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+{"text": "The plastic response of fine roots to a changing environment is suggested to affect the growth and form of a plant. Here we show that the plasticity of fine root growth may increase plant productivity based on an experiment using young seedlings (14-week old) of loblolly pine. We use two contrasting pine ecotypes, \"mesic\" and \"xeric\", to investigate the adaptive significance of such a plastic response.The partitioning of biomass to fine roots is observed to reduce with increased nutrient availability. For the \"mesic\" ecotype, increased stem biomass as a consequence of more nutrients may be primarily due to reduced fine-root biomass partitioning. For the \"xeric\" ecotype, the favorable influence of the plasticity of fine root growth on stem growth results from increased allocation of biomass to foliage and decreased allocation to fine roots. An evolutionary genetic analysis indicates that the plasticity of fine root growth is inducible, whereas the plasticity of foliage is constitutive.Results promise to enhance a fundamental understanding of evolutionary changes of tree architecture under domestication and to design sound silvicultural and breeding measures for improving plant productivity. In anPhenotypic plasticity is the potential of an organism to alter its phenotype in changing environments -19. PhenLoblolly pine is the most important tree species for fiber production in the southern US . BecauseP < 0.001). These values increased to 102% and 199% for these two ecotypes, respectively, when the trees were treated for 14 weeks (P < 0.001).After 4 weeks of treatment, trees receiving the high nutrient treatment displayed 22% (\"xeric\") and 47% (\"mesic\") greater stem biomass than those under the low fertilizer treatment (P < 0.01), as compared to the significance level when the proportion was not held constant (P < 0.0001). These dependent relationships suggest that increased stem biomass due to better nutrient supplies was attributable to both increased foliage investment and decreased energetic costs of fine root construction.Allometric analysis was used to evaluate the influences of foliage and fine-root biomass partitioning on stem growth which arise from differences in nutrient supply. On both harvesting dates, the proportion of foliage biomass to total plant biomass increased markedly, whereas the proportion of fine root biomass decreased significantly, with better nutrient supplies. As an illustration, we use Figure py\u21901 = -0.09), whereas the plasticity of fine-root biomass partitioning, i.e., decreased partitioning of biomass to fine roots under higher fertilization, had a significant impact on the corresponding increase of stem biomass . Plasticity between different growth stages indicates the dependence of plastic responses on the timing and sequences of developmental events. Ecotypic variation in developmental plasticity represents different genetic bases involved in relevant developmental events [Foliage and fine roots have complementary roles in uptake of resources; the former in energy and carbon uptake and the latter in water and nutrient uptake ,22. Mechl events .Ecotypic differentiation of loblolly pine could be explained by limits of plasticity. Quantitative evolutionary genetic models predict that the phenotypic plasticity of a trait is costly or physiologically limiting when the trait is forced to respond to environmental variation (\"passive\" response). DeWitt et al.  delineatOur analysis suggests that the plasticity of fine-root biomass proportion is physiologically limiting, whereas the plasticity of foliage biomass proportion is not. The relationship of the shoot biomass residuals was positive with the degree of plasticity of foliage biomass proportion Fig. , but negForest tree form (biomass partitioning) is highly plastic in response to changes in nutrient levels. The carbon budgets for forest trees show a surprisingly large role of roots. Under low nutrient conditions that predominate in natural forests, 60\u201380% of photosynthate is allocated below ground, compared with 30% for high nutrient levels [Progress towards domestication in trees will be slowed by long generation times, but is likely to be based on the exploitation of interactions between genotypes and yield associated with various types of agronomic methods , as have been shown for herbaceous crop plants. However, the environmental uncertainties during the long life span of trees have caused some breeders to consider the value of plasticity as a trait itself. And plasticity could obscure the relationship between phenotype and genotype, making selection less efficient. Efforts to domesticate forest trees will be enhanced by a deeper knowledge of phenotypic plasticity .Our study of biomass partitioning in relation to varying nutritional levels in loblolly pine supports the previous hypothesis, proposed by Linder and Axelson , that thPinus taeda L.). We used two contrasting loblolly pine ecotypes from regions that differ in soil resource availability. One of the ecotypes, known as the \"Lost Pines\" of Texas, is adapted to droughty conditions and low soil fertility and is denoted by \"xeric\", whereas the other, Atlantic Coastal Plain, is adapted to more moderate conditions and is denoted by \"mesic\". Adaptive differentiation between the contrasting \"xeric\" and \"mesic\" ecotypes has been previously characterized [Phenotypic plasticity was evaluated for fine roots and biomass partitioning of a commercially important forest tree species, loblolly pine  and fine root biomass (py2\u2190) to stem biomass through the change of nutritional levels were estimated by solving the following regular equations:Path analysis was used to identify the cause-effect relationships in a complex system . Path anpy 1\u2190+ r12py 2\u2190= ry1r12py 1\u2190+ py 2\u2190= ry2ry 1and ry 2are the family correlation coefficients of the plasticity of foliage biomass and fine-root biomass with the plasticity of stem biomass, respectively, and r12 is the family correlation coefficient between the plasticity of foliage biomass and fine-root biomass. Residuals were estimated to evaluate the degree of determination for the path analysis [where analysis . All datRW designed the study, carried out the experiment, analyzed the data and drafted the manuscript. JEG participated in the experiment. SEM participated in the design of the study. DMO participated in the design and coordination. All authors read and approved the final manuscript."}
+{"text": "Primary care physicians underestimate the prevalence of domestic violence and community violence. Victims are therefore at risk of further episodes of violence, with psychological and physical consequences. We used an interview to assess the prevalence of domestic and community violence among Swiss natives and foreigners. In a follow-up study, we evaluated the consequences of the interview for the positive patients.We evaluated the prevalence of violence by use of a questionnaire in an interview, in an academic general internal medicine clinic in Switzerland. In a follow-up, we evaluated the consequences of the interview for positive patients. The participants were 38 residents and 446 consecutive patients. Questionnaires were presented in the principal language spoken by our patients. They addressed sociodemographics, present and past violence, the security or lack of security felt by victims of violence, and the patients' own violence. Between 3 and 6 months after the first interview, we did a follow-up of all patients who had reported domestic violence in the last year.Of the 366 patients included in the study, 36 (9.8%) reported being victims of physical violence during the last year (physicians identified only 4 patients out of the 36), and 34/366 (9.3%) reported being victims of psychological violence. Domestic violence was responsible for 67.3% of the cases, and community violence for 21.8%. In 10.9% of the cases, both forms of violence were found.Of 29 patients who reported being victims of domestic violence, 22 were found in the follow-up. The frequency of violence had diminished (4/22) or the violence had ceased (17/22).The prevalence of violence is high; domestic violence is more frequent than community violence. There was no statistically significant difference between the Swiss and foreign patients' responses related to the rates of violence. Patients in a currently violent relationship stated that participating in the study helped them and that the violence decreased or ceased a few months later. Violence is defined by the World Health Organization (WHO) as \"the intentional use of physical force or power, threatened or actual, against oneself, another person, or against a group or community, that either results in or has a high likelihood of resulting in injury, death, psychological harm, maldevelopment, or deprivation.\" Violence can be divided into three broad categories, which are self-directed violence, interpersonal violence, and collective violence. Our focus is on interpersonal violence, which is divided into two subcategories: family and intimate partner violence, and community violence; that is, violence between unrelated individuals who may or may not know each other. The nature of violent acts can be physical, sexual, or psychological, or involve deprivation or neglect . UNICEF According to the WHO, an estimated 1.6 million people from around the world died as a result of violence , and 520,000 were killed as a result of interpersonal violence, in 2000. This is the \"tip of the iceberg,\" as it is impossible to estimate non-fatal violence precisely . The LawAs for domestic violence, which we know is frequent, especially towards women, 48 population-based surveys from around the world found that 10 to 69% of women reported being physically assaulted by an intimate male partner at some point in their lives . In SwitAn estimate of the direct costs of violence concluded that 400 million Swiss francs are annually spent on violence against women by public authorities, through the health care system, police and judicial interventions, social services, shelters or specialized centers for battered women, and for research on the subject, most of it for intimate partner violence . HoweverEven though battered women seek more hospital care than other women ,11, physThe aim of our study was to assess the prevalence of domestic and community violence among the patients (Swiss natives and foreigners) of an academic general internal medicine clinic. A follow-up was carried out to assess the consequences of the interview for patients who had reported domestic violence.Our study limits itself to psychological and physical abuse experienced during the last 12 months and during the patient's lifetime.We conducted a study that allowed us to screen for violence among our patients, with a follow-up for patients who were screened as being victims or perpetrators of domestic violence. It took place in an academic general internal medicine clinic in Lausanne, Switzerland. The site is a primary care clinic where residents provide adult ambulatory care for approximately 22,000 outpatient visits per year, at walk-in and follow-up visits. We focused on walk-in patients, as they are not known beforehand by the residents. We included all physicians working at the clinic at the time of the study.We based our sample-size calculation on a recent study using a similar design . We baseThe physicians were 22 women and 16 men. The majority of the residents were at the end of postgraduate training in general internal medicine or family medicine .We tried to minimize a Hawthorne effect; that is, a change in attitude in response to attention from the investigators (15). To blind them to the purpose of the study, we only informed them that a study focusing on the principal health problems brought up during consultations would take place among the patients. The physicians received a questionnaire through which they were asked about the main problems they identified during the consultation.Consecutive patients (men and women) consulting for an emergency visit at the clinic were approached during business hours (8 h-18 h) on weekdays. Eligible patients were over 18 years of age and spoke French, or were able to read French, English, Spanish, or Albanian, the languages into which the questionnaire was translated, and agreed to be questioned alone. Patients who were severely ill and needed to lie down were not included in the study, as they could not be interviewed in a private place. A research psychologist informed the recruited patients that the study was aimed at improving the medical follow-up of patients. For safety reasons recommended by the World Health Organization , we toldThe psychologist interviewed patients before the medical visit. Patients were asked for demographic information, which included their age, sex, nationality, spoken language(s), level of education, profession, marital status, household composition, and family composition. The questionnaire itself comprised a modified version of the partner violence screen (PVS), a tool tested and validated in 1994 in two urban emergency departments in Denver, Colorado . This quBetween 3 and 6 months after the intervention, we phoned the patients who had been screened as victims or perpetrators of violence to learn whether their situation had changed. The questionnaire assessed the frequency and form of violence suffered or perpetrated. We also asked them if they had talked about it with their partner or with someone else.t-test or Wilcoxon rank-sum test for continuous data. We considered p values < 0.05 to be significant.We used the chi-square or Fisher's exact test for categorical data and a To look for confounding and interaction in a multivariable analysis , we introduced age, sex, nationality, spoken language, level of education, profession, composition of the patient's household circle, and composition of the patient's family circle into the model.The physicians were similar in their sociodemographic characteristics and professional achievements: they were 22 women and 16 men; their mean age was 32.9 years old. The majority of them were at the end of the postgraduate training in general internal medicine (median duration of training: 6 years). Among all residents, one of them came from Africa, another from Eastern- Europe, and all the others were Caucasian.We approached 446 patients in total. The demographic characteristics of the patients were similar and are summarized in Table We found that 9.8% (36/366) of the patients had suffered physical violence (only 4 patients of these 36 were identified by physicians) and that 9.3% (34/366) had suffered psychological violence in the last 12 months, with 15 patients declaring both forms. Domestic violence was responsible for 67.3% (37 patients), and community violence for 21.8% (12 patients). In 10.9% (6 cases), both domestic violence and community violence were reported. Patients said they had suffered violence at another time in their lives in 49% (126/257) of the cases; the forms were domestic violence in 50.8% of the patients, community violence in 38.1%, and both forms in 11.1%.The patients who were victims of physical or psychological violence were asked more questions. Some of them  said they did not feel safe with their partner or in their family circle, and 28.4% (27/95) said they currently felt unsafe, because of a past or present partner/member of their family circle. The request for a consultation was due to these circumstances in 10.3% (9/87) of the cases. Three questions about the perpetrator's position were asked. To the first question, 30.3% (111/366) of the patients answered in the affirmative. To the second question, 55.7% (204/366) of the patients said \"yes\" for shouting and 36.9% (135/366) for hitting. To the last one, 17.2% (63/366) answered \"yes.\"There was no significant difference between the responses of Swiss and foreign patients, except for one regarding the patients' own violence. More foreign patients than Swiss patients said they were afraid of not being able to control themselves . We also found a significant difference between men and women. Men suffer more community violence and women more domestic violence, as present and past violence experienced . These results are summarized in Table The patients who were screened as being victims or perpetrators of domestic violence were re-contacted by phone. They were 18 victims and 16 perpetrators of violence; 5 were both. The victims were 5 men and 13 women; their mean age was 29.44 ; 10 were from Europe (8 being Swiss), 5 were from Latin America , and 4 were from Africa. The perpetrators were 8 men and 8 women; their mean age was 33.12 ; there were 7 from Europe (3 being Swiss), 1 from North America, 5 from Latin America, and 4 from Africa. We were able to make contact with 22 of the 29 patients involved; 3 victims and 4 perpetrators could not be found. More than half the victims  and perpetrators  said they had been able to talk about their problem of violence with the person responsible for the violence or with the victim; 73.3% (11/15) of the victims and 41.7% (5/12) of the perpetrators spoke with someone else. The violence totally ceased for 73.3% (11/15) of the victims and 75% (9/12) of the perpetrators. It diminished in frequency for 20% (3/15) of the victims and 16.7% (2/12) of the perpetrators, and in the case of one victim/perpetrator, it occurred once between her participation in the study and the follow-up interview. The form of violence changed for 46.7% (7/15) of the victims and 45.5% (5/11) of the perpetrators, mostly becoming verbal . One victim lost the possibility of seeing his children anymore. Interestingly enough, some patients  told us, without us asking, that the study helped them by allowing them to talk about the violence or in becoming conscious that the situation should be changed, etc. Finally, it seems important to state that for 7 of the patients (31.8%), the relationship they had at the time of the study had ended by the time we did this follow-up. The results are summarized in Table The originality of this study lies in our decision to include consecutive patients consulting for an emergency visit, so that we had women and men, including Swiss and foreign patients, who took part in the study. We found rates of violence similar to those reported in other studies -8. Our rWe did not find a statistically significant difference between the Swiss and foreign patients' responses related to rates of violence. Not many studies have compared rates of violence among women and men, or among natives and foreigners. A United States national survey on intimate-partner violence  revealedThe detection of the domestic violence/community violence by physicians is low in our study (8%) as documented in the literature. One study found screening rates of only 13% for victims of acute intimate partner violence presenting to an emergency department . LimitedAt follow-up, we noted that domestic violence had diminished or ceased for the majority of the patients we contacted. Victims and perpetrators did not differ in their responses, except that perpetrators tended to have more difficulty in talking about their violent behavior to people than the victims had in talking about what happened to them. It seems that the interview by the psychologist made it possible for the patients to initiate changes in their family life, resulting in decreased violence.Patients were free to not participate, so we probably missed some who had a problem of violence. For example, one woman said she did not want to participate in the study because the subject concerned her and she was afraid that it would be too disturbing to talk about it. Also, as she was seeing a psychologist for this problem, she did not want to talk about it again.We did not have a group control. This is a methodological limitation, but also an ethical necessity when working with people affected by violence. Victims of violence cannot be left without anyone to intervene.The rate of detection of domestic violence by physicians is insufficient, as is generally documented in the literature. The low physician suspicion/detection rate can be related to the lack of awareness that their patients are exposed to violence and their impression that it is not a relevant issue for that particular unit. Another reason could be the barriers to disclosure by the victim's feelings of shame, loyalty to the partner or intimidation by the perpetrator and fear of being not believed .The originality of this study is that this is one of the few papers to assess prevalence of violence experiences in a primary care population clinic in Switzerland, including both Swiss nationals and foreign patients, and the assessment of the provider's understanding and recognition of his or her patient's exposure to violence.The prevalence of violence is high, and domestic violence is more frequent than community violence. There was no statistically significant difference between the Swiss and foreign patients' responses related to rates of violence.Finally, we noted that domestic violence had diminished or ceased at follow-up for the majority of the patients we contacted. Study participation was considered to be helpful by victims because they felt that the provider recognized their real problem and that they were not alone in this situation; the victim could also feel that the violence perpetrated against him or her was not deserved .The author(s) declare that they have no competing interests.Claire Morier-Genoud: conceived and designed the study, prepared the training and gave the lecture, collected the data by interviewing the patients, participated in the statistical analysis and the interpretation of the data, and wrote the manuscript.Patrick Bodenmann: participated in the design and coordination of the study in the collection and interpretation of the data, and helped to draft the manuscript.Bernard Favrat participated in the design of the study, did the statistical analysis, and participated in the interpretation of the data.Marco Vannotti participated in the design of the study, gave the lecture, and participated in the interpretation of data.All the authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Switzerland is confronted with the problem of interpersonal violence. Violence is in the increase and the potential for aggression seems to be rising. Observations by hospitals discern an appalling increase of the severity of the injuries. The aim of this study is to collect accurate information about the social environment, the motivation and possible reasons for violence. We also intend to investigate whether sociocultural, or ethnic differences among male victims exist.For the first time in Switzerland, this survey employed a validated questionnaire from the division of violence prevention, Atlanta, Georgia. The first part of the questionnaire addressed social and demographic factors which could influence the risk of violence: age, gender, duration of stay in Switzerland, nationality and educational level. Beside these social structural factors, the questionnaire included questions on experience of violent offences in the past, information about the most recent violent offence and intra and interpersonal facts. The questionnaire itself consists of 27 questions, translated into German and French. In a pilot study, the questionnaire was checked with adolescents for feasibility and comprehensibility.69 male VIVs were interviewed at two hospitals in the Canton of Bern. Most of the adolescents emphasised that weapons were not used during their confrontations. It is astonishing that all of the young men considered themselves to be victims. Most of the brawls were incited after an exchange of verbal abuse and provocations with unfamiliar individuals. The rivals could neither be classified with the help of ethnic categories nor identifiable groups of the youth scenes. The incidents took place in scenes, where violence was more likely to happen. Interestingly and contrary to a general perception the offenders are well integrated into sport and leisure clubs. A further surprising result of our research is that the attitude towards religion differs between young men with experience of violence and non-violent men.Youth violence is a health issue, which concerns us globally. The human and economic toll of violence on victims and offenders, their families, and on society in general is high. The economic costs associated with violence-related illness and disability is estimated to be millions of Swiss francs each year. Physicians and psychologists are compelled to identify the factors, which cause young people to be violent, to find out which interventions prove to be successful, and to design effective prevention programs. The identification of effective programs depends on the availability of reliable and valid measures to assess changes in violence-related attitudes. In our efforts to create healthier communities, we need to investigate; document and do research on the causes and circumstances of youth violence. Like other European countries, Switzerland is also struggling with the problem of interpersonal violence . Not onlInterpersonal violence can be defined as a conscious physical attack, with the intention to cause physical and psychological harm ,11,12. AMost of the measures in this compendium intend to assess such factors as serious violent and delinquent behaviour, conflict resolution strategies, social and emotional competencies, peer influences, parental monitoring and supervision, family relationships, exposure to violence, collective efficacy, and neighbourhood characteristics. The compendium also contains a number of scales and assessments to measure factors such as aggressive fantasies, beliefs supportive of aggression, attributional biases, and unsocial and aggressive behaviour .Based on our personal experience we hypothesised that VIVs in Switzerland, a country with a very high standard of living and education, may not belong to socially deprived parts of the community, like in other European countries, but can be found within higher educational and social levels too.Therefore the aim of the study was to collect detailed information about the social environment, the motivation and possible reasons for violent behaviour. We also intended to investigate whether there are sociocultural or ethnic differences among VIVs. To the best of our knowledge, this type of study, with interviews in an emergency shortly after the violent incident, is unique.The University Emergency Department of the Inselspital Berne is the only major emergency department in the city of Berne providing its services 24 hours a day and 7 days a week. Berne is the capital of Switzerland, with over 128,000 inhabitants. The official spoken and written language in Berne is German. 78% of the population of Berne are Swiss nationals, and about 22% hold a foreign passport [The regional hospital of the city of Biel is a Level II emergency department and was chosen as the second study site. Biel/Bienne is officially considered bilingual (french-german), with about 70,000 residents. It is the largest bilingual city in Switzerland. In December 2006, 27% of Biel residents were foreigners .Each of these units sees about 30,000 patients a year,1000 of whom are VIVs.The first part of the questionnaire addressed social and demographic factors which could influence the risk of violence: age, gender, duration of stay in Switzerland, nationality and educational level. Beside these social structural factors, the questionnaire included questions on experience of violent offences in the past, information about the most recent violent offence and intra and interpersonal facts.Original Questions selected from the questionnaire.For the following questions, indicate how many times you did something during the last 7 days.1. I got angry very easily with someone.2. I got into a physical fight because I was angry.3. I slapped or kicked someone.4. I made someone look stupid with words.5. I have threatened to hurt or to hit someone.6. In the past six months, I have attacked or threatened someone with a weapon.7. In the past six months, I was involved in a gang fight.8. How often were you involved in a fight in the last twelve months?9. During the past 12 months, how many times were you in a physical fight in which you were injured and had to be treated by a doctor or nurse? (excluding the current one)?10. Where did most of your fights take place within the last 12 months?11. How did the last fight happen?12. When was the last time that you hurt someone in a fight?13. What kinds of feelings were caused by this?14. When was the last time that you have seen a fight without participating in it (excluding the current one)?15. Who did you fight with most often?16. Do you have a special type of fighting?17. Do you attend to be active (participating) or passive (witness) in fights?18. On which level are most of your confrontations with others?19. Where I come from is important.20. I have a clear sense of my religion and what it means for me21. If I go out, I like to meet people of other nationalities.22. People of other nationalities make me aggressive and angry.23. Did you experience violence in childhood?24. Do you and your environment consume alcohol or drugs before you are involved in fights?25. Do you identify yourself with an association, a group or a party of which you are a member?26. What do you do in your free time?27. For completing this questionnaire, you will receive a CD-coupon amounting to CHF 20. What kind of music are you going to buy with this coupon?We decided with the help of a psychologist specialized in youth violence (AG) to identify question directly related to hypothesis.The questionnaire consisted of 27 questions, translated into German and French. In a pilot study, the questionnaire was checked with adolescents for feasibility and comprehensibility.Included in this prospective survey were VIVs (>16 years) treated at the emergency departments of the two participating hospitals. During the recruitment of the participants, 189 VIVs were approached. The final decision on inclusion was at the discretion of the consultant on call. In total, 100 VIVs agreed to complete the questionnaire, including 69 men and 19 women . The interview costs more time than expected . Therefore the number of patients recruited for this pilot study, has been limited to 100. As a bonus, the interviewed patients received either a CD or a cinema coupon for 20 Swiss Francs (USD 20). This had been found to be useful in previous studies [The physicians interviewing the VIVs were trained in advance according to the requirements of the text. After medical treatment, the VIV was asked if he/she was interested in participating. The required coupon was handed over if the patient agreed and filled out the questionnaire. VIVs who could not speak German or French and who were physically and psychologically unable to complete the questionnaire were excluded.48 (70%) of 69 male patients were Swiss citizens. 30% had completed a public school; 35% had finished an apprenticeship; 6% had obtained a university entrance certificate; 4% held a college degree and 1% attended a university.Question 1-5: For the following questions, indicate how many times you did something during the last 7 days.I got angry very easily with someone.I got into a physical fight because I was angry.I slapped or kicked someone.I made someone look stupid with words.I have threatened to hurt or to hit someone.Answers 1-5: Verbal confrontations and threats occur more often than fights ?Answer 9: 20% of VIVs stated that they had been involved in an additional brawl in the course of the previous year ?Answer 14: 20% stated that they had witnessed a fight during the last half year and 33% during the last month.Question 15: Who did you fight with most often?Answer 15: In most cases, the opponents were strangers (50%); only about 10% were colleagues or adherents of a different political opinion. Family members were not mentioned as adversaries. 42% stated that their opponents were people of other nationalities  in fights?Answer 17: 13% asserted that they are active in fights; 50% believed they tended to be passive and for 37% stated this depended on the situation.Question 18: On which level are most of your confrontations with others?Answer 18: Most of the confrontations remained at the verbal level (88%),Question 19+20: Where I come from is important. I have a clear sense of my religion and what it means for me.Answer 19+20: The personal origin was important for 39% of the VIVs. 18% stated that their religion was significant for them  considered themselves a member of a distinct group of friends or a sports club (22%).Question 26: What do you do in your free time?Answer 26: The VIVs spent their spare time in many different ways. They had casual meeting with friends (70%), played videogames (25%), played sports (63%) or listened to music (65%).Question 27: For completing this questionnaire, you will receive a CD-coupon amounting to CHF 20. What kind of music are you going to buy with this coupon?Answer 27: Most VIVs were fans of black hip hop or rap music.Violence among adolescents has become a public concern. Are there any distinct behaviour patterns and personality traits recognisable in VIVs? Do they choose to spend their time in distinct surroundings? Do they pursue specific interests?It is interesting that most of the adolescents emphasised that weapons were not used and were of no importance during their confrontations. Instead of guns and knives, they relied on fists. Fortunately for Switzerland, there is no gradual armament of the scene. In contrast to other countries, weapons are still considered a taboo . This isIt is astonishing that all young men considered themselves as victims! From their point of view, they were only defending themselves. They seemed convinced, that they had to act, after having been verbally abused, provoked or attacked. They seemed unable to see their part in the incidence. These findings reveal a widely known unconscious defence mechanism. After having been involved in a violent incidence, even the most aggressive perpetuators tend to clear themselves of any personal responsibility. Psychologically they have to see themselves as innocent victims, because any other self-description might arise tension. The vile behaviour creates a conflict with the self-image. Self-images don't contain actual features and character traits, but serve a distinct psychological purpose. Self-images need to be whitewashed from any disturbing features or deeds. Self-descriptions need to be in compliance with the values and attitudes of the social environment we belong to, actions, which contradict these values, must be suppressed. When we reflect our actions, we naturally tune in our habitat. This phenomenon raises several questions about violence prevention. It means, that it is not sufficient to work on the conscious level, when we want to help young people at risk. They might honestly declare, that they abhor violence and believe in peaceful conflict resolutions, while their actual behaviour and reactions tell a different story. In conflict situations unconscious motives prevail. Violent features and thoughts are spontaneously suppressed, because on wants to tune in to a society, which focuses on victims and has great difficulty in accepting violence as a behaviours pattern in ourselves. This means, that prevention work might be of no avail, when we work on a conscious, cognitive level. Promoting peaceful gaols, handing out leaflets against violence or organizing workshops in order to encourage peaceful communication, might not have a big effect. The very first step in violence prevention might be the acceptance of our problematic features. These questions need to be investigated in future studies . How canunfamiliar and who also could not be classified into an ethnic group or known sociological sub-group. The incidents happened in scenes in which violence is more likely to happen. This indicates, that the social context plays an important role. Violent behaviour is influence by the social surroundings of the young men, might even have certain function with respective social context. These findings are consistent with the existing literature [Most of the fights occurred after verbal provocations from people who were terature .In contrast to a generally held opinion, young men who are engaged in violence participate in sport club. This could be a sign, that they are integrated into society and enjoy acceptable leisure activities. Engaging in sportive activities does not seem to reduce the likelihood of violence. The engagement in sport clubs does not deter young men from being violent. Sportive activities don't seem to be an outlet for aggression either. Our finding also indicate, that violent criminals may not belong to a marginalised stratum and .A further interesting difference between young men with experience of violence and non-violent men is their attitude towards religion. Men without experience of violence value religion more than violent men. Is religion therefore a deterrent? This could mean, that a religious attitude does not foster violence but might even impede it . This fiThe data confirms that there is a small group of persistently violent men. According to our data, they have experienced violence during their childhood and adolescence. However, we could not discern whether they were victims or perpetrators .Our conclusions are preliminary, as they have been recorded for a small group of patients. In order to be validated, they have to be confirmed in larger groups of patients. Furthermore: this analysis might be biased because the inclusion of the patients was not consecutive and intoxicated or violent patients were not asked to participate. Some VIVs might have been reluctant to answer sincerely, fearing reprisals or the instigation of interrogations by the police, although they were assured this would not be the case. It is difficult to determine whether the answers are accurate or distorted.Youth violence is a serious global public health problem. Despite low violence rates across Switzerland in comparison to other European countries, interpersonal violence is rising and claims the health of many young people. The human and economic toll of violence on victims and offenders, their families, and society in general is high. The ecoThe authors declare that they have no competing interests.AKE has been the principal investigator and drafted the manuscript, AE and FE collected data and interviewed patients, UK reviewed and analysed data, KD coordinated study at both hospital sites, AR supervised study at Biel Hospital, VJ helped revising the manuscript, HZ co-designed study, UL co-designed study, AG designed study and drafted the questions, co-drafted the manuscript. All authors read and approved the final manuscript."}
+{"text": "Both violence and depression during pregnancy have been linked to adverse neonatal outcomes, particularly low birth weight. The aim of this study was to investigate the independent and interactive effects of these maternal exposures upon neonatal outcomes among pregnant adolescents in a disadvantaged population from Sao Paulo, Brazil.930 consecutive pregnant teenagers, admitted for delivery were recruited. Violence was assessed using the Californian Perinatal Assessment. Mental illness was measured using the Composite International Diagnostic Interview (CIDI). Apgar scores of newborns were estimated and their weight measured.21.9% of mothers reported lifetime violence (2% during pregnancy) and 24.3% had a common mental disorder in the past 12 months. The exposures were correlated and each was associated with low education. Lifetime violence was strongly associated with Common Mental Disorders. Violence during pregnancy (PR = 2.59(1.05\u20136.40) and threat of physical violence (PR = 1.86(1.03\u20133.35) and any common mental disorders (PR = 2.09 (1.21\u20133.63)  were independently associated with low birth weight.Efforts to improve neonatal outcomes in low income countries may be neglecting two important independent, but correlated risk factors: maternal experience of violence and common mental disorder. Both the experience of violence and antenatal depression may be risk factors for adverse neonatal outcomes, particularly low birth weight. A review of studies conducted in the USA and Europe showed aThe study was carried out in the Hospital Maternidade Mario de Moraes Altenfelder, the only public hospital providing obstetric care to people living in a poor neighbourhood in the north of S\u00e3o Paulo. Consecutive adolescents (11 to 19 years old) admitted to the hospital for obstetric care between 24/7/2001 and 27/11/2002 were invited to participate.Data were collected through interviews in hospital after the women had recovered from labor and the effects of anaesthesia. This period varied between 4 to 48 hours after delivery.Violence was assessed using the relevant section from the Californian Perinatal Assessment,12. The 1. Sometimes women (girls) are physically attacked by another person. Have you ever been attacked with a gun, knife or other weapon, either by a family member or a lover or friend, or by a stranger? 2. Have you ever been attacked by anyone without a weapon but with the intent to seriously injure you?3. Have you ever been threatened with the intent to seriously harm or injure you?4. Has anyone ever made you have any kind of sex by using physical force or by threatening to harm you?5. Did any of these incidents occur during your pregnancy?6. Did you ever ask for police help or for a restraining order due to domestic violence?The first two questions were combined to generate the variable 'any physical violence' and questions one to four were combined to generate the variable \"any lifetime violence\". Item 4 was used to define \"any sexual violence\". Item 5 was used to define the exposure of any type of violence experienced during pregnancy.th version (DSM-IV).Mental disorders were assessed using the Composite International Diagnostic Interview (CIDI 2.1 version). The interview has been validated for use in Brazil,14 and ath percentile for expected weight according to gestational age[Five outcomes were considered: still-birth; pre term birth, Small for gestagional age (SGA), low birth weight and low Apgar scores. Babies were weighed immediately after delivery by a pediatrician using a digital scale with a precision level of 10 grams. Low birth weight was defined as < 2500 grams. A low Apgar score was defined as below 7 at 5 minutes . Gestatiional age, using, ional age populatiWe also asked about the participants' age, education, socio-economic status and living arrangements. A Brazilian classification of social-economic class  was usedWritten informed consent was sought after explanations about the aims, potential risks and benefits of the research. Interviewers offered referral to the social and mental health team of the hospital or other agencies, as appropriate. The study was approved by the ethical committee of the hospital  and the ethical committee of Federal University of Sao Paulo.Prevalence ratios (PR) and 95% confidence intervals were calculated for associations of violence exposures with maternal and newborn health outcomes. Poisson regression with robust variance was usedOne thousand and two pregnant adolescents were admitted to the hospital during the study period, representing 24.4% of the 4108 women admitted for obstetric care. One thousand adolescents agreed to participate, of whom 70, admitted for miscarriage were excluded entirely from this study (n = 930). There were eight twin pregnancies, and ten still births; Apgar scores, pre term births and small-for-gestational-age births were studied only in singleton live births leaving 912 mother/child dyads for these analyses. Low birth weight was restricted to singleton live births with 37 weeks of gestational age or above leaving 795 mother/child dyads for these analyses.A third of the mothers were 16 or younger Table . One in 203 participants (21.8%) had experienced one or more types of violence at some time in their lives. The most common category was actual physical violence, experienced by 14% (n = 130) (evenly distributed between with and without a weapon), with threats of physical violence reported by 10% (n = 95) and 5% (n = 48) reporting sexual violence. Most of the violence went unreported; only 4% of those who suffered violence with a weapon and 17% of those reporting sexual violence had sought police help. Only 2% of mothers (n = 19) experienced violence during pregnancy; 26% of these had asked for police help.Two hundred and twenty six participants (24.3%) were diagnosed with a Common Mental Disorder in the previous 12 months. The most common diagnosis was depression (13.0%) followed by PTSD (9.8%), anxiety disorders (5.7%), somatoform disorder (1.8%) and dissociative disorder (0.3%). There was much comorbidity between depression, anxiety and PTSD: 32.0% of those with PTSD and 39.6% of those with anxiety also had depression.Low education and higher alcohol intake in pregnancy were associated with lifetime violence and CMD tables  and 2. HAfter adjusting for age and years of education, violence was strongly associated with CMD: lifetime physical violence, PR 2.17 (95%CI 1.72\u20132.73); lifetime threats of physical violence, PR 2.39 (95%CI 1.89\u20133.02); lifetime sexual violence, PR 2.60 (95%CI 2.00\u20133.41); and any violence during pregnancy, PR 2.29 (95%CI 1.50\u20133.50). We examined the association between violence and specific types of CMD. The risk for depressive disorder was higher for mothers who had experienced lifetime physical violence , but associations with threats of physical violence , sexual violence  and violence during pregnancy  were not statistically significant. Post-traumatic stress disorder (PTSD) was more strongly associated with all of the categories of violence; actual physical violence , threats of violence , sexual violence  and violence during pregnancy . Anxiety disorders were strongly associated with actual physical violence ; threats  and sexual violence .Ten babies (1.1%) were still born. Only one of these mothers had experienced lifetime violence (not during the pregnancy). There was no association between CMD and still birth, crude PR = 1.33 (95%CI 0.35\u20135.12). Apgar scores at five minutes were low (below 7) in 146 babies (16%). Neither lifetime violence  = 1.11, 95%CI 0.77\u20131.60), nor violence during pregnancy , nor CMD  was associated with low Apgar scores. 14.2% (131) were born with less than 37 weeks of gestational age. After adjustment for all potential confounders and mediators (*), pre term birth was found to be associated with common mental disorders (PR = 1.45 95% CI 1.00\u20132.09) but not with experience of violence. 239 babies (22.6%) were small for gestational age (SGA). After controlling for all potential confounders and mediators (*) only violence during pregnancy was associated with SGA .One hundred and forty one babies (15.5%) had low birth-weight (LBW). Among those over 37 weeks of gestational age (n = 795) there were 62 babies with LBW (7.8%). There was a statistically significant trend for the risk of LBW to decrease with the number of ante-natal consultations . LBW was also strongly associated with pregnancy complications .Table The effects of violence during pregnancy and CMD in the last 12 months on birth weight were additive rather than multiplicative; with no statistical interaction in the final model (p = 0.31) (interaction term PR = 0.35 (95%CI 0.11\u20132.00). One hundred and seventy seven adolescents had a CMD and did not suffer violence during pregnancy , seven had only been exposed to violence during pregnancy  and 10 had suffered both exposures .Brazilian adolescents (10\u201319 years old) comprise 21% of the population. We founThe main limitation of our study is the timing of the interviews with mothers. The measurement of mental health shortly after childbirth may be confounded by emotional experiences common after childbirth. Also, recall bias is a possibility when exposures are ascertained after the outcomes have occurred. Arguably, this is more likely for still birth than for the less striking outcomes of low Apgar scores and low birth weight. However, we used a structured mental health diagnostic interview, delivered by trained interviewers who ensured that interviews were only carried out after the mother had fully recovered from childbirth. This method has been used in other studies,23. AnteThe 22% prevalence of lifetime physical violence in our study is consistent with other estimates; 25% in India, and 13.et al's review[et al [Very few studies have examined the impact of violence on newborn outcomes in developing countries. Nasir 's review reported's review-4 , substance abuse (such as smoking), low socioeconomic status (leading to hunger), maternal medical problems and maternal mental illness ,31. The The effect of violence on young mothers who have limited personal and social resources can be devastating. Appropriate interventions are urgently required to avoid or minimize the effects of violence on the health of the mothers and the babies. The implication for clinical practice is that all adolescent mothers should be routinely screened for the experience of violence, both lifetime and during pregnancy, as well as for mental disorder. Identification of the one should alert clinical teams to the possibility of the other, given the strong correlations that we and others have reported. Exposed mothers should be treated as 'at risk', supported intensively during the pregnancy and monitored for fetal growth. Antenatal mental disorder seems particularly likely to be associated with adverse obstetric factors . At the social policy level, concerted efforts are need to combat gender-based violence not only as a human rights issue but as a major risk factor for poor maternal and newborn health; health professionals must actively engage in this advocacy.Further research, preferably utilizing longitudinal designs, is needed to tease out the causal mechanisms linking violence with maternal and newborn outcomes. Such studies should examine not only the role of physical violence but also the influence of behaviors that cause harm without the use of physical force including neglect, humiliation and non-violent coerced sexual acts. The role of protective factors, such as social support, also merits investigation.The author(s) declare that they have no competing interests.CPF, VP and MP participated in the study concept and design, analysis and interpretation of data, drafting the manuscript and reviewing the manuscript for important intellectual content. MB, EC, RG, RL and SM participated in the study concept and design, acquisition of data, obtaining funding and critically reviewed the paper for important intellectual content. All authors approved the version submitted.The pre-publication history for this paper can be accessed here:"}
+{"text": "Bacillus megatherium D01 biomass have once been investigated, still the mechanism active in the platinum biosorption remains to be seen and requires further elucidating.Platinum nanomaterial is one of the significant noble metal catalysts, and the interaction of platinum with microbe is one of the key factors in influencing the size and the distribution of the platinum nanoparticles on the microbial biomass. Some properties of Pt(IV) adsorption and reduction by resting cells of Bacillus megatherium D02 biomass on a molecular level has been obtained. The image of scanning electron microscopy (SEM) of the D02 biomass challenged with Pt(IV) displayed a clear distribution of bioreduced platinum particles with sizes of nanometer scale on the biomass. The state of Pt(IV) bioreduced to elemental Pt(0) examined via X-ray photoelectron spectroscopy (XPS) suggested that the biomass reduces the Pt(IV) to Pt(II) followed by a slower reduction to Pt(0). The analysis of glucose content in the hydrolysates of D02 biomass for different time intervals using ultraviolet-visible (UV-vis) spectrophotometry indicated that certain reducing sugars occur in the hydrolyzed biomass and that the hydrolysis of polysaccharides of the biomass is a rapid process. The infrared (IR) spectrometry on D02 biomass and that challenged with Pt(IV), and on glucose and that reacted with Pt(IV) demonstrated that the interaction of the biomass with Pt(IV) seems to be through oxygenous or nitrogenous chemical functional groups on the cell wall biopolymers; that the potential binding sites for Pt species include hydroxyl of saccharides, carboxylate anion and carboxyl of amino acid residues, peptide bond, etc.; and that the free monosaccharic group bearing hemiacetalic hydroxyl from the hydrolyzed biomass behaving as an electron donor, in situ reduces the Pt(IV) to Pt(0). And moreover, the binding of the Pt(IV) to the oxygen of the carbonyl group of peptide bond caused a change in the secondary structure of proteins; i.e. a transformation, in polypeptide chains, of \u03b2-folded to \u03b1-helical form; it might be expected to be more advantageous than \u03b2-folded form to the platinum nanoparticles under shelter from gathering although the both special conformations of proteins could be much probably responsible for the stabilization of the particles.A further insight into the biosorption mechanism of Pt(IV) onto resting cells of That knowledge could serve as a guide in the researches for improving the preparation of highly dispersive supported platinum catalyst and for fabricating new advanced platinum nanostructured devices by biotechnological methods. Bacillus megatherium D01 biomass were described previously [Bacillus megatherium D02 biomass using IR and other spectroscopic techniques.The roles the microorganisms play in biotechnological applications including effective recovery of noble metal ions -3, createviously , and yetBacillus megatherium D02 and easy to obtain and culture. It was cultured in an aqueous solution containing beef gels, peptone, salt, etc. [2PtCl6\u00b76H2O) aqueous solution at pH 3.5 and 37\u00b0C for 2 hours (h).The strain D02 was screened out from different bacterial strains that were isolated from soils and waters of mining areas, because it has a relatively strong ability to adsorb and reduce Au(III), Ag(I), Pt(IV), Pd(II) and Rh(III). The strain is Gram-positive and identified as lt, etc. . The ads62- anion, i.e. six-coordinate, octahedral complex. While the contact with D02 biomass suspension at pH 3.5, the liberation of chloride ion from the platinum complex occurred and the rate of the release was essentially as fast as that of the adsorption of the metal [62- anion due to electrostatic interactions. The process therefore is believed to involve the initial formation of an ion pair between negatively charged PtCl62- and positively charged oxygenous or nitrogenous functional groups on the biomass, followed by elimination of chloride [6-2 anion at early stage. As the interaction of the D02 biomass with the platinum occurred mainly between the functional groups of the biomass and their adsorbate, i.e. the Pt(IV) cation, it therefore was described to replace the PtCl6-2 in the text. Similarly Pt(II) for PtCl4-2.The chloroplatinic acid in an aqueous solution is generally in the form of PtClhe metal . The intchloride ; thus thThe appearance of Pt(IV) challenging D02 biomass was observed by SEM examination; the reducing ratios of Pt(IV) to Pt(II) and Pt(II) to Pt(0) by the biomass were determined using XPS and the hydrolysates of the biomass for different time intervals were analyzed for glucose content via UV-vis spectrophotometry. The interaction of chemical functional groups from the cell wall surfaces of the biomass with Pt(IV) was further studied by means of IR spectroscopic technique.The biosorbent was prepared after a reported method . The harSEM examination: Two samples of (I) blank D02 biomass powder and (II) that challenged with Pt(IV) at pH 3.5 and 37\u00b0C for 48 h followed by drying under vacuum at 80\u00b0C and then by grinding were sprayed with gold prior to examination. The specimens were then examined under a LEO-1530 scan electron microscope (Germany).K\u03b1 radiation with hv of 1 486.60 eV and using the binding energy of 284.7eV of the amorphous carbon C1s as a reference point.XPS examination: Two samples of the D02 biomass powder challenged with Pt(IV) at pH 3.5 and 37\u00b0C, respectively, for 12 and 24 h were dried under vacuum at 80\u00b0C to be pressed into pellets prior to examination. The specimens were then determined on a PHI Quantum 2000 X-ray photoelectron spectrometer (USA). The spectra were recorded by monochromatized Al Two 30 mg samples of the D02 biomass powder were suspended separately in 6 ml of diluted HCl with final biomass concentration of 5 mg/ml and the pH adjustment at 3.5. Two milliliters of the biomass suspension (10 mg biomass) was placed separately into 6 test tubes followed by centrifugation to remove the supernatants. A 2 ml of deionized water at pH 3.5 was added to each of the six samples of the biomass followed by shake at 130 r/min in an incubator at 37\u00b0C; two sections of three samples each for the respective reaction times of 10 min and 24 h were centrifuged at 3500 r./min for 8 min. The supernatant of each tube, i.e. the hydrolysate of the biomass, was analyzed for glucose content by a phenol-sulfuric acid method ,12 at 48-1 at a resolution of 4 cm-1 with 32 scans.Generally, biological samples contain much water that strongly interferes with IR absorptions; in order to get rid of the water disturbing, the samples must be dried as much as possible to thoroughly eliminate the liquid water prior to examination. Two samples of (I) the hydrolysate of D02 biomass on hydrolysis for 24 h, just from the above sample for analyzing the glucose content and (II) that challenged with Pt(IV) at pH 3.5 and 37\u00b0C for 2 h; and six samples of (III) D02 biomass powder (IV) that challenged with Pt(IV) at pH 3.5 and 37\u00b0C for 2 d (V) 4 d (VI) 6 d (VII) 8 d and (VIII) 10 d were analysed. Three other samples of (IX) chloroplatinic acid powder (X) anhydrous glucose powder and (XI) a close to saturated aqueous solution of chloroplatinic acid powder fully mixed with anhydrous glucose (1.4: 1 in gravimetric ratio) were heated respectively in an oil-bath at 100\u00b0C for 60 min followed by drying prior to examination. The samples for IR analysis were prepared by pressing powdered KBr pellets mixed intimately with about 5% \u2013 10% of finely ground powder of the each sample, and then determined on a Nicolet 740SX FTIR spectrophotometer with a MCT-B detector (USA). The spectra were recorded in the range 4 000 ~ 625 cmUnder the scan electron microscope, the blank D02 biomass hadn't any small metallic particles to be seen Figure , but thaThe UV-vis spectra of the hydrolysates of D02 biomass on hydrolysis for 10 min and 24 h respectively displayed an absorption band near 488 nm arising from glucose  and free hydroxyl (C-O-H) of the cell walls, an IR comparative study between the hydrolysate of D02 biomass and that challenged with Pt(IV) was performed. The use of the hydrolysate in this case would avoid any insoluble impurities that could interfere with the IR spectra studied. The spectrum of the hydrolysate displayed absorptions near 1 593 and 1 404 cm-1  [-1 and a decrease in the intensity of the symmetric at 1 404 cm-1  . The binric band , which crmis R08  and so f-1 corresponding to the \u03b4N-H + \u222aC-N, a coupled vibration including N-H in-plane bending and C-N stretching modes of the amideIIband originating from the C-N-H group of the peptide bond (HNC = O) [N-H + \u222aC-N from 1 551 to 1 537 cm-1  ,17  and 1 053 cm-1 (\u03b4O-H + \u222aC-O) can be found in Figure -1 (\u222aC = O) and 979 cm-1 (\u03b4O-H) in IR must have resulted from the oxidation of reducing sugars to their corresponding acids by platinum cations [As indicated earlier, two clear increases in the intensities of absorptions of the saccharide hydroxyl at 3 410 cm cations ,16.-1 range, it can be observed that the spectra of chloroplatinic acid and glucose showed only a single band of H2O at 1 622 and 1 645 cm-1 respectively, and the glucose reacted with Pt(IV) displayed a spectrum with the occurrence of the \u222aC = O of the carboxyl at 1 715 cm-1 besides one absorption of H2O at 1 642 cm-1. The result meant that the free aldehyde group shifted from the cyclic hemiacetalic hydroxyl of the glucose had already been oxidized to the carboxyl by the platinum cation. Further analysis of this sample using X-ray powder diffractometry gave a pattern with peaks corresponding exactly to those of the elemental Pt(0), which proved that the Pt(IV) had been reduced to the Pt(0) by the glucose under the reaction conditions. The redox reaction of the Pt(IV) with the glucose can be expressed as follows:In order to verify the above inference; glucose, the commonest reducing sugar, was examined by IR for the interaction with Pt(IV). To be in comparison with each other; IR spectra of chloroplatinic acid, glucose and that challenged with Pt(IV) were shown in Figures As a matter of fact, this is a model reaction of the Pt(IV) with the D02 biomass, the mechanism of the bioreduction of Pt(IV) to Pt(0) by the biomass can be assumed to be the same as that by the glucose. The biomass fulfilled the roles as a catalyst as well as an electron donor in this redox reaction. Both UV-vis and IR analyses testified that in the system some polysaccharides of the biomass had been hydrolyzed to reducing sugars; so when they met the Pt(IV) adsorbed on the cell wall surfaces, the reduction of Pt(IV) to Pt(II) followed by a slower reduction to Pt(0) occurred:-1  being reduced, hence two clear increases in the respective intensities of the carboxyl absorptions at 1 726 and 979 cm1 Figure , curve 2rmis R08 , etc. It-1 disappeared after the contact with Pt(IV) [C = O (1 726 cm-1) and \u03b4O-H (979 cm-1) from the carboxyl absorptions were found to change with prolonging the exposure time of the D02 biomass to Pt(IV). The ratios of the intensity of the \u222aC = O of the carboxyl at 1 726 cm-1 to that of the \u222aC = O (the amideIband) of the peptide bond at 1 652 cm-1  for different time intervals are shown: 0.637 (2 d), 0.706 (4 d), 0.739 (6 d), 0.581 (8 d) and 0.423 (10 d). As seen from the values, the carbonyl absorption of the carboxyl becomes the most intense as the biomass challenged Pt(IV) for 6 days, and it becomes lower for 8 days and lowest for 10 days. Fourest et al. [Sargassum biomass showed the IR spectra with an obvious decrease in the intensity of the free \u222aC = O of the carboxyl at 1738 cm-1 after the contact with Cd(II), the absorption became weak and finally disappeared with increasing the concentration of Cd(II). The result reflected that the carboxyl is also an active group for binding Cd(II) and there is not the occurrence of the redox reaction but only the binding action between Sargassum biomass and Cd(II). Based on electronegativity and steric effect points of view, the carboxyl could successfully compete with the peptide bond for binding Pt species [-1, which caused a decrease in the intensity of this band.It has been reported that the carbonyl absorption of the carboxyl at 1726 cmh Pt(IV) , this ret et al.  noted th-1  hydrogen bond linking neighboring peptide chains to destroy the original pleated structure of \u03b2-folded and led to the occurrence of \u03b1-helical conformation that has the regular arrays of intra-chain (i.e. intra-molecular) hydrogen bond. So a change in the secondary structures of proteins, namely, a transformation of \u03b2-folded to \u03b1-helical conformation, took placed. In general; \u03b1-helical form, the most content and the commonest secondary structure in proteins, bearing 169 atoms of both carbon and nitrogen in each of the helical rings closed by intra-molecular hydrogen bonding, has a fixed nanometer diameter and a pitch of 0.54 nm between the helices [6-2 anion, i.e. an octahedral coordinated complex with diameter of about 0.65 nm, was quickly attracted on the surface of the proteins of the biomass and the release of chloride ion from the platinum complex took place simultaneously with the adsorption of PtCl6-2 [It is of interest to note that the amideIband, due to the carbonyl stretching absorption of polypeptides, can be found to split into two peaks at 1 658 and 1 635 cm1 Figure , curve 11 Figure , curve 2 helices . And \u03b2-f PtCl6-2 , so thatBacillus megatherium D02 biomass on a molecular level has further been investigated mainly by infrared spectrometry in this report. The findings of the Pt(IV) bioreduced by the biomass to elemental Pt(0) at near normal temperature followed by the formation of platinum nanoparticles show that the biomass must have behaved as a catalyst as well as a role in sheltering the particles from gathering besides as an electron donor in this redox reaction. Further analysis suggests that the binding of the Pt(IV) to proteins led to a change, in polypeptide chains, of \u03b2-folded to \u03b1-helical form in that the latter might be expected to be more advantageous than the former to the nanoparticles under shelter from gathering, although the both special secondary structures of proteins could be much probably responsible for the stabilization of the particles. At all events, both patterns of the secondary constructions of proteins  and pores of the net-like structural polysaccharides on peptidoglycan layers of cell walls of the biomass may perform a significant function for stabilization and uniformity of the particles by having the pH under control in process of the biosorption. A better understanding of the biosorbent mechanisms responsible for Pt(IV) binding and reduction could contribute to the development of a method for fabrication of platinum nanoscale devices, and for an improvement of preparation of highly dispersive supported platinum catalysts by biotechnological methods.The biosorption mechanism of Pt(IV) onto RX carried out the SEM examination, participated in the interpretation of the SEM images. YY carried out the XRD examination, participated in analysis and interpretation of XRD patterns. JZ and ZX carried out biosorption examinations, participated in the analyses of both adsorptive efficiency and capacity of the biomass. ZL conceived of the study and carried out the IR examinations and performed further synthetic analyses and drafted the manuscript. All the authors read and approved the final manuscript."}
+{"text": "Violence against women is now widely recognised as an important public health problem, owing to its health consequences. Violence against women among many Indian communities on a regularly basis goes unreported. The objective of this study is to report the prevalence and other related issues of various forms of domestic violence against women from the eastern zone of India.It is a population-based study covering both married women (n = 1718) and men (n = 1715) from three of the four states of Eastern India selected through a systematic multistage sampling strategy. Interviews were conducted using separate pre-piloted structured questionnaires for women (victimization) and men (perpetration). Women were asked whether their husband or any other family members committed violent acts against them. And men were asked whether they had ever perpetrated violent acts against their wives. Three principle domestic violence outcome variables  were determined by response to a set of questions for each variable. In addition, data on socio-economic characteristics were collected. Descriptive statistics, bi- and multivariate analyses were done.The overall prevalence of physical, psychological, sexual and any form of violence among women of Eastern India were 16%, 52%, 25% and 56% respectively. These rates reported by men were 22%, 59%, 17% and 59.5% respectively. Men reported higher prevalence of all forms of violence apart from sexual violence. Husbands were mostly responsible for violence in majority of cases and some women reported the involvement of husbands' parents. It is found that various acts of violence were continuing among majority of women who reported violence. Some socio-economic characteristics of women have significant association with the occurrence of domestic violence. Urban residence, older age, lower education and lower family income are associated with occurrence of domestic violence. Multivariate logistic regressions revealed that the physical violence has significant association with state, residence , age and occupation of women, and monthly family income. Similar associations are found for psychological violence  and sexual violence .The prevalence of domestic violence in Eastern India is relatively high compared to majority of information available from India and confirms that domestic violence is a universal phenomenon. The primary healthcare institutions in India should institutionalise the routine screening and treatment for violence related injuries and trauma. Also, these results provide vital information to assess the situation to develop public health interventions, and to sensitise the concerned agencies to implement the laws related to violence against women. Violence against women is widely recognised as an important public health problem, owing to its substantial consequences for women's physical, mental and reproductive health -5. This India possessed several communities which are distinct in their geography, language and culture. In several places of India, violence faced by women on a regularly basis goes unreported even in newspapers, where as newspapers often carry reports about young women being burnt alive or dying due to unnatural causes in unnatural circumstances . EstimatWe hypothesize that domestic violence is wide-spread phenomenon and variation in its prevalence occur across the eastern Indian states, as these states differ from each other in overall development. Also, it is hypothesized that differences occur within the population of these states based on some socio-economic characteristics such as habitation , age, religion/caste affiliation, education, occupation and income. The purpose of the present study is to report the prevalence of various forms of domestic violence against women and to examine various related issues from the eastern zone of India. The term domestic violence is usually taken to mean partner abuse, specifically violence perpetrated by male partner. However, it may also be used to refers to violence perpetrated by any member of the household towards the women . HoweverThe eastern zone of India possessed four states namely, Orissa, West Bengal, Bihar and Jharkhand. Of these four states, three states  were selected to have a wider representation of the zone. The population of these states was 31.7 million, 80.2 million and 26.9 million in the year 2001 ,28. The All the interviews were held in local language of the state. Interviews took place in a private place in or outside the respondents' home, and care has been taken to avoid presence of other family/community members during interviews. If some one comes nearer during interview, the discussion on general health was made and the interview was restarted after the third person has retired. Interviewers stressed that honest responses were needed during interview to gain insight into the issue. Participants were assured of the confidentiality of their responses. To attain all these, care has been taken to establish rapport with every participant prior to interviews. Women and men were interviewed by women and men investigators, respectively. Individual verbal informed consent was obtained from all participants by explaining the purpose of the study. These field works were carried out during September 2004\u2013July 2005.The sample size was calculated based on the available estimated prevalence of domestic violence for these states . Based oAfter selecting the village/urban pocket, the research team met village/community heads and elders before initiating the data collection, and the purpose of the survey was explained. Rapport is established with the community and especially the women were taken to the confidence. The sample to be collected from each village was determined by dividing total rural sample required for that state by total number of villages (eight). In each village, eight random points were identified from all corners and care has been taken to include all communities. From each point, required number of sample was collected from households spread in four directions of the point. Similarly, in each urban pocket, participants were selected from the households spread in all the four directions. A married woman up to the age of 50 years of sampled household was sampled from each household. Corresponding to the women sample, married men aged below 50 years were selected in the similar way from the neighbouring village/urban pocket. Initially, 1753 women and 1730 men were contacted; however, 35 women and 15 men have refused to participate, yielding a refusal rate of 2% and 0.8% among women and men, respectively. Thus, samples of 1718 women and 1715 men were obtained.Three principle domestic violence outcome variables considered in our analysis are: physical violence, psychological violence and sexual violence. They were determined by response to a set of questions for each outcome variable. If a woman (as a victim)/man (as a perpetrator) gave a positive response to any of the questions in a set, it is considered as violence of that category. The questions used for women and men were listed in Annexure 1a and 1b, respectively in Additional file Data were collected on a number of community-level and individual-level variables that have been linked to domestic violence. The community-level variables included are the state of residence , residence , religion  and caste. During the survey, individual caste of the respondent was collected and they were categorized subsequently during analysis. The Government of India had categorised some ethnic groups (castes and tribes) into scheduled castes, scheduled tribes and backward castes, and these categories are entitled for positive discrimination in educational, employment and other developmental opportunities for their upliftment. The uncategorized castes, which form the majority of the population, are often referred to as forward castes. The individual-level variables were: age in years , education, which was categorized in to illiterate (those who can neither read nor write), functional literate , school education (1\u201310 years of schooling) and, college education and above (those having more than 10 years of education). The occupation of the participant was recorded and the responses were categorized into salaried jobs , farming and small business , labourer (daily-waged skilled and unskilled labourers), housewives (only women) and other occupations. The monthly income of the family was calculated during data analysis based on the information collected on income of all members as well as from common sources of the family. The income details were collected in Indian Rupees (INR). One INR was equivalent to 0.02 United States Dollars (US$). For logistic regression, these variables were used as categorical variables, except the age. The categories under each variable were explained above. The age was taken as continuous variable.p value of less than 0.05 was considered as the minimum level of significance.The data were computerized through Epi-Info 6. The database of Epi-Info was exported to SPSS and further analysis was carried out. The prevalence with 95% confidence intervals (CI) of different forms of domestic violence reported by women and men were computed for each of the states. For the domestic violence prevalence reported by women, the associations with socio-economic variables  were examined by using both bivariate and multivariate procedures. For each of the group under a variable, the prevalences in the form of percentages were presented and bivariate logistic regressions were carried out. In addition, multiple logistic regression analysis was used to model the presence or absence of physical, psychological and sexual violence, and any form of domestic violence by all of the aforementioned socio-economic variables. For these logistic regression analyses, the dependent variables were dichotomised (presence or absence of violence). The independent variables were categorised into different groups as described under measurements. While calculating odds ratios (OR), the category with the lowest weight was taken as the reference category. The OR is the value by which odds of the event (occurrence of violence) change when the independent variable increases by one unit/step. And it has been calculated by adjusting for all other independent variables in multivariate models. A The study protocol has been approved by the Human Ethical Committee of Regional Medical Research Centre. Individual informed consent was obtained from all participants, as mentioned above. Guidelines of World Health Organization, including the importance of ensuring confidentiality and privacy, both as means to protect the safety of study participants and field staff, and to improve the quality of the data were followed .The details of socio-economic characteristics of sampled women and men participants were presented in Table The prevalence of physical, psychological, sexual and any form of domestic violence in the life time of women were presented in Table Similarly, men were also interviewed to know whether they perpetrated any violence during their life time against their wives Table . The perIt was probed from the women about the person, who actually perpetrated different violent behaviour. Table It was probed to know whether or not the reported behaviours of violence are continuing currently among the women, who reported the experience of different acts of physical, psychological and sexual violence during their lifetime. If it is continuing, it was further probed for each act to know the periodicity of their occurrence. It is probed to know whether they are experiencing these acts daily. It is found that, almost all acts of violence were still continuing among majority of women Table . For exaTable The above associations were further examined through bivariate logistic regressions by taking presence or absence of violence as a dependent variable and women's socio-economic characteristic as a covariate (independent variables). OR along with levels of significance of regression models for all types of violence are shown in Table Further, multivariate logistic regressions were carried out to examine these associations, separately for each type of domestic violence Tables  and 10. In the present study, women reported as high as 56% of some form of violence against them in Eastern part of India. The levels of physical, psychological and sexual violence against women were also considerably high. These data along with the world-wide literature confirm that domestic violence is a universal phenomenon existing in all communities ,12,31. AThe present data demonstrated that in Eastern India, the domestic violence is persisting considerably across all socio-economic strata. Some characteristics of women namely, residence, age, education, occupation and family income have influence on the prevalence of domestic violence. The prevalence of violence decreased along with the increase of women's education and family income. However, no comprehensive studies are available from this part of India to compare these findings. One nation-wide study from India revealed that higher socio-economic status as a protective buffer against domestic violence . The datSome of the earlier studies from India revealed that though inadequate and failure of timely payment of dowry has been focused as an important reason for domestic violence in India, several other triggers of domestic violence such as negligence or failure in performing duties expected of women in the family also led to violence against women . These cThis study, along with the domestic violence rates based on the reporting of women, presented the prevalence of domestic violence reported by men, as perpetrator. These rates are in corroboration with those reported by women. Almost all research on domestic violence has relied on women's rather than men's report of their experiences . Few stuThere are limitations in this study, as usual to this type of research topic. The topic of interview is very sensitive and participants may not express their views openly, as they think that their responses may damage the reputation of themselves and their families. Sometimes in this type of research, participants may also report the behaviour that is believed to be consistent with their culture, rather than the actual . HoweverThe study confirms the high prevalence of all forms of violence against women across all socio-economic settings in eastern zone of India. However, urban residence, older age, lower education and lower family income are associated with occurrence of domestic violence. Women are at risk of violence from the husband than any other type of perpetrator. This situation has public health implications as public health can have a role in preventing the violence and its health consequences. Also, the primary healthcare institutions in India should institutionalise the routine screening and treatment for violence related injuries and trauma. These results also provide vital information to assess the situation to develop interventions as well as policies and programmes towards preventing violence against women. As India has already passed a bill against domestic violence, the present results on robustness of the problem will be useful to sensitise the concerned agencies to strictly implement the law.The authors declare that they have no competing interests.Both the authors contributed to the conception of the study design and development of study instruments. BVB involved in field works; coordinated in the data collection; computerized and analysed the data; interpreted the results; prepared the manuscript. Both the authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Annexure 1. A: Questions posed to women in this study to consider physical, psychological and sexual violence against women. B: Questions posed to men in this study to consider physical, psychological and sexual violence against their wivesClick here for file"}
+{"text": "India and Pakistan have disputed ownership of the Kashmir Valley region for many years, resulting in several conflicts since the end of partition in 1947. Very little is known about the prevalence of violence and insecurity in this population.We undertook a two-stage cluster household survey in two districts (30 villages) of the Indian part of Kashmir to assess experiences with violence and mental health status among the conflict-affected Kashmiri population. The article presents our findings for confrontations with violence. Data were collected for recent events (last 3 months) and those occurring since the start of the conflict. Informed consent was obtained for all interviews.510 interviews were completed. Respondents reported frequent direct confrontations with violence since the start of conflict, including exposure to crossfire (85.7%), round up raids (82.7%), the witnessing of torture (66.9%), rape (13.3%), and self-experience of forced labour (33.7%), arrests/kidnapping (16.9%), torture (12.9%), and sexual violence (11.6%). Males reported more confrontations with violence than females, and had an increased likelihood of having directly experienced physical/mental maltreatment , violation of their modesty  and injury . Males also had high odds of self-being arrested/kidnapped .The civilian population in Kashmir is exposed to high levels of violence, as demonstrated by the high frequency of deliberate events as detention, hostage, and torture. The reported violence may result in substantial health, including mental health problems. Males reported significantly more confrontations with almost all violent events; this can be explained by higher participation in outdoor activities. The British rule over Jammu and Kashmir terminated in 1947. During partition, the Kashmiri population \u2013 the majority of whom is Muslim \u2013 was promised a choice of joining either India or Pakistan through a popular vote but this plebiscite never took place. Instead, partition was the start of a long history of conflict affecting the roughly 8 million inhabitants of Kashmir . Respondents reported having an average household of nine persons . Nearly all respondents were originally from the Kashmir area . The majority of respondents were married  and half  had no formal schooling. A quarter of respondents  reported high or total dependence on financial/material assistance from the authorities or from charity.Confrontation with violence was reported both in the past since 1989) and more recently (three months prior to the survey). Exposure to crossfire  reported being physically or mentally mistreated themselves , forced labour , violation of modesty  and injury , and had a higher odds of being arrested/kidnapped .This paper presents findings related to confrontation with violence among the conflict-affected Kashmiri population. We did not assess who was responsible for the violence because it was not relevant for our medical needs assessment. We found a high exposure to violence (being in the vicinity but not witnessing or self-experiencing) among the civilian participants in our survey, reflecting a pervasive climate of violence in which the population is living. The frequency of exposure to violence on multiple occasions (>5 times) since the start of the conflict Table  is high High levels of confrontation with violence have been reported in another recent study from Kashmir. In this study, no substantial differences between males (59.51%) and females 57.39%) were found for lifetime prevalence of traumatic experiences. 9% were f, and mayThe high level of people reporting being tortured while detained or taken hostage is a particular concern, indicating that the violence against civilians is not simply circumstantial. We used \"violation of modesty\" as the local equivalent for sexual violence . The facThe completion rate of the survey was good (93%), and the design was adapted to the purpose and the context. However, there are a number of potential limitations. First, there is a possible selection bias in the fact that only people who were home during the time of the survey were interviewed. This methodology was deemed necessary for security reasons. The selection of one person per household may lead to a bias as individuals in large households are under represented. However, we do not think this bias influenced our findings since the overall household size in our sample was large (9). Second, retrospective study designs are subject to recall bias, and we cannot exclude recall bias in the participants' answers on confrontations with violence. However, a recent study  has demoThis survey aimed to determine exposure to violence and mental health impact as part of a routine programme assessment. We found that the Kashmiri population is confronted with high levels of violence committed by all parties to the conflict, with potentially substantial implications for mental health. This confrontation with violent events is not simply an environmental effect of living in a conflict-affected area, as demonstrated by the high frequency of deliberate events as detention, hostage, and torture. The conflict continues with no end in sight, with civilian deaths reported as this article goes to print .The authors declare that they have no competing interests.KJ designed and co-ordinated the study and wrote the first draft of the paper. NF supported the conceptual framing of the findings, assisted with the analysis, and led subsequent drafts. SK and KL provided statistical support for the design and analysis, and helped with the writing of the paper. SF, RG and BR oversaw the implementation of the survey, managed data collection in the field, and contributed to the writing of the paper. RK provided conceptual oversight and contributed to the writing of the paper."}
+{"text": "Spousal violence against women is a serious public-health issue. Although there is a growing body of literature on this subject, there are still many unanswered questions regarding the prevalence of this violence, the risk factors, the consequences, and how to address the issue. The purpose of this literature review is to organize and synthesize the empirical evidence on spousal violence against women in Bangladesh and to provide direction for both researchers and practitioners for future work in this area. The review suggests that spousal violence against women is high in Bangladesh. The list of correlates is long and inconclusive. Although there is evidence on adverse consequences of this violence on health of women and their children, more research is needed to explore the multifaceted consequences of violence for women, children, families, and communities. Action research is needed to develop and test preventive and curative interventions. Over the past 15 years, the growing body of research on violence against women has brought the issue from a position of near-invisibility to being recognized as having far-reaching health and economic impacts for women and societies , The Prevention of Women and Child Repression Act (2000), and The Cruelty to Women Ordinance (1983). Furthermore, Bangladesh has ratified the Convention on the Elimination of All Forms of Discrimination against Women (1979), which calls for the protection of women from gender-based abuse and negligence. The Government is committed to eliminating violence against women by 2015 in Bangladesh, as stated by the Minister of Health and Family Welfare and as detailed in Target 6 of Millennium Development Goal 5 to improve maternal health . Although domestic violence, particularly violence perpetrated by husbands, is the most common violence against women in Bangladesh  among women of reproductive age in Bangladesh; and original analysis of ICDDR, B surveillance data on deaths from intentional injury. We included data on deaths from intentional injury since spousal violence can result in either suicide or homicide; furthermore, intimate partner homicides may be labelled as suicides, and actual suicides may be an indicator of mental instability resulting from violence and other causes . Data onThe growing body of research suggests that spousal violence is highly prevalent in Bangladesh. In 2001, about 60%  of women reported having ever-experienced physical or sexual spousal violence. Consistent with the global trend, the overwhelming majority of physical and sexual violence against women was perpetrated by husbands, not by other persons . While det al. found that 47% of women of lower socioeconomic status in six villages in three districts of Bangladesh reported ever having experienced physical domestic violence  of rural and urban Bangladeshi women who experienced physical violence within the past year reported that the violence was severe, including having been hit with a fist, dragged, kicked, or threatened with a weapon . Eighty-Available data indicate strong associations between violence by intimate partners and physical health of women. While similar to other countries, most injuries reported to a large 2001 WHO study on spousal violence in rural Bangladesh were minor , and the majority of ever-injured women reported losing consciousness due to a violent incident, which was quite uncommon for other countries covered by the WHO study. Eighty percent of rural women who reported ever having been injured by their husbands reported having needed healthcare for an injury  Table)..Table).Spousal violence can result in death, but as the social stigma associated with homicide are strong and official penalties are very severe, deaths attributable to and associated with domestic violence are difficult to enumerate even in surveillance systems. In a study of deaths from intentional injury to women aged 15-44 years registered in the ICDDR, B's Health and Demographic Surveillance System in Matlab from 1992 to 1998, verbal autopsy data linked violence by husbands and/or other relatives to 10% of homicides. The investigators suggested that this is probably an underestimate of spousal violence-related homicide .While physical violence is well-documented in Bangladesh, a very few studies have investigated sexual violence. The WHO study of 2001 found that 37% of women in the urban study site and 50% of women in the rural study site reported having been sexually violated by their husbands at some point in the past, and 20% of urban and 24% of rural Bangladeshi women reported having been sexually violated by their husbands in the previous year  to end their lives compared to women who had not experienced spousal violence . Sexual Suicide is an issue of growing concern in Bangladesh with some areas reporting 37% of all deaths of women aged 15-44 years attributable to suicide. Longitudinal data suggest that the percentage of deaths attributable to suicide among women, particularly young women of reproductive age, is increasing , the number and sex of children, whether or not the mother of the wife was abused by her father, whether or not the mother of the husband was abused by his father, membership of wife in savings and/or microcredit groups, and income generation of women.The strongest factor associated with violence of husbands towards their wives in both urban and rural areas was a history of abuse of the husband's mother by his father, according to analysis of Naved and Persson . The relper se but the patriarchal attitude of the families demanding the dowry that is positively associated with physical violence against women \u2014impact on spousal relations and deserve to be taken into account in future quantitative research .Attitudes of women towards spousal violence vary by whether or not they have experienced spousal violence. An association between experience of physical or sexual violence and agreeing that violence is sometimes justified has been identified, but the direction of the association is not known. Sixty-four percent of urban women and 86% of rural women who have ever experienced spousal violence reported that a husband has the right to beat his wife under certain circumstances. These circumstances range from not completing housework adequately to refusing sex, to disobeying her husband, to being unfaithful. In contrast, of women who have never experienced spousal violence, 45% of urban women and 73% of rural women agreed that violence was sometimes justified .There are official channels of conflict resolution, but these are not yet well-understood or accessible to women who need to use them. The Government has established One-stop Crisis Centres in different districts in Bangladesh which facilitate person-centred counselling to abused women. A substantial barrier to institutionalizing counselling in all health-delivery points is the lack of trained professionals in this field, and this problem is more acute in rural areas as psychologists are reluctant to work in rural areas for extended periods. In an operations research context, ICDDR, B trained paramedics in spousal violence counselling. Preliminary findings from a study comparing the quality of counselling delivered by psychologists and paramedics in Matlab suggested that, with careful training and close supervision, primary-level healthcare professionals were able to provide some spousal violence counselling services ; expectations of sexual relations within marriage; and marital conflict and communication within marriage ,11,26.Research is required to explore the multifaceted consequences of violence for women, their children, their families, and communities. One of the consequences highlighted in this paper is suicide. Suicide is the number one cause of death among women of reproductive age in some age-groups in certain areas of Bangladesh. Suicide and associated factors need to be better understood as does the relationship between spousal violence and suicide. The very high and increasing rates of suicide among young unmarried women suggest that the relationships among premarital intimate partnerships, unwanted pregnancy, and suicide should be explored.To facilitate the measurement trends of spousal violence and its correlates over time, spousal violence should be included in the existing public-health surveillance systems. One important lesson from this paper is that, without consistent methods applied to the same population over time, it is impossible to document the trends over time.Action research to develop and test interventions (preventive and curative) involving the Government of Bangladesh, health systems, women, men, communities, NGOs, and private-sector businesses is required to facilitate means of preventing spousal violence and of providing care, counselling, shelter, and rehabilitation for women who experience spousal violence and their children. For these activities to have a maximum impact, monitoring, evaluation, and sharing results efficiently are critical.Programmes can address spousal violence on tertiary, secondary, and primary levels. Tertiary and secondary-level programmes aim at providing care and resources to women who have experienced spousal violence or are identified to be at risk of spousal violence. Primary prevention aims at behaviour change to prevent spousal violence .Tertiary or secondary prevention measures can be developed and/or streamlined based on the information already available. Tertiary prevention measures include facility- or community-based screening of women with visible signs of abuse. Health systems are ideally positioned to screen such women for spousal violence, but to be effective on such a socially- and culturally-sensitive issue, these require staff competent in counselling, a working referral system, and active support of the social and legal systems. Systems need to be in place to demonstrate the benefits to woman of using such spousal violence support services. Because women often cite \u2018shame to family\u2019 as a barrier to seeking care for spousal violence, such programmes might demonstrate how they can have a positive impact on the family.Bangladesh currently has a very few counsellors trained on issues around spousal violence. Operations research on a counselling initiative might include testing the impact of individual and group therapy counselling for victims.Secondary prevention measures include identifying women most at risk of being exposed to spousal violence and providing them with the tools to seek help and support before they require assistance. Mechanisms for secondary prevention include facility- or community-based screening of women who may be most at risk\u2014for example, young newly-married women, pregnant women, women with minimal or no formal education, and women who have recently joined the paid labour force.Primary prevention to reduce spousal violence requires changing societal attitudes and behaviour to prevent spousal violence from occurring at all. One possible mechanism for primary prevention is universal screening of women. Such screening will facilitate the identification of women who have experienced spousal violence who may not otherwise have been identified and also will give the message to all women screened that spousal violence is an unacceptable behaviour, and a violation of women's rights for which women have recourse. Family-life education courses, including strategies for spousal communication and conflict resolution, particularly community-based, are important forums for spreading messages aiming at reducing spousal violence.Primary prevention activities should also target men and boys who perpetrate or are at risk of perpetrating violence. Punitive measures against men who perpetrate spousal violence must be accompanied by rehabilitation and reintegration programmes. There are no systems in place in Bangladesh to help perpetrators of spousal violence develop or turn to strategies other than violence. Such systems need to be developed and carefully implemented and monitored.Unfortunately, there are minimal data on the training received by medical service providers in responding to persons who have been victims of spousal violence or the perpetrators of spousal violence. Few resources are available from the health systems to provide counselling or otherwise help women and their husbands to address the abuse. Much research is needed to understand how to help clinicians provide proper services to these patients, and how the health and legal systems should cooperate effectively and efficiently to reduce the incidence of spousal violence.The issue of spousal violence against women is complex and is culturally and socially sensitive. Nonetheless, the Government has demonstrated its commitment to eliminating violence against women in a short time. Research institutions, non-governmental and civil society organizations have critical roles to play in working with the Government of Bangladesh as a united front to reduce violence against women and the associated and unnecessary physical and mental anguish, death, and disability."}
+{"text": "Early family-level and social-level stressors are both assumed to be the components of two main path models explaining the association between exposure to interparental violence in childhood and its long-term consequences on mental health explored through life-course epidemiological studies.To investigate the association between exposure to interparental violence in childhood and mental health outcomes in adulthood when taking into account early family and social stressors.A retrospective French cohort study of 3023 adults representative of the general population in the Paris metropolitan area was conducted in 2005 through at-home, face-to-face interviews. The outcomes measures were current depression and lifetime suicide attempt, intimate partner violence, violence against children and alcohol dependence.The adults exposed to interparental violence during childhood had a higher risk of psychosocial maladjustment. After adjusting for family- and social-level stressors in childhood, this risk was, respectively, 1.44 (95% CI 1.03 to 2.00) for depression, 3.17 (1.75 to 5.73) for conjugal violence, 4.75 (1.60 to 14.14) for child maltreatment and 1.75 (1.19 to 2.57) for alcohol dependence.The adult consequences of parental violence in childhood\u2014and this independently of the other forms of domestic violence and the related psychosocial risks\u2014should lead to intensifying the prevention of and screening for this form of maltreatment of children. Domestic violence, including exposure to interparental violence, has been identified and reported in US surveys over the last three decades. They showed the extent of this societal problem,5\u2013Despite different methodological concerns,10Intimate partner violence has been described in an integrated, ecological framework where personal, situational and sociocultural factors interact.The main limitations of the studies conducted in this research field are the lack of large-scale longitudinal datasets including relevant social factors. Moreover, very little is known about how interparental violence combines with other adversities in childhood to promote adult psychosocial maladjustment.The aim of our study was to investigate the association between interparental violence exposure in childhood and mental health outcomes in adulthood when simultaneously taking into account other family and social stressors in childhood. Our hypothesis was that this adverse early life event has a negative impact on different mental health outcomes throughout the individual\u2019s lifetime, over and above both coexisting familial and social stressors during childhood.The SIRS  cohort study is a longitudinal epidemiological survey of the general adult population of the Paris metropolitan area, conducted since 2005 by the National Institute of Health and Medical Research (INSERM). This cohort study was approved by the French authority that protects privacy and personal data .This survey, which has been described elsewhere,Of the 4560 selected adults, 28.6% refused to participate, and 3.1% who did not speak French and 1.8% who were too sick or unable to answer the survey were excluded from the study population. Overall, 3023 subjects were included in the cohort, for a participation rate of 71.4%. No individual data were collected from the non-respondents, but according to the four-level socioeconomic categorisation of neighbourhoods used for sample stratification, the response rates were higher in both extreme categories, ie, the richest and poorest neighbourhoods (73.9% and 76.5% respectively), than in the two intermediate categories .We used the retrospective data on childhood circumstances and the cross-sectional data on adulthood indicators collected at that time.et al.Witnessing interparental violence in childhood before the age of 18 years was our main independent variable. It was defined according to the answer to the following question: \u201cDid you witness interparental violence before the age of 18?\u201d (yes/no). Five mental health outcomes were studied: depression, lifetime suicide attempt, two violent intrafamilial conduct disorders and alcohol dependence. Depression was investigated by the Mini-International Neuropsychiatric Interview. The depression index was evaluated by a seven-item scale for measuring the occurrence of depressive symptoms during the last 15 days. The scale\u2019s internal and external validity has been demonstrated in the French population by Lecrubier Family-level stressors included parent\u2013child relationships and childhood adversities. The perceived quality of parent\u2013child relationships was assessed separately for both the mother and the father by the following question: \u201cBefore the age of 18, how would you rate your relationship with (1) your father and (2) your mother?\u201d, reported and then dichotomised as very good or good vs poor or very poor. Childhood adversities before the age of 18 were the following: parental separation or divorce, parental death, parental incarceration, the occurrence of a parental suicide attempt, parental alcoholism and other forms of domestic violence, with respect to which the respondents were asked if they had been physically abused and/or sexually abused before the age of 18. The questions used to assess these characteristics were standard questions about childhood living arrangements used in previous French sociological and health surveys. Social-level stressors during childhood were poor parental health status, housing problems, prolonged parental unemployment and family financial problems during childhood.In addition to age and gender, we adjusted for socioeconomic factors in adulthood recorded at the time of the survey. Individual socioeconomic status (SES) was assessed on the basis of the following variables: level of education, monthly household income per consumer unit and occupational status .We investigated the association between exposure to interparental violence during childhood prior to age 18 and psychosocial adjustment outcomes in adulthood. The first step in the analysis consisted of examining the past social and family characteristics of the adults who had been exposed to interparental violence during childhood compared with those who had not, and in analysing the bivariate association between having witnessed interparental violence and mental health outcomes in adulthood by age group. Second, we used a binary logistic regression model for each of these outcomes (baseline model) while controlling for age, gender and adult SES. Third, we introduced in the baseline model: (1) the family-level stressors after adjusting for child sexual abuse and/or physical maltreatment; and (2) the social-level stressors in childhood. Interactions between exposure to interparental violence and significant predictive variables in the final model were systematically tested. The data were weighted in order to take into account the sampling design (which combined multistage random clustering and oversampling of poor neighbourhoods during stratification) and then the post-stratification adjustment according to the general population census data. SPSS 15.0 procedures  were used to perform the statistical analysis.The study population included 3023 subjects (53% women and 47% men), 29 of whom  did not provide information about past parental violence. Among the respondents, 16% reported having witnessed interparental violence before the age of 18. This prevalence decreased with subject age, from 18.8% in the 18\u201336 age group to 13.5% in the 55 and over age group. There were neither gender- or nationality-related differences in interparental violence exposure, except in the oldest age group, where French people reported more exposure to interparental violence than did foreigners. The current SES was the same in both groups, except for the level of education. The respondents with the lowest level of education were more likely to have witnessed interparental violence than those with an intermediate or the highest level of education.Exposure to interparental violence during childhood was significantly associated with all five adulthood mental health outcomes that were studied, with higher odds ratios for intimate partner violence and perpetrating child maltreatment, but also a significant association with depression, lifetime suicide attempt and alcohol dependence , the risk of depression was higher in women exposed to interparental violence in childhood. On the other hand, the men had a 15-fold higher risk of committing violent acts against their children, women a two-fold higher risk. But intimate partner violence was the same for both genders. Finally, the risk of alcohol dependence was higher in the male respondents from high-conflict families and/or with a parental history of alcoholism, and it was also higher after adjusting for depression. This risk was not observed in the women.Interparental violence is now widely documented as an adverse childhood experienceHowever, the limitations of our study warrant some consideration. First, the marital discord variable was not quantified by means of a standardised scale. Nonetheless, the odds ratios were strong enough to suggest that even mildly violent acts, such as verbal rather than physical acts, have negative consequences in adulthood. In the present study, our goal was not to determine a prevalence rate for this event in the general population, but rather to assess the adulthood consequences of a climate of parental violence perceived by the respondents when they were children. Second, with regard to potential non-response bias in such a population-based cohort study, we indicated above that the response rates were higher in the neighbourhoods in both extreme social categories. If one assumes that negative early life events are more frequent in families living in the poorest neighbourhoods, such differences in response rates would not have biased our estimates. On the other hand, we cannot ascertain the number of subjects who did not respond for reasons associated with parental violence during childhood . Third, retrospective reports of adverse childhood experiences could be subject to recall bias. In the literature, the analysis of the validity of retrospective studies has shown that the prevalence of past events is underestimated.We found that adults who had witnessed interparental violence during childhood had had numerous other adverse familial and social experiences and had a higher risk of negative mental health outcomes in adulthood. These associations could be explained by the emotional security hypothesis developed by Davies and Cummings.Numerous types of adverse childhood experiences, such as parental divorce,Our second indicator of psychosocial functioning was intrafamilial conduct disorders. The strength of the association between parental discord and both child and intimate partner violence partially concurs with the notion of intergenerational transmission of violence. Indeed, physical abuse and/or sexual abuse during childhood were independently associated, respectively, with both outcomes and with child maltreatment. This intergenerational cycle of maltreatment and violence has been explained by different theoretical approaches, most of which are based on a general family of cognitive/behavioural paradigms.Our study sought to demonstrate the potential harm of interparental violence to children for their future adulthood life, and this independently of other forms of domestic violence and related psychosocial risks. Such a demonstration warrants further research studies so that these consequences become a cornerstone of any risk\u2013benefit analysis of screening interventions towards intimate partner violence.Children\u2019s exposure to interparental violence is a form of maltreatment and has consequences on child\u2019s development.Early family-level and social-level stressors are both assumed to be the components of two main hypotheses explaining the association between exposure to interparental violence and negative health outcomes in children and later in adulthood.The adults exposed to interparental violence in childhood have a higher risk of mental health disorders in adulthood than those not exposed, and even after adjusting for other forms of domestic violence.Family- and social-level stressors in childhood do not explain this association.Intensification of prevention of and screening for domestic violence including interparental violence is a public health issue for the well-being of future generations."}
+{"text": "This is possibly due to exogenous polyamine salvage from the host, which replenishes the intracellular polyamine pool. This implies that disruption of polyamine metabolism as an antimalarial chemotherapy strategy may require targeting both polyamine biosynthesis and transport simultaneously. In the absence of a clear understanding of the uptake mechanism of polyamines into Plasmodium falciparum parasites, polyamine transport into both the infected erythrocytes and parasites isolated from the erythrocyte were investigated using radioisotope flux techniques. While the characteristics of transport of putrescine into infected erythrocytes (iRBC) were similar to those of transport into uninfected erythrocytes (RBC) spermidine uptake occurred via the new permeability pathways (NPP) induced by the parasite in the erythrocyte membrane. Once inside the erythrocyte cytoplasm, both putrescine and spermidine are taken up by the parasite via a temperature- and glucose-dependent, saturable process. The uptake of both these polyamines was competed for by other polyamines and biosynthesis inhibition led to increased total uptake of both putrescine and spermidine. Polyamine uptake was pH dependent with uptake increasing with increasing pH, but did not appear to be coupled to the Na+ or K+ gradients. Membrane potential perturbations influenced polyamine uptake, consistent with the transport being an electrogenic process.Polyamines are present at high levels in proliferating cells, including cancerous cells and protozoan parasites and the inhibition of their synthesis has been exploited in antiproliferative strategies. Inhibition of the malaria parasite's polyamine biosynthetic pathway causes cytostatic arrest in the trophozoite stage but does not cure"}
+{"text": "The lack of conceptual references for these expressions complicates the organization of literature. Furthermore, the absence of clear concepts may be an obstacle to clinical treatment because the use of these words by the patients does not necessarily point to a diagnosis of posttraumatic stress disorder.Several terms in the scientific literature about posttraumatic stress disorder are used with different meanings in studies conducted by different authors. Words such as A critical review of scientific literature showed that stress can be divided in stages to facilitate specific terminological adjustments to the event itself, to the subject-event interaction and to psychological responses. Moreover, it demonstrated that the varying concept of trauma expands into fundamental psychotherapeutic definitions and that the meanings of violence associated with barbarism are an obstacle to resilience. Therefore, this study updates the etymological origins and applications of these words, connects them to the expansions of meanings that can be operated in the clinical care of patients with posttraumatic stress disorder, and analyzes them critically according to the criterion A of DSM-IV and ICD-10.trauma led to a broader understanding of this phenomenon in its psychic dimensions, that a barbarian type of violence constitutes an obstacle to resilience, and that the criterion A of the DSM-IV and ICD-10 shows imprecision and conceptual fragilities.The terminology in the literature about posttraumatic stress disorder includes a plethora of terms whose meanings are not fully understood, and that, therefore, limit this terminology. The analysis of these terms suggested that the transformation of the concept of conceptual approach in any part of the paper. Researches that relied solely on empirical indicators, like psychopathological, neurobiological or pharmacological aspects, were excluded. The focus here was in conceptual aspects, even when some few empirical studies were included.To develop this debate article, a current specialized literature review was achieved by searching and retrieving the key terms from two major databases: PubMed and PsycINFO. The key terms included \"disaster\", \"catastrophe\", \"barbarism\", \"terrorism\", \"trauma\", \"psychic trauma\" and \"violence\", also in combination with the terms \"PTSD\", \"concept\" and \"conceptual aspects\". The data were captured specially from review articles. The included studies were those mostly identified by the authors as relevant by the presence of a As it was noted a paucity of medical references related to conceptual aspects of these terms, a wider literature needed to be included, including chapters, books and articles proceeded from the Humanities areas. \"Interdisciplinary research is needed in this area to include perspectives from a range of different disciplines\" once that \"to promote public health (...) new dimensions of such interactions and the implications thereof should be pursued in collaboration with researchers from broader areas\" . Posttraumatic stress disorder (PTSD) was defined as a psychiatric disease in 1980 and included in the third edition of the DSM by the American Psychiatric Association (APA) . ConsideThe plethora of terms used in PTSD literature, such as aggression, violence, disaster, catastrophe, barbarism, stressful event and trauma, do not point to a corresponding understanding of the meanings, applications and limits of these constructs. These terms are often vaguely used, meanings change in different studies, and there is a lack of conceptual references to guide their use in scientific literature. Some of the most common expressions define events according to consequences, such as in the case of the word \"stressor\", and not according to the characteristics of that single event ,8.An initial attempt to undo such terminological confusion is to refer to etymology and to retrace the path of the concept into clinical practice. Rather than to establish definitive positions about this topic, the purpose of this study is to promote a discussion of conceptual questions about PTSD terms. Therefore, this study presents definitions of catastrophe, disaster, trauma, violence and barbarism from a clinical perspective to clarify their scope and limitations.disastro  [The word disaster has its origin in the Italian word d star\") , and refd star\") . \"Disastd star\") .Disasters tend to expose unselected populations to trauma, randomly. Within a given community, individuals can be directly, indirectly or remotely exposed to the event. The medical model focuses on specific intervention for each of these groups, aiming to prevention, healing and recovery of PTSD and other psychiatric disorders. On the other hand, the wellness models comprehend disasters from the distress challenge, focusing on restoring homeostasis . Therefocatastrophe is Greek (kata + strophein) and its literal meaning was \"overturn\". According to its definition, it is an event that causes trauma [Ecological disasters may also be called catastrophes, events of great proportions usually associated with natural phenomena that cause death and destruction. The origin of s trauma  due to iA careful examination of the words used by patients is fundamental to understand the psychological impact of events that may trigger traumatic responses. Situations described as \"catastrophic\" may not necessarily indicate that the situation that the patients experienced was a \"catastrophe\", although it may have an equally devastating psychological representation.The event alone has importance to the mental healthcare professional only when it is a situation that can produce psychopathological responses from the patient. The idea of a \"disaster taxonomy\" is based on the principle that there are variable emotional responses that depend on the type of disaster, the degree of personal impact, the size of the group affected, and the geographical and temporal range of the event .The \"disaster taxonomy\" stresses the importance of avoiding overlapping or confusing terms to define events because their different aspects may trigger different psychological responses. The use of an adequate terminology requires the understanding of how a traumatic situation causes and becomes part of a stressful process, which may be divided intro three major stages: 1) environmental input in the form of an event; 2) immediate apprehension of the event; and 3) psychological responses after the event .The first stage is restricted to the event itself, and may be objectively measured in number of victims, degrees in the Richter scale, or square kilometers of affected area. \"Pure\" words, without qualifiers, such as \"event\", \"stimulus\", \"loss\", \"disaster\" or \"catastrophe\" are used for such description. The second stage goes beyond the isolated event and incorporates the initial perceptions of the victim. Words such as \"danger\", \"shock\", \"risk of death\", \"threat\" or \"stressor\" illustrate the interaction between the stimulus and the person that experiences it. The third stage corresponds to the psychological response to the event, and words such as \"mourning\", \"response to stress\" or \"trauma\" are used . Intensetrauma, originally used in medicine, has an Indo-European root with a double meaning: a) to rub, grind, perforate; and b) overcome, to go through [Trauma is a violent shock that is capable of producing an impact that the individual cannot resist. Therefore, trauma that \"perforates\" is the same that makes \"go through\", which describe the two possible psychic developments seen in a traumatic situation: the development of PTSD or of resilience \u2013 the ability to go through trauma and to introject meaning into one's own life.The word  through ,14. Trautrauma. In the 19th Century, except in the psychoanalytic literature, the word trauma referred primarily to wounds or violent tissue rupture and had no psychological connotations. The hypothesis that a terrible event might cause effects other than those merely physical was developed in the 1860's, with the description of the \"Railroad Spinal Syndrome\" by John Eric Erichsen [The formal recognition of PTSD in 1980 changed the conceptual understanding of Erichsen . Since tErichsen di\u00e1these\". Therefore, he described the \"n\u00e9vrose traumatique\" or \"hyst\u00e9rie traumatique\" to classify these cases. Charcot concluded that a physical trauma could produce emotional disorders [In 1882, Jean-Martin Charcot studied patients whose psychic symptoms appeared after severe trauma, such as train crashes or wars, and served as traumatic triggers in individuals that had a certain inherited predisposition or \"isorders .On the psychical mechanism of hysterical phenomena (1893), Freud expanded on the concept of traumatic neurosis. The conceptual fluctuations of the word trauma in Freudian works suggest that the relative difficulties to establish this definition are much older than the conceptual confusion observed in PTSD. In his early papers, Freud used trauma as a key to explain the etiology of neurosis. From 1897 on, the concept loses importance as the concept of fantasy develops and takes the place previously held by traumatic events. However, the infamy of the First World War brought back the problems of \"traumatic neurosis\" to Freudian works, particularly in Introduction to psychoanalysis and the war neurosis (1919) [Beyond the pleasure principle (1920) [In henomena 93, Freudneurosis 19 [BeyonTrauma was understood by psychoanalysis as an unspeakable experience, not elaborated, not signified, that was incorporated but could not be introjected, according to Nicolas Abraham (1919\u20131975) and Maria Torok (1925\u20131998) [introjected trauma. In contrast, the paralyzing expansion of the incorporated trauma perpetuates the symptomatic reliving of suffering disconnected from language and, thus, far from the attribution of meaning to experience. Therefore, the way the event is treated or elaborated may become the traumatic element itself [25\u20131998) . The bart itself .Violence may be understood as the action of force or the act of violation. From Latin, violentia carries the broad meaning of behaviors with an origin in vis  and refers to \"vehemence; passionate and uncontrolled force.\" Acts of excessive violence may result in the violation of rules, rights and norms, in which cases violence is understood as violare  [raction) . The defviolentia), which is restricted to an act that should meet three conditions: deliberate attitude of the perpetrator, physical force, and destructive intent. Episodic violence corresponds to this concept and is characterized by the fact that it is direct and perpetrated fast and intermittently as an acute insult to a person's well being by means of a dramatic form of violence.Violence may be initially understood according to a minimalist concept (violence as violentia) includes psychic and subjective elements and stresses the victim's perspective. Its standard form is structural violence, an indirect form of violence whose norms are established socially and that is defined as a chronic insult to well being that kills or harms people slowly by continuous deprivation of basic human needs [On the other side, the comprehensive concept : \"Violence is the intentional use of physical force or power, threatened or real, against oneself, another person, or against a group or community, that either results in or has a high likelihood of resulting in injury, death, psychological harm, maldevelopment, or deprivation\" . Accordiintention adds complexity to this concept because intention is not always identifiable in a violent act [The WHO definition validates the concept of violence as an international, and not only local, problem, and prescribes the protection of vulnerable populations. However, the incorporation of the notion of lent act . The reslent act .The depth and breadth of the WHO definition are adequate to that organization's purposes, which require an ecological model of violence centered on multiple levels. However, when the breadth of what is denoted in a term expands, its descriptive power is retracted . A comprThe application of this comprehensive concept of violence to PTSD would result in very permissive boundaries for the definition of a phenomenon as violent. Traumatic situations that have social, political or economic origins are beyond the reach of psychiatric and psychological treatments. Moreover, evil-minded individuals could distort the use of this broader sense of the word to claim medical benefits and secondary gains based on this \"happy combination of a vague descriptive content and a negative emotional connotation\" .The concept of violence as force  was refuted by Hannah Arendt, who established the distinction between power, potency (vigor), force, authority and violence. In the common sense, these terms are usually misunderstood or mistaken because are comprehended as a whole from the aspects of the domination of someone or something over others -30. ArenArendt's theory of violence, although developed in the field of political science, also enriches the clinical care to victims of violence. The idea of denaturalization of violence destroys positivist references and inspires practices that go farther then a merely organicistic or psychologizing understanding of traumatic phenomena.The notion of depersonalization of violence adds new meanings to the psychotherapy of domestic violence victims, who usually have ambiguous feelings for the perpetrators, which complicates their psychotherapeutic implication in the process of elaborating trauma. To understand violence by means of its instrumental character and not by personification of evil allows the victims to elaborate on their suffering based on the functions that violence operates in that relationship, and not based on a moral judgment of the aggressor.Finally, demythification of violence indicates the \"banalization of evil\" and has clear implications on psychotherapeutic care because a barbarian type of violence is a barrier to the process of understanding the traumatic experience, as will be seen next.Iliad. This term was used by ancient Greeks to refer to foreign peoples. The term, originally not disqualifying, referred to those that did not understand Greek and pronounced inarticulate and incomprehensible sounds, such as onomatopoeias: \"bar-bar-bar\". Barbarians only became dangerous and culturally inferior enemies after the Greco-Persian Wars (5th Century B.C.). The notion of barbarism as a clear opposition to civilization is assigned to Romans, who borrowed the term \"barbarism\" from the Greek and for the first time raised an insurmountable barrier between Romans and Barbarians [The first appearance of the word \"barbarism\", associated with rude, brutal and unintelligible speech, is found in Homer's rbarians .socialization; (b) culture; or, more importantly, (c) in a pre-human (savage) stage in relation to those that called them barbarians [Romans started using the term not only to describe peoples beyond their borders, but also for those in their own world who did not belong to the Greek-Roman cultural world . Thereforbarians .It is no longer startling that a highly refined and educated civilization may reach the worst of barbarism, such as in Nazi Germany, which used the advancement of their techniques and knowledge to exterminate human beings rationally and in an industrial scale. Therefore, the simple and single definitions of barbarism and civilization as opposites do not exist . The terThis dialectical trap may be avoided by defining a culture as barbarian if it lacks structures to recognize the alterity of things, by defining customs as barbarian if their effects deny a specific form of human existence, and by describing individuals as barbarian if they are incapable of tolerating diversity. The meaning of barbarism is more clearly defined when a culture is analyzed in relation to itself, not by classifying as barbarian those who were left out of any civilization process, but by recognizing barbarism when people fall behind, in a peculiarly hideous way, their own civilization even though this civilization has achieved the highest levels of development .In general, terrorist attacks can be considered as one specific form of barbarism, once \"attackers tend to use horrific violence to cause massive destruction and death and to use other tactics  to terrify the public\" , and mosThe terrorism and other barbarian aspects of violence imply a lack of meaning rather than some symbolic construction that \"justifies\" the traumatic event that affected the victims or their relatives. Finding a \"justification\" for violence outlines a symbolic shield against terror, the initial mechanism of assigning meaning to traumatic experiences. Conversely, lack of meaning is the trademark of barbarian violence and an obstacle to the elaboration of trauma and its consequent symbolic integration in the victim's life. This significant gap is filled by the abundance of symptoms that result from the persistence of the traumatic memory.The tenth edition of the International Classification of Diseases (ICD-10) and the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) describe similar criteria to diagnose PTSD. According to criterion A of DSM-IV, a diagnosis of PTSD should be made when \"the person has experienced, witnessed, or been confronted with an event or events that involve actual or threatened death or serious injury, or a threat to the physical integrity of oneself or others\" (criterion A1), and whose response to the event \"involved intense fear, helplessness or horror\" (criterion A2) . Accordideath, serious injury or a threat to physical integrity. In regard to the first stage of the process of stress \u2013 the isolated environmental input \u2013 the criterion A1 indicates a return to the older meaning of the word \"trauma\", which refers exclusively to a bodily wound, as originally used in medicine.The historical trajectory of the concept of PTSD, from DSM-III to the revised edition of DSM-IV, attests to \"the centrality of the stressor criterion in the definition of this disorder\" . In the The second stage in the process of stress can be found in criterion A2 of the DSM-IV, and reflects the psychological dimension of trauma through the immediate apprehension of the event by the victim in the form a response of \"intense fear, helplessness or horror.\" Some authors question the restriction of criterion A2 to only these trauma-related emotions, \"when there is recent evidence that both anger with others and shame\" are also \"strong predictors of PTSD symptoms longitudinally\" .The criterion A1 excludes the situations that do not involve direct physical violence or \"threat to physical integrity\", but that are capable of producing psychopathological responses of reliving, avoidance, and hyperarousal. The point here is not to artificially expand the criterion to include the more questionable dimensions of a broader concept of violence. The purpose is to rectify, from a nosographic perspective, the conclusion that, if an event can be simply characterized as a \"threat\", such threat may be defined not only as physical threat, but also as moral coercion, psychic intimidation, or symbolic coercion. A good example of such is the development of PTSD by some victims of bullying, a type of violence that is perpetrated not necessarily and only by means of threat to physical integrity, but that may occur by means of systematic psychological attacks that take the form of verbal offense, acts with the intent to ridicule in front of others, and social isolation from youth groups with certain physical or psychic characteristics.indirect exposure, largely through the media, questions the \"perhaps outdated stimulus-response model\" based on the \"conventional assumptions that a disaster should be defined as local and must be directed experienced to cause psychopathology\" [Furthermore, in the context of ongoing terrorist threats, especially after the 9/11 attacks, the presence of widespread posttraumatic stress disorder symptoms among individuals not directly exposed to the attacks was documented and replicated in independent cross-sectional and longitudinal studies . This inthology\" .stress disorder, the patient should have \"been exposed to a stressful event or situation.\" Besides, the description of the nature of this stressor as \"something exceptionally threatening or catastrophic\" evokes the conceptual weakness of this criterion rather than is an attempt to outline or characterize the type of event. After all, if the notion of exceptionality refers to something that is \"out of the ordinary\" and that \"occurs beyond the limits of what is usual, normal, frequent or ordinary\" [The fragility of the ICD-10 approach to the event itself is shown, initially, in the circularity of its definition, once it is based on the response elicited, which leads to a tautology: to develop posttraumatic rdinary\" , what woexceptionally threatening a situation that might not cause pervasive distress \"in almost anyone.\" The perspective adopted in the ICD-10 is in disagreement with a decisive factor in clinical practice: for patients that present typical PTSD symptoms, the traumatic factor is not necessarily identified in the reality that they can share, but maybe only in their personal and subjective experiences.Moreover, this construct becomes even more complex when analyzed according to an individual perspective. A person with PTSD symptoms may have perceived as The conceptual inaccuracy of criterion A of the ICD-10 may also be found in the specification of the nature of the event as \"catastrophic\" or \"threatening\". How to discuss, then, the barbarian forms of violence that are not a catastrophe or do not constitute a direct threat, but that are capable of triggering PTSD? Barbarism may be described as a type of violence that, in some cases, prescinds from the association with \"exceptional threat\" for its perpetration. Barbarism may simply be a paroxysmal form of violence, an act without warning, without threat, whose only objective is destruction and death of another human being.Not infrequent are cases of people that get together to perpetrate acts of violence against homeless, black or native people, gays or members of other minorities by ambushing the victims, sometimes when asleep, without any type of previous threat. Because of the barbarian death of one of their members, these minorities may develop chronic hyperarousal, avoidance and intrusive thoughts of situations associated with that barbarism, and, therefore, meet all the criteria for a PTSD diagnosis although they have not concretely experienced a direct threat. In such situations, something that should be perceived as normal by most anyone, such as belonging to a minority group , is experienced as an \"exceptionally threatening\" situation.In the same manner, \"there are now replicated findings that PTSD symptoms related to the September 11, 2001, attacks occurred in large numbers of persons who did not fit the traditional definition of exposure to a traumatic event\" . It is lA careful analysis of the criteria currently used by the APA and WHO psychiatric guidelines shows inaccurate definitions of violence, disaster, catastrophe, trauma and barbarism. Such critical appraisal should inspire new studies to improve diagnostic and therapeutic parameters used in clinical practice.physical aspect of trauma. According to the considerations above, violence prescinds from force and physical damage, and the psychological dimension of a threat may be the most devastating face of violence. The restriction of this criterion to the minimalist concept of violence may exclude diagnoses and treatment of a significant number of patients with PTSD. At the same time, a broad understanding of violence exceeds the limits of clinical practice, as demonstrated in the discussion of the concept established by the WHO. Between these two concepts, there is an alternative: to include the aspect of psychological violence as one more possible trigger of PTSD without expanding the concept to the point of introducing aspects that are too broad for clinical practice.In the analysis of the DSM-IV criteria, such improvement should focus on the fact that this classification limits the real event or threat to the The ICD-10 also shows misconceptions in its criterion A, such as the generalizing definitions discussed above. New definitions of this diagnostic category should necessarily include the \"impossibility of symbolic elaboration\" as a condition for the development of this disorder. This is a dimension that includes the senses of \"trauma\", \"barbarism\" and, many times, \"catastrophe\", and replaces vague expressions such as \"exceptionally threatening.\"The difficulty in understanding and elaborating an event adds an important diagnostic value to this criterion because it assigns priority to a person's individual psychic response rather than to the effects found in \"almost anyone.\" This latter expression is not only imprecise, but may also exclude PTSD cases that, although originated in individual situations, may result in psychic sequelae of traumatic characteristics.In summary, the aspects discussed may be organized as a blueprint for the changes in criterion A of the diagnostic classifications as follows:A) Delayed psychic response to a situation or event (either short- or long-lasting) whose understanding or symbolic elaboration is not possible for the person because of the magnitude of physical or psychological threat or the actual presence of death, injury or severe psychological distress, with an immediate response of intense fear, helplessness, dissociation or horror.The examination of PTSD terminology defined two completely different tasks: the understanding of the meaning of the expressions used by the patient; and the search for terms that translate such expressions into medical language. The \"disaster\" described by the patient may be only an unexpected experience that requires an intense existential implication for which this person does not feel prepared. A situation referred to as \"traumatic\" raises the examiner's interest in obtaining details of the circumstances of the event described because the extension and intensity of the stimulus may reflect differently in psyche. A more severe psychopathological presentation is marked by silence and difficulty in elaboration, which may indicate experiences of severe contact with a form of violence, such as barbarism.The purpose of clarifying the terms used in the field of PTSD is to provide a resource for conceptual clarity, terminological precision, and understanding of meaningful nuances of the words used in clinical practice. Patients seen after an experience of imminent death do not need therapists that operate according to a good-versus-evil system, or who assign all evil to the perpetrator and reinforce the patient's role as a victim. These patients have already \"faced death\" and, therefore, therapy should not be conducted through the personalization of violence; it is not the positivist naturalization of violence that provides the theoretical basis for the clinical treatment of PTSD; and, finally, it is not the mythification of violence that will enable society to overcome this phenomenon.1. The plethora of terms used in PTSD literature does not reflect the understanding of meanings, applications and limits of these concepts.2. The \"disaster taxonomy\" indicates event characteristics that may generate different psychic responses and, therefore, requires precise terms to describe the three stages of the process of stress.3. Because of changes in the concept of trauma along time, it can now be understood in its integrative or paralyzing psychic dimensions depending on how the traumatic experience is elaborated.4. The use of a broad concept of violence in PTSD leads to questions out of the range of psychotherapy, and the barbarian type of violence functions as a barrier to resilience.5. The criterion A of the ICD-10 and DSM-IV show conceptual imprecision and fragilities that may have effects on the diagnosis and treatment of victims of violence.The authors declare that they have no competing interests.LLB conceived of the study, performed the literature search, participated in the interpretation and discussion of data, drafted and wrote the manuscript. JPF performed the literature search, participated in the interpretation and discussion of data, coordinated the multiple revisions of the manuscript. MFM conceived of the study, participated in the interpretation of data and coordinated the multiple revisions of the manuscript. JJM participated in the interpretation of data and critically revised the first versions of the manuscript. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Biomass has been studied as biomarker to evaluate the effect of heavy metals on microbial communities. Nevertheless, the most important methodological problem when working with natural and artificial microbial mats is the difficulty to evaluate changes produced on microorganism populations that are found in thicknesses of just a few mm depth.in situ the effect of Pb and Cu stress in cyanobacterial populations.Here, we applied for first time a recently published new method based on confocal laser scanning microscopy and image-program analysis to determine Microcoleus sp.  was detected within a week. According to the data presented in this report, this biomass inspection has a main advantage: besides total biomass, diversity, individual biomass of each population and their position can be analysed at microscale level. CLSM-IA could be a good method for analyzing changes in microbial biomass as a response to the addition of heavy metals and also to other kind of pollutants.The results showed that both in the microcosm polluted by Cu and by Pb, a drastic reduction in total biomass for cyanobacterial and  Microcoleus chthonoplastes is dominant in marine intertidal microbial mats, hypersaline environments and hot deserts Benthic stratified sediments  that cover kilometer long areas of coastal territory are made up of a dense biomass of microorganisms Nevertheless, the most important methodological problem when working with microbial mats is the difficulty to evaluate changes produced on cyanobacterial populations that are found in thicknesses of just a few mm depth. One of the main goals of assessing the effects of heavy metals on microbial communities in natural and artificial ecosystems in the laboratory is the search for an effective and reliable bioindicator. Changes in diversity or biomass can usually be considered good indicators for evaluating the effect of metals on bacterial populations. Amongst those most used are molecular techniques and biochemical analysis Optical microscopy techniques such as fluorescence microscopy have also been used \u22123 mgC/cm3 of sediment. This method was especially suitable for the quantitative analysis of a large number of CLSM images generated from benthic sediments in which complex populations of cyanobacteria were abundant, such as unpolluted an oil-polluted microbial mats We have recently published a new method based on determining cyanobacterial bimass by the Confocal Laser Scanning Microscopy- Image Analysis (CLSM-IA). This method has made it possible to characterize and identify the photoautotrophic microorganisms and also, with the aid of image-analysis computer systems, to determine their biomass In this study, we applied CLSM-IA for first time to cyanobacterial populations living in microcosms in order to determine the effect of heavy metals on these microorganisms, specifically in terms of biomass.Microcoleus sp. in microcosms. In order to do so, we have used Confocal Laser Scanning Microscopy (CLSM), which detects the natural fluorescence of photosynthetic pigments, and we have determined the total and differentiated biomass of the cyanobacteria by means of an image analysis program, Image J v1.37 (CLSM-IA) In this paper, we have studied the effect of lead (Pb) and copper (Cu) on the cyanobacteria population, and mainly on Microcoleus sp. and Halomicronema-like were the most abundant filamentous cyanobacteria. Microcoleus sp. presents its trichomes oriented in parallel, enclosed in a common homogenous sheath and the mature end cells are conical. Cells (diameter 3\u20136 \u00b5m) composing the trichomes are longer than they are wide  and end cells are rounded  from the first mm of depth, as it was in this layer that the greatest cyanobacteria growth was observed  to 0.507\u00b10.25 mgC \u00b7cm\u22123 of sediment (day 10). Microcoleus sp. was not detected at t\u200a=\u200a112, therefore, the biomass determined at the end of the study was probably mainly due to Halomicronema-like. In the microcosm polluted by Cu, the same effect was noticed, a reduction of the total biomass (from 3.813\u00b13.20 to 0.409\u00b10.38 mgC \u00b7cm\u22123 of sediment), and Microcoleus sp. biomass (from 3.622\u00b13.14 to 0.199\u00b10.25 mgC \u00b7cm\u22123 of sediment) 7 days after beginning the experiment.In the microcosm polluted by Pb, a drastic reduction in total biomass from 9.275\u00b14.62 to 1.265\u00b10.38 mgC \u00b7cmMicrocoleus sp. after only one week. In Microcoleus sp. biomass values are expressed in mg C\u00b7 cm\u22122 of sediment. This figure shows the lethal effect of Pb and Cu in the above mentioned populations, similar to those represented in Microcoleus consortium, a degradation of the photosynthetic pigments, which can be seen through the absence of fluorescence in the emission spectra, and alterations of the cell ultrastructure, such as expanded thylakoids and large intrathylakoidal spaces, in cultures treated with Pb and Cu respectively .In both cases it is shown that, in the doses of metals used, both Pb and Cu are lethal for cyanobacterial populations and a quantification Certain reports use changes in community composition and biomass as useful bioindicators for determining the effect of heavy metals on different ecosystems in situ cyanobacterial biomass determination, especially in the application to various benthic sediment samples. According to the data presented in this report, this biomass inspection has two main advantages: first, in addition to total biomass, diversity, individual biomass of each population and their position can be analysed; second, this method can be applied to different kind of natural and artificial ecosystems, including polluted mats. The overall results shown in this paper indicate that Pb and Cu in the concentrations used in this study have a drastic effect on cyanobacterial biomass and mainly on Microcoleus sp. in only one week, and that CLSM-IA is a good method for analyzing changes in microbial biomass as a response to the addition of heavy metals and also to other kind of pollutants. Although the method applied would not be suitable to analyse the physiologic state of the cells, we are currently applying the lambda scan function of CLSM to analyse the physiological state of the photosynthetic pigments.In conclusion, we believe that CLSM-IA is the best method for estimating cyanobacterial biomass in microbial mats. The method takes advantage of the accurate and nondestructive optical sectioning of confocal microscopy, and autofluorescence of cyanobacteria, presenting a good technique for Samples from microbial mats were obtained from the Ebro delta, located in the northeast of Spain , near the Salines de la Trinitat, in the Alfacs Peninsula, on May 2006. Samples were taken in 17 cm\u00d712 cm\u00d78 cm polypropylene boxes and carried to the laboratory. Once in the laboratory, different microcosm experiments were prepared, one of them unpolluted and used as a control experiment, and the other two polluted with 25 mM Pb and 10 mM Cu, respectively. The latter microcosms were maintained in polluted conditions for three days, and afterwards metal solutions were removed. Samples from microcosms were taken with glass cores of diameter of 6 cm on days 0 (before contamination), 7, 10 and 112 for further analysis.3)2 and CuSO4  with deionized water and were sterilized by filtration with polycarbonate membrane filters. Heavy metal stock solutions were prepared 24 hours before to be used and kept in darkness at 4\u00b0C.We selected copper and lead on the basis of their biological functions and effects. Copper is an essential metal with known biological functions (micronutrient) in all organisms. In cyanobacteria, copper is a component of plastocyanin in the photosynthetic electron-transport chain; however, at high concentrations, it has toxic effects both on animals and plants. Lead is a toxic metal with no biological function. Lead and copper stock solutions were prepared as Pb oil immersion objective lens, excited with a diode 561 nm, and were viewed in a Leica TCS SP2 AOBS . The emission fluorescence was captured between 590 and 800 nm (autofluorescence). Natural fluorescence of chlorophyll xyz function of CLSM was used. Images were acquired in 512\u00d7512 pixel and 8 bit format. Each stack consisted of 21 optical sections (image xy) on the z dimension, whose value corresponded to a thickness of 20 \u00b5m. The samples from microcosms were taken at depth intervals (at each 250 \u00b5m) from the first mm of depth and four replicates of each depth were done. The images obtained were used to determine cyanobacterial biomass in the first mm of depth of the microcosms, where cyanobacteria were dominant.In order to obtain optical series (stacks of images), the ImageJ v1.37, was used to determine cyanobacterial and Microcoleus sp. biomass from mat samples, according to Sol\u00e9 et al. \u22123 to convert it to biomass Once the images of cyanobacteria were obtained by CLSM, a free image-processing and analysis program, Microcoleus sp. biomass determination, the binary stacks were obtained using the same optimum threshold as in total biomass determination. In this case, Microcoleus sp. was selected from the stack of images, whilst the remaining genera of cyanobacteria were erased. Finally, the Microcoleus sp. biomass was determined following the same protocol used for total cyanobacterial biomass estimation.For Microcoleus sp. biomass profiles were represented using KaleidaGraphs software .Total cyanobacterial biomass and the"}
+{"text": "Currently the majority of cancer patients are considered ineligible for intensive care treatment and oncologists are struggling to get their patients admitted to intensive care units. Critical care and oncology are frequently two separate worlds that communicate rarely and thus do not share novel developments in their fields. However, cancer medicine is rapidly improving and cancer is eventually becoming a chronic disease. Oncology is therefore characterized by a growing number of older and medically unfit patients that receive numerous novel drug classes with unexpected side effects.All of these changes will generate more medically challenging patients in acute distress that need to be considered for intensive care. An intense exchange between intensivists, oncologists, psychologists and palliative care specialists is warranted to communicate the developments in each field in order to improve triage and patient treatment. Here, we argue that \"critical care of cancer patients\" needs to be recognized as a medical subspecialty and that there is an urgent need to develop it systematically.As prognosis of cancer improves, novel therapeutic concepts are being introduced and more and more older cancer patients receive full treatment the number of acutely ill patients is growing significantly. This development a major challenge to current concepts of intensive care and it needs to be redefined who of these patients should be treated, for how long and how intensively. The discovery of the human genome and the development of high-power bioinfomatics tools have led to an exponential growth of biomedical knowledge. One of the areas that has benefited the most over the last few years has been cancer research which is now translating into a dramatic development of clinical oncology. As a result targeted therapy and personalized medicine will eventually transform cancer into a chronic or even curable disease.While improved overall survival and reduced acute toxicities are already visible in certain, defined clinical scenarios -3, seconHere, we propose that one of the most striking consequences is the urgent need for new critical care concepts for cancer patients. This hypothesis is supported by the following arguments:chronic disease. However, in accordance with current recommendations advanced stage cancer patients are frequently denied admission by intensive care units that are normally run by non-oncologists [Some cancers are likely to become a ologists -6. When palliative care need to step back or even come closer? In terminally ill lung cancer patients for example the frequency of end-of-life-admissions to hospices as well as ICUs have been reported to increase substantially suggesting that both specialities are needed for this patient cohort [When critical care comes into play does t cohort . Early it cohort . This emscoring systems appears to be difficult as they are not developed for this cohort and often lead to wrong predictions. Therefore, they are the subject of an ongoing debate and further improvement appears necessary [In cancer patients neutropenia, autologous bone marrow transplantation and the characteristics of the underlying malignancy now have limited influence on the acute outcome while the number of organ failures and severity of infection are correlate with survival -12. ICU ecessary -22. Evenecessary ,22.physician's perception of a critically ill patient's prognosis has been shown to influence survival more strongly than the baseline disease severity, development of organ dysfunction or use of vasopressors [This is of particular importance as the treating pressors . The propressors . A reappinternal and geriatric medicine into the world of cancer treatment [Life expectancy is rising globally and in 2025 1.2 billion people will be older than 60 years of age [reatment . So far These issues can only be addressed when medical oncology and geriatric as well as intensive care medicine start working closely together to develop common, interdisciplinary algorithms.novel drug classes is entering the market and novel, unexpected serious adverse events (SAE) are encountered [An increasing number of ountered -29. A clpersonal relationships with the treating nurses and physicians are common. If such patients enter the ICU the physician-patient relationship becomes extraordinarily difficult as the ICU days mean much more suffering and pain than regular treatment. It is hard to imagine that this stress can be tolerated by the caregivers without long-term psychological damage or at least specialized psychological guidance. ICUs specialized in the challenges of cancer medicine would have the opportunity to develop psychological support strategies so far not existent in critical care.To date future intensive care patients frequently enter the hospital via the emergency room in critical conditions necessitating sedation and respiratory support. A personal relationship to the ICU staff is therefore only developed in select cases. Cancer patients are treated long-term and The dynamic of this development is underlined by the fact that substantial progress has already been made both in intensive care and hematology/oncology which already results in an improved survival of critically ill cancer patients -32.To best address the above challenges we propose a four step strategy:early and aggressive treatment has been reported to be beneficial for cancer and non-cancer patients alike [reevaluation after a limited treatment phase appears to allow for more solid judgment.1. Permissive ICU admission policies for cancer patients and ts alike ,33,34. Dts alike ,30,35. Taddress ICU admission and code orders early to learn the patient's and family's opinion and to give them time to consider. This information should help define preliminary treatment goals upon admission and refine them after an early treatment phase of 4-6 days.2. Oncologists spend more time talking to the patients and their families than colleagues from many other specialties. However, the aspect of intensive care and resuscitation are rarely addressed in these discussions. Therefore, end-of-life decisions and do-not-resuscitate orders are often issued by the treating intensivist alone ,37. This3. How can oncological knowledge best be brought to the ICU and how can critical care be optimized for cancer patients? The already growing number of cancer patients undergoing critical care currently necessitates a 'distribution' of these patients to IC wards of various specialties. This development is viewed with concern, not only by the patients and their families, but also by the responsible physicians, and is certainly not a desirable development.Oncologists and intensivists are usually distinct types of physicians that clearly know why they do not want to perform their colleagues' work. While oncologist have significant time to research and identify the optimal strategies for their patients, intensivists sometimes need to make quick decisions based on little or -on the contrary-, extremely complex, but inconclusive information. Oncologists spend a lot of their time talking with patients and families while intensivists focus on delicate, invasive procedures. Therefore, they share little common ground and patients either benefit from the one set of skills or the other, rarely both at the same time.The most obvious solution would be to have common rounds and prime different mind sets by common rotations during training. Intensivists should be encouraged to acquire practical and theoretical knowledge of haematology and oncology as part of their specialty training and vice versa.An alternative approach is being pursued at our institution where a medical ICU (MICU) is integrated into the oncology service. The department of medical oncology is part of a regional comprehensive cancer center and the MICU part of a portal for emergency medicine and intensive care. Thus there are closest interactions with neighboring specialties e.g. radiotherapy, gastroenterology, palliative care, surgery, psychology on the one hand and anesthesia, emergency medicine, general as well as specialized surgical, neurological and cardiac intensive care on the other. The ICU is directed by physicians that are trained medical oncologists and intensive care specialists. An increasing number of interns that were once dedicated to become oncologists have now discovered their interest and talent for the other specialty and are pursuing such a double qualification. This is a development we have anecdotally seen in the field of bone marrow transplantation where haematologist had become familiar with the treatment of their critically ill patients due to isolation policies. However, in this scenario few doctors treat highly selected, fit patients for a limited number of ICU diagnoses such as sepsis or neutropenic colitis. A third approach would be a core training in one specialty and an supplementary education in the other. Best such training should be implemented in major medical centers as these often have a culture of intensive training and sufficient resources to develop and implement novel structures. If successful this process can spread to other tertiary and even secondary medical centers. \"Critical Cancer Care\" can only be developed as a joint effort by intensivists and oncologists. Depending on the national system it could be a two year secondary subspecialty training after internal medicine/hematology/oncology or internal medicine/intensive care or anesthesiology/intensive care. Training should be organized by both specialities and should consist of theoretical instruction in the new subject and a common practical training including rotations of e.g. six months to non critical cancer care e.g. oncology clinic for intensivists and anesthesiology for the oncologists. This is in contrast to other subspecialties such as neurosurgical or cardiac critical care that are based on skills taught in the \"mother\" specialty and that are linked to acute medicine and critical care on a daily basis. Also knowledge relevant to the treatment of cardiologic patients is more familiar to intensivists than the treatment of solid tumors.To measure the impact of such a structure pilot medical centers should determine the ICU length of stay, respirator days and in hospital mortality as well as patient and referring oncologist satisfaction. The period prior to the implementation of this specialty program should be compared to the period thereafter.'critical care of cancer patients' needs to be recognized, defined and developed. The foreseeable development of cancer medicine necessitates that qualified critical care will eventually be available for a rapidly growing number of cancer patients.Whichever approach will demonstrate successful it is clear that the specialty of physicians specialized in defined organ systems such as urologists, pulmologists, gastroenterologists, gynecologists etc. It appears to be most beneficial for the critically ill cancer patients to bring these specialties into the above scenario e.g. by regular consultations. First, they are needed to participate in the treatment as referring oncologists and second they can contribute expertise concerning organ-specific complications such as airway stenosis, bleeding from gastrointestinal tumors, urinary tract obstruction etc.4. Most patients with advanced cancers are treated in an outpatient setting by medical oncologists. However, inpatient treatment is often performed by Taken together, the number of cancer patients needing critical care is dramatically rising and as a consequence the array of open questions regarding the optimal management of such patients is growing. These uncertainties can only be addressed successfully when the oncology and intensive care communities start developing 'critical cancer care' concepts as a long-term, joint effort.Cancer medicine is rapidly improving and cancer is eventually becoming a chronic disease. Oncology is therefore characterized by a growing number of older and medically unfit patients that receive numerous novel drug classes with unexpected side effects. These changes will most likely generate more medically challenging patients in acute distress that need to be considered for intensive care. An intense exchange between intensivists, oncologists, psychologists and palliative care specialists is warranted to communicate the developments in each field in order to improve triage and patient treatment. We postulate that \"critical care of cancer patients\" needs to be recognized as a medical subspecialty and that there is an urgent need to develop it systematically.The authors declare that they have no competing interests.This manuscript has not previously been presented nor is it currently under consideration elsewhere.MK, ASV, MBB and MH all wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/612/prepub"}
+{"text": "Plasmodium falciparum-infected erythrocytes (IE) plays a crucial role in anti-malarial immunity by promoting clearance of blood-stage infection by monocytes and macrophages. The effects of phagocytosis of opsonized IE on macrophage pro-inflammatory cytokine responses are poorly understood.Antibody opsonization of Phagocytic clearance, cytokine response and intracellular signalling were measured using IFN-\u03b3-primed human monocyte-derived macrophages (MDM) incubated with opsonized and unopsonized trophozoite-stage CS2 IE, a chondroitin sulphate-binding malaria strain. Cytokine secretion was measured by bead array or ELISA, mRNA using quantitative PCR, and activation of NF-\u03baB by Western blot and electrophoretic mobility shift assay. Data were analysed using the Mann\u2013Whitney U test or the Wilcoxon signed rank test as appropriate.Unopsonized CS2 IE were not phagocytosed whereas IE opsonized with pooled patient immune serum (PPS) were (Phagocytic index (PI)=18.4, [SE 0.38] n=3). Unopsonized and opsonized IE induced expression of TNF, IL-1\u03b2 and IL-6 mRNA by MDM and activated NF-\u03baB to a similar extent. Unopsonized IE induced secretion of IL-6  but no IL-1\u03b2 or TNF, whereas PPS-opsonized IE induced secretion of IL-1\u03b2 (18.6 pg/mL [34.2-14.4]) and TNF (113 pg/ml [421\u201317.0]) and increased IL-6 secretion . Opsonized, but not unopsonized, CS2 IE activated caspase-1 cleavage and enzymatic activity in MDM showing that Fc receptor-mediated phagocytosis activates the inflammasome. MDM attached to IgG-coated surfaces however secreted IL-1\u03b2 in response to unopsonized IE, suggesting that internalization of IE is not absolutely required to activate the inflammasome and stimulate IL-1\u03b2 secretion.It is concluded that IL-6 secretion from MDM in response to CS2 IE does not require phagocytosis, whereas secretion of TNF and IL-1\u03b2 is dependent on Fc\u03b3 receptor-mediated phagocytosis; for IL-1\u03b2, this occurs by activation of the inflammasome. The data presented in this paper show that generating antibody responses to blood-stage malaria parasites is potentially beneficial both in reducing parasitaemia via Fc\u03b3 receptor-dependent macrophage phagocytosis and in generating a robust pro-inflammatory response. Unprimed MDM exposed to human erythrocytes as a negative control secreted no IL-1\u03b2 and this was not increased following incubation with CS2 IE  activation by the inflammasome . Since IP. falciparum IE are well understood, however the pathways regulating the subsequent inflammatory response, important for control of parasitaemia, are not. Here it is shown that unopsonized CS2 IE, although not internalized by human MDM, stimulate pro-inflammatory cytokine mRNA expression. This is associated with a low level of IL-6 secretion but no IL-1\u03b2 or TNF secretion. Opsonization by immune serum increases IE internalization via Fc\u03b3 receptors without increasing cytokine mRNA levels, and activates components of the inflammasome leading to IL-1\u03b2 secretion. These data are consistent with recently published studies [The mechanisms of phagocytosis of  studies  in whichP. falciparum strain, obtained by selection for CSA binding [The present study investigated cytokine responses to purified CS2 trophozoite-infected erythrocytes. CS2 is a laboratory-derived  binding  similar et al. demonstrated that cytokine production by murine bone marrow-derived dendritic cells, in response to schizont bursts, were mainly due to recognition by a TLR9-dependent mechanism of protein DNA complexes released from merozoites [These observations may suggest that surface pattern recognition receptors, in addition to endosomal receptors, play a role in cytokine elicitation in response to IE. It has been shown that toll-like receptor 2 (TLR2) stimulates pro-inflammatory cytokine production in response to parasite-derived glycosylphosphatidylinositol  and TLR4rozoites ,49 and trozoites . In the rozoites . The lacrozoites .Additional signalling required for robust pro-inflammatory responses may be downstream of Fc\u03b3 receptors and/or be activated by internal pattern recognition receptors recognizing ligands in the IE that are released following ingestion of the cells. Whether there is a qualitative difference in the cytokines produced in response to these different pathways remains to be determined. Recent studies have suggested that murine dendritic cells require the presence of multiple Toll-like receptors to induce TNF secretion in response to schizont stage murine malaria parasites since NF-\u03baB nuclear translocation, TNF secretion and dendritic cell activation are abrogated in bone marrow-derived dendritic cells obtained from TLR9, TLR4 and MyD88 knockout mice . These dData reported herein, using the CS2 parasite strain, differ from those reported using CD36-binding isolates . In the The data presented in this paper show that generating antibody responses to blood-stage malaria parasites is potentially beneficial both in reducing parasitaemia via Fc\u03b3 receptor-dependent macrophage phagocytosis and in generating a robust pro-inflammatory response.CSA: Chondroitin sulphate A; IE: Trophozoite-stage infected erythrocytes; IE-PPS: IE opsonized with pooled immune serum; EMSA: Electrophoretic mobility shift assay; MDM: Monocyte-derived macrophages; IQR: Interquartile range; IRAK4: Interleukin-1 receptor-associated kinase-4; IL-1\u03b2: Interleukin-1\u03b2; TNF: Tumour necrosis factor ; IL-6: Interleukin-6.The authors declare that they have no competing interests.JZ and LEL performed experiments and participated in data analysis. WH performed experiments. SJR designed the study, participated in data analysis and helped to draft the manuscript. AJ designed the study and drafted the manuscript. All authors read and approved the final manuscript."}
+{"text": "Brassica plants. We also specify the advantages and disadvantages of PTR-MS analyses and new technological developments to overcome their limitations.Root herbivores are notoriously difficult to study, as they feed hidden in the soil. However, root herbivores may be traced by analyzing specific volatile organic compounds (VOCs) that are produced by damaged roots. These VOCs not only support parasitoids in the localization of their host, but also may help scientists study belowground plant-herbivore interactions. Herbivore-induced VOCs are usually analyzed by gas-chromatography mass spectrometry (GC-MS), but with this off-line method, the gases of interest need to be preconcentrated, and destructive sampling is required to assess the level of damage to the roots. In contrast to this, proton-transfer-reaction mass spectrometry (PTR-MS) is a very sensitive on-line, non-invasive method. PTR-MS already has been successfully applied to analyze VOCs produced by aboveground (infested) plant parts. In this review, we provide a brief overview of PTR-MS and illustrate how this technology can be applied to detect specific root-herbivore induced VOCs from  We can approximately convert emissions by the following formulaA conversion of the unit commonly used for VOC emissions, in ng\u00b7gZ)-3-Hexenol, for example, was emitted at a rate of 153.6\u00a0ng\u00b7g\u22121 [dw]\u00b7h\u22121, which is 43\u00a0% of all GLVs in the herbivore treatment, but when expressed as a mixing ratio (ppbv), the same compound constitutes 53\u00a0% of all GLVs , which are one of the most widespread VOC classes in the plant kingdom, at least above ground Table\u00a0. BecauseDue to low emission rates, the VOCs sampled from the plant headspace usually need to be pre-concentrated on adsorbents before they can be analyzed on gas chromatography (GC) platforms. Most commonly, plant VOCs are sampled on tubes filled with polymer materials, such as Tenax, Porapaq, Carbopack, and charcoal, or on solid phase micro-extraction fibers , which results in extensive fragmentation providing rich ion fragments, PTR-MS relies on chemical ionization (CI), a soft ionization method with few or no ion fragments in the mass spectra. A detailed description of the PTR-MS technology has been published elsewhere . Typically, only molecules with a proton affinity higher than that of water  will be ionized by proton-transfer-reactions with H3O+ ions. Organic compounds such as aldehydes, ketones, alcohols, oxygenated aromatic and aliphatic compounds will be readily protonated .In addition to the normal proton-transfer-reaction, the Hber Fig.\u00a0, no. 3. via dehydration, whereas acetaldehyde or acetone is less likely to dissociate , each at a concentration of 1 ppmv . The calibration factors obtained for the fixed set of compounds in the certified gas mixture can be used to calculate the calibration factors of other compounds, by taking into account their collision rate constants, transmission efficiency factors, and fragmentation ratios. In this way, ion intensities  can be converted to absolute concentrations as gas mixing ratios . PTR-MS can operate in two modes, namely the full mass scan and selective ion monitoring (SIM). The first scans the relative abundance of all detectable masses, and should be regarded as a fingerprint of a given trace gas sample . Association processes with water are quite sensitive to higher collision energies (E/N), thus, if the intensity of a signal decreases with higher E/N, the signal is contributed by a compound associated with one or more water molecules.13C incorporation into a molecule rises in a linear fashion with the number of carbon atoms in that molecule. For example, with the natural 13C abundance of 1.1\u00a0%, a molecule containing 5 carbon atoms, such as isoprene (M\u2009=\u200968) has a chance of 5.5\u00a0% to contain exactly one 13C. With PTR-MS, isoprene is detected as C5H9+ at m/z\u2009=\u200969, however, this signal also can be attributed to a water\u2013methanol cluster-ion, CH3OH (H2O)H+. If the ratio between m/z\u2009=\u200969 and its isotope at m/z\u2009=\u200970 indicates a 13C abundance close to the expected value for a 5-carbon compound (5.5\u00a0%) the signal measured at m/z\u2009=\u200969 is more likely to be derived from isoprene. Additionally, in complex gas mixtures, such as the ones derived from plant headspaces or human breath, it is common practice to proceed along these lines for compound identification . To overcome these limitations, several new technologies have been developed. Combining PTR-MS with a GC, in which the VOCs are first separated by their retention time in the GC and then detected one by one by PTR-MS, avoids the overlap of different compounds and fragments  is commonly employed to ionize compounds. In addition, other ions such as NO+ and O2+ can be produced in the ion source with the switchable reagent ions (SRI) technology  from different compounds can be superimposed on one Since its development, PTR-MS has found many applications in a wide range of fields, including medicine  immediately after leaf wounding. With PTR-MS, this process has been studied at a high time-resolution, which yielded new insight into the regulation of this pathway  during infestation with a belowground herbivore, the larvae of the cabbage root fly (Delia radicum). The root headspace of infested and non-infested plants was sampled from a cuvette fitted around the base of the stem  and dimethyltrisulfide  displayed considerable increases in emission rates due to herbivore feeding. In addition, we also found that the biosynthetically related compound methanethiol (m/z\u2009=\u200949) was emitted at higher rates when root fly larvae were feeding. Previous GC analyses have not detected methanethiol, which might originate from the compound selectivity of the adsorbents that have been used for collection. Interestingly, we also found a considerable increase in m/z\u2009=\u200960, which is related to glucosinolate breakdown products  converts the glucosinolates that are stored in the vacuoles into toxic and volatile products, such as isothiocyanates and nitriles  or of jewel beetles (Phaenops cyanea), demonstrates that insect antennae are capable of detecting, for instance, the GLV (Z)-3-hexen-1-ol at around 1 ppmv and 1 pptv, for the two species, respectively. Compared to that, the detection limits in PTR-MS already are in a similar range of several parts per trillion volume, depending on the properties of the instrumentation.As outlined, PTR-MS has in the recent years opened an avenue for new insight into fast changing, highly dynamic processes involved in plant VOC emissions caused by plant-environment interactions. Here, we show that, due to its sensitivity and the ability to record real-time responses, PTR-MS is an excellent technique to non-invasively trace the feeding activities of cryptically feeding root herbivores by measuring VOC emissions from the root headspace. Certainly, PTR-MS also has its practical and technical limitations. Besides difficulties in linking masses without doubt to compounds, many quadrupole-based systems lack sensitivity in the higher mass range (above 120\u00a0amu), which is relevant for plant-herbivore interactions, as many biologically important compounds, such as several isothiocyanates or generally hemiterpenoids and sesquiterpenoids are difficult to detect. These shortcomings can be overcome partially by combining on-line sampling with PTR-MS and off-line GC-MS methods, or by use of high sensitivity PTR-MS instruments with mass analyzers, such as distinctive quadrupoles, triple quadrupole technology, ion trap, or time-of-flight, which can provide sensitivity also in the higher mass range (Tani et al., Finally, time-resolved and sensitive on-line sampling of root-induced volatiles with PTR-MS will certainly contribute to our understanding of the role of VOCs in belowground multi-trophic interactions. In particular, linking real-time responses in the emission of VOCs to immediate behavioral responses of herbivores, and to the higher trophic levels of parasitoids and predators will unravel further details of the VOC \u2018language\u2019 among plants and between plants and insects. This may be achieved by \u2018sniffing out\u2019 the VOCs in parallel with olfactometer assays, a prime example of which is the development of a six-arm olfactometer, simultaneously equipped with a VOC sampling unit (Turlings et al.,"}
+{"text": "R2 is =0.616. In this erratum we report corrected In the paper titled \u201cReference Values of Pulse Wave Velocity in Healthy People from an Urban and Rural Argentinean Population\u201d there was an error in Figure\u20091. The correct value of In the paper entitled \u201cReference Values of Pulse Wave Velocity in Healthy People from an Urban and Rural Argentinean Population\u201d a mistake was introduced in Figure\u20093. In this erratum we report modified"}
+{"text": "Batrachochytrium dendrobatidis (Bd), and for which there is an urgent need to develop mitigation methods. We treated tadpoles of the common midwife toad (Alytes obstetricans) with antifungal agents using a capture-treat-release approach in the field. Antifungal treatment during the spring reduced the prevalence of Bd in the cohort of tadpoles that had overwintered and reduced transmission of Bd from this cohort to the uninfected young-of-the-year cohort. Unfortunately, the mitigation was only transient, and the antifungal treatment was unable to prevent the rapid spread of Bd through the young-of-the year cohort. During the winter, Bd prevalence reached 100% in both the control and treated ponds. In the following spring, no effects of treatment were detectable anymore. We conclude that the sporadic application of antifungal agents in the present study was not sufficient for the long-term and large-scale control of Bd in this amphibian system.Emerging infectious diseases can drive host populations to extinction and are a major driver of biodiversity loss. Controlling diseases and mitigating their impacts is therefore a priority for conservation science and practice. Chytridiomycosis is a devastating disease of amphibians that is caused by the fungal pathogen  An increasing number of emerging fungal diseases is causing host mass mortality, extirpating local populations, and threatening biodiversity2. These diseases can cause population declines because all host individuals can become infected before host population size falls below a threshold that would halt an epizootic2. Chytridiomycosis is an emerging infectious fungal disease of amphibians which has become a model system for the study of disease-induced host population decline and extinction3. The etiological agent of chytridiomycosis is the chytrid fungus Batrachochytrium dendrobatidis (Bd) (Chytridiomycota), which can infect larval, metamorph, and adult amphibians4 (a second pathogenic chytrid fungus was recently described5). Bd infects a wide range of amphibian species, some acting as a reservoir for the pathogen6. Even though Bd is responsible for amphibian mass mortality, population extirpations, global extinctions of species, and the loss of phylogenetic diversity8, there is no established method to mitigate the effects of the disease in wild populations12. However, it is known from agriculture that fungal pathogens of plants can be controlled using fungicides. Fungicides are known to reduce Bd infection13 and two proof-of-concept studies have shown that fungicides can be used to control or even eradicate Bd in the wild15.Emerging infectious diseases have been recognized as important drivers of the loss of biodiversityBd infection prevalence of the different host life history stages and the sensitivity of the host population growth rate to stage-specific disease-induced mortality16. Bd does not seem to affect the survival of tadpoles, which allows this stage to act as an intraspecific reservoir for Bd19. In contrast, Bd reduces survival of post-metamorphic individuals21, the life history stage to which the amphibian population growth rate is highly sensitive24. Fungal treatments that clear the infection or that reduce the infection load in tadpoles before metamorphosis may therefore reduce mortality in post-metamorphic juveniles and increase population viability16. Such treatments may further reduce disease prevalence in amphibian species where Bd transmission occurs between overlapping generations of tadpoles.The efficacy of mitigation depends on the Alytes obstetricans) is a good model system for studying the epidemiology and control of Bd because this host is highly susceptible and commonly infected with this pathogen26. This species has suffered Bd-driven population declines and extirpations and is red-listed in some European countries29. The tadpoles of this species overwinter so that in the summer there are often multiple cohorts of tadpoles present in the same pond. After overwintering, Bd prevalence is often very high, which may lead to pathogen transmission to young-of-the-year tadpoles30. In a previous laboratory study, we had shown that treatment with an antifungal agent  could reduce Bd prevalence and load in A. obstetricans tadpoles31. The purpose of the present study was to use antifungal agents to reduce the prevalence and infection intensity (zoospore load) of Bd in natural populations of the common midwife toad. We predicted that antifungal treatment of tadpoles would reduce Bd prevalence and load, allow tadpoles to metamorphose into a disease-free state, and reduce Bd transmission between overlapping cohorts of tadpoles.The common midwife toad  or the young-of-the-year cohort (n\u2009=\u20091484), hereafter referred to as the 2010 and 2011 cohort, respectively. The tadpoles in the 2010 cohort were captured in the months of May, June, and July 2011, whereas the tadpoles in the 2011 cohort were captured in the months of August 2011 to April 2012. The 2010 and 2011 cohorts were analysed separately because they occurred in the pond at different times of the year.A total of 2096 Bd if it had a Bd zoospore load >0.01. The Bd prevalence refers to the proportion of tadpoles that were infected with Bd. In the statistical analysis of micro-parasite infections, it is standard practice to separately analyse pathogen prevalence and pathogen load (or pathogen burden). The analysis of Bd prevalence was based on all of the tadpoles in the data set (n\u2009=\u20092096 tadpoles), whereas the analysis of Bd zoospore load (see below) was based on the subset of tadpoles that were classified as being infected with Bd.A tadpole was classified as infected with Bd in the tadpoles was lowest in August and September and highest in the winter in most ponds . Similarly, for most of the statistical analyses below, the treatment status of a tadpole (fungicide versus control) depends upon its pond of origin and not whether that particular individual was treated with fungicide or not. For this reason, these statistical analyses are labelled as occurring at the pond level. This approach is justified if fungicide treatment of a fraction of the tadpole population in the treatment ponds reduces the risk of Bd transmission to the untreated tadpoles (i.e. herd immunity).The prevalence of nds Fig.\u00a0. In the Bd as a function of the fungicide treatment , the covariate time, and their interaction. Here all tadpoles belonging to the same pond were assigned the same fungicide treatment status . The 2010 and 2011 cohorts were analysed separately because they occurred at different times of the year. The interpretation of the parameter estimates is as follows: the intercept refers to the prevalence of Bd in the control ponds on the intercept date (see below for the definition of the intercept date). The fungicide treatment is the difference in Bd prevalence between the treated ponds and the control ponds on the intercept date. The covariate time is a slope that indicates whether the prevalence of Bd increases or decreases over time. A significant interaction between fungicide treatment and time indicates that the difference in Bd prevalence between the control ponds and treated ponds changes over time. In the case of a significant fungicide treatment:time interaction, it is not possible to make a general statement about the effect of the fungicide treatment.We used generalized linear mixed effects models with binomial errors to model the prevalence of 2) to account for the curvature of the relationship between Bd prevalence and time , it became necessary to include a quadratic effect of time , the Bd prevalence was lower in the treatment ponds (compared to the control ponds) and therefore it had to increase rapidly over the fall to reach the same high level  in the winter . The seasonal pattern in the Bd zoospore load resembled the seasonal pattern of the Bd prevalence , the covariate time, and their interaction. All tadpoles belonging to the same pond were assigned the same fungicide treatment status . The 2010 and 2011 cohorts were analysed separately because they occurred at different times of the year. The interpretation of the parameter estimates is the same as described for the prevalence of Bd. The intercept date for the 2010 cohort is May 5, 2011, whereas the intercept date for the 2011 cohort is December 27, 2011  with normal errors to model the log10-transformed Bd-infected tadpoles in the 2010 cohort, examination of the model selection results , July 2011 , and October 2011 to April 2012 (recapture of 2011 cohort tadpoles marked 0 or 1 times in September 2011).In the three treatment ponds , tadpoles were marked with visible implant elastomer (VIE) tags to indicate whether they had been treated with fungicide once or twice. The VIE tags allowed us to test whether the number of times that an individual tadpole had been treated with fungicide influenced its 2, 113\u2009=\u200918.29, p\u2009<\u20090.001). The fungicide treatment decreased the Bd zoospore load of the tadpoles in the SagO and Herg ponds but increased the Bd zoospore load in the SagU pond . Similarly, for the 2011 cohort tadpoles, the fungicide reduced the Bd zoospore load in individual tadpoles but the effect was not significant . We point out that in Fig.\u00a0Bd zoospore load follows the expected pattern in 5 of the 6 panels (the SagU pond in the top row is the exception). In summary, tadpoles that had been treated with fungicide once or twice, generally had lower zoospore loads than the tadpoles that had never been treated but the effect was not significant.For the 2010 cohort tadpoles captured in June 2011, the interaction between pond and fungicide treatment was significant  and it is therefore a challenge to separate their effects. We tried to model the temporal dynamics of Bd infection as thoroughly as possible to make the analysis as conservative as possible with respect to detecting an effect of temperature .We had originally wanted to include pond temperature as a covariate in the preceding analyses. Unfortunately, the temperature data logger failed for the SchU pond. In the preceding analysis, we decided that it was more important to include the SchU pond than temperature. The purpose of the present analysis was to estimate the effects of temperature on Bd prevalence of the 2011 cohort, there was strong support for a positive linear effect of time  rather than clear infection.Developing treatments against fungal diseases is a priority for both humans and wildlifeBd zoospore load was weaker and inconsistent , the Bd zoospore load was actually higher in the treated ponds than the control ponds. Overall, the results are consistent with the interpretation that the antifungal treatment cleared weak infections (low Bd zoospore load) but had no measurable effect on the heavy infections (high Bd zoospore load). The analysis at the individual level generally found that the median Bd zoospore load declined with the number of fungicide applications, but the results were not statistically significant . By December, almost 100% of all the tadpoles were infected with Bd in both the control ponds and the treated ponds. The monotonic rise in the Bd prevalence from summer to winter suggests that there is a very high force of infection in these tadpole populations. The observation that Bd prevalence and Bd zoospore load remained high throughout the winter . In the present study, the Bd infection spread rapidly during the months of August, September, and October, when pond temperatures were in the range of the optimal growth conditions  and winter (December and January), the Bd zoospore load remained high but did not change in the tadpole population 33. Lam et al.45 assumed that p\u2009\u2248\u20090.8 which led Woodhams et al.9 to suggest that R0\u2009\u2248\u20095 for Bd. Since R0 for Bd is unknown, it is hard to predict whether successful control of Bd might be achieved (reservoir hosts and persistence of zoospores in the environment make control even more difficult). Thus, estimating R0, a basic epidemiological parameter, would be of great importance to better inform the science and practice of disease control.The present study has shown that a capture-treat-release approach using antifungals can be used to transiently decrease the prevalence of Bd treatments have been tested in the laboratory12 but the number of successful attempts at Bd mitigation is still very low12. It is therefore important to do more field trials of mitigation methods15 that are ethically and legally acceptable12. Since the Bd-host interaction is strongly context-dependent46, mitigation strategies should be tested under a variety of environmental conditions to determine whether a method can be used only locally or whether it is transferrable to other systems. Last but not least, even if recent studies suggest that Bd mitigation in natural populations may be possible, prevention is better than cure47. Thus, preventing the emergence of pathogens in na\u00efve wildlife populations is the best way to protect biodiversity against emerging infectious diseases48.Many potential anti-A. obstetricans and Bd30 were selected: Sagerh\u00fcsli upper (SagO), Sagerh\u00fcsli lower (SagU), Hergiswald (Herg), Schauensee upper (SchO), Schauensee lower (SchU), and Hinter Rohren (HiR). These ponds were similar in elevation (range\u2009=\u2009558\u2013798\u2009m), surface area (6\u201330\u2009m2), and depth (0.5\u20132.5\u2009m). The water temperature of each of the six ponds was measured six times per day over the duration of the study using HOBO H8 temperature data loggers . The tadpoles in SagO, SagU, and Herg were treated with an antifungal agent whereas SchO, SchU, and HiR served as control ponds.The study was carried out in canton Lucerne in central Switzerland. At four different sites, six ponds that were known to contain \u00ae antifungal agent for 8 days in 100\u2009L cattle tanks that were filled with 50\u2009L of tap water. We used a concentration of 0.6\u2009ml of General Tonic\u00ae per litre of water31. After 4 days, the tanks were filled with a fresh solution of General Tonic\u00ae water. Cattle tanks were placed near the ponds and covered with a shade cloth. Fungicide-treated tadpoles were marked with visible implant elastomer (VIE) tags before being released into their original ponds49. A different VIE tag colour was used for the fungal treatments of May, June, July and September 2011. Tadpoles were tagged every time that they were captured and treated with antifungals.The study took place from May 2011 to April 2012 and involved two different cohorts of tadpoles: 2010 and 2011. For the 2010 cohort, most of the tadpoles (98.2%\u2009=\u2009612/623) were captured in the months of May, June, and July  and this cohort left the ponds as metamorphs by August 2011. For the 2011 cohort, most of the tadpoles (98.5%\u2009=\u20091484/1507) were captured from August 2011 to April 2012 . Tadpoles in the 2010 cohort were treated with fungicide on three separate occasions , whereas tadpoles in the 2011 cohort were treated once (September). Field-captured tadpoles were treated with General TonicA. obstetricans tadpoles were captured each month except in February 2012 because the ponds were frozen. Tadpoles were captured using dip nets and tested for Bd infection by swabbing the mouthparts with sterile rayon-tipped plain swabs using a plastic applicator . In the treatment ponds, the surveyors swabbed a maximum of 30 previously treated tadpoles (VIE tag present) and 30 untreated tadpoles (no VIE tag) each month. In the control ponds, the surveyors swabbed a maximum of 30 untreated tadpoles each month. We obtained a total of 2132 mouthpart swab samples that were stored at \u221220\u2009\u00b0C until analysis.Over the duration of the study (May 2011 to April 2012), \u00ae Ultra Sample Preparation Reagent Protocol (Applied Biosystems by Life Technologies). The tip of each Bd swab (1\u20132 mm) was cut off using a sterile and single-use surgical carbon blade (Paramount\u00ae). The tips were placed into 60\u2009\u00b5L of PrepManTM Ultra (Applied Biosystems by Life Technologies) with 0.03\u20130.04\u2009g Zirconial/Glas-beads . The samples were shaken at 30\u2009Hz for 45\u2009seconds, using a TissueLyser Adapter Set 2\u2009\u00d7\u200924 (QIAGEN\u00ae) then centrifuged at 13,000\u2009rpm for 1\u2009minute using an ALC Multispeed refrigerated centrifuge. The last two steps were repeated before placing the samples into an Eppendorf Thermomixer Comfort\u00ae at 99\u2009\u00b0C for 10\u2009minutes followed by centrifugation at 13,000\u2009rpm for 3 minutes47. The resultant supernatant was placed into an Eppendorf tube and stored at \u221220\u2009\u00b0C.DNA was extracted from the tadpole mouthpart swabs using the PrepManBd in the tadpole mouthpart swabs following the protocol of Boyle et al.50. The specific primers ITS1\u20133 Chytr and 5.8S Chytr and the minor groove binder probe Chytr MGB2 were used to amplify and detect the 5.8S rRNA gene and the flanking internal transcribed spacer (ITS) of the Bd pathogen50. Each qPCR reaction had a total reaction volume of 20\u2009\u00b5L and contained 10\u2009\u00b5L of 2x Master Mix , 0.9\u2009\u00b5M of each primer, 0.25\u2009\u00b5M of the MGB2 probe, and 5\u2009\u00b5L of DNA (diluted 1:10 in water). The thermocycling conditions were as follows: a preincubation step at 95\u2009\u00b0C for 10\u2009minutes followed by 50 cycles at 95\u2009\u00b0C for 15\u2009seconds and 60\u2009\u00b0C for 1 minute50. The qPCR was performed using a LightCycler\u00ae 96 Real-Time PCR System (Roche Applied Science). All DNA samples were run in duplicate. Each 96-well plate contained 3 negative controls, 3 positive controls, and 4 standards  run in triplicate to quantify the Bd zoospore load. The four standards contained 101, 102, 103, and 104 copy numbers of the target gene.Quantitative PCR (qPCR) was used to estimate the zoospore load of Bd zoospore load was very high for a sample of 606 DNA extractions that had been run in duplicate for the qPCR assay .The repeatability of the Bd zoospore load\u2009>\u20090.01 were defined as being infected with Bd. We analysed two different response variables: the proportion of tadpoles infected with Bd (n\u2009=\u20092096 tadpoles), and the log10-transformed Bd zoospore load for the subset of infected tadpoles (n\u2009=\u20091638 tadpoles). We used generalized linear mixed effects (GLME) models with binomial errors and linear mixed effects models (LME) with normal errors to model the Bd prevalence and the log10-transformed Bd zoospore load, respectively. The 2010 and 2011 cohorts were analysed separately because they occurred at different times of the year . The fixed effects structure included the fungicide treatment , the covariate time, the covariate time2 (only for the 2011 cohort), and their interactions. For the 2011 cohort, the covariate time2 was included because the temporal change in the Bd infection resembled a negative quadratic function. The random effects structure included the categorical factors pond, month, and their interaction. In this study, we expected that there would be no difference between ponds at the beginning of the experiment. The fungicide treatment was expected to gradually reduce the Bd prevalence over time in the tadpoles. Thus, the treatment-by-time interaction was expected to be more informative than the main effect of treatment.The 36 tadpoles that were captured outside the dominant sampling period for their cohort (13 tadpoles for 2010 cohort and 23 tadpoles for the 2011 cohort) were eliminated from the analysis. The final sample size was therefore 2096 tadpoles: 612 and 1484 tadpoles for the 2010 and 2011 cohorts, respectively. Tadpoles with a 2 were highly correlated, which caused problems with model convergence and parameter estimation. These variables were therefore transformed using the poly function in R so that they were orthogonal to each other. The origin (t\u2009=\u20090) of this transformed time scale is day 136 , and day 148 . After transformation, time2 was a negative quadratic (concave down). A positive slope for the transformed time2 variable therefore meant that the Bd prevalence (or Bd zoospore load) was highest in the winter and lower in the fall and spring.Each cohort had its own time scale: day 1  to day 68  for the 2010 cohort, and day 1  to day 246  for the 2011 cohort. For each cohort, the covariate time was expressed in months by dividing the number of days by 30. For the 2011 cohort, time and timeBd infection  over time. We expected the dynamics of the Bd infection to be different between the treatment ponds and the control ponds. In other words, we expected an interaction between the fungicide treatment and time, which indicates that the rate at which tadpoles acquire or lose Bd infections is different between the treatment ponds and the control ponds. In this study, we expected that the interaction term would be more important than the main effect of the fungicide treatment. For each cohort, the meaning of the contrast in the Y-intercept between the control and treated ponds depends on how the time covariate was rescaled. For the 2010 cohort, this contrast measures pre-existing differences in Bd infection between the two types of ponds on May 5, 2011, which was prior to the application of the fungicide treatment. For the 2011 cohort, this contrast compares the Bd prevalence (or log10-transformed Bd zoospore load) between treated and control ponds on December 15, 2011 .We monitored the epidemiology of the 51.We used a model selection approach based on the Akaike information criterion (AIC) to find the most parsimonious model. Models were ranked according to their AIC values and the Akaike weights were calculated for each model. The support for a given explanatory variable of interest is the sum of the Akaike weights of all the models in the set that include that particular explanatory variable. The support for a given explanatory variable can range from low (0.0%) to high (100.0%). We also used the Akaike weights to calculate the model-averaged parameter estimates and their 95% confidence limitsBd zoospore load. We used linear models to test whether the number of fungicide treatments applied to an individual tadpole influenced its log10-transformed Bd zoospore load. This analysis was restricted to the tadpoles in the three experimental ponds . The 2010 and 2011 cohorts were analysed separately. For the 2010 cohort, the analysis was further split for the months of June and July because the number of fungicide applications differed between these two months. For the 2010 cohort, tadpoles captured in June (n\u2009=\u2009172) had been treated never or once, whereas the tadpoles captured in July (n\u2009=\u2009167) had been treated never, once, or twice. For the 2011 cohort, tadpoles captured in the months following the single fungicide treatment in September (n = 569) had been treated never or once.In the three treatment ponds , the marking of the tadpoles with VIE tags allowed us to test whether the number of times that an individual tadpole had been treated with fungicide influenced its Bd prevalence and the log10-transformed Bd zoospore load as a quadratic function of the pond temperature (\u00b0C) and a quadratic function of time (days). The random effects structure was similar to the previous models. We restricted these analyses to the 2011 cohort because there were only 3 time points for the 2010 cohort. Due to a data logger failure, the analysis was restricted to five ponds because mean monthly pond temperature data were not available for SchU. As before, time (range: day 1 to day 246) and time2 were rescaled as orthogonal contrasts, and the same was done for temperature (range: 2.2\u2009\u00b0C to 18.2\u2009\u00b0C) and temperature2. For the 2011 cohort, the origins of the transformed time scale and t\ufeffhe transformed temperature scale are day 137  and 9.48\u2009\u00b0C , and day 151  and 8.75\u2009\u00b0C . After transformation, time2 was a negative quadratic  was highest in the winter and lower in the fall and spring (the reverse was true for the transformed temperature2 variable).We used GLME and LME models with binomial and normal errors to model \u00b0C Table\u00a0 for all \u00b0C Table\u00a0 for the c Figure\u00a0 whereas c Figure\u00a0. A posit52. We used the glmer function and the lmer function in the lme4 package to run the generalized linear mixed effects models with binomial errors\u00a0and normal errors, respectively. We used the model.sel function and the model.av function in the MuMIn package to create the model selection tables and the model-averaged parameter estimates. We used the confint function in the base package to calculate the\u00a095% confidence intervals (CI) for the model-averaged parameter estimates. We used the poly function in the base package to rescale time and time2 (or temperature and temperature2) so that they were uncorrelated with each other.We used R for all statistical analysesExperiments were performed in accordance with the relevant regulations . The study was conducted under the animal experimentation permit number 75/2009 by the veterinary office of the canton Z\u00fcrich; the permit included an approval of the methods. Capture permits (as required by the nature conservation law) were provided by the nature conservation office of canton Lucerne.Supplemental Material"}
+{"text": "Passer domesticus) were exposed to a chronic stress protocol. We took physiological and behavioral measurements before, during, and after the protocol. Blood samples were used to assess four aspects of hypothalamic-pituitary-adrenal (HPA) axis function: baseline corticosterone, stress-induced corticosterone, negative feedback, and the maximal capacity to secrete corticosterone. We also assessed bacterial killing capacity and changes in uric acid concentration. Neophobia trials were used to assess behavioral changes throughout the protocol. We found no significant changes in HPA axis regulation in any of the four aspects we tested. However, we found that uric acid concentrations and neophobia significantly decreased after only four days of the chronic stress protocol, while bacterial killing capacity did not decrease until after eight days of exposure. These results indicate that different components of the stress response can be impacted by chronic stress on different timescales. Our results further indicate the importance of assessing multiple aspects of both physiology and behavior in order to understand how exposure to chronic stress may influence ability to cope with future challenges.Despite decades of research, we still lack a complete understanding of what factors influence the transition of the necessary and adaptive acute stress response to what has become known as chronic stress. This gap in knowledge has illuminated the necessity for studies that examine the thresholds between these two sides of the stress response. Here, we determine how repeated exposure to acute stressors influences physiological and behavioral responses. In this repeated measures study, house sparrows ( Passer domesticus) to this threshold in order to identify key transition points in physiological and behavioral indices.In recent decades, the stress response has become widely studied across many disciplines including ecology e.g.,\u00a0, conservThe stress response involves several behavioral and physiological systems e.g.,\u00a0. We focuCORT helps regulate downstream effects of the stress response, including metabolic, immunological, and behavioral effects . CORT isGallus gallus domesticus) (Sturnus vulgaris)  , but remulgaris) . Additioulgaris) , while oulgaris) . Finallyulgaris)  and incrulgaris)  in rodenThe goal of this study was to expose house sparrows to repeated acute stressors over the course of several days to push them just to the edge of experiencing serious symptoms of chronic stress. Prior studies have shown that different aspects of HPA axis function and regulation are significantly altered after eight to ten days of simulated chronic stress in laboratory and field settings . While wTo test the physiological and behavioral effects of repeated exposure to stressors, 22 wild free-living house sparrows  were caught June 1\u20133 2016 in Medford, MA, USA. Individual birds were randomly assigned to one of two treatment groups and housed in cages in separate rooms according to this distinction. Randomization occurred such that each group contained both males and females and such that no group contained birds that were all captured on the same day. Birds were housed in pairs and/or in view of other birds in cages (45 cm \u00d7\u00a037 cm \u00d7\u00a033 cm) and kept on a natural 15L:9D light cycle. An acclimation period occurred for at least 2 weeks during which birds were disturbed only for routine husbandry. This period was determined based on previous research involving house sparrows, which showed hormonal acclimation occurring after 2 weeks in captivity .This study was a repeated measures design in which the responses of individual birds during the experiment were compared to pre-stress samples. At the end of the initial 2-week acclimation period, a pre-experiment blood sample was taken . Assay sensitivity was 1 ng/ml; baseline and negative feedback samples were often below the level of detection (20 samples) and therefore assigned this floor value. The inter and intra-assay CVs were 7.6% and 3.5% respectively.Radioimmunoassays (RIA) were used to quantify CORT concentrations in the baseline, stress-induced, negative feedback, and Max plasma samples . Brieflyin vitro analyses of bacterial killing capacity. All samples were assayed within 10 days of sampling as longer periods of freezing have been shown to dramatically affect killing  at 37\u00a0\u00b0C for 30\u00a0min. Then, 250 \u00b5L of tryptic soy broth (TSB) was added to each tube, followed by an additional 12-hour incubation at 37\u00a0\u00b0C. Positive controls with only E. coli and TSB were also created. Finally, a NanoDrop spectrophotometer  was used to quantify absorbance of each sample at 300 nm. Killing capacity was calculated as 1 \u2013(absorbance of sample / absorbance of positive control). All samples were run in triplicate when possible; only 4 samples out of all those that were assayed had to be run in duplicate. The inter-assay CV, based on the positive controls, was 11.5%.Baseline plasma samples were also used for  killing . This as killing . Briefly\u00ae Red Uric Acid/Uricase Assay Kit . This assay relies on the conversion of uric acid to hydrogen peroxide, which subsequently reacts with Amplex\u00ae Red reagent in the presence of horseradish peroxidase (HRP) to form a red-fluorescent product. Briefly, plasma samples were diluted with 200 \u00b5L of the provided reaction buffer and a uric acid standard curve was generated using the provided reagents. To have enough reagents to run all samples, the Mastermix was slightly altered to include 150 \u00b5L of Amplex\u00ae Red reagent and 85 \u00b5L of the reaction buffer. To account for this change, spectrophotometric readings were taken at 30 and 45\u00a0min at 560 nm. No statistical differences were found between these samples and therefore all further analysis was completed using the 30-minute sample. A negative control, lacking uric acid, and a maximum-reading positive control were also included on the 96-well plates. Four separate assays were completed and samples were run in triplicate. The inter-assay CV from the positive controls was 7.3%.Baseline plasma samples were used to quantify uric acid using the AmplexIn addition to the physiological data gathered from plasma sampling, changes in behavior were assessed by measuring the birds\u2019 responses to novel objects. Three neophobia trials were conducted prior to any stimuli, after five days of the chronic stress protocol, and after seven days of the chronic stress protocol. Because blood and behavioral samples could not be taken the same day, we were limited which days the neophobia trials could occur. Beginning five days prior to the first neophobia trial, food dishes were removed each night (within 1\u00a0h before lights off) and replaced each morning (within 1\u00a0h after lights on) to acclimate the birds. At this time, the cage liners were also replaced to remove any excess food was removed from the bottom of each cage; this was to ensure that birds would reliably return to their food in the morning.On the night directly before a trial, food dishes were removed and opaque blinders were placed between each cage to prevent the birds from seeing the neighboring trial, which could alter their own behavior . The folThree control trials were also conducted (one each day) prior to each neophobia trial to ensure that the stress treatments did not affect the birds\u2019 normal feeding behavior. During these control trials, food dishes were returned in the morning without a novel object. Neophobia and control trials were conducted on days separate from blood sampling.All statistical analyses were conducted in RStudio . Before F-tests with Kenward-Roger adjusted degrees of freedom. Interactions were checked using the same method, but with an interaction term between group or sex and sample.Before conducting further analyses, we tested whether there was an effect of group or sex in any of the datasets. This was done in order to check if differences in sex or stimulus order (whether cage rolling or cage tapping was experienced first) caused significant effects to physiological or behavioral responses. We used linear mixed effects models (LMM) with individual bird identity as a random effect and group and sample as main effects in separate models  and three negative feedback samples (n\u00a0=\u00a063) did not contain enough plasma to be run. One sample from the stress-induced data was dropped because that bird did not mount an acute stress response to the restraint stimulus and a second sample did not contain enough plasma (n\u00a0=\u00a064). The different CORT responses measured here  play complicated activation, inhibition, permissive, and preparative roles and interact with different physiological systems  while two samples did not contain enough plasma to be run in the uric acid assay (n\u00a0=\u00a064). A single LMM was used to assess changes in killing capacity and uric acid concentrations over the course of the chronic stress protocol; again, individual bird identity was included as a random effect. Tukey\u2019s multiple comparison post-hoc analyses were conducted as indicated above.One sample did not contain enough plasma to be run in the BKA (n\u00a0=\u00a059). Interactions between total perch hops and group, sex, and object were checked with individual LMMs and again a separate LMM was completed to compare activities across sampling periods. Separate models were used to assess changes in the control trials for both the feed latency and perch hopping data sets.Birds that failed to approach the novel object in the allotted time were assigned a ceiling value of 20\u00a0min. Interactions between feed latency and novel object were assessed with separate LMMs and a final LMM was run to compare feed latencies across the sampling protocol. Perch hops were counted during the last 15\u00a0min of the neophobia videos. The first 5\u00a0min were discounted to avoid confounding effects of experimenter presence. All videos were coded by the same person. The movements of two birds were obscured from where the camera was placed and one sample from an additional bird was removed from analysis because they did not display any perch hopping in the allotted time, making log transformation impossible . There were also no significant interactions between group and sample . Therefore, stimulus order was removed from any further analyses of those datasets. We conducted the same check for sex effects and interactions and found none ; sex was therefore removed from any further analyses in all datasets, except for the perch hopping data where we did detect a significant effect of sex.There was no main effect of group in the datasets for stress-induced, negative feedback, and maximum capacity CORT, bacterial killing, uric acid, or neophobia datasets  or group B  across the chronic stress protocol. We also did not detect any statistically significant differences in CORT levels at any of the other three bleed types  over the course of the chronic stress protocol . There was, however, a significant main effect of object . Regardless of object type, feed latency decreased significantly after 4 days of the chronic stress protocol  and sample  nor were there significant interactions between object and perch hops . Perch hopping significantly decreased after the initiation of the stress protocol and remained reduced through the remainder of the protocol , however neither females  nor males  significantly changed their activity during these control trials. We also found a significant interaction between group and perch hopping  during the control trials when no novel objects were present. Further inspection of graphs revealed that this interaction was driven by birds in group A (experienced cage rolling first) increased their perch hopping at the final sample point, while those in group B (experienced cage tapping first) decreased their perch hopping  significantly reduced feedback strength  . Birds tAlthough our original hypotheses for the behavioral measurements were based on prior studies connecting CORT and behavior, there is an alternative interpretation arising from the reactive scope model that could explain the decreases. The data suggest that chronic stress induced house sparrows to take additional risk when foraging. Rather than delaying their approach towards a novel, potentially dangerous, object, these birds more readily risked potential harm to forage. In certain circumstances this could be quite dangerous, particularly in the wild where novel objects might not be so innocuous as a colored food dish, ribbon, or plastic egg.Many behavioral ecology studies have attempted to link CORT to broader indices of behavior, including explorative behavior and foraging activity e.g., . The resWe have shown that both physiology and behavior can significantly change over the course of a short chronic stress protocol. Most importantly, there are different timescales at which the effects of chronic stress are being reflected in the different indices measured here. Additionally, birds became less neophobic and more active. These results suggest that the overall physiology of the birds is entering a new state, perhaps indicative of \u201cchronic stress.\u201d It is possible that these metrics indicate that these birds are closer to the overload threshold of the reactive scope framework. Interestingly, eight days of the chronic stress protocol did not appear sufficient to significantly alter HPA function, although a longer period of stress may have elicited an effect. Furthermore, these results provide important support for the idea that CORT is not the best indicator of chronic stress, particularly considering its complicated relationship with so many other physiological and behavioral systems. Despite decades of research on stress, we still lack the ability to predict under what circumstances pathological symptoms may develop. We hope that these data provide initial steps towards being able to better interpret results pertaining to the vertebrate stress response and their relation to overall individual animal health and fitness.10.7717/peerj.4961/supp-1Data S1Click here for additional data file."}
+{"text": "Food-based dietary guidelines are promoted to improve diet quality. In applying dietary recommendations, such as the MyPlate, the number of servings in a food group is the unit of measure used to make food selections. However, within each food group, different foods can vary greatly in their nutritional quality despite often having similar energy  values. This study aimed to develop a novel unit of measure that accounts for both the quantity of energy and the quality of nutrients, as defined by caloric and micronutrient density, respectively, in foods and to demonstrate its usability in identifying high quality foods within a food group.A standardized unit of measure reflecting the quality of kilocalories for nutrition  was developed through a mathematical function dependent on the energy content  and micronutrient density of foods items within a food group. Nutrition composition of 1806 food items was extracted from the USDA nutrient database. For each food item analyzed, qCaln ratios were calculated to compare qCaln to its caloric content. Finally, a case example was developed comparing two plates adapted from the MyPlate.Examples of food items with highest and lowest qCaln ratios were displayed for five food groups: vegetables, fruits/fruit juices, milk/dairy products, meats/meat alternatives, and breads/cereals. Additionally, the applicability of the qCaln was presented through comparing two plates, adopted from the USDA MyPlate, to show differences in food quality.The newly developed qCaln measure can be used to rank foods in terms of their nutrient density while accounting for their energy content. The proposed metric can provide consumers, public health professionals, researchers, and policy makers with an easy-to-understand measure of food quality and a practical tool to assess diet quality among individuals and population groups.The online version of this article (doi:10.1186/s12937-016-0215-4) contains supplementary material, which is available to authorized users. Identifying best methods to measure food and diet quality is an ongoing scientific and policy-level challenge facing public health professionals, researchers, and decision-makers. Over the years, several approaches and methods have been proposed to assess food and diet quality, including diet quality indices and various nutrient profiling methods.Diet quality indices were developed to measure conformance of individuals and population groups to international and national dietary guidelines and recommendations, such as the U.S. dietary guidelines (MyPlate) . The HeaThe nutrient profiling method, defined as the science of ranking foods based on nutrient composition , 5, reprChallenges are faced when interpreting currently used diet quality indices and nutrient profiling measures, particularly in non-research settings, when consumers are requested to meet food-based dietary guidelines (FBDGs) and nutrition recommendations. For example, in applying dietary recommendations, such as the US MyPlate , the numIn order to address this problem, the present study aimed to create a novel unit of measure that accounts for both the quantity of energy  and the quality of nutrients, as defined by micronutrient density, in foods and to demonstrate the usability of this novel measure to facilitate the assessment of food quality. Specific objectives of the study included 1) developing a standardized unit of measure reflecting the quality of kilocalories for nutrition  that can account for the caloric and nutrient density per food item, 2) comparing food items per food groups based on the newly developed qCaln measure, and 3) testing the applicability of qCaln measure through demonstrating different food plates with various qCaln ratio scores.Findings from this study can provide a new measure that allows for merging multiple nutritional parameters into one simple, standardized unit. In addition, this newly proposed measure could provide researchers and public health professionals with a relatively easier tool to assess the diet quality of individuals and population groups in comparison to other nutrient profiling tools and diet quality indices.Generally, food energy has been measured in kilocalories , which accounts for the energy in the macronutrients  included in the sum total of ingredients in any food item. The quality of the food, however, is more often than not, determined by the amount and distribution of key micronutrients in food. Energy, macronutrient and micronutrient content of all food items within the Nutritionist Pro software  were included in the computations. The food composition database in this software was based on the United States Department of Agriculture (USDA) National Nutrient Database for Standard Reference, release 27 . In addii is the weighting for each micronutrient I, \u201cSi\u201d is the micronutrient score for each micronutrient \u201cI\u201d, and n is the amount of micronutrients included in the calculation. The value wi is the relative weighting for importance to each of the micronutrients.The qCaln is a function of the caloric content (per 100\u00a0g) from macronutrients and the amount and distribution of micronutrients (per 100\u00a0g) in a particular food such that:Equation\u00a0wi of 0.10 as the weighting scores indicate that each micronutrient is equally important in a diet. For the purposes of this study, weightings for the selected micronutrients were considered identical, and weightings for micronutrients that were not included in the calculation were assumed to be \u201c0\u201d. Saturated fat, total sugar, and sodium were negatively weighted in the qCaln calculation, indicating that these micronutrients need to be limited to prevent cardio-metabolic risk factors, including elevated blood sugar, cholesterol, or blood pressure levels [Thus, each micronutrient was assigned an equal weighting e levels \u201333.Equation\u00a0i\u201d was calculated. The calculation is based on the amount of the selected micronutrient in the food item relative to other food items in the same food group to ensure proper comparison. The distribution of each selected micronutrient among all foods within each food group was tested for normality and were found to be close to a normal distribution. Given a normal distribution, the value of Si will depend on the standard score  of the micronutrient. The equation for the standard score of a micronutrient amount relative to the rest of the food group is given by:For each food item, and for each micronutrient \u201ci\u201d, a micronutrient score \u201cSi of a micronutrient i was estimated based on z-score tables such that (0\u2009\u2264\u2009Pi\u2009\u2264\u20091). Then, for each micronutrient i,Equation\u00a0i was calculated for each micronutrient i, Eq.\u00a0th percentile (0.5\u2009<\u2009Pi\u2009\u2264\u20091.0) will have a greater qCaln value than the listed value of Cal indicating higher quality food, and foods with lower than the 50th percentile (0.0\u2009\u2264\u2009Pi\u2009<\u20090.5) have qCaln values less than the listed Cal, indicating less quality. Also, it follows from Eqs.\u00a0max\u2009=\u20092 * Cal).Once Si for each micronutrient, a qCaln value per 100\u00a0g was calculated for 1806 different food items from five different food groups. These 5 main food groups were: 1.) breads and cereals, 2.) dairy products, 3.) fruits and juices, 4.) vegetables, 5.) and meat and meat alternatives. Using the weightings from Eq.\u00a0After calculating STo show the applicability of the qCaln ratio, two separate plates were compared. Each plate was based on FBDG recommendations from USDA MyPlate  and consOf the 1806 total food items analyzed, 534 had qCaln ratio values\u2009>\u20091.0 when compared to kilocalorie  content, and 1272 had ratios\u2009<\u20091.0. Ratios that are\u2009>\u20091.0 can be interpreted as higher quality food per calorie, whereas qCaln ratios\u2009<\u20091.0 represent lower micronutrient content per food item compared to its caloric content. Figures\u00a0Figure\u00a0Figure\u00a0Figure\u00a0Figure\u00a0Figure\u00a0In the case of vegetables, the highest 10 qCaln ratios included spinach, leeks, green peppers, collards, and turnip (greens), whereas those of the lowest qCaln ratios, reflecting lower micronutrient content to caloric content, were sweet pickles, pickled eggplants, canned capers, and red cabbage. For fruits, food items with the highest qCaln ratios were dried mixed fruits , dry cranberry beans, wild raspberries, cherries, and blackberries, and those of the lower qCaln ratios were canned juices and fruits canned in syrup such as papaya, plums, and loquats. These results are in line with dietary recommendations that highlight the high nutrient yet low energy density of green leafy vegetables and other fresh fruits and vegetables that are not preserved in salt or sugar. It is worth noting that the sugar content of canned fruits and fruit juices was a major contributor to lower qCaln ratios for fruit items. When analyzing the dairy products, food items with the highest quality were low-fat, non-fat, and low sodium varieties of milk and cheese as these items score lower in terms of fat and thus caloric content and have lower sodium levels, which is found in abundance within dairy products and could contribute to elevated sodium intake levels.Figure\u00a0Finally, two separate meals were compared that would be considered good diet choices for each plate according to the MyPlate guidelines . Note thThis study aimed to develop a new unit of measure that accounts for both the caloric and micronutrient density of foods and to demonstrate the usability of this novel measure in assessing food quality. The qCaln was the standardized unit developed based on the caloric density and the amount and distribution of micronutrients per 100\u00a0g in a particular food. Results showed that food items in any food group can be analyzed based on their qCaln values. In addition, through calculating a qCaln ratio for each food item, the caloric and micronutrient contents of a food were compared with the food\u2019s energy density  in one single measure. Using the qCaln ratio, higher quality foods could be differentiated easily from lower quality foods. More specifically, the qCaln ratios of food items within each of the food groups, including vegetables, fruits/fruit juices, milk/dairy products, breads/cereals, and meats/meat alternatives, were compared to each other in this paper.The advantages of the proposed approach over other existing food quality measures and nutrient profiling approaches lies in simplifying complex nutrient composition data into a singular based score that reflects both nutrient and energy density. The definition of nutrient dense foods have expanded over the years due to numerous attempts by researchers, regulatory agencies, and food companies to identify adequate criteria for ranking foods based on their nutrient composition. Some of the models highlighted the importance of limiting certain nutrients, such as total fat, saturated fat, sugar, or sodium, that were associated with adverse health outcomes, when defining nutrient-density , 35. OthThe selection of the 10 micronutrients that were included in the calculation of the qCaln accounted for two groups of nutrients: those that are not consumed in sufficient amounts and contribute, in part, to micronutrient deficiencies across various population groups \u201317 and lIt is worth noting that the percent daily value (%DV) of each micronutrient was not included in the calculation for qCaln within the present study for a number of reasons. First, Eqs.\u00a0The proposed qCaln allows for a better interpretation of the food quality not only by researchers but also consumers. The qCaln can be linked to food-based dietary guidelines depicted visually through the MyPlate, MyPyramid, and the Mediterranean Diet Pyramid among other education models used by health care professionals to educate consumers about healthy dietary choices. For example, promoting the consumption of low-fat and low-sodium dairy products that have higher qCaln ratios , can assist consumers in meeting their beneficial nutrient requirements while avoiding less desirable nutrients. Similarly, promoting the consumption of fresh fruits like oranges rather than orange juice from frozen concentrate or sweetened fruit juices can help in making smarter food choices.Training consumers to select foods with higher qCaln values, where appropriate, can help improve diet quality and meet dietary recommendations. Currently, dietary guidelines are presented to the public as recommendations in terms of serving sizes of food groups that are promoted to be consumed, such as whole grains, low fat dairy products, and foods to avoid including those high in sugar and fat. These recommendations are usually based on studies highlighting the daily optimal intakes of energy, and macro- as well as micro-nutrients among humans . Despite the extensive efforts exerted by nutrition professionals to relay dietary guidelines in simple and interactive manners, consumers still face challenges in interpreting these recommendations. Difficulties arise when consumers are asked to make healthy dietary choices from a wide array of food products available on the market and with various nutrition facts labeling systems. In addition, deciphering the labeling systems, which include the amount of calories, macronutrient, and micronutrient content of foods, requires health and nutrition literacy at the consumer end, which may be lacking by various population groups and in numerous contexts. This is further complicated with the diversity of units, serving sizes, and nutrients listed on nutrition facts labels in differing countries, in addition to front-of-package labels along with other competing marketing slogans and techniques. Furthermore, fresh produce and certain types of meats and dairy products lack nutrition labels and the choice among the myriad of produce on the market leaves consumers confused as to which of the food items within each of the food groups are the most nutritious. The qCaln measure can merge the multiple nutritional measures including, caloric content of food, and the macro- and micronutrient content of food into one simple, standardized unit. In addition, the qCaln can be linked to optimal daily nutrient intakes of individuals and allows for daily meal construction. This linkage will allow consumers to make food purchases with conscious dietary knowledge rather than just preference, taste, convenience, and cost. The qCaln, if deemed valid in various contexts, could be used in addition to food labeling either on the front or the side of products, and on menus at various points of purchase with the potential for expanding nutrition education and supporting nutrition labeling systems that are already in place. Future research should evaluate the experiences of consumers towards the use of the qCaln and their success in attempting to make healthier dietary choices at different points of sales .Using the qCaln, as a composite score that takes into consideration energy and nutrient density, is not only valuable for consumers but also researchers and health professionals who seek to assess the diet quality of individuals and population groups. The proposed measure can provide a simple method to assess nutrient profiles of individual food items within the same food category while also providing investigators with a relatively simple tool to assess the overall nutrient profiles of individuals . The latter measure can be then linked to other global measures of diet quality, such as the HEI and diet diversity scores.One of the main benefits and applications of the qCaln is to help individuals make better food item choices and improve their overall dietary intake. In fact, nutrition programs that attempt to increase awareness and proper consumption of micronutrients achieve some of the highest benefit-cost ratios, even in the short term . HoweverFindings from this paper need to be interpreted in light of a number of limitations. First, equal weights were assumed for all included micronutrients in the calculation for the qCaln. Although the use of equal-weighted scores has been used extensively by other researchers when developing nutrient profiling methods , targeteAnother limitation of the qCaln calculations in the present study is the lack of inclusion of bioavailability data and the effect of food preparation on micronutrient content of foods. For example, animal-based protein sources are more readily digested and absorbed allowing for higher bioavailability of essential micronutrients compared to plant-based sources, and cooked vegetables that are prepared through steaming have different micronutrient contents compared to boiled or baked vegetables . To addrThe proposed unit of measure reflects a novel nutrient profiling approach that accounts for both the quantity of energy  and the quality of nutrients in foods into one standardized metric and demonstrates the usability of this new measure to facilitate the assessment of food quality. Results show that the qCaln can be used to rank foods in terms of their nutrient and energy content simultaneously, making food selections within a food group easier to understand. This easy-to-use tool can be applied by public health professionals, regulatory agencies, and the food industry to promote the selection of healthier nutrient-dense yet low in energy content foods and assist consumers in meeting dietary guidelines and nutrition recommendations. In addition, this novel metric could be further validated by researchers in comparison to other nutrient profiling and diet quality measures to test its usability in assessing diet quality of individuals and population groups. This metric can have other applications and can be further expanded beyond the nutrition research serving as the impetus for nutrition-sensitive agriculture through tracking of food quality from production to consumption."}
+{"text": "SCN5A is expressed in cardiomyocytes and gastrointestinal (GI) smooth muscle cells (SMCs) as the voltage-gated mechanosensitive sodium channel NaV1.5. The influx of Na+ through NaV1.5 produces a fast depolarization in membrane potential, indispensable for electrical excitability in cardiomyocytes and important for electrical slow waves in GI smooth muscle. As such, abnormal NaV1.5 voltage gating or mechanosensitivity may result in channelopathies. SCN5A mutation G615E \u2013 found separately in cases of acquired long-QT syndrome, sudden cardiac death, and irritable bowel syndrome \u2013 has a relatively minor effect on NaV1.5 voltage gating. The aim of this study was to test whether G615E impacts mechanosensitivity. Mechanosensitivity of wild-type (WT) or G615E-NaV1.5 in HEK-293 cells was examined by shear stress on voltage- or current-clamped whole cells or pressure on macroscopic patches. Unlike WT, voltage-clamped G615E-NaV1.5 showed a loss in shear- and pressure-sensitivity of peak current yet a normal leftward shift in the voltage-dependence of activation. In current-clamp, shear stress led to a significant increase in firing spike frequency with a decrease in firing threshold for WT but not G615E-NaV1.5. Our results show that the G615E mutation leads to functionally abnormal NaV1.5 channels, which cause disruptions in mechanosensitivity and mechano-electrical feedback and suggest a potential contribution to smooth muscle pathophysiology. In all, the current-clamp data suggest that loss of mechanosensitivity in G615E NaV1.5 would lead to a loss of mechanically induced excitability in WT NaV1.5.Shear stress accelerated the spiking upstroke for WT Na faster) . Howeverest (I)] . The effV1.5 mechanosensitivity and its role in cellular mechano-electrical feedback. We used a unique SCN5A mutation G615E that is associated with acquired long-QT syndrome [V1.5 had mostly intact voltage-dependent function \u2013 normal voltage-dependence of activation, and either no or minor changes in voltage-dependence of inactivation.The goal of the current study was to examine Nasyndrome , sudden syndrome , and irrsyndrome . In prevsyndrome ,17 and iV1.5. We tested both constructs in whole cell and patch, using shear stress and patch pressure as mechanical stimuli, respectively. For WT NaV1.5, we saw force produce several changes to voltage-dependent function, similar to previous studies on NaV1.5 [V1.4 [V1.5. Thus, G615E NaV1.5 joins previously identified disease-associated mutations with abnormal NaV1.5 mechanosensitivity. These associated with long QT syndrome [V1.5 with abnormalities in voltage-dependent function that were further accentuated by mechanical forces. However, to our knowledge, this is the first NaV1.5 mutation that has disrupted mechanosensitivity, while voltage-sensitivity remained mostly intact. Our findings may be relevant for understanding channelopathy mechanisms in cases when NaV1.5 mutations fail to reveal functional changes using voltage-dependence protocols. In such cases, and as we see with G615E NaV1.5, the functional impact may be on mechanical [On the other hand, we found dramatic differences in mechanosensitivity of voltage-dependent gating between WT and G615E Nan NaV1.5 ,12,16,22.5 [V1.4 , but thesyndrome  in the hchanical  sensitivV1.5 mechanosensitivity. First, G615E is located on the intracellular linker connecting DI and DII, which is a novel location for a missense mutation to impact mechanosensitivity. Other NaV1.5 channelopathies like G298S have loss-of-mechanosensitivity and are in linkers as well. However, how these linkers contribute to the mechanism of NaV1.5 mechanosensitivity remains unclear. Second, our findings suggest that mechanisms of voltage- and mechano-sensation by NaV1.5 may be distinct. This is surprising since mechanical stimuli are well established to modulate voltage-sensitivity of voltage-gated channels [V1.5 lost one but not both mechanosensitive responses \u2013 it lacked a perfusion-induced current increase but maintained a negative shift in the voltage-dependence of activation. This would suggest that mechanosensitive increases in peak Na+ current may be mechanistically distinct from mechanically induced shifts in voltage-dependence of activation and availability. Fourth, the findings suggest that the mechanosensitivity of the voltage-dependence of activation may be separate from that of availability. Previous and current studies show that mechanical stimuli accelerate the kinetics of activation and inactivation by the same constant. If the acceleration of activation by force is the rate-limiting step [V1.5 demonstrates an intact mechanosensitivity of activation but a loss of mechanosensitivity of inactivation, suggesting separate mechanisms. In all, our results shed important light on the mechanism of NaV1.5 mechanosensitivity and show that NaV1.5 mechano- and voltage-sensitivity may be targeted separately.Our results provide intriguing mechanistic insights on Nachannels ,12,25 buchannels ,12. Thiring step , it may V1.5 mechanosensitivity impacts SMC excitability, also called mechano-electrical feedback [V1.5 channels at the upstroke. But given the acceleration of inactivation, NaV1.5 stretch also results in a more significant current decrease during inactivation [V1.5 mechanosensitivity on SMC function is difficult to judge only from the voltage-dependent operation. We designed current-clamp protocols in a reductionist system, a HEK-293 cell that expressed only WT NaV1.5 or G615E NaV1.5. Similar to previous studies [V1.5 had decreased mechanosensitivity of both spontaneous and elicited firing, suggesting that NaV1.5 mechanosensitivity may play an important excitatory role in SMC mechano-electrical feedback. However, a note of caution is required. We set the resting potential hyperpolarized to allow for full NaV1.5 availability, but this system limits our ability to determine whether sub-threshold events by either channel can result in firing, as may happen in excitable cells. In a set of preliminary studies, we investigated the potential influence of G615E-NaV1.5 on mechano-electrical feedback in GI SMCs by computational modeling. In silico modeling simulated NaV1.5 voltage- and current-clamp in vitro behavior, and the resulting SMC electrical activity and cytoplasmic Ca2+ concentrations were affected by mechanical forces for WT but not G615E-NaV1.5 .We are ultimately interested in understanding how Nafeedback . These ctivation \u201313 and stivation . Therefo studies ,28, we fV) [V1.1 mechanosensitivity leads to \u201cmechanical braking\u201d of neuronal excitability [in vivo and to replicate these for studies in vitro. It is unclear how the current in vitro mechanostimulation protocols correlate with in vivo physiology. To fully understand mechano-electrical coupling we will need to integrate mechanosensitivity of NaV1.5 and other mechanosensitive ion channels into cell models and to place these models into physiologically relevant mechanical contexts.In addition to mechanosensitive voltage-gated sodium channels, cells have other mechanosensitive voltage-gated ion channels, such as potassium (KV)  and mechV) . The efftability . FurtherV1.5 missense mutation G615E disrupts NaV1.5 mechanosensitivity without a significant impact on voltage-dependent function, which may have important consequences for mechano-electrical coupling in myocytes. This raises the possibility of directly targeting NaV1.5 mechanosensitivity in disease with a drug such as ranolazine [V1.5 peak current but can inhibit mechanosensitivity [In summary, the disease-associated Nanolazine ,22, whicsitivity ,31."}
+{"text": "We propose two generalized Haldane models on laser-coupling optical lattices. Laser-assisted nearest neighbour tunnelings generate artificial staggered magnetic flux, facilitating the realization of topological nontrivial band structures. As generalizations of Haldane model, these models support topological insulator and semimetal phases featuring high Chern numbers. We show simple rules for computing Chern numbers of our models and display the phase diagrams. Moreover, numerical calculations of energy spectra are in perfect agreement with our theoretical expectations. Our models may serve as two new family members for generalizing Haldane model on optical lattices. In lattice systems, the topologically nontrivial band structures are currently attracting a great deal of interest3. The band topology is characterized by a topological invariant-Chern number-taking4 integer values and signaling topological order. Nontrivial topological order has experimental consequences, such as quantized Hall conductivity5 and the existence of edge states3. In two-dimensional lattice systems, one of the simplest models supporting topological bands was proposed by Haldane6. This model features real nearest-neighbor (NN) hopping and complex next-nearest-neighbor (NNN) hopping on a honeycomb lattice. Haldane showed that this two-band model is topologically equivalent to integer quantum Hall state.Topologically ordered phases of matter have been a hot topic in condensed matter physics since the discovery of the integer quantum Hall effect10, topological superconductor13 and topological semimetal19 have been widely explored. To simulate these fascinating quantum phenomena, ultracold atoms in optical lattice is an ideal playground, for its versatile control of interactions which are hard to adjust in traditional solid state systems20. Several methods have been proposed to realize topological nontrivial band structures with cold atoms, such as lattice shaking23, rotation24, laser coupling28, or using synthetic dimensionality29. Recently, Haldane model has been realized experimentally through lattice shaking30. Measurement of Chern number of Hofstadter bands has been successfully performed31. The quantized conductance in neutral matter system has also been observed32. Besides, there are many other developments on Chern number detection34, models design that support nontrivial topology37 and so on. Recently, some three-band tight-binding models have been proposed40, because of their high Chern numbers and easy access to topological semimetal phases.In recent years, topological insulatorIn the present paper, we propose two simple lattice models that support topological insulator and semimetal phases featuring high Chern numbers. Our tight-binding model is defined on laser-coupling optical lattice, on which complex NN tunnelings are laser assisted. The three-band model is defined on state-dependent lattice whereas the four-band model is on state-independent lattice, both pierced by effective staggered magnetic fields. As generalizations of Haldane model, these models support topological nontrivial energy bands. Chern numbers are computed in a simple way and rich phase diagrams are exhibited. Moreover, the topological edge states show time-reversal (TR) symmetry for a special case, although TR symmetry breaking in the bulk is required for a nonzero Chern number.i of three sublattices. Normal NNN tunnelings of amplitude t take place within the same triangular sublattices. Laser-assisted NN tunnelings43 of amplitude \u03bba(b) are accompanied by recoil momentum p  of the laser coupling processes behave like onsite potentials. To eliminate the explicit spatial dependence of our Hamiltonian, we perform a unitary transformation36In our three-band model, fermionic ultracold atoms are trapped on honeycomb-like lattice. Atoms of three different hyperfine states are located on inequivalent sites, labeled by A, B and C Fig.\u00a0. In the m p Fig.\u00a0, generatThus giving a transformed Hamiltonian of real NN tunnelings and complex NNN tunnelingslux Fig.\u00a0. The preui is the NNN vector and \u03bdi is the NN vector.\u03c8n,k . The topological order of each band is characterized by the Chern numberz component of curl Given the momentum-space Hamiltonian (4), one easily knows there are three energy bands featured by Bloch wave functions x, y, z} of three bands. Setting t as unit, high Chern number phases a(b) becomes large, it reduces to an effective two-band model with Chern numbers \u03d5 affect the nontrivial scale of detuning \u0394, allowing for the largest value |\u0394|\u2009=\u20099 when it approaches \u00b12\u03c0/3. The coupling strength \u03bba(b) has no influence on Chern numbers, but it turns out to modify the energy gaps and lead to topological semimetal phases as shown in the following numerical calculations.We are more interested in two symmetric cases and show their phase diagrams. For the isotropic case \u03d5. For the isotropic case of phase diagram Fig.\u00a0b with fixed \u0394a. Specially, there is a symmetry kx by \u2212i\u2202x and integrating over x, one can find effective edge Hamiltonian \u03d5 is presented in Fig.\u00a0\u03d5, two edge states show up between the two energy gaps. Here the upper gap is indirect zero, signaling topological semimetal phase. Comparing Fig.\u00a0\u03bba(b) modifies the energy gap and results in topological insulator-semimetal phase transitions.The defining characteristic of topological nontrivial band structure is the existence of gapless edge states. We numerically calculate the energy spectra for 1D ribbons with zigzag and armchair edges, which is periodic in x and y direction respectively. The displayed edge states within the bulk energy gaps address different topological nontrivial phases. To demonstrate previous analytical results, we show two kinds of topological phase transitions driven by resonance detuning \u0394 or artificial magnetic flux H\u2009=\u2009HT\u2009+\u2009HL\u2009+\u2009HV:HT is the NNN hopping term on the honeycomb lattice  and . We find topological insulator phases characterized by one or two pair of edge states when resonance detuning \u03d5\u2009=\u2009\u03c0/2, the two nontrivial intervals are  and . The two topological insulator phases become semimetal phases at the same detuning  at the two Dirac points. We also show the analytical phase diagrams of two symmetric cases, verified by numerical calculations of energy spectra. For the four-band model, the Peierls phase depend on the sublattice indices instead of internal states. Different topological nontrivial phases characterized by one pair or two pairs of edge states, can be easily transformed to each other by changing the resonance detuning \u03b4 and artificial magnetic flux \u03d5. The effects of laser coupling on topological band structures are discussed. Compared with Haldane model, topological properties of our models are much richer, featuring high Chern number and easy control of topological insulator-semimetal phase transitions. For the staggered artificial magnetic flux, our models can be viewed as two new family members for generalizing Haldane model on optical lattice.In conclusion, we have proposed and studied two generalized Haldane models on laser-coupling optical lattice. Laser-assisted NN tunnelings generate artificial staggered magnetic flux, facilitating the realization of topological nontrivial band structures. For the three-band model, we show a simple rule for computing Chern numbers, which are obtained from the values of energy Hk in equation (En(k) the energy of n-th band. If wave function is analytic in the whole Brillouin zone (BZ), the contour integral in equation  and (8) on 1D ribbons of width 25 and 30, respectively. The symmetric and non-symmetric energy spectra correspond to different boundary conditions of armchair and zigzag edges, respectively."}
+{"text": "The full width at half maximum (fwhm) is 103 nm, the internal quantum efficiency (IQE) exceeds 88%, and the external quantum efficiency (EQE) is 69%. According to Rietveld refinement analysis and density functional theory (DFT) calculations, Bi3+ ions randomly occupy all\u00a0La sites in orthorhombic La4GeO8. Importantly, the oxygen-vacancy-induced electronic localization around the Bi3+ ions is the main reason for the highly efficient orangish-red luminescence. These results provide a new perspective and insight from the local electron structure for designing inorganic phosphor materials that realize the unique luminescence performance of Bi3+ ions.Phosphor-converted white-light-emitting diodes (pc-WLED) have been extensively employed as solid-state lighting sources, which have a very important role in people\u2019s daily lives. However, due to the scarcity of the red component, it is difficult to realize warm white light efficiently. Hence, red-emitting phosphors are urgently required for improving the illumination quality. In this work, we develop a novel orangish-red La Phosphorescent substances that give white-light-emitting diodes a more natural reddish hue can be made by tuning the electron structure of their activator ions. Jun Lin of China\u2019s Changchun Institute of Applied Chemistry, Chinese Academy of Sciences and colleagues combined germanium, lanthanum and bismuth oxides to manufacture a reddish phosphorescent substance that, when added to blue and green phosphors on an LED chip, produced more natural-looking light compared to conventional phosphor-converted white LEDs. LEDs are constantly being improved as an energy-efficient light source, but the colour of the light they emit can be unnaturally white or blue. Fabricating high quality red-emitting phosphors to change this has been a challenge. The researchers developed a substance with a unique reddish photoluminescence caused by electrons localizing around bismuth ions. Their approach offers a new perspective for exploring luminescence in inorganic materials. Conventional WLEDs are composed of a blue LED chip and yellow YAG:Ce3+ phosphor; however, they emit cold white light because the emission spectra do not cover the red region5. Therefore, red phosphor is very important for producing warm white luminescence and improving the luminous efficiency. Many previous works have explored the use of red-emitting phosphor to enhance pc-WLED lighting quality. Eu3+-doped inorganic phosphor materials are the most frequently investigated red phosphor materials due to the typical 4f\u20134f partial spin and forbidden transition7. However, Eu3+ is rarely utilized in warm pc-WLEDs because its excitation spectra do not fit well with near-ultraviolet (n-UV) and blue light8. The most widely commercially available red phosphors are Eu2+-doped nitride phosphors12, such as CaAlSiN3:Eu2+ and Sr2Si5N8:Eu2+. Although these phosphors realize high quantum efficiency, harsh synthesis conditions (high temperature and high pressure) and deep-red emission limit their large-scale application \u00a0in the production of warm\u00a0white light. In addition, Zhang et al. reported a narrow-band red-emitting SrLiAl3N4:Eu2+ phosphor that was obtained via facile atmospheric pressure synthesis, which could easily compensate the red component for YAG:Ce3+ and potentially serve as an alternative phosphor for n-UV pc-WLEDs. However, the spectral overlap still remains a large problem13. To date, linear red-emitting Mn4+-doped fluoride phosphors have attracted substantial attention, as their quantum yield can exceed 98% under blue light irradiation15. However, they have two serious drawbacks: low thermal stability and massive HF acid use. Hence, exploiting high-quality red-emitting phosphor materials remains challenging.Phosphor-converted white-light-emitting diodes (pc-WLED) have become the next-generation solid-state lighting source in both indoor and outdoor lighting areas3+-activated phosphors have been extensively investigated due to their unique luminescence performance16. Their excitation spectra are located in the n-UV area; thus, spectral overlap can be efficiently avoided. However, Bi3+ ions typically emit blue and green light and rarely emit red light, except in a suitable matrix, such as ScVO4 and ZnWO420. Bi3+ contains a naked 6s6p energy level; hence, the surrounding coordination environment is very sensitive22. The luminescence performance of Bi3+ can be easily influenced by tuning the surrounding electron structure. Recently, it was demonstrated that the formation of a vacant defect could contribute to the spectral adjustment. For instance, Zhang et al.23 reported a giant enhancement of Bi3+ luminescence that was realized by generating an oxygen vacancy, which is a new strategy for exploring novel Bi3+-doped phosphor materials.Recently, Bi4GeO8:Bi3+ (denoted as LGO:Bi3+) phosphor. Under 397 nm n-UV excitation, LGO:Bi3+ displays an orangish-red emission for which the peak is at 600 nm, fwhm\u2009=\u2009103 nm, IQE\u2009=\u200988.3%, and EQE\u2009=\u200969%. Via experimental and theoretical studies, we demonstrate that the unique photoluminescence performance is caused by oxygen-vacancy-induced electron localization around the Bi3+ ions. This finding provides a new insight into the design of novel luminescence materials by changing the local electron structures of activator ions. The fabricated pc-WLED devices realize a high color rendering index (CRI\u2009=\u200995.1) and a low correlated color temperature (CCT\u2009=\u20095323 K), thereby indicating that LGO:Bi3+ is a superb red-emitting candidate in the field of solid-state lighting.In this work, we develop a novel and high-quality red-emitting La3+ phosphor is evaluated in detail through diffuse reflectance (DR) spectra. The DR spectra of the LGO matrix show only one prominent band, which is centered at 280 nm , in addition to the prominent band in the region of 232\u2013321 nm, a shoulder band appears at ~400 nm. The former is mainly attributed to the matrix\u00a0absorption of LGO and the 1S0\u21921P1 transition of Bi3+, while the latter originates from the 1S0\u21923P1 transition of Bi3+,24. The optical bandgap value can be obtained via linear extrapolation based on the DR spectra according to the following equations:25A represents the absorption constant, R is the reflectance coefficient (%), F(R) is the absorption coefficient, Eg is the optical bandgap value, and hv represents the photon energy. The bandgap values for the LGO matrix and LGO:0.007Bi3+ are 4.89 and 4.95 eV, respectively . This emission can be attributed to the characteristic 3P1\u21921S0 transition of Bi3+. In the PL spectrum of LGO:Bi3+, an emission redshift of ~50 nm and a broader fwhm compared to those of YAG:Ce3+ (maximum at 550 nm and fwhm\u2009=\u200987.5 nm) are observed, thereby demonstrating that LGO:Bi3+ could cover more of the red component in PL spectra. Simultaneously, LGO:Bi3+ shows no reabsorption between the PLE and PL spectra, the IQE value can reach 88.3%, and EQE\u2009=\u200969%. The calculated Commission Internationale de l\u2019Eclairage (CIE) diagrams for LGO:0.007Bi3+ (\u03bbex\u2009=\u2009397 nm) and YAG:Ce3+ (\u03bbex\u2009=\u2009460 nm) are located in the orangish-red region  and yellow region   is consistent with the RT spectra (298 K)  Fig.\u00a0, respect) Figure\u00a0 and S1b,7 Figure\u00a0-S1f. The27. In Fig.\u00a0xBi3+ (x\u2009=\u20090.01 and 0.03) samples and standard \u03b1-Bi2O3 powder are plotted. All samples show the typical Bi3+ peaks at ~159 and 164.6 eV, which are assigned to Bi 4f7/2 and Bi 4f5/2, respectively. In addition, when the LGO:Bi samples are\u00a0treated under a N2/H2 (90%/10%) reducing atmosphere, no luminescence is\u00a0observed. PL spectra that are\u00a0treated in a different environment are shown in Figure\u00a03+ ions.Since the valence of Bi has a critical influence on its luminescence properties, it is necessary to define its valence in the LGO matrix29. Fig.\u00a03+. Although its emission intensity gradually declines as the temperature increases from 298 K to 573 K, it maintains 78.4% emission intensity at 398 K of the original intensity at 298 K. The quenching temperature, which is denoted as T50 , is 488 K. The quenching process is ascribed to the thermally excited nonradiative transition. Furthermore, the emission peak position and shape show almost no shift  could be indexed with the standard La4GeO8 (PDF No. 40\u20131185), thereby demonstrating the formation of a pure phase . All the as-prepared samples crystalize in orthorhombic unit cells with space group P1. The refined lattice parameters for LGO are a\u2009=\u20097.6642(8) \u00c5, b\u2009=\u20095.8470(9) \u00c5, c\u2009=\u200918.2897(4) \u00c5, and V\u2009=\u2009819.629(3) \u00c53 and the detailed structural information is listed in Table\u00a0c and cell volume V as functions of the Bi3+ ion concentration x. The lattice parameters gradually decrease as x increases, which results from the smaller ionic radius of Bi3+  compared to La3+ . Furthermore, the lattice parameters a and b gradually increase with x, thereby indicating that the La lattices may be locally distorted with the incorporation of Bi3+  system. Figure\u00a0b-axis direction, LGO exhibits a highly symmetric structure, which consists of three types of [LaOn] polyhedra  and two types of [GeO4] tetrahedra. The six crystallographic La sites can be sorted into three categories: La1\u2013La3. The La1 site is connected with six O atoms to form a distorted octahedron, while the La2 and La3 sites are coordinated with seven O atoms to form decahedra. The O atoms in LGO can also be divided into three types: Oi-Oiii. The structural information that is obtained via DFT calculations well agrees with the Rietveld refinement results. The calculated average La\u2013O bond lengths are summarized in Fig.\u00a0Density functional theory (DFT) calculations are used to further investigate the electron properties and structural configuration of the LGO:xBi3+  is determined from FT-IR spectra . The peak splitting degree at ~400 cm\u22121 also differs between the two samples (marked by a pink dashed rectangle). These variations in the Raman spectra demonstrate that the local lattice coordination environment in the LGO matrix changes slightly with Bi3+ ion incorporation and may result in the formation of lattice defects. XPS analysis is an effective method for elucidating the presence of vacancies. With the doping of Bi3+ into the LGO matrix, the 3d orbital of La and the Ge binding energy remain unchanged  maps around the Bi atoms at the La1\u2013La3 sites , 2.08 eV (600 nm), and 2.24 eV (550 nm). The crystal splitting field energy (Dq) is expressed as follows:34Z is the charge of the anion, e is the charge of one electron, r is the radius of the d wave function, and R is the average La\u2013O bond length. As the bond length decreases, the crystal field splitting increases. On the basis of Fig.\u00a03+ at the La1, La2, and La3 sites, respectively. The oxygen-vacancy-induced electron localization around the Bi atoms is the crucial factor for generating orangish-red emission in the LGO:Bi3+ phosphor.To further investigate the influence of oxygen vacancies on the Bites Fig.\u00a0 without tra Fig.\u00a0 and life3+ phosphor in a pc-WLED device, we fabricated a pc-WLED device\u00a0by using LGO:0.007Bi3+, commercial green Ba3Si6O12N2:Eu2+ phosphor, blue BAM:Eu2+ phosphor, and a 400 nm n-UV LED chip. For comparison, the pc-WLED is fabricated similarly by\u00a0using commercial YAG:Ce3+ phosphor with a 460 nm LED chip. The electroluminescence (EL) spectra are presented in Fig.\u00a03+ covers more of the red component in the visible light region than YAG:Ce3+. With a 3 V, 20 mA current driving, the CCT (correlated color temperature) and CRI (color rendering index) of the as-fabricated pc-WLEDs for LGO:0.007Bi3+ are 5323 K and 95.2, respectively, which are superior compared to the commercial YAG:Ce3+ phosphor (6015 K and 72.2). The luminous efficiency of the as-fabricated pc-WLEDs for LGO:0.007Bi3+ is 6.4 lm/W, which should be optimized via proper process treatment. Moreover, the CIE color coordinates of the former  correspond to a more suitable white-emitting position than those of the latter . The fabricated pc-WLED of LGO:Bi3+ exhibits much warmer white light than that of YAG:Ce3+  and the fwhm\u2009=\u2009103 nm. Its IQE could reach 88.3%, EQE\u2009=\u200969%. Rietveld refinement confirms that the as-prepared samples crystalize in orthorhombic unit cells with space group P1, and its lattice parameters are a\u2009=\u20097.6642(8) \u00c5, b\u2009=\u20095.8470(9) \u00c5, c\u2009=\u200918.2897(4) \u00c5, and V\u2009=\u2009819.629(3) \u00c53. The XRD spectra and DFT calculation results demonstrate that Bi3+ ions randomly occupy La1\u2013La3 sites in LGO, which will emit deep-red (1.93 eV), orange (2.08 eV), and yellow (2.24 eV) light when substituting at La1, La2, and La3 sites, respectively. Interestingly, the unique orangish-red luminescence behavior should be ascribed to the electron localization surrounding Bi3+ ions with the presence of adjacent O vacant defects through ELF maps analysis. The fabricated n-UV-based pc-WLED with a hybrid of LGO:Bi3+ phosphor, blue BAM:Eu2+ and green Ba3Si6O12N2:Eu2+ phosphor achieves high CRI (95.2) and low CCT (5323 K), indicating a superb candidate in lighting field. The concept of focusing on the electron localization properties surrounding activators ions offers a new perspective and insight for exploring unique luminescence behavior in inorganic luminescence materials.In summary, we have successfully exploited an orangish-red-emitting LGO:Bix4-GeO8:xBi3+ (x\u2009=\u20090\u20120.03), which is denoted as LGO:xBi3+, were prepared via a conventional high-temperature solid-state approach. The raw materials of La2O3 (99.99%), GeO2 (99.999%) and Bi2O3 (99.99%) were all purchased from Aladdin. First, stoichiometric raw materials were weighed, put into agate, and ground together for 1 h. The obtained mixtures were transferred into corundum crucibles and annealed in a horizontal tube furnace at 1100\u20121300\u2009\u00b0C for 4 h in air. After naturally cooling to room temperature, the annealed samples were ground again. In addition, Lax4-GeO8:xBi3+ (x\u2009=\u20090\u20120.03) samples were prepared under an N2/H2 (90%/10%) reduced atmosphere with the same other conditions for comparison.Samples of La3+ phosphor, blue BAM:Eu2+ phosphor, green Ba3Si6O12N2:Eu2+ phosphor, and a 400 nm LED chip. In a typical fabrication process, the LGO:0.007Bi3+, BAM:Eu2+ and Ba3Si6O12N2:Eu2+ phosphors were evenly blended with silicone resins A and B (A:B\u2009=\u20091:1) in the agate mortar and the resulting mixture was coated on a 400 nm LED chip. The packaged devices were cured in an oven at 120\u2103 for 12 h to form the resulting pc-WLED devices. The commercially available yellow YAG:Ce3+ phosphor and a 460 nm LED chip were also fabricated to pc-WLED via the same method for comparison.pc-WLED devices were fabricated by\u00a0using the as-prepared orangish-red LGO:0.007BiPowder X-ray diffraction (XRD) were collected on a D8 Focus diffractometer with Ni-filtered Cu-K\u03b1 . Rietveld refinements were performed with General Structure Analysis System (GSAS) software based on XRD data. Photoluminescence excitation (PLE) and photoluminescence emission (PL) spectra were collected by a fluorescence spectrometer  whose excitation source is a 450 W xenon lamp. Diffuse reflectance (DR) spectra were collected with using a UV\u2013vis-NIR spectrophotometer (Hitachi U-4100). Photoluminescence decay curves were obtained with using a Lecroy Wave Runner 6100 Digital Osilloscope (1 GHz) (Contimuum Sunlite OPO), the excitation was a tunable laser (pulse width\u2009=\u20094 ns and gate\u2009=\u200950 ns). Fourier-transform infrared spectra (FT-IR) were performed on spectrophotometer  by using KBr pellet technique. Raman spectra were obtained on Raman spectrometer (JYT6400) using a 512 nm laser. The photoluminescence quantum yield (QY) was obtained on an absolute PL quantum yield measurement system . The electroluminescence performances of pc-WLED devices were measured analyzer system (tarspec SSP6612.) by using an integrating sphere. All the above measurements were performed at room temperature. Temperature-dependent PL spectra (10\u2013300 K and 298\u2013573 K) were recorded on a fluorescence spectrophotometer (Edinburgh Instruments FLSP-920) with a temperature controller.s25p65d16s2), Ge (4s24p2), O (2s22p4) and Bi (6s26p3) electrons were treated as the valence electrons. The exchange and correlation functional were described via the Perdew\u2013Burke\u2013Ernzerhof (PBE) generalized gradient approximation. To investigate the electronic properties and obtain an accurate description of the density of states, we employed the Heyd\u2013Scuseria\u2013Ernzerhof (HSE06) method. A plane-wave cutoff energy of 400 eV was applied in our calculations and 4\u00d74\u00d72 Monkhorst\u2013Pack k grids were used during the optimization. The iterative process was considered to have converged when the force on the atom was less than 0.01 eV\u2009\u00c5\u22121 and the energy change was <10\u20135 eV per atom.The first-principle density functional theory (DFT) calculations were performed with the Vienna ab initio simulation package (VASP) code. The electron\u2013ion interaction was treated with the projector augmented wave (PAW) method. La (5supplemental materials"}
+{"text": "Dispersion of APD was assessed in each sample by pacing at a cycle length of 1,000 ms, followed by a premature beat with a coupling interval of 400 ms. Vulnerability to re-entry was assessed with an aggressive pacing protocol with pacing cycle lengths decreasing from 450 to 250 ms in 25 ms intervals for normal tissue and 300\u2013150 ms for remodeled tissue. Simulated fibrosis at smaller spatial scales tended to lengthen APD, increase APD dispersion, and increase vulnerability to sustained re-entry relative to fibrosis at larger spatial scales. This study shows that when fibrosis is represented by smoothly varying tissue diffusion, the spatial scale of fibrosis has important effects on both dispersion of recovery and vulnerability to re-entry.Fibrosis in atrial tissue can act as a substrate for persistent atrial fibrillation, and can be focal or diffuse. Regions of fibrosis are associated with slowed or blocked conduction, and several approaches have been used to model these effects. In this study a computational model of 2D atrial tissue was used to investigate how the spatial scale of regions of simulated fibrosis influenced the dispersion of action potential duration (APD) and vulnerability to re-entry in simulated normal human atrial tissue, and human tissue that has undergone remodeling as a result of persistent atrial fibrillation. Electrical activity was simulated in a 10 \u00d7 10 cm square 2D domain, with a spatially varying diffusion coefficient as described below. Cellular electrophysiology was represented by the Courtemanche model for human atrial cells, with the model parameters set for normal and remodeled cells. The effect of fibrosis was modeled with a smoothly varying diffusion coefficient, obtained from sampling a Gaussian random field (GRF) with length scales of between 1.25 and 10.0 mm. Twenty samples were drawn from each field, and used to allocate a value of diffusion coefficient between 0.05 and 0.2 mm Although atrial fibrillation (AF) is a common cardiac arrhythmia, the mechanisms that initiate and sustain it are complex and remain incompletely understood. Circulating waves of electrical activation and recovery, often called re-entrant waves or rotors, are believed to sustain AF Nattel, . The anaFibrosis is hard to study in experimental preparations because it is difficult to control the extent of fibrosis and the size of individual lesions, and so computational models of cardiac cell and tissue electrophysiology have been used to examine how regions of simulated fibrosis affect activation and recovery in cardiac tissue, as well as vulnerability to arrhythmias  with exponential covariance, which is specified by a mean, a variance, and a correlation length. In 2-dimensions (2D), a GRF can be sampled to produce a series of random fields. In each sample, the values of the field will be correlated in both principal directions, and will be normally distributed. However the configuration of fluctuations in the field will vary from sample to sample. This approach has been used in models of groundwater flow with varying hydraulic conductivity , vulnerability to re-entry, and dynamics of re-entry.Electrical activation and recovery were simulated in a 2D sheet of atrial tissue, to enable the effect of cell and tissue electrophysiology to be examined without the additional complexity of anatomical structure. To represent the effect of fibrosis, the diffusion coefficient was set to vary smoothly from a low value representing regions of fibrosis, up to higher levels representing normal excitable tissue. The length scale of fluctuations in diffusion coefficient was varied by taking samples from GRFs with different correlation lengths. The effects of heterogeneities in diffusion at different length scales were examined by simulating normal beats during pacing at a steady cycle length, a single premature stimulus, vulnerability to re-entry during rapid decremental pacing, and behavior of an imposed spiral wave. These simulations were run using model parameters set to represent normal human atrial cells, and cells that have undergone remodeling as a consequence of persistent AF.+]i and [K+]i were fixed at their default initial values of 11.17 and 139.00 mM, respectively. The CRN model was implemented in C, based on code automatically generated from the CellML repository (www.cellml.org).Cardiac cellular electrophysiology was modeled using the Courtemanche-Ramirez-Nattel (CRN) model of human atrial cells , IK, ur was decreased by 49%, and the maximum conductance of IK1 was increased by 110%. These changes are based on those described in a previous study  can represent a smoothly varying field in space, and are composed of a mean and a covariance. The covariance function describes correlations between the value of the field at a single point and within its neighborhood. A squared exponential covariance function includes a correlation length \u03b4, which can be used to vary the length scale of fluctuations in the field.2ms\u22121, respectively. The value of the diffusion coefficient was capped at 0.2 mm2ms\u22121.Stationary GRFs with mean of 0, variance of 1.0, and an exponential covariance function were generated with length scales of 1.25, 2.50, 5.0, and 10.0 mm using the method of circulant embedding, which is detailed in Kroese and Botev . Samples2ms\u22121 were designated areas of fibrosis, and were set to be coupled but inexcitable, with a diffusion coefficient of 0.05 mm2ms\u22121. Inexcitability was imposed by setting the membrane current term in the monodomain equation to zero, while retaining the term describing voltage diffusion.Regions with a diffusion coefficient less than 0.05 mm2ms\u22121 as expected, and the effect of truncation at 0.05 and 0.2 mm2ms\u22121 can be seen.Figures CRNnormal and CRNremodelled variants.Since the configuration of fibrotic regions would be expected to affect simulated tissue behavior, 20 samples of the GRF were obtained at each of the four length scales. The procedure described above was used to generate a set of 20 diffusion fields, and these were then used for simulations with the different pacing protocols described in the next section. Multiple samples enabled the effect of length scale to be assessed independent of the specific configuration of a single sample relative to pacing sites. Each sample was then used to simulate the different pacing protocols specified below, with both 2ms\u22121 was retained, but elsewhere it was set to 0.2 mm2ms\u22121.To compare the effect of the smooth variations in diffusion coefficient provided by the GRF with abrupt changes, a further set of diffusion fields were generated. For each length scale, a copy was made of each of the 20 diffusion fields obtained by sampling the GRF. These copies were then further processed so that they contained only two values of diffusion coefficient, with an abrupt change between the two. In the fibrotic regions, the diffusion coefficient of 0.05 mm2ms\u22121, and the cell model was set to be ineexcitable) was 18.7, 18.0, 19.1, and 17.0% at length scales of 1.25, 2.50, 5.0, and 10.0 mm, respectively. The size and configuration of these regions was different in each of the 20 samples. At a length scale of 10 mm, the proportion of simulated fibrotic tissue ranged between 4.9 and 25.1% across the 20 samples. With a length scale of 1.25 mm, which was a smaller length scale relative to the size of the simulated tissue, the range was 17.9\u201319.6%.Within the 20 diffusion fields generated at each length scale, the average proportion of simulated fibrotic tissue  had a sharp upstroke, and an amplitude and recovery that were comparable in slope to the other time series shown. In contrast, the corresponding voltage time series shown for a length scale of 10 mm in Figure CRNremodelled variant produced a lower amplitude deflection in the inexcitable regions.A possible explanation for these observations is explored in Figure CRNremodelled variant resulted in a lower amplitude excursion. The lower amplitude excursion in the CRNremodelled variant in turn shortened the recovery of neighboring excitable regions by acting as a current sink. At longer length scales, these neighboring regions were smaller in extent, which could explain in the skewed distribution toward shorter APD seen in the right hand panels of Figure CRNnormal variant could act as a current source during recovery, acting to prolong APD, an effect that would have had more impact on neighboring regions. This could explain the skew toward longer APD values seen in the left hand panels of Figure The interaction of local APD, heterogenous diffusion, and the length scale of heterogeneities therefore appears to be complex, and this is emphasized by the range of action potential shapes seen in Figure CRNnormal variant, some configurations of heterogeneity resulted in one or more of the pacing stimuli being blocked, and so the number of beats recorded was less than 9. However, this effect was not observed with the CRNremodelled variant, which could be attributed to the greater prolongation of APD in heterogenous tissue with the CRNnormal variant described above. Overall, the prevalence of additional beats and re-entry was greater with the CRNremodelled variant, and with shorter length scales. Additional simulations were run using diffusion fields where the smoothly varying diffusion obtained from the GRF was replaced by an abrupt transition, and the maximum number of beats elicited was 10. These data are included as Supplementary Figure The response of this complex tissue model to decremental pacing with 9 stimuli is illustrated in Figure CRNremodelled model variant is illustrated in Video The precise configuration of regions with lower diffusion was important for eliciting re-entry, and this is illustrated in Figure CRNremodelled variant resulted in sustained re-entry of more than 2 beats, whereas simulations with the CRNnormal variant tended to result in non-sustained re-entry  is an important tissue property, and excitable tissue must be large enough to accommodate the wavelength in order to sustain re-entry. Heterogeneous diffusion modulates wavelength by slowing conduction velocity (delaying activation) and modifying APD, and so the behavior of re-entry was found to depend on the configuration of heterogeneities. Figure t Figure , and oney Figure , whereasy Figure , and a mIt is possible to represent heterogeneity in cardiac tissue conductivity at different length scales using samples of a GRF.2ms\u22121, but larger length scales of 5.0 and 10.0 mm tended to produce a wider spread of activation delays.The length scale of heterogeneity did not markedly affect the median delay in activation time relative to simulations with a uniform diffusion coefficient of 0.2 mmCRNnormal variant rather than CRNremodelled.A delay in recovery relative to simulations with a uniform diffusion coefficient was found. This tended to be greater in simulations with heterogeneity on a smaller length scale, and greater with the CRNnormal variant and negatively skewed toward lower APD with the CRNremodelled variant. This difference was attributed to inexcitable regions of simulated fibrosis acting as a current source during repolarization in simulations with the CRNnormal variant, and as a current sink in simulations with the CRNremodelled variant.Distributions of APD tended to be positively skewed toward higher APD with the CRNremodelled variant were most likely to result in sustained re-entry. However, the configuration of regions of simulated fibrosis also played an important role.With decremental pacing, simulations with smaller length scale heterogeneity and the CRNremodelled variant were also most likely to result in sustained re-entryWith a spiral wave as an initial condition, simulations with smaller length scale heterogeneity and the It is well-known that electrical heterogeneity is an important factor for determining vulnerability to arrhythmias in cardiac tissue, and this has been investigated in experimental preparations (Allessie et al., The interaction of heterogenous diffusion, length scale, simulated fibrosis, and APD is complex and merits further investigation. The relationship of this study to previous work is discussed below.The findings of the present study depend on the way that cellular electrophysiology and regions of fibrosis have been represented. In previous studies, regions of fibrosis have been represented in many different ways including inexcitable obstacles (Ten Tusscher and Panfilov, 2ms\u22121. The slowed upstroke and lower amplitude of voltage excursion observed in simulated fibrosed regions compared to normal tissue was comparable to other studies that have used more detailed models of fibroblast electrophysiology (Ashihara et al., In the present study regions of diffuse fibrosis were represented by smoothly varying but reduced diffusion, and focal fibrosis was represented as inexcitable tissue with a fixed diffusion coefficient of 0.05 mm2ms\u22121 in fibrosed regions and 0.2 mm2ms\u22121 elsewhere (see Supplementary Figure CRNnormal variant, simulations with abrupt changes in diffusion had a longer APD and greater APD dispersion, especially at shorter length scales, compared to simulations with smoothly varying diffusion. With both variants, the delay in activation time was much shorter compared to simulations with smoothly varying diffusion. The overall response to decremental pacing was also similar for the CRNnormal variant, but it was not possible to elicit more than one additional beat with the CRNremodelled variant (see Supplementary Figure An additional set of simulations was done to compare the effect of simulations that used smoothly varying diffusion fields with simulations where the diffusion coefficient was set to 0.05 mmThe length scale of heterogeneity was found to play a role in the prolongation and dispersion of APD, as well as in determining vulnerability to re-entry and rotor stability. A shorter length scale favored a longer APD, greater APD dispersion, and sustained re-entry. The configuration of simulated fibrotic regions was also important because a small isthmus of tissue often formed part of the activation pathway during re-entry as illustrated in Figure 2ms\u22121, there were discontinuities at the boundary of these regions as illustrated in Figures One of the aims of the present study was to investigate the effect of smooth changes in diffusion. Since diffusion was capped at 0.2 and 0.05 mmCRNnormal representing normal atrial myocytes, and CRNremodelled representing remodeled atrial myocytes (Wilhelms et al., CRNremodelled variant tended to modulate the effects of inexcitable fibrotic regions, and to increase vulnerability to re-entry. A further consequence of the shorter APD was the shorter wavelength of the CRNremodelled variant, which favored sustained re-entry in almost all simulations with the CRNremodelled variant and a spiral wave as an initial condition (Figure Persistent or permanent AF leads to changes in the electrophysiology of atrial myocytes, which act to shorten APD and hence wavelength, and these changes are consistent with the observed progression of AF from a short-lived paroxysmal arrhythmia to a permanent state Nattel, . In the n Figure .2+ handling mechanisms, which are affected by remodeling and also feed back into action potential shape (Grandi et al., It is possible that more complex effects come into play in real cardiac tissue. Isolated atrial myocytes from canine right atrium show a wide range of action potential shapes (Feng et al., The anatomy and configuration of fibrosis has recently been shown to be more important in determining rotor location than cellular electrophysiology (Deng et al., This study used a very simple representation of fibrosis, as discussed above. There was no attempt to represent the detailed anatomy of the human atria, which would add a further layer of complexity to the findings. In particular, diffusion was assumed to be isotropic, although fibrosis is known to have a stronger effect on lateral connections between myocytes, acting to increase anisotropy (Kohl and Gourdie, RC designed the study, wrote and implemented the computational model, ran the simulations, prepared the figures, and wrote the manuscript.The author declares 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": "Following publication of the original article , the autThe correct versions of the figures are given below:"}
+{"text": "In the original publication figures The correct figures are provided in this correction.The original article has been corrected."}
+{"text": "CS  and peripartum antibiotics were also associated with greater alpha diversity of the meconium microbiome . Meconium from infants born by CS  had lower relative abundance of the genus Escherichia (p < 0.001). Prenatal antibiotic use and peripartum antibiotic use  were associated with differential abundance of several bacterial taxa in the meconium. Bacterial taxa in the meconium microbiome were also differentially associated with infant excess weight at 12 months of age, however, sample size was limited for this comparison. In conclusion, prenatal and peripartum antibiotic use along with CS delivery were associated with differences in the diversity and composition of the meconium microbiome. Whether or not these differences in the meconium microbiome portend risk for long-term health outcomes warrants further exploration.The meconium microbiome may provide insight into intrauterine and peripartum exposures and the very earliest intestinal pioneering microbes. Prenatal antibiotics have been associated with later obesity in children, which is thought to be driven by microbiome dependent mechanisms. However, there is little data regarding associations of prenatal or peripartum antibiotic exposure, with or without cesarean section (CS), with the features of the meconium microbiome. In this study, 16S ribosomal RNA gene sequencing was performed on bacterial DNA of meconium samples from 105 infants in a birth cohort study. After multivariable adjustment, delivery mode ( The human gut microbiome undergoes rapid dynamic development in the early years of life, reaching a stable state with a diversity and complexity resembling adult gut microbiota generally by the age of three . These dAberrant early intestinal microbiome development has been associated with specific childhood diseases, such as obesity. Childhood obesity has become a critical public health problem ,8, with Early life antibiotic use, including during the prenatal and peripartum period, has been associated with higher obesity risk in studies ,15,16,17The study reported in this manuscript was undertaken to investigate the association of prenatal and peripartum antibiotic exposure and delivery mode on the meconium microbiome, and to determine if the meconium microbiome was associated with excess weight of the infant at 1 year of life.Mothers were recruited prenatally in an ongoing longitudinal childhood health cohort study within the Inova Health System \u201cThe First 1000 Days of Life and Beyond\u201d. Informed consent was obtained from all subjects. All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by the Inova Health System Institutional Review Board . Infants had meconium collected . Meconium was stored in Eppendorf Tubes at \u221280\u00b0C within 12 hours of collection. Only full term infants  were included.Demographic information was collected including infant sex, maternal ethnicity, and gestational age at delivery by questionnaire and the electronic medical record. Detailed information was recorded regarding delivery  and emergency CS . Pregnancy details collected from the electronic medical records included the use of prenatal antibiotics (defined as antibiotics given to mother from conception to 2 days prior to delivery) and peripartum antibiotics (defined as antibiotics given to the mother from 2 days prior to delivery to during the delivery). Weight gain during pregnancy was calculated with pre-pregnancy weight and admission weight at delivery, noted in electronic health records . The Heag for 2 min. Supernatant was removed, avoiding the pellet, and placed in a new ET with one Inhibit X tablet (Qiagen). Samples were vortexed until the tablet was dissolved and incubated at room temperature for 3 min. Samples were centrifuged at 20,000\u00d7 g for 2 min; supernatant was removed and put into a new Eppendorf Tube and centrifuged again at 20,000\u00d7 g for 3 min. The Qiagen QIAmp DNA Stool Mini kit protocol then followed (Qiagen). Meconium samples stored at \u221280 \u00b0C were thawed on ice and suspended in ASL buffer  at a ratio of 2.5 mL ASL to 0.5 g of meconium . Tube-stStaphylococcus aureus and Escherichia coli were also included.Sequencing libraries were prepared using a Nextera XT kit  according to the 16S Metagenomic Illumina Library Preparation Protocol for sequencing the variable V3 and V4 regions of the 16S rRNA gene. If samples failed QC, library preparation was completed again with the \u201cPCR 1\u201d using Hemo KlenTaq\u00ae  for the PCR reaction. Hemo KlenTaq\u00ae is known to work well with sample containing PCR inhibitors, especially bilirubin, which is highly present in meconium samples. Each specimen/condition passing QC was then sequenced on the Illumina MiSeq platform (Illumina) with paired-end reads 301 bp (600 cycles). Only 3 meconium samples failed sequencing, leaving a total of 105 for analysis. Sequencing of negative controls of lysis buffer undergoing the above DNA extraction process was performed. Positive control samples of Demuliplexed reads from each sample were treated as single-ended reads and bacterial operational taxonomic units (OTUs) were picked using the open reference method using QIIME 1.9  with GreThe OTU counts table and the phylogenetic tree created by QIIME were imported into R bioconductor package phyloseq (Version 1.25.2) for data analysis. All the figures in the manuscript were generated by R Version 3.4.0. A single rarefaction at 7324 was performed  to normaBeta diversity was measured by the unweighted unifrac distance in order to capture the differences in the low abundance features. PERMANOVA was performed using the adonis function in the R package Vegan 2.5-3 to compare the beta distances between multiple clinical factors. The adonis function in R package Vegan 2.5-3 was also used to perform permutational multivariate analysis of variance using the unweighted unifrac distance matrices. Alpha diversity was measured by the number of OTUs, and the Fisher, Shannon, and Simpson indices. Two sample Wilcoxon test was used to access differences in alpha diversity between groups. Normalized counts for each sample were used to calculate the abundance of bacteria at the phylum, class, order, family, and genus levels. A two sample Wilcoxon test was used to compare the differences in relative abundance between groups at the phylum and genus levels using the normalized counts. Raw read counts for each OTU for each sample were analyzed by R Bioconductor package EdgeR 3.20.9 to discover differentially abundant OTUs according to clinical factors of interest. Benjamini and Hochberg adjustment was performed for multiple testings . The 16S rRNA sequence data are available in NCBI Sequence Read Archive (SRA), PRJNA600283.p < 0.001). Of those receiving peripartum antibiotics for VD, all were for detected Group B Streptococcus (GBS) prophylaxis. Women without a penicillin allergy undergoing a scheduled CS received a first generation cephalosporin within 60 minutes prior to the start of CS . Those who were penicillin allergic (2/27) received clindamycin (900 mg intravenously) and gentamicin (5 mg/kg intravenously). Women undergoing emergency CS also received a first generation cephalosporin; additional azithromycin was given in 12/16 mothers (500 mg intravenously). All 19 women who had VD and received antibiotics for GBS prophylaxis received penicillin . Dosing for peripartum antibiotics was confirmed in the medical administration record, as these were inpatient medications. Seventeen out of 105 (16%) of mothers received prenatal antibiotics; these were given for urinary tract infections (10/17) and respiratory infections (7/17). Penicillins, cephalosporins, or macrolides were used in all 17 cases. One out of 17 received antibiotics in the 1st trimester, 7/17 in 2nd trimester, 8/17 in 3rd trimester, and 1/17 in both the 1st and 2nd trimester. While these prenatal antibiotics were documented in the electronic medical record, dosing and duration could often not be confirmed, as they were not part of the medical administration record due to being outpatient medications.Meconium was collected within 48 hours of life and was sequenced successfully from 105 infants. Forty-five (43%) were male; 62 (59%) were born by VD and 43 (41%) by CS. Of those delivered by CS, 27/43 (63%) were by a scheduled CS and 16/43 (37%) by emergency CS. p < 0.05). No significant differences were found in the sex, maternal pre-pregnancy BMI, maternal weight gain during pregnancy, weight status of the infant at 12 months of age, and scheduled CS versus emergency CS . In multivariable analyses adjusted for covariates that were significant in univariable models, delivery mode (p = 0.044), prenatal antibiotic use (p = 0.005), and peripartum antibiotic use (p < 0.001) remained significant.In the 105 meconium samples, each of the clinical variables was examined in relation to beta diversity using the Adonis test with 9999 permutations. Beta diversity was significantly associated with delivery mode (CS versus VD), prenatal antibiotic use, and peripartum antibiotic use  and Simpson (p = 0.002) indices compared to VD, but no difference in the number of observed OTUs (p = 0.55) or Fisher (p = 0.58) diversity. Use of peripartum antibiotics was associated with higher Shannon and Simpson diversity, but a lower number of observed OTUs and Fisher diversity . Shannon diversity was also higher in samples from non-Hispanic mothers (p = 0.04), but sample size for ethnicity analysis was limited. No significant differences in any of the alpha diversity measures were observed based on sex, maternal weight gain during pregnancy, and weight status of the infant at 12 months of age.When analyzing the alpha diversity of meconium, infants delivered born by CS had higher Shannon (iversity a. While p < 0.05) among VD infants. Observed OTUs, and Shannon and Fisher indices were also higher in samples from non-Hispanic mothers (p < 0.05), however, the peripartum antibiotic use in infants born by VD was highly skewed between the Hispanic and non-Hispanic populations, with 9 out of 20 (45%) non-Hispanic mothers using peripartum antibiotics compared with only 2 out of 13 (15%) Hispanic mothers. Therefore, whether there was a difference in alpha diversity in the samples from mothers that did not use peripartum antibiotics was further examined (11 Hispanic mothers and 11 non-Hispanic mothers). Two sample t-tests demonstrated that, while the average alpha diversity in the four alpha diversity measures were still higher in samples from infants born from non-Hispanic mothers, the differences were no longer significant. It was therefore concluded that the difference observed between ethnicity in the mothers may be driven by the peripartum antibiotic use.Next, whether these alpha diversity associations remained significant when considering only infants born by VD was investigated b. PrenatEscherichia had the highest mean abundance (28.5%), followed by Methylobacterium (15.7%) and Streptococcus (7.2%).Overall, in full term meconium samples, Proteobacteria had the highest mean abundance at the phylum level (65.2%), followed by Firmicutes (26.3%), and Bacteroidetes (4.2%). At the genus level, Escherichia and Bacteroides were higher in VD (FDR adjusted p < 0.05). Escherichia in particular was markedly higher in VD (38%) vs. CS (15%) infants (FDR adjusted p < 0.001), Methylobacterium and Veillonella were higher in CS meconium samples (adjusted p < 0.05), with Methylobacterium being markedly higher in CS compared to VD . At the genus level, the relative abundance of Streptococcus . Among the top 10 most significant OTUs, 8 were from the genus tococcus , which w infants . SignifiClostridium and 2 OTUs from the family Enterobacteriaceae with unspecified genus were enriched in meconium samples from infants born to mothers who used prenatal antibiotics. Next, antibiotic use was examined. A total of 126 bacterial OTUs were differentially abundant between meconium samples from infants delivered to mothers with vs. without prenatal antibiotic usage. The number of OTUs decreased to 75 if only infants born by VD were considered . The topEnterococcus was lowes in the meconium of infants born to mothers that used peripartum antibiotics, whereas the OTUs belonging to Agrobacterium and Streptococcus were higher.A total of 214 OTUs were significantly different in relative abundance between samples from mothers with vs. without peripartum antibiotics use; the number of significant OTUs decreased to 53 for infants born by VD only . The topStreptococcus, Klebsiella, Escherichia, Veillonella, and Acinetobacter. There were more OTUs from the genera Pseudomonas and Enterococcus and fewer OTUs from Escherichia and Propionibacterium in the meconium of infants born to mothers with excess weight gain. Differentially abundant OTUs were also examined between other clinically relevant factors. Escherichia. Prior studies support the notion that the microbiome present in the first passage of newborn meconium may reflect the infant\u2019s intrauterine and perinatal environmental exposures [In our birth cohort of full-term mother-child dyads, it was found that prenatal antibiotic use, peripartum antibiotic use, and delivery mode were associated with features of the meconium microbiome. The beta diversity of meconium, after multivariable adjustment, was associated with delivery mode, with prenatal antibiotic use and with peripartum antibiotic use. CS and peripartum antibiotics were associated with greater alpha diversity (Shannon and Simpson), and CS samples had a lower relative abundance of the genus xposures . This stPeripartum antibiotics are given to \u226540% of pregnant women in the USA and have successfully decreased the rate of neonatal GBS infection and maternal post CS infection ,35,36. IEscherichia were seen in the meconium from infants born by CS. This is a shift from the expected \u201cnormal\u201d, very early colonization pattern where there is a predominance of facultative anaerobes, such as species from Escherichia, as seen in infants born by VD [In examining the association of peripartum antibiotics with the meconium microbiome, it is important to account for delivery mode, which can result in a lack of exposure to maternal vaginal and perineal microbes . Indeed,rn by VD . Prenatal antibiotic exposure was also associated with changes in the meconium microbiome, although to a lesser extent, with differences in beta diversity and relative abundance of certain taxa and a trend towards lower alpha diversity. Prenatal antibiotic exposure has been associated with later obesity in children , howeverPrenatal and peripartum antibiotics, in addition to CS delivery, have been associated with obesity ,18,19,20There are several limitations of our study. The sample size was limited to rigorously examine prenatal antibiotics as well as other factors such as excess infant weight at 1 year of age and ethnicity. Furthermore, although in general there was homogeneity in the type of peripartum antibiotics used, there was a lesser degree of prenatal antibiotics used, and a larger sample size would be needed to determine whether specific antibiotics and doses of antibiotics, broad versus narrow spectrum antibiotics or the specific timing in the prenatal period in which they were received, impacted the microbiome to a greater degree. Additionally, the dosing and duration of all of the prenatal antibiotics could not be confirmed. A further limitation is that meconium samples generally had a low abundance of bacteria and as with any low abundance microbiomes, there was a potential for contamination; our inclusion of both positive and negative controls serves to minimize this concern. Additionally, anthropometrics used were parent reported, which are known to have varying reliability. While measured anthropometrics were preferred, the accuracy of parent-reported anthropometrics was improved in this study by the removal of outliers, which has been previously validated in this cohort .In conclusion, through examination of the meconium, which may provide an insight into intrauterine events and the very earliest pioneering gut microbes, it can be seen that prenatal and peripartum antibiotic use, in addition to CS delivery, were associated with differences in the structure and composition of the very early infant gut microbiome. Prospective longitudinal studies are needed to follow how early life antibiotic exposure may continue to affect microbiome development trajectories and association with diseases, such as obesity, thought to be related to aberrant gut microbiome development."}
+{"text": "Despite our understanding of the principal factors that shape bird migration strategies, there is conflicting evidence regarding the role of habitat in shaping migration routes and schedules, including day and night activity and differences between autumn and spring. For fly-and-forage migrants, we predict that habitat characteristics might guide migration speed, route selection and migrating schedules.We use solar-powered GPS transmitters, obtaining high accuracy data, to monitor the migratory movements of Eleonora\u2019s falcon breeding in Cyprus, which is the easternmost breeding population of the species. We tested for potential preferences in habitat characteristics along the migration routes, separately for the northern, drier part and the more vegetated southern part of the trips. We also examined the relationship between migration speed and vegetative cover during day and at night, accounting for wind support.We found that tagged individuals repeatedly exhibited an anticlockwise loop migration pattern with spring routes being more easterly than autumn ones. We identified a preference for migration through vegetation-rich areas, where during daytime tagged individuals travel at slower migration speeds compared to vegetation-poor areas, indicating fly-and-forage activity. Birds roosted during most nights, combining refueling stopovers at selected vegetation-rich areas before or after crossing ecological barriers. Conversely, both during day and night, tagged individuals overflew unsuitable habitats more quickly.Our results suggest that habitat is an important factor in Eleonora\u2019s falcon migratory strategies. Active selection of vegetation rich areas in combination with reduced migration speeds there, allows the migrating falcons to combine migration during the day with fly-and-forage refueling, while roosting most nights. Migration is part of the annual cycle of many bird species that has evolved over millennia \u20133. The mAvian migration strategies are guided by pre-migratory fuel deposition, but refueling en route is equally critical . IntegraFalco eleonorae) is a complete, long-distance, trans-equatorial migrant species [Eleonora\u2019s falcon  was used as a decoy [Birds were caught using mist nets at nesting and foraging locations . Mist ne a decoy . In addi a decoy  and mark a decoy , while b a decoy . Sex and a decoy . Transmi a decoy . Two dif a decoy , 50, 51. a decoy , with in a decoy .The positional error of GPS locations was less than 20\u2009m for 80% of the retrieved points , we thusBecause of the lower accuracy of PTT obtained locations, we decided to rely only on GPS positions for path metric estimates using all available segments. Segment length between two successive telemetry fixes was calculated based on the geodesic distance , i.e. thBird activity and flying mode between two successive telemetry fixes  and related tortuosity of the tracks affect path metrics . TherefoAs stopovers we considered areas with a 25\u2009km radius where an individual stayed for at least 24\u2009h without exhibiting directional migratory movement, allowing for one outlier point per stopover , 65. ThioN. We used the Corrected Akaike\u2019s Information Criterion (AICc) to select the best models for each test, considering all models with \u0394AICc <\u20094 compared to the best supported model.In order to determine whether Eleonora\u2019s falcon selects specific habitat characteristics within its seasonal migration corridors, we compared actual bird positions with random locations generated within a 50\u2009km buffer either side of each trip . This diBecause migration speed is related to the activity of tracked individuals, for example slower migration speeds are expected when birds are foraging, we investigated which factors affect the migration speed of Eleonora\u2019s falcon during migration with the use of GLMMs with a Gaussian distribution with identity link function using lme4 in R 3.5.1 . Specifio degrees for autumn and 350o in spring, based on the shortest direct line potential migratory route from Akrotiri colony in Cyprus to the point of the narrowest crossing over the Mozambique Channel to Madagascar and vice versa.Knowing that wind will have an effect on migration speeds, and to make sure that the wind effect is accounted for when estimating the independent effect of habitat characteristics, we included wind as a fixed effect in our models. The loggers that we used did not provide flying altitude data, so to calculate tailwind at each GPS location we used winds at 850\u2009hPa pressure level, corresponding to a mean altitude of 1500\u2009m\u2009a.s.l.. In previous studies on raptors migrating through Africa a 925\u2009hPa pressure level  has been used, but most of those studies were conducted on the western African flyway e.g. , 68, 79), 7968, 7From the eight Eleonora\u2019s falcons tagged that provided migration data Table\u00a0, we obtaN\u2009=\u20098, mean\u2009=\u200924.75\u2009days, SD\u2009=\u200911.52) than spring migration , though we note that the confirmed durations in spring came from only one individual (Farofylakas) for which the comparative results indicate the same average duration . In autumn, departure from Cyprus occurred between 4th October and 6th November, with the peak around 26th October . Arrival in Madagascar occurred between 8 and 28th November without any obvious peak. In spring, departure from Madagascar was between 9 and 15th April, and arrival in Cyprus between 25th April and 9th May , compared to 7245\u2009km (SD\u2009=\u2009401) in spring. The shortest path distance was 5749\u2009km (SD\u2009=\u2009209) in autumn and 5845 (SD\u2009=\u2009626) in spring, so actual distances traveled were about 24% further than the minimum possible distance. The longest apparently continuous flight, recorded for Ifigeneia, was 3530\u2009km, covered in 85\u2009h with an average migration speed of 40\u2009km/h (maximum single segment migration speed 73\u2009km/h). We found that during the crossing of the drier (northern) part of their migration routes, the falcons used areas with higher NDVI in lower elevations compared to generated random points within the 50\u2009km buffer zone either side of their path  and 6.41\u2009km/h  respectively. The highest migration speeds though were recorded at night, both in autumn and spring, over the Mediterranean, Sinai and the Red Sea with a maximum migration speed of 89\u2009km/h recorded. Overall, migration speeds were significantly slower over areas with higher NDVI and higher tree cover and also significantly slower in spring compared to autumn because of the relative effect of tailwinds  compared with the day  (N\u2009=\u2009901): a) full list of candidate models; b) full model; c) candidate model (\u0394AICc <4); d) candidate model (\u0394AICc <4); e) candidate model (\u0394AICc <4); f) candidate model (\u0394AICc <4); g) best supported model. In the drier north (e.g. Sahara desert), the falcons used areas with higher NDVI compared to randomly generated points, avoiding higher elevations. Table S2. GLMM results for comparison between actual positions and random points generated within a 50\u2009km buffer zone either side of the flying path, in relation to NDVI, tree cover and elevation for the south part of the migratory routes (latitude 16\u2009N southwards) (N\u2009=\u20092811): a) full list of candidate models; b) full model, c) candidate model (\u0394 AICc <\u20094); d) best supported model. In the more vegetated south they occurred , in areas with higher tree cover, and subsequently higher elevations (particularly in autumn) than what was available to them alongside their routes. Table S3. GLMM results for the effect of all GPS telemetry locations (N\u2009=\u20091435) on migration speed, of NDVI, tailwind, tree cover and season, with interactions of season with the other fixed effects, and individual and year as crossed random factors: a) full list of candidate models; b) full model; c) Candidate model (\u0394AICc <\u20094); d) Candidate model (\u0394AICc <\u20094); e) Candidate model (\u0394AICc <\u20094); f) best supported model. Migration speeds were significantly slower over areas with higher NDVI and tree cover and also significantly slower in spring compared to autumn because of wind effect. Table S4. GLMM results for the effect on migration speed, of NDVI, tailwind, tree cover, season and day/night (excluding transitioning segments between day and night) with the interactions of day/night with the other fixed effects and individual and year as crossed random factors (N\u2009=\u2009984): a) full model; b) best supported model (Delta AICc <\u20094). In this dataset, slower migration speeds were recorded at night, and over areas with higher NDVI. Differences in migration speed between day and night were greater in autumn compared with spring, with significantly slower migration speeds at night during spring. Table S5. GLMM results for the effect on active migratory movements\u2019 migration speed (>\u20095\u2009km/h), of NDVI, tailwind, tree cover, season and day/night , with interactions of day/night with the other fixed effects and individual and year as crossed random factors: a) full list of candidate models; b) full model; c) Candidate model (\u0394AICc <\u20094); d) Candidate model (\u0394AICc <\u20094); e) best supported model. In this dataset, migration speeds were higher with increasing tailwind. Migration speeds were still slower over higher NDVI, but conversely, higher during the night compared with the day. Also, the effect of increasing tree cover percentage on migration speed was higher during the day compared to the night. A seasonal effect was also identified here, with the difference in migration speed between day and night being again greater in autumn compared to spring with significantly slower migration speeds at night during spring. Figure S1. Migratory routes during (a) autumn 2013 and (b) spring 2014 migration seasons overlaid on to the respective average monthly east \u2013 west winds (U wind component) at 850 hPa (c1500 m a.s.l.). Line colors represent the different individuals. East-west wind component is weaker compared to north-south component, facilitating north-south primary movement direction to and from wintering grounds. Data downloaded from ERA5 atmospheric reanalysis product of the European Centre for Medium-Range Weather Forecasts (ECMWF)."}
+{"text": "The mammalian Notch family ligands delta\u2010like 3 (DLL3) is reported to be a potential therapeutic target for large cell neuroendocrine carcinomas (LCNEC). The effect of DLL3 expression on LCNEC prognosis has not yet been elucidated.We reviewed the medical records of 70 LCNEC patients undergoing surgical resection between 2001 and 2015 using a prospectively maintained database. We performed immunohistochemistry for DLL3 and investigated the correlation between the sensitivity of LCNEC to platinum\u2010based adjuvant chemotherapy.P = 0.36, five\u2010year RFS: 41.7% vs. 35.7% P = 0.74). In contrast, among patients with DLL3\u2010negative tumors, significantly greater five\u2010year OS and RFS rates were observed for patients with adjuvant chemotherapy than for those without it . A multivariate analysis for the RFS revealed that adjuvant chemotherapy was a significant independent prognostic factor among patients with DLL3\u2010negative tumors , although it was not a factor among patients with DLL3\u2010positive tumors .DLL3 expression was positive in 26 (37.1%) LCNEC patients. A total of 23 patients (32.9%) received platinum\u2010based adjuvant chemotherapy. Among patients with DLL3 expression\u2010positive tumors, no difference was found in the five\u2010year overall survival (OS) or recurrence\u2010free survival (RFS) between patients with and without adjuvant chemotherapy (surgery + chemotherapy vs. surgery alone, five\u2010year OS: 58.3% vs. 35.7% Our results revealed that DLL3 is a predictive marker of sensitivity to platinum\u2010based adjuvant chemotherapy for LCNEC.DLL3 was a predictive marker of sensitivity to platinum\u2010based adjuvant chemotherapy for LCNEC. Among patients with DLL3 expression\u2010negative LCNEC, platinum\u2010based adjuvant chemotherapy significantly improved the OS and RFS, although it did not do so among patients with DLL3 expression\u2010positive LCNEC.Our results suggest that DLL3 expression\u2010positive LCNEC may be better treated with other types of adjuvant chemotherapy, such as the anti\u2010DLL3 therapies if these effects are confirmed by ongoing clinical research. DLL3 was a predictive marker of sensitivity to platinum\u2010based adjuvant chemotherapy for LCNEC. Among patients with DLL3 expression\u2010negative LCNEC, platinum\u2010based adjuvant chemotherapy significantly improved the OS and RFS, although it did not do so among patients with DLL3 expression\u2010positive LCNEC. Large cell neuroendocrine carcinoma (LCNEC) is categorized as high\u2010grade neuroendocrine carcinoma (HGNEC) in the fourth edition of the World Health Organization Classification of Lung Tumors.et al. reported that pulmonary LCNEC comprise two genomic subgroups with specific transcriptional patterns, defined as \u201ctype I LCNEC\u201d and \u201ctype II LCNEC\u201d in the first comprehensive molecular analysis of LCNEC.Delta\u2010like protein 3 (DLL3) is an atypical member of the Notch receptor ligand family and reported to inhibit Notch signaling.These previous findings suggest that DLL3 expression will play a key role in clinical practice of LCNEC in the near future; however, the association of DLL3 expression with the clinicopathological features of LCNEC has not yet been elucidated. In the present study, we analyzed the effect of DLL3 expression on overall survival (OS) and recurrence free survival (RFS) in LCNEC and further analyzed the predictability of this expression for the efficacy of adjuvant chemotherapy.n = 5), synchronous multiple lung cancers (n = 3), performing induction chemotherapy or radiotherapy (n = 3), and insufficient medical records or pathological samples (n = 6). The 70 remaining patients were analyzed  staging system.Contrast\u2010enhanced chest and abdominal computed tomography (CT), positron emission tomography (PET)\u2010CT, and brain magnetic resonance imaging (MRI) were performed for preoperative staging. Patients were evaluated postoperatively at three\u2010month intervals for two years, at six\u2010month intervals for the subsequent three years, and annually thereafter. Follow\u2010up examinations included chest radiography, contrast\u2010enhanced CT, brain MRI, and bone scintigraphy as well as hematologic and biochemical analyses, including the measurement of the tumor markers. The OS and RFS were calculated according to the Kaplan\u2010Meier method, and the log\u2010rank test was used to evaluate differences in the distributions. OS was defined as the time interval between the date of surgery and the date of death. RFS was defined as the time interval between the date of surgery and the date of death without recurrence or the date of the first recurrence detected by a radiological examination.LCNEC was diagnosed based on the histopathological criteria described by the World Health Organization in 2015Immunohistochemical staining was performed on 4 \u03bcm thick formalin\u2010fixed, paraffin\u2010embedded sections. Sections were deparaffinized in xylene and rehydrated in graded alcohol baths. Antigen retrieval was performed by immersing the sections in 10\u2009mM sodium citrate buffer  for 20\u2009minutes at 98\u00b0C in a water bath. Endogenous peroxidase activity was blocked in 3% hydrogen peroxide in water. Immunostaining was performed using the avidin\u2013biotin complex technique . Sections were incubated with anti\u2010DLL3  at a dilution of 1:300 at 4\u00b0C overnight and then incubated with biotinylated goat\u2010anti\u2010rabbit IgG, followed by peroxidase\u2010conjugated streptavidin complex. Complexes were visualized with 3,30\u2010diaminobenzidine, and the slides were counterstained with Mayer's hematoxylin, dehydrated and mounted. For the neuroendocrine markers, the sections were immunostained with the streptavidin\u2010biotin technique with an automated immunostainer  according to the manufacturer's instructions. Antibodies against chromogranin A , synaptophysin , and CD56 (NCAM)  were used.All samples were evaluated by an expert pathologist (Y.S.) without knowledge of the patient's outcome. In this study, the cutoff value for the percentage of positive tumor cells was set at 1% for DLL3 Fig. . We set t\u2010test and the chi\u2010square test were performed to assess the significance of the differences in age, sex, smoking status, lung function, surgical procedure, adjuvant chemotherapy, pathological stage (p\u2010stage) and the other pathological factors between the DLL3 expression\u2010positive and DLL3 expression\u2010negative groups. The OS and RFS were calculated according to the Kaplan\u2010Meier method, and differences in the distributions were evaluated by the log\u2010rank test. The Cox proportional hazards model was used to evaluate the association between prognostic factors and the RFS after pulmonary resection, with the hazards ratio (HR) and 95% confidence intervals (CIs). P\u2010values of <0.05 were considered statistically significant.Statistical analyses were carried out using the JMP 14 software program . Student's P <\u20090.01), patients with a better lung function (P\u2009<\u20090.01), and more patients with lymphatic permeation than the DLL3 expression\u2010negative group (P\u2009<\u20090.01). The five patients with combined LCNEC were all included in the DLL3 expression\u2010negative group. There was no marked difference in the sex, smoking status, surgical procedure, pathological stage, pathological tumor size, or rate of lymph node metastasis, pleural invasion, or vascular invasion between the patients with DLL3 expression\u2010positive and expression\u2010negative LCNEC. Patients who received adjuvant chemotherapy were significantly younger in both DLL3 expression positive and negative groups (P <\u20090.01). In DLL3 expression\u2010positive group, significantly more patients who underwent surgery alone were performed sublobar resection (P =\u20090.04). In the DLL3 expression\u2010negative group, significantly more patients with pleural invasion received adjuvant chemotherapy (P =\u20090.04).The median follow\u2010up period of all patients was 37.8 months. DLL3 expression was positive in 26 37.1%) LCNEC patients. Adjuvant chemotherapy was administered to 12 (46.2%) of the 26 patients in the DLL3 expression\u2010positive group, and 11 (25.0%) of the 44 patients in the DLL3 expression\u2010negative group. The patient characteristics are summarized in Table % LCNEC pThe regimens of adjuvant chemotherapies are shown in Table We next examined the correlation between DLL3 expression and three neuroendocrine markers. The comparison of immunohistochemical staining for the three neuroendocrine markers between DLL3\u2010positive and DLL3\u2010negative LCNEC are summarized in Table P = 0.73, five\u2010year RFS: 38.5% vs. 36.4% P = 0.91)   Fig. . We then.01) Fig .P = 0.36, five\u2010year RFS: 41.7% vs. 35.7% P = 0.74)   Fig. . In cont01) Fig. .P <\u20090.01), although it was not a significant independent prognostic factor among patients with DLL3 expression\u2010positive LCNEC   that were considered to have an influence on the recurrence of patients with LCNEC.8) Table .In the present study, we demonstrated that DLL3 expression was a strong predictor of the efficacy of platinum\u2010based adjuvant chemotherapy for pulmonary LCNEC patients. Among patients with DLL3 expression\u2010negative LCNEC, platinum\u2010based adjuvant chemotherapy significantly improved the OS and RFS, although it did not do so among patients with DLL3 expression\u2010positive LCNEC. These results were confirmed by a multivariate analysis.DLL3 is a unique Notch receptor ligand that inhibits the Notch pathway.There is increasing evidence that surgical resection alone is insufficient for treating LCNEC,et al. revealed that LCNEC could be divided into two subtypes (type I and type II LCNEC) based on the genetic background according to a transcriptome analysis conducted with a next\u2010generation sequencer.We previously reported that the combination of three neuroendocrine markers was predictive of the sensitivity to platinum\u2010based chemotherapy.et al. compared the performance of four DLL3 antibodies in HGNEC samples and cell cultures and found poor results concerning the overall agreement.The rate of DLL3 expression by immunohistochemistry was reported to be 65% among LCNEC patients,et al. reported that LCNEC responded better to an SCLC\u2010targeting regimen (platinum\u2010etoposide).Several limitations associated with the present study warrant mention. First, this study was a retrospective, nonrandomized single\u2010center study with a small sample size. There may be some selection bias with regard to the performance of adjuvant chemotherapy, and its selection criteria have not been clarified. Second, the chemotherapy regimens were not standardized in this study. An NSCLC\u2010targeting regimen (platinum\u2010vinorelbine) was administered to patients with LCNEC before Rossi There are no potential conflicts of interest."}
+{"text": "Somatic cell nuclear transfer (SCNT) has broad applications but is limited by low cloning efficiency. In this review, we mainly focus on SCNT-mediated epigenetic reprogramming in livestock and also describe mice data for reference. This review presents the factors contributing to low cloning efficiency, demonstrates that incomplete epigenetic reprogramming leads to the low developmental potential of cloned embryos, and further describes the regulation of epigenetic reprogramming by long non-coding RNAs, which is a new research perspective in the field of SCNT-mediated epigenetic reprogramming. In conclusion, this review provides new insights into the epigenetic regulatory mechanism during SCNT-mediated nuclear reprogramming, which could have great implications for improving cloning efficiency. Somatic cell nuclear transfer (SCNT) is an assisted reproduction technology for the generation of cloned mammals that involves the culture of donor somatic cells and oocytes, transplantation of donor cell nuclei into enucleated oocytes, activation of reconstructed embryos, and transfer of cloned embryos into surrogates . SCNT enThe first SCNT mammal was a sheep known as Dolly that was born in 1997 , and sinAlthough SCNT has been successfully used to clone many species of mammals with significant improvements in cloning efficiency in more than 20 years since the birth of Dolly, the proportion of cloned embryos that develop to full term remains very low, greatly limiting the application of SCNT technology . To imprDuring development, totipotent embryos differentiate into pluripotent stem cells and subsequently into differentiated cells. Cell fate determination is largely achieved by activating some genes while suppressing other genes through epigenetic modification such as DNA methylation, histone modification, genomic imprinting, and X chromosome inactivation (XCI) . These hde novo DNA methylation  and histone deacetylase (Hdac). Hat opens up chromatin, which allows transcription factor binding and leads to activation of gene transcription, whereas Hdac promotes gene inactivation . After fGenomic imprinting, an epigenetically regulated phenomenon that shows monoallelic parent-specific gene expression, is controlled by the differentially methylated region (DMR) or specific histone modifications. The DMR is protected by DNA-binding complexes composed of Dnmt1, zinc finger protein 57, and tripartite motif-containing 28, and the H3K27me3 mark . In geneFemale mammals have two X chromosomes, whereas only one is present in males. In order to balance gene dosage, female mammals silence one X chromosome through the activity of the Xist gene product, a long non-coding (lnc)RNA on the inactive X chromosome that recruits transcriptional repressors such as PcG proteins . During As epigenetic modification regulates gene expression, disrupted epigenetic modification during SCNT leads to the abnormal transcription of genes related to development in cloned embryos. The persistently high expression of donor somatic cell-specific genes and failure to activate genes related to embryo development are against SCNT-mediated nuclear reprogramming . TherefoPresently, the aim of SCNT-mediated nuclear reprogramming-related research is to improve epigenetic reconstruction in cloned embryos, as the degree of epigenetic reprogramming determines the developmental competence of cloned embryos .Improving DNA methylation reprogramming has been applied in cloned embryos . One wayModifying histone marks is another approach for increasing the development competence of cloned embryos. Hdac inhibitor treatment increases histone acetylation and opens up the chromatin structure, which facilitates the binding of transcription factors that activate genes involved in early embryonic development. Hdac inhibitors have been used to improve the developmental ability of cloned embryos. For example, trichostatin A (the class I and II Hdac inhibitor), scriptaid (a synthetic Hdac inhibitor with low toxicity), and valproic acid all increase histone acetylation levels, especially H3K9ac and H3K14ac, improve gene expression levels in cloned embryos, and thus enhance SCNT-mediated nuclear reprogramming . These rImportantly, with the enhanced development of cloned embryos induced by histone modification improvements, genome imprinting in cloned embryos, fetuses, and offspring is also effectively maintained, suggesting that epigenetic modifications form mutually regulatory networks . H19 knoTargeting XCI is another strategy for improving developmental potential in SCNT. When Xist is deleted, the pattern of X-linked gene expression is corrected in cloned embryos, and the birth rate of cloned mammals is improved . The inhWith the understanding of epigenetic modifications during SCNT, researchers have successfully cloned macaque monkeys . TherefoAn increasing number of studies have shown that the degree of epigenetic reprogramming determines the developmental potential of cloned embryos . HoweverLncRNAs are gene transcripts longer than 200 nucleotides that do not encode proteins but nonetheless play a critical role in gene regulation in nearly all physiologic processes, as well as in cell fate determination during development . A comprRecent studies have shown that lncRNAs participate in many epigenetic modification processes, such as DNA methylation, histone modification, genome imprinting and XCI, and regulate the activation or silencing of genes according to cell function requirements . During After SCNT, epigenetic and gene expression profiles undergo substantial changes in cloned embryos. Naturally, lots of differentially expressed lncRNAs exist during the development of cloned embryos. However, research on key lncRNAs during the SCNT process is very limited. Encouragingly, the existing studies have suggested that lncRNAs can regulate the developmental competence of cloned embryos . StudiesIt is known to be difficult to produce cloned embryos, and their developmental competence remains poor. Moreover, studies on the factors that regulate epigenetic reprogramming during SCNT, especially those investigating lncRNAs, are still limited. To enhance the developmental potential of cloned embryos, a systematic analysis of the factors and mechanisms involved in SCNT is required. In recent years, with technological advancements, particularly the application of single-cell transcriptome sequencing, great progress has been made in discovering and identifying the reprogramming factors related to the development of cloned embryos. To date, lots of novel genes and lncRNAs have been revealed . HoweverEfforts to improve cloning efficiency have also promoted the application of SCNT technology. Presently, a series of agriculturally and economically important animals can be cloned, which could not only enable the protection of endangered species but also accelerate the utilization of livestock. Gland bioreactors can be created through SCNT to produce therapeutic proteins. Animal models can also be generated through SCNT to investigate the pathogenesis of human diseases. Moreover, with the CRISPR/Cas9-mediated genome editing technology, SCNT can produce desired animals or models for specific applications. In short, if cloning efficiency is greatly improved, the application of SCNT technology will be more extensive.SCNT has important theoretical and practical research value. In this review, we present our understanding of SCNT-mediated nuclear reprogramming, especially the factors contributing to low cloning efficiency, and that incomplete epigenetic reprogramming leads to the low developmental potential of cloned embryos. We further demonstrate that the application of epigenetic modification methods can improve cloning efficiency. We also describe the regulation of epigenetic reprogramming by lncRNAs and provide a new research perspective in the field of SCNT-mediated epigenetic reprogramming. The elucidation of these mechanisms has enhanced cloning efficiency and expanded the application of SCNT technology in agriculture, regenerative medicine, and other areas. We believe that with further advances in technology, more molecular mechanisms will be revealed to enhance the development of cloned embryos, and improved cloning efficiency will promote the extensive application of SCNT technology.YH designed the manuscript. XW, JQ, and JL wrote the manuscript. YH, HH, and ZL provided the writing guidance and revised 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."}
+{"text": "Both excipients were added in the composition for their mucoadhesive properties. The formulative space was rationalized based on the drug physicochemical stability and the main critical quality attributes of the formulation, e.g., rheological properties, syringeability, mucoadhesiveness and in vitro penetration of budesonide in porcine esophageal tissue. The obtained results demonstrated that gums allowed a prolonged residence time. However, the concentration of the mucoadhesive polymer has to be rationalized appropriately to permit the syringeability of the formulation and, therefore, easy dosing by the patient/caregiver. Eosinophilic esophagitis (EE) is a chronic immune/antigen-mediated esophageal inflammatory disease for which off-label topical corticosteroids  are widely used in clinic. In general, thickening excipients are mixed with industrial products to improve the residence time of the drug on the esophageal mucosa. The compounding procedures are empirical and the composition is not supported by real physicochemical and technological characterization. The current study aimed to propose a standardized budesonide oral formulation intended to improve the resistance time of the drug on the esophageal mucosa for EE treatment. Different placebo and drug-loaded (0.025%  Eosinophilic esophagitis (EE) is a chronic immune/antigen-mediated esophageal inflammatory disease associated with esophageal dysfunction resulting from severe eosinophil-predominant inflammation ,2. EE trWhen an authorized medicinal product is not available on the market, the compounding of extemporaneous preparations by the community and hospital pharmacists is crucial to meet the special needs of patients ,17. The In the case of the BU for EE, a certain number of studies in the literature suggested the clinical efficacy of viscous preparations. Frequently, they have been prepared by mixing a commercial sterile suspension of BU to be nebulized, which is indicated in the treatment of bronchial asthma and in infants and children with croup, with a thickening agent  ,20,21. AThe development of standardized formulations can be a valid strategy to support pharmacists in their activities, reducing heterogenicity and uncertainty in compounding procedures, improving the quality of the final preparation, at the same time. Indeed, the availability of well set up and validated operating procedures is instrumental in assuring the quality of the magistral preparation.The aim of this work is to propose a standardized BU oral formulation to improve the residence time of the drug on the esophageal mucosa. Starting from a formulation already in use in hospital pharmacies, based on xanthan gum, six pharmacists were enrolled in the compounding process to verify its reproducibility. On the bases of these results, the formulation and the compounding procedures were optimized. Considering the evidence, reported in the literature, on the combined use with galactomannans, guar gum was selected to compare its performances, when in ratio 1:1, as opposed to xanthan gum alone. The formulative space was rationalized based on the drug\u2019s physicochemical stability and the main critical quality attributes of the formulation, such as rheological properties, mucoadhesiveness and in vitro penetration of BU in porcine esophageal tissue. Micronized Budesonide (BU) was obtained from Farmabios, Gropello Cairoli, PV, Italy. Guar Gum  was kindly gifted by Lamberti spa, Albizzate, VA, Italy. All other materials were obtained from the named supplier: Xanthan Gum , (ethylenedinitrilo)tetraacetic acid disodium salt dihydrate (EDTA), sodium benzoate, sodium saccharin , glycerin, sodium dihydrogen phosphate, orthophosphoric acid, ethanol chemical grade .Acetonitrile was HPLC-gradient grade. Purified water was obtained from the purification system Milli-Q, according to Ph. Eur. 10.0 ed.w/w was added. All substances were mixed to form a homogeneous mixture, then, the exact weighted amount of BU (1 mg/4 mL) was added and carefully mixed. Purified water was weighed and added, then stirred until a uniform system was obtained. Different percentages of gums were used: F1P was prepared using XG 2% w/w, F2P with XG:GG 2% w/w, F3P with XG 1.5% w/w and F4P with XG:GG 1.5% w/w. Placebo formulations were prepared for rheological and technological evaluation. BU was added only to obtain final loaded formulations F1, F2 and F4. The composition of the formulations is reported in The exact amount of glycerin (23.6 mL) was weighed on an analytical balance and poured into a beaker. Sodium saccharin, EDTA and sodium benzoate were crushed to a fine powder with mortar and pestle, then, the exact amount of each was weighted and the powders were transferred into the same beaker. Then, XG or the mixture of gums (XG:GG) 1/1 The pH was measured at time T = 0, using a pHmeter CyberScan 1100 .v/v/v) and analyzed [About two grams of each formulation were exactly weighed and transferred into a 10 mL amber glass volumetric flask, bringing up to volume with ethanol. Then, the flask was placed in an ultrasound bath for 20 min. The sample was then centrifuged for 5 min at 3500 rpm. A portion of the supernatant was diluted to 2:5 with the mobile phase composed of phosphate buffer pH 3.2:acetonitrile:ethanol  at 40 \u00b0C. The drug content was measured at time T = 0, T = 10, 20, 60 days. Evaluation of BU content was also performed after T = 30 days at room temperature exposed to light.The steady and dynamic shear rheological properties of the formulations were carried out using a controlled stress/strain rheometer Anton Paar MCR 302  equipped with a plate-plate geometry (25 mm diameter and 500 \u03bcm gap). The temperature was controlled by a Peltier system on the bottom plate. Each sample was transferred to the rheometer plate and was then equilibrated at 25 \u00b0C for 5 min before steady and dynamic shear rheological measurements were taken.\u22121 at 25 \u00b0C. The shear stress-shear rate data were fitted to the well-known power law and Casson models [Flow behavior was evaluated at controlled strain mode to obtain flow rheological data (shear stress and shear rate) over a shear rate range of 0.1\u2013100 sn models  to descr\u22121 at 2% strain using a small-amplitude oscillatory rheological measurement. The applied 2% strain was confirmed within the linear viscoelastic region by strain sweep measurement. Frequency sweep tests were also performed at 25 \u00b0C. The RheoCompass software  was used to obtain the experimental data and to calculate the storage (or elastic) modulus (G\u2032), loss (or viscous) modulus (G\u2033) and loss factor (tan \u03b4 = G\u2033/G\u2032).Dynamic rheological data were obtained from frequency sweeps over the range of 0.628\u201362.8 rad s\u00ae, VWR International, Milan, Italy) filled with 9 mL formulation was positioned in the dynamometer holder, downward needle. The plunger end of the syringe was placed in contact with a 50 N loading cell. Testing was carried out at the crosshead speed of 0.5 mm/s. The loading force required to displace the plunger was measured as a function of plunger displacement. The following parameters were also determined from the force-displacement plot:Plunger-stopper break loose force : the force required to initiate the movement of the plunger;Maximum force (MF): the highest force measured before the plunger finishes its course at the front end of the syringe;Dynamic glide force (DGF): the force required to sustain the movement of the plunger to expel the content of the syringe.The measurement of the injection force was performed in compression mode by using a software-controlled texture analyzer . A 10 mL syringe  in A dose of the selected formulation was deposited onto a 3 \u00d7 2 cm mucosal surface corresponding to a total amount of about 500 mg. Then, the porcine esophageal membrane was placed on the sample support and pH 6.8 phosphate buffer solution (PBS) was dropped at a rate of 1 mL/min to simulate the physiological environment and the saliva swallowing. The test was performed at room temperature.The residence time was qualitatively estimated by checking the time required for each formulation to be thoroughly washed away from the surface of the mucosa exposed to the washing medium. In all cases, after 30 min from the beginning of the experiment, the apparatus was dismantled, and the applied test sample was peeled away using an adhesive tape strip . The muc2 = 0.99930; 22S: R2 = 0.99999). The analysis was carried out by HPLC-DAD  using an RP-C18 column  with pre-column . The mobile phase was composed of phosphate buffer pH 3.2:acetonitrile:ethanol . The elup < 0.05 level.Tests for significant differences between means were performed by the one-way ANOVA followed by Turkey-Kramer post-analyses. Differences were considered significant at the As the number of patients suffering from EE is increasing, among both adults and young population, the need to have a standardized topical dosage form has grown. Moreover, the widespread habit of mixing the industrial inhalation product with sweeteners  should not be the first choice. Instead, the use of a viscous preparation as a vehicle is well established in improving the efficacy of topical corticosteroid administration. The proprietary blend Mucolox\u2122 gave good results , but it XG is a polysaccharide, largely applied in the food industry. It is highly stable in a wide range of pH and ionic strength, and, dispersed in water at moderate temperatures, it increases its viscosity even at low concentration ,31. Tempw/w, according to the Italian Pharmacopoeia  or the handling during the application, F1P was modified and a mixture of XG and GG was used F2P, . The add\u22121, which is the highest that can be reached with the experimental setup used. In addition, samples loaded with the active principle were investigated and it was concluded that the behavior was not influenced by the presence of BU  and XG:GG (F2P and F4P), mixed at room temperature. They have different behaviors: formulations containing the mixture of the gums have higher shear stress in comparison to those containing only XG, as already reported in the literature , but theA different behavior among the formulations was also evidenced from the performed dynamic shear measurements. In particular, as shown in p < 0.0001).To identify elastic or viscous behavior, loss factor values can be calculated. When this value is smaller than the unit, a sample is more elastic than viscous, and this has been suggested as a rheological criterion for safe-swallow foods meant for dysphagia . All proThe administration of this preparation in very young patients was performed by caregivers, normally the parents, by means of a big syringe containing 60 mL of the formulation. During this treatment, some difficulties due to the hard extrusion of the formulation from the syringe were described by the users. The values measured for the injection force measured for placebo formulations are reported in In the force vs. displacement plot of low-viscosity formulations , two difp = 0.3890), suggesting that the force required to initiate the movement of the plunger was independent from the formulation.PBF values were not statistically different among the tested products (p = 0.1565). Moreover, in the shear stress plots, F4P showed also a pseudoplastic behavior that could not be detected with the syringeability test, probably because the applied force felt below the useful range of stress. In the case of this pseudoplastic formulation, taking into consideration the loss factor value, another rheological parameter, we can observe that it was higher if compared to the other formulations, suggesting that F4P has a more pronounced viscous component, even if its overall behavior was mainly elastic. However, overall results show that in any case low forces are needed to extrude the formulations from a syringe, despite the differences highlighted in the rheological studies. The measured extrusion forces showed that F4P required a slightly higher extrusion force (in terms of DGF) with respect to the other formulations, even if the differences were not statistically significant  can interact more strongly with mucin glycoprotein [The good mucoadhesive properties can be due to the presence of free \u2013COO\u00af groups of XG. As a matter of fact, it is well known that polymers that exhibit a high density of available hydrogen bonding groups (\u2013COOoprotein . MoreoveAs it is known, penetration of BU into the mucosa is the final combination of many events; the physicochemical characteristics of the molecule, and the mucoadhesive properties and viscosity of the formulation. The advantage of increased viscosity of the formulation is a reduced outflow from the mucosal surface, with a consequent increase in the contact time of the drug with the mucous membrane, and therefore, in its penetration into the tissue. On the other hand, an excessive viscosity could obviously create problems in the extrusion from a syringe. The results showed that the use of the two gums in combination can help to modulate the viscosity of the formulation, however, remaining within an optimal range such as to be retained onto the mucosal surface for enough time. Moreover, thanks to its pseudoplastic behavior, the extrusion of such formulations through a syringe can take place in a feasible way. Unfortunately, this aspect could not be fully investigated through the experiments carried out in this work, since the viscosity data should be collected in a higher shear range to highlight a correlation between syringeability test and viscosity measurements.The prepared formulations showed suitable technological and rheological characteristics for the treatment of EE. As the addition of GG to XG caused an increase in viscosity, the choice of a mixture of gums allows the use of thickening agents in a reduced amount. In this case, the obtained results were not significantly different from those measured with the formulation already in use. Considering the limited percentage of BU absorbed in the in vitro penetration experiments, further evaluations should be carried out to rationalize the drug content and reduce the risk of systemic side effects."}
+{"text": "The resulting continuous \u03b2-sheet nanocrystal network exhibits improved compressive strength and stiffness over the initial network lacking \u03b2-sheets of up to 30\u2009MPa  and 6\u2009MPa  respectively. The network demonstrates improved resistance to strong acid, base and protein denaturants over 28 days.The high toughness of natural spider-silk is attributed to their unique \u03b2-sheet secondary structures. However, the preparation of mechanically strong \u03b2-sheet rich materials remains a significant challenge due to challenges involved in processing the polymers/proteins, and managing the assembly of the hydrophobic residues. Inspired by spider-silk, our approach effectively utilizes the superior mechanical toughness and stability afforded by localised \u03b2-sheet domains within an amorphous network. Using a grafting-from polymerisation approach within an amorphous hydrophilic network allows for spatially controlled growth of poly and poly(valine- It is known the \u03b2-sheet structures in silk-inspired materials generate increased mechanical properties. Here, the authors report on a method of creating silk-inspired materials using in situ formation of \u03b2-sheets in an amorphous polymer to replicate the structure of silk and increase the mechanical properties. The ability to form these secondary structures in response to various environmental cues , as well as their diverse functionality, has made them a cornerstone of biochemical research. The importance of how these secondary structures affect material properties is exemplified in naturally occurring dragline spider-silk; a natural polypeptide-based structure which displays a high tensile strength comparable to high tensile steel10. Specifically, its excellent mechanical properties are attributed to the spatial arrangement of the amino acids within the polypeptide, which arrange to form higher-order \u03b2-sheet architectures through hydrogen bonding primarily\u00a0from the hydrophobic amino acid residues 14. Surrounding these \u03b2-sheet architectures is an arrangement of semi-amorphous, highly extendable glycine-rich regions. This specific arrangement  leads to spider silk displaying incredible toughness\u2014a tensile strength between 0.88\u20131.5\u2009GPa coupled with an extension at break of 21\u201327 %13. This incredible mechanical potential garners significant interest towards the utilisation of polypeptides in synthetic materials, such as hydrogels, films and fibres19 for a wide range of applications, including tissue engineering and drug delivery22.Polypeptides are the fundamental building blocks of many naturally occurring materials due to their ability to fold into specific yet complex molecular architectures, including \u03b1-helices and \u03b2-sheets29. N-Carboxyanhydride ring-opening polymerization (NCA ROP) is comparatively a cheaper and a more simple method towards the synthesis of large quantities of polypeptides with a greater potential to create long chain polypeptides, although the method lacks the same level of sequence control observed in solid-state synthesis and genetic engineering35. Despite this lack of control, we have recently shown the star-shaped polymers synthesised from NCA ROP \u2018arms\u2019 are able to observe equivalent, if not better anti-microbial activity when compared to sequence-controlled polypeptides )36. Specifically, when designing polypeptides for \u03b2-sheet formation, the control of hydrophobic association is fundamental, yet difficult to achieve\u00a0in the laboratory. This generally leads to ill-defined, uncontrolled and poorly soluble aggregates37, which greatly impacts the mechanical potential of the formed structures (e.g. bulk hydrogels)37.When synthesising \u03b2-sheet forming peptides, both the synthetic technique and the molecular components need to be carefully considered. Synthesis via genetically modified microbes and solid-state synthesis are both common methods for producing peptides, but can require acute knowledge of the peptide sequences that the genes are derived from in the case of the former, and in the case of the latter, solid-state synthesis is limited to the fabrication short chain peptides due to decreasing reaction yield with increased coupling cycles for each amino acid22. A grafting-to approach may then be used to subsequently incorporate peptides into a hydrogel39. For example, Clarke, Pashuck40 have reported the use of grafted \u03b2-sheet forming peptide sequences to the polymeric backbone of hydrogels to induce self-healing properties, however because the peptides remained short with a high hydrophilic residue content, mechanical performance was limited. When employing NCA ROP to synthesise linear polypeptides, hydrophilic blocks such as poly(ethylene glycol)35 or hydrophilic polypeptides33 have often been used to prevent this uncontrolled association from occurring, but their inclusion is highly detrimental to the polypeptides mechanical potential due to the disruption of the hydrophobic association35. Natural spider-silk uses blocks of alanine to form \u03b2-sheets during the natural silk fiber production process which involves specific environmental changes to encourage the formation of the \u03b2-sheets10. However, it should be noted that although it is well known that alanine forms \u03b2-sheets at low molecular weight , high molecular weight (larger blocks) alanine has been shown to form \u03b1-helices43. Our group has had previous success using valine NCA ROP to form \u03b2-sheets, making valine a viable option for constructing spider-silk inspired materials44.Generally, attempts commonly used to allay the uncontrolled association of hydrophobic residues and subsequent unusable aggregates introduce a high ratio of hydrophilic components to form polypeptidesHerein, we report a simple method to mimic spider-silk by creating localised \u03b2-sheet domains, which endow the formed bulk hydrogels with superior mechanical performance. A grafting-from approach is used to incorporate \u03b2-sheet forming polypeptides into a pre-fabricated three-dimensional hydrophilic network, which acts as a template to guide \u03b2-sheet assembly to mimic the amorphous matrix found in spider-silk. Free amine groups embedded in the networks are subsequently used as a site for NCA ROP of valine and glycine resulting in the spatial and controlled formation of crystalline \u03b2-sheet nanocrystals. The physical characteristics of the \u03b2-sheet reinforced networks are reported in terms of compressive characteristics, revealing a three-orders of magnitude increase in toughness attributed to the overall \u03b2-sheet network rather than the general effect of increased hydrophobicity. Given the natural ease and versatility of polypeptide synthesis via NCA ROP, this synthetic approach can facilitate the incorporation of \u03b2-sheets in a far broader range of materials moving into the foreseeable future.L-valine)  chains are known to result in \u03b2-sheet formation, resulting in crystalline and rigid \u03b2-sheet regions being observed45. Thus, glycine was introduced to act as a \u03b2-sheet destabilizing residue12 and being responsible for amorphous \u03b2-sheet regions in spider-silk10. Thus, L-valine NCA  and glycine NCA (Gly NCA) were synthesised and then used to form crystalline and amorphous \u03b2-sheet regions respectively.To demonstrate a proof of concept, an initial network was synthesised by free radical polymerization with pendant amine groups followed by directed \u03b2-sheet incorporation via NCA ROP Fig.\u00a0. The iniThe polymerization of \u03b2-sheet forming polypeptides via NCA ROP is grown from the available amine groups of the polymer backbone as initiating sites. Hence, Val NCA monomer concentration was varied from 30 to 360\u2009mg/mL to yield increasing pVal chain lengths Table\u00a0. As expe12, it is used to replace small quantities of Val NCA at 5 and 10\u2009mol % within a constant total molar concentration at 2.51\u2009mmol/mL  in a solution  and insufficient swellability to take on enough overall mass in water  was performed for both hydrogels and cryogels. Examination of the initial networks showed smooth surfaces for both hydrogels and cryogels as expected with highly swellable polymeric networks   binding assay under fluorescence microscopyage Fig.\u00a0, the labage Fig.\u00a0, resultiage Fig.\u00a0. Howeverne) Fig.\u00a0. In the ne) Fig.\u00a0. At closne) Fig.\u00a0, the \u03b2-s\u22121 , indicating successful polymerization48. The amide A band is shown to have a small shoulder at 3300\u2009cm\u22121 once glycine is introduced, which is more evident in samples containing greater amounts of glycine  has been identified as the most sensitive spectral region corresponding to different polypeptide secondary structures and used to determine their conformations49. Hereby, the strong amide peak around 1627 cm\u22121 with a weak shoulder at 1690 cm\u22121 suggests the formation of \u03b2-sheet secondary structure adopted in the mixture of antiparallel and parallel conformations, respectively49. The shoulder assigned to the parallel conformations slightly increases with increasing glycine content  as the amide V bond is not diminished in the glycine containing gels51. Other distinguishing features of the \u03b2-sheet incorporated networks include amide II band (N\u2013H in-plane bending) near 1530\u2009cm\u22121, amide V band (N\u2013H out-of-plane bending) around 710\u2009cm\u22121, and the pVal aliphatic side-chain C\u2013H stretch peaks around 2965\u2009cm\u22121 (which is diminished slightly one glycine is introduced)50. Also, the characteristic peaks of the initial network are still clearly present in the spectra of \u03b2\u2013sheets-incorporated networks, including C\u2013H stretch on the polymer backbone, C\u2009=\u2009O stretch of ester group and C\u2013O\u2013C stretch in the side chain. The increase in \u03b2-sheet content with increasing Val NCA concentrations in the ROP process was also highlighted with the intensity of the \u03b2-sheet band (amide I) relative to the polymer backbone stretch in the initial network (C\u2013O\u2013C stretch)  spectroscopy and X-ray Diffraction (XRD). The FTIR spectra Fig.\u00a0 shows a h) Table\u00a0. The high) Table\u00a0. XRD pat35. The stress-strain curves obtained indicate hydrogel stiffening and strength consistently increased with increasing pVal formed \u03b2-sheet content with the Young\u2019s Modulus increasing by three orders of magnitude (from 2 kPa to 9.4\u2009MPa)  Fig.\u00a0. These fPa) Fig.\u00a0 provides3, implying that the overall integrity of the gel remains intact despite the introduction of \u03b2-sheet forming residues. However, a loss in strength and subsequent increase in compressibility defines the tunable nature of these materials  (PMMA), a typical hydrophobic polymer that is hard to crystallize, was synthesised using RAFT  Fig.\u00a0 shows thThe \u03b2-sheets-incorporated networks also exhibited remarkable stability at harsh pH conditions (2\u2009M HCl and 2\u2009M NaOH solution) and in the presence of protein denaturants (6\u2009M GdnCl) Fig.\u00a0. After 462. Using our grafting-from strategy, \u03b2-sheet regions can be loaded into 3D-printed networks, endowing them with the enhanced physical properties defined above  grafts of up to 79 repeat units on average which would proceed to form a continuous \u03b2-sheet nanocrystal network. Subsequently, compressive strength was found to increase by 3 orders of magnitude up to 9.9\u2009MPa for the hydrogels and 2 orders of magnitude up to 30\u2009MPa for the cryogels. Furthermore, the properties of these networks can be easily tuned using glycine, with poly grafts introducing an increased compressibility from 40 to 60% without a significant loss in toughness. This easily employable approach provides a powerful tool in the preparation of unique hybrid networks furnished with \u03b2-sheets and can be further used to tune the mechanical and degradation properties in other hydrogels for biomedical functionality and materials science.L-valine (\u226598%) and glycine (\u226598%) were purchased from Merck and used as received. Triphosgene (98%), (+)-\u03b1-pinene (99%), anhydrous n-pentane (>99%), anhydrous N,N-Dimethylformamide (DMF) (>99.8%), 2-aminoethyl methacrylate hydrochloride (97%), triethylamine , oligo(ethylene glycol) methylether methacrylate , oligo(ethylene glycol) dimethacrylate , ammonium persulfate , N, N, N\u2019, N\u2032-Tetramethyl ethylenediamine , Diphenylphosphine oxide  Thioflavin T (ThT), Guanidine hydrochloride , phosphate buffered saline (PBS) tablets, protease from streptomyces griseus (Type XIV), N-(3-Dimethylaminopropyl)-N\u2019-ethyl carbodiimide (EDCI), 4-(Dimethylamino)pyridine  and N, N, N\u2019, N\u201d, N\u201d-pentamethyl diethylenetriamine  were all purchased from Sigma Aldrich and used as received. Anhydrous and deoxygenated Tetrahydrofuran (THF)  was obtained by distillation from benzophenone and sodium metal under argon. AR grade hydrochloric acid (HCl), sodium hydroxide (NaOH) and other solvents were purchased from Chem-Supply Pty. Ltd. and used without further purification. L-valine (\u226598%) was purchased from Merck and used as received. Anhydrous and deoxygenated Tetrahydrofuran (THF)  was obtained by distillation from benzophenone and sodium metal under argon. Deuterated chloroform (CDCl3) was purchased from Cambridge Isotope Laboratories and used as received. 2-(((butylthio)carbonothiolyl)thio)propanoic acid (TTC-1) was received from Dulux Group Australia and used as received. Methyl methacrylate  was de-inhibited by basic alumina prior to use.L-valine NCA was synthesised using a modified procedure from previously reported literature44. Briefly, L-valine  was suspended within 80\u2009mL of anhydrous THF in a two-necked round bottom flask under argon. Triphosgene  was added and the mixture was continuously stirred 60\u2009\u00b0C for 2\u2009h or until all valine had dissolved. The reaction mixture was sparged with argon into a saturated NaOH solution for 1\u2009h after which solvent was removed in vacuo  until equilibrium. The reduced mixture was redissolved in anhydrous 50\u2009mL ethyl acetate which was chilled in 5\u2009\u00b0C and then washed with saturated brine at 5\u2009\u00b0C and 0.5\u2009w/v % sodium bicarbonate aqueous solution at 5\u2009\u00b0C with the organic phase being washed after separation each time. The resulting organic phase was dried using magnesium sulphate with the filtrate being reduced in vacuo  until equilibrium. The residue was recrystallised using n-pentane overnight. The resulting crystals were filtered and reprecipitated to afford a white powder (~60% Yield). 1H NMR : \u03b4H 0.92 , 2.00\u20132.18 , 4.08 , 8.84 .63. Glycine  was suspended within a mixture of 250\u2009mL of anhydrous THF and 21.2\u2009mL (+)-\u03b1-pinene (133\u2009mmol)in a two-necked round bottom flask under argon. Triphosgene  was dissolved and added dropwise over 1.5\u2009h while mixture was continuously stirred 70\u2009\u00b0C and stirred for a further 1\u2009h. The reaction mixture was sparged with argon into a saturated NaOH solution for 1\u2009h after which the suspension was filtered and the filtrate was reduced in vacuo  until equilibrium. The resulting suspension was redissolved in THF and recrystallized using n-pentane overnight. The supernatant was removed and recrystallization was repeated two more times. The resulting white solid was filtered and washed with n-pentane . 1H NMR : \u03b4H 4.33 , 8.84 .Glycine NCA was synthesised using a modified procedure from previously reported literature2 for a few minutes. After addition of APS (20\u2009mg) and TEMED (25\u2009\u00b5L) , the solution was sonicated and separated into 1\u2009mL batches in 3\u2009mL syringes and allowed to stand at room temperature for 3 days. The resulting gels were swelled in DI water and washed six times to remove unreacted reactant. They were dehydrated in vacuo  for 2 days and stored in vacuo at room temperature for further use.2-aminoethyl methacrylate hydrochloride  was first deprotonated with TEA  in 20\u2009mL of DI water overnight. The mixture was mixed with co-monomer OEGMEMA  and crosslinker OEGDMA  and degassed with NThe gel precursor solution was prepared and transferred to the syringes in the same procedures as described above. Then the solution in the mould was frozen at \u221218\u2009\u00b0C and kept at this temperature for 3 days. After thawing and being washed with water, the cryogel sample was dried and stored in vacuo for further use.The following synthesis of HB360 is used as an example of the formation of \u03b2-sheet incorporated networks  was then switched on, and the reaction mixture was left at room temperature with occasional agitation under positive N2 pressure for 3 days. A small aliquot (~100\u2009\u03bcL) of the supernatant was taken for 1\u2009H NMR analysis to check monomer conversion rate, according to an established calibration curve as described below. After the reaction, the supernatant was removed, and the network was washed with DMF (3\u2009\u00d7\u20093\u2009mL) and sonicated in DMF (3\u2009mL) for 30\u2009min. To further remove potential non-grafted monomers/polymers, the network was then washed with THF (6\u2009\u00d7\u20093\u2009mL). Finally, the network was dried and stored under vacuum.The initial network hydrogel or cryogel was swollen in 3\u2009mL of TTC-1 (RAFT agent)  dissolved in anhydrous DMF. EDCI  and DMAP  was then added in the mixture with the reaction sealed at room temperature with occasional agitation under vacuum for 2 days to the RAFT agent. Afterwards, the network was washed with anhydrous DMF for several times to remove the free RAFT agent and remaining catalyst until the network colour did not fade. The network attached with RAFT agent was then transferred to the MMA monomer solutions (in 3\u2009mL anhydrous DMF) at different concentrations  in presence of PMDETA . The reaction mixture was sealed and degassed using nitrogen for approximately 1\u2009h. The UV light source  and TEA (600\u2009\u00b5L) overnight and then washed with anhydrous DMF (6\u2009\u00d7\u200910\u2009mL).2-Aminoethyl methacrylate hydrochloride , co-monomer OEGMEMA  and crosslinker OEGDMA  were dissolved in 25.8\u2009mL of DMF: deionised water (7:3), with TPO photoinitiator  dissolved into the solution just prior to printing. The 3D printing was performed on an ANYCUBIC Photon 3D Printer with a \u03bbThe swollen networks were transferred to vials containing 50\u2009mL of valine NCA in anh. DMF (120\u2009mg/mL). The vials were then sealed and agitated at room temperature under vacuum for 3 days. The resulting networks were then washed with six aliquots of DI water, followed by subsequent washing with DMF (3\u2009\u00d7\u20093\u2009mL) before being sonicated in DMF (3\u2009mL) for 30\u2009min.1H) Nuclear Magnetic Resonance (NMR) spectroscopic analysis was performed on a Varian Unity Plus 400\u2009MHz spectrometer using deuterated chloroform (CDCl3) as the solvent. Attenuated total reflection- Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy was carried out using a Bruker Tensor 27 FTIR, with GladiATR ATR attachment obtained from Pike Technologies. The FTIR was equipped with OPUS 6.5 spectroscopy software from Bruker Optik GmBH. X-ray diffraction (XRD) spectroscopy was carried out on a Bruker D8 Advance Diffractometer, using standard Ni-filtered Cu k\u03b1 radiation. Fluorescent images of ThT stained gel networks were acquired on a Leica TCS SP2 confocal laser scanning microscope (CLSM), using excitation wavelength of 490\u2009nm. Scanning electron microscope (SEM) images were acquired using a FEI Quanta 200 ESEM FEG. Samples were pre-coated with gold using a Dynavac Mini Sputter Coater prior to imaging. Thermogravimetric analysis (TGA) was performed on a TGA/SDTA851e, Mettler Toledo in air with heating rate of 10\u2009\u00b0C\u00b7min\u22121.Proton  at 5 different concentrations  diluted with CDCl3 (900\u2009\u00b5L). Integration of the Val NCA doublet peak at 0.92 ppm relative to the methyl DMF signals at 2.88 ppm and 2.96 ppm determined for the 5 Val NCA concentrations, afforded a calibration curve as shown in Supplementary Fig.\u00a0A calibration curve was generated from 3 (900\u2009\u00b5L). These solutions were analysed by 1H NMR by normalising against the same peaks of DMF, to determine Val NCA concentrations of each solution. This was then used to calculate the monomer conversion , the network was dried under heat in vacuo  for 24\u2009h and then swollen in water until it reached equilibrium swelling. The weight of dry and fully swollen samples was determined by analytical balance and denoted as Wd and Ws, respectively. The equilibrium swell ratio (Q) is defined by Eq. . The samples were removed from each vial at certain time point and washed in deionised water. Then the samples were dehydrated by soaking in ethanol for 1\u2009h followed by drying in vacuo  for 24\u2009h. Finally, the samples were weighed, and the mass values obtained were plotted against time to obtain the degradation profiles.The compressive strain-stress curves were obtained using an Instron Microtester 5848 equipped with a 2 kN static load cell and Bluehill material testing software. cylindrical hydrated network samples  were compressed to their maximum strain between two parallel plates at a crosshead speed of 0.1\u2009mm/second. Engineering stresses and strains were recorded.Supplementary InformationPeer Review File"}
+{"text": "Objectives:\u00a0We aimed to validate the vasoactive-ventilation-renal\u00a0(VVR) score and to compare it with other indices as a predictor of outcome in neonates recovering from surgery for critical congenital heart disease. We also sought to determine the optimal time at which the VVR score should be measured.Methods: We retrospectively reviewed neonates recovering from cardiac surgery between July 2017 and June 2020. The VVR score was calculated at admission, 24, 48, and 72 hours postoperatively. Max values, defined as the highest of the four scores, were also recorded. The main end result of interest was a composite outcome which included prolonged intensive care unit stay and mortality. Receiver operating characteristic curves were generated, and areas under the curve with 95% confidence intervals were calculated for all time points. Multivariable logistic regression modeling was also performed.Results: We reviewed 73 neonates and 21 of them showed composite outcomes. The area under the curve value for VVR score as a predictor of composite outcome was greatest at postoperative 72-hour max . On multivariable regression analysis, the VVR max 72 hours remained a strong independent predictor of prolonged ICU stay and mortality .Conclusions: We validated the utility of the VVR score in neonatal cardiac surgery for critical congenital heart disease. The VVR follow-up in postoperative 72 hours is superior to other indices and especially the maximum VVR value is a potentially powerful clinical tool to predict ICU stay and mortality. Newborns operated for critical congenital heart disease (CHD) have a high risk of postoperative mortality and morbidity -4. TheseFor this purpose, it has been shown that the vasoactive inotrope score (VIS) and serum lactate levels, which are used recently in clinical practice, are correlated with prognosis -9. VIS iHowever, newborns undergoing cardiac surgery often have multiorgan dysfunction and especially pulmonary and renal systems are affected ,11. NeitIn order to overcome this limitation of serum lactate and VIS, Miletic et al. introduced the vasoactive-ventilation-renal (VVR) score in 2015 and used markers of the cardiovascular, pulmonary, and renal systems, which are the most frequently affected systems in CHD surgery . In the In the most recent multicenter retrospective study involving only newborns, it was found that the postoperative twelfth-hour VVR score was more effective in predicting the prolonged mechanical ventilation (MV) duration compared to other indices, Society of Thoracic Surgeons - European Association for Cardio-Thoracic Surgery Congenital Heart Surgery (STAT) Mortality Categories and cardiopulmonary bypass (CPB) duration .However, in the vulnerable neonatal populations, the possibility to have temporary cardiovascular, pulmonary, and renal dysfunction in the early postoperative period is higher.\u00a0Therefore, it would be rational to follow up these patients with VVR scores for a longer period not to miss out on the sustained dysfunction which is more likely to influence the outcome.The aim of our study is to evaluate the potential of predicting prolonged intensive care unit (ICU) stay and/or mortality by calculating the VVR score for 72 hours postoperatively, and to compare it with other indices and, if possible, to find an appropriate cut-off value and to provide an idea for future studies.https://authorea.com/users/371116/articles/489571-validation-of-the-vasoactive-ventilation-renal-score-as-a-predictor-of-prolonged-intensive-care-unit-stay-and-mortality-after-critical-congenital-heart-surgery-in-neonates).This article was previously posted to the Authorea preprint server on October 29, 2020 . Informed written consent were obtained from the parents of all participants for surgical and intensive care procedures.Study populationAll neonates, defined as less than or equal to 28-days-old, who underwent pediatric cardiac surgery (with or without CPB) were reviewed.Exclusion criteria were as follows: (i) premature babies who underwent ligation of patent ductus arteriosus (PDA); (ii) cases that placed on extracorporeal membrane oxygenation (ECMO) support in the operating room or those requiring ECMO support within the first 72 hours postoperatively were excluded from the study.Data collectionData were collected via medical record review. Demographic and preoperative data collection included sex, birth weight, gestational week, being premature , being low birth weight (<2500 g),\u00a0antenatal diagnosis status, postnatal admission day, underlying cardiovascular diagnoses, presence of noncardiac anomalies, need for MV\u00a0and inotropic support at admission, need for MV and inotropic support at operation day, preoperative sepsis and need of dialysis, and postnatal day of surgery were recorded.Perioperative data collection included surgical procedure performed, STAT Mortality Category , use andPostoperative data collection included the need for ECMO, need for inhaled nitric oxide therapy, presence of postoperative open sternotomy, the occurrence of arrhythmias, postoperative MV and inotropic duration, duration of ICU stay, and mortality were recorded.\u00a0We recorded MV variables, arterial blood gas\u00a0and lactate measurements, and doses of inotropic and vasopressor medications at four postoperative time points: ICU admission, 24 hours, 48 hours, and 72 hours after ICU arrival. We also recorded preoperative serum creatinine and daily serum creatinine measurements obtained within the first 72 postoperative hours.Derivation of the VVRVVR scores were calculated as follows: VI (Ventilation Index) + VIS + \u0394Cr.VIS = dopamine dose [\u03bcg/kg/min] + dobutamine dose [\u03bcg/kg/min] + 100 \u00d7 epinephrine dose [\u03bcg/kg/min] + 10 \u00d7 milrinone dose [\u03bcg/kg/min] + 10,000 \u00d7 vasopressin dose [U/kg/min] + 100 \u00d7 norepinephrine dose [\u03bcg/kg/min]).2))/1,000).VI =  and median (25th-75th percentile) for quantitative data. It is presented as frequency (percentage) for categorical data. The significance level was taken as p<0.05.All statistical analyses were performed using IBM SPSS version 23 . Shapiro-Wilk tests were used to determine normal distribution. The main outcome of interest was the composite outcome . The study group was dichotomized as upper 25th percentile of ICU stay and/or mortality (defined as having composite outcome) versus lower 75th percentile for ICU stay.\u00a0Bivariate comparisons were performed for demographic and perioperative characteristics of patients with and without composite outcome using Chi-square or Fisher exact test for categorical variables. In the comparison of quantitative variables according to paired groups, an independent two-sample t-test was used for normally distributed data and the Mann-Whitney U test was used for non-normally distributed data. Repeated analysis of variance was used to compare normally distributed data over three or more times, and the Friedman test was used to compare non-normally distributed data. Variables that attained a bivariate significance of 0.2 or less were considered for inclusion in a multivariable logistic regression model.\u00a0Receiver operating curve (ROC) analysis was used to determine cut-off values for VIS, VI, VVR, VVRThe medical files of 95 newborns who were operated on in the first 28 days of life during the study period were examined. Sixteen of these patients were preterm babies who had bedside PDA ligation. Six patients were excluded from the study because they were placed on ECMO support in the operation room. As a result, the study group consisted of 73 patients. The anatomical diagnoses of the patients included in the study and the surgical procedures performed are summarized in Table The mean gestational week and birth weight of the study group was 38.6 \u00b1 1.6 weeks and 3179 \u00b1 528 g, respectively. Over 72.6% of the cases were male, 12.3% were premature, and 13.7% had an additional non-cardiac anomaly. The antenatal diagnosis rate was 28.8%. 38.4% of the cases had single ventricle anatomy and 82.2% were STAT category 4 or 5. None of the cohorts needed ECMO support in the first 72 hours postoperatively.The postoperative prolonged ICU stay of the cohort was defined as 29 days or longer. Eight cases died in the postoperative period. Two of these cases died before 29 days (15th and 20th days). Thus, 21 cases met the composite outcome criterion, which is the main outcome of our study. The study population was divided into two according to the composite outcome, and bivariate analysis of demographic, anthropometric, and perioperative features of cases with and without composite outcome are presented in Table ROC analysis of serum lactate, VIS, VI, and VVR scores was performed, and area under curve (AUC) values, cutoff values, sensitivity, and specificity data at all measurement points are presented in Table 72max score, the odds of a composite outcome increased by 45% . The remaining variables that were significant on bivariate analysis were not significant on multivariable analysis and did not appreciably affect the model. The best multivariable model for composite outcome including VVR 72 hour max, lactate 72-hour max, and single ventricle physiology is presented in Table All variables in Tables 72max, we dichotomized the variable into high and low. A VVR72max cutoff value of 46.5 was chosen to maximize total accuracy and minimize weighted error ratios, and correctly classified 90% of cases. On multivariable logistic regression analysis, a high VVR72max remained strongly associated with the composite outcome .To simplify the interpretation and use of the VVRWe have validated the VVR score as a multiorgan system severity of illness index for neonates recovering from surgery for critical congenital heart disease. We demonstrated that the VVR strongly predicts prolonged postoperative ICU stay and/or mortality (composite outcome) more so than VIS, VI, and serum lactate.In a limited number of prior studies, the VVR calculated at 48 hours  and at 12 hours ,15 was f72max) of each case in the first 72 hours with the maximum values of other indices, and unlike other studies, we reached the highest AUC value and highest accuracy in prediction of results. Specifically, if the postoperative VVR72max of a neonate is 46.5 and above, this case was found to be 23 times riskier in terms of prolonged ICU stay and mortality.In order to include all cases in the analysis, we compared the highest measurements  and hospital stay better than VIS and serum lactate level. The cutoff value in this study was found to be 22.5 .In the next prospective study of Miletic et al., the patient population was expanded as under 18 years of age and those entering CPB. The VVR index at postop 48th hour gave better results than all measurements, and the cutoff value was found to be 13 [Scherer et al. included pediatric and adult congenital heart patients in their study, and within 48-hour postoperative measurements; it was stated that the predictivity of the 12th-hour VVR score was the most effective, and the cutoff value was determined as 25 .In a recent multi-center study involving only newborns, like our study, score calculations were made in the postoperative 12-hour period, and the VVR score was found to be superior in determining prolonged MV duration (determined as > 96 hours in this study) compared to VIS, VI, and serum lactate. In this study, the cutoff value was expressed as 35 .As it can be seen, as the study age group descends into complex patient groups such as the neonatal period, the cutoff values increase, but the VVR score maintains its strong predictivity compared to other indices.In our study, 82% of the patient group was in STAT mortality categories 4 and 5 and 80% had undergone CPB.\u00a0Also, the rate of antenatal diagnosis was as low as 28.8%, so an important part of the study group applied to the hospital with cardiopulmonary decompensation, and 44% required preoperative MV. So our cohort consists of more severe and complex cases according to the literature. We think this is the reason why we found the VVR score cutoff value to be higher.Although VVR is a powerful marker in determining outcomes like postoperative ICU stay and mortality, there are many additional factors that affect outcomes, especially in neonates. For example, even if our study could not be demonstrated due to the limited number of cases, airway anomalies and gastrointestinal anomalies can prolong hospitalization and cause deaths even if the cardiac operation is successful. In addition, cases without antenatal diagnosis or with delays in postnatal diagnosis and treatment admit to the hospital with cardiopulmonary decompensation, and the multiorgan injuries that occur because of this, affect the postoperative process negatively. The next logical step will be increasing the predictivity of the VVR score by adding well-defined preoperative variables and other organ system markers in future studies.Longer scoring time in vulnerable populations like the neonatal period will enable us to predict persistent dysfunction leading to poor prognosis rather than acute dysfunction in the postoperative transition period. As our study shows, 72-hour values have the best predictivity after the maximum value in neonatal cardiac surgery. With multi-center and larger heterogeneous patient cohorts, specific cutoff values should be determined according to anatomical diagnosis, age group, surgical procedure, and postoperative measurement time.Study limitationsOur study has been designed retrospectively and includes only newborn cases. Therefore, multicenter and comprehensive prospective studies are needed regarding the applicability of the VVR score to all congenital heart diseases. We acknowledge that the VVR score cannot reliably be calculated for patients requiring ECMO support and peritoneal dialysis. Studies are needed which focus on this subgroup of patients to identify risk factors for poor outcomes.We validated the utility of the VVR score in neonatal cardiac surgery for critical congenital heart disease. The VVR follow-up in postoperative 72 hours is superior to other indices, and especially, the maximum VVR value is a potentially powerful clinical tool to predict ICU stay and mortality. Neonates with a peak VVR greater than or equal to 46.5 within the first 72 postoperative hours should be considered at increased risk for poor prognosis."}
+{"text": "Acacia nilotica pods\u2019 extract and the hydrothermal method to prepare nanoparticles of pure zinc oxide and pure copper oxide and nanocomposites of both oxides in different ratios. Five samples were prepared with different ratios of zinc oxide and copper oxide; 100% ZnO (ZC0), 75% ZnO: 25% CuO (ZC25), 50% ZnO: 50% CuO (ZC50), 25% ZnO: 75% CuO (ZC75), and 100% CuO (ZC100). Several techniques have been applied to characterize the prepared powders as FTIR, XRD, SEM, and TEM. The XRD results confirm the formation of the hexagonal wurtzite phase of zinc oxide and the monoclinic tenorite phase of copper oxide. The microscopy results show the formation of a heterostructure of nanocomposites with an average particle size of 13\u201327 nm.This work represents a novel combination between  Zinc oxide has been widely applied as photocatalysts ,2,3,4, a3O4-ZnO gas sensor towards triethylamine [2+ = 50 at.% [2O3, which changed the bandgap to 3.17 eV, 3.28 eV and 3.16 eV, respectively [Q. Zeng et al. reported enhanced gas sensing properties of NiO-ZnO nanocomposites, which they attributed to the formation of a p-n heterojunction . The samhylamine . Y. Wang 50 at.% . M. Toe ectively . Copper Various methods are applied to prepare metal oxide nanoparticles and nanocomposites as simple precipitation , electroTagetes spp. petals in the presence of CTAB surfactant [Sambucus nigra L. extract [R. Mohamed et al. prepared CuO-ZnO heterojunction photocatalyst through a simple wet method in the presence of F-127 surfactant . A. Prajrfactant . Moreoverfactant . ZnO-CuO extract .Acacia nilotica, also known as gum arabic tree, belongs to the Fabaceae family. It is popular in Africa and Asia and the tree has roots, pods, stems, and leaves. The extract has many phytochemical components as flavonoids, phenols, and tannins [Acacia nilotica have been used to prepare Ag-TiO2 nanocomposites [ tannins ,39. The  tannins , the lea tannins , the aermposites .Acacia nilotica Pods\u2019 extract and the hydrothermal method. Pristine zinc oxide and copper oxide nanoparticles were synthesized in addition to copper oxide-zinc oxide nanocomposites with different ratios. The functional groups and the formed phases were determined by Fourier Transform Infrared spectroscopy (FTIR) and X-ray diffraction (XRD), respectively. The morphology and particle size were followed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM).The recent work reports for the first time the combination between a green synthesis that employs 2.6H2O), zinc chloride (ZnCl2), potassium hydroxide (KOH) were bought from Sigma-Aldrich. All the primary chemicals used in this work were of analytical grade. All solutions were prepared with double-distilled water. The Acacia nilotica pods were bought from a local store, Al-Ahsa, Saudi Arabia.Copper chloride hexahydrate  (Philips XL30) was used, accelerating voltage of 30 kV and the magnification up to 400,000\u00d7. A high-resolution, JEOL JEM-1011 Transmission Electron Microscope was used for TEM imaging.Acacia nilotica pods were rinsed with tap water three times and with distilled water for the last time. To prepare the extract, 10 g of dried pods were boiled with 100 mL of distilled water at 60 \u00b0C for 15 min under magnetic stirring. The prepared extract was centrifuged at 8000 rpm for 5 min and then filtered via Whatman No. 1 filter paper, to remove all fine plant debris and stored at 4 \u00b0C [The  at 4 \u00b0C . The pH 2.6H2O) and a 0.2 M solution of zinc chloride (ZnCl2) were prepared in two different containers. The compositions of samples are mentioned in Acacia nilotica extract is added to a 100 mL of 0.2 M zinc chloride salt solution to prepare sample ZC0. A 1 M KOH was added to the previous mixture while under continuous stirring until the pH reaches 10.A 0.2 M solution of copper chloride hexahydrate  after precipitation, (c) after hydrothermal treatment and drying and (d) after calcination. The FTIR of Acacia nilotica pods\u2019 extract shows a very broad band between 2800\u20133600 cm\u22121 which may correspond to the stretching vibration of aromatic and aliphatic OH groups (3200\u20133500 cm\u22121) or stretching of aromatic and aliphatic C-H (3000\u20133100 cm\u22121). A sharp finger-like peak around 1500\u20131700 cm\u22121 stands for carbonyl groups or bending of aromatic C=C. A very broad band below 1000 cm\u22121 may refer to the bending of aromatic C-H or C-O-C groups. According to the literature, the FTIR results of Acacia nilotica pods\u2019 extract confirm the presence of flavonoids, tannins and terpenoids in the extract [\u22121 [ extract ,39. The ract [\u22121 .Samples ZC25, ZC50, and ZC75 show the peaks corresponding to wurtzite hexagonal zinc oxide and tenorite monoclinic copper oxide with different intensities. However, the intensity of peaks corresponding to wurtzite zinc oxide is decreasing gradually in the samples ZC25, ZC50, and ZC75 with decreasing the amount of zinc precursor in the starting materials. The same can be observed in an opposite way for the monoclinic tenorite and the intensity of copper oxide peaks increases gradually in samples ZC25, ZC50, ZC75 with an increasing copper precursor in the initial compositions  2,26,48,4826,48.Acacia nilotica extract, which seems to be more effective as a capping agent than ethanol as a solvent in decreasing the crystallite size [Calotropis gigantea, and the average crystallite size was about 35 nm [The crystallite size of zinc oxide and copper oxide is estimated using the Debye\u2013Scherrer formula and presented in ite size . C. Kumaut 35 nm . M. Mansut 35 nm . Again, Acacia nilotica phytoexctract acted as capping agents which enhances the growth of CuO bipyramid.Adding 25% copper oxide into zinc oxide resulted in changing the morphology from stacked layers/aggolmerated particles into homogenous elongated particles, as can be depicted in In the present study, ZnO NPs, ZnO-CuO NCs, and CuO NPs have been prepared by an inexpensive and environmentally friendly method; the green-hydrothermal method. The proposed mechanism is presented in the following equations;Acacia nilotica pods\u2019 extract act as capping agents. The capping agents play an important role in decreasing the agglomeration and in controlling the growth during the hydrothermal step.The presence of phytoconstituents, i.e., flavonoids, tannins, and terpenoids; in the Acacia nilotica pods\u2019 extract with the hydrothermal method resulted in the synthesis of ZnO NPs, ZnO-CuO NCs, and CuO NPs. The prepared ZnO NPs have a hexagonal wurtzite phase and the CuO NPs have a monoclinic tenorite phase. Interestingly, ZnO-CuO NCs have a hexagonal phase for ZnO and a monoclinic phase for CuO with no secondary phases. The average crystallite size of NPs and NCs is in the range of 17\u201330 nm. The morphology of the particles depend on the ratio ZnO-CuO and the prepared particles showed different morphologies as agglomerated particles, nanorods, intermeshed flakes together with irregularly shaped particles.In the recent work, successful combination of"}
+{"text": "High-shear mixer coatings as well as mechanofusion processes are used in the particle-engineering of dry powder inhalation carrier systems. The aim of coating the carrier particle is usually to decrease carrier\u2013drug adhesion. This study comprises the in-depth comparison of two established dry particle coating options. Both processes were conducted with and without a model additive (magnesium stearate). In doing so, changes in the behaviour of the processed particles can be traced back to either the process or the additive. It can be stated that the coarse model carrier showed no significant changes when processed without additives. By coating the particles with magnesium stearate, the surface energy decreased significantly. This leads to a significant enhancement of the aerodynamic performance of the respective carrier-based blends. Comparing the engineered carriers with each other, the high-shear mixer coating shows significant benefits, namely, lower drug\u2013carrier adhesion and the higher efficiency of the coating process. The increasing prevalence of respiratory diseases has been reported frequently . Due to In this study, an assessment of additive-free and additive-containing processed powders was conducted. This enables the comparison of both approaches on different levels, allowing conclusions on whether changes can be attributed to the additive or the process itself. Since both methods are already established and lead to distinct increases in fine particle fraction (FPF), it is important to investigate the differences. Moreover, this study can help in deciding which particle coating process shall be preferred for adding magnesium stearate or similar additives.\u00ae 230  a crystalline, sieved inhalation grade lactose monohydrate was used. Magnesium stearate  served as the model coating material. For the sake of simplicity, the materials are shortened to the following: InhaLac 230 (IH230), magnesium stearate (MgSt), InhaLac 230 processed in the mechanofusion reactor , InhaLac 230 processed in the high-shear mixer . Micronised ipratropium bromide served as the model drug .As the model carrier in the coating processes, InhaLac\u00ae using the Picoline\u00ae platform . The carrier and coating materials (2% (w/w) MgSt) were premixed in a Turbula\u00ae blender  at 42 rpm for 5 min. Pre-blends were then transferred to the AMS. The AMS coating started by increasing the rotor speed up to 4000 rpm within one minute and keeping it constant at 4000 rpm over 10 min.The mechanofusion process was conducted with the Angmill Mechanofusion System (AMS) unit of the Picobond\u00ae unit of the Picoline platform. A rotor speed of 500 rpm was kept constant for 15 min.The high-shear mixer (HSM) coating was conducted using the PicomixAll raw materials were sieved before processing (mesh size: 250 \u00b5m), as well as the coated samples after processing. To assess the energy input to the powder, the data transfer tool of the Picoline was used. The instrument performance over the whole processes using the same powder mass was averaged and then multiplied with the respective process duration to obtain the total energy input. The procedure was then repeated without powder. The idle running energy was subtracted from the total energy input to calculate the energy input into the powder sample. All coatings were conducted at monitored relative humidity (RH) of 45% \u00b1 15% and room temperature (20 \u00b0C to 25 \u00b0C).A power compensation DSC  was used for each measurement, and data were evaluated with the PYRIS\u2122 Software (PerkinElmer Inc.). Approximately 5 mg of the respective sample was weighed into an aluminium pan, which was sealed and pierced afterwards to allow gas to escape. A pierced, empty aluminium pan was used as reference. During analysis, the sample was flushed with nitrogen to avoid any oxidation processes. All samples were heated at a rate of 10 \u00b0C/min up to 240 \u00b0C. Results are plotted as the heat flow (mW) over temperature. Furthermore, the thermogravimetric properties of the sample were analysed , using the same heating rate and heating range as that of the DSC. This provides additional information on what happens at the events observable in the DSC .Dynamic vapour sorption (DVS) was carried out using the DVS Resolution . For these experiments, approximately 70 mg of the respective sample was weighed into the sample pan. Measurements were carried out at 25 \u00b0C. The method consisted of two cycles of increasing the partial pressure of bidistilled water (aq. bidest.) from 0\u201390% in 10% steps and returning to 0%. At the beginning of every measurement, the sample was conditioned for 180 min at 0% RH. Every step of the method was not limited by time but by a change in mass of below 0.005%/min.\u00ae, Sympatec GmbH, Clausthal-Zellerfeld, Germany) was equipped with a RODOS\u00ae dispersion module and a R4 lens with a measuring range of 0.5 to 350 \u00b5m. With compressed air (2 bar), the automatically fed  material was dispersed in an aerosol jet towards the measuring zone.Laser diffraction was applied to determine the particle size distribution (PSD) of the initial and processed material. For this, a Helium Neon Laser Optical System  based on the Fraunhofer enhanced equation.0 0.05\u20130.35).The assessment of specific surface area (SSA) using the BET method and the surface energy of solids (SE) was carried out using the surface energy analyser . The powder samples were weighed into silanised glass columns (4 mm inner diameter) and fixed with silanised glass wool on both ends. To avoid hollows and cracks in the powder bed, the filled columns were compressed by tapping for 10 min using the SMS column packer accessory. Prior to every measurement, all columns were conditioned at 0% RH and 10 sccm carrier gas flow (nitrogen) for one hour to get rid of any volatile contamination. For SSA measurements, a series of octane injections was performed, providing an adsorption isotherm. The calculation of the SSA was based on the linear section of the adsorption isotherm  for dispersive SE as well as chloroform and toluene for determination of acid-base surface properties. All injected concentrations were based on the respective monolayer capacity, leading to surface coverages from 0.5% up to 10%. For the determination of the dead volume, a double injection of methane was performed at the beginning and at the end of every experiment. All experiments were carried out at 0% RH and 30 \u00b0C with a carrier gas flow of 10 sccm.The raw data were analysed using the SEA Analysis Software . Calculations were based on the DellaVolpe scale in combination with the Dorris and Gray approach and the polarisation method ,19. For The imaging of particle morphology was conducted with scanning electron microscopy (SEM) using Phenom XL . To guarantee sample grounding and to minimise charging effects, samples were fixed onto carbon stickers and gold-sputtered with BAL-Tec SCP 050 Sputter Coater . All images were taken at 1000\u00d7 or 2500\u00d7 magnitude with an acceleration voltage of 10 kV and with the use of a backscatter detector.\u00ae 100 CN, Merck), using a solvent mixture  adjusted to pH 3.2 as mobile phase. The method was validated in terms of system suitability, specificity, precision, repeatability and linearity. The limit of quantification was calculated based on the corresponding ICH guideline (CPMP/ICH/381/95) as 0.08 \u00b5g/mL. The quantification calculations were based on an external standard calibration curve (R2 > 0.99) covering a concentration range from 0.21 \u00b5g/mL to 104.8 \u00b5g/mL. All values used were within the calibrated range. All solvents used were chromatographic grade and supplied by Honeywell Riedel-de Ha\u00ebn .The quantification of drug content was carried out using high-performance liquid chromatography . The analytical procedure was based on a cyanopropyl-substituted stationary phase (LiChrospherw/w active ingredient) using the Picomix high-shear mixer module with two mixing steps at 500 rpm and one sieving step (mesh size: 250 \u00b5m) in between. All mixing steps were conducted at a monitored RH of 45% \u00b1 15% and room temperature (20 \u00b0C to 25 \u00b0C). Every mixture comprised a batch size of 30 g and was tested for homogeneity by analysing 10 randomly picked samples via high performance liquid chromatography. A blend was considered homogeneous at a relative standard deviation below 5% and a recovery of 90\u2013110%.All adhesive mixtures were prepared  with the fast-screening impactor . The FSI allows rapid determination of fine particle dose < 5 \u00b5m (FPD) and fine particle fraction (FPF). All experiments were operated at a flow rate corresponding to a 4 kPa pressure drop over the inhaler (78.3 L/min). All results are shown as an average of five measurements with five doses per measurement. All FSI experiments took place in a climate chamber with constant environmental conditions of 21 \u00b0C and 45% RH. High performance liquid chromatography  was used for drug quantification.The aerodynamic assessment was carried out using the Novolizerw/w). All results are displayed as the average of three measurements at a monitored temperature of 20 \u00b0C (\u00b15 \u00b0C) and 45% (\u00b115%) RH.For the adhesion strength screening, an e200LS air-jet sieve  was used. A specified mesh-sized analytical sieve was inserted (20 \u00b5m for adhesion strength screening). For every repetition, 5 g of sample was placed on the sieve, covered with a lid and then dispersed via a rotating pressurised air nozzle. A negative pressure of 4 kPa was created by a pump, sucking particles below 20 \u00b5m through the sieve. The process was stopped at 6 s, 30 s, 1 min, 2 min, 5 min and 10 min for sampling. Per sampling time, 10 randomised powder samples were picked from above the sieve. From each of the resulting 60 samples, a specific mass of 7 mg (\u00b10.5 mg) was weighed into an Eppendorf tube, dissolved in 2 mL of solvent (aq. bidest.) and analysed in the high-performance liquid chromatography. The resulting drug content was put in in relation to the nominal drug content of the adhesive mixture . All samples were dissolved using diluted nitric acid in combination with sonication (20 min) and shaking (15 min). After centrifugation , the samples were diluted and measured using a flame atomisation (air\u2013acetylene flame) AAS system . The calculation of the content was based on a six-point calibration curve from zero up to 1 ppm magnesium concentration.All statistical calculations were carried out with Microsoft Excel 2016 .The in-depth investigation of two established dry particle coating processes was conducted with a broad spectrum of physico-chemical analysis tools. Since those particle coating processes are used for formulation optimisation attempts ,11 in thThe energy input in both preparation procedures was determined by recording the performance data of the instrument for both conducted processes using the same sample mass. The energy input into the powder was calculated to be 1234 J for the AMS and 1318 J for the HSM process. This corresponds to approximately 6% higher energy input in the HSM coating attempt. Even though both processes show no major differences in energy input, both methods introduce considerable amounts of mechanical energy into the sample. As a comparison: preparing an interactive blend as described introduces approximately 250 J of mechanical energy. Since the mechanical stress during dry particle coating could already cause fundamental changes in the particle properties, such as amorphisation or fragmentation, both processes were conducted with and without additives. This allows the evaluation of whether changes in powder behaviour can be attributed to processing or to the additive itself.When applying mechanical stress to a powder sample, an expectable change is in particle size distribution. To make sure no particle fragmentation occurs, particle size distributions were measured via laser diffraction. The results are displayed in The results demonstrate that even though considerable mechanical stress was applied during both processes, the particle size distribution is unchanged, which indicates that particles were not fragmented significantly when processed without the additive.Since particle size distributions have a huge impact on the performance as a carrier platform in dry powder inhalation formulations , not decOtherwise, one would observe increasing surface areas after processing due to decreased particle sizes.Another alteration, which could have been introduced even without particle fragmentation, is the formation of crystal defects, leading to partial amorphisation. Since the starting material, InhaLac 230, is a highly crystalline lactose, even small amounts of process-induced amorphous content are detectable. As analytical tools, two different methods were used: differential scanning calorimetry (DSC) in combination with thermogravimetric analysis (TGA) and dynamic vapour sorption (DVS). Both techniques were applied to the additive-free samples. Thus, solely the process-induced changes in solid state properties can be tracked. All DSC graphs  show essDVS was chosen as an orthogonal method to verify the conclusion made based on the DSC experiments. For comparability, only additive-free, processed lactose samples were investigated. A slight difference between processed and non-processed samples can be seen. The processed samples reproducibly gained more mass than the non-processed, which can be explained by little differences in surface appearance or the creation of non-recrystallisable defects, which is substantiated by no identifiable differences between both cycles. This combination of methods showed that in both used dry particle coating methods, no introduction of major changes in the solid state of the particles had occurred. This is important not only for stability issues but also for tracing back new particle properties after the process with the additive\u2014not to the process itself, but to the additive.p-value > 0.05 when comparing d10, d50 and d90 values). Following the investigation of the addition of mechanical stress, the determination of particle specifications, namely PSD  and BET 2/g while the AMS sample only increased to 0.4 m2/g. A general increase in surface can be substantiated by the SEM images (A and B) in This observation was supported with the specific surface area results. Both processed samples showed a difference between the BET-specific surface area depending on if processed with or without the additive. The specific surface area of the HSM sample increased to 0.8 mw/w) MgSt was added in both coating strategies. The measured MgSt content of 1.31% and 1.23% before air-jet sieving indicates a loss of more than 30% due to processing. After air-jet sieving, the additive content of the HSM sample decreased by 18.0%, while the content of the AMS sample decreased by over 60% . Comparing the surface energy distribution of the starting material with the material after processing without the additive, the dispersive part of the surface energy showed no significant changes. The acid-base (polar) part, however, decreased after processing. Considering the standard deviations, there was no significant change . At low surface coverages of the probe gases, the total surface energy decreased by up to 15.3% (HSM). Comparing both coating processes, it can be observed that coating in a high-shear mixer led to lower surface energies at low surface coverages  as well as a significant difference between both processes . Such decreases in surface energy can be linked to the decreased tendency of the surface to interact with surroundings  [Processing with the model additive MgSt caused significant changes in surface area and surface energy  .p-value < 0.001), but the difference between both processed carriers was not . Decreases in interaction strength can therefore lead to significant changes in aerodynamic performance, as attachment and detachment of micronised drug particles will be altered. The impact of processing with the additive on resulting fine particle fractions (FPF) of the respective interactive blends assessed in the FSI is displayed in 2 > 0.95). The mean residual drug concentration (starting concentration defined as 100%) after 10 min of dispersing and removing particles below 20 \u00b5m is defined as carrier residue (CR). The slope of the linearised function describes the speed of decrease (SOD) of drug content in the dispersed system. In a real setting, the drug has to be detached in a split second [To gain further insight into the adhesion of drug particles on either non-processed or processed carriers, an adhesion force screening of the blends was carried out. The air-jet sieve (AJS) allows the simulation of a situation that is exemplary of a blend that is intended to be dispersed in the airstream. With a mesh size of 20 \u00b5m and a negative pressure of 4 kPa, mainly drug particles are sucked through the sieve and therefore eliminated from the dispersed (using pressurised air) system. Sampling at defined times and quantification of residual API in the blend allow conclusions of adhesion strength; the higher the adhesion strength, the less API will detach from the lactose particles. All prepared interactive blends were assessed with this method. The gathered data  were dest second  during ip-values < 0.001; SOD: p-values < 0.05). This was confirmed by the results of the SEA analysis. Another indication for reduced adhesion between the carrier and drug was the share of drug deposited in the pre-separator of the FSI. It also represents an approximation of the amount of drug, which did not detach and, hence, impacted the lactose carrier in the pre-separator. The shares are shown in p-value < 0.001). All blends fulfilled the mentioned requirements to be considered homogenous before the testing, so the data point at zero seconds is 100% \u00b1 10% . It can be observed that the HSM blend showed less adhesion forces between carrier and API, leading to higher detachment and drug content decreases in the dispersed system. The resulting CR and SOD values of all investigated blends differ in statistical significance  lead to substantial alterations in particle behaviour. The investigation of coating attempts with and without coating materials allowed conclusions on the origin of the introduced changes. Using additive-free processes, the alterations in particle appearance, aerodynamic performance or adhesive properties were traced back to the additive, not the process. Even if AMS and HSM coatings both led to significant increases in FPF, the HSM coating showed superiority in terms of carrier residues and speed of detachment (of drug content). In addition, the SEM images before and after air-jet sieving showed a more complete coating for the HSM process. This was substantiated by the determination of firmly bound additive content. Even though SE decrease and therefore the decrease in the work of adhesion were superior for the HSM surface, the aerodynamic performance showed no significant difference between both options. This supports the theory of energetic levelling of higher energy binding sites on the DPI carrier surface. Although the AMS coating showed inferior coating efficacy, the high-energy sites on the lactose surface decreased for both coating strategies, which therefore substantiates the positive effect of using dry particle coatings in DPI formulation development. Furthermore, this work supports the hypothesis that adhesion strength between drug and carrier may be crucial but not solely decisive for the aerodynamic performance of the respective interactive blend. In conclusion, the HSM strategy should be preferred in a comparable experimental setup due to its reported benefits combined with less processing time."}
+{"text": "Cellulose polymer insulation material is widely used in oil immersed bushing. Moisture is one of the important reasons for the deterioration of cellulose polymer insulation, which seriously threatens the safe and stable operation of bushing. It is significant to study the polarization and depolarization behavior of oil-immersed cellulose polymer insulation with different moisture condition under higher voltage. Based on polarization/depolarization current method and charge difference method, the polarization/depolarization current, interfacial polarization current and electrical conductivity of cellulose polymer under different DC voltages and humidity were obtained. Based on molecular-dynamics simulation, the effect of moisture on cellulose polymer insulation was analyzed. The results show that the polarization and depolarization currents become larger with the increase in DC voltage and moisture. The higher applied voltage will accelerate the charge carrier motion. The ionization of water molecules will produce more charge carriers. Thus, high DC voltage and moisture content will increase the interface polarization current. Increased moisture content results in more charge carriers ionized by water molecules. In addition, the invasion of moisture will reduce the band width of cellulose polymer and enhance its electrostatic potential, so as to improve its overall electrical conductivity. This paper provides a reference for analyzing the polarization characteristics of charge carriers in cellulose polymer insulation. The oil-immersed transformer is the core equipment of power transmission in the power grid, wherein safety and stability are significantly important to energy security and social stability. According to statistics, fault of bushing is one of the main causes for transformer faults, and the cellulose polymer paper, as the main insulation material of bushing, is the main factor contributing to the fault of bushing after moisture invaded ,2,3. To To date, some scholars have studied the effect of moisture on cellulose polymer paper. Wenyu Ye, et al. found that the structure of liquid-solid interface is determined by the interaction between insulating oil and cellulose polymer paper, which is based on the Van der Waals effect, and water molecules will gather at the interface because of the interaction of the liquid-solid interface at certain electric field value . GuanweiIn order not to damage the sealing performance of bushing, nondestructive testing methods based on dielectric relaxation theory have been widely used by scholars, among which polarization and depolarization current (PDC) method and frequency domain spectroscopy (FDS) method are the most popularly used ,8,9. BotAlthough some scholars have studied the correlation between the dielectric characteristic parameters of bushing insulating paper in time domain and moisture, most of the research is based on low excitation voltage, resulting in low signal-to-noise ratio, which is easy to be interfered by environmental noise. Additionally, and yet worse, the cellulose paper in the bushing works at a higher voltage, which means the traditional low-voltage measurement results and rules are not applicable to a higher excitation voltage. In addition, there is little research on analyzing the charge accumulation at the interface of oil-paper insulation in bushing based on PDC measurement results. Therefore, it is necessary to analyze the time-domain relaxation behavior of the bushing insulating paper with various moisture contents at high voltage.This paper studies the time domain relaxation behavior of charge carriers for cellu-lose polymer insulation used in oil immersed bushing under higher voltage. Firstly, the polarization and depolarization currents of cellulose polymer insulation with different moisture conditions are obtained. Then, the influence of moisture and applied voltage on the characteristics of interface polarization behavior is studied; the motion of the interface charge carriers during the process of polarization and depolarization and the relationship between the steady state time of electrical conductivity are analyzed. Finally, based on molecular dynamics simulation, the effect of moisture on the insulation properties of cel-lulose polymers is analyzed.Cellulose is a natural polymer compound, its chemical structure is a linear polymer composed of many \u03b2-D-glucopyranosyl groups connected to each other by 1, 4-\u03b2 glycoside bonds. Cellulose polymer insulation is mainly composed of cellulose macromolecular chains composed of cellulose monomers, as shown in In this paper, an oil immersed bushing with cellulose polymer insulation, where the maximum voltage is 40.5 kV, is tested. The structure diagram of the bushing is shown in PDC tests in different DC voltages are carried out; the schematic diagram and physical diagram of PDC test is shown in By applying and removing DC voltages to the insulating medium, the dielectric response characteristics in time domain from the polarization and depolarization current can be extracted based on a PDC test. The schematic diagram is shown as For the insulating medium, the polarization process mainly includes dipole polarization and interface polarization. Due to the difference of dielectric constant and electrical conductivity of composite insulating mediums, the free charge will accumulate at the interface and will not dissipate easily, resulting in the asymmetry of polarization process and depolarization process. Polarization current consists of dipole relaxation current Electrical conductivity can reflect the degree of moisture content of insulating materials. Charge difference analysis method (CDA) can effectively calculate the electrical conductivity of insulating materials by analyzing the change characteristics of the charge amount difference in charge and discharged process ,16,17,18Thus the charge amount difference at any time is as Equation (5), and the difference between polarization current and depolarization current at any time is as Equation (6):k of charge amount difference function with time is approximately constant, as shown in Equation (7).The charge amount difference is numerically the integral of the conductance current over time. As the conductance current changes little with time, the slope From Equations (6) and (7), r\u03c3 of the composite insulating medium can be obtained from the conductance current, as shown in Equation (10).When the time of polarization and depolarization process is equal and long enough, the current difference at the final moment is equal to the conductance current, as shown in Equation (9). The electrical conductivity X-Y model when the electrical conductivity of the whole insulating material is known [The composite insulation structure in the bushing is composed of aluminum foil and oil immersed cellulose polymer wrapped closely, as shown in is known .X model, the electrical conductivity of the composite insulating structure is shown as Equation (11), where X is the thickness ratio.As for simplified X equals to 1, the electrical conductivity of cellulose polymer insulation can be simplified, and the electrical conductivity of the cellulose polymer is as shown in Equation (12).Since the electrical conductivity of aluminum foil is much greater than that of cellulose polymer and the thickness of aluminum foil is negligible compared with that of cellulose polymer insulation, which means In order to study the effect of moisture on the insulation properties of cellulose polymer, the models of cellulose without moisture and after moisture were constructed by using Materials Studio software .Three cellulose chains with a degree of polymerization of 10 were used as the cellulose polymer insulation model without moisture, while 3.5% water molecules were added into three cellulose chains with a degree of polymerization of 10 as the cellulose polymer insulation model with moisture. The two cellulose polymer insulation models are shown in First, 10,000 steps of geometric optimization were carried out for the two models by using the Steepest descent method. Then, the two models were annealed to make the model reach the most realistic condition; the annealing temperature was 300\u2013500 K. Under Compass force field, constant-pressure and constant-temperature ensemble (NPT ensemble), with a constant number of molecules, pressure, and temperature, was used to balance each model and to make the model more reasonable with 500 ps. Based on the density functional theory (DFT), the calculations for two cellulose models were analyzed by DMol3 Tool in Materials Studio. The geometry optimization, molecule orbitals and electrostatic potential were calculated by employing the PBE function under the generalized gradient approximation (GGA) exchange-correlation term. The double numerical plus polarization (DNP) basis set was applied for setting the parameters of C, H, O atoms in this computational work. The all-electron method was adopted for core electron calculation.The polarization and depolarization current of bushings with different moisture conditions and applied voltages are shown in This phenomenon can be explained by the differing hydrophilic qualities of oil and cellulose polymer. Since the hydrophilic quality of cellulose polymer is much higher than that of insulation oil, when water is present, most water molecules in the insulation oil will migrate to the cellulose polymer with the invasion of moisture. As polar molecules, the more moisture infiltrates into the cellulose polymer insulation, the more polar molecules are involved in the polarization reaction, leading to a larger polarization intensity difference. The moisture content, on one hand, can increase the electrical conductivity of the insulation system, whereas, on the other hand, it can enhance the response speed of the interface polarization, aggravating the dielectric asymmetry of the oil-immersed cellulose polymer insulation system at the same time. Finally, the insulation resistance and capacitance increase, and the polarization process is aggravated ,23,24. TWhen the applied voltage is lower than 1000 V, the current fluctuates significantly with time. This is because the polarization process of oil-immersed cellulose polymer insulation system inside the bushing cannot be fully stimulated by low DC voltage in such a large size. Therefore, the polarization and depolarization currents of the insulation are small and susceptible to the interference of environmental noise, which has an obvious influence on the accuracy of the test results. When the applied DC voltage increases, the corresponding current becomes larger and the fluctuation of that decreases. The development trend of polarization and depolarization current is not affected by applied DC voltage, which means the applied DC voltage has little influence on the general trend of the dielectric polarization process. Thus, a high applied DC voltage test can not only improve the signal-to-noise ratio, but also reflect the polarization process of bushing more clearly, resulting in a more accurate and effective test result.The interface polarization current of the three bushings can be calculated by Equations (1)\u2013(3), as shown in The insulation system of the bushing is composed of aluminum foil and cellulose polymer, and the dielectric constants of both are different. When applying DC voltage in the insulation system, the interface polarization process occurs, and an interface polarization current is generated. When the voltage increases, the electric field becomes larger, accelerating the motion of the charge carriers. When the moisture infiltrates into the insulation medium, the water molecules are ionized to produce more charge carriers under the effect of electric field. This explains the characteristic of interface polarization current under the influence of moisture content and applied DC voltage. The migration process of charge carriers at the cellulose polymer interface is shown in in The charge difference spectrum of normal bushing, dampened oil bushing and dampened cellulose polymer paper bushing under different voltages are shown in The slopes of the polarization/depolarization charge amount difference at 200 V, 1000 V and 4000 V are shown in \u03c3. The geometric capacitance of the insulation in the bushing is 228 pF, thus the real electrical conductivity of normal, bushings with dampened oil and dampened cellulose polymer insulation are 7.9748 \u00d7 10\u221213 S/m, 9.7884 \u00d7 10\u221213 S/m and 1.1672 \u00d7 10\u221212 S/m, respectively. The results indicate that electrical conductivity increases when the moisture content in the insulation material is higher, leading to the deterioration of the insulation performance.The electrical conductivity change behavior of cellulose polymer insulation with different moisture conditions is shown in For normal bushing, electrical conductivity is stabilized the fastest because the interface polarization current dissipates faster with time, resulting in the electrical conductivity current taking less time to stabilize. However, the polarization process can be influenced by the invasion of moisture when the insulation material is dampened, resulting in a longer time that the interface polarization current dissipates and the higher value of the interface polarization current. Additionally, moisture content can also lead to a longer time that the conductance current takes to be stable and can decrease the initial value of polarization and depolarization charge amount difference. The polarization current increases significantly while the depolarization current is less affected under the influence of moisture content, leading to the increase in slope of polarization/depolarization charge amount difference. Because the electrical conductivity is linear with the slope of the polarization/depolarization charge amount difference curve, the electrical conductivity of the bushing with dampened oil and dampened cellulose polymer in the stable state is larger than that of normal bushing. These reasons explain that normal bushing has a larger electrical conductivity than that of bushing with dampened oil and cellulose polymer insulation, and that the electrical conductivity of normal bushing takes less time to stabilize.E) of unmoistened cellulose is 5.657 eV, while the band gap width of moistened cellulose is 5.546 eV. The wider the band width, the weaker the electrical conductivity. The energy band width of cellulose is narrowed after the water content of cellulose is increased, so the moistened cellulose polymer insulation electrical conductivity is stronger.The band structure of unmoistened cellulose and moistened cellulose is shown in The electrostatic potential of unmoistened cellulose and moistened cellulose is shown in Due to the existence of a large number of hydroxyl groups in cellulose, these hydroxyl groups can easily form hydrogen bonds with each other. It is precisely because of the existence of hydrogen bonds that cellulose has strong intermolecular and intramolecular interaction forces, and the ability of cellulose to resist external damage is also closely related to the concentration of hydrogen bonds. The molecular simulation results show that the presence of water destroys the intramolecular and intermolecular hydrogen bonds in the cellulose chain, and forms a new hydrogen bond interaction with the oxygen atom on the cellulose hydroxyl group. Water mainly acts on the hydroxyl and glycoside bonds of cellulose and destroys their stability. Therefore, the motion of charge carriers in the insulating paper cellulose is more intense under the action of the electric field. This is consistent with the conclusion in  that chaIn conclusion, with the invasion of moisture, the electrical conductivity and polarity of cellulose polymer insulation will increase. Under the excitation of DC electric field, the polarization/depolarization process is more intense, which is specifically reflected in the experiment by the polarization/depolarization current being greater. In addition, the electrostatic potential of cellulose polymer is stronger after moisture invasion, indicating that its electrical conductivity characteristics are better under the action of electric field, that is, the electrical conductivity is greater, which verifies the calculated electrical conductivity of cellulose polymer insulation in different oil-immersed bushings above.The polarization/depolarization current of bushings increases when the applied voltage becomes higher. High applied voltage can decrease the influence of noise. Moisture can aggravate the polarization/depolarization process, leading to higher polarization/depolarization current.When applied voltage is higher, the speed of charge carriers increases, leading to the increase of interface polarization current. Water molecules are ionized under the effect of electric field, producing more charge carriers, thus increasing the interface polarization current as well. With the increase of interface current, the relaxation behavior will be further intensified.The charge amount difference of polarization and depolarization current is linearly aligned with the time, and the slope becomes larger with the increase in moisture, which is more obvious under high applied voltage. Water molecules produce more charge carriers after being ionized and provide more paths for charge movement.With the invasion of moisture, the band width of cellulose polymer insulation becomes narrower, and its electrostatic potential increases, which improves the electrical conductivity of cellulose polymer insulation, and the polarization characteristics under DC electric field are more significant.This paper studies the time domain relaxation behavior of charge carriers for cellulose polymer insulation used in oil-immersed bushing, and the characteristics of dielectric response and motion of charge carriers have been analyzed. The conclusions drawn are as follows:"}
+{"text": "Bioprinting is increasingly used to create complex tissue constructs for an array of research applications, and there are also increasing efforts to print tissues for transplantation. Bioprinting may also prove valuable in the context of drug screening for personalized medicine for treatment of diseases such as cancer. However, the rapidly expanding bioprinting research field is currently limited by access to bioprinters. To increase the availability of bioprinting technologies we present here an open source extrusion bioprinter based on the E3D motion system and tool changer to enable high-resolution multimaterial bioprinting. As proof of concept, the bioprinter is used to create collagen constructs using freeform reversible embedding of suspended hydrogels (FRESH) methodology, as well as multimaterial constructs composed of distinct sections of laminin and collagen. Data is presented demonstrating that the bioprinted constructs support growth of cells either seeded onto printed constructs or included in the bioink prior to bioprinting. This open source bioprinter is easily adapted for different bioprinting applications, and additional tools can be incorporated to increase the capabilities of the system. Such constructs present opportunities to study cell biology and pathogenic processes in complex, well-defined environments. Bioprinted constructs also hold potential to become widely used model systems and assays for drug screening5, and their viability as transplantable materials for patient care is an area of intense research interest9.Additive manufacturing is an umbrella term for a variety of manufacturing techniques usually based on building three-dimensional (3D) objects in a layer-by-layer fashion. Additive technologies are gaining widespread popularity in a variety of research fields, as well as in industry, as 3D printing enables rapid prototyping and generation of objects with complex geometries from a very wide range and combination of materials. Bioprinting describes the application of additive manufacturing to reproducibly create tissue-like 3D constructs composed of biomolecules and/or biomaterials and cells. As a tissue engineering technology, bioprinting permits cells to be incorporated into printed constructs post-production or during the printing process, and the ultimate goal is often to recapitulate the architecture and function of living tissues and organs10, laser-assisted bioprinting, stereolithography, and inkjet-based bioprinting11. To date extrusion-based bioprinting is one of the most widespread techniques, which has borrowed innovations from the analogous fused filament fabrication (FFF) technology that is extensively used in plastic additive manufacturing. Compared to other bioprinting technologies, extrusion based bioprinting has several advantages such as low cost, a relatively high printing speed, and compatibility with bioinks in a wide range of viscosities. However, extrusion-based bioprinting does suffer from a relatively low spatial resolution, and cell viability can be compromised due to the mechanical stress generated during extrusion of cells through the deposition needle or nozzle12.Developments in the field of bioprinting encompasses many established fields of research such as material science, cell and tissue engineering, regenerative medicine, as well as robotics, automation and biotechnology. This requirement for interdisciplinary collaborations presents a number of challenges to progress in the bioprinting field; however, by increasing the availability of user-friendly solutions, more researchers are likely to adopt the technology and in doing so advance the field as a whole. As with standard additive manufacturing, many different technologies are capable of constructing bioprinted 3D-objects in a layer-by-layer fashion. The most commonly used techniques for the printing of cell-laden bioinks are extrusion-based bioprinting14. However, this strategy employing sacrificial materials typically demands a bioprinting platform with the capacity to perform multimaterial printing. Freeform reversible embedding of suspended hydrogels (FRESH) is an innovative solution that provides a 3D structurally supportive print environment, without the need for multimaterial printer functionality16. During FRESH bioprinting collagen-based bioinks (or other bioinks of interest) are extruded directly into a support bath consisting of gelatin microparticles, and this permits complex 3D constructs to be printed in an essentially suspended state, without the need for printed support structures. The gelatin bath can subsequently be melted away by increasing the temperature, allowing the construct to be retrieved. The FRESH bioprinting strategy has been widely adopted throughout the bioprinting research community17; therefore, designing bioprinting platforms that are compatible with FRESH bioprinting will likely be of great general interest.A challenge that confronts many additive manufacturing techniques, especially those using soft materials such as hydrogels, is the low structural stability of the construct during the printing process, which can negatively impact the geometry and architecture of the intended construct design. Strategies that have been employed to circumvent this limitation include the use of sacrificial support materials. This allows for a construct to be printed in one or more bioinks of choice, while it is simultaneously embedded in a structurally supportive scaffold material printed in parallel. Once the construct is complete and stabilized through post-printing reactions or treatments, the sacrificial material can be removed to release the construct. This technique can also be used to introduce design features such as channels that facilitate the perfusion of printed constructs19, and while they may be limited to the extrusion of a single type of bioink at a time, previous studies demonstrate the benefits of leveraging existing additive manufacturing technologies for bioprinting. In the present study we present an open source extrusion-based bioprinter built around the E3D motion system and tool changer, with the potential to produce constructs using up to four syringe pump tools and bioinks. The bioprinter features a transparent polycarbonate cabinet with an integrated HEPA filter and air intake fan to enable contamination-free bioprinting, and automatic tool offset calibration and bed leveling, making it easy to use multiple syringe pump tools to combine different bioinks in order to create complex bioprinted 3D constructs. Due to the open source nature of the components used, the system is highly adaptable to allow for incorporation of new tools and functionalities. FRESH bioprinting as well as multimaterial printing with collagen and laminin was performed to demonstrate the capacity of the system.Efforts to bring bioprinters to a wider audience have included the repurposing of affordable FFF-printers for bioprinting applications20. E3D provides free access to the systems build files, which facilitated the design process when adapting the existing system for the bioprinting purpose described here21. A polycarbonate box compatible with ethanol-based surface sterilization and fitted with an integrated HEPA filter and air intake fan was constructed to enclose the E3D system to reduce the risk of contamination of bioprinted constructs. The polycarbonate enclosure could easily be adapted to allow for temperature and humidity control should this be required for future bioprinting applications  printing or extrusion through the syringe and 1\u00a0week later  into a hydrogel construct, which is removed after printing, can provide perfusion channels, which can increase cell viability and survival in larger constructsink Fig.\u00a0d. Col-F ink Fig.\u00a0e. Unexpeink Fig.\u00a0e.35, the analysis of multi-cell type behaviors36, and is important for future efforts to facilitate and scale up bioprinting of constructs for use in research and drug screening. The open source bioprinter is easily adapted for different bioprinting applications, and the possibility of incorporating additional tools should accelerate the development of novel bioprinting approaches and applications.The open source bioprinter presented here, based on the E3D motion system and tool changer, enables a variety of bioprinting tasks including FRESH bioprinting, multimaterial prints, and the extrusion of cell-laden bioinks. In the presented configuration only two of the four tool positions were used. The remaining tool positions present opportunities for further automation of protocols to reduce user intervention, and to increase reproducibility and throughput. For instance, we are currently developing a grip tool and protocol to manipulate the 3D printed basket in which the FRESH collagen constructs are bioprinted, to allow for automated construct transport from the gelatin support bath to a heated washing station, which melts the gelatin support material. The grip tool can subsequently move the construct through additional washing solutions, effectively automating key steps of the post-processing procedure. The benefits of automating biofabrication protocols have been outlined for the formation of tissue spheroids21, and enclosed in a custom-built polycarbonate cabinet with an integrated a HEPA filter with an air intake fan. The syringe pump extrusion tool  program originally developed by Danal Estesess Fig.\u00a0. This is.factory file format, which is native to Simplify3D. Further information about software configuration and slicing settings can be found in the The toolpath generation software  was used to generate G-code files for bioprinting of the different constructs. Bioprinting was typically performed at a speed of 10\u00a0mm/s, as printing at this speed produced reproducible constructs of both the collagen and laminin bioinks. The printing parameters are available in a \u03c0r2h), where r is the inner radius of the Hamilton 0.25\u00a0ml syringe (1.15\u00a0mm). This yielded a value of 0.39\u2009\u00b1\u20090.02\u00a0\u03bcl (mean\u2009\u00b1\u2009S.D.). To assess the practical lower limits of the resolution of the bioprinter we used a nozzle with a 50\u00a0\u03bcm cross-sectional diameter to extrude strings of collagen using FRESH bioprinting. The strings were imaged by differential interference contrast using a Zeiss LSM710 confocal microscope (Zeiss), and the average cross-sectional width of the finest collagen strings that we could produce and recover was calculated from multiple measurements along the length of the printed string  was mounted beneath the lead screw and adjusted such that its contact point was in contact with the plunger press. The stepper motor was programmed to take 10\u00a0mm, 1\u00a0mm, or 0.1\u00a0mm steps sizes and for each step the measured displacement distance was recorded from the dial indicator. These measurements were repeated 10 times for each step size, and a linear plot was prepared  according to the instructions provided by the supplier ATCC.The MDA-MB-231 breast cancer cell line was obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco\u2019s Modified Eagle Medium (DMEM) Glutamax , supplemented with 10% Fetal Bovine Serum , referred to hereafter as culture medium, in a humidified incubator  bioink and LaminInk\u2009+\u2009(Cellink) laminin bioink were first transferred to a 5\u00a0ml plastic syringe with male-male LuerLock. To reduce the presence of bubbles in the bioink, the loaded syringe was centrifuged for 4\u00a0min at 1200\u00d716, using LifeInk 240 (Advanced Biomatrix). The FRESH LifeSupport powder (Cellink), from which the gelatin microparticle support bath was prepared, was used as per the manufacturer\u2019s instructions. Following print completion, the tool was left at the tool dock and the build plate lowered. All constructs generated using FRESH bioprinting was made in a custom-designed and easily moveable basket. After a completed print, the basket holding the collagen construct was placed at 37\u00a0\u00b0C for 1\u00a0h to melt the gelatin support material and to thereby release the printed construct. Constructs were washed three times in 1\u00d7\u2009phosphate buffered saline  and stored in PBS at 4\u00a0\u00b0C until further use. For fluorescent imaging, collagen constructs were stained for 1\u00a0h at 37\u00a0\u00b0C with 10\u00a0\u03bcM Col-F  prepared in 1\u00d7\u2009PBS, and then washed twice (5\u00a0min each) with 1\u00d7\u2009PBS at room temperature. Constructs were imaged by confocal microscopy performed on a Zeiss LSM700  instrument and images were captured using Zen imaging software . For cell seeding, FRESH constructs were first typically stored overnight at 4\u00a0\u00b0C in 1\u00d7\u2009PBS supplemented with the antibiotics penicillin and streptomycin . Individual constructs were next moved to separate wells in a \u00b5-slide 8-well coverglass-bottomed chamber (Ibidi) and a cell suspension of 30\u2009\u00d7\u2009103 MDA-MB-231 cells diluted in 250\u00a0\u00b5l culture medium supplemented with 1\u00d7\u2009PenStrep per well was deposited directly onto the collagen construct or into an adjacent, construct-free wells, and cultured for 20\u00a0h under standard cell culture conditions. Cell staining and analysis of viability and cell morphology by microscopy are described below.FRESH bioprinting was performed according to the protocol by Lee et al.38. For cell-laden collagen constructs, the z-plane containing the highest number of cells was selected for analysis. Individual cells were identified as regions of interest (ROI) by applying a threshold to the NucBlue signal. The ImageJ particle analysis function was applied to this thresholded image to include particles within a size range of 15\u2013600 \u03bcm2. This range excluded non-specific signals from small debris and very large clusters of cells. However, it included smaller cell clusters that could be delineated as individual cells by applying the automatic watershed function to the thresholded image. This ROI mask of individual cells was then overlaid on the PI and Cell event caspase-3/7 channels and the mean fluorescence was recorded in each ROI for each channel. This data was exported to Excel, and mean intensity per ROI and channel was background adjusted using an average from three ROIs measured in cell-free areas of the collagen construct. Data was statistically analyzed using ordinary one-way ANOVA with Tukey\u2019s multiple comparisons in GraphPad Prism (Prism).To assess cell viability in FRESH bioprinted collagen constructs, cell-seeded constructs were incubated with the NucBlue nuclear stain to detect individual cells , propidium iodide (PI) to identify dead cells , and the Cell event caspase-3/7 stain that reports on the activity of caspase 3 and 7 in apoptotic cells . Staining solutions were diluted in OptiMEM , supplemented with 1\u00d7\u2009PenStrep. To control for the effectivity of the viability stains, cells were treated for 5\u00a0h with staurosporine , a potent protein kinase inhibitor known to induce apoptosis. Fluorescent signals for the respective stains were captured by confocal microscopy with an LSM700 instrument and Zen software (Zeiss). Multiple z-planes were captured for cell-laden constructs, while a single in-focus z-plane was captured for cells attached directly to the glass in construct-free wells. These experiments were conducted in duplicate on three independent occasions. Image analysis was conducted in the Fiji version of ImageJTo visualize cells adhering to FRESH bioprinted collagen constructs the cells were labelled overnight with SiR-actin , which permits fluorescence imaging of the actin cytoskeleton. Samples were washed twice in OptiMEM cell culture medium, which was then exchanged for OptiMEM containing the NucBlue nuclear stain. The cells were imaged as described above by confocal microscopy.FRESH bioprinting of constructs for one week culture was performed as described above, however to reduce unnecessary handling, constructs were printed directly on a Labtek 8 well chamber coverslip. Onto each construct 24,000 cells/well were seeded in DMEM . Half the media was exchanged every 1\u20132\u00a0days. Live/dead staining was performed on day 1 after cell seeding, and in separate constructs 1\u00a0week after cell seeding. Briefly, cells were stained with fluorescein diacetate (Sigma) 8\u00a0\u00b5g/ml and PI, diluted 1:1000 in OptiMEM, for 10\u00a0min. Then constructs were washed once with OptiMEM. Images were taken using an LSM 700 confocal microscope (Zeiss) and a 10\u00d7\u2009objective. For each construct two images were taken of a z-plane with high cell density and used for analysis in ImageJ. In ImageJ both channels were thresholded manually and the automatic watershed function applied. Subsequently, the particle analysis tool was used to count the number of live or dead cells. Using Excel, the percentage of viable cells was calculated for each image. To determine statistically significant differences between groups, data were imported to GraphPad Prism and analyzed by Student\u2019s t-test. Experiments were performed in three independent repetitions with two timepoints and four to five constructs per timepoint and repetition.6 cells/ml was mixed with 1\u00a0ml Laminink\u2009+\u2009Bioink (Cellink) by passing back and forth between two 1\u00a0ml syringes. For control, the cell-laden bioink was directly deposited into wells from the plastic syringe without extrusion through a thin needle or nozzle. The remaining ink was loaded into a 250\u00a0\u00b5l Hamilton glass syringe and bioprinting performed with an 18 G needle. For crosslinking of all constructs, including non-printed controls, the recommended Crosslinking Agent (Cellink) was applied for 1\u00a0min. Subsequently, the constructs were washed once in warm (37\u00a0\u00b0C) DMEM Glutamax, supplemented with 10% FBS and 1\u00d7\u2009PenStrep, and then either cultured for one week in 100\u00a0\u00b5l of media, with half the media being exchanged daily, in a cell incubator  or directly used for live/dead staining, described above. Experiments were conducted in three independent repetitions with two timepoints and a total number of three to five printed constructs and non-printed control hydrogels for each group and repetition. For each construct two images were taken of a z-plane with high cell density and used for particle analysis in ImageJ, and statistical analysis in GraphPad Prism (Prism), as described above.Cell-laden laminin bioink was prepared as follows; 100\u00a0\u00b5l of cell suspension containing 11\u2009\u00d7\u2009102, to induce gelation of collagen and laminin, respectively.Multimaterial constructs were bioprinted using acidified collagen (Advanced Biomatrix) and Laminink\u2009+\u2009(Cellink) containing cells as described above. Printing was done directly onto a glass slide, followed by incubation in 50\u00a0mM HEPES (pH 7.4) containing 50\u00a0mM CaClSupplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4."}
+{"text": "Hypertension is a major cause of morbidity and mortality and its control has important clinical and socio-economic benefits to the family and community. Unfortunately, the extent of blood pressure (BP) control and its potential predictors in hypertensive patients in many rural communities in low-resource settings are largely unknown. This study assessed the extent of uncontrolled BP and its predictors amongst hypertensive patients accessing primary health care in a rural community in South Africa.This cross-sectional study included 422 randomly selected hypertensive patients. Demographic and clinical data were collected using structured face-to-face questionnaire supplemented by respondents\u2019 clinical records.n = 286, 67.8%) and diabetes  were the most common comorbidities. Treatment adherence was achieved in only 36.3% and BP was controlled to target in 50.2% of the respondents. Significant predictors of uncontrolled BP were poor treatment adherence , obesity compared with normal weight and overweight  and being a diabetic .Obesity plus overweight . We also propose the use of Community-Based Physical and Electronic Reminding and Tracking System (CB-PERTS) to address poor treatment adherence. This clinic serves a predominantly rural population of over 132 000 inhabitants and accepts patient referrals from 10 PHC clinics in the district for various primary health care services. Data collection was from November 2015 to January 2016.19 All newly diagnosed hypertensive patients with no previous follow up, too ill to answer questions or needed urgent treatment were excluded. According to the clinic records, the estimated number of hypertensive patients seen yearly is approximately 12 000. The minimum required sample size (n) was estimated using the following equation:17All hypertensive patients 18 years and above, accessing care at the study setting, were eligible participants. Hypertension was defined as systolic BP \u2265 140 and diastolic BP \u2265 90 mmHg following repeated measurements.Z is the critical value for a 95% confidence level, e is the considered acceptable margin of error of 5% and P is the assumed proportion of 50% of those with poorly controlled BP in the population of patients with 422 eligible participants who were systematically selected at random.20 Thus, on each day of the data collection, every eligible third hypertensive patient was recruited.where 21 but with some modifications to the specific context of the setting and to supplement the respondents\u2019 clinical notes in line with the study objectives. Data on demography , lifestyle choices , duration of treatment and adherence 21 were collected.The survey instrument was a validated questionnaire that had been used previously2), BMI category ,22 comorbid illness , antihypertensive drugs therapy  and BP measured in each occasion during at least two different clinic visits. The BP, height and weight were measured during clinic visit on the day of the data collection by the triaging healthcare staff as part of the routine process of care. Specifically, BP was measured using a validated and regularly calibrated automated device, and this is in line with the current South African Hypertension Practice Guidelines.10 The measurements were taken from the arm of each study participant at rest using an appropriate size cuff. During the measurement, the patient was seated, his or her shoulders supported on the backrest of the chair, legs not crossed, feet placed on the floor and neither the patient nor the healthcare professional was talking. The average of the BP reading from the previous visit and that obtained on the day of data collection/interview were recorded and used as a measure of BP control. The BP measurement were in line with the existing local and international hypertension protocols and guidelines.19 The BP control targets were based on both the guidelines of the International Society of Hypertension and the South African Hypertension Society.19 These target BP in the study setting at the time of the study were < 140/90 mmHg for uncomplicated hypertension and \u2264 130/80 mmHg for those with comorbidity such as diabetes and chronic kidney disease (CKD).20 Data on measures of CKD, diabetes and dyslipidaemia, as documented in the patients\u2019 files at the time of initial diagnosis were also extracted. In line with the South African Hypertension Guidelines, CKD was defined as estimated glomerular filtration rate (eGFR) \u200a< \u200a60\u200a mL/min, which was obtained from the laboratory values of creatinine and calculation of the estimated GFR using MDRD formula. Dyslipidaemia was defined as total fasting cholesterol > 5.1 mmol/L.10Data were collected from Patients\u2019 clinical notes on body mass index  version 24.0 . All categorical data were presented as frequencies and percentages. Normality check was performed on continuous variables to identify patterns of data distribution. Normally distributed continuous data were presented as mean and standard deviation (s.d.). Independent variables were initially tested for association with BP control using the Chi-square test. Statistically significant variables found in the bivariate analyses were included stepwise in a multivariate logistic regression analysis to determine the factors that remained associated with control of BP when entered into the model alongside the other variables that were shown to be significant. Statistical significance was set at Permission to conduct the study was given by the Sedibeng District health services. Ethical approval was granted by the Ethics Committees of the University of the Witwatersrand, South Africa (certificate number: M150859) and University of Liverpool, United Kingdom (certificate number: 7-16-2015-01). Study participants gave informed consent before they were enrolled. All personal patient information was de-identified at source and codes used for all participants, thereby, preserving their anonymity.n = 229) were men of mean age 59.4 (s.d. = 12.0) years. Whilst women  had a mean age 59.7 (s.d. = 12.3) years. Over half of the participants were unemployed  and the majority had either primary  or secondary school education . The details of the demographic characteristics of the participants are shown in Of the 422 hypertensive patients\u2019 who participated in the study, all were black people, slightly over half  kg/m2. A total of 54% (n = 228) and 50.9% (n = 215) had diabetes and dyslipidaemia, respectively. Target BP control, irrespective of comorbidities, was achieved in 50.2% of patients. Mean systolic BP was 138.7 mmHg (s.d. = 15.9) for men and 140.3 mmHg (s.d. = 17.5) for women. Mean diastolic BP was 84.4 mmHg (s.d. = 9.8) for men and 84.8 mmHg (s.d. = 10.9) for women.n = 71, 16.8%), 83.1% did this less than three times a week with an exercise duration of < 30 min in 47 (66.2%). Most of the respondents  did not drink alcohol, but of those 32.0% (n = 135) who did, 104 (77.0%) .0 only occasionally on less than a weekly basis. The majority of participants  reported that they smoked or used tobacco products.n = 405, 96.0%) with the commonest pattern being three or more . However, the majority of patients  were not fully adherent with the hypertensive treatment, with the mean duration of treatment being 10.0 (s.d. = 7.6) years in women and 8.5 (s.d. = 6.8) years in men.21 In this table, the reply of \u2018none of the time\u2019 to 13 of the 14 items on this scale was achieved by over 90% of the respondents in only six of the 13 items or questions in the scale, signifying poor adherence. However, adherence to follow up appointments was good because almost all the respondents (97.5%) answered \u2018all of the time\u2019 to the remaining one question on the scale: \u2018how often do you make the next appointment before you leave the clinic\u2019.p = 0.019) and their BMI category (p < 0.001). Uncontrolled BP was also associated with the presence of certain comorbid conditions: bronchial asthma (p = 0.011), diabetes (p < 0.001) and CKD (p = 0.041). In addition, the number of drugs prescribed (p = 0.01), the correct combination of drugs (p = 0.025) and adherence to treatment (p < 0.001) were found to be associated with uncontrolled BP.Preliminary analysis using the Chi-square test indicated that uncontrolled BP was significantly associated with two demographic factors: the sex of the patient (p < 0.001) and the presence of diabetes  clearly contribute to the association with uncontrolled BP, the major factor was the lack of adherence to treatment .These factors were therefore included in the multivariate logistic regression analysis to identify which ones were most likely to indicate possible uncontrolled BP. Body mass index, diabetes and treatment adherence remained in the model following stepwise elimination of those factors that were not significant in the model. The odds ratios (ORs) of obesity when compared with normal weight and overweight independently were not significantly different, and the BMI category was reconsidered as a dichotomous variable normal weight and overweight as compared with obesity. The multivariate logistic regression analysis showed the association between the determinant factors and uncontrolled BP . Althoug24 in one of the first nationally representatives estimates of the burden of hypertension and extent of hypertensive care in South Africa. Similar to what we found, almost half of the treated patients are not controlled.24 It is important to emphasise that although most studies of BP control in South Africa have reported lower control rates,12 with the exception of a 57.0 % BP control rate reported by Onwukwe and Omole,9 we are of the opinion that our control rate is still unsatisfactory. This is because of the fact that almost half of the respondents whose BP was not controlled to recommended levels could be predisposed to various cardiovascular complications and these have important public health implications such as stroke, congestive heart failure, peripheral vascular disease and CKD.8 This lack of BP control occurred despite the observation that the prescribed antihypertensive medications combinations were appropriate in most patients (96%) and in line with the existing treatment guidelines and consistent with previous studies.12This study found that BP was controlled to target in 50.2% of the hypertensive patients, all of whom were black people and accessing PHC. Our finding is in line with the BP control report by Berry et al.19 Specifically, the Creole Clinical Trial that was performed on black patients in sub-Saharan Africa emphasises the compelling need to use appropriate combination therapy to treat hypertension.25 In that study, amlodipine combined with hydrochlorothiazide or perindopril was found to be more efficacious than perindopril combined with hydrochlorothiazide in reducing BP to optimal levels.25 In African Americans, achieving optimal BP control goals with these antihypertensive drugs combinations is very hard according to the evidence from the Reasons for Geographic and Racial Differences in Stroke (REGARDS) study.27 This is because of a combination of factors such as dietary pattern, predisposition to increased salt sensitivity and treatment resistant hypertension, which effectively raises their BP medication requirements resulting in suboptimal control of the BP.27 In those with resistant hypertension, the use of spironolactone have been found to improve BP control, and when a thiazide or thiazide such as diuretic is indicated, chlorthalidone or indapamide have been shown to be more efficacious than hydrochlorothiazide in lowering BP.28 A total of 84% of the patients in this study had comorbid diabetes, heart failure and CKD either alone or in combination, thus, requiring a compelling use of an ACE-I for effective and optimum treatment.10 Apart from its recommended use in the control of BP preferably in combination with amlodipine in uncomplicated hypertension, the compelling rationale for the use of ACE-I is to slow or halt the progression of microvascular complications associated with diabetes, heart failure or CKD.29In this study notably, the participants were essentially black patients. The frequency of use of the first-line antihypertensive drugs were enalapril, an ACE-I (87.9%); amlodipine, a CCB (71.3%), and hydrochlorothiazide, a thiazide diuretics (60.2%). We found that these drugs were used in appropriate combinations in 96% of the patients and appropriate drug combination is recommended in the current South African hypertension treatment and other international guidelines.19 In this study, we do not have full data on the optimisation of doses of the antihypertensive medications used by the patients or the actual reasoning for the preference to use a particular antihypertensive drug.Optimisation of prescribed antihypertensive medication is currently supported by evidence because patients who are not receiving the correct dose and combination of their medications finds it difficult to achieve optimal BP control.18 Specifically, in terms of behaviour, the HBM is a health behaviour theoretical framework that can be used to explain why people take actions to control their illness such as hypertension and to suggest/design interventions against it.18 In practice, the constructs of the HBM are perceived susceptibility, severity, benefits, barriers, cues to actions and self-efficacy.17 Although this study was not designed to use these constructs to predict BP control, however, it is assumed that it can be used to design interventions against the predictors of BP control emanating from the study.18 This is because evidence from the use of the HBM as a behavioural change framework for disease conditions indicates that people\u2019s beliefs about whether or not they are susceptible to a disease such as hypertension and their perceptions of the benefits of trying to avoid it, influences their readiness to act, which includes accepting interventions directed at risk reductions for the disease.18Risk perception to uncontrolled BP and poor adherence to recommended treatment for hypertension may be a health behavioural issue, which is well rooted in the HBM and can be used to design interventions to address it.30 The subsequent observation that lack of adherence to treatment is the most significant factor associated with uncontrolled BP  also confirms previous findings.31 Factors affecting adherence to treatment includes pill burden (resulting from lack of use of fixed or single dose pill combinations) and medication side effects especially cough and angioedema when an ACE-I such as enalapril is used.33 Specifically, in this study, we did not directly assess the side effect of drugs but have a direct and quantifiable data on pill burden because almost 70% of the patients were prescribed three or more different antihypertensive pills that were not in fixed dose combinations: a major failure in the public health sector in South Africa. It is important to emphasise that in line with other international treatment guidelines, current local guidelines on treatment of hypertension recommends the use of fixed or single dose pills to help patients deal with the issue of poor medication adherence and control of BP.19 Unfortunately, fixed dose combination of antihypertensive pills are presently not available in the South African Public health sector, therefore, patients are made to depend on multiple pills, which potentially predisposes them to poor adherence. Furthermore, in our study, as previously expressed, we assessed adherence including its underlying contributors such as medication side effects (indirect assessment) and pill burden (direct assessment) by the use of the 14-item or questions in the validated Hill-Bone compliance (adherence) scale.21 This adherence scale is relatively subjective, non-invasive and cost-effective tool that also has its limitations.21 However, it is important to emphasise that the difficulty in accurately assessing adherence to hypertensive treatment is well-documented in literature and this has led to the suggestion for the use of combination of methods.33 Based on this, direct measurement of ingested antihypertensive drugs in blood or urine as performed in a study in the United Kingdom and Czech Republic appears to be the best method to accurately assess and document adherence and its effect on BP control.34 Unfortunately, because of economic cost and invasive nature of this biochemical method of assessing adherence, we were unable to fit it into our study methods.33 Furthermore, given that the relatively subjective method that we used appears to be less accurate compared with the biochemical method, our reported level of adherence in relation to BP control needs to be interpreted with caution. This becomes more imperative as many of the questions in the 14-item Hill-Bone compliance scale actually assesses hypertension treatment behaviour rather than adherence based on actual ingestion of the antihypertensive drug.21 This may be an indication of why our reported level of adherence to treatment is very poor whilst actual BP control reached a modest 50.2% threshold. However, as already indicated, our reported BP control rate is in line with a previous finding in South Africa.24In our study, only 36.3% of the respondents were fully adherent to hypertensive treatment and this is consistent with previous reports.36 In line with previous studies, both are associated with prolonged poor BP control8 and in practice has socio-economic consequences of increasing the healthcare costs of treatment and productive workforce disability.35 Specifically, we found that obesity compared with normal weight and overweight was a strong predictor  of uncontrolled BP, which is consistent with observations from previous studies.37 Also, consistent with the findings of Reboldi et al.,38 54% of the patients in this study had comorbid diabetes. This was also shown to be a risk factor for uncontrolled BP  and is similar to the reports by Duggirala et al.39 and Morgado et al.31 Diabetes is a regularly implicated risk factor for nephropathy, retinopathy and neuropathy,39 indicating the need for regular screening for these conditions in primary care. Also, approximately 10% of patients in our study had comorbid renal disease, which was not associated with uncontrolled BP. However, this finding needs to be interpreted with caution as a previous study revealed that silent renal disease not usually diagnosed/identified at screening was present in 25% of hypertensive men and 6% of women.40 This calls for concern, especially, if the number of patients with renal disease (CKD) in this study is extrapolated to a population level as there are usually large costs and other public health consequences associated with managing these patients.Comorbid obesity and diabetes observed in this study, appear to be common in South Africa and tend to be important factors for cardiovascular disease.42 In this study, it was observed that a few participants (16.8%) were meeting exercise goals and alcohol intake was extremely low (32%). Surprisingly, we found that 82.5% either smoke or use tobacco products, which is a leading risk factor for cardiovascular events including hypertension. However, it is important to note that the link between smoking/use of tobacco and BP is debatable and according to the recommendations of the South African Hypertension guideline, in people who are already hypertensive, cessation of use of tobacco products does not readily translate to a drop in systolic BP.10 This is in contrast to other life style modifications such as moderate weight loss, use of Dietary Approaches to Stop Hypertension (DASH) diet, reduction in salt/sodium use, physical activity and moderation of alcohol intake, which result in varying degrees of reduction in systolic BP in people who are already hypertensive.10 On the strength of our findings, the historical role that lifestyle modifications play in the adjunctive management of cardiovascular diseases including hypertension needs to be re-enforced during patients\u2019 encounters with attending PHC clinicians, and the HBM may be useful in this circumstance.18There is overwhelming evidence showing that lifestyle choices associated with inadequate physical activity, habitual smoking and excessive alcohol intake , are major public health concerns because of their associations with raised BP and its exacerbations.15 The interview aspect of the study including the responses to the items in the Hill-Bone compliance (adherence) scale was mainly based on self-report, which may not only create a form of information bias but being a cross-sectional design, cannot infer causation.43 This may be a plausible explanation for the disparity between the level of BP control and treatment adherence. However, the use of evidence-based BP control targets and measurements taken in the clinic from patients, and the combination of face-to-face interview and patients\u2019 clinical records for data collection, may have helped to ensure greater certainty in these aspects of the data.Our findings may not be generally applicable to other racial groups in South Africa, as only black patients were studied and there may be peculiar demographic and clinical differences in BP control that would need to be tested and compared with other racial groups.10 In addition, apart from the constructs of the HBM that can be used to screen for risk perceptions for hypertension and barriers to treatment adherence, which is necessary for the design of interventions, an additional measure to prevent poor adherence to treatment in patients with chronic conditions such as hypertension is Community-Based Physical and Electronic Reminding and Tracking System (CB-PERTS). This is for use in patients identified in the clinic to have poor treatment adherence. The components of CB-PERTS in authors\u2019 view will include: (1) scheduled home visits by community healthcare workers to encourage adherence, (2) supportive interview and (3) establishing an electronic alert system such as mobile phones that may be used by a patient to notify the healthcare professional about any difficulty with treatment adherence or used by healthcare professional to remind a patient about the need for treatment adherence . Incidentally, our proposal on the use of digital system to assist in managing chronic hypertension is supported by WHO recommendation.44 We recognise that a previous study (STAR) in South Africa that used the digital system (mobile-phone short message system) strategy produced minimal changes in adherence.45 In contrast, our proposal is a combination of three strategies, which include the use of the mobile-phone digital system, and we are of the view that it may produce a different result if it is given an opportunity for a trial in primary care. Additional study is underway to implement/assess the effectiveness of the CB-PERTS in improving treatment adherence.We align with the recommendations of the South African Hypertension treatment guidelines for the use of fixed dose or single pill combinations to reduce pill burden in patients.This study, which used a combination of interview supplemented by clinical records, found that BP was controlled to target in approximately half of the hypertensive patients. Adherence to treatment may be the most important objective in the PHC setting for hypertensive patients. Whilst it is known that the HBM can be used to design interventions against observed predictors of uncontrolled BP including poor adherence to treatment, CB-PERTS as proposed in this report should be tested and brought to use if found effective in improving treatment adherence. Hypertensive patients with diabetes and or obesity require special consideration, as these conditions appear to be an independent risk factor for uncontrolled BP. Therefore, there is a need for regular screening, health education and promotion initiatives including behavioural change interventions aimed at the identified risk factors and predictors of uncontrolled BP in primary care, and this is in line with the health belief model."}
+{"text": "Home blood pressure measurement (HBPM) has the advantage of measuring blood pressure (BP) multiple times over a long period. HBPM effectively diagnoses stress-induced transient BP elevations , insufficient BP control throughout the day , and even BP variability. In most cases, HBPM may increase self-awareness of BP, increasing the compliance of treatment. Cumulative evidence has reported better improved predictive values of HBPM in cardiovascular morbidity and mortality than office BP monitoring. In this position paper, the Korean Society of Hypertension Home Blood Pressure Forum provides comprehensive information and clinical importance on HBPM. Home blood pressure monitoring (HBPM) has three characteristics: (1) monitoring is performed by the patients themselves; (2) measurements are performed at the home; (3) an automatic blood pressure (BP) device is used rather than a conventional manometer. These traits result in several advantages and limitations.First, self-monitoring means that healthcare professionals do not take part in the measurement. BP may be elevated due to conscious or unconscious anxiety in the presence of healthcare providers. This phenomenon is known as the white coat effect, which is the reason for diagnosing hypertension in some persons and leading to unnecessary medication. The white coat effect can be avoided if the patients measure their own BP. During this process, the patient should take the initiative, and this includes purchasing a device to self-monitor. This may eventually improve disease awareness and treatment compliance. However, a correct understanding of the monitoring method and continuous training require non-healthcare professionals to self-monitor their BP. Since most people consider purchasing a BP manometer only after being advised by a physician, it is necessary for physicians to firstly recognize the importance of HBPM. In addition, if reliable education on HBPM is not provided to healthcare professionals and patients, reliable BP data may not be obtained.Second, monitoring BP at home has the advantage of obtaining accurate BP measurements while avoiding the anxiety that patients may experience when they visit a medical facility. Also, mean BP and trends in BP fluctuation can be obtained with repeated monitoring. However, whether the BP at rest represents a person's overall risk of cardiovascular events remains debatable. The disadvantage of only using only BP monitored at rest is that it is impossible to obtain high BP measurements during work or outdoor activities involving exercise or stress. As a result of these challenges, HBPM may not be an optimal alternative.Lastly, there are advantages and disadvantages associated with using an automatic BP manometer. Most automatic BP manometers use oscillometric measurements to measure diastolic BP using a constant formula that considers the pressure where oscillations begin and where they have maximum amplitude. Therefore, concerns have been raised regarding accuracy as a single BP formula cannot function accurately for every patient.This statement for HBPM were prepared to provide comprehensive information on HBPM, considering the above benefits and limitations. A survey of 330 physicians on HBPM was conducted in 2016. In the survey, 89.4% of the respondents said that HBPM is necessary or very necessary, but 30% of the respondents said that HBPM is more important than clinic BP monitoring. Meanwhile, 49.7% of the respondents said that they recommend HBPM to patients, thereby showing that it is necessary to improve physicians\u2019 thinking in the role and usefulness of HBPM.The purpose of this statement is to share more professional knowledge about HBPM. In addition, we hope to avoid the white coat effect, and will be able to identify more patients with masked hypertension.Since BP monitoring using a mercury BP manometer has been available, BP levels measured at home are usually lower than those measured in an office setting. However, auscultatory measurement using a mercury-based BP manometer at home is not widely used because it is difficult to perform and requires professional training. In 1981, Donald Nunn invented a completely automatic BP manometer, making ambulatory BP monitoring (ABPM) and HBPM possible. Following this invention, many studies highlighting the importance of ambulatory BP have been conducted in Europe. In Japan, it was reported that HBPM is not less significant compared to ABPM. Active research has led to the emergence of a field called chronologic medicine regarding BP changes, hormonal changes, and differences in occurrence of cardiovascular diseases in a day. The concept of morning hypertension, nocturnal hypertension, and nighttime dipper/nondipper were introduced and their clinical significance was supported by the ABPM study results.Nocturnal hypertension or nighttime nondipper status has been linked to an increased risk of cardiovascular events. Although short-term BP variability throughout the day can be assessed using ABPM, variability assessment is not feasible with HBPM. This is cited as a limitation of HBPM; however, the clinical significance of short-term BP variability is uncertain. Several studies have monitored home BP and observed its relationship to cardiovascular events. These studies have shown that patients with white coat hypertension have higher future hypertension and cardiovascular events than people with normal BP.Japan has had many research achievements related to home BP. In 2003, a guideline on HBPM was published. A comparable guideline was published in Korea by the working group for BP monitoring of the Korean Society of Hypertension (KSH) in 2007, specifically as a chapter of the Guideline of Self-Measuring of BP. However, the 2007 version lacked expertise because the contents were too simple and only covered recommendations on monitoring methods and their effectiveness without any background explanation. Professor Imai's second guideline on HBPM was published in Japan in 2012. The clinical significance of HBPM, selection of a BP monitor, reliability of BP monitors, monitoring method, and recording and archiving of monitoring data is described; however, there were no substantial changes compared to the previously published guideline.In Europe, a guideline on home BP was published in 2000 and revised in 2008. Europe has focused on ABPM and did not take much initiative in recommending HBPM, possibly because the majority of home BP monitors have been produced by companies of Asian countries, including Japan and Taiwan, home BP devices have become widely available and affordable, but ABPM still requires expensive machinery and testing fees. Over time, the importance of home BP has increased in Europe as studies have reported that the association between home BP and cardiovascular events is not weaker than the association between ambulatory BP and cardiovascular events. With these findings, the 2008 guideline recommended HBPM more strongly. However, no further guideline has been published since 2008, and the existing content of the guideline is covered in the Guideline on Hypertension published by the European Society of Hypertension.P\u2009<\u20090.001) , 2322, 2The period when the compression band pressure is higher than the pressure at maximum oscillation is called the escalating phase, and when it is lower, the declining phase. Systolic pressure is the BP at which the amplitude of oscillations observed is 50% of the maximum oscillation during the escalating phase. The diastolic pressure is the BP at which the amplitude of oscillations observed is 75% of the maximum oscillation during the declining phase. The oscillation ratio is an essential algorithm obtained empirically in oscillometric BP monitoring. This empirical ratio is sensitively affected by pulse pressure and aortic stiffness Fig.\u00a0.Fig. 2A\u00a0Measuring BP using this fundamental principle has its shortcomings. For example, from a physiological point of view, a comprehensive model should include the concept of dynamic compliance. In addition, the effect of the so-called transluminal pressure applied directly to the artery due to the difference between the pressure of the compression band artery must be considered. A comprehensive simulation that takes all of these factors into account shows a significant difference depending on the dynamic compliance and spasticity of the artery. This means that even in an oscillometric automated device with an optimized algorithm, there can be significant variation in accuracy between individuals. Furthermore, it is difficult to obtain a stable BP value in patients with high BP variability unless BP monitoring is repeated a sufficient number of times.In the early 1990s, auscultatory automatic BP devices had already passed the British Hypertension Society (BHS) validation standards . These dIn conclusion, oscillometric automated devices operate by deriving systolic and diastolic pressure from mean BP using the maximum oscillation amplitude, oscillation ratio, and several standard indicators. However, the specific algorithms used by devices have not been disclosed. Therefore, the accuracy of a device in actual clinical practice should be evaluated according to the international oscillometric automated device validation protocol. In particular, certain groups, such as the elderly, pregnant women, pediatric patients, patients with a very thin or thick upper-arm circumference, and patients with arterial stiffness, who were highly likely not to have been taken into account when the oscillometric BP manometer algorithms were designed, should be cautious of measurement errors. Auscultatory ABPM was partially limited to ABPM, but an auscultatory automated device that can be used in the clinic or home setting will be developed as hardware further improves.Home BP is monitored using oscillometric automated devices. Auscultatory or semi-automatic BP devices are not recommended. There are three types of devices available based on measurement location: finger, wrist, and upper arm.Devices that measure BP using a finger are not recommended because of their inaccuracies due to measurement distortion with peripheral vasoconstriction, BP alteration as the finger is too far away from the heart, and the effect of limb position on BP.Devices that measure BP at the wrist have the same last two problems as the finger devices. Inaccurate measurements may be obtained if the wrist device is not placed at the heart level or if the wrist is flexion or hyperextension during measurement. Also, if the radial artery is not fully compressed, both the radial and ulnar artery oscillation waves influence BP values, making it difficult to produce an accurate algorithm to estimate systolic and diastolic BP and, ultimately, they lead to obtaining inaccurate BP values. However, wrist devices may be considered in special populations, such as obese or elderly individuals, in whom the upper arm is more challenging to perform BP measurement. Because the correct placement of the cuff at heart level is essential for an accurate measurement, some wrist devices have built-in sensors to indicate the correct position. Wrist devices are commonly used among patients despite their limitations because the measurement is readily performed without removing the clothes. Approximately 20 wrist devices that passed validation protocol with established protocols are currently available in the market.Devices that measure BP in the upper arm are the most reliable in both clinical practice and major hypertension trials for HBPM. Most international recommendations for oscillometric automated office BP measurement devices also apply to automated home BP devices. For accurate BP measurement, it is important to use validated home BP devices according to established protocols and to select a cuff to fit the upper-arm circumference. Many new HBPM devices have a built-in memory that automatically stores BP readings. Commonly, home BP devices are inappropriate in patients with arrhythmias. Recently, some devices can detect BP during irregular heart rhythm, such as atrial fibrillation. The majority of HBPM devices assess daytime BP, but some devices become available to measure during sleep thus they can detect sleep BP. Also, some devices are available to transmit BP data to the patients and their healthcare providers via Bluetooth. Approximately 200 devices that passed the validation protocol with established protocols are available on the market.In an auscultatory BP sphygmomanometer, the role of the BP cuff is to compress the artery with the inflatable part of the BP cuff (bladder), whereas, in an oscillometric automated device, the cuff is at the signal sensor of oscillation of the artery. Thus, it is also important to document the cuff's type, shape, and material characteristics under the device validation procedure. The cuff size is selected by upper arm circumferences according to the device\u2019s instructions Table . FurtherA proper device should be selected to measure BP accurately Table .Table 3Phttp://www.dableducational.org, and a list of internationally validated BP devices can be found on the KSH website.For the accurate diagnosis of hypertension, the BP device is validated according to established protocols. However, unfortunately, less than 20% of devices on the market have been validated. Validation of BP devices began in ad hoc protocols in the 1980s, and then reputable organizations published validation procedures. The US Association for the Advancement of Medical Instrumentation (AAMI) and the BHS published more systemized standard protocol for clinical validation of automated BP devices against a mercury sphygmomanometer as a reference value. In 2002, the European Society of Hypertension (ESH) Working group developed the ESH-International Protocol (ESH-IP), and it was stringently revised in 2010. ESH-IP 2002 was widely used and more practical because the sample size and pass criteria were simplified compared to previous protocols. In 2018, the universal protocol (Association for the AAMI/ESH/Organization for Standardization [ISO]), single protocols for global acceptance, was developed. This protocol recommends that reference BP measurements for validation can be obtained using mercury sphygmomanometers or non-mercury sphygmomanometers (aneroid or other) that fulfill the ISO 81060\u20131 requirement for accuracy  because of concern with mercury toxicity in many countries Table . AAMI, Ehttp://www.dableducational.org/ and the universal protocol for device validation is currently used for the device validation .Fig.Fig50].FMasked hypertension is defined as BP that is less than 140/90\u00a0mmHg when measured in the clinic while it is hypertensive when measured with HBPM or ABPM. The frequency of masked hypertension is 15%\u201330%. It is associated with factors such as male sex, smoking history, and alcohol consumption. It is also common in patients with high occupational stress, and those with a high BP variability are at a higher risk of masked hypertension , 52. SinWhite coat hypertension and masked hypertension are observed in the normal population that have never received antihypertensive treatment and in patients who are already on antihypertensive treatment. According to data from an ABPM registry, the percentage of patients taking antihypertensive medication exhibiting a white coat effect with high clinic BP but normal ambulatory BP  was 13.5% of all treated patients. Also, 13.8% of patients on antihypertensive medication had uncontrolled hypertension misinterpreted due to the masked effect  .P\u2009=\u20090.86) in WUCH patients compared to patients with controlled hypertension [According to a cohort study performed in Spain involving approximately 60,000 patients, there was no significant increase in total mortality  or cardiovascular mortality /ESH Guidelines for the Management of Arterial Hypertension, which includes recommendations on HBPMs , 60 The Based on home BP, the diagnostic criteria for hypertension is a BP\u2009\u2265\u2009135/85\u00a0mmHg (Table ACC/AHA (American College of Cardiology/American Heart Association) published a new version of the Guideline for the Prevention, Detection, Evaluation, and Management of High Blood Pressure in Adults in 2017 and published the \u201cSelf-measured blood pressure monitoring at home: a joint policy statement from the American Heart Association and American Medical Association\u201d in 2020 , 63. TheIn 2020, ACC/AHA published their Clinical Practice Guideline for Self-Measured Blood Pressure Monitoring At Home . This inIn Korea, the Guidelines for Blood Pressure Monitoring was published primarily by the working group for BP monitoring of KSH and home BP was covered in the \"Self-Measurement of Blood Pressure\" section . ThereafThe main recommendations include the following: (1) HBPM is recommended for the diagnosis and assessment of prognosis of hypertension, white coat hypertension, and masked hypertension ; and (2) for accurate home BP measurements, accurate measurement method should be educated to all patients . Home BP is more useful in predicting the incidence of cardio-cerebrovascular disease than clinic BP in patients with hypertension. Since its usefulness is high in terms of the medical economy, the importance of home BP in the diagnosis and the management of hypertension, is emphasized in the hypertension guidelines . And HBPIn the Guidelines for the Management of Hypertension published by the KSH in 2018, hypertension using HBPM is defined as\u2009\u2265\u2009135/85\u00a0mmHg and \u201cmorning hypertension\u201d is defined as cases in which BP measured in the morning is\u2009\u2265\u2009135/85\u00a0mmHg and is higher than the BP before sleep. However, a home BP level that corresponding to normal BP and a target BP for treatment could not be presented.In 2012, the JSH published \"Self-Monitoring of BP at Home (2nd edition),\" a guideline for HBPM, with Professor Imai as the lead author . Many seIn this guideline, the diagnostic criteria for hypertension using home BP were defined as\u2009\u2265\u2009135/85\u00a0mmHg, which was mostly selected based on previous cross-sectional studies and guidelines . AccordiIn 2008, the AHA published a statement with the American Society of Hypertension. Based on previous studies, target home BPs were set at\u2009<\u2009135/85\u00a0mmHg for the general population,\u2009<\u2009130/80\u00a0mmHg for high-risk patients and\u2009<\u2009125/75\u00a0mmHg for diabetic patients. In addition, based on the results of a previous meta-analysis, normal home BP  was reported to be\u2009<\u2009120/80\u00a0mmHg . Based oThe Guidelines for the Management of Hypertension published by JSH in 2019 also included HBPM as \u201cmeasurement and clinical evaluation of BP\u201d . In addiThe 2019 JSH Guidelines strongly recommend antihypertensive treatment based on home BP rather than office BP , as home BP is more reproducible and correlates with cardiovascular disease. Furthermore, home BP monitoring is useful for diagnosing white coat hypertension and masked hypertension. It is also actively recommended for patients undergoing antihypertensive treatment as it helps to evaluate hypotension during antihypertensive treatment and improves patient adherence to antihypertenive medication.Intervention studies using home BP have been performed previously, but they did not provide sufficient evidence. Based on the Ohasama and HOMED-BP studies, it was proposed that the target home BP should be set 5\u00a0mmHg lower than that for clinic BP for both systolic and diastolic pressures \u201375. TherHBPM using an automated device enables patients with hypertension to obtain more BP values by monitoring BP at home without limitations on time while avoiding the whitecoat effect. In addition, there are several advantages for hypertension treatment, such as enhanced compliance to antihypertensive drugs, since the patients can directly check the change of BP with medication or lifestyle modification. In particular, the use of home BP is gradually increasing as the number of validated BP manometers has increased in recent years, and the devices have become more popular as the price of manometers has gone down.However, there are still limitations with HBPM. If these limitations are not addressed, it may cause unnecessary concerns to patients and delay appropriate actions. For this reason, health care providers should be fully aware of this and provide education to patients. There are several limitations of HBPM that may become clinically problematic.Measurement of home BP using oscillometric method may be inaccurate in the presence of severe arrhythmia. Accordingly, devices capable of detecting arrhythmia such as atrial fibrillation have been developed, which may help the diagnosis of paroxysmal atrial fibrillation, which is challenging to diagnose. In the case of arrhythmias, errors in the BP measurement using automated devices are inevitable. According to a study by Pagonas et al.  in 2013,Xie et al.  investigGestational BP monitoring is critical to the diagnosis of gestational hypertension. BP during pregnancy directly impacts the mother's health and fetal development, and BP must be accurately monitored using a proper method to determine whether to treat hypertension and maintain proper target BP. In other words, receiving unnecessary treatment due to incorrectly detected high BP resulting from a white coat effect and missing the optimal treatment period by depending on clinic BP and not recognizing masked hypertension may cause problems, including progression of eclampsia. Therefore, BP must be monitored accurately through HBPM or ABPM. However, due to the increase in blood volume and decrease in peripheral vascular resistance, the pulse pressure widens during pregnancy. Thus, oscillometric HBPM may be inaccurate. It should also be considered that there is not much research-validated using conventional BP manometers in pregnant women. Even though a BP manometer is validated, it cannot be considered suitable during pregnancy. According to a meta-analysis done by Bello et al.  in 2018,HBPM may be superior to ABPM in terms of convenience and capability for repeated measurement; however, its limitation is the difficulty in BP monitoring during sleep. Importantly, this limitation lies in that assessment of nocturnal hypertension due to sleep apnea, nighttime dipping, and morning surge is not feasible Fig.\u00a0.Fig. 8FaThe 2019 International Expert Group of Nocturnal Home Blood Pressure has reported that home nocturnal BP monitoring using automated devices, including the Microlife Watch BPN  and Omron HEM7252G-HP, showed a comparable level of accuracy with ambulatory nocturnal BP monitoring . HoweverAccurate BP monitoring is the most critical aspect of hypertension management. Accurate home BP monitoring better reflects prognosis than clinic BP and can enhance medication compliance and treatment response. Smartwatches and smartphones are essential personal mobile devices. It is estimated that at least 5 billion people possess mobile devices and more than half of these people have smartphones. According to a report by the Korea Information Research and Development Institute in 2018, 37.8% of Koreans\u2009\u2265\u200970\u00a0years old had smartphones, tenfold the percentage in 2013, which was 3.6% , from 19Although prior research has reported that the accuracy of BP monitoring using smartphones is 95\u2013 100%, actual measurements are presented as a range rather than specific values as a result may fluctuate depending on the measurement method . Even thBP monitoring using a smartwatch has two challenges. First, the accuracy of photoplethysmographic sensors is not yet validated. If there is a near-infrared light source around the site of BP monitoring, spot monitoring can be less accurate. Second, self-monitoring is prone to error despite proper training. The error can be even more prominent with unstable resting posture.BP obtained through a conventional monitoring device should be entered periodically into the BP monitoring app in the smartphone to monitor BP with a smartwatch. Paradoxically, monitoring BP with a mobile device has collateral benefits. BP should be monitored periodically with a conventional BP manometer and the mobile device and eventually check the BP more accurately. It is recommended to perform the BP measurement three times, 2\u00a0min apart for the calibration, and it must be done again if switching the smartwatch to the other wrist.The core problem in the adjustment process is the difference in BPs between the left and right arms. It has been reported in many epidemiological studies that the gap between the systolic/diastolic pressures of the two arms is approximately 3.3/2.0\u00a0mmHg, respectively. Also, the BP difference between the two arms widens as BP increases. Therefore, if the calibration is performed using BP values measured in the opposite arm, it can cause an error of at least 3\u00a0mmHg, which cannot be addressed by any internal calibration mechanism.Moreover, the clinical studies on smartwatches show that the patient can measure BP on the opposite arm with a general BP manometer while measuring BP with a smartwatch, thus compensating for the difference between the two arms and enabling a simultaneous BP measurement in both arms. However, in real life, BP cannot be measured in both arms without assistance, so the benefit of measuring BP in one arm with a smartwatch while measuring in the other arm with a general BP manometer is not practically obtainable. Therefore, a position paper from the KSH, smartwatch-based cuffless BP measurement recommends calibration by obtaining a BP measurement signal with a smartwatch and then measuring BP in the same arm with a general BP manometer . Also, wDevices certified as medical devices are not recommended for patients with a systolic BP that is considered very high\u2009\u2265\u2009160\u00a0mmHg or very low\u2009\u2264\u200980\u00a0mmHg since their accuracy is not yet validated. They are also not recommended for patients with aortic insufficiency, atrial fibrillation with high heart rate variability, peripheral vascular disease with weak blood flow, glycosuria, cardiomyopathy, end-stage renal failure, hand tremor, or blood coagulation disorder. In addition, they are not recommended for patients taking antiplatelet/anticoagulant drugs or states with hormone fluctuation, such as pregnancy, since their vascular characteristics differ from the normal range.Therefore, accurate self-measured BP monitoring using a smartwatch is achieved only when a standardized procedure is followed. However, it is not recommended for patients with systolic BP considered very high\u2009\u2265\u2009160\u00a0mmHg or very low\u2009\u2264\u200980\u00a0mmHg since it is not well established in these populations.So far, BP monitoring using mobile devices has been primarily effective in raising awareness of the importance of BP management among the general public and the early diagnosis of hypertension rather than monitoring patients with hypertension. Implementation in the geriatric population may be limited as they may find it difficult to adjust the device using a general BP manometer periodically. However, its significance lies in raising awareness of hypertension in tech-savvy adults in their 30\u00a0s to 40\u00a0s, triggering early hypertension management.On the other hand, periodic BP monitoring  with mobile devices can facilitate routine BP monitoring in patients with hypertension. However, we cannot exclude the risk that patients may self-adjust medication based on a BP result inappropriately measured in an unrelaxed state. In addition, various studies have reported that mobile devices' BP monitoring results indicated significant differences over 24-h periods, between days of the week, and even from season to season . Most imLastly, BP monitoring using a mobile device can open up a new field for assessing the dynamic changes in BP . Only stThis position paper is an official opinion of Korean Society of Hypertension Home Blood Pressure Forum. HBPM has advantages, including effectively diagnoses stress-induced transient BP elevations , insufficient BP control throughout the day , and even BP variability. Additionally, HBPM may increase self-awareness of BP, increasing the compliance of treatment. Cumulative evidence has reported better improved predictive values of HBPM in cardiovascular morbidity and mortality than office BP monitoring."}
+{"text": "Sixty\u2010six PvCO2 samples were obtained, and there was a high correlation between PvCO2 and PaCO2 . Regarding type 2 respiratory failure, there was a high correlation between EtCO2 and PaCO2 (r = 0.81). The Bland\u2013Altman analysis between PaCO2 and EtCO2 revealed a bias of 5.7 mmHg, with limits of agreement ranging from \u22125.1 mmHg to 16.5 mmHg. In contrast, the analysis between PaCO2 and PvCO2 revealed a bias of \u22126.8 mmHg, and the limits of agreement ranged from \u221222.13 mmHg to 8.53 mmHg.A total of 100 samples were included that comprised 67 men (67%). The mean age of the subjects was 77\u2009\u00b1\u200913 years. Chronic obstructive pulmonary disease (COPD) (43%) was the most common disease. There was a high correlation between EtCO2 measured by CapnoEye\u00ae was significantly correlated to PaCO2 levels in patients with respiratory diseases. Moreover, CapnoEye\u00ae may be more useful for predicting hypercapnia conditions in which respiratory diseases are compared with measure PvCO2.EtCO 2 measured by CapnoEye\u00ae was significantly correlated to PaCO2 levels in patients with respiratory diseases. While type 1 patients displayed a moderate correlation, type 2 patients demonstrated a high correlation between EtCO2 and PaCO2. In contrast, the correlation between PvCO2 and PaCO2 was moderate for both type 1 and type 2. CapnoEye\u00ae may be more useful for predicting hypercapnia conditions in which respiratory diseases are compared with measure PvCO2.EtCO ABGarterial blood gasCOPDchronic obstructive pulmonary disease2EtCOend\u2010tidal carbon dioxideICUintensive care unit2PaCOpartial pressure of arterial carbon dioxide2PaOpartial pressure of arterial oxygen2PvCOpartial pressure of venous carbon dioxide12) is <60\u2009mmHg and/or the partial pressure of arterial carbon dioxide (PaCO2) is >45\u2009mmHg. Respiratory failure is classified according to blood gas abnormalities into types 1 and 2. Type 1 (hypoxemic) respiratory failure is a disorder of the respiratory system in which the PaO2 and PaCO2 are \u226460\u2009mmHg and <45\u2009mmHg, respectively, due to respiratory dysfunction. A PaCO2\u2009>\u200945\u2009mmHg is defined as type 2 (hypercapnic) respiratory failure. Hypoxemia is a common condition and can be attributed to ventilation failure.Respiratory failure is a clinical condition characterized by the failure of the respiratory system to maintain its major function, that is, gas exchange, in which the partial pressure of arterial oxygen  assessment is essential to confirm the diagnosis of respiratory failure, and frequent measurements are necessary to confirm the respiratory status during the course of treatment. An American Thoracic Society/European Respiratory Society position paper reported on ABG analysis as the preferred method for determining the need for oxygen, as it includes acid\u2013base information.The monitoring of respiratory condition necessitates the most non\u2010invasive technique in the perioperative period and the intensive care unit (ICU).2) is another non\u2010invasive method for estimating PaCO2. EtCO2 is generally measured as biological information in patients in the operating room or in the ICU with controlled ventilation. However, it is not usually measured in general wards or laboratories.2.The measurement of end\u2010tidal carbon dioxide  can non\u2010invasively measure EtCO2 levels. CapnoEye\u00ae was used to measure EtCO2 and PaCO2 in patients with type 1 and 2 respiratory failure. We aimed to calculate and evaluate the correlation coefficients and their measurement accuracies. We also intended to compare differences between venous and arterial carbon dioxide levels only in patients who simultaneously underwent venous blood gas measurements. Furthermore, we also aimed to compare the efficacy of EtCO2 and PvCO2 in predicting PaCO2 levels.We aimed to evaluate the accuracy and efficacy of the novel portable capnometer for predicting PaCO22 was measured from February 2017 to December 2019. Simultaneously, we collected arterial blood gas sampling to measure the PaCO2. Moreover, we only compared the venous carbon dioxide (PvCO2) and PaCO2 values in patients who had underwent the sampling concurrently. These samples were collected under similar conditions at a particular time. EtCO2 was measured using the CapnoEye\u00ae . The measured data were compared overall and between the two groups. We collected 66 samples of PvCO2 (34 patients with type 1 and 32 patients with type 2 respiratory failure). The PvCO2 and EtCO2 data were also compared overall and between the groups.We collected a total of 100 samples of EtCO2 and PaCO2 and between PvCO2 and PaCO2 using Pearson's moment correlation coefficient in patients with type 1 and type 2 respiratory failure and both groups overall. We also analyzed the agreement between EtCO2 and PaCO2 and between PvCO2 and PaCO2 using Bland\u2013Altman analysis. Moreover, we calculated the estimated bias  and precision  in subjects with type 1 and type 2 respiratory failure and both groups overall. Data are presented as the mean (standard deviation). Descriptive data are presented as the median, frequency, and percentage. Statistical analyses were performed using JMP 14 .We analyzed the correlation between EtCO2.2This study was conducted in accordance with the tenets of the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Clinical Investigation for the National Center for Global Health and Medicine . All subjects provided informed consent for their inclusion in this study.32, EtCO2, and PvCO2 were 74\u2009\u00b1\u200913\u2009years, 22.0\u2009\u00b1\u20094.6, 47.2\u2009\u00b1\u200911.8\u00a0mmHg, 41.5\u2009\u00b1\u200910.1\u00a0mmHg, and 53.4\u2009\u00b1\u200913.2\u00a0mmHg, respectively. Table\u00a0Table\u00a02 and PaCO2 in all patients . The correlation coefficient was 0.883, and we observed a high positive correlation. . Figure\u00a02 and PaCO2 in all patients , with a correlation coefficient of 0.814, which demonstrated a high positive correlation . Next, we separated the data into two categories, Type 1 and Type 2, and illustrated the correlations between them in the figures  displayed a moderate correlation (r\u2009=\u20090.47), type 2 patients (n\u2009=\u200949) demonstrated a high correlation (r\u2009=\u20090.81) between EtCO2 and PaCO2. In contrast, the correlation between PvCO2 and PaCO2 was moderate for both type 1 (n\u2009=\u200934) and type 2 (n\u2009=\u200932) .No specific technical problems were encountered in these measurements. Figure\u00a0s Figure\u00a0. While t2 and EtCO2 and between PaCO2 and PvCO2. The Bland\u2013Altman analysis between PaCO2 and EtCO2 revealed a bias of 5.7\u00a0mmHg, with limits of agreement (bias\u2009\u00b1\u20091.96 SD) ranging from \u22125.1\u00a0mmHg to 16.5\u00a0mmHg. However, it also revealed a bias of \u22126.8\u00a0mmHg between PaCO2 and PvCO2, with limits of agreement (bias\u2009\u00b1\u20091.96 SD) ranging from \u221222.13\u2009mmHg to 8.53\u2009mmHg.Figure\u00a042 measured using the novel portable capnometer was significantly correlated with PaCO2 levels in patients with respiratory diseases. The correlation coefficient between EtCO2 and PaCO2 was 0.883, thus indicating a positive correlation . Moreover, the correlation coefficient between PvCO2 and PaCO2 was 0.814 and also indicated a positive correlation . The Bland\u2013Altman analysis demonstrated good agreement and tolerance between the two variables. Furthermore, EtCO2 measured using the novel tool may be more useful for predicting hypercapnia conditions in which respiratory diseases are compared with measure PvCO2. However, the limits of agreement against both groups were wide. Therefore, care providers must pay attention to the characteristics and errors of these devices.In our study, EtCO2 has been measured for the non\u2010invasive estimation of PaCO2. EtCO2 is commonly measured in the closed circuit in patients undergoing mechanical ventilation. Therefore, it has been most often used as a non\u2010invasive substitute for PaCO2 in anesthesia, anesthetic recovery, and intensive care.2 would more accurately predict PaCO2. Without significant changes in cardiac output or in the ventilation/perfusion ratio, PaCO2 can be estimated with clinically acceptable sensitivity and specificity than EtCO2.2 is challenging in patients who are not intubated. This can be attributed to the difficulty to collect pure respiratory gas.Conventionally, EtCO2) for determining hypoxemia, measuring SpO2 alone is not sufficient to detect hyperventilation or hypoventilation. Particularly with the administration of supplemental oxygen, patients can display adequate arterial saturation during hypoventilation.2 retention in patients with respiratory failure. Nonetheless, no devices have been identified to easily monitor CO2 retention in general wards and outpatient clinics. Recently developed transportable capnometers can measure EtCO2 easily and non\u2010invasively in patients who are not intubated. Moreover, a recent study demonstrated that they could provide reliable EtCO2 values compared to PaCO2 in patients undergoing general anesthesia.Despite the usefulness of monitoring oxygen saturation  lower than PaCO2. We also confirmed a high correlation between EtCO2 and PaCO2 in patients with type 1 and 2 respiratory failure. In other words, the non\u2010invasive estimation of PaCO2 by measuring EtCO2 using CapnoEye\u00ae is clinically useful. The measurement of EtCO2 was not associated with any complications. In addition, the patient burden was extremely small. It is necessary to wait for few minutes for a 0\u2010point correction before the measurement. However, the measurement process is easy. The measurement only required disposable mouthpieces, and CapnoEye\u00ae was able to measure EtCO2 at a low price. However, its disadvantage was the inability to continuously monitor and record the parameter. While this measurement device is simple, based on a proprietary algorithm, and is easily measured and carried around, it does not enable offline storage of measurement data; therefore, it requires the user to take notes or pictures of the displayed data on the screen. It could not be used for patients with impaired consciousness or cognitive decline who were unable to respond to our instructions.CapnoEye\u00ae can measure EtCO2 was correlated with PaCO2 higher than PvCO2, particularly in type 2 respiratory failure. The measurement of PvCO2 instead of PaCO2 would result in data discrepancies in patients with chronic respiratory failure, as in our patient cohort. The effects of cardiac output and tissue oxygen consumption may have caused differences in the venous and arterial CO2 levels in patients with chronic respiratory failure, compared with known reports. Therefore, our findings suggest that the predicate of arterial blood gas analysis likely preferred EtCO2 to PvCO2 in patients with chronic respiratory failure, predominantly type 2 respiratory failure. Type 2 monitoring is more important in actual clinical practice. This highlights the significance of performing non\u2010invasive monitoring in patients with a tendency for chronic CO2 retention.A previous study reported that arterial blood gas analysis during the acute exacerbation of chronic obstructive pulmonary disease could be substituted by venous blood gas analysis.52 in patients who were alert and breathed spontaneously. Thus, it was unsuitable for patients who did not spontaneously breathe, were unable to respond to our instructions, or demonstrated poor spontaneous breathing, such as those with neuromuscular diseases. Fourth, some patients with type 2 respiratory failure underwent repeated measurements, which may be a source of bias. However, it is unknown whether EtCO2 measurements using this novel device in patients with refractory failure and spontaneous breathing can be considered an alternative to PaCO2 measurements that use arterial blood gas analysis in clinical settings.Our study had several limitations. First, the Bland\u2013Altman analysis demonstrated that the limits of agreement against both groups were wide. Therefore, care providers must pay attention to the characteristics and errors of these devices. Second, this was a single\u2010center retrospective study comprising a small sample size. Third, the CapnoEye\u00ae measurement changes depending on the patient's breathing pattern. Therefore, it could only evaluate EtCO62 measuring instruments have displayed a high correlation with PaCO2 and are clinically applied as a substitute for PaCO2. The novel portable capnometer can measure EtCO2 in a simple and non\u2010invasive way. EtCO2 measured using CapnoEye\u00ae demonstrated a positive, strong, and statistically significant correlation with PaCO2 levels in patients with respiratory diseases. Furthermore, EtCO2 measured using the novel tool may be more useful for predicting hypercapnia conditions in which respiratory diseases are compared to measure PvCO2.Considering the improved accuracy of measuring instruments, EtCOWe intend to conduct future studies to confirm the usefulness of these measurements, particularly for type 2 patients in primary care.None.This study was conducted in accordance with the tenets of the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Clinical Investigation for the National Center for Global Health and Medicine . Written informed consent was obtained from all patients.MS conceptualized and designed the study, drafted the initial manuscript, and reviewed and revised the manuscript. MS, SF, KS, and KT entered and collected data into the database. MS and SF contributed significantly to managing the data and MS to the statistical analysis. All authors revised the article critically for important intellectual content, gave their final approval of the version to be published, and agreed to be accountable for all aspects of the work."}
+{"text": "The purpose of this study was to investigate vaccine effectiveness in relieving symptoms in patients with the SARS-CoV-2 delta (B.1.617.2) variant.In this retrospective study, 31 patients did not receive any vaccine , 21 patients received 1-dose of inactivated vaccine , and 60 patients received at least 2-dose inactivated vaccine . The baseline data, clinical outcomes and vaccination information were collected and analyzed.p\u2009=\u20090.001), but there was no significant difference in any of the other baseline data among the three groups. The TV group showed higher IgG antibody levels and cycle threshold values of SARS-CoV-2 than the NV and OV groups (p\u2009<\u20090.01), and time to peak viral load was shorter in the TV group (3.5\u2009\u00b1\u20092.3 d) than in the NV (4.8\u2009\u00b1\u20092.8 d) and OV groups . The patients in the TV group (18%) showed a higher recovery rate without drug therapy (p\u2009<\u20090.001). Viral clearance time and hospital stay were significantly shorter in the TV group than in the NV and OV groups (p\u2009<\u20090.01), and there were no significant differences in these parameters between the OV and NV groups, but IgG values were higher in the OV group (p\u2009=\u20090.025). No severe complications occurred in this study.Patients in the OV group were younger than those in the other two groups (in\u00a0vivo.In this study, our results shows that two-dose vaccination can reduce viral loads and accelerate viral clearance, and two-dose vaccination enhance the protection of IgG antibodies in\u00a0vivo; however, one-dose vaccination did not confer protective effectiveness.Our results suggest that 2-dose vaccination can reduce viral load and accelerate viral clearance in patients with the delta variant and enhance the protection afforded by IgG antibodies  Coronavirus disease 2019 (COVID-19) is oneof the most serious global public health crisis in this century, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreaks have caused millions of deaths and placed heavy burdens on individuals and societies . In addiPrevious reports have shown that vaccination is effective in reducing household transmission of the alpha variant by 40\u201350%, with viral load in the upper respiratory tract significantly lower in vaccinated patients ,15. OtheEvaluating the vaccines against SARS cov-2 variants is the focus of the current pandemic, and it is equally important to take preventive measures to prevent infection . In the via SARS-CoV-2 nucleic acid amplification tests, and the results were confirmed by the Xi\u2019an Center for Disease Control and Prevention. This study was approved by the Ethics Committee of the First Affiliated Hospital of Xi\u2019an Jiaotong University, and all patients provided written informed consent. All research methods were carried out in compliance with the relevant declarations of medical ethics and the Declaration of Helsinki.All patients were treated by the medical team of the First Affiliated Hospital of Xi\u2019an Jiaotong University between 20 December 2021, and 20 January 2022. The eligible discharged patients were screened in this retrospective study , and patThe collected data included demographic characteristics, epidemiological data , clinical data, chest computed tomography (CT), SARS-CoV-2 detection information, antiviral antibody information, complications and outcomes. All data were obtained from the electronic medical record system, and no follow-up data were included in this study. Vaccination information was collected from the patients, including vaccine name, dose, and date of administration. Two-dose vaccination was defined as having received a second or third dose of vaccine at least 14\u2009days prior; one-dose vaccination was defined as having received the first dose of the vaccine at least 21\u2009days ago but not the second dose. Unvaccinated patients did not receive any SARS-CoV-2 vaccine. Antibodies against SARS-CoV-2 (anti-Spike IgG and IgM) were detected by laboratory tests. All data were extracted by two independent physicians, and any disputed data were resolved in consultation with a third independent physician.via quantitative reverse transcription polymerase chain reaction  according to the national guideline and the manufacturer\u2019s instructions, and all patients were examined daily during hospitalization. The conditions for SARS-CoV-2 nucleic acid amplification were 50\u2009\u00b0C for 15\u2009min, 95\u2009\u00b0C for 15\u2009min, followed by 45 cycles of 94\u2009\u00b0C for 15\u2009s and 55\u2009\u00b0C for 45\u2009s. Positive detection was defined as a cycle threshold  less than 40(500 copies/ml), and the test procedure was carried out in strict accordance with the protocol. A patient\u2019s viral load was defined as the patient\u2019s lowest Ct value during hospitalization, and the time to peak viral load was defined as the time from the first positive SARS-CoV-2 test to the lowest Ct value during hospitalization. The incubation period was defined as the number of days from contact exposure to the onset of a positive nucleic acid result.SARS-CoV-2 nucleic acid expression was detected with SARS-CoV-2 virus kits via a commercial ELISA kit  according to the manufacturer\u2019s instructions, and the antibodies were detected at admission, day 7, day 10, day 14 and at discharge. Briefly, 96-well plates were coated with purified SARS-CoV-2 antigen in phosphate buffer overnight at 4\u2009\u00b0C in physiological saline (PBS). We added 10\u2009\u00b5l of each serum sample to a reaction plate, mixed it with 50\u2009\u00b5l magnetic beads and 50\u2009\u00b5l buffer, incubated it in the reaction plate for 10\u2009min and then washed it. Then, we added 100\u2009\u00b5l acridine ester-labeled recombinant magnetic beads and incubated them in the reaction plate for 10\u2009min. After washing, we added the substrate solution and mixed it well to detect the luminescence signal value. The assay detects antibodies to the spike protein of SARS-CoV-2 with a cut-off (CO) value of <1.0 for negative results and the value \u22651.0 were defined as positive, the final value of the test was calculated with the optical density value of the sample/CO value.Serum antibodies (anti-Spike IgG and IgM) against SARS-CoV-2 were detected All patients were treated in an isolation hospital according to the national guidelines. Briefly, therapeutic strategies were selected according to the severity of the symptoms and comorbidities of the patients. Patients with mild symptoms and no pulmonary imaging changes were given prone position ventilation and drug therapy for underlying diseases, and patients with obvious symptoms (with pulmonary imaging changes) were selectively given thymalfasin  and lopinavir/ritonavir (250\u2009mg twice daily). Neutralizing antibodies or convalescent plasma were selectively used for severe patients.SARS-CoV-2 patients were classified into four types: mild, common, severe and critical, the clinical classification of SARS-CoV-2 was defined previously by the National Health Commission of China ,22. A pat tests and analysis of variance (ANOVA). Categorical variables were presented as frequency and percentages and were analyzed using chi-square tests or the Kruskal\u2013Wallis test, Mann\u2013Whitney U test or Fisher exact test. p values <0.05 were considered statistically significant.All data were collected with Excel file  and analyzed by using SPSS v. 22.0  software. Quantitative variables were presented as mean and standard deviation and were tested for normality first, and then hypothesis testing was performed with paired p\u2009=\u20090.001), and no significant differences were confirmed for the other baseline characteristics. Except for the incidence of intestinal symptoms, which was higher in the NV group than in the other two groups (p\u2009=\u20090.02), there were no significant differences in the other symptoms and comorbidities, and the most common symptoms included cough, sputum production, sore throat, anosmia and dysgeusia (p\u2009>\u20090.05); however, the incidence of any symptoms in TV group was significantly lower than NV and OV groups (p\u2009<\u20090.05).There were 112 patients with the SARS-CoV-2 delta variant included in this retrospective cross-sectional study , and thep\u2009>\u20090.05). The TV group showed a similar incubation period of SARS-CoV-2 as the NV and OV groups. However, the cycle threshold value of SARS-CoV-2 was higher in the TV group (32.4\u2009\u00b1\u20095.1) than in the NV (28.4\u2009\u00b1\u20095.7) and OV groups , and time to peak viral load were shorter in the TV group (3.5\u2009\u00b1\u20092.3 d) than in the NV (4.8\u2009\u00b1\u20092.8 d) and OV groups  and higher in the OV group than in the NV group (p\u2009=\u20090.025). Most patients underwent the therapeutic strategies described in the Methods section, and the patients in the TV group (18%) showed a higher recovery rate without drug therapy .All clinical and epidemiological data are listed in p\u2009>\u20090.05). During therapy, SARS-CoV-2 viral clearance time was significantly shorter in the TV group (11.2\u2009\u00b1\u20091.9 d) than in the NV (14.3\u2009\u00b1\u20093.6 d) and OV groups . Moreover, the hospital stays of patients in the TV group (14.4\u2009\u00b1\u20092.2 d) were shorter than those of patients in the NV (17.6\u2009\u00b1\u20094.4 d) and OV groups . There were no significant differences in these parameters between the NV and OV groups (p\u2009>\u20090.05). No severe adverse events  occurred in the three groups.The outcome parameters after isolation therapy are shown in The spike protein of the delta variant shows a higher binding affinity with the human angiotensin-converting enzyme 2 receptors than that of the spike proteins of earlier variants, suggesting that the delta variant has a high transmission and rapid cell infectivity ,24. ThusA previous study revealed that vaccination shows a low protective effect against the delta variant except for a satisfactory protective effect against severe infections . There wOur results confirmed that only two-dose vaccinated patients had lower viral load and higher IgG antibody levels, and the IgG value may be one of the indicators reflecting the protective effect of vaccination. These data indicate that the vaccines remain effective against the delta variant. Previous studies have demonstrated that serum IgG in patients with SARS-CoV-2 infection was closely related to its protective effect, and if the IgG antibody value was less than 50 BAU/mL, the protective effect of the vaccine was very limited . In our Despite vaccine neutralization of the delta variant, the symptom with the slowest recovery is anosmia/dysgeusia; indeed, anosmia/dysgeusia may be a good predictor of SARS-CoV-2 infection . One stuThis study has the following limitations. First, due to the vaccination policy, there are increasingly fewer subjects who are not vaccinated; therefore, the numbers of unvaccinated and one-dose vaccinated patients were low in this study, which could cause selection bias and affect the conclusions. Second, this study had a small sample size; we only analyzed the hospitalization data of patients in three specific groups and did not include the data from each patient\u2019s entire disease process, which could also affect the accuracy of the conclusions. Third, we only analyzed the effectiveness of inactivated vaccines and did not include live attenuated vaccines or mRNA vaccines, which may have affected the results. Finally, this study did not analyze the protective effects of vaccines in different populations, which may affect the generalizability of the conclusions.In this retrospective study, our results suggest that two-dose vaccination could alleviate symptoms of the SARS-CoV-2 delta variant, reduce viral load and accelerate viral clearance in patients. The two-dose vaccinated patients showed higher IgG antibody levels than the unvaccinated and one-dose vaccinated patients, and the two-dose vaccinated group exhibited a higher recovery rate without the need for medications than the other two groups."}
+{"text": "This is followed by immobilization of streptavidin. Streptavidin serves as a universal anchor for biotinylated antibodies, enabling simple preparation of tailor-made affinity samplers. The functionality of the device was tested using a model protein  and biotinylated anti-hCG antibodies for affinity capture. In a laboratory setting, the performance was demonstrated, and a 14-fold increase of target binding compared to binding without bmAb was achieved. The recovery of hCG captured with bmAb-treated samplers was determined to be 33% and comparable to previously described affinity capture approaches. Application of the smart affinity samplers to human serum containing hCG showed an R2 of 0.98 (200\u20131000\u00a0pg\u00a0mL\u22121), precision of\u2009\u2264\u20099.1% RSD, and estimated limit of detection of 65\u00a0pg\u00a0mL\u22121. Although further optimization and validation are necessary prior to application to real samples in clinical settings, the potential of the device for use in determination of low abundant biomarkers in complex samples has been demonstrated.The modification of an easily available resource like paper to circumvent expensive or intensive sample pretreatment could be the answer to sample analysis in resource-poor regions. Therefore, a novel on-paper device combining sample collection with affinity sample pretreatment is introduced here. Universal smart affinity samplers are produced by a simple KIOThe online version contains supplementary material available at 10.1007/s00216-022-04161-w. That is why recently, sampling cards for blood and other matrices have been facilitated as smart samplers in several ways. These smart samplers allow the pretreatment to start already upon blood collection, thus saving valuable time and material in the preparation of the sample later on. Integration of sample pretreatment steps as, for example, tryptic digestion for bottom-up protein analysis ) was used as internal standard (IS) and purchased from Sigma-Aldrich. The AQUA\u2122 peptide was reduced and alkylated in-house according to the protocol from Lund et al. ). For the \u03b2T5 peptide, the following two transitions were monitored: m/z\u00a0964.2\u2009\u2192\u20091036.3 and m/z\u00a0964.2\u2009\u2192\u20091317.8. Similarly, for the internal standard peptide, the transitions m/z\u00a0969.2\u2009\u2192\u20091046.3 and m/z\u00a0969.2\u2009\u2192\u20091327.8 were monitored. Data acquisition and processing were carried out using Thermo Fisher Scientific Xcalibur\u2122 software.Chromatographic separation was carried out with a Dionex  Ultimate 3000 RSCL Nano system equipped with an Acclaim PepMap\u2122 100 C18  on the fluorescence was investigated , paper which was oxidized and immobilized with streptavidin, and paper that was oxidized and treated with BSA. For the latter two, a volume of only 0.5\u00a0\u00b5L of protein (SA or BSA) was applied in order to leave a distinct spot on the discs. Figure\u00a0\u22121 bmAb. The modified paper was then incubated with 5\u00a0\u00b5L hCG  was applied and incubation with the target analyte performed using 5\u00a0\u00b5L of 100\u00a0ng\u00a0mL\u22121 hCG in 50\u00a0mM ABC. Firstly, it was tested if hCG could be captured with oxidized paper discs immobilized with BSA (5\u00a0\u00b5L of 2% (w/v) in 1\u00a0mM HCl) instead of SA . These conditions were compared to the proof-of-principle procedure  SA and subsequently treated with 250\u00a0\u00b5L of 2.3\u00a0mg L\u22121 in 50\u00a0mM ABC with 5% (w/v) BSA). Theoretically, all three volumes of bmAb contained sufficient amounts of antibody to capture the entire amount of hCG added  BSA  bmAb.In order to evaluate whether the amount of bmAb was limiting the hCG binding and to ensure high recoveries of the target analyte, paper discs were treated with varying volumes of bmAb  while the hCG concentration remained constant  BSA, respectively; Fig.\u00a0\u22121 in 50\u00a0mM ABC, 5% (w/v) BSA) on SA paper discs not treated with bmAb, but otherwise following the standard procedure . Figure\u00a0\u22121) of hCG. The correlation value (R2) for the polynomial regression equals 0.98. The relative standard deviation (RSD) of each concentration was 7.0%, 7.0%, 9.1%, and 8.7% for 200\u00a0pg\u00a0mL\u22121, 500\u00a0pg\u00a0mL\u22121, 750\u00a0pg\u00a0mL\u22121, and 1000\u00a0pg\u00a0mL\u22121, respectively. The calibration curve in Fig.\u00a0\u22121) for the used amount of bmAb . This could be a possible explanation for the modest correlation value. However, it is shown that different hCG concentrations can be extracted from human serum when using the tailor-made affinity smart samplers. The average signal to noise ratio (S/N) for the 200\u00a0pg\u00a0mL\u22121 samples (n\u2009=\u20093) is 6.1. Assuming a S/N\u2009=\u20093 as definition of the limit of detection (LOD), then an estimate of 65\u00a0pg\u00a0mL\u22121 can be determined for SA-bmAb paper discs Below is the link to the electronic supplementary material."}
+{"text": "Malnutrition among adolescents is a persistent problem with a profound impact on different dimensions of health. The objective of this analysis is to assess the burden of malnutrition  and their associated socio-demographic factors among Indian adolescents (10\u201319 years) from the Comprehensive National Nutritional Survey (CNNS 2016-18) data.We used Individual-level data of 35,831 adolescents from the CNNS conducted in 2016\u201318 for this analysis. CNNS collected data on the nutritional status of adolescents along with socio-demographic variables from all states of India. Burden of stunting , thinness , overweight (BAZ > 1 SD) and obesity (BAZ > 2 SD) were estimated for the entire country and individual states. A multivariable logistic regression analysis was used to assess the socio-demographic factors associated with stunting, thinness, and overweight.CNNS collected data from 35,831 adolescents, of which 31,941 with BAZ scores, and 32,045 with HAZ scores were included in the final analysis. The burden of stunting and thinness among Indian adolescents was 27.4%  and 24.4% , respectively. The burden of overweight and obesity was 4.8%  and 1.1% , respectively. Adolescents in the age group of 15\u201319 years  compared to 10\u201314 years, females  compared to males, were at increased odds of getting stunted. Adolescents from lowest wealth index families  were at increased odds of thinness compared to peers of higher wealth index families. Adolescents of 10\u201314 years  compared to 15\u201319 years, urban residents  compared to rural residents, were at increased odds of overweight.Indian adolescents face the double burden of malnutrition that is undernutrition (stunting and thinness) alongside overnutrition (overweight and obesity) that are linked with socio-demographic factors. The National Nutritional Programs (POSHAN Abhiyan) should prioritize high-risk groups specifically older age group (15\u201319 years), females, and low wealth Index quintile families identified in this analysis. Adolescence (aged 10\u201319 years) is an essential phase in a child's growth with rapid physical, psychological, and cognitive development . The 1.22) among adolescent boys and girls of 15\u201319 years age was 45 and 42%, respectively (2) among adolescent girls of age 10\u201314 as 77% and that of 15\u201318 years age group as 45% , the National Nutrition Monitoring Bureau (NNMB), the District Level Health Surveys (DLHS), the Annual Health Surveys (AHS), and the Rapid Survey of Children (RSoC). National Family Health Survey-4 reported the nutritional status of the 15\u201319 years age group using body mass index (BMI) as an indicator but not BMI-for-age z-scores (BAZ) . As per ectively . The RapIn India, there are no nationally representative data on nutritional status of adolescents (10\u201319 years of age) that use comprehensive and appropriate anthropometric indicators. For adolescents, using the standard BMI-for age (BAZ) scores by World Health Organization (WHO) . The ComThe CNNS was conducted from February 2016 to October 2018 by the Ministry of Health and Family Welfare, Government of India, collaborating with United Nations Children's Fund (UNICEF) and the Population Council. The methodological details of the survey are available in detail in the CNNS report . BrieflyThe CNNS used a household and an individual questionnaire for all participants using the Computer-Assisted Personal Interviewing (CAPI) method in the principal languages of the state and/or English after obtaining informed consent from the caregiver and assent from the adolescent . For eveIndividual-level data for 10\u201319 years collected from the CNNS was accessed for this secondary analysis. The age group 10\u201319 years included children who had completed 120 months till 228 months. The age group was classified as early adolescents (120\u2013179 months) and late adolescents (180\u2013228 months). World Health Organization (WHO) Anthro Plus software was used to derive the BMI-for-age z-score (BAZ) and Height-for-age z-score (HAZ) . WHO stap-value < 0.25 on univariate analysis, variables of contextual and clinical importance were included in the final model  was seen in 8,877 Indian adolescents with a burden of 27.4% . The burden of thinness was 24.4%  as seen in 7,896 adolescents. The double burden of stunting and thinness in the same individual was seen in 8.6%  adolescents, while the prevalence of co-existing stunting and overweight was seen in 0.8% . The burden of overweight and obesity among Indian adolescents was 4.8%  and 1.1% , respectively .A higher prevalence of stunting was seen in north-eastern states\u2014Assam , Meghalaya , Nagaland  and Jharkhand  compared to the national average . Seven states have thinness more than the national average , two of which are from the Southern region\u2014Telangana , and Karnataka . Overweight  was seen to be more prevalent in Tamil Nadu , Delhi , Goa , Kerala , Sikkim , Arunachal Pradesh , and Tripura . States with a prevalence of overweight lower than the national average include Madhya Pradesh , Uttar Pradesh , Bihar , and Jharkhand . Obesity was found to be higher than the national average  in states like Goa , Delhi , Tamilnadu , Punjab , and Manipur  .Adolescents in the age group of 15\u201319 years , female gender , Muslim religion , scheduled caste  and scheduled tribes  were at increased odds of stunted growth compared to their counterparts . SimilarAdolescents from other backward classes , lowest wealth index families , western India  were at increased odds of thinness compared to their counterparts at a statistically significant level. The age group of 15\u201319 years  compared to 10\u201314 years age group, Female gender  compared to males were at decreased odds of thinness. Adolescents from eastern India  and north-eastern India  were at decreased odds of thinness compared to adolescents from northern India .Adolescents in the age group of 10\u201314 years , residence in urban areas , others  category of social class , mothers' education more than 12 years  highest wealth index families  were at increased odds of overweight compared to their counterparts. Similarly, adolescents residing in north-eastern India , eastern India , and southern India  were at increased odds of overweight compared to adolescents residing in north India .Our analysis has shown that among Indian adolescents, 27.4% were stunted, 24.4% were thin, 4.8 and 1.1% were overweight and obese, respectively. While stunting was higher in girls and the late adolescent age group (15\u201319 years), thinness was higher in boys and early adolescence (10\u201314 years). Overweight was marginally higher in boys than girls and early adolescence compared to late adolescence. The socio-demographic factors of adolescent malnutrition  in India from our analysis include gender, wealth index, social class, religion, and maternal education.In the present study, the prevalence of stunting was higher in late adolescence (30% in late adolescence vs. 25.6% in early adolescence). According to the Global School-Based survey conducted in 57 Low-Middle-Income countries (LMICs) on children aged 12\u201315 years, 14.6% of adolescents in India were stunted . Our estThe burden of stunting among girls was higher than boys, corroborating with previous estimates by Bhargava et al. on NFHS data . Indian In the current study, thinness was higher in boys (29.9%) than girls (18.9%). In NFHS-3, the reported prevalence of thinness was 22.3% (boys) and 9.9% (girls), which subsequently decreased to 16.5 and 9% in NFHS-4 in boys and girls, respectively. In many neighboring countries, thinness among boys is higher than girls, viz Indonesia (11 vs. 5%), Bangladesh 19.6 vs. 15.4%), and Nepal (37.8 vs. 26.2%) \u201325. This.4%, and In the present study, the prevalence of thinness was 27.6% in early adolescence and 20.9% in late adolescence, which was similar to findings by Kumar P et al. observed in adolescents of Bihar and Uttar Pradesh . This coOur analysis has shown a marginally higher prevalence of overweight (BAZ > 1SD) among boys (4.9%) compared to girls (4.7%), with no statistically significant difference. The prevalence rates of overweight reported in NFHS-4 were slightly higher than CNNS data, and there was a marginal increase between NFHS-3 (3.0% in boys and 4.3% in girls) and NFHS-4 (6.2% in boys and 4.9% in girls) . The burThe literature about coexisting stunting with thinness or obesity is sparsely available amongst adolescents in India. The UDAYA study conducted in Uttar Pradesh and Bihar states reports that the co-existence of stunting with thinness was seen in 11 percent of boys and 8 percent of early adolescent girls .The strength of the present analysis is the use of CNNS data, a large, quality-controlled, recent, and representative national sample. We have used BAZ scores, an appropriate indicator for reporting nutritional status among adolescents unlike BMI which is appropriate for adults and used in NFHS surveys. We have not analyzed and triangulated other forms of malnutrition, including the micronutrient deficiencies and co-existence of different forms of malnutrition which is a limitation of this analysis. Another limitation of this analysis is that the socio-demographic predictors of malnutrition among adolescents needs to be triangulated with clinical characteristics and dietary intakes. Since adolescent malnutrition especially stunting and thinness, are high, correcting these in this age group could be the game-changer in breaking the intergenerational cycle of malnutrition. With the identified burden of overweight and obesity, interventions focusing on the early lifestyle changes in this age group must be taken up through already existing national health programs. Identification of state-specific determinants of malnutrition coupled with effective interventional strategies can benefit in a targeted reduction in the burden of malnutrition. Growth monitoring and nutritional surveillance like quarterly anthropometric assessment can contribute to the real-time correction of malnutrition among adolescents. School health programs and POSHAN Abhiyan (Prime Minister's Overarching Scheme for Holistic Nourishment) must be linked with the periodic anthropometric assessment of this age group.Among Indian adolescents, there is the burden of thinness, stunting, and overweight, indicating the double burden of malnutrition. Socio-demographic factors of malnutrition identified in this analysis include gender , religion , social class (Scheduled castes and scheduled tribes), not attending school, no/less maternal education, poor wealth index families. These factors can prioritize interventional strategies at the district and state levels to eliminate malnutrition. Adolescence presents a final opportunity for correction of malnutrition before the child enters adulthood. Nutritional surveillance in this age group coupled with targeted nutritional programs for undernutrition and lifestyle changes for overweight and obesity will correct malnutrition in this crucial age group.The original contributions presented in the study are included in the article/Ethical approvals for the CNNS were obtained from the Population Council's Institutional Review Board, New York, and the Institutional Ethical Committee of Post Graduate Institute of Medical Education and Research, Chandigarh, India (PGI/IEC/2015/1508).RPa, MM, and SC conceptualized the article, interpreted the results, and contributed to manuscript preparation. UT and VK was involved in manuscript writing. NR was involved in conceptualizing the article, performing statistical analysis, interpreting the results, and writing the manuscript. CK performed the statistical analysis. JG, RPu, and RH critically reviewed the manuscript and had the final decision in processing the manuscript. 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.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": "Correction to: Virchows Archiv10.1007/s00428-022-03444-yIn the original published version of the above article contained incorrect Table legend. The Table The original article has been corrected."}
+{"text": "The original version of this article unfortunately contained a mistake. Two rows are missing in the Table\u00a02.The corrected Table\u00a0The original article has been corrected."}
+{"text": "The original article has been corrected."}
+{"text": "The original version of this article unfortunately contained errors in Table\u00a02 in the rows 1-year and 7-year-change of the section White meat, mean (SD) (g/day).The corrected Table"}
+{"text": "The original version of this article unfortunately contained a mistake. Figures\u00a0The corrected captions with figures are given in the following page.The original article has been corrected."}
+{"text": "The original article has been corrected."}
+{"text": "For patients with large tumors palliative radiotherapy often is the only local treatment option. To prevent toxicity the administered doses are low. Dose escalation to the tumor could be an option to better smyptom control and prolong local control rates. In this prospective study we used a\u00a0very pragmatic approach with a simultaneously integrated boost (SIB) to an almost geometrically defined tumor core to achieve this. The primary endpoint was to demonstrate feasibility.Patients with solid tumors >\u202f4\u202fcm in diameter of different histologies were eligible in this single arm, prospective, multi-institutional clinical feasibility trial with two treatment concepts: 5\u202f\u00d7\u20095\u202fGy with an integrated boost to the tumor core of 5\u202f\u00d7\u200910\u202fGy or 10\u202f\u00d7\u20093\u202fGy with a\u00a0boost of 10\u202f\u00d7\u20096\u202fGy. The objective of dose escalation in this study was to deliver a\u00a0minimum dose of 150% of the prescribed dose to the gross tumor volume (GTV) tumor core and to reach a\u00a0maximum of at least 200% in the tumor core.3 (range 49.4\u20131179.6\u202fcm3). The corresponding core volumes were 72.85\u202fcm3 on average (range 4.21\u2013338.3\u202fcm3). Dose escalation to the tumor core with mean doses of 167.7\u2013207.7% related to the nonboost prescribed isodose led to PTV mean doses of 120.5\u2013163.3%. Treatment delivery and short-term follow-up was successful in all patients.In all, 21\u00a0patients at three study sites were recruited between January 2019 and November 2020 and were almost evenly spread (9 to 12) between the two concepts. The treated planning target volumes (PTV) averaged 389.42\u202fcmPalliative radiotherapy with SIB to the tumor core seems to be a\u00a0feasible and well-tolerated treatment concept for large tumors. The applied high doses of up to 50\u202fGy in 5\u00a0fractions (or 60\u202fGy in 10\u00a0fractions) did not cause unexpected side effects in the 42\u00a0day follow-up period. Further research is needed for more information on efficacy and long-term toxicity.The online version of this article (10.1007/s00066-022-01976-5) contains supplementary material, which is available to authorized users. Patients in a\u00a0palliative setting are usually treated with the intent of rapid symptom control and low side effects. Therefore, radiotherapy is often the only suitable local treatment option. Typical doses and fractionation schedules are 5\u00a0fractions of 4\u20135\u202fGy or 10\u00a0fractions of 3\u202fGy, corresponding to a\u00a0dose equivalent of 40\u202fGy or less in conventional fractionation . HoweverPalliative patients with large tumors often show symptoms like shortness of breath or pain. These patients are not good candidates for high-precision techniques which require either strict immobilization or are time-consuming; thus, the inclusion of a\u00a0SIB for dose escalation should require standard techniques with robust plans. Whether optimization and administration of such plans in a\u00a0wide spectrum of clinical situations is possible requires further investigation of optimal concepts.We report the results of a\u00a0prospective feasibility study in which robust volumetric modulated arc therapy (VMAT) plans in standard settings were used to achieve the announced dose escalation in the palliative treatment of patients with large (>\u202f4\u202fcm) tumors in different clinical situations.The tumor core boost study was a\u00a0single arm, prospective, multi-institutional clinical feasibility trial with a\u00a0planned sample size of 25\u00a0patients treated with palliative intent for tumors (primary or metastases) larger than 4\u202fcm in diameter. Treatment consisted of palliative radiotherapy with 30\u202fGy in 10\u00a0fractions or 25\u202fGy in 5\u00a0fractions. Which regimen to use was the investigator\u2019s choice and depended on the general assessment of the patient\u2019s overall clinical situation. The center of the target volume (the \u201ctumor core\u201d) received an additional boost dose and the dose to the core was escalated by 100% as compared to the standard prescription dose; thus, the integrated boost volume received a\u00a0total dose of 60\u202fGy in 10\u00a0fractions or 50\u202fGy in 5\u00a0fractions. The aim of the study was to demonstrate that dose escalation with this SIB concept is feasible in a\u00a0non-SBRT setting without high rates of side effects because of a\u00a0significantly increased dose to surrounding organs at risk.Strahlenschutzverordnung or \u00a728a R\u00f6ntgenverordnung was needed.The study complied with the Declaration of Helsinki, and the protocol was approved by ethics committee of the Christian-Albrechts-University Kiel (D535/18) and listed on German Clinical Trials Register (DRKS00015763). As decided by DEGRO Expert Panel (inquiry 116), no approval analog \u00a723 Patients were eligible if they had a\u00a0large solid tumor (\u2265\u202f4\u202fcm in diameter), almost irrespective of histology , and were scheduled for palliative radiotherapy to this lesion. Further inclusion criteria were age above 18\u00a0years and informed consent. Moreover, a\u00a0tumor core of at least 2\u202fcm in spherical diameter with a\u00a01\u202fcm margin in all directions inside the GTV was to be definable.Patients were excluded if critical normal tissue structures (CNTS) were inside the GTV  to avoid major complications due to overdosage to such critical volumes. Moreover, patients were excluded in case of pregnancy, high grade renal failure (glomerular filtration rate <\u202f30\u202fml/min), and legal incapacity.The GTV included the whole macroscopic tumor as defined in the planning computed tomography (CT). Additional information from other imaging modalities like magnetic resonance imaging (MRI) or positron emission tomography CT (PET/CT) was also used, if available. The SIB volume, called GTV tumor core, was defined as a\u00a0rotund volume measuring at least 20\u202fmm in diameter and lying completely inside the GTV with an additional inner margin of at least 10\u202fmm in all directions; this inner margin was chosen to safely avoid overdosage outside the GTV even in case of relatively larger motion during treatment. Further information on tumor biology  uptake in PET scanning or visible necrosis) was not considered for target volume definition in this study. Other macroscopic manifestations of tumor-like nodal metastases measuring <\u202f4\u202fcm could be included in the clinical target volume (CTV), but not in the high-dose GTV tumor core. The additional margin for the planning target volume (PTV) depended on the internal standards of the participating institutions.mean) and max dose (Dmax) were documented and compared with the doses of an additional standard radiotherapy (RT) plan with homogenous dose distribution without SIB. To simplify this plan comparison of the different OARs with the different fractionation regimes, we defined new patient-specific parameters: the maximum increase of any OAR\u2019s Dmean and Dmax calculated in Gy and %.The listed OARs were to be contoured if they were within 5\u202fcm in cranial-to-caudal orientation of the GTV: brain, pituitary gland, optic nerves, optic chiasm, eyes, lenses, parotid glands, spinal cord, lungs, heart, esophagus, liver, kidneys, bladder, rectum, bowel bag. For every OAR of every patient, mean dose , and Dmax to the PTV and GTV tumor core were documented. In addition, a\u00a0standard plan with homogeneous dose distribution must be calculated for all patients; this could be, depending on the clinical situation, an appropriate VMAT, intensity modulated RT (IMRT) or a\u00a03-dimensional conformal RT plan (3D-CRT).The prescribed dose was the dose on the PTV-surrounding isodose. The objective of dose escalation in this study was to deliver a\u00a0minimum dose of 150% of the prescribed dose to the GTV tumor core and to reach a\u00a0maximum of at least 200% in the tumor core. DRadiation was to be applied with daily image guidance via cone beam CT (CBCT) or stereoscopic X\u2011rays, if possible. Due to the palliative setting, exceptions were allowed for patients with special impairments. Overall treatment time (from the first to the last treatment fraction) should not exceed 3\u00a0weeks for patients receiving 10\u00a0fractions and 2\u00a0weeks for patients with 5\u00a0fractions.Patients were clinically investigated at baseline before start of radiation treatment (d0), weekly during radiotherapy at treatment days d1, d7, d14, and at the day of last treatment (RT+0d). Early follow-up visits at day RT+7d and day RT+14d were done via phone; at day RT+42d, all patients were clinically investigated. Because of the estimated short overall survival of the patients and the short follow-up period with focus on feasibility, an investigation of tumor response (RECIST criteria) was not performed.Optimization of treatment plans with SIB with protocol-defined maximum doses to the tumor core andSuccessful administration of the planned RT dose in at least 80% of patients.The primary endpoint of the study was feasibility of a\u00a0SIB with 100% dose escalation to the tumor core in palliative RT. Feasibility criteria were defined asSecondary endpoints were toxicity and side-effects of radiotherapy with SIB. Side effects and SAEs (serious adverse event) were documented according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v4.03 . It was Recruitment of patients was lower than expected. We included a\u00a0total of 21\u00a0patients from January 2019 until November 2020 until the study was closed at the end of 2020. All patients gave their informed consent. The patients were almost evenly spread over both fractionation regimes. Treated locations differed a\u00a0lot. In 7 of 21\u00a0cases, tumors were located in the head and neck region, in 5\u00a0cases in the thoracic region , and in 9\u00a0cases the tumors were located in the abdomen or pelvis. In all, 7\u00a0primary tumors, 5\u00a0nodal metastases, and 10\u00a0distant metastases were treated. Non-small cell lung cancer (NSCLC) was the most common histology . Other histologies included endometrial cancer, head and neck squamous cell carcinoma (HNSCC), rectal cancer, melanoma, breast cancer, fallopian tube cancer, sarcoma, and squamous cell carcinoma of unknown primary (SSC-CUP). More patient characteristics are listed in Table\u00a0The prescribed treatment was successfully delivered in all patients. More than 95% of the patients (20 of 21) completed treatment with SIB to the tumor core in the scheduled treatment time . Treatment of one patient in 5\u2011fractionation regime was completed within 17\u00a0days. No patient\u2019s treatment was abandoned.Rates of acute toxicities at any point of the monitored period were 100% grade \u2265\u202f1, 33.3% grade \u2265\u202f2, and 9.5% grade \u2265\u202f3, respectively. No grade 4 toxicities were observed. Corrected for toxicities documented at d0 or d1 , the estimated rate of acute toxicities grade \u2265\u202f2 was 28.6%. Considering only those cases where the toxicities lasted until the last follow-up time-point (RT+42d), the rates were 61.1% for toxicities of grade \u2265\u202f1 and 5.6% grade \u2265\u202f2 (Table\u00a0Six adverse events (AE) and three severe adverse events (SAE) were reported. Two SAEs resulted from unplanned hospitalization due to a\u00a0systemic tumor progression and both patients died in the hospital; there was no evidence for radiation-related side effects. The third SAE was related to death of a\u00a0patient during palliative care in a\u00a0hospice. Before death, 2\u00a0of the 3\u00a0patients showed maximum documented acute toxicity grades of\u00a01, while 1\u00a0patient showed grade\u00a02. The reported AEs were moderate pneumonia , moderate local inflammation in the irradiated region , pancytopenia after chemotherapy requiring blood transfusion and granulocyte colony stimulating factor (G\u2011CSF), serious hemorrhage of a\u00a0tumor before start of radiation treatment , moderate colitis after immunotherapy (treated with prednisolone), and repeated cardiac decompensations caused by known heart failure with delay of radiation treatment.The postulated feasibility requirements, namely the calculation of robust treatment plans with sparing of OARs and successful delivery of the prescribed treatment in more than 80% of the patients, were both met (treatment adherence 100%). With regard to the secondary endpoints, the appearance of toxicities greater than grade\u00a02 on CTCAE scale was 28.6% and thus higher than the expected rate of 20%. However, the vast majority of the observed toxicity derived not from radiotherapy treatment but was rather caused by tumor-specific complications or related to other cancer-specific treatment, mainly chemotherapy.The objectives of dose escalation were a\u00a0minimum dose to GTV tumor core of 150%  and a\u00a0maximum dose of at least 200% of the prescript PTV enclosing isodose. These study objectives were reached in more than 90% of the patients. In two cases, minor protocol deviations with a\u00a0slight underdosage of GTV tumor core were detected.In the main testing center  VMAT plans were generated with the treatment planning system Eclipse\u00a0v.13.7.14  and the associated AAA algorithm for a\u00a0TrueBeam STx linear accelerator (LINAC). VMAT techniques were used in all patients. In comparison with other modern technologies available in radiotherapy , VMAT is known to be more monitor unit (MU) efficient \u201319. Due At least half coplanar double arcs were used for all these plans. While using additional \u2018shell\u2019 structures, it was possible to modulate the dose gradient within the target volume. The aim was a\u00a0preferably uniform dose fall from the integrated boost area in the core of the tumor to the target volume enclosing isodose. The number of shells used was based on the size and shape of the target volume.Normalization was performed typically on the maximum dose value, which was 50\u202fGy or 60\u202fGy, respectively, at the target maximum . To achieve a\u00a0higher mean dose in the tumor core, it was also possible to normalize to the tumor core target mean . For the different normalization methods, the variability of several dose parameters  were then consecutively analyzed.3 (range 49.4\u20131179.6\u202fcm3). This conformed to a mean idealized spherical diameter of 9.06\u202fcm. The corresponding core volumes averaged 72.85\u202fcm3 . The relation of core volume and PTV for all patients is shown in Fig.\u00a0The treated PTVs had a\u00a0mean of 389.42\u202fcmMaximum doses to GTV tumor core and PTV were in all cases identical . At the main test center Kiel and in study site\u00a0b , plans show a\u00a0switch from maximum doses of 200% of the prescribed PTV dose to mean doses of exactly 200%. This resulted from the above-mentioned differences in plan normalization and led to higher maximum doses. In study site\u00a0c , a\u00a0third approach was used with significantly higher doses than at the other sites. The participating physicists focused on dose escalation as high as possible. First, they optimized the VMAT plans for around 200% dose maximum. Second, the resulting OAR doses were used and increased to obtain a\u00a0higher dose maximum inside the GTV tumor core, while maintaining the prescript PTV surrounding isodose level. Treatment planning was performed with Monaco 5.10 . The different planning approaches are illustrated in Fig.\u00a0The benefit of escalated PTV doses had one drawback\u2014a\u00a0significant increase of doses delivered to surrounding OARs. Altogether dose statistics of 136 delineated OARs were measured. Because the treated sites were located throughout the human body, not all to-be-documented OARs were delineated in every treatment plan. The spinal cord was the most common (delineated in 18 of 21\u00a0patients), optic chiasm the least (1 of 21). Doses to OARs in the tumor core boost plan were compared to doses in standard RT plan (without core boost).As an example, patient 21 (Tab.\u00a0A1 supplement) with a\u00a0PTV Dmax of 100.5\u202fGy was selected. Dmax to OAR spinal cord was 33.4\u202fGy in the core boost treatment plan and 11.1\u202fGy in the standard plan (both 10\u00a0fractions). This was an increase of 22.3\u202fGy or 200.9%. For another OAR (bowel bag) Dmax was 46.1\u202fGy (core boost), and 24.6\u202fGy . This was an increase of 21.5\u202fGy or 87.4%. Both parameters were higher for OAR spinal cord than for bowel bag and higher than for any other OAR of this patient. Consequently, 22.3\u202fGy and 200.9% were recorded for patient\u00a021. Results of all patients are depicted in Fig.\u00a0To our knowledge, an integrated boost to partial parts of large GTVs with the objective of radical dose escalation has not yet been investigated in prospective clinical trials although some case reports have described this approach , 22. TheThe primary endpoint of this feasibility study, a\u00a0combination of technical planning criteria and treatment adherence, was met. Creation of treatment plans according to the protocol was possible in all patients and treatment adherence was excellent. Acute toxicity grade \u2265\u202f3 was 28.6% and therefore slightly higher than the estimated 20% but none of the treated patients experienced radiotherapy-related severe toxicity; all severe adverse events were related to the underlying disease and were caused by tumor progression. Thus, this concept of inhomogeneous dose prescription to the GTV with a\u00a0simultaneous integrated boost to the tumor core was feasible and well tolerated and might offer a\u00a0tool for dose escalation in palliative treatment of large tumors.The study has on the other hand disclosed a\u00a0variety of difficulties with this approach. Although the predefined protocol-specific dose-escalation concept could be adopted in all patients, plan statistics differed a\u00a0lot. We achieved ablative radiation doses in the center of the GTV with significant increase in PTV mean dose (20\u201363%), even though sizes of the boost volumes were only 6\u201331% of the whole PTVs. As expected, relatively larger core volumes led to a\u00a0higher increase of PTV mean dose. Due to the fact that the inner margin around the core boost volume was set to 1\u202fcm for safety reasons , the boost volume in small tumors was relatively smaller compared to larger ones. To overcome this problem, smaller safety margins would be required for smaller tumors to increase the core volume to the relatively same size as in larger tumors. For example, in a\u00a0spherical tumor with 5\u202fcm in diameter, the core boost volume makes up only 22% of the whole GTV in case of a\u00a0safety margin of 1\u202fcm around the core. An increase of the core volume to more than half of the GTV would require reducing the safety margin to about 4\u202fmm. Smaller margins like this require more precise image guidance (as used in SBRT) than were specified in this study .mean) might have no clinical effect in palliative situation and is consequently tolerable. On the other hand, one must be very careful with high absolute dose increases such as the outliers in Fig.\u00a0max in 10\u00a0daily fractions compared to a\u00a0well-established treatment regime (10\u202f\u00d7\u20093\u202fGy) could lead to unpredictable toxicity. These extreme outliers were a\u00a0consequence of the dose-maximizing approach of study center\u00a0c. Another crucial impact to OAR statistics was the location of the treated tumor. Chan et\u00a0al. had shown that dose escalation to lung tumors with an inhomogeneous dose distribution has no significant impact on the mean dose to the lung [Finally, the individual physical treatment planning also influences plan quality and the level of dose escalation a\u00a0lot. The used and established techniques of the different planning systems  allow complex dose distributions. Each of the three planning systems used in this study had its advantages and the planning physicists had different ideas about how to deal with the study requirements and we did no central quality assessment of the different treatment plans. Study sites were responsible for their approaches. The use of additional shell structures in study site\u00a0a might offer more influence in the arrangement of dose hotspots than the stricter dose-maximizing approach in study site\u00a0c Fig.\u00a0. The keythe lung . SubgrouFor more precise guidelines for treatment of large tumors, further studies are needed. To overcome the shortcomings of this study , an investigation with a\u00a0much more homogenous study cohort  or different set-ups for different tumor sizes and locations is needed. In addition, more precise guidelines for GTV/PTV ratio, dose coverage, and dose gradients should be included. Finally, to validate clinical benefits of the concept different endpoints, more cases and consequently more participating institutes are required. Therefore, an additional multicenter study to implement the SIB concept in the treatment of large tumors is under development.Radical dose escalation with more than 50\u202fGy in 5\u00a0fractions or 60\u202fGy in 10\u00a0fractions to the tumor core was feasible in the small subgroup of palliative patients with large tumors. The delivery of the demonstrated SIB technique remains safe, and with use of large safety margins it is simple to implement in daily routine with standard treatment settings. Efficacy needs to be investigated in an additional trial.Table A1 in the supplementary material shows detailed characteristics of GTV tumor core and PTV for all patients treated with tumor core boost. The patient count is not chronological, instead it is sorted by treatment groups  and study sites . Deviations to protocol are marked fat."}
+{"text": "N = 301, the Leipzig Study for Mind-Body-Emotion Interactions, N = 220), to replicate published findings and expand them by investigating diffusivity in the LC\u2019s ascending noradrenergic bundle. In younger adults, LC fractional anisotropy was significantly lower, compared to older adults. However, in the LC\u2019s ascending noradrenergic bundle, we observed significantly higher fractional anisotropy in younger adults, relative to older adults. These findings indicate that diffusivity in the LC versus the ascending noradrenergic bundle are both susceptible to structural changes in aging that have opposing effects on fractional anisotropy.The noradrenergic locus coeruleus (LC) is a small brainstem nucleus that promotes arousal and attention. Recent studies have examined the microstructural properties of the LC using diffusion-weighted magnetic resonance imaging and found unexpected age-related differences in fractional anisotropy - a measure of white matter integrity. Here, we used two datasets (Berlin Aging Study-II,  Fractional anisotropy is widely used as a measure of structural integrity  and has a strong inverse correlation with mean or radial diffusivity . BASE-II participants signed written informed consent and received monetary compensation for participation. MRI acquisitions were approved by the ethics committees of the German Psychological Society , for which extensive details can be found elsewhere . The Dec2.1.(v6.3) eddy-current and motion correction, brain extraction tool, and resampling to isotropic resolution of 2mm3 (v3.1) to compute fractional anisotropy (FA) and eigenvalue images  Pipeline, we applied FSL\u2019s  of 2mm3 . We usede images . With die images  and the e images , which rMATLAB ver. R2019a). After accurate atlas registration to individual subject space was confirmed with visual inspection, mean and radial diffusion images were created from eigen value images in MATLAB with custom scripts. Atlases were then converted into a binary mask and multiplied by the diffusion image to provide fractional anisotropy, mean, and radial diffusivity values along the atlases, per voxel, within the native space. Diffusivity values were then averaged to provide one diffusivity value per participant within each ROI.Both fractional anisotropy and atlas images were registered into MNI152 linear, 1 mm brain space. Using ANTS nonlinear registration  the atlaSince the noradrenergic bundle overlaps with a portion of the LC atlas, we conducted an along-tract analysis of fractional anisotropy of the noradrenergic bundle. 50 equidistant points were imposed along the noradrenergic bundle as discussed elsewhere . Each po2.2.All statistical analyses were conducted using the R software  with tidt-tests were conducted for fractional anisotropy at each of the 50 equidistant points between younger and older adults. P values were false-discovery rate adjusted and surviving points of significant FA differences between age groups are plotted in To clarify the significant interactions of age and the 3-level ROI factors, we conducted two follow-up ANOVAs with the ROI factor reduced to 2 levels to separately contrast the control frontopontine tract with each of the other two ROIs  = 27.18, p < .001, F = 26.07, p < .001, F = 6.49, p = .003, F = 5.50, p = .008, Complete ANOVA tables for fractional anisotropy across datasets are displayed in ectively . We alsoF(299) = 26.16, p < .001, F(299) = 9.72, p = .002, F(216) = 24.34, p < .001, F(216) = 5.23, p = .023, Separate ANOVAs with the \u201cROI\u201d factor either contrasting the frontopontine tract and noradrenergic bundle fractional anisotropy or contrasting the frontopontine tract and LC fractional anisotropy both yielded significant Age X ROI effects  = 0.24, p < .001 and r(243) = 0.19, p < .001, respectively. However, only three percent of the variance was accounted for (R2adj = 0.03). We did not observe any significant relationship in the BASE-II young adult cohort. We were also not able to replicate these findings in the LEMON dataset.Finally, we observed significant positive correlations between LC fractional anisotropy and noradrenergic bundle fractional anisotropy within the BASE-II older adult cohort in the left and right hemispheres, 4.Unmyelinated neurons and numerous innervations to blood capillaries may expose the LC to toxins throughout aging . During Fractional anisotropy has been observed to correlate with white matter integrity, increasing until the age of about 35\u201340 and decreasing into late life or with disease . AdditioWhile fractional anisotropy tended to be higher in older than younger adults within the LC itself, older adults typically showed lower fractional anisotropy than younger adults along the noradrenergic bundle white-matter ascending tract, a typical age-related pattern in white matter . The lacIn the BASE-II and LEMON datasets, age differences in the noradrenergic bundle contrasted with a lack of age differences in the right and left control white-matter frontopontine tracts, suggesting that the age effects in the noradrenergic ascending tract reflect more than just a global change in white matter. Thus, together, these data indicate that diffusivity properties of the LC and its ascending noradrenergic tract are affected by aging in opposite ways. Our findings of age differences in fractional anisotropy in the LC and its ascending tracts extend a growing set of observations of age differences in the structure of the LC in aging .To date, most in vivo findings of LC structure have relied on LC-MRI sequences that show a cross-sectional increase in LC-neuromelanin sensitive contrast from young adulthood until around age 57, at which point it levels off or declines , potentiIn the BASE-II dataset, there were no significant correlations between LC-MRI contrast from those scans and diffusivity measures from the LC or noradrenergic bundle. This suggests that the diffusivity differences reflect different structural changes than the LC-sensitive scans. An important future research objective should be to examine the relationship between LC diffusivity measures and cognition, or markers of brain health, as has been done for LC-MRI contrast . One iniOur results raise the question of what properties of the LC are changing to lead its tissue to show higher fractional anisotropy with age. One possibility could be an increase in inflammation that restricts fluid flow, as animal research has demonstrated that increases in microglial density affect diffusivity, as measured using an orientation dispersion index . AnotherMean and radial diffusivity in the LC also showed some age differences . However, given the opposite findings in the ascending white matter tract, we were still able to extract meaningful signal.Crossing fibers may indicate opposite or unexpected relationships with diffusivity values that may be related to our unexpected findings . Despite5.in-vivo in the locus coeruleus versus noradrenergic bundle (In this study, we identified unique associations of LC diffusivity in the context of healthy adults across two different data sets. We consistently observed lower fractional anisotropy in the locus coeruleus of younger adults, compared to older adults but higher fractional anisotropy in the ascending noradrenergic bundle of younger adults, compared to older adults. Fractional anisotropy is a measurement of structural integrity, and these age findings add to a growing literature highlighting age-related differences involving the locus coeruleus. To our knowledge, this is the first study to compare diffusivity differences c bundle .1"}
+{"text": "What are the characteristics of meta-analyses published in oncology journals, and what are the study characteristics associated with favorable outcomes?This scoping review of 93 meta-analyses found that many meta-analyses report on a drug, include randomized studies, and often have an author funded by industry. Methodologic quality was no different between studies with study or author industry funding and those with independent research, but industry funding was associated with the tone of the authors\u2019 conclusion.The multiple factors associated with having a positive study conclusion suggest that future research should be performed to elucidate reasons for more favorable conclusions among studies with study or author industry funding. This scoping review examines meta-analyses published in oncology journals to identify the factors associated with having a positive study conclusion. Many meta-analyses have been conducted on a wide array of topics, and many of these have focused on treatment efficacy of drugs or bias in interventional studies on a specific topic.To examine the factors associated with having a positive study conclusion in meta-analyses in the field of oncology.All meta-analyses published between January 1, 2018, and December 31, 2021, on 5 oncology journal websites were identified and study characteristics, study results, and information on study authors were abstracted. The meta-analysis authors\u2019 conclusions were coded as positive, negative, or equivocal, and each article subject matter was coded as one that could affect profits and marketing of a company. Whether an association existed between study characteristics and authors\u2019 conclusions was also examined.Database searches resulted in 3947 potential articles, of which 93 meta-analyses were included in this study. Of the 21 studies with author funding from industry, 17 studies (81.0%) reported favorable conclusions. Of the 9 studies that received industry funding, 7 (77.8%) reported favorable conclusions, and of the 63 studies that did not have author or study funding from industry, 30 (47.6%) reported favorable conclusions. Studies that were funded through nonindustry sources and authors who had no relevant conflict of interest had the lowest percentage of positive conclusions and the highest percentage of negative and equivocal conclusions compared with studies with other sources of potential conflict of interest.In this cross-sectional study of meta-analyses published in oncology journals, multiple factors were associated with having a positive study conclusion, which suggests that future research should be performed to elucidate reasons for more favorable conclusions among studies with study or author industry funding. When meta-analyses are conducted, authors should be aware of potential biases. For example, bias can occur when the meta-analysis is authored by people who are experts in the topic area and who have authored studies that could be included in the review, thus resulting in a situation that promotes their work or because of findings that are in alignment with specialty-specific recommendations.1 Many meta-analyses have been conducted on a wide array of topics. In the medical specialty of oncology, many of these meta-analyses have focused on treatment efficacy of drugs or bias in interventional studies on a specific topic.10 To our knowledge, there have been no scoping analyses evaluating the general characteristics of meta-analyses in oncology journals and factors associated with bias. Therefore, we performed a scoping review to examine the factors associated with having a positive study conclusion in meta-analyses in the field of oncology.Preregistration of a meta-analysis protocol may help reduce bias occurring from post hoc decision-making and provide greater transparency in study methods, and the use of multiple databases can help to identify all available evidence rather than a possibly biased selection.STROBE) reporting guidelines.11We sought to systematically assemble a list of oncology meta-analyses by searching common oncology journal websites for all meta-analyses published between January 1, 2018, and December 31, 2021. In accordance with 45 CFR \u00a746.102(f), this scoping review was not submitted for University of California, San Francisco Institutional Review Board approval because it involved publicly available data and did not involve individual patient data. We followed the Strengthening the Reporting of Observational Studies in Epidemiology  among oncology journals that publish meta-analyses. We searched each journal\u2019s website by using the term meta-analysis in the search bar, allowing search results from all oncology journals affiliated with the parent journal, and limiting to the predefined dates. When there was the option, we also limited the search to research and review articles. For this review, we did not include studies that were systematic reviews only. Because we were assessing the general landscape of meta-analyses in oncology, we included all studies that had a meta-analysis design, as described by the author of the meta-analysis, and we had no further inclusion or exclusion criteria.We selected I2 for the primary outcome (or first primary outcome mentioned), heterogeneity categorization, whether the meta-analysis had been preregistered, whether an assessment of study quality or bias was performed, tool used for study quality or bias assessment, whether the data were dual reviewed, source of study funding, author conflict of interest, country of first author, and names of first and last authors. Outcome type was grouped into overall survival, tumor response , safety and adverse events, behavior, fertility, pain, risk, testing, or other. For years of included trials, if the meta-analysis included studies from the inception of PubMed or MEDLINE, we determined that this was from 1966. For heterogeneity categorization, we defined an I2 of less than 25% as low, 25% to 49% as low to moderate, 50% to 74% as moderate to high, and 75% or above as high. For each first and last author, we searched PubMed for the number of meta-analyses on which the individual was a coauthor. If we were unable to find the specific author, we searched Google Scholar for data on the number of coauthored meta-analyses. We only used Google Scholar data if there was a user profile with a verified affiliation. We then categorized the number of author publications into less than 10, 10 to 24, and greater than 24. However, for logistic regression, this was used as a continuous variable.For all studies, we abstracted data on journal, year of publication, intervention type, tumor type, outcome type, data source, years of included trials, number of studies, total number of participants, study design included, main outcome, whether the pooled results were calculated with random or fixed effects or both, rationale explained for type of modeling used (random vs fixed), We coded the overall tone of the authors\u2019 conclusion, based on the abstract and at the end of the discussion, as positive, negative, or equivocal. We coded a study as positive if the authors\u2019 conclusion promoted the intervention or exposure being studied. We coded a study as negative if the authors\u2019 conclusion was negative toward an intervention or exposure . We coded a study as equivocal if we were unable to determine whether the authors\u2019 conclusion supported or refuted an intervention or exposure. Examples of how studies were coded are provided in eTable 1 in We evaluated potential conflicts of interest in 2 ways. First, we categorized funding generally\u2014whether the study or author received any funding from industry or not. These variables were coded as industry, nonindustry, none, or not reported. Second, we coded each article subject matter as one that could affect profits and marketing of a company  or not . We then coded each study as having potential conflicts of interest for the author , study , or independent . If a study had industry funding, we assumed that at least 1 of the authors had industry funding to conduct the study. If there was no study funding, we then looked to see if the authors reported receipt of funding from industry. Information on conflicts of interest was only obtained from information reported in the published meta-analysis.We calculated numbers (percentages) or medians (IQRs) for characteristics overall and stratified results by type of meta-analysis. We used a Fisher exact test to examine the difference in the type of potential conflicts of interest and the number of studies with positive, negative, or equivocal conclusions. We used logistic regression to examine factors associated with a positive conclusion or not. For the regression model, we included variables on study  and author characteristics  and manually removed variables one at a time if their removal resulted in a lower Akaike information criterion value. We used a 2-sided \u03b1\u2009=\u2009.05 for statistical significance. The analyses were performed in R statistical software, version 4.2.1 .Journal of Clinical Oncology (31 [33.3%]) or JAMA Oncology (28 [30.1%]). The year with the greatest number of meta-analyses published was 2020 (28 [30.1%]). The median number of years included in a meta-analysis was 51 . The median number of included studies was 23 , and the median number of participants was 7584 .Our searches resulted in 3947 potential articles , of whicThe most common tumor type was multiple (36 [38.7%]), but the most common single tumor type was breast (12 [12.9%]). The most common intervention type was drug (35 [37.6%]), and the most common outcome type was overall survival, with or without another outcome (36 [38.7%]). The most common type of study design was randomized clinical trials only (39 [41.9%]), and the most common analysis type was a random-effects model (52 [55.9%]). The number of studies that provided justification for using a random- or fixed-effects model was 35 (37.6%). Most studies were study-level (aggregate) meta-analyses (65 [69.9%]).I2 >75%) in 27 studies (29.0%), moderate to high in 7 (7.5%), low to moderate in 7 (7.5%), low in 15 (16.1%), and not indicated in 31 (33.3%). Only 35 studies (37.6%) were preregistered before being conducting, and 45 (48.4%) evaluated the quality of included studies. Sixty-one studies (65.6%) had dual review of the abstracted data.Heterogeneity was high (The most common funding source was a nonindustry source (38 [40.9%]), but many did not disclose funding (33 [35.5%]). Sixty-three studies (67.7%) had authors who reported industry conflict of interest. The US was the most commonly represented geographic area (22 [23.7%]), but 54 studies (58.1%) had authors from multiple countries. Sixty-seven (72.0%) of the first authors and 51 (54.8%) of the last authors had published fewer than 10 previous meta-analyses, but it was also common for last authors to publish 25 or more meta-analyses (17 [18.3%]). The \u03ba statistic for study conclusion was 0.59, indicating moderate agreement. Of the 21 studies with author funding from industry, 17 studies (81.0%) reported favorable conclusions. Of the 9 studies that received industry funding, 7 (77.8%) reported favorable conclusions, and of the 63 studies that did not have author or study funding from industry, 30 (47.6%) reported favorable conclusions . StudiesIn the adjusted logistic analyses , having 12 exploring the role of financial conflict of interest on the conclusions of cost-effectiveness research.In this scoping review, we found that for meta-analyses in top oncology journals, drug interventions were the most studied intervention type, but biomarkers and patient characteristics were also commonly studied intervention types. This study also found that intervention-related industry payments to authors or study sponsorship were associated with a higher probability of a meta-analysis finding favorable results for an intervention, when compared with non\u2013industry-funded studies. These reports are consistent with results from a prior study13 Although it is often accepted that registration trials are funded by and written by employees of industry, meta-research, including meta-analyses and cost-effectiveness studies that inform health policy, may be more susceptible to industry influence (either by choice of topic or by methodologic choices within topics). Moreover, a prior Cochrane review14 found that conflicts of interest are common and associated with favorable conclusions for general medicine drug and device studies. A previous report12 found that meta-analyses on cost-effectiveness studies in oncology resulted in a 40-fold greater likelihood of concluding that a drug is cost-effective. However, potential conflicts of interest are often not reported in general or oncology-specific meta-analyses.15Potential conflicts of interest are well recognized for being a factor for biasing study results.16 rather than important clinical end points, which may be more likely to be positive. Another explanation is that in contrast with randomized clinical trials with prespecified statistical plans, cost-effectiveness research and meta-analyses have more analytic flexibility. Indeed, analytic choices can lead to widely divergent outcome estimates.17Although there are several possible explanations for why industry-funded studies are more often positive, one may be that industry is incentivized to focus on avenues of research that they know will be successful. Conversely, industry-funded studies may also rely more on surrogate end points19We found that many meta-analyses were traditional meta-analysis, with a study as the unit of observation, but one-third are other types. Network meta-analyses are becoming popular because they allow researchers to compare effectiveness of multiple interventions. Still, approximately 20% of meta-analyses used patient-level data. Besides the obvious deciding factor of whether the researcher has access to individual-level data, the advantages and disadvantages of each type of meta-analysis has led to debate about which one is better, although patient-level meta-analysis may be more preferred when determining efficacy.20 However, if there was no heterogeneity in study results, there would be little reason to conduct a meta-analysis because the answer would be known. An advantage of a patient-level meta-analysis is the ability to investigate heterogeneity due to individual-level effect modifiers and not only study-level factors or ecologic summaries of individual-level factors . To balance the expectation of equipoise and conducting appropriate tests that accommodate heterogeneity in study results, researchers have suggested alternative methods of statistical analysis21 and ways in which to present the data, such as using prediction intervals in addition to the traditional I2 value.22 Although we did not perform an exhaustive review of reporting quality, we found that, for the items we evaluated, the quality of reporting in studies was moderate at best, with most studies (65.6%) having dual review of study data, but fewer studies reporting the quality of included studies (48.4%), providing a rationale for type of model used (37.6%%), or preregistering their study (37.6%).We found that most meta-analyses had high heterogeneity in their pooled analysis, yet, although most studies used a random-effects model, there was no justification provided for using this method of analysis. Some authors have suggested that even with the use of a random-effects model, if there is a high degree of heterogeneity, the results should not be pooled.Our analysis was limited by several factors. First, we included a search of only 5 oncology journal websites over the course of 4 years, and our results may not be applicable to the meta-analysis literature at large. These journals may have a better editorial process than other journals often selecting higher quality manuscripts and the results are likely a better-case scenario because of better study methods. Second, our analysis was purely descriptive, and we did not examine factors associated with our findings. Third, we made several assumptions with the years of included studies , so our years of study inclusion may be overestimated. Fourth, we were not able to determine numbers of meta-analyses that some authors had published, so data are limited for those variables.In this scoping review of meta-analyses in oncology journals, we found that most studies either did not report on heterogeneity or had a high degree of heterogeneity, and most studies used a random-effects model but did not provide justification for its use. We also found that study funding or author conflicts of interest and having a first author with more published meta-analyses were associated with more favorable conclusions. The multiple factors associated with having a positive study conclusion suggest that future research should be performed to elucidate reasons for more favorable conclusions among studies with study or author industry funding."}
+{"text": "Increasing NAD+ is becoming a therapy for metabolic dysfunction; however, the impact of daily NAD+ fluctuations remains unknown. Here, we demonstrate that time-of-day determines the efficacy of NAD+ treatment for diet-induced metabolic disease in mice. Increasing NAD+ prior to the active phase in obese male mice ameliorated metabolic markers including body weight, glucose and insulin tolerance, hepatic inflammation and nutrient sensing pathways. However, raising NAD+ immediately before the rest phase selectively compromised these responses. Remarkably, timed NAD+ adjusted circadian oscillations of the liver clock until completely inverting its oscillatory phase when increased just before the rest period, resulting in misaligned molecular and behavioral rhythms in male and female mice. Our findings unveil the time-of-day dependence of NAD+-based therapies and support a chronobiology-based approach.The circadian clock is an endogenous time-tracking system that anticipates daily environmental changes. Misalignment of the clock can cause obesity, which is accompanied by reduced levels of the clock-controlled, rhythmic metabolite NAD The timing of NAD\u2009+\u2009supply determines its efficacy to treat metabolic disease. Here, the authors show that increasing NAD\u2009+\u2009at the early active phase maximizes weight loss and glucose regulation in mice. NAD\u2009+\u2009can displace the phase of the liver clock which can cause circadian misalignment. The main cause appears as combined inappropriate nutrition and sedentary lifestyles. Overweight, insulin resistance, \u03b2-cell dysfunction, increased circulating glucose and lipids and non-alcoholic fatty liver disease (NAFLD) characterize the pathophysiology of T2D2. Countless research efforts have explored pharmacological treatments for T2D and associated pathologies leading to promising compounds, which together with lifestyle interventions constitute first-line treatments3. During the last few years, the circadian system has been increasingly recognized as a key actor for development and treatment of diet-induced metabolic dysfunction. Yet, circadian rhythms in the clinical practice remain largely overlooked and time-of-day is hardly considered in treatment decisions8.In the last few decades, the prevalence of obesity has become epidemic through the world and is a major risk factor for type 2 diabetes (T2D)9. Aligned synchrony between all body clocks maintains homeostasis and health, for example, by adjusting metabolic performance to daily environmental fluctuations. Conversely, persistent circadian misalignment is a cause of severe diseases, including obesity and metabolic syndrome, T2D, or cardiovascular disease, amongst others12. At the molecular level, the circadian machinery is expressed in almost all cell types and consists of transcriptional-translational autoregulatory feedback loops. The positive loop is driven by the CLOCK:BMAL1 transcriptional activator, which rhythmically binds to E-box genomic elements, thereby activating transcription of many genes including the circadian repressors, Period (Per1-3) and Cryptochrome (Cry1-2). PER:CRY complexes directly repress CLOCK:BMAL1 leading to transcriptional silencing. A number of interlocked regulatory loops, such as the one governed by RORs/REV-ERB\u03b1 to regulate Bmal1 expression, intertwine to confer complexity, redundancy, and robustness to circadian rhythms13. Consequently, a set of clock-controlled genes (CCGs) ranging from 5\u201325% depending on the tissue or cell type, display transcriptional circadian rhythms14. Notably, rhythmic transcripts are functionally related, including rate-limiting enzymes, hence providing means to adjust the pace of many metabolic pathways around the day and driving rhythms in the tissue metabolome18. A paradigmatic example is illustrated by daily rhythms in nicotinamide adenine dinucleotide (NAD+) bioavailability, imposed by circadian oscillations in the clock-controlled gene Nampt, the rate-limiting enzyme for the NAM salvage pathway to NAD+20. Several lines of evidence demonstrate that the molecular clock and NAD+ oscillations sustain mitochondrial function and bioenergetics, manifested in daily rhythms in respiration, fatty acid oxidation, or nutrient utilization25. Indeed, it is considered that clock-controlled NAD+ biosynthesis occupies a fundamental position connecting circadian metabolic pathways28.Circadian rhythms are evolutionary conserved 24-h cycles in physiology dictated by an intrinsic circadian clock. In mammals, the suprachiasmatic nucleus (SCN), a master timekeeper in the hypothalamus, receives photic cues from the retina to align internal and external time. The SCN distally synchronizes ancillary oscillators in peripheral tissues. Importantly, certain cues such as nutritional inputs effectively synchronize peripheral clocks+ and its phosphorylated and reduced forms NADH, NADP+, and NADPH, are coenzymes for hydride transfer enzymes, crucial to biological redox reactions. NAD+/NADH ratio is a basic determinant of the rate of catabolism and energy production30. In fed state or nutrient overload NAD+/NADH ratio falls, and a prolonged redox imbalance potentially leads to metabolic pathologies, such as diabetes31. Along these lines, extensive research demonstrates that NAD+ levels significantly decline in metabolic tissues of mice and patients with obesity37. NAD+ decay itself may contribute to metabolic dysfunction by distinct mechanisms, including increased oxidative stress and ROS production, disbalance in the oxidative-reductive capacity, disrupted Ca2+ homeostasis, or reduced activity of sirtuins39; a class of deacetylase enzymes using NAD+ as cofactor and known to influence mitochondrial function and metabolism. In recent years, NAD+ has emerged as a target for the treatment of metabolic diseases, as boosting endogenous NAD+ levels has been proven effective against diet-induced metabolic pathologies, including insulin resistance, hyperglycemia, hypertriglyceridemia and NAFLD45. All these studies aim to increase NAD+ levels either genetically or pharmacologically, yet they mostly overlook the circadian trait of NAD+ bioavailability. Consequently, the implications of circadian rhythms in the function and effectiveness of NAD+ boosters as a therapy for diet-induced metabolic dysfunction remain largely obscure.NAD+ levels. To approach this question, we used a mouse model of diet-induced obesity (DIO), which is known to present decreased, non-rhythmic levels of NAD+17, and pharmacologically recovered daily rhythms of NAD+ with a peak at the onset of the active phase. To do so, we used a daily timed intraperitoneal (IP) injection with NAD+ itself at ZT11. We show that obese mice with enforced NAD+ oscillations improved metabolic health, significantly lost weight, and corrected NAFLD. Our analyses revealed that hepatic transcriptional signatures of inflammation disappeared in these mice. Indeed, hepatic signaling involving AMPK, AKT, mTOR was rewired after restoring rhythmic NAD+ in obese mice, providing increased insulin sensitivity during the active period. Together, we demonstrated that a single daily injection with NAD+ treats the pathophysiology of diet-induced obesity, with comparable efficiency to NAD+ precursors. Remarkably, these metabolic and molecular improvements were not recapitulated by obese mice with antiphase increase of NAD+, at the onset of the rest phase, which showed only selective recovery of metabolic health. Further analyses demonstrated that lipid oxidative pathways and the molecular clock are central mediators for phase-dependent, differential effects of NAD+. Particularly, NAD+ provided at the onset of the rest phase uncoupled oscillations between central and peripheral clocks, by means of inverting the phase of the hepatic clock while food intake and activity remained rhythmic. Collectively, our findings reveal that timed NAD+ supply can shape the oscillatory phase of the hepatic molecular clock in vivo and expose a previously unappreciated time-dependent effect of NAD+ as a treatment for metabolic dysfunction, paving the way for chronotherapy and personalized medicine.In this work, we aimed to characterize the metabolic consequences of rhythms in NAD+ administration improves metabolic fitness in obesity, we used a mouse model of diet-induced obesity (DIO) where instead of increasing NAD+ by chronic supplementation with metabolic precursors, we directly supplied the metabolite itself in a daily single IP injection scheduled at ZT11, corresponding to an hour before the normal circadian rise of hepatic NAD+46. Hence, after 8 weeks of high-fat diet (HFD) feeding, mice were treated for 22 days with saline solution (HF group) or 50\u2009mg/Kg of NAD+  with respect to their obese non-treated littermates (HF), which was sustained after 22 days . At the end of the treatment, hepatic NAD+ content was measured by HPLC, showing the expected oscillation with a peak at ~ZT12 in control mice .At week 8 on HFD, mice displayed expected increase in body weight which was accompanied by significantly higher caloric intake during both light and dark periods47 Fig.\u00a0. NotablyCD, Fig.\u00a0 which isCD, Fig.\u00a016,21,36,P\u2009<\u20090.001 Two-way ANOVA, Tukey post-test) and a six-hour phase delay in the oscillatory pattern . After 20 days of treatment, this improvement was also apparent at ZT4 . As both insulin and glucose levels were lower in NAD+-treated mice, insulin sensitization might occur. Accordingly, glucose clearance upon insulin IP injection was largely enhanced by NAD+ chronotherapy . Interestingly, NAD+ treatment at ZT11 promoted a slight, albeit not significant, improvement in insulin tolerance with respect to control lean mice when tested at ZT16  was used to semi-quantitatively assess hepatic steatosis Fig.\u00a0, revealie16 Fig.\u00a0. Additioels Fig.\u00a0 and augmels Fig.\u00a0. Togethe50 was overexpressed across the day in the livers from HFD-fed mice, while those treated with NAD+ showed markedly reduced PPAR\u03b3 levels  and dark (ZT18) phases in mouse livers from CD, HF, and HFN groups. 76 common genes were differentially expressed (DE) between day and night in all groups , Cry1, Nr1d2 (Rev-Erb\u03b2), Rorc, Tef, Nfil3 or Ciart . Out of these, 322 transcripts were exclusively fluctuating in the CD group, 1327 fluctuated solely in the HF, interestingly, 306 newly fluctuating transcripts appeared in the HFN group  or night (ZT18). At ZT6, 724 hepatic transcripts were significantly DE between CD and HF mice, while 936 transcripts varied when comparing HF and HFN groups, with 182 (12%) overlapping transcripts  mice to control conditions  showed that IL6-JAK-STAT3 and TGF\u03b2 signaling were the highest enriched hallmarks  . As shown, these hepatic expression changes at ZT18 were accompanied by improvement of hyperlipidemia and fatty liver traits after restoring NAD+ oscillations in obese mice  are previously described direct targets of FOXA257  Fig.\u00a0, with siice Fig.\u00a0. Pathwayrks Fig.\u00a0; and de rks Fig.\u00a0. Accordi+ oscillations in obese mice. To confirm this at the molecular level, we first evaluated phosphorylation of AKT1, a key kinase effector of insulin signaling59, along the day. As previously described, AKT1 phosphorylation at Ser 474 (p-AKT(S473)) appeared cyclic in CD fed mice60, with a peak at ZT18  at ZT12 , hence imposing daily oscillations to insulin signaling. Furthermore, diurnal rhythms in AMPK phosphorylation at T172 were also restored by NAD+ treatment in obese mice, although with a unique peak at ZT12, which was six hours phase delayed compared to their control, lean littermates  and HNF4\u03b1 TFs, both implicated in maintaining lipid homeostasis in the liver77. Also, a motif recognized by IRF3 and NR4A1 (Nur77) appeared significantly enriched (P\u2009=\u20091e-5), and interestingly, Nur77 has been shown to regulate the cytoplasmic shuttling of LKB1, hereby phosphorylating and activating AMPK78. Together, these data indicate that oscillatory NAD+ in obese mice activates a gene expression program favoring processes highly demandant for vesicle trafficking, such as translocation of membrane receptors or autophagy, and reinforce the idea of pharmacological supply of NAD+ preferably targeting activation of AMPK even in the context of high caloric feeding.A search for de novo motif enrichment within the promoters of NAD+ oscillations depend on the time of the day, we supplied NAD+ in opposite phase to its natural rhythmicity, hereby at the end of the active phase in mice, ZT23 (HFN23 group). In these HFN23 mice, oscillations of hepatic NAD+ were induced with antiphase respect to CD and HFN mice, showing a peak at ZT0 and decreasing at ZT12-18 , glucose and insulin tolerance showed non-significant improvement compared to the HF-fed mice. Actually, the NAD+ treatment at ZT11 was significantly more favorable to improving glucose homeostasis than at ZT23 , NAD+ supply at ZT11 showed still a significantly better performance than at ZT23 . Opposite, hepatic steatosis was reduced to a similar extent in HFN and HFN23 groups , the macrophage recruiter gene Csf2,  and macrophage markers   Fig.\u00a0, evidencBP\u03b1 Fig.\u00a0, which t+ at ZT23 to obese mice did not recapitulate hepatic AKT phosphorylation and activity, as did at ZT11 , but not at ZT23 (HFN23). Indeed, fatty acid oxidation-related genes were highly expressed at the end of the rest period (~ZT12)86; yet, unique to the HFN group was that the breadth of transcriptional activity further extended through the active period, reaching significantly higher levels than in the non-treated, obese mice (HF) at ZT18 . Hereby, expression of these genes at ZT18 was altered depending on the time of NAD+ treatment, in a way that the treatment at ZT11 significantly enhanced their expression, whereas in mice treated at ZT23, expression was significantly reduced to levels largely comparable to the CD littermates  at Ser79 and ACC2 at Ser212. These in turn downregulate the production of malonyl-CoA, the major substrate for fatty acid synthase (FAS) and a strong inhibitor of carnitine palmitoyl transferase 1 (CPT-1). Consequently, fatty acid synthesis is suppressed in favor of lipid oxidation, partially through activation of the rate-limiting step sustained by CPT-1T18 Fig.\u00a0. Hence, ups Fig.\u00a0. As expe+ at ZT23 presented some metabolic ameliorations mostly consisting of improved basal circulating glucose levels and reduced hepatic steatosis and inflammatory markers, we did not find consistent changes in gene expression or nutrient sensing signaling. Intriguingly, our microarray data showed that transcripts with highest fold-change after NAD+ treatment were Metallothionein 1 and 2 (Mt1 and Mt2), two antioxidants and longevity regulators known to protect from HFD-induced obesity89, and these transcripts were significantly more overexpressed in obese mice treated with NAD+ at ZT23 . A similar case was found for the gene lipocalin 2 (Lcn2), which encodes for a secreted protein protective against NAFLD90. Hence, while NAD+ chronotherapy works optimally at ZT11, its supply at ZT23 induces distinct protective pathways responsible for a mild, albeit noticeable, improvement of HFD-induced metabolic disease.While obese mice treated with NAD+ at ZT11 and ZT23 led to significantly different consequences in metabolic fitness and daily gene expression in the liver of obese mice. Hence, we reasoned that the molecular clock might be responsible for daily variations in the effectiveness of the treatment. Thereby, we compared the hepatic clock protein expression along the day between obese mice treated at ZT11 and at ZT23  and repressors  . To determine whether the observed antiphase dynamic of the clock transcriptional regulators was functional, we selected the genes Dbp, Tef, Nfil3, and Noct, whose expression is directly and mostly controlled by the core clock machinery, and analyzed their hepatic expression around the day  or at ZT17 (HFN17). We found expected phase advance in clock protein oscillations in livers from HFN5 mice, while treatment at ZT17 resulted in phase delayed oscillations  Fig.\u00a0. Indeed,day Fig.\u00a0. Coincidion Fig.\u00a0. To furt95, and the NAD+ precursor NR increases BMAL1 recruitment to chromatin in livers from aged mice46, we hypothesized that inverted expression of clock genes in HFN23 might be driven by time-specific recruitment of BMAL1 to chromatin. To test this, we performed ChIP analyses to measure BMAL1 binding to regulatory E-boxes of clock and clock-controlled genes . A non-related region at the 3\u2019 UTR region of Dbp gene was used as a negative control. We further evaluated the effect of NAD+ supplementation in the expression of NAD+ biosynthesis and salvage genes Nmrk1, Nampt, Nmnat3, and Nadk which also showed inverted phase specifically in HFN23 mice  or 30\u2009min (HFN_23) windows , which was previously described to boost hepatic NAD+ after IP injection in one hour101. Hence, three weeks with NAM chronotherapy performed best when applied at ZT11 to improve body weight, GTT and ITT , nicotinic acid (NA) or nicotinamide mononucleotide (\u03b2-NMN), while some tissues such a liver produce NAD+ de novo from tryptophan, in a much less efficient biosynthetic pathway104. At this regard, the NAD+ precursors NAM, NMN and NR have been preferentially used as NAD+ boosters; however, we set up a therapy with NAD+ because the limited data tracing metabolic fluxes suggest distinct, tissue-specific effects of NR and NMN105. Moreover, NAD+ uptake appears fast and effective in cells, and a mitochondrial active transporter has been recently described109. Yet, to gain insights into the bioavailability of NAD+ precursors in our study, it would be necessary to unravel the hepatic NAD+ metabolome in all tested conditions, as for example, the possibility that time-dependent decline in NADPH and NADP+ levels in livers from obese mice110 contributes to differences between HFN and HFN23 mice cannot be ruled out, constituting a limitation of our study. However, we demonstrated that hepatic NAD+ levels raised within an hour after IP injection in obese mice, and followed a circadian turnover when administered at ZT11, at the onset of the active phase 135. Indeed, both NAD+ consumers SIRT1137 and SIRT321 provide reciprocal regulation to the clock machinery to modulate circadian transcription and metabolism in the liver. Furthermore, recent research shows that a NAD+-SIRT1 interplay mediates deacetylation and nuclear translocation of PER2 and, in line with our results, shapes BMAL1 function, while this control is altered in livers from aged mice46. Through activation of SIRT1 and SIRT3, it is also possible that rising NAD+ at ~ZT12 might contribute to rhythmic lipid oxidation and mitochondrial function driven by protein acetylation, including PPAR\u03b3138, while keeping the hepatic clock aligned to the external time. Yet, the regulation of the circadian system by sirtuins in health and disease remains to be fully disentangled. Circadian misalignment imposed by antiphase NAD+ in our HFN23 and HFNAM_23 mice might obstruct metabolic improvements, through uncoupling of the central light-synchronized and peripheral NAD+-synchronized clocks. Although hepatic neutral lipid content was reduced independently of time-of-treatment . Food intake and body weight were measured once a week. For daily food intake measurements, mice were single housed, and measurements were recorded for 1 week. Female mice were used for the experiments using NAM as a NAD+ precursor, and the rest of the experiments were performed in male mice.Four-week-old C57Bl/6\u2009J mice were obtained from the Biological Models Unit at the Instituto de Investigaciones Biom\u00e9dicas . The mice were kept under a 12:12-h light:dark cycles. Food and water were provided ad libitum. Temperature and humidity were constantly monitored. Mice were randomly distributed to four groups. The control group was fed during 11 weeks with normal chow , bearing 24% calories from protein, 18% calories from fat, and 58% calories from carbohydrates. The other three experimental groups were fed a high-fat diet , consisting of 15% calories from protein, 53% calories from fat and 38% calories from carbohydrates, and customized to match NADAll animal experimental procedures were reviewed and approved by the Internal Committee for the Care and Use of Laboratory Animals (CICUAL) at the Instituto de Investigaciones Biom\u00e9dicas, , and are registered under protocol no. ID240.+ and NAM were purchased from SIGMA  and were dissolved in 0.9% NaCl isotonic saline solution and filter sterilized. To determine the NAD+ dose, we wanted to keep two premises: (1) to keep NAD+ levels into the physiological range, and (2) avoid undesirable secondary effects of high doses. To do so, we chose the range of tested doses based on previous reports151, and treated mice with IP injection of 800, 100, 50, or 10\u2009mg/kg body weight, while keeping a constant volume of approximately 180 \u03bcl. Control mice were injected with isotonic saline solution. C57Bl/6\u2009J male mice (n\u2009=\u20093) were IP injected, and sacrificed one hour later. NAD+ was measured by HPLC as described below. Because we planned on a chronic treatment, the minimum dose inducing a statistically significant increase in hepatic NAD+ with respect to control livers was selected as the experimental dose . Hence, for all experiments, mice were IP injected with 50\u2009mg/kg of NAD+ for 20 consecutive days, either at ZT11 (one hour before lights off), or at ZT23 (one hour before lights on). Of note, we didn\u00b4t find differences in hepatic NAD+ at a dose of 10\u2009mg/kg, a reason why we did not try lower concentrations. The dose for NAM treatment in female mice (200\u2009mg/kg) was selected based on previous reports154.NAD+ measurements were performed according to Yoshino and Imai 2013155, with subtle modifications. 100\u2009mg of frozen tissue were processed in a final volume of 2\u2009ml of 10% HClO4 with a Polytron homogenizer (Kinematica CH-6010 Kiriens-Lu) and centrifuged at 12,000\u2009\u00d7\u2009g for 5\u2009min at 4\u2009\u00b0C. The supernatant was neutralized adding a one-third volume of 3\u2009M K2CO3, and vortexed. After 10\u2009min of incubation on ice, samples were cleared by a 12,000\u2009g centrifugation at 4\u2009\u00b0C during 5\u2009min. The supernatant was diluted at 30% with 50\u2009mM phosphate buffer . 50\u2009\u03bcl of the samples were analyzed using a 1260 infinity quaternary LC VL HPLC system (Agilent) attached to a diode array detector. Analytes were separated on a ZORBAX Eclipse XDB-C18 4.6\u2009\u00d7\u2009150\u2009mm, 5\u2009\u03bcm column (Agilent p/n 993967-902). For the HPLC, the gradient mobile phase was delivered at a flow rate of 1\u2009ml/min, and consisted of two solvents: (A) 50\u2009mM phosphate buffer pH 6.8 and (B) methanol 100%. The initial concentration of A was 100%, the solution was held into the column for 5\u2009min and then B was progressively increased to 5% over 1\u2009min, held at 5% B for 5\u2009min, followed by an increase to 15% B over 2\u2009min, held at 15% B for 10\u2009min and returned to starting conditions of 100% A in 1\u2009min, and held at 100% A for 6\u2009min. NAD+ was detected using a sample wavelength of 261\u2009nm and reference wavelength of 360\u2009nm. Adequate standards including NAD+ were used for calibration and identification of the retention/migration time of NAD+ within the samples. Instrument control, data acquisition, and analysis were performed using the Agilent ChemStation system for LC, according to manufacturer\u2019s instructions. NAD+ levels were quantitated based on the peak area in the chromatograms compared to a standard curve and normalized to tissue weight.NADAt 8, 10, and 11 weeks of experimental paradigms, mice were subjected to either 12\u2009h or 5\u2009h of fasting, followed by a glucose tolerance test (GTT) or an insulin tolerance test (ITT) respectively. For the GTT, IP injection of D-glucose (SIGMA cat no. G7021) at 2\u2009mg/kg was used, while for ITT, human insulin (Eli Lilly cat. HI0210) at 0.6\u2009U/kg was IP injected. Circulating glucose was measured from a tail-tip blood drop, using an ACCU CHEK active glucometer (ROCHE) at time points 0 (before injection) and 15, 30, 60, and 120\u2009min after IP injection of either glucose (GTT) or insulin (ITT). Experiments were performed per triplicate, using 5\u20136 mice per experiment.Blood serum was collected postmortem by cardiac puncture. Triglycerides (TG) in serum and liver were measured using the Triglyceride Colorimetric Assay Kit . Free fatty acid content was determined with the Free Fatty Acid Fluorometric Assay Kit . Serum insulin and leptin levels were measured by ELISA, using the Ultra-Sensitive Mouse Insulin ELISA Kit  and the Mouse Leptin ELISA Kit  according to the manufacturer\u2019s instructions. Hepatic cholesterol was determined using a Cholesterol Quantitation Kit . Absorbance/fluorescence was measured using a Synergy H1 microplate reader (BioTek).n\u2009=\u200910 mice, and three technical replicates) was registered using a portable digital thermometer (BIOSEB) every 3\u2009h throughout 24\u2009h. For the acquisition of infrared thermography, mice were placed inside an acrylic box in darkness. Thermal images were acquired at ZT12 using an Inframetrics C2 Thermal Imaging System Compact Pocket-Size camera (FLIR Systems) with a frequency of 9\u2009Hz, thermal sensitivity <0.10\u2009\u00b0C, resolution 80\u2009\u00d7\u200960 (4800 pixels) and temperature range of 14 to 302 \u00b0 F. . Images processing was performed using FLIR-Tools software v 5.13.17214 (2015 FLIR\u00ae Systems).Rectal temperature in mice (156. Oil Red O working solution (3.75\u2009mg/ml) was applied on OCT-embedded liver sections for 5\u2009min at RT. Slides were washed twice during 10\u2009min. in water, and mounted in vectashield mounting media . The images were captured with the Olympus camera DP70 system using the DPController v 1.1.1.65 software, coupled to a Olympus BX51 microscope with the DPManager software v. 1.1.1.71, using a \u00d740 magnification. The background was corrected by white balance and was selected as a blank area outside the section. For representative images, some sections were stained with Gil I haematoxylin. Surface of lipid droplets was quantified using the ImageJ software (v 1.53), by converting RGB to 8-bit grayscale images, and then using the \u201canalyze particles\u201d plug-in to measure the area and size of the lipid drops157. Three frames per biopsy were used for image analyses and quantification .Frozen OCT-embedded liver tissues were cut into 10-\u03bcm sections using a Leica cryostat and air dried for 10\u2009min at room temperature. Slides were briefly washed with PBS and fixed for 2\u2009min with 4% fresh paraformaldehyde. Preparation of Oil Red O  working solution and staining of slides was performed according to Mehlem et al.3 region above the optic chiasm was dissected out using microscissors. Tissues were placed in microcentrifuge tubes in 100\u2009\u00b5l of Trizol and kept at \u221280\u2009\u00b0C until use. Total RNA was subsequently extracted and resuspended in 12\u2009\u00b5l of water.For gene expression analysis from the SCN, frozen brains were placed on ice, and the 1\u2009mmTM Reagent, Invitrogen, cat. no. 15596018). The homogenate was incubated for 5\u2009min at RT, then 0.1\u2009ml of chloroform was added, shaken, and incubated at RT for 3\u2009min followed by a centrifugation during 15\u2009min at 12,000\u2009\u00d7\u2009g at 4\u2009\u00b0C. The upper phase was extracted, and 0.25\u2009ml of isopropanol was added. After a 10\u2009min incubation at RT, RNA was precipitated by centrifugation for 10\u2009min at 12,000\u2009\u00d7\u2009g and 4\u2009\u00b0C. The RNA was washed with 1\u2009ml of 75% ethanol and resuspended in 20\u2009\u00b5l of molecular biology grade water . 2\u2009\u00b5l of the sample were used to quantify its concentration and assess its quality in a NanoDrop (Thermo Scientific).20\u2009mg of liver tissue or the dissected SCN, were homogenized  for 30\u2009seconds with 0.5\u2009ml of Trizol . 500\u2009ng of RNA were mixed with 2\u2009\u00b5l of 5X iScript Reaction Mix and 0.5\u2009\u00b5l of the enzyme iScript Reverse transcriptase in a volume of 10\u2009\u00b5l. The thermal cycler (Axygen MaxyGeneTM II) was programmed as follows: Alignment for 5\u2009min at 25\u2009\u00b0C, reverse transcription for 20\u2009min at 46\u2009\u00b0C and inactivation for 1\u2009min at 95\u2009\u00b0C. The reaction was cooled to 4\u2009\u00b0C and diluted to 5\u2009ng/\u00b5l.It was performed using the kit iScriptB2m, Ppia, and Tbp. The geometric mean was used to determine Ct values of the housekeeping genes and expression values for the genes of interest were calculated using \u0394CT methodology. Primer sequences are available in Supplementary Data\u00a0The reactions were performed in a final volume of 10\u2009\u00b5l, adding 5\u2009\u00b5l of the Universal SYBR Green Super Mix reagent , 1\u2009\u00b5l of 2.5\u2009\u00b5M forward and reverse primers, and 7.5\u2009ng of cDNA per reaction. The thermal cycler  was set to the following program: 30\u2009s at 95\u2009\u00b0C followed by 40 cycles of 5\u2009s at 95\u2009\u00b0C and 30\u2009s at 65\u2009\u00b0C. Single-product amplification was verified by an integrated post-run melting curve analysis. Values were normalized to the housekeeping genes (\u0394CT),where \u0394CT is the difference of CT values between S18 gene and mtCOX1 gene158.10\u2009mg of liver were used to extract DNA with the DNeasy Blood & Tissue Kit , according to the manufacturer\u2019s instructions. Quantitative PCR was performed using 7.5\u2009ng of DNA and 2.5\u2009\u03bcM of S18 and mtCOX1 primers as described for cDNA quantification, with a program of 20\u2009min at 95\u2009\u00b0C, followed by 50 to 55 cycles of 15\u2009s at 95\u2009\u00b0C, 20\u2009s at 58\u2009\u00b0C and 20\u2009s at 72\u2009\u00b0C. Single-product amplification was verified by an integrated post-run melting curve analysis. 5\u20136 mice were analyzed for each time point and condition, with two technical replicates. mtDNA content using the formula: 2\u2009\u00d7\u20092n\u2009=\u20093 biological replicates). The Clariom\u2122 D Array consists of 66100 genes (transcript clusters), 214900 transcripts, 498500 exons and 282500 exon-exon splice junctions from Mus musculus. Sequences are mapped to the National Center for Biotechnology Information (NCBI) UniGene database. The arrays were scanned in the GeneChip Scanner 3000 7\u2009G (Affymetrix) and the GeneChip Command Console Software (v 4.0.3) was used to obtain the.CEL intensity files. Normalized gene expression data (.CHP files) were obtained with the Transcriptome Analysis Console (TAC v4.0.1.36) software using default parameters. Changes in gene expression  were subjected to functional analyses using the \u201cCompute Overlaps\u201d tool to explore overlap with the CP  and the GO:BP  gene sets at the MSigDB (molecular signature database) v7.0. The tool is available at: https://www.gsea-msigdb.org/gsea/msigdb/annotate.jsp, and estimates statistical significance by calculating the FDR q-value. This is the FDR analog of the hypergeometric P value after correction for multiple hypothesis testing according to Benjamini and Hochberg. Gene set enrichment analysis (GSEA) was performed using GSEA v. 4.0.3.54 to determine the enrichment score within the Hallmark gene set collection in MSigDB v7.0159, selecting the Signal2Noise as the metric for ranking genes. The findMotifs.pl program in the HOMER software v 2.0160 was used for motif discovery and enrichment, searching within the genomic regions encompassing 300 Kb upstream and 50 Kb downstream the TSS, and selecting 6\u20138\u2009bp for motif length. Motif enrichment is calculated by findMotifs.pl using the cumulative hypergeometric distribution.Liver RNA samples for microarray analysis were prepared using our previously described procedures, with slight modifications. Briefly, total RNA was first extracted with TRIzol Reagent (Invitrogen), then cleaned with RNeasy Mini purification Kit (QIAGEN cat. no. 74106) according to the manufacturer\u2019s RNA CleanUp protocol. RIN values (\u22657.0) were validated with an Agilent Bioanalyzer 2100. 900\u2009ng of total RNA per sample was used as a template to obtain cDNA with the GeneChip cDNA synthesis Kit . Microarray experiments were conducted by the Microarray Unit at the National Institute of Genomic Medicine  using the mouse Clariom\u2122 D Assay (Applied Biosystems\u2122), as per manufacturer\u2019s instructions. Microarray experiments were performed in triplicate  for 10\u2009min at RT. The reaction was stopped by adding a NaOH (6\u2009M) for 10\u2009min. The absorbance of the samples was read in a spectrophotometer  at 370\u2009nm and the mean absorbance of control tubes (RIPA buffer) was then subtracted. To calculate the PCO concentration expressed as nmol PCO/mg protein, we used the following equation:The determination of the carbonyl content was performed from total hepatic protein extracts (0.5\u2009mg/ml), following a previously published protocol3VO4 1\u2009mM, NaF 0.5\u2009mM). Total protein was quantified with Bradford reagent , and 25\u2009\u03bcg of extract were suspended 1:6 (v/v) in 6\u00d7 Laemmli buffer , separated on sodium dodecyl sulfate\u2013polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto PVDF membranes (Merck-Millipore), using the Mini-PROTEAN electrophoretic system (Bio-Rad). Membranes were blocked using non-fat milk in PBST buffer for one hour and incubated with the corresponding primary antibody overnight at 4\u2009\u00b0C. Membranes were washed three times with PBST and incubated with the secondary antibody for 5 hrs at RT. Antibodies used in this study were: From Cell Signaling: PPAR\u03b3 (2443), AKT (9272), Phospho-AKTSer473 (9271), AMPK\u03b1 (5831), Phospho-AMPK\u03b1Thr172 (50081), mTOR (2893),), Phospho-mTORSer2448 (5536), Phospho-p70 S6KThr389 (9234), Phospho-4E-BP1Thr37/46 (2855), RSK1/RSK2/RSK3 (9355), Phospho-p90RSKSer359 (8753), REV-ERB\u03b1 (13418), ULK1 (8054), Phospho-ULK1Ser555 (5869), all diluted 1:1000; from Santa Cruz: C/EBP\u03b1 ; from Abcam: BMAL1 ; from Alpha Diagnostics International: PER2 (PER21-A 1:2000); from Bethyl Laboratories: CRY1 (A302-614A 1:1000); from Sigma: \u03b1-Tubulin ; from Genetex: GAPDH-HRP  and P84  The secondary antibodies were Anti-rabbit IgG  or Anti-mouse IgG , conjugated to horseradish peroxidase. For detection, the Immobilon Western Chemiluminescent HRP Substrate  was used and luminescence was visualized and documented in a Gel Logic 1500 Imaging System (KODAK). Protein bands were quantified by densitometric analysis using Image Studio Lite Version 5.0 software (LI-COR biosciences). 3\u20135 biological replicates were used for each quantification. Uncropped, unprocessed scans of the blots are available in the Source Data file.Livers were lysed in 1\u00d7 RIPA buffer supplemented with a protease/phosphatase inhibitor cocktail  was used for profiling inflammation cytokines, following the manufacturer\u2019s instructions. Absorbance at 450\u2009nm was measured using a Synergy H1 microplate reader (BioTek).48, with subtle modifications. Briefly, 20\u201360\u2009mg of fresh liver from mice sacrificed at ~ZT16 were weighed, and samples were minced and homogenized in 300\u2009\u03bcl homogenization buffer  at 4\u2009\u00b0C. Then, 5\u2009\u03bcl palmitic acid [14\u2009C] 100\u2009\u03bcC/ml (Perkin Elmer) was added, and these lysates were incubated for 2\u2009hr at 37\u2009\u00b0C. Eppendorf tubes were prepared to contain small pieces of Whatman paper in the cap of the tube, which were wet with 20\u2009\u03bcl NaOH 3\u2009M, while 150\u2009\u03bcl 70% perchloric acid was placed inside the tube. Lysates were added to these tubes and incubated 1\u2009h at 37\u2009\u00b0C. The trapped 14CO2 was determined transferring the filter discs to a scintillation vial with 4\u2009ml of scintillation liquid and measuring in a Beckman LS6500 scintillation counter.Fatty acid oxidation was quantitated ex vivo following our previous protocols162 with modifications as follows. Mice were sacrificed by cervical dislocation at ~ZT16, and ~100\u2009mg of liver was dissected and placed on ice on mitochondrial isolation buffer  and centrifuged at 800\u2009g for 10\u2009min at 4\u2009\u00b0C. The tissue was homogenized using the Polytron tissue homogenizer at low potency for 8\u2009seconds and centrifuged at 800\u2009g (10\u2009min at 4\u2009\u00b0C). The supernatant was collected in 15\u2009ml falcon tubes and centrifuged at 8000\u2009\u00d7\u2009g (10\u2009min at 4\u2009\u00b0C). The resulting pellet containing the mitochondria was washed three times with 1\u2009ml of MIB1 and resuspended on 0.5\u2009ml of mitochondrial assay solution  with the addition of substrates . Total protein was determined using the Qubit\u00ae 3.0 Fluorometer and 14\u2009\u00b5g of isolated mitochondria were diluted in MAS1 buffer with substrates with or without the presence of the CPT-1 inhibitor, etomoxir (3\u2009\u00b5M) and loaded per well in the XFe96 plate. The plate containing mitochondria was centrifuged at 2000 g for 20\u2009min at 4\u2009\u00b0C. The oxygen consumption rate (OCR) was measured with seven technical replicates for each mouse, as the following compounds were injected to final concentrations per well: ADP (4\u2009mM), oligomycin (2.5\u2009\u00b5M), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone known as FCCP (2.0\u2009\u00b5M) and antimycin A (1\u2009\u00b5M) / rotenone (1\u2009\u00b5M). OCR was measured in the absence or presence of etomoxir, with sequential addition of ADP (ATP precursor), oligomycin (complex V inhibitor), FCCP (a protonophore), and Rotenone/antimycin A . Four mitochondrial respiration states were calculated: basal respiration (respiration of mitochondria with substrates but without ADP), ATP production or phosphorylating respiration (rate of ATP formation from ADP and inorganic phosphate), proton leak or non-phosphorylating respiration (rate of oxygen consumption while ATP synthase is inhibited with oligomycin), and Maximal respiration state after the addition of FCCP. The instrument control, data analysis and file management were performed with the Agilent Seahorse Wave v2.6 software.The Palmitoyl-CoA oxidation depends on the activity of Carnitine palmitoyltransferase-1 (CPT-1), the rate-controlling enzyme for long-chain fatty acid oxidation. Measurements of CPT-1-mediated mitochondrial respiration was determined in mitochondria freshly isolated from mouse liver using the Seahorse XFe96 Extracellular Flux Analyzer (Agilent Technologies), as previously describedg for 30\u2009min at 4\u2009\u00b0C. The DNA was washed with 70% ethanol, and resuspended in 50\u2009\u03bcL of molecular-grade water. 1.5\u2009\u03bcl were used for subsequent qRT-PCR reactions with specific primers designed using Primer3web v4.1.0, within regulatory regions previously identified as BMAL1 binding sites in mouse liver, as reported in the ChIP-Atlas database163. Primer sequences are available in Supplementary Data\u00a0100\u2013200\u2009mg of liver tissue were homogenized with a pestle in PBS. Dual crosslinking was performed in a final volume of 1\u2009ml using 2\u2009mM of DSG  for 10\u2009min at RT on a rotary shaker. DSG was washed out and a second crosslink was performed using 1% formaldehyde  in PBS for 15\u2009min at RT on a rotary shaker. Crosslinking was stopped with 0.125\u2009M glycine for 5\u2009min at 4\u2009\u00b0C. After two washes with ice-cold PBS, nuclei were isolated by resuspending the tissue in 600\u2009\u03bcL of ice-cold nuclei preparation buffer  and incubating at 4\u2009\u00b0C for 5\u2009min in rotation. Nuclei were collected by centrifugation at 1,500\u2009g for 12\u2009min at 4\u2009\u00b0C and, and resuspended in 600\u2009\u03bcL of cold nuclear lysis buffer  for 30\u2009min on ice. Nuclear lysates were stored at \u221280\u2009\u00b0C. 300\u2009\u03bcL of lysates were sonicated using a Bioruptor Pico Sonicator (Diagenode) for 15 cycles (30\u2009s ON/30\u2009s OFF). Chromatin fragments (100\u2013500\u2009bp) were evaluated on agarose gels using 10\u2009\u03bcL of sonicated chromatin for DNA purification using the phenol method. 600\u2009\u03bcL of ice-cold ChIP-dilution buffer  was added to the fragmented chromatin, and 10% volume was recovered as the Input. Immunoprecipitation was set up overnight at 4\u2009\u00b0C, by adding 20\u2009\u03bcL of magnetic beads  and a combination of two anti-BMAL1 antibodies: 1.25\u2009\u03bcL rabbit anti-BMAL1  and 2.5\u2009\u03bcL rabbit anti-BMAL1  in 900\u2009\u03bcL final volume. Immunoprecipitations with 4\u2009\u03bcL of normal mouse IgG  were performed simultaneously in 900\u2009\u03bcL final volume. Sequential washes of the magnetic beads were performed for 10\u2009min at 4\u2009\u00b0C, as follows: Wash buffer 1 , Wash buffer 2 , Wash buffer 3  and TE buffer . Chromatin was eluted by adding 400\u2009\u03bcL of fresh elution buffer  to the magnetic beads and incubating overnight at 65\u2009\u00b0C. A treatment with RNase A at 0.1\u2009mg/ml for 30\u2009min at 37\u2009\u00b0C was performed. The DNA was purified from the IPs and Inputs by adding one volume of phenol:chloroform:isoamamyl alcohol (25:24:1). After mixing and centrifugation, the aqueous phase was recovered, and DNA was precipitated by adding 1/10 volumes of sodium acetate (0.3\u2009M\u2009pH 5.2), 20\u2009\u03bcg of glycogen  and 2 volumes of ice-cold ethanol, at \u221280\u2009\u00b0C overnight. DNA was pelleted by centrifugation at 12,000\u2009\u00d7\u2009164. Activity profiles were obtained averaging 5 consecutive days prior to the NAD+ treatment, and 5 consecutive days after the start of the treatment. Activity profile data from 30\u2009min were averaged for statistical comparisons.Mice were individually housed in a light-tight, ventilated cabinet, under a 12\u2009h light:12\u2009h dark cycle, and ad libitum access to food and water. At the appropriate time for each treatment, animals were removed from their cages to receive IP injections for less than 2\u2009min each. Cages were equipped with two infrared motion sensors . Beam break data were continuously recorded and compiled with the OASPAD20 (OMNIALVA) software, v2019, and files containing the number of beam breaks per 6-min bin were exported. Double-plotted actograms were generated using RhythmicAllyP\u2009<\u20090.05. GraphPad Prism version 8.4.2.679 for Windows  and Excel  were used for statistical analyses and plotting. 24-h period rhythms were assessed employing CircWave version 1.4165, CircWave uses a forward linear harmonic regression to calculate the profile of the wave fitted into a 24\u2009h period. Daily rhythms were confirmed when the null amplitude hypothesis was rejected by running an F test that produced a significant value (P\u2009<\u20090.05). CircWave provides the calculation of the Centre of Gravity (CoG), representing the acrophase of the curve, with SD. Double-plotted data (ZT24) for visualization proposes are indicated in Figure legends, and were not included in the statistical analyses. Data from live mice were replicated in two independent experiments with similar results. Figures were assembled using Adobe Illustrator CC 2015 .All data were presented as the mean\u2009\u00b1\u2009standard error of the mean, and two-way analysis of variance (ANOVA) followed by Tukey\u2019s test for multiple comparisons was used for statistical analyses except when otherwise noted in the Figure legends. Differences between groups were rated as statistically significant at Further information on research design is available in the\u00a0Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Reporting Summary"}
+{"text": "Sepsis is the most common cause of admission to intensive care units worldwide. Sepsis patients frequently suffer from sepsis-associated encephalopathy (SAE) reflecting acute brain dysfunction. SAE may result in increased mortality, extended length of hospital stay, and long-term cognitive dysfunction. The diagnosis of SAE is based on clinical assessments, but a valid biomarker to identify and confirm SAE and to assess SAE severity is missing. Several blood-based biomarkers indicating neuronal injury have been evaluated in sepsis and their potential role as early diagnosis and prognostic markers has been studied. Among those, the neuroaxonal injury marker neurofilament light chain (NfL) was identified to potentially serve as a prognostic biomarker for SAE and to predict long-term cognitive impairment. In this review, we summarize the current knowledge of biomarkers, especially NfL, in SAE and discuss a possible future clinical application considering existing limitations. Sepsis is a potentially life-threatening organ dysfunction caused by a dysregulated host response to severe infection , 2. Due Sepsis-associated encephalopathy (SAE) is one of the most common organ dysfunctions in sepsis and is also associated with significantly higher mortality rates , 16\u201318. Due to inconsistent diagnostic criteria in clinical studies and diverging daily clinical practice, the estimated prevalence of delirium in SAE varies from 9 to 71% in sepsis patients . As an eIn addition to acute changes in mental state during the acute phase, sepsis is associated with long-term cognitive dysfunction following hospital discharge. According to a study of long-term cognitive outcomes in patients admitted to ICUs with acute respiratory failure, septic or cardiogenic shock, 40% (after three months) and 34% (after twelve months) of patients had persisting cognitive impairment after hospital discharge . The sevIn the ICU, delirium severity is routinely measured using the CAM-ICU and Intensive Care Delirium Screening Checklist (ICDSC), both showing high reliability and validity in patients who are able to interact with the investigator. According to CAM-ICU, a patient is rated as delirious when (a) the mental status acutely changes or fluctuates and (b) the patient fails to pay attention and either (c) the patient exhibits disorganized thinking or (d) has an altered level of consciousness. To evaluate the level of consciousness, the Richmond Agitation-Sedation Scale (RASS) is used . AnotherThe major disadvantage of the CAM-ICU and the other scores is the reliance on patient interaction. Therefore, these clinical tools cannot be used in deeply sedated patients . Furthermore, patients with hypoactive delirium might also be underrecognized. Thus, the delirium rates in the ICU are likely to be underestimated in severely ill patients.Besides challenges in the practical use of delirium tests itself in SAE patients, a recent study evaluated the current practice of clinical SAE diagnostics in the ICU and demonstrated a great heterogeneity in the application of diagnostic tests in Germany .in-vivo data indicate neuronal and synaptic damage in sepsis alongside with activation of immune cells in the CNS. Analyses of brain autopsies from patients who died of sepsis showed diffuse cerebral damage with neuronal apoptosis, axonal damage and ischemic lesions ..21].Biomarkers should provide objective and quantitative results. In SAE, an ideal biomarker would provide high sensitivity and specificity independent of the effects of sedatives. It should enable an early diagnosis as well as a reliable outcome assessment. Several potential biomarkers have been proposed and studied in the course of SAE, such as neuron- and glia-derived proteins , 56, butIn the last decade, neurofilament light chain (NfL) has been intensively investigated as a biomarker in several neurological diseases . NfL is Neurofilaments are cylindrical proteins found in the neuronal cytoplasm . TogetheDue to its proven role as a biomarker directly reflecting neuronal damage with the opportunity of blood measurements, NfL has been considered an ideal candidate for the diagnosis and prognostic assessment in SAE . HoweverVery recently, in a gender and age-matched series of patients with community-acquired pneumonia we demonstrated that serum NfL levels were associated with the occurrence of SAE as determined by confusion or delirium but not with overall disease severity, thus supporting the specificity of Nfl as a marker for CNS involvement in infectious and inflammatory disease . These fConsidering these results, blood NfL levels could serve as a biomarker for SAE and may have potential to predict long-term cognitive impairment after sepsis. In addition to the evidence provided by the above-mentioned first studies with rather small sample size there are several arguments that NfL might have a larger potential as compared to other biomarkers. It has already proven to be directly associated with long-term cognitive impairment and even predicting worsening of cognition over time in other neurological diseases, such as multiple sclerosis, cardiac surgery, Alzheimers\u2019\u00a0disease and mild cognitive impairment \u2013101. MorTogether, an increase of NfL levels at a defined time point during sepsis or changes in serum NfL levels over time during SAE might be suitable and clinically useful to predict long-term cognitive outcome in patients with sepsis. An NfL increase might enable ICU clinicians to identify patients at high risk for structural brain damage and therefore to prioritize brain imaging and protection during the hospital stay , 56. TheThere are several factors that may influence serum levels of NfL in the setting of severely ill sepsis patients Fig.\u00a0. Here, NIn general, NfL values show also interindividual variability in healthy individuals and in patients with certain comorbidities , 106. ThSeveral immunoassays  are currently available to reliably quantify NfL in blood at very low concentrations . To dateIn this regard, NfL might be valuable by providing information on long-term prognosis, as repeated NfL measurements might help to differentiate between reversible brain dysfunction  and structured brain damage  . FurtherIn 2001, Pepe and colleagues published a five-phase framework for cancer biomarker development, which has been modified by the Geneva Task Force for the Roadmap of Alzheimer\u2019s Biomarkers for the development of biomarkers , 115. Ac"}
+{"text": "Sepsis-associated encephalopathy is a severe neurologic syndrome characterized by a diffuse dysfunction of the brain caused by sepsis. This review provides a concise overview of diagnostic tools and management strategies for SAE at the acute phase and in the long term. Early recognition and diagnosis of SAE are crucial for effective management. Because neurologic evaluation can be confounded by several factors in the intensive care unit setting, a multimodal approach is warranted for diagnosis and management. Diagnostic tools commonly employed include clinical evaluation, metabolic tests, electroencephalography, and neuroimaging in selected cases. The usefulness of blood biomarkers of brain injury for diagnosis remains limited. Clinical evaluation involves assessing the patient's mental status, motor responses, brainstem reflexes, and presence of abnormal movements. Electroencephalography can rule out non-convulsive seizures and help detect several patterns of various severity such as generalized slowing, epileptiform discharges, and triphasic waves. In patients with acute encephalopathy, the diagnostic value of non-contrast computed tomography is limited. In septic patients with persistent encephalopathy, seizures, and/or focal signs, magnetic resonance imaging detects brain injury in more than 50% of cases, mainly cerebrovascular complications, and white matter changes. Timely identification and treatment of the underlying infection are paramount, along with effective control of systemic factors that may contribute to secondary brain injury. Upon admission to the ICU, maintaining appropriate levels of oxygenation, blood pressure, and metabolic balance is crucial. Throughout the ICU stay, it is important to be mindful of the potential neurotoxic effects associated with specific medications like midazolam and cefepime, and to closely monitor patients for non-convulsive seizures. The potential efficacy of targeted neurocritical care during the acute phase in optimizing patient outcomes deserves to be further investigated. Sepsis-associated encephalopathy may lead to permanent neurologic sequelae. Seizures occurring in the acute phase increase the susceptibility to long-term epilepsy. Extended ICU stays and the presence of sepsis-associated encephalopathy are linked to functional disability and neuropsychological sequelae, underscoring the necessity for long-term surveillance in the comprehensive care of septic patients. Sepsis-associated encephalopathy (SAE) is a severe neurologic syndrome characterized by a diffuse dysfunction of the brain caused by sepsis, a life-threatening condition resulting from the dysregulated response of the body to an infection. At the acute phase, patients with SAE typically present with an acute onset of encephalopathy, ranging from delirium to coma . The patIn the present article, we review recent findings in the field of SAE focusing on its epidemiology, diagnosis, and management at the acute phase. We also provide an update on long-term effects of SAE observed in sepsis survivors.SAE is most frequently defined as an acute encephalopathy occurring during sepsis or septic shock, and not attributable to any other cause than sepsis itself . SAE is Most of the available data on acute encephalopathy in the ICU has been generated from studies conducted in the general population. Few specific epidemiological studies have been conducted in patients with sepsis, and risk factors for SAE identified in these studies are described in Table ~10% of cases) and dysautonomia are less frequent [~2% of cases) and focal signs (~1% of cases) are uncommon and should trigger investigations to rule out brain injury. Thus, underdiagnosis of SAE is probable in the absence of systematic screening with validated tools [Clinical evaluation of SAE is challenging in the ICU because neurologic assessment can be confounded by several factors, including fever, metabolic derangements, and the use of hypnotic agents in mechanically ventilated patients. SAE manifests as a rapid change from baseline cognitive status or level of consciousness, and presents with a wide range of symptoms, from mild delirium 19%) to coma (40%) 9% to com. Coma orfrequent . Convulsed tools . ConversData on the usefulness of brain CT studies in patients with SAE is limited. In a meta-analysis conducted in adults with acute non-traumatic encephalopathy, CT abnormal findings were observed in 11% of cases . In mediBrain magnetic resonance imaging (MRI) is indicated in presence of focal signs, brainstem symptoms, new-onset seizures, and in case of persistent encephalopathy in the absence of common confounders (i.e. metabolic/toxic factors and sedation). It is recommended to include a diffusion-weighted imaging (DWI) sequence in the MRI protocol, which is the most sensitive sequence for detection of cerebral ischemia and inflammatory changes. MRI alterations diagnosed at the acute phase of SAE include parenchymal lesions and atrophy, that are reported in about 55% and 16% of cases, respectively , 18. IscWhite matter lesions (WML) are observed in a significant percentage (14\u201381%) of SAE patients presenting with persistent encephalopathy , 21. TheBrain atrophy is more pronounced in patient with SAE than in healthy controls . This atAcute neuroimaging changes have prognostic significance as predictors of disability and survival in the first year following SAE. In a single-center study, ischemic stroke was found to be independently associated with increased ICU mortality and poor functional status at 6 months . In anotEEG can be a valuable tool in the positive diagnosis of SAE, for excluding non-convulsive status epilepticus, and for prognostication Fig.\u00a0. In non-One of the main challenges is the impact of sedation on EEG background, which is dependent on the dosage and the specific sedative used. Sedatives can lead to a dose-dependent slowing of the EEG background. Benzodiazepines often produce diffuse rapid rhythms >\u200913Hz), whereas propofol and barbiturates may result in low voltage, discontinuous patterns at moderate doses, and burst suppression or suppression patterns at higher doses , 26  introduced specific terms for critical care EEG to address this issue .Early EEG abnormalities may precede clinical neurologic impairment and correlate with the severity of encephalopathy , 29. In The pathophysiology of electrographic seizures (ESz) in SAE remains debated, but could be related to the increased neuronal excitotoxicity and epileptogenic factors, including neurotoxic antimicrobials, metabolic disturbances, and severe acute kidney injury. Most ESz are non-convulsive, highlighting the interest of continuous EEG recording . ESz areSome EEG patterns are associated with delirium, including slow delta rhythm, absence of EEG reactivity, discontinuity, and presence/burden periodic discharges (PDs) , 34, 37.Some EEG patterns are also associated with ICU mortality. The absence of reactivity has been shown to be independently associated with ICU and 1 year mortality , 32, 36.Evoked potentials (EPs) are neural responses time-locked to some stimulus and differ from EEG signals as they are stimulus-induced. EPs reflect the combined activity of many neurons firing together and necessitate averaging multiple sensory or auditory stimulations. These components are labeled based on their polarity (negative as \"N\" and positive as \"P\") and their latency (measured in milliseconds) from the stimulus. . Somato-Blood biomarkers associated with neuronal injury, specifically neuron-specific enolase (NSE) and neurofilament light (NfL), as well as biomarkers linked to glial injury, such as protein S100 beta (PS100), were evaluated in sepsis patients to anticipate the onset of SAE and predict outcomes. NSE is the most accessible biomarker, and the prevalence of elevated NSE levels (i.e. >\u200912.5\u00b5g/L) in sepsis varies between 28 and 53% , 46. PreIn a single-center study, a NSE threshold\u2009>\u200924.15\u00b5g/L (AUC 0.66) had a specificity of 83% and a sensitivity of 54.2% for the diagnosis of SAE . One proThe prognostic value of glial injury biomarkers was examined in several studies, highlighting conflicting results. Previous prospective cohort studies indicated that high PS100 levels were associated with hospital mortality  , but alsOne study investigated the link between serum NfL levels and SAE outcomes . Among sOne prospective study found that serum concentrations of Glial Fibrillary Acidic Protein (GFAP), a protein expressed by astrocytes, were higher in SAE patients compared to non-SAE patients . Serum GUsing blood biomarkers for SAE diagnosis faces challenges due to undefined optimal assessment timing, uncertainty about the precise SAE onset, inconclusive findings about prognostic benefits and definitive thresholds, and limited access to certain biomarkers, impeding their routine clinical use. The profile of biomarkers differs between SAE, sepsis, and delirium patients, suggesting that pathways related to SAE are different from those related to delirium and sepsis itself.Multidisciplinary care is frequently necessary for the comprehensive management of SAE. Prompt identification and treatment of the infection are vital, typically involving the administration of antibiotics and supportive measures such as fluid resuscitation and vasopressors. Moreover, it is essential to control factors contributing to secondary brain injury, which includes maintaining adequate levels of oxygenation and blood pressure, addressing metabolic imbalances, and detecting/treating seizures. Delirium prevention is of paramount importance and implementation of the ABCDEF bundle is associated with improved survival and a reduction in the number of days of delirium and coma . A simplTo prevent secondary insults resulting from agitation, or dysautonomia in severe cases, the use of sedatives  or antipsychotics may be necessary. In two randomized controlled trials conducted in patients with sepsis, the use of dexmedetomidine compared to standard sedation did not result in lower rates of delirium or coma , 56. TheSystemic causes of secondary brain injury are frequent at sepsis onset and are linked to poorer outcomes . These cThree distinct phenotypes of antibiotic-associated encephalopathy have been identified: First, an acute-onset encephalopathy commonly accompanied by clinical seizure (mostly stereotyped clonus or myoclonus) or non-epileptic myoclonus, typically manifesting within days of antibiotic administration ; second, an encephalopathy characterized by psychosis that arises within days of antibiotic administration ; and third, a subacute encephalopathy linked to cerebellar signs and MRI abnormalities that develop weeks after initiating antibiotic therapy (commonly associated with metronidazole) . The mosCefepime remains the most frequently reported molecule associated with neurologic events, with renal dysfunction being the primary risk factor for cefepime-induced neurotoxicity \u201373. The Although most studies on long term outcomes have focused on the general ICU population, current data suggest that sepsis survivors experience a wide range of cognitive, psychiatric, physical, and social impairment after ICU discharge . A generInflammatory processes are thought to participate in early brain alterations but also in long-term cognitive impairment . In a prSepsis survivors face a higher long-term risk of seizures than other hospitalized patients. In a large cohort, the annual incidence of seizure after sepsis was 1.29%, with incidence rate ratios of 4.98 and 4.33, compared to the general population and hospitalized patients without sepsis, respectively . Among sSepsis survivors experience long-term emotional and behavioral changes, including depressive symptoms, anxiety and post-traumatic stress disorder (PTSD) . A studyCompared to mechanically ventilated patients of similar acuity and length of stay without sepsis, patients with sepsis have an increased risk of mortality and a similar risk of new disability at 6 months . CriticaICU-acquired weakness is another frequent complication associated with sepsis resulting from alterations of small nerve fibers . TypicalSAE is a complex condition that requires a multidisciplinary approach for its diagnosis and management. Timely identification and treatment of the underlying infection are paramount, along with effective control of systemic factors that may contribute to secondary brain injury. Upon admission to the ICU, maintaining appropriate levels of oxygenation, blood pressure, and metabolic balance is crucial. Throughout the ICU stay, it is important to be mindful of the potential neurotoxic effects associated with specific medications like midazolam and cefepime, and to closely monitor patients for non-convulsive seizures. A multimodal approach based on clinical evaluation, neuroimaging and bedside available non-invasive tools may have important prognostic implications both at the acute phase and in the long term. The potential efficacy of targeted neurocritical care during the acute phase in optimizing patient outcomes deserves to be further investigated.SAE may lead to permanent neurologic sequelae. Seizures occurring in the acute phase increase the susceptibility to long-term epilepsy. Extended ICU stays and the presence of sepsis-associated encephalopathy are linked to functional disability and neuropsychological sequelae, underscoring the necessity for long-term surveillance in the comprehensive care of septic patients."}
+{"text": "Peronophythora litchii accounts for severe losses in the field and during storage. While ample quantitative studies have shown that 6-pentyl-2H-pyran-2-one (6PP) possesses antifungal activities against multiple plant pathogenic fungi, the regulatory mechanisms of 6PP-mediated inhibition of fungal pathogenesis and growth are still unknown. Here, we investigated the potential molecular targets of 6PP in the phytopathogenic oomycetes P. litchii through integrated deployment of RNA-sequencing, functional genetics, and biochemical techniques to investigate the regulatory effects of 6PP against P. litchii. Previously we demonstrated that 6PP exerted significant oomyticidal activities. Also, comparative transcriptomic evaluation of P. litchii strains treated with 6PP Revealed significant up-regulations in the expression profile of TOR pathway-related genes, including PlCytochrome C and the transcription factors PlYY1. We also noticed that 6PP treatment down-regulated putative negative regulatory genes of the TOR pathway, including PlSpm1 and PlrhoH12 in P. litchii. Protein-ligand binding analyses revealed stable affinities between PlYY1, PlCytochrome C, PlSpm1, PlrhoH12 proteins, and the 6PP ligand. Phenotypic characterization of PlYY1 targeted gene deletion strains generated in this study using CRISPR/Cas9 and homologous recombination strategies significantly reduced the vegetative growth, sporangium, encystment, zoospore release, and pathogenicity of P. litchii. These findings suggest that 6PP-mediated activation of PlYY1 expression positively regulates TOR-related responses and significantly influences vegetative growth and the virulence of P. litchii. The current investigations revealed novel targets for 6PP and underscored the potential of deploying 6PP in developing management strategies for controlling the litchi downy blight pathogen.The litchi downy blight disease of litchi caused by  Litchi chinensis Sonn.) is an economically important evergreen fruit tree crop cultivated across selected subtropical and tropical regions, including southern provinces of China, South Africa, Australia, Hawaii, and Israel [P. litchii is one of the most destructive diseases of Litchi and accounts for 20\u201330% onfield and post-harvest losses per year [P. litchii heavily relies on the application of synthetic agrochemicals [P. litchii [Litchi . However, the action mechanism of 6PP against P. litchii is unclear, and TOR pathway-related genes of response to 6PP stress in P. litchii are not understood.Previous studies have shown that 6-Pentyl-2H-Pyran-2-One , a bioactive secondary metabolite isolated from  glaucus ,13,14,15Botrytis cinerea, V. dahliae, F. oxysporum, and Fusarium graminearum [Magnaporthe oryzae [Arabidopsis, the TOR pathway is associated with the response to sugar and the maintenance of root tip homeostasis [P. litchii and its influence on essential pathways associated with microbial pathogenesis, including the TOR pathway, remain unclear.Core cellular pathways, including the conserved Ser/Thr protein kinase target of the rapamycin (TOR) pathway, have been identified as a key regulator of cellular growth, survival, metabolism, ribosome biogenesis, translation, and transcription, processes ,17,18. Sminearum ,27,28,29e oryzae . In Arabeostasis . Also, Teostasis . HoweverP. litchii treated with 6PP and identified 6PP-responsive genes, including the YinYang1 (YY1) transcription factor. Additional results from molecular docking analyses and phenotypic characterization of YY1 gene deletion strains indicated that 6PP possibly suppresses vegetative growth, sporulation, cyst formation, and pathogenesis of P. litchii by regulating the activation of the TOR pathway. These findings highlight the critical role of 6PP-mediated regulation of YY1 expressions on the TOR pathway and the impact on cellular/vegetative development and the pathogenesis of phytopathogenic oomycetes.Here, we monitored the transcriptomic dynamics in Tender litchi leaves (cv. Baitangying) were collected from a Hainan University orchard . Leaf shapes and sizes were uniform and disease-free. Fresh and healthy Litchi fruits were harvested from orchards in Haikou, China, for infection assays. All fruits were healthy, uniform in shape, and at 80% ripe.Carrot juice agar (CJA) medium was prepared by incorporating the juice from 0.2 kg of crushed carrots into the water to attain a final volume of 1 L, adding 15 g agar/L for solid media.The 6PP was an analytical-grade standard purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd., Shanghai, China.P. litchii by air fumigation. The fumigation device is composed of two petri dish bottoms. One bottom of the petri dish contains P. litchii, and the other bottom of the petri dish contains 6PP, then wrapped with parafilm. The fumigation device allows air exchange, but 6PP has no direct contact with P. litchii. P. litchii was cultured on CJA medium for 3 days, and fumigated with 6PP (86 \u03bcg/mL) for another day, then the biomass was collected and frozen in liquid nitrogen. The 6PP concentration (86 \u03bcg/mL) was selected based on our preliminary results, which are the 2.0 \u00d7 EC50 values of 6PP against P. litchii mycelial growth  in the P. litchii database we have established. After searching and matching to transcriptome data, TOR pathway genes regulated by 6PP were identified based on their differential expression pattern and predicted domains, molecular weight, and isoelectric points using Simple Modular Architecture Research Tool (SMART) database, NCBI Batch CD-Search  and online program ExPASy ProtParam . Additionally, we deployed MEME suit for further DNA-biding motif analyses  online website.Genome (ID: PRJNA290406) and proteome (GWHAOTU00000000.1) data for https://services.healthtech.dtu.dk./service.hph?SignalP-4.1, accessed on 22 December 2021), and then homology modeling was processed and analyzed for applicability by SWISS-MODEL  and SAVES v.6.0 . Furthermore, autodock software (version 1.5.6) and python software (version 2.5) was used for docking analyses using the default parameters to calculate the binding affinities between ligand and receptors, and 50 interaction models were accepted to establish with default docking parameter [Ligand 6PP structure, obtained from PubChem database, was converted from a two-dimensional model to mol 2 format by Openbable (version 3.1.1) process. Target protein structures were required for signal peptide prediction by SignalP-4.1 , using primer pairs listed in PlYY1 were inoculated in liquid SD-Trp media and diluted into different concentration solutions to compare growth volumes . Consecutively, pGBKT7-PlYY1 was cultured at SD-Trp-Leu, SD-Trp-Leu-His-Ade, and SD-Trp-Leu-His-Ade+X-\u03b1-gal medium separately to ascertain whether the target vector had been transferred into yeast AH109 correctly and has a Transcriptional activation ability. In order to determine the accuracy of the experiment, positive co-transformed control pGBKT7-p53+pGADT7-ADT and negative control pGBKT7 were conducted. This experiment was performed by three times.Transcriptional activation is a ubiquitously essential process that regulates gene modification. Additional tests were performed to examine the activation abilities of the HPH) and two homologous flanking sequences (about 1kb) were connected by overlap extension PCR and cloned to linearized plasmid pBluescript II KS+. According to the previous gene knock-out technique [P. litchii, and transformants were separated by CJA medium supplemented with G418 at concentration 20 \u03bcg/mL. The conventional cetyltrimethylammonium bromide (CTAB) method was used to extract genomic DNA. Moreover, transformants were verified and identified by polymerase chain reaction (PCR) in three rounds. Also, PCR products were sequenced if transformants identification was verified, and all primers are provided in To knock out the gene efficiently, two fragments of sgRNA were synthesized and cloned into an \u201call-in-one\u201d pYF515 vector, respectively. Donor DNA hygromycin B resistance gene , the wild-type, and the complementation (\u25b2PlYY1-2) strains were inoculated in CJA medium at 25 \u00b0C for 5-days in darkness, and mycelial growth rate, sporangia production, and zoospore release rate were investigated. The total harvest volume of sporangia was 30 mL for each plate, and the release of zoospore was induced by low temperature (12 \u00b0C). To determine the oospores production, P. litchii was inoculated on CJA medium, and the 10th and 15th day, plugs (1 cm \u00d7 1 cm) were cut from 1 cm around the inoculation site, and oospores were counted under a microscope. All experiments were repeated five times, with three replicates in each experiment.For phenotypic examinations, the two independent 4 sporangia/mL. Lesion size and pattern were analyzed at 48 hpi and 60 hpi at room temperature. Results were generated from three independent biological experiments, with each experiment comprising three technical replicates. Each biological replicate consists of 27 leaves and 30 fruits. To investigate whether the pathogenic capacity of mutants was affected, both the leaves and lychee fruits were used for testing. Three strains, WT, \u2206PlYY1-1, and \u2206PlYY1-2, were incubated as described in Air fumigation was utilized, and both WT and mutant strains\u2019 discs (\u03a6 = 6 mm) were inoculated in CJA medium, which was fumigated with 6PP for 5 days at 25 \u00b1 1 \u00b0C. The concentration of 6PP is 1.0 \u00d7 EC50. Mycelial growth was measured and used in deriving the inhibition rate. Negative control (NK) was set up without 6PP. This experiment was replicated three times with five copies each time.p < 0.05, while double asterisks (**) represented p < 0.01, and ns represented no significant difference.Statistical computations were performed using the one-way ANOVA  with Tukey\u2019s multiple comparisons test, and all results exhibited mean \u00b1 SEM. Significance differences recorded between treatments and control groups are denoted with an asterisk (*). A single asterisk (*) represented p-value < 0.05 and |log2Fold change| \u2265 1. A comprehensive analysis of 17,959 genes was conducted in the Transcriptome data, comparing the treatment and control groups. Of these, 1428 genes exhibited differential expression, with 406 genes up-regulated and 1022 genes down-regulated.  were identified from P. litchii through homologous gene comparison. Furthermore, by conducting transcriptome differential expression gene analysis, it was observed that two TOR pathway-related genes, g1902.t1  and g12953.t1 , were significantly up-regulated, while two other TOR pathway-related genes, g125.t1  and g2677.t1 , were significantly down-regulated. whereas no autophagy gene was not affected  were identified using a threshold gulated. A. This saffected B,C. Protectively D. Furtheectively E, suggesTo elucidate the potential action mechanism of the TOR pathway and 6PP at the molecular level, we conducted molecular docking analyses of the ligand (6PP) to the binding sites of PlYY1.t1, g12953.t1, g125.t1, and g2677.t. Our findings revealed that the main forces involved in the interactions were hydrogen bonding, van der Waals forces, and conjugate bonding A\u2013D. The A binding energy below \u22125.0 kcal/mol signifies a strong binding capability, with lower binding energy implying a more stable interaction between the ligand and molecule. The outcomes of the docking analysis indicate that four proteins possess potential binding sites for 6PP.PlYY1 versus control pGBKT7 indicated that the pGBKT7-PlYY1 construct did not impact yeast growth, as the undifferentiated growth was performed in the same diluted concentration solutions on the SD-Trp medium. Meanwhile, the pGBKT7-PlYY1 cassette had been successfully transferred to the yeast strain . The findings revealed a marked reduction in the mutants\u2019 pathogenicity compared to the WT strain.The contribution of 2 at 48 hpi, while the lesion areas infected by mutant strains \u2206PlYY1-1 and \u2206PlYY1-2 were approximately 0.40 and 0.42 cm2, respectively. Similarly, at 60 hpi, the lesion area infected by the WT strain increased to 1.71 cm2, whereas the lesion areas infected by mutant strains \u2206PlYY1-1 and \u2206PlYY1-2 were approximately 0.57 and 0.70 cm2 respectively , and Arabidopsis thaliana, YY1 functions have been linked to vegetative growth [P. litchii and other phytopathogenic oomycetes, including P. litchii. In this study, we demonstrated using transcriptional activation assays to confirm that the transcription factor YY1 in P. litchii binds to regulatory proteins associated with the TOR pathway, but further experiments are needed to confirm this speculation. Genetic deactivation of the PlYY1 gene triggered significant inhibition in vegetative growth, sporangia production, encystment, zoospore release, and virulence of P. litchii without altering oospore production. These findings indicated that PlYY1 is essential in vegetable growth and asexual reproduction in P. litchii. The current observations partly and independently validate previous reports linking the YY1 transcription factor to embryonic (peri-implantation) survival in mice [Previous studies showed that YY1 promotes cancer and tumor development in humans. Also, in e growth ,45,46,47 in mice  and as a in mice .PlYY1 defective strains to 6PP. These observations revealed PlYY1 as a probable novel target for 6PP. Additionally, we reasoned that 6PP influences vegetative development, cellular differentiation, and pathogenesis in P. litchii via PlYY1-mediated regulation of the TOR pathway.Similarly, we demonstrated that 6PP treatments triggered a significant enhancement in the expression pattern of the PlYY1 gene and compromised the sensitivity of P. litchii by identifying and analyzing TOR pathway-related genes expression, molecular docking, knocking out the PlYY1 gene, and the sensitivity of the PlYY1 gene to 6PP. These results will help improve our understanding of the mechanism response to 6PP stress and promote more effective disease management strategies in P. litchii. However, the function of TOR is complex in eukaryotes and provides clues into the pathological significance of the TOR in P. litchii.In the study, we preliminarily explored the response of the TOR pathway to 6PP stress in"}
+{"text": "Background: Despite the development of patient-centred or patient-reported outcome measures (PCOMs or PROMs) in palliative and end-of-life care over recent years, their routine use in practice faces continuing challenges.Objective: To update a highly cited literature review, identify and synthesise new evidence on facilitators, barriers, lessons learned, PCOMs used, models of implementation, implementation outcomes, costs, and consequences of implementing PCOMs in palliative care clinical practice.Methods: We will search MEDLINE, PsycINFO, CINAHL, Embase, Emcare, SCI-Expanded, SSCI, ESCI, and BNI. The database search will be supplemented by a list of studies from the expert advisory committee, hand-searching of reference lists for included articles, and citations of the original review. We will include primary studies using a PCOM during clinical care of adult patients with advanced disease in palliative care settings and extract data on reported models of implementation, PCOMs, facilitators, barriers, lessons learned, costs, and implementation outcomes. Gough\u2019s Weight of Evidence Framework will be used to assess the robustness and relevance of the studies. We will narratively synthesise and tabulate the findings. This review will follow PRISMA, PRISMA-Abstract, PRISMA-P, and PRISMA-Search as the reporting guidelines.Source of funding: Marie Curie. The funder is not involved in designing or conducting this study.Protocol registration: CRD42023398653 (13/02/2023) The importance of using outcome measures to understand the effect and effectiveness of health interventions has been growing as they are increasingly credited as an essential component of evidence-based clinical practice, specifically, outcome measures that the patient completes - so-called patient-reported outcome measures or PROMs - because of their specificity to patients\u2019 individual needs. Data collected at the individual patient level can be used immediately for patient-centred care, as it allows healthcare professionals to act on any distressing symptoms or palliative needs they might have. It can also inform shared decision-making and advance care planning . AdditioIn palliative and end-of-life care, it is equally important to learn about physical effects and psychological, existential, emotional and practical outcomes. However, implementing PROMs as a routine part of palliative care clinical practice has been slow and challenging in a number of countries. Compared to other settings and conditions, measuring outcomes in palliative care faces unique challenges. One reason is that patients\u2019 health is expected to deteriorate, and symptoms may worsen; deterioration will make the outcome measurement challenging, as well as changes in cognitive abilities towards the end of life. PROMs are impossible to use closer to the time of death once the patient becomes unable to communicate .Our previous review summarised barriers, facilitators, and lessons learned from the published palliative care literature and provided recommendations for implementing outcome measures at the patient, healthcare professional, and management and policy-makers levels for three timepoints: preparation, implementation, and assessment/improvement  to include both patient-reported and proxy-reported measures that focus on the concerns important to patients.Palliative Medicine and included the literature published between 1985 and 2013. In the last decade, there has been a plethora of publications demonstrating the exponential growth in palliative care outcome measurement. An update on these developments is warranted.The original systematic review (cited 307 times on 16 January 2023) was published in1.To identify the patient-centred outcome measures implemented in palliative care clinical practice;2.To identify the facilitators of implementing patient-centred outcome measures in palliative care clinical practice;3.To identify the barriers to implementing patient-centred outcome measures in palliative care clinical practice;4.To identify lessons learned on implementing patient-centred outcome measures in palliative care clinical practice;5.To identify the implementation models used when implementing patient-centred outcome measures in palliative care clinical practice;6.To identify what implementation outcomes were measured and how, when implementing patient-centred outcome measures in palliative care clinical practice;7.To assess the financial costs of implementing patient-centred outcome measures in palliative care clinical practice;8.To update the recommendations from our previous literature to inform the implementation process in palliative care clinical practice for stakeholders at different levels.th February 2023.This systematic literature review and narrative synthesis was reported based on PRISMA-P (Types of studiesPrimary research studies reporting information related to one of the eight objectives of this review outlined above. All types of evidence synthesis will be considered research studies and included.Types of participantsAdult patients (18 years old and over) with advanced diseases and receiving palliative and end-of-life care, their informal carers, and healthcare professionals.Types of settingAll settings in which palliative care is provided. As in the original review, we will include studies conducted both in specialist and non-specialist palliative care settings, provided that in the non-specialist settings, a palliative care measure was implemented.Implementation of outcome measures as the interventionThe included articles will report on the implementation of PROMs. PROMs are a form of outcome measure and comprise standardised, validated questionnaires that patients complete to measure their perceptions of their own health status and well-being .Types of studiesNarrative reviews, editorials, commentaries, and case reports will be excluded. If we identify relevant narrative reviews, we will check their references for eligible studies.Types of participantsStudies with a paediatric or mixed population where fewer than 50% of participants are 18 years old or over.Types of settingStudies with non-palliative care settings or mixed settings when fewer than 50% of participants are from palliative care settings.Implementation of outcome measures as the interventionStudies reporting data from routine outcome measurement without information on the implementation process as they relate to settings in which implementation has already taken place.Studies reporting exclusively on the development, testing and feasibility phases of new measures or validation of the linguistic translation of measures, as they do not relate to implementing measures in clinical practice.Electronic searchesMEDLINE,Embase, andEmcare via Ovid SP,CINAHL andPsycINFO via EBSCOhost,British Nursing Index via ProQuest,Science Citation Index-Expanded,Social Science Citation Index, andEmerging Sources Citation Index via Web of Science without any limitations to language, date, document type, or publication status.An information specialist will systematically searchThe searches were designed by an expert information scientist and peer-reviewed by another information scientist in collaboration with two clinical experts. The search strategies were tailored based on the included studies in the original review and tested against a new set of relevant studies supplied by the experts in MEDLINE before the agreement on the final search strategy.Extended data -Outcome reporter -Factors  related to the implementation-Implementation models used-Outcomes of implementation -Country-Date of research and publication-Age groups-Healthcare conditions -Setting -Scale of the study -Study design-Objectives of the study-Limitations and strengths of the study-Sample size-Notes (including relevant information not matching other data points)-Potentially relevant references-Lead author's name and email addressThe data extraction form will be designed, piloted and revised based on the following data points:We will initially use the form in Google Docs document format for piloting, and when finalised, we will use Google Sheets to extract data.Qualitative data will be extracted and analysed following the Guidance on the Conduct of Narrative Synthesis in Systematic Reviews  describing/guiding the process of translating effective interventions and research evidence into practice (process frameworks); (b) analysing what influences implementation outcomes (determinant frameworks); and (c) evaluating implementation efforts (outcome frameworks) . We willWe will consider and, where feasible, extract the PROGRESS plus : A generic and non-review-specific judgement about the coherence and integrity of the evidence in its own terms, i.e. the generally accepted criteria for evaluating the quality of this type of evidence.Weight of Evidence B (WoE B): A review-specific judgement about the appropriateness of that form of evidence for answering the review questions, i.e. the fitness for the purpose of that form of evidence.Weight of Evidence C (WoE C): A review-specific judgement about the relevance of the focus of the evidence for the review question, e.g. the relevance of the type of sample, evidence gathering or analysis in relation to the review question.These three sets of judgements can then be combined to form an overall assessment Weight of Evidence D (WoE D) of the extent that a study contributes evidence to answer a review question.Two reviewers will independently make the assessments, with any disagreement resolved by discussion and, if needed, by a third assessor.We have replaced the terminology patient-reported outcome measures (PROMs) with patient-centred outcome measures (PCOMs).Four new objectives in this update were not part of the previous review: implementation outcomes, costs, implementation models, and updating the recommendations. As a result of adding an objective to extract implementation models, we may have enough information to report on implementation processes using the multilevel framework for health program evaluation   outcome measures in palliative and end-of-life care. The study objective is the identification of new evidence on facilitators, barriers, lessons learned, used instruments, models of implementation, implementation outcomes, costs, and conseqences of implementing PCOMs. Therfore, the authors describe the search tools, types of studies, participants and settings. Proxy outcome measures will be considered in the presented review to include patients in bad conditions. The study protocol describes the exclusion criteria and search methods in detail. The description of the underlying disease (e.g. cancer vs non-cancer) would be helpful in the \"Data extraction\" section. The changes from the original systematic review are clearly described and relevant. In sum, the authors deal with an important update of the systamatic review for PROMs in palliative care. Therefore, I would accept this manuscript for indexing with only the described little minor revision.Is the study design appropriate for the research question?YesIs the rationale for, and objectives of, the study clearly described?YesAre sufficient details of the methods provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?YesReviewer Expertise:Palliative Care, patient reported outcome measurementI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This is a protocol describing a planned update of a systematic review. The justification for updating the review is sound, and the objectives are clear. In the Design section of the methods, four PRISMA reporting guidelines are referenced, including PRISMA-P (for systematic review protocols). I would imagine that this protocol should be reported according to PRISMA-P, with the review itself reported according to PRISMA and PRISMA-S. Other changes from the previous review were reported, however not consistently. I would like to understand which things changed, and also which remained the same. For example, in 'Data synthesis', you are using an implementation framework published after the original review. What did you previously use to guide data synthesis? The contrast would be helpful here. Overall, this will be an interesting update, which has been well-justified and well described.Is the study design appropriate for the research question?YesIs the rationale for, and objectives of, the study clearly described?YesAre sufficient details of the methods provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?Not applicableReviewer Expertise:Epidemiology, biostatisticsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above."}