silastic splint in preventing adhesions after bilateral endoscopic sinus surgery (ESS) for chronic rhinosinusitis with nasal polyps (CRSwNP), and to assess nasal symptoms and endoscopic findings in splinted and non-splinted sides.After completion of ESS, silicon silastic splints were randomly inserted in the middle meatus of one nasal side, while no stent in the other side (control). The surgeon was blinded to the side selection, and splint insertion until removal after 1 week. Patients were followed -up after 1 week, 1 and 6 months. Each side of the nasal cavity was assessed for adhesions, crusting, pus, pain, nasal obstruction, and nasal discharge by endoscopic examination and visual analogue scale.Forty-nine patients  were included. At the 1st week visit, there was no significant difference between the splinted and non-splinted sides for all investigated parameters.After 1- month, adhesions were seen in 10% of the splinted sides, while it was in 26% of the non-splinted sides (P\u00a0=\u00a00.037).P\u00a0=\u00a00.007). All other examined parameters remained statistically insignificant between both sides throughout the follow -up visits.At the 6 -month follow-up visit, the adhesions rate remained 10% in the splinted sides, however the rate increased to 32% in the non-splinted sides (Middle meatal silastic splint is significantly reducing middle meatal adhesions with low complication rate in CRSwNP patients undergoing ESS. Our results support its usage when the middle turbinate is unstable or traumatized during surgery. \u2022Unstable middle turbinate after endoscopic sinus surgery can cause middle meatal adhesions leading to surgical failure.\u2022A silastic splints were inserted in the middle meatus of one nasal side, while no stent in the other side.\u2022The technique is simple, and keeps the middle turbinate lateralized until healing occurs.\u2022The splint is well tolerated by all patients with no adverse effects.\u2022Middle meatal silastic splint is significantly reducing middle meatal adhesions with low complication rate. The disease is estimated to affect approximately 13% of the population with a significant impact on patients\u2019 quality of life. It is classified into two phenotypes: CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP) .Patients who fail to respond to medical treatments  are candidates for endoscopic sinus surgery (ESS) which is considered the gold standard surgical treatment . HoweverAdhesions often form between middle turbinate and lateral nasal wall after ESS ,6. LaterNumerous absorbable and nonabsorbable materials, like; Floseal, Merocel, Gelfoam, fibrin glue, hyaluronic acid, and mitomycin C have been evaluated for their role in preventing middle meataladhesions with variable success rates. However, no method has been accepted as a gold standard technique to fill the middle meatal space after ESS .The aim of this prospective double-blind randomized controlled study was to investigate the efficacy of a silastic splint in preventing synechiae formation between middle turbinate and lateral nasal wall after bilateral ESS for CRSwNP patients, and to assess nasal symptoms and endoscopic middle meatal findings in splinted and non-splinted sides. We also sought to evaluate the patient tolerance of the middle meatal silastic splint.2We designed a prospective, double-blind, randomized controlled study for patients with CRSwNP undergoing bilateral ESS. Approval form our institutional review board committee was obtained. All patients were consented before surgery, and they had detailed history, clinical examination including nasal endoscopy and sinonasal CT scan.Inclusion criteria were adult patients (\u226518 years of age), CRwNP who failed to respond to medical treatment , bilateral sinus disease, unstable middle turbinate and/or debrided polypoidal middle turbinate mucosa during surgery, and no previous history of endoscopic sinus surgery.Exclusion criteria included; resected middle turbinate, asymmetric disease involvement, allergic fungal sinusitis, known cases of primary immunodeficiency, granulomatous diseases, cystic fibrosis, primary ciliary dysfunction, patients with unilateral disease, sinonasal malignancy, and patients who did not complete 6 months follow-up time.http://www.random.org) was done preoperatively to choose in which side of the nose the silastic splint would be placed, which is referred to as the \u201csplint side\u201d, while the other side (the control side) is referred to as the \u201cnon splint side.\u201dComputer-generated randomization  guidelines .Fig. 1CoAll patients were operated by the same surgeon (H\u00b7O.). A silicone sheet of 1\u00a0mm thickness was used . After cutting the splint to appropriate size, it was folded back on itself and sutured by Vicryl 3/0  to form inverted U- shape similar to Gall and Witterick technique  but withAll patients were discharged home on the first post-operative day on oral antibiotic for 2 weeks, 20\u00a0mg prednisolone/day for 1 week, and saline nasal irrigation q. i.d.The splint was removed by senior resident at the first postoperative visit after 1 week under local anesthesia for all patients by grasping the long ends of the suture or the anterior border of the splint. Endoscopic examination was performed in the clinic by the same senior surgeon on day 7, 1 month, and 6 months after surgery observing the presence of any synechia, crusting, and pus. Furthermore, the middle meatus of each side was described as \u201cwidely open\u201d when the ethmoid cavity easily seen by endoscope compared to the other side.Each side of the nasal cavity was assessed for pain/discomfort, nasal obstruction, and nasal discharge by using 10 points visual analogue scale (VAS) ranging from 0 (no symptoms) to 10 ; this was done by ENT resident who was blind to the splinted side.Sample size was calculated based on the observed middle meatal adhesion rate after ESS in our department of approximately 25%. Recently, Baguley et al.  reportedt-test, while the Mann-Whitney U test was used for nonparametric data analysis. For categorical data analysis; the Chi square test or Fisher's exact test was used as appropriate. A P value of <0.05 was considered statistically significant.Parametric data was analysed using the unpaired 3Overall, 60 patients with CRSwNP were recruited but 11 patients were excluded; 8 patients because they did not undergo surgery, and 3 patients did not complete 6 -month follow-up. Forty-nine patients (98 sides) who underwent bilateral ESS for CRSwNP and fulfilled our inclusion and exclusion criteria were included in this study. The mean age was 35 years . Thirty-one patients (63%) were male and 18 patients (37%) were female. The silastic splint was inserted in the right middle meatus in 22 patients (45%) and in the left side in 27 patients (55%), Interestingly, the patients did not complain of pain/discomfort difference between the splinted and non-splinted sides while the splint was in place. Also, splint removal was well tolerated by all patients and no patient had vaso-vagal attack or significant bleeding necessitated nasal pack. Splint complications such as movement from its place or aspiration were not reported.The average preoperative VAS for nasal obstruction in the splinted sides was 7.5 (SD\u00a0\u00b1\u00a02.3), while in the non-splinted sides it was 6.3 (SD\u00a0\u00b1\u00a02.7). For nasal discharge, the VAS was 3.9 (SD\u00a0\u00b1\u00a03.2) in the splinted sides versus 3.1(SD\u00a0\u00b1\u00a03.1) in the non-splinted sides.st week visit after surgery, there was no statistically significant difference between the splinted and non-splinted sides for all investigated parameters, At the1P\u00a0=\u00a00.037). Pain, crusting, pus in the middle meatus, nasal discharge and obstruction were statistically not significant between both sides, After 1- month of surgery, adhesions were seen in 10% of the splinted sides, while it was in 26% of the non-splinted sides with a significant difference (P\u00a0=\u00a00.007). All other examined parameters remained statistically insignificant between both sides, At the 6- month follow-up visit, the adhesions rate remained 10% in the splinted sides, however the rate increased to 32% in the non-splinted sides  compared to 13 non-splinted sides (26%) after 1-month of surgery (P\u00a0=\u00a00.037). This incidence remained steady for the splinted sides, while it increased to 32% for the non-splinted sides after 6- months follow-up time.Although different techniques and packing materials to prevent middle meatal adhesions after ESS have been employed, no universal agreement on their significant efficacy .Making mucosal abrasions between the middle turbinate and nasal septum for permanent medialization of the middle turbinate has been proposed by Friedman and his colleagues . This maSilastic sheet has been used in the middle ear for prevention of adhesions between tympanic membrane and middle ear mucosa with high success rates. It has been found that silastic sheet promotes middle ear mucosa healing without apparent foreign body reaction, rejection, or chronic inflammatory response . The proThe ease of the technique with high success rate, and few complications, encouraged other authors to conduct a prospective study using various shapes of silicon stent. Lee and Lee  conducteA prospective double-blind randomized controlled study was conducted by Baguley et al.  to invesAll authors who conducted studies on middle meatal silastic splints with various shapes found significant reduction in adhesions rate between splinted and non-splinted sides. Similarly, silastic splints were very well tolerated inside middle meatus and the reported complications rate was very low. Manji and his colleagues  conducteWe agree with lee and lee  that theThe difference in success rates could be explained by: (1) disease selection as all authors included both CRSwNP and CRSsNP in their inclusion criteria, (2) status of the middle turbinate because stable and/or non-traumatized middle turbinate affect synechiae formation, (3) shape of the splint may play a role. In this study, we tried to overcome these factors since all of our patients had CRSwNP and had unstable and/or had debrided polypoidal middle turbinates. Fortunately, we a achieved a high success rate of adhesions free middle meatus in 90% of cases which are close and coincide with reported ones. The splint was well tolerated by all patients with low pain score on VAS which was comparable to the non-splinted side. We did not find a statistically significant difference for pus in the middle meatus, crusting, nasal discharge, and nasal obstruction between the splinted and non-splinted sides throughout the follow-up time of 6 months.We preferred to use inverted U-shape splint because it takes the curvature of the root of the middle turbinate. An opening in the superior part of the splint was not made as we do not think that the dome of the splint reaches up to level of the frontal recess and interfere with mucus drainage. In addition, a small crust can block the superior opening. The long suture ends in our splint allows easy removal in cases with congested nose with narrow space and even when the ends were retracted, they were pulled anteriorly by suction.Splint was removed 2 weeks after surgery in all previous reports depending on Lee and Lee  descript5Middle meatal silastic splint is significantly reducing adhesions between the middle turbinate and lateral nasal wall with low complications rate in CRSwNP patients undergoing ESS.It is well tolerated by patients with insignificant pain and it is cost effective compared to other packing materials. Our results support its usage when the middle turbinate is unstable or traumatized during surgery.This work was approved by the institutional review board committee of King Abdullah University Hospital (Jordan university of Science and Technology), IRB number 26/75/2014.The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guidelines on human experimentation of King Abdulla University Hospital and with the Helsinki Declaration of 1975, as revised in 2008.No fund was received.Haitham Odat.Informed consents were obtained from all patients.All authors contributed significantly in study design, data collection, data analysis, drafting the manuscript, and revising the study.Not commissioned, externally peer reviewed.1Name of the registry: Research Registry.2Unique Identifying number or registration ID: researchregistry5855.3Hyperlink to your specific registration (must be publicly accessible and will be checked):https://www.researchregistry.com/browse-the-registry#home/No conflict of interest to declare."}
+{"text": "The formation of fluid-filled microvacuoles, termed glistenings, is a common complication of intraocular lenses (IOLs) made from hydrophobic acrylate. Using our well-established in-vitro laboratory method, we evaluated a new IOL material\u2019s resistance to glistening formation.2) was evaluated in five sections of each IOL. The results for each model were compared and rated on a modified Miyata Scale for grading glistening severity.An in-vitro stress test for glistening induction was performed on 20 samples of hydrophobic acrylic IOLs: ten of the new Eyecryl ASHFY600  compared with ten samples of AcrySof IQ SN60WF . The number of microvacuoles per square millimetre  MVs/mm2. The lowest number of glistenings was found in the five sections of the Eyecryl ASHFY600 with 0.52 (\u00b10.24) MVs/mm2. Mean value of the Eyecryl ASHFY600 IOL, using the Miyata Scale, was Zero.In all cases, glistening number was higher in the central section of the IOL optic than in the periphery. Mean number of MV/mmIn this in-vitro laboratory study, the new hydrophobic acrylic IOL showed a high resistance to microvacuole formation. Results from this in-vitro study suggest that glistening numbers will be low in clinical use in the Eyecryl ASHFY600. Hydrophobic acrylic intraocular lenses (IOLs) can develop a whitish, opaque material change under certain environmental conditions or over time , 2. ThisThe Acrysof hydrophobic acrylic IOL material  has become increasingly popular since the 1990s and is now a widely used IOL material that is approved by all regulatory authorities around the world. Since its introduction, increasing light scattering due to glistening formation has been observed in lenses made from Acrysof IOL material . Miyata We evaluated, using an established in vitro laboratory method, the formation of glistenings of a new hydrophobic IOL, one which the manufacturer claims is more resistant to glistening formation: the Eyecryl Plus ASHFY600, and compared it to the well-established and accepted AcrySof IQ SN60WF.Ten monofocal Eyecryl Plus ASHFY600 IOLs  and ten monofocal AcrySof IQ SN60WF IOLs  were tested for their resistance to glistening formation. All IOLs had the same refractive power of +\u200921.0 dioptres. The Eyecryl Plus ASHFY600 IOL and AcrySof IQ SN60WF are both single-piece IOLs, made from a hydrophobic acrylic material (Table\u00a0Microvacuoles (glistenings) were induced in-vitro by temperature changes using an established accelerated aging protocol as previously described in our earlier studies , 10. In All samples were examined under an EMZ-8TR Trinocular Zoom Stereo microscope . Microscopic images of all IOLs were taken immediately after aging process using an Infinity-2CB digital camera  Fig.\u00a0. After pImage analysis was performed using the ImageJ software 1.49v.  Prior to2. Given number of glistenings was divided by 1.63 to obtain the number of microvacuoles per square millimetre (MVs/mm2).A 1200\u2009\u00d7\u20091600 pixels area of the images in 90-fold magnification was selected to evaluate the number of glistenings. The central section was observed to correspond to the region with the highest glistening density. An image of a micrometer in 90-fold magnification was used to calibrate results with the dimensions of the lens to determine the density of glistenings. As 1\u2009mm corresponds to 1086 Pixels and the original image size was 1200 Pixels\u2009\u00d7\u20091600 Pixels, total image size was 1.63\u2009mm2), grade 1 (25\u2013100 MVs/mm2), grade 2 (100\u2013200 MVs/mm2), grade 3 (>\u2009200 MVs/mm2).The number of glistenings of the central part of the lenses was compared to a modification of the Miyata scale : Grade 02 in the central part and from all five sections was averaged for ten IOLs from each group and given as mean (\u00b1standard deviation). Statistical analysis was performed using Excel V.14.7.7  performing two-sided student\u2019s t-tests. A P-value less than 0.05 was considered statistically significant.The number of MVs/mm2. The lowest amount of glistenings was obtained averaging the five sections of the Eyecryl Plus ASHFY600, with 0.52 (\u00b10.24) MVs/mm2. For the AcrySof IOLs the glistening number in the central part was higher compared to the value of all 5 sections (p\u00a0<\u2009\u00a00.05), for the ASHFY600 both values were very similar, without a statistically significant difference (p\u00a0=\u20090.32) .In general, hydrophobic acrylate has some advantages over other IOL materials. Lenses made of hydrophobic acrylate show a lower tendency to develop posterior capsule opacification in comparison to those made of polymethyl methacrylate (PMMA) or hydrophilic acrylate . Complic2 at a special frequency of 100 lp/mm and a 3-mm-aperture [2 showed elevated straylight levels of 19.3\u2009deg2/sr, which would result in difficulties for patients while driving [Despite these benefits, hydrophobic acrylic IOLs are prone to develop glistenings. This long-term change in the material can worsen the lens\u2019 optical performance , 9. In raperture . Thus, g driving . Fortuna2) compared to lenses produced in 2003 (315.7\u2009\u00b1\u2009149.4 MV/mm2). Our results showed similar values for Acrysof produced in 2017, with a mean number of central glistenings of 41.84 (\u00b127.67) MVs/mm2 suggesting a maintenance of the improved process that leads to the reduced glistening formation.In 2013, Thomes and Callaghan reported on the continuous improvements (for which they unfortunately do not provide details) in manufacturing process of the Acrysof copolymer intended to reduce the incidence of glistening formation. They compared AcrySof lenses manufactured in 2003 with those made in 2012 [The Eyecryl ASHFY600 IOL is made from a hydrophobic acrylate polymer Table .The Eyecryl lens is manufactured by lathe-cutting the polymer which is different to the way Acrysof IOL is made, which is cast-moulding manufactured. Possibly the Eyecryl lens retains a more homogenous copolymer distribution within the final IOL whereas the cast-moulding procedure of the Acrysof lens might be rearranging the polymer distribution. In cast-moulding, care must be taken to avoid the development of inhomogeneities that can re-distribute co-polymers, chances which would make the lens susceptible to further material changes such as microvacuole formation . In a coAfter shaping the lens by lathe-cutting or cast-moulding, a subsequent step in manufacturing usually includes a polishing process. This stage has been shown to be the potential cause of postoperative material changes in hydrophilic acrylic lenses from a series of lenses affected by opacification, the residual polishing materials, like Aluminium Oxide, might have remained on the lens surface and provoking the postoperative clouding of the lenses .Thus, the IOL production process as well as the polymer are crucial elements in providing a lens with a resistance to material changes. Our results suggest that lathe-cutting a lens is superior to cast-moulding and we consider the new technologies, such as laser-cutting the lens, might further improve IOL manufacturing.Another approach to reduce the tendency for glistening formation is to improve the polymer by introducing hydrophobic IOL polymer compositions that have increased hygroscopy. Hygroscopy describes a material\u2019s ability to absorb and hold water inside the material. Water entering the material connects with the hydrophilic groups, thus avoiding water accumulation in vacuoles or pockets and forming glistenings . The morAs described above, even though improvements in the Acrysof material between 2003 and 2012 led to an increasing resistance to glistening formation, one can still induce glistenings in these lenses [2 all over the IOL and 0.74 (\u00b10.54) MV/mm2 in its central part after accelerated aging, the corresponding grade on the Miyata Scale was 0 for all tested lenses. Resistance against glistening formation was superior to the well-established AcrySof IQ SN60WF IOL, which in comparison showed values of 19.89 (\u00b110.57) MV/mm2 all over the IOL and 41.84 (\u00b127.67) MV/mm2 in the centre of the lens optic.The new Eyecryl ASHFY600 IOL has low tendency towards glistening formation. With a mean value of 0.52 (\u00b10.24) MV/mm"}
+{"text": "This collection presents an array of articles addressing the non-dopaminergic mechanisms in Parkinson\u2019s disease (PD). The dopaminergic system has been the main focus for almost 70 years and dopaminergic-based strategies still remain the best symptomatic medication to improve quality of life of parkinsonian patients. However, the interest in non-dopaminergic systems, even beyond neurotransmission itself, has grown with the accumulation of data showing that PD cannot be considered as a pure motor disease and understood only through the prism of the nigrostriatal dopaminergic pathway deficiency. Indeed, the disease progresses through different stages and damages to other areas and neurochemical systems precede the degeneration of dopaminergic neurons in the substantia nigra compacta (SNc). The other affected systems participate in the so-called prodromal or pre-symptomatic phases and contribute to the malfunctioning of the motor and non-motor circuits. Meanwhile, the non-motor symptoms are increasingly recognized in PD. They severely alter the quality of life of the patients since they are often more debilitating than the motor ones and poorly respond to dopaminergic-based therapies. Finally, the studies addressing the mechanisms of action of antiparkinsonian drugs have revealed that their benefits/side effects involved mechanisms other than the dopaminergic ones.Paredes-Rodriguez et\u00a0al. review the situation of the noradrenergic system in PD. The noradrenergic system issued from the locus coeruleus can display severe damages in the disease, presumably before the dopaminergic neurons according to the hypothesis that the disease progresses from the caudal to the rostral parts of the brainstem. In fact, the authors recall that the noradrenergic system exerts anti-inflammatory and neuroprotective effect on the dopaminergic degeneration, and noradrenergic damage can consequently favor the progress of the disease. Even though the noradrenergic system participates in the mechanism of action of L-DOPA, this interaction is still unclear and needs further investigation. Mallet et\u00a0al. review another classical non-dopaminergic neurotransmitter that has been considered in PD even before dopamine, namely acetylcholine. The authors focus their analysis on the striatal cholinergic interneurons, which display specific electrophysiological features but are still difficult to apprehend due to their sparse distribution. Based on recent literature, the authors report the advantages of using optogenetic approaches possibly combined with computational studies to investigate the role of cholinergic interneurons in the physiology and pathophysiology of motor behavior and cognition. On the other hand, the serotonergic system is one of the most studied non-dopaminergic neurotransmitter systems during the last decade in view of the demonstration of its primary participation in dyskinesia induced by L-DOPA. Schintu et\u00a0al. rendered hemiparkinsonian inbred depression-like flinders sensitive line (FSL) rats and studied their motor responses to chronic L-DOPA. In contrast to control animals, the hemiparkinsonian FSL rats did not display sensitization in the turning response to apomorphine or L-DOPA and only a weak increase in dyskinesia. The marked differences in L-DOPA-induced dyskinesia were not associated with modification of the striatal expression of deltaFosB, a supposed marker of this side effect.Among the classical non-dopaminergic systems involved in PD, Chambers et\u00a0al. recall that some neurons in the pedunculopontine nucleus, a complex structure housing GABAergic, glutamatergic, and cholinergic neurons, degenerate in PD. The neuronal degenerative process in the pedunculopontine nucleus likely participates in motor disability, notably gait control, and in non-motor symptoms including sleep deficits, likely due to the innervation of brain regions different from the SNc. Murueta-Goyena et\u00a0al. cover the non-motor symptoms in PD and the GABAergic system alterations. They recall the participation of GABAergic mechanisms in various pre-symptomatic disturbances including olfactory loss, gastrointestinal abnormalities, visual alterations, cognitive and mood disorders, and make a special emphasis on rapid eye movement sleep behavior disorder. In this line, Meideiros et\u00a0al. describe how neurotoxic and genetic manipulations of rodents have been utilized to reproduce some of the major sleep disturbances associated with PD. They further discuss how these abnormalities can be linked to noradrenaline, serotonin, and orexin transmission. Several neurotransmitter imbalances operate during the disease and may participate in the complexity and heterogeneity of the neuropsychiatric symptoms exhibited by PD patients. Dujardin and Sgambato propose a thorough examination of the neuropsychiatric symptoms, including depression, anxiety, apathy, psychosis, and impulse control disorders, their prevalence and pathophysiology. The authors deepened their analysis by looking at the relative contribution of the neurotransmitter systems dopamine, noradrenaline, serotonin, acetylcholine, GABA, and glutamate in each of the above-mentioned neuropsychiatric symptoms in both PD patients and animal models.The imbalance of neurotransmitters in the brain is multiple in part due to degenerative processes away from the SNc. Ingallinesi et\u00a0al. focused on GPR88, almost exclusively expressed by the medium spiny GABAergic neurons of the striatum. They used a lentiviral-mediated knock-down approach of the receptor in the 6-hydroxydopamine neurotoxic rat model of PD and reported reduced amphetamine- but increased L-DOPA-induced turning behavior. These behavioral effects were paralleled by a normalization of some striatal tissue markers such as GAD67 expression, although the striatal expression of deltaFosB did not parallel L-DOPA-induced dyskinesia. Yoshio et\u00a0al. also recall the interest of another orphan receptor, the GPR143. This receptor has been proposed as the target of L-DOPA, which would be its endogenous ligand at least in the retina. The authors review the possible contribution of GPR143 in PD with a special emphasis on its colocalization with the protein \u03b1-synuclein in Lewy bodies.Beyond the neurotransmitter systems, there are some opportunities to develop treatments on specific targets, as the orphan receptors GPR88 and GPR143. Gomez-Benito et\u00a0al. discuss and provide detailed comparative analysis of two models based on \u03b1-synuclein: the \u03b1-synuclein pre-formed fibril model and the recombinant adeno-associated virus vector-mediated \u03b1-Synuclein overexpression models. The multiplicity and development of novel models are necessary for progressing in the understanding of the disease and help to decipher specific mechanisms. In any case, far beyond the consideration of dopaminergic neurons of the SNc, PD is a multifactorial disease evolving before the onset of motor symptoms.The availability of new animal models conditions the progress of research in the involvement of different neurotransmitters in the motor and non-motor symptoms of the disease. 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": "Introduction: Newborns are subject to many painful procedures. Pharmacological and non-pharmacological approaches alone are not enough, and it is necessary to consider other contributing elements such as the environment, interprofessional collaboration and parental involvement. The aim of this feasibility study was to explore interprofessionality and the role of parents in improving the management of painful procedures in newborns and pain management strategies.Materials and Methods: a pre-post feasibility study using a mixed method approach was conducted. Questionnaires, interviews and focus groups were used to describe the parents' views on their child's pain management and involvement in care as well as to explore the level of interprofessionality and feasibility.Results: Collaboration between physicians and nurses improved following the implementation of a complex interprofessional intervention involving professionals, parents and newborns. In spite of improving professional collaboration in procedural pain management, parents were attributed a passive role or only marginally involved in in the infant's pain management. However, parents stated\u2014as elicited by the questionnaires and interviews\u2014that they wished to receive more information and be included in painful procedures executed on their infant.Discussion: Management of painful procedures in neonates needs to be changed. Interprofessional collaboration contributes to improved procedural pain management in neonates. It is essential to include parents as active members in the interprofessional healthcare team. Newborns, especially premature newborns, are a patient group that is highly vulnerable to pain. Numerous studies have shown that newborns are subjected to many painful procedures in a stressful environment during their neonatal hospitalization \u20136. DespiIn procedural pain management, many interfaces related to skills and tasks can be identified. Healthcare professionals' attitudes and beliefs, and parents' lack of knowledge and information can influence and limit optimal pain management. Pain relief constitutes an ethical foundation of the nursing profession, yet it is not always given adequate priority \u201311. NursThe aim of this feasibility study, was to explore interprofessionality and the role of parents in improving the management of painful procedures in newborns and pain management strategies.A pre-post study with a mixed-method approach was used. A mixed method design was chosen because it has the advantage of increasing the understanding of how interventions should be structured, describing the context and promoting better interpretation of results \u201336. We fThis feasibility study was conducted in a neonatal unit level IIb at a regional hospital in Southern Switzerland. This study was approved by the local ethics committee (CE TI 330). The neonatology unit includes a total of six beds with three intermediate care beds and three specialized care beds. According to unit statistics, 128 newborns were admitted in 2018 representing 878 hospital days.In a previous study, we reported the development of the NEODOL intervention in accordance with phase one of the MRC guidelines  . The NEOThe study is divided into 3 phases: pre-intervention (T0), implementation of the NEODOL intervention (T1), and post-intervention (T2). The total duration of the T1 intervention was 5 months .The NEODOL intervention was implemented for a feasibility study. First, the healthcare professionals  received a structured interprofessional education program  . A booklThe Italian version of the questionnaire \u201cAssessment of Interprofessional Team Collaboration Scale\u201d (AICTS-II) was used to assess the level of interprofessional collaboration \u201346. AICTThe questionnaire \u201cParent Attitudes about Infant Nociception\u201d (PAIN)  was usedFor both questionnaires, permission for use was obtained from the authors , 46, 47.Focus groups with healthcare professionals were conducted to determine intervention acceptability and feasibility (barriers and facilitators to the implementation) and to gather suggestions for refining the NEODOL intervention to render it more user-friendly. An interview-guide was developed addressing the experience with the NEODOL intervention and difficulties encountered. Participants were invited to provide feedback on interprofessional collaboration between physicians and nurses as well as on the parents' involvement. These focus groups were conducted at the end of the 5-month pilot period.Semi-directed interviews with parents were conducted and recorded. The parents had a child that was hospitalized in the neonatology unit. In order to facilitate the organization of the interviews, they were conducted only with the mothers and in their homes after the infant was discharged. An interview guide was developed that addressed the experience of painful procedures, concerns about pain and parental involvement in care.In order to partially minimize social desirability bias, the focus groups and interviews were conducted by persons who were not involved in the research but were trained in qualitative methods.Data analysis were performed using SPSS software . For each of the questionnaires, due to the small sample, only descriptive analyses were performed.U-tests were used for evaluating differences in AICTS scores between nurses and physicians.For AICTS-II, scores for the different domains  were calculated by the mean of the items in each domain, where values less than or equal to four indicate an inadequate perception of the domain of interprofessional collaboration , 45. ManData analysis were managed using MAXQDA software (version 18). The interviews and focus group were transcribed, and thematic analysis was performed . This anN = 47) and in T2 was 60% (N = 37)  . AverageThe use of AICTS-II questionnaire provided baseline information on staff attitudes and perceptions of interprofessional collaboration. Staff perceptions of team functioning before and after the NEODOL intervention was also determined. The results of the AICTS total and subscales among all participants as well as in subgroups are presented in A total of three focus groups were conducted. In two focus groups doctors and nurses both participated. One focus group consisted of nurses only with different levels of professional experience. In total, 20 healthcare professionals (13 nurses and 7 physicians) participated in the focus groups. The mean age was 36.2 years (range 28\u201356).Increasing awareness of pain,\u201d \u201cInterprofessional collaboration,\u201d \u201cParental involvement,\u201d \u201cCulture change,\u201d \u201cFacilitators to using NEODOL\u201d and \u201cBarriers to using NEODOL.\u201d Physicians and nurses generally reported positive outcomes from implementing the NEODOL intervention.A total of six categories emerged: \u201cInterprofessional education was highly regarded and considered to increased knowledge among both nurses and physicians. A more reflective and collaborative practice has developed as a result of this awareness, as one nurse commented that the NEODOL intervention has given more attention than before to perform certain types of care. A physician noted: \u201cNEODOL (.) has helped many people to understand that there are various ways of working. There is one way, as I have often heard, out of habit, and there is, in contrast, a wise way of working. So, when I enter an environment where you work out of habit, I learn to work out of habit, and I do not take into account other factors. NEODOL raises awareness to consider: \u201cevery time I prescribe something, every time I take action, I have to rationalize why I do it, how I do it, and when I do it'.\u201dNurses and physicians reported positive experiences of interprofessional collaboration with better communication and collaboration in organizing pain care. One nurse stated: \u201cA positive experience was also working in collaboration with physicians. Maybe, you had a blood test in the morning, (.) at 7.30 am. And you knew your newborn that all care was planned for 9 am. And if it was not an urgent blood test, of course, I always encountered positive collaboration.\u201dIn each focus groups, the participants spoke about their work in a way that revealed their commitment to managing procedural pain better. This was a common thread throughout. In one of the focus groups of nurses, the importance was described as follows: \u201cI feel like I am doing many things that we used to do before anyway, but without even realizing it. The fact is, they are now documented.\u201d Another nurse commented: \u201cWith NEODOL, care is more patient-centered (.) It is no longer based on the organization of the ward (.) There is clinical reasoning (.) And you realize it is feasible. It is not impossible.Differences in professional attitudes toward parental involvement in pain management were observed. A few nurses practiced parental involvement and found it to be a positive experience. For others, parental involvement was difficult because the professionals were afraid of parental judgment, of not succeeding or simply out of routine/habit. \u201cSo my perception is that sometimes (.) the parents are not involved (.), because you are a little afraid of the judgment, or the pressure that a parent can eh rightly have against you because you are doing a painful procedure on the child, to their child (\u2026) In my opinion, when there is too little experience, there is a tendency perhaps not to call the parent, to do the procedure on your own, without feeling the additional pressure from the parents.\u201d Other nurses reported that parents were too stressed, and that it was their duty as nurses to protect the parents from their child's pain. Parental involvement was controlled by the nurses and not in partnership with the parents. A nurse said: \u201cIf the parent is already anxious and we say: \u2018hold your child in your arms and we will do the bloodwork from the heel', the parents start to get more anxious. Then, the babies become agitated as they feel the agitation and anxiety of their mom or dad. And in that case, maybe it is better that the parents were not involved (laughter). Honestly, it would be better in situations like these that I give the baby a pacifier with glucose and swaddle him/her in the blanket, so the baby stays calm and quiet. In times like these, it is more difficult and time-consuming to involve the parents.\u201d One physician reported that parental involvement depended on the type of procedure performed.The reminders were considered to be facilitators for the implementation. The pain champion promoted change. This role was considered invaluable for promoting the use of the NEODOL intervention in practice. The pain champion was locally based, accessible and also understood the contextual complexities of the unit such as the workload, culture and challenges specific to the patient population.Organizational resources constituted the main barriers, namely time, resources and workload. \u201cThere are situations, in which things have gone very well but (.) it depends a lot on both the parents and the time you have (.) especially (.) the number of patients you have, in order to devote time to the babies. It is true that there have been situations where painful procedures have been done and the child has not felt anything. This (.) in my opinion really needs time to do everything.\u201dBefore the intervention was implemented, 10 parents completed the PAIN questionnaire. A total of 14 other parents responded to the questionnaire after the implementation. Parents rated their infant's most severe pain on a scale of 1 to 10\u2013no pain to severe pain\u2014with an average of 4.4 (SD 2.55) before the introduction of NEODOL and 3.93 (SD 2.81) after. Parents expected infants to experience less pain with 2.8 (SD 2.35) before implementation and 3.31 (SD 1.75) after. However, they also expected that a high degree of pain relief was provided to their infant. Parents, therefore, understood pain better after the NEODOL intervention.n = 7/10; 70%) reported that healthcare professionals had never or not often asked them about their preferences to be present or not during painful procedures. After the intervention,  of the parents had not been asked about their preferences. The presence of parents during painful procedures seemed higher post-intervention. In pre-intervention, only 30% (n = 3/10) of parents reported that they were shown how to identify their infant's signs of pain but 64% (n = 9/14) in post-intervention. A total of 18 parents out of 24 (75%) had responded to the PAIN questionnaire (pre and post intervention). Most of these comments  indicated that they wanted to be involved \u201calways\u201d or \u201cas much as possible\u201d and receive more information about their baby's pain.Before the NEODOL intervention, parents , that of the fathers was 39 years (range 28\u201351). The newborns were two girls and two boys with a mean gestational age of 36.25 (range 30\u201340). Their mean birth weight was 2,531 g (range 1400\u20133175).Analysis yielded five main themes. The first concerns the experience of being a parent in neonatology. The second is the appreciation of professional skills. The third relates to infant procedural pain as a source of parental stress. The fourth describes the parental involvement in painful procedures and the last one concerns the NEODOL intervention.Parenting in neonatology is a major challenge because there is much uncertainty. Parents are destabilized by this difficult experience and experience guilt. One mother explains: \u201cThe hardest moment, (.) is when they took me out and I had to leave the baby. I had a knot in my throat. That was the only time I started crying and (.) I had a hard time (bitter laughter). Because (\u2026) it is very hard to have to go home and leave the baby there.\u201d The neonatal unit was described as having limited space and privacy. A strict schedule for care existed, which did not make the parents' experience any easier. Another mother reported: \u201cIt was clearly not (.) easy for us parents, because it is the first child. The first experience so (.)we did not know how to behave.\u201dThe mothers described their distress at the sight of various procedures or technical acts such as insertion of nasogastric tubes or heel punctures. Observing their baby being treated was difficult: \u201cThe first time I saw (.) the catheter. Putting the tube in is the hardest part (.) It really bothered the baby. I interpreted it as painful for the baby.\u201dParents had confidence in the clinical staff and were satisfied with the care. The parents were grateful for the professionalism of the nurses and doctors. Professional support and assistance were reassuring to parents. One mother stated: \u201cI must say that they always took their time, for example they warmed her foot, they really tried to be as minimally invasive as possible; they waited until she was calm. They did everything calmly, with a lot of patience. I appreciate that.\u201dParents wanted to be involved in their child's care. However, they needed adequate information in order to support their child during a painful procedure. Generally, parents had a passive role such as simply being present at their child's side: One mother recounted: \u201cAnd then he was crying a lot, so I stood there just to make him feel the warmth. (.) They gave him shots (\u2026) almost every morning (.)he was in pain, suffering (.) that is what I thought.\u201d Another mother pointed out that she was not present during various painful procedures: \u201cThen, they put it [the nasogastric tube] in his nose. They did not call me in, because I am sure he will have cried (bitter laughter). I only wondered about that afterwards when [the nurse] told me, eh, he [baby] was pricked to have a venous line, (.) I said to myself, no, poor thing! They did not let me be of assistance. That is to say they would not let me come in because she would be crying very loudly. Poor thing, only just born (sighs). Maybe in these moments, they do not wish for parents to be present, and so they did not tell me. (\u2026) I was not present, I did not see her cry.\u201d Parents did not know what they could do to help or what their child needed. The parents wished for better information. Healthcare professionals should provide information and education on painful procedures, preferably in a calm environment. Such information should be offered without parents having to ask for it.Parents appreciated this intervention as it increased the sensitivity of the healthcare professionals. Through NEODOL, parents saw a chance to become more involved in their child's painful procedures. \u201cThe thing, at least from my point of view, \u2026 to know what they [healthcare professionals] are doing, to know how they [children] may feel while undergoing these treatments, to be involved as much as possible, to feel that I as a parent can help.\u201d To increase parental involvement, it was suggested: \u201cJust this: [as parents] to be better informed about the procedures. That it is it: to know when they do it, and at what time to be present.\u201dThe purpose of this study was to explore interprofessionality in a pediatric neonatology ward, and the role of parents in improving the management of painful procedures in newborns. For the development of the NEODOL intervention, Craig's Social Communication Model of Pain proved to be an invaluable guide to understand the interactions between various dimensions. This model provided insight into the influence of biological, psychological and social factors as well as of the context on pain experience. Drawing on this model, these elements were included in NEODOL, thereby providing a multifaceted approach to improve procedural pain management in neonates.In reporting this feasibility study, we followed the phase two of revised MRC guidelines , 38. ThuA competent, collaborative, patient-centered, interprofessional team is needed to ensure the pain management related to procedures . HoweverWith the bundle of care, healthcare professionals reported being able to communicate and collaborate more effectively as a team . The preNEODOL introduced parents as members of the interprofessional team as advocated by this recent systematic review . ParentsAlthough parents are not present 24 h a day, they want to be better informed about their baby's care and to be involved in the management of neonatal pain as much as possible , 29, 60.Craig's Social Communication Model of Pain, which has informed this study, highlights that pain must be approached in a multi-dimensional manner. All relevant stakeholders need to be included. The model provides a basis for conceptualizing interprofessional collaboration, which not only refers to healthcare professionals but also includes parents. For optimal management of painful procedures, collaboration between healthcare team members and parents is essential.Several limitations of this study can be identified. Only one neonatology unit was involved in this feasibility and acceptability study. The participating pediatric unit consists of 19 beds, including six for neonatology. Recruitment was challenging. As the objective of this study was to determine feasibility and acceptability of the intervention , 50, smaWe found that the NEODOL intervention targeting all groups  constitutes an innovative way to demonstrate the importance of interprofessional collaboration in the management of painful procedures. This feasibility and acceptability study shows that we are moving in the right direction and highlights the need for further research.The datasets presented in this article are not readily available because of patient confidentiality and participant privacy. Requests to access the datasets should be directed to corresponding author Colette Balice-Bourgois.The studies involving human participants were reviewed and approved by Ethics Committee Tessin, Bellinzona (Swissethics). The patients/participants provided their written informed consent to participate in this study.CB-B is the main author of the manuscript, organizing all aspects of the article including data extraction, drafting of the initial manuscript, and making revisions. MZ-S guided the conceptualization of the article and supervised drafting and revising of the manuscript. MZ-S, GS, and CN all provided guidance critically reviewed the manuscript. All authors contributed to manuscript revision, read 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": "The goal of resuscitative fluid therapy is to rapidly expand circulating blood volume in order to restore tissue perfusion. Although this therapy often serves to improve macrohemodynamic parameters, it can be associated with adverse effects on the microcirculation and endothelium. The endothelial surface layer (ESL) provides a protective barrier over the endothelium and is important for regulating transvascular fluid movement, vasomotor tone, coagulation, and inflammation. Shedding or thinning of the ESL can promote interstitial edema and inflammation and may cause microcirculatory dysfunction. The pathophysiologic perturbations of critical illness and rapid, large-volume fluid therapy both cause shedding or thinning of the ESL. Research suggests that restricting the volume of crystalloid, or \u201cclear\u201d fluid, may preserve some ESL integrity and improve outcome based on animal experimental models and preliminary clinical trials in people. This narrative review critically evaluates the evidence for the detrimental effects of resuscitative fluid therapy on the ESL and provides suggestions for future research directions in this field. The importance of microcirculatory function and health of the endothelium has become a large area of interest for criticalists in the last two decades. Over this time, fluid resuscitation strategies have experienced a shift in perspective. Aggressive \u201cclear\u201d fluid resuscitation was once considered vital for stabilization of macrohemodynamic variables. However, in certain patient cohorts this approach has now been shown to either not improve outcome or worsen outcome \u20134. ThougEG when referring to specific components of the structure, or biomarkers of these components, whereas ESL is used when referring to the layer as a whole. In regards to shedding of the ESL, the term ESL will be used when the evidence supports loss of the whole layer and the term EG used when the evidence only supports that isolated components have been shed. Use of the term glycocalyx refers to any type of cell surface glycocalyx and is not restricted to the endothelium, such as during discussion of glycocalyx shedding biomarkers.The ESL includes a structural scaffold, the endothelial glycocalyx (EG), and associated molecules suspended within a plasma layer. We use the term Resuscitative fluid therapy includes a range of fluid choices, including isotonic crystalloid fluid, synthetic colloid fluid, and hyperosmolar crystalloid fluid, with the former being the most common type of fluid used . In geneBox 1Proposed mechanisms of ESL sheddingDilution of plasma proteinsRelease of natriuretic peptidesInflammatory cytokine release (certain fluid types)Possible downstream effectsExacerbation of inflammationMicrocirculatory dysfunctionIncreased vascular permeabilityIncreased interstitial edemaProthrombotic conditions*Proposed strategies that may mitigate ESL sheddingReduction in dose of clear fluidsSlowing down administration of resuscitative fluids\u201cEarlier\u201d vasopressor therapy for vasodilatory shockAdjunctive protein administration (such as plasma)*These proposed strategies are not based on evidence from clinical veterinary research and require further investigation. These guidelines are opinion of the authors only, after considering the breadth of evidence available. It is currently unknown if \u201cglycoprotective\u201d strategies benefit patients. Therapy should always be tailored to individual patient needs, with prioritization of reestablishing adequate perfusion and a thorough cost to benefit analysis.Most cells in the body are covered in a protective layer of carbohydrate scaffold, which houses many different molecules that serve a variety of functions. This surface layer is called a glycocalyx. The general structure of the glycocalyx has similarities between cell types, with only small variations in individual proteins or carbohydrate molecules. The EG coats the luminal surface of the endothelium and is a vital structure for cell signaling and transvascular permeability. It is composed of proteoglycans, glycosaminoglycans (GAGs), and glycoproteins . TogetheThe EG serves a range of functions, many of which are still being characterized. These functions include maintenance of a surface barrier that buffers circulating leucocytes, inhibits coagulation, regulates fluid flux, and communicates changes in vascular wall shear forces. The sulfated GAGs on the surface of the endothelium generate and maintain a net negative charge, which repels similarly charged cells from the endothelial surface . AlthougThe traditional Starling hypothesis describes a relationship between the intravascular and the interstitial colloid osmotic pressure and implies the importance of this relationship in determining net transvascular fluid filtration. Many studies in the last four decades, however, have built a body of evidence stating that it is mainly the low COP in the sub-glycocalyx area that creates the pressure gradient opposing capillary hydrostatic pressure , 26. TheThe EG also plays an important role to changing pressures and flow within the intravascular space. Proteoglycans, especially those with heparan sulfate chains, play an important role in responding to changes in vascular wall shear stress or changes in intravascular pressure , 30, 31.ex vivo models leads to increased expression of adhesion molecules, increased leucocyte adhesion, and increased cytokine production , detection of EG components on the surface of the endothelium, direct visualization of the ESL via tissue fixation and microscopy, and indirect visualization via real-time videomicroscopy. Clinical studies usually rely on measurement of circulating components of the EG, or EG biomarkers, in serum or plasma samples to assess systemic shedding. The most frequently reported EG biomarkers are syndecan-1, heparan sulfate, and hyaluronan. There are several limitations to relying on this kind of assessment of the ESL. Firstly, studies often only measure a single component of the EG at a single point in time. That particular component may have other sources of shedding. For example, syndecan-1 is not only present on the surface of the endothelium but also on epithelial cells and leucocytes \u201368. Alsoin vivo rather than interpreting circulating biomarker concentrations. These techniques are mostly reserved for use in laboratory models rather than clinical use. Specialized tissue fixation techniques can be used to directly visualize the ESL via electron microscopy, often employed in non-survival rodent models  are released from the cardiac atria and ventricles during stretch of the myocardium . These pn = 26) showed no significant rise in BNP or biomarker concentrations (syndecan-1 and heparan sulfate) after 25 mL/kg of LRS given over 30 min during surgery (n = 15 per group) (n = 6) and baseline variability may have hampered the ability to detect a difference  . A recenfference .One of the challenges of interpreting studies that measure EG biomarker concentrations as the primary assessment of EG shedding is the variability in choice of biomarker. Across multiple studies investigating the effect of fluid loading on the EG, it appears that hyaluronan is consistently increased immediately after a large volume of crystalloid, or synthetic colloid fluid in the setting of hypervolemia , 126. SyMuch of the evidence concerning EG or ESL shedding after fluid therapy concerns the use of large volumes of crystalloid fluid. Administering other fluid types that have less redistribution to the interstitium may theoretically be associated with less EG shedding. This section assesses the evidence for the comparative effect of crystalloid, synthetic colloid, and hypertonic fluids on the EG or ESL.Multiple rodent studies have shown that administration of large volumes of crystalloid fluid for hemorrhagic shock is associated with more EG shedding, compared to fluids containing protein , 130\u2013133n = 6) with those that received 80 mL/kg of Plasmalyte-148\u00ae (n = 6) . Detection of between-group differences was also likely affected by small sample size in this study (n = 8 per group), similar to the study in dogs.There is a scarcity of \u201clarge animal\u201d models, or those including pigs, sheep, and dogs, that have assessed ESL shedding after fluid resuscitation. This may be partially due to a limitation on validated assays available for measuring glycocalyx biomarkers across these species. Current commercially available validated options for assessing EG shedding are restricted to circulating hyaluronan concentration. A canine hemorrhagic shock model did not detect a significant difference in plasma hyaluronan concentrations when comparing dogs that received 20 mL/kg of FWB ( (n = 6) . Other d (n = 6) . Althougn = 6 per group)  . This stin vitro and animal model studies, including decreased leucocyte response to pro-inflammatory stimuli or circulatory shock states, when compared to other fluid types  volumes of crystalloid fluid intravenously as rapidly as possible, or as a bolus , 160. ThIt is much less known if restricting crystalloid fluid in other types of shock may be beneficial. In the setting of human trauma, discussion concerning restrictive crystalloid fluid strategies focuses on delayed resuscitation or permissive hypotension in order to avoid clot disruption and dilutional coagulopathy before definitive hemostasis , 165. AlFast fluid administration serves to rapidly improve clinical signs and macrohemodynamic variables, and has been the bread-and-butter of emergency and critical care medicine for decades. However, normalization of macrohemodynamic variables in people does not always translate to improved microcirculatory flow \u201349, 51. Intense ongoing debate surrounds the choice of fluid for critically ill patients, including crystalloid, synthetic colloid, and protein-containing solutions. These conversations surround relative fluid effectiveness and adverse effects, many of which are still yet to be fully elucidated for clinical relevance in certain species or disease subsets. Adding in the potential benefit or detriment of certain fluid types to preservation of the ESL and optimizing microcirculation is still premature. Bearing that in mind, the current evidence supports mitigating hemodilution and avoiding hypervolemia in order to optimize ESL recovery. There is theoretical benefit for the ESL to the use of protein-containing solutions, such as plasma and albumin, rather than \u201cclear\u201d fluids, but this must be carefully considered against any detrimental effects of blood products, including financial cost. At this stage, no clear recommendation can be made for veterinary medicine, and more clinical research is required.Many drugs have been explored as to their protective or resurrecting actions for the ESL and have been reviewed in detail elsewhere , 176. Thin vivo. Further work is required on developing validated, reliable EG biomarker assays and determining the relationship between these biomarkers and shedding of the ESL in vivo, especially for dogs and cats. Despite these current limitations, future research directions should also focus on strategies limiting crystalloid fluid volumes, especially in the setting of sepsis and early vasopressor drug therapy.The coating of endothelium by the EG and its associated molecules serves many functions in the body, both intact and when denuded. Infusion of large volumes of fluid causes disruption of this layer, which may propagate interstitial edema and inflammation. There is some evidence that slow fluid administration or restricting the volume of crystalloid fluid for shock resuscitation may benefit the patient. Exactly how to do this and for which patient subset this strategy benefits in veterinary medicine remains to be identified. Clinical veterinary research assessing the effect of glycoprotective therapy or strategies is currently limited by a lack of validated EG biomarker assays and affordable, reliable technology for estimating ESL thickness LS: preparation of manuscript and final approval. DH: revision of manuscript and final approval. Both 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": "Flexible composites as sensors are applied under a small voltage, but the effect of the external electrical field on the resistance is always ignored and unexplored by current research. Herein, we investigate the electrical resistance relaxation of anisotropic composites when they are subjected to an external electric field. The anisotropic composites were 3D-printed based on carbon-fiber-filled silicon rubber. Constant DC voltages were applied to the composites, and the output electrical current increased with time, namely the electrical resistance relax with time. The deflection and migration of carbon fibers are dominantly responsible for the resistance relaxation, and the angle\u2019s evolution of a carbon fiber, under the application and removal of the electrical field, was well observed. The other factor hindering the resistance relaxation is the increased temperature originating from the Joule heating effect. This work provides a new understanding in the working duration and the static characteristics of flexible composites. Flexible conductive composites have been widely fabricated as various sensors due to their response of electrical signals to various loadings ,2. VarioThe work duration and static characteristics of flexible sensors are of great significant to evaluate the sensing stability and reliability, especially referring to the piezoresistive sensors . ConstanRecently, our group 3d-printed an anisotropic composite based on carbon-fiber-filled conductive silicon rubber , and theOccasionally and interestingly, we found the characterized inherent resistance (>100 \u03a9) of various conductive polymer composites to be unstable when tested with a multimeter, and it was further proved in our printed composites , in whicHerein, the behavior of the printed composites under the external electric field is studied. The roles of Joule heating effect and the migration of fibers played on the resistance relaxation were experimentally characterized, in-situ observed and schematically revealed. The effect of the electrical field on the piezoresistivity was finally tested. All the experimental results would do a favor in better understanding on the working duration of conductive composites and sensors.The printing process was based on an extrusion device cooperated with a desktop printer and it was controlled by a computer. The printed composites were then pre-cured at 150 \u00b0C for 5 min and post-cured at 170 \u00b0C for 10 min. The polymer matrix material (methyl vinyl rubber) and vulcanizing agent were obtained from Blue-star Silicone Co. Ltd. Shanghai, China. The fillers of short carbon fibers purchased from Zhongli New Material Technology Co. Ltd. Cangzhou, China, whose nominal length was180 \u03bcm and diameter was 8 \u03bcm. The 3D printing device for the tested samples is shown in \u2225 and \u22a5 direction, respectively. \u2225 and \u22a5 represent test parallel and perpendicular to the orientation direction of the anisotropic composites, respectively.The method to characterize the resistance relaxation behavior of the printed composites under the external electric field is depicted in \u2225 direction and one sample in \u22a5 direction, with the dimension of 50 mm \u00d7 10 mm \u00d71 mm, was respectively inserted into the test fixture. Pressure was loaded on the test fixture by the counterweights, and the pressure interval was 20 kPa. Multimeter (Victor 86E) was connected to both ends of the printed composite to record the resistances.The piezoresistivity test of the printed composites is depicted in \u22a5 direction are displayed in dI) 13%, 12% and 10%, respectively, which indicate the resistance relaxation (dR) of 13%, 12% and 10%, respectively, according to Formula (1).The resistance relaxation of the printed composites under the external electric field is studied, and the experimental results in \u22a5 direction.The results demonstrate the stable resistance value is about 85~90% of the starting original test value. Our finding proves the thoughtless of related researched on the resistance creep in piezoresistive composites. Comprehensive consideration, of the combined effect of compressive load and electric field, is greatly helpful for recognizing and redefining the piezoresistive effect. What is emphasized is the current value is small (<6 mA) in the conductive path of \u2225 direction, as shown in \u2225 direction is similar to that in \u22a5 direction: namely, the electrical resistance relaxes with time under the external electric fields of 3~10 V. The curves slightly changed when the applied voltage further increased to 6 and 10 V, with the resulted electrical currents over 20 mA. To the contrary, the electrical current decreases (resistance creep) rapidly in the 0~3 min instead of the resistance relaxation in a low resulted current, which is highlighted with a yellow ellipse in The experimental curves are a bit different in the R = U/I) is calculated, with resistance relaxation from 2155 to 1845 \u03a9 after the 12 h of applied voltage, and it recovers to 2120 \u03a9 after the removal of electrical field for 6 h, which demonstrates the reversibility of the resistance relaxation after a long time (>6 h) removal of applied electrical field and further refers to the reversible transformation of the conductive pathway in the printed composites after the removal of electrical field.To judge whether the resistance relaxation is reversible, a printed sample was applied under a DC voltage of 3 V for about 12 h, and then the electric field was removed for a short time and a long time, respectively, and the electric field was applied again. The demonstrations are described in Two factors may weigh for the unusual electrical resistance relaxation as well as the rough curves, i.e., (1) according to the Joule heating effect, the temperature of composites increased during power-on, resulting in the positive temperature coefficient effect of the electrical resistance, and the resistance further increased. (2) The filler of carbon fibers may deflect and migrate under the external electric field, bringing about the rearrangements of conductive paths of the composites, and the resistances are therefore influenced.The temperatures changes are characterized with an infrared thermal image (FLIR-E6390). Joule\u2019s law expresses the relationship of heat generated by the applied voltage (Formula (2)), and the temperature of the composites increases when the generated heat (Q) is higher than the lost heat.\u22a5 direction substantially decreases the generated heat, and the corresponding temperature stays unchanged. The tests of the composites in \u2225 direction under 3 and 6 V are therefore depicted in \u2225 direction) for the temperature increase from 25 to 29 \u00b0C (The high resistance (\u22482 k\u03a9) in to 29 \u00b0C . The var\u2225 direction  were recorded. As shown in the blue circle in \u2225 direction. The compressive load together with the electrical field are responsible for the downward trend of \u22a5 direction with a higher relative resistance in \u22a5 direction. The other need to be highlighted is described in The aforementioned experimental results inspired us to verify the role of electrical field on the piezoresistivity; the result is demonstrated in \u22a5\u00a0direction, while slightly different in \u2225 direction. Two factors\u2019, including the Joule heating effect and interaction between fibers, weight for the resistance relaxation are experimentally discussed and verified. Our work will be beneficial for the better understanding on the working duration and static characterizations of flexible composites.In conclusion, this paper stresses the ignored effect of the electric field on the resistance relaxation/creep of flexible composites. The resistance relaxation of the printed composites under the external electric field (3\u201310 V) is well revealed. Resistance relaxes sharply in the 0~5 min and becomes stable later in the"}
+{"text": "The effect of aerobic exercise interventions on episodic memory performance among older adults without dementia remains a matter of intense debate. Prior meta-analyses examining this association have reported minimal improvements in episodic memory performance following exercise training but have also been plagued by several limitations, including restrictive inclusion criteria, combined sample populations, and infrequent examination of the effect of exercise parameters . To address these gaps, we conducted a meta-analysis of randomized controlled trials (RCTs) to determine if aerobic exercise interventions influence episodic memory performance in older adults without dementia and to examine potential moderators of these effects . Included studies met the following criteria: (1)Studies of adults (M\u226555 years) with normal cognition, subjective cognitive decline, or mild cognitive impairment; (2)Aerobic exercise RCTs; and (3)Assessment of episodic memory. Intervention effects were represented by Hedges\u2019 g and combined into pooled effect sizes using random- and mixed-effects models. Thirty-three studies met inclusion criteria, representing data from 2,488 participants. The primary analysis yielded a significant positive effect of aerobic exercise on episodic memory . Mixed-effects analyses demonstrated a positive effect on episodic memory among studies with a high percentage of females (>66%), participants with normal cognition, studies reporting intensity, studies with a no-contact or nonaerobic physical activity control group, and studies prescribing 2,100\u20133,900 total minutes of activity . These results suggest that aerobic exercise may act as an accessible, non-pharmaceutical intervention to improve episodic memory in late adulthood before changes in cognition are detected."}
+{"text": "CFTR gene, is associated with chronic infection/inflammation responsible for airway epithelium alteration and lung function decline. Modifier genes induce phenotype variability between people with CF (pwCF) carrying the same CFTR variants. Among these, the gene encoding for the amino acid transporter SLC6A14 has been associated with lung disease severity and age of primary airway infection by the bacteria Pseudomonas aeruginosa. In this study, we investigated whether the single nucleotide polymorphism (SNP) rs3788766, located within SLC6A14 promoter, is associated with lung disease severity in a large French cohort of pwCF. We also studied the consequences of this SNP on SLC6A14 promoter activity using a luciferase reporter and the role of SLC6A14 in the mechanistic target of rapamycin kinase (mTOR) signaling pathway and airway epithelial repair. We confirm that SLC6A14 rs3788766 SNP is associated with lung disease severity in pwCF , with the minor allele G being deleterious. In bronchial epithelial cell lines deficient for CFTR, SLC6A14 promoter activity is reduced in the presence of the rs3788766\u00a0G allele. SLC6A14 inhibition with a specific pharmacological blocker reduced 3H-arginine transport, mTOR phosphorylation, and bronchial epithelial repair rates in wound healing assays. To conclude, our study highlights that SLC6A14 genotype might affect lung disease severity of people with cystic fibrosis via mTOR and epithelial repair mechanism modulation in the lung.Cystic fibrosis (CF), due to pathogenic variants in  CFTR) gene , the most common lethal autosomal recessive genetic disease in the Caucasian population, results from pathogenic variants in the CF transmembrane conductance regulator (TR) gene . Manifesithelium .CFTR variants and living in the same environment involves genetic modifiers, which are expected to contribute to almost 50% of CF lung phenotype , we previously identified five CF lung disease modifier loci by genome-wide association studies (GWAS) . SLC6A14 is located on chromosome X and encodes for the neutral and cationic amino acid transporter SLC6A14 that concentrates amino acids into cells  by using a sodium and chloride electrochemical gradient  , includigradient . This trgradient  and to bgradient , pancreagradient , and colgradient  cancer cSLC6A14 gene has pleiotropic effects in pwCF (SLC6A14 single nucleotide polymorphisms (SNPs) being associated with different phenotypes such as meconium ileus (MI) occurrence, a severe neonatal intestinal obstruction . Informed written consent was obtained from each participant and/or parents or guardians. For the analysis, we excluded pancreatic-sufficient participants since they had milder disease (n = 820), non-genotyped participants for the SLC6A14 rs3788766 SNP (n = 436), and participants without forced expiratory volume in 1\u00a0s (FEV1) measurements (n = 177). We also excluded participants under 6\u00a0years of age, since their spirometry data was less reliable, and those over 40\u00a0years of age, in order to limit selective survival bias (n = 285).As of 31 January 2021, 4,975 pwCF had been included in the French CF modifier gene study . The stu1 were collected on a quarterly basis according to international CF care recommendations (1) using Global Lung Function Initiative (GLI) equations  CF-specific lung phenotype. SaKnorm is a quantitative phenotype that allows direct comparison of lung phenotypes between pwCF and accounts for differential survival  chemistry .Measurements of FEVndations  and exprquations . To assesurvival . Lung fuwww.legifrance.gouv.fr). The institution informed the participant and made sure that he was not opposed to the use of surgical samples for research purposes. Staining was performed using 5-\u03bcm-thick paraffin sections from formalin-fixed paraffin-embedded lung biopsies. Immunolabeling for SLC6A14 was performed on a Bond-III\u00ae automat  using anti-SLC6A14 antibody .Human lung biopsy was obtained from the lung explant of a 29-year-old male with CF (homozygous for the F508del variant), after lung transplantation that occurred in the H\u00f4pital Foch, Suresnes 92150, France. Biopsy was collected and processed in compliance with the current French public health legislation  was solubilized in 100% methanol (MeOH) and then diluted in the respective culture media to achieve 1, 2.5, or 5\u00a0mM (used concentrations are specified in figure legends). Equivalent volumes of MeOH alone were used for control conditions, thus reaching 0.87, 2.18, and 4.36%, respectively.CFTR-WT and Calu-3 CFTR-KD, kindly provided by Dr. Marc Chanson  in MEM-Glutamax (Invitrogen) medium supplemented with SVF 10% , penicillin-streptomycin , sodium pyruvate 5% (Invitrogen), essential amino acids 1\u00a0mM (Invitrogen), and HEPES buffer 10\u00a0mM . Calu-3 cells were then seeded in plates with 12 (3\u20133.5\u00d7105 cells/well) or 24 (2\u00d7105 cells/well) wells (TPP) and maintained at 37\u00b0C in a humidified atmosphere with 5% CO2. Non-CF and CF primary human bronchial epithelial cells (HBECs)  (characteristics in 5 cells/well) until confluent in hAEC culture medium supplemented with antibiotics (Epithelix).Human bronchial epithelial cell lines Calu-3- Chanson   were used . Calu-3-CFTR-KD cells were seeded in 24-well plates and transfected at 60% confluence with 1\u00a0\u03bcg/ml of the GLuc SLC6A14 promoter reporter using Lipofectamine 3,000 (Invitrogen). After 24\u00a0h of transfection, culture media were collected and centrifuged for 10\u00a0min at 10,000x g, and supernatants were stored at \u201320\u00b0C. SLC6A14 promoter activity was quantified by measuring Gaussia luciferase and secreted embryonic alkaline phosphatase (SEAP) used as endogenous reporter and was measured using a Secrete-Pair\u2122 Dual Luminescence Assay kit (GeneCopoeia). SLC6A14 promoter activity is represented as the ratio of GLuc normalized by SEAP.Gaussia luciferase (GLuc) reporters driven by the 2, 0.8\u00a0mM MgSO4, 5\u00a0mM glucose, pH = 7.4) for 30\u00a0min at 37\u00b0C. Cells were then incubated with 300\u00a0\u00b5l of HEPES buffer supplemented with 100\u00a0\u00b5M Arginine and Arginine Monohydrochloride L-  for 15\u00a0min and washed 3 times on ice with ice-cold HEPES with 10\u00a0mM Arginine (inhibition of uptake). Finally, cells were lysed with 400\u00a0\u00b5l 0.5M NaOH for 15\u00a0min while shaking on ice. Radioactivity levels were measured in 300\u00a0\u00b5l of the sample in 7\u00a0ml of a scintillation liquid, Ecolite Plus , and using Hidex 300SL  equipment.SLC6A14 amino acid transport was studied as described by others . Brieflyhttps://imagej.nih.gov/ij/index.html) to measure areas of the wounds at t = 0\u00a0h and t = 6\u00a0h (end-point assay), and mean wound closure (% of the area at t = 0\u00a0h) was calculated (wound closure = T0-T6/T0). Statistical analysis is performed on raw data. Data illustrated are reported to one MeOH  control.Cell monolayers grown on plastic supports were injured mechanically (3 wounds per well) as previously described . Afterwa\u00ae Non-Radioactive Cytotoxicity Assay, Promega, Madison, WI, United States).Toxicity of \u03b1-MT was verified using a commercially available assay  and transferred onto nitrocellulose membranes using an iBlot2\u2122 Gel Transfer Device and iBlot2\u2122 Nitrocellulose Regular Stacks . Membranes were incubated with specific primary antibodies followed by corresponding secondary-HRP antibodies. Mouse anti\u2013\u03b2-actin antibody (A2228)  was from Sigma-Aldrich. Rabbit anti\u2013phopho-mTOR , rabbit anti-mTOR , anti-mouse HRP , and anti-rabbit  antibodies were from Cell Signaling Technology . Immunodetection was carried out using Clarity\u2122 Western ECL Substrate . Image acquisition was performed using Las-3000 . Densitometric quantification was performed using ImageJ software.Protein extracts (20\u00a0\u00b5g) in RIPA buffer supplemented with antiprotease-antiphosphatase  were reduced and size-separated on 4-15% Mini-PROTEANSLC6A14 rs3788766 genotypes was evaluated by linear regression. We applied additive SNP coding, and the reference allele  was taken from annotations of the human genome (http://www.ensembl.org). Fisher\u2019s exact test was used to test conformance of the allele frequencies with the Hardy-Weinberg equilibrium. A p-value of less than 5% was interpreted as evidence of a statistically significant association. Analyses were carried out using R software .Descriptive statistics are reported as mean \u00b1 standard deviation (SD) or percentages. Association between lung disease severity and t-tests were used to compare two groups. One-way ANOVA was used for comparison of more than two groups and was followed by appropriate post hoc tests as indicated in the figure\u2019s legends. Values of p < 0.05 were considered to be significant. In figures, statistical differences are indicated as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) or non-significant (NS).All data are presented as mean \u00b1 SD and the number of repeated experiments is indicated in the figure legends. GraphPad Prism version 7.05  was used to analyze all data. Paired or unpaired SLC6A14 rs3788766 SNP in our cohort was 0.38, similar to that reported in Europeans (0.36). As SLC6A14 is located on the X chromosome, the Hardy-Weinberg equilibrium p-value was computed by Fisher\u2019s exact test among females. Results showed that our cohort does not significantly diverge from the Hardy-Weinberg equilibrium . We also found that SLC6A14 rs3788766 SNP was associated with lung function with the G allele being deleterious. Linear regression models estimated that pwCF carriers of the minor allele G had a significant increase in lung disease severity, which was measured by an average loss in the SaKnorm Z-value of 0.038 \u00b1 0.016 for each G allele (p = 0.020)  and after application of the exclusion criteria, 3,257 pwCF were analyzed in the genotype/phenotype association study. Demographic characteristics of the participants are summarized in = 0.020) . OverallG allele .SLC6A14 rs3788766 SNP, located within SLC6A14 promoter (SLC6A14 mRNA expression. Quantitaive-trait Loci (QTL) expression data extracted from GTEx  show that rs3788766\u00a0G is associated with a decrease in SLC6A14 transcript expression in different tissues  and the minor salivary gland . In the lung, a diminished but not statistically significant expression is observed (AA vs. GG genotypes). It is worth mentioning that for the GTEx project, the prefered location for the lung tissue collection is in the inferior segment of the left upper lobe, 1\u00a0cm below the pleural surface, avoiding any large arteries, veins, and bronchi. We observed by immunohistochemistry that SLC6A14 is predominantly expressed in the bronchial epithelium of pwCF  than cells transfected with the A allele , that pwCF carrying at least one minor allele G of the SLC6A14 rs3788766 SNP exhibit reduced lung function compared to those carrying two major allele A, confirming prior studies  in vitro (via the JAK2/STAT3 pathway. SLC6A14 involvement in cell proliferation has also been shown in other cancer cells such as pancreatic cancer cells (Here, we report, for the first time, that SLC6A14 is involved in bronchial epithelial repair. In healthy epithelia, repair processes involving cell proliferation, migration, and differentiation facilitate epithelial integrity restoration and function. In CF, repair mechanisms are altered and chronic infections with various pathogens and exacerbated inflammation induce progressive epithelial damage . Previou pathway . Whetherell line . In addiin vitro . They aler cells .In addition to its role in epithelial repair, we highlighted that it may be involved in the mTOR pathway. The mTOR pathway balances anabolism and catabolism in order to control key cellular processes such as cell growth or proliferation. It is very sensitive to amino acid starvation, especially leucine and arginine . SLC6A14SLC6A14 rs3788766 genotype influences the lung disease severity of pwCF. This study also showed that SLC6A14 might influence CF lung phenotype via the mTOR signaling pathway and epithelial repair process modulation.To conclude, we confirmed that"}
+{"text": "Triggering receptor expressed on myeloid cells 2 (TREM2) signaling is considered to regulate anti-inflammatory responses in macrophages, dendritic cell maturation, osteoclast development, induction of obesity, and Alzheimer\u2019s disease pathogenesis. However, little is known regarding the effect of TREM2 on natural killer (NK) cells.\u2212CD122+NK1.1+ precursor NK (pNK) cells expressed TREM2 and their population increased in TREM2-overexpressing transgenic (TREM2-TG) mice compared with that in female C57BL/6\u2009J wild type (WT) mice. Both NK cell-activating receptors and NK cell-associated genes were expressed at higher levels in various tissues of TREM2-TG mice than in WT mice. In addition, bone marrow-derived hematopoietic stem cells (HSCs) of TREM2-TG mice (TG-HSCs) successfully differentiated into NK cells in vitro, with a higher yield from TG-HSCs than from WT-HSCs. In contrast, TREM2 signaling inhibition by TREM2-Ig or a phosphatidylinositol 3-kinase (PI3K) inhibitor affected the expression of the NK cell receptor repertoire and decreased the expression levels of NK cell-associated genes, resulting in significant impairment of NK cell differentiation. Moreover, in melanoma-bearing WT mice, injection of bone marrow cells from TREM2-TG mice exerted greater antitumor effects than that with cells from WT control mice.Here, we demonstrated for the first time that CD3Collectively, our data clearly showed that TREM2 promoted NK cell development and tumor regression, suggesting TREM2 as a new candidate for cancer immunotherapy.The online version contains supplementary material available at 10.1186/s12865-021-00420-0. Natural killer (NK) cells play key roles in both innate and adaptive immunity  by killi+ lineage\u2212 HSCs) leads to their differentiation into precursor NK (pNK) cells that express CD122 receptor (IL-2R\u03b2+/CD15R\u03b2+) \u2009\u00d7\u2009100.The lactate dehydrogenase-release assay kit  was used to measure the cytotoxicity of NK cells, according to the manufacturer\u2019s instructions. In brief, NK cells were stimulated with 20\u2009ng/mL of recombinant murine IL-2 for 48\u2009h, washed twice with phosphate-buffered saline, and seeded into 96-well round-bottom microtiter tissue culture plates at various effector:target cell ratios. Target cell samples . Differences were considered statistically significant at P\u2009<\u20090.05.All values are expressed as mean\u2009\u00b1\u2009standard error of the mean (SEM). All experiments were repeated at least three times, independently. Student\u2019s Additional file 1: Fig. S1. The number of NK1.1+ cells, from TREM2-TG mice, expressing NK cell receptors is higher than in WT mice. Graphs show the absolute number of cells that express the NK cell receptors in the spleen, BM, and liver of WT (opened bar) and TREM2-TG mice (solid bar). Data are shown as mean\u2009\u00b1\u2009SED of three independent experiments. *P\u2009<\u20090.05, **P\u2009<\u20090.01, and ***P\u2009<\u20090.001 by Student\u2019s t-test. Fig. S2. TREM2-TG mice show enhanced NK cell cytotoxicity. (A) Real-time qPCR analysis to determine expression of the TREM2, Ifng, Prf1, and Gzmb (granzyme B) mRNAs in splenic NK1.1+ cells of WT or TREM2-TG mice. (B) LDH assay to measure cytotoxicity of NK1.1+ cells purified from splenocytes harvested from WT (open square) and TREM2-TG (solid circle) mice. Three independent experiments were performed (A-B). *P\u2009<\u20090.05, **P\u2009<\u20090.01, and ***P\u2009<\u20090.001 by Student\u2019s t-test. Fig. S3. CD4+T cell, CD8+T cell, and B220+ B cell frequency and absolute number in WT and TREM2-TG mice are similar. Percentage (A) and absolute number (B) of CD4, CD8, and B cells in spleen of WT and TREM2-TG mice determined by flow cytometry (N\u2009=\u20095). Three independent experiments were performed, but no significant differences were observed between WT and TG mice group. Fig. S4. Inhibition of TREM2 signaling reduces the NK cell pool in vivo. Representative flow cytometry plots of expression of NK cell-specific receptors  on surface of cells isolated from the spleen, BM, and liver of WT mice injected (i.p.) with 100\u2009\u03bcg of TREM2-Ig or hu-Ig (control) twice per week for 4\u2009weeks. Fig. S5. TREM2 signaling enhances NK cell-related gene expression in differentiated NK cells in vitro. Quantitative real-time PCR analysis of NK cell-associated genes using mRNA isolated from mNK cells derived from WT or TREM2-TG HSCs. Data are shown as mean\u2009\u00b1\u2009SED of three independent experiments. *P\u2009<\u20090.05, **P\u2009<\u20090.01, ***P\u2009<\u20090.001 vs. WT+ hu-Ig, and \u2020P\u2009<\u20090.05, \u2020\u2020P\u2009<\u20090.01, \u2020\u2020\u2020P\u2009<\u20090.01 vs. TG\u2009+\u2009hu-Ig, based on two-way ANOVA with Bonferroni post-hoc test. Fig. S6. The development and cytotoxicity of NK cells is regulated by TREM2 via PI3K signaling. (A) Representative flow cytometry plots showing the relative ratio of mNK cells developed in vitro from pNK cells of WT-HSCs and TREM2-HSCs treated with DMSO or LY294002 (1\u2009\u03bcM). (B) Quantitative real-time PCR analysis of the indicated genes using mRNA isolated from mNK cells differentiated from WT-HSCs or TREM2-TG-HSCs treated with DMSO (opened bar) or LY294002 (solid bar). *P\u2009<\u20090.05, **P\u2009<\u20090.01, ***P\u2009<\u20090.001 vs. WT+ DMSO, and \u2020P\u2009<\u20090.05, \u2020\u2020P\u2009<\u20090.01 vs. TG\u2009+\u2009DMSO, based on two-way ANOVA with Bonferroni post-hoc test. Fig. S7. The CD3\u2212NK1.1+Mac-1+CD27+ NK cell subset is reduced in peripheral blood of TREM2-K/O mice. Representative flow cytometry plots show the NK cell subsets  in the peripheral blood (A) or BM (B) of WT and TREM2-TG mice. Three independent experiments were performed. Fig. S8. TREM2 regulates NK cell differentiation via PLC-\u03b3 signaling pathway. (A) Representative flow cytometry analysis showing NK1.1 and NKG2A/C/E expression on the differentiated NK cells derived from TG-HSCs treated with DMSO (control) or U-73122 (PLC-\u03b3 inhibitor) during pNK to mNK cell differentiation stage. (B) RT-PCR analysis of the indicated genes on mNK cells differentiated from WT-HSCs or TREM2 TG-HSCs treated with DMSO (control) or U-73122 (PLC-\u03b3 inhibitor). \u03b2-actin (housekeeping gene) was used as a control. The cropped images from the different parts of the gel were displayed. The full-length gels are presented in Fig. S9. Fig. S9. The CD45.2+NK1.1+ cells persist in the tumor site in the recipient mice that received TREM2-TG-BM transplantation. Representative flow cytometry analysis of the expression of CD45.2+ NK1.1+ cells derived from TREM2-TG-HSCs in the tumor model (N\u2009=\u20093). Data from day 41 after the 4\u2009weeks adaptive transplantation following period. Fig. S10. Full-length gel images of cropped gels. (A) Full-length gel corresponding to Fig. S8B; E4bp4 (left), Id2 (middle), TREM2 (right). (B) Full-length gel corresponding to Fig. S8B, \u03b2-actin."}
+{"text": "Deinococcota, Halobacterota, and Latescibacterota in zone A; Crenarchaeota, Fusobacteriota, and Deltaproteobacterium Sva0485 in zone B; and Fervidibacteria and Campilobacterota in zone C, which indicates taxon-specific preferences in deep soil habitats. Differences in the microbiota between the zones were rather abrupt, which is concomitant with abrupt changes in the physical-chemical parameters. Overall, moisture content, total carbon (TC), pH, and electric conductivity (EC) were most predictive of microbial richness and diversity, while total sulfur (TS) and total phosphorous (TP) contents were additionally predictive of community composition. We also found statistically significant associations between taxa and soil properties, most of which involved moisture and TC contents. Our findings show that under-explored habitats for microbial survival and existence may prevail at greater soil depths near water or within water-bearing layers, a valuable substantiation also for the ongoing search for biosignatures on other planets, such as Mars.The extreme environmental conditions and lack of water on the soil surface in hyperarid deserts hamper microbial life, allowing only highly specialized microbial communities to the establish colonies and survive. Until now, the microbial communities that inhabit or have inhabited soils of hyperarid environments at greater depths have been poorly studied. We analyzed for the first time the variation in microbial communities down to a depth of 3.4 m in one of the driest places of the world, the hyperarid Yungay region in the Atacama Desert, and we related it to changes in soil physico-chemical characteristics. We found that the moisture content changed from 2 to 11% with depth and enabled the differentiation of three depth intervals: (i) surface zone A (0\u201360 cm), (ii) intermediate zone B (60\u2013220 cm), and (iii) deep zone C (220\u2013340 cm). Each zone showed further specific physicochemical and mineralogical features. Likewise, some bacterial phyla were unique in each zone, i.e., members of the taxa  This region is surrounded by mountain ranges that limit its extension to the Coastal Cordillera to the west and the Cordillera de Domeyko to the east . The mea\u20131 , with pr\u20131 . While t\u20131 . At pres\u20131 .18O) isotopes. Others works have reported the existence of unexpectedly large microbial populations in the hyperarid soils, especially in very particular and extreme habitats, such as the underside of quartz rocks  basin . It is situated southeast of Antofagasta within the hyperarid core of the Atacama Desert. The positional coordinates are 24\u00b003\u203249.6\u2033S, 69\u00b052\u203249.8\u2033W . Note th3 (pH 8.5), 0.1 M NaOH, and 1 M HCl. P not extracted by these solutions was classified as residual P. The amount of inorganic P (Pi) in each extract was determined by Soil samples were taken in March 2017. The soil profile was sampled at 10 cm depth intervals until a final depth of 340 cm. Before the sampling procedure, the surface area was cleaned and cut back into its exposure. We took soil samples immediately after excavating a specific depth layer to avoid effects of the exposure of the soil to ambient air and temperature. Soil samples were manually taken and stored in plastic bags. Additionally, soil samples for microbiological analyses were collected in sterile tubes by using plastic gloves and shovels properly cleaned with ethanol to avoid contamination. All samples were taken to the laboratory in Antofagasta and Germany to conducted physicochemical, mineralogical, and microbiological analyses. The moisture content (MC) of the soil samples was measured immediately in the laboratory in Antofagasta by drying soil at 105\u00b0C in an oven and calculating the loss of weight after drying. Electric conductivity (EC) and pH were measured in a soil-distilled water suspension with a solution ratio of 1:2.5. The total contents of C, N, S, and TOC were determined by elemental analysis employing approx. 20 mg of sample material . For the determination of TOC contents, inorganic carbon was removed with 20% HCl for 2 h. Total P, reactive P that corresponds to inorganic P, and unreactive P (URP) (which includes organic P and polyphosphates in soils) were determined by the extraction method proposed by n < 50 for each defined zone). As the distribution was not normal, a non-parametric Friedman test for related samples was used to determine significant variations (p < 0.05) in the distribution of MC, TC, TOC, TN, TS and TP between the proposal zonification. All statistical analyses were performed with IBM SPSS Statistical software version 21.Based on the MC distribution along the soil profile, it was possible to distinguish and propose three distinct zones in the soil profile: surface zone A (0\u201360 cm), intermediate zone B (60\u2013220 cm), and deep zone C (220\u2013340 cm). For each zone, a linear regression was calculated for moisture content depending on depth. All data obtained were tested for normality of distribution by using a Shapiro\u2013Wilk test  with a slope of 0.073% moisture cm\u20131 R2: 0.80; and deep zone C  with a slope of 0.143% moisture cm\u20131 R2: 0.77. Moreover, soil MC increased significantly  in the three proposal zones. In surface zone A, the MC increased from 0.66 to 5%. Additionally, we identified an evaporation layer between 0 and 20 cm within zone A, reflected by the lowest moisture content values registered in the soil profile (0.66%). In intermediate zone B, the moisture content increased to 15% at the 220 cm soil depth. In deep zone C, the moisture content reached a maximum of 21% at 330 cm soil depth. A local minimum of 3% moisture content between 220 and 240 cm depth was noted.According to the World Reference Base for Soil Resources, the soil profile can be classified as a Gypsisol. The profile and the complete description are given in p < 0.05, Friedman test). The average pH values were 8.3 in surface zone A, 9.0 in intermediate zone B, and 8.9 in deep zone C . The average EC value in surface zone A was 107 mS cm\u20131, 22.1 mS cm\u20131 in intermediate zone B and 14.3 mS cm\u20131 in deep zone C . Concerning the TOC, the values were low along the soil profile . However, there were no significant differences between the means of TOC in the three proposal zones . On the other hand, the TN concentrations generally decreased with increasing soil depth (\u20131 at 50 cm depth), while in zones B and C, TN decreased from 628 to 39 \u03bcg N g\u20131 and 628 to 134 \u03bcg N g\u20131, respectively. There were no significant differences between TN means in the three-zone intervals . Similarly, high TS contents were measured in the first 50 cm of soil depth (surface zone A), ranging from 23.9 to 35.3 mg S g\u20131 soil . There were significant differences between TS means in the three-zone intervals .The contents of TC in surface zone A varied between 190 and 1125 \u03bcg C g0\u2013330 cm . There w profile . In zoneil depth . Thus, Tg\u20131 soil . These v\u20131 at 30\u201350 cm depth) (\u20131). In deep zone C, PT decreased at 230-240 cm depth to increase to 783 \u03bcg Pg\u20131 at 340 cm depth. There were significant differences between TP means in the three-zone intervals .In contrast to TC, TN, and TS, TP increased in the soil profile. TP showed minimal values in surface zone A (122\u2013216 \u03bcg Pgm depth) . Overall\u20131 at 140, 210, and 320 cm soil depths along with the soil profile. The latter two signals coincided with increasing TOC concentrations in depth. The easily extractable P (the sum of Pi-H2O and Pi-NaHCO3) only accounted for 1.0\u20135.6% of the total P (2O were observed in intermediate zone B and deep zone C (8.1\u201320.6 \u03bcg Pg\u20131) than in surface zone A (1.9\u20132.2 \u03bcg Pg\u20131). A similar trend was observed for the Pi-NaOH fraction, with high values found in zones B and C (0.8\u20133.7 \u03bcg Pg\u20131) compared with surface zone A (0.3\u20131.1 \u03bcg Pg\u20131).Reactive P (or inorganic P) dominated the soil profile , represe total P . The hig total P . Some P \u20131 soil. Amplicon sequencing revealed that the phyla Proteobacteria, Actinobacteria, Bacteroidota and Firmicutes had the highest prevalence in the soil profile . In general, Firmicutes exhibited a slightly greater detection frequency in surface zone A than in zone C, while it was not detected in zone B. Within Firmicutes, the occurrence of the families Lachnospiraceae and Bacillaceae was limited to zone A, while Oscillospiraceae and Salisediminibacter incerta sedis occurred only in zone C (wetter conditions). Similarly, the actinobacterial families had different niches, with Sporichthyaceae being only detected in layers of zones B and C, while Illumatobacteraceae were only present in different layers within zone A. Verrucomicrobiota and Planctomycetota were exclusively present in deep zone C  and Marinobacteraceae (320\u2013330 cm). The family Sphingomonadaceae of Proteobacteria showed a high abundance in several layers of zone B but a lower abundance in zone C  in intermediate zone B; and Campilobacterota and Fervidibacteria in deep zone C . These zones also showed significant variations in pH, EC, TC, TS and TP (p < 0.05 Friedman test). Instead, at least two zones were similar in terms of TOC and TN (P > 0.05 Friedman test). The increase in MC in the depth soil was observed in the soil profile (Climatic factors (mainly low precipitation and high temperature) limit biological productivity and activity, chemical reactions, and weathering . Consequ profile . Recentl profile . Althoug profile . It has  profile . Whether\u20131); however, in surface zone A, the soil is exceptionally saline, reaching values over 100 mS cm\u20131. The highest EC value of 185 mS cm\u20131 was recorded at 40 cm depth and was related to salt minerals such as halite, albite, calcite, anorthite, and darapskite . Anhydrous sulfate phases were typical in the surface layers, and the uppermost units had the least water-soluble phases. In the 0\u201310 cm surface layer, sulfur minerals such as Bassanita (11.6%), anhydrite (4.4%) and gypsum (1.3%) were predominant. Similar results for TOC and S contents were reported for the 0-80 cm soil depth by The highest TN concentrations in surface zone A originated from N minerals such as darapskite (37.6%) and nitratine (2.8%) . It has 2O (representing inorganic labile P) were observed in intermediate zone B and deep zone C (8.1\u201320.6 \u03bcg Pg\u20131)  was dominant around the soil profile, which is in agreement with \u03bcg Pg\u20131) , reflect\u03bcg Pg\u20131) .Increased aridity and temperature may also decouple the spatial variability of soil nutrient stocks and cycling in global drylands . In the Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes in all studied depth zones . Thus, this microbial description is in accordance with the results of our study. Recently, bacterial, and archaeal communities from hypolithic microhabitats were analyzed in the Atacama Desert to specifically identify the potentially viable microbiota (intracellular DNA versus extracellular DNA), showing habitat-specific communities dominated by bacteria. Proteobacteria were almost exclusively identified in the extracellular DNA pool in Yungay Salar halite nodules and gypsum crusts (representing an indicator for a previously existing community in this location), and Cyanobacteria showed the most abundant intracellular DNA in these hypolithic environments  in surface zone A, while in intermediary zone B and depth zone C, the EC values fluctuated between 5 and 24 mS cm\u20131. However, we did not observe a particular accumulation of halophilic taxa in zone A; rather, they were present in all zones. Nevertheless, EC had a significant effect on the bacterial community structure , Actinobacteria (Bifidobactericeae and Sporichthyaceae), and Firmicutes , which have been detected in different saline environments , arid biological crusts (United States), and hypersaline lagoons (Chile)  . InteresBacillus .2O .https://www.ncbi.nlm.nih.gov/genbank/, PRJNA638921.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 below: BF, FR, and RB conceived and designed the study. BF, AC, FG, FA, FR, GM, DC, and RB performed the field work. AC, FG, DC, GM, JP, and WK performed the experimental procedures and laboratory work. JA, EC-N, CK, and FR analyzed sequencing data. BF, EC-N, FR, and RB wrote the manuscript. RM, WA, CK, EK, and CS reviewed and edited the manuscript. All authors read and approved the final 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": "A monolithic three-dimensional integrated static random access memory containing a feedback field effect transistor (M3D-FBFET-SRAM) was proposed. The M3D-FBFET-SRAM cell consists of one metal oxide semiconductor field effect transistor (MOSFET) and one FBFET, and each transistor is located on the top tier and one on the bottom tier in a monolithic 3D integration, respectively. The electrical characteristics and operation of the NFBFET in the M3D-FBFET-SRAM cell were investigated using a TCAD simulator. For SRAM operation, the optimum doping profile of the NFBFET was used for non-turn-off characteristics. For the M3D-FBFET-SRAM cell, the operation of the SRAM and electrical coupling occurring between the top and bottom tier transistor were investigated. As the thickness of interlayer dielectric decreases, the reading \u2018ON\u2019 current decreases. To prevent performance degradation, two ways to compensate for current level were suggested. Over the past few decades, computing systems have followed the von Neumann architecture . An impoA monolithic three-dimensional M3D) integration is one of fabrication technologies for overcoming Moore\u2019s Law D integra,6,7,8,9.To improve the performance, density, and power consumption of SRAM, the configurations of SRAM with novel devices are introduced ,24,25. AIn this study, the M3D-FBFET-SRAM cell is proposed, and its electrical characteristics are investigated using technology computer aided design (TCAD). First, the simulation structure of the M3D-FBFET-SRAM cell is described in In this section, the electrical characteristics of the M3D-FBFET-SRAM cell will be discussed. First, the operation and electrical characteristics of the NFBFET were investigated with respect to the energy band diagram. Moreover, during the writing operation of the M3D-FBFET-SRAM cell, the role of NFBFET was investigated. Based on the NFBFET operation, the M3D-FBFET-SRAM cell operation was investigated, particularly, due to the M3D structure, the electrical coupling occurring between the top and bottom tier transistors . This coBLV) and word line 2 voltage (WL2V) are applied with 1.9 V and 1 V, respectively. At this state, the electrons from the source region cannot be injected into the channel region, due to the potential barrier at the gated channel region, as shown in WL1V), the potential barrier is lowering, and the electrons drift into the ungated channel region by the drain-source field. The injected electrons accumulate at the potential well at the ungated channel region; thereafter, the potential well is eliminated by accumulated electrons. The holes from the drain region can diffuse by the lowered potential barrier at the ungated channel region, and accumulate at the gated channel region, as shown in DS-NFBFETI) versus WL1V. There is an abrupt increment of the NFBFET current at WL1V = 0.17 V. The hysteresis characteristic, which is the threshold voltage difference between forward and reverse, can be controlled by the doping profile of the channel region [l region . For theDS-NFBFETI) for writing the \u2018ON\u2019 and \u2018OFF\u2019 operation in the M3D-FBFEET-SRAM cell, respectively. The red line and black square-lines denote the operation current and DC characteristics of the NFBFET, respectively. When the writing \u2018ON\u2019 operation begins, BLV, WL1V, and WL2V are applied for 1.9 V, 0.6 V, and 1.0 V, respectively. When the writing \u2018ON\u2019 pulse is applied to the M3D-FBFET-SRAM cell, the NFBFET currents change following the blue arrows, as shown in BLV, WL1V, and WL2V are applied for 0.7 V, 0.6 V, and 1.0 V, respectively. When the writing \u2018OFF\u2019 pulse is applied to the SRAM cell, the NFBFET current changes following the blue arrows, as shown in BLV, WL1V, and WL2V, and the current pulse of DS-NFBFETI, respectively. DS-NFBFETI are approximately 15 \u03bcA and 0.2 nA, respectively. The writing \u2018ON\u2019 and \u2018OFF\u2019 speeds are about 0.4 ns, as shown in 2 (F = feature size) [re size) , and thire size) .DS-NFBFETI, which can be controlled by holding voltages of WL2V [ of VWL2 . As showILDT). As ILDT decreases, the electric field occurring at the bottom tier transistor effects the top tier transistor. This coupling effect causes unexpected changes in system performance. In order to design the system with intended performance, the investigation for changing the electrical characteristics must be proceeded with respect to ILDT. In this section, the electrical coupling occurring between the top and bottom transistors was investigated. Furthermore, the optimum voltages were suggested for short ILDT.For the M3D structure, the electrical characteristics of the top tier transistor can be changed by the thickness of the interlayer dielectric (DS-NMOSFETI), which is located in the top tier, at ILDT = 3 nm. The red and black lines denote DS-NMOSFETI at WL1V = 0.6 and 0 V, respectively. As ILDT decreases, the electric field applied to the top tier transistor is stronger. When WL1V is applied for 0.6 V, the threshold voltages of DS-NMOSFETI changes from 0.67 V (for WL1V = 0 V) to 0.50 V, as shown in ILDT. As ILDT decreases, the threshold voltage of DS-NMOSFETI decreases and DS-NMOSFETI increases at the same WL2V. The current level (DS-NFBFETI) must be lower to match the current levels of the NFBFET and the NMOSFET by their serial connection; thus, AV must also be reduced, as shown in ILDT decreases, the reading \u2018ON\u2019 currents decrease in the direction of the orange arrow, as shown in ILDT = 3 nm at modified BLV and WL2V, respectively. The black and red lines denote DS-NFBFETI with original and modified voltage levels, respectively. To design the M3D-FBFET-SRAM with shorter ILDT, it is necessary to solve the problem of lowering the reading \u2018ON\u2019 current, because the low reading \u2018ON\u2019 current is a critical problem in SRAM operation. To solve this problem, higher BLV and WL2V can be chosen to create a high reading \u2018ON\u2019 current, as shown in ILD can achieve a lower critical delay due to shorter monolithic inter-tier via the ILDT. However, higher voltage levels of BLV and WL2V are required, and power consumption will be high. In order to design the M3D-FBFET-SRAM, the appropriate ILDT must be investigated for an acceptable trade-off regarding the performance.ILDT. As ILDT decreases, the reading \u2018ON\u2019 current decreases. To compensate for the reading \u2018ON\u2019 current, a higher BLV and WL2V can be applied; however, the power consumption will be higher. Therefore, in order to design the M3D-FBFET-SRAM, the investigation of the appropriate ILDT must be conducted.In this study, the M3D-FBFET-SRAM cell was proposed. The M3D-FBFET-SRAM cell consists of one NFBFET and one NMOSFET, and the NFBFET and NMOSFET in the M3D structure are located on the bottom and top tier, respectively. The transistors are stacked vertically; therefore, the M3D-FBFET-SRAM cell area achieved up to a 50% reduction compared with 2-D planer cell structure. The electrical characteristics of the M3D-FBFET-SRAM were investigated. First, for the NFBFET, the DC characteristics and role were investigated during the writing operation. In order to achieve the non-turn-off characteristics, the optimum doping profile of the NFBFET was used. Based on the NFBFET operation, the M3D-FBFET-SRAM cell operation was investigated. The reading \u2018ON\u2019 and \u2018OFF\u2019 current are approximately 15 \u03bcA and 0.2 nA, respectively. For the retention characteristics, the M3D-FBFET-SRAM cell can maintain the data for at least 30,000 s. In particular, the electrical coupling occurring between the top and bottom transistors was investigated with respect to"}
+{"text": "The management of animals on our planet is being scrutinised as never before. We face unprecedented loss of our animal species, from which it will take millions of years to recover, depending on how much damage is done before we start to reverse the disastrous mass extinction we have engineered. As well as the extinctions, the anthropogenic pressure on wildlife is depleting populations and causing them widespread stress, which depletes their immune system and makes them prone to new diseases. The human population has just come through a bad pandemic, which originated in wildlife under pressure from humans, in this case bats. Some of the blame must lie in the encroachment of humans into the territory of bats. Expansion of tourism into bat caves, widespread use of pesticides that accumulate in bats, demolition of old buildings providing roosting sites and the harvesting of bats for human consumption are all evidence of the pressure that bats are under in China and elsewhere . WildlifAs well as the anthropogenic assault, wildlife needs to cope with rapid climate change, with few resources being devoted to assisting them. Their opportunities for migration to climatic zones for which they evolved are extremely limited, as many wildlife species are confined to small pockets of indigenous fauna in the midst of intensive agriculture to feed humans. Livestock too face the ravages of climate change, which reduces their growth and milk production, and again causes stress: stress of escaping floods and fires, stress from depleted feed resources and restricted water supplies. Nowhere is the frantic attempt by humans to preserve their food resources more evident than in the livestock sector. Rapidly increasing demand for meat is evident as a result of the growing world population of humans and the greater affluence in parts of the world where meat consumption was hitherto uncommon. As human affluence grows, the affluence of other (non-human) animals shrinks alarmingly \u2013 their resources are squandered for our benefit. With limited land and labour resources, increasingly intensive animal production systems are being developed, using precision livestock farming to substitute for human care. The most extreme development in this field is the quest for a commercially available lab grown meat. In 2023 we will see new laboratories established for meat culture, with meat companies investing in the products, and perhaps one or more of the food companies offering their first artificial meat product to the market. Animals is becoming a force for good. Publications in Animals are mostly, although not uniquely, about improving the sustainability of our management of animals or predicting the effects of the current threats to our animals. For example, in 2021, Amira Gomez and I published a paper [It is clear from these growing challenges to the animals with which we share this planet, that we urgently need a strategy to manage all animals on planet earth more sustainably. To develop such a strategy, we need good scientific information, and that\u2019s where Animals. Now that we have achieve that, our next challenge is to make sure it is used effectively to address the most serious challenges to animal life on our planet, including humans, that we have ever faced. About 200 articles were published in Animals in 2022 on the themes of biodiversity, climate change and intensification of animal production. What are you doing to address these challenges?Eleven years ago, I strove to make scientific information on animal issues available to all, through our new open access journal, This special issue welcomes contributions, especially original articles and reviews."}
+{"text": "Teneurin C-terminal associated peptide (TCAP) is an ancient bioactive peptide that is highly conserved in metazoans. TCAP administration reduces cellular and behavioural stress in vertebrate and urochordate models, yet despite numerous studies in higher animals, there is limited knowledge of its role in invertebrates. In particular, there are no studies on TCAP\u2019s effects on the heart of any metazoan, which is a critical organ in the stress response. We used the Sydney rock oyster (SRO) as an invertebrate model to investigate a potential role for sroTCAP in regulating cardiac activity, including during stress. sroTCAP is localized to the neural innervation network of the SRO heart, and suggested binding with various heart proteins related to metabolism and stress, including SOD, GAPDH and metabotropic glutamate receptor. Intramuscular injection of sroTCAP (10 pmol) significantly altered the expression of heart genes that are known to regulate remodelling processes under different conditions, and modulated several gene families responsible for stress mitigation. sroTCAP (1 and 10 pmol) was shown to cause transient bradycardia (heart rate was reduced by up to 63% and for up to 40\u00a0min post-administration), indicative of an unstressed state. In summary, this study has established a role for a TCAP in the regulation of cardiac activity through modulation of physiological and molecular components associated with energy conservation, stress and adaptation. This represents a novel function for TCAP and may have implications for higher-order metazoans. A lack or imbalance in cellular stress-mitigating responses is known to promote cellular damage, disease, and mortality , 2, withSaccostrea\u00a0glomerata), are continuously exposed to changing environments , pollutants , and pathogens, resulting in stress  according to the manufacturer\u2019s instructions.Adult SROs used in this study were obtained from Port Stephens Fisheries Institute, NSW Department of Primary Industry and transferred to the University of the Sunshine Coast aquaculture lab, where they were housed in a closed recirculating tank containing natural seawater at 23\u00b0C \u00b1 1\u00b0C and 34 \u00b1 1 ppt and acclimated for 5-7 days before the experiments. Animals were routinely fed commercial microalgae concentrates  under the animal ethics committee regulations, nonetheless, the animals were housed and handled with care to avoid unnecessary stress.2.2Freshly excised whole SRO hearts from four adult animals were fixed in a 4% paraformaldehyde solution in phosphate-buffered saline (PBS) at 4\u00b0C overnight with gentle shaking. The fixed hearts were dehydrated with increasing concentrations of ethanol  for 10\u00a0min in each solution. The dehydrated heart tissues were immersed in xylene twice for 15\u00a0min each before being embedded in paraffin three times (at 65\u00b0C for 40\u00a0min each). Paraffin infiltrated heart tissues were embedded in paraffin blocks, and tissue sections (8-10 \u00b5m thick) were subsequently prepared using a microtome , as previously done . Section2.3via the PRIDE  into the adductor muscle using a 27 G needle. Oysters were divided into 4 groups (n=6) Table\u00a01 \u00ae Ultra RNA Library Prep Kit for Illumina\u00ae and sequenced using NovaSeq 6000 PE150 by Novogene (Hong Kong), which generated over 18 million reads (~ 6 GB). Raw reads were deposited in the NCBI SRA database (accession PRJNA804582). The raw transcriptome data were processed using CLC genomic version 21 (Qiagen); QC check, trimming, mapping, and pair-wise differential gene expression (DEG) using default parameters except for mapping  before being snap-frozen in liquid nitrogen and stored at -80\u00b0C. Two heart samples from each treatment were pooled (n=3 heart pairs/treatment) and processed for total RNA using a TRIzol reagent (ThermoFisher), according to the manufacturer\u2019s protocol. Non-directional poly-A libraries were prepared using NEBNextO genome ), in whiP-value were used in R Studio as previously described  value of \u22640.05 and log2 fold change of > 1 and < -1. The filtered DGEs were used to obtain the enriched gene ontology (GO) terms using Omics-box  with the default parameters. REVIGO was used to obtain the most representative GO terms from the enrichment list . A dispeescribed .Teneurin relative gene expression was evaluated by the reads per kilobase million (RPKM) between the four treatments and graphed.Additionally, a list of genes associated with stress; heat shock proteins (HSPs), antioxidants, apoptosis, metabolism and osmoregulation were retrieved from the literature , 40, and2.5via pericardial or intramuscular (IM) routes. For pericardial administration, SROs were shucked, and the pericardium membrane was cut to allow placement of a hook under the heart between the atrial and ventricular chambers and the hook was connected to a force transducer (ADInstruments), modified from Ha Park, Kim  of FSSW (negative controls) or 50 \u00b5L sroTCAP (10 pmol in FSSW). At least 24\u00a0h before the experiment, a small gap was created in the shell using a bone cutter to allow treatment injection directly into the adductor muscle. Following injections, oysters were kept outside the water for 10\u00a0min to allow sroTCAP/FSSW to circulate. Then, oysters were shucked, and the heart was connected to the force transducer as described above and continuously recorded for 2\u00a0h. Different animals were used for each treatment, therefore, no washing steps were required.The contraction time of each beat (time between peak contractions) was measured. Ten to 20 contraction time-points were extracted for each treatment per animal (n=3-4) at ~20 min post sroTCAP/FSSW delivery and/or during the peak of the sroTCAP effect and HR was calculated as beats per minute (BPM).2.6A priori hypothesis of a-P-value < 0.05 was considered statistically significant.All results are expressed as mean \u00b1 SEM. Statistical significance was assessed by paired t-test using SPSS software version 26 . 33.1TCAP immunoreactivity (ir-TCAP) was detected on the epithelial cell layer on the SRO heart periphery of both atrial chambers and to a lesser degree in the ventricle. In some regions of the atria (but not in the ventricle), cells with ir-TCAP were observed to penetrate from the peripheral epithelial tissue deeper into the heart connective tissue and muscle fibres (Atrium 1 and 2)  identified a total of 128 DEGs (97 upregulated and 31 downregulated). A comparison of ST vs SS (stress with and without sroTCAP administration) identified that sroTCAP had the lowest number of differentially expressed genes, 22 upregulated and 33 downregulated genes. The highest number of DEGs was observed for ST vs AT, where 245 genes were differentially expressed, 102 upregulated and 143 downregulated.To assess the impact of sroTCAP on the heart under stress conditions, the oysters were injected with sroTCAP or FSSW (control) before being exposed to either ambient or stress  conditions Table\u00a01.vs AS and ST vs AS, 20 in ST vs SS and 24 in SS vs AS. Comparison of ST vs AT had 27 GO terms, the highest number among the comparison groups. Several GO terms were found both in stressed SRO with and without sroTCAP, which could be considered stress-specific gene changes. These included \u2018axonemal central apparatus assembly\u2019, \u2018gamma-aminobutyric acid biosynthetic processes\u2019, \u2018response to stimuli\u2019, \u2018signalling\u2019, \u2018protein folding\u2019, and \u2018regulation of transcription from RNA polymerase II promoter\u2019 in response to stress. \u2018Triglyceride mobilization\u2019 was downregulated with sroTCAP at ambient but upregulated with sroTCAP and stress  but downregulated under stress with sroTCAP administration (ST vs SS). \u2018Reactive oxygen species metabolic process\u2019 was also downregulated when sroTCAP was delivered at ambient compared to ambient control (AT vs AS). \u2018T-helper 1 cell activation\u2019 was upregulated in stress sroTCAP compared to FSSW at ambient (ST vs AS) and sroTCAP at ambient (ST vs AT). \u2018Inclusion body assembly\u2019 was downregulated in sroTCAP stress compared to sroTCAP at ambient (ST vs AT) and stress FSSW (ST vs SS)  Figure\u00a03extracellular superoxide dismutase [Cu-Zn]-like were downregulated in sroTCAP at ambient (AT vs AS) and stress sroTCAP (ST vs SS) and were not differentially expressed in the other comparisons. Three downregulated cytochrome P450 isoforms were found only in AT vs AS, and a single isoform was upregulated in ST vs AT. NADPH oxidase 5-like isoform X2 was upregulated in both stress control (SS vs AS) and in ST vs AT and is therefore considered stress-specific.Following a search for genes of interest related to heart function, stress, and environmental adaptation, their DEG was evaluated in all comparisons Table\u00a03.vs AS). However, two upregulated and one downregulated heat shock 70 kDa protein 12A-like isoforms were specific to ST groups, while in the sroTCAP ambient control group (AT vs AS), one isoform was up and downregulated.Of the 13 differentially expressed HSPs detected, 8 were upregulated in the stress control group (SS vs AS) and ambient sroTCAP control (AT vs AS)  Table\u00a03.myosin-16- like isoform X4 was downregulated in all ST comparisons, while myosin heavy chain, striated muscle isoform X1 was downregulated in AT vs AS (Paramyosin-like isoform X3 was downregulated in the stress control (SS vs AS) and in two ST comparisons therefore, the changes are considered stress-specific and unrelated to sroTCAP.Genes encoding structural heart proteins were evaluated and AT vs AS Table\u00a03.putative zinc finger protein) was found in the stress control group (SS vs AS). Zinc finger BED domain-containing protein 1-like was downregulated in ambient sroTCAP control (AT vs AS) while the rest were found in ST comparisons. Of those, zinc finger CCCH domain-containing protein 13 isoform X1, NFX1-type zinc finger-containing protein 1-like and probable transcription-associated protein 1 were upregulated, while putative zinc finger protein, helicase with zinc finger domain 2-like and cotranscriptional regulator FAM172A isoform X2 were downregulated.Transcription regulators were evaluated to assess their differences among the different groups Table\u00a03.Excitatory amino acid transporter 1 isoform X3 and solute carrier family 23 member 2-like were both found in the stress control (SS vs AS) and are considered stress-related. Zinc transporter 5, solute carrier family 28 member 3-like, sodium- and chloride-dependent neutral and basic amino acid transporter B(0+)-like isoform X2 and proton channel OtopLc-like isoform X10 were upregulated in at least one comparison having an ST group, while iron transport multicopper oxidase fetC isoform X1 and voltage-dependent calcium channel subunit alpha-2/delta-3-like were downregulated in ST vs AT.Transporters and osmoregulation-related genes were evaluated to assess the heart\u2019s response to the low salinity stress Table\u00a03.inositol 1,4,5-trisphosphate receptor (IP3R) was differentially expressed and found to be downregulated in ST vs AT comparison (ryanodine receptor (RYR), L-type\u00a0Ca2+ channel and\u00a0stromal interaction\u00a0molecule 1 (STIM1).Membrane proteins involved in calcium influx in heart tissue were investigated, and only mparison Table\u00a03.teneurin transcript expression compared to ambient and stress controls (teneurin significantly increased when sroTCAP was delivered to oysters under stress (ST) compared to oysters under stress administered with FSSW (SS) (P = 0.038). Additionally, ST group had a significant increase in teneurin expression compared to the ambient control (AS) (P = 0.0167).Meanwhile, sroTCAP delivery to oysters maintained under stress conditions increased controls Figure\u00a043.4via the pericardial cavity, and changes in HR were analyzed. No change in heart rate was observed when FSSW was applied, while the positive controls, 5-HT and ACh (10 mM) caused a short spike in the amplitude of heart contraction and increased HR (P < 0.0001 (P<0.0001) reduced the HR, inducing bradycardia  (The administration of sroTCAP (10 pmol) by IM injection caused bradycardia 15\u00a0min post-injection and continuing until ~40 min post-injection, after which arrhythmia occurred (<0.0001) Table S54teneurin orthologues were known to be expressed in the developing hearts of vertebrates  and invertebrates   , 44, the tissues , which lThe spatial distribution of TCAP in the SRO heart was analyzed using IHC, demonstrating that TCAP is mainly localized in the atria with lower abundance in the ventricle. TCAP was localized to several areas of heart epithelia in both the atria and the ventricle, and in some regions of the atria, ir-TCAP was interspersed from the peripheral epithelial tissues to the inner walls of the heart chambers. GABA, tyrosine hydroxylase and FMRF-amide are recognized neural markers \u201347, and \u00ae 6600+), low abundance binding proteins have been detected. Several proteins involved in energy metabolism, protein chaperones and structural proteins were also identified in the pull-down assay, suggesting they may interact with sroTCAP or are accessory proteins that bind to the binding partners of sroTCAP. Metabotropic glutamate receptor 1 (mGluR), detected in one of the pull-down replicates (LC-MS/MS spectra of mGluR peptides are shown in P = 0.0109), AT (P = 0.0109), and AS (P = 0.0078)  and GAPDH in the heart, as Abramov, Suwansa-ard  previous 0.0078) . The act 0.0078) \u201351, simi 0.0078) , 32, 52. 0.0078) \u201355. LatrPrevious studies predicted that TCAP might be intrinsically unstructured and able to change its conformation as it lacks a well-defined 3D structure , 56, 57.Given that sroTCAP appears to interact with a diverse range of SRO proteins and is also present in the heart, a comparative transcriptome analysis was performed to further understand the molecular changes induced by sroTCAP administration under stress conditions. In the heart, only a small proportion  were considered significantly DEG between the treatment groups, with sroTCAP administration under stress compared to sroTCAP at ambient group showing the largest difference. This suggests that sroTCAP stimulated extensive transcriptional changes during stress and ambient conditions, emphasizing that the effects of sroTCAP on SRO are significantly different when delivered under different conditions.vs AS), demonstrating that sroTCAP may modulate ROS abundance in cardiac tissue under ambient conditions. These results are consistent with previous studies, which showed that TCAP could reduce ROS abundance under ambient and stress conditions and modulate apoptosis in response to stress in SRO hemocytes and a mouse hypothalamic cell line   and SRO ell line , 26.teneurin expression in SRO hemocytes under ambient or stress conditions (Oncorhynchus mykiss) TCAP-3 which was shown to modulate the expression of its precursor teneurin, where 1 nM attenuated expression and 100 nM increased expression in vitro  but not in ambient TCAP (AT vs AS) and stress control (SS vs AS). In rats, TCAP-1 administration upregulated the myosin heavy chain expression in skeletal muscle and remodelled the muscle tissue .Several key genes related to heart function and stress were differentially expressed among the treatment groups. Downregulation of e tissue . Specifie tissue . The redryanodine receptor (RYR), L-type\u00a0Ca2+ channel and\u00a0stromal interaction\u00a0molecule 1 (STIM1), IP3R was the only DEG detected when sroTCAP was delivered during stress compared to ambient conditions. IP3Rs are cation channels that facilitate Ca2+ release in cardiomyocytes and the delivery of Ca2+ to the mitochondria  but significantly downregulated when sroTCAP was administered under stress (ST\u00a0vs\u00a0SS). These results indicate that, given the same stress treatment, the gene expression profile of the heart was considerably different when FSSW control or sroTCAP were injected.Transcription factors are a diverse group of molecules that can react to internal and external stimuli and alter gene expression, which can be induced under stress . sroTCAPvs\u00a0AS) can be seen as an adaptive response to stress, and sroTCAP seems to have a modulating effect under similar stress conditions. For example, several apoptosis inhibitor genes and HSP 70 B2-like, 20 and 68-like genes were upregulated under stress but remained unchanged post sroTCAP delivery under the same stress condition. Similarly, a few transcription factors were downregulated under stress but were unchanged in the stress with sroTCAP group.The gene expression profile in the stress control  was still cardiac bioactive, having similar effects to the 10 pmol sroTCAP. Nonetheless, both delivery methods and concentrations showed that sroTCAP induced transient bradycardia.Helix pomatia, mechanical stress led to a 20% increase in HR and tachycardia-like effects , hypoxia or anoxia consistently induced bradycardia  and the proteomics data are deposited in the ProteomeXchange Consortium, accession number PXD039155 (https://www.ebi.ac.uk/pride/archive/projects/PXD039155).The datasets presented in this study can be found in online repositories. The transcriptome data presented in the study are deposited in the NCBI SRA database, accession number PRJNA804582 (AE, LP, and DL conceptualized the manuscript. TA, SS-A, PD, FR, SC, DL, and AE contributed to the design of the manuscript. TA, SS-A, and PD conducted the experiments. TW contributed to the LC-MS/MS analysis. TA, SS-A, SC, and AE wrote the manuscript. MD and WO\u2019C provided the experimental animals. All authors contributed to the article and approved the submitted version."}
+{"text": "Non\u2010coding RNA (ncRNA) regulatory networks are emerging as critical regulators of gene expression. These intricate networks of ncRNA:ncRNA interactions modulate multiple cellular pathways and impact the development and progression of multiple diseases. Herpesviruses, including Kaposi\u2019s sarcoma\u2010associated herpesvirus, are adept at utilising ncRNAs, encoding their own as well as dysregulating host ncRNAs to modulate virus gene expression and the host response to infection. Research has mainly focused on unidirectional ncRNA\u2010mediated regulation of target protein\u2010coding transcripts; however, we identify a novel host ncRNA regulatory network essential for KSHV lytic replication in B cells. KSHV\u2010mediated upregulation of the host cell circRNA, circHIPK3, is a key component of this network, functioning as a competing endogenous RNA of miR\u201030c, leading to increased levels of the miR\u201030c target, DLL4. Dysregulation of this network highlights a novel mechanism of cell cycle control during KSHV lytic replication in B cells. Importantly, disruption at any point within this novel ncRNA regulatory network has a detrimental effect on KSHV lytic replication, highlighting the essential nature of this network and potential for therapeutic intervention. Lytic replication of Kaposi\u2019s sarcoma\u2010associated herpesvirus in B cells depends on the dysregulation of a host non\u2010coding RNA regulatory network. The circular RNA circHIPK3 interferes with the miR\u201030c/DLL4 axis to enhance viral lytic replication. Even within ncRNAs, there is a wide repertoire of species generally categorised based on their size. Small ncRNAs are less than 200 nucleotides in length, and include microRNAs (miRNAs), small\u2010interfering, small nuclear, small nucleolar and PIWI\u2010interacting RNAs  are a novel class of lncRNAs, characterised by a covalently closed loop lacking a 5' end cap or 3' poly (A) tail. Originally thought to be rare splicing errors, recent breakthroughs have shown circRNAs are highly abundant and play key roles in gene regulation , are particularly proficient at utilising ncRNA regulatory pathways , using specific probes against the unique backsplice site within circHIPK3. Results demonstrated a clear cytoplasmic localisation in TREx\u2010BCBL1\u2010RTA cells  highlighted circHIPK3 (hsa_circ_0000284) contained complementary sequences to both miR\u201029b and miR\u201030c, with both software identifying two high score binding sites for miR\u201029b, and two and three, respectively, for miR\u201030c  , where binding to highly complementary RNAs induces miRNA degradation  and 20\u2009nM antagomiRs (ThermoFisher) were transfected using Lipofectamine RNAi Max (ThermoFisher) and left for 24\u2009h before addition of doxycycline and further experiments. Plasmids were transfected in a 1:2 ratio with Lipofectamine 2000 (ThermoFisher). CircHIPK3 LNA GapmeRs (Qiagen) were incubated at 100\u2009nM for 24\u2009h prior to lytic induction in DLL4 KD TREx\u2010BCBL1\u2010RTA cells for double\u2010KD experiments.TREx\u2010BCBL1\u2010RTA cells, a B\u2010cell lymphoma cell line latently infected with KSHV engineered to contain a doxycycline\u2010inducible myc\u2010Rta, were a gift from Professor JU Jung . TREx\u2010BCBL1\u2010RTA cells were cultured in RPMI1640 with glutamine (Gibco), supplemented with 10% foetal bovine serum (FBS) (Gibco), 1% P/S (Gibco) and 100\u2009\u00b5g/ml hygromycin B (ThermoFisher). HEK\u2010293T cells were purchased from ATCC and cultured in Dulbecco\u2019s modified Eagle\u2019s medium with glutamine (DMEM) (Lonza), supplemented with 10% FBS and 1% P/S. BC\u20103 cells were purchased from the ATCC and cultured in RPMI1640 with glutamine (Gibco) supplemented with 20% FBS and 1% P/S. HEK\u2010293T\u2010rKSHV.219 were kindly provided by Dr Jeffery Vieira (University of Washington) and were cultured in Dulbecco\u2019s modified Eagle\u2019s medium with glutamine (DMEM) (Lonza), supplemented with 10% FBS, 1% P/S and 3\u2009\u03bcg/ml puromycin (Gibco). All cell lines tested negative for mycoplasma. Virus lytic replication in TREx\u2010BCBL1\u2010RTA cells was induced via addition of 2\u2009\u00b5g/ml doxycycline hyclate (Sigma\u2010Aldrich). HEK\u2010293T\u2010rKSHV.219 cells and BC\u20103s were induced via addition of 20\u2009ng/ml TPA and 4\u2009mM sodium butyrate. All cells were cultured at 37\u00b0C at 5% COet\u2009al, Total RNA was extracted using Monarch Total RNA Miniprep kit (NEB) as per manufacturer\u2019s protocol. One microgram RNA was reverse transcribed using LunaScript RT SuperMix Kit (NEB). qPCR was performed using synthesised cDNA, GoTaq qPCR MasterMix (promega) and the appropriate primer. qPCR was performed on Rotorgene Q and analysed by the \u0394\u0394CT method against a housekeeping gene as previously described before plotting as fold change  as per manufacturer\u2019s protocol. RNA was then DNase treated using DNase I (18068015 Thermo Fisher) as per protocol. One microgram RNA was reverse transcribed using miScript II RT kit (Qiagen) as per protocol before qPCR using miScript SYBR Green kit (Qiagen) and analysis by the \u0394\u0394CT against SNORD68.Antibodies used in western blotting are listed below: ORF65 , ORF57 , GAPDH , GFP  and DLL4 (Proteintech 21584\u20101\u2010AP 1/200). Ago2 (abcam ab186733) was used in RIPs.DLL4 and circHIPK3 cloned into it. psiCheck2 was a gift from Dr James Boyne (Leeds Beckett University). GFP, GFP\u2010ORF50, GFP\u2010ORF57 and GFP\u2010ORF57 RGG1/2 have been described previously . PLKO.1 TRC cloning vector was purchased from Addgene  with shRNAs against Cell lysates were separated using 8\u201312% polyacrylamide gels and transferred to Amersham Nitrocellulose Membranes  via Trans\u2010blot Turbo Transfer system (Bio\u2010Rad). Membranes were blocked in TBS + 0.1% tween with 5% wt/vol dried skimmed milk powder. Membranes were probed with appropriate primary antibodies and secondary horseradish peroxidase conjugated IgG antibodies at 1/5,000 (Dako Agilent). Proteins were detected with ECL Western Blotting Substrate (Promega) or SuperSignal\u2122 West Femto Maximum Sensitivity Substrate (ThermoFisher) before visualisation with G box (Syngene).TREx\u2010BCBL\u20101\u2010RTA cells were seeded onto poly\u2010L\u2010lysine (Sigma\u2010Aldrich)\u2010coated coverslips and 2\u2009\u00b5g/ml doxycycline hyclate (Sigma\u2010Aldrich) was added 3\u2009h later. FISH was performed 24\u2009h later using ViewRNA Cell Plus Assay (ThermoFisher) as manufacturer\u2019s protocol. Coverslips were mounted using Vectashield Hardset Mounting Medium with DAPI (Vector laboratories). Images were obtained using a Zeiss LSM880 Inverted Confocal Microscope and processed using ZEN 2009 imaging software (Carl Zeiss) as previously described  was added 3\u2009h later. Cells were fixed with 15\u2009min with 4% paraformaldehyde and permeabilised with PBS + 1% Triton. All further incubation steps occurred at 37\u00b0C. Coverslips were blocked for 1\u2009h with PBS and 1% BSA before 1\u2009h incubation with the appropriate primary antibody and followed by 1\u2009h with Alexa\u2010Fluor conjugated secondary antibody 488 (Invitrogen 1/500). Coverslips were mounted using Vectashield Hardset Mounting Medium with DAPI (Vector laboratories). Images were obtained using a Zeiss LSM880 Inverted Confocal Microscope and processed using ZEN 2009 imaging software (Carl Zeiss) as previously described  as per manufacturer\u2019s instructions before qPCR analysis.For biotinylated RIPs, HEK\u2010293T cells were seeded and transfected with 20\u2009nM biotinylated LNA miR\u201029b or miR\u201030c (Qiagen) in combination with 2\u2009\u03bcl Lipofectamine RNAi Max (ThermoFisher). Twenty\u2010four hours post\u2010transfection, RIPs were performed as per manufacturer\u2019s directions using Dynabeads MyOne Streptavidin T1 (ThermoFisher). RNA was extracted and purified using TRIzol LS (Invitrogen) as per manufacturer\u2019s instructions before analysis via qPCR. Samples were analysed using fold enrichment over per cent inputs.Ago2 RIPs were performed on TREx\u2010BCBL\u20101 RTA cells using EZ\u2010Magna RIP RNA\u2010binding Immunoprecipitation kit (Merck millipore) as per manufacturer\u2019s instructions. RNA was extracted and purified using TRIzol LS (Invitrogen) as per manufacturer\u2019s instructions before analysis via qPCR. Samples were analysed using fold enrichment over per cent inputs.For GFP RIPs, HEK\u2010293Ts were transfected with 2\u2009\u03bcg GFP or GFP\u2010ORF57 plasmids with DNA in a 1:2 ratio with Lipofectamine 2000 (Thermo Fisher Scientific). Cells were lysed before incubated with GFP\u2010Trap Agarose beads overnight at 4\u00b0C (Chromotek) and washed as manufacturer\u2019s protocol. Samples were incubated with Proteinase K buffer  for 30\u2009min at 55\u00b0C before RNA extracted via TRIzol LS (Invitrogen) as per manufacturer\u2019s instructions before analysis via qPCR. Samples were analysed using fold enrichment over per cent inputs.DLL4 was identified using NCBI AceView before cloned into psiCheck2 plasmid. HEK\u2010293T cells were transfected with 50\u2009nM miR\u201030c or a scrambled control alongside psi\u2010check2 plasmid. Luciferase reporter assays were performed 24\u2009h post\u2010transfection using Dual Luciferase Reporter Assay System (Promega) as per the manufacturer\u2019s directions.The 3\u2019UTR of g at room temperature, in the presence of 8\u2009\u03bcg/ml of polybrene (Merck Millipore). Three \u00b5g/ml Puromycin (Gibco) was added 48\u2009h after transduction before KD analysed via qPCR and western blot if appropriate.Lentiviruses were generated by transfection of HEK\u2010293T cells seeded in 12\u2010well plates using a three\u2010plasmid system. Per 12\u2010well, 4\u2009\u00b5l of Lipofectamine 2000 (Thermo Scientific) was used together with 1.2\u2009\u00b5g of pLKO.1 plasmid expressing shRNA against the protein of interest, 0.65\u2009\u00b5g of pVSV.G and 0.65\u2009\u00b5g psPAX2. pVSV.G and psPAX2 were a gift from Dr. Edwin Chen . Eight hours post\u2010transfection, media were changed with 1.5\u2009ml of DMEM supplemented with 10% (v/v) FCS. Two days post\u2010transfection, viral supernatants were harvested, filtered through a 0.45\u2009\u00b5m filter (Merck Millipore) and immediately used for transductions of TREx BCBL1\u2010RTA cells. Cells  in 12\u2010well plates were infected by spin inoculation for 60\u2009min at 800\u2009Site\u2010directed mutagenesis of GFP\u2010ORF57 for W292A mutant was carried out using QuikChange site\u2010directed mutagenesis kit (Agilent technologies) as per manufacturer\u2019s instructions.Cells were seeded, harvested as required and followed by fixing overnight in 70% ethanol at \u221220\u00b0C. Cells were washed in PBS containing 0.5% BSA, before incubation for 30\u2009min at room temperature in PBS containing 0.5% BSA, 5\u2009\u00b5g/ml RNase A (ThermoFisher) and 25\u2009\u00b5g/ml propidium iodide (Sigma). Samples were analysed by CytoFlex S Benchtop Flow Cytometer (Beckman Coulter). For cell cycle drug treatment, TREX\u2010BCBL\u20101 cells were pre\u2010treated for 16\u2009h with 10\u2009\u00b5M RO\u20103306, 0.5\u2009\u00b5M nocodazole or 2\u2009mm thymidine before addition of doxycycline. Viral load was measured after 48\u2009h.Total RNA was extracted from TREx\u2010BCBL\u20101s at 0, 16 and 24\u2009h post\u2010lytic induction. Small RNA libraries were prepared using the TruSeq Small RNA Library Prep kit (Illumina). Samples were assessed using Agilent High Sensitivity D1000 Screen Tape Station and gel electrophoresis and size\u2010based extraction performed. cDNA libraries were analysed via Illumina HiSeq (Illumina) by University of Leeds NGS facility and data deposited at Gene Expression Omnibus (GSE186652).Total RNA was extracted from TREx\u2010BCBL\u20101s at 0 and 18\u2009h post\u2010lytic induction. mRNA sequencing libraries were created using an Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina). cDNA libraries were generated and analysed using an Agilent Technologies 2100 Bioanalyze. Libraries were sequenced using a HiSeq (Illumina) 75 sequencing platform.et al, et al, et al, P\u2010value\u2009<\u20090.05. To reduce rate of false discovery rate in the DE analysis, we included only transcripts with at least 1 CPM in three samples.Raw reads were processed for RNA expression using standard bioinformatics pipeline. Quality filtered (Q\u2009<\u200920) and adapter trimmed reads (Trimmomatic v0.39)  using three or more biologically independent experiments. Differences between means were analysed by unpaired Student\u2019s\u2009Katherine L Harper: Conceptualization; Data curation; Formal analysis; Investigation; Writing\u2014original draft; Writing\u2014review and editing. Timothy J Mottram: Data curation; Investigation; Writing\u2014review and editing. Chinedu A Anene: Data curation; Formal analysis; Writing\u2014review and editing. Becky Foster: Investigation; Writing\u2014review and editing. Molly R Patterson: Resources; Writing\u2014review and editing. Euan McDonnell: Data curation; Writing\u2014review and editing. Andrew Macdonald: Resources; Writing\u2014review and editing. David Westhead: Funding acquisition; Writing\u2014review and editing. Adrian Whitehouse: Conceptualization; Formal analysis; Supervision; Funding acquisition; Writing\u2014original draft; Project administration; Writing\u2014review and editing.CRediT author contributions listed above, the contributions in detail are:In addition to the Conceptualisation (KLH and AW); Data curation ; Formal Analysis (KLH and CAA); Funding acquisition (AW and DW); Investigation ; Reagents (MRP and AM); Writing \u2013 original draft (KLH and AW) and Writing \u2013 review & editing .The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Source Data for AppendixClick here for additional data file.Source Data for Figure 1Click here for additional data file.Source Data for Figure 2Click here for additional data file.Source Data for Figure 3Click here for additional data file.Source Data for Figure 4Click here for additional data file.Source Data for Figure 5Click here for additional data file.Source Data for Figure 6Click here for additional data file.Source Data for Figure 7Click here for additional data file."}
+{"text": "Prolonged alcohol intake for many years has been known to cause serious ailments in human beings since time memorial. Even after knowing that this dangerous addiction paves the way to one\u2019s own grave, there isn\u2019t much difference in the way the community sees this deadly habit. Time and again history has proven that this fatal addiction could make the life of those who consume it terrible. Also, the lives of the dear ones of alcoholic people are affected as alcohol not only affects those who consume them but also kin and friends. Various research studies conducted over many years clearly show the association of prolonged alcohol intake in the causation, aggravation, worsening, and deterioration of the health of its consumers. Moreover, chronic alcohol intake single-handedly is one of the major etiological factors in various serious diseases. Through the ages, alcoholism has been undisputedly maintaining its position in the list of risk factors for preventable diseases in the world. According to a WHO report, 5.3% of all deaths that occurred worldwide in the year 2016 were because of harmful alcohol use [Alcohol clearly plays a very important role in making many other diseases progress to their advanced stages. It has been also noted that alcohol intake and its related disorders are often associated with many other manifestations; for example, patients with alcoholic neuropathy often have associated nutritional deficiencies. Recent studies have clearly proved that alcoholism is associated with many types of cancers too and this understanding of alcoholism has spurred research minds all over the globe to find out the exact pathophysiology behind the same. Alcohol is a very easily available source of addiction, which is one of the main reasons why it remains a serious threat to the community. There is a huge variety that is available as far as alcoholic drinks are concerned. Alcohol is also one of the cheaply accessible means of addiction; this explains why alcoholism is so prevalent. A person may initially start consuming alcohol in very low amounts most probably with just a desire to try it, but once he or she gets addicted, then getting rid of the habit becomes extremely difficult. Even if a person is mentally resolute enough to quit alcoholism, his or her body, which has been modified because of the chronic use of alcohol, won\u2019t be up to the challenge anytime soon; he or she has to overcome many hurdles put forward by the body, which could in an umbrella term be referred to as alcohol withdrawal syndrome.There are many social stigmas associated with alcohol intake. Most people get into this addiction by getting inspired by the people whom they admire, like actors, celebrities, role models, etc. Also, exposure to the sight of family members, relatives and friends drinking alcohol has a huge impact on one\u2019s mindset as he or she may take it to be something that is normal. In the long run, most of the time, even without their realization, people get pathetically trapped in this dangerous fatal habit of alcoholism, which eventually makes their lives pitiful in almost all aspects. Studies have shown that alcohol is also a key player in many other domains too like accidents, suicide, depression, hallucinations, violence, memory disturbances, etc.The main purpose of this review article is to enable any person reading this article to get a comprehensive insight into the effects of alcohol on the various systems of the human body, and for the same, many recognized research articles published in numerous well-acknowledged journals across the globe are reviewed. The article is written using very basic and simple terminologies so that even a layperson who reads it would be able to understand it. For the easy acceptability and understanding of the reader, the discussion is written in such a way that almost every major system is reviewed one by one and the effect of alcohol on these systems put forward in very simple language. The strategies used for the establishment of this review article are summarised in Figure Impact of alcohol on the central nervous system (CNS)Alcohol exerts various effects on our CNS in various ways, the common ones being depression of the CNS, destruction of the brain cells, contraction of the tissues of the brain, suppression of the excitatory nerve pathway activity, neuronal injury, etc . AlcoholAlcohol use disorder (AUD) is chronic in nature and is characterized by uncontrolled drinking and also a preoccupation with alcohol. The severity of AUD is a crucial factor in how it is going to affect the human body. AUD can be mild, moderate, or severe according to the symptoms a person experiences. The clinical manifestations of AUD include signs and symptoms such as inability to control the amount of alcohol intake, spending a lot of time drinking, feeling an uncontrollable craving for alcohol, loss of interest in social activities, failure to fulfil tasks within the time provided, etc. Most of the time, along with the person who consumes alcohol, several other factors are also to be taken care of in order to effectively manage alcohol-related health conditions. These factors can be social, environmental, genetic, psychological, etc, which make a considerable impact on how alcohol affects the behaviour and body of those consuming it. Binge drinking, i.e., drinking to such an extent on a single occasion that the blood alcohol concentration level becomes 0.08% or more, is a very relevant aspect of alcohol intake, which has to be dealt with, with utmost urgency. Certain research studies suggest that mild to moderate alcohol intake provides a certain sort of protection against a few CNS disorders like dementia, ischemia of neurons, etc, but this in no way should encourage the community in promoting alcohol intake as in reality, it is very difficult to remain within the limits of mild to moderate alcohol intake, and thus, eventually, people do end up as full-time severe alcohol abusers. Epilepsy, a seizure disorder caused by disturbed nerve cell activity in the brain, aggravates on excessive alcohol intake as alcohol increases the frequency of seizures in patients of epilepsy . The issImpact of alcohol on the cardiovascular system (CVS)Chronic alcohol intake is undoubtedly a very important risk factor as far as\u00a0cardiovascular diseases are concerned and several clinical trials do point out this fact. The results of several research studies conducted in various settings clearly indicate that increased intake of alcohol has increased adverse effects on our heart and its vasculature. Alcohol exerts its action on the cardiovascular system both directly and indirectly. Blood pressure, a very vital player in the domain of cardiovascular diseases, is in turn itself affected by increased alcohol consumption. Blood pressure gets increased on regular consumption of alcohol in a manner which is dose-dependent, which in turn increases the risk of hypertension and eventually leads to various cardiovascular complications. How exactly alcohol causes hypertension is still unclear with many pathophysiological theories out there. Atrial fibrillation, one of the most common causes of arrhythmia, is associated with the high-volume chronic intake of alcohol and above 14 g alcohol/day, the relative risk dramatically increases by 10% for each extra standard drink (14 g ethanol) .Cerebrovascular accidents are increased to a great extent at almost all levels of alcohol intake . AlcoholImpact of alcohol on the digestive systemChronic alcoholism is found to have a very strong relationship with both acute pancreatitis and chronic pancreatitis. Chronic alcohol intake impairs the repair ability of the structures of the exocrine pancreas, thereby leading to pancreatic dysfunctioning . Most ofImpact of alcohol on the causation of cancerAlcohol has much to do with cancers too and continuous research studies are conducted in order to find out the relationship between the two in detail. In a meta-analysis, it was found that women consuming alcohol had a later menopause onset, which is found to be associated with reduced cardiovascular disease risk and also all-cause mortality, but unfortunately, the happiness of this advantage gets compromised by the ironic fact that it has an increased risk of cancer (including ovarian and breast cancers) ,22. LargImpact of alcohol on other systemsApart from the systemic manifestations which do affect a particular system of the body, there are various disorders in which alcohol indirectly provides its crucial contribution. It is a common finding that one could perceive that alcohol is most of the time in the list of risk factors for various diseases. Alcohol has been found to adversely affect our immune system and the matter of concern as far as this issue is concerned is that immune responses are influenced by even moderate amounts of alcohol intake . AlcoholAlcohol seldom leaves any system untouched as far as leaving its impression is concerned, spanning from single tissue involvement to complex organ system manifestations. Almost all the major organs that make up a human\u2019s physiological being are dramatically affected by the overconsumption of alcohol. There is an enormous overall economic cost that is paid for alcohol abuse all over the world."}
+{"text": "The number of nonagenarians is growing globally. The promotion of mental wellbeing is increasingly important. The aim of this study was to explore mental wellbeing and psychological experiences of older adults in their early 90\u00a0s who were living at home.We conducted a qualitative study using semi-structured face-to-face interviews with 20 older adults in their early 90\u00a0s. A thematic analysis, according to Braun and Clarke, was used to analyze data.An inner process of older adults in their early 90\u00a0s was revealed; its three themes were the \u201creality of aging,\u201d \u201cseeking emptiness of the mind,\u201d and \u201cstill moving on.\u201d Older adults in this study experienced functional decline, regret, and loneliness. They were tired of life and nearly gave up. Emptying their minds helped them reset their attitudes and find a way to move on. After realizing that negative thinking did not help anything, they focused on what they could do and their daily routines. Perceived social usefulness validated participants\u2019 self-worth. However, a few were consistently active without negative perceptions of aging.Understanding the psychological process and mental wellbeing in later life aids in the development of practical healthcare policies to assist the growing oldest-old population in cope with age-related challenges and improve their mental wellbeing. The ongoing demographic transition constitutes a challenge for healthcare providers and policymakers to help the oldest old live not only longer but also better lives by making wise use of resources \u20133. The wIn later life, people are more likely to experience bereavement, loss of social networks, and health problems, all of which can negatively affect mental wellbeing , 6. FurtBy contrast, a high level of mental wellbeing positively affects mortality, morbidity, and life satisfaction. Mental wellbeing has a greater impact on life satisfaction than on physical health in old age . The wilAs the aging population is heterogeneous, the concept of segmenting the older adult group has emerged in gerontology research . In JapaIn our previous qualitative study, which was a part of the Arakawa study, near-centenarians, and centenarians (mean age:\u00a098.6\u00a0years) maintained autonomy by making small decisions despite their functional decline, and found joy in the little things that made life worth living . What prThis study is part of the 85\u2009+\u2009Arakawa study, a cohort study of older residents  in Arakawa City Ward, Tokyo, Japan, with the aim of identifying contributing factors to a healthy and long life in older adults. The cohort study collected a variety of data, including physical and cognitive functions, mental health, lifestyle, and physical activities. As our previous studies  reportedParticipants were older adults who met the inclusion criteria and were purposively selected from the Arakawa cohort registry. At the three-year follow-up survey of the Arakawa 85\u2009+\u2009study, we asked older participants aged 90\u201394\u00a0years to determine whether they would be interested in a qualitative in-person interview. The inclusion criteria were as follows: 1) age in their early 90\u00a0s (to differentiate from the sample in our previous study of older adults aged 95\u00a0years and older) , 2) thosDuring the recruitment process, a research assistant called 21 candidates to explain the outline of the interview survey and asked them to participate in the study. To maintain the voluntary nature of the interviews, the 21 interviewees decided to participate in the study on their own, and visits were scheduled via telephone. One of the candidates declined from the study because of ill health a few days prior to the scheduled interview. Then, a nursing researcher (HK or KY) made home visits and explained the purpose and procedures of the study to the participants and their family members , both orally and in writing. Informed consent was obtained from those who agreed to participate.An interview guide was developed for this study. We began with open-ended questions that allowed older adults to express themselves freely, including \u201cHow do you spend a typical day and what do you think about it?\u201d \u201cWhat is the same or different between now and when you were in your 80\u00a0s?\u201d \u201cAre you happy that you have exceeded the average life expectancy? Why?\u201d and \u201cWhat do you expect, look forward to, or worry about in your life?\u201d Then, based on their responses, we asked exploratory questions on their mental wellbeing and psychological experiences, such as \u201cHow did you feel about it?\u201d \u201cCould you share your feelings and concerns?\u201d \u201cDoes your family care about you?\u201d and \u201cWould you talk to me in more detail?\u201d For relatively shorter interviews, we obtained supplementary information from the families who sat in the interview for a better understanding and deeper interpretation after the interview.Data were collected between October 2020 and November 2021. The survey was suspended from January to August 2021 due to the COVID-19 pandemic, except in April and July. Twenty participants were interviewed at their homes. The interviews were audiotaped with permission from the participant (and family) and transcribed verbatim by a transcription service specialist. Personally identifiable information was anonymized.A thematic analysis was performed according to the following multistep process described by Braun and Clarke : 1) KY iWhen no new analytical information emerges and the maximum amount of information on the phenomenon is provided, the data are deemed saturated . AccordiWe conducted investigator triangulation to ensure the credibility and trustworthiness of the qualitative research . After cTwenty older adults (11 women and 9 men) participated in this study. The mean age was 91.1\u00a0years. Table Three phases in the process of inner change among older adults in their early 90\u00a0s were identified Fig.\u00a0. In the The study participants were aware of the aging process and generally experienced a decline in physical and cognitive functions, feelings of loneliness, and regrets over what they could not achieve by reviewing their lives. They were also losing the energy to find meaning in life. They were tired and seemed burdened with living.Although some participants had expected to experience a decline in physical activity as they aged, the actual decline in a variety of functions made them feel that something was missing, as if they were losing who they had been:Since my legs have stopped working, I wonder [if] this is what it is. I see. This is what happens to us as we get older. Now, I'm sad, sad. What I should say\u2014something like, I'm stunned? .You know, gradually, speech impediment, stammering....Even when I'm talking, I start to lose track. Sometimes I don't know what I'm talking about. That's why I was so shocked when I started to lose control of my body. I thought I was fine until then.Furthermore, one of the male participants was shocked to notice a deterioration in his own cognitive and language functions, including losing track of conversations and having difficulty retrieving words:I've been feeling less and less strong in past years. Several participants realized that they had to acknowledge the reality of irreversible aging and progressive physical and functional decline when they were over 90, and they felt helpless that they had nothing to do but just grow old, as expressed below;I'm afraid I have no choice. .I didn't regret it until I was 80 or 90 years old. When I was younger, I didn't know anything. As the participants reached the end of their lives, many of them looked back on the past rather than to the future, expressing regret over what they had wanted to do and failed to do:Life goes by so fast. So fast. I wanted to do it right when my husband was gone. I did what I did right, but I wanted to do it better. Then, sad memories came to mind, and they suffered from unresolved psychological pain, as illustrated here:I couldn't let my child[ren] live a childlike life. I really feel sorry for them, my son and daughter. One of the male participants, whose house had burned three times during the war, regretted that his children had to put up with economic hardship, although he had financial responsibility for his family:I used to go out with my friends, go out drinking, but all my friends are gone. I have no friends anymore. Almost none. There are almost no people my age in the town council. They've all gone before me. Due to bereavement over the loss of many family members and friends, participants gradually decreased their interactions with others, experienced a greater sense of loneliness, and felt closer to their own deaths every time they lost a family member or friend, as expressed below:Well, there are very few people I can reveal my heart [to]. Even [when] I talk to my children\u2026they don\u2019t understand it. If they studied something like this [this qualitative study] or theme, it might be different. But I don\u2019t have the chance to talk about this kind of story to my children or any other people, and they never understand me. I can only talk about specific matters, like I feel sick, I have trouble with my legs, I want you to do this\u2026.Well, no deep conversation, I think it is going to happen gradually. Furthermore, multiple participants felt lonely because there was no one they could talk to about their deepest feelings or who understood them. Conversations were superficial, even with their family members; the participants were often only asked about their health. They felt that they could talk to no one in depth or that no one would understand their true feelings:I am losing more and more friends. In these two years, three more friends passed away (\u2026) I talked to my friends that we could have gotten together again if there were no COVID-19 pandemic (\u2026) The impact was huge. It has been a heartless two years for older people. Due to behavioral restrictions on COVID-19 for almost two years, one of the female participants felt regret and loneliness for not being able to say goodbye to her friends who died:Life was long and fragile for the study participants in their 90\u00a0s. Most of them repeatedly used negative expressions such as \u201cbother\u201d and \u201csick and tired.\u201d Their lives were narrowing in terms of independence and relationships. It was difficult to find pleasure and hope. They were negative about themselves and their future. Life seemed meaningless and wasted, as expressed below:I can't do anything anymore. It's so boring. .What a bother! When you can't move yourself, it bothers me. It got boring. Life is fast. It goes by before I can do anything. I am now 93 years old, and my legs are weakening so much. I am convinced that I will not be able to walk even if I live to be 100. So, I think that it is a question of living a long life. Some of the participants said that even daily living was a burden and that they hoped a late spouse would bring them to heaven:I wish my late husband would come to pick me up. .It\u2019s really annoying\u2026I live in front of a cemetery. I visit his (my husband\u2019s) grave and complain to him, \u201cWhy are you forgetting me (to bring to the heaven)?\u201d Many of the study participants used the terms \u201cbother\u201d and \u201csick and tired\u201d to represent voluntarily shutting down their thoughts. In difficult situations, they tried to separate themselves from their thoughts and emotions to avoid falling into despair. They sought to empty their minds to find inner peace from nothingness. They unconsciously tried to detach themselves from the negative perceptions of self and the future by clearing their minds of distracting thoughts and concentrating on following a daily routine.Many participants did not think about their future and recognized that there was no point in doing so. Since thinking about the future could not solve their problems, they talked about the benefits of thinking about nothing and staying free of distractions:Just so so. I feel nothing that probably good for me. .I don\u2019t think [about the future]. Nothing starts to happen even if I think about it. When I'm gardening, I focus on that. When I have free time, I feel lonely somehow. When I'm moving, I don\u2019t have such thoughts. Concentrating on what they were doing also helped them dismiss their feelings of anxiety and loneliness:No, I don\u2019t think (about the future). In that way, my mother lived until the age of 103. So, I don\u2019t think about age. I believe it naturally ends. I don\u2019t think about it much. One of the participants wanted to behave like his mother because his mother had been happy until her death at the age of 103 without seriously thinking about her age and future.Regardless of being satisfied or not, what should I say? Something like passing by\u2026.Well, we are living, so we have to do so anyway. You know. It\u2019s annoying\u2026everything is burdensome. I just get up in the morning and have breakfast, something like that\u2026.I have no goals. I am just living\u2026.Yes, I am just living\u2026.I just eat, drink, and sleep. That\u2019s all. Most participants valued a routine. They spent their time in a regular routine and adopted an attitude of indifference toward other matters. This passed the time, as described below:I have a routine that I follow almost every day, so I mostly live by that routine. I repeat it...because a year is a set number of days, right? [Life is] a series of them. It's hard to say how long we can live, and I don't have much faith in it myself. So, the age doesn\u2019t matter. I don\u2019t think about my age. It\u2019s a simple routine, but I think it\u2019s good if we repeat it. One woman was not aware of the average life expectancy but believed that longevity was the result of a regular daily routine. Thus, she placed importance on repeating the routine, as explained here:Most of the participants accepted death as a natural occurrence. They realized whether they wanted it to or not, life would go on, and thus, they had to move on. The acceptance of reality helped them take action. They were not waiting for death but striving to move forward and grow, even if only a little. They focused on what they could do, and their ultimate desire was to remain useful to others.That's how it is with everyone. No matter how healthy you are, you'll all die in the end. When I was young, I was scared of death and sickness. So, I thought I should try to maintain my fitness many times. Somewhere along the way, I forgot all about it. The fear of dying is gradually disappearing. The fear of dying...I think it can\u2019t be helped anymore. I\u2019ve given up. It\u2019s\u2026something. We\u2019re gradually giving up, aren\u2019t we? Negative thinking helps nothing\u2026.There is no point in resisting. Being older than 90\u00a0years meant that all participants were coming closer to the end of life. Some realized that death was natural and could not be resolved by resisting it. Accepting it helped ease their fear:I have to think for myself and live by myself. Others will not help you. I am grateful that I am healthy enough to talk like this and eat what I want and that I am not sick. Life ends someday. It is not useful to spend to maintain a negative mindset. Eventually, the participants in this study shifted to thinking positively in order to live full lives:Every day is full of life. It\u2019s a mindset. Yes. I think it is a blessing to be able to think that way. I try to interpret things in a positive way. People who are dissatisfied are unhappy. It is better to live with satisfaction. Furthermore, she continued to say the importance of the mindset:When the participants had a new mindset, they started to actively find out what they could do. They were self-disciplined and concentrated on doing what they could, as exemplified below:If I stay in a daze, I feel like I\u2019m going to develop dementia, so I have to do something. I\u2019m trying to use my head to match the numbers in Yomiuri [newspaper]. .So, to not to forget, writing and reading printed texts are something I always do. I\u2019m 93 years old. I guess it\u2019s normal for my body to deteriorate like this. It's natural. On the contrary, I say, \u201cI don\u2019t know what I\u2019m going to do if I don\u2019t have strong legs.\u201d I hold onto the refrigerator there and do 10 squats. It\u2019s hard. I do 10 squats two times a day. Children and grandchildren are around. I feel they are stimulating me\u2026.When they come, (my children) bring their children\u2026.Well, it\u2019s my role to read a picture book to them in the New Year. First, I love flowers. People buy flowers or get small potted plants from other people and they die within a few days, but I make them last. Yes, I make them last\u2026.I make flower arrangements that are displayed somewhere special. Even if it is not particularly beautiful, when this flower blooms, I\u2019ll cut it and give it to my friends. The study participants still desired to be useful to others, even if only in a small manner. Playing their roles helped them recognize the value of their own existence. Helping others enhanced their self-esteem, motivation, and personal development, as demonstrated below:When I make them, everyone says, \u201cMs. I makes a beautiful one\u201d and \u201cbeautiful stitch marks.\u201d I made about 60 masks for other people. I gave them to various people. People asked me, \u201cDid you make it?\u201d and when I said \u201cyes,\u201d they said, \u201cI want it. Please make it for me\u201d (laugh). One participant had been sewing since she was young. She could sense her own value because others had asked her to make masks during COVID-19, and when she did, they praised her and were pleased with her work. Such interactions with others illuminated the lives of older adults and made their lives worthwhile. She happily talked about her masks:While some participants were able to pursue active living by overcoming the negative phases, a few participants had never tired of life and were consistently active. One of them talked about her wish to travel abroad at the age of 90 after the COVID-19 pandemic.Yes, I would like to travel abroad. It must be boring to spend time like this at home with nothing to do. .I think that people who have hobbies can live a long life without getting bored\u2026.A man with nothing, for example, he worked for a company and retired, but working was his hobby. Such kinds of people often get sick soon after their retirement. These participants not only enjoyed their hobbies but also continued to make some progress, such as learning. Thus, their lives were fulfilling, and they experienced a sense of enjoyment and hope, as expressed below:This study revealed the psychological processes of older adults in their early 90\u00a0s. Three themes emerged: \u201cthe reality of aging,\u201d \u201cseeking emptiness of the mind,\u201d and \u201cstill moving on.\u201d In their early 90\u00a0s, some older adults nearly gave up on life with negative perceptions of the self and future. Emptying their minds helped them reset their attitudes. After realizing that negative thinking did not help anything, they focused on their abilities and desires. Perceived social usefulness helped them identify their strengths and validate their self-worth.With an increase in the oldest-old population, the focus is shifting from negative aspects, such as mortality, morbidity, and disability, to positive perspectives, such as promoting mental wellbeing , 6, 22. Social engagement builds the basis of social relationships by providing a sense of belonging, social identity, and fulfillment . MeaningLoneliness and isolation negatively affect mental wellbeing . ExistenA content analysis by Newall et al.  revealedPurpose in life, which refers to having goals, a sense of direction, and meaning, is associated with improved mental and physical health outcomes . Older aIn the second phase of the process, \u201cseeking emptiness of the mind,\u201d the participants focused on spirituality, peace of mind, and personal development in the personal dimension. When participants in this study were overwhelmed with negative thoughts and faced an identity crisis, they sought to empty their minds. Many participants tried to ignore all their distressing thoughts and shut down their thoughts because they wanted to be free from heavy psychological burdens and despair. They sought inner peace with \u201cno distractions.\u201d They attempted to focus on their daily routines and surrendered to the flow of life. As routines should be performed regularly, they help older adults save energy with minimal cognitive effort and perform everyday tasks, resulting in a sense of control , 40.Once emptying the mind brought peace of mind, the participants realized that aging was a natural process and there was no point lamenting what was lost. They began to gain a new perspective of the world. As people age, they are likely to orient toward deeper psychological issues rather than superficial or materialistic ones . PreviouFinally, in the last phase, \u201cstill moving on,\u201d the improvement of mental wellbeing of the oldest old was observed in all functional, social, and personal dimensions. After resetting their mindset, their personal wellbeing improved. They accepted the reality of aging, showed gratitude for life, and turned optimistic and active. They were positive and proactive in maintaining their fitness and were happy to help others. Psychological flourishing is possible with experience of purposeful life and personal growth even in very late life .Social engagement is \u201ca potential health-promoting factor\u201d . Social The environmental dimension is described as natural surroundings and living conditions . Home anIn this study, a few participants were consistently positive and did not experience a negative phase of the aging process. However, it remains unclear whether they underwent a different process. The differences between older adults with and without a negative phase deserve further attention in future research. As the oldest-old population is highly heterogeneous, differences across subgroups, including various levels of physical health, functional impairment, and contextual factors, should be considered . QualitaThis study had some limitations. First, the participants were relatively healthy older residents in a municipality in an Asian country with a long-life expectancy. Their cultural backgrounds may have affected the findings. Second, coding was conducted by one researcher (KY), not by two independent researchers. Consensus was reached by KY and HK after reviewing and refining all codes, subthemes, and corresponding passages. Third, most interviews were conducted during the COVID-19 pandemic, and lengthy interviews were not feasible. The participants might have become nervous with preventive measures such as wearing masks and maintaining social distancing. Only one participant had a 15-min interview, which was very short, and we could not obtain supplementary information from his family because he lived alone. We admit that the depth of the data is limited. In addition, comments made by family members might have influenced participants\u2019 statements. Despite the short interview time, many participants were glad to be part of the study, talk about themselves, and help the younger generations with their experiences.The growing oldest-old population requires more resources; this is an urgent health policy agenda. Healthcare professionals and policymakers should shift away from traditional stereotype models and focus on improving wellbeing and capacities \u20133. Physi"}
+{"text": "The present data set contains self-report data of German individuals participating in a longitudinal data assessment via online surveys conducted in the year preceeding the general elections in Germany.Data of N\u00a0=\u00a0122 individuals are included in the data set. Those individuals participated in an initial, extensive survey between November 2020 and February 2021 (T1) as well as in a final survey after the general German elections, thus, between the end of September 2021 and October 2021 (T3). Of those individuals, n = 93 additionally participated in an intermediate survey in between the previously mentioned ones between the end of May and the end of June 2021 (T2).Next to the assessment of sociodemographic variables, information on  news consumption, such as the frequency of being confronted with counter-attitudinal news, and on political attitudes, for example via current voting intentions for one of the major German parties, were assessed in the initial survey (T1).In the intermediate survey (T2), participants provided information on recent political news consumption habits including the frequency of being confronted with counter-attitudinal news, current voting intentions for one of the major German parties, as well as on extraordinary events that happened recently and impacted their voting intentions.In the final survey (T3), sociodemographic variables and actual voting decisions in the general German elections in 2021 were assessed. Moreover, variables on recent political news consumption habits, including the frequency of being confronted with counter-attitudinal news, and extraordinary events that happened recently and impacted voting decisions were assessed. Finally, a detailed self-report questionnaire retrospectively assessing political news consumption for the time between participation in the initial survey (T1) and the final survey (T3) was completed by participants. Not only did this questionnaire assess which online and offline news channels  participants used. Besides, the questionnaire included items on how many outlets per channel were used and the frequency of being confronted with counter-attitudinal news within each channel.This data set is provided alongside the present article to be used for further investigations of the stability of voting intentions, thus, political attitudes. Moreover, a content analysis of the open responses on which extraordinary events happened and impacted voting intentions/decisions can provide further knowledge on factors influencing voting intentions and their variability versus stability. These associations provide insights of importance to estimate the necessary sample sizes for forthcoming research projects.\u2022Additionally, content analyses on which kinds of extraordinary events impact voting intentions, thus, political attitudes, can be conducted.\u2022Finally, the present data on a German sample might contribute to cross-cultural comparisons of associations between various variables related to news consumption and political attitudes when combined with additional data sets from other countries.1The relations between information consumption, more specifically political news consumption, and political attitudes gained increasing attention during the last decade. More specifically, some experts fear that a high degree of homogeneity  of political news consumption is contributing to extremization and polarization of political attitudes; see, for example, discussions on \u201cfilter bubble\u201d and \u201cecho chambers\u201d. Oftentimes especially algorithms on the internet are being criticized for information filtering and contributing to extremization and polarization of opinions However, empirical research on the existence of absolutely homogeneous information environments is inconclusive After data cleaning (explained in detail below), the final data set comprises data of N\u00a0=\u00a0122 individuals  who participated at least in the survey at T1 and the survey at T3. The mean age of the sample at T1 was M\u00a0=\u00a030.09 (SD\u00a0=\u00a012.53) years, and the mean age at T3 was M\u00a0=\u00a030.85 (SD\u00a0=\u00a012.61). Most participants indicated either high school degree/A-level  or a university, including university of applied sciences, degree  as their highest educational degree. Participants came from all federal states in Germany, except the Saarland and Saxony-Anhalt.Items presented to all participants in the surveys at T1, T2, and T3 and for which data are included in the data set are presented in https://osf.io/4hsxe/.Data of all surveys and all items are included in the excel file (sheet: \u201cdata\u201d) provided alongside this article. The excel file does also include detailed descriptions of the wording of each item and its response options in both German and English language (sheet: \u201ccodebook\u201d). Additionally, the wording of each item in English language is provided in the PDF file uploaded to the OSF available from: https://osf.io/r6yfv/.2The present data set was collected from a sample of German volunteers eligible to vote in the general German elections in 2021. More specifically, all participants included in the data set participated in an initial survey (T1) between November 2020 and February 2021. After participating in this survey, participants were re-invited to participate in monthly surveys starting in the end of February until after the general German elections in September 2021. Invitations were sent to all participants who provided their email address after participating in the initial survey (T1) on a monthly basis via email. Unfortunately, due to issues related to data matching and issues when programming the survey, only data from two additional surveys after the initial survey (T1) can be provided: Data from the survey (T2) conducted from end of May to end of June 2021 and data from the final survey (T3) after the general elections in Germany in 2021, which was conducted between end of September 2021 and October 2021. A summary of the data collection is depicted in Participation in the initial survey was advertised in various ways both offline  and online : When the first author gave an interview on topics related to the content of the survey, the link was presented. Additionally, Twitter ads were used, and students writing their theses on data of this research project supported data collection. Lastly, some influencers on Instagram and YouTube provided the link to the survey to their audiences and followers.https://www.surveycoder.com/; https://ckannen.com/). As an incentive for participation, all participants received anonymous feedback on their scores in several of the self-report measures assessed. The local ethics committee at Ulm University, Ulm, Germany approved the data collection including all monthly surveys. The data set including the exact wording of all items and all response options in English and German language is provided in the excel data file. Aside from sociodemographic variables, which were used in previous studies, items on party affiliation All surveys were programmed on the SurveyCoder tool , and n\u00a0=\u00a02 additional participants were excluded due to providing a higher educational degree in the initial compared to the final survey. Finally, n\u00a0=\u00a09 participants were excluded due to failing the attention check item. This led to the final sample size of N\u00a0=\u00a0122 participants.The present research project was approved by the local ethics committee of Ulm University, Ulm, Germany (247/20). The data collection procedure followed the latest revision of the Declaration of Helsinki. All participants provided informed electronic consent prior to participation in each of the surveys. Data were collected in a pseudonymous way to match data across surveys. Data were anonymized before the upload.Cornelia Sindermann: Conceptualization, Methodology, Software, Validation, Formal Analysis, Investigation, Data Curation, Writing \u2013 original draft, Visualization, Supervision, Project Administration, Funding Acquisition; Christopher Kannen: Software, Writing \u2013 review & editing; Christian Montag: Writing \u2013 review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.Nevertheless, the authors declare the following financial interests/personal relationships:https://about.fb.com/de/news/h/gespraechskreis-digitalitaet-und-verantwortung/) debating ethical questions linked to social media, digitalization and society/democracy at Facebook. In this context, he receives no salary for his activities. Finally, he mentions that he currently functions as independent scientist on the scientific advisory board of the Nymphenburg group . This activity is financially compensated. Moreover, he is on the scientific advisory board of Applied Cognition , an activity which is also compensated.For reasons of transparency Dr. Montag mentions that he has received (to Ulm University and earlier University of Bonn) grants from agencies such as the German Research Foundation (DFG). Dr. Montag has performed grant reviews for several agencies; has edited journal sections and articles; has given academic lectures in clinical or scientific venues or companies; and has generated books or book chapters for publishers of mental health texts. For some of these activities he received royalties, but never from gaming or social media companies. Dr. Montag mentions that he is part of a discussion circle (Digitalit\u00e4t und Verantwortung:"}
+{"text": "Carpobrotus modestus S.T.Blake; crassulacean acid metabolism (CAM) plants) to 7.01\u00a0MPa2 , suggesting that C. modestus was more isohydric and R. spinescens was more anisohydric. More isohydric species C. modestus, Carpobrotus rossii (Haw.) Schwantes and Disphyma crassifolium (L.) L.Bolus (CAM plants) had greater leaf succulence, lower root allocation, used stored water and ceased transpiration at higher predawn leaf water potential, shortly after reaching their turgor loss point. The remaining nine species that are not CAM plants had larger hydroscape areas and ceased transpiration at lower predawn leaf water potential. Greater leaf succulence was not related to cumulative water loss until transpiration ceased in drying soils. All 12 species had high turgor loss points (\u22121.32 to \u22120.59\u00a0MPa), but turgor loss point was not related to hydroscape area or leaf succulence. Our data suggest that overall greater leaf succulence was related to isohydry, but this may have been influenced by the fact that these species were also CAM plants.Succulence describes the amount of water stored in cells or organs, regardless of plant life-form, including woody and herbaceous plants. In dry environments, plants with greater survival often have greater leaf succulence. However, it is unclear how leaf succulence relates to plant drought resistance strategies, including isohydry (closing stomata to maintain leaf water status) and anisohydry (adjusting cell turgor to tolerate low leaf water status), which exist on a continuum that can be quantified by hydroscape area (larger hydroscape area indicates more anisohydric). We evaluated 12 woody species with differing leaf succulence in a glasshouse dry-down experiment to determine relationships among leaf succulence  and plant drought response . Hydroscape areas ranged from 0.72 ( Agave and Opuntia spp., have succulent tissues that store water and ensure survival in drying soils, and these plants often have thickened leaves and stems adopting the crassulacean acid metabolism (CAM) photosynthesis pathway (3 plants), the conifer Pinus radiata had a longer desiccation time than angiosperms due to greater leaf succulence, early stomatal closure and lower cuticular transpiration , clones with higher values of leaf succulence maintained higher leaf relative water content and less negative leaf water potential under drought conditions  has been shown to assist in coping with salinity  and midday leaf water potentials (\u0470md) where stomata control leaf water potential in drying soil (TLP) are also considered to be more anisohydric  are more isohydric as they close stomata, have low cuticular transpiration and metabolic activity and use stored water to maintain water status in drying soils  will be more isohydric; (ii) will cease transpiration at higher leaf water potentials in drying soils; and (iii) will avoid drought stress by using stored water to maintain water status .Carpobrotus modestus S.T.Blake, C. rossii (Haw.) Schwantes and Disphyma crassifolium (L.) L.Bolus; and nine halophyte shrubs  Paul G.Wilson, Rhagodia candolleana Moq., R. spinescens R.Br. and Tetragonia implexicoma (Miq.) Hook.f. (We selected 12 woody shrubs and subshrubs from the families Aizoaceae and Amaranthaceae that varied according to their leaf succulence. These included three CAM plants with thickened leaves and/or stems\u2014e shrubs \u2014A. ciner Hook.f. .R. spinescens) of the same age were obtained as tube stock (plugs) from three commercial nurseries  in December 2019. We propagated R. spinescens from cuttings in December 2019. We transplanted tube stock plants on 23 June 2020 (winter) into pots  filled with 6-kg potting mix , 1.5-kg granular soil wetter SaturAid and 1000-g dolomite lime; air-filled porosity\u00a0=\u00a09.3\u00a0\u00b1\u00a00.3%, bulk density\u00a0=\u00a00.47\u00a0\u00b1\u00a00.007\u00a0g\u00a0cm\u22123, water-holding capacity\u00a0=\u00a054\u00a0\u00b1\u00a00.5%, permanent wilting point\u00a0=\u00a09.1% of soil water content and electrical conductivity\u00a0=\u00a01.05\u00a0\u00b1\u00a00.02 dS m\u22121; Plants (except Five replicate pots per species were moved to an enclosed rainout shelter in Burnley Campus for the dry-down experiment (9 March 2021) and arranged in a complete randomized block design. Initially, all pots were watered three times a day with an automatic irrigation system . In the evening (19:00\u00a0h) before the dry-down commenced, all pots were watered by hand to field capacity. We stopped watering on 17 March 2021 and started the dry-down experiment, which finished on 16 August 2021. All pots were not covered, and water loss included evaporation and transpiration. Over this period, the daytime (07:00\u201319:00\u00a0h) average temperature was 19.5\u00a0\u00b1\u00a00.03 \u00b0C, average humidity was 90.8\u00a0\u00b1\u00a00.0006%; the night-time (19:00\u201307:00\u00a0h) average temperature was 13.3\u00a0\u00b1\u00a00.03 \u00b0C and average humidity was 95.4\u00a0\u00b1\u00a00.0006%. The vapor pressure deficit  was calculated as  Cumulative VPD (KPa\u00a0h) was determined as the addition of VPD multiplied by time (hours).\u22121) was determined as  every weekday from 18 March to 2 April 2021; every second day from 3 April to 12 April 2021; and once a week from 13 April to 16 August 2021. Daily evapotranspiration  was measured on terminal shoots sampled from pots weighed at predawn (05:30\u00a0h) every weekday from 18 March to 2 April 2021; every second day from 3 April to 12 April 2021; and once a week from 13 April to 16 August. Midday leaf water potential (\u03a8md) was measured on terminal shoots sampled from each pot at 13:00\u00a0h on the same days. Both \u03a8pd and \u03a8md were measured using a Scholander-type pressure chamber .Predawn leaf water potential (\u03a8TLP (MPa) from PV curves measured on well-watered plants from February to August 2021. One terminal shoot from each well-watered replicate (six replicates per species) was collected in the early morning (06:30\u201307:00\u00a0h) and recut under distilled water. We then placed the shoots in 50-ml centrifuge tubes with distilled water to rehydrate the shoots for 2\u20133\u00a0h or until leaf water potentials were above \u22120.1\u00a0MPa. Rehydrated shoots were weighed before measuring leaf water potential with the Scholander-type pressure chamber. We repeated the weighing and leaf water potential measurements as samples dried in the laboratory. After finishing the measurements, samples were oven-dried at 80 \u00b0C until reaching a constant weight to determine the dry mass. \u03a8TLP was determined using the PV curve fitting Excel v5.6 developed by K.P.Tu and J.B.Fisher (http://landflux.org/Tools.php) based on We determined \u03a8Plant morphological traits were measured on well-watered plants with five pots per species when dry-down started. Soil was removed from roots, and shoots were separated into leaves and stems to determine fresh and dry weight of leaves, stems and roots. Leaf areas were measured using a LI3100 area meter . Samples were placed in an 80 \u00b0C oven for a week until reaching a constant weight, after which dry weights were measured. Leaf ash weight was determined by burning oven-dried leaves in a 500 \u00b0C muffle furnace for 2\u00a0h, and this process was repeated until they reached constant weight.2O cm\u22122 leaf area g\u22121 organic matter cm\u22122 leaf area) was determined as  was measured on the midpoint of freshly picked leaves using electronic Vernier calipers. Leaf succulence quotient (g Hmined as 2O cm\u22122 leaf area) was calculated as (Degree of leaf succulence (g Hlated as \u22121 plant dry mass) was determined as  was determined asLeaf mass per area  against cumulative VPD (x-axis). Gamma distribution with the inverse link was selected for generalized linear regression models (GLMs) based on the comparison of AIC scores (Model 1). The point where evapotranspiration ceased (E0) was where the curve crossed the axis of symmetry (y\u00a0=\u00a01/x is symmetric to y\u00a0=\u00a0x). Daily ET at E0 (kg d\u22121) was determined as the y value of E0, and cumulative VPD at E0 (KPa\u00a0h) was determined as the x value of E0.We plotted daily ET  against their corresponding \u03a8pd . Gamma distribution was used for GLMs. We compared the AIC scores for models using different link functions and selected the exponential function. We then used the \u03a8TLP  and the exponential model (Model 2) to predict the daily ET at \u03a8TLP . We then used daily ET at \u03a8TLP (kg day\u22121) as y value for Model 1, the GLM model we built for daily ET (kg day\u22121) and cumulative VPD (KPa\u00a0h), to determine cumulative VPD when \u03a8TLP was reached (KPa\u00a0h).To determine the time when plants reached their \u03a8y-axis) for each predawn measurement and cumulative VPD  using the same approach described above (Model 3). The cumulative VPD at E0  and the fitted Model 3 were used to determine the pot weight at E0 . The cumulative ET at E0 (kg) was calculated as the initial pot weight (kg) \u2212 the pot weight at E0 (kg). We then adjusted cumulative water loss before plants ceased evapotranspiration by initial shoot dry mass  and leaf area , as plants did not lose many leaves when plants ceased evapotranspiration.We fitted an inverse function to the relationship between pot weight  was predicted by the daily ET at E0  using Model 2; the GLM exponential model we built using the relationship between daily ET  and \u03a8pd . The predawn leaf water potential difference between E0 and \u03a8TLP (MPa) was then calculated as \u03a8pd at E0 (MPa) \u2212 \u03a8TLP (MPa).The predawn leaf water potential at 2) to quantify isohydric or anisohydric strategies  were used to quantify the hydroscape area. This approach was similar to one approach taken by We used hydroscape area , and the optimal number of clusters was selected using the \u2018elbow\u2019 method  to investigate variation in multiple traits among the 12 species based on morphological traits including LMA, RMF, degree of leaf succulence, leaf thickness and leaf succulence quotient, and on traits associated with drought resistance strategies including hydroscape area, \u03a8\u2019 method . IndividE0; Tree Physiology Online). Disphyma crassifolium ceased evapotranspiration first (cumulative VPD at E0\u00a0=\u00a0102.6\u00a0KPa\u00a0h) and A. semibaccata and A. paludosa took the longest time to cease evapotranspiration .The 12 species varied in the time it took to cease evapotranspiration (cumulative VPD at E0 (cumulative ET at E0) also varied among the 12 species (Tree Physiology Online). The highest water spenders were R. candolleana (cumulative ET at E0\u00a0=\u00a02.7\u00a0kg), C. rossii (2.6\u00a0kg), A. paludosa (2.6\u00a0kg), R. spinescens (2.5\u00a0kg) and T. implexicoma (2.5\u00a0kg). The lowest water spenders included E. tomentosa (cumulative ET at E0\u00a0=\u00a01.8\u00a0kg), D. crassifolium (1.9\u00a0kg), A. nummularia (2.0\u00a0kg), A. cinerea (2.0\u00a0kg), A. semibaccata (2.0\u00a0kg), C. modestus (2.1\u00a0kg) and M. oppositifolia (2.1\u00a0kg). However, the cumulative water loss per shoot dry mass for A. semibaccata was the highest (cumulative ET at E0/shoot dry mass\u00a0=\u00a042.33\u00a0kg H2O kg\u22121 shoot dry mass) and that for A. nummularia was the lowest . The ranks changed when adjusting the cumulative water loss by leaf area, where C. modestus was the highest water spender (cumulative ET at E0/leaf area\u00a0=\u00a0139.05\u00a0kg H2O m\u22122 leaf area) and M. oppositifolia was the lowest water spender (30.99\u00a0kg H2O m\u22122 leaf area).The amount of water loss before plants reached TLP), and some plants closed stomata at much lower \u03a8pd compared with \u03a8TLP , A. paludosa (1.93\u00a0MPa), R. spinescens (3.47\u00a0MPa) and T. implexicoma (2.37\u00a0MPa). Although C. rossii was also one of the greatest water spenders, it ceased evapotranspiration very close to its \u03a8TLP (\u2212\u0394\u03a8pd between E0 and \u03a8TLP\u00a0=\u00a00.45\u00a0MPa). The lowest water spenders differed in their maintenance of water status. Both C. modestus and D. crassifolium ceased transpiration at \u03a8pd close to their \u03a8TLP (\u2212\u0394\u03a8pd between E0 and \u03a8TLP for C. modestus\u00a0=\u00a00.38\u00a0MPa and D. crassifolium\u00a0=\u00a00.22\u00a0MPa), whereas A. nummularia and E. tomentosa had the largest changes in water status between E0 and \u03a8TLP (\u2212\u0394\u03a8pd between E0 and \u03a8TLP for A. nummularia\u00a0=\u00a02.85\u00a0MPa and E. tomentosa\u00a0=\u00a01.90\u00a0MPa).All plants were still transpiring at their turgor loss point (\u03a82. Carpobrotus modestus (0.72\u00a0MPa2), D. crassifolium (0.83\u00a0MPa2) and C. rossii (1.10\u00a0MPa2) had the smallest hydroscape areas (Tree Physiology Online), which indicated that they were more isohydric. Rhagodia spinescens (7.01\u00a0MPa2) and R. candolleana (4.83\u00a0MPa2) had the largest hydroscape areas and were more anisohydric. Based on their hydroscape areas, we divided the 12 species into two groups; a group including C. modestus, C. rossii and D. crassifolium  and at much lower leaf water potentials than their \u03a8TLP (\u0394\u03a8pd between E0 and \u03a8TLP). However, there were no significant relationships between hydroscape area and either water use (cumulative ET at E0) or turgor loss point (\u03a8TLP) (Tree Physiology Online).There was a strongly significant relationship between hydroscape area and both \u03a8E0; C. modestus, C. rossii and D. crassifolium) were removed from the analysis.All measures of succulence, the root mass fraction (RMF) and leaf mass per area (LMA) were strongly correlated with hydroscape area among the 12 species but not water use (cumulative ET at R\u00a0=\u00a0\u22120.38), degree of leaf succulence (R\u00a0=\u00a0\u22120.40), leaf thickness (R\u00a0=\u00a0\u22120.39), LMA (R\u00a0=\u00a0\u22120.37), \u03a8pd at E0 (R\u00a0=\u00a0\u22120.34) and \u03a8TLP (R\u00a0=\u00a0\u22120.05), and positively correlated with RMF (R\u00a0=\u00a00.37). Therefore, species with smaller PC1 values  had a greater leaf succulence quotient, degree of leaf succulence, leaf thickness, LMA, \u03a8pd at E0 and \u03a8TLP, and smaller RMF. The second axis (PC2) explained 16.8% of the variation and was positively correlated with hydroscape area (R\u00a0=\u00a00.43) and cumulative ET at E0 (R\u00a0=\u00a00.62) and negatively correlated with cumulative VPD at E0 (R\u00a0=\u00a0\u22120.53). Thus, species with larger PC2 values  had greater cumulative VPD at E0 and smaller hydroscape area and cumulative ET at E0. Succulence traits  and LMA were highly correlated, and RMF was negatively related to succulence measures and LMA (Tree Physiology Online). Hydroscape area was negatively correlated with \u03a8pd at E0 (Tree Physiology Online). Cluster analysis revealed two groups, with the three CAM plants C. modestus, C. rossii and D. crassifolium grouped together and the other nine species in the second group.To explore variation in morphological and physiological traits among the 12 species, we conducted PCA and cluster analysis. The first two principal components explained 77.8% of the variation among species . The firC. modestus, C. rossii and D. crassifolium; and (ii) more anisohydric species: A. cinerea, A. nummularia, A. paludosa, A. semibaccata, E. tomentosa, M. oppositifolia, R. candolleana and R. spinescens and T. implexicoma. The hydroscape areas of the three CAM plants (0.72\u20131.10\u00a0MPa2) were the smallest among the 12 species; these plants were more isohydric and ceased transpiration at higher leaf water potentials. This is consistent with our hypothesis and other studies that have shown that CAM plants with thickened leaves or stems are more isohydric (A. semibaccata (2.60\u00a0MPa2), which was a C4 plant, was also relatively isohydric compared with other species (2.60\u00a0MPa2) and closed its stomata at a relatively high predawn leaf water potential (\u22121.86\u00a0MPa). Other plants in the Amaranthaceae that were more isohydric included E. tomentosa , M. oppositifolia  and A. cinerea , whereas R. candolleana (C3 plant) and R. spinescens (C3 plant) were the most anisohydric species in our study with large hydroscape areas  and ceased evapotranspiration at the most negative \u03a8pd . The CAM plants with thickened leaves or stems in our study were strongly isohydric, ceased transpiration at higher leaf water potentials and maintained higher water status during drought which likely confers higher drought resistance.We hypothesized that woody plants with greater leaf succulence (i) will be more isohydric; (ii) will cease transpiration at higher leaf water potentials in drying soils; and (iii) will avoid drought stress by using stored water to maintain water status . We were able to distinguish two groups within the 12 species, based on degree of isohydry and anisohydry: (i) more isohydric species that all were CAM plants: sohydric . IsohydrE0) was not significantly related to drought resistance. Since anisohydric species maintain transpiration until lower \u03a8, it is reasonable to expect greater water use  than most other plants. This is consistent with Teucrium fruticans, Jacobaea maritima and Rosmarinus officinalis) and isohydric behavior (Olearia axillaris). Therefore, we cannot assume that isohydric species are more conservative in their water use, or vice versa, and therefore should not assume drought resistance based on water use alone.However, ceasing transpiration to maintain a higher leaf water potential may not necessarily improve the ability of species to survive a drought cycle. The volume of water transpired per unit decrease in leaf water potential differs among species , and theater use ; howeverTLP; \u22121.32 to \u22120.59\u00a0MPa), indicating low tolerance of tissue dehydration. All species experienced \u03a8pd much lower than \u03a8TLP, and most species also ceased transpiration at much lower \u03a8pd than their \u03a8TLP. We measured \u03a8TLP on well-watered plants, rather than droughted plants, and therefore the measured \u03a8TLP on well-watered plants may be higher due to the accumulation of cell solutes or osmotic adjustment  had larger differences in leaf water potential from \u03a8TLP to when evapotranspiration ceased compared with more isohydric species  in our study. There was no relationship between \u03a8TLP and predawn leaf water potential when transpiration ceased (\u03a8pd at E0) or hydroscape area (degree of iso-anisohydry). In contrast, the relationship between \u03a8TLP and hydroscape area was significant in eight woody species  with the greatest leaf succulence were the most isohydric  and ceased transpiration at less negative leaf water potentials (\u0470pd at E0) that were closer to their \u03a8TLP . This is consistent with our hypotheses, as greater leaf succulence means that there is a larger pool of internally stored water in leaves  dropped to ~\u221235\u00a0MPa . This suggests that for the nine other woody species, greater leaf succulence is not associated with isohydric strategies, although these nine species were far less succulent and had more anisohydric behavior with larger hydroscape areas and ceased transpiration at more negative \u03a8pd compared with the three CAM plants with thickened leaves and/or stems. These nine species may use their succulent tissues to accumulate sugars for osmotic adjustment to maintain the stomata open at a low \u03a8 during drought , leaf succulence quotient (g H2O cm2 leaf area g\u22121 organic matter cm\u22122 leaf area) and leaf thickness (mm) were highly correlated to each other. We expected that there would be differences in succulence measures as the leaf succulence quotient describes the stored water per unit organic matter, while the degree of leaf succulence describes the stored water per unit leaf area  and therefore, the leaf succulence quotient significantly correlated with degree of leaf succulence.The three measures of leaf succulence in our experiment including the degree of leaf succulence , and there were no relationships between \u03a8TLP and either \u03a8pd when transpiration ceased or in hydroscape area. The three CAM plants, C. modestus, C. rossii and D. crassifolium were more isohydric with greater leaf succulence and lower root allocation. These CAM plants with thickened leaves and/or stems had smaller hydroscape areas, ceased transpiration at less negative \u03a8pd, shortly after \u03a8TLP, and used the stored water to maintain high water status. In contrast, the nine other woody plants ceased transpiration at more negative \u03a8pd and had larger hydroscape areas compared with the three CAM plants. Besides, there was a significant relationship between hydroscape area and leaf succulence among all 12 species, but when the three CAM plants were removed, the relationship was no longer significant. This suggests that greater leaf succulence can be related to isohydry, but this may have been influenced by the fact that these species with the greatest leaf succulence were also CAM plants.Our results showed a continuum of isohydry and anisohydry, with hydroscape areas ranging from 0.72 to 7.01\u00a0MPaSupplementary_Tables_tpad066Click here for additional data file."}
+{"text": "Vector control tools, long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS), have significantly contributed to malaria prevention efforts in sub-Saharan Africa. However, insecticide resistance has seriously hampered their efficacy in recent years and new tools are essential to further progress. In2Care\u00ae EaveTubes (ETs) are an inexpensive, new resistance-breaking vector control product under World Health Organization (WHO) evaluation informed by mosquito ecology to efficiently target malaria vectors. By installing ETs in the walls of the house at the eave level that funnel the natural airflow, mosquitoes are drawn in by the same heat and odor cues that typically attract them through open eaves. Once inside an ET, mosquitoes are exposed to insecticide-treated netting placed inside the ET. The aim of this study is to test whether ETs as stand-alone tool have an effect on the epidemiology of malaria in villages where houses have been modified with the ET intervention.A two-armed, cluster randomized controlled trial will be conducted to evaluate the effect of ETs on clinical malaria incidence in children living in C\u00f4te d\u2019Ivoire. Thirty-four villages will be selected based on population size and the proportion of houses suitable for modification with ETs ). Based on the population census, 55 households per cluster with eligible children  will be randomly selected for recruitment into the active detection cohorts. In the treatment arm, we will enroll eligible children who reside in ET-treated houses. The intervention and control cohorts will be followed for 4\u00a0months for baseline covariate measurements and 24\u00a0months with intervention. During case detection visits, blood samples will be taken from all febrile children and tested for malaria infection with rapid diagnostic tests (RDTs). All positive clinical malaria infections will be treated. To estimate the impact of the ET on malaria vector densities, entomological measurements (indoor sampling with CDC traps) will be conducted monthly in 20 clusters  in 10 randomly selected households per cluster. To estimate the infectiousness of malaria vectors, sporozoite rates will be measured in subsets of the collected mosquito samples.Findings will serve as an efficacy trial of ETs and will be submitted to the WHO Vector Control Advisory Group (VCAG) for assessment of public health value. Entomological outcomes will also be measured as proxies of malaria transmission to help develop guidelines for the evaluation of future In2Care\u00ae ETs products.ClinicalTrials.gov NCT05736679. Registered on 10 February 2023.The online version contains supplementary material available at 10.1186/s13063-023-07639-9. The epidemiological monitoring visits are expected to last approximately 30\u00a0min, with an additional 30\u00a0min twice a year for anemia and respiratory testing.The entomological monitoring will require 3 nights (6\u00a0pm\u20138 am) per house each month. ET installations will require approximately 3\u00a0months for an estimated 3000 households in 17 village clusters. Retreatment of netting inserts takes approx. 10\u00a0min per house and is done from the outside of the house.The number of villages for the cRCT is estimated at 17 per treatment arm based on the power analysis which is detailed in the Statistical Analysis Plan.True PE/Minimum effect size\u2009=\u200935%Baseline first-time malaria infection hazard rate\u2009=\u20091.5 cases of falciparum malaria per person-year (conservative estimate based on control arm data from first SET cRCT)Coefficient of variation (k)\u2009=\u200940% (based on first SET cRCT)Loss to follow-up (LTFU) rate\u2009=\u200920%Monitoring\u2009=\u20092\u00a0years to capture 2 peak transmission seasonsOne interim analysis for efficacy and non-binding futility with the O\u2019Brien-Fleming error spending function when 50% information is collectedThe sample size determination on the required number of households per cluster for testing the primary hypothesis on PE is based on the hazard rate comparison in the proportional hazards regression model. With the following specifications: power\u2009=\u200980%, 2-sided type-I error rate\u2009=\u20095%.With 17 clusters per treatment, 55 children (aged 0.5 to 10\u00a0years old) from 55 different households per cluster are expected to yield 1315 independent first-time malaria events with a 24-month follow-up period to yield 80% power in testing the primary hypothesis on PE. Since the sample size already factors in a 20% LTFU rate, there is no need for replacement subjects.Local workers will be recruited from the trial village to enhance community engagement and study participation. Community sensitization procedures will include meetings with village leaders and inhabitants to introduce the study and each of its components.The unit of randomization for the intervention and control will be a village cluster. For baseline, recruitment of participants for enrollment will be based on random HH selection following census and mapping of the study area. The study statistician will analyze baseline data to inform potential stratification criteria, i.e., baseline malaria incidence levels and/or adult entomological endpoints. Following stratification (as needed), allocation of individual villages to trial arms will be conducted using randomization. One of the eligible randomization allocations will be selected at a public ceremony in the presence of community leaders.Cluster allocation was randomly conducted using a lottery mechanism whereby pieces of paper will be marked ET and Control, placed in a basket, mixed, and then a randomly selected child will be asked to draw from the marked papers. Each drawn paper must correspond to a given village, until they are all listed out. This event will be a public ceremony in the presence of community leaders and the Ministry of Health.The study statistician will use a random number generator to generate the allocation sequence and assign clusters to treatment or control arms. Trained study staff will enroll participants to study clusters.Given the nature of the intervention, it is impossible to conduct this study in a fully blinded manner but those parts of the data collection that can be blinded will be. Observer bias will be reduced where feasible. All laboratory work will be blinded. Mosquito collector bias will be reduced by using standard CDC light traps which do not rely on the ability of the fieldworker to collect specimens. Trap catches will not be examined and analyzed by those who collected them but by different technicians who will not know the trap location. We will use codes to identify any clinical samples. Electronic records will not carry the name of the research participants, only an alphanumeric code. Primary analysis by the project statistician will be conducted on blinded data .Datasets will only be unblinded once they have been locked.The pre-trial \u201cramp up phase\u201d will be used to recruit and train local staff, and obtain ethical approval for the trial. During ramp up, forty candidate villages will be identified based on size (100\u2013300 households) and proximity to Bouak\u00e9, C\u00f4te d\u2019Ivoire, i.e., within a 50-km radius of B\u00e9oumi town in the B\u00e9oumi district of the Gb\u00eak\u00ea region. We will select 34 of the 40 candidate villages based on ET suitability  and indications of participatory willingness based on meetings with the community leaders.In each of the 34 trial villages, project personnel will meet with the authorities in each community (\u201cchef du village\u201d) to obtain permission to talk to the village residents about the trial. Village outreach will begin with town hall meetings to explain the trial and the possibility of a village being assigned to the treatment or the control arm, to outline all trial activities that will take place in the villages, and to answer any questions from the audience. A few days after the town hall meeting, the chef du village will be contacted to find out whether the community agrees to participate in the trial.During ramp-up, a Population Census will be conducted in all the villages. Householders will be asked for information about their households  and given a new bed net if they have fewer than 2 per HH so that universal coverage is guaranteed in both trial arms. HH owners will be asked a short series of questions regarding their use of vector control tools, the availability of health care in the village, and markers of socio-economic status . For each HH, we will record the number of structures/dwellings, their construction , number of windows, and number of doors. Following the local customs and local health ministry procedures in C\u00f4te d\u2019Ivoire, each household  will be assigned a letter-number code that will be written in chalk on the door-jam of the house. During these activities, every eligible child in the village (between the ages of 6\u00a0months and 8\u00a0years old) will be assigned a unique identifier and their parents given a card with that unique identifier.Community health workers will receive refresher training from a clinician before the onset of the trial baseline period.Based on the Population Census data, 55 children in each village will be randomly selected for active monitoring of malaria infection. Informed consent will be obtained, and these children will be given ID cards for presentation to study nurses and health workers.The baseline prevalence of malaria infection in the cohorts will be measured by taking blood samples from all children and confirming parasite infection using rapid diagnostic tests (RDTs). At the end of the baseline period, at the onset of the clinical phase, the entire study cohort will be cleared of malaria parasites, regardless of infection status, using a standard dose of first-line antimalarials.Baseline entomological sampling will be done in all clusters in both arms, in 10 randomly selected HH per cluster. Once informed consent is obtained indoor mosquito collections will be done using CDC light trap collections during 3 consecutive nights per house. Subsets of 10% of baseline collected anophelines will be identified at species level and tested for sporozoites to quantify the baseline EIR in each trial arm.Once census and baseline prevalence data have been obtained, randomization will be done to allocate the selected villages to the trial arms which will potentially be stratified by epidemiological, environmental, and/or demographic factors if it is necessary. At the end of the randomization process, there will be 17 villages in the control arm and 17 in the treatment arm. In the 17 treatment arm villages, heads of households that are suitable for ET installation will be offered the option of having ETs installed.Following randomization, trial personnel will go door-to-door in the 17 selected intervention villages to obtain consent from the owners of eligible (ET suitable) houses for the installation of ETs. ET installation will start in parallel: commencing in village clusters as soon as\u2009>\u200950% consent in that particular village has been obtained. It is estimated that 3-month installation time is needed for the 17 ET clusters. There is an expectation of\u2009>\u200970% of HHs with ET installation in the intervention clusters, with 8\u201310 ETs per house on average. Standard Operational Manuals (developed from the first SET cRCT) will be used. It is estimated that 4-month installation time is needed for the 17 ET clusters.At the start of the clinical follow-up period, the clean ET netting inserts will be replaced with the deltamethrin-treated ET inserts in all treatment clusters. This is estimated to take 2\u20133\u00a0weeks\u2019 time.Clinical follow-up will run for 2\u00a0years to cover two high transmission seasons  and the remaining lower transmission periods.Epidemiological monitoringThe incidence of malaria infection and clinical malaria will be determined by active case detection in febrile children in the study cohorts. Clinical monitoring and treatment will be performed by trained nurses from the Institut Pierre Richet, who will collaborate with the community health workers present in the trial villages. On the first visit, in the trial baseline period, parasite prevalence will be measured in all cohort children by RDT . Immediately following this initial blood sampling, every child will be treated with a 3-day course of standard, first-line antimalarials  to clear any malaria parasite infections.Plasmodium infection.Children enrolled in the active detection cohort will be visited every 2\u00a0weeks during the peak malaria transmission season (May\u2013October) and monthly during the rest of the year when transmission rates are low (November\u2013April). At each visit, the clinical team will record the axillary temperature of each child. If the child is febrile or has a history of fever in the past 48\u00a0h or the parents report that their child was sick, the child receives a physical examination and a record will be made of symptoms. A finger prick blood sample will be taken from all febrile children. RDTs will be used to detect Children who are RDT-positive and are diagnosed with uncomplicated malaria by the study nurse will be treated immediately with first-line antimalarials for 3\u00a0days. This is the standard procedure for diagnosing and treating malaria in C\u00f4te d\u2019Ivoire. Because it is a malaria-endemic region, only symptomatic children are typically treated. ACT treatment for malaria will be provided free of charge through the NMCP system. The community health worker who normally provides diagnostic and treatment in the village will be responsible for monitoring the child until he or she is cured. If the child exhibits any symptoms of severe illness, he or she will be sent immediately to the closest health clinic for treatment. Children who have been treated for malaria will be considered not at risk for 2\u00a0weeks following treatment and there will be no data collection for these individuals during this time. Nurses and community health workers will capture history of travel away from the household to flag any cases that might be a result of infection while traveling outside the village.Clinical malaria incidence will also be monitored continuously by passive case detection using the existing clinical system: Community Health Workers and local health facilities. Any enrolled child between 6\u00a0months and 10\u00a0years old will be counted whenever they are brought to a community health worker or local health clinic with a body temperature of\u2009\u2265\u200937.5\u00a0\u00b0C. These data are planned to be provided weekly to ET Trial personnel.Twice a year (at the beginning and end of the peak transmission season), all cohort children of 5\u00a0years of age or younger will have a blood sample taken for immediate measurement of anemia. Blood samples will be checked for hemoglobin levels using a spectrophotometer (Hemocue Hb diagnostic system).Entomological evaluationsDeltamethrin will be the AI deployed in ET. In addition, the standard pyrethroid-only net distributed in the study area is PermaNet 2.0, which contains deltamethrin. Long-term efficacy of the insecticide-treated ETs will be evaluated using periodic WHO cone bioassays to validate continued impact against resistant wild-type mosquitoes.Anopheles mosquitoes indoors on a monthly basis: in 20 clusters  in 10 randomly selected households per cluster. Per trial arm, 10 new clusters will be selected each month so that all study villages will be sampled bimonthly, as recommended by the WHO VCAG. In the 10 selected treatment clusters, we will select only ET-treated households. Mosquitoes will be captured indoors overnight using miniature CDC light traps placed next to a sleeper under an LLIN for 3 consecutive nights. These traps will be placed inside the house by Vector Control Product Evaluation Centre/Institut Pierre Richet (VCPEC-IPR) entomology technicians at 6\u00a0pm in the evening and collected by the technicians at 8 am the following morning. Mosquitoes will be sorted by Household ID and date of collection. Indoor temperature and relative humidity will be recorded in enrolled HHs during entomological sampling using data logging devices.We will assess the impact of ETs on entomological measures of malaria transmission by measuring the density of An. gambiae s.l. and other species recorded. In subsets of 10% of all caught anophelines, we will type to species level using PCR. Results will be used to estimate mean vector density and species composition per trial arm. For the An. gambiae and An. funestus samples in these subsets, we will assess the presence of sporozoites using CSP-ELISA. Sporozoite prevalence will be measured from a random sample of up to 60 anopheline females per cluster per sampling night.A subset of 10% of all caught mosquitoes will be identified by microscopy and the numbers of .e., mean number of sporozoite infective per bites per cluster per month) where we assume that a mosquito caught indoors and actively searching is able to bite the host.We will estimate the EIR in each study arm  in the treatment villages and general house condition in all villages will be monitored by quarterly village walk-throughs every 4\u00a0months by project staff in both study arms. In addition, a designated member of the study team will be available for householders to report ET-related construction problems and get them fixed during the trial.The persistence of the chemical insecticide used on the inserts will be monitored bimonthly by taking a sample of inserts from the treated villages to the lab (these will be replaced with fresh inserts) and exposing mosquitoes to them in a controlled bioassay. We will use F1 adult female anopheline mosquitoes reared from field-collected eggs in WHO cone tests with a 3-min exposure and mortality monitored 1\u00a0day post-exposure. Mortality will be compared against equivalent mosquitoes (wild-type resistant) exposed to untreated \u201ccontrol\u201d inserts. Inserts will be replaced once the mortality post-exposure falls below 70% .Data formsA combination of standardized paper-based or digital forms (under Android tablets) will be used. VCPEC-IPR and University of Notre Dame (UND)/Center for Research Computing (CRC) will work together to develop the quantitative forms to be uploaded on Android tablets. Entered data  will be automatically assessed for quality using established quality control rules, then reviewed, and appended to the data already present. Data forms can be made available upon request submitted to UND.The final clinical survey of the child cohort will take place at the end of the 24-month follow-up. The final light trap collection will take place at about the same time.At the end of the trial, HH owners will be offered to have the ETs blocked with a closed insert or plastic cap to block the tubes. However, we intend to liaise with stakeholders throughout the trial to gage interest in providing continued retreatment for ET inserts after the trial, as is now taking place in C\u00f4te d\u2019Ivoire.The sub-studies described in this section are not part of the WHO-approved clinical study design as they are secondary endpoints and not part of the primary evaluation of public health impact of the intervention.We will conduct an assessment of key user acceptance indicators and cost-effectiveness of the ETs intervention vis-a-vis other alternative technologies or practices available. These sub-studies will be critical for successful scaling of the product, as they will help identify the most socially acceptable and sustainable way of achieving and maintaining high coverage of ETs. User acceptance does not always translate into adoption unless the intervention is found to be affordable and more cost-effective compared to other available options for malaria protection. In this regard, cost-effectiveness analyses are planned in this study.Cost-effectiveness studiesA Global Health Economist within UND\u2019s Keough School of Global Affairs will conduct an economic evaluation to estimate the costs and cost-effectiveness of the ETs intervention. The cost analysis will take the gold-standard, societal perspective which includes both provider and community costs. Implementation costs will be carefully monitored during ETs installation and maintenance activities. Data on incremental costs of the ETs product, house modifications during install, and their sources will be collected from project expenditure records. Household costs will be collected at cohort enrolment shortly after intervention installation, and the unit costs (cost per house and person) will be calculated. This cost study will demonstrate the costs of using ETs as a stand-alone tool compared to the previously combined intervention of SET.The cost-effectiveness analysis will have the primary endpoint of cost per disability-adjusted life years (DALYs) averted by ETs. Incremental cost per DALY averted for ETs (Intervention arm) relative to LLINs alone (control arm) will be calculated using the epidemiological data collected during the trial. Cost-effectiveness ratios will be presented as point estimates and ranges  on the epidemiological data and reflecting any uncertainty in costs) and interpreted against a range of willingness to pay thresholds and in relation to the cost-effectiveness of other malaria control interventions.Cost modeling will assess the cost-effectiveness of ETs compared to the previously applied SET intervention, and compared to standard vector control interventions such as LLINs and IRS. The potential cost-effectiveness of ETs at scale over time will be simulated to facilitate comparison with other malaria control interventions and cost-effectiveness benchmarks. We will measure the cost of ETs\u2019 implementation in relation to manufacturing, efficacy, and coverage to model projections of cost-effectiveness to incentivize potential procurers.User acceptance studiesSocial scientists from the Institut Pierre Richet will use quantitative methods to assess ETs\u2019 adoption, adherence, and acceptability among study participants. Combined, endpoints from these assessments will inform potential bottlenecks to product access, uptake, and implementation post-trial.Ethnographic data will be collected during the baseline population census to inform socio-economic status of households and participants. Adoption will be assessed using indicators of informed consent rates and ETs coverage rates in the trial. Adherence will be assessed via quality control monitoring of ETs\u2019 integrity during the trial. User acceptance will be measured based on inhabitants\u2019 willingness to pay and continue using ETs after the trial through an endline survey.To design potential strategies for scale-up, we will identify and engage with key local, national, and international level stakeholders, involving them in discussions and creating a driving team to identify mechanisms for expansion and institutionalization while acting as advocates for the innovation.Participant retention strategies include fostering relationships with participants, provision of community health worker and nurse contact information for easy communication to alleviate concerns, and periodic generation of retention rates to evaluate strategies.UND will achieve a systems approach to protocol adherence, implementation, data gathering, and sharing by installing a program monitoring plan  that will support rigorous data collection for policy recommendation. The UND CCRC will host the data server and will be responsible for data management, data form processing, and provision of support for training in-country collaborators\u2019 data entry personnel.Hardcopies of study and study-related documents  will be stored at VCPEC-IPR in a locked filing cabinet under the supervision of a designated archivist. Electronic data will be stored on UND servers with restricted access.After study files have been held in the VCPEC-IPR for 5 years, the archivist will contact the sponsor to determine their future storage requirements. At the request of the Sponsor, it will either continue to be kept at VCPEC-IPR, sent to the Sponsor for storage, or destroyed. All hardcopies of documents will be destroyed by shredding and data on disks will be wiped, and the disks broken prior to disposal in the VCPEC-IPR waste.Study subjects will be identified only by their study identification number and any electronic database will only contain their study identification number. Personal identifiers will be removed from the transcripts of interviews and discussions with participants being identified only through a study identification number. Thus, all data will be anonymized on data entry. The UND CRC databases will be password-protected and accessible only to authorized personnel. All hardcopy documents will be securely stored in locked filing cabinets and accessible only to authorized personnel.The PI will maintain appropriate medical and research records for this study in compliance with the principles of good clinical practice and regulatory and institutional requirements for the protection of confidentiality of participants.The authorized representatives of the sponsor, the ethics committee(s), or regulatory bodies may inspect all documents and records required to be maintained by the investigator, including but not limited to, medical records  for the participants in this study. The clinical study site will permit access to such records.Not applicable. Study has no planned future uses for the biological specimens.The baseline characteristics of the enrolled subjects, households, and clusters will be summarized by treatment arm . Specifically, we will examine subject age and gender at the individual level, wall type and roof type, house open eaves, number of windows, number of doors at the household levels, and cluster population and baseline prevalence rate at the cluster level.In the following analysis that involves malaria incidence, a child will not be considered at risk for malaria for 2\u00a0weeks after any treatment for malaria.Protective efficacy (PE) against clinical malaria infection will be determined by comparing hazard rates of malaria clinical cases between the two treatment arms based on an intention to treat (ITT) analysis. The primary hypothesis on PE against overall malaria case incidence will be tested by comparing the hazard rates of the overall malaria case incidence between the control and ET in the ITT population using a proportional hazards model. The model will include relevant individual-level, household-level, and cluster-level baseline covariates, treatment assignment, and follow-up visits and random effects to account for correlations among the subjects within the same cluster and among multiple infections within the same individual. The hazard ratio function \u201310, withz-score\u2009>\u20092.7965, the trial would stop for definitive harmful effects of ETs though the chance this would occur is close to 0 if not 0), the study can stop for futility. Since we adopt the non-binding futility boundary the study can continue to collect more data even if we cross the futility boundary at the interim look, there will be no inflation of type I error. In other words, the trial does not need to stop to accept the null hypothesis when the test statistic falls in the futility region at the interim stage. If the interim z-score\u2009<\u2009\u2009\u2212\u20092.7965 then the study can stop for efficacy. The study may also continue even if the efficacy boundary is crossed at the interim look, and there will be no inflation of type I error as efficacy is already established at the interim. If the interim z-score falls within , the study continues.If the z-score of the log(hazard ratio) from the comparison between ETs and control at the interim look is\u2009>\u2009\u2009\u2212\u20090.7288 then the study can stop for futility A similar proportional hazard model used for analyzing the primary endpoint above will be applied. The malaria hazard ratio between ET and control will be estimated, along with 95% CI.PE of ETs against malaria infections A similar proportional hazard model used for analyzing the primary endpoint above will be applied. The malaria hazard ratio between ET and control will be estimated, along with 95% CI.PE analysis without baseline covariatesA PE analysis of clinical malaria infection will be also performed by removing all the baseline covariates from the proportional hazards presented above and keeping the treatment arm as the only covariate in the proportional hazard model. The hazard ratio between ET and control will be provided, along with a 2-sided 95% CIs. A similar analysis will be performed for clinical and asymptomatic malaria infections combined.AnemiaThe first study on ET  suggestsEffect of ETs on malaria prevalenceMalaria prevalence data will be collected at baseline and at the end of Year 1 and Year 2. A mixed-effects logistic regression as used in the anemia analysis above will be used to compare ETs and control on malaria prevalence over time, with the outcome anemia status being replaced malaria status (Y or N).Incidence rateThe malaria incidence rate is defined as the ratio of the number of new malaria cases during the follow-up period vs the sum of the time at risk (in year) across the individuals within the same cluster. The incidence rates per person-year during the whole intervention follow-up will be calculated by cluster in the ET and the control arms respectively. Summary statistics of the cluster-level incidence rate will be provided by treatment arm; the incidence ratio between the two arms will be also calculated.Effects of ETs on entomologyThe endpoints in the entomological analysis include anopheline density collected by light-trap, anopheline sporozoite rate, and anopheline EIR.We will report the frequency and proportion of each anopheline mosquito Genus and species collected using a light trap for each cluster and by treatment arm. The time profile plots of overall anopheline density will be obtained at the baseline and during the intervention period. An appropriate statistical model for anopheline density will be identified after examining the distributional characteristics of the density data, which are likely to follow (zero-inflated) Poisson distribution or (zero-inflated) negative binomial distribution if there is over-dispersion. The covariates in the models will include fixed effects of treatment, time, cluster population size, number of houses in a cluster, and a random effect for cluster. The ratio between ET and control in anopheline density will be estimated along with a 95% CI; the %reduction in anopheline density by ET is given by (1\u2009\u2212\u2009density ratio)\u2009\u00d7\u2009100%.The analysis for sporozoite rate will be similar to that for analyzing mosquito density If the data on sporozoite positivity are highly unbalanced in the sense that the marginal distribution of the variable , then the model might lead to unstable estimates or the model might not even converge. The ratio between ET and control in sporozoite rate will be estimated along with a 95% CI; the %reduction in sporozoite rate by ET is given by (1\u2009\u2212\u2009sporozoite rate ratio)\u2009\u00d7\u2009100%. In such cases, only summary statistics will be provided. EIR is the product of the anopheline vector density and the sporozoite rate and its analysis is similar to what\u2019s used for analyzing sporozoite rate. Summary statistics will be provided on sporozoite rate and EIR at baseline and per year during the intervention period by treatment group.Analysis of the relationship between malaria hazard rate and entomological endpointsTo explore the relationship between the malaria hazard rate and the entomological endpoints, a similar model as the proportional hazards regression model used to address the primary objective on the malaria infection will be applied to the clusters from which the entomological data are collected, with similar random effects specification. For the covariates in the model, in addition to those used for analyzing the primary endpoint, we will also include a covariate that captures the entomological information; whether it is a linear or non-linear term will be informed by the exploratory data analysis of the relationship between the cluster-level incidence rate and each of the entomological endpoints . The regression coefficient associated with the entomological covariate quantifies how the entomological covariate affects the malaria hazard rate.Safety assessmentMean, minimum and maximum frequency, and percentage of AEs and SAEs across clusters among enrolled subjects will be summarized by treatment arm. The AE/SAEs summary will be provided for both clinical diagnosis and symptoms. In addition, they will be labeled as Probable, Possible, Plausible , or Unlikely  due to ETs.A formal interim analysis during the intervention period will be conducted to test the primary hypothesis as outlined above. The interim analysis will be performed by an independent statistician on the DSMB, and the interim outcome report will be shared only with In2Care and VCAG, not with UND or Cote d\u2019Ivoire investigators to mitigate unintentional bias in data collection in the remaining period of the study after the interim analysis. The final decision to stop should always rest with the DSMC, not the investigators, or the funder.The analysis outlined in the primary and secondary analysis will be based on the intent-to-treat (ITT) dataset. The study will also examine the per-protocol (PP) dataset. If the PP dataset differs significantly from the ITT dataset for a particular analysis, the analysis will also be performed in the PP dataset.Significant effort will be made to avoid having missing values on outcome . When missing values occur for an outcome for reasons not related to the outcome, reasons for missingness and the missing fraction by treatment arm and cluster will be reported. Per protocol, the subjects are screened actively on the malaria status (the outcome) every 4\u00a0weeks.If a subject misses one or more scheduled visits due to reasons not related to the ETs product or the outcome, the subject will have missing values on the outcome that can be regarded as ignorable missingness (MAR or MCAR ).If a subject drops out study due to reasons unrelated to the ETs product and/or malaria infection, then the missing observations from the subject can be regarded as ignorable missingness (MAR or MCAR ).In both cases, all the available data from the subject will be included in the primary and secondary analysis, without employing any specific missing data analysis techniques, due to the ignorability of the missing mechanisms .Missing baseline covariates  that are a part of the regression models for the outcome of interest will be imputed using simple hot-deck imputation methods if the missing fraction for the covariate is\u2009<\u20095%. If the missing fraction for a covariable is\u2009\u2265\u20095%, appropriate multiple imputation approaches will be applied. If\u2009\u2265\u200950% of the subjects have missing values on a covariate (due to missing at random or missing completely at random), that covariate will be excluded in the model.The SAP and analytic code will be made open access. Data and supporting information will be made available 12\u00a0months following the completion of data analysis and will remain open access in the public domain.In2Care will serve as the lead organization for this program and will assume the overall responsibility for management, oversight, and administration for the program. The coordinating personnel at UND will include the Lead PI, Co-Investigators, Program Manager, and Finance Manager. UND will communicate on a day-to-day basis with In2Care and VCPEC-IPR. VCPEC-IPR will be responsible for running the cRCT on a day-to-day basis which includes but will not be limited to conducting a baseline survey, deploying ETs, entomological monitoring, and subject follow-up. Representatives from VCPEC-IPR and UND will all serve on the data management team to oversee the development and implementation of data collection, recording, and cleaning.A Trial Steering Committee (TSC) will be established. Members on the committee will have the clinical, epidemiological, and statistical expertise to monitor study progress and safety of participants, and the committee will have access to the study data. SOPs will be developed, adhering to Good Clinical Practice and Good Field Entomology Practice.The trial will not consist of a data monitoring committee as the TSC will serve in this capacity. In addition, routine data monitoring and management for Quality Assurance and Quality Control will be conducted in partnership between UND and VCPEC-IPR data management teams.Safety oversight will be carried out by the TSC. Children of the study cohorts will have access to the current standard of care. Serious adverse events (SAEs), whether attributed or not to ETs or LLINs, will be recorded throughout the trial. The host institution medical expert (Dr. Serge Assi) and his clinical team will be responsible for recording, reporting, and managing SAEs, including follow-up, in accordance with national guidelines. A summary table of all SAE will be provided at regular intervals to the TSC.The Chief Investigator will permit study-related monitoring, audits, and inspections by the study sponsor, IRB, TSC, and government regulatory bodies, of all study-related documents . The investigator will ensure the capability for inspections of applicable study-related facilities .All amendments to the protocol will be documented. Substantial amendments will require a report and approval by the TSC and/or the ethical review boards.Dissemination of results includes submission to WHO VCAG, workshop with study partners, on-site meetings in Cote de Ivoire, and presentations at scientific meetings and/or peer-reviewed publications.Plasmodium falciparum [Control efforts in sub-Saharan Africa over the past 15 years have prevented millions of clinical cases of malaria caused by lciparum ; howeverIn2Care\u00ae ETs could help meet the pressing need for new vector control tools. ETs are an inexpensive, field-ready technology, informed by mosquito ecology to efficiently target and kill malaria vectors. In a previous cRCT conducted in C\u00f4te d\u2019Ivoire, ETs\u2009+\u2009window screenings were shown to reduce malaria case incidence by 38% and by 47% in villages with\u2009>\u200970% coverage . The objProtocol version 4.0 from July 17, 2022. The study has currently completed the baseline prevalence survey , whereby participants were recruited, screened, and enrolled for confirming parasite infections using RDTs. Baseline data analyses are ongoing to verify underlying assumptions of malaria prevalence and coefficient of variation. Recruitment, screening, and enrolment of subjects for follow-up with intervention are scheduled to commence in August 2023.Additional file 1."}
+{"text": "This manuscript is a master statistical analysis plan for each of three-cluster randomized controlled trials to evaluate the efficacy of attractive targeted sugar baits (ATSB) described in an already published protocol. The master SAP contains an overarching plan for all three trials, which can be adapted to trial-specific circumstances. The primary objective of the trials is to evaluate the efficacy of ATSB in the presence of universal vector control coverage with insecticide-treated nets (ITN) or indoor residual spraying (IRS) after two transmission seasons on clinical malaria incidence as compared with universal vector control coverage with ITN or IRS alone. The primary outcome measure is the incidence rate of clinical malaria, assessed in cohorts aged 12\u00a0months to less than 15\u00a0years  during monthly follow-up visits. The primary unadjusted analysis will be conducted on the intention-to-treat analysis population without adjustment for any anticipated confounding variables. The primary outcome will be analyzed using a multi-level model constructed on a generalized linear model framework with a Poisson likelihood and a log link function. Random intercepts will be included for each study cluster and a fixed effect for study-arm. The analyst will be blinded to study arm assignment. Several secondary outcomes will be analyzed, as well as a pooled analysis  across the three trial sites. Additionally, a standard meta-analysis is expected to be conducted using combined data from all sites.The online version contains supplementary material available at 10.1186/s13063-023-07762-7. Highly effective interventions against malaria vectors that preferentially feed on humans late at night and rest inside houses have been developed and implemented at scale. Their effectiveness is a function of the fact that they specifically target indoor-biting and indoor-resting mosquitoes, which are often the same mosquito species comprising the bulk of the vectorial system.However, several mosquito species have evolved high levels of resistance to the insecticides used in long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS) as a result of prolonged exposure through the scale-up of these interventions. There is increasing concern that this insecticide resistance is undermining the effectiveness of these interventions. Furthermore, malaria vectors exhibit different behavioral characteristics, such as outdoor and daytime biting, that compromise the effectiveness of existing vector control strategies.Anopheles species to take a blood meal to obtain the protein necessary for egg production, all Anopheles must feed regularly and frequently on liquid and carbohydrates (sugars) to survive. Mosquitoes are guided to sugar sources by chemical attractants. The ATSB (Attractive Targeted Sugar Bait) is designed specifically to attract the mosquito with a source of liquid and sugar and includes an ingestion toxicant to then kill the mosquito. Using sugar sources to attract mosquitoes to an ingestion toxicant is a relatively simple and inexpensive strategy that has been shown to be highly efficacious for mosquito control in a limited number of trials.In addition to the biological need for female https://www.bitrex.com/en-us) that deters mammalian consumption of the bait. The bait station has a protective membrane that covers and protects the bait from rain and dust, but that allows mosquitoes to feed through it  to kill the foraging vectors. The bait additionally contains a commonly used bittering agent called Bitrex  established that large proportions >\u200925%) of the mosquito population were marked daily by the food dye . Proof o5% of theModeling of entomological ATSB study data suggests that ATSBs could markedly reduce mosquito populations across a range of different transmission intensities and should have great potential when used in combination with other indoor vector control tools.The World Health Organization Vector Control Advisory Group (VCAG) reviewed these data and recommended the evaluation of the potential of the Westham ATSB to reduce clinical malaria incidence in different transmission settings in sub-Saharan Africa. This SAP is intended to serve as a master SAP for each of three trial sites in Kenya, Mali, and Zambia. Three harmonized clinical trials will use this master plan as the basis for site-specific SAPs which may contain minor modifications to adhere to site-specific nuances, including but not limited to, changes in covariables included in analysis or definitions and cutoffs of said variables and summary measures. While the intent of the harmonization is to largely ensure that the trial analysis is conducted comparably and identically, the site-specific SAPs will require minor modifications .Is outdoor deployment of ATSBs plus universal vector control coverage (LLIN or IRS) more effective than universal vector control coverage alone at reducing cohort-based clinical malaria incidence over a 2-year period?(2)Is deployment of ATSBs associated with a reduction in community parasite infection prevalence?(3)Is deployment of ATSBs associated with a reduction in passively detected confirmed malaria case incidence?(4)Is deployment of ATSBs associated with a decline in malaria vector abundance , longevity of vector mosquitoes (parity status), sporozoite rates, and EIR?(5)What are the barriers to high ATSB coverage?(6)Does ATSB deployment affect LLIN use?(7)What is the cost and cost-effectiveness of outdoor ATSB deployment as a vector control intervention?To evaluate the efficacy of ATSB deployment in the context of universal vector control coverage (IRS or LLIN) coverage after two transmission seasons in population-based cohort clinical malaria incidence as compared with universal coverage with standard vector control alone.(2)To evaluate the efficacy of ATSB deployment in the context of universal vector control coverage (IRS or LLIN) on community parasite infection prevalence as compared with universal coverage of vector control alone.(3)To evaluate the efficacy of ATSB deployment in the context of universal vector control coverage (IRS or LLIN) on passively detected confirmed malaria case incidence as compared with universal coverage of vector control coverage alone.(4)To assess a minimum set of entomological outcomes  that measure ATSB efficacy in reducing the target vector population and transmission.(5)To assess the acceptability of ATSBs by communities and other stakeholders. This includes the identification of potential barriers to uptake and consistent ATSB coverage, together with an assessment of ATSB impact on coverage and use of existing malaria control interventions .(6)To estimate the cost and cost-effectiveness of deploying ATSBs for malaria control.(7)To assess the safety of ATSBs on humans by monitoring adverse effects in communities where ATSBs are deployed compared to the control.An open-label two-arm cluster randomized controlled trial (CRCT) design will be used comparing ATSB\u2009+\u2009universal coverage with a WHO core VC intervention vs universal coverage with VC alone . The trial will follow a group-sequential design  with onenot responsible for trial implementation. Randomization was conducted independently for each trial. The steps for randomization are as follows:Establish balance criteria. The factors described in Table Generate a list of at least 100,000 randomizations (Allocation sequences)Check randomizations  against balance criteria and drop those that do not meet balance criteriaAssess the number of randomizations  remaining. If fewer than 10,000 acceptable randomizations (sequences) remain, stop and relax restriction criteria. If a high proportion of allocation sequences remain , consider tightening balance criteria.Test the remaining set of potential randomizations  for validity, specifically that all clusters are being independently assigned to study arms .Randomly choose a randomization .Flip a coin to determine if arm A or arm B is ATSB or control.Restricted randomization was used to allocate study clusters to intervention and control arms with a balance between study arms on key baseline characteristics, including the primary outcome. Steps one through seven below were carried out by an independent statistician in collaboration with a member of the study team who was After allocation, the intervention will be implemented in the entire ATSB arm according to the assignment. Allocation of study arms will not be blinded to the participants, the deliverers of the intervention, or the main investigators (but will be to lab workers carrying out tests on blood samples and mosquitos). Sham bait stations will not be used in control areas.Full details of the sample size calculations are contained in the trial master protocol and study site-specific protocols; the sample size determination is presented here in summary form Tables , and 4.The sample size calculations for the case incidence cohort were calculated using the formula for cluster randomized trial event rates with a person-time denominator . AssumptThe sample size calculations for the parasite prevalence surveys were calculated using the formula for cluster randomized trial proportions  using PAhttps://about.scanform.qed.ai/) registers at facilities and by community health workers. Individually identifiable data will not be extracted or collected by the trial staff for this outcome.Data from all health facilities regarding people of all ages will be used to calculate confirmed malaria case incidence in the intervention and control clusters in Mali and Kenya. In Kenya, specific dates for data collection will be specified in the study site-specific SAP and protocol in terms of timing of data collection/analysis start after ATSB deployment . In Mali, facility-based assistants use electronic tablets with a custom application for collecting case data. Data will be transmitted weekly to the field data manager. In Kenya, health care data are entered into ScanForm  was estimated by analysis of 1000 simulated trial data sets. The effect of the intervention was assumed to be a reduction of daily survival probability from 80% in the control arm to 75% in the intervention arm, equivalent to an increase in NPR from 48.8 to 57.8% based on the formula from Davidson . Each data set was simulated from a generalized linear mixed-effects model (GLMM) with a binomial response, which was the number of non-parous females counted out of the total number of females collected. Variation in NPR between clusters, households, and months was simulated as normally distributed random effects on the logit scale. The household random effect variance of 0.18 was estimated from pilot data collected in Mali. The cluster random effect was estimated from the same data, but the upper 50% confidence limit of 0.04 was used in preference to anti-conservatively using the point estimate of zero. The inter-month variance was set at 1, giving a monthly mean NPR range of approximately 0.2 to 0.8. The total number of female mosquitoes trapped per household per night was sampled from a simulated Poisson distribution with a mean catch of 2.5 females. Inter-cluster and inter-month variation in the number trapped was simulated as normal random effects on the log scale with variances of 0.16  and 0.5 (giving a monthly mean catch range of approximately 0.5 to 6) respectively. The number of clusters to be included per arm of the study to achieve at least 80% power with a 0.05 two-tailed alpha is shown in the table below .The trials are planned under a superiority framework. The comparisons will consist of two-sided tests of the null hypothesis of no difference in efficacy between the ATSB (intervention) arm and the control arm. All primary comparisons will consist of comparisons of the outcome in the intervention arm vs. the outcome in the control arm.One interim analysis is planned in Mali and Zambia. In Kenya, an additional (second) interim analysis is planned because this trial has a 6-month-longer follow-up than the other two trials because transmission occurs throughout the year in Kenya. In Kenya, the interim analyses will be event, rather than time, driven. In Mali and Zambia, the interim analysis will be conducted at the end of the first transmission season in the first year.n\u2009=\u2009415) and 75% (n\u2009=\u2009622) of the total number of expected primary outcome events over 2\u00a0years in the control arm (n\u2009=\u2009829) have occurred (whichever comes first). The number of events will be tracked by an independent statistician. In Zambia and Mali, an interim analysis will be conducted after the first transmission season regardless of the total number of events.In Kenya, interim analyses will occur either after 50% and 75% of person-time have been completed , or after 50%  at the World Health Organization. This recommendation is only expected after the results of two trials show significant benefit, either in the interim or in the final analysis. As such the independent statistician working in collaboration with each trial site will advise the DSMB of each trial site on the results of the interim analysis as well as results of interim or final analyses of the other trials. The DSMB can thus include the interim results from other sites in their consideration of recommending an early referral to the VCAG.The DSMBs of each trial may consider recommending an early submission of the ATSB dossier for overwhelming benefit if a test of the null hypothesis that the cumulative clinical incidence of malaria in the intervention arm in the intention to treat analysis population is lower than the cumulative clinical incidence in the intention to treat analysis population of the control arm. The null hypothesis was equal incidence between the two arms; the interim analysis assumes a two-sided test of the null hypothesis at a significance level of A DSMB recommendation to discontinue the trial will not be based on the results of this statistical test. The DSMB can advise continuing the trial even if statistically the boundary is crossed, e.g., in order to continue collecting more epidemiological, entomological, or safety information or data for further sub-group analyses. It is the intent of the investigators to continue the trial even in the case of an early efficacy demonstration across more than one site since there is an expectation of significant heterogeneity in the effect of ATSB across entomological settings.The trials do not include formal stopping rules based on harm, because the intervention is not targeted to humans and the expected risk to trial participants is expected to be minimal; thus, formal harm-based stopping rules are not needed. However, this does not preclude the DSMB from stopping the trial for harm should unforeseen consequences of the ATSB or trial procedures lead to harm. For example, deliberate abuse or misuse of the ATSB products or unforeseen non-target insect impacts could lead to harm which causes trial stoppage.Should no early stopping rule be invoked and the trials continued after each interim analysis, then the final analysis per trial (country) will be conducted collectively at the end of two seasons/years. This analysis will occur at the site  level. A final pooled individual participant data (IPD) analysis and meta-analysis of trial outcomes will be conducted collectively after the termination of the trials in all sites.The primary outcome measure is the incidence rate of clinical malaria defined as history of fever or a measured temperature\u2009\u2265\u200937.5\u00a0\u00b0C and a positive malaria rapid diagnostic test (RDT) (the definition is specified in full in a later section). This will be assessed among people aged 12\u00a0months to less than 15\u00a0years . These outcomes will be ascertained through follow-up visits. Visits will be conducted within\u2009\u00b1\u20095\u00a0days for true monthly intervals and specific follow-up time between visits will be computed to the nearest one day.Prevalence of malaria infection among participants aged 6\u00a0months and older, detected by RDT. This outcome will be assessed annually cross-sectionally . For the cross-sectional analyses , measurement will occur in each member of the study sample within an approximate 1-month (30-day) observation window.Incidence rate of passively reported clinical malaria among participants of all ages, defined as the number of malaria confirmed cases (by RDT or microscopy), linked to study clusters by place of residence, per 1000 population per year, using routine data from health facilities serving the study population  and cluster population sizes for the denominator. This outcome is assessed daily at routine health facilities and dispensaries .Anopheles spp. mosquitoes captured during Human Landing Catches which have never been gravid as determined by the method of Detinova. The outcome is assessed in a sub-sample of clusters, houses, and nights on a monthly basis.Parity as a proxy for daily female vector mosquito survival\u2014this outcome is defined as the non-parous proportion, e.g., the proportion of freshly-caught, non-blood-fed female adult Anopheles spp. captured in CDC UV Light traps.Mosquito abundance\u2014The number of adult Anopheles spp. captured via HLC or CDC UV light traps found to be sporozoite positive by anti-circumsporozoite protein (\u03b1-CSP) enzyme-linked immune-sorbent assay (ELISA) divided by the number of adult female Anopheles spp. tested in ELISA assay.Sporozoite rate\u2014The number of adult female Anopheles spp. captured via HLC divided by the number of person-nights (days) of HLC collection.Human landing/biting rate\u2014The number of adult female Entomological inoculation rate\u2014The human landing/biting rate (6) multiplied by the sporozoite rate (5) multiplied by 365\u00a0days per year to yield annualized EIR. It should be noted that all EIR estimates will be expressed in terms of annual rates.p-values.The trial is generally intended to control type-I error to less than 5%. As such, given the planned interim analyses at each trial site, type-I error will be controlled using an Haybittle-Peto boundaries as discussed above. The main trial results (treatment efficacy estimates) will be presented with 95% confidence intervals and two-sided Since the intervention is deployed on a group basis rather than individually, adherence definitions will take account of this. Standard adherence will be defined as the intention to treat a cluster of residences with ATSBs, as randomized. Individual adherence will be defined based on ATSBs present at individuals\u2019 households. Both individual and cluster-level adherence measures will be defined and pre-categorized prior to final analysis and used to categorize the per-protocol trial population.The per-protocol analysis populations will be defined as those living in intervention clusters where ATSB was deployed and replaced according to the planned schedule. Clusters where more than 1-month delay in ATSB deployment occurred or where substantial deployment of ATSB into control areas occurs  will be removed from the per-protocol analysis population.Standard protocol deviations will be considered reportable/summarizable when clusters refuse placement of ATSB or have been treated/not treated contrary to their randomization assignment and providing initial study consent. Additionally, protocol deviations will be considered to have occurred if ATSB replacement visits for an entire cluster by the study team are delayed by more than 3\u00a0weeks from the expected timeline according to study planning.Protocol deviations related to failure to deliver or replace ATSB will be summarized in the final trial reports as well as incorporated into the calculation of adherence.There are two analysis populations for the primary outcome assessment: These are the intention-to-treat population and the per-protocol analysis population. The intention-to-treat population consists of all eligible individuals recruited and consented to participate in the study. The primary analysis will be conducted on the intention-to-treat population. Per-protocol analysis populations will be those eligible, recruited, and consented individuals whose adherence at cluster level meets the adherence standard. Additional household-level per-protocol analysis may be conducted consisting of ATSB deployment at the household level consistent with randomization assignment.While the trial tests multiple secondary outcomes, no adjustment will be made for multiplicity because the trials each have two arms and a single primary outcome. Additionally, each trial is powered and run independently and as such no adjustment for multiplicity on account of the three trials is being made. Secondary outcomes are assumed to be on the same causal pathway as the primary outcome and as such are also not adjusted for multiplicity of testing since these are expected to relate to the same hypothesis.de jure residents present in intervention and control clusters (and associated buffer areas where applicable) during the study period. The population to be sampled for outcome assessment considers several additional criteria for inclusion in the cohort studies as outlined below. The clusters for the trials are circumscribed geographic areas usually representing from one to a few villages or in some cases in Zambia, geographically identified parts of villages. Clusters generally represent somewhere from 100 to 400 households in size and widely vary in geographic area. Individuals greater than 18 years of age will provide individual consent. For individuals aged 6 months to less than 18 years of age, consent will be sought from the parent or guardian of the child. For children greater than 6 years and less than 18, oral assent will be sought from the child.The trial population, as a whole, consists of all de facto and Since the trial is conducted as a cluster randomized study, no individual screening is conducted. Trial areas will be enumerated prior to cohort enrollment and the enumeration will identify households with residents that meet eligibility criteria for cohort participation and for eligibility in cross-sectional household samples . Cluster-level screening is anticipated to be conducted during a baseline period in each study site. A larger number of clusters than planned for the final study power will be included in each site (~\u200910% extra clusters). These clusters will be included in baseline data collection but excess clusters will be excluded prior to randomization. Exclusion will consider the following criteria: malaria prevalence and incidence defined as per primary and secondary trial outcomes with a specific aim to exclude any clusters found to have zero or near zero malaria incidence or prevalence in the baseline period or those with dramatically higher incidence/prevalence as compared to other study clusters . Additionally, logistical feasibility of implementation will also be considered with clusters in which implementation of intervention or data collection is determined to be impracticable, to be considered for exclusion, or where community-level consent for participation in the trial is refused.Household residentAt least 12\u00a0months of age and less than 15\u00a0years of age at the time of enrollment .Eligibility for participation is described in detail in the protocol but in short, the cohort monitoring requires that the individual resides in the study areas within the core sampling areas and additionally is a:Resident whose home is located within a buffer zonePregnant at the time of cohort enrollment.Pregnant at any time during the cohort study.And is not a:Recruitment into the cohort study will be conducted by first completing an enumeration of all households and their members in the study clusters. This enumeration will be used as a sampling frame to select households with eligible individuals for the cohort study. Within each study cluster, a simple random sample of households with eligible individuals will be selected. In Mali and Kenya, a simple random sample of individuals will be selected from census lists. Within clusters, sampling for the cohort study will exclude people living in households within a geographic buffer zone around the perimeter of the cluster. Further details of recruitment are contained in the master trial protocol.The CONSORT diagram will include at minimum the following elements shown in Table It is anticipated that there will be approximately 20% LTFU withdrawal from each cohort. This is accounted for in the sample size calculations. Level of non-participation in the cross-sectional household surveys is expected to be 10\u201320%. LTFU will be summarized by arm and by cluster.The study anticipates summarizing a number of baseline participant characteristics at the individual, household, and cluster levels. Table Plasmodium falciparum infection will be used to resolve if the positive RDT is a result of persistent antigenemia or a true infection (reinfection/recrudescence). In Mali, only microscopy will be used to resolve such cases. In Kenya and Zambia, PCR results will be used where available, and otherwise microscopy. Where the RDT and either the PCR or microscopy results are both positive in month two and the patient meets the other clinical criteria (patent fever or history of fever in the previous 48\u00a0h), these observations will be treated as new clinical cases. To keep field procedures unambiguous, a blood slide will be taken whenever a positive RDT is recorded in Mali. Temporary absences from the study area not resulting in failure to ascertain monthly outcomes will not be considered as reducing individual exposure time. Absences greater than the testing interval (1\u00a0month\u2009\u00b1\u20095\u00a0days) and/or resulting in the failure to ascertain a monthly test result will be removed from the exposure time\u2014meaning that exposure will only be considered to start 1\u00a0month prior to the most recent test result.The primary outcome measure is the incidence rate of clinical malaria cases assessed among people aged 12\u00a0months to less than 15\u00a0years . A clinical case is defined as having an axillary temperature of\u2009\u2265\u200937.5\u00a0\u00b0C or self-reported fever within the past 48\u00a0h, plus a positive malaria RDT. Incidence rate is defined as the total number of incident malaria cases divided by the total person-time observed among each cohort. Outcome assessment will be conducted on each cohort participant monthly. As malaria treatment drugs will be administered to all positive clinical cases (fever\u2009+\u2009positive RDT) after monthly case ascertainment, each positive (treated) participant will have 2\u00a0weeks of the following month of observation time subtracted from their at-risk person-time to account for the prophylactic effect due to sustained antimalarial drug concentration and hence not being at risk of infection. In individuals who are symptomatic and have a positive RDT test in the month following a positive diagnosis of malaria via RDT and treatment, a positive RDT in the following month may indicate persistence of antigen in the blood after effective treatment rather than true reinfection. In such cases, PCR or microscopy results for a If a participant is symptomatic and positive by RDT, they are treated and the subsequent 2\u00a0weeks of follow-up time are censored.If in the next month the participant is also symptomatic and again positive by RDT, they will be treated and PCR or microscopy will be used to determine if they are considered a case of persistent antigenemia or a true new clinical caseIf PCR or microscopy in month two is positive, they are considered to have contributed the person-time between the previous visit and this visit less than 2\u00a0weeks and they are considered to contribute a second case to the numerator; two more weeks of follow-up will be censored following the second positive. In Mali, a person who is a malaria case on the day they re-enter the study does not contribute to the number of cases as no follow-up is associated with the case, i.e., they contribute neither to the numerator nor the denominator until they have contributed follow-up.If PCR or microscopy is negative, then contributed follow-up time between the previous visit and the second visit with the second positive RDT is included (minus 2\u00a0weeks) and only one case is included in the numerator; however, two more weeks of follow-up time are censored after the second RDT positive (due to the required treatment).In summary:1. RDT infection prevalence2. Passive incidencePrevalence of patent malaria infection detected by RDT among participants aged 6\u00a0months and older is calculated as the number of eligible, consenting participants with positive RDT results divided by the number of eligible, consenting participants with valid RDT results, collected during the cross-sectional survey (rolling survey in Kenya).3. ParityIncidence rate of passively reported clinical malaria among participants of all ages, defined as the number of malaria confirmed cases (by RDT or microscopy), linked to study clusters by place of residence, per 1000 population per year, using routine data from health facilities with patients linked to study clusters  and cluster population sizes for the denominator. Cluster population sizes will be calculated based on the number of HH residents identified in the cluster area (core only where possible/relevant) during the census/enumeration. Malaria-confirmed cases will include only those given a diagnosis of blood test (RDT or microscopy) confirmed malaria (ICD-10-M B50-54 and subcodes).Anopheles spp. mosquitoes captured during human landing catch which have never been gravid (are parous) as determined by the method of Detinova (1962). The outcome is assessed in a sub-sample of clusters, houses, and nights. Data will be disaggregated by species (and/or sub-species) with species determination made by taxonomic key and PCR where necessary. Mosquitoes will be classified as parous or non-parous. Mosquitoes with inconclusive results will be excluded from the analysis of parity.4. Mosquito abundanceThe parity outcome is the non-parous proportion: the proportion of freshly caught, non-blood-fed female adult Anopheles spp. captured in CDC UV light traps per night per trap. The outcome is assessed in a sub-sample of clusters, houses, and nights. Data will be disaggregated by species (and/or sub-species) with species determination made by taxonomic key and PCR where necessary. Mosquitoes with inconclusive speciation will be included in total Anopheles spp. abundance calculations but excluded from any species-specific analyses.5. Sporozoite rateThe number of adult Anopheles spp. captured via HLC or CDC UV light trap found to be sporozoite positive by anti-circumsporozoite protein (\u03b1-CSP) enzyme-linked immune-sorbent assay (ELISA) divided by the number of adult female Anopheles spp. tested in ELISA assay. The outcome is assessed in a sub-sample of clusters, houses, and nights. Data will be disaggregated by species (and/or sub-species) with species determination made by taxonomic key and PCR where necessary. Mosquitoes with inconclusive speciation will be included in total Anopheles spp. sporozoite rate calculations but excluded from any species-specific analyses. Mosquitoes with inconclusive \u03b1-CSP ELISA results will be excluded from all calculations.6. Human landing/biting rateThe number of adult female Anopheles spp. captured via HLC divided by the number of person-nights (days) of HLC collection. The outcome is assessed in a sub-sample of clusters, houses and nights. Data will be disaggregated by species (and/or sub-species) with species determination made by taxonomic key and PCR where necessary. Mosquitoes with inconclusive speciation will be included in total Anopheles spp. landing rate calculations but excluded from any species-specific analyses. Data will also be disaggregated by indoor versus outdoor collection location.7. EIRThe number of adult female For each month, the month-specific human landing/biting rate will be multiplied (6) by the month-specific sporozoite rate (5) to yield month-specific EIRs. Month-specific EIRs will be summed over the months of the year to yield the number of infectious bites expected in each year. This is a calculated outcome and will be disaggregated by species (and/or sub-species). Results will always be presented in terms of annualized EIR  even when the EIR estimate is made for specific months or other periods.yij is incidence at the individual level , \u03b1 is the global intercept, Xij is the arm assignment for individual i in cluster j, \u03b2arm is the arm effect to be estimated, uj are random intercepts for the cluster and exposureij is the person time at risk for individual i in cluster j, \u03bbij refers to the E(yij|uj), and \u03c3 is the standard deviation of the random intercept distribution:The primary unadjusted analysis will be conducted on the intention-to-treat analysis population without adjustment for any anticipated confounding variables as these are considered to be balanced due to randomization. The analysis of the primary outcome, cumulative clinical incidence of malaria, will be analyzed using a multi-level (variance components model) constructed on a generalized linear model framework with a Poisson likelihood and a log link function. Random intercepts will be included for each study cluster and study arm will be included as a fixed effect coded categorically as 0 for arm A and 1 for arm B. The analyst will be blinded to the true assignment until the allocation code is broken. The model will take the form below where And the random intercepts are assumed to follow a normal distribution:p-value based on the z-statistic. The primary outcome will also be checked for the distributional assumption that the mean and variance of the outcome are similar after conditioning on cluster . If variance is substantially larger, a negative binomial likelihood will be considered.Results will be presented as the incidence rate ratio (IRR), corresponding 95% confidence interval, and Adjusted analyses will be carried out on the primary and secondary outcomes to determine whether the estimate of treatment-effect is affected by the inclusion of additional covariables. The prespecified covariates will be developed and tested prior to final analysis but specific to each site. For the primary and secondary outcomes, one additional analysis will include all covariables which are used in restricted randomization with variables treated exactly as specified in randomization. Because these variables cannot be fully prespecified until the restricted randomization is complete, the full specification of these covariables cannot yet be made. However, these analyses will be prespecified for the primary outcome prior to data lock and the statistical analysis plan for each trial site will be updated to reflect these analyses. Examples of prespecified covariates that may be included in the adjusted analyses are described in Table . Imputation for these baseline missing covariates , \u03b1 is the global intercept, Xij is the arm assignment for individual i in cluster j, \u03b2arm is the arm effect to be estimated, uj are random intercepts for the cluster, and \u03c3 is the standard deviation of the random intercept distribution:The prevalence of malaria infection among participants aged 6\u00a0months and older, detected by RDT, will be analyzed using a multi-level (variance components model) constructed on a generalized linear model framework with a Bernoulli likelihood and a logit link function. Random intercepts will be included for each study cluster, and the study arm will be included as a fixed effect coded categorically as 0 for arm A and 1 for arm B. The analyst will be blinded to the true assignment until the results are presented. The model will take the form below where And the random intercepts are assumed to follow a normal distribution:e\u03b1 and the odds ratio above and the standard deviation or variance of the random effects distribution. 95% confidence intervals for the odds ratio and e\u03b1 estimates as well as z-statistics and p-values for each coefficient will be presented.Model results will be presented as the estimates of yi is the total incidence at the cluster level where only aggregated data is available (i indexes clusters), \u03b1 is the global intercept, Xi is the arm assignment for cluster i, \u03b2arm is the arm effect to be estimated, exposurei is the person time at risk for cluster i, and \u03bbij refers to the log E(yij|uj).:The incidence of clinical malaria obtained from passive case detection will be analyzed as total incidence using a generalized linear model framework with a Poisson likelihood and a log link function. The incidence will be summed for all months of follow-up within each study cluster, and the study arm will be included as a fixed effect coded categorically as 0 for arm A and 1 for arm B. Exposure will be the population of the cluster as assessed during enumeration. The analyst will be blinded to the true assignment until the results are presented. The model will take the form below where where the likelihood is of the form:z-statistics and p-values for each coefficient will be presented. Results will be presented as incidence rates and incidence rate ratios along with their associated 95% confidence intervals, and p-values.Model results will be presented as the estimates of The outcome will also be checked for the distributional assumption that the mean and variance of the outcome are similar after conditioning on a cluster ; if the variance is substantially larger, a negative binomial likelihood will be considered.yij is incidence at the individual , \u03b1 is the global intercept, Xij is the arm assignment for individual i in cluster j, \u03b2arm is the arm effect to be estimated, uj are random intercepts for the cluster and exposureij is the person time at risk for individual i in cluster j, \u03bbij refers to the log E(yij|uj), and \u03c3 is the standard deviation of the random intercept distribution:Where individual-level data is available for this outcome, a similar approach will be followed but instead focused on cumulative incidence and using a variance components model. The model will take the form below where where the likelihood is of the form:And the random intercepts are assumed to follow a normal distribution:p-value based on the z-statistic. The primary outcome will also be checked for the distributional assumption that the mean and variance of the outcome are similar after conditioning on a cluster  and if variance is substantially larger a negative binomial likelihood will be considered.Results will be presented as the incidence rate ratio (IRR), corresponding 95% confidence interval, and pij is the probability of parity at the individual mosquito level , \u03b1 is the global intercept, Xarmij is the arm assignment for individual i in cluster j, \u03b2arm is the arm effect to be estimated, Xindoorsij represents the individual mosquito being caught indoors, \u03b2indoors is the effect of being indoors on parity relative to collection happening outside, Xtimeij represents a measure of the continuous time since the start of the trial, and \u03b2time is meant to capture an overarching time trend; this variable can also be interacted with the study arm fixed effect to produce an estimate of the difference in time trend by study arm. Xmonthij represents a series of monthly dummy variables in which individual mosquitoes were caught, and \u03b2month represents the series of monthly intercepts, intended to capture seasonal variation in parity. uj are random intercepts for the cluster, \u03c3 is the standard deviation of the cluster random intercept distribution, hk are random intercepts for houses, and \u03c3h is the standard deviation of the household random intercept distribution:Daily female vector mosquito survival determined by parity is the main entomological outcome of the trial. The primary analysis will be conducted using parity data at the individual mosquito level with a multi-level (variance components model) constructed on a generalized linear model framework with a Bernoulli likelihood and a logit link function. Random intercepts will be included for each entomological study cluster and for each sampling household, and the study arm will be included as a fixed effect coded categorically as 0 for arm A and 1 for arm B. A simple model will first be considered as an unadjusted analysis which only includes fixed effects for study arm as described, and nested random effects for household and study cluster and an intercept. A more fully adjusted model will also be used for analysis to account for the complex sampling design by which mosquitoes are captured for parity analysis. This model will include fixed effects for collection location (indoors vs. outdoors), time since intervention, and calendar month as a seasonality adjustment. Additional random effects will be considered for the catch team/HLC individual. The models will generally take the form below where where the likelihood is of the form:And the random intercepts are assumed to follow a normal distribution:\u03b1 and the odds ratio for the arm above and the standard deviation or variance of the random effects distributions. 95% confidence intervals for the odds ratio and \u03b1 estimates as well as z-statistics and p-values for each coefficient will be presented.Model results will be presented as the estimates of t-test. Results will be presented as mean parity and standard deviation of parity as well as t-statistic and p-value. 95% CIs for mean parity will also be presented for each arm.Analysis based on cluster summaries will also be considered. All parity measurements within each cluster will be summarized as a single proportion. The cluster estimates of the proportion parous will be compared across arms using Student\u2019s Anopheles spp. mosquitoes via CDC UV light traps placed indoors and outdoors near houses overnight will be constructed on a generalized linear model framework with a Poisson likelihood and a log link function. Random intercepts will be included for each entomological study cluster and study arm will be included as a fixed effect coded categorically as 0 for arm A and 1 for arm B. A simple model will first be considered an unadjusted analysis which only includes fixed effects for study arm as described, and cluster-level random effects and an intercept. Autoregressive terms may also be considered with appropriate lags determined by temporal partial auto-correlation functions. The model will take the form below where yij is the count of adult Anopheles spp. mosquitoes caught at the individual trap night , \u03b1 is the global intercept, Xarmij is the arm assignment for individual i in cluster j, \u03b2arm is the arm effect to be estimated, Xindoorsij represents the trap-night observation being indoors, \u03b2indoors is the effect of being indoors on mosquito density/abundance relative to collection happening outside, Xmonthij represents a series of monthly dummy variables in which individual mosquitoes were caught, and \u03b2month represents the series of monthly intercepts. uj are random intercepts for the cluster and exposureij is the number of trap nights corresponding to the particular yij observation  term may be omitted)) for trap night i in cluster j, \u03bbij refers to the log E(yij|uj), and \u03c3 is the standard deviation of the random intercept distribution:The analysis of data on mosquito abundance derived from capture of adult where the likelihood is of the form:And the random intercepts are assumed to follow a normal distribution:p-value based on the z-statistic. This outcome will also be checked for the distributional assumption that the mean and variance of the outcome are similar after conditioning on cluster ; if the variance is substantially larger, a negative binomial likelihood will be considered.Results will be presented as the incidence rate ratio (IRR), corresponding 95% confidence interval, and Anopheles spp. which are sporozoite positive and captured during the trial will be analyzed using a multi-level (variance components model) constructed on a generalized linear model framework with a Bernoulli likelihood and a logit link function. Random intercepts will be included for each study cluster and the study arm will be included as a fixed effect coded categorically as 0 for arm A and 1 for arm B. A simple model will first be considered an unadjusted analysis which only includes fixed effects for the study arm as described and cluster-level random effects and an intercept. A more fully adjusted model will also be used for analysis to account for the complex sampling design by which mosquitoes are captured for \u03b1-CSP ELISA. This model will include fixed effects for the capture method (HLC vs. CDC light trap), collection location (indoors vs. outdoors), time since intervention, and calendar month as a seasonality adjustment. Additional random effects will be considered for the catch team/HLC individual. The models will generally take the form below where pij is the probability sporozoite positivity at the individual mosquito level , \u03b1 is the global intercept, Xarmij is the arm assignment for individual i in cluster j, \u03b2arm is the arm effect to be estimated, XHLCij represents the individual mosquito being caught by HLC, \u03b2HLC is the effect of HLC catch on sporozoite rate relative to CDC light trap, Xindoorsij represents the individual mosquito being caught indoors, \u03b2indoors is the effect of being indoors on parity relative to collection happening outside, Xtimeij represents a measure of the continuous time since the start of the trial, and \u03b2time is meant to capture an overarching time trend; this variable can also be interacted with the study arm fixed effect to produce an estimate of the difference in time trend by study arm. Xmonthij represents a series of monthly dummy variables in which individual mosquitoes were caught, and \u03b2month represents the series of monthly intercepts, intended to capture seasonal variation in sporozoite rate. uj are random intercepts for the cluster and \u03c3 is the standard deviation of the random intercept distribution:Sporozoite rate or the proportion of adult female where the likelihood is of the form:And the random intercepts are assumed to follow a normal distribution:\u03b1 and the odds ratio above and the standard deviation or variance of the random effects distribution. 95% confidence intervals for the odds ratio and \u03b1 estimates as well as z-statistics and p-values for each coefficient will be presented. Sporozoite rate will be directly estimated as the predicted probability of being sporozoite positive in each month when captured via HLC and in each study arm and indoors and outdoors. 95% prediction intervals for sporozoite rate will also be presented.Model results will be presented as the estimates of Anopheles spp. mosquitoes via HLC conducted indoors and outdoors near houses overnight will be constructed on a generalized linear model framework with a Poisson likelihood and a log link function. Random intercepts will be included for each study cluster, and the study arm will be included as a fixed effect coded categorically as 0 for arm A and 1 for arm B. A simple model will first be considered an unadjusted analysis which only includes fixed effects for the study arm as described, and cluster-level random effects and an intercept. Additional random effects will be considered for catch date, household, and/or HLC \u201ccatcher\u201d and autoregressive terms may also be considered with appropriate lags determined by temporal partial auto-correlation functions. The model will take the form below where yij is the count of adult Anopheles spp. mosquitoes landing on an individual catcher during a specific night , \u03b1 is the global intercept, Xarmij is the arm assignment for individual i in cluster j, \u03b2arm is the arm effect to be estimated, Xindoorsij represents the catch-night observation being indoors and \u03b2indoors is the effect of being indoors on human landing relative to collection happening outside, Xmonthij represent a series of monthly dummy variables in which individual mosquitoes were caught and \u03b2month the series of monthly intercepts. uj are random intercepts for the cluster and exposureij is the number of catch-nights corresponding to the particular yij observation  term may be omitted)) for catch-night i in cluster j, \u03bbij refers to the log E(yij|uj) and \u03c3 is the standard deviation of the random intercept distribution:The analysis of data on human landing/biting rate derived from the capture of adult where the likelihood is of the form:And the random intercepts are assumed to follow a normal distribution:p-value based on the z-statistic. This outcome will also be checked for the distributional assumption that the mean and variance of the outcome are similar after conditioning on cluster ; if the variance is substantially larger, a negative binomial likelihood will be considered. Human landing rate will be taken to be the predicted mean landing catch per day in each month disaggregated by arm, and indoors vs. outdoors. 95% prediction intervals will also be calculated.Results will be presented as the incidence rate ratio (IRR), corresponding 95% confidence interval, and Anopheles spp. mosquitoes caught via HLC or CDC light trap indoors or outdoors only and will follow similar principles to the analysis of total sporozoite-positive mosquitoes. The analysis will be based on Student\u2019s t-test. For this analysis, estimates of EIR will be made independently for each cluster by calculating an estimated annual EIR within each cluster according to the following formula.n represents the number of months of the year. Where collections are not made during the full calendar year because the malaria transmission season is assumed to be short and infectious bites are not expected outside of the transmission season, zero will be substituted for the estimated number of infectious bites per person-day during these months as shown in the formula above. In the formula above, b represents the number of mosquitoes captured via HLC on a catch person-night j during month i, s represents the estimated sporozoite rate for each cluster in month i, d represents the number of person catch-days for person catch night j in month i , and finally, m represents the total number of observations (person catch-nights) of HLC conducted. EIR within each cluster will be summarized as a single annualized EIR estimate post-intervention. The cluster estimates of the EIR will be compared across arms using a Student\u2019s t-test. Results will be presented as mean annualized EIR and standard deviation of annualized EIR as well as t-statistic and p-value. 95% CIs for mean parity will also be presented for each arm. Should the distribution of EIR be substantially non-normal a non-parametric test such as the Mann\u2013Whitney U-test may be considered.The analysis of the entomological inoculation rate will utilize data derived from capture of adult Individual pooled analysis across the three trial sites (countries) will be conducted collectively following the completion of all three trials. This analysis will follow similar statistical principles to each analysis specified above. The pooled analysis will likely include a study site specified as a \u201cfixed\u201d effect to allow for examination of effect modification by site. Factors related to malaria prevalence such as housing density, and density of ATSB coverage in addition to others will be examined as possible determinants of the outcomes or modifiers of ATSB effect. Additionally, a standard individual patient data meta-analysis is expected to be conducted using combined data from all sites. Heterogeneity of results from each site will be examined and this will be used to determine if pooling data and joint estimation of effect size are appropriate or if data should be treated only independently by trial site.Significant effort will be made to reduce missing outcome data by revisiting cohort households multiple times and pre-scheduling follow-up visits where possible. When missing data does arise due to failed monthly outcome assessment, no imputation will be used. Missing outcomes due to participant absence will result in censoring . They will also have 2\u00a0weeks of the next period follow-up time removed as per the definition of the primary outcome. Two sensitivity analyses will be carried out for the primary outcome. These will be the last observation carried forward  at each missing time point or that the absence of a clinical case at last observation would indicate no clinical cases observed at any missing time points and full follow-up time). This analysis is consistent with a true intention to treat protocol. A second sensitivity analysis will be to assume that all missing values would have resulted in negative findings thus imputing zero extra unobserved clinical cases across both study arms and assuming full follow-up time. These analyses will only be applied to the intention-to-treat analysis population because the per-protocol study population already assumes that full follow-up  occurred. Full reporting of the fraction of missing outcome assessments by study arm will be conducted for the intention-to-treat study population.P1, P2, \u2026 Pk from the sample. Seed for the imputation will be pre-set as an 8-digit number based on the date of analysis and documented in all scripts relying on pseudo-random number generators. If the missing values for a covariate are\u2009\u2265\u20095%, then they will be imputed using Markov chain Monte Carlo (MCMC) methods [Missing baseline covariates (as defined in the SAP prior to data lock) will be imputed using simple imputation methods in the covariate-adjusted analysis based on the covariate distributions, should the proportion of missing values for a particular covariate be less than 5%. For a continuous variable, missing values will be imputed from random values from a normal distribution with mean and standard deviation calculated from the available sample. For a categorical variable, missing values will be imputed from random values from a uniform distribution with probabilities  methods .The main risks associated with the intervention are the risk of ingestion of the bait\u2009+\u2009toxicant by humans, animals, and/or non-target arthropods\u2014particularly the local pollinator insect (bee) population. To mitigate ingestion risk for humans and other mammals bittering agents (Bitrex\u2122) have been added to ATSBs to reduce likelihood of ingestion of ATSBs. Pre-trial studies suggest that interaction between pollinators and ATSBs is insignificant and therefore ATSBs are not a risk to NTOs. As the main harms are not expected to be encountered by study participants there is no formal plan for statistical analyses of harms to study participants. Continued monitoring of trial sites for misuse or product loss will be conducted and these data will be reviewed by the DSMB but they will not be formally analyzed statistically. Unexpected harm may occur during the course of trial and will be considered in reviews and by DSMB though no formal analysis is planned.Statistical software and hardware platforms may vary by trial site. Reporting of statistical analysis will include specific details of software platform, including language, version, and details of any additional libraries used in analysis. Each trial will also develop a trial-specific SAP and maintain trial-specific standard operating procedures, trial master files, and statistical master files.Additional file 1.Additional file 2."}
+{"text": "Ionic liquid 1-butyl-3-methylimidazolium chloride [BMIM][Cl] was used to prepare cellulose (CELL), cellulose/polycaprolactone (CELL/PCL), cellulose/polycaprolactone/keratin (CELL/PCL/KER), and cellulose/polycaprolactone/keratin/ground calcium carbonate (CELL/PCL/KER/GCC) biodegradable mulch films. Attenuated Total Reflectance Fourier-Transform Infrared (ATR-FTIR) spectroscopy, optical microscopy, and Field-Emission Scanning Electron Microscopy (FE-SEM) were used to verify the films\u2019 surface chemistry and morphology. Mulch film made of only cellulose regenerated from ionic liquid solution exhibited the highest tensile strength (75.3 \u00b1 2.1 MPa) and modulus of elasticity of 944.4 \u00b1 2.0 MPa. Among samples containing PCL, CELL/PCL/KER/GCC is characterized by the highest tensile strength (15.8 \u00b1 0.4 MPa) and modulus of elasticity (687.5 \u00b1 16.6 MPa). The film\u2019s breaking strain decreased for all samples containing PCL upon the addition of KER and KER/GCC. The melting temperature of pure PCL is 62.3 \u00b0C, whereas that of CELL/PCL film has a slight tendency for melting point depression (61.0 \u00b0C), which is a characteristic of partially miscible polymer blends. Furthermore, Differential Scanning Calorimetry (DSC) analysis revealed that the addition of KER or KER/GCC to CELL/PCL films resulted in an increment in melting temperature from 61.0 to 62.6 and 68.9 \u00b0C and an improvement in sample crystallinity by 2.2 and 3.0 times, respectively. The light transmittance of all studied samples was greater than 60%. The reported method for mulch film preparation is green and recyclable ([BMIM][Cl] can be recovered), and the inclusion of KER derived by extraction from waste chicken feathers enables conversion to organic biofertilizer. The findings of this study contribute to sustainable agriculture by providing nutrients that enhance the growth rate of plants, and hence food production, while reducing environmental pressure. The addition of GCC furthermore provides a source of Ca The continuous growth of the world population in the 21st century inevitably led to increased food demand and enhanced production. Taking into account the limited availability of arable land, both efficient and sustainable agricultural practices become big challenges ,4. Altho3), a readily available natural functional filler that promotes the crystallization of the polymer by seeding many small nuclei. In parallel, it can impart whiteness that will reduce soil warming due to the resulting albedo effect and itself constitutes a plant micronutrient calcium source as well as providing a soil pH buffer action. Besides the addition of CaCO3, the tensile properties of the blends can be adjusted by incorporating various amounts of the most common polyester, partially crystalline polycaprolactone (PCL) [Cl] ,6. Imidane (PCL) , into mune (PCL) . It has ne (PCL) . Additione (PCL) .The above considerations prompted us to initiate this study, which aims to prepare mulch films that can be valued for their low cost, relative biodegradability, and processability. To achieve this, as described above, readily available naturally occurring materials such as cellulose, keratin, and calcium carbonate, as well as biodegradable polycaprolactone, were employed. Ionic liquid [BMIM][Cl] was used to prepare cellulose, cellulose/polycaprolactone, cellulose/polycaprolactone/keratin, and cellulose/polycaprolactone/keratin/ground calcium carbonate mulch films. ATR-FTIR spectroscopy, optical microscopy, and FE-SEM were used to verify the surface chemistry and morphology of the resulting films. Thereafter, the films with their various component blends were characterized in terms of their mechanical and optical properties. Simulated degradation in the soil was then applied to verify the environmental feasibility of adopting them as mulch films. The outcomes of this study will be beneficial in the ongoing development of biodegradable, micronutrient-delivering mulch films and their potential for commercialization, especially since, to the authors\u2019 knowledge, the combination of cellulose/polycaprolactone/keratin/ground calcium carbonate has not been reported elsewhere.Mw = 262.9 kDa, polydispersity of 3.6, Stora Enso Enocell, Finland). PCL  was purchased from Sigma-Aldrich, Saint Louis, MO, USA. 1-butyl-3-methylimidazolium chloride [BMIM][Cl]  was obtained from IoLiTec Ionic Liquids Technologies GmbH . Ground CaCO3 (GCC) (ISO brightness \u2265 96%) made from Norwegian marble without the use of dispersing agent, and so chemical-free, was supplied by Omya Hustadmarmor AS . The GCC particle size distribution was quoted according to the proportional volume of sample below a given particle diameter determined by time-average light scattering, d%(vol), as follows: d10(vol) \u2264 3.33 \u00b5m; d50(vol) \u2264 4.61 \u00b5m; d90(vol) \u2264 6.05 \u00b5m. To remove moisture, before the experiments, CELL and [BMIM][Cl] ionic liquid were dried at 70 \u00b0C under vacuum for about 12 h, while ground PCL pellets were dried for the same time at room temperature.Cellulose (CELL) was obtained by grinding prehydrolyzed Kraft birch pulp sheets  through the extraction process detailed in a previously published procedure , Figure Five different neat and biocomposite films with the following chemical compositions  were preCellulose (1000 mg) was dissolved in 20 mL [BMIM][Cl] at 110 \u00b0C under constant stirring for 2 h.The PCL sample was similarly prepared by the dissolution of 1000 mg of ground PCL pellets in 20 mL [BMIM][Cl], heated up to 90 \u00b0C, and mechanically stirred at that temperature for 24 h.To prepare CELL/PCL and CELL/PCL/KER blends, PCL or PCL and KER were added into the previously prepared CELL solution, and the respective mixtures were mechanically stirred for 24 h at 90 \u00b0C, as described in the literature .The inclusion of GCC to form the CELL/PCL/KER/GCC blend was performed in two steps. The first step included ultrasonic dispersion of GCC in [BMIM][Cl] for 7 min using an ultrasonic homogenizer  with a 19 mm diameter probe tip at 20 kHz and 750 W output power to deagglomerate the as-delivered dewatered crumble-like press cake. After that, CELL was added to the GCC dispersion, heated up to 110 \u00b0C, and mechanically stirred at that temperature for 2 h. Thereafter, PCL and KER were added, and the mixture was mechanically stirred for 24 h at 90 \u00b0C.The resulting homogenous blends were cast into thin layer films using a glass Petri dish. After casting, the samples were then immersed in deionized water to remove the water-miscible [BMIM][Cl] by extraction. After several washing cycles following immersion in water for a total of 24 h, the biocomposite films were air dried. Before testing, they were additionally dried for a further 24 h in a vacuum chamber.w/w)  at 80 \u00b0C and 80 mbar for 4 h, each from 600 mL batches, until the majority of the water had been removed from both the immersion and washing solution as collected from the biocomposite gel regeneration stage, and secondly, after storing overnight at \u221220 \u00b0C, by freeze-drying at \u221260 \u00b0C under 0.011 mbar for 24 h until constant weight was reached (19.7 g from the 600 mL batch). The concentration of cellulose biopolymer in the ionic liquid (22.0 g) was fixed at 4.35% (w/w) . The recThe morphology of the samples was studied using a Field Emission Scanning Electron Microscope operating at 20 kV, together with a microanalysis Tescan Mira3 XMU system . Polymer configuration and spherulite properties were visualized using a Biological LED Polarization Optical Microscope (POM) . Thermal infrared images were taken with a Fluke PTI120-9HZ Pocket Thermal Imager, and data handling was performed by the in-built thermal imaging software Fluke SmartView 4.3 .\u22121. The spectral resolution was 4 cm\u22121 following 32 scans, and the results obtained from extracted keratin were analyzed using the spectral analysis software OMNICTM  in the range 1600 cm\u22121\u20131700 cm\u22121.Attenuated Total Reflection Fourier-Transform Infrared (ATR\u2013FTIR) spectrometry, Nicolet iS10 , was used to detect the surface chemistry of the prepared biocomposite materials. Absorbance spectra were measured in the range 4000\u2013500 cm\u22121. Using a starting sample weight of 5 \u00b1 0.5 mg, the heat flow was monitored during heating under a nitrogen purge gas flow of 50 mL min\u22121.Thermal behavior of the biocomposite films was examined using a differential scanning calorimeter . The temperature was increased from room temperature to 150 \u00b0C at a heating rate of 10 \u00b0C minElemental analysis of powdered keratin was performed using an elemental analyzer , and the measured contents of N and S were found to amount to 12.78% and 3.72%, respectively .The thickness of three specimens for each formulation  was measured at five points using an electronic caliper .The light transmittance characteristic of blend films was investigated in the wavelength range of 200\u2013800 nm using an ultraviolet\u2013visible spectrophotometer .\u22121. The tests were performed under 55% relative air humidity at 23 \u00b0C. The resulting stress\u2013strain curves were used to determine tensile strength, elongation at break, and modulus of elasticity using the software Trapezium X .Film tensile behavior was analyzed using a texture analyzer  operating in tensile mode with a 500 N load cell and a gauge length of 20\u2009mm. The specimens (35 mm \u00d7 10\u2009mm) were stretched in triplicate at a crosshead speed of 10\u2009mm min3) kept in a climate chamber  conditioned at 23 \u00b0C and 50\u201355% relative humidity for 28 days (\u22121 nitrogen (N), 60\u2013250 mg L\u22121 phosphorus (P2O5), and 80\u2013400 mg L\u22121 potassium (K2O). Cellulose and biocomposite films were cut into a size of 20 \u00d7 10 mm2. The weight of each sample was recorded before burial (labeled as W1). After that, the films were buried in the soil at a depth of 25 mm from the surface.The biodegradability of the mulch films was tested in an in-soil degradation experiment carried out in an open polyethylene container (22 \u00d7 15 \u00d7 10 mm 28 days . The soiW2). Weight loss (WL) was measured and taken as a percent of the biodegradability of each corresponding sample. The weight loss was calculated using the following Equation:Samples were removed from the soil at specific times , washed with deionized water, and dried to a constant weight . ICP-OES measurement was performed using a Thermo Scientific iCAP 6500 Duo ICP  spectrometer equipped with RACID86 charge injector device (CID) detector, standard glass concentric nebulizer, quartz torch, and alumina injector. Before ICP measurements, microwave digestion  of samples was performed in a microwave digester, Advanced Microwave Digestion System , using an HPR-1000/10S segmented rotor operating under high pressure.\u2212) interacts through hydrogen bonding with the hydroxyl groups present in the cellulose , the viscosity of the solution is too high to enable satisfactory mixing, and the dissolution of cellulose takes a long time. Operating at 110 \u00b0C provided a suitable balance between [BMIM][Cl] viscosity reduction and thermal stability with ease in achieving complete cellulose dissolution. To establish the conditions for the efficient dissolution of cellulose in ionic liquid, the process was monitored using a polarization optical microscope. As is evident from ormation .\u22121, and 1364 and 2892 cm\u22121, respectively [Recorded FTIR spectra of cellulose before and after dissolution and regeneration from [BMIM][Cl] , i.e., tectively .The visual appearance of the powdered keratin and its FE-SEM micrographs are presented in \u22121) was then predicted and deconvoluted with a Lorentzian line shape function, and the deconvoluted spectrum was fitted with individual Gaussian bands using Origin Pro 8.5 software .The secondary structure of extracted keratin was identified using ATR-FTIR spectroscopy . First, Furthermore, based on the performed deconvolution of its ATR-FTIR spectrum , it can 3 filler  of keratin, which suggests that by adding a higher content of keratin, we can make films with low transparency. Such mulch films can also reduce weed growth due to their antimicrobial properties [Cl] dissolution of cellulose, the destruction of inter- and intramolecular hydrogen bonding was observed in the amorphous region, and the existence of glycosidic linkage at 896 cm\u22121 cm was detected [Cl]) as a green solvent. Miscibility; morphological, physicochemical, thermal, and mechanical properties; and biodegradability were evaluated in terms of the respective film composition. The structural properties of the \u03b1-helix and \u03b2-sheets of the extracted KER were investigated using a Fourier transform spectroscope in an IR absorption mode, applying deconvolution methods to determine the secondary structure of the polypeptide. The mechanical strength and elastic properties of the resulting biocomposites were less than those of the pure cellulose biopolymer alone due to the structural segregation arising from the limited partial miscibility within the polymer blends. The opposite effect was observed upon the addition of KER and GCC, with an increased modulus of elasticity and tensile stress response compared to the dual-polymer CELL/PCL blends. The light transmittance of the mulch film at different ratios of keratin addition was noticeably lower than that of the single CELL and CELL/PCL films. The biodegradation test also indicated that the incorporation of KER enhanced the biodegradability of the biocomposites, while GCC addition slightly inhibited degradation, partly due to an increased hydrophobicity and crystallization growth of semi-crystalline biopolymers. This work illustrates a successful design path, including the use of environmentally friendly recoverable ionic liquid for cellulose dissolution and regeneration, toward obtaining functional biodegradable mulch films that can be integrated into the soil during application and contribute to its enrichment with organic/inorganic fertilizing activity after biodegradation. The incorporation of natural cellulose biomass and keratin from chicken feather waste in the formulation has several advantages, such as a reduction in costs and the valorization of agricultural waste, which promotes a healthy environment and a circular bioeconomy. For this reason, in the future, continuing research on mulch films, different polysaccharides, and polypeptides will be applied for the preparation of agricultural mulch entirely on a biological basis, without the use of synthetic biopolymers."}
+{"text": "Atherosclerosis is one of the most fatal diseases in the world. The associated thickening of the arterial wall and its background and consequences make it a very composite disease entity with many mechanisms that lead to its creation. It is an active process, and scientists from various branches are engaged in research, including molecular biologists, cardiologists, and immunologists. This review summarizes the available information on the pathophysiological implications of atherosclerosis, focusing on endothelium dysfunction, inflammatory factors, aging, and uric acid, vitamin D, and miRNA expression as recent evidence of interactions of the molecular and cellular elements. Analyzing new discoveries for the underlying causes of this condition assists the general research to improve understanding of the mechanism of pathophysiology and thus prevention of cardiovascular diseases. Cardiovascular disease (CVD) is the biggest cause of mortality in the world ,2. AtherRecent observations established that atherosclerosis is the leading cause of vascular disease globally ,8. AccorAtherosclerosis is considered to be caused by multiple factors, including genetic and environmental factors. There are many known risk factors for atherosclerosis, including hypercholesterolemia, hypertension, diabetes mellitus, kidney diseases, and cigarette smoking .Symptoms of CVD have a significant impact on one\u2019s quality of life. They are examined as patient-reported outcomes in terms of their genesis, expression, similarities, and differences between illnesses. Atherosclerosis could be latent and symptomless, but the consequences are quite diverse. The symptoms of acute coronary syndrome (ACS), heart failure, valve problems, stroke, rhythm abnormalities, and peripheral vascular disease are considered to be the most common consequences of atherosclerosis . Chest dThis chronic disease can escalate for many years and can lead to serious complications due to ongoing inflammatory processes. Atherosclerosis results in CVD, which can take the form of ischemic heart disease, stroke, or other vascular diseases .Moreover, chronic kidney disease (CKD) is also a complication of atherosclerosis. The composition of atherosclerotic plaques is responsible for the severity and advancement of CKD. For this very reason, it is necessary to start treatment at an early stage of atherosclerosis to achieve the best results .The pathogeneses of atherosclerosis and diabetes are closely related. Diabetes has been shown to be a triggering factor for atherosclerosis due to dyslipidemia, hyperglycemia, oxidative stress, and chronic inflammation. These components are common in both diseases .The most crucial recommendations for the prevention of atherosclerosis include a proper diet, physical exercise, smoking cessation, adequate stress management, and good quality of sleep .Meat consumption, especially that of red meat and processed meat products, should be limited to 0.5 kg per week ,25. FishAs part of the prevention of cardiovascular diseases (CVDs), healthy people are recommended to undertake physical activity, namely either over 150 min/week of moderate intensity aerobic exercise, 75 min/week of high-intensity aerobic exercise, or an equivalent to either of these .2) [Atherosclerosis is chronic arterial inflammation caused by both conventional  and unco2) . This lo2) . Further2) .Afterward, the active NLRP3 inflammasome triggers the recruitment and activation of caspase-1, which participates not only in the activation of the precursors of IL-1\u03b2 and IL-18 but also in the breaking of gasdermin D . IL-1\u03b2 aInflammation starts with the activation of NLRP3 inflammasomes, which results in the production of proinflammatory cytokines IL-1 and IL-18, acting via the autocrine, paracrine, or endocrine pathways . IL-1 haAge increases the aortic plaque burden and the size and severity of the aortic root plaques in the AD-fed ApoE mice independent of the number of weeks on the diet. However, there was no effect from age on the size or severity of oscillatory shear-induced carotid artery atheromas after PCL.Treatment with PCSK9 increased the total and LDL cholesterol in the young mice, but the older mice had a larger aortic root atheroma size and morphology grade. Age did not affect the size or degree of the carotid artery lesions in the mice overexpressing PCK9 after PCL. Atherosclerotic plaques will expand spontaneously in the atheroprone regions of the aorta of the AD-fed mice after PCSK9 overexpression by an AAV. The transgenic ApoE mice also overexpressed human PCSK9, demonstrating larger atherosclerotic lesions with greater monocyte infiltration compared with the PCSK9 wildtype ApoE mice .Aging is one of the strongest risk factors for atherosclerosis  which inThe vascular endothelium, as an integral component of the cardiovascular system intimately interfacing with the blood, plays a crucial role in maintaining systemic homeostasis. It acts as a lining of the cardiovascular system, forming a particular barrier to various molecules ,69,70. IHowever, the process begins with the structure of the endothelium and the function of its cells. They produce a series of different cytokines and chemokines like C-X-C motif chemokine 12 (CXCL12), which initiates inflammation and the recruitment of cells which attend to this process. Endothelial cell receptors such as C-X-C chemokine receptor type 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) can be introduced as new targets for the treatment of atherosclerosis. However, the role of arterial ACKR3 is still not fully known, especially in reference to the human system . In termBoth vasoconstrictors and vasodilators play a significant role in the proper functioning of the cardiovascular system ,79.In addition to nitrogen oxide (NO), molecules that stimulate vasodilation include hydrogen sulfide, carbon monoxide, arachidonic acid metabolites, and hydroperoxide. The endothelium also produces opposing compounds . These substances are essential in maintaining vascular homeostasis ,80,81.Endothelial dysfunction occurs under the influence of many factors, including local hemodynamic changes. Endothelial dysfunction may be considered a change in endothelial phenotype, which significantly affects, inter alia, the regulation of homeostasis, coagulation processes, fibrinolysis, or vascular tone. The epithelium is more susceptible to damage during turbulent blood flow and increased shear stress gradients. Sites such as vascular bifurcations or branches will therefore be more vulnerable . When thFor vascular endothelial dysfunction in the context of atherosclerosis, we should mainly consider the activation of the endothelium. If the right conditions are present, such as the previously mentioned hemodynamics of blood flow being altered, and the endothelium is damaged, then molecular activation takes place in the form of expression of chemokines, cytokines, and adhesion molecules that will interact with leukocytes, platelets, and other immune cells . Under tA characteristic feature of the initial stages of atherosclerosis is the local accumulation of monocytes and lymphocytes in the dysfunctional inner membrane of the vessel wall. For this to occur on the surface of this membrane, adhesion molecules involved in the interactions of vessel wall cells with blood cells appear. Adhesion molecules in the endothelium include E-selection, P-selection, intercellular adhesion molecule-1, or VCAM-1. Their recruitment under appropriate stimuli leads to accumulation of the previously mentioned monocytes and lymphocytes ,83.Recruited monocytes differentiate into macrophages and transform into foam cells through the uptake of extracellularly modified lipoproteins . ActivatCurrently, efforts are being made to identify novel therapeutic targets for atherosclerosis treatment. Many of these targets revolve around vascular endothelial damage, as it constitutes an essential part of its pathogenesis. Activation of purinergic signaling is among the emerging therapeutic avenues. ATP is released by endothelial cells in response to shear stress, exerting its effects through receptors such as P2X4, P2X7, and P2Y2, thereby modulating the vascular tone. Purinergic activation, mediated by A2A, P2X4, P2X7, and P2Y1 receptors, participates in inflammatory responses and leukocyte adhesion to endothelial cells. Further research is warranted to discern the actions of individual P2 receptors, rendering them effective therapeutic targets . Recent A serum UA level higher than 6.8 mg/dL is considered to be an abnormally high level of UA . HyperurFirst, morphological plaque characteristics appear to differ among patients with high UA levels. In accordance with the analysis which examined the morphological features of atherosclerotic plaques, in patients with high UA levels, the plaque had a wider maximum lipid arc, longer calcification length, and thinner minimum fibrous cap thickness. According to a correlation analysis, the UA value was inversely associated with the minimum fibrous cap thickness and positively associated with the maximum lipid arc, average lipid arc, and calcification length . AdditioOxidative stress, inflammation, and endothelial dysfunction are the pathologic processes linked to higher serum UA levels. Therefore, it is not unexpected that elevated serum UA is linked to various negative consequences, such as hypertension, chronic kidney diseases, and CVDs .A series of recent studies discussed the relationship between the renin\u2013angiotensin\u2013aldosterone system (RAAS) and UA, together with their role in atherosclerosis. Angiotensin II (AngII), a component of the RAAS, affects multiple cellular processes and can cause vasoconstriction, regulate acid-base balance, modulate immune and inflammatory pathways, and stimulate the release of chemokines. UA has been found to activate the RAAS and promote the expression of angiotensinogen, angiotensin-converting enzyme, and AngII receptors, as well as stimulate oxidative stress, inflammation, and smooth muscle cell proliferation. Additionally, UA and AngII together may lead to a more complex inflammatory and oxidative response compared with their individual effects ,106,107.These studies discovered that UA might increase the amount of inflammatory components and intensify intercellular adhesion molecule formation, which would exacerbate inflammatory responses and accelerate the development of atherosclerosis ,109,110.Interestingly, Hu et al. investigated the effect of HDL-C on carotid atherosclerosis at different serum UA levels. Elevations in the UA levels were associated with HDL-C dysfunction .Yu et al. established that a high level of UA promotes atherosclerosis by targeting nuclear factor erythroid 2-related factor 2 (NRF2)-mediated autophagy dysfunction and ferroptosis. Ferroptosis is a recently identified type of controlled cell death which is primarily driven by iron-dependent lipid peroxidation and has played a role in several pathogenic diseases such as CVDs, cancer development, and acute kidney disease. A high UA level potentially accelerates the development of atherosclerosis by influencing the ferroptosis of the foam cells. Moreover, a high UA level inhibits NRF2, which is a key antioxidant regulator critical to supporting metabolic and redox balance by controlling cellular antioxidants, thus promoting the formation of atherosclerotic plaques .In conclusion, UA exerts a pro-atherogenic effect on plaque development . UA enhaThese findings might contribute to a deeper understanding of the role of UA in the pathogenesis of atherosclerosis. The potential of lowering the UA levels in patients with hyperuricemia and CVDs could result in a better clinical treatment strategy.Vitamin D plays an important role in the human body. The first studies on vit. D and its impact on the human body concerned rickets and date back to the turn of the 20th century. They led to the identification of a deficiency of vit. D as the etiology of this disease . Vit. D Vit. D needs to be activated by 25-hydroxylation in the liver, yielding 25OHD, and through 1\u03b1-hydroxylation in the kidney yielding the vit. D hormone 1,25(OH)2D. It is now believed that almost all of the biological effects of vit. D are mediated by its active form, 1,25(OH)2D, signaling mainly through the intracellular vit. D receptor (VDR) . As a liVit. D metabolism is highly regulated by levels of calcium, phosphate, fibroblast growth factor 23, and parathyroid hormone .The level of 25(OH)D is influenced by the method of ingesting and the source of foods rich in vit. D. It is also dependent on the exposure to sunlight and fresh air during physical activities. Race and ethnicity also influence differences in the markers of vit. D metabolism . HoweverOver the years, numerous publications have included both case studies and experimental findings that have linked vit. D levels to CVD risk in its origin and occurrence. As a result, further research into vit. D supplementation for the clinical improvement of patients has been conducted. However, some studies did not confirm this correlation and did not prove the effectiveness of such treatment .The focus of this article concerns atherosclerosis and innovative approaches to treating it by means of research on new therapeutic solutions and pathophysiological factors. Due to the complexity of this condition, interactions between environmental and genetic factors are still being studied.Recent experimental and clinical studies showed evidence for the effect of vit. D signaling on the modulation of atherosclerosis pathogenesis. Vit. D signaling reduces the expression of TNF\u03b1, IL-6, IL-1, and IL-8 in isolated blood monocytes. IL-6 suppression leads to a decrease in the synthesis of acute phase C-reactive protein. This may contribute to the formation of atherosclerotic changes ,136. VitA link has been found between vit. D deficiency or supplementation and the development of a myocardial infarction or a stroke. Lower levels of vit. D are associated with a higher risk of CVD and the development of hypertension. Unfortunately, conclusions regarding the relief of symptoms in CVDs are still unclear and may, with further research, counter many prejudices . Using cSince obesity accelerates the aging process of cells, its relationship with vit. D deficiency should also be carefully considered. One study conducted on this topic showed that obesity may be an important pathogenetic factor in subclinical atherosclerosis, and vit. D supplementation may have a protective role against the occurrence of comorbidities .On the other hand, vit. D supplementation has not been shown to reduce the CVD burden in older adults. A 2020 randomized controlled trial (RCT) found that vit. D supplementation at 2000 IU/day for 3 years did not reduce elevated systolic and diastolic blood pressure, which is often increased due to atherosclerotic disease. This suggests that vit. D supplementation for less than 3 years does not influence SBP or DBP. Elevated arterial blood pressure is a known cause of atherosclerosis, and high blood pressure is a risk factor for developing atherosclerosis . A 2017 MicroRNAs (miRNAs) are small non-coding RNAs that have diverse cellular roles  but are By regulating the expression of genes that are important in the pathogenesis of processes leading to atherosclerosis, miRNAs can affect the level of synthesized protein in cells. This protein may be significant in dysregulation, which then contributes to the intensification of atherosclerotic processes , and miRNM-Exos helps promote vascular smooth muscle migration and proliferation, and miR-21-3p plays a key role in regulating the NM-Exos-induced effects. Therefore, exosomal miR-21-3p from nicotine-treated macrophages can accelerate the development of atherosclerosis by supporting this relation . Sharma Liu X. et al. demonstrated that the NFATc3 macrophage upregulates miR-204 to reduce the SR-A and CD36 levels, thereby preventing foam cell formation and atherosclerosis. The NFATc3/miR-204 axis may be another target in the development of individual therapy against atherosclerosis . MiR-320DNA methylation is a key epigenetic modification in regulating cell function by silencing the relative gene expression. DNA and miRNA methylation are important epigenetic factors in AS. This study reviews recent insights into the role of miRNA and DNA methylation and their interaction in AS progression . DNA metAtherosclerosis is a common vascular aging condition. Nevertheless, a variety of factors could have an impact on this process, leading to an increase in cardiovascular risk and a high rate of morbidity and mortality.In this review, we focused on the important molecular aspects of arteriosclerosis. We paid attention to the role of the immune system, its dysfunctions, and its influence on the development of this disease. Endothelial dysfunction, its pathomechanism, and its influence on the development of CVDs were also considered to be of interest. Moreover, as advancing age is a significant risk factor for atherosclerotic CVD, we discussed the vascular intrinsic and extrinsic mechanisms of how aging influences the pathology of atherosclerosis. Furthermore, we drew attention to the influence of several factors, such as UA, vit. D, and miRNA, since their key role in the pathogenesis of atherosclerosis has been established in research in recent years.These findings might shed new light on the cellular mechanisms of atherosclerosis and prospective targets for prevention and treatment in the future. The factors presented in this review are summarized in However, the significant scientific achievements of recent years and the many new discoveries and mechanisms still require careful attention and additional studies."}
+{"text": "Staphylococcus aureus bacteremia (SAB), including right-sided infective endocarditis (NCT03138733). We reported molecular characteristics of methicillin-resistant S. aureus (MRSA) along with the clinical outcomes.A Phase 3 clinical trial evaluated ceftobiprole (BPR) for the treatment of spa, and SCCmec types.94 patients had MRSA isolated from baseline blood cultures, 90 of which were available for molecular characterization. Susceptibility testing used the CLSI broth microdilution method. Total genomic DNA was extracted and sequenced. Assembled genomes were used to determine multilocus sequence typing (MLST), clonal complex (CC), A total of 10 CC were present within the 90 isolates. 71 (78.9%) MRSA fell into 3 predominant clones: CC8, CC22, and CC5 (Table). Among CC8 , 4 ST8-like  from USA (3) and Ukraine (1) were presumptively designated as USA300, whereas 12 ST239  from Bulgaria (1), Georgia (1), Russia (1), Serbia (2), and Ukraine (7) were designated as the Hungarian clone. Other ST8-like MRSA  from Ukraine (15), Russia (2), Spain (1), and Argentina (1) were identified as USA300LAV. Nineteen  MRSA belonged to CC22/ST22. Fifteen  MRSA were CC5, and included ST5 and related single- (ST225 and ST2704) or double-locus (ST1827) variants. CC5 MRSA were designated as USA100  or USA800 . Other strains were represented by 7 CC, including those associated with livestock infections. Overall, clinical responses (CR) and microbiological eradication (ME) were similar between treatment arms (Table). CR and ME rates were higher in the BPR arm among patients infected with CC5 strains, whereas these rates in the comparator arm were higher in patients infected with CC22 and other CC types.This global SAB trial included patients with MRSA strains belonging to pandemic lineages and those causing infections in United States hospitals. This strain set includes clones previously associated with hospital- and community-acquired infections as well as strains associated with livestock infections. In general, CR and ME were comparable between the 2 study arms with small differences in outcomes by CC type.Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|Cipla: Grant/Research Support|Entasis: Grant/Research Support|GSK: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Leonard R. Duncan, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|CorMedix: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support John H. Kimbrough, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Thomas L. Holland, MD, Aridis: Advisor/Consultant|Basilea Pharmaceutica: Advisor/Consultant|Karius: Advisor/Consultant|Lysovant: Advisor/Consultant Vance G. Fowler, MD, MHS, Amphliphi Biosciences, Integrated Biotherapeutics; C3J, Armata, Valanbio; Akagera, Aridis, Roche, Astra Zeneca: Advisor/Consultant|Genentech, Regeneron, Deep Blue, Basilea, Janssen;: Grant/Research Support|Infectious Diseases Society of America: Honoraria|MedImmune, Allergan, Pfizer, Advanced Liquid Logics, Theravance, Novartis, Merck; Medical Biosurfaces; Locus; Affinergy; Contrafect; Karius;: Grant/Research Support|Novartis, Debiopharm, Genentech, Achaogen, Affinium, Medicines Co., MedImmune, Bayer, Basilea, Affinergy, Janssen, Contrafect, Regeneron, Destiny,: Advisor/Consultant|Sepsis diagnostic: Patent pending|UpToDate: Royalties|Valanbio and ArcBio: Stock Options Mark E. Jones, PhD, Astellas Pharma Global Development, Inc: Support for the present publication|Basilea Pharmaceutica International Ltd: Employee of Basilea Pharmaceutica International Ltd|Basilea Pharmaceutica International Ltd: Stocks/Bonds Marc Engelhardt, MD, Astellas Pharma Global Development, Inc.: Support for the present publication|Basilea Pharmaceutica International Ltd: Employee of Basilea Pharmaceutica International Ltd|Basilea Pharmaceutica International Ltd: Stocks/Bonds Jennifer Smart, PhD, Basilea Pharmaceutica International Ltd, Allschwil, Switzerland: Stocks/Bonds Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|bioMerieux: Grant/Research Support|Cipla: Grant/Research Support|CorMedix: Grant/Research Support|Entasis: Grant/Research Support|Melinta: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support"}
+{"text": "Burkholderia species can cause chronic and often severe respiratory tract infections in cystic fibrosis (CF) patients. The treatment of infections Burkholderia isolates is complicated by low cell permeability to most antimicrobial agents, presence of beta-lactamases and expression of efflux systems. We evaluated the activity of meropenem-vaborbactam (MEV) and comparator agents tested against Burkholderia spp. isolates collected during a surveillance study.Burkholderia spp. isolates were received as part of a global surveillance program from 2014 to 2022. Isolates were identified using MALDI-TOF MS and included B. cenocepacia (11), B. cepacia species complex , B. gladioli (28) and B. multivorans (21). Isolates were susceptibility (S) tested by reference broth microdilution methods and CLSI interpretative criteria was applied for comparator agents for BCC isolates. Meropenem (MEM) alone breakpoints published by CLSI for BCC or EUCAST for Pseudomonas aeruginosa breakpoints were applied for MEV for comparison purposes.A total of 328 50/90, 1/2 mg/L) inhibited 95.1% of the BCC isolates at \u22644 mg/L and 97.0% at \u22648 mg/L. Trimethoprim-sulfamethoxazole (T/S), MEM and ceftazidime (CAZ) were active against 87.7%, 86.9% and 84.3% of the BCC isolates, respectively and levofloxacin and minocycline inhibited 63.4% and 82.5% of BCC isolates, respectively. Against B. multivorans (21), MEV  inhibited 95.2% of the isolates at \u22644 mg/L or \u22648 mg/L while MEM inhibited 71.4% of the isolates at \u22644 mg/L and 95.2% of the isolates at \u22648 mg/L. T/S and CAZ were active against 71.4% and 81.0% of the B. multivorans isolates. T/S MEV, MEM and CAZ were active against all 11 B. cenocepacia isolates. MEM and MEV  exhibited the same activity against 28 B. gladioli isolates.MEV (MICBurkholderia spp. isolates can lead to respiratory failure which is the primary cause of mortality and morbidity in CF patients. Despite the elevated susceptibility rates for the therapies of choice for these isolates, T/S, CAZ and MEM resistant isolates have been reported. MEV demonstrated good activity against BCC isolates that are commonly recovered from CF patients.Chronic pulmonary infections caused by Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|bioMerieux: Grant/Research Support|Cipla: Grant/Research Support|CorMedix: Grant/Research Support|Entasis: Grant/Research Support|Melinta: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Dee Shortridge, PhD, Melinta: Grant/Research Support|Shionogi: Grant/Research Support Kelley Fedler, BS, Melinta: Grant/Research Support|Paratek: Grant/Research Support Cecilia G. Carvalhaes, MD, PhD, AbbVie: Grant/Research Support|bioMerieux: Grant/Research Support|Cipla: Grant/Research Support|CorMedix: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support"}
+{"text": "Fluconazole (FLC) resistance (R) is a raising concern to treat IC, and ECHs are often used as first-line therapy. We evaluated the C. albicans , C. glabrata , C. parapsilosis , C. tropicalis , C. krusei , C. dubliniensis , and C. auris  isolates were collected (1/patient) in 2022 from 52 medical centers located in Europe , North America , Asia-Pacific , and Latin America , identified by MALDI-TOF MS and/or sequencing and tested by CLSI broth microdilution. CLSI breakpoints (BP) were applied where available. CARS CDC tentative BPs were applied.RFZ exhibited activity against CA (Table) inhibiting 99.1% of the isolates overall, and 98.6%, 99.0%, 100%, and 100% of isolates from NA, EU, AP, and LA. Only 1 CA isolate (from US) displayed a FLC MIC value within the susceptible-dose dependent (SDD) category. CSP, ANF, and MCF showed S rates of 99.1% against CA. All but 1 CG isolate was susceptible (S) to RZF (99.0%S). CG S rates to CSP, ANF, MCF, and FLC were 98.0%, 98.0%, 97.1%, and 96.1% (SDD), respectively. All CP isolates were S to RZF, CSF, and MCF. However, only 84.1% of CP isolates were S to FLC. RZF and other ECHs inhibited all FLC-R isolates . All CT and CK isolates were S to RZF and other ECHs. All CT were also S to azoles, and 94.4% of CK were S to VRC. Only RZF BPs are available by CLSI against CD (86.7%S). All CARS isolates  were FLC-R; but 77.8% and 88.9% were S to RZF and other ECHs, respectively.in vitro activity against invasive candidiasis isolates regardless of the region, and remained active against FLC-R CP and most of the FLC-R CARS isolates. Based on MIC50 and MIC90 results, RZF had similar activity to the other ECHs overall.RZF demonstrated potent Cecilia G. Carvalhaes, MD, PhD, AbbVie: Grant/Research Support|bioMerieux: Grant/Research Support|Cipla: Grant/Research Support|CorMedix: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support Paul Rhomberg, BS, MT(ASCP), bioMerieux: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support Abby Klauer, BS, Melinta: Grant/Research Support Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|bioMerieux: Grant/Research Support|Cipla: Grant/Research Support|CorMedix: Grant/Research Support|Entasis: Grant/Research Support|Melinta: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support"}
+{"text": "Microbial cell-free DNA (mcfDNA) sequencing can establish the etiology of multiple infectious syndromes by identifying pathogen DNA from the plasma of infected patients. Here, we describe the potential impact of a positive mcfDNA result on clinical decision making among immunocompromised adults with suspected pneumonia.This prospective observational study evaluated the potential utility of mcfDNA sequencing in adults with active hematological malignancies undergoing a diagnostic bronchoscopy for pneumonia at 10 US Medical Centers as part of the PICKUP Study (Abstract # 544 IDWeek 2022). Plasma mcfDNA was collected on all participants at the time of bronchoscopy. Clinical impact of the mcfDNA sequencing results vs usual care (UC) testing \u2013 including bronchoscopy- were adjudicated and then compared for: 1) identification of probable cause of pneumonia or clinically significant non-pulmonary infection and 2) potential changes to antimicrobial therapy if mcfDNA sequencing results were available to treating clinicians.Of 223 participants analyzed, median (IQR) age was 62 (50-69) years and 72 (32.3%) were female. Plasma mcfDNA identified a probable cause of pneumonia in 57/223  participants and could have changed antimicrobial therapy in 21/57 . A probable cause of pneumonia was identified by mcfDNA in 23/223  participants when no cause was identified by UC, and these detections could have resulted in an antimicrobial change in 17/23 . A clinically relevant non-pulmonary infection was identified in 88/223  participants and antimicrobial therapy could have changed in 22/88 . Collectively, antimicrobial therapy could have changed for 41/223  participants if mcfDNA results were available to treating clinicians.Positive plasma mcfDNA sequencing results could have supported changes in clinical management for pneumonia and non-pulmonary infections among immunocompromised patients undergoing bronchoscopy. Further studies are needed to refine the optimal timing of mcfDNA in relation to UC testing and establish the impact of real-time mcfDNA results on patient outcomes.Deng B. Madut, MD, Karius: Advisor/Consultant Roy F. Chemaly, MD/MPH, Eurofins-VViracor: Grant/Research Support|Karius: Advisor/Consultant Sanjeet S. Dadwal, MD, FACP, FIDSA, Allovir: Advisor/Consultant|Allovir: Grant/Research Support|Ansun Biopharma: Grant/Research Support|Aseptiscope, Inc: Stocks/Bonds|Astellas: Honoraria|Karius: Grant/Research Support|Matinas Biopharma: Stocks/Bonds|Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer/Amplyx: Grant/Research Support|Takeda: Advisor/Consultant|Takeda: Honoraria|Viracor: Honoraria Joshua A. Hill, MD, Allovir: Advisor/Consultant|Allovir: Grant/Research Support|Century Therapeutics: Advisor/Consultant|Covance/CSL: Advisor/Consultant|Deverra: Grant/Research Support|Eversana Life Science Services, LLC: Advisor/Consultant|GeoVax: Grant/Research Support|Gilead: Advisor/Consultant|Gilead: Grant/Research Support|Karius: Advisor/Consultant|Karius: Grant/Research Support|Merck: Grant/Research Support|Moderna DSMB: Advisor/Consultant|Octapharma AG: Advisor/Consultant|OptumHealth: Advisor/Consultant|Oxford Immunotec: Grant/Research Support|Pfizer (previously Amplyx/Medpace): Advisor/Consultant|Senti BioSciences, Inc: Advisor/Consultant|Symbio: Advisor/Consultant|Takeda: Advisor/Consultant|Takeda: Grant/Research Support|Up-to-Date: Advisor/Consultant Yeon Joo Lee, MD, MPH, AiCuris: institutional research support for clinical trials|Karius: institutional research support for clinical trials|Merck: Grant/Research Support|Scynexis: institutional research support for clinical trials Ghady Haidar, MD, Allovir: Grant/Research Support|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|Karius: Advisor/Consultant|Karius: Grant/Research Support|NIH: Grant/Research Support Alfred Luk, MD, Bill & Melinda Gates Foundation: Grant/Research Support|Karius: Advisor/Consultant|Karius: Grant/Research Support Fareed Khawaja, MBBS, MEDSCAPE: Honoraria|Viracor: Grant/Research Support Genovefa Papanicolaou, MD, Allovir: Advisor/Consultant|Amplyx: Advisor/Consultant|Astellas: Advisor/Consultant|Cidara: Advisor/Consultant|CSL Behring: Advisor/Consultant|DSMC: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Merck: institutional research support for clinical trials|MSD: Advisor/Consultant|Octapharma: Advisor/Consultant|Partners Rx: Advisor/Consultant|Shire/Takeda: institutional research support for clinical trials|Symbio: Advisor/Consultant|Symbio: Advisor/Consultant|Takeda: Advisor/Consultant|Vera Pharma: Advisor/Consultant Micah T. McClain, MD, PhD, Biomeme Inc: Methods to diagnose and treat acute respiratory infections Eileen K. Maziarz, MD, Karius, Inc: Advisor/Consultant Robert Bigelow, PhD, Covidien: Stocks/Bonds|Elixir Medical: Advisor/Consultant|Johnson & Johnson: Stocks/Bonds|Mckesson: Stocks/Bonds|Merck: Stocks/Bonds|Organon: Stocks/Bonds|Pfizer: Stocks/Bonds|Sanofi: Stocks/Bonds|Viatris: Stocks/Bonds Daniel Lupu, MD, PHD, Karius Inc: Employee|Karius Inc: Stocks/Bonds Sivan Bercovici, PhD, Karius: Stocks/Bonds Bradley A. Perkins, MD, Karius, Inc: Stocks/Bonds Timothy A. Blauwkamp, PhD, Karius: Board Member|Karius: Ownership Interest Vance G. Fowler, MD, MHS, Amphliphi Biosciences, Integrated Biotherapeutics; C3J, Armata, Valanbio; Akagera, Aridis, Roche, Astra Zeneca: Advisor/Consultant|Genentech, Regeneron, Deep Blue, Basilea, Janssen;: Grant/Research Support|Infectious Diseases Society of America: Honoraria|MedImmune, Allergan, Pfizer, Advanced Liquid Logics, Theravance, Novartis, Merck; Medical Biosurfaces; Locus; Affinergy; Contrafect; Karius;: Grant/Research Support|Novartis, Debiopharm, Genentech, Achaogen, Affinium, Medicines Co., MedImmune, Bayer, Basilea, Affinergy, Janssen, Contrafect, Regeneron, Destiny,: Advisor/Consultant|Sepsis diagnostic: Patent pending|UpToDate: Royalties|Valanbio and ArcBio: Stock Options Thomas L. Holland, MD, Aridis: Advisor/Consultant|Basilea Pharmaceutica: Advisor/Consultant|Karius: Advisor/Consultant|Lysovant: Advisor/Consultant Stephen P. Bergin, MD, Karius, Inc.: Grant/Research Support"}
+{"text": "In March 2023, the Clinical and Laboratory Standards Institute (CLSI) lowered the Enterobacterales susceptible (S)/resistant breakpoints for amikacin from \u226416/\u226564 mg/L to \u22644/\u226516 mg/L and gentamicin and tobramycin from \u22644/\u226516 mg/L to \u22642/\u22658 mg/L. As aminoglycosides are frequently used to treat infections caused by multidrug-resistant (MDR) and carbapenem-resistant Enterobacterales (CRE), we evaluated the activity of plazomicin and the impact of CLSI breakpoint changes on the susceptibility rates of Enterobacterales collected from US medical centers.5,725 Enterobacterales isolates were consecutively collected (1/patient) from 36 US medical centers in 2020\u20132022 and susceptibility tested by broth microdilution against amikacin, gentamicin, tobramycin, and plazomicin. Susceptibility rates were calculated using both current CLSI/USFDA breakpoints and the recently revised CLSI breakpoints. Aminoglycoside-nonsusceptible isolates were screened for genes encoding aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methyltransferases (16RMT).Plazomicin was active against 96.3% of isolates and retained potent activity against MDR (93.1%S) and CRE (92.1%S) isolates. The highest variations in susceptibility rates due to breakpoint changes were observed with amikacin, especially among MDR  and CRE (77.8%S to 61.9%S). Against all Enterobacterales, amikacin susceptibility rates decreased from 99.6%S to 95.0%S; susceptibility to gentamicin and tobramycin decreased 0.8% and 2.6%, respectively. Gentamicin and tobramycin showed limited activity against MDR and CRE with both 2022 and 2023 breakpoints. AME-encoding genes were observed in 474 (8.3% of isolates) and 16RMT was found in 7 isolates (0.1%). Plazomicin was active against 97.3% of AME producers.Amikacin\u2019s spectrum of activity against CRE and MDR Enterobacterales was drastically reduced when interpretative criteria based on PK/PD parameters currently used to establish breakpoints to other antimicrobials were applied. Plazomicin is markedly more active than amikacin, gentamicin, or tobramycin against CRE and MDR Enterobacterales causing infections in US medical centers.Helio S. Sader, MD, PhD, FIDSA, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|Cipla: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|Cipla: Grant/Research Support|Entasis: Grant/Research Support|GSK: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support S J Ryan Arends, PhD, Cipla: Grant/Research Support|GSK: Grant/Research Support Cecilia G. Carvalhaes, MD, PhD, AbbVie: Grant/Research Support|bioMerieux: Grant/Research Support|Cipla: Grant/Research Support|CorMedix: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|bioMerieux: Grant/Research Support|Cipla: Grant/Research Support|CorMedix: Grant/Research Support|Entasis: Grant/Research Support|Melinta: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support"}
+{"text": "P. aeruginosa (PSA) and A. baumannii-calcoaceticus complex (ABC) as part of the SENTRY Antimicrobial Surveillance Program.Cefiderocol (CFDC) is a siderophore-conjugated cephalosporin with activity against Gram-negative bacteria. CFDC and comparator activities were evaluated against resistant and molecularly characterized Acinetobacter spp. (979 ABC) were consecutively collected from 64 US sites in 2020\u20132022. Susceptibility testing was performed by broth microdilution and CFDC testing used iron-depleted media. FDA and CLSI breakpoints were used for CFDC. CLSI criteria were applied to comparators, except for imipenem-relebactam . Isolates with a resistance phenotype to \u2265 3 classes were defined as multidrug resistant (MDR). ABC and PSA with imipenem and/or meropenem (MER) MICs \u2265 4 \u03bcg/mL or ceftazidime (CAZ) and/or cefepime MICs \u2265 16 \u03bcg/mL were subjected to genome sequencing for screening of acquired carbapenemase genes.3,384 PSA and 1,186 50/90, 0.12/0.5 \u03bcg/mL) that did meet the MIC screening criteria and against those that did not . CFDC also had similar MIC50 values (0.12 \u03bcg/mL) against the MDR and carbapenemase-positive PSA populations, whereas other agents had compromised activity. 47.3% (463/979) of ABC met the MIC screening criteria, while 37.1% (363/979) had an MDR phenotype, and 28.4% carried carbapenemase genes. In general, CFDC, IMR, meropenem-vaborbactam, colistin (COL), MER, ceftazidime, and CAZ had activity against ABC that did not meet the MIC screening criteria, but only CFDC  and COL were active against the resistant ABC subsets.31.3%  and 14.7%  of PSA met the MIC screening criteria for molecular characterization and showed an MDR phenotype, respectively (Table). Carbapenemase genes were detected in 10 PSA (< 1%). CFDC had similar MICs against PSA (MICMany PSA had a MDR phenotype but acquired carbapenemase genes remained rare in this subset. Resistance and presence of carbapenemase genes were high in ABC. CFDC showed potent activity against PSA and ABC subsets in US hospitals, including across resistant and molecularly characterized subsets, where treatment options are limited.Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|Cipla: Grant/Research Support|Entasis: Grant/Research Support|GSK: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Cory Hubler, BS, AbbVie: Grant/Research Support|Shionogi: Grant/Research Support Valerie Kantro, BA, AbbVie: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Dee Shortridge, PhD, Melinta: Grant/Research Support|Shionogi: Grant/Research Support Helio S. Sader, MD, PhD, FIDSA, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|Cipla: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|bioMerieux: Grant/Research Support|Cipla: Grant/Research Support|CorMedix: Grant/Research Support|Entasis: Grant/Research Support|Melinta: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support"}
+{"text": 
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