diff --git "a/deduped/dedup_0353.jsonl" "b/deduped/dedup_0353.jsonl"
new file mode 100644--- /dev/null
+++ "b/deduped/dedup_0353.jsonl"
@@ -0,0 +1,45 @@
+{"text": "Within the peer-reviewed literature, associations between two things are not always recognized until commonalities between them become apparent. These commonalities can provide justification for the inference of a new relationship where none was previously known, and are the basis of most observation-based hypothesis formation. It has been shown that the crux of the problem is not finding inferable associations, which are extraordinarily abundant given the scale-free networks that arise from literature-based associations, but determining which ones are informative. The Mutual Information Measure (MIM) is a well-established method to measure how informative an association is, but is limited to direct (i.e. observable) associations.Herein, we attempt to extend the calculation of mutual information to indirect (i.e. inferable) associations by using the MIM of shared associations. Objects of general research interest  found within MEDLINE are used to create a network of associations for evaluation.Mutual information calculations can be effectively extended into implied relationships and a significance cutoff estimated from analysis of random word networks. Of the models tested, the shared minimum MIM (MMIM) model is found to correlate best with the observed strength and frequency of known associations. Using three test cases, the MMIM method tends to rank more specific relationships higher than counting the number of shared relationships within a network. This is reflected by the growth in the number of scholarly journals published every year as well as the number of total records indexed in biomedical literature reference databases such as MEDLINE[Most scientific fields are data-intensive, but perhaps even more so for biology and medicine. Sequencing efforts have generated billions of base pairs of genetic information across hundreds of thousands of species, and ushered in the relatively recent completion of the Human Genome Project. Microars MEDLINE. In any transitive\", \"implicit\", \"indirect\" or \"inferable\" relationships. Deciding that no relationship exists when no co-mentions exist is somewhat of an over-simplification, but a necessary one. Realistically, several co-mentions between terms could be observed without a definitive relationship present. However, if one uses a greater-than-zero cutoff to define when a relationship exists, false-negatives become a problem: Some co-mentions below the cutoff will constitute a real relationship. Using zero co-mentions as a cutoff is a convenience to avoid this problem even if the end result is that some relationships are declared \"known\" when they really are not.Most scientific knowledge comes from peer-reviewed articles and is written in free-form text, which is difficult to analyze algorithmically. However, the idea that novel relationships within text could be computationally identified based upon existing relationships has its roots in an approach developed by a researcher named Don Swanson, who used software to identify words shared between article titles . Using tWhile pioneering, a keyword-based method such as Swanson and Smalheiser's is both limiting and highly burdensome, especially where a large body of literature is concerned, because the number of unique keywords grows quickly per record analyzed. Neither is the method amenable to open-ended querying \u2013 that is, telling a user what is implicitly related to a query term. Rather, one must essentially begin by postulating a relationship between a query term, A, and another term, C, where a set of intermediate terms, B, can be found that connect the two. Even improvements in visualizing or exploring records that share commonalities and/or define entities of interest ,9 are liIt was previously demonstrated that, because the number of associations between terms follows a scale-free, or inverse power-law, distribution, the number of inferable associations with any given term rapidly approaches the maximum number of possible associations as the number of direct associations grows. That isimplied mutual information content of two unrelated terms.Previous work demonstrated that it was feasible to identify pertinent implicit relationships by ranking inferred relationships and preferentially examining those at the top of the list. One of The general approach to associating objects by searching for their co-occurrence within text has been used in many fields as a simple, yet comprehensive way to identify potential associations. In biology and medicine, co-occurrence has been used to identify potential relationships between genes ,14, protimplicitly associated with each other, and new associations can be potentially inferred by evaluation of their shared associations. Since there are many implicitly associated objects, the relevance of each one is also evaluated using the MIM. However, a MIM can be calculated to evaluate the relevance of an association between A and B and between B and C, but it is not clear how each of these individual scores extends to the inference of an association between A and C. Therefore, we explore and evaluate different methods.To begin a search for novel, inferable associations within the literature, relevant \"objects\" of interest in scientific research were first defined by assimilating database entries from relevant databases into one central database. By doing this, both words and phrases can be identified within text, and it permits synonymous terms to be mapped to primary terms. All electronically available literature was then analyzed for associations between objects of interest by searching for their co-occurrence within MEDLINE records (titles & abstracts), summing the total number found. The significance of this collective set of co-occurrences is evaluated using the mutual information measure (MIM), which was originally based upon Shannon's Entropy theory , but hasCode was written in Visual Basic 6.0 (SP5) using ODBC extensions to interface with an SQL-based database, with database queries written in SQL. Programs were executed on a Pentium 4 3.06 GHz machine with 1 GB of RAM and two ultra-fast SCSI hard drives. The National Library of Medicine graciously provided an electronic archive of MEDLINE records in XML format. To obtain a set of common words for analysis, the Merriam-Webster dictionary was parsed into individual words and each word summed by the number of times it was observed within the dictionary. 10,000 words were chosen with dictionary frequencies ranging from 322 to 28. This range was selected so that no extremely common or rare words would be within the list. To create a database of random word associations, only 100,000 titles/abstracts were used. This was done to avoid network saturation (i.e. having a significant number of objects related to every other object) and to ensure that the distribution in the number of associations between words resembled the same power-law distribution observed for biomedical objects.The occurrence of such objects within scientific text is identified by comparing phrases within MEDLINE records to entries in the object recognition database (ORD). This ORD is built by inputting terms found in several different biomedical databases, all freely available for download. Objects classified as diseases, disorders, syndromes or phenotypes were obtained from Online Mendelian Inheritance in Man (OMIM) ; chemicaAcronyms for entries, if not explicitly stated within the assimilated database, were obtained from an acronym database. SimilarA scoring scheme based upon the Mutual Information Measure (MIM)  is used AB is the measured probability that A and B will be observed together in the same abstract, while PA and PB are the probabilities of observing A or B, respectively, in a given abstract. Furthermore, because scientific research and discovery is a time-dependant process, prior information can be incorporated to refine the probabilities in Equation (1). The describing of a disease or discovery of a gene, for example, will occur at a given point in time  represents the larger of the two values between Af and Bf. And At is the total number of records processed.Where TMIM in Equation 1 then evaluates to 1 and the log value to zero \u2013 the information gained on one object by observing the other. If the probability of observing A increases when B is mentioned, then MIM > 0. If A and B are rarely mentioned together, then MIM < 0. When considering scientific writing style with reference to biomedical objects such as genes, diseases and chemical compounds, there is a probability that two of them might be mentioned together in the same record without having an established association. For example, one of the objects may be very commonly used in many studies , or one of the objects may be of great scientific/medical interest and authors may make an extra effort to speculate how their results might relate to such objects . The MIM provides a way of quantifying literature-based object dependencies. However, taking the log value can provide a negative weighting to an association when two frequent terms are mentioned together. Optimally, irrelevant or uninformative associations  would be ignored entirely rather than penalized. Therefore, the log function is removed and the equation becomes:As an example of how the MIM score is used, assume that the probability A will appear in any given record within a database of records is 10% and the probability of B appearing is the same. If the appearance of A is completely independent of the appearance of B then no information about one can be gained by observing the appearance of the other. The probability both A and B will be observed in the same record is thus 0.1*0.1 = 0.01. The value of The possibility remains that rare associations might receive a very high MIM score , but it t objects in this set, each individual object can be given the symbol \"Bn\", where 0 <n <t. For each Bn, there is another set of objects related to it by literature co-occurrence, called \"C\". Each object in the set C may or may not be connected to the primary object, A. That is, an association may consist of A\u2194B\u2194C where an object in the set C also belongs to the set B. The symbol \"\u2194\" is used here to represent the existence of a non-directional association between two objects.Figure n and Bn-C associations. Two models are thus proposed and evaluated, the numeric score obtained by any one of them will only be relevant in terms of how well it assigns a relative importance to each A-C connection within a list.Objects in the set C having no literature-based association with the primary object, A, represent associations that have not been previously made, or at least documented, by others. These represent new associations that can potentially be inferred by virtue of their shared associations. Because the number of implicit associations rapidly increases with each established association, the goal here is to provide a quantitative measure of the strength of an implicit association based solely upon the associations shared by two objects. After all, if no known relationship is documented, then these shared associations will be the only way to understand the nature of an relationship between A and C. Since directly associated objects also share associations with other objects, it is reasoned that the strength of known associations can be used to benchmark how well the scores from implicit associations correlate with the relative importance of an association. However, it is not clear how A-C relationships are best evaluated given a set of component A-Bn and Bn-C MIM association is averaged over a total of t shared connections and then finally divided by t to normalize the total score by the total number of connections. The function for the normalized averaged MIM (AMIM) model is:The first model to be tested assumes that the total information content of an implied A-C association can be approximated by the mutual information measure of each component connection. Thus, the MIM scores for each A-BAs model by which A-B and B-C values were summed was also considered, but it would be functionally indistinguishable from the AMIM model in terms of ranking implicit relationships, so it was not included.n association as well as the Bn-C connection, and the information potential an A-C connection will be no greater than the least mutual information given by A-Bn or Bn-C. This is equivalent to assuming that a chain can be no stronger than its weakest link. The equation for the normalized minimum MIM (MMIM) model is:The second model views the process of inferring an A-C connection as function of each of its component processes, limited in its potential by the mutual information in each step of the inference process. That is, inferring an A-C connection depends upon how much information is in the A-BA total of 12,899,016 MEDLINE records recorded from 1967 to May 2003 were processed in chronological order to create a network of 10,873,926 associations between a total of 112,805 unique objects assimilated from the databases mentioned. When including synonyms, the total number of recognizable phrases for these unique objects was 223,540 .The distribution of objects found in MEDLINE ranges from more general categories  that are found in a higher percentage of records  to the more specific. The frequency of objects when plotted follows a power-law distribution and resembles that of a scale-free network, which is reasonable given that new objects are typically studied in terms of their relationship to known objects .Records were chosen for analysis due to their electronic availability and are also because they are a good source of pertinent information due to their brief, focused nature that presumably contains a summary of the most important findings in each report. Several objects were examined to see if associated objects with high MIM scores correlated with the relative importance of the association. This was done by obtaining summary descriptions of an object from various authoritative sources such as review articles, glossaries or biomedical databases. Table Table known, relevant associations are ranked within the set of all inferable associations, the better the ranking method is.When ranking inferred associations, the goal is for the score assigned to an inference to correlate well with the amount of mutual information gained from any given association. The only reference basis for this is the mutual information contained within established associations. Since they too will frequently contain shared associations, they can be evaluated independently using only their shared associations Figure . Severalknown relationships is added together, in order from highest MIM score to lowest, to reflect the fastest rate by which they could be discovered. When plotted, this curve is what would be observed were mutual information preserved exactly (the \"exact\" curve). Because it's neither expected that all possible relationships are known, nor that mutual information is static as the scientific discovery progresses, it is not anticipated that this curve would or even should be followed exactly . However, it is reasonable to expect established relationships with high mutual information content to retain a relatively high mutual information content when evaluated on the basis of its shared relationships. Thus, it is expected that the implicit MIM curve follow the \"exact\" MIM curve.To evaluate this, a graph is drawn to reflect the rate that established relationships are discovered within the set of all objects analyzed. The total of all MIM scores for Figure To gain a better quantitative estimate of performance, 50 objects were chosen at random from both the MEDLINE and random word databases. Each object was analyzed to identify and rank other objects that shared relationships with it as described and the area under the curve (AUC) was taken for each of the ranking methods shown in Figure A peculiar effect was noted with the average MIM-based scoring model: Some implicitly associated objects received higher MIM scores than the primary object itself, which is also analyzed as a control. There tend to be relatively few, sometimes none, such instances per analysis, but it occurs when a relatively rare object shares several or more associations with the primary object. This effect was not present in the minimum MIM model.The scores assigned by inference methods so far have no meaning by themselves, but only as a means of ranking the potential relevance of an inference. Because the majority of database objects will be present in the list of implicit connections, the question naturally arises as to where a significance cutoff can be drawn. A range of significance for a given MIM score can be estimated by analysis of a random word network in which we would expect that meaningful relationships are only encountered by chance.t random associations and dividing by t would also be expected to have an average  MIM score of 1. This is true for any set of A-B associations as well as a corresponding set of B-C associations, thus an average value of 1 should still be obtained when calculating the average minimum MIM score. Figure Since the MMIM model performed slightly better than the AMIM model, we evaluated it using the random words database. Words in the random network were effectively chosen at random from the Merriam-Webster dictionary , and so relevant associations between these words co-occurring within MEDLINE records should occur predominantly by chance. An uninformative association  would have an average MIM score of 1 (e.g. see Equation 5). Thus, summing a set of ileum is frequently used to test capsaicin effects because of its contractile response. Tachykinins Substance P and Neurokinin A as well as the neurotransmitter acetylcholine[tetrodotoxin[atropine[Using the example of capsaicin brought up earlier, we analyzed it using the methods described to identify and rank objects sharing relationships with it Table . When raylcholine are resprodotoxin or atrop[atropine. These iper se. To do so presumes that Swanson's initial study was the \"correct\" way of finding relevant implicit relationships and Swanson did not employ the open-discovery model in these examples anyway. It would be useful to know, however, where Swanson's predictions rank among others using models in which implicit relevance is judged by counting the number of shared relationships and where it is evaluated by the MMIM. Both Raynaud's and Migraine headaches were analyzed as starting objects (A), the goal being to find all C objects that share relationships and rank them by their relevance. Both known and implicit relationships were displayed. The top 10 results are summarized in Table Finally, we also sought to re-evaluate some of Swanson's original hypotheses as has been done in other text-mining studies ,11,30. IWhen ranking implicit relationships by the number of shared relationships, fish oil scored #1025 in the Raynaud's list and magnesium (the link Swanson found with migraines ) scored sumatriptan is a drug used to treat migraines and other items ranking highly on the list such as nausea, vomiting, and dizziness typically accompany migraines. Notably, one of the important links that Swanson used to surmise the role of magnesium is also on this list: Seizures, which cause migraines.Examining the relationships that tend to rank highly in both models it is apparent that, when ranking by the number of shared relationships, the higher-scoring entries tend to be more general and vague in nature . When ranked by the MMIM, their relevance to the object in question is more readily apparent. For example, Information retrieval (IR) methods are limited to querying what is known; yet often the most valuable information is what is not directly known. Mutual information measures have been used successfully in many IR applications, and a method has been presented here to extend it to inferable associations. We find that the normalized MMIM method of ranking inferences based upon their shared associations correlates best the level of currently established mutual information. A good correlation is suggestive that mutual information is being captured even though evaluation proceeds indirectly, through intermediates. For simplicity, we have used a cutoff of zero co-occurrences to suggest that no association between objects has been made, but it is quite possible that a number of co-occurrences could be noted between two objects yet no specific relationship between them documented. Or additionally, a certain relationship may be known between the two, but other important relationships still remain to be inferred. At this point, however, it is not clear how this would effectively and quantitatively be taken into account.The method reported was applied to biomedical research, but could ostensibly be applied to any domain in which the goal is to identify undiscovered relationships. Importantly, this method of automated inference ranking provides a quantitative way of prioritizing inferred associations when available literature is growing rapidly in size and scope."}
+{"text": "The effect of selective PDE-I (vinpocetine), PDE-III , PDE-IV , and PDE-V (zaprinast) isozyme inhibitors on TNF-\u03b1 and IL-1\u03b2 production from LPS stimulated human monocytes was investigated. The PDE-IV inhibitors caused a concentration dependent inhibition of TNF-\u03b1 production, but only partially inhibited IL-1\u03b2 at high concentrations. High concentrations of the PDE-III inhibitors weakly inhibited TNF-\u03b1, but had no effect on IL-1\u03b2 production. PDE-V inhibition was associated with an augmentation of cytokine secretion. Studies with combinations of PDE isozyme inhibitors indicated that PDE-III and PDE-V inhibitors modulate rolipram's suppression of TNF production in an additive manner. These data confirm that TNF-\u03b1 and IL-1\u03b2 production from LPS stimulated human monocytes are differentially regulated, and suggest that PDE-IV inhibitors have the potential to suppress TNF levels in man."}
+{"text": "Despite the potential impact on treatment adherence and recovery, there is a dearth of data on the extent and correlates of internalized stigma in patients with schizophrenia in low income countries. We conducted a study to determine the extent, domains and correlates of internalized stigma amongst outpatients with schizophrenia in Ethiopia.The study was a cross-sectional facility-based survey conducted at a specialist psychiatric hospital in Addis Ababa, Ethiopia. Consecutive consenting individuals with a diagnosis of schizophrenia were recruited and assessed using an Amharic version of the Internalized Stigma of Mental Illness (ISMI) scale.Data were collected from 212 individuals, who were mostly single (71.2%), unemployed (70.3%) and male (65.1%). Nearly all participants (97.4%) expressed agreement to at least one stigma item contained in the ISMI; 46.7% had a moderate to high mean stigma score. Rural residence , single marital status  and having prominent psychotic symptoms  were associated independently with a higher stigma score. Almost half of those who discontinued their treatment reported that they had done so because of perceived stigma. Those who had attempted suicide (45.3%) were more likely to have a high stigma score . Over 60% of the variation in the experience of stigma was explained by four factors: social withdrawal (16.7%), perceived discrimination (14.1%), alienation (13.9%) and stereotype endorsement (12.7%).Internalized stigma is a major problem among persons with schizophrenia in this outpatient setting in Ethiopia. Internalized stigma has the potential to substantially affect adherence to medication and is likely to affect the recovery process. Whilst most mental illnesses may be associated with some degree of stigma, schizophrenia has been described as \u201ca modern day equivalent of leprosy\u201d, conferring unparalleled social ostracism to the person with schizophrenia and their family . Stigma Furthermore, two major classes of stigma are recognized: public stigma and personal stigma. Public stigma  typically describes the endorsement of stereotypes and enacted behaviours against people with mental illness by professionals and the general population ,3. Perso(i) Perceived stigma: what the individual thinks are society\u2019s beliefs about the stigmatized group(ii) Experienced stigma: actual discrimination experienced(iii) Self-stigma: a product of the internalization of public stigmaIn internalized stigma or self-stigma, a gradual process of psychological assimilation of the public stereotypes towards mental illness is said to occur such that the person with mental illness progressively loses what they think they are and wish to be in the future . In esseStigma is a major problem across different societies, but the particular manifestations of stigma may vary. Variation in the interaction between stigma and mental illness is evident in different settings . Stigma Most of our understanding about internalized stigma originates from high-income countries. Yet the few available studies have demonstrated high levels of internalized stigma amongst patients with schizophrenia in some African countries: Ghana , NigeriaSystematic investigation of the extent and correlates of internalized stigma is an essential part of planning for recovery programs for schizophrenia. This research is timely for the Ethiopian setting and other low-income country settings as mental health services are undergoing a shift towards primary health care and community settings . CommuniThe primary objective of the study was to assess the extent of internalized stigma among patients with schizophrenia attending the outpatient department of a psychiatric hospital in Ethiopia. Related to this we aimed to determine which socio-demographic and clinical factors are associated with the experience of stigma and to explore the potential impact of stigma on medication adherence and risk behaviour; important factors that may affect the recovery process.The study was a facility-based cross-sectional survey conducted at a psychiatric hospital located in the capital city, Addis Ababa. The hospital is the oldest and largest public psychiatric hospital in Ethiopia. Patients from all over the country come to the hospital to receive treatment for severe mental disorders. The hospital service is organized under specialist programs or specialist case teams. Each case team is expected to have expert knowledge about the disorders it treats. Patients with schizophrenia are treated by psychoses case teams and this case team manages all individuals with schizophrenia referred or seen by the hospital. The hospital has an inpatient service with 280 bed and a large out-patient service, with around 115,000 outpatients visiting the hospital each year.Participants of this study were patients with a clinical diagnosis of schizophrenia, aged 18 years and above. Potential participants were selected by reviewing the medical records of patients attending regular follow-up appointments. Individuals with clinically established impairment of insight, a significant level of cognitive impairment or significant level of substance abuse in the previous three months were excluded based on a review of clinical records and clinical assessment by senior psychiatric residents. We focused on schizophrenia in order to have a relatively uniform group of participants. Individuals with substance abuse were excluded because of the potential impact of substance abuse on the experience of stigma.The Internalized Stigma of Mental Illness (ISMI) Scale for the assessment of stigma  was tranIn addition to the ISMI, participants were asked simple questions in relation to whether they experienced stigma within their family, in the neighbourhood and whether they felt disrespected by the mental health staff because of their illness.Furthermore, we asked two questions on medication adherence and one on suicide attempts. The medication adherence question asked about history of non-adherence with psychotropic medications and whether the non-adherence behaviour was linked to stigma. Specifically we asked \u201cHave you ever discontinued medication\u201d? and \u201cDid your experience of stigma contribute to your decision to discontinuing medication?\u201d. The question on suicide simply asked whether the participant had felt so desperate that they had attempted suicide (\u201cHave you ever felt so desperate that you even attempted to harm yourself or end your life?\u201d). This question is similar to the single question used in asking about suicide attempt in the widely used structured diagnostic interview instrument of the World Health Organization, the Composite International Diagnostic Interview . The occThe 24-item ISMI scale, which has been validated and used in an outpatient setting for patients with schizophrenia , was traAmharic) by senior psychiatric residents. Although the ISMI is self-administered instrument, the ISMI questions were directly asked by the residents. Because of the variable level of literacy it was deemed necessary to use interviewers who read the questions to the participants.After inclusion criteria were met, all instruments were administered in the local language  and from urban areas (79.7%). The mean age of the participants was 33.3 years (SD 8.9). Nearly three-quarters (70.3%) were unemployed although most had some level of education, and 20.8% were college graduates. Only 25.9% were married (Table\u20092 (276)\u2009=\u20092496.0, p\u2009<\u20090.001). Finally, the communalities were all above 0.43. Only factors with Eigen values of one or above were included. Four factors were identified from the factor analysis: social withdrawal, perceived discrimination, alienation and stereotype endorsement. These factors explained 61.9% of the total variance: social withdrawal 16.7%, perceived discrimination 14.1%, alienation 37.9% and stereotype endorsement 12.4%  expressed strong agreement to at least one internalized stigma item  were rural residence , single marital status  and having prominent psychotic symptoms  . The association between medication non-adherence and suicide attempt was of borderline statistical significance . For those discontinuing their medication, stigma was the most important factor associated with discontinuation of their medication  after controlling for the experience of extrapyramidal side effects, educational status, symptom level, residence, employment, age and sex.This study demonstrates the high burden of internalized stigma among patients with schizophrenia attending a specialist hospital in Ethiopia. Almost all participants had experienced at least some level of internalized stigma and about three-quarters endorsed strongly at least one internalized stigma item. The experience of stigma in each stigma domain was also high (50 to 72%) with no significant difference within categories.This is the first study in Ethiopia to investigate internalized stigma among patients with schizophrenia and the second addressing the issue of stigma among patients with schizophrenia. This study is also one of the very few studies dealing with internalized stigma from sub-Saharan Africa.The current study replicates findings from previous studies of stigma relating to mental illness from sub-Saharan Africa -35 and AOne of the explanations for the apparently favourable outcome of schizophrenia in low-income countries, is the presumed low prevalence of stigma in these settings . This asFindings from this study support the hypothesis that stigma is an important obstacle in the recovery process. Nearly half of all patients who were non-adherent to their medication attributed their decision to discontinue treatment to stigma. This group was predominantly among those with higher level of stigma score. Treatment adherence is the most important predictor of positive outcome in severe mental disorders in low-income countries ,44. StigAdditionally we found that a higher stigma score was associated robustly with a history of suicide attempt, an important negative outcome of severe mental disorders. Therefore, tackling stigma should be an important public mental health intervention priority in low income countries. Strategies to address stigma should be incorporated into initiatives to scale up mental health services in low and middle-income countries .A higher level of internalized stigma was associated with three factors: having rural residence, single marital status and prominent psychotic symptoms. Rural residence has been found previously to be associated with higher stigma , and wasThe association of internalized stigma with psychotic symptoms may be attributed to \u201creverse causality\u201d. Patients with higher levels of psychotic symptoms may be more likely to perceive negative attitudes from others, related to their psychopathology. On the other hand, uncontrolled symptoms may attract more attention to the affected person and this may lead to discrimination.There are two pathways in which uncontrolled symptoms may act as barriers to recovery: through reduced adherence to treatment because of denial and fear of stigma, and the psychological impact of stigma. Although not robustly associated with internalized stigma, the trend in this study is that education may be protective against stigma, particularly stereotyped endorsement. Again this is in line with findings of studies in other settings -54.The study design has inherent weaknesses, which were difficult to avoid because of feasibility issues. The cross-sectional design meant that it was not possible to infer causality from the findings. The lack of longitudinal analysis in relation to factors associated with internalized stigma has been highlighted previously . The stuThis study suggests that a significant burden of stigma is borne by patients with schizophrenia in Ethiopia. There is sufficient data now from low-income countries to suggest that a low prevalence of stigma cannot explain the presumed favourable outcome of schizophrenia in low-income countries. The current report, along with other similar data from Ethiopia and other low-income countries, supports the need to incorporate culturally appropriate methods of addressing internalized stigma into rehabilitation packages for this group. Particular attention should be paid to the potential for self-stigma to inhibit help seeking and treatment adherence.All the authors declared no competing interest.DA, TS and AF were involved in the design of the study and all authors were involved in the analysis of data and preparation of 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/1471-244X/12/239/prepub"}
+{"text": "Dental caries is a chronic disease which can affect us at any age. The term \u201ccaries\u201d denotes both the disease process and its consequences, that is, the damage caused by the disease process. Dental caries has a multifactorial aetiology in which there is interplay of three principal factors: the host , the microflora (plaque), and the substrate (diet), and a fourth factor: time. The role of sugar (and other fermentable carbohydrates such as highly refined flour) as a risk factor in the initiation and progression of dental caries is overwhelming. Whether this initial demineralization proceeds to clinically detectable caries or whether the lesion is remineralized by plaque minerals depends on a number of factors, of which the amount and frequency of further sugars consumption are of utmost importance. This paper reviews the role of sugar and sugar substitutes in dental caries. To most people the term \u201csugar\u201d refers to the common household foodstuff table sugar (sucrose). Yet sucrose is only one of many naturally occurring sugars used in the human diet. Technically the term \u201csugars\u201d applies to two classifications of carbohydrates: free-form monosaccharides (simple sugars) which include the more common glucose, fructose, and galactose and disaccharides (two simple sugar molecules linked together) which include the most common sucrose, lactose, and maltose. Naturally occurring sugars are available in fruits, vegetables, grains, and dairy foods. Sweeteners are added sugars that are used as ingredients to both satisfy our taste and in some cases provide added energy. Grouping sweeteners as \u201cnutritive\u201d or \u201cnonnutritive\u201d acknowledges a difference in the amount of energy provided by the sweetener. Nutritive sweeteners may be referred to as caloric and include sugars and sugar alcohols. Nonnutritive sweeteners offer no energy and can sweeten with little volume. Both sugar alcohols and nonnutritive sweeteners can replace the sugars and are sometimes referred to as sugar substitutes, sugar replacers, or alternative sweeteners . We searOf the many factors that contribute to the development of dental caries, diet plays an important role. Fifty years ago dietary issues relevant to dental caries were largely concerned with dietary sugars. Although sugars are undoubtedly the most important dietary factors in the etiology of dental caries, today's diet contains an increasing range of fermentable carbohydrates, including highly processed starch-containing foods and foods that contain novel synthetic carbohydrates such as oligofructose, sucralose, and glucose polymers. Coupled with this, there now exists a wide range of noncariogenic sweeteners that have an important role to play in caries control. Research continues to identify foods and factors that protect against dental caries and those that have a practical dietary application and can help to make dietary advice more positive, which aids compliance .Sucrose for years was billed as the \u201carchcriminal\u201d of dental caries because it was considered to be so much more cariogenic than other sugars . HoweverStreptococcus miutans have reported increased cariogenicity of sucrose compared to other sugars; however, this effect appears to be bacterial strain-specific and not consistent across different animal models. More recent clinical studies have indicated that the caries-associated virulence of glucan may have more to do with an alteration in plaque ecology by increasing the porosity of plaque, permitting deeper penetration of dietary sugars and greater acid production adjacent to the tooth surface [Sugars can be readily metabolized by many bacteria involved in dental biofilm formation, generating acid byproducts that can lead to demineralization of the tooth structure. Lactose (milk sugar) has been shown to be less acidogenic than other sugars and less cariogenic, based on animal studies . Sucrose surface .It is generally accepted that the prevalence of caries is related to the form in which sugar is ingested and the frequency of its consumption. By \u201cform\u201d we meant the physical consistency of the sugar-containing foods. Distinctions are made between liquid and adhesive (sticky) foods as well as foods which vary in adhesiveness between the extremes. The term \u201cfrequency\u201d refers to the number of times per day that sugary foods are eaten. It is clear that both form and frequency affect the length of time that teeth are exposed to sugar . HoweverThe relationship of the physical consistency of food to caries is not entirely clear. Several studies have incriminated the stickiness of foods as prime factor in the initiation of caries , 12. OthAlthough a high intake frequency increases the overall length of time that the teeth are exposed to sugars, it does not give complete information on the total time of exposure. The total cariogenic load is also determined by the form of the food product; that is, the physical consistency of the sugar-containing foods affects their retention time in the mouth. Distinctions can be made between liquids that are cleared rapidly and adhesive (sticky) foods that vary widely in retentiveness. Particularly high retention rates have been found for products such as sweet biscuits, crackers, and potato chips (crisps) . Other aSaliva plays a major role in protecting the teeth, owing to its cleaning actions as well as its acid neutralizing, antisolubility, and antimicrobial properties. A high secretion rate, together with mastication, helps to eliminate sugars and food particles from the oral cavity. A short clearance time reduces the length of time that sugar is available for acid production by the bacteria in the dental plaque. During hyposalivation, which can be found in relation to irradiation in the head and neck area, Sjogren's syndrome, surgery or medication, or in older age groups with poor health, an increased caries rate is often found. Lanke  performeThe development of noncaloric sugar substitutes, marketed for weight control, is big business in the United States. Commercial development of these products, from the laboratory to marketing, is time consuming and expensive. Aspartame, a dipeptide composed of two naturally occurring amino acids, became available in the United States in 1982 . One of It is imperative to remember that the usefulness of a sugar substitute has to be looked upon not only from a cariological but also from a nutritional, toxicological, economic, and technical point of view. When evaluating a nonsugar sweetener in relation to dental caries, it is important to consider the potential for metabolism by oral microorganisms and dental plaque, the influence of consumption on cariogenic microorganisms, and the risk of microbial adaptation to the sweetener. Sugar substitutes can be categorised into two major groups: intense sweeteners  like aspartame, saccharin, sulfame, glycyrrhizin, and so forth and bulk sweeteners  like sorbitol, xylitol, mannitol, and so forth . IntenseAlbeit sugar is associated with the dental diseases like dental caries, we emphasize the fact that sugar alone is not the sole determinant of these diseases. To prevent dental diseases, oral health care workers should persuade their patients to adopt special dietary programs and educate patients and motivate them to alter their customary dietary behavior. Furthermore, such health education must compete with the food manufacturers marketing techniques to significantly reduce dental caries in the population at large."}
+{"text": "Self stigma among people with mental illness results from multiple cognitive and environmental factors and processes. It can negatively affect adherence to psychiatric services, self esteem, hope, social integration and quality of life of people with mental illness. The purpose of this study was to measure the level of self stigma and its correlates among people with mental illness at Jimma University Specialized Hospital, Psychiatry clinic in southwest Ethiopia.Facility based cross-sectional study was conducted on 422 consecutive samples of people with mental illness using interviewer administered and pretested internalized stigma of mental illness (ISMI) scale. Data was entered using EPI-DATA and analysis was done using STATA software. Bivariate and multivariate linear regressions were done to identify correlates of self stigma.On a scale ranging from 1 to 4, the mean self stigma score was 2.32 (SD\u2009=\u20090.30). Females had higher self stigma  than males. Patients with a history of traditional treatment had higher self stigma . There was an inverse relationship between level of education and self-stigma . Perceived signs  and supernatural causes of mental illness  were positively correlated with self stigma. Higher number of drug side effects were positively correlated  while higher self esteem was negatively correlated  with self stigma.High feeling of inferiority  but less agreement with common stereotypes (stereotype endorsement) was found. Female showed higher self stigma than male. History of traditional treatment and higher perceived supernatural explanation of mental illness were associated with higher self stigma. Drug side effects and perceived signs of mental illness were correlated with increased self stigma while education and self esteem decreased self stigma among people with mental illness. Patient empowerment psychosocial interventions and strategies to reduce drug side effects can be helpful in reducing self stigma among people with mental illnesses. Stigma against people with mental illness is a complex public health problem which exists in different forms and many actors like the public, family members, media, patients themselves and even sometimes the health providers are involved-3. StudiWhen people with mental illness bear high level of self stigma, they may have less resistance capability to public stigma, and thus submissive to discriminatory behaviors so that it negatively affects the rehabilitation and treatment processes of the patients. For instance, more than two thirds of people with mental illness in England reported that they have stopped doing things they wanted to do because of self stigma. Two thirds of people with mental health problems live alone about four times more than the general population.Systematic reviews of stigma identified that combating wrongly held beliefs about mental illness, improving self-esteem, empowerment (education), help seeking behavior, protesting stigma and advocacy for mental health as the most important self stigma reduction strategies concerning the patients. In these reviews, targeting high risk groups was suggested to combat self stigma among people with mental illness,14. HighStudies conducted using the internalized stigma of mental illness (ISMI) scale in Europe and Iran reported high prevalence of self stigma among people with schizophrenia,17. A stThose patients who receive modern psychiatric treatment are expected to have lower self stigma if they pass through a systematic psychosocial approach beside the biomedical treatment model process. But the level and correlates of self stigma among new and follow-up psychiatric patients in southwest Ethiopia particularly in Jimma University Specialized Hospital (JUSH) Psychiatry clinic attendants has not been investigated. The main purpose of this study was hence, to measure the level of self stigma and its correlates among JUSH, Psychiatry clinic attendants of people with mental illness in southwest Ethiopia.Hospital based cross-sectional study was conducted from June to August 2012 in Jimma University specialized hospital (JUSH) among psychiatric service attendants. JUSH is a teaching and referral hospital located in Jimma city 352\u00a0km southwest of Addis Ababa, Ethiopia. Each year, the hospital serves for approximately 9, 000 inpatients and 80,000 outpatients with a catchment population of about 15 million. PsychiaRepresentative sample of 422 consecutive new and follow-up psychiatric services attendants were included in this study. The sample size was determined using single population proportion formula by assuming 50% level of self stigma to get the maximum sample size, at 95% confidence level and considering and a 5% margin of error and non-response contingency. Respondents were screened using the Clinical Global Impression (CGI) scale to assess their eligibility to participate in the interview for the study. The scaData was collected by trained psychiatric nurses at JUSH, Psychiatry clinic through interviewer-administered questionnaires and a patient chart review to identify their diagnosis and other medical information. The data collection was supervised by specialist mental health workers. Data collectors and the supervisors were trained on the contents and procedures of the data collection.Alienation is \u201cthe subjective experience of being less than a full member of society\u201d. The Stereotype Endorsement is \u201cthe degree to which patients agreed with common stereotypes about people with a mental illness\u201d. The Discrimination Experience measures \u201crespondents\u2019 perceptions of the way they tend to be treated by others\u201d. The Social Withdrawal measures the self exclusion from social events/situation due to mental illness\u201d. The Stigma Resistance subscale is \u201ca person\u2019s ability to resist stigma\u201d[self stigma score was obtained by summing the scores of the five subscales. Higher score showed higher self stigma.To measure self stigma among the patients, the Internalized Stigma of Mental Illness (ISMI) Scale was used stigma\u201d. Unlike A study in Iran showed that the ISMI subscales had reliability values  of . In the current study, the following reliability values  were found: alienation\u2009=\u20090.84, stereotype endorsement\u2009=\u20090.73, discrimination experience\u2009=\u20090.79, social withdrawal\u2009=\u20090.77, stigma resistance\u2009=\u20090.65, over all self stigma\u2009=\u20090.89.self esteem was measured using the Rosenberg self-esteem scale[In addition to the ISMI scale, em scale. The scaThe whole questionnaire was translated and administered in local languages (Affan Oromo and Amharic) and it was back translated to English to ensure semantic equivalence. The questionnaire was also pre-tested in the psychiatric clinic before the main study. Based on the pre-test, some items were modified and more clarifications were given to the data collectors on items which were not understood well.t test and ANOVA) of stigma scores and other variables were done for each of the five subscales of ISMI separately and for the overall self stigma scores. Six separate multivariate linear regression models were developed using variables which had significant statistical associations with the respective subscales and the overall self stigma scores during bivariate analysis. Unadjusted and adjusted standardized regression coefficients were presented for each variable in each model. A P-value <0.05 was used to declare significant statistical association. Multicollinearity between variables was checked using tolerance analysis (variance inflation factor).After checking for the completeness of each questionnaire, data entered was done using EPI-DATA version 3.1 and then exported to STATA version 10.0 for analysis. A frequency table was computed for socio-demographic and other variables. Stigma scores were checked for normal distribution. Tests of significant mean differences  were urban residents. Two hundred and ninety six 70.14%) of the respondents were male. The mean age was 33.11 (SD\u2009=\u200911.37) years. Two hundred and nine (49.53%) of them were single in marital status. Majority were Muslim religion followers (59.24%) and Oromo ethnic groups (60.43%). One hundred and eighty six (44.08%) of them were in secondary educational status and majority were farmers (28.44%) and private enterprise workers (25.12%). Average family size was 5.37 (SD\u2009=\u20092.72). The average family monthly income was about 74.70 (SD\u2009=\u2009120.15) USD (Table\u00a04% of theThe majority of the patients were diagnosed with mood (49.05%) and psychotic (36.02%) disorders. The remaining 9.00% and 5.92% were diagnosed with anxiety and other disorders  respectively. Beside their psychiatric diagnosis, 19 (4.50%) had co-morbidities and 194 (45.97%) reported some kind of side effects of their medication. In addition, the health providers identified a mean of 2.53 (SD\u2009=\u20090.97) number of side effects attributed to the patients\u2019 medications. The mean time since the onset of the patients mental illness was 5.87 (SD\u2009=\u20094.80) years while the mean time since the start of medical follow up was 4.55 (4.25) years. Mean number of visits to the psychiatric hospital was 21.51 (SD\u2009=\u200922.56). Two hundred and sixteen (51.18%) ever had experience of traditional treatment before seeking help at the psychiatric clinic.Seventy six 18.01%) had family/relative with a history of mental illness episodes. Regardless of the contents of the messages, 16.59%, 15.17% and 3.08% watched and heard about mental illness on television, radio and in religious places respectively in the period of one year before the time of data collection. Stress, rumination and drug addiction were the leading perceived causes of mental illness and sleep disturbance, talking to oneself and showing strange behaviours were the top three perceived signs of mental illnesses  score was 2.46(SD\u2009=\u20090.50). Females had significantly higher alienation  than males. Those patients who ever had traditional treatment had also higher alienation . Higher education level was significantly correlated with lower alienation  while higher scores of perceived supernatural causes of mental illness was significantly correlated with increased alienation . As the duration treatment increased, alienation score decreased significantly . This model explained 15% of the variance of alienation.The mean score for agreeing on the common stereotypes about people with a mental illness was 2.20 (SD\u2009=\u20090.34). Compared with farmers, private enterprise workers , government employees  and students  had significantly lower stereotype endorsement scores. Patients with higher education  and higher self esteem  had lower stereotype endorsement. Higher perceivement of supernatural causes of mental illness was correlated with higher stereotype endorsement . The model explained 13% of the variance in stereotype endorsement.Mean perceived discrimination score was 2.28 (SD\u2009=\u20090.42). As education level increases, discrimination experience score decreases significantly . Respondents with higher score in perceived supernatural causes  and higher number of drug side effects  had higher discrimination experience scores. The explained variance of this model was 10%.Mean score for self exclusion from social events was 2.27 (SD\u2009=\u20090.38). Experiences with traditional treatment were significantly associated with an increase in social withdrawal score . Significant decrease in social withdrawal was observed as the educational status of individuals increased  while there was a significant increase in social withdrawal when the score in perceived supernatural causes of mental illness increased . The model explained only 7% of the variance in social withdrawal.The mean score for stigma resistance subscale was 2.41 (SD\u2009=\u20090.40). Patients with substance related disorders and personality disorders  had significantly better stigma resistance than patients with diagnosis of mood disorder. Patients with higher education  and higher self esteem  had better stigma resistance compared with their counterparts. This model explained 20% of the variance in stigma resistance . Among the total respondents, 25.12% of them showed 2.5 and above self stigma score. Compared with males, females had higher self stigma . Private enterprise workers had significantly lower self stigma  than farmers. Patients who ever had traditional treatment had higher self stigma  than patients without a history of traditional treatment. Higher education was significantly correlated with lower self stigma . Increase in perceived signs  and perceived supernatural causes of mental illness  was significantly correlated with an increase in self stigma among patients with mental illness. Higher number of drug side effects positively correlated  while higher self esteem negatively correlated  with self stigma. The multivariate model explained 18% of the variance in self stigma among people with mental illness , marital status, age and income status. These factors were usually identified as important predictors of stigma in other studies,18,24,26In contrast to the educational status of the patients, those individuals with higher perceived supernatural explanation of mental illness had significantly higher overall self stigma and higher scores in all the five subscales. Such association could have existed since patients with high perceived supernatural causes of mental illness may have had more self blaming explanation or that such patients possibly attended to western treatment in the hospital after trials and exhaustion of unsuccessful traditional and religious healings. Similarly, a higher score of perceived sign of mental illness were associated with higher alienation and discrimination experience subscales and overall self stigma scores. In addition, as the number of drug side effects increased, there was a significant increase in discrimination experience subscale and overall self stigma. These positive associations of higher perceived signs and number of drug side effects with self stigma can be related to the visible nature of the perceived signs and drug side effects  to other people.The inverse relationship between self esteem and self stigma was reported in previous studies-9,28 andHigh feeling of inferiority  but less agreement with common stereotypes (stereotype endorsement) about people with mental illness was found. Females showed higher self stigma than males. History of traditional treatment and higher perceived supernatural explanation of mental illness were associated with higher self stigma. An increased educational status was one of the important factors which was inversely related to self stigma among people with mental illnesses. Higher number of drug side effects and perceived signs of mental illness were significant predictors of higher self stigma while high self esteem was correlated with lower self stigma. Psychosocial patient empowerment interventions with stronger emphasis on females, who ever had traditional treatment and who keep supernatural explanations of mental illness and who have les education, is recommended. Strategies which can reduce drug side effects can be helpful in reducing self stigma among people with mental illnesses. Further studies needs to be done whether self stigma is attached to gender roles.The authors declare that they have no competing interests.EG, MT and SD designed the study, involved in the data collection, analysis and drafting of the manuscript. GF, AML, NM were involved in the design of the study, analysis of the data and critically reviewed the manuscript. All authors read and approved the final manuscript."}
+{"text": "To what degree does being male or female influence the development of manual skills in pre-pubescent children? This question is important because of the emphasis placed on developing important new manual skills during this period of a child's education . We investigated age and sex-differences in the ability of 422 children to control a handheld stylus. A task battery deployed using tablet PC technology presented interactive visual targets on a computer screen whilst simultaneously recording participant's objective kinematic responses, via their interactions with the on-screen stimuli using the handheld stylus. The battery required children use the stylus to: (i) make a series of aiming movements, (ii) trace a series of abstract shapes and (iii) track a moving object. The tasks were not familiar to the children, allowing measurement of a general ability that might be meaningfully labelled \u2018manual control\u2019, whilst minimising culturally determined differences in experience (as much as possible). A reliable interaction between sex and age was found on the aiming task, with girls' movement times being faster than boys in younger age groups (e.g. 4\u20135 years) but with this pattern reversing in older children (10\u201311 years). The improved performance in older boys on the aiming task is consistent with prior evidence of a male advantage for gross-motor aiming tasks, which begins to emerge during adolescence. A small but reliable sex difference was found in tracing skill, with girls showing a slightly higher level of performance than boys irrespective of age. There were no reliable sex differences between boys and girls on the tracking task. Overall, the findings suggest that prepubescent girls are more likely to have superior manual control abilities for performing novel tasks. However, these small population differences do not suggest that the sexes require different educational support whilst developing their manual skills. Large population-based studies of children reliably find sex differences for specific cognitive functions In particular, there is currently insufficient clarity over the role that sex may play in the development of children's manual motor skills. This is despite the topic being of fundamental educational importance. For example, children's handwriting development is heavily influenced, amongst other factors, by their underlying manual dexterity There is some evidence for sex differences in motor-skill development if \u2018motor skill\u2019 is treated as a homogenous ability. Epidemiological studies have found that development coordination disorder (DCD) is at least twice as common in boys than girls Fine-motor skills can be considered a specialised sub-category of motor behaviour A technologically innovative approach to overcoming these limitations is to use digital tablets to record manual movements see . This meIt is clear that the issue of pre-pubescent sex differences in fine motor manual control has yet to be directly investigated. In order to address this issue, we measured performance in children aged 4\u201311 years as this age range can be considered pre-pubescent with reasonable confidence. Moreover, this age range corresponds to \u2018primary schools\u2019 within the UK educational system \u2013 schools where the focus is on developing core manual skills such as handwriting and drawing. To allow us to examine manual control objectively, with respect to both its speed and quality, in a large community based sample, we employed a portable digital tablet system capable of providing detailed kinematic information on how children interacted, using a stylus, with visual stimuli presented on a tablet PC's screen.In deciding upon the particular battery of tasks we presented to participants via the tablet system, we were mindful of the variety of assessment methods used in the previous research. A very broad range of tasks can be used to assess fine-motor control , with performance on any of them dependent in large part on prior experience in the specific task. Nevertheless, a common feature of many of these canonical \u2018fine-motor\u2019 tasks is that they require precise \u2018hand-eye coordination\u2019. Such visuo-manual control is often discussed as being particularly important in manual tasks requiring object manipulation We reasoned that testing a large number of children on this task battery would allow us to draw some solid conclusions regarding the degree to which sex influences the development of \u2018manual control\u2019 abilities within pre-pubescent children, in particular those relevant to learning to manipulate a stylus manually. On the basis of the \u2018gender similarities hypothesis\u2019 Participants were recruited from two primary schools in West Yorkshire, UK. A total of 422 out of 484 students agreed to participate . The test battery was designed and presented using the Clinical Kinematic Assessment Tool (CKAT), a custom software package specialised for presenting interactive visual stimuli on a tablet laptop computer screen, whilst simultaneously recording participant's kinematic responses to these stimuli via interactions with the screen using a handheld stylus , a measure of the spatio-temporal accuracy of participants' tracking, provided an index of performance on the tracking task. RMSE was calculated as the straight-line distance in millimetres between the centre of the moving target and the tip of the stylus for each sampled point during the time-series. For each Tracking trial (i.e. without and with guide-line) a mean value for RMSE with respect to each speed condition  was calculated and statistically analysed.This sub-test comprised of two trials. In the first, participants began by placing the stylus tip on a static dot (10 mm diameter) presented in the centre of the tablet's screen. After a second's delay the dot moved across the screen and participants were instructed to keep the tip of the stylus as close as possible to the dot's centre for the remainder of the trial. The motion was described by two oscillating sinusoidal waveforms in the axes of the screen. The frequencies and amplitudes of these waveforms were in a 2\u22361 ratio, resulting in a repeating \u2018figure-8\u2019 spatial pattern see  with heith target, after which the finish position (a circle with the letter \u2018F\u2019 within it) appeared on screen , the rapid succession of responses to be made and the fact participants were instructed that upon arriving at a target-dot the next one could appear anywhere on the screen, it was expected that participants would treat each aiming as a discrete response . Movement time (MT) was calculated for each of the 75 discrete aiming movements and defined as the time between arrival at one target location and arrival at the next one (i.e. a composite measure of the time taken to prepare and then execute each aimed movement), in seconds. MT was calculated with respect to the final target position (i.e. after the dot had jumped) for Jump events. Fast MTs were indicative of an optimal task response). For statistical analysis, a median value for the MT of aiming movement made during Baseline experimental conditions was calculated. This was compared to two further median MT values derived from responses during the Online Correction condition. Within this condition a median MT value was calculated for the six aimed movements made in response to the \u2018Jump\u2019 events and a separate median was calculated for responses made to the interspersed normal stimuli presentations (termed the \u2018Embedded-Baseline\u2019).The aiming subtest required 75 successive aiming movements to target-dots on the tablet's screen. Participants started by placing their stylus on the start position (a circle with the letter \u2018S\u2019 within it), triggering a target-dot (5 mm diameter) to appear at location 1 see . Particireen see . The ovePath accuracy (PA) for each trial was defined as the arithmetic mean (in mm) across all samples within each trial for the distance from the stylus to an idealised reference path (i.e. path A or B). Initial exploration of the data suggested that there was a degree of individual variation in the movement time (MT) within each of the Tracing trials  score. A median pPA value for participant's performance on the three Tracing trials presenting Path A and a separate one for the trials presenting Path B were calculated and analysed statistically .The Tracing subtest comprised six trials. In each trial the participant was required to place their stylus on the start position on an otherwise blank screen. After one second a tracing path (4 mm width) would appear, adjoining the start position to a finish position marked at the other end of the path see . To compials see . A compoRMSE, MT and pPA) were initially explored using graphs, skew and kurtosis values and Shapiro-Wilks tests of normality. Prior to statistical analysis reciprocal transformations were applied to all three outcome variables to normalise their distributions and resolve outliers. Performance on each of the transformed outcomes was then analysed separately using multi-level linear modelling (MLM) techniques ; see Field RMSE (the primary outcome measure for the Tracking subtest) two additional repeated measures, both nested within participants, were also included to examine the influence of Trial Type (With- or Without-Guide) and Speed  respectively. Equivalently, for MLM analysis of MT (the primary outcome for Aiming subtest) a repeated measure of Response-type  was included. Whilst modelling of pPA, the outcome measure for Tracing, specified a repeated measure of Path Type (i.e. Tracing Path A or B) instead.All analyses were conducted in R . Primary outcomes for each subtest \u200a=\u200a86.24; p<.001), depicted in 2(8)\u200a=\u200a10.21; p\u200a=\u200a.251). No 3- or 2-way interactions involving Sex as a factor, or the main effect of Sex, were significant .For two participants a recording error on this subtest meant their response had to be excluded from this portion of the analyses (leaving n\u200a=\u200a420). MLM analysis of the reciprocal RMSE does not improve for the youngest Age Group when in the second trial the additional \u2018guide-line\u2019 is provided, irrespective of the speed of the dot. For older age groups their RMSEs do improve on the guide-line trial , with this benefit increasing with age but also diminishing as the target moves faster. This interpretation is supported by d>.20; \u2018Moderate\u2019 d>.50; \u2018Large\u2019 d>.80) In relation to the significant 3-way interaction, MT outcome revealed a significant 3-way interaction (depicted in 2(6)\u200a=\u200a14.79; p\u200a=\u200a.022). Subordinate main effects for Age Band and Response Type and 2-way interactions for Sex \u00d7 Age Band and Age Band \u00d7 Response Type were also significant . All remaining main effects and interactions were non-significant (p>.857). MT during Baseline and Embedded-Baseline trials but not during \u2018jump\u2019 events. In both these conditions a similar pattern is shown: a consistent female advantage in the youngest two age groups (4\u20135 and 6\u20137 year olds) which shows signs of reversing with age. In the older two age groups (8\u20139 and 10\u201311 year olds) there was either no significant sex difference within age-group or a significant male advantage. d<0.5).One participant had only partial data recorded for the Jump condition and therefore their responses were excluded from this portion of the analyses. For the remainder of the sample (n\u200a=\u200a421), MLMs of the reciprocal 2(3)\u200a=\u200a259.57; p<.001) and Sex (\u03c72(1)\u200a=\u200a15.25; p<.001) upon reciprocal pPA but no additional significant main effect for Path type (A or B) (\u03c72(1)\u200a=\u200a1.95; p\u200a=\u200a.165) or any significant 2- or 3-way interactions . Inferring from descriptive statistics, the main effect of sex indicated Girls' mean reciprocal pPA score was significantly higher (better) than boys . Post-hoc tests also showed that from one Age band to the next 6-to-7 year olds out-performed 4-to-5 year olds , 8-to-9 year olds were better than 6-to-7 year olds  and 10-to-11 year olds outdid the 8-to-9 year olds . Effect sizes suggested \u2018Moderate\u2019 to \u2018Large\u2019 sized improvement with Age but only a \u2018Small\u2019 sized effect for Sex. See Multilevel linear modelling found significant main effects of both Age Band (\u03c7Our study explored pre-pubescent (4\u201311 years old) children's manual control, using a set of tasks that reflected the basic eye-hand coordination challenges commonly encountered when engaging in such behaviour. We restricted our attention to the control of a stylus held in the hand and explored three separate tasks that had different control demands: aiming movements, tracking and tracing. Our findings provide the first detailed evaluation of the degree to which sex differences influence the development of manual control within this age range. Girls exhibited better performance on the aiming and the tracing tasks but the higher performance observed in the aiming task was restricted to the youngest age groups. These female advantages are broadly consistent with previous studies, which used non-computerised assessments and reported girls outperforming boys on tests of manual dexterity (7 to 12 years old) The emergence of a male advantage in the older age groups for aiming suggests that the magnitude and direction of sex-differences has the potential to change over the course of development. The interaction observed is consistent with other studies that have shown a male advantage from adolescence onwards for producing fast, simple manual responses (e.g. finger-tapping and simple reaction times) The causes of these emerging male-advantages cannot be resolved from our study. Parsimonious explanations for why male advantages might emerge include: increasing anatomical differences in males and females (e.g. greater musculature) In contrast to the aiming and tracing task, there were no sex differences in the tracking task. It is always difficult to interpret a null finding but the fact that differences emerged on the other two tasks suggests that any diversity between the sexes on the tracking task must be very small if it exists at all. Tracking tasks are known to be sensitive indicators of certain neurological deficits because they rely on corticocerebellar and visuomotor control systems to generate accurate predictions of an external target's motion most manual behaviours involving a handheld stylus but it would be overly reductive to claim it should be taken as a proxy for all manual behaviours. Specifically, our battery is likely to be most relevant to understanding the underlying functions that contribute to an individual's proficiency in educationally important activities requiring manual stylus use, such as handwriting, drawing and touch-screen computer use.Our findings sound a note of caution for past and future studies that explore sex differences using complex \u2018fine motor tasks\u2019 (e.g. handwriting) in part because such tasks become more prone to the effects of experience but also because such tasks contain different control elements The fact that we have found sex differences in manual control raises the issue of whether the disparities warrant different educational approaches to handwriting tuition. This requires consideration of the sizes of the sex-differences observed: the absolute differences and associated standardised effect sizes we report are generally \u2018small\u2019, when judged against conventional thresholds Finally, we should emphasise that the present study has focussed exclusively on population differences . There are good reasons to suppose that at an individual level there will be more boys than girls who have specific problems with eye-hand coordination"}
+{"text": "The CAQK peptide selectively binds to injured mouse and human brain, and systemically injected CAQK specifically homes to sites of brain injury in mouse models. The CAQK target is a proteoglycan complex upregulated in brain injuries. Coupling to CAQK increased injury site accumulation of systemically administered molecules ranging from a drug-sized molecule to nanoparticles. CAQK-coated nanoparticles containing silencing oligonucleotides provided the first evidence of gene silencing in injured brain parenchyma by systemically administered siRNA. These findings present an effective targeting strategy for the delivery of therapeutics in clinical management of acute brain injuries.Traumatic brain injury (TBI) is a major health and socio-economic problem, but no pharmacological agent is currently approved for the treatment of acute TBI. Thus, there is a great need for advances in this field. Here, we describe a short peptide (sequence CAQK) identified by  Accurate treatment of traumatic brain injuries, a leading cause of neurological disability and death in young people, is hampered by poor accumulation of drugs in the brain. Here, the authors describe a tetrapeptide that can efficiently target brain injuries and deliver therapeutic or diagnostic payload. Acute brain injury such as traumatic brain injury (TBI) disrupts the normal function of the brain and generally has a poor prognosis for functional recovery and survival. Termed a \u2018silent epidemic', TBI is a leading cause of mortality and morbidity in children, teens and active adults from ages 1 to 44, with an annual incidence of 2.5 million in the US (ref. The blood\u2013brain barrier (BBB) is considered a major impediment to systemic treatment of central nervous system (CNS) diseases. As a result, localized delivery of drugs within the brain has been explored, but it has limitations in clinical settings. In acute brain injury and several cerebrovascular diseases, including stroke, hypertension and ischaemia, the BBB is transiently disrupted, which allows extravascular access for macromolecules and neuroprotective drugs from the systemic circulation. In fact, the leakage of serum proteins into brain parenchyma is used to test for BBB integrityin vivo phage display as a powerful and unbiased method to probe tissues in situ for specific molecular signatures and discovered homing peptides specific for different pathologies including tumours, atherosclerotic plaques and wounds56We previously employed To isolate peptides that specifically target brain injury, we inflicted unilateral puncturing stab wound injuries to the right hemisphere of adult male mice . The pen5\u2009p.f.u.) of total recovered phage pool To validate the selectivity of CAQK, a fluoresceinamine (FAM)-labelled CAQK peptide was chemically synthesized. In agreement with the phage screening results, the synthetic FAM-CAQK peptide, when injected intravenously, showed selective homing to the injured brain on macroscopic examination , and immTo investigate if CAQK targets other types of brain injuries, we tested peptide homing in a controlled cortical impact injury (CCI) model. Without penetrating injury, this model mimics cortical tissue loss, axonal injury, concussion and BBB dysfunction of TBI ref. . CAQK hoCAQK homing to PBI was observed up to 5 days after the injury , indicatTo further characterize the binding of CAQK to the injured brain area, we carried out overlay binding experiments with silver nanoparticles (AgNPs) conjugated with CAQK (CAQK-nanoparticles (NPs)) on mouse brain sections . CAQK-NPTo identify the potential protein targets of CAQK in the brain tissue, we performed mass spectrometry proteomics analysis of proteins separated from extracts of injured brain by affinity chromatography on immobilized peptides . Table 113We confirmed the increase in expression of ECM-associated CSPGs at sites of brain injury by immunostaining. Versican, tenascin-R, and the hyaluronan and proteoglycan link protein (hapln4), all of which are components of the brain ECM complex upregulated following an injury, showed high expression in the injured but not the uninjured hemisphere of the brain . The sigin vitro binding of CAQK phage to ECM produced by U251 human astrocytoma cells was tested. These cells express high levels of versican and other members of the brain ECM , administered intravenously, showed significantly greater accumulation in injured brain tissue than control NPs . The locTo demonstrate the versatility of CAQK system, we tested delivery of oligonucleotides loaded into porous silicon NPs (PSiNPs) as a carrier19ex vivo binding of CAQK-conjugated silver NPs on human cortical sections obtained from a head trauma patient. The CAQK-NPs showed intense binding to the injured brain sections from the cortex and the corpus callosum areas, whereas binding to normal brain sections was minimal  was used during the entire procedure and afterwards, the scalp was closed with sutures, anaesthesia discontinued and mice were administered buprenorphine i.p. for pain control. For the first 2\u2009h after injury, mice were closely monitored in their cages.All animal experiments were conducted under an approved protocol of the Institutional Animal Care and Use Committee of Sanford Burnham Prebys Medical Discovery Institute. Eight- to ten-week-old male BL6 mice were anaesthetized with 4% isoflurane  in 70% NE. coli BLT5403 and purified for input for second round of screening. The colonies recovered from second round were sequenced using Sanger sequencing . Alternatively, after first round, the phages in the lysate were rescued by amplification in E. coli and peptide-encoding portion of the phage genome was sequenced using Ion Torrent high-throughput sequencing.Six hours after brain injury, mice were intravenously injected with 1e10\u2009p.f.u. of a CX7C na\u00efve phage library in 100\u2009\u03bcl of PBS. The library was allowed to circulate for 30\u2009min, after which mice were anaesthetized with 2.5% avertin and perfused with PBS intracardially. Brains were extracted, and the tissue surrounding the injury and the corresponding region from the contralateral side was isolated. Tissues were homogenized in LB-NP 40 (1%) and phage was processed as describedN,N,N\u2032,N\u2032-Tetramethyl-O-(1H-benzotriazol-1-yl)uranium hexafluorophosphate (OR) O-(Benzotriazol-1-yl)-N,N,N\u2032,N\u2032-tetramethyluronium hexafluorophosphate) activator, collidine activator base and 5% piperazine for deprotection. Fluorescein and biotin tags were incorporated during synthesis at the N-terminus of the sequence. Cleavage using a 95% trifluoro acetic acid followed by purification gave peptides with >90% purity. Peptides were lyophilized and stored at \u221220\u2009\u00b0C.The peptides were synthesized on a microwave-assisted automated peptide synthesizer  following Fmoc/t-Bu  strategy on rink amide resin with HBTU  in both directions along the midline. Once good exposure to liver was obtained, the artery was clamped ) and excision wound 2\u2009mm in depth on the surface of one of the livers lobes was made. The clamp was removed from the artery allowing blood to flow. The incisions on peritoneum, muscle and skin were closed. The mice were then placed on a heating pad in their cage and monitored closely until they recovered from anaesthesia.For skin wounds, induction of anaesthesia was done and the skin was cleaned with alcohol and betadine and four 6- or 8-mm skin biopsies were made to the back skin of the mouse. None of the skin wounds were covered or sutured closed to guarantee optimal and infection-free healing. To test peptide homing, FAM-labelled peptide (50\u2009nmoles) was injected i.v. 6\u2009h after injury and allowed to circulate for 30\u2009min. Mice were perfused intracardially with saline and organs were isolated and analysed by immunostaining.Animals were intravenously injected, 6\u2009h after injury, with 50\u2009nmoles of peptide dissolved in PBS, and allowed to circulate for 30\u2009min. Mice were perfused intracardially with saline and organs were isolated and imaged using the Illumatool Bright Light System LT-9900 (Lightools Research). Brains and organs were placed in 4% paraformaldehyde (PFA) at pH 7.4 overnight, washed with PBS and placed in graded sucrose solutions overnight before optimal cutting temperature compound (OCT) embedding. Ten-micrometre-thick sections were cut and analysed by immunofluorescence. For a complete histological analysis, sections were stained with the Movat pentachrome kit (American Mastertech Inc.) following the manufacturers instructions.\u22121 in PBS for 5\u2009min, washed with PBS, mounted using mounting media (Vector Biolabs) and imaged on a confocal microscope (Zeiss LSM-710). Staining was done using the following antibodies and reagents: Fluorescein (Invitrogen A889), Versican , hapln4 , tenascin0-R , Wisteria floribunda agglutinin , NG2 and olig2 , GFAP  and MBP (Millipore - MAB386).Frozen sections were permeabilized using PBS-Triton (0.2%), blocking was carried out using 5% blocking buffer: 5% bovine serum albumin, 1% goat serum, 1% donkey serum in PBS-T. Primary antibodies were incubated in diluted (1%) blocking buffer overnight at dilutions 1/100 or 1/200 at 4\u2009\u00b0C, washed with PBS-T and incubated with secondary antibodies diluted 1/200 or 1/500 in 1% diluted buffer for 1\u2009h at room temperature, subsequently washed with PBS-T, counterstained with 4,6-diamidino-2-phenylindole 1\u2009\u03bcg\u2009mlCLARITY was performed on freshly extracted brain tissues as describedn-octyl-beta-D-glucopyranoside and protease inhibitor cocktail (Roche) as describedD-glucopyranoside and protease inhibitor cocktail in PBS) followed by washing with 0.5\u2009mM control peptide in wash buffer to remove nonspecifically bound proteins. The bound proteins were eluted with 2\u2009mM-free CAQK peptide. The eluted factions were pooled, their protein concentration determined by using bicinchoninic acid protein assay (Thermo Fischer) and the samples were digested using the filter-aided sample preparation methodMus musculus Uniprot protein sequence database (version July 2014).For identifying CAQK binding proteins, mouse brains with brain injury were collected 6\u2009h after injury. Using liquid nitrogen, the brains were crushed and ground into powder using a mortar and pestle. Next, brain tissue was lysed in PBS containing 200\u2009mM 2SO4 stopped the reaction and the plate was read at 495\u2009nm . For enzymatic digestion, chondroitinase ABC  or hyaluronidase (500\u2009U\u2009ml\u22121) was added to the plate for 3\u2009h at 37\u2009\u00b0C. The plate was then washed with PBST three times before incubation with phage.Cells grown as confluent monolayer in a 96-well plate were gently removed by a enzyme-free cell dissociation buffer (Thermo Fisher Scientific) and plates blocked with 200\u2009\u03bcl of 0.5% bovine serum albumin in PBS with 0.005% T20 for 1\u2009h at 37\u2009\u00b0C. Phage was incubated in the plate at 4\u2009\u00b0C for overnight and unbound phage was removed by washing three times with 200\u2009\u03bcl of PBST. The bound phage was detected by incubating with an in-house generated anti-T7 phage antibody for 1\u2009h at 4\u2009\u00b0C. Following washing, horseradish peroxidase-labelled anti-rabbit IgG (Sigma-Aldrich) was diluted 1:1,000 in PBS, and 100\u2009\u03bcl was added to the wells, followed by 30\u2009min incubation at room temperature and washing three times. Next, 100\u2009\u03bcl of o-phenylenediamine dihydrochloride (OPD) silver and gold substrate (Sigma-Aldrich) was added to the wells and incubated at room temperature until visible colour was observed (<30\u2009min). Adding 50\u2009\u03bcl of 1\u2009M H3 (252\u2009mg) dissolved in 2.5\u2009l water was stirred and heated to 60\u2009\u00b0C, then 50\u2009ml water containing tannic acid (6.1\u2009mg) and trisodium citrate dihydrate (1\u2009g) was added. After 3\u2009min, the solution was brought to a boil for 20\u2009min. Final optical density at 400\u2009nm was \u223c10. Lipoic PEG amine  was reduced in 84\u2009mM tris-carboxylethyl phosphine  in 4.1\u2009ml water for 3\u2009h. AgNPs were portioned to 500\u2009ml and heated to 50\u2009\u00b0C, then lipoic PEG amine solution (0.79\u2009ml) was added, followed by 0.25\u2009ml 0.5\u2009M TCEP. After 30\u2009min, the solution was cooled to room temperature. Tween 20  and 20\u2009ml 2\u2009M NaCl were added, and incubated overnight at 4\u2009\u00b0C. AgNPs were concentrated 50-fold and purified by stirred cell (Millipore) with a 100\u2009kDa membrane into 0.5 \u00d7 PBS with 0.005% T20 and 5\u2009mM TCEP, then passivated with 0.03\u2009mM N-acetyl-L-cysteine methyl ester (Sigma), and 0.10\u2009mM tetracysteine peptide , washed at 20 kRCF and resuspended at 300 OD at 405nm in 0.05\u2009M phosphate buffer with 0.005% T20. This product could be stored at least 6 months at 4\u2009\u00b0C. A bifunctional linker was reacted with the amine to introduce maleimide groups , washed by centrifugation, and reacted with cysteine peptide (FAM-x-CAQK-NH2) or a control thiol-containing peptide, or L-cysteine (Sigma). The product was washed in PBS with 0.005% T20 (PBST), filtered (0.22\u2009\u03bcm), with a typical final optical density of 150 at the Ag plasmon peak of 400\u2009nm. We estimated this concentration to be \u223c30\u2009nM in AgNPs using an extinction coefficient of 5 \u00d7 109\u2009M\u22121\u2009cm\u22121 for spherical silver obtained from ref. AgNPs with PEG coating and peptide functionality were prepared as reported previously with some modificationsex vivo binding were carried out on frozen brain tissue sections following the same protocol for immunofluorescence staining described above taking the nanoparticles as if they were the primary antibody and using no secondary antibody. Peptide-coated AgNPs in PBS-T at a 1nM concentration were used and the incubation time was 1\u2009h at 37\u2009\u00b0C. Sections were imaged with fluorescent microscopy by looking at intrinsic emission from the FAM tag on the peptide.Overlay experiments to analyse p-type silicon wafers  were purchased from Virginia Semiconductor, Inc. PSiNPs were prepared by electrochemical perforation etching of the silicon wafers, as described previously\u22122 was applied for 1.8\u2009s, followed by a higher current density of 400\u2009mA\u2009cm\u22122 for 0.36\u2009s. This waveform was repeated for 140 cycles, generating a perforated porous silicon film with alternating layers of high and low porosity. The resulting porous nanostructure was removed from the silicon substrate by applying a current density of 3.7\u2009mA\u2009cm\u22122 for 250\u2009s in an electrolyte consisting of 1:30 (by volume) of 48% aqueous HF:ethanol. The free-standing porous silicon films were then fractured to the desired size  by ultrasonication, and the resulting nanoparticles were oxidized by immersion in aqueous borax solution to activate photoluminescenceSingle-crystalline highly doped \u22121 in ethanol) was mixed with 20\u2009\u03bcl of 3-(ethoxydimethyl)-propylamine silane by vortex overnight at room temperature. The amine-terminated nanoparticles were rinsed three times with ethanol and then further reacted with 1\u2009ml of succinimidyl carboxy methyl ester-polyethylene glycol-maleimide (10\u2009mg\u2009ml\u22121 in ethanol) for 2\u2009h, followed by rinsing with ethanol and deionized water (three times each). An aqueous solution of the peptides  was added to the resulting nanoparticles and mixed by vortexing for 2\u2009h to conjugate the peptide via the free cysteine residue at the terminal group of the peptide\u22121 (peptide/PSiNPs). The oligonucleotide siGFP was electrostatically loaded to the positively charged porous inner structure of the nanoparticles by mixing with siRNA solution (200\u2009\u03bcM) at 4\u2009\u00b0C for 24\u2009h. The loading amount of siGFP was\u223c7\u2009wt% (siGFP/PSiNP), which was determined by measuring absorbance at 260\u2009nm. Note that the PEG linkers were attached only onto the outer surface (not the inside pores) of PSiNPs due to their large molecular weight and long-chain length relative to the pore size (\u223c12\u2009nm). 3-(ethoxydimethyl)-propylamine silane and succinimidyl carboxy methyl ester-polyethylene glycol-maleimide  were purchased from Sigma-Aldrich and Laysan Bio, respectively, and used as received without further purification. RNase-free water was purchased from Thermo Fischer . Small interfering RNA against GFP (siGFP) was purchased from Dharmacon. The sequences for the sense and antisense strands of siGFP are: 5\u2032-GGCUACGUCCAGGAGCGCACCdTdT-3\u2032 (sense) and 5\u2032- UGCGCUCCUGGACGUAGCCTTdTdT-3\u2032 (antisense).An aliquot of PSiNPs  was used to determine the hydrodynamic size and Zeta potential of the nanoparticles. Photoluminescence and fluorescence spectra were obtained using a QE pro spectrometer (Ocean Optics). Concentration of siRNA was determined by measuring absorbance at 260\u2009nm using a spectrometer  based on the ODn=3). Three days after injury, mice were perfused, organs harvested and fixed for downstream analysis. The tissues were imaged under a time-gated imaging set-up and the GFP expression was analysed by researchers blinded to the experimental groups.Transgenic CAG-GFP mice were purchased from The Jackson Laboratory (stock #006567). Brain injuries were done as described above. Peptide-conjugated and siRNA-loaded PSiNPs (300\u2009\u03bcg) were administered twice via tail-vein injections at 6 and 24\u2009h post injury (\u03bbex=410\u2009nm) images were taken.Gated luminescence images were acquired from a custom-built time-domain imaging system using an intensified CCD camera , as reportedFormalin-fixed human brain tissues were obtained from the Brain Tissue Repository maintained by the Center for Neuroscience & Regenerative Medicine (CNRM) at the Uniformed Services University of the Health Sciences (USU) in Bethesda, MD. The TBI case is from a patient with moderate TBI (automobile accident) who died at age 72. The control case is from a 63-year-old male without any neurologic diagnosis or any signs of TBI on detailed neuropathologic evaluation. Fixed tissues were cryopreserved and sectioned for overlay binding with AgNPs as described above. For immunohistochemistry, an antigen retrieval step was done before incubation with anti-hapln4 and anti-versican antibodies.t test. The details of the statistical tests carried out are indicated in respective figure legends.All data represents mean value\u00b1s.e.m. All the significance analysis was done using Statistica 8.0 software, using one-way ANOVA or two-tailed heteroscedastic Student's The data that support the findings of this study are available from the corresponding author on request.How to cite this article: Mann, A. P. et al. A peptide for targeted, systemic delivery of imaging and therapeutic compounds into acute brain injuries. Nat. Commun. 7:11980 doi: 10.1038/ncomms11980 (2016).Supplementary Figures 1-11"}
+{"text": "Neural connections remain partially viable after stroke, and access to these residual connections provides a substrate for training-induced plasticity. The objective of this project was to test if reflex excitability could be modified with arm and leg (A & L) cycling training. Nineteen individuals with chronic stroke (more than six months postlesion) performed 30 min of A & L cycling training three times a week for five weeks. Changes in reflex excitability were inferred from modulation of cutaneous and stretch reflexes. A multiple baseline (three pretests) within-subject control design was used. Plasticity in reflex excitability was determined as an increase in the conditioning effect of arm cycling on soleus stretch reflex amplitude on the more affected side, by the index of modulation, and by the modulation ratio between sides for cutaneous reflexes. In general, A & L cycling training induces plasticity and modifies reflex excitability after stroke. The arms and the legs are coupled in the human nervous system such that activity in the arms affects activity in the legs and vice versa. In quadrupeds, forelimb\u2013hindlimb coordination is well documented and has been attributed to propriospinal linkages between cervical and lumbosacral spinal central pattern-generating networks ,3,4,5,6.The modulation of reflex amplitudes can be used to probe for changes in interlimb neural activity ,7. InvesExploiting the neural and mechanical linkages between the arms and legs, which are inherent parts of human locomotion, could enhance the recovery of walking for those who have suffered neurological damage such as a stroke ,5,23,24.A complication of stroke is alterations in interneuronal pathways, stemming from damage to supraspinal centers, which disrupts some of the descending regulation ,29. The Although neural pathways are corrupted bilaterally after stroke, residual connectivity in spinal networks provides a substrate for training-induced plasticity arising from A & L cycling training . The extNineteen individuals with chronic stroke (more than six months postinfarct) were enrolled in the study. To assist with determining a participant\u2019s functional status and the clinical features of this population, clinical assessments were performed by a licensed physical therapist before and after A & L cycling training see   score. This is in line with many other post-stroke treadmill training protocols where training volume was increased . ParticiAll exercise sessions were supervised by a CSEP (Canadian Society for Exercise Physiology) certified exercise physiologist and several laboratory assistants to ensure appropriate monitoring. Exercise sessions were not initiated if a participant\u2019s blood pressure (BP), measured with a digital blood pressure cuff placed over the LA arm, exceeded 140/90 mmHg, in accordance with Canada\u2019s Physical Activity Guidelines . This onA multiple baseline within-subject control design was used for this study ,52. FiguSoleus stretch reflexes were evoked using an electrodynamic shaker , placed over the triceps surae tendons of the LA and MA legs, in separate trials as described previously ,56. ConsStretch reflexes were collected under two conditions: (1) with the arms and legs not moving (static) and (2) with the arms cycling rhythmically at 1 Hz (conditioned) while the legs remained static. Arm cycling frequency was set to 1 Hz and participants maintained cycling frequency with the use of visual feedback. All reflexes were evoked at the \u201c7 o\u2019clock\u201d position for the LA hand for both the static and conditioned trials. This position was previously shown to have the largest modulatory effect on H-reflex amplitude . The samD\u201d is the peak-to-peak displacement of the tip of the shaker in contact with the tendon, \u201cG\u201d is a constant (the acceleration due to gravity), \u201cA\u201d is the acceleration measured by the accelerometer in units of gravity, and \u201cF\u201d is the frequency of the sinusoid (100 Hz).As a proxy for the intensity of the pulse between conditions, an accelerometer , mounted to the tip of the shaker, recorded stimulation amplitude, as in previous studies ,58,59. TArmCycle \u2212 StretchReflexStatic)/StretchReflexStatic] \u00d7 100). Negative values indicate a decrease in reflex amplitude during the arm cycling conditioned trial compared to the static trial. To compare modulation between the LA and MA sides, the difference in stretch reflex amplitudes was calculated by subtracting values for arm cycling from static control amplitudes. A negative value indicates a greater degree of modulation on the LA side and a positive value indicates a greater degree of modulation on the MA side. Background EMG (bEMG) was assessed to monitor possible effects of heteronymous and contralateral muscle activity on reflex amplitudes. For the contralateral SOL and TA bilaterally, bEMG was calculated as the average value of background activity from a 20 ms prestimulus period. Data were normalized to the peak EMG recorded during A & L cycling for each muscle for each session.Modulation of stretch reflexes due to arm cycling was evaluated by calculating the index of modulation as the change in stretch reflex peak-to-peak amplitude between the static and conditioned trials and then expressed as a percentage  and superficial peroneal (SP) nerve stimulation during A & L cycling was used to assess neurophysiological changes in reflex excitability arising from locomotor training. Cutaneous reflexes were evoked via simultaneous combined surface stimulation of the nerves innervating the dorsum of the hand (SR) and foot (SP) . ElectroEMG data from the soleus (SOL), tibialis anterior (TA), flexor carpi radialis (FCR), and posterior deltoid (PD), from the LA and MA limbs, were collected with surface electrodes placed in bipolar configuration over the muscle bellies of interest. Muscles from all four limbs bilaterally that have been previously associated with interlimb reflex effects were chosen ,33,61,62Participants performed A & L cycling on the same cycle ergometer used for training. max \u2212 bEMGmin)/bEMGmax] \u00d7 100) and this measure provides an index of overall amplitude modulation independent of the pattern of modulation across A & L cycling phases [max \u2212 Reflexmin)/bEMGmax] \u00d7 100). The ratio between the LA and MA modulation index for each muscle was also determined.To investigate phase-dependent modulation within each movement cycle, data were broken apart into 8 equidistant phases. Phases 1\u20134 represent the arm and leg power phase, corresponding to the LA arm at top dead center (0 deg) to full extension of the arm and leg (180 deg) . Evoked g phases . CutaneoUsing commercially available software  pretest and post-test data were compared with two different methods; a within-subjects and a between-subjects analysis to evaluate the extent to which arm and leg cycling training altered reflex modulation. Two statistical methods are provided as a means of increasing statistical testing procedures for multiple baseline designs. For the within-subject analyses, post-test data were compared to the 95% confidence interval (CI) created from three pretest sessions. To establish the 95% CI for each measure, variability was computed from three pretest sessions and used to create a data range with which the post-test value was compared. If the post-test value fell outside the 95% CI range, it was considered statistically significant for that participant . A graphp \u2264 0.05. As an additional measure of the magnitude of any differences between pre\u2013post, the observed effect size for post-test differences for stretch and cutaneous reflex parameters is also reported using Cohen\u2019s d. In this calculation we used the conventional small effect as d = 0.2, a medium effect as d = 0.5, and a large effect as d = 0.8 [For the between-subject analysis, using group data, we used repeated-measures ANOVA to evaluate the extent to which arm and leg cycling training altered reflex modulation. For stretch reflex and modulation index parameters, a 1 \u00d7 4  repeated-measures ANOVA was used. For cutaneous reflex parameters an 8 (phase) \u00d7 4  repeated-measure ANOVA was used and only main effects and interaction effects for time are reported and considered. For comparison between baseline and post-test data, a repeated-measures ANOVA was first performed in a planned contrast to examine differences across the three pretest sessions. If no difference was found, data were pooled together to create an average pretest value, which was compared to the post-test. Assumptions for ANOVA were evaluated for parametric tests for a within-subject design with statistical significance set at  d = 0.8 .No significant differences for input trigger amplitude between the static and conditioned trials for the pretests and the post-test were found. A constant displacement of the pulse was maintained during all stretch reflex trials within each experimental session. For the LA side, the shaker displacement was 0.51 \u00b1 0.23, 0.52 \u00b1 0.15, 0.47 \u00b1 0.13, and 0.52 \u00b1 0.12 mm for pre1, pre2, pre3, and post-tests, respectively. For the MA side the shaker displacement was 0.48 \u00b1 0.17, 0.49 \u00b1 0.11, 0.44 \u00b1 0.13, and 0.48 \u00b1 0.16 mm for pre1, pre2, pre3, and post-tests, respectively. There was no significant difference in shaker displacement between the LA and MA sides. Therefore, differences in stretch reflex amplitude were not caused by changes in input size and modulation due to arm cycling.  = 3.497, p = 0.025, d = 0.733). When comparing the baseline soleus stretch reflex was reduced with arm cycling compared to static. For the MA SOL, there was average with the post-test, the MA SOL shows an 80.8% increase in the inhibitory effect of arm cycling on soleus stretch reflex excitability in 15 of the 19 participants  = 8.983, p = 0.011, d = 1.034) after training. = 9.558, p = 0.008, d = 1.012), where pretest stretch reflex amplitudes for all participants were reduced with arm cycling by 38.4% for the LA side compared to 10.9% for the MA side. After training, the post-test revealed there was no longer a significant difference between the sides in stretch reflex inhibitory modulation, where now both LA and MA stretch reflexes for all participants are reduced by 36.4%. When comparing the absolute difference in the degree of modulation between the LA and MA sides after training, there was a significant main effect of time across all sessions  = 3.770, p = 0.020, d = 0.763). With the baseline average and post-test values there was a significant effect  = 10.190, p = 0.007, d = 0.839) such that the relative effect of modulation during arm cycling for the pretests was 27.8% stronger for the LA side and for the post-test 14.2% stronger on the MA side in 10 of the 19 participants.At the pretest sessions there was a significantly larger degree of modulation on the LA side compared to the MA side (Fy-axes represent the power (solid line) and recovery (dotted line) phases of A & L cycling. As there were no significant differences between pretest data samples, for simplification the average value across the three tests is shown. = 3.947, p = 0.001, d = 0.976), LA TA  = 2.720, p = 0.045, d = 0.768), and MA FCR  = 2.620, p = 0.016, d = 0.873). For the LA PD there was a significant interaction for phase and time  = 1.699, p = 0.031, d = 0.966). When looking at specific phases for reflex amplitudes, there are significant differences between the baseline average and post-test values for muscles with significant main effects. For the MA TA, significant effects were found for phase 2 and 3  = 8.561, p = 0.012, d = 0.435 and F = 5.049, p = 0.043, d = 0.400, respectively). For the LA TA, significant effects were found for phase 3 and 5  = 5.813, p = 0.031, d = 0.254 and F = 4.486, p = 0.045, d = 0.362, respectively). For the MA FCR, there were significant differences found when comparing the baseline average and post-test values for phase 6  = 5.094, p = 0.040, d = 0.204) and 7  = 4.601, p = 0.042, d = 0.215). For the LA PD, significant effects were found for phase 2  = 3.561, p = 0.041, d = 0.379). In summary, there were significant main effects of training on cutaneous reflexes at different phases for the MA and LA TA, MA FCR, and LA PD. The results of omnibus ANOVA for all phases and tests reveal main effects of time and phase. As main effects of phase are not of interest, we only addressed main effects of time. Results revealed that there were main effects of time for the MA TA  = 3.149, p = 0.037, d = 0.682). When comparing the pretest average to post-test values for bEMG at specific phases of A & L cycling, there were significant differences between baseline and post-test values for several muscles. For MA TA, A & L cycling training increased activity for phase 2  = 5.373, p = 0.039, d = 0.555), 3  = 5.871, p = 0.032, d = 0.494), and 6  = 5.044, p = 0.044, d = 0.564). For the MA FCR, bEMG was decreased for phase 2 following training  = 5.956, p = 0.046, d = 0.371) and was increased for the LA PD bEMG for phase 7 following training  = 5.063, p = 0.043, d = 0.464). In summary, there were significant main effects of training on background EMG at different phases for the MA TA and FCR, and LA PD.Investigating background EMG levels between conditions allows for comparison of reflex amplitudes that cannot be explained by simple gain scaling with motoneuronal pool excitability. The results of omnibus ANOVA for all phases and test sessions reveal main effects of time for background EMG in the LA TA  = 3.809, p = 0.025, d = 0.631). When comparing the pretest average to the post-test, we found that modulation increased by 30.3% in 11 out of 19 participants for the MA FCR  = 5.026, p = 0.039, d = 0.771) following training. Although no significant main effect was found, we saw a 9.8% increase in 8 out of 19 participants  = 4.999, p = 0.040, d = 0.359) for the LA TA when comparing with the pretest average. There was a significant main effect of time for the ratio of modulation for the TA  = 3.889, p = 0.014, d = 0.631). Following training, modulation was decreased by 54.6% in 10 out of 19 participants  = 5.345, p = 0.034, d = 0.613). When comparing the pretest average to the post-test we found that modulation was also decreased by 14.2% in 12 out of 19 participants for the SOL  = 4.566, p = 0.048, d = 0.317). In summary, the modulation index for cutaneous reflexes was changed after training for the MA FCR and LA TA and the amplitude ratio between LA and MA reflexes was changed for the SOL and TA.  = 4.095, p = 0.013, d = 0.804), LA PD  = 3.656, p = 0.043, d = 0.731), and MA FCR  = 3.396, p = 0.028, d = 0.719). When comparing the pretest average to the post test, we found that modulation decreased by 23.1% in 9 out of 19 participants following training in the MA TA  = 8.372, p = 0.013, d = 0.732) and decreased by 20.2% in 8 out of 19 participants in the LA PD  = 5.219, p = 0.041, d = 0.518). For the ratio of modulation for background EMG, there was a significant main effect of time for the TA  = 3.311, p = 0.046, d = 0.697) and the FCR  = 3.150, p = 0.037, d = 0.789). When comparing the pretest average to the post test, we found that the ratio increased by 21.2% in 7 out of 19 participants for the TA  = 5.685, p = 0.034, d = 0.617) and decreased by 20.2% in 10 out of 19 participants for the FCR  = 7.988, p = 0.015, d = 0.737) following training. In summary, the modulation index for background EMG was changed after training for the MA TA and LA PD, and the modulation index ratio was changed for the TA and FCR.For bEMG, there were significant main effects of time for the MA TA  to cause the difference in modulatory effects of arm cycling on MA SOL stretch reflex excitability seen after training here. Modulation of Ia PSI by rhythmic activity in cervical spinal cord oscillators is an important mechanism for reflex modulation in the legs in response to arm cycling ,22. ThisThe A & L cycling training produced a change in modulation of stretch reflex amplitude for the LA and MA sides. The amount of suppression on the MA side was restored to the relative levels seen on the LA side, and showed an increased depth of modulation after A & L cycling training. Initial differences between sides in stretch reflex modulation during arm cycling could be due to asymmetrical differences of presynaptic regulation following stroke. Arm and leg muscles show reduced Ia PSI  and onlyResponses to cutaneous stimulation showed altered modulation patterns following training. When examining specific functional phases for A & L cycling, reflexes in the LA SOL, LA TA, MA TA, MA FCR, and LA PD are differentially modulated following A & L cycling training. There were also some instances where there were differences in background EMG as a result of training, which could contribute to changes in reflex activity\u2014see, for example, MA TA. Additionally, in a few cases the background changes were in line with changes in reflex activity in the LA TA, MA FCR, and LA PD, for example. Thus, for these muscles, only phases where there were no concomitant changes in bEMG were considered further. In some cases, the modulation following training represents a return to what one \u201cnormally\u201d observes in these networks. For example, cutaneous reflex activity in the LA TA is mainly suppressive during locomotion after stroke  or heredIn the wrist flexor FCR muscle on the MA side, reflexes were facilitated specifically in the power phase, following A & L cycling training, seen with both the within-participant and repeated-measures analysis. This is evidence for increased response to stimulation from the contralateral (LA) side. Increased reflex activity from contralateral stimulation could represent an increase in crossed responses following training, where access is to be best gained by stimulating cutaneous fields in the LA hand . MeasuriThese results should be interpreted in light of several methodological considerations that must be acknowledged. Because of the linked motion of the cranks, it is unknown how much propulsion was powered from the legs versus the arms. It is possible that participants who imparted the most force via the arm-crank were most likely to respond to the intervention. Future research should focus on measuring the relative participation from the arms versus the legs during training, and how this correlates to improvement. Additionally, features of the stroke, including the time since injury, location of the stroke infarct, or degree of impairment, could have affected the likelihood of A & L cycling training inducing plasticity. However, we did not find an association between these characteristics and our outcomes, instead observing some aspects of plasticity induced irrespective of time after injury or location of stroke. For example, even a participant who was 29 years post-stroke showed plastic changes alongside participants who were 2 years post-stroke. Nevertheless, future research on this topic is warranted.Concomitant changes in bEMG, as a result of the training, could affect both stretch and cutaneous reflex amplitudes between tests. As a result, only trials and phases without changes in bEMG were considered in detail. In addition, evoked responses may vary somewhat from one session to the next, making comparison across multiple tests difficult. However the measures used here have been previously shown to have high reliability across multiple baseline points , and forLastly, statistical design for multiple baseline designs is limited, and little consensus exists across the literature on the best statistical design to use. Therefore, we offer two types of statistical assessments: a within-participant design, whereby we use the confidence interval from three pretest sessions to compare the post-test value, and a design using repeated-measures ANOVA to evaluate significant post-test effects with a planned contrast. For many of our outcome variables, results from both statistical designs support the same conclusion, therefore, we are confident in our observations.In response to motor training, neural circuits have the plastic ability to alter their structure and function ,83,84,85Here we have evidence of neural plasticity in the portion of neural linkages that remain accessible after stroke arising from A & L cycling training affecting muscle afferent and cutaneous reflex pathway excitability after stroke. The focus of future research should be to determine exactly how these changes in reflex excitability are associated with the recovery of motor function. We have recently shown that A & L cycling does indeed transfer to improved walking ability after stroke  and the Plasticity within reflex pathways can be used to probe changes in neural connectivity as a result of rehabilitation interventions for patient populations. For example, testing the conditioning effects of arm cycling on the soleus stretch reflex could be used as a proxy for measuring the change in strength of excitability in neural connections between the arms and the legs. Stretch reflex testing, a protocol based on a relatively simple procedure (a tendon tap), might be useful for application in stroke populations, compared to testing the electrically evoked H-reflex pathway, as allodynia is not uncommon in this group. In addition, hyperreflexia, as measured by stretch reflexes, would more clearly reveal reflex modulation associated with the fusimotor system . TestingA & L cycling training for stroke participants modifies reflex excitability. This was determined as an increase in the conditioning effect of arm cycling on soleus stretch reflex excitability on the MA side, and as a change in modulation, a change in the index of modulation, and a change in the modulation ratio between the LA and MA sides of cutaneous reflexes. Testing for plasticity in reflex pathways may be used to probe changes in neural connectivity resulting from rehabilitation interventions for clinical populations."}
+{"text": "Akt signaling plays a central role in many biological processes that are key players in human immunodeficiency virus 1 (HIV-1) pathogenesis. The persistence of latent reservoirs in successfully treated patients, mainly located in macrophages and latently infected resting CD4+ T cells, remains a major obstacle in HIV-1 eradication. We assessed the in vitro effects of an HIV protease inhibitor (PI) and a non-nucleoside reverse transcriptase inhibitor (NNRTI) on HIV-1 Nef-induced Akt activation in macrophages and on HIV-1 reactivation in U1 monocytoid cells. Ex vivo, we investigated the impact of combination antiretroviral therapy (cART) on Akt activation, as measured by flow cytometry, and on the viral reservoir size, quantified by qPCR, in monocytes and autologous resting CD4+ T cells from HIV-infected individuals . We found that, in myeloid cells, both Akt activation and HIV-1 reactivation were inhibited by PI but not by NNRTI in vitro. Our results indicate that cART decreases Akt activation and reduces the size of the HIV reservoir in both monocytes and resting CD4+ T cells. Our study indicates that Akt activation could play a role in HIV reservoir formation, indicating that drugs which target Akt could be efficient for limiting its size in aviremic chronically infected patients. The serine/threonine kinase Akt, also known as protein kinase B, plays important roles in the cellular processes involved in HIV-1 pathogenesis. As a key regulator in the phosphoinositide 3-kinase (PI3K) pathway, it impacts cell survival, metabolism, growth, and proliferation ,3,4,5.HIV-1 Nef is an accessory viral protein known to be abundantly expressed in the early stages of infection. Several aspects of the viral pathogenesis have been showed to be affected by this multifunctional protein, which plays an important role despite lacking enzymatic activity ,7. BesidThe use of combination anti-retroviral therapy (cART) has allowed for the reduction of the viral load of infected individuals to undetectable levels . HoweverInitiation of cART during primary HIV infection may limit the establishment of viral reservoirs, and very early cART limits the seeding of the HIV reservoir in long-lived central memory CD4+ T cells ,33,34. BWe report here a study indicating that Akt activation by Nef in Monocyte-Derived Macrophages (MDMs) can be blocked by the PI lopinavir/ritonavir, but not by the NNRTI nevirapine. We observed the important role of Akt activation in HIV-1 reactivation from latently infected U1 monocytoid cells in vitro, which could be blocked by PI but not by NNRTI. Finally, in vivo cART-blocked Akt activation in monocytes, but also in resting CD4+ T cells, meanwhile limited the size of the viral reservoir in both cell types isolated from aviremic chronically HIV-1 infected patients.\u2212, CD69\u2212, and HLA-DR\u2212 CD4+ T cells (resting CD4+ T cells) were obtained from the peripheral blood of HIV+ patients using a negative selection assay using magnetic beads  as described previously [+ patients by Ficoll gradient centrifugation as described previously [v/v) fetal calf serum. PBLs in suspension and adherent monocytes were collected after 2 h as described previously [Human PBMCs, purified PBLs, and MDMs were prepared from the peripheral blood of healthy donors and cultured as described previously . Purifieeviously . PBMCs weviously  and culteviously .6 cells) were treated with recombinant myristoylated Nef protein (rNef) from the SF2 HIV-1 strain (1\u2013100 ng/mL) . Cell pellets were collected at various times after rNef treatment, washed extensively, and either lysed before Western blot analysis or fixed with BD Cytofix  for 20 min before flow cytometric analysis. MDMs were treated with the Akt inhibitor VIII (cat # sc-202048A)   for 2 h before addition of rNef (100 ng/mL) for 30 min.MDMs  for 30 min using an RNeasy mini kit . A total of 2 \u00b5g of RNA was reverse transcribed into cDNA with Superscript IV RT  using oligo (dT) primers. The 5 \u00b5L of reverse transcription reaction product was amplified using primers against Akt . The beta-globin gene was amplified as an internal control . The PCR product was electrophoresed on a 2% agarose gel containing SYBR Green I Stain .6 cells were thoroughly resuspended in 250 \u00b5L of BD Cytofix/Cytoperm solution for 20 min at 4 \u00b0C. Cells were washed two times in 1X BD Perm/Wash solution. The cells were pelleted by centrifugation. The cells were thoroughly resuspended in 500 \u00b5L of 1X BD Perm/Wash containing PE-conjugated anti-pAkt (cat# 558275)/anti-Akt (cat# 560049) antibodies (BD Biosciences) or isotype control antibody (cat# 551436) (BD Pharmingen). The cells were incubated at 4 \u00b0C for 30 min in the dark. The cells were washed three times in 1X BD Perm/Wash solution and resuspended in 1X PBS prior to flow cytometric analysis.PBLs, monocytes, and resting CD4+ T cells were fixed and permeabilized (BD Cytofix/Cytoperm kit) . Briefly, 106/well in triplicates in 96-well plates in a final volume of 100 \u00b5L medium containing lopinavir/ritonavir, nevirapine, and Akt inhibitor VIII at 50 \u00b5M for 0\u20132 h. MDMs were left untreated or treated with rNef (1\u2013100 ng/mL) for 30 min. After 0\u20132 h, 10 \u00b5L MTT reagent was added to each well and the plates were incubated for another 4 h in the cell culture incubator at 37 \u00b0C. The plate was centrifuged at 500 g for 10 min. The cell culture media was aspirated, 100 \u00b5L of crystal-dissolving solution was added to each well, and the absorbance was measured at 570 nm using Multiskan Ex .Cell viability was measured using the MTT assay kit . Where specified, MDMs and monocytoid U1 cells stimulated or not with TNF were seeded at a density of 0.1 \u00d7 10The monocytoid U1 cell line was obtained from the AIDS Research and Reference Reagent Program , National Institute of Health (NIH)). Cells were mock-treated or treated with TNF 10 ng/mL in the absence or presence of lopinavir/ritonavir, nevirapine, and Akt inhibitor VIII at 50 \u00b5M. At 24 h post treatment, culture supernatants of U1 cells were harvested and HIV-1 production was measured by determining p24 antigen concentration by ELISA  . A WST 16 cells monocytes and resting CD4+ T cells using a QIAamp DNA Blood kit  as per the manufacturer\u2019s instructions. HIV-1 proviral DNA was quantified using a Biocentric Generic HIV DNA Cell kit  on an MX3005p real-time PCR instrument  as per the manufacturers\u2019 instructions.DNA was extracted from 10n = 8; NNRTI, n = 23) were studied for levels of Akt activation and HIV-1 proviral DNA in monocytes and autologous resting CD4+ T cells. In addition, we enrolled four HIV-1-infected individuals at the Besan\u00e7on University Hospital . These patients were na\u00efve from cART treatment and were studied for HIV-1 proviral DNA in monocytes and autologous resting CD4+ T cells.We enrolled 31 chronically HIV-1-infected individuals at the Besan\u00e7on University Hospital . The patients were treated with cART (treatment range: 1.4\u201327.2 years) and had undetectable plasma HIV-1 RNA levels (<40 copies/mL) for at least 1 year. These 31 patients treated with cART  or Wilcoxon t test. Differences were considered significant at a value of p < 0.05.The figures show the means and standard deviations for independent experiments. Plotting and statistical analysis were performed using Excel. Results from in vitro reactivation studies and HIV proviral DNA using patient cell cultures of monocytes and resting CD4+ T cells are shown as medians and quartiles. Data sets were analyzed using an unpaired nonparametric All of the patients who were enrolled at the Besan\u00e7on University Hospital (France) gave their written informed consent to participate in the study according to the Helsinki declaration. The Human Protection Committee East Area II (CPP EST-2) from France was consulted and approved the study (CPP14/455) ; URL of registry: clinicaltrials.gov; Date of registration: 29 July 2016; Date of enrolment of the first participant to the trial: 9 June 2015; \u201cRetrospectively registered\u201d). This study did not rely solely on medical records. The authors did not have any contact with the study subjects and performed tests on patient blood samples that were part of routine care. The blood samples were anonymized before being used by the authors.473 and threonine308 in MDM treated with rNef as determined by Western blotting and flow cytometry (We studied the impact of Nef on both Akt expression and activation in MDMs. We observed that treatment of MDMs with rNef led to enhanced Akt expression in a dose-dependent manner (ytometry A. The inytometry C. We didytometry D.Since HIV-1 protease inhibitors (PI), but not reverse transcriptase inhibitors (RTI), have been reported to block Akt activation in several cell types, such as peripheral blood mononuclear cells (PBMCs) and monocytes/macrophages ,17,18,19Since Akt activation participates in immune activation and favours resistance to apoptosis, controlling the Akt pathway using PIs could have an impact on HIV-1 recovery from latently infected monocytoid cells ,43. Ther6 versus 288 \u00d7 106 cells/L, p = 0.734), median CD4 counts at the initiation of treatment , median CD4 counts at the last point , and median HIV RNA load at zenith  between PI-treated and NNRTI-treated patients (p = 0.694) and the duration of therapy . The duration with undetectable plasma HIV-1 RNA levels was not significantly different in the PI-treated arm compared to the NNRTI-treated arm   and with undetectable plasma HIV-1 RNA levels (<40 copies/mL) for at least 1 year were included in the study between 2015 and 2017. Of these thirty-one patients  treated with cART, twenty-three were treated with NNRTI-based cART and eight with PI-based cART (as their most recent treatment) and for more than one year . We did patients . We did = 0.804) .In HIV-1-positive patients under cART, Akt activation was lower in monocytes compared to autologous resting CD4+ T cells with the lowest activation in the bulk of PBLs A Table . The lowp = 0.114) (p = 0.095)   and resting CD4+ T cells  compared to cART-na\u00efve patients  A. A simi= 0.095) B. Under ctively) C,D. In Ppatients A,B. We dpatients A,B (Tablpatients .HIV-1 Nef, a pleiotropic early viral protein, has been demonstrated to activate several key signaling molecules, including Akt, MAPK, and ERK ,46. In tExogenous HIV-1 Nef is internalized by MDMs as reported previously for CD4+T cells ,47. The Latency represents the major obstacle in the complete cure of HIV-1 ,54,55,56Although the use of cART has been extensively studied in regard to residual viremia, only little is known on their impact on cellular reservoirs of HIV-1 especially in monocytes isolated from patients. We determined the levels of Akt activation and HIV-1 proviral DNA in monocytes and autologous resting CD4+ T cells from cART-treated patients. In cART-na\u00efve patients, we observed that the amount of HIV-1 proviral DNA was 0.5 log smaller in monocytes than in resting CD4+ T cells . In agreIn addition to its role in HIV-1 reactivation and latency, Akt activation is critical for cell survival . In factOur study shows that exposure of MDMs to HIV-1 Nef leads to enhanced Akt expression and activation. Importantly, we observed that the immunomodulatory effects of PIs, by blocking Akt activation especially triggered by Nef and TNF, limit HIV-1 recovery from latently infected monocytoid U1 cells in vitro. Finally, in aviremic chronically HIV-1-infected patients, our study indicates that PI-based cART decreases Akt activation and limits the size of the monocyte reservoir. Altogether, our results support the need to further assess the therapeutic use of HIV-1 protease inhibitors in view of their impact on persistence of myeloid HIV-1 reservoirs and might provide and/or accompany new therapeutic strategies for a remission."}
+{"text": "We test here the impact of HIV protease inhibitor (PI) based combination anti-retroviral therapy (cART) over nonnucleoside reverse transcriptase inhibitor (NNRTI)-based cART on HIV-1 reactivation and integration in resting CD4+ T cells. This is a prospective cohort study of patients with chronic HIV-1 infection treated with conventional cART with an undetectable viremia. We performed a seven-year study of 47 patients with chronic HIV-infection treated with cART regimens and with undetectable plasma HIV-1 RNA levels for at least 1 year. Of these 47 patients treated with cART, 24 were treated with a PI-based regimen and 23 with a NNRTI-based regimen as their most recent treatment for more than one year. We evaluated the HIV-1 reservoir using reactivation assay and integrated HIV-1 DNA, respectively, in resting CD4+ T cells. Resting CD4+ T cells isolated from PI-treated patients compared to NNRTI-treated patients showed a limited HIV-1 reactivation upon T-cell stimulation (p\u2009=\u20090\u00b7024) and a lower level of HIV-1 integration (p\u2009=\u20090\u00b7024). Our study indicates that PI-based cART could be more efficient than NNRTI-based cART for limiting HIV-1 reactivation in aviremic chronically infected patients.A latent viral reservoir that resides in resting CD4 Usually, a better virologic performance of nonnucleoside reverse transcriptase inhibitor-(NNRTI)-based cART compared to protease inhibitor-(PI)-including regimens has been reported10+ T cells613+ T cells in aviremic chronically infected patients is so far unknown. We report here a study indicating a higher efficiency of PI-based cART over NNRTI-based cART for limiting HIV-1 reactivation in CD4+ T cells from aviremic chronically HIV-1 infected patients.In contrast to the measurement of viremia in patients on cART, the impact of cART on the size of the cellular reservoirs of HIV-1 has been much less studied. Initiation of cART during primary HIV infection may limit the establishment of viral reservoirs, and very early cART limits the seeding of the HIV reservoir in long-lived central memory CD46 versus 333\u00b7106 cells/l, P\u2009=\u20090.579), median CD4 counts at initiation of treatment , median CD4 counts at the last point , and median HIV RNA load at zenith  between PI-treated and NNRTI-treated patients (P\u2009=\u20090\u00b7985) and the duration of therapy . The duration with undetectable plasma HIV-1 RNA levels was not significantly different in the PI-treated arm compared to the NNRTI-treated arm  (Forty-seven patients with chronic HIV-1 infection treated with cART (treatment range: 2 years to 16 years) and with undetectable plasma HIV-1 RNA levels (<40 copies/ml) for at least 1 year were included in the study between 2008 and 2014. Of these 47 patients  treated with cART, 24 were treated with PI-based cART and 23 with NNRTI-based cART (as their most recent treatment) for more than one year  and S2. patients . We did =\u20090\u00b7581) . At the =\u20090\u00b7581)  and S2.ex vivo reactivation capacity of the HIV-infected cells depending of cART treatment, we analyzed the effect of a known HIV inducer (anti-CD2+anti-CD28 antibodies)15ex vivo cultures of purified resting CD4+ T cells isolated from HIV+ patients treated with PI-based cART and with NNRTI-based cART. Since latently infected resting CD4+ T cells that harbour integrated replication-competent viral DNA represent the primary long-lived source of persistent HIV-1 in patients under cART5+ T cells isolated from HIV+ patients. We observed, following reactivation of HIV-1 from latency in purified resting CD4+ T cells, that HIV-1 recovery was 0\u00b792\u2009log of HIV RNA copies/ml  lower in HIV-1-infected patients treated with PI compared to those treated with NNRTI as their lastly administered treatment  lower in purified resting CD4+ T cells isolated from HIV-1 patients treated with PI-based cART compared to HIV-1-infected patients treated with NNRTI-based cART  and the Besan\u00e7on University Hospital (France) gave their written informed consent to participate in the study according to the Helsinki declaration. The study was approved by the local ethics committees of the Saint-Pierre University Hospital  and the Besancon University Hospital  (CPP EST-2). To perform our study, we used blood samples that were part of a routine care. It was an observational study and not a clinical trial. According to the French Regulatory Authority for clinical studies, prospective and retrospective studies with observation analysis only are not evaluated by Human Protection Committees. The Human Protection Committee East Area II from France was consulted and issued a formal waiver of approval. This study did not rely solely on medical records. The authors did not have any contact with the study subjects and performed tests on patient blood samples that were part of a routine care. The blood samples were anonymized before being used by the authors.We enrolled 47 chronically HIV-1 infected individuals at the St-Pierre Hospital  and the Besan\u00e7on University Hospital . The patients were treated with cART (treatment range: 2 years to 16 years) and had undetectable plasma HIV-1 RNA levels (<40 copies/ml) for at least 1 year. Characteristics of these patients were well documented and presented in \u2212, CD69\u2212, HLA-DR\u2212 CD4+ T cells (resting CD4+ T cells) were obtained from the peripheral blood of HIV+ patients using a negative selection assay using magnetic beads  as described previouslyPurified CD255 purified CD25\u2212, CD69\u2212, HLA-DR\u2212 CD4+ T cells (resting CD4+ T cells) were mock-treated or treated with anti-CD2+ anti-CD28 antibodies as a positive control for global T-cell activation15+ T cells isolated from the peripheral blood of HIV-infected patients, six days after treatment, culture supernatants were tested for quantitative HIV-1 RNA levels using the COBAS AmpliPrep/COBAS Amplicor HIV-1 Monitor Test, according to the manufacturer\u2019s instructions.One day after isolation, 5\u2009\u00d7\u200910Alu-gag PCR with some modificationsAlu elements that are present in the human genome. The level of HIV-1 integration was measured by comparing the detection signal to an integration standard curve that was prepared from U1 cells. ACH2 cells were used for calibration. The sequences of first step PCR amplification primers were as follows: genomic Alu forward, 5\u2032-GCCTCCCAAAGTGCTGGGATTACAG-3\u2032; and HIV-1 gag reverse, 5\u2032-GCTCTCGCACCCATCTCTCTCC-3\u2032. The reaction conditions were 1x Tfi PCR reaction buffer, 1.5\u2009mM MgCl2, 1\u2009mM dNTPs, 100\u2009nM Alu forward primer, 600\u2009nM gag reverse primer and 5 U of Tfi DNA polymerase (Invitrogen). The thermal cycler  was programmed to perform a 2\u2009min hot start at 94\u2009\u00b0C, followed by 20 cycles of following: denaturation at 94\u2009\u00b0C for 30\u2009sec, annealing at 50\u2009\u00b0C for 1\u2009min, and extension at 72\u2009\u00b0C for 1\u2009min 40\u2009sec. The second round of real time quantitative PCR was performed using 21\u2009\u03bcl of Nested Alu-gag PCR along with standard curve amplification products. The sequences of the primers were as follows: LTR forward, 5\u2032-GCCTCAATAAAGCTTGCCTTGA-3\u2032; and LTR reverse, 5\u2032-TCCACACTGACTAAAAGGGTCTGA-3\u2032. The LTR Taqman probe, labeled at its 5\u2032 terminus with the reporter fluorophore 6-carboxyfluorescein (FAM) and at its 3\u2032 terminus with the quencher 4-(4-dimethylamino-phenylazo)-benzene (DABCYL), had the following sequence: 5\u2032-FAM-GCGAGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGCTCGC-DABCYL-3\u2032. The reaction was carried out in a volume of 50\u2009\u03bcl containing 1 X Taqman Master mix , 250\u2009nM concentration of LTR forward and reverse primers, and 200\u2009nM Taqman probe. The reactions were performed on Stratagene Mx2005\u2009P. The thermal program was 2\u2009min hot start at 95\u2009\u00b0C, followed by 40 cycles of denaturation at 95\u2009\u00b0C for 15\u2009sec and annealing and extension at 60\u2009\u00b0C for 1\u2009min.Integrated HIV-1 DNA was measured by using a previously described x vivo reactivation studies and HIV integration using patient cell cultures of resting CD4+ T cells are shown as medians. Data sets were analyzed using an unpaired nonparametric t-test (Mann-Whitney test). Differences were considered significant at a value of p\u2009<\u20090.05.The program used for plotting and statistical analysis was Prism version 6.0 (GraphPad Software). Results from eHow to cite this article: Kumar, A. et al. Limited HIV-1 Reactivation in Resting CD4+ T cells from Aviremic Patients under Protease Inhibitors. Sci. Rep.6, 38313; doi: 10.1038/srep38313 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."}
+{"text": "The 5\u2009at.% Fe doped In2O3 nanodot arrays are shown to consist of the cubic bixbyite structure of In2O3. The nanodot arrays are demonstrated to be doped by Fe ions with mixed valences of\u2009+2 and\u2009+3, ruling out the presence of cluster and secondary phase related to Fe. The nanodot arrays exhibit the ferromagnetism at room temperature, where the magnetic moment increases as the dot size is reduced, rising to a maximum of about 230\u2009emu/cm3 . This indicates an effect due to the surface of the nanodot arrays. The optical band width is also increased to 4.55\u2009eV for the smallest dot array, thus indicating that the surface states are responsible for the magnetism and also enhance the band gap due to Burstein-Moss effect. Our results will be benefit for understanding the physical properties of oxide semiconductor nanostructures in the application of nano-spintronics devices.Ordered Fe-doped In Since Dietl et al. theoretically predicted the room temperature (RT) ferromagnetism in Mn doped ZnO and GaN semiconductors via Zener\u2019s p-d exchange model2, many efforts have been carried out on a series of transition metal (TM) elements doped wide-band-gap oxide semiconductors, such as ZnO5, TiO27, In2O313 and SnO215. Among them, Fe-doped In2O3 magnetic semiconductor is very attractive mainly due to its excellent electrical conductivity, high optical transparency, and high solubility of Fe ions in In2O3 host lattice11. In the past decade, lots of studies have mainly focused on the thin film, bulk, and polycrystalline powders of Fe-doped In2O310. As advanced devices call for smaller nanostructured systems, the fabrication of lower-dimensional DMS structures is drawing increasing interest due to their advantages of small size, unique magnetic and optical properties17.Dilute magnetic semiconductors (DMSs), combining both semiconductor and ferromagnetic properties, have gained great attention due to their potential applications in spintronic devices20. In these studies, the self-assembly Stranski-Kranstanov (SK) growth method is adopted. However, this method results in randomly distribution, easily aggregate, and applies only to growing the material which has a large lattice mismatch with the substrate, such as MnGe and GaMnAs quantum dots on Si. Another method used to fabricate the well-patterned semiconductor nanostructures is the lithography technique or electron beam lithography, but they are impractical for large array sizes and cost relatively high22. Recently, ultrathin porous anodized aluminum (PAA) was used as contact templates to fabricate the large-areas, highly ordered nanostructures because of their low cost, more flexibility for pore size and separation control, easy fabrication, and so forth26. This bottom-up template can be used as the mask which excellently adheres to the substrate for direct deposition, and the highly ordered nanodot arrays can be obtained after removal the PAA templates.Actually, the fabrication and magnetic properties of Mn or Cr doped Ge, GaAs and InAs DMS nanostructured materials, such as quantum dots, have been reported2O3 nanodot arrays by a comprehensive analysis of structure and magnetic properties of the arrays. The band gap dependence on the size of Fe-doped In2O3 nanodot arrays was also investigated by the experiment.In this paper, we presented the growth of DMS nanodot arrays by pulsed laser deposition (PLD) using the PAA templates, and provided the insights into the origin of the RT ferromagnetism in Fe-doped In0.95Fe0.05)2O3 nanodot arrays with average diameters of 80, 65 and 50\u2009nm, respectively. The dot height and inter-dot distance of these arrays are 40\u2009nm and 105\u2009nm, respectively. The highly ordered nanodot arrays are the same hexagonally lattice pattern as the PAA templates. The diameter distribution of the (In0.95Fe0.05)2O3 arrays is homogenous, and the diameters of these dot arrays are approximately equal to those of the corresponding templates. Moreover, the smaller diameter of nanodot arrays than that of the PAA templates can also be obtained by reducing the height of nanodot arrays. Figure\u00a00.95Fe0.05)2O3 nanodot arrays with the height of 10\u2009nm and diameter of 35\u2009nm, where the PAA template with the pore diameter of 50\u2009nm is left behind intentionally. The AFM profiles 2O3 nanodot arrays with different diameters are shown in Fig.\u00a02O3 and exhibit a preferred (222) orientation. No extra diffraction peaks of secondary phases, such as metallic Fe and Fe oxides, could be detected. A slight shift in the position of the (222) peaks to higher angles is observed, indicating a reduction in the lattice constant  full width at half maximum (FWHM) peak of the nanodot arrays is large, and the FWHM becomes broader with the decrease of the diameter. This reveals the weak crystallinity and the small grain size of nanodot arrays. The grain size of (In0.95Fe0.05)2O3 nanodot arrays is also calculated by the Scherrer formula of d\u2009=\u20090.89\u03bb/(\u03b2 \u00b7 cos\u03b8) . As shown in Fig.\u00a0The diffraction patterns of the 2O3 nanodot arrays. As shown in Fig.\u00a03/2 and Fe 2p1/2 are located at 710.3 and 723.9\u2009eV, respectively, which excludes the formation of metallic Fe clusters, the binding energy of Fe 2p3/2 and Fe 2p1/2 in which are respectively 707.0 and 720.0\u2009eV29. Furthermore, comparing with the binding energies of Fe2+ 2p3/2 (709.9\u2009eV) and Fe3+ 2p3/2 (711.0\u2009eV)29, we can conclude that the Fe element exists in the ion form with the mixed valence of\u2009+2 and\u2009+3 in the (In0.95Fe0.05)2O3 nanodot array, which is likely facilitated by the oxygen vacancies due to the very low deposition oxygen pressure (5\u2009\u00d7\u200910\u22123 mTorr). To further study the local structure of Fe in the In2O3 lattice, we performed the XAS measurements at the Fe L-edge. Figure\u00a0L2,3 XAS spectra of the samples. It can be found that the L3 edge is composed of two peaks (707\u2009eV and 709\u2009eV) and 720\u2009eV and 722\u2009eV are related to the L2 edge. The split in the Fe L2,3 edges is usually attributed to the Fe ions with mixed valence of Fe2+ and Fe3+, further ruling out the appearance of metallic Fe31, which is consistent with the above XPS analysis. Moreover, as presented in the Fig.\u00a0L3 edge shoulder peaks to lower energies with the decreased diameter of nanodot arrays, indicating an increase of the Fe2+/Fe3+ ratio. This increase of the Fe2+ ions in smaller nanodot arrays may be ascribed to the more oxygen vacancies due to the increase in surface-to-volume ratio as the diameter decreases28.The XPS measurement was performed to investigate the valence state of Fe element in the 2O3 nanodot arrays, indicating a strong ferromagnetism above RT. As shown in Fig.\u00a0s) of nanodot arrays sharply increases from 1.85 to 15.30 \u00b5B/Fe as the nanodot size decreases , which is much larger than that of the 5\u2009at.% Fe doped In2O3 films11. Such huge Ms in zero dimensional nanodot arrays may be attributed to the abundant defects associated with the larger surfaces since the defects are reported to play an important role in the magnetic behavior of DMSs31. It is known that the vacancies and interstitials in oxide semiconductors can occur during the growth32. For zero-dimensional nanostructures, such defects are more prevalent as compared to films because of the increase in the surface area33. As the nanodot size decreases, the surface defects are increased due to the increase of the surface-to-volume ratio, and therefore, the enhanced magnetic moments is observed. Plot of Ms (\u00b5B/Fe) vs 1/D shown in the inset of Fig.\u00a00.95Fe0.05)2O3 nanodot arrays.All above structural and compositional results approve that the Fe element incorporates into the In34. This agrees with our results of XRD, XPS and XAS.The zero-field cooled (ZFC) and field-cooled (FC) curves for the 50 nm-diameter nanodot array is shown in Fig.\u00a00.95Fe0.05)2O3 nanodot arrays with different diameters. With the decrease of the nanodot arrays in diameter, the absorption edge of nanodot arrays shows an obvious blue-shift, indicating an increase of the band gap. Figure\u00a035:\u03b1 is the absorption coefficient; h\u03bd is the photo energy; Eg is the optical band gap; and A is a constant. The Eg of the samples is obtained by extrapolating the linear region to zero absorption. It can be seen that the band gap increases from 3.54 to 4.55\u2009eV as the diameter decreases from the 80 to 35\u2009nm. The increased band gap in (In0.95Fe0.05)2O3 nanodot arrays can be possibly attributed to the Burstein-Moss effect (BM effect). The BM shift of the Eg can be obtained by the following equation:36Figure\u00a0ne, and h are the effective electron mass in the conduction band, carrier concentration and Plank\u2019s constant, respectively. As the diameter of nanodot arrays gets smaller, as mentioned above, there will be more oxygen vacancies which act as donors and contribute to carriers. Obviously, an increase in the carrier density will ultimately induce a blue-shift of the band gap. The decrease of the lattice constant and grain size shown Fig.\u00a038.where 2O3 nanodot arrays with different sizes. The Fe-doped In2O3 nanodot arrays exhibit the obvious size-dependent magnetic and optic behaviors. Moreover, the surface defects play a crucial role in the unique huge magnetic moment of the Fe-doped In2O3 nanodot arrays. The increased band gap in (In0.95Fe0.05)2O3 nanodot arrays is possibly attributed to the Burstein-Moss effect. These results are very promising for future spintronic devices.In summary, we have observed the RT ferromagnetism in the highly ordered Fe-doped In2C2O4 at 0\u2009\u00b0C. After the two-step anodization, a thin polymethylmetacrylate (PMMA) layer was spin-coated onto the top surface of the PAA templates, and then the aluminum was etched away by immersing the template in a mixture of CuCl2 and HCl. The thin nonporous barrier layer was removed by floating the PAA templates in 5\u2009wt% H3PO4 at 30\u2009\u00b0C for 40\u2009min. The template was transferred to the Al2O3 substrate, and the PMMA layer was dissolved by C3H6O at 60\u2009\u00b0C. In this work, the average pore diameter of the PAA templates can be tuned from 50 to \u200980\u00a0nm by immersing the PAA templates in H3PO4 for different durations, and the thickness and inter-pore distance of the mask was about 200 and 105\u2009nm, respectively. The Fe-doped In2O3 nanodot arrays was subsequently deposited on the PAA/Al2O3 (0001) substrate from the ceramic target of nominal composition (In0.95Fe0.05)2O3 by the PLD using a KrF excimer laser (\u03bb\u2009=\u2009248\u2009nm) with a pulse repetition rate of 10\u2009Hz and an energy density of 250 mJ/pulse. The target-to-substrate was 7\u2009cm and the ceramic (In0.95Fe0.05)2O3 target was prepared from high-purity In2O3  and \u03b1-Fe2O3  powders by a conventional solid-state reaction technique. During deposition, the substrate temperature was maintained\u00a0at 600\u2009\u00b0C, the chamber oxygen pressure was 5\u2009\u00d7\u200910\u22123 mTorr, and the height of nanodots were controlled by the deposition time. After deposition, the samples were cooled down to room temperature slowly at the same oxygen pressure. The PAA template was then dissolved using a 5\u2009wt% NaOH solution at 35\u2009\u00b0C and highly ordered (In0.95Fe0.05)2O3 nanodot arrays were obtained.In our experiment, the ultrathin PAA templates with various pore diameters were fabricated by a two-step anodization at a constant voltage of 40\u2009V in 3.6\u2009wt% H0.95Fe0.05)2O3 nanodot arrays and PAA templates were investigated by scanning electron microscope (SEM) and atomic force microscopy (AFM). The crystal structures of the nanodot arrays and the films were characterized by x-ray diffraction (XRD) using Cu K\u03b1 radiation (\u03bb\u2009=\u20090.15406\u2009nm). The composition of the samples was determined by x-ray photoelectron spectroscopy (XPS) and x-ray absorption spectra (XAS). Optical transmission spectra of the samples were measured by an UV-vis spectrophotometer. The magnetic properties measurements were performed by a superconducting quantum interference device (SQUID) magnetometer.The morphology of the (InSupplementary Information"}
+{"text": "Background: Juvenile dermatomyositis (JDM) is a systemic, autoimmune, interferon (IFN)-mediated inflammatory muscle disorder that affects children younger than 18 years of age. JDM primarily affects the skin and the skeletal muscles. Interestingly, the role of viral infections has been hypothesized. Mammalian 2\u2032-5\u2032-oligoadenylate synthetase (OAS) genes have been thoroughly characterized as components of the IFN-induced antiviral system, and they are connected to several innate immune-activated diseases. The main purpose of the paper is to define the potential interrelationship between the OAS gene family network and the molecular events that characterize JDM along with double-stranded RNA (dsRNA) molecular pathways. Methods: We analyzed three microarray datasets obtained from the NCBI in order to verify the expression levels of the OAS gene family network in muscle biopsies (MBx) of JDM patients compared to healthy controls. Furthermore, From GSE51392, we decided to select significant gene expression profiles of primary nasal and bronchial epithelial cells isolated from healthy subjects and treated with polyinosinic-polycytidylic acid (poly(I:C)), a synthetic analog of double-stranded RNA (dsRNA), a molecular pattern associated with viral infection. Results: The analysis showed that all OAS genes were modulated in JDM muscle biopsies. Furthermore, 99% of OASs gene family networks were significantly upregulated. Of importance, 39.9% of modulated genes in JDM overlapped with those of primary epithelial cells treated with poly(I:C). Moreover, the microarray analysis showed that the double-stranded dsRNA virus gene network was highly expressed. In addition, we showed that the innate/adaptive immunity markers were significantly expressed in JDM muscles biopsies. and that their levels were positively correlated to OAS gene family expression. Conclusion: OAS gene expression is extremely modulated in JDM as well as in the dsRNA viral gene network. These data lead us to speculate on the potential involvement of a viral infection as a trigger moment for this systemic autoimmune disease. Further in vitro and translational studies are needed to verify this hypothesis in order to strategically plan treatment interventions. Juvenile dermatomyositis (JDM) is a serious pediatric systemic autoimmune disease primarily affecting the proximal muscles and skin. JDM cutaneous manifestations can be difficult to treat and may progress to ulcerative diseases or subcutaneous calcifications . The incPrevious studies indicate that innate immunity plays a pivotal role in the pathogenesis of JDM . Mature The oligoadenylate synthase (OAS) proteins are double-stranded (ds) RNA-activated enzymes which are induced by IFNs. The OAS family consists of four members, OAS1, OAS2, OAS3, and OAS-like protein (OASL) . These pIn this paper, we explored the possible role of OAS genes antiviral response in JDM. The analysis of the OAS gene family and the genes related to it suggests that dsRNA viral infections could be involved in the pathogenesis of this disease. n = 20), acute quadriplegic myopathy  (n = 3), JDM (n = 21), amyotrophic lateral sclerosis (ALS) (n = 9), spastic paraplegia  (n = 4), fascioscapulohumeral muscular dystrophy (FSHD), Emery Dreifuss muscular dystrophy  (n = 22), Becker muscular dystrophy  (n = 5), Duchenne muscular dystrophy (complete loss of dystrophin) (n = 10), Calpain 3 (LGMD2A) (n = 9), dysferlin (LGMD2B) (n = 10), and fukutin related protein FKRP  (n = 7). The GSE3307 contained 13 groups of datasets of several muscle diseases. The available groups were: normal human skeletal muscle , OAS2 (p < 0.001), OAS3 (p < 0.001), and OASL (p < 0.001) were highly expressed in JDM MBx compared to healthy controls . Furthermore, none of the expression values of OAS1 and OAS3 genes in the healthy group fell in the expression range of the JDM group A. To validate these results, we decided to perform a new analysis of two more different microarray datasets (GSE11971 and GSE48280) composed by the MBx of healthy donor and children affected by JDM. The analysis confirmed the results obtained for GSE3307 .In order to verify if the OASs genes could be involved in the JDM pathogenesis, we performed a gene network analysis. For this reason, we interrogated the GIANT and STRING online software.For the GIANT online tool, we set the tissue menu as skeletal muscles, the network filter with a minimum relationship confidence of 0.18, and the maximum number of genes as 50. The list of 54 genes (including the four OASs genes) obtained was loaded into the multiple sequences search set of the STRING software in order to obtain a graphical network representation A. STRING analysis showed a large number of genes belonging to a specific biological process (response to the virus). These results were confirmed by a gene ontology analysis performed with FunRich online tools .p < 0.0001) (p < 0.0001)  B. Among  0.0001) B. No sig 0.0001) . All these results confirmed a potential role played by the OAS gene family network in the muscles of patients with JDM.p-value <0.0001) . Starting from this result, we decided to overlap the transcriptome of GSE51392 composed by primary epithelial cells treated with polyinosinic-polycytidylic acid (poly(I:C)) (20 \u00b5g/mL for 24 h) and GSE3307 microarray dataset, based on JDM MBx. The FDR analysis obtained by GEO2R, produced for GSE51392 a large number of significantly upregulated genes (5515 genes) . The ove<0.0001) .Inside the list of 1736 significantly upregulated genes that overlapped between GSE51392 and GSE3307, the OAS genes family was still present. Also, the dsRNA genes belonging to the transduction pathway, such as RIG-I , IFIH1, IPS1 , LGP2 , CD14, TLR3, TBK1, and IRF, were part of those genes that were significantly modulated in common, in the two GSE  Supplem. The expp < 0.0001), IFIH1 (p < 0.0001) (p < 0.0001) and TLR3 (p < 0.0001) (p < 0.01) was significantly downregulated (p < 0.001), TBK1 (p < 0.001), and IRF3 (p < 0.001) were significantly upregulated in JDM vs. healthy controls (p < 0.001) (p < 0.0001) in JDM vs. healthy controls , or DDX58 or IFIH1 (Cytoplasmic pathways), respectively . The mic 0.0001) A, and al 0.0001) B, in MBxegulated , whereascontrols B. As forcontrols A in JDM < 0.001) A. Similacontrols A. This fFurthermore, the expression levels of OAS genes family were related to DDX58  and IFIH1 , the principal pattern recognition receptor (recognizing dsRNA), and a sensor for viral infection .Similar results were obtained for GSE51392. Indeed, RIG-I (DDX58), IFIH1, OAS1, OAS2, OAS3, and OASL resulted significantly modulated and correlated to each other .In order to verify the role played by the innate immunity and its correlation with OAS gene family expression in JDM patients\u2019 biopsies, we performed an expression analysis of the main markers involved.With regards to the innate immunity involvement in JDM, we analyzed cyclophilin A  , CD14, CThe expression levels of all markers, excepting CD14, were significantly correlated with OAS genes family expression levels . FurtherThe correlation analysis revealed that IFNGR1 was significantly correlated with OAS gene family expression. This result was probably due to the fact that the production of IFNG is a prerogative of T lymphocytes, and not of innate immunity ,33. OverIn order to present a complete framework of the situation on the innate immunity activity, we decided to analyze the expression levels of monocyte activation markers such as IL18 , CD163 , LAMP3 , and CHIAll these findings would seem to confirm the involvement of innate immunity in the biopsies of patients with JDM, and a possible correlation with the OAS family gene expression.In order to complete the immunological pathways potentially involved in JDM, we decided to analyze the involvement of adaptive immunity.In recent years, the role played by Major Histocompatibility Complex MHC class I/II in JDM has already been studied ,39,40. IFurthermore, the MHC class I expression levels were significantly correlated with OAS gene expression . No corrWith regard to the analysis of genes referable to recruitment and activation of T and B lymphocytes, we selected CCL19 , CCL3 4, CCL4 4, CCL5 44, and CXThe correlation analysis between OAS gene expression levels and CCL19 showed significant results. With regard to CCL4, CCL5, and CXCL13, we observed a significant correlation with OAS1, OAS2, and OAS3. CCL3 was only positively correlated with OASL .These results could explain the possible recruitment and subsequent activation of adaptive immunity in the muscular biopsies of patients affected by JDM.In this manuscript, we analyzed three different microarray datasets of MBx from patients with JDM, and we showed that almost 98% of the OAS genes family network was significantly upregulated. Surprisingly, the microarray analysis showed that the dsRNA virus gene network was highly expressed in the MBx of JDM patients . These dIt has been demonstrated that type 1 IFNs are linked to the pathogenesis of some forms of myositis , althougSeveral papers have shown that excessive keratinocyte apoptosis can be responsible for the development of skin lesions in subjects with dermatomyositis . One of Furthermore, it is important to highlight that innate immunity could play a predominant role in the progression of JDM, before the induction of adaptive immune responses . One of In addition, we found upregulation of the genes produced during extracellular or intracellular dsRNA viral replication. Recently, several papers have documented the modulation of MDA5 in JDM muscle biopsies . This moThe hypothesis that the onset of autoimmune rheumatic diseases could be triggered by virus infection is controversial and based on case reports ,65,66,67This paper proposes to explain the role of innate and adaptive immunity in JDM through OAS gene family action. Our observations led us to think that a plausible explanation of JDM etiogenesis could be that monocytes that are locally attracted in the muscle of those that are affected by JDM could transform into activated macrophages by proceeding with the elimination of the causative etiological agent . Subsequently, the release of chemokines specific for T and B lymphocytes could determine the polarization and activation (IFNG) of T lymphocytes. Moreover, the cytotoxic activity of CD8 T lymphocytes mediated by the major histocompatibility complexes of class I expressed by myocytes could determine the propagation of the disease.These results are not conclusive. Further studies are needed to investigate the role of the OAS gene family in the disease mechanism, and whether they contribute to muscle tissue damage in patients with JDM disorders. http://www.ncbi.nlm.nih.gov/geo/) (access on 18 January 2018) databank under accession numbers GSE3307 (GPL96/97), GSE11971, GSE48280, and GSE51392 in order to delineate a correlation between the OAS gene family network and the molecular events that characterize JDM.In this manuscript, we evaluate microarray datasets from the NCBI GEO (n = 20) from volunteers participating in exercise physiology studies, and MBx from patients with JDM . Complete experimental details can be retrieved in the publication by Bakay et al. and Dadgar et al. [From the GSE3307 microarray dataset, we sorted the data of normal MBx . From the GSE11971 microarray dataset, we selected the data of MBx from 16 girls with JDM not receiving any nonsteroidal or immunosuppressive therapy, and four healthy age- and sex-matched controls. As for the GSE48280 microarray dataset, we analyzed the data of MBx from five patients with JDM  and five controls obtained from individuals during hip replacement surgery. The complete experimental details of the two microarray datasets can be found in the publications by Chen et al. and Su\u00e1rez-Calvet et al. ,71.From GSE51392, we decided to select significant gene expression profiles of primary nasal and bronchial epithelial cells isolated from healthy subjects and treated with Polyinosinic-polycytidylic acid (poly I:C) 20\u00b5g/mL for 24 hr. This molecule is a synthetic immunostimulant mimicking a double-stranded RNA (dsRNA) structure. The poly(I:C) cell treatment is able to activate antiviral receptors such as TLR3, RIG-I/MDA5, and PKR, with subsequent induction of multiple inflammatory pathways, such as NF-kB and IRF. For more details refer to the publication by Wagener AH et al. .p < 0.01, for the GPL96 Platform of GSE3307, we identified 3348 upregulated and 6480 downregulated genes in JDM MBx vs. healthy MBx; for the GPL97 Platform, we found 1313 upregulated and 4693 downregulated genes. In order to exclude the genes redundant in the two platforms GPL96/97, we overlapped the significant up- and downregulated genes obtained during the analysis. The number unique upregulated genes obtained was 4353, and the unique downregulated genes was 8018. With regard to GSE11971, we identified 5395 upregulated and 2942 downregulated genes, comparing JDM MBx vs. healthy MBx. For GSE48280, we identified 1511 upregulated and 1621 downregulated genes. The analysis of GSE51392 produced the following results: 5515 upregulated and 6146 downregulated genes (The Multi-Experiment Viewer (MeV) software was chosen to identify differentially expressed genes. In cases where multiple probes insisted on the same NCBI GeneID, we selected those with the highest variance. By restricting the threshold level of significance to ed genes .http://www.bioinformatics.lu) (access on 18 January 2018). Weighted Gene Networks were obtained for the commonly modulated genes using STRING software (http://string-db.org/) (access on 18 January 2018) and GIANT software (http://giant.princeton.edu/) (access on 18 January 2018). The STRING combined score was determined with data from Neighborhood in the Genome, Experimental and Biochemical Data Co-occurrence Across Genomes [In order to identify genes that are commonly modulated in OAS gene network and JDM MBx, Venn diagrams were drawn using the online utility Venn Diagram Generator ( Genomes .http://giant.princeton.edu/) (access on 18 January 2018), setting the tissue menu to skeletal muscles, the network filter to a minimum relationship confidence of 0.18, and the maximum number of genes to 50.The OASs gene pathway was obtained from the GIANT database  (access on 18 January 2018) is an online tool that is able to provide a global view of genes interactions. This information is derived from published experimental evidence and bioinformatics analysis predictions. Using the available online graphical user interface, we set the filters to 0 related genes and no related attributes, in order to restrict the gene network. The results for each gene were obtained by linear regression [GeneMANIA (gression .http://www.funrich.org) (access on 20 January 2018) against the human FunRich background database [Biological pathway, biological process, expression site heatmap, and analysis of KEGG pathways were drawn with the FunRich tool (database .p < 0.05). A Chi-square with a Yates correction analysis was performed by GraphPad Prism software for a 2 \u00d7 2 contingency table . The analysis of the microarray data by a Z-score transformation was used in order to allow for the comparison of microarray data independent of the original hybridization intensities. [For statistical analysis and graphical representation, we used the Prism 7 software . The Shapiro\u2013Wilk test was used as the normality test. Significant differences between groups were assessed using the Mann\u2013Whitney U test, and the Kruskal\u2013Wallis test was performed to compare data between all groups, followed by Dunn\u2019s post hoc test. Correlation analyses were determined using Spearman\u2019s \u03c1 correlation . All tesIn conclusion, our datasets analysis shows that OAS genes are upregulated in the muscle biopsies of JDM patients .OASs gene expression could be induced by infiltrating monocytes, activation by dsRNA viruses, or by necrotic/apoptotic events in muscle myositis. Moreover, the activation of IFNs pathway induced by viral infections could represent the link between the upregulation of both the OAS gene family and the dsRNA virus network that we observed in our study."}
+{"text": "Healthy young women (n\u00a0=\u00a029) were allocated to a group (n\u00a0=\u00a014) receiving an amino-acid supplement  and a placebo group (n\u00a0=\u00a015) receiving a supplement not-containing the amino acids. The amino-acid supplement and placebo were given twice/day for 6 weeks. After a wash-out (2 months) from the 1st test, the amino-acid group received the placebo and the placebo group the amino-acid supplement. The body compositions/skin conditions were measured 4 times  in each test. Percentage-change of muscle mass in the amino-acid group increased up to 4 weeks (p\u00a0=\u00a00.05) and was higher than that in the placebo group (p\u00a0=\u00a00.09). Skin texture estimated by the image processing of neck skin replica tended to increase in the amino-acid group at 6 weeks compared with that at 0 week, though there was no significant intergroup difference. In conclusion, the young adult women having no fitness habit showed the significant increase of the muscle amount and improvement tendency of the skin texture by the continuous intake of the amino-acid supplement.The objective of this study is to elucidate the effect of a supplement enriched with  In 2000 the World Health Organization (WHO) has defined the healthy life expectancy (HALE) at birth as the average number of years during which one can enjoy healthy life without any care, and clearly stated its significance in human life. Since then, many people in the world have come to recognize that expanding HALE is important together with expanding average life expectancy. Health risks/problems including osteoporosis, skin damage, perimenopausal symptoms, and presbyopia have increased with aging in any people. It has been, however, thought that we can prevent various diseases caused by aging by suppressing or delaying the decline of body functions and as the result extend both HALE and average life expectancy. This concept is called \u201canti-aging medicine\u201d and we have thought that daily habits of eating, fitness, and sleep, and cosmetic cleaning are included as the important factors that are essential for anti-aging. Consciousness about health and anti-aging is high especially in middle-aged and elderly, while most women of broad generation tend to have common desires to become beautiful and highly interest in weight control. Thus, the number of population which habitually takes health supplements has increased to result in the expansion of a worldwide market of healthy foods whose main purpose is to attain the anti-aging, slim figure, health maintenance, beautiful skin, and the like. Mainstream of the past may need beauty cosmetics, but healthy supplements having effects from the inside of a body have attracted more attention recently. As a result, sales target of various beauty supplements includes all generations and the consumption of amino-acid supplements has been expanding in the supplement market worldwide; thus, the growth of the demand for such supplements is not temporally but continuously.l-leucine, l-arginine, and l-glutamine on body compositions/skin conditions, because physiological significance of these amino acids is quite important as described below. Therefore, the supplement used in this study was enriched with the three amino acids in addition to some vitamins.The objective of this study is to elucidate the effect of a supplement enriched with l-leucine, which is one of branched chain amino acids (BCAAs). A study on aged rats confirms that l-leucine stimulated muscle protein synthesis. Pasiakos et al. describe that muscle protein synthesis was accelerated with a supplement enriched with l-leucine in convalescing person who had exercised and presume that the intake of l-leucine after exercise is effective for the promotion of synthesis of body proteins. Indeed, l-leucine affects a signal pathway involved in metabolic control. Metabolic syndrome is a worldwide social problem at present and a possibility that intake of l-leucine is effective in the prevention of overweight and diabetes was indicated.The signature of the physical aging appears as the change in body compositions including the decrease of muscle mass and the increase of fat amount. The decrease of muscle mass will cause the drop of the physical capacity and the increase of risks such as falling. Muscle synthesis responding to assimilation of food taken decreases with aging probably due to the reduction of susceptibility in protein synthesis. At least a part of the reduction of susceptibility will be ascribed to the decrease of physiological concentration of l-Arginine is an essential amino acid for infants/children and under certain conditions for adults. When one suffers from an injury and an infectious disease, sufficient supply of l-arginine is desirable, because this amino acid is important in certain biological systems and is known to have a function to stimulate immune reactions through activation of T-cells.l-Arginine is metabolized in vivo to nitric oxide (NO) which has diverse physiological functions such as vasodilation, the improvement of exercise ability, the improvement of capacity of nutrition supplement to each organ, the suppression of accumulation of fatigue substances, and the improvement of antioxidative property.,8 Increase of growth hormone was also observed in blood after intake of l-arginine supplement.l-Glutamine is an amino acid whose amount is the highest in human muscle and plasma accounting for approximately 60% of total free amino acids in a body and is the major source of energy in intestinal mucosal cells, fibroblasts, lymphocytes, and macrophages. Though this amino acid is not an essential amino acid, it is an indispensable ingredient for the normal growth of human culture cells. This amino acid is involved in the DNA synthesis as a precursor of nucleic acids and nucleotides. In addition, l-glutamate is the precursor of l-arginine which generates NO as described above and glutathione, the major biological antioxidant.l-Glutamate is supposed to have an anti-oxidation capacity and reported to resolve diarrhea and improve an intestinal barrier function after its oral administration in infants with diarrhea or malnutrition.l-Glutamine additionally participates in the immune function and is supposed to elevate the ability of anti-oxidation, organ protection, and immunity, and to support the metabolism, and the synthesis and degradation of proteins.l-leucine, l-arginine, and l-glutamine on health. Each amino acid possesses specific functions described above, while combination of these amino acids is presumed to give different functions from those observed in the single amino acid. There are, however, few in-depth reports on the functions obtained by combining amino acids though we can access reports on the functions observed in the mixture of BCAAs. In addition, subjects in those studies were person with specific disease, aged people, or athletes though there are a few reports on the healthy young subjects.,19 Based on these situation and backgrounds, we performed a clinical trial using healthy young Japanese women who have no fitness habits to make clear the function(s) caused by an amino acid-containing supplement enriched with the three amino acids, i.e., l-leucine, l-arginine, and l-glutamine. The trial is a placebo-controlled, double-blind, crossover study in which the subjects ingested the supplement enriched with the amino acids for a prescribed period, and cosmetic and health effects were examined by skin tests and observations of body composition change, respectively.There are many studies on the effects of amino acids including This clinical trial was registered with the University Hospital Medical Information Network (UMIN) on May 19, 2015 (Registration Number: UMIN000017615).Twenty nine Japanese female students and staff aged 20 years or older in Kobe College from whom consent was obtained in written form in advance participated in this trial voluntarily. The subjects included in this trial did not suffer from any problematic diseases under treatment, not regularly take pharmaceutical products or supplements, and not smoke cigarettes. This study was performed in conformity with the purport of Helsinki Declaration (issued in 1964 and modified in 2008), and under the full consideration of medical ethics. The study was reviewed and approved by Ethical Review Board of Kobe College Graduate School of Human Sciences in 2015. All measurements were conducted in facilities in Kobe College. Even if a subject once agreed to participate in the study, it was decided that the subject was able to retire from the participation at any time including duration of the study without any disadvantage according to her free will.The study was conducted based on a placebo-controlled, double blind, randomized, cross-over design. Prior to implementation of the study, we explained the experiment contents orally and by using text to the participants. Table\u00a0l-leucine, l-asparagine, and l-glutamine shown in Table\u00a0The controller assigned each subject into the 2 groups in the order of decreasing values of the skin moisture content perfectly randomly. To the 2 groups, 14 and 15 subjects each were assigned; the subjects (21.5\u00a0\u00b1\u00a02.1 years) in one group (AA group) took a supplement containing amino acids mixture of l-leucine 600\u00a0mg, l-arginine 250\u00a0mg, and l-glutamine 300\u00a0mg, citric acid (acidifier), maltitol and sucralose (sweetener) as excipients, and multivitamins . The placebo sample was prepared by substituting only the amino acids by maltitol in the supplement without any alteration of other ingredients  in a bag (Table\u00a0The subjects took 1 package each of the test supplement or placebo after dinner and before bedtime (2 packages in a day) for 6 weeks. The measurement was conducted 4 times during the test at 0 week (before ingestion), 2, 4, and 6 weeks. After a washout period for 2 months from the end of the 1st test period, the test supplement containing amino acids was replaced with the placebo and vice versa in the 2nd test period. The first test was performed from May 26 to July 9 in 2015, and the second one in which the groups were rearranged from October 21 to December 17 in 2015.The subjects were instructed to avoid overeating, excessive alcohol consumption, excessive exercise, and poor sleep during the test as to living habits. A meal frequency was examined before and after the study to confirm the eating style of the subjects during the test.\u00ae TM300 . Skin color (redness and yellowness) at each specified region at the neck was measured 5 times to calculate the average values with a colorimeter . Skin brightness was measured by a specular component excluded (SCE) method to correctly observe the targeted position by eliminating specular reflection light. The skin viscoelastic character at the inner side of the forearm was measured by using a skin viscoelasticity measuring instrument . R2 (skin elasticity: recovery ratio of skin length), R6 (skin sagging: ratio of viscosity and elasticity when elongated), and R7 (skin firmness: ratio of elasticity during constriction) were used as the main parameters of Cutometer to assess skin elasticity and to measure elasticity of the upper skin layer using negative pressure which deforms the skin mechanically. Replicas of the skin surface of the designated regions at the inner side of the forearm and the neck were collected using silicone impression material kit, ASB-01 .After the subjects changed into prescribed clothes (sweat suit of top and bottom set), a body component was measured with a body composition analyzer . After measurement of the body composition, the subjects washed the inner side of the forearm and the neck lightly, measuring points, using a designated detergent, and were allowed to sit for 20\u00a0min for the acclimation to the measurement environment. A skin test was conducted in a temperature/humidity-controlled room . The measurement points for skin moisture content were at the inner side of the forearm determined using an exclusive ruler and at the fixed point of the neck where the line taken down from the joint of an ear and the line pulled to an Adam\u2019s apple crossed vertically. Moisture content of the skin was measured with a skin surface hygrometer Skicon-200EX . Transepidermal water loss (TEWL) was measured with Tewametert test. Two-way (group\u00a0\u00d7 time) repeated-measures ANOVA was used to determine the significance of differences between and within the groups. Differences were considered statistically significant when p value is less than 0.05 (p<0.05). All data were analyzed by using SPSS .Experimental data are represented as the means\u00a0\u00b1 SD. The intergroup and intragroup differences of mean values were interpreted by Student\u2019s paired p value was <0.1 (vs 0 week). The % change of muscle mass from the baseline increased in the AA group, but decreased in the P group; as a result the % change in the AA group was significantly higher than that in the P group at 4 weeks \u00a0=\u00a07.8].Table\u00a0p<0.05) and at 2 and 6 weeks in the P group . The TEWL tended to decrease with time in the skin of the forearm and the neck in both groups; the decrease of TEWL in the forearm skin was significant at 6 weeks in the P group (data not shown). Table\u00a0p<0.05 and p<0.01, respectively) and at 2, 4, and 6 weeks in the P group . Neither skin moisture nor TEWL in the skin of the neck were significantly different between the AA group and the P group . Brightness of the skin at the neck in the P group was higher than that in the AA group at 6\u00a0weeks without significant difference (Table\u00a0p\u00a0=\u00a00.05). The slight skin redness in the AA group was slight, but significantly higher than that in the P group at 6 weeks.Next, the skin color was examined using a colorimeter according to the SCE procedure Table\u00a0. In the ce Table\u00a0. RednessA replica of the skin was picked followed by estimating skin texture by an image processing of the skin replica. Uniformity of the skin texture is estimated by the ratio of volume rate of the skin texture to the rate of whole skin volume. The higher the ratio became, the higher the uniform texture achieved.p\u00a0=\u00a00.03), while the forearm skin texture did not change in both AA and P groups  for 6 weeks. In the study, the body constitutions and the texture of skin were improved in the subjects belonging to the AA group though the change of weight was similar in both AA and P groups. In the AA group, body fat tended to decrease, while body muscle amount increased up to 4 weeks in comparison with the starting time. Thus, there observed a tendency of intergroup difference in the increase of the body muscle amount which was higher in the subjects in the AA group than those in the P group. Based on these results, we concluded that the continuous intake of the amino acid supplement containing l-leucine, l-arginine, and l-glutamine for at least 6 weeks tended to increased the muscle amount of the young adult women. Paddon-Jones et al. report similar result in which the intake of essential amino acids stimulated muscle protein synthesis in the young and elderly people.In this study, we examined the changes in the body constitutions and the skin conditions in healthy adult women having no habitual exercise during and after taking either supplement not containing amino acids (P group) or that containing Hereafter we will discuss the respective effects of the three amino acids on the present results.l-leucine in several studies hitherto conducted: (1) l-leucine is an amino acid indispensable for protein synthesis  and a potent activator in the protein synthesis in white adipose fat-cells via a similar pathway, (2) the carbon skeleton of l-leucine is utilized for the production of ATP, and (3) l-leucine is an activator of the mechanistic target of rapamycin (mTOR) whose signaling is important in vivo to control metabolism and the multiply, growth, and survival of cells. Based on these results, the effects of intake of l-leucine have been extensively studied in the various situations such as aging, muscle disorders, protein/energy deprivation, overweight, and genuine diabetes;,24 overall these results indicating that l-leucine is most likely to affect both muscle mass and fat mass in the various physical situations. Pasiakos et al. performed a clinical study in which the following two groups were compared after physical exercise; one group took essential amino acid supplement and another group l-leucine-enriched essential amino acid supplement. The latter supplement elevated the plasma concentration of l-leucine followed by activation of the muscle cells and muscle protein synthesis during convalescence of fatigue in certain types of exercises requiring stamina. In our study, similar results were obtained though the enrolled subjects did not perform habitual exercise. Katsanos et al. report that the promotion of a muscle protein synthesis by ingestion of l-leucine was different between young and elderly person; in young adults the enhancement of muscle protein synthesis by l-leucine occurred regardless of its ingested amount, whereas in elderly adults high concentration of l-leucine was required for the enhancement. There is a report that the intake of l-leucine did not change the body protein amount in aging model mice and elderly people, though l-leucine stimulated body proteosynthesis.,26 Our result found in the healthy young women taking 1,200\u00a0mg of l-leucine per day in the AA group was well compatible with the above results. Thus, the present results observed in the young adult may not be accepted in elderly people.The followings have been elucidated as the role of l-leucine on the body fat reduction, the increase of energy expense in an obese model mouse, and the increase of the adipose tissue by 25% without increasing food consumption in a young rat. However, since a survey using a simplified-food frequency questionnaire implemented during the experimental period of this study confirms no significant change in the amount and contents of diets taken by the subjects (data not shown), the change of body compositions in this study was supposed not to attribute to the change of dietary amounts and contents. When l-leucine was orally taken as a supplement, no change was observed in the intake of the diet, though there is a report describing that direct injection of l-leucine into the brain caused the restriction of intake of food. In conclusion, l-leucine was likely to increase the muscle amount and decrease the body fat without significant changes in the weight and dietary habits.There are several reports describing the effects of l-Glutamine has the following effects or rolls in vivo: (1) control of manifestation of many genes related to metabolism, signal transduction, cell defense, and repair, (2) activation of intracellular signaling pathways, (3) an energy source of cells of the immunity and digestive systems with rapid turnover, (4) a precursor of glutathione, an important anti-oxidant in vivo, and (5) control and stimulation of the turnover of proteosynthesis. Furthermore, restraint of proteolysis is reported in culture cells of the mouse skeletal muscle. Therefore, other than l-leucine, l-glutamine may be involved in at least a part of the increase of the muscle amount through proteosynthesis in the AA group. Many researchers have tried to verify the effect of l-glutamine from the viewpoints of medical nutrition,34 and elucidated that this amino acid is important in maintaining functional status of the bowel and good physical conditions, and acts as an energy source of cells in the immune and digestive systems. Actually, oral administration of 30\u00a0g of l-glutamine to obese and overweight adults for 14 days resulted in the decrease of the ratio of Firmicutes to Bacteroides, a good biomarker for obesity. Though the amount of l-glutamine taken in this study was much smaller and 1,200\u00a0mg/day, there was a possibility that the intake of l-glutamate changed the composition of intestinal flora followed by reduction of the body fat. A natural moisturizing factor (NMF) consisting of various components including amino acids holds the key for the moisture content of the skin by maintaining water in the corneocytes, and thereby moisturization of the stratum corneum (SC) of the epidermal keratinocyte is achieved. Thus, the decrease of NMF is accompanied by the decrease of moisture in the corneocytes, disturbance of the arrangement of the corneocytes, formation of the dried and flaking skin, and aggravation of skin conditions. Main components of NMF are amino acids (approximately 40%) formed by degradation of filaggrin (filament aggregating protein). In our study, TEWL (indicator of decrease of moisturization) tended to decrease by the intake of amino acids in the skin of the forearm and the neck in both AA and P groups. The decrease of TEWL indicated the improvement of the barrier function of the SC and skin conditions are closely related to gastrointestinal functions and status. Constipated state will increase corruption products in the gut which in turn aggravate skin functions and conditions.l-Glutamine will maintain the gut in desirable conditions and is likely to have a function to maintain the homeostasis of organs including the gut and the skin. These results and observations are consistent with the improvement of skin texture (smoothness) at the crista and sulcus cutis examined by dermoscopic images in our study. l-Glutamine by which the environment in the gut was arranged and nutritional absorption efficiency was elevated is potentially associated with the skin texture improvement. In addition, l-glutamine is converted to glutathione whose strong antioxidant property is probably contribute to the improvement of skin conditions. Contrary to our expectation, however, the improvement extent of the skin conditions including the moisture content, TEWL, and viscoelasticity was approximately the same in the AA and P groups. This resultmay be explained by the effect of multi vitamins as suggested by Boyera et al.l-Arginine plays the central role in several biological systems including immune functions and the intake of l-arginine-enriched supplement enhanced the potency of self-antitumor immunity, prolonged survival time, and inhibited the growth of tumor in a cancer model mouse by inhibiting the number of myeloid-derived suppressor cells.l-Arginine is a substrate of both arginase and nitric oxide synthase (NOS) and is converted to urea and l-ornithine by arginase and to NO by NOS. l-Ornithine is a precursor of l-proline and important in the growth and restoration of cells. l-Proline biosynthesized from l-arginine via l-ornithine is the main constituent amino acid of collagen proteins. There is a possibility that collagen regeneration in the dermis is promoted by l-proline converted from ingested l-arginine, and thereby viscoelasticity of the skin improved. NO expands the blood vessel and improves blood stream. In addition, a study on the effects of intake of l-arginine supplement in rats shows a significant increase of NO level in blood, a decline of an oxidation stress, and a prolongation of an exercise time. Shan et al. demonstrate that l-arginine supplementation caused the promotion of the bioavailability of NO and the elevation of the anti-oxidative potency. The present study has confirmed the improvement of skin texture and skin viscoelasticity in the AA group. This observation can be explained by the follows: (1) the enhancement of NMF production in the epidermis and collagen regeneration in the dermis caused by the increase of blood stream by NO produced from l-arginine and the increase of supply of nutritious food matters, (2) the restraint of an oxidation stress of cells caused by anti-oxidative activity of l-arginine, and (3) the promotion of collagen regeneration by l-proline biotransformed from l-arginine.l-leucine, l-glutamine, and l-arginine for 6 weeks changed the body constitutions such as the increase of the muscle mass and the decrease of the fat amount. The highest muscle amount was achieved at 4 weeks of the intake with a statistically significant difference in comparison with that of the P group, followed by a slight reduction at 6 weeks, though the reason(s) for this remain unclear at present. The improvement of skin texture was attained in the AA group. These findings were attributable to the additive effects produced by the combination of the three amino acids. Astaxanthin has high antioxidant and anti-inflammatory activities, and long-term astaxanthin supplementation suppresses the deterioration of wrinkles, moisture content, and interleukin-1\u03b1 levels generated by environmental factors, such as UV and dryness, in a clinical study. In addition, the oral administration of a dried whole egg powder of farmed soft-shelled turtles, Pelodiscus sinesis, containing various proteins and amino acids inhibits the formation of advanced glycation end-products in the serum and skin of diabetic rats. Based on these reports, it is expected that the supplements used in this study can be combined with ingredients with high anti-glycation and anti-oxidant properties to develop more functional supplements to the skin.The results described above indicate that the intake of the supplement containing l-leucine, l-glutamine, and l-arginine on the body components and skin conditions in younger women, several limitations have to be discussed. First, there are no placebo arms for the single amino acid, which enable to clearly demonstrate the role of the amino-acid combination. Second, since the sample size was small and only younger women were enrolled, generalizability of the data needs to be regarded with caution.Although this study provides the comprehensive and in-depth insight into the effect of the combination of In conclusion this study shows that the consumption of the amino acid mixture affected the body compositions and the skin conditions of the healthy young women, and we can expect novel utilities of amino acid supplement based on these effects."}
+{"text": "The conventional chemotherapeutic agents, used for cancer chemotherapy, have major limitations including non-specificity, ubiquitous biodistribution, low concentration in tumor tissue, and systemic toxicity. In recent years, owing to their unique features, polymeric nanoparticles have been widely used for the target-specific delivery of drugs in the body. Although polymeric nanoparticles have addressed a number of important issues, the bioavailability of drugs at the disease site, and especially upon cellular internalization, remains a challenge. A polymer nanocarrier system with a stimuli-responsive property , for example, would be amenable to address the intracellular delivery barriers by taking advantage of pH, temperature, or redox potentials. With a greater understanding of the difference between normal and pathological tissues, there is a highly promising role of stimuli-responsive nanocarriers for drug delivery in the future. In this review, we highlighted the recent advances in different types of stimuli-responsive polymers for drug delivery. Cancer is a common cause of death every year worldwide ]-co-glycolide]-PEG-poly coglycolide] copolymer capped with N-Boc-histidine  (PEG) and hydrophobic poly(\u03b3-benzyl L-glutamate) as a carrier to release DOX at mildly acidic conditions , whereas DOX is released significantly more under acidic conditions (pH 5.0). Interestingly, DOX-loaded pH-sensitive PNPs exhibited higher toxicity to SCC7 cancer cells than DOX-loaded micelles without the pH-sensitive linker. Heller et al. prepared biodegradable polyacetal biodegradable carriers, which hydrolyze at acidic pH. Etrych et al. synthesized an HPMA drug carrier with paclitaxel (PTX) and docetaxel (DTX) via the hydrolytically unstable hydrazone linkage , in which DOX was conjugated to hydrophobic segments through an acid-sensitive hydrazone linker  and click chemistry for site-specific sustained delivery of the antitumor drug DOX  (PVD) was attached to doxorubicin (DOX) via an acid-sensitive linker that could release free drug under mild acidic conditions -polycaprolactone (PCL) block copolymer . Due to its amphiphilic nature, the copolymer formed PNPs in aqueous conditions. DOX was loaded into PNPs with 75% loading efficacy. The micelle released DOX at 10 mM GSH, mimicking intracellular conditions. In vitro data revealed that DOX-loaded reduction-sensitive micelles were toxic to SCC7 cells compared to reduction-insensitive control micelles.The same group also attempted to prepare carboxymethyl dextran (CMD) with lithocholic acid (LCA) through a disulfide linkage  triblock copolymer as a DOX drug carrier -Hypoxia, a pathological state of inadequate oxygen, is involved in the pathogenesis of intractable diseases such as cancer, cardiopathy, ischemia, rheumatoid arthritis, and vascular diseases Harris, . Oxygen In vitro and in vivo experiments indicated high tumor accumulation and anti-tumor efficacy. In another study, the same group reported hypoxia-sensitive block copolymer composed of PEG and poly(\u03b5-(4-nitro)benzyloxycarbonyl-l-lysine) \u2013azobenzene\u2013poly(ethylene glycol) can self-assemble to form polymersomes in an aqueous medium , singlet oxygen (1O2), superoxide (OBy utilizing the high accumulation of reactive oxygen species (ROS) in some disease tissues, ROS-response polymer nanoparticles are also an effective mechanism to control targeted drug release. It has been reported the mucosal ROS concentrations in inflammatory tissues and colon cancer were 10- to 100-fold higher than that of normal tissues. In this section, we will discuss recent progress in reactive oxygen species (ROS)-responsive carrier systems. ROS refers to a class of oxygen-derived chemical species produced by the body. Typical ROS species include hydrogen peroxide  cross-linking (RG-MSN). This drug delivery system facilitates the monitoring of the bio-distribution of the drug carrier by magnetic resonance imaging  cross-linking to act as a gatekeeper. Tumor growth can be effectively inhibited after treatment with DOX-RG-MSN in combination with PDT-assisted chemotherapy. 1O2 is created upon irradiation with light and is utilized for photodynamically triggered drug release. Using this 1O2-responsive nanocarrier delivery system, DOX can easily reach the tumor site and be accumulated in the nuclei to kill the cancer cells, therefore decreasing the side effects of chemotherapy.Tumors with high interstitial pressure and a dense extracellular matrix affect the uptake of nanoparticles and tumor penetration. Photothermal therapy (PTT) has overcome these barriers for hyperthermia damage to cancer cells, enhanced tumor penetration of nanoparticles, and triggered release of cargo  and exogenous stimuli . The endogenous stimuli-responsive system relies on the abnormal environments in diseased tissues for target-specific drug delivery, while the exogenous stimuli-responsive one needs prior knowledge on the location of the target site for effective therapy. Because of the heterogeneity in the physiological conditions, exogenous stimuli-responsive drug delivery systems would be more favorable. A number of polymers have been explored for preparation of different stimuli-responsive drug delivery systems. Nonetheless, a number of challenges remain and future studies will need to address several issues.First, the biocompatibility and biodegradability of the polymers must be improved. Second, it is essential to understand how stimuli-responsive polymers interact with the biological components. Upon administration, stimuli-responsive polymers encounter a variety of biological molecules, cells, and tissues. The surface of stimuli-responsive polymers would be quickly covered with biological molecules such as serum proteins which play a vital role in determining the subsequent bio-distribution and cellular responses of polymers. Next, to protect their cargo molecules from biological degradation and lower harmful side-effects in healthy cells and tissues, the polymeric nanocarriers must have the ability to access the target cells while passing the biological barriers and escaping from the reticuloendothelial system (RES). The polymeric nanocarriers need to escape from the RES to improve their circulation time, which is the significant parameter to accomplish efficient delivery of cargo molecules to the target site. For this purpose, along with surface architecture, the various parameters  should be tuned. The further understanding of cancer biology and polymer chemistry will catalyze optimization pathways to attain more efficient anti-tumor systems. Additionally, the good permeability of tumor vasculature is mostly constructed in experimental animal models. However, in practice, the growth rate of tumors in the human body is relatively slower than that in animal models, leading to an unsatisfied EPR effect. As a result, it is urgent to improve the progression of active targeting to tumors via ligand-mediated or exogenous stimulus. Getting insights into the differences between normal and pathological tissues will also result in the development of new selective triggers, targeted therapies and a highly promising role for stimuli-responsive nano-assemblies. The complicated synthesis and difficulties in the scale-up of stimuli-responsive polymers are likely to hinder their clinical translation. This is one of the significant reasons why many stimuli-responsive polymers have been reported, whereas only a few of them could enter the clinical stage.Future opportunities for nanomedicines are looking toward combinations of different types of stimuli to develop multifunctional drug delivery nanosystems. The combing diagnostic and therapeutic agents may be introduced to enable the visual tracking of cancer therapy. In the near future, we anticipate that multifunctional drug delivery nanosystems for cancer therapy will be developed for actual clinical applications.NR, HK, JL, and JP wrote and edited the paper.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": "Mutations that target the ubiquitous process of ribosome assembly paradoxically cause diverse tissue-specific disorders (ribosomopathies) that are often associated with an increased risk of cancer. Ribosomes are the essential macromolecular machines that read the genetic code in all cells in all kingdoms of life. Following pre-assembly in the nucleus, precursors of the large 60S and small 40S ribosomal subunits are exported to the cytoplasm where the final steps in maturation are completed. Here, I review the recent insights into the conserved mechanisms of ribosome assembly that have come from functional characterisation of the genes mutated in human ribosomopathies. In particular, recent advances in cryo-electron microscopy, coupled with genetic, biochemical and prior structural data, have revealed that the SBDS protein that is deficient in the inherited leukaemia predisposition disorder Shwachman-Diamond syndrome couples the final step in cytoplasmic 60S ribosomal subunit maturation to a quality control assessment of the structural and functional integrity of the nascent particle. Thus, study of this fascinating disorder is providing remarkable insights into how the large ribosomal subunit is functionally activated in the cytoplasm to enter the actively translating pool of ribosomes. SDS is also characterised by multiple developmental anomalies including poor growth, exocrine pancreatic insufficiency, skeletal abnormalities  and cognitive impairment. However, registry data indicate that the phenotypic spectrum of this rare disease  is broad . Thus, loss-of-function TP53 mutations appear to be biologically important for clonal progression and transformation to haematological malignancy in SDS. Although the incidence is unknown, a limited number of case reports have highlighted a range of solid tumours presenting at a young age in individuals with SDS  in SDS  in SDS  reveals ) in SDS .The aim of this review is to discuss the considerable expansion in our understanding of the molecular basis of SDS and to illustrate how elucidation of the function of the genes mutated in this disorder is providing important new mechanistic insights into the fundamental conserved process of ribosome assembly and its quality control. A key remaining challenge is to explain how mutations that affect protein synthesis in all cells cause such tissue-specific abnormalities. Finally, we need to harness new mechanistic insights to develop novel therapeutics that will improve the outcomes for SDS patients and their families.2SBDS gene in the majority of individuals with SDS . Although most parents of children with SDS are carriers, about 10% of SBDS mutations arise de novo  that introduces an in-frame stop codon, suggesting that complete absence of functional SBDS protein is incompatible with life . Consistscoideum  and is rscoideum  and micescoideum . Thus, twith SDS .SBDS mutations, it was suspected that one or more additional genes might be linked to this disorder. The molecular pathology of SDS has indeed now been expanded to include biallelic mutations in DNAC21  at 12 years of age  processome) . EndonucThe majority of the transiently interacting assembly factors are released prior to nuclear export. Completion of large subunit maturation in the cytoplasm involves recruitment of the last remaining ribosomal proteins, assembly of the peptidyl transferase centre (PTC), removal the final assembly factors and release of the nascent 60S subunit into the actively translating pool of ribosomes . The AAAThe eIF6 protein is a nuclear shuttling assembly factor that is highly conserved in archaea and all eukaryotes. Originally identified as an anti-association factor that inhibits joining of the 40S and 60S ribosomal subunits in wheat germ extracts , eIF6 is4TIF6 alleles could rescue the fitness defect of Efl1 deleted cells  recruits PKC\u03b2II to the ribosome to promote eIF6 eviction by phosphorylating eIF6 residue S235 . Howeverpe cells . Thus, c6To better understand the function of the SBDS protein at a molecular level, its structure and dynamic properties have been investigated in detail. The SBDS protein is widely expressed and highly conserved, with orthologues in prokaryotes and all eukaryotes, but not in eubacteria . X-ray cEFL1 is a cytoplasmic GTPase that is homologous to prokaryotic elongation factor G  and the ribosomal translocase EF-2 in eukaryotes. Like EF-G, EFL1 has a five-domain architecture , but it Methanothermobacter thermautotropicus SBDS , human SBDS, human EFL1 and non-hydrolysable GTP analogue (GMPPCP) as the substrate for single-particle cryo-EM experiments  were determined, revealing that the SBDS protein binds to the ribosomal P-site on the intersubunit face of the 60S subunit A and B iUpon EFL1 binding, SBDS undergoes a dramatic conformational rearrangement in which domain III rotates by 180\u00b0 away from the P-stalk base (closed state) to interact with helix 69 (open state), while SBDS domain I remains anchored in the P-site. The conformational changes in SBDS visualised by cryo-EM are consistent with the global dynamic motion of the SBDS protein observed off the ribosome using NMR spectroscopy . Interes8Based on structural, biochemical, and genetic data, a conformational switching mechanism for eIF6 release by SBDS and EFL1 has been proposed . AlthougIn summary, SBDS functions as a multi-tasking 60S subunit assembly factor that 1) promotes allosteric conformational switching of the EFL1 GTPase and 2) protects and proofreads the P-site and the base of the P-stalk. SBDS domain I protects and proofreads the structural integrity of the peptide exit tunnel and the PTC; domains I and II couple dynamic interdomain motion to EFL1 conformational switching; SBDS domain III interrogates the translational GTPase-binding site at the P-stalk base in the closed state.Dictyostelium cells overexpressing a dominant negative SBDS variant reflected that of a mature 60S subunit enriched in eIF6 but not Nmd3, with uL16 already incorporated. Furthermore, yeast cells carrying the T-ALL-associated uL16-H123P allele as the sole copy of uL16 lacked detectable expression of uL16 that is required both for Nmd3 release and for Sdo1 recruitment to the 60S subunit in\u00a0vivo  alter the length of the flexible linker between domains I and II, highlighting a key role for the domain I-II linker in allowing conformational switching of the SBDS protein on the 60S subunit.NMR spectroscopy was used to interrogate the impact of 29 disease-associated missense variants on the structural integrity of the SBDS protein and classify them into two discrete groups: those affecting the overall stability or fold of SBDS and those that alter surface epitopes without altering the stability or fold of the protein . The cry10Why use such a complex strategy to evict eIF6 during the final step in maturation of the large ribosomal subunit? One explanation is that it provides a mechanism to couple eIF6 release to a final quality control assessment of the structural integrity of key functional sites on the nascent 60S subunit. Together, SBDS and EFL1 proofread the integrity of the polypeptide exit tunnel, the P-site, the GTPase centre, the base of the P-stalk and the SRL. The six N-terminal residues of SBDS insert into the proximal end of the exit tunnel, while the C-terminus of the zinc finger assembly factor Rei1 probes the distal part of the tunnel . Thus, R11DNAJC21 in SDS patients who are negative for mutations in the SBDS gene.A fundamental conserved role for the SBDS protein in late cytoplasmic maturation of the large ribosomal subunit has been established using a wide range of genetic, biochemical and structural approaches in multiple independent species. Nevertheless, an increasing array of functions has been proposed for SBDS, including roles in rRNA processing , chemota12DNAJC21 gene (GenBank: NM_001012339.2) have recently been identified in eight individuals with SDS who were negative for mutations in the SBDS gene. Four individuals presented similarly with bone marrow failure, intra-uterine growth retardation and/or short stature, dental abnormalities, hyperkeratosis, skin pigmentation and retinal dystrophy, one developing acute megakaryocytic leukaemia at 12 years of age , a splice variant (c.983+1G\u00a0>\u00a0T) and a missense mutation . Another four patients from three unrelated families who had been diagnosed clinically with SDS carried biallelic mutations in DNAJC21, including two nonsense mutations  and a missense variant   . InteresE. coli DnaJ) that interact with client heat shock protein 70 (Hsp70) chaperones to stimulate their ATPase activity . Depletion of ribosomal protein rpl3 or the 60S subunit assembly factors sbds, pescadillo , particularly when the proliferative rates of affected tissues are high during specific stages of developmental. Perturbation of both global and tissue-specific mRNA translation (uORFs and complex 5\u2032UTRs) may also play a role. Tissue-specific stress responses to impaired translation and the downstream consequences of p53 activation also influence the phenotypic outcome. Elucidating the mechanisms underlying the differing molecular responses to p53 activation is a key challenge for the future.15The poorly understood mechanism of clonal progression to MDS/AML in SDS is encapsulated by Dameshek's riddle: how does attenuated translation and a hypoproliferative state (bone marrow failure) promote the transition to a hyperproliferative disorder (leukaemia) ? HaematoSbds gene from osterix-expressing mesenchymal stem and progenitor cells (MPCs) induced mitochondrial dysfunction, oxidative stress, increased generation of reactive oxygen species and activated the DNA damage response in HSPCs downstream of p53-S100A8/9-Toll like receptor inflammatory signalling  is a rare, clinically and genetically heterogeneous autosomal dominant disorder that classically presents within the first year of life with normochromic, macrocytic anaemia, reticulocytopenia and reduced or absent erythroid precursors in the bone marrow. Around 50% of individuals have associated short stature and physical anomalies including craniofacial, thumb, cardiac and urogenital defects  (POLR1C and POLR1D (The  154500)  encodes  154500) . The disd POLR1D  in TCS sEMG1 (yeast NEP1) cause autosomal recessive Bowen-Conradi syndrome (OMIM: 211180)  family of methyltransferases (Mutations in  211180) . NEP1 ensferases . Howeversferases .604901) that is found exclusively in Ojibway-Cree children from a First Nations Canadian population , caused by mutations in MRP RNA, is characterised by short stature, sparse hair, malabsorption, haematological and immunological defects and predisposition to cancer  (Box C/D snoRNAs are evolutionarily conserved non-protein-coding RNAs involved in ribosome biogenesis . Interests (LCC) . LCC is ts (LCC) . The U8 ts (LCC) . U8 is tts (LCC) . The conts (LCC) . A consets (LCC) . In summ17SBDS and DNAJC21 mutations, while prospective sequencing of serial samples from SDS patients will be important to identify the key genetic changes that promote clonal progression to MDS and leukaemia. The generation of viable SDS animal models will be important both for elucidating mechanisms of disease and for testing novel therapeutics, but significant progress may also come from cellular and induced pluripotent stem cell disease models (For SDS patients and their families, it will be important to identify informative prognostic biomarkers to predict who is at highest risk of progressing to MDS/AML and who may benefit from early therapeutic intervention. International registries will help expand the range of clinical phenotypes in SDS and define the natural history of the condition. Exome sequencing will likely reveal further gene variants among the 5\u201310% of individuals clinically diagnosed as SDS who are negative for e models . The devNone."}
+{"text": "This has hindered the process of making a more refined description of the Acb using non-invasive neuroimaging technologies and approaches. In this study, high-resolution ex vivo macaque brain diffusion MRI data were acquired to investigate the tractography-based parcellation of the Acb. Our results identified a shell-core-like partitioning in macaques that is similar to that in humans as well as an alternative solution that subdivided the Acb into four parcels, the medial shell, the lateral shell, the ventral core, and the dorsal core. Furthermore, we characterized the specific anatomical and functional connectivity profiles of these Acb subregions and generalized their specialized functions to establish a fine-grained macaque Acb brainnetome atlas. This atlas should be helpful in neuroimaging, stereotactic surgery, and comparative neuroimaging studies to reveal the neurophysiological substrates of various diseases and cognitive functions associated with the Acb.Limited in part by the spatial resolution of typical  The nucleus accumbens (Acb) is an integral part of the striatal complex  and exhiHistorically, the boundaries of the Acb were never well-defined histochemically, e.g., the Acb core fades into the ventral caudate and putamen nuclei, especially far up the medial caudate border , but havin vivo neuroimaging, partially limited by the spatial resolution of the MRI data, connectivity studies have only identified a shell-core-like partitioning of the Acb in humans  scanners. Then, the striatum was recursively parcellated based on probabilistic diffusion tractography to generate a relatively accurate connectivity-based Acb region. Then this connection unit was used as the region of interest (ROI) to detect the parcellation of the macaque Acb and to test the hypothesis that high-resolution tractography can enable a finer subdivision of the Acb than the conventional dichotomy. We further characterized the anatomical and resting-state functional connectivity (rsFC) profiles of these Acb subregions. Their unique connectivity profiles together with earlier research findings were then used to generalize about the possible specialized functionality of these subregions to establish the final fine-grained macaque Acb brainnetome atlas.ex vivo rhesus macaque MRI dataset (MDS1) consisted of 8 adult macaque brain specimens  and 4 non-diffusion gradients acquisition . The data quality and availability of these low b-value diffusion images in tractography-based parcellation were checked  was used in this study to characterize the rsFC profiles of the Acb subregions. Their functional MRI data were acquired on a 3T Siemens Magnetom Verio MR scanner under anesthesia  and have been preprocessed and used in an earlier study  approved by the animal care and use committee of the Institute of Automation, Chinese Academy of Sciences (CAS). The in vivo individuals in MDS2; calculation of the transformations between individual brains and a macaque brain template  FSL FLIRT boundary-based registration BBR; . The braSCR\\_014815; p > 0.04%  as was done in earlier studies (The rhesus macaque striatum excluding the tail was chosen as the seed to perform a tractography-based parcellation. This procedure is similar to the Automatic Tractography-based Parcellation Pipeline program (ATPP RRID:  studies  to limit studies  and was  studies . 7) The  studies . In this studies .To avoid spending large amounts of time and resources on the dorsal striatum, which was not the focus of this study, a recursive parcellation procedure was used to parcellate the striatum as follows. In the first parcellation, the cluster number of the spectral clustering algorithm was preset to range from 2 to 8 and the average Cramer\u2019s V (CV) was used to judge the consistency of the spatial distribution of these subregions among individuals as in earlier studies . The optThe Acb region extracted from the MPM produced by tractography-based parcellation, but not the microanatomically defined Acb, was used as the seed for the subsequent tractography-based parcellation, to identify the fine subdivisions of the macaque Acb. To take advantage of detailed connectivity heterogeneity within the Acb detected by high resolution tractography, the tractography-based parcellation of the macaque Acb at a relative fine-grained resolution was performed using those original tractographic images have not yet been down-sampled  diffusion space, the whole-brain connectivity probability map  was generated for each Acb subregion and then thresholded to reduce false positive connections. All individual maps and their binary images were transformed into MNI monkey space. We used these individual maps to generate a group-level averaged connectivity probability map and used their binary images to generate a probability fiber tract map. As in previous studies , to furtz-values using Fisher\u2019s z-transformation and transformed into MNI monkey space. All the normalized z-valued rsFC maps were fed into a random effects one-sample t-test to determine the regions that had significant correlations with the given subregion. A statistical threshold of p < 0.05 (uncorrected) was set to achieve a corrected cluster-wise statistical significance of p < 0.05. The cluster size was estimated on the basis of the GM mask and the group-averaged Gaussian filter width. Then, a minimum statistic test for conjunction (For each subregion in subject (MDS2) function space, the Pearson correlation coefficients between the mean time series of the given subregion and the time series of each voxel in the GM mask was calculated to generate the rsFC map. Note that the mean time series of these Acb subregions were calculated using fMRI data without smoothing. This map was converted to junction  was perfA set of target regions was used to characterize the anatomical and functional connectivity profiles of the Acb subregions. These targets were extracted from a histological rhesus macaque brain atlas , which wWe used a set of connectivity ratios to build the connectivity fingerprint and calculated the connectivity ratio as follows: given a target, the connectivity strength  between this target and each Acb subregion was first calculated using the above-mentioned averaged connectivity probability map or significant rsFC map. Then, the connectivity ratio of one of the Acb subregions was defined as:i) is one of the n Acb subregions; CS) is the connectivity strength between the given target and seed (i); CR) is the connectivity ratio of seed(i).where, seed and the holistic connective architecture are indicated to be complementary and correlative cf. , which aA recursive parcellation procedure, proposed and validated by previous studies , was useex vivo macaque diffusion images were chosen to retest it. Gratifyingly, this dataset confirmed our hypothesis by providing the 4-cluster solution as a second optimal solution along with the 2-cluster one. The shell-like and core-like divisions in the 2-cluster solution have similar morphological distributions and connectivity profiles to those of found in previous anatomical and histochemical studies  are believed to possess multiple functional roles, e.g., in switching the global mode of behavior away from free-feeding when necessary and in computing the value of stimulus-outcome pairs . In addiA tractography-based recursive parcellation of the striatum was performed in macaques to delineate a relatively accurate connectivity-based Acb region. Using this region, we identified connectivity-based shell-core-like partitioning in macaques that is similar to that in humans and verified the hypothesis that high-resolution tractography enables the identification of finer Acb subdivisions, beyond the well-documented dichotomous shell-core architecture. We further characterized the unique anatomical and functional connectivity profiles of these Acb subregions, generated their possible specialized functionalities, and finally established a fine-grained macaque Acb brainnetome atlas.All datasets generated for this study are included in the manuscript and/or the All experimental procedures were performed in strict accordance with the recommendations in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All of the animals were handled according to the protocol (#IA-2016-05) approved by the animal care and use committee of the Institute of Automation, Chinese Academy of Sciences.TJ and HL conceived and designed the study. BH, BZ, DZ, and XZ acquired and checked the data. XX, LF, CC, HD, and YD performed research and analyzed the data. XX, LF, HL, and TJ wrote the manuscript. All authors reviewed and approved the submitted 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": "Objective assessment of prosthetic paravalvular leak (PVL) is complex and challenging even in transesophageal echocardiography (TEE). Our aim was to assess the value of cardiovascular magnetic resonance (CMR) in quantifying PVL in aortic (AVR) or mitral valve (MVR)\u00a0replacement.Thirty-one patients  with a preliminary diagnosis of significant PVL  were recruited. The TEE PVL grading was based on the semi-quantitative (SQ) TEE according to the VARC II PVL classification . Non-contrast CMR studies were acquired at 1.5\u00a0T with a quantitative approach (phase-contrast velocity encoded imaging). The CMR PVL severity was classified according to regurgitant fraction (RF: (1) mild\u2009\u2264\u200920%, (2) moderate 21%\u201339%, or (3) severe\u2009\u2265\u200940%).All patients revealed symptoms of heart failure  and typical cardiovascular disease risk factors. The SQ-TEE results revealed several categories: (1) mild , (2) moderate , and (3) severe  PVL. However, CMR PVL RF reclassified the severity of PVL: (1) mild to moderate (in 80%), (2) moderate to severe (in 47%), and (3) severe to moderate (in 40%). The receiver operating characteristic analysis showed that SQ-TEE and CMR PVL-vol and -RF predicted the upper tertile of NT-proBNP (>\u20092000\u00a0pg/ml) with the best sensitivity for CMR parameters.The SQ-TEE showed moderate agreement with CMR and underestimated a considerable number of AVR or MVR-PVL. Prosthetic valve paravalvular leak (PVL) is a rare finding in patients with valve surgical prostheses. PVL is usually an early complication of cardiac surgery or a consequence of infective endocarditis. PVL is detected in up to 10% of patients after aortic valve replacement (AVR) and 17% of patients after mitral valve replacement (MVR) . AlthougTransthoracic echocardiography (TTE) is the primary imaging tool used in the follow-up of patients with valve prostheses. However, TTE has a high interobserver variability and it is considered a screening method in patients with PVL . DespiteCardiovascular magnetic resonance (CMR) is a modern imaging tool with high accuracy and reproducibility in the evaluation of cardiac chamber volume, function, and mass , 9. CMR Therefore, our aim was to compare TEE and CMR in PVL quantification related to either AVR or MVR.All the patients with either AVR or MVR and TTE diagnosis of PVL were referred to our center for further diagnostics and treatment. All of the patients had undergone a comprehensive TEE to confirm a PVL versus a transvalvular regurgitation, which was the main inclusion criterion. The exclusion criteria included: (1) any form of prosthesis degeneration resulting in transvalvular regurgitation on TEE; one patient was excluded for transvalvular regurgitation instead of PVL, and one patient was excluded for coexistence of trans- and paravalvular regurgitations, (2) TAVI, (3) contraindications to CMR, and/or (4) high probability of incomplete or nondiagnostic CMR images , infectious diseases in the previous month.Finally, 31 patients  with either AVR-PVL (n\u2009=\u200923) or MVR-PVL (n\u2009=\u20098) were enrolled (2018\u20132019). A detailed medical history and additional laboratory tests were collected in all the patients, including a complete blood count (CBC), serum lactate dehydrogenase (LDH) and N-terminal pro-B-type natriuretic peptide (NT-proBNP). Hemolysis was identified by serum LDH\u2009>\u2009460 U/l, blood hemoglobin\u2009<\u200913.8\u00a0g/dl  or\u2009<\u200912.4\u00a0g/dl , and reticulocyte count\u2009>\u20092% with no cause of hemolysis other than PVL .All of the subjects were scheduled for a TEE and non-contrast CMR performed within 1\u00a0week at our center. None of the patients were pre-medicated or sedated before or during the TEE and CMR, which would have affected the severity of PVL. All patients had routine clinical follow-up 6 months after their enrollment.This study was designed as a prospective single-center study and conducted in accordance with the principles of the Declaration of Helsinki and the local ethics committee. All patients provided signed informed consent. This work was supported by the STRATEGMED II grant .All of the TEE studies were performed with a commercially available imaging system , and each of the examinations followed the European Association of Cardiovascular Imaging (EACVI)/American Society of Echocardiography (ASE) recommendations , 16. TheThe CMR images were acquired on the 1.5\u00a0T system  with a dedicated phased-array cardiac coil and analysed using a cardiac software . The CMR study protocol included a non-contrast examination with a multi-planar cine balanced steady-state free precession (bSSFP) acquisitions and flow visualization using phase contrast (PC) flow imaging. Cardiac chambers volumes and functions were analysed by bSSFP in several planes, including 2- and 4-chambers, orthogonal left ventricular (LV) outflow track, and parallel short-axis planes covering both the atria and ventricles. The typical scan parameters used were time to echo/time of repetition (TE/TR) of 1.9/4.3\u00a0ms, slice thickness 4\u20138\u00a0mm (no inter-slice gap), and temporal resolution 30\u201340\u00a0ms. The cine bSSFP planes for the AV prosthesis and ascending aorta were placed perpendicular to the aortic root. Through-plane PC flow imaging was obtained at the slices perpendicular to the axis of flow with the positions just above the AVR and velocity encoding maximum values starting at 200\u00a0cm/s. PC imaging was repeated with the modified maximum velocity value and/or the position of the slice to avoid aliasing or artifacts . The AVRThe severity of either MVR or AVR-PVL was based on regurgitant volume (PVL-vol) and the regurgitant fraction (PVL-RF). The final grading of CMR PVL was classified according to the RF: (1) mild\u2009\u2264\u200920%, (2) moderate 21%\u201339%, or (3) severe\u2009\u2265\u200940%. No uniform cut-offs and guidelines for the quantification of PVL in CMR can be found, and we used the same criteria as in the most of the previous papers evaluating the utility of CMR for assessing PVL  versus 482\u00a0pg/ml [303\u20131933]; p\u2009<\u20090.01).Patients with PVL-RF in CMR above the median also revealed significantly increased NT-proBNP compared to subjects with PVL-RF below the median .The NT-proBNP serum levels showed significant associations with CMR PVL-RF , CMR PVL-vol , and TEE SQ-PVL . However, neither CMR PVL-RF nor PVL-vol revealed any associations with the vena contracta of the PVL on TEE (p\u2009=\u2009ns).The study group was divided into subgroups of patients with PVL around mechanical or biological prostheses. No differences in the associations between SQ-TEE %PVL and CMR PVL-vol or CMR PVL-RF in either mechanical or biological prostheses were found Table .Table 3PThe association between SQ-TEE and CMR PVL-vol showed no significant differences in patients with MVR-PVL compared to AVR-PVL . However, a significant association between SQ-TEE and CMR PVL-RF was found only in AVR  and not in MVR-PVL . The rates of concordant severity of PVL assessed in SQ-TEE and CMR were similar between MVR and AVRs .Eight cases of hemolytic anemia (25%) were analyzed, and those patients\u2019 PVLs were classified in SQ-TEE as mild (patient\u2009=\u20091), moderate (n\u2009=\u20096), and severe (n\u2009=\u20091). The CMR quantifications in those patients revealed mostly severe (n\u2009=\u20095) or moderate (n\u2009=\u20093) PVL.LV dimensions and systolic function were assessed in all patients in CMR. The median for LV ejection fraction (LVEF) was 59% (52%\u201365%), and five patients with systolic dysfunction (LVEF\u2009<\u200950%) were included in the study. The LV end-diastolic volume (LVEDV) ranged from 111 to 673\u00a0ml (mean 183\u00a0ml [145\u2013258]) and LV mass from 60 to 343\u00a0g (mean 168\u00a0g [135\u2013213]). Both CMR and TEE showed significant associations in LV parameters . However, significant differences between CMR and echocardiography in LVEDV were noted  and LV mass (mean 168\u00a0g [135\u2013213] versus 247\u00a0g [200\u2013318] p\u2009<\u20090.001).LVEDV quantified by CMR showed a strong association with CMR PVL-vol , a moderate correlation with CMR PVL-RF , and also an association with %PVL obtained in TEE .Analysis of receiver operating characterisitic (ROC) curve was performed to find the optimal parameters to predict the greatest cardiac overload and hemolysis.The ROC analysis showed that %PVL and CMR PVL-vol and PVL-RF predicted the upper tertile of NT-proBNP (>\u20092000\u00a0pg/ml) in our study group with the best sensitivity for CMR parameters Table . HoweverMoreover, the ROC analysis revealed that all the imaging parameters used in our study, including TEE (%PVL) and CMR , failed to provide a statistically significant prediction for PVL-related hemolysis.Our prospective study presents important findings with respect to the added value of CMR for diagnostic grading and risk stratification in symptomatic patients with AVR or MVR and PVL. First, SQ-TEE showed a moderate association with CMR PVL-vol and RF and underestimated the CMR severity of PVL in half of these cases. Those observations were not dependent on the type  or position (AVR or MVR). Both SQ-TEE and CMR PC quantification parameters of PVL were associated with NT-proBNP. However, CMR PVL-vol and PVL-RF, but not LV parameters, showed the strongest correlation and the best prediction for the severely increased NT-proBNP. As expected, the CMR PVL-vol revealed the strongest association with LV enlargement. Finally, none of the TEE or CMR parameters predicted PVL-related hemolysis.To the best of our knowledge, we present the first study evaluating the added value of CMR to SQ-TEE in patient quantification and stratification with a PVL related to MVR or AVR.Pflaumer et al. published a first case report 2005) of a severe AVR PVL confirmed on CMR, which was underestimated on echocardiography . There a05 of a sUnderestimation of PVL in echocardiography may also underestimate our interpretation of patient symptoms, result in suboptimal pharmacotherapy, and limit the number of patients scheduled for percutaneous transcatheter closure or repeat surgery. This misclassification of AV-PVL severity may explain worse outcomes in patients with even mild PVL in TTE , 25. In We do not have a clinical follow-up in our study group yet, so we cannot relate our imaging parameters to patients\u2019 prognosis. Instead, we used NT-proBNP, which is an important marker of clinical prognosis, cardiac overload and wall stress, especially in patients with PVL. While parameters acquired in both modalities were associated with NT-proBNP, CMR-derived quantification parameters showed stronger correlation coefficients with this natriuretic peptide. Schewel et al. found that an increased in NT-proBNP that was greater than 1640\u00a0ng/l in patients with post-procedural PVL was associated with significantly increased rate of death in a follow-up . In our Eight (25%) patients had laboratory evidence of hemolytic anemia related to PVL. Although CMR led to an upgrade of the class of PVL in most of them, none of the imaging parameters of PVL severity or LV remodeling predicted the PVL-related hemolysis. No other reference reports describe this prediction. This finding suggests that the mechanism behind this phenomenon is complex and that there is no straight association between the severity of leak, shear stress, and the degree of red blood cell damage .The CMR PC technique is based on the assessment of velocities in the selected image plane. Different lengths of valve prostheses used in SAVR or TAVI require using different levels of assessment in the ascending aorta, which might impact the grading of PVL. Therefore, our results are not strictly comparable to the studies assessing PVL related to TAVI .Finally, the great majority of studies used only TTE in comparison to CMR, which has a modest agreement with TEE in the degree of PVL . AlthougOur study group included mostly patients with AVRs-related PVL with a minority of MVR-PVL. The total number of study patients was too low to provide a separate analysis for both subgroups. The clinical characteristics of the study patients is complex and reflects the real clinical practice. However, although a prosthetic PVL was a main cause of HF symptoms and LV remodeling, it was not possible to separate the minor effects of other factors  or comorbidities. We only had a 6-month clinical follow-up with vital status of study patients , and this period was not enough to relate our results to clinical prognosis. We did not use three-dimensional echocardiography parameters in this study, which could have improved the compatibility in measurements with CMR. Finally, although CMR is a reference tool for volumetric assessment, no true gold standard for measuring the severity of PVL exists. Moreover, the PC method may also be susceptible to artifacts in close proximity to prosthesis. Finally, quantification of MVR function is always an indirect method that is more prone to errors.SQ-TEE shows moderate agreement with CMR, and the Valve Academic Research Consortium II classification underestimated a considerable number of AVR or MVR-PVL cases. The LV cavity enlargement assessed by CMR reflects the PVL-vol, and a significant increase in NT-proBNP is related to PVL severity and not to LV remodeling. While there is no true gold standard for the severity of PVL, a non-contrast CMR did appear to show superior prediction for the upper tertile of NT-proBNP  compared to SQ-TEE . Neither TEE nor CMR parameters are helpful in predicting PVL-related hemolysis.TEE is a necessary imaging modality for screening, grading, and guiding the percutaneous interventions. However, CMR is a complementary tool, which should be implemented in routine practice in patients with at least moderate PVL and/or difficult, incoherent cases. Finally, a need for evidence supporting new cut-off values for CMR quantification of PVL and optimal time for intervention exists."}
+{"text": "Conclusions: Characteristics of mitral PVL and adjacent structures are associated with the severity of hemolysis and pulmonary hypertension. Evaluating the structural and functional characteristics of mitral PVL by complementary multimodal imaging would be important for understanding the clinical presentation and deciding optimal treatments for individual patients.Objective: Clinical presentation of patients with mitral paravalvular leakage (PVL) varies from asymptomatic to heart failure related with hemolytic anemia or pulmonary hypertension. We aimed to investigate the structural and functional characteristics of mitral PVL by multimodal imaging and their association with the severity of hemolysis and hemodynamic significance. Methods: A total of 74 patients with mitral PVL who underwent both cardiac computed tomography (CT) and echocardiography from March 2010 to December 2017 was investigated. Location and size of PVL, degree of left atrial (LA) calcification as measured by CT, and hemodynamic variables as measured by echocardiography were comprehensively analyzed. To investigate the degree of hemolysis and pulmonary hypertension, level of lactate dehydrogenase (LDH) and Doppler estimated systolic pulmonary artery pressure (SPAP) were used respectively. Results: Level of LDH was not related to PVL perimeter and was variable, especially in patients with a small PVL. However, it was positively correlated with mean mitral regurgitation velocity. Additionally, SPAP was significantly correlated with PVL perimeter and LA calcium score. In multivariable analysis, mean mitral regurgitation velocity was significantly correlated with levels of LDH (\u03b2 = 0.345;  Paravalvular leakage (PVL) after mitral valve (MV) surgery is not uncommon, with rates varying from 7% to 17% after surgical implantation of prosthetic valves or annuloplasty rings . DespiteIn this study, we aimed to investigate (1) the structural and functional characteristics of mitral PVL and nearby structures by echocardiography and cardiac CT and (2) their associations with hemolysis and pulmonary hypertension, which can possibly result in heart failure.We retrospectively probed the clinical data for patients with prior MV surgery, repair, or replacement who underwent both cardiac CT and echocardiography between March 2010 and December 2017. Excluding 43 patients that had a time interval between CT and echocardiography that was greater than 90 days, and 36 patients without multiphase CT data, a total of 327 patients was included in our study. After excluding another 253 patients without PVL confirmed by CT and echocardiography, we finally enrolled 74 patients diagnosed with mitral PVL. In the 74 patients with PVL, cardiac CT was usually performed by clinician\u2019s discretion when prosthetic valve dysfunction was suspected. Demographic data and information on surgical findings were collected from electronic medical records. The institutional review board of our institution approved this retrospective study, and informed consent was waived.2 + right atrial pressure]. Right atrial pressure was estimated by measuring inferior vena cava diameter and its response to inspiration. A comprehensive evaluation was performed to evaluate prosthetic or repaired MV function using routine and modified views. Valvular function was evaluated using Doppler measurements, such as peak or mean diastolic pressure gradients for prosthetic or repaired MV. A high-velocity, eccentric turbulent jet, with its origin beyond the edge of the sewing ring, was considered to indicate PVL [All subjects underwent standard two-dimensional transthoracic echocardiography (TTE) and/or transesophageal echocardiography (TEE) within 90 days from CT. Conventional two-dimensional and Doppler measurements were performed as recommended by the American Society of Echocardiography guidelines using commercially available equipment . The calcate PVL ,11. The cate PVL . Mitral All CT scans were performed with a dual-source CT scanner  or a wide coverage, 256-detector row CT scanner  with an electrocardiographically gated data-acquisition mode. CT scans were performed with the triple-phase injection method .From raw data sets, image reconstruction was performed with a medium kernel and iterative reconstruction, where the reconstruction slice thickness was 0.625 to 0.75 mm with 0.5- to 0.625-mm increments between slices. For all patients, 10 transverse datasets were reconstructed for every 10% of the cardiac cycle (0\u201390%), and reconstructed images were transferred to an image server and analyzed with dedicated three-dimensional (3D) software .A dedicated radiologist who was blinded to clinical information and the results of TTE and TEE performed all the CT analyses. Assessment of the mitral PVL was performed using multiplanar reformatted images in cine mode. The short-axis view of the MV was used to assess the presence and location of the PVL. If PVL was present, the location was recorded using a localization system based on the surgical view and classified as anterolateral, anteromedial, posteromedial, or posterolateral. Mitral PVL size was assessed by measuring the area and perimeter on the short-axis view. The size of the mitral PVL was assessed in mid-systolic phase  by measuring the area and perimeter on the short-axis view. These measurements were confirmed to be concordant between two radiologists in a previous study by our group .To add structural information from around the prosthetic MV, the left atrial (LA) calcium score was measured on a noncontrast-enhanced electrocardiography-gated scan. After identifying LA calcification greater than 130 Hounsfield units, the degree of LA calcification was quantified using the Agatston score system. Visual grading of LA calcification was performed according to extent of calcification within LA circumference and classified into four groups based on the corresponding score as follows: no LA calcification (0), mild LA calcification (1\u2013500), moderate LA calcification (501\u20135000), and severe LA calcification (>5000).Presence of hemolytic anemia was defined as hemoglobin level <13 g/dL in women and <15 g/dL in men with one or more laboratory signs of hemolysis , haptoglobin, or reticulocytosis abnormalities in peripheral blood smears) . Level ot-test for continuous variables and \u03c72 statistics for categorical variables. Mean values of LDH between groups of four different PVL locations were compared by analysis of variance (ANOVA). Simple linear regression analysis was performed to determine the associations between PVL parameters and the degree of hemolysis and heart failure. The variables that showed p < 0.1 during univariate analysis as well as age and sex were entered into a multivariable linear regression model and described with standardized \u03b2- and p-values. A value of p < 0.05 was considered significant. All statistical analyses were performed using the Statistical Package for the Social Sciences version 22.0 software program .Continuous variables are presented as mean \u00b1 standard deviation, while categorical variables are expressed as percentages. The patient groups were compared using Student\u2019s The baseline clinical characteristics and data of valve surgery are presented in 2, and mean diastolic pressure gradient through the MV was 6.4 \u00b1 2.5 mmHg. In terms of PVL severity, proportions of mild, moderate, and severe PVL were 54.1%, 32.4%, and 13.5%, respectively. Average peak MR velocity through the PVL was 5.4 \u00b1 0.9 m/s, and mean MR velocity was 3.7 \u00b1 0.7 m/s. The estimated SPAP was 55.2 \u00b1 15.2 mmHg.2 and 41.3 \u00b1 24.8 mm, respectively. The most common PVL location was the posterolateral, followed by posteromedial, anteromedial, and anterolateral. Patients without LA calcification composed only 27.0% in this population. The prevalence rates of mild, moderate, and severe LA calcification were 33.8%, 23.0%, and 13.5%, respectively. The average LA calcium score was 3507 \u00b1 8057. Intriguingly, there was no difference in hole area and perimeter of PVL in three groups, which were divided into mild, moderate, and severe PVL according to the current guideline  (p = 0.794). Notably, levels of LDH were highly variable when PVL hole area was smaller than 1.0 cm2 (p = 0.016) (p = 0.062); which was consistent in the analysis of TEE (p = 0.060) (p = 0.016)  A. Simila 1.0 cm2 . Regardi 1.0 cm2 B. There = 0.016) C, howeves of TEE . There ws of TEE . LV ejec= 0.060) D. In mul= 0.016) . A reprep = 0.032 and r = 0.272; p = 0.037, respectively) (p = 0.045)   A. There ctively) B. Intrigctively) C, and SP= 0.045) D. In mulctively) . A repreThe principal findings of the present study are as follows: (1) degree of hemolysis in mitral PVL was not related to PVL size and was particularly variable in patients with a small PVL; (2) degree of hemolysis was closely associated with mean MR velocity; and (3) The hemodynamic index of pulmonary hypertension was significantly associated with PVL size and severity of LA calcification. From the study findings, we suggest a complementary role for multimodal imaging in the evaluation of structural and functional characteristics of mitral PVL and adjacent chambers .PVL is defined as abnormal communication between the sewing ring of a surgical prosthesis and the native annulus ,12. SeveA variety of diagnostic tests should be performed to evaluate PVL. Echocardiography is the initial choice of imaging modality for PVL diagnosis and offers valuable information related to hemodynamic consequences of PVL, such as valve function, heart chamber size and function, and pulmonary artery pressure. However, grading and quantification of PVL using conventional echocardiographic parameters, such as vena contracta, or the effective regurgitant orifice area and regurgitant volume measured by the proximal isovelocity surface area method, is a considerable challenge because PVL is usually observed as eccentric regurgitation. In our study, there was no significant difference in PVL hole area and perimeter on cardiac CT between three groups of PVL severity according to the current guideline. Transesophageal echocardiography with 3D reconstruction can provide more precise information regarding the location of PVL and is a crucial imaging modality for intraprocedural guidance. Some recent studies using multiplanar reconstruction of 3D TEE imaging reported acceptable accuracy in the quantitative measurement of PVL size on 3D color Doppler image ,26.Cardiac CT demonstrates excellent spatial resolution and can provide data on the aforementioned structural characteristics of PVL, which is helpful when planning and providing guidance for treatment . RecentlIn the current study, using cardiac CT and echocardiography, we demonstrated that the degree of hemolysis was closely associated with mean MR velocity, and that degree of pulmonary hypertension was closely related to size of PVL and surrounding structures such as LA calcification.The size of PVL was not associated with the degree of hemolysis, but functional factors such as MR velocity through the PVL and LV ejection fraction were related to degree of hemolysis. Hemolytic anemia results from shear stress on red blood cells. Besides, the interaction of blood with a foreign prosthetic material, and higher velocity through the PVL may facilitate a higher degree of hemolysis . A previThe degree of pulmonary hypertension was closely related to PVL hole area and perimeter, which are structural characteristics of PVL. Recently, several studies reported additional value of CT in the evaluation of PVL ,31. ThesFirst, this study was retrospective and cross-sectional in design, which has inherent potential limitations. We only count heart failure or hemolytic anemia episodes occurring at the time of CT and echocardiography imaging, not later during the follow-up period, and compared their association with various parameters on multimodal imaging. And, as cardiac CT was usually performed by clinician\u2019s discretion when prosthetic valve dysfunction was suspected, there would be a relatively small number of patients without symptom or patients with renal insufficiency in our study population which resulted in selection bias. Second, although pulmonary hypertension can be induced or aggravated by hemodynamic consequence of mitral PVL, the chronic pulmonary artery remodeling caused by preoperative MV pathologies may affect the level of SPAP. In addition, we did not assess SPAP and other hemodynamic parameters by cardiac catheterization in this study. As for the hemolysis, there has been no single parameter recommended for evaluating the degree of hemolysis and each parameter of hemolysis, as each parameter can be affected by various clinical conditions. The level of LDH is thought to be a reliable marker for quantifying red cell damage in patients with valve replacement , and it Patients with mitral PVL may present with hemolytic anemia and pulmonary hypertension, which can result in clinical heart failure. In this analysis, characteristics of mitral PVL and adjacent structures were associated with the severity of hemolysis and pulmonary hypertension. The degree of hemolysis was related to the mean mitral regurgitation velocity rather than the size of PVL, and the hemodynamic index of pulmonary hypertension was significantly related to PVL size and severity of LA calcification. From these results, we would like to highlight the complementary role of multimodal imaging in the evaluation of mitral PVL for understanding its clinical presentations and treatment response. Considering high mortality and morbidity in patients with PVL , we beliCardiac CT has an advantage in evaluating structural characteristics of mitral PVL, and surrounding structures such as LA calcification whereas echocardiography is beneficial in assessing functional and hemodynamic characteristics related to the mitral PVL. The imaging modalities have a complementary role in evaluation of mitral PVL and are crucial for understanding the clinical presentation of patients with mitral PVL. Understanding the characteristics of mitral PVL with multimodal imaging is important to improve clinical outcome."}
+{"text": "Non-governmental organisations (NGOs) in Ukraine have provided HIV testing, treatment, and condom distribution for men who have sex with men (MSM). HIV prevalence among MSM in Ukraine is 5.6%. We estimated the impact and cost-effectiveness of MSM-targeted NGO activities in Ukraine.A mathematical model of HIV transmission among MSM was calibrated to data from Ukraine (2011-2018).The model, designed before the 2022 Russian invasion of Ukraine, evaluated the impact of 2018 status quo (SQ) coverage levels of 28% of MSM being NGO clients over 2016-2020 and 2021-2030 compared to no NGO activities over these time periods. Impact was measured in HIV incidence and infections averted. We compared the costs and disability adjusted life years [DALYs] for the SQ and a counterfactual scenario  until 2030 to estimate the mean incremental cost-effectiveness ratio .Without NGO activity over 2016-2020, the HIV incidence in 2021 would have been 44% (95%CrI: 36%-59%) higher than with SQ levels of NGO activity, with 25% (21-30%) more incident infections occurring over 2016-2020. Continuing with SQ NGO coverage levels will decrease HIV incidence by 41% over 2021-2030, whereas it will increase by 79% (60-120%) with no NGOs over this period and 37% (30-51%) more HIV infections will occur. Compared to if NGO activities had ceased over 2016-2020 (but continued thereafter), the SQ scenario averts 14,918 DALYs over 2016-2030 with a mean ICER of US$600.15 per DALY averted.MSM-targeted NGOs in Ukraine have prevented considerable HIV infections and are highly cost-effective compared with a willingness-to-pay threshold of 50% of Ukraine\u2019s 2018 GDP . The Eastern Europe and Central Asia region has a fast growing HIV epidemic, with UkMost funding for HIV treatment and prevention services in Ukraine, including among MSM, goes to non-governmental organisations (NGOs). NGOs taTo help policymakers allocate their limited resources, information is needed on the benefits and costs of different health interventions. This has become particularly important in middle-income countries such as Ukraine because funding for NGO activities is transitioning from international funding agencies to governments. AlthougWe developed a dynamic, deterministic model of HIV transmission among MSM  and not accessing NGOs, with a constant proportion being high-risk and the remainder low-risk. Individuals exit due to age-related or HIV-related deaths, with only the former being replaced with new entrants. Transitions occur between the low- and high-risk groups at rates that differ for ages 18-39 and \u226540 years but balance the flows between these groups. MSM become clients of NGOs at time-varying rates from 2003 and cease contact with NGOs at a constant rate. HIV-positive MSM become NGO clients at a higher rate due to NGO efforts to link HIV-positive MSM to care; our previous analyses show a higher proportion of NGO clients are HIV-positive than for non-NGO clients.We assume all HIV transmission is within the MSM population as few MSM  have ever injected drugs and most HIV-infected women are female sex workers, 13, whiFollowing HIV infection, individuals progress through acute, latent, pre-AIDS, and AIDS stages of HIV disease. Transmissibility is heightened during acute infection and pre-AIDS HIV disease, whereasPre-exposure prophylaxis (PrEP) is not readily available in Ukraine. A full The model is primarily parameterised using four national IBBS among MSM implemented in 2011 , 2013 , 2015 , and 2018 \u201318. Key Age-related mortality data comes from UN databases and we uThe model is seeded so that the age distribution of MSM in 1990 matches Ukraine\u2019s male population, with MSM then entering and aging through the model. Although the HIV epidemic probably started later in Ukraine than Western Europe, there iIncreased condom usage during their last anal intercourse ,Increased HIV testing in last year ,Increased coverage of ART .In our previous analyses of the MSM IBBS data, being an NGO client was associated with: IncreasWe therefore assumed the rate of HIV-diagnosis and ART initiation are higher among NGO clients than non-NGO clients, and that they have higher condom use. This assumption is supported by NGO activities for MSM in Ukraine being focussed on providing HIV testing and counselling services, condom distribution, and linking HIV-positive people to ART services. NGOs also offer post-diagnosis case-management to MSM that test positive for HIV, incorporating counselling and linkage to ART. A detailed description of the assumptions made around the effects of NGOs is in the supplement .NGOs targeting MSM initiated in Ukraine in 2003. Numbers from Alliance for Public Health (APH) indicate that there were 22,824 MSM registered with NGOs in 2013, 50,615 in 2018 (~28% of MSM), and stable thereafter, except for a COVID-19-related dip in 2020 . These numbers of MSM registered as NGO clients include those that receive at least the minimum package of services . They do not include other MSM that would have benefitted from NGO services such as condom distribution. The model was fit to these trends by assuming different rates of joining NGOs over time, with the rate increasing between 2003-2012 and 2013-2019 and then being constant thereafter, except in 2020 where a lower rate is assumed. Overall levels of condom use are assumed to have increased over 1990-2007, with rath-97.5th percentile range.We calibrated the model using an Approximate Bayesian computation sequential Monte Carlo (ABC SMC) scheme to summaThe baseline model fits were cross-validated against overall HIV incidence  andWe modelled the impact of existing coverage levels of NGOs (status quo) over 2016-2020, and if they were continued over 2021-2030, both compared to counterfactual scenarios where there was no effect of NGOs on condom use, HIV testing and initiating ART over these specific time periods, equivalent to NGOs ceasing activities and their effects stopping over these time periods. Although the recent invasion by Russia will have changed the status quo situation, it is too early to understand to what degree, with the counterfactual situation now also being relevant to show what could occur if services are disrupted compared to them being maintained. For the 2021-2030 scenario, we also varied the counterfactual to estimate the impact of each beneficial effect of the NGOs. We also estimated the impact over 2021-2030 of increasing the coverage of NGOs from 28% to 60% of MSM by 2025. Impact was estimated in terms of the number and percentage of HIV infections averted, percentage reduction in HIV incidence and HIV prevalence, and number and percentage of HIV deaths averted.To investigate our assumptions about the effectiveness of NGOs, we also performed a sensitivity analysis in where we assumed not all the differences in condom use, HIV testing and ART coverage between NGO and non-NGO clients were due to NGO activities. For these, the counterfactual scenario incorporated different percentages of the difference in condom use, HIV testing, and linkage to HIV care between NGO and non-NGO clients. The status quo scenario was then compared to these scenarios. This can be interpreted as different percentages of the effects we are currently prescribing to NGOs being due to other causes.Unit cost estimates for ART and NGO services for MSM  came from published reports\u201329 and uDue to the ongoing nature of NGO activities, we estimated the cost-effectiveness of current NGO activities by comparing the status quo scenario over 2016-2030 with a counterfactual scenario where NGO activities are stopped for 5 years (costs and effects of NGOs are removed) from 2016 and then restarted in 2021 until 2030. This allows us to capture some of the longer-term benefits of the NGO activities that occurred over 2016-2020. This waWith existing levels of NGO activity , where 2If there had been no NGO activity over 2016-2020 then HIV incidence would have been 44% (95%CrI: 36%-59%) higher in 2021, at 1.05 (95%CrI: 0.84-1.27) per 100 person-years . There wWhen we consider the effect of removing specific beneficial effects of NGOs over 2016-2020, we find the number of HIV infections increases by 3% (95%CrI: 2%-6%) compared with the status quo scenario if we remove the increase in HIV diagnosis; 9% (95%CrI: 6%-14%) if we remove the increase in ART initiation; and 12% (95%CrI: 11%-14%) if we remove the increase in condom usage.Lastly, If NGO activities were to cease over 2021-2030, then the number of incident HIV infections over this period would increase by 37% (95%CrI: 30%-51%) compared to the status quo scenario, HIV incidence in 2030 would be 79% (95%CrI: 60%-120%) higher, and 15% (95%CrI: 12%-20%) more deaths would occur . ImportaAlternatively, if NGO activities were scaled-up from 2021 such that double  the number of MSM were clients of NGOs in 2025 than the status quo scenario , then incidence decreases by half  of what it would be in 2030 with the status quo scenario, to 0.23 (95%CrI: 0.14-0.30) per 100 person-years. This is 69% lower than it is currently estimated to be in 2020. HIV prevalence also decreases further to 3.9% (95%CrI: 3.0%-4.6%) in 2030, and there would be 29% (95%CrI: 25%-37%) fewer HIV infections and 12% (95%CrI: 10%-16%) fewer HIV deaths over 2021-2030 than for the status quo scenario.Compared to the counterfactual scenario with no effects of NGOs over 2016-2020 (but with NGO activities continuing over 2021-2030), the status quo scenario with NGOs in place over 2016-2030 is estimated to avert 3,025  HIV infections, 1,895  HIV deaths and 14,918 DALYs over 2016-2030. The status quo scenario costs US$35,600,695 , whilst the counterfactual scenario costs US$26,627,922 . The status quo scenario therefore incurs additional costs of US$8,952,733 over 2016-2030, resulting in a mean ICER of US$600.15 per DALY averted . The meaThe various one-way sensitivity analyses showed the cost-effectiveness results were robust, with the greatest mean ICER per DALY averted occurring when we assume higher costs for ART : US$863.95/DALY averted, which is still lower than the 0.5xGDP threshold, and so highly cost-effective. Conversely, the intervention becomes even more cost-effective (US$114/DALY averted) when a longer time horizon to 2050 is assumed. In the additional sensitivity analysis where different percentages of the effects prescribed to NGOs are assumed to be due to other causes , NGOs arOur modelling analyses suggest that without current NGO activities among MSM in Ukraine (over 2016-2020), the HIV incidence in 2021 would have been 44% higher and 25% more HIV infections would have occurred from 2016-2020. Going forward to 2030, continuing current NGO activities will reduce incidence by 40% and prevalence by 25% compared to what they are in 2021, whereas they would be 79% and 21% higher by 2030, respectively, if NGO activities cease. Importantly, most of the increase in HIV incidence occurs in the first year highlighting the importance of maintaining NGO activities despite the war with Russia because the public health implications of not doing so are stark. Doubling coverage of NGO activities from 2021 will further halve incidence by 2030. Projections suggest much of the ongoing benefit of NGO activities is through improving condom distribution and linkage to ART, with current NGO activities estimated to have increased the coverage of ART among HIV-positive MSM from 25% to 42% in 2021. Lastly, our analyses suggest that current NGO activities are highly cost-effective, costing US$600.15 per DALY averted, with all model projections and sensitivity analyses being lower than available willingness-to-pay thresholds for Ukraine.To our knowledge, this is the first study to examine the impact and cost-effectiveness of MSM-targeted NGOs in Ukraine. Wirtz et al previously modelled the impact of HIV prevention and treatment interventions among MSM in various low- and middle-income countries (LMIC), including Ukraine, and found that little impact on HIV incidence would be achieved unless ART was expanded. UnfortuThere has been limited research on Ukraine\u2019s HIV epidemic among MSM. Our previous study used in this analysis found that NGOs were associated with beneficial outcomes among MSM, agreeinThe ICER per DALY averted for our study compares favourably with ICERs for other health interventions in Ukraine, particularly when considering this study\u2019s ICER over a longer time horizon to 2050 (US$114). Other interventions in Ukraine with published ICERs include treatments for pneumonia  scaling The strength of our modelling includes using four rounds of national IBBS data to parameterise and calibrate the model within a Bayesian framework. Another strength is its novelty, with there being few evaluations of MSM-targeted interventions for Eastern Europe. LimitatThe IBBS surveys are also limited because they are observational, and, so, could only be used to look at associations rather than causality in the epidemiological analyses that fed into this modelling. It is possible that some of our modelled intervention effects may result from more health-conscious people attending NGOs or from risk-behaviours differing between HIV-negative and HIV-positive MSM (HIV-positive MSM attended NGOs more than HIV-negative MSM), resulting in our projections possibly being overestimates. To counter this, we showed using stratified analyses that the beneficial associations of being an NGO client remained for both HIV-negative and HIV-positive MSM . Also, mOur analyses suggest that NGOs targeting MSM in Ukraine are highly cost-effective and have been preventing considerable HIV infections and deaths. This beneficial impact has been achieved through condom distribution, while quickly diagnosing HIV-positive MSM and linking them to ART. UnfortuSupplemental Data File"}
+{"text": "Optimum configuration of a micromixer with two-layer crossing microstructure was performed using mixing analysis, surrogate modeling, along with an optimization algorithm. Mixing performance was used to determine the optimum designs at Reynolds number 40. A surrogate modeling method based on a radial basis neural network (RBNN) was used to approximate the value of the objective function. The optimization study was carried out with three design variables; viz., the ratio of the main channel thickness to the pitch length (H/PI), the ratio of the thickness of the diagonal channel to the pitch length (W/PI), and the ratio of the depth of the channel to the pitch length (d/PI). Through a primary parametric study, the design space was constrained. The design points surrounded by the design constraints were chosen using a well-known technique called Latin hypercube sampling (LHS). The optimal design confirmed a 32.0% enhancement of the mixing index as compared to the reference design.  The recent advancements in miniaturized research in biochemistry and biomedicine demand development in the area of microfluidic systems. Micromixers are one of the fundamental components of micro total analysis systems (\u03bc-TAS) and microfluidic systems ,2. A micOn the basis of their working principles, micromixers have been classified into passive micromixers and active micromixers. Mixing performance is increased by employing external energy sources within an active micromixer. There are various methods available for flow manipulation to enhance mixing, such as pressure, acoustic, thermal, magnetic, electrokinetic, etc. In an active micromixer, higher mixing efficiency can be achieved in a very short mixing length. However, these micromixers require complex manufacturing methods, and their integration into a microfluidic system is also difficult. In a passive micromixer, the mixing is simply increased with the help of a modification in the geometrical structure of the microchannel. Recently, passive micromixers have been the superior preference for researchers over active micromixers because of their easier fabrication and their incorporation technique into microfluidic systems ,5,6,7. Commercial software using computational fluid dynamics (CFD) is one of the most consistent and popular tools for fluid flow structural analyses, as well as for evaluating the performance of microfluidic devices ,9,10,11.For performance development of the available micromixers, the design optimization technique using the CFD tool has progressively and increasingly become the method preferred by researchers. Structural optimization of a micromixer with a slanted groove microchannel was performed ; using eThrough a literature survey, we have established that a numerical optimization process using three-dimensional CFD has been a constructive tool for improvement of existing micromixers. This study performed single-objective optimizations for the further enhancement of micromixer performance in terms of mixing index, as proposed by Ahmed et al. . The optIn our previous study , to insp\u22124 kg/m s and 997 kg/m3, respectively [\u221211 m2/s [The present section formulates the numerical model used in this study for the proposed design of the microchannel. As discussed, the flow inside micro devices is characterized by the omnipresence of laminar flow; therefore, the flow was considered as laminar. In this study, numerical simulations were performed by assuming flow as laminar, steady, and incompressible. The detailed analysis of the mixing between the fluids was investigated by solving, continuity, mass diffusion, and three-dimensional Navier\u2013Stokes equations. Under the considered assumptions, the equations are mathematically articulated as:ariation  of the fectively . The dif\u221211 m2/s . The num\u221211 m2/s  for prin\u221211 m2/s . Additio\u221211 m2/s  was used\u221211 m2/s  was used\u22126) at each node of the computational domain.For numerical simulations, diverse boundary conditions were applied at the outlet, inlets, and side walls. Normal inlet velocity was calculated on the basis of the properties of water and specified as the inlet boundary condition at both inlets. Atmospheric pressure (zero) was applied at the outlet section, and the frictionless wall was specified. The convergence criteria of numerical solution were taken in terms of the root mean square value  on the cross-sectional plane, and the number of sample points is denoted by N. Subsequently, the following mathematical expression was used to calculate the mixing index:2 is the variation of the dye mass fraction, and the maximum variance value on the sample plane is denoted by the On the basis of variance of the dye mass fraction across a cross-sectional plane, the mixing efficiency was determined. The plane was taken at the end of the last mixing unit, and the mass fraction variation was calculated using following formula:MI) of the micromixer was considered for objective function values. The primary and very fundamental process during the optimization process is to select the appropriate design constraints that influence the objective function values. A detailed parametric investigation was carried out to select the sensitive parameters for structural optimization. Three dimensionless geometric parameters: the ratio of the main channel thickness to the pitch length (H/PI), the ratio of the thickness of the diagonal channel to the pitch length (W/PI), and the ratio of the depth of the channel to the pitch length (d/PI) were chosen to optimize the proposed micromixer. The design ranges of the parameters were restricted based on a preliminary study, shown in u and xl represent the upper and lower limits of the design variable x, respectively. In this study, the optimization procedure maintained the following steps; initially effective design parameters and proper design constraints were determined through the preliminary study to enhance the performance of the micromixer. To formulate the surrogate model, design of experiments (DOE) was applied to select the uniformly distributed design points within the design constraint. Two types of DOE methods are found in the literature: random design and orthogonal design. In the orthogonal design technique, model parameters are self-determining and represent that the factors are not related  and can be varied separately. There are some limitations of orthogonal designs. Firstly, they are quite unconvincing when it comes to determining the important factor. As the fundamental function is determined, the probability of reproducing the design points is high, generally termed as the collapse problem . TherefoIn the present work, a random design based on LHS  was chosNext, a surrogate was formulated depending on the (twenty-eight) objective function values. To determine the optimal points within the design constraint, the algorithms needed a large number of estimations of objective function. Therefore, for the sake of computational time saving, it was obligatory to formulate a surrogate model on the basis of distinct numerical analysis within the design constraints. In this study, to estimate the objective function values, the RBNN technique was used . j,and yj is the basis function. Various techniques can be applied to select the functions. The function can be classified as nonlinear or linear. For the linear model, the parameters, including the basis function, are stable throughout the iteration progression. On the other hand, if the basis function differs through the iteration, it calls for a nonlinear model. The iteration procedure, corresponding to a search for the best plane within the multidimensional space, offers a suitable match to the learning data. The parameters of the surrogate model are termed as a user-defined error goal (EG) and the spread constant (SC) [The RBNN is an artificial neural network (ANN) that considers the radial basis functions (RBF) as activation functions, constructed by three layers , which contain linear or nonlinear neurons. Each activation function depends on the distance from the center vector to the input layer; thus, the function becomes symmetric along the center vector . The hidant (SC) . The finant (SC) . 6 to 1.9 \u00d7 106 were tested for mixing index evaluation down to the microchannel length at Reynolds numbers (Re = 40), as shown in 6 and 1.6 \u00d7 106, Re = 40. Additionally, tiny variation was found in the mixing development (0.27%) at the exit of the micromixer between the two grid systems, represented by the mesh element sizes 3.5 \u03bcm and 3.0 \u03bcm, respectively  was considered to be the most favorable grid system. In any computational study to ensure a high-quality grid system, it is a predominant criterion to diminish the numerical inaccuracies generated during the discretization process. In this study a tetrahedral grid was built for the computational domain. To establish an optimum number of nodes as well as mesh size, an investigation of grid-dependency was executed. A grid system containing a number of nodes (five) starting from 0.5 \u00d7 10ectively b. TherefThe numerical model was validated qualitatively and quantitatively with experimental findings , as demo1\u2013A6) at the crossing nodes, shown in Consequences of the Reynolds numbers on mixing performance were examined both quantitatively and qualitatively through a numerical procedure. To demonstrate the mixing developments, the mass fraction variations were captured on six cross-sectional planes  on the behavior of the mixing performance at Re = 1.0 and Re = 40 was performed as shown in For qualitative evaluations of mixing performance values, mass fraction distributions were plotted (on x\u2013y planes) at Re = 40, shown in In this study, the well-known surrogate models, RBNN, were usedto achieve the optimized micromixer.The developments of objective function (MI) value along the down way length of the micromixer for optimized geometry and reference design are represented at Reynolds number 40. Developing rates of the mixing index increase for both designs along the channel length, depicted in x) is articulated by incorporating the streamwise vorticity on y\u2013z planes, mathematically represented as:y represents the y direction velocity components and vz represents the z direction velocity component. Along the channel length, the value of the circulation increases in both micromixers. MI) was considered as an objective function to find the most efficient design.Geometric optimization of a proposed two-layer crossing micromixer was carried out using 3D Navier\u2013Stokes formulas. To estimate the objective function, the well-known RBNN model was used. The optimization study was performed with three design variables; viz., the ratio of the main channel thickness to the pitch length (H/PI), the ratio of the thickness of the diagonal channel to the pitch length (W/PI), and the ratio of the depth of the channel to the pitch length (d/PI). The mixing index for the micromixer  and the objective function (MI = 0.86) for the optimum micromixer. The objective function values of the reference design and predicted optimal design were 0.65 and 0.86, respectively, which confirms a 32% relative increase in the objective function through the surrogate-based optimization procedure. The calculated objective function value using Navier\u2013Stokes equation was compared with the predicted value. The RBNN model represented a 6.2% relative deviation of objective function value from the optimum point. The study represents that the single-objective optimization procedure is favorable for the improvement of micromixer performance. The optimum micromixers could be incorporated into a micro-total analysis system and lab-on-chip (LOC) systems to facilitate the study of reaction kinetics, dilution of fluid samples, and enhancement of reaction selectivity."}
+{"text": "Enterococcus faecium in tyramine production and its response to fermentation temperature in a traditional Korean fermented soybean paste, Cheonggukjang. Tyramine content was detected in retail Cheonggukjang products at high concentrations exceeding the recommended limit up to a factor of 14. All retail Cheonggukjang products contained Enterococcus spp. at concentrations of at least 6 Log CFU/g. Upon isolation of Enterococcus strains, approximately 93% (157 strains) produced tyramine at over 100 \u00b5g/mL. The strains that produced the highest concentrations of tyramine (301.14\u2013315.29 \u03bcg/mL) were identified as E. faecium through 16S rRNA sequencing. The results indicate that E. faecium is one of the major contributing factors to high tyramine content in Cheonggukjang. During fermentation, tyramine content in Cheonggukjang groups co-inoculated with E. faecium strains was highest at 45 \u00b0C, followed by 37 \u00b0C and 25 \u00b0C. The tyramine content of most Cheonggukjang groups continually increased as fermentation progressed, except groups fermented at 25 \u00b0C. At 45 \u00b0C, the tyramine content occasionally exceeded the recommended limit within 3 days of fermentation. The results suggest that lowering fermentation temperature and shortening duration may reduce the tyramine content of Cheonggukjang, thereby reducing the safety risks that may arise when consuming food with high tyramine concentrations.The study evaluated the role of  Cheonggukjang is a traditional Korean soybean paste produced by fermenting soybeans with Bacillus subtilis. Traditional methods of Cheonggukjang production utilize rice straw added to steamed soybeans for a short fermentation period of approximately 2\u20133 days, while starter cultures are used instead of rice straw for modern methods of production [Cheonggukjang is a process involving microbial enzymatic proteolysis resulting in uniquely characteristic savory aromatic and flavor properties [Cheonggukjang has been reported to be associated with numerous benefits such as antioxidative, antihypertensive, thrombolytic, and antimicrobial properties [Cheonggukjang, potentially hazardous biogenic amines (BAs) may be produced during fermentation of the proteinous food rich in precursor amino acids.oduction ,2. Fermeoperties ,5. Howev\u03b2-phenylethylamine, 100 mg/kg for histamine, and 100\u2013800 mg/kg for tyramine in foods.The majority of BAs are formed through the reductive amination of ketones and aldehydes, as well as the decarboxylation of amino acids by microbially produced enzymes . Though Cheonggukjang have shown that tyramine in particular has been detected in high concentrations up to 1913.51, 251.66, and 905.0 mg/kg, respectively. Ibe, et al. [Enterococcus faecium may be largely responsible for the BA content of Miso (a Japanese fermented soybean paste). Notably, numerous studies have reported that Enterococcus spp. possess the tyrosine decarboxylase gene (tdc) [E. faecium in Cheonggukjang, while a previous study by Jeon, et al. [Enterococcus spp. isolated from Cheonggukjang exhibited tyramine production at concentrations of at least 351.59 \u03bcg/mL. Taken together, the previous reports imply that E. faecium may also be responsible for the BA content of Cheonggukjang. Meanwhile, the growth of Enterococcus spp. has been reported to occur at temperatures ranging from 10 \u00b0C up to 45 \u00b0C that overlap with Cheonggukjang fermentation temperatures ranging from 25 to 50 \u00b0C [E. faecium growth and tyramine production during the fermentation of contaminated Cheonggukjang products. Furthermore, a previous study reported that tyramine content increases in fermented soybeans as fermentation progresses [E. faecium strains may be affected by incubation conditions such as temperature and time. Therefore, the current study assessed the safety risk of BAs (particularly tyramine) in Cheonggukjang, clarified the microorganism responsible for tyramine accumulation, and evaluated the effect of fermentation temperature/duration on E. faecium growth and subsequent tyramine production in the food.Previous studies by Ko, et al. , Jeon, e, et al.  suggestene (tdc) ,24. Morene (tdc)  confirmeto 50 \u00b0C ,28,29,30ogresses , the proCheonggukjang products were purchased from various retail markets in the Republic of Korea and stored at 4 \u00b0C until further experimentation. Within a day of storage, the BA content of Cheonggukjang products was measured, followed by physicochemical and microbial analyses.Six representative, but different Cheonggukjang, the physicochemical properties of Cheonggukjang samples (retail Cheonggukjang products purchased and Cheonggukjang groups fermented in this study) were measured as described below. Samples weighing 10 g using an analytical balance  were homogenized with 90 mL of distilled water using a stomacher . The pH of the homogenates was measured using a pH meter , while salinity was measured using the procedure described by the Association of Official Analytical Chemists  [To investigate the influencing factors such as pH, salinity, and water activity on BA content in  960.29) . The watCheonggukjang samples was conducted using Plate Count Agar ; de Man, Rogosa, and Sharpe  agar; and m-Enterococcus Agar  for the enumeration of total mesophilic viable bacteria, lactic acid bacteria, and Enterococcus spp., respectively. Samples weighing 10 g were homogenized with 90 mL of sterile 0.1% peptone saline using a stomacher. The homogenates were 10-fold serially diluted with 0.1% peptone saline up to 10\u22125, and 100 \u03bcL of each dilution was spread on PCA, MRS agar, and m-EA in duplicates. Incubation conditions were set according to the manufacturer\u2019s instructions: PCA at 37 \u00b0C for 24 h and m-EA at 37 \u00b0C for 48 h under aerobic condition; MRS agar at 37 \u00b0C for 48 h under anaerobic condition. Anaerobic condition was achieved using an anaerobic chamber  containing an atmosphere of 95% nitrogen and 5% hydrogen. After incubation, the bacterial concentrations of the Cheonggukjang samples were calculated by enumerating the colony-forming units (CFU) on the plates of respective media with approximately 10 to 300 colonies [The analysis of the microbial community in colonies  and adjuEnterococcus strains were isolated from retail Cheonggukjang products according to the method described by Marekov\u00e1, et al. [v/v).A total of 169 , et al. , with miEnterococcus strains that displayed the highest tyramine production were further investigated through sequence analysis of 16S rRNA gene amplified with the universal bacterial primer pair 518F (5\u2032-CCAGCAGCCGCGGTAATACG-3\u2032) and 805R (5\u2032-GACTACCAGGGTATCTAAT-3\u2032) . The identities of sequences were determined using the basic local alignment search tool (BLAST) of the National Center for Biotechnology Information .The identities (at species level) of the individual E. faecium, several temperatures were set for in situ Cheonggukjang fermentation experiments. The temperature for Cheonggukjang fermentation (intermediate-temperature group) was determined based upon previous studies in which 37 \u00b0C was reported as the temperature commonly used for Cheonggukjang production [Cheonggukjang fermentation were utilized for the low and high temperature groups, respectively [To investigate the effect of fermentation temperature on tyramine production by oduction ,35,36. Iectively ,30.Glycine max Merrill) were purchased from a retail market in the Republic of Korea. The soybeans were soaked in distilled water at 4 \u00b0C for 12 h, and subsequently drained for 1 h. Approximately 200 g of soybeans were adjusted to a final salinity of 2.40% according to the salinity of Cheonggukjang outlined in the 9th revision of the Korean food composition table [B. subtilis KCTC 3135  and 4 Log CFU/g of E. faecium KCCM 12118  or E. faecium CJE 216 (strain isolated from Cheonggukjang and selected owing to both strong tyramine production and tdc gene expression). The control group (without any E. faecium strains) was inoculated with only B. subtilis KCTC 3135 to a final concentration of 6 Log CFU/g. The sizes of inocula were selected with consideration of the cell count of each microorganism in Cheonggukjang products determined in our previous study [White soybeans  were homogenized by vortex  and stored at 4 \u00b0C for 2 h. The mixture was then centrifuged at 3000\u00d7 g for 10 min at 4 \u00b0C , and the supernatant was collected. Upon resuspension of the pellet with 20 mL of 0.4 M perchloric acid, the mixture was stored at 4 \u00b0C for 2 h and centrifuged again at 3000\u00d7 g at 4 \u00b0C for 10 min. The supernatant was combined with the previously collected supernatant and adjusted to a final volume of 50 mL with 0.4 M perchloric acid. Then, the extract was filtered through Whatman paper No. 1 .Quantification of the BA content of , et al. . Five grEnterococcus spp. based on Marcobal, et al. [g at 4 \u00b0C for 10 min. The extract was filtered through Whatman paper No. 1.The bacterial production of BAs was measured using the procedures described by Eerola, et al. , modifie, et al. . A loopf\u03b2-phenylethylamine hydrochloride, putrescine dihydrochloride, cadaverine dihydrochloride, histamine dihydrochloride, tyramine hydrochloride, spermidine trihydrochloride, and spermine tetrahydrochloride . Internal standard solution with the same concentrations was prepared using 1,7-diaminoheptane (Sigma-Aldrich).Standard solutions with concentrations of 0, 10, 50, 100, and 1000 ppm were prepared for tryptamine, g for 5 min. After filtration using 0.2 \u03bcm pore-size filters (Millipore), the filtered supernatant was kept at 4 \u00b0C until further analysis using HPLC.Derivatization of BAs was conducted according to the method described by Eerola, et al. . One mil18 4 \u03bcm column  held at 40 \u00b0C was utilized. The mobile phases were 0.1 M ammonium acetate dissolved in deionized water  and acetonitrile  adjusted to a flow rate of 1 mL/min with a linear gradient starting from 50% of solvent B reaching 90% by 19 min. A 10 \u03bcL sample was injected and monitored at 254 nm. The limits of detection were approximately 0.1 \u03bcg/mL for all BAs in standard solutions and bacterial cultures, and about 0.1 mg/kg for all BAs in food matrices [Chromatographic separation of BAs was conducted according to the method previously developed by Eerola, et al.  and modimatrices .tdc) involved RNA extraction from bacterial cultures (for in vitro experiments) and Cheonggukjang samples  with a Ribo-Ex Total RNA isolation solution . The extraction was conducted according to the manufacturer\u2019s instructions with minor modifications as follows. To prepare bacterial culture for in vitro gene expression analysis, a loopful (10 \u00b5L) of glycerol stock of each enterococcal strain was inoculated in 5 mL of MRS broth supplemented with 0.5% L-histidine monohydrochloride monohydrate, L-tyrosine disodium salt hydrate, L-ornithine monohydrochloride, L-lysine monohydrochloride (pH 5.8), and 0.0005% pyridoxal-HCl  and incubated at 37 \u00b0C for 48 h. Approximately 100 \u03bcL of the broth culture was then transferred to another tube containing 5 mL of the same medium and incubated under the same conditions. As for Cheonggukjang samples, 10 g of Cheonggukjang were gently mixed with 40 mL of phosphate buffer in a sterile bag, and the liquid part was collected. Subsequently, 3 mL of the bacterial culture or all liquid part of the mixture were immediately transferred into a 50 mL conical tube and centrifuged at 10,000\u00d7 g at 4 \u00b0C for 5 min. After removing the supernatant, the pellet was suspended with 7 mL of phosphate buffer and centrifuged under the same conditions. Then, the pellet was homogenized with 800 \u03bcL of Ribo-Ex reagent in a bacterial lysing tube  using a Precellys 24 homogenizer  with two cycles for 30 s at 6800 rpm, pausing for 90 s between cycles. Approximately 200 \u03bcL of chloroform were added to the lysate, vortexed, and centrifuged at 10,000\u00d7 g for 1 min. Approximately 400 \u03bcL of the supernatant were mixed with 600 \u03bcL of chilled absolute ethanol. The mixture was reacted at \u221270 \u00b0C for 15 min and purified with a Nucleospin RNA kit  according to the manufacturer\u2019s instructions. The quality of the extracted RNA was evaluated using a NanoDrop 1000 spectrophotometer .Expression analysis of tyrosine decarboxylase gene  containing reverse transcriptase, RNase inhibitor, oligo (dT) primers, random primers, and deoxynucleoside triphosphates (dNTPs) was used to synthesize cDNA from 1 \u03bcL of extracted RNA according to the manufacturer\u2019s instructions. Reverse transcription was conducted under the following conditions: 37 \u00b0C for 15 min, 50 \u00b0C for 5 min, and 98 \u00b0C for 5 min. After the reaction, the resulting cDNA was stored at \u221270 \u00b0C until quantitative PCR analysis.q-tdc F (5\u2032-AGACCAAGTAATTCCAGTGCC-3\u2032) and q-tdc R (5\u2032-CACCGACTACACCTAAGATTGG-3\u2032) primers were used for the quantitation of tdc gene expression by E. faecium. The primers for reference genes including q-gap F (5\u2032-ATACGACACAACTCAAGGACG-3\u2032) and q-gap R (5\u2032-GATATCTACGCCTAGTTCGCC-3\u2032) [tufA-RT F (5\u2032-TACACGCCACTACGCTCAC-3\u2032) and tufA-RT R (5\u2032-AGCTCCGTCCATTTGAGCAG-3\u2032) [tdc gene expression. The efficiency of each set of primers for reverse transcription quantitative polymerase chain reaction (RT-qPCR) was determined by the following equation: E = 10 S) values of serial dilutions of cDNA obtained from reverse-transcription of RNA from E. faecium KCCM 12118.As designed by Kang, et al. , q-tdc FCGCC-3\u2032) , along wGCAG-3\u2032)  were usetdc gene expression in bacterial cultures and Cheonggukjang samples, 5 \u03bcL of a 10-fold diluted cDNA were added to 15 \u03bcL of a master mix containing 10 \u03bcL of Power SYBR Green PCR Master Mix , 3 \u03bcL of RNase free water, and 1 \u03bcL of each primer . Subsequently, thermal cycling was conducted using an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems) with the thermal cycling conditions programmed as follows: initial denaturation at 95 \u00b0C for 10 min; 40 cycles at 95 \u00b0C for 15 s (denaturation step), and 60 \u00b0C for 60 s . Annealing and elongation conditions for primer tufA-RT were set at 55 \u00b0C for 60 s. Melting curve analysis was conducted using the RT-PCR system to confirm the specificity and to analyze the amplified products. Ct values were detected when the emissions from fluorescence exceeded the fixed threshold automatically determined by thermocycler software. Relative expression of tdc genes was further calculated by the 2\u2212(\u0394\u0394ct) method, normalized to the expression levels detected in E. faecium KCCM 12118  or Cheonggukjang groups fermented at 37 \u00b0C , and expressed as n-fold differences to compare gene expression levels in different bacterial cultures and Cheonggukjang samples.For the RT-qPCR analysis of p) value of <0.05 were considered statistically significant.Data were presented as means and standard deviations of duplicates or triplicates. All measurements on retail products were performed in triplicates, while the other experiments were conducted in duplicate. The significance of differences was determined by one-way analysis of variance (ANOVA) with Fisher\u2019s pairwise comparison module of the Minitab statistical software, version 17 , and differences with probability  in Cheonggukjang . The results were similar to the study conducted by Lee, et al. [Cheonggukjang to be 7.0 \u00b1 0.8. Jeon, et al. [Cheonggukjang to be pH 6.07 \u00b1 0.72 (range of pH 4.62\u20138.14) and pH 7.21 \u00b1 0.59 (range of pH 5.89\u20137.95), respectively. Such differences in the pH of the Cheonggukjang products may be owing to different fermentation conditions [Cheonggukjang products ranged from 1.95 to 9.36% with an average salinity of 5.16 \u00b1 2.78%. In comparison, Ko, et al. [Cheonggukjang to be 2.12 \u00b1 1.66% (0.12\u201311.51%), 1.56 \u00b1 1.19% (0.10\u20135.33%), and 3.51 \u00b1 2.45 (1.64\u20138.39%), respectively. Though the salinity of the Cheonggukjang products was found to vary substantially, Ko, et al. [Cheonggukjang salinities may be traced to the production process, as some methods utilize the addition of different amounts of salt to preserve the fermented soybean product. The water activity of retail Cheonggukjang products ranged from 0.919 to 0.973 with an average of 0.951 \u00b1 0.019. In a previous study by Kim, et al. [Cheonggukjang products analyzed in the current study were mostly similar to the values reported in previous studies. Although the results of the current study did not show any correlation between physicochemical properties and BA content  based on linear regression analyses (data not shown), it is noteworthy that the ranges of the physicochemical parameters were within the specific conditions for the growth of E. faecium, which are as follows: pH, from 4 to 10 [Physicochemical and microbial properties as well as BA content in retail ggukjang . Table 1, et al. , which r, et al.  and Yoo,, et al.  also repnditions  and/or fnditions . The sal, et al. , Jeon, e, et al. , and Kan, et al.  reported, et al.  suggeste, et al. , the ave 4 to 10 ; salinit 4 to 10 ; water a 0.12\u201311.%, 1.56 \u00b1Cheonggukjang products. The number of total mesophilic viable bacteria ranged from 8.54 to 9.81 Log CFU/g, with an average of 9.27 \u00b1 0.45 Log CFU/g. Comparatively, Ko, et al. [Cheonggukjang products to be 7.50 \u00b1 1.01 Log CFU/g (5.30\u20139.98 Log CFU/g) and 9.65 \u00b1 0.77 Log CFU/g (8.23\u201311.66 Log CFU/g), respectively. The wide range of total mesophilic viable bacteria may result from an insufficient standardization of Cheonggukjang manufacturing processes such as different fermentation materials and conditions [Enterococcus spp. were detected at concentrations of 6.64\u20137.99 Log CFU/g, with an average of 7.17 \u00b1 0.49 Log CFU/g. The number of lactic acid bacteria was found to be approximately 6.66\u20138.12 Log CFU/g, with an average of 7.09 \u00b1 0.58 Log CFU/g . Evaluation of \u03b2-phenylethylamine content in two Cheonggukjang products revealed that concentrations exceeded the recommended limit of 30 mg/kg, with one product exceeding the limit by a factor of approximately 3. Though the histamine content of all Cheonggukjang products was found to be below the recommended limit of 100 mg/kg, the tyramine content of three products exceeded the 100 mg/kg limit by factors of 2, 9, and 14, respectively. Other studies have reported similarly high concentrations of tyramine in Cheonggukjang. Ko, et al. [Cheonggukjang products, exceeding the recommended limit by factors of 19 and 9, respectively. Furthermore, Cho, et al. [Cheonggukjang products contained high concentrations of tyramine, which exceeded the recommended limit by up to factors of 5, 5, and 3, respectively. Altogether, as \u03b2-phenylethylamine and tyramine content of several Cheonggukjang products exceeded the recommended limits, overconsumption of such fermented soybean products may occasionally result in adverse effects on the body. Moreover, Cheonggukjang was found to contain other BAs enhancing the toxicity of \u03b2-phenylethylamine and tyramine. Therefore, further research remains necessary for precautionary measures and remedial methods to reduce the BA content of Cheonggukjang to ensure the safety of the fermented soybean food.lalanine . The higectively . A previ, et al. , Han, et, et al. , and JeoBacillus, Clostridium, Enterobacteriaceae, enterococci, Lactobacillus, and Pseudomonas, are capable of producing the decarboxylases responsible for the conversion of amino acids into BAs [Enterococcus spp. have been suggested to be responsible for tyramine accumulation in Chinese and Japanese fermented soybean products [Cheonggukjang products using an MRS broth-based assay medium. As shown in \u03b2-phenylethylamine, putrescine, cadaverine, spermidine, and spermine was observed at concentrations lower than 10 \u00b5g/mL. Histamine production by 168 of the 169 strains was detected at quantities lower than 2 \u00b5g/mL; however, only one strain was capable of producing histamine at 96.06 \u00b5g/mL. Though tyramine production ranged from ND (not detected) to 315.29 \u00b5g/mL, 157 strains (about 93%) produced over 100 \u00b5g/mL. Through 16S rRNA sequencing, the seven strains  that produced the highest levels of tyramine  products may partially result from tyramine production by E. faecium. Jeon, et al. [Enterococcus spp. exhibited strong production of tyramine ranging from 0.41 \u03bcg/mL to 351.59 \u03bcg/mL in assay media. The author also found tyramine-producing Bacillus spp. (up to 123.08 \u03bcg/mL) and suggested that the species is one of the major tyramine producers in Cheonggukjang along with Enterococcus species based on the in situ fermentation experiment. Consequently, the present results suggest that Enterococcus spp. (particularly E. faecium) may be largely responsible for high tyramine concentrations in Cheonggukjang.Microbial decarboxylation of free amino acids is one of the main causing factors in the production of BAs, and various microorganisms, including into BAs ,61,62. Cproducts ,53, the , et al.  suggeste, et al.  demonstr, et al. , high leq-tdc (for the quantitation of tdc gene expression) along with q-gap and tufA-RT  was calculated to be 100.71%, 94.84%, and 95.03%, respectively. An amplification efficiency between 90 and 110% indicates that the results of gene expression obtained using RT-qPCR are reproducible [In the current study, the efficiency of primer sets oducible .tdc gene expression by the seven E. faecium strains  with the highest tyramine production in vitro as described in the previous section . Among the strains, E. faecium strain CJE 216 showed the highest expression level of tdc gene . Each group was further divided into three groups based on fermentation temperatures of 25 \u00b0C, 37 \u00b0C, and 45 \u00b0C . As shown in Cheonggukjang were measured at 24-hour intervals for 3 days of fermentation. The pH of all Cheonggukjang groups was lowest on day 2, with progressively lower pH depending on the fermentation temperature, independent of which inoculum was used. The pH on day 2 of Cheonggukjang fermentation at 25 \u00b0C, 37 \u00b0C, and 45 \u00b0C ranged from pH 6.34 to 6.36, pH 5.90 to 6.09, and pH 5.49 to 5.88, respectively (Cheonggukjang groups fermented at 45 \u00b0C (high-temperature group) resulted in a lower pH than other groups fermented at 37 \u00b0C and 25 \u00b0C , regardless of inoculum, the BA content was expected to be detected at the highest concentration among all Cheonggukjang groups. As for water activity, all Cheonggukjang groups remained within 0.95\u20130.97 during fermentation  increased by 1.63 Log CFU/g, 3.52 Log CFU/g, and 4.06 Log CFU/g after 3 days of fermentation at 25 \u00b0C, 37 \u00b0C, and 45 \u00b0C, respectively (Cheonggukjang co-inoculated with E. faecium CJE 216 at 4 Log CFU/g (and B. subtilis KCTC 3135), enterococcal count increased at all fermentation temperatures of 25 \u00b0C, 37 \u00b0C, and 45 \u00b0C by 3.48 Log CFU/g, 4.78 Log CFU/g, and 4.80 Log CFU/g, respectively, by day 3. Enterococcus spp. were not detected in the control group for the duration of the fermentation period. The results displayed progressively higher enterococcal counts that increased alongside rising fermentation temperatures with the highest enterococcal counts in Cheonggukjang fermented at 45 \u00b0C (high-temperature group). The findings were comparable to a previous study by Morandi, et al. [E. faecium growth as the reported generation time at 25 \u00b0C was nearly two times longer than at 37 \u00b0C. E. faecium has been reported to display active growth within the temperature range of 37\u201353 \u00b0C, with an optimal growth temperature of 42.7 \u00b0C [E. faecium growth, thereby increasing the potential for high tyramine production during Cheonggukjang fermentation.The enterococcal count in ectively . In Cheo, et al. , which s 42.7 \u00b0C ,70. The Cheonggukjang co-inoculated with either E. faecium KCCM 12118 or E. faecium CJE 216, along with B. subtilis KCTC 3135, was measured during fermentation, as seen in E. faecium was detected at concentrations that did not exceed 10 mg/kg in all fermentation conditions  during the fermentation period, and consequently exceeded the recommended limit for consumption. Jeon, et al. [Enterococcus spp. during soybean fermentation at 37 \u00b0C. The report demonstrated that tyramine concentrations continued to increase as fermentation progressed. Given the results, safety precautions regarding the limitation of fermentation duration and temperature appear to be necessary as extended periods of fermentation as well as high fermentation temperatures may increase tyramine content in Cheonggukjang beyond the recommended safe limit for consumption. Besides, the results showing a lower tyramine content in Cheonggukjang during fermentation at lower temperatures coincide with the results in the previous section that displayed a reduction in enterococcal count alongside a decrease in fermentation temperature. Taken together, the present study indicates that lower fermentation temperatures inhibit enterococcal growth, thereby limiting acid production and maintaining low levels of tyramine in Cheonggukjang. Therefore, utilizing lower temperatures for Cheonggukjang fermentation may reduce the risks associated with Enterococcus growth and tyramine accumulation.The tyramine content of nditions a. In conectively b. As forentation c. All Chnd 45 \u00b0C b,c. The , et al. , E. faec, et al. . In the , et al.  also reptdc gene expression by tyramine-producing E. faecium strains were detected and quantified during fermentation of Cheonggukjang at 25 \u00b0C, 37 \u00b0C, and 45 \u00b0C. As Cheonggukjang is mostly fermented at 37 \u00b0C, the tdc gene expression detected in Cheonggukjang groups fermented at 45 \u00b0C was normalized to that detected in the corresponding groups fermented at 37 \u00b0C according to the E. faecium strains used as inoculants. In Cheonggukjang fermented at 25 \u00b0C, tyramine content continuously remained at concentrations lower than 10 mg/kg, and tdc gene expression by E. faecium KCCM 12118 and E. faecium CJE 216 was not detected in all Cheonggukjang groups. In contrast, the highest tdc gene expression by E. faecium KCCM 12118 was detected in Cheonggukjang fermented at 45 \u00b0C and was upregulated in the range of 1.90- to 7.15-fold throughout Cheonggukjang fermentation, compared with that in Cheonggukjang fermented at 37 \u00b0C . Nonetheless, the variation in detected tdc gene expression levels during fermentation did not necessarily reflect the tyramine content observed for Cheonggukjang. After one day of fermentation, the Cheonggukjang group with E. faecium CJE 216 fermented at 37 \u00b0C contained a lower tyramine content than at 45 \u00b0C; however, tdc gene expression was slightly higher at 37 \u00b0C as described right above. The results showed that there may be differences between gene expression level and enzyme activity (and products thereof). Glanemann, et al. [E. faecium strains in an assay medium appeared to be insignificantly related to tdc gene expression level (data not shown). Therefore, utilizing HPLC analysis appears to be essential for the quantification of BA content and/or bacterial BA production in food samples [Cheonggukjang at high temperatures results in increased tdc gene expression and tyramine production. Therefore, low-temperature fermentation appears to be necessary to minimize both tdc gene expression and tyramine production by Enterococcus spp. and thereby ensure the safety of fermented soybean products.The changes in at 37 \u00b0C a\u2013c, left, et al.  reported, et al. , while t samples ,73,74. W samples ,31. In tCheonggukjang, diagnosed the microbial causative agent  responsible for high tyramine levels, and evaluated the impact of fermentation temperature on enterococcal growth (as well as acid production and tdc gene expression) and tyramine production. Of the retail Cheonggukjang examined, half of the products contained tyramine content that exceeded the recommended limit for safe consumption by up to a factor of approximately 14. E. faecium strains isolated from the retail Cheonggukjang products were highly capable of producing tyramine in assay media, which indicated that the species is principally, or at least partly, responsible for tyramine accumulation in the food.The current study assessed the safety risk of tyramine in Cheonggukjang groups co-inoculated with B. subtilis (used as an inoculant to ferment soybeans) and E. faecium (either isolated in this study or designated previously as the type strain) strains was highest at 45 \u00b0C, followed by 37 \u00b0C and 25 \u00b0C. On the other hand, the control group inoculated with only B. subtilis strain (without any E. faecium inoculants) had the lowest tyramine content at all fermentation temperatures, which supported the notion that E. faecium may be a key producer of tyramine in Cheonggukjang. Another implication of the results was that a lower fermentation temperature leads to a lower tyramine content below the recommended limit in Cheonggukjang, even though the tyramine content continually increases during fermentation. Therefore, low temperatures and a short fermentation duration may reduce the accumulation of tyramine caused by E. faecium growth in Cheonggukjang, thereby reducing the safety risks associated with consuming food with high BA concentrations.During in situ fermentation at different temperatures, the tyramine content of"}
+{"text": "Lymphoblastoid cell lines (LCLs) are generated by transforming primary B cells with Epstein\u2013Barr virus (EBV) and are used extensively as model systems in viral oncology, immunology, and human genetics research. In this study, we characterized single-cell transcriptomic profiles of five LCLs and present a simple discrete-time simulation to explore the influence of stochasticity on LCL clonal evolution. Single-cell RNA sequencing (scRNA-seq) revealed substantial phenotypic heterogeneity within and across LCLs with respect to immunoglobulin isotype; virus-modulated host pathways involved in survival, activation, and differentiation; viral replication state; and oxidative stress. This heterogeneity is likely attributable to intrinsic variance in primary B cells and host\u2013pathogen dynamics. Stochastic simulations demonstrate that initial primary cell heterogeneity, random sampling, time in culture, and even mild differences in phenotype-specific fitness can contribute substantially to dynamic diversity in populations of nominally clonal cells. Lymphoblastoid cell lines (LCLs) are immortalized cells prepared by in vitro transformation of resting primary B cells from peripheral blood with Epstein\u2013Barr virus (EBV) . LCLs arEBV is a double-stranded oncogenic gammaherpesvirus infecting over 90% of humans . In vivoIn vitro, the process of LCL production also necessarily involves multiple transitions in viral transcriptional programs. In the immediate-early stage of infection (the pre-latent phase), expression from the W promoter yields EBNA-LP, EBNA2, and several noncoding RNAs . A brief burst of lytic gene transcription (without lytic replication) is also observed during pre-latency . EBNA-LPAlthough studied extensively, complete characterization of the viral and host determinants of growth arrest versus immortalization of early-infected cells remains elusive . As one In this study, we characterize the transcriptomic profiles of five different LCLs with single-cell resolution to assess inter- and intra-sample heterogeneity. Four of the sampled LCLs  were transformed with the prototypical B95-8 strain of EBV derived from an infectious mononucleosis patient , while aThree LCLs were prepared in-house by infection of PBMCs from two donors (sample numbers 461 and 777) with one of two different EBV strains (B95-8 or M81). Each of these three samples  was prepared and processed using standard single-cell RNA sequencing workflows . Two additional, publicly available data\u00a0sets were obtained for commercially available samples of the GM12878 and GM18502 LCLs, which were generated as previously reported by Osorio and colleagues . These fCount matrices for the five samples exhibited similar feature, total RNA count, and mitochondrial gene distributions  and 2 an+ and class-switched IgA+ and IgG+ subpopulations, whereas two samples (LCL 461 B95-8 and GM18502) almost exclusively expressed IgG , where the gene\u2019s expression was constrained to (and varied inversely with expression levels of) IgM+ cells (The five LCL populations exhibit distinct immunoglobulin (Ig) profiles with respect to both gene expression levels and isotype frequencies . Three oIGHG1-4; . Additioal blood . The immal blood . SignifiM+ cells .IGHG1, although low levels of IGHM transcripts are observed in up to half of the population. The proportion of IgM+, IgA+, and IgG+ subpopulations in LCL 777 B95-8 were 69%, 7%,\u00a0and 24%; in LCL 777 M81 were 1%, 35%, and\u00a064%; and in GM12878 were 6%, 73%,\u00a0and 18%. Abundance of Ig light chain gene (kappa or lambda) and heavy chain isoform expression are generally correlated with variable heavy chain expression in each of the five samples , typically within the first four PCs. Consequently, differences in Ig isotype are effectively captured in dimensionally reduced data\u00a0sets generated from PCs using t-distributed stochastic neighbor embedding (tSNE) even at low clustering resolution. In samples with more homogenous isotype expression (LCL 461 B95-8 and GM18502), the relative Ig expression level is a significant factor in distinguishing clusters.NFKB2, NFKBIA,\u00a0and EBI3) central to B cell activation and survival ; however, these transcripts are present more broadly (>80% of cells) and at higher levels than NF\u03baB-related genes (20\u201330% of cells) in each sample  displays strong anticorrelation with Ig expression level. However, MCL1 and BCL2 mRNAs are more broadly expressed across cells within each LCL, while BCL2L2/BCL-W is only modestly expressed in LCLs   are markers of a transitional phenotype; and PRDM1 (BLIMP1) and XBP1 promote the ASC state. As cell cycle marker expression was regressed out, mitotic phase has negligible influence on the observed trends.Ig gradients are closely related to expression of differentiation and maturation markers  , which ad PRDM1) . The appd PRDM1) . The ortd PRDM1) . In thisWhereas distinctions in Ig isotype class expression tend toward discrete partitioning, intra-isotype expression of differentiation and maturation genes reflects a continuum of transcriptomic states and cellular functions. Thus, within a given isotype, elevated Ig heavy chain expression is negatively correlated with activation/anti-apoptotic gene expression and positively correlated with maturation/differentiation gene expression. These relationships are most readily evident in LCL samples consisting of a single class-switched population, such as GM18502 .RUNX3 and FCER2/CD23 correlated with NF\u03baB expression  exhibited modestly faster growth in culture than ICAM-1lo cells (ASC phenotype) between 1\u00a0and\u00a04 days post-sorting. Notably, metabolic activity was elevated in ICAM-1hi cells than ICAM-1lo cells across all three LCLs, as indicated by higher rates of glycolysis and oxygen consumption  aligned against the human reference genome containing the viral genome as an extra chromosome  were found to co-express MIER2 and NFATC1. Negligible expression of either gene was observed in GM18502  contain clusters that exhibit metabolic transcriptional profiles in stark contrast with typical expression in each population . Cells wMT-CO1 and 2, complex IV) and NADH-ubiquinone oxidoreductase subunits  as well as a lack of canonical markers of lymphoid , B cell-specific lineage , and in some cases, MHC class I and II antigen presentation , unfolded protein responses (PPIA and\u00a0HSP90AB1), metabolic shunt pathways , and cytoskeletal rearrangements (ACTB and\u00a0TUBB) is enriched consistently in this subset relative to the bulk population in each of the three LCLs  as a function of initial phenotype frequencies, population sampling (with replacement), and potential differences in phenotypic fitness captured as fixed, (un)equal isotype-specific proliferation probabilities. The model assumes a fixed cell death rate across all isotypes in any given division round. The number of simulated trials can be adjusted to capture individual stochastic realizations or probabilistic outcome distributions. Each parameter and assumption can be adjusted by the user for tailored applications.Three randomly selected realizations and averaged outcomes  of the model for a fixed sample size (n\u00a0=\u00a01000 cells) demonstrate the effects of intrinsic stochasticity on the evolution of phenotype proportions over many rounds of cell division (rounds\u00a0=\u00a0300), even when each phenotype confers equivalent fitness . In the The effect of sample size on inter-trial variance can be substantial, even when cell populations are sampled with replacement to maintain phenotype proportions in each round . Mean phIt is notable that minor differences in relative fitness (1\u20132%) can lead to dramatic changes in isotype distributions over time . The ratCluster simulation was implemented by random sampling from four arbitrary, isotype-specific 2D normal distributions based on empirical observations that Ig isotypes yield distinct clusters in dimensionally reduced single-cell RNA-seq data . Simulat+ cells present across the samples can be explained by the isotype\u2019s low frequency in the peripheral blood . The only notable difference between LCL 461 B95-8 and LCL 777 B95-8 is that the former sample was in culture substantially longer prior to single-cell library preparation. Given that the GM18502 line was derived more than a decade ago, these observations implicate the influence of culture period in significantly altering the isotype proportions present within LCLs, which is altogether consistent with known (and profound) challenges associated with cell culture . In thisMultiple isotypes within an LCL sample guarantee clonal diversity, but the presence of a single isotype does not necessarily ensure the inverse (intra-sample homogeneity). While not in the scope of the present study, B cell receptor (BCR) 5\u2019 single cell sequencing of LCL samples could provide insights into variable regions and whether subpopulations of a given isotype are the progeny of one or multiple founder cells (and whether this changes over time).n transcripts in a population at any given time is roughly constant  developed as early as the pre-latent phase of infection  represent a limited subset of transcription factors associated with B (and T) lymphocyte activation  ostensibly may result from factors including naturally low transcript abundance, reduced transcript capture efficiency, and/or reduced efficiency of reverse transcription to cDNA owing to RNA secondary structural motifs  on average are memory cells of various classes. If the rate of transformation leading to LCL outgrowth is 10%, then \u2248750 memory cells out of 1 million PBMCs define the initial isotype frequency of the eventual LCL. This sample size is small relative to the donor\u2019s total memory B cell compartment and may lead to founder cell effects. Consequently, B cell population undersampling may be a foregone conclusion in the context of LCL preparation.Additional studies that utilize time-resolved single-cell sampling from original B cells through early infection and long-term LCL outgrowth in culture will be essential to explore further the factors contributing to longitudinal stability and variation in transcriptional profiles of B cells immortalized by EBV infection. Moreover, while the transcriptomic profiles we report provide a valuable resource, additional molecular layers must be interrogated through parallel -omics techniques  across individual cells to understand deeply the mechanistic underpinnings of transcriptional heterogeneity.Single-cell RNA sequencing reveals that LCLs including widely used commercial lines exhibit substantial phenotypic diversity. During the early stages of LCL generation, EBV infection drives cell proliferation by mimicking the process of B cell activation. After successful LCL outgrowth, infected B cells occupy a range of phenotypic states along a continuum between activation and plasma cell differentiation and, in some cases, exhibit signs of lytic reactivation. The diversity observed within LCLs  can originate from intrinsic heterogeneity within primary cells, transcriptional programs of the viral transformant, and the realization of inherently stochastic processes (including certain gene expression programs) over time. The data reported herein enable extensive hypothesis generation and interrogation of aspects of B cell biology, EBV pathogenesis, and host\u2013virus interactions. Moreover, this work highlights the importance of considering the possible sources and experimental consequences of cell population heterogeneity when using cultured cell lines.+ B cells were extracted from each PBMC sample through magnetic separation . Purified B cells were cultured in RPMI 1640 media supplemented with 15% fetal calf serum , 2 mM L-glutamine, penicillin (100 units/mL), streptomycin , and cyclosporine A (0.5 \u03bcg/mL).Whole blood samples from two normal donors (777 and 461) were obtained from the Gulf Coast Regional Blood Center. PBMCs were isolated from each sample by Ficoll gradient . CD192 to produce the following cultures: 777_B95-8, 777_M81, and 461_B95-8. After virus incubation, cells were rinsed in 1\u00d7 PBS and resuspended in R15 media. LCL outgrowth was achieved from each of these three samples, resulting in LCL_777_B95-8, LCL_777_M81, and LCL_461_B95-8.B95-8 and M81 strains of EBV were generated from the B95-8 Z-HT and M81 cell lines, respectively, as described previously . SeparatLCL 777_B95-8, 777_M81, and 461_B95-8 were generated in our laboratory by infection of primary human B cells obtained from the Gulf Coast Regional Blood Center with EBV strains B95-8 and M81. These lines were confirmed to be mycoplasma negative using the Sigma Lookout PCR kit.All three in-house LCL samples were cultured in supplemented RPMI media as described above, substituting 10% FCS instead of 15% FCS. Prior to single-cell sample preparation, LCL_777_B95-8 and LCL_777_M81 were maintained in culture for approximately 1\u00a0month, whereas LCL_461_B95-8 was cultured for longer than 6 months. Immediately prior to single-cell sample preparation, LCLs were resuspended and disaggregated.LCL_777_B95-8, LCL_777_M81, and LCL_461_B95-8 were created as described above. LCLs GM_12878 and GM_18502 were obtained, prepared, sequenced, and aligned as described by Osorio and colleagues . BrieflySingle-cell RNA samples for LCL_777_B95-8, LCL_777_M81, and LCL_461_B95-8 were prepared using the General Sample Preparation demonstrated protocol from 10\u00d7 Genomics  adapted from the original published methods . BrieflySingle-cell libraries for sequencing were prepared from each sample using the methods described in the 10\u00d7 Genomics Single Cell 3\u2019 Reagent Kit Protocol . In brief, GEMs were prepared using the 10\u00d7 Chromium Controller, after which cDNA synthesis and feature barcoding were performed and sequencing libraries for each sample were constructed. Sequencing runs were performed on an Illumina HiSeq 3000/4000 . Samples for LCL_777_B95-8 and LCL_777_M81 were sequenced in a pooled run in a single HiSeq lane.Raw base call files (*bcl.gz) from sequencing runs were processed using CellRanger v.2.0.0 to generate fastq files (*fastq.gz) via CellRanger\u2019s \u2018mkfastq\u2019 command. CellRanger\u2019s \u2018count\u2019 command was then used to align reads from the three in-house LCL samples to the human reference genome (hg38) with the Type 1 EBV reference genome (NC_007605) concatenated as an extra chromosome . This process yielded gene-barcode matrices (UMI count matrices) for subsequent analysis.https://github.com/esorelle/ig-evo-sim; copy archived at https://archive.softwareheritage.org/swh:1:dir:8c47b2c0202aa8f255380c742a3cda3ff777abc7/).UMI count matrices for all five LCL samples were analyzed using the Seurat single-cell analysis package for R .Cell pellets were collected for each of the five LCLs, and total mRNA was extracted from each pellet using the Promega SV96 Total RNA Isolation Kit  and quantified using a NanoDrop 2000 spectrophotometer (Thermo). Total mRNA was then used to create cDNA pools for each sample using a High-Capacity cDNA Reverse Transcription Kit . Previously reported primer sequences flanking each heavy  and light chain (Ig kappa\u00a0and Ig lambda) gene of interest (LCLs were stained with CD54 (ICAM-1) antibody  according to the supplier\u2019s manual. Then, cells were sorted on a Beckman Coulter Astrios cell sorter by anti-CD54 fluorescence, with ICAM-1-high and ICAM-1-low being defined as the top 15% and bottom 15%, respectively. 24 hr after sorting of ICAM-1-high and ICAM-1-low LCLs, extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured using the Seahorse XF24 extracellular flux analyzer (Agilent Technologies) Cell Energy Phenotype Test. Suspension LCLs were attached to culture plates by using Cell-Tak (BD Bioscience). ECAR and OCR were measured in Seahorse XF Base Medium supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose (Sigma Aldrich). ECAR and OCR values were normalized to cell number. For stress measurements, ECAR and OCR were measured over time after injection of oligomycin and FCCP. Metabolic potential measures the ability of cells to meet energetic demands under conditions of stress and is the percentage increase of stressed over baseline ECAR or OCR.th cell division round). The simulation was implemented in Python, and the code used to generate the simulated data is provided as a supporting file. The code is also available at (add as public GitHub repo) and may be freely implemented and modified.The concept of a discrete-time Markov chain was adapted to simulate the evolution of phenotype frequencies, using immunoglobulin heavy chain isotype distributions within LCLs as an example. Briefly, the simulation takes as input a cell population of size n comprising B cells of different Ig heavy chain isotype classes at user-defined initial frequencies, fixed probabilities of proliferation in synchronous rounds of cell division, and a constant cell death rate assumption . Within the scope of computational feasibility, users can specify the number of rounds of cell division to simulate and the number of simulation trials to run. Additionally, users may choose to generate simulated cluster data modeled from distinct 2D normal distributions for each isotype for a specified number of trials at fixed intervals  are deposited in the NCBI Sequence Read Archive (SRA) and can be accessed along with processed data from the NCBI Gene Expression Omnibus . In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.[Editors\u2019 note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Erik K Flemington (Reviewer #2).Thank you for submitting your work entitled \"Single-cell characterization of transcriptomic heterogeneity in lymphoblastoid cell lines\" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.The reviewers clearly appreciated your work which, by including five LCL cell lines from different donors and two different virus strains, generates a valuable resource to understand the dynamics between latent and lytic infection in EBV-infected B cells and B cell differentiation versus activation. However, the results and conclusions were not really validated and the work remains quite descriptive. We came to the conclusion, especially taking into regard the comments of reviewer #3, that this work in its current state is not of profound general interest to the readers in the non-EBV field and therefore not suited for publication in Reviewer #1:The authors report single cell RNA sequencing of five LCL lines. Three of them are recently derived, two with the B95-8 and one with the M81 EBV, and two have been in culture for prolonged periods of time. Two LCLs are from the same donor, one B95-8 and one M81 transformed. Three are predominated by IgG switched memory, one by IgA switched memory and only one by IgM, originating from na\u00efve or unswitched memory B cells. Interestingly, B cell activation and NF-kappaB expression correlates inversely with both antibody isotype class switching and plasma cell differentiation. Lytic EBV gene expression can only be observed in a small subset, primarily in the LCLs from the same donor transformed with B95-8 and M81 viruses but is not higher in the M81 transformed LCL. Finally, the authors model how heterogenous LCL composition might evolve, but the GM12878 cell line demonstrates that this does not necessarily drive to clonality in all instances. The authors suggest that LCLs go through a founder bottleneck that renders possibly every LCL different and that this should be taken into account in using these cellular models.The study described the comprehensive analysis of five LCLs and generates a valuable resource to understand the dynamics in EBV infected B cells between latent and lytic infection and B cell differentiation versus activation. However, some more information on the correlation of LCL proliferation with gene expression, latent EBV gene expression and antibody isotype composition from uninfected to LCL to lytic reactivation should be provided.1) The gene expression analysis suggests inverse correlation of B cell activation and differentiation. Is this reflected by LCL proliferation in vitro? Do the LCLs with higher frequencies of differentiated LCLs proliferate slower than for example LCL777 B95-8.2) The authors report lytic EBV gene expression, but presumably latent transcripts were rarely sequenced due to their low transcript number per cell. Nevertheless, it would be interesting if LMP1 expression frequency is elevated in LCLs with higher frequencies of activated cells and diminished in IgA or IgG expressing cells. The authors should attempt to address these questions by alternative means like flow cytometry or immune fluorescence microscopy.3) Does lytic EBV reactivation occur in all antibody isotype carrying subpopulations similarly, or is it enriched in XBP1 positive IgG and IgA carrying B cells? Is there any preference between IgA and IgG expressing differentiated B cells?4) Do the LCLs reflect peripheral B cell composition of the donors at all? Does for example donor 777 have a higher percentage of IgA positive B cells in the peripheral blood than 461.5) GM12878 might argue that LCL composition could be stable over time in some LCLs and not necessarily drift towards monoclonality. Do the authors have any longitudinal information on BCR isotype composition in their investigated LCLs?Reviewer #2:This paper demonstrates fairly wide diversity of cell transcriptomes within EBV derived LCL populations. This intra-cell population diversity extends even to cell lines that have been in culture for many years. This likely speaks to the principles driving transcriptional activation which derives from chance intermolecular interactions that are albeit favored or disfavored based on changes in chromatin and chromatin domain structures. The relevance is that the certain level of randomness of these principles can lead to dynamic changes in entire transcription and differentiation programs even within a single cell population. While there is already evidence for these kinds of issues in cell populations, this work brings out these principles in the context of LCLs/EBV , it would be helpful if the authors could provide additional evidence that these are true lytic cells vs abortive lytic cells (the latter of which would itself would be an interesting finding). It would be helpful if the authors could plot distributions of the percentage of viral lytic transcript reads to cell transcript reads in these populations of cells . Since herpesviral lytic infection typically results in a substantial proportion of lytic transcripts , this would help determine whether most of these cells are truly lytic or abortive lytic .Reviewer #3:Summary: Lymphoblastoid Cell Lines (LCLs) are induced by infecting primary B cells with Epstein-Barr Virus (EBV) and constitute a widely used cell line model in molecular biology, oncology and immunology. Understanding the intra- and inter-heterogeneity of these cell lines using single-cell analysis is key to design research and interpret experimental results. The authors induced three cell lines using two EBV strains and they performed single cell RNA-seq using the 10x platform and conducted a very classical analysis using Seurat. They added two datasets from the literature . They describe that each cell line has a certain level of intra-heterogeneity with different Ig expression patterns, different maturation stages , as well as exhibit different viral lytic/latent stages  and mitochondrial gene expression .eLife. No functional analysis is provided to indeed demonstrate that intra- and inter-variability has implications in study design.General comments: This study is needed and interesting as a resource for the community of scientists using these cell lines but I see major problems in the design of the study and the analysis. Beyond doing scRNA-seq and displaying clustering analysis, the final aim and the novelty of the study are not clear to me. It is neither clear how one can use such analysis to guide his research. As presented, the data analysis is very preliminary  and the study needs substantial improvements before publication in a top-tier journal such as 1) Design of the experiments: the overall scope of the study is very limited with only five different donors and it is not clear what is the contribution of the different viral strains. The rational of the study design is not outlined. The authors have chosen to look at the transcriptome only; we would have expected to have more -omics for a resource paper such as ATAC-seq and methylome to document the underlying heterogeneity of the cells.2) The description of the variability of LCLs is not novel. Ozgyin et al., 2019 have already provided a genotype-independent functional genomic variability of the LCLs. This study is not quoted.3) The variability between the five LCLs is not clearly delineated. Each cell line is heterogenous  but the authors have studied the cell lines independently without attempting to merge the data. The inter-heterogeneity is very poorly documented. The data analysis to perform a merge would require substantial computational work that goes beyond the scope of a two-month revision. It would be very important to explain the contribution of the inter-individual genomic variability between donors.eLife, we expect that such a claim is documented with experiments. The authors claim that \"primary cell heterogeneity, random sampling, time in culture, and even mild differences in phenotype-specific fitness\" can contribute to such heterogeneity. These are very general claims that do not help and are not supported with experiments.4) The authors claim in the Abstract that \"This heterogeneity is likely attributable to intrinsic variance in primary B cells and host-pathogen dynamics.\" In a publication in a top journal such as eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and P\u00e4ivi Ojala as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Erik K Flemington (Reviewer #2).Thank you for submitting your article \"Single-cell characterization of transcriptomic heterogeneity in lymphoblastoid cell lines\" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:Lymphoblastoid Cell Lines (LCLs) are induced when primary B cells are infected with the human herpesvirus Epstein-Barr Virus (EBV) and are a widely used model in molecular biology, oncology, and immunology. The authors have used single cell transcriptomics to demonstrate substantial phenotypic heterogeneity within and across LCLs. Hence, this work is important for researchers working with LCLs for the design and interpretation of experiments.Revisions:Please include in your discussion the points raised by reviewers #1 and #3 about the limitations of this study:Reviewer #1: Due to lack of donor material prior to transformation by EBV the authors could not assess if the heterogeneity of their cell lines was donor dependent. Furthermore, they could not provide any information on the longitudinal stability of the reported heterogeneity.Reviewer #3: It remains to uncover the origin of this variability using other layers of -omics and longitudinal sampling of the cells along the transformation process. [Editors\u2019 note: The authors appealed the original decision. What follows is the authors\u2019 response to the first round of review.]The reviewers clearly appreciated your work which, by including five LCL cell lines from different donors and two different virus strains, generates a valuable resource to understand the dynamics between latent and lytic infection in EBV-infected B cells and B cell differentiation versus activation. However, the results and conclusions were not really validated and the work remains quite descriptive. We came to the conclusion, especially taking into regard the comments of reviewer #3, that this work in its current state is not of profound general interest to the readers in the non-EBV field and therefore not suited for publication in eLife.We thank the reviewers and editors for their consideration and recognition of our work. Based on the unanimous conclusion by the reviewers and editors that the work \u201cgenerates a valuable resource,\u201d we request that the submission be reconsidered as a Tools and Resources article rather than a Research Article.We have revised the manuscript with data from three additional experiments that directly address reviewer requests for validation and functional implications of our findings. Further, we have provided responses and references to address other questions raised during the review. These are described further in the point-by-point responses to each review comment. We believe that these revisions have strengthened our manuscript substantially and adequately resolve the reviewers\u2019 concerns.We strongly believe this work will be of interest to readers outside of the EBV field because of the widespread use of LCLs in genetic and immunological research . The diversity in viral transcription observed within the datasets will certainly be relevant to EBV researchers; the effects of viral transformation on host cell phenotypic heterogeneity for these widely used models are likewise relevant to a more general readership.Reviewer #1:[\u2026]1) The gene expression analysis suggests inverse correlation of B cell activation and differentiation. Is this reflected by LCL proliferation in vitro? Do the LCLs with higher frequencies of differentiated LCLs proliferate slower than for example LCL777 B95-8.We have provided new data to address this question. We sorted LCLs by ICAM (a marker for activated cells) expression and found that ICAM-high populations exhibit more rapid proliferation than ICAM-low populations in the days immediately after sorting . This highlights a functional implication of the observed transcriptomic heterogeneity with respect to cell population growth dynamics.2) The authors report lytic EBV gene expression, but presumably latent transcripts were rarely sequenced due to their low transcript number per cell. Nevertheless, it would be interesting if LMP1 expression frequency is elevated in LCLs with higher frequencies of activated cells and diminished in IgA or IgG expressing cells. The authors should attempt to address these questions by alternative means like flow cytometry or immune fluorescence microscopy.As recently reported by our lab , FACS sorting by ICAM-1 expression serves as a reliable proxy for viral LMP-1 expression due to tight correlation (via EBV induction of NF-\u03baB pathways)  and correlates directly to the extent of DNA replication in LCLs [see Figure 3D]. Additional RNA-FiSH data from the cited paper [see Figure 6] confirms that only a fraction of cells within LCL populations express LMP1. These findings are consistent with the newly provided cell proliferation and metabolic data sorted by ICAM status, which collectively indicate the functional role of elevated LMP1 in activated cell subpopulations.3) Does lytic EBV reactivation occur in all antibody isotype carrying subpopulations similarly, or is it enriched in XBP1 positive IgG and IgA carrying B cells? Is there any preference between IgA and IgG expressing differentiated B cells?The limited number of lytic cells preclude a definitive answer to this question; however, we note that cells expressing each of the three heavy chain isotypes are observed within the lytic cluster in LCL 777 B95-8 at isotype frequencies roughly corresponding to those of the whole cell population . This suggests similarity in lytic reactivation rates across isotypes .4) Do the LCLs reflect peripheral B cell composition of the donors at all? Does for example donor 777 have a higher percentage of IgA positive B cells in the peripheral blood than 461.https://journals.plos.org/plospathogens/article/comments?id=10.1371/journal.ppat.1002697], we would expect that this does reflect B cell composition of donor, however such composition can change over time.The diversity among LCLs almost certainly reflects different donor peripheral B cell compositions . However, we did not retain  primary cell samples prior to transformation, so it is not possible to answer this question dispositively for the reported lines. As noted by Heath and Rickinson The authors have also been able to identify unique transcriptome signatures for reactivating cells which is potentially interesting from the standpoint of informing us on the nature of apparently stochastic events that trigger this transition to the EBV lytic phase. Nevertheless, given the lack of detection of BZLF1 (and possibly other lytic transcripts?), it would be helpful if the authors could provide additional evidence that these are true lytic cells vs abortive lytic cells (the latter of which would itself would be an interesting finding). It would be helpful if the authors could plot distributions of the percentage of viral lytic transcript reads to cell transcript reads in these populations of cells . Since herpesviral lytic infection typically results in a substantial proportion of lytic transcripts , this would help determine whether most of these cells are truly lytic or abortive lytic .Thank you to reviewer #2 for making this important suggestion. We have re-analyzed the level of host and viral gene expression in the lytic cell clusters and have found that the percentage of lytic transcripts in cells varies across the lytic cluster . In the highest-expressing cells, the 20 most prevalent lytic genes account for 10-15% of all transcripts, which is consistent with truly lytic cells. Interestingly, about half of the cells in the cluster have less than 10% lytic transcript content. As the reviewer has noted, this could indicate abortive lytic replication; alternatively, these could be cells that had only recently initiated lytic replication at the time of sample preparation. We have added this analysis and corresponding discussion to the manuscript.Reviewer #3:Summary: Lymphoblastoid Cell Lines (LCLs) are induced by infecting primary B cells with Epstein-Barr Virus (EBV) and constitute a widely used cell line model in molecular biology, oncology and immunology. Understanding the intra- and inter-heterogeneity of these cell lines using single-cell analysis is key to design research and interpret experimental results. The authors induced three cell lines using two EBV strains and they performed single cell RNA-seq using the 10x platform and conducted a very classical analysis using Seurat. They added two datasets from the literature . They descibe that each cell line has a certain level of intra-heterogeneity with different Ig expression patterns, different maturation stages , as well as exhibit different viral lytic/latent stages  and mitochondrial gene expression .General comments: This study is needed and interesting as a resource for the community of scientists using these cell lines but I see major problems in the design of the study and the analysis. Beyond doing scRNA-seq and displaying clustering analysis, the final aim and the novelty of the study are not clear to me. It is neither clear how one can use such analysis to guide his research. As presented, the data analysis is very preliminary  and the study needs substantial improvements before publication in a top-tier journal such as eLife. No functional analysis is provided to indeed demonstrate that intra- and inter-variability has implications in study design.We thank reviewer #3 for acknowledging the utility of our reported work for a broad community of researchers beyond the EBV community. We believe that the newly added data and analysis signify substantial improvements to the manuscript with respect to validation and functional implications for LCLs.Respectfully, it is unclear why the reviewer cites 9 out of 38 total figures and figure supplements to broadly assert that the presented data is \u201cvery preliminary\u201d \u2013 we provided these specific supplements  for completeness and analytical transparency . This assertion does not substantively challenge the key data and findings presented in the main figures.Regarding aim and novelty, we note several key aspects of the work including: 1) the first (to our knowledge) evidence of multiclonality in LCLs at single-cell resolution, 2) single-cell characterization of inverse expression patterns for B cell activation and differentiation programs , 3) the first single-cell dataset revealing EBV lytic replication at appreciable levels, and 4) the first report of LCL subpopulations with distinct metabolic profiles.With respect to how such analysis might allow one to guide her own research, the datasets clearly identify numerous genes of interest and correlations that can inform (to name just a few areas):1) Subsequent targeted studies in B cell activation and metabolism; modeling of host-pathogen dynamics2) Guides to developing molecular reporters and other tools for live-cell studies3) Transcriptome-wide evaluation of the effects of chemical perturbations including potential therapeutics .This work clearly opens new avenues of research, including refinement of a model for B cell functional states and dynamics in the context of EBV infection and associated lymphomas.We provide further detailed addresses to reviewer #3\u2019s concerns below, which we believe have resulted in a substantially improved manuscript.1) Design of the experiments: the overall scope of the study is very limited with only five different donors and it is not clear what is the contribution of the different viral strains. The rational of the study design is not outlined. The authors have chosen to look at the transcriptome only; we would have expected to have more -omics for a resource paper such as ATAC-seq and methylome to document the underlying heterogeneity of the cells.a priori, defining such a standard would have proven challenging. Whether 100% of LCLs uniformly exhibit the B cell activation / differentiation continuum or what percentage of LCLs display polyclonality are questions best addressed by future studies.A previously uncharacterized inverse relationship between activation and differentiation transcriptional signatures at the single cell level is consistently shown in 5 LCLs from different donors prepared and analyzed in different labs. At the same time, notable and meaningful clonal differences are found to exist within and across the LCLs, demonstrating that nominally identical cell populations should not be assumed to be monolithic with respect to identity or fitness. We cite data linking some of these differences to variation in viral transcriptional states and demonstrate the effects on cell proliferation. In and of themselves, these are valuable findings. While inclusion of additional samples may offer further confirmation or refinement of the generalizability of either finding, it is not immediately clear to us that further samples are necessary in this case. The reviewer contends that five samples constitute a limited study but provides no rationale or statistical basis for what defines a study of sufficient size \u2013 this is necessarily dependent on the nature and magnitude of what is being investigated/tested. Because the findings of the study were not known The contributions from testing different viral strains (B95-8 and M81) in cells from the same donor are articulated throughout the manuscript.27 As one consequence, it is unclear whether or to what extent viral transformation may influence the resulting LCL cell populations. The possibility of significant phenotypic diversity within and across LCL samples warrants consideration, given the intrinsic variance of the human primary B cell repertoire28,29 and the multiplicity of viral transcription programs active in the journey to immortalization.\u201dWe believe the rationale and aim of the study is clearly defined in the manuscript Introduction: \u201cAlthough studied extensively, complete characterization of the viral and host determinants of growth arrest versus immortalization of early-infected cells remains elusive.Reviewer #3 is correct that additional -omics data would add layers of functional detail such as epigenetic profiling to the study. While we appreciate the reviewer\u2019s focus and expertise in multi-omics analyses and the importance of these techniques, this work was conceived and executed with a different scope and focus. We feel that the work in its present form provides a rich resource and compelling findings that will be sufficiently valuable to the readership.2) The description of the variability of LCLs is not novel. Ozgyin et al., 2019 have already provided a genotype-independent functional genomic variability of the LCLs. This study is not quoted.We thank reviewer #3 for providing this reference. We have cited and discussed it within our revised manuscript. The statement that \u201cvariability of LCLs is not novel\u201d is rather vague and reductive \u2013 the work by Ozgyin characterizes certain aspects of variability within LCLs, and our work characterizes aspects of variability that are distinct from that paper\u2019s scope. Below, we highlight several key differences of our manuscript relative to the work by Ozgyin and colleagues:1) Ozgyin et al. provide a thorough exploration of bulk transcriptomic and epigenetic heterogeneity in multiple LCLs cultured from a single donor. By contrast, we report a conserved continuum of transcriptomic heterogeneity across LCLs from multiple donors.2) Ozgyin et al. show that heritable epigenetic signatures persist across different samples from the same donor and that these differences may have pharmacological implications. Our work shows that transcriptional differences within LCLs are correlated to different functional states intrinsic to B cell biology and are influenced by heterogeneity in viral programs.3) Ozgyin et al. present FACS data showing light chain variability across samples from the same donor . Our work presents single-cell sequencing data showing heavy chain clonality within and across donors, as well as light chain data.4) Ozgyin et al. focus on host cell transcription as influenced by chromatin accessibility. Our work focuses on the effects of host-pathogen interactions by reporting both host cell and viral reads and their relative expression in individual cells within LCLs.5) Our work describes a stochastic modeling framework to explore the effects of differential phenotypic fitness and other experimental considerations on LCL evolution.6) Notably, both works highlight the broad utility of and interest in LCLs across research disciplines3) The variability between the five LCLs is not clearly delineated. Each cell line is heterogenous  but the authors have studied the cell lines independently without attempting to merge the data. The inter-heterogeneity is very poorly documented. The data analysis to perform a merge would require substantial computational work that goes beyond the scope of a two-month revision. It would be very important to explain the contribution of the inter-individual genomic variability between donors.The revised manuscript provides a characterization of the merged datasets . It shows that donor-specific differences are a dominant source of LCL heterogeneity. The fact that donor-specific genomic differences dominate relative to conserved intra-sample trends is unsurprising. This analysis reinforced discussion related to the stochastic model of cell phenotype evolution \u2013 that the contribution of inter-individual genomic variability is significant.4) The authors claim in the Abstract that \"This heterogeneity is likely attributable to intrinsic variance in primary B cells and host-pathogen dynamics.\" In a publication in a top journal such as eLife, we expect that such a claim is documented with experiments. The authors claim that \"primary cell heterogeneity, random sampling, time in culture, and even mild differences in phenotype-specific fitness\" can contribute to such heterogeneity. These are very general claims that do not help and are not supported with experiments.These are general and foundational principles. In the case of the effects of host-pathogen dynamics, please refer to the substantial body of research describing EBV LMP1-mediated upregulation of NF-\u03baB signaling pathways and the newly-provided data showing higher proliferation of ICAM-1 \u2013 high (an LMP1 proxy) cells within LCLs. In the case of the effects of random sampling, time in culture, and phenotype-specific fitness, we were referring to results of stochastic simulations. While not so dispositive as clean results from wet lab experiments, models and simulations can be valuable tools for research and guide subsequent studies.[Editors\u2019 note: what follows is the authors\u2019 response to the second round of review.]Revisions:Please include in your discussion the points raised by reviewers #1 and #3 about the limitations of this study:Reviewer #1: Due to lack of donor material prior to transformation by EBV the authors could not assess if the heterogeneity of their cell lines was donor dependent. Furthermore, they could not provide any information on the longitudinal stability of the reported heterogeneity.We have added the following sentences to the Discussion section to address these points:\u201cA notable limitation of this study is the lack of access to (GM12878 and GM18502) or retention of (LCL_461 and LCL_777) original donor primary B cells and longitudinal sampling, which would have provided direct insight into donor-dependent cellular heterogeneity.\u201d\u201cAdditional studies that utilize time-resolved single-cell sampling from original B cells through early infection and long-term LCL outgrowth in culture will be essential to explore further the longitudinal stability and variation in transcriptional profiles following EBV infection.\u201dReviewer #3: It remains to uncover the origin of this variability using other layers of -omics and longitudinal sampling of the cells along the transformation process.We have added the following sentences to the Discussion section to address these points:\u201cAdditional studies that utilize time-resolved single-cell sampling from original B cells through early infection and long-term LCL outgrowth in culture will be essential to explore further the longitudinal stability and variation in transcriptional profiles following EBV infection. Moreover, while the transcriptomic profiles we report provide a valuable resource, additional molecular layers must be interrogated through parallel -omics techniques  across individual cells to understand deeply the mechanistic underpinnings of transcriptional heterogeneity.\u201d"}
+{"text": "We present a novel hybrid localization algorithm for wireless sensor networks in the absence of knowledge regarding the transmit power and path-loss exponent. Transmit power and the path-loss exponent are critical parameters for target localization algorithms in wireless sensor networks, which help extract target position information from the received signal strength. In the absence of information on transmit power and path-loss exponent, it is critical to estimate them for reliable deployment of conventional target localization algorithms. In this paper, we propose a simultaneous estimation of transmit power and path-loss exponent based on Kalman filter. The unknown transmit power and path-loss exponent are estimated using a Kalman filter with the tentatively estimated target position based solely on angle information. Subsequently, the target position is refined using a hybrid method incorporating received signal strength measurements based on the estimated transmit power and path-loss exponent. Our proposed algorithm accurately estimates transmit power and path-loss exponent and yields almost the same target position accuracy as the simulation results confirm, as the hybrid target localization algorithms with known transmit power and path-loss exponent. Simulation results confirm the proposed algorithm achieves 99.7% accuracy of the target localization performance with known transmit power and path-loss exponent, even in the presence of severe received signal strength measurement noise. Wireless sensor networks (WSNs) are networks comprising spatially spread sensor (or anchor) nodes equipped with sensors for detecting physical environments ,2. RecenA majority of these localization algorithms, except for those based solely on AOA measurements, assume that the distance between an anchor and the target can be determined precisely using the measured RSS, which is tantamount to assuming perfect knowledge regarding the transmit power (TP) and path-loss exponent (PLE). However, in practice, prior knowledge of the TP and PLE is not always feasible. In the absence of exact information on TP or PLE, RSS measurements cannot be used reliably for target localization, as they can result in significant performance degradation of RSS-based algorithms. Hence, several studies have focused on RSS-based target localization algorithms for unknown TP or PLE ,27,28,29Simultaneous estimation of target position and unknown TP from RSS data was proposed in  using seOn the contrary, several iterative approaches for estimating target position and TP or PLE for RSS-based algorithms have been proposed with various optimization criteria ,28,29). ,29. 28,2Hybrid target localization algorithms for unknown TP or PLE have also been developed ,31,32,33These existing hybrid target localization algorithms, in the absence of information on TP or PLE, rely on exhaustive maximum likelihood estimator (MLE) approximation or iterative updates of the target position and TP or PLE; however, this approach requires significant computational resources. Furthermore, as RSS measurements generally suffer from severe noise, the overall performances of existing hybrid target localization algorithms with unknown TP or PLE are inferior to that of the cutting-edge AOA-only target localization algorithm. In practice, it is difficult to precisely determine both TP and PLE; however, all existing hybrid algorithms dealing with imperfect knowledge regarding TP or PLE assume that either TP or PLE is unknown. Therefore, the development of a hybrid target localization algorithm that does not depend on prior knowledge of both TP and PLE is essential.In this paper, we propose a novel hybrid target localization scheme for unknown TP and PLE. In the proposed approach, TP and PLE are simultaneously estimated and updated using a Kalman filter (KF) based on the tentatively estimated target position based on AOA-only measurements. The Kalman filter is famous for its robust estimation performance under severe dynamic noise environments ,35.Subsequently, RSS measurements are incorporated to obtain the refined target position using the error covariance weighted least squares (EC-WLS) algorithm . Unlike The remainder of the paper follows. In this section, the system model and problem formation for target localization based on RSS and AOA in wireless sensor networks are described. A general linear target estimation approaches with known TP and PLE are introduced and exiting related studies on target localization schemes for unknown TP or PLE have been formally described.i-th sensor (anchor) node for This section introduces the mathematical formulation of the hybrid target localization scheme utilizing both RSS and AOA measurements in WSNs. Let ectively .A1.There is only a single sensor node with unknown position (target).A2.All anchor nodes are equipped with a device that can measure the AOA  and RSS.A3.All anchor nodes are fixed at known positions and can transfer RSS/AOA measurements to a central node (processor).A4.All measurement errors are i.i.d. zero-mean Gaussian noise with unknown variances which are the same for all anchor nodes.A5.All nodes are in line-of-sight with respect to the target.A6.All nodes are located at a homogeneous environment so that the path-loss exponent (PLE) is the same for all nodes.These RSS and AOA information are sent to a central node (processor) where the target position is estimated. We assume the following assumptions for the target localization in WSNs based on RSS and AOA measurements:The received power is related to distance by a path-loss model; in the absence of noise, the RSS, deled as ,(1)Pi=Pobtained:P^i=P0\u221210In the presence of noise, the azimuth angle, When the TP and PLE are known, the MLE for the target position based on RSS/AOA measurements,  follows :(5)x^ML=(i)From the relation between Cartesian and spherical coordinates, a direct estimation of the target position From the maximum likelihood Function , the folThe WLS solution based on range-based weights, referred to as the target-range WLS (TR-WLS) solution, is presented in , while tAs a closed-form solution of this ML equation is not feasible, several sub-optimal linear estimators have been proposed. All linear estimates are derived from one of the following relations between the target position and the RSS/AOA measurements:The existing RSS/AOA localization algorithms estimate the target position when TP and PLE are known. However, as di in  and \u03bbi aand \u03b7 in  are not i-th node, When the target position is known, TP and PLE can be easily estimated from the received signal power using a linear equation. Given the target position, Using the relation between the distance ( (Pi) in , we obtaHowever, since the goal of the proposed algorithm is to estimate target position, it is not a valid assumption that the target position is known. Furthermore, even if an acceptable estimate of the target position is given, due to the severe RSS noise, the simple LS solution cannot be used for a hybrid target localization algorithm, as shall be shown later. Therefore, there have been several partial solutions for this challenging problem of estimating target position using RSS measurement under unknown TP and PLE, as summarized in the following.Several hybrid target localization algorithms dealing with imperfect knowledge on TP or PLE have been reported ,31,32,33N ones) andIn , a hybriTo obtain the optimal PLE  algorithm, is simple, practical, and achieves the best target position accuracy among the above-mentioned hybrid target localization schemes without TP or PLE. However, even TT-WLS exhibits inferior performance in comparison with the AOA-only target localization algorithm derived from the state-of-art EC-WLS algorithm in the presence of severe RSS measurement, as discussed in the simulation section. Therefore, it is important to develop a hybrid target estimation algorithm with unknown TP or PLE that exceeds the performance of AOA-only target localization algorithms.This section presents the proposed hybrid localization with unknown TP and PLE based on a KF. The proposed algorithm comprises three steps: To estimate the initial target estimation without TP or PLE, only the equations related to AOA measurements are considered from :(21)ciT in k-th iteration. The system matrix in the state-space model, H, is replaced with the time-varying model, In the above example, let us consider the target is linearly moving with a velocity Equation .Once the KF reaches the steady state, the TP and PLE estimates in lgorithm  is used given in  and(36)Algorithm 1 Proposed hybrid target localization algorithm under unknown TP and PLE.1. Estimate the target position from AOA measurements.Solve2. Estimate the TP and PLE using the KF.i. Initialize ii. Predict and update forPredict:Update:end3. Estimate the target position using EC-WLS with the estimated parameters Solve1BTCA\u22121c 2. Estima and Qk. fork=1,\u22ef1ATC\u00af\u22121b In summary, the proposed hybrid target localization algorithm, summarized in Algorithm 1, does not depend on the prior information on the unknown parameters, i.e., TP and PLE.K be the total number of iterations. The complexity of the proposed method for target localization is In this section, we compare the computational complexities of existing mentioned target localization algorithms with unknown TP and PLE. The proposed method has three parts for target localization; estimation of initial target, estimation of TP and PLE, and estimation of final target. The complexity for initial target estimation using only AOA measurements is M denotes the mesh size K, is set to 1000. The performance is measured by the root-mean-square error (RMSE), which is defined asi-th run.This section details the performance of our proposed algorithm in numerical simulations under experimental settings utilized in most of previous works. Target and anchor nodes are arbitrarily placed inside a box with an edge length We compare the RMSEs of the proposed algorithm with those of the AOA-only algorithm based on EC-WLS , SR-WLS N, increases; the measurement noises are modeled as uniform distributions over the nodes as N, increases; In practice, measurement noises typically feature inhomogeneous noise variance among the anchor nodes. Finally, we compare the performance of the proposed algorithm on a large scale. Since most hybrid localization algorithms estimate the target position at the central node (processor) using the gathered RSS/AOA data from all nodes in the sensor network via a communication link such as IEEE 802.11 (refer related references in ), the prHowever, the proposed algorithm relies on relatively reliable initial estimation of the target position. When the initial estimation is corrupted by severe angle noise due to possible multipath propagation (non-line-of-sight) or multiple inference, the proposed algorithm may not recover from the bad initialization of the TP and PLE. However, in such cases, all existing hybrid algorithms are subject to fail as well. Furthermore, possible TP/PLE acquisition delay due to the convergence time of Kalman filter in the presence of poor initialization can be problematic for a certain application requiring rapid target estimation.Nonetheless, the proposed algorithm enhances the accuracy of the estimated target. The proposed algorithm produces almost the same performance with the state-of-the-art algorithm with known TP and PLE , while cIn order to verify the robust performance of the proposed algorithm in realistic adversary situations, we have considered a multipath propagation scenario and a multiple interference environment.We also consider the multiple interference. The multiple interference are caused by additive noises from other communication networks and statistically modeled as inverse Gaussian distribution as studied in ,38, wherSince the conventional methods and the proposed algorithm are developed under the zero mean Gaussian noise assumption, non-Gaussian interference with nonzero mean would degrade the performance.Herein, we presented a novel hybrid localization algorithm under unknown TP and PLE. The initial target position was estimated via the AOA-only EC-WLS method, and the TP and PLE were estimated by using a KF. After the TP and PLE are estimated, the EC-WLS algorithm is used to incorporate the RSS measurements. The proposed algorithm outperforms the conventional hybrid target localization methods for unknown TP as well as the AOA-only EC-WLS method. Furthermore, we prove that the proposed method almost achieves a performance comparable to that of the EC-WLS algorithm with known TP and PLE. The proposed algorithm can be further extended for the case where each node has a different PLE value or multiple targets have different TP values, which is left for future research."}
+{"text": "The current study describes the implementation of an online Future Problem Solving (FPS) program in the field of Health education and set out to explore its contribution to students' eHealth Literacy identity, by using two levels of teacher guidance: minimal vs. frequent. FPS was employed in two groups of Health students. In the research group, frequent weekly guidance was provided to the students centered on the enhancement of eHealth Literacy skills, whereas in the control group minimal guidance was offered by the lecturer. Data for the analysis were gathered from 113 Israeli undergraduate students of a Management of Health Service Organizations program, of whom 62 comprised the research group. Data were gathered twice, pre- and post-program implementation from both groups. Findings showed significant differences between the tests only for the research group, with increased levels of eHealth Literacy skills detected between the tests. The perception of the FPS program as meaningful contributed to students' perceived eHealth Literacy skills only in the research group whereas non-significant results were shown for the control group. This study mainly shows that the enhancement of skills in online educational environments requires frequent and personalized guidance. Faculty must recognize the role of the instructor as a facilitator of learning and design successful scaffolding strategies to nurture students' lifelong learning skills during distance learning. Coping with the wide array of urgent needs raised by the COVID-19 pandemic outbreak requires staff in the healthcare system to demonstrate diverse adaptive capabilities. These include skills such as solving ill-structured problems and digital literacy. These lifelong learning skills are becoming an essential part of the array of tasks that characterize healthcare professionals, and designers of higher education settings must assume an important role in honing these skills by employing innovative instructional approaches  learning and instruction method aided by technology during an online course. FPS allows students to think creatively and imaginatively to address contemporary challenges that might get worsen in the future  Which teaching guidance style  would be more effective in promoting Health students' eHealth Literacy? To assess the effectiveness of teaching styles in this aspect, a quasi-experimental, pre/post-test design was used to examine students' perceived eHealth Literacy before and after the program implementation. It was expected that participants who received frequent assistance from the teacher (research group) would report attaining higher levels of eHealth Literacy skills, following the implementation of the FPS program compared to its onset, than the control group (H1).(Q2) How the perception of the FPS program as meaningful (in terms of advancing future thinking) would impact students' reported eHealth Literacy skills? It was expected that the research group students would tend to attribute the increase in eHealth Literacy skills to the program (H2). This trajectory was also expected in the control group, yet we anticipated a relatively lower impact of the program on the eHealth Literacy skills compared to the research group (H3).lack of education, 1 = elementary school, 2 = high school, 3 = BA degree, 4 = MA degree, 5 = doctoral degree. Non-significant between-group differences were found on all the measured variables. Researchers emphasized prior to obtaining consent that the questionnaires were both anonymous and voluntary. Finally, participants were assured that no identifying information about the courses would be processed. The research was approved by the college's Ethics Committee.Data for the analysis were gathered from 3rd-year 113 Israeli undergraduate students of a Health Management program , of whom, 62 comprised the research group. Participants were enrolled in two courses dealing with quality in health systems. Both groups (research and control) were taught by the same instructors. strongly disagree to 5= strongly agree). Cronbach's alphas ranged from 0.88 to 0.92.This eight-item scale . Cronbach's alphas ranged from 0.86 to 0.93.Based on the theoretical framework, this scale was constructed for the purpose of the current study. This six-item scale corresponds to the six steps of the FPS program  instructions and examples of how to navigate digital media, access information by locating and sharing materials; (2) how to analyze messages in a variety of forms, obtain information critically, identify the source of information, and evaluate the quality and credibility of the content; (3) ways to create content in a variety of forms, which enable making use of language, images, sound, and new digital tools and technologies, and effectively collaborate to construct new knowledge or digital artifacts using technology and media. Students in the control group received an asynchronous session regarding eHealth Literacy and were encouraged to seek the instructor's feedback via email or zoom meetings, yet synchronous lessons were not scheduled for this purpose due to a lack of institutional resources.This step included devising criteria to evaluate the solutions the students raised. After small group discussions, a Google Doc was created in which each group could upload five ideal criteria. Next, the lecturer with the help of student suggestions selected the five most appropriate criteria to assess the solutions. The criteria were: the solution is creative, easy to adapt, applicable, inexpensive in terms of manpower and funds, safe, has a long-term effect, does not require complicated technology, and contributes to the entire society.not at all to 6 = absolutely.Each group evaluated their four solutions according to the criteria using a grid that indicated the quality of each criterion on a Likert-type score ranging from 1 = Finally, each group devised a plan to pitch their desired solution. In this plan, they specified their suggested steps for implementation and described how the solution might work, who could help them implement it, who might oppose it, and how such challenges could be mitigated. The students had to present their action plan in a plenary meeting using online platforms such as \u201cThinglink\u201d. In terms of eHealth Literacy, they constructed new knowledge by creating media expressions and communicating them with others. Exemplary solutions were: developing biometric security measures for preserving medical confidentiality, creating safe areas in open spaces for conducting online discussions, preparing a support system for the elderly population based on one-on-one solutions within distancing regulations for instructing them in medical literacy and increasing their digital abilities, developing equipment for long-distance diagnosis and treatment, etc.The different treatments given to the research and the control groups stemmed from the limited institutional resources available during the pandemic outbreak. This study was designed as pilot research with the goal of evaluating the impact of minimal vs. frequent guidance in an online FPS program on students' eHealth Literacy skills. The objective was to implement the program more widely depending on this study's results.Analysis of variance was applied to allow the characterization of differences between the pre- and post- interventions within the research and control groups. Data were also analyzed using Partial Least Squares Structural Equation Modeling . It was postulated that participants who received frequent assistance from the teacher (research group) would report attaining higher levels of eHealth Literacy skills, following implementation of FPS compared to the onset of the program, than the control group.F = 12.664, p < 0.01, \u03b72 = 0.110] for the research group, with increased levels of eHealth Literacy skills detected between the tests . Non-significant results were shown between the tests for the control group . H1 was corroborated.A univariate analysis was applied to allow the characterization of differences between the pre- and post- test in each group (research/control) on eHealth Literacy skills. As shown in The second research question dealt with students' perception of the FPS program as a precursor of eHealth Literacy skills. It was postulated that the research group students would tend to attribute the increase in eHealth Literacy skills to the efficiency of the program (H2).To assess H2, Model 1  was consp < 0.01), namely, the Future Thinking independent variable was positively and moderately connected to the eHealth Literacy dependent variable among the research group. This confirms H2. The model evaluation included the examination of the coefficient of determination (R2) value. R2 (0.226) can be considered moderate  of the path model. Values larger than 0 suggest that the model has predictive relevance for the endogenous construct , namely, the connection between the Future Thinking independent variable and the eHealth Literacy dependent variable was found non-significant for the control group. Hence H3 was confirmed. R2 (0.079) can be considered very low (Hair et al., Q2-value indicated for our model was close to zero 0.001.According to H3, a positive link was expected to be obtained in the analysis of the control group's data, with a relatively lower impact of the program on the skills compared to the research group. The same model as in In the current study, an FPS program was employed in two groups of Health students distinguished by the level of teacher guidance: minimal vs. frequent. It mainly sought to assess the program's impact on Health students' eHealth literacy skills. The first research question related to the potential effect of teaching guidance styles, minimal vs. frequent guidance, practiced during the FPS program, on students' perceived levels of eHealth Literacy skills. According to the findings, increased levels of eHealth Literacy skills were detected after the intervention in the research group compared to its beginning, whereas non-significant results were shown between the tests for the control group. In addition, students in the research group tended to attribute the increase in eHealth Literacy skills to the efficiency of the program and its ability to raise their level of awareness of social problems that might arise in the future and ways to solve them. This trajectory was found non-significant in the control group.It appears that the manner of guidance and the role of the lecturer were essential for developing eHealth Literacy skills among the students. The research group received extensive guidance through workshops and active learning to develop their abilities to locate and use material within the framework of eHealth Literacy and to practice and develop those abilities collaboratively. The online instruction that the control group received consisted of only general guidelines about the requirements of the assignment in a non-synchronized manner with no active guidance from the lecturer.The findings of this study emphasize the importance of the lecturer's mediation in the form of guidance while devoting time to the processes of developing eHealth Literacy abilities among students in the health professions. They also emphasize the changes that need to be implemented in the role of academia in medical education. These changes involve training graduates to adapt to the changing environment of the medical world and the demands of the health care system and working world of the twenty-first century. Updated instructional approaches are required to support the adaptation to the changes that the health care system is undergoing due to the fourth industrial revolution and its long-term impact upon numerous areas of life (Mullan et al., These changes necessitate an updated mission statement of the academia in line with the overall trend that marks the transformation of its role from an agent of knowledge to a leader of change and an agent of culture, whose responsibility is to develop abilities that are suitable to the twenty-first century and its demands. The role of academia and its institutions was previously perceived as providing of knowledge. Today, however, their role is perceived as to teach and develop new abilities among students and to prepare them for today's work market (Alt and Naamati-Schneider, The present work features limitations and directions for future research that warrant mention. The study assessed students' perceptions of their awareness of FPS and digital literacy skills using a self-reporting survey. Some studies find substantial biases in self-report measures and strong divergence between subjective and objective assessments thus data gained by such measures should be interpreted cautiously Bowman, . HoweverThis research provides a useful constructivist pedagogical tool of FPS accompanied by effective guidance and illustrates how this method might be utilized online to advance students' eHealth Literacy skills. Whereas, previous research assessed students' problem-solving skills and digital literacy abilities, suggesting that learners possess only basic competencies of digital literacy skills, and basic knowledge on how to use information and communication technologies (Ozdamar-Keskin et al., Based on our findings, Model 3  is suggeOther suggestions for improving online teaching effectiveness are to tailor constructivist learning methods and strategies to online teaching. Such methods enable interaction and collaboration with students to support their higher-order thinking abilities and other important twenty first-century learning outcomes such as digital skills. Methods such as FPS engage students with thought-provoking problems and spur them to collaboratively offer viable solutions in a safe environment. Teachers might benefit from having training sessions on how to leverage advanced online pedagogical methods to advance their students' lifelong learning skills.Datasets are available on request to the corresponding authors.The studies involving human participants were reviewed and approved by Kinneret Academic College. The patients/participants provided their written informed consent to participate in this study.DA: conceptualization, data curation, writing-original draft preparation, and writing-reviewing and editing. LN-S: data curation, methodology, writing-original draft preparation, and reviewing and editing. AM: data curation, writing-original draft preparation, and reviewing and editing. 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": "Triticum aestivum L., also known as common wheat, is affected by many biotic stresses. Root diseases are the most difficult to tackle due to the complexity of phenotypic evaluation and the lack of resistant sources compared to other biotic stress factors. Soil-borne pathogens such as the root-lesion nematodes caused by the Pratylenchus species and crown rot caused by various Fusarium species are major wheat root diseases, causing substantial yield losses globally. A set of 189 advanced spring bread wheat lines obtained from the International Maize and Wheat Improvement Center (CIMMYT) were genotyped with 4056 single nucleotide polymorphisms (SNP) markers and screened for root-lesion nematodes and crown rot resistance. Population structure revealed that the genotypes could be divided into five subpopulations. Genome-Wide Association Studies were carried out for both resistances to Pratylenchus and Fusarium species. Based on our results, 11 different SNPs on chromosomes 1A, 1B, 2A, 3A, 4A, 5B, and 5D were significantly associated with root-lesion nematode resistance. Seven markers demonstrated association with P. neglectus, while the remaining four were linked to P. thornei resistance. In the case of crown rot, eight different markers on chromosomes 1A, 2B, 3A, 4B, 5B, and 7D were associated with Fusarium crown rot resistance. Identification and screening of root diseases is a challenging task; therefore, the newly identified resistant sources/genotypes could be exploited by breeders to be incorporated in breeding programs. The use of the identified markers in marker-assisted selection could enhance the selection process and cultivar development with root-lesion nematode and crown rot resistance. Triticum aestivum L.) is one of the most important cereal crops for human consumption around the world. Wheat provides around 20% of human daily caloric intake. It is the main source of carbohydrates and is a significant source of proteins, inorganic ions, and vitamins [Bread wheat  are plant-parasitic nematodes that feed on wheat roots [Pratylenchus species are the most common RLN species in wheat and usually coexist in fields [Based on economic impact, parasitic nematodes and Fusarium are among the main soil-borne pathogens attacking plant roots. The root-lesion nematodes (RLN) at roots ,5. Theseat roots ,7. The tn fields . They can fields .Another economically important wheat root disease is Fusarium crown rot (CR), which is caused by various Fusarium species ,11,12. TIdentifying genetic resistance in wheat could provide a valuable alternative to the conventional control methods. Genetic approaches are potentially the most important strategies to effectively control CR and nematodes ,17,18,19P. neglectus and P. thornei) and CR (F. culmorum) diseases to identify novel sources of resistance to soil-borne diseases.One way to screen large numbers of genetic resources is to take advantage of molecular markers. Marker-trait associations (MTAs) can be discovered using genome-wide association study (GWAS) ,22. In G2) ranged between 0.79 (CR_Y) and 0.98 (PN2). The results suggest a high level of heritability for the evaluated traits in wheat.The results of the variance analysis (ANOVA) revealed significant differences among all experiments and growth conditions . AccordiPratylenchus sp. (P. neglectus and P. thornei) for resistance assessment (P. thornei (PT1), the nematode count (nematodes/plant) ranged from 25 (GID6176558) to 1925 (GID6181747), with a mean value of 506.56 while in the second experiment (PT2), the range of nematodes/plant were 92 (GID6176334) to 3104 (GID6624543), and the mean value was 849.75. Among the checks, the cultivar \u2018Seri\u2019 had the highest nematode count (890 and 944) while the line Croc_1/Ae.squarrosa(224)//Opata had the lowest nematode count (247 and 264) in the PT1 and PT2 trials, respectively. The number of P. neglectus nematodes in PN1 and PN2 ranged from 25 (GID6176409) to 3149 (GID6417653) and 75 (GID6280393) to 3653 (GID6417653), respectively. The mean values for PN1 and PN2 were 789.95 and 948.05, respectively. The lines with the lowest PN count according to GR1 and GR2 mean values were GID6278849 (82.5 plant\u22121), GID6176409 (85 plant\u22121), and GID61781747 (90 plant\u22121). For the resistance against PT, the lines with the lowest mean nematode counts in GR1 and GR2 were GID6176334 (77 plant\u22121), GID6174858 (93.5 plant\u22121), and GID6174847 (97.5 plant\u22121). Overall means for PN were higher than for PT. Out of the 189 lines tested, 113 (59.8%) and 115 (60.8%) had nematode counts below average in PN1 and PN2, respectively. For the PT experiments, 104 (55%) and 105 (55.6%) lines had nematode counts below the average. The range for PN and PT counts is given in Ae.squarrosa(224)//Opata had the lowest nematode count for both PN1-2 and PT1-2 experiments (Ae.squarrosa(224)//Opata. Overall, out of the panel we evaluated for CR, PN, and PT resistance, six genotypes were resistant against CR, PT, and PN. Genotypes GID6181748, GID6278812, GID6279005, GID6424861, GID6424874 and GID6487731 showed moderate to complete resistance against crown-rot and root-lesion nematodes.Two consecutive GR experiments were conducted with sessment . The scrsessment . In the eriments . AccordiCrown rot scoring for seedling resistance was conducted in two growth room experiments (CR1 and CR2), and scores ranged from 1.2 (GID6175213) to 4.4 (GID6417653) in CR1 and between 1.1 (GID6279632) and 4.6 (GID6174901) in CR2 . The cheCorrelation analysis was performed to observe similarities between GR, GH, and field experiments and between different seasons. Thus, we carried out a correlation analysis between CR, PN, and PT experiments . Based oGenotyping-by-sequencing (GBS) yielded a total of 4056 SNP markers after initial filtering for missing data. The distribution of markers was the highest in the B genome with 2013 SNPs, followed by the A genome with 1518 SNPs and the D genome with 429 SNPs. The highest marker density was observed in B, and the lowest was observed in the D genomes, as expected in wheat. This is due to the recent addition of the D genome to the bread wheat genome compared to A and B genomes, and relatively lower marker information is available in the GBS databases. A total of 96 SNPs out of 4056 were not assigned into any genome, and they were classified as unknown. Of the 21 linkage groups, chromosome 2B had the highest number of markers (434 SNPs), while 4D had the least number of markers (17 SNPs).The kinship matrix of the 189 bread wheat lines, excluding the checks, is shown in r2 values were plotted to the physical distance in the base pair (bp) to infer the extent of LD decay. LD decayed with genetic distance, and it reached 50% of its initial value at just below the 5 Mb. The statistically significant threshold for R2 for the whole genome LD was kept at 0.2 (data not shown).Linkage disequilibrium was estimated between the mapped SNPs on each chromosome. The pairwise P. neglectus and remaining with P. thornei resistance. One marker, \u2018S5B_84880275\u2019, showed significant association in the PN1 and PN2 experiment, located on chromosome 5B. Chromosome 5B had the highest number of markers associated with PT1, CR1, CR2, PN1, and PN2. Only one marker (S3A_501892003) was identified for PT2.Association analysis was carried out using the mixed linear model (MLM). Nineteen different SNPs were significantly associated with CR, PT, and PN resistance . Nine SNWheat is one of the main crops grown globally as a staple food and source of income for many people. A decrease in wheat yields could result in global food security issues and may lead to hunger in some cases. Wheat production is limited by several abiotic and biotic stresses. Biotic stress factors are a continuous thread to crop production. Although considerable efforts have been undertaken in many breeding programs to increase biotic stress tolerance/resistance, with the newly evolving strains, the race for releasing new cultivars with resistance to biotic stresses should be elevated. Despite the importance of root diseases such as crown rot and root-lesion nematodes, they have received little attention in most breeding programs. Therefore, the knowledge in resistance or tolerance sources against such diseases and molecular understanding is quite limited. In many cases, root diseases are mistakenly recorded as nutrient deficiencies, and proper control measures are not applied.The development of nematode-resistant wheat was first carried out by Brown and Ellis . ResistaAe. squarrosa (224)//Opata) in the PT1 experiment, 21% in the PT2, 22% in the PN1, and around 27% PN2 experiment. Similarly, for Fusarium culmorum, the GR1 and GR2 experiments had 17%, and 24% of genotypes had lower CR scores compared to the best checks. In the case of the CR_GH experiment, about 11% of genotypes demonstrated a lower CR score, for the field experiment in Yozgat (CR_Y), 18% and Konya (CR_K), 21% of the total germplasm had a lower CR score compared to the best checks.A panel of 198 elite spring wheat accessions, including checks, were evaluated against CR, PT, and PN, and significant genetic variation for resistance was obtained. The panel was well balanced, with 31.2% of the lines resistant, 15.3% moderately resistant, 14.8% moderately susceptible, and 38.6% susceptible to CR . In totaGWAS is an efficient approach for identifying molecular markers linked to important agronomic traits and disease resistance in bread wheat ,26,27,28P. neglectus resistance, while four markers were associated with PT resistance. There were several reports of crown rot and root-lesion nematode resistance. Collard et al. [F. pseudograminearum and F. culmorum) in Kukri  and Janz  DH mapping population. They identified a QTL on chromosome 4B, linked to the dwarfing gene Rht-B1. However, the dwarfing gene Rht-B1 is very much fixed in the CIMMYT germplasm. Therefore, the QTL identified in this study on chromosome 4B is most likely not linked to the dwarfing gene. Several reports [We identified 19 significant MTAs with the root disease resistance on chromosomes 1A, 1B, 2A, 2B, 3A, 4A, 4B, 5B, 5D, and 7D. Of the 19 markers, 8 markers showed association with CR resistance and 11 with PT and PN resistance . There wd et al.  reportedd et al.  reportedd et al.  reported reports ,39 ident reports . In a si reports  reported2 mean 8.36) was associated with resistance against P. neglectus (PN1 and PN2), and S4B_539004405 (R2 mean 5.1) was associated with resistance to CR (CR1 and CR2). These two markers were consistently observed across the two different experiments. The favorable allele at S5B_84880275 and S4B_539004405 were found in 36 and 24 lines, respectively. Consistency in these two markers with the acceptable R2 values . In both cases, phenotyping is very important. Results from our study could help to expand our knowledge of the source of resistance in germplasm and genomic regions controlling resistance against crown rot and root-lesion nematodes in wheat. Of the SNPs associated with resistance against CR, PT, and PN, two were consistent across experiments or seasons. The SNP marker S5B_84880275  and P. thornei (PT), 189 lines and 4 check cultivars were evaluated under growth room (GR) conditions, while for resistance to F. culmorum (CR), 189 lines and 5 checks were phenotyped in the GR, greenhouse (GH), and under field conditions. Wheat cultivars \u201c2-49\u201d, \u201cSunco\u201d, and \u201cAltay\u201d were used as resistant checks, whereas the lines \u201cSeri\u201d and \u201cSuzen\u201d were used as susceptible checks for F. culmorum. In addition, the lines \u201cCroc_1/Ae. squarrosa (224)//Opata\u201d and \u201cGs50a\u201d were used as a moderate resistant check for P. thornei, whereas the cultivars \u201cGatcher\u201d and \u201cSuzen\u201d were used as susceptible checks for both P. thornei and P. neglectus (Ae. squarrosa (224)//Opata\u201d was used as moderately resistant for both nematodes species.A total of 198 CIMMYT  spring bread wheat lines, including nine check varieties, were evaluated in this study . The lineglectus  while thP. thornei and P. neglectus were evaluated under GR conditions with two consecutive independent experiments . A single pre-germinated seed was planted in a standard small tube (2.5 cm in diam \u00d7 16 cm in length) filled with a sterilized mixture of sand, field soil, and organic matter . The field soil and sand were sieved and sterilized at 110 \u00b0C for two h on two consecutive days, whereas the organic matter was sterilized at 70 \u00b0C for 5 h. One week after planting, each plant was inoculated with 400 nematodes of either P. thornei or P. neglectus originating from nematodes reared on carrot discs as described by Dababat et al. [P. thornei and P. neglectus individuals were extracted from the roots and soil using the modified Baermann funnel [P. thornei and P. neglectus nematodes per plant was calculated based on the number of nematodes in 1 mL suspension multiplied by the total volume and counted under a microscope. Genotypes were divided into five groups based on the number of nematodes per plant, considering the reaction of check varieties used with their known resistant responses to both nematodes species.Resistance against t et al. . Trials n funnel . The totF. culmorum isolate was obtained from an infected wheat plant in K\u0131r\u015fehir, Turkey . A monosporic isolate was transferred to nutrient agar and cultured at 23 \u00b1 1 \u00b0C with a 12 h photoperiod for 10 days for spore formation. Bags (35 \u00d7 48 cm) filled with wheat bran were moistured and sealed with cotton and then autoclaved at 121 \u00b0C for 20 min for three consecutive days. The spore suspension was prepared by adding sterilized distilled water to each Petri dish containing F. culmorum culture. The autoclaved bags filled with wheat bran were inoculated with spore suspension under sterilized conditions and were incubated for 2\u20133 weeks with a 12 h photoperiod and 23 \u00b1 1 \u00b0C by shaking until the bran was sufficiently colonized by the fungus [The e fungus . The funF. culmorum was evaluated in the GR with two consecutive experiments (CR1 and CR2). Fungus inoculated wheat bran was suspended with distilled water and filtered using two layers of cheesecloth. Spore concentration was adjusted to 1 \u00d7 106 spores mL\u22121, and methylcellulose (0.1%) was added to the spore suspension prior to use. A total of ten seeds per accession were placed on moist blotting papers in sterilized Petri dishes and left to germinate at 22 \u00b0C for 2\u20133 days to obtain similar phenological development. Each seedling was sown in a separate plastic tube (2.5 cm in diam \u00d7 16 cm in height)  filled with (62 g) potting mix of sterilized sand, soil, and organic manure  and covered with the same substrate. After one week of growth, each seedling  was inoculated from the base of the stem with F. culmorum spore suspension. Tubes inoculated with F. culmorum were kept at 23 \u00b1 1 \u00b0C for 48 h under high humidity (80\u201390%) by covering them with plastic sheeting. Following incubation, seedlings were kept in the GR for 42 days ), with a day/night photoperiod of 16/8 h, at 23 \u00b1 1 \u00b0C, and relative humidity of 60%/80% (\u00b15%). The plants per each experiment were placed in a randomized complete block design (RCBD) with five replications (1 plant per replicate), and each experiment was repeated twice.Resistance against F. culmorum in the CR_GH, two seeds of each wheat line were planted in one plastic tube (3.8 cm in diam. \u00d7 21 cm in length)  using the same potting mix as described above, along with 0.5 g fungus-colonized wheat bran (as an inoculum source). Tubes were then put in a stand  placed on the sand to facilitate root growth. Experiments were sufficiently irrigated during the growing season. Plants were exposed to drought stress at maturity to promote disease development. The experiment was set up using RCBD with six replications and two plants per replicate.To evaluate the resistance in the spring wheat panel against Field experiments were conducted at Yozgat (CR_Y) and Bahri Da\u011fda\u015f International Agricultural Research Institute in Konya (CR_K), Turkey, under naturally infested field conditions during the 2013\u20132014 and 2014\u20132015 growing seasons (October to June). For each line, 5 g of seeds were sown in 1 m rows and infested with an addition of 2 g of fungus-colonized wheat bran. Experiments were arranged in RCBD with three replications. Disease symptoms were scored by randomly picking up 15 individual tillers from each row at the full maturity stage.Seedling resistance (Zadoks growth stage 14) was evaluated from the GR experiments, while for GH and field experiments, plants were evaluated for adult plant resistance. When the diseases were scored, crown rot was scored by one person. While root-lesion nematode scoring was performed by three well-trained people using binoculars. Plants were harvested at the end of each experiment, after 7 weeks in GR, and at full maturity in GH and field experiments, following common practices. Disease scoring was carried out using a modified method of Wildermuth and McNamara , followi2) for all environments was estimated according to Lewien et al. [Data analyses were performed using QTL Icimapping software . Experimn et al. . DescripGenomic DNA was extracted from bulked leaves of 10 two-week-old seedlings leaves using a cetyltrimethylammonium bromide procedure  modified2 value equal to 0.5 using PLINK [Population structure was assessed using ADMIXTURE software with 10 cross-validations and k ranging between 2 and 20 . Overallng PLINK .r2). GWAS was conducted using the mixed linear model (MLM) implemented in the Genome Association and Prediction Integrated Tool (GAPIT) in R [Linkage disequilibrium (LD) was calculated using polymorphic markers with allele frequency higher than 5% using a custom R script . LD was IT) in R . The genIT) in R . This me"}
+{"text": "BP may provide a new therapeutic approach for HGSOC.High-grade serous ovarian cancer (HGSOC) constitutes 80% of ovarian cancer. Cancer stem cells (CSCs) are responsible for most of the tumor metastasis and chemoresistance. n-Butylidenephthalide (BP) is a potential anti-tumor agent for treating a variety of cancers. The aim of this study was to evaluate the effect of BP on CSCs of HGSOC. CSCs were isolated using the CSC marker  from KURAMOCHI and OVSAHO cells (HGSOC cell lines). The cell proliferation, IC50 , cell migration and invasion, TUNEL  dUTP nick end labeling) assay, western blot of ovarian CSC were evaluated. The animal xenograft studies were evaluated on an immunodeficient mouse model. The results showed the proliferation of ALDH+ cells was greater than that of ALDH- cells. The dosage of IC50 of BP was higher in ALDH+ cells than in mixed cancer cells (317.2 vs. 206.5 \u03bcg/ml) in KURAMOCHI cells, but not in OVSAHO cells (61.1 vs. 48.5 \u03bcg/ml). BP could inhibit the migration and invasion of both cancer stem cells. BP treatment could activate apoptosis signaling, as indicated by the TUNEL assay and the increased expression of cleaved caspase-3, -7, and -9 but not cleaved caspase-8. A low dose of BP (20 and 25 \u03bcg/mL) treatment could increase the toxicity of taxol and cisplatin. In the animal model, BP (200 mg/kg) treatment also decreased the KURAMOCHI and OVSAHO tumor growth rate and induced tumor apoptosis. In conclusion, BP could kill ALDH+ CSCs of HGSOC  Ovarian cancer is the deadliest cancer in women. Although its incidence is not high, ranking 7th in Taiwanese women, more than 1400 new cases are diagnosed each year Tumors are composed of cells with varying degrees of malignancy. Tumor development is mediated by specialized, pluripotent, and self-proliferating cells that have tumorigenic properties and are known as cancer stem cells (CSCs) The current treatment for ovarian cancer involves debulking surgery and adjuvant chemotherapy with platinum and paclitaxel Angelica sinensis is a common Chinese herbal medicine used for the treatment of cough, headache, and angina and to strengthen muscles A. sinensis was identified to be n-butylidenephthalide (BP). BP has been used to treat a variety of cancers, including the brain However, the effect of BP on HGSOC has not yet been determined. This study aims to determine whether BP can kill HGSOC CSCs and whether BP co-treatment with common chemotherapeutic agents increases their effectiveness.2.The HGSOC cell lines (KURAMOCHI and OVSAHO cells) were used in this study and purchased from Japan Cell Bank. Both cell lines were confirmed gene expressions mimicking HGSOC oC. Cells were incubated with inhibitor DEAB as control cells to identify for ALDH+ and ALDH- cell populations. Then BD FACSVerse flow cytometer  was used to examine and analyze all the stained cells. The BD FACSAria Fusion flow cytometer (BD Biosciences) was used for sorting ALDH+ cells. After sorting, ALDH+ and ALDH- cells propagated using the above culture medium for no more than five passages. According to our analysis, the percentage of ALDH+ cells was more than 80% within 5 passages.We used cell fluorescence-activated cell sorting (FACS) to isolate ALDH+ cells from the KURAMOCHI and OVSAHO cell lines BP (Sigma-Aldrich) was dissolved in vitamin K solution (Sigma-Aldrich). The stock solution was with a concentration of 200 \u03bcg/\u03bcl. The following antibodies were used: caspase 3, 7, 8, 9 ; tubulin and beta-actin . The secondary antibodies goat anti-rabbit and -mouse were purchased from Cell Signaling Technology. The FITC conjugated secondary antibody was purchased from Sigma.3 cells/cm2 in 96-well plates with different concentrations of BP  for 48h. Then the half-maximal inhibitory concentration (IC50) of both types of cells was obtained. The four-parameter logistic regression (4PL) method described in the previous literature was used b), where Y is the response, and X is the concentration. The variable a is the bottom of the curve, and d is the top of the curve. The variable b is the slope factor, and c is the concentration corresponding to the response midway between a and d Cell viability was determined by XTT assay  used as instructed by the manufacturer. We seeded 2 \u00d7 104 cells) were seeded into the top well of a Boyden chamber (24-well transwell) with a pore size of 8 \u03bcm . The ALDH+ cells migrate toward the lower well filled with the same culture medium with or without BP . After 48 hours of migration, crystal violet (Sigma) was used to stain the migrated cells. The stained cells were counted using a bright-field microscope. Each experiment was repeated three times.Tumor cells . Treatments were added to both upper and lower chambers as indicated. After 24 hours, the free cells were removed gently with a cotton swab. The invading cells were fixed with 4% formaldehyde and crystal violet (Sigma) stained. The slides were air-dried and photographed, and the cells were counted using a bright-field microscope.Invasion assays were carried out in Matrigel-coated Boyden chambers  in 24-well plates (BD). In the top wells, the culture medium without serum was seeded with 5 \u00d7 10Tumor cell and tissue lysates were loaded onto a gradient 5-20% sodium dodecyl sulfate-polyacrylamide gradient gel. After electrophoretic separation, the proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membrane was blocked at room temperature in a solution of 3% nonfat dry milk in PBS and 0.1% Tween-20 and then rinsed in PBS/0.1% Tween-20. Blots were incubated with diluted solutions of polyclonal anti-caspase 3, 8, or anti-cleaved caspase 3, 7, 8, or 9 antibodies  and treated with 1:5000 diluted anti-rabbit immunoglobulin G horseradish peroxidase (HRP) for staining . Beta-actin proteins  were used as internal controls. HRP signals were detected using an electrochemiluminescence kit .+ cells/well) in 96-well plates. BP (25\u03bcg/ml in KURAMOCHI and 20 \u03bcg/ml in OVSAHO) with or without cisplatin (5\u03bcM or 10 \u03bcM) or taxol (10 nM or 50 nM). Each experiment was conducted in triplicate. Plates were incubated at 37\u02daC with 5% CO2 for 72 hours. Viable cells were counted by the XTT assay as described.To determine the killing ability of BP alone or in combination with cisplatin or taxol, we measured the viability of KURAMOCHI and OVSAHO cells . All procedures were performed in compliance with the National Institutes Health Guide for the Care and Use of Laboratory Animals. Non-obese, diabetic-severe combined immunodeficiency mice (NOD-SCID) (strain NOD.CB17-Prkdcscid/JTcu) purchased from Tzu Chi University were used for these experiments.+ cells (1\u00d7 106) were injected subcutaneously into the backs of 4-5-week-old female mice. After tumors had grown to a volume of 50 mm3, mice were separated into two groups . Controls were treated with vehicle alone , and the experimental group was treated with different doses of BP (100 or 200 mg/kg) for 5 days. Tumor dimensions (length and width) were measured with calipers and the tumor volume determined using the following formula: volume = 1/2 (length \u00d7 width2).KURAMOCHI and OVSAHO ALDHFor histological examination, tumor tissues were fixed in 4% paraformaldehyde. Tumors were cut into 6-\u03bcm thick sections and stained with hematoxylin and eosin. Tumor tissues were observed under a microscope at 200\u00d7 magnification. The morphology and cell density were observed and recorded.5 cells in one well of 12-well culture plates. Cultured cells were allowed to attach for 24 hours. Then we treated BP  for 48 hours. Adherent tumor cells were fixed in 4% paraformaldehyde. For ALDH+ KURAMOCHI and OVSAHO cells xenograft, tumor samples were fixed in formalin and embedded in paraffin, and sectioned with a 3 \u03bcm of thickness. TUNEL probes were used to detect breaks in DNA strands, followed by incubation in permeabilization solution for 2 min on ice. Cells were washed twice in PBS and the TUNEL reaction mixture added, followed by incubation at 37\u00b0C for 60 min in a humidified atmosphere in the dark. Samples were washed twice in PBS twice and observed under a fluorescence microscope.Cell apoptosis was assayed using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Assay Kit  according to the manufacturer's instructions. For ALDH+ KURAMOCHI and OVSAHO cells, they were seeded with 1\u202f\u00d7\u202f10Data are presented as the mean \u00b1 SD of at least three independent experiments. The Mann-Whitney U test was used to compare two independent variables, and one-way ANOVA with post-hoc analysis with the Bonferroni test was used to compare three independent variables. Statistical analysis was performed using GraphPad Prism 6 . P < 0.05 was considered a significant difference.+ cells composed 3.6% in KURAMOCHI cells and 40% in OVASAHO cells   Fig. B. The molls Fig. E. The AL+ cells was 317.2 \u03bcg/ml  and OVSAHO ALDH+ cells with BP (20 \u03bcg/ml) in combination with cisplatin  resulted in cell viability significantly lower than that of controls  treatment (p < 0.001)  in KURAMOCHI and OVSAHO cells, the tumor volumes were smaller than those of control mice treated with the vehicle  Fig. A, 9A. Ho01) Fig. C-D, 9C-Drol Fig. E, 9E.Taken together, these results suggest that BP inhibited xenograft growth by activating apoptosis of type 2 ovarian cancer stem cells.A. Sinensis. Our results show that BP significantly inhibited ovarian CSC proliferation in vitro and in vivo. BP also inhibited ovarian CSC migration and invasion. BP treatment resulted in the death of ovarian CSCs via activation of the apoptosis signaling pathway. BP increased the toxicity of the chemotherapeutic drugs cisplatin and taxol on ovarian CSCs. BP also inhibited the tumorigenicity of CSCs via induced apoptosis in NOD-SCID mice. These findings suggest that BP may be useful for ovarian cancer therapy.In ovarian cancer, tumor aggressiveness, resistance to therapy, and disease relapse may be determined by a small population of CSCs in vivo.ALDH+ cells are considered to be ovarian CSCs Apoptosis is a targeted cell death program regulated by the caspase protein cascade to prevent inflammatory reactions and damage to surrounding cells Cisplatin and taxol are standard treatments in adjuvant chemotherapy for ovarian cancer in vitro was higher than we expected. Previous studies showed that the effective BP concentration ranged from 15-67 or 50-84 \u03bcg/mL in vitro and 300-700 mg/kg in vivoin vitro and 100-200 mg/ml in vivo. We speculated that survival signals may be upregulated during BP treatment in KURAMOCHI cells. This possibility requires further investigation. However, in OVASAHO cells, the IC50 of BP was 48.5 \u03bcg/ml in ALDH+ cells which is compatible with previous reports This study addresses the possible clinical use of BP for killing ovarian CSCs. However, the concentration of BP required to kill KURAMOCHI cells There were two xenograft models that could be used in our study, the subcutaneous xenograft model instead of the orthotopic implantation model. The orthotopic transplantation model could provide a suitable microenvironment and enable tumor cells to develop their malignant behavior in vivo tumor formation experiment to study the in vivo effect which might be reflected in the in vivo status. Another limitation was the lack of normal cells and other cancer cells as controls. We only used the cells of type 2 ovarian cancer which occupied almost 80% of ovarian cancer. Therefore, the results of these experiments might be applied to 80% of ovarian cancer.The limitation of this study was to use ALDH as a marker for isolating ovarian CSC. In HGSOC, there are several CSC markers in ovarian cancer such as CD24, CD44, CD117, CD133, and ROR1 In conclusion, BP kills CSCs of HGSOC via activation of the apoptosis signaling pathway. BP may also be an additive to conventional chemo drugs to lessen the side effect. BP may prove useful in treatments to slow ovarian cancer progression."}
+{"text": "Acrolein is a known pro-inflammatory toxic aldehyde, propagating cellular damage and tissue inflammation in humans and animal models of various diseases. Osteoarthritis (OA) has a significant inflammatory component; however, presence of acrolein in synovial fluid of joints with OA has not been previously reported. The first aim of this study was to evaluate evidence of acrolein in the synovial fluid of dogs with OA as well as in Control joints. The second aim was to determine if evidence of acrolein can be detected in synovial fluid samples that have been in a frozen state for long periods of time.n\u2009=\u20095) and dogs with OA  and frozen until the time of analysis. Additionally, frozen synovial fluid samples from a biobank  were used to evaluate ability to detect evidence of acrolein in long-term stored samples (median of 4.89\u2009years) in Old Control (n\u2009=\u20095) and Old OA (n\u2009=\u20095) samples. Measurements of acrolein in all synovial fluid samples was based on detection of its major protein adduct, N \u03b5 - lysine (FDP-lysine), using the western blot method. Synovial fluid matrix metalloproteinase 2 (MMP2) was measured in all samples using the western blot method as a positive control of OA inflammation.In this pilot clinical study, synovial fluid samples were prospectively collected  from a single joint of both clinically healthy  and OA (n\u2009=\u200921) groups in both Old and New samples. Acrolein-lysine adduct and MMP2 were detectable at a lower level in the Old compared to New synovial fluid samples; however, the differences were not statistically significant (p\u2009>\u00a00.1). The measured MMP2 levels were significantly higher in the OA compared to Control group samples (p\u2009=\u00a00.033), but not for acrolein-lysine adduct (p\u2009=\u00a00.30).Acrolein-lysine adduct was detected in both Control (This study confirmed evidence of acrolein in canine synovial fluid of both OA and Control groups. Freezing of synovial fluid for up to 5 years does not appear to significantly affect the ability to detect acrolein-lysine adduct and MMP2 in these samples. Endogenous acrolein is one of the cytotoxic aldehydes that is a byproduct of lipid peroxidation or enzymatic oxidation of polyamine metabolites . AcroleiOsteoarthritis (OA) is defined as a disorder involving movable joints characterized by cell stress and extracellular matrix degradation initiated by micro- and macro-injury that activates maladaptive repair responses including pro-inflammatory pathways of innate immunity . The disn\u2009=\u200910; 5 OA and 5 Control) that had been frozen for 4\u20135\u2009years (referred to as Old samples) to evaluate the effect of long-term freezing on the ability to detect acrolein.This study was approved by the Purdue Animal Care and Used Committee (protocol #1804001744111). Informed owner-signed consents were obtained from owners of all dogs enrolled in the study. Due to lack of previously established measurements for acrolein in dogs and the pilot nature of the study the target sample size was to enroll a minimum of 15 dogs in the OA group and 5 dogs in the control group. Prospectively collected synovial fluid samples that were frozen for less than 6\u2009months prior to analysis (referred to as New samples) from this cohort were compared to a bank of samples  for OA of one or more joints. Diagnosis of OA was based on initial orthopedic examination by a board-certified surgeon , radiographic evidence of OA based on orthogonal views or computed tomography of the joint, and intraoperative confirmation of OA during joint exploration via arthrotomy or arthroscopy. If more than one joint was affected, the most clinically affected joint that underwent surgical intervention at the time of enrollment was included in the study for synovial fluid sampling. Exclusion criteria for the OA group included OA secondary to trauma or non-OA forms of arthritis in any joint , systemic diseases including neurological abnormalities, history of surgical intervention in the affected joint, or use of corticosteroids within a month of enrollment in the study. Inclusion criteria for the New Control group were adult, medium to large breed healthy dogs that were deemed free of any abnormalities based on complete physical, orthopedic and neurological examinations. Exclusion criteria for the New Control group was presence of any systemic illness or history of surgical intervention in the sampled joint.The Old samples for both OA group and Control groups were randomly selected from a surplus of available stored frozen samples from a completed study at University of Prince Edward Island (UPEI). The Animal Care Committee of the University of Prince Edward Island in accordance with the Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care (#11\u2013062) had approved the completed UPEI study and surplus of the synovial fluid samples were available for the current project. The inclusion criteria for Old samples in the OA group and Control group dogs were the same as the fresh samples of OA and Control groups with the exception that OA group dogs were only affected with uni- or bilateral knee OA secondary to CrCLR.Medical record information for age, breed, gender, body condition score (BCS) based on a 9-point scale  were obtDogs in the OA group underwent general anesthesia for surgical intervention relevant to the underlying cause of OA in each joint. The anesthesia protocol was unrelated to the study and based on the discretion of the attending board-certified anesthesiologist for each case. Aseptic arthrocentesis was performed using a 3\u2009ml syringe and 22-gauge needle prior to surgical intervention for each joint. Dogs in the New Control group were recruited from an in-house research colony Purdue University College of Veterinary Medicine. The Control dogs were anesthetized for reasons unrelated to this project and arthrocentesis was performed from both knees using the previously described technique. The knee joint was selected due to ease of access to the joint and ability to obtain higher synovial fluid volume compared to other joints in healthy dogs. The synovial fluid samples obtained from dogs in both OA and Control groups enrolled at Purdue University were centrifuged in EDTA-free tubes at 4\u2009\u00b0C for 10\u2009min at 14,000 RPM to remove cells and debris. Thereafter, theses samples were aliquoted in 2\u2009ml cryovials, labeled as New OA and New Control samples, and frozen at -80\u2009\u00b0C until analysis. The dog synovial fluid samples used from the UPEI study for the OA group had been collected in the same manner as the PUVTH dogs and Control group samples had been obtained via arthrocentesis of knees immediately after humane euthanasia (for reasons unrelated to the project). The synovial fluid samples from the UPEI study had been maintained in the frozen state at -80\u2009\u00b0C after acquisition until analysis, when they were centrifuged, aliquoted and labeled as Old OA and Old Control samples based on their group designation at the time of batch analysis.N \u03b5 -lysine (FDP-lysine) is a major protein adduct of acrolein, which is detectable by western blot method [Given the reactivity and short half-life of acrolein, direct in vivo quantification of the molecule is difficult \u20135. Howevt method \u201329. In tt method , 31. Synt method . Matrix t method \u201336.Briefly, protein concentrations were measured using the Bicinochoninic Acid protein assay kit  and SPECTRAmax . Thirty micrograms of protein with 20% Sodium dodecyl sulfate, \u03b2-mercaptoethanol, and 2x Laemmli buffer were loaded to a 15% Tris-HCL gels and electrophoresed at 80\u2009V for 2\u20133\u2009h providing adequate time for band separation. Proteins were then transferred to a nitrocellulose membrane by electro blotting on ice in 70\u2009V for 1\u20132\u2009h depending on the protein size in 1x transfer buffer with 20% methanol . The membrane was incubated in Ponceau stain . The membranes for MMP2 and A1AT experiments were cut with a razor blade horizontally into strips within the known and desired kDa ranges. This previously used method , 37, wasDescriptive data for normally distributed variables was reported as mean (\u00b1standard deviation: SD) and as median (range) for those without a normal distribution. Statistical analysis was performed using STATA SE (v. 15.1) software. Data analyses for all variables  were accomplished using student\u2019s t-test, Wilcoxon rank sum test, Fisher\u2019s exact test. Evaluation of correlations of MMP2 and acrolein levels with other patient variables  was performed using Spearman\u2019s correlation test.n\u2009=\u200910) were available from knees of five OA and five Control dogs . Old samples from the bank of frozen synovial fluid samples (p\u2009<\u00a0.001). The OA group had eight neutered females, 12 neutered males and one intact male, while the Control group included four intact females, five intact males and one neutered male. Overall, the Control group had a significantly higher number of sexually intact dogs compared to the OA group (P\u2009<\u2009.001). The New OA synovial fluid samples collected from PUVTH cohort were from elbow , knee  and shoulder  joints. In four dogs , samples were also obtained from the joint contralateral to that included in the study that were also affected with OA. This was due to concerns that the primary joint that had been sampled may not have yielded adequate samples for the western blot. These samples were run but not included in the analysis since the primary sampled joint had adequate sample volume and the study required only one sample per dog to be included in the analysis. These excluded samples are highlighted in Fig.\u00a0n\u2009=\u200914) three of which had concurrent medial meniscal tear in the knee followed by elbow dysplasia (n\u2009=\u20095) being most common. The components of elbow dysplasia in these dogs are reported in Table Dogs in the OA group were significantly heavier in weight compared to controls p\u2009<\u00a0.00. The OA Acrolein-lysine adduct and MMP2 were detected in both OA and Control group samples. The western blot raw data for acrolein-lysine adduct, MMP2 and A1AT are presented in Fig. p\u2009=\u20090.033) in the OA compared to Control samples but not for acrolein-lysine adduct (p\u2009=\u00a00.299)   and Control (n\u2009=\u200910) groups (p\u2009=\u00a00.041) but not for acrolein-lysine adduct (p\u2009>\u00a00.1).Comparison of samples, post normalization to A1AT, showed significantly higher levels of MMP2 p\u2009=\u20090.03 in the 05) Fig.\u00a0. Due to rs\u2009=\u20090.335) but not statistically significant correlation (p\u2009=\u00a00.061) between measured MMP2 and reported chronicity of clinical signs in the OA group.The reported chronicity of clinical symptoms associated with OA in dogs was 3 months . Correlations between the biomarkers (MMP2 and acrolein-lysine adduct) and disease chronicity were evaluated. There was only a weak positive (n\u2009=\u200915) based on previously reported grading scheme [n\u2009=\u20095) and shoulder (n\u2009=\u20091) joints, only radiographic grading of knee joints in the OA group were used in evaluating correlations between measured biomarkers and individual grades. Spearman\u2019s rank order correlation showed the radiographic knee OA grade categories  to be significantly correlated with each other (p\u2009<\u00a00.05) with the exception of joint effusion and intra-articular mineralization. However, none of these grades had a significant correlation with the measured acrolein-lysine adduct or MMP2 measurements in the OA group.Radiographic scoring of knees with OA (g scheme  had scorThis is the first study demonstrating evidence of acrolein based on acrolein-lysine adduct (FDP-lysine) detection in synovial fluid of dogs in naturally occurring secondary OA as well as control joints. The indirect measurement of acrolein using acrolein-lysine adduct has been used in other studies , 21, 39 Acrolein is capable of initiating and propagating both pain and inflammation by perpetuating oxidative stress and cell death , 8, 29. Elevation of synovial fluid MMP2 as a positive control was demonstrated in osteoarthritic joints as a biomarker of inflammation compared to control joints in this study. The use of synovial fluid MMP2 as diagnostic biomarker for canine knee OA has been controversial with some studies showing a positive correlation with OA similar to the findings in this study , 50 and Limitations of the current study include the pilot nature with limited sample size, as well as heterogenicity of sampled joints with regards to location  and etiology of OA. The clinical model of OA in this study was strictly non-traumatic in etiology; therefore, evaluation of presence of acrolein in post-traumatic OA warrants further investigation. In future studies with larger sample size, acrolein levels can be evaluated in specific joints only  to evaluate the acrolein profile in a range of OA severity while accounting for additional etiologies of OA in that specific joint  and possible response to medical or surgical interventions. Additionally, levels of acrolein in other forms of arthritis  warrants evaluation before acrolein can be considered as a candidate diagnostic biomarker of OA. Measurement of acrolein lysine-adduct in this study was based the western blot method that relies on a software-based semi-subjective quantification of the biomarker levels. Use of other quantitative methods such as enzyme-linked immunosorbent assay (ELISA) methodology may provide a basis for developing objective reference ranges for acrolein levels in healthy and disease states.This study documented evidence of acrolein in synovial fluid of dogs with OA as well as in control healthy joints by measuring its acrolein-lysine adduct. It is also a proof of concept for using the western blot methodology used herein for detection of acrolein and MMP2 in canine synovial fluid samples, and supports use of samples that have been in a frozen state for an average of 4 years. Future studies are required to investigate the role of acrolein as an inflammatory biomarker in osteoarthritis in dogs."}
+{"text": "Breast cancer is a global health issue and a leading cause of death among women. Early detection through increased awareness and knowledge on breast cancer and breast cancer screening is thus crucial. The aim of the present study was to assess the effect of an educational intervention program on breast cancer knowledge and the practice of breast self-examination among young female students of a university in Bangladesh.t-tests were performed to investigate the differences between pre- and post-test stages.A quasi-experimental (pre-post) study design was conducted at Jahangirnagar University in Bangladesh. Educational information on breast cancer and breast self-examination (BSE), demonstration of BSE procedure and leaflets were distributed among 400 female students after obtaining written informed consent. The stepwise procedures of BSE performance were demonstrated with images. Pre-intervention and 15\u2009days post-intervention assessments were conducted to assess the changes in knowledge on breast cancer and practices of BSE. Mc-Nemar\u2019s tests and paired sampled p\u2009<\u20090.001), risk factors , treatment , prevention , screening of breast cancer  and process of BSE . Likewise, a significant percentage of change in BSE practices was obtained between pre-test and post-test .A total of 400 female university students aged 18-26\u2009years were included in the sample. Significant changes were found in knowledge and awareness about breast cancer and BSE practices after the educational intervention. The significant differences were measured in the mean scores of pre-test vs. post-test: breast cancer symptoms (2.99\u2009\u00b1\u20091.05 vs. 6.35\u2009\u00b1\u20091.15; Study findings confirm that the study population had inadequate awareness and knowledge at baseline which was improved significantly after educational intervention. A nationwide roll-out with community-based interventions is recommended for the female population in both rural and urban areas.The online version contains supplementary material available at 10.1186/s12885-022-09311-y. Breast cancer is a worldwide health concern and one of the most prominent causes of mortality among women. In 2018, approximately 2 million new breast cancer cases were detected, which is approximately 23% of all cancers, the most occurring cancer among women . Breast Recommended screening methods like mammograms, clinical breast examination, ultrasounds and MRIs are not financially feasible to implement as a nationwide screening program in low-resource countries like Bangladesh. Moreover, lack of knowledge and awareness about breast cancer has been reported from some studies conducted with females in Bangladesh which may contribute to less adherence of women to receive recommended screening \u201313. TherA pre-post quasi-experimental interventional study was conducted among female university students residing in dormitories of Jahanginagar University in Dhaka, Bangladesh from December 2019 to March 2020. Jahanginagar University is the largest and only fully residential university in Bangladesh.The study was conducted among 400 female respondents of aged 18-26\u2009years corresponding to Honours 1st year to master\u2019s students. Inclusion criteria included: being female students residing in the university\u2019s dormitories and being aged 18-26\u2009years old. Exclusion criterion was being female students who didn\u2019t reside within residential dormitories. Initially, Yamane\u2019s simplified sampling formula was employed to determine a sample size and a total of 386 participants were estimated. However, we gathered 400 responses in order to make sure that our final sample size was large enough to detect statistical differences pre-post intervention. The proportionate stratified random sampling technique was conducted to calculate the study sample from each dormitory. In this approach, each stratum sample size was directly proportional to the population size of the entire population of the strata. The study was carried out in three phases: first phase (pre-intervention phase), second phase (intervention phase) and third phase (post-intervention phase).A pre-designed structured interview questionnaire was used to collect the following data from the respondents: socio-demographic data, respondent\u2019s knowledge, attitude and practice regarding breast cancer, screening and BSE.Materials  were used during the interventional phase. All the sessions were conducted in the respective dormitories of the respondents. Participants were divided into groups of 10-15 people to conduct the sessions so that the educational intervention could be clearly delivered to and understood by the participants. Each session took 45-60\u2009min. After the pre-test session, the participants were given a short break to rearrange themselves into divided groups and get prepared for the educational session. Both pre-test and intervention sessions were conducted on the same day. Each participant was assigned a unique ID number so that they could be traced back for the post-test session. To ensure that the respondents could understand the educational materials, in every session one or two respondents from each group were encouraged to demonstrate and share what they had learned. This was also chosen randomly among the participants who were willing to perform this task.Fifteen days after the education session, participants were re-contacted for a post-test survey. During post-test phase, participants were exposed to the same questions in the pre-test questionnaire to assess any changes in knowledge about breast cancer and practices of BSE.A pre-tested and semi-structured questionnaire including informed consent, socio-demographic information and questions related to knowledge towards breast cancer and BSE practices, was prepared for the study through extensive literature review \u201317.The questionnaire was reviewed by an external reviewer who was an oncologist and had sound knowledge about breast cancer. Likewise, a pilot test was conducted with 20 participants to assess the readability of the questionnaire. The questionnaire was finalized after incorporating minor amendments based on participant feedback during the pilot study. A paper-pen-based survey was conducted among participants. Additionally, a few post-test surveys were undertaken via telephone from respondents who could not be present during the post-test session.Socio-demographic information was recorded during the survey including age, study year (1st/2nd/3rd/4th/Master\u2019s), marital status (unmarried/married), family history of breast cancer (yes/no), and relationship with breast cancer affected patient (mother/sister/cousin/aunt/grandmother).To assess participant knowledge of breast cancer, a total of 43 questions  were asked during the survey. All questions were answered with three possible responses . During analysis, \u2018yes\u2019 responses were coded as \u20181\u2019; while \u2018no\u2019 or \u2018don\u2019t know\u2019 responses were scored as \u20180\u2019. To get the total score of a construct , the raw scores from each question were summated. The greater scores indicate the more knowledge. The distributions of all questions (both pre-test and post-test) are presented in Have you ever self-examined your breast for breast cancer?\u201d was used to assess the BSE with binary responses (yes/no).A single question \u201cp-value less than 0.05 was deemed as statistically significant.The SPSS version 25.0 was used for processing and analyzing data. Descriptive statistics were performed. To assess the differences between pre-test and post-test, Mc-Nemar tests and paired sample t-tests were computed as appropriate. Before performing the Mc-Nemar tests, each question was transformed into dichotomous . A The study was conducted in accordance with the Institutional Research Ethics guidelines and ethical principle involving human participation . Formal ethics approval was granted by the Biosafety, Biosecurity, and Ethical Clearance Committee, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh. At first, all participants were informed about the purpose and objectives of the study. Participants were informed that it was a three-phase study, and also about the duration of the study and the approximate time that would be taken from them. Then, written informed consents were taken from each of them who agreed to participate in the study. All information related to participants was kept confidential.The sample comprised of a total of 400 female university students aged 18-26\u2009years (see Table\u00a0p\u2009<\u20090.001), risk factors , treatment , prevention , screening of breast cancer  and process of breast self-examination . Likewise, a significate percentage of change in BSE practices was obtained between pre-test and post-test  , and Sivas  \u201321. In tp\u2009<\u20090.001). This finding is consistent with a previous study by Ceber et al. where the knowledge level on BSE was higher in the experimental group who received an educational session  [p\u2009<\u20090.05)) [p\u2009<\u20090.001) (Table p\u2009<\u20090.001) [t\u2009=\u20099.84, p\u2009<\u20090.001) [In the present study, the knowledge of BSE also increased significantly. The mean difference in the knowledge about process of BSE  was 2.37\u2009\u00b1\u20092.00 (\u2009\u00b1\u20090.84) . The stu\u2009\u00b1\u20090.84) . Another<\u20090.05)) . These s<\u20090.001) . Similar<\u20090.001) . In a di<\u20090.001) .All these studies discussed above that showed that educational interventions can lead to positive changes in knowledge, awareness and practice towards breast cancer and BSE. However, the time interval between the educational intervention and post-test survey, as well as the number of educational sessions, can impact the outcome. The difference in knowledge and practice were statistically higher in our study after educational intervention but the changes in percentage were not satisfactory (ranging from 12.5 to 47.4%.) if we compare with some other studies conducted on women worldwide. Studies in Iran, Egypt and Pakistan have showed higher percentage of changes in knowledge and practice than our study , 19, 20.Given the fact that this study was conducted with university students (1st year undergraduates to post-graduates) in combination with an efficient, flexible and attractive educational session, this is justifiable that they understood the information provided at the educational session on breast cancer and practice of BSE easily, indicate the successful outcome of the educational session that was conducted in this study. Though all changes in the knowledge and practice of breast cancer and BSE were statistically significant, the percentages of changes were not satisfactory at all. It was expected to changes more than 50%. However, our findings demonstrated the changes ranging from 12.5 to 47.4%.Even though our study was with university students, all women regardless of their socio-economic or demographic conditions need to be educated about breast cancer and breast cancer screening methods. This education should be culturally appropriate and targeted towards individual population so that it can create greater impact.The study had only 15\u2009days interval between the pre-test, educational session and the post-test assessment, increasing the chance of recall bias. If more time interval could be given, that might have impacted the outcomes. No follow-up session of the educational intervention on breast cancer and BSE could be given, also due to time constraints. Respondents were given no reminder to practice BSE during the interval phase. All the data were self-reported by the respondents and no verification could be done to assess the accuracy of the data given by respondents whether the claim of practicing BSE were true or not. The quality of BSE practice could not be assessed. So, it is unknown that if the respondents who are claiming to actually practicing BSE were being able to do it properly or not. Moreover, we also cannot claim any generalizability to other groups of women in Bangladesh given that our sample are highly educated.The findings indicated that women\u2019s knowledge regarding breast cancer warning symptoms, risk factors, treatment, prevention, effective screening methods and practice of BSE were sub-optimal at baseline. The results of the post-test of this study suggest that women\u2019s knowledge was significantly increased after providing an educational intervention. However, educational sessions should be continued because increased knowledge level is important to change behavior about early diagnosis for breast cancer. This study concludes that the educational program on breast cancer and BSE has been effective in improvement of knowledge and BSE practice levels of women. A future study with larger and diversified population is recommended to assess the effectiveness in different population groups of women and monitor the changes in awareness and practice of breast cancer and breast cancer screening.Additional file 1: Table S1. Knowledge about symptoms of breast cancer. Table S2. Knowledge about risks of breast cancer. Table S3. Knowledge about treatment of breast cancer. Table S4. Knowledge about prevention of breast cancer. Table S5. Knowledge about screening of breast cancer. Table S6. Knowledge about process of breast cancer."}
+{"text": "The influences of the P/Al molar ratio and P123 addition on catalyst structure and surface acid-base characteristics were investigated in detail. Increasing the P/Al molar ratio more favored crystalline aluminophosphate. The P123-assisted Al3+ and PO43\u2212 are known to be stabilized through weak steric force so that the formation of crystalline aluminophosphate could be inhibited at higher P/Al molar ratios. The results showed that the prepared Al-P-O catalysts possessed appropriate weak acid and weak base sites, which was beneficial to the reaction of catechol and methanol. The Al-1.1P-O catalyst synthesized with the assistance of P123 exhibited superior catalytic performances, with 52.5% catechol conversion and higher guaiacol selectivity of 97.6%.A series of Al-P-O catalysts (Al- As an important fine chemical raw material and intermediate ,2,3, guareaction ,14,15. IO-methylation of catechol to synthesize guaiacol, which is an economically and environmentally friendly route for industrial applications, has attracted extensive attention from scientific researchers [O-methylation of catechol due to its low toxicity and low cost. The heterogeneous catalysts used for vapour-phase selective O-methylation of catechol and methanol is the main factor affecting the yield and quality of the guaiacol. It has been widely believed that solid acid-base catalysts were active catalysts for O-methylation of catechol and methanol. Lots of solid acid-base catalysts, such as supported catalysts [O-methylation of catechol and methanol. Among various solid acid-base catalysts, phosphate catalysts, especially aluminophosphates, have been widely used for vapour-phase selective O-methylation of catechol and methanol because of their high activity and low cost.Recently, vapour-phase selective earchers ,18,19,20atalysts ,17,21, matalysts , phosphaatalysts ,25,26,27atalysts ,28,29,301.1O showed the highest performance (88.4% conversion of catechol and 84.1% selectivity of guaiacol). These reported aluminophosphate catalysts usually suffer a serious problem of lower guaiacol selectivity and bad stability, increasing the cost of product purification or limiting the application in actual production. Hence, it is still important to develop a simple approach to fabricate aluminophosphates with higher activity, selectivity, and stability for vapor-phase O-methylation of catechol with methanol. It has been reported that the catalytic performance of aluminophosphate catalysts could be significantly improved by optimizing catalyst compositions, improving catalyst preparation processes, using additional ligand, carefully selecting operating conditions, etc. [Liao et al.  found thns, etc. ,25,26.xP-O) were prepared by a P123-assisted one-pot method at different P/Al molar ratios, which exhibited excellent catalytic activity, guaiacol selectivity, and stability for vapour-phase selective O-methylation of catechol and methanol. The effects of the P/Al molar ratio and P123 addition on catalyst structure and surface acid-base characteristics were investigated in detail.In this work, a series of Al-P-O catalysts (Al-20PO70EO20) was bought from Sigma-Aldrich Reagent . Other chemicals were supplied by Sinopharm Chemical Reagent Co., Ltd. .All chemicals were purchased and used without further purification. Pluronic P123 (EO3 (99.0%) was used as the aluminum source, H3PO4 (85%) was used as the phosphorus source, and the block copolymer P123 (Mav = 5800) was used as the additional ligand. A series of Al-P-O catalysts with different P/Al molar ratios were prepared using a P123-assisted one-pot method. Typically, 44.5 g AlCl3 and 16.9 g P123 were firstly dissolved in 200 mL of deionized water respectively. After that, the aqueous solution of AlCl3 and P123 were directly mixed with vigorous magnetic stirring and heated to 60 \u00b0C in a water bath. Then, a required H3PO4 was poured into the mixed aqueous solution. Following this, 6 mol/L ammonium hydroxide solution were dropped into the above solution until pH value of the mixture was ca. 9. The mixture was further stirred for 1 h. Then, the mixture was evaporated at 80 \u00b0C, dried at 100 \u00b0C for 12 h, and calcined with a heating rate of 2 \u00b0C/min in air at 550 \u00b0C for 12 h in sequence. The prepared materials were labelled as Al-xP-O, where x represented Al/P molar ratio.AlCla was also prepared by the same approach as Al-1.1P-O without P123 addition.For comparison, Al-1.1P-O\u03b8) ranged from 10\u00b0 to 70\u00b0, and the scanning speed was 8\u00b0/min. The crystallite size of AlPO4 was calculated using the full-widths at half maximum (FWHM) of the AlPO4 (0 2 0) peak through the Scherrer equation.Powder X-ray diffraction (XRD) measurements of the samples were performed with a Germany Bruker D8 Advance ray diffractometer using Cu K\u03b1 radiation at 40 kV and 40 mA. The diffraction angle (22 adsorption-desorption isotherms were performed on the Micromeritics ASAP 2020 sorptometer at liquid nitrogen temperature (\u2212196 \u00b0C). Before the measurement, each catalyst was degassed at 250 \u00b0C for 5 h. The specific surface area (SBET) was calculated by the Brunauer-Emmett-Teller (BET) method within the range of relative pressure P/P0 = 0.10~0.30. The average pore size (Da) was calculated using the BJH method. The pore volume (Vp) was set as the single point value when P/P0 was 0.990.NSEM images were acquired by employing a FEI Nova nanoSEM 450 electron microscope. The samples were sprayed with metal layer before testing.FT-IR spectra were performed on a Germany Bruker TENSOR 27 Fourier transform infrared spectrometer. The structure of the sample and the vibration of the skeleton were made using a KBr support chip. .3 (CO2) as the probe molecule and TCD as the detector. Prior to the measurement, a 100 mg sample was accurately weighed and placed in a quartz tube and then heated to 400 \u00b0C at a rate of 10 \u00b0C/min under Ar flow (30 mL/min) for 1 h to remove the moisture in samples and the residual gas in the test system. Then it was cooled to 50 \u00b0C and exposed to NH3 (CO2) for 0.5 h. Ar flow (30 mL/min) was purged from the quartz tube to remove the physical adsorption of NH3 (CO2) from the sample surface. Finally, the sample was heated at a rate of 10 \u00b0C/min to the specified temperature in Ar flow (30 mL/min), and the desorption temperature of NH3 (CO2) was detected using a thermal conductivity detector (TCD). The total surface acidity and basicity of the samples were determined from the TPD of NH3 and CO2, respectively.Temperature programmed desorption (TPD) was performed on a Biode PCA-1200 chemisorption analyzer for the surface acid (base) center strength and number of the catalysts by using NHThe TGA of the catalysts was investigated on a Netzsch STA 4449 F3, which was used to measure the carbon deposition of the catalyst after reaction.O-methylation synthesis of guaiacol by catechol and methanol was performed in a self-made fixed-bed reactor at atmospheric pressure with a length of 750 mm and an inside diameter of 14 mm quartz tube. The catalyst evaluation system was shown in Vapour-phase selective Ccatechol), selectivity of guaiacol (Sguaiacol), selectivity of 1,2-dimethoxybenzene , selectivity of other byproducts (Sother byproducts), and yield of guaiacol (Ycatechol) were calculated using the normalization method, with catechol as the reference substance. The details are shown in Equations (1)\u2013(5):f1, f2, and f3 represented the correction factor of guaiacol; 1,2-dimethoxybenzene; and other byproducts, respectively. In view of the similar properties and low content of these byproducts, we calculate the other byproducts as a whole. The calibration factor of 2-methoxy-6-methylphenol is used to calculate other byproducts, where A, A1, A2, and A3 represent chromatographic peak areas of catechol; guaiacol; 1,2-dimethoxybenzene; and the sum of peak areas of all byproducts.The conversion of catechol  catalysts, together with that of Al-1.1P-Oa for comparison. The Al-0P-O sample showed three strong diffraction peaks of \u03b3-Al2O3 around 2\u03b8 = 37\u00b0, 46\u00b0, and 67\u00b0. When the P/Al molar ratio increased to 0.25, it was interesting to note that the diffraction peaks intensities of \u03b3-Al2O3 decreased significantly, and a new weak and broad diffraction peak corresponding to amorphous aluminum phosphate around 2\u03b8 = 24\u00b0 appeared [2O5 were observed. These results demonstrated that the addition of P resulted in forming amorphous aluminum phosphate rather than a mixture of Al2O3 and P2O5. With the increasing of the P/Al molar ratio to 0.5, the diffraction peak around 2\u03b8 = 24\u00b0 was observed clearly. Concurrently, the diffraction peaks corresponding to \u03b3-Al2O3 completely disappeared. With further increase of the P/Al molar ratio to 1.1, the intensity of aluminum phosphate diffraction peaks kept no obvious change. When the P/Al molar ratio further increased to 1.15, four sharp diffraction peaks assigned to aluminum phosphate around 2\u03b8 = 20.4\u00b0, 21.6\u00b0, 23.2\u00b0, and 35.8\u00b0 were observed, corresponding to lattice planes of (0 2 0), (2 1 1), and (2 1 2) of tridymite and a lattice plane of (2 6 0) of \u03b1-cristobalite, respectively [4AlP2O7 were also noticed around 2\u03b8 = 29.8\u00b0, 31.1\u00b0, and 38.7\u00b0. The intensity of AlPO4 crystalline phase and NH4AlP2O7 crystalline phase became stronger in the Al-1.20P-O sample than in Al-1.15P-O. 4 calculated using the Scherrer equation. AlPO4 crystallite sizes were 30.9 nm for the Al-1.15P-O and 34.3 nm for the Al-1.20P-O, respectively. These results suggest that AlPO4 crystalline phase and NH4AlP2O7 crystalline phase formed when the P/Al molar ratio reached 1.15. Larger crystalline sizes are easier to form on Al-xP-O with higher P/Al molar ratios. Compared with the Al-1.1P-O, the Al-1.1P-Oa sample showed distinct crystalline phases of AlPO4 and NH4AlP2O7. These results demonstrated that the presence of P123 in the precursor solutions could restrain the formation of crystalline phases in regard to Al and P species.appeared ,30,31. Nectively . In addiSBET) of 319 m2 g\u22121, a pore volume (VP) of 2.03 cm3 g\u22121, and an average pore size (Da) of 21.6 nm, whereas those of the Al-1.10P-O were 147 m2 g\u22121, 1.08 cm3 g\u22121, and 23.9 nm, respectively. However, Al-1.15P-O only had a SBET of 30 m2 g\u22121, VP of 0.10 cm3 g\u22121 and Da of 14.2 nm. Compared with the Al-1.1P-O sample, the Al-1.1P-Oa sample-prepared absence of P123 showed smaller specific surface area, pore volume, and average pore size. Combined with XRD results, it can be speculated that P addition can bond with Al3+ to form aluminum phosphate, further altering the basic textural properties of the Al-xP-O samples. Higher P/Al molar ratios decreased specific surface area, pore volume, and average pore size. The existence of P123 favors a higher specific surface area, pore volume, and average pore size.The basic textural properties of the prepared Al-P-O samples are summarized in xP-O samples are showed in x > 1.1) were easy to condense to particles with crystal phase structure, leading to a prominent decrease in the specific surface area of samples, which was consistent with XRD and BET results. As for the Al-1.1P-Oa sample, a dense and less porous morphology was observed.The microscopic morphology of Al-xP-O samples are presented in \u22121 and 1634 cm\u22121, which were attributed to the stretching vibration the O-H bond and bending vibration of physically adsorbed water, respectively [\u22121, 730 cm\u22121 and 490 cm\u22121. The vibration signal at 1139 cm\u22121 could belong to the asymmetric stretching vibration of the P-O bond in the (PO4)3\u2212 tetrahedron. The vibration signal located around 490 cm\u22121 was attributed to the bending vibration of O-P-O in the (PO4)3\u2212 tetrahedron. The vibration signal around 730 cm\u22121 was assigned to the vibration of Al-O bond combined with P-O bond [x \u2264 1.1, Al-xP-O samples had a weak vibration signal near 730 cm\u22121, this indicated that Al-xP-O samples (x < 1.1) had an amorphous aluminum phosphate structure instead of a simple mixture of Al2O3 and P2O5. When x > 1.1, the vibration signal of the Al-xP-O samples at 730 cm\u22121 was significantly enhanced. This strong vibration signal should be caused by the formation of crystalline aluminum phosphate phase, which is consistent with the results of XRD characterization.FT-IR spectra of Al-ectively ,33,34,35P-O bond ,31,36,373-TPD profiles and surface acidic and basic parameters of Al-xP-O and Al-1.1P-Oa samples are displayed in 3 desorption temperature with the increasing P/Al molar ratio, indicating that the addition of P weakened the strength of the acid site. The area of the NH3 desorption peak increased when the P/Al molar ratio increased from 0 to 0.75. With the increase of the P/Al molar ratio to 1.10, the area of the NH3 desorption peak decreased slightly. However, when the P/Al molar ratio further increased to 1.15 and 1.2, a sudden decrease of the area of the NH3 desorption peak appeared, indicating that the number of acid sites of catalysts with higher P/Al molar ratio (x \u2265 1.15) was very low. It could be speculated that the P-OH group was the main source of the weak acid center. The catalyst with the higher P/Al molar ratio (P/Al > 1.1) eased the formation of aluminum phosphate crystal phase, resulting in a low number of acid centers. Compared with the Al-1.1P-O sample, Al-1.1P-Oa had a lower NH3 desorption temperature and a smaller area of NH3 desorption peak, proving that P123 addition could boost the formation of stronger acid sites.NH2-TPD profiles of Al-xP-O samples are shown in 2 desorption temperature in the range of 100\u2013350 \u00b0C. The desorption peak in the ranges of 100\u2013200 \u00b0C and 200\u2013350 \u00b0C were assigned to weak basic site and medium-strength basic site [2 desorption peaks shifted towards low temperature with the increasing P/Al molar ratio, indicating that the strength of the basic sites gradually decreased. However, the area of the CO2 desorption peak for the catalysts with a P/Al molar ratio between 0.25 and 1.1 showed no obvious changes, whereas the area of the CO2 desorption peak decreased significantly in Al-xP-O samples (P/Al > 1.1). In the case of Al-1.1P-Oa, a lower CO2 desorption temperature and a smaller area of the CO2 desorption peak than those of the Al-1.1P-O sample indicate that P123 addition could promote the formation of stronger basic sites.COsic site . The CO2xP-O catalysts for vapour-phase selective O-methylation of catechol and methanol to guaiacol was investigated under optimized reaction conditions, and the results are listed in x = 0.75. As the P/Al molar ratio increased further, the catalytic activity started to decrease, and the catechol conversion decreased to 58.0% at x = 1.10, further increasing the P/Al molar ratio to x = 1.15, only a 25.2% conversion. These results were in accordance with the number of acid sites measured by NH3-TPD shown in a catalysts without P123 showed lower catechol conversion of 43.6% compared to Al-1.1P-O (catechol conversion of 58.0%) but also showed a high selectivity of 97.7%. Combined with the characterization results, the Al-xP-O catalysts which possessed weak acid-base sites exhibited excellent guaiacol selectivity with higher catalytic activity. Although the Al-0.75P-O catalyst had the highest yield, it was worth noting that multiple byproducts were generated, including 3-methylcatechol; 2-methoxy-6-methylphenol; 2,3-dimethoxytoluene; etc. Poor selectivity certainly would increase the cost of product separation. Therefore, Al-1.10P-O catalysts, which exhibited a higher selectivity of 97.6% with a comparatively higher conversion of 58.0%, was considered to be chosen for further stability study.The catalytic performance of Al-cat = 6 g, Reaction temperature = 275 \u00b0C, LHSV = 0.6 h\u22121, methanol/catechol = 6 mol. To understand the effect of P123 on the stability of the catalyst, a stability test was also carried out on an Al-1.10P-Oa sample under the same conditions. As can be seen in a, catechol conversion and guaiacol selectivity decrease from ca. 43.6% and ca. 97.7% to ca. 18.0% and ca. 95.9%, respectively, during the 100-h reaction time.The catalytic stability test was conducted on the Al-1.1P-O catalyst under the reaction conditions ma catalyst (denoted as Al-1.1P-Oa-S100) after 100 h for O-methylation of catechol. The samples exhibited a first weight loss in the low temperature range of ca. 50\u2013250 \u00b0C, corresponding to physically adsorbed commands. This process was followed by the removal of the carbon deposited on the catalyst surface in the range of ca. 250\u2013650 \u00b0C, which was used to quantify the amount of deposited carbon. When the temperature reached 650 \u00b0C, negligible weight loss was detected. The results of the amounts of carbon depositions are given in a-S100 were three times as high as those on Al-1.1P-O, indicating that the addition of P123 was indeed beneficial to inhibit and eliminate the carbon deposition. Combined with the catalyst characterization results, we speculated that smaller pore volume suppressed the large molecular size byproducts detaching from the catalyst surface. As the reaction went on, the active center of the reaction was gradually occupied, leading to the gradual decline of catechol conversion.It has been established that carbon deposition is mainly responsible for the deactivation of acid/base catalysts. Thus, TG was employed to investigate the amount of carbon depositions on the spent Al-1.1P-O catalyst after 300 h (denoted as Al-1.1P-O-S300) and the spent Al-1.10P-OxP-O samples were prepared through a P123-assisted one-pot method and used for O-methylation of catechol and methanol to produce guaiacol. Al and P species were mainly in the aluminum phosphate phase. When x \u2264 1.1, the aluminum phosphate phase was amorphous, and the aluminum phosphate in crystalline phase with large particle size formed in Al-xP-O samples with higher P/Al molar ratios (x > 1.1). P123 addition could inhibit the formation of aluminum phosphate crystal phase to a certain extent. P/Al molar ratio and P123 addition had a significant influence on crystal structure, acid-base sites, and basic textural properties. All prepared samples showed weak acid-base sites, which were beneficial to producing guaiacol with catechol and methanol. The Al-1.1P-O catalyst prepared with P123-assistance exhibited superior catalytic performances with ca. 58.0% catechol conversion, ca. 97.6% guaiacol selectivity, and 300 h stability.In summary, Al-"}
+{"text": "Both linguistic and extralinguistic consultations are essential in translation practice and have been commonly investigated as an integral topic in previous studies. However, since extralinguistic information is usually longer in extent and not specifically designed for a linguistic purpose, extralinguistic consultations involve different search strategies compared with linguistic consultations. Drawing on eye-tracking and screen-recording data, this study compares linguistic and extralinguistic consultations in terms of cognitive resources allocation and information processing patterns in English\u2013Chinese translation. It also explores the differences among 17 language learners, 20 student translators, and 21 professional translators, and the effect of extralinguistic consultation on their translation quality. The findings are as follows: (1) all participants allocate more attention and lower cognitive load to extralinguistic consultations than to linguistic consultations; (2) participants\u2019 translation experience levels and their attention allocated to extralinguistic consultation show an inverted U-shaped relationship; and (3) participants who consult extralinguistic information before drafting or devote more attention to extralinguistic consultation produce target texts with significantly higher scores. Online consultation behavior in translation can normally involve two types of information from external resources: linguistic information, such as entries in an online dictionary, and extralinguistic information, such as cultural and subject-domain information provided by a general website. Linguistic information is more commonly consulted by translators than extralinguistic information, as is indicated by translators\u2019 greater reliance on using dictionaries  and a loBelonging to different information search tasks, linguistic and extralinguistic consultations are triggered by different information needs and involve different search processes. Linguistic consultation is normally triggered by more specific information needs, such as the meaning of a particular term. It belongs to the fact-finding task type, which is defined as \u201ca task in which you are looking for specific facts or pieces of information\u201d , p. 1005Against this background, this study seeks to uncover the nature of extralinguistic consultation and its impact on translation process and product. Eye-tracking (supported by screen-recording) as the main data collection method will be used, as this approach gives \u201ca detailed picture of the complex processing involved in constructing meaning from a string of words or characters and representing that meaning in the words or characters of a new language\u201d , p. 33. Behavioral measures and cognitive load have been widely applied in exploring the differences between fact-finding and information-gathering tasks. Behavioral measures include the amount of time spent and the number of pages viewed during information-seeking tasks. Previous studies have consistently found that information-gathering tasks tend to be significantly longer and involve more webpages when compared with fact-finding tasks . CognitiSince Secondly, when encountering the need for extralinguistic information, inexperienced translators are prone to consulting linguistic information, while experienced translators tend to seek help from extralinguistic information. In sum, compared with experienced translators, inexperienced translators show a heavier reliance on external resources and prefer to search for extralinguistic information in linguistic resources. It is worth noting that most existing studies have only analyzed the differences in extralinguistic consultation between experienced and inexperienced translators at the text/document level, without giving attention to the analysis of processing units (chunks or segments). Recognizing this limitation, the present study compares the differences in extralinguistic consultation not only at the text level but also at the processing unit level.The effect of extralinguistic consultation on translation quality has been investigated in previous studies from two aspects: (1) the impact of extralinguistic consultation on translation quality and (2) the quantity and quality of extralinguistic information consulted during translation.Regarding the first aspect, it has been found that translators with extralinguistic knowledge of the source text produce better translation products. Regarding the effect of the quantity and quality of extralinguistic information on translation quality, To summarize, previous studies have found that translators who have access to extralinguistic knowledge on ST produce significantly better translation products. However, translators who consult a greater amount of extralinguistic information do not necessarily produce better translations. The present study moves a step further by investigating the impact on translation quality from two perspectives: (a) comparing the impact of extralinguistic consultation before and during the drafting phase and (b) improving the method of measuring the information quantity by applying eye-tracking data.Sixty-eight native Mandarin Chinese speakers with English as their second language were recruited as participants on a voluntary basis. None of them had been brought up in a bilingual environment. They were all touch typists and had normal or corrected-to-normal vision. They were told that anonymity and confidentiality would be ensured, asked to sign a consent form, and rewarded with a supermarket gift card. The experiment was approved by the research ethics committee of a United Kingdom University.SD\u2009=\u20090.78). They were undergraduates in their junior or senior year majoring in English language and literature at Chinese universities with an IELTS overall score \u22657.0. The group of student translators (S1 to S23) consisted of 23 participants  with an average age of 23.45\u2009years . They all had an IELTS overall score \u22657.0, finished a 1-year United Kingdom-based MA program in Translation Studies but with no professional translation experience. The group of professional translators (P1 to P23) consisted of 23 participants  with an average age of 44.09\u2009years . They were all full-time university lecturers on translation courses, with at least 5\u2009years of freelancing translation experience  and more than 200,000 words translated. In sum: the language learners had neither translation training nor professional work experience; the student translators only had translation training experience; and the professional translators had both types of experience.Based on their training and work experience, the participants were categorized into three groups: language learners, student translators, and professional translators. The group of language learners (L1 to L22) consisted of 22 participants  with an average age of 21.55\u2009years  tools. After the translation task, participants were asked to complete a questionnaire about their educational and professional background and web-searching experience. They were also asked to rank their familiarity with the source text using a five-point Likert scale . A ranking of five meant that the participant\u2019s extralinguistic consultation might be influenced by their being \u201cvery familiar\u201d with the subject matter, and their data would then be eliminated. In this study, the data from P23 was discarded as the participant reported themself to be very familiar with Jewish culture.All experiments were prepared and run in a lab with an eye-tracker connected to a 23\u201d LCD monitor which functioned as the presentation screen. The screen resolution was set at 1280*1024 pixels, and the fixation radius was the default setting of the Tobii system, 35 pixels per inch. To suit the eye-tracker-based design, we purposely split the screen into two equal areas shown in , with thThe process data were collected by eye-tracking and screen-recording methods. Eye-tracking data provided the following three fixation-related measurements.This measurement was indicated by total fixation duration (TFD) on a selected Area of Interest AOI; . In the In reading and translation studies, this measurement has commonly been indicated by average fixation duration , with loThis measurement refers to the ways in which participants scan and read the webpages under consultation, and is reflected in two visualization tools: gaze plots and heat maps ,b. Gaze Screen-recording videos were transcribed for further investigation with the aim of creating a script \u201cresembling \u2018stage directions\u2019\u201d , p. 76 aProduct data include the quality assessments of the target texts. In the past two decades, a variety of assessment methods have been developed and applied in translation teaching and practice, such as calibration of dichotomous items , comparaIn the present study, we used the Multidimensional Quality Metrics (MQM) framework. Developed by the QT Launchpad project , this frTwo professional translators were invited to assess all target texts. They had prior experience of using the MQM framework and were thoroughly familiarized with the official guidelines and assessing procedure. Each of them was given a portfolio which includes the source text, a reference translation, and 58 target texts. They were asked to identify problematic issues into categories of accuracy penalty (AP), fluency penalty (FP), or verity penalties (VP), and to annotate them as minor, major or critical.After receiving all the evaluation reports, we calculated the penalty points and, subsequently, the scores of translation quality (TQ), using the following formulae , p. 6:p\u2009<\u20090.001, indicating an almost perfect agreement between the raters  the amount of attention indicated by TFD; (b) cognitive load indicated by average fixation duration; and (c) the reading path reflected in heat maps and gaze plots.t-tests were utilized for the comparison \u201cin which the same participants are tested at two different times or for two different treatments\u201d (t(16)\u2009=\u20091.597, p\u2009<\u20090.05, d\u2009=\u20090.81] and student translators , but not for professional translators .As can be seen from atments\u201d , p. 104.t-tests confirm that the differences were statistically significant for all three groups .Regarding cognitive load, As for viewing patterns, we examined the heat maps and gaze plots of the linguistic and extralinguistic consultations. In general, for linguistic consultations, regardless of their experience levels, the participants focused on target pages from online dictionaries or the lexical information sections from search engines. Their fixations formed a horizontal path and were concentrated on the relevant information, which was located in a small area of the webpage. Whereas for extralinguistic consultations, the participants tended to read SERPs, content from encyclopedias, or relevant information portals. Their fixations were scattered over a larger area including more information, and followed a vertical path.In summary, linguistic and extralinguistic consultations possess the characteristics of fact-finding and information-gathering tasks, respectively. Compared with linguistic consultations, extralinguistic consultations attracted significantly larger amounts of attention, and required lower cognitive load and more scanning.In this section, we will explore the differences in extralinguistic consultations across the three groups of participants from two levels: (a) when they translate the entire text and (b) when they translate selected Rich Points. It is important to note that a comparison between the language learners and student translators, and a comparison between the student and professional translators, are carried out separately. This is because the language learners and the student translators mainly differ in their training experience, while the student and professional translators in their professional experience. The language learners and professional translators are different in both aspects of translation experience, training, and work experience, so that the comparison between these two groups is not considered to be reliable.t-tests were the parametric tests to determine \u201cwhether the means of the two independently measured groups differ at a statistically significant level\u201d (t(35)\u2009=\u2009\u22121.860, p\u2009>\u20090.05], but the difference between the student and professional translators was statistically significant .When translating the entire text, the reliance on extralinguistic consultations was indicated by the proportion of TFD allocated to extralinguistic consultations over the amounts of TFD on the entire consultation process see . Among tt level\u201d , p. 91. Another difference at the text level was whether the participants performed a pre-translation preparation, which was reflected in the timestamp associated with the extralinguistic consultations. For those participants who conducted preparation before drafting, they followed almost the same procedure: read through the source text and then use search engines to find subject information. But for those without preparation, we noticed an intriguing difference across the three groups. A large number of language learners  and student translators  who did not conduct pre-translation preparation consulted extralinguistic information during drafting. However, this kind of postponed extralinguistic consultation was rarely seen among the professional translators .We further compared the differences across the three groups of participants in extralinguistic consultations when translating selected Rich Points. The investigation was based on two metrics: (a) the proportion of attention (indicated by TFD) distributed to extralinguistic consultations over the amount of attention distributed to the entire consultation process and (b) translators\u2019 consultation styles and their preference toward linguistic or extralinguistic consultations.t-tests . Student and professional translators, on the other hand, did not change their consultation style much when translating this Rich Point. Student translators always sought help from extralinguistic information no matter whether linguistic consultations were successful or not, whereas professional translators tended to rely on their internal knowledge when they failed with linguistic consultations.The effect of extralinguistic consultation on translation quality was investigated from three perspectives: (a) comparing the quality with or without preparation before drafting; (b) comparing the quality with pre-task preparation or with consultation during drafting; and (c) examining the correlations between the quantity of extralinguistic information consulted (indicated by the amount of TFD) and translation quality.t-tests show that the differences were statistically significant .t(13)\u2009=\u20093.098, p\u2009<\u20090.05, d\u2009=\u20090.80 for language learners; t(16)\u2009=\u20091.589, p\u2009<\u20090.05, d\u2009=\u20090.67 for student translators; and t(11)\u2009=\u20092.568, p\u2009<\u20090.05, d\u2009=\u20090.78 for professional translators].We then compared the translation scores of the participants who had extralinguistic consultations before and during drafting see . The resThe Spearman correlation coefficients were conducted to measure the strength of the linear relationship between the amount of attention allocated to extralinguistic consultations and translation quality . The resOur results confirm that linguistic and extralinguistic consultations possess the characteristics of fact-finding and information-gathering tasks, respectively. Three differences between these two types of consultation can be summarized as follows. Firstly, translators allocate a larger amount of attention to extralinguistic consultations than to linguistic consultations. This difference is in line with previous findings on general information tasks: information-gathering tasks take longer to accomplish than fact-finding tasks . SecondlBased on these findings, we propose two implications for translation pedagogy and further research. Firstly, translation teachers should consider ways of improving the efficiency of extralinguistic consultations. For example, they could demonstrate the use of built-in search functions, which could highlight keywords on the SERPs and accelerate the scanning process. Secondly, since linguistic and extralinguistic consultations reveal different characteristics and require different search techniques, further studies should consider extralinguistic consultation as a sole research object rather than an integral part of consultation, for an optimal result.In translating an entire text, two differences in extralinguistic consultations are found across three groups of translators. Firstly, translators\u2019 experience levels and the proportion of attention allocated to extralinguistic consultations forms an inverted U-shaped relationship: language learners devote insignificantly less attention to extralinguistic consultation than student translators, while professional translators have a significantly lower reliance on extralinguistic consultation than student translators. This finding is consistent with Another difference reported in the present study relates to translators\u2019 consultation styles regarding how they search for subject knowledge. Non-professionals (language learners and student translators) who do not consult extralinguistic information before drafting tend to search for extralinguistic information during drafting, whereas professional translators without extralinguistic consultation before drafting do not conduct similar behaviors. In translating selected Rich Points, translators with different experience levels perform different styles of extralinguistic consultation. Based on this model, translating \u201clatke\u201d and \u201cthe \u2018attendant\u2019 candle\u201d without extralinguistic consultation could be considered as two different tasks: one with a low chance of non-cooperation, and the other with a high chance of non-cooperation. Facing the difference in uncertainty between the two tasks, student translators and professional translators exercise the same level of effort. Students always devote a high level of effort even though this may be inefficient labor; whereas professionals, who are more confident in their internal knowledge, devote a lower level of effort to consulting external information. This finding is in line with This study shows that translators who prepare with extralinguistic knowledge before drafting produce significantly better target texts. This finding is consistent with The present research also reports that translators who consult a greater amount of extralinguistic information produce better target texts. This differs to the finding reported by Using a combination of eye-tracking and screen-recording data, this study uncovered the nature of extralinguistic consultation by exploring the differences between linguistic and extralinguistic consultations. It further provided pedagogical suggestions for translation training by comparing extralinguistic consultation behaviors across translators with different experience levels and through investigating the effect of extralinguistic consultation on translation quality. In summary, this study shows that: (a) translators, irrespective of their translation experience, allocate a larger amount of attention, a lower cognitive load, more scanning and less deep processing to extralinguistic consultations than to linguistic consultations; (b) translators\u2019 experiences and the proportion of attention allocated to extralinguistic consultations forms an inverted U-shaped relationship; (c) non-professionals who do not conduct pre-translation preparation tend to consult extralinguistic information during drafting, whereas professional translators rarely conduct extralinguistic consultation during drafting phase; (d) translators with different experience levels adopt different risk management strategies: non-experts have a heavier reliance on external information and tend to confirm search outcomes by consulting multiple information sources; while professional translators have a lower level of dependency on extralinguistic information and prefer solving problems with their internal knowledge; and (e) among translators with the same level of experience, those who prepare with subject knowledge before drafting or who devote more attention to extralinguistic consultations produce target texts with significantly higher scores.Our research evidences the importance of extralinguistic consultation in producing high-quality translation and provides implications for improvements in translation pedagogy and for future research. We suggest that pre-task preparation, which has been widely studied in sight translation and interpreting, should receive more attention in written translation from translation instructors and researchers.This study has certain limitations since it has been conducted using one source text under one experimental environment. Consequently, this study could be replicated with multiple specialized source texts, such as legal documents and medical reports, and with more language directions. It could be designed as a longitudinal study on translation students regarding the development of their extralinguistic consultation skills. In this way, it would be possible to (a) collect data on more diverse extralinguistic consultations; (b) investigate how the unique linguistic features of Mandarin could have an effect on translators\u2019 consultation behaviors; and (c) provide more suggestions for translation pedagogy based on translation students\u2019 attitude toward the training.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the ethics committee of Durham University. The patients/participants provided their written informed consent to participate in this study.YC designed the study, collected, annotated, and analyzed the data, and wrote the original draft. BZ conceptualized and designed the study, reviewed and edited the manuscript, and supervised the project. The two authors contributed equally to the article and approved the submitted version.The work was supported by the National Social Science Fund of China (no. 20BYY014). YC is funded by the China Scholarship Council (CSC no. 201808060310).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": "In this study, virtual product development method for reducing vibration and noise is proposed for designing at the concept development stage of a vehicle. To this end, the vibration characteristics of the system are predicted through the Lagrange-multiplier frequency-based substructuring technique. The concepts of contact, blocked and transmitted force, and force transmissibility were used for determining the improvement subsystem or combination of subsystems when using the modular platform. Moreover, after the subsystems to be improved were determined, Artificial Neural Network was used as a method of predicting vibration characteristics according to the change of design variables. To verify this, the prediction of the blocked force was performed by changing the young\u2019s modulus of the simplified substructure. Finally, the reduction in response was confirmed by applying the blocked force of the simplified subframe to the simplified structure, and a vehicle development process using a database at the concept setting stage is proposed. A general vehicle development process can be summarized into three steps: concept-design-prototype testing. Engineers try to solve many problems in the concept development stage of a vehicle, but after the production of the prototype, many unexpected problems are identified. Thus, the engineers need to go back to the design stage again and try to solve the problems. Such a general development process is time-consuming and cumbersome, thereby increasing inefficiency and making it difficult to respond quickly to the market.Studies have been conducted on the product development process to respond quickly to the market. Among them, the set-based design (SBD) process, which finds a new combination set of subsystem based on previous data, is representative.1. Through SBD, engineers determine and limit the range of the design variables2. A study in which SBD was applied to vehicle design by proposing these alternative sets was useful in the preliminary stages of detailed design3. Durward et al. claimed that if vehicle development teams and marketing teams participated in the development process in the manner of point-based serial design process, all organizing teams must review according to all changes, and there is no guarantee that the results would be converged4. However, in the proposed set-based concurrent engineering, assorted designs are proposed in advance, and the final draft is drawn out through collaboration with other teams. The proposed SBD method establishes a map of the performance space. In other words, from the perspective of full vehicles, each team determines the range of major performance. In addition, SBD builds feasible sets by integrating the space considered by each team. And SBD derives results by gradually narrowing the established sets through inter-team communication. Furthermore, uncertainty according to the process progress is controlled through these sets. However, this method makes it difficult to derive appropriate results if the vehicle\u2019s performance map is tightly integrated.SBD presents a series of alternatives instead of a single solution6. Volkswagen\u2019s modular platform fixed specific sections in engine rooms with high design complexity and focused on applying standard parts. In terms of the fixed sections, the distance between the pedals in the front axle was fixed. Through this, it is easy to achieve significant cost reduction. Toyota\u2019s modular platform fixed the length of the underbody and focused on applying standard parts within the underbody. It is characterized by standardizing large parts that have excellent commonization deployment effects such as engines, transmissions, and suspension parts. In order to maximize these advantages, it is necessary to convert the results of previously developed common parts into databases and use them in the preliminary stage of vehicle development. This is because if the performance of the product is preemptively predicted at the vehicle development concept stage, decision-making based on reasonable data is enabled, and time and effort to improve the product can be saved in the design stage.In summary, the set-based design process is a method that is used in the concept stage based on previous data. Also, it is possible to proactively respond to the problems of the prototype testing stage since it helps the researchers know many problems in advance through preceding development. Moreover, it provides flexibility in the occurrence of issues in the development process. Recently, automobile manufacturers have established a modular platform commonization strategy by using the advantages of this set-based design process. In particular, Volkswagen and Toyota have continuously deployed new models and expanded their lineups to respond to customer needs in various regions, and at this time, they are pursuing a component commonization strategy to curb rising vehicle costs8.In general, the process of predicting characteristics in the preceding stage of vehicle detailed design is called Virtual Product Development (VPD), and CAD/CAE are used. Starting with the development of the algorithm in the 1970s, it has been widely studied in CAD/CAE design, and various application software has been developed9.VPD from a manufacturing and development perspective involves numerical analysis of 3D modeling. Substructuring, which is widely used among these analysis methods, is a technique that analyzes the system using dynamic characteristics of the structure. The advantage of the method is that it is modeled and analyzed in the most suitable domain. For example, it is possible to freely combine the experimentally measured results and analyzed dynamic characteristics. Moreover, models of various development groups can be shared and combined. Because the shared information requires only dynamic information (e.g. Frequency response function), the details  of the component model can be kept confidential. Therefore, it may be useful in joint development with partner companies11.Substructuring methods include the modal synthesis method, component mode synthesis method, and frequency response function based synthesis (FBS) method. Among these, FBS is a method widely used in recent years; it uses transfer functions in the frequency domain. To predict characteristics with FBS, the transfer function matrices and modal information are requiredA strategy for the combination of subsystems are required. When combining subsystems, it should be possible to find an appropriate combination from the perspective of a vehicle through strategies. This is because the characteristics of subsystem may be changed by connection with the other subsystem.A quantitative indicator to manage characteristics of subsystem in the database are needed. Since the characteristics of a subsystem change after the subsystem are connected to different subsystems, it is difficult to identify the characteristics unrelated to the connection. Therefore, it is necessary to find a quantitative indicator independent of the characteristics of the connection.It is necessary to predict the characteristics of subsystems that are not in the database. When design variables are changed, it is necessary to predict the characteristics of the system based on experimental and analysis data. Previously, analysis has been performed on the number of all cases by changing design variables. However, to perform analysis on many subsystems in the database is time-consuming.The following should be considered to use the substructuring methods in modular platform strategy:Methods for solving the aforementioned considerations are described below.12.Since forces of the system is transmitted through connection parts, it is necessary to compare forces transmitted to the subsystem to select a combination set of subsystems that satisfy the target performance. Therefore, it is natural to use the contact force, which is the force generated at the connection, as an indicator of combination selection. The contact force is obtained using Lagrange-Multiplier-Frequency Based Substructuring (LM-FBS). LM-FBS is a technique that predicts the responses of the multi-level subsystems through a Boolean matrix containing connection informationGeneral substructuring methods predict the characteristics of the system from the perspective of substructure impedance by first satisfying the compatibility condition of the displacement on the contact surface, then satisfying the equilibrium condition. However, this method directly obtains only the displacement between the contact surfaces. That is, LM-FBS establishes a formula to first satisfy the equilibrium condition from the perspective of substructure admittance, then the compatibility condition. LM-FBS has the advantage of directly obtaining contact force using a formula representing the magnitude of the contact force by the Lagrange Multiplier (LM), and easily performing assembly using a boolean matrix. Therefore, it is appropriately used for determining a combination of subsystems when developing a vehicle with a new concept based on data of various existing subsystems.13. It is used to predict the system\u2019s response when the subsystem to which the exciting vibration is connected changes in the experiment. Therefore, the blocked force presents characteristics independent of connection; thus, it is suitable for the performance management of product in the database.Moreover, it is necessary to manage the characteristics of subsystems in the database to combine subsystems based on the database, and because the characteristics change depending on the connection between subsystems, it is difficult to use the frequency response function or contact force. The blocked force, which has been widely studied in recent years, is a force that is independent of the characteristics of the connection to which subsystems are connected and is not subordinate to the interrelationship between the source and receiver14 derived the sensitivity calculation equation and design parameters for the eigenvalues and eigenvectors of the linear structure. Thereafter, Rogers15 and Garg16 improved Fox\u2019s research results and promoted the development of sensitivity methods. Nelson17 and Lim and Junkins18 simplified the sensitivity formula of feature vectors. However, model prediction by parametric or numerical study takes a lot of effort and time.The characteristics of a subsystem that is not in the database is predicted through a numerical method. In general, in complex subsystems, numerical characteristics prediction has been performed through the sensitivity analysis of models by performing parametric model modification. Variables commonly used in parameter correction include mass, stiffness, and damping matrices. By using the orthogonal criterion and solving the model\u2019s characteristic equation, Fox and Kapoor19 modified the model using a multilayer perceptron neural network and an error inversion algorithm. Atalla and Inman20 and Levin et al.21 modified models of one-dimensional frame structure and two-dimensional plate structure, respectively, through structural frequency response functions measured using the RBF neural network. ANNs are gradually expanding their scope of application22 and building a pretrained model has the advantage of taking less time than predicting using existing finite element methods when predicting new data. Therefore, it is necessary to examine the applicability of ANN to predict data that is not in the database when improving parts in the concept development stage of the vehicle development stage. If it is possible to learn using ANN and predict new improvement plans in advance, efficient improvement in product development time and effort is expected. If the improvement of subsystem is preemptively predicted in the concept stage from the perspective of vehicle characteristics, it is expected that the time and effort of detailed-level design and performance verification stage are reduced.In recent years, researchers have increasingly preferred using artificial neural network (ANN) algorithms. Typical types of neural networks are feedforward neural networks, multilayer perceptron neural networks, and radial basic functional (RBF) neural networks. Pandey and BarariThe rest of the paper is structured as follows. \u201cTraditional substructuring methods were based on the dynamic equations of motion. LM-FBS, which has been widely studied recently, has similarly been derived based on the dynamic equations of motion and is easily combined with other models using the Boolean matrix. This section introduces LM-FBS as a representative substructuring technique and summarizes it.To explain this, Fig.\u00a0Z(s)(\u03c9) is the system impedance, u(s)(\u03c9) the node displacement, F(s)(\u03c9) the external force, and Fc(s)(\u03c9) the contact force between connections.If the system is modeled in the frequency domain, it is presented as outlined in Eq.\u00a0.1\\documeB represents Boolean matrix containing connection information.The conditions of the system\u2019s connection (interface compatibility) are summarized as Eq.\u00a0 and expr\u03bb (LM) representing magnitude, it was outlined as presented in Eq.\u00a0  5) were Using admittance,Multiplying both sides of Eq.\u00a0 by BooleWhen Eq.\u00a0 was summY of substructures and B containing connection information is known, the final response point can be calculated according to Eq.\u00a0, E2 (105\u00a0*\u00a00.51 GPa), E3 (105\u00a0*\u00a00.52 GPa), E100 (105\u00a0*\u00a01.49 GPa). The blocked force to be predicted was set in the range of 1 to 250\u00a0Hz at 1\u00a0Hz intervals. In terms of the expression methods of blocked force, the two methods of Magnitude/Phase and Real/Imaginary part were compared. Therefore, the output is configured to predict the Magnitude/Phase and Real/Imaginary part of the Blocked force every hertz, and the prediction was repeatedly performed 250 times every hertz to predict the 1 to 250\u00a0Hz band. K-fold cross validation was used for data training. K-fold cross validation is a statistical analysis method in which the collected samples are cross-validated by making k-folds, and it can improve accuracy in cases where the total number of data is small. In addition, the early stop was applied to prevent overfitting. For the configuration of the neural network algorithm, the fitnet of Matlab, a commercial software, was used, and Bayesian regularization backpropagation was used for the training function. Training parameters consisted of the following: Marquardt adjustment parameter was 0.005, decrease/increase factor for Marquardt adjustment parameter was 0.1 and 10, respectively and minimum performance gradient was 1e\u22127.Here, subframe was selected as the part to be predicted. Moreover, the input of the neural network model was set to be Young's modulus, one of the design variables, and the output was set to predict the blocked force, an indicator of subsystem characteristics management Fig.\u00a0. Young'sThe number of nodes in Hidden layers was set differently for magnitude/phase and real/imaginary part of blocked force data, respectively. In predictions using Magnitude/Phase, hidden layers consist of two layers, each consisting of 10 and 8 nodes. Similarly, two hidden layers consist of 8 and 5 nodes. For each hertz, the input data was 1\u2009\u00d7\u2009100 vectors and the output data was composed of 1\u2009\u00d7\u2009100 matrices. That is, since the frequency band of interest is 1 to 250\u00a0Hz, the 250 by 100 matrix becomes the final output. The number of datasets is 100, which are randomly divided into training sets and test sets at an 8:2 ratio. Data preprocessing of input/output dataset was performed using MinMax normalization method.An example of mean squared error versus number of epochs for real/imaginary part of blocked force data is shown in Fig.\u00a0Based on the training results, the mean square error for the pretrained model is tabulated in Table On the basis of the results, it was found that training the real/imaginary part predicted more accurate results than training the magnitude and phase of the blocked force. The representative blocked force prediction result is expressed in Fig.\u00a0Based on the prediction results, it was identified that the magnitude of the blocked force was predicted with the error within 2.0% on average for the data, indicating that the results were similar to the results of analyzing the actual FEM. Therefore, if there were pre-trained model built with ANN, the blocked force of a new substructure could be easily predicted when developing another vehicle, and the characteristics of the system could be identified in advance; thus, an efficient development could be conducted.Finally, the lowest root sum square of blocked force at the interval of interest (1\u2013250\u00a0Hz) was selected and the response of simplified structure when the blocked force of the optimal subframe was connected to the frame was presented in Fig.\u00a0In this paper, substructuring and ANN based VPD method for designing at the concept stage was proposed. Vibration characteristics of the system according to the combination of subsystems were predicted through a dynamics-based substructuring technique. A simple vehicle frame structure was modeled and verified using an LM-FBS. Through this, it was confirmed that it was difficult to identify the characteristics of the system through the frequency response function of the subsystems when determining improved subsystems or combinations of subsystems. Because the frequency response function of the subsystems didn\u2019t reflected the connection characteristics, the contact force, an indicator that reflected the connection characteristics, was used as an indicator related to the combination of subsystems. Moreover, the transmitted force was introduced at the response point. Force transmissibility was used to select improved subsystems. This Force transmissibility methodology has the advantage of being able to clearly compare the transmission of forces between subsystems rather than the existing contribution evaluation or transfer path analysis, making it easier to identify the improvement subsystem. For subsystem management in the database, a blocked force that did not reflect the connection characteristics was introduced. It was confirmed through simulation that a blocked force independent of the connection characteristics was essential when applying the modular platform strategy because various combinations were made between subsystems.Finally, when subsystems in the database were subject to improvement, a blocked force that was not in the database was predicted using the ANN. In terms of the data representation methods for training the blocked force, it was confirmed that the method of expressing it with real and imaginary parts showed a higher accuracy than the method of expressing it with magnitude and phase. Finally, it was confirmed that the final response was improved by connecting the improved subframe with the original substructure. Such a method of improving subsystem using an ANN had the advantage in that it took less time and effort than the existing method using the finite element method; thus, it could be applied at the concept stage of vehicle development. Finally, it was confirmed that the response was reduced by connecting the optimal subframe to the original system, and a vehicle development process using a database at the concept stage was proposed."}
+{"text": "Scientific Reports 10.1038/s41598-021-93326-1, published online 02 July 2021Retraction of: The Editors have retracted this Article.After publication, concerns were raised about the nature of the samples used in this study, in particular that the cytokine and antibodies are diluted beyond the point at which any active molecules are expected to be present. Post-publication peer review confirmed that some of the methods used in this study are not sensitive enough to provide interpretable results at these concentrations. This means that without further corroborative evidence, the data presented in the paper are not sufficient to attribute the differences in the signal to the sample preparation method. The Editors therefore no longer have confidence in the results reported in this Article.Kristina N. Woods disagrees with this retraction."}
+{"text": "Plant roots have important functions, such as acquiring nutrients and water from the surrounding soil and transporting them upwards to the shoots. Simultaneously, they must be able to exclude potentially harmful substances and prevent the entry of pathogens into the roots. The endodermis surrounds the vascular tissues and forms hydrophobic diffusion barriers including Casparian strips and suberin lamella. Suberin in cell walls can be induced by a range of environmental factors and contribute to against biotic and abiotic threats. Tremendous progress has been made in biosynthesis of suberin and its function, little is known about the effect of its plasticity and distribution on stress tolerance. In field conditions, biotic and abiotic stress can exist at the same time, and little is known about the change of suberization under that condition. This paper update the progress of research related to suberin biosynthesis and its function, and also discuss the change of suberization in plant roots and its role on biotic and abiotic stresses tolerance. Arabidopsis, soybean, oats, barley and wheat , monounsaturated \u03c9-hydroxyacids, \u03b1,\u03c9-dicarboxylic acids and glycerol are the major monomers of suberin, followed by alcohols and unsubstituted fatty acids . SuberinThe biosynthetic machinery responsible for suberin productions is complex due to the chemical diversity of the suberin polymers. Chemical analysis and biochemical studies three decades ago were the initial steps to elucidate the biosynthesis pathways and structure of suberin . After t(ABC) transporters, lipid transport proteins and secretion through vesicles are thought to be involved in the export of these monomers or building blocks out of plasma membrane to the site of polymerization  toxicity and ammonium stress, which suggest that the function of suberin is to block the entry of toxic elements into the cytoplasm . Suberizt stress . In Arabt stress . The dect stress . In the t stress .Moreover, suberin might act as a barrier to prevent penetration by pathogens and nematode Figure\u00a02The growth of the human population will increase the demand for food in the future. However, the change of climate will intensify extreme weather conditions, for example drought, which might lead to decreased crop production. It is important to develop crops with increased yield and these crops should also be adapted to the specific soil conditions and climatic environment. The CSs and suberin lamellae in the endodermis of plant roots seem to play pivotal roles in controlling the uptake of water and nutrients and protect plants against biotic and abiotic threats . UnderstSuberin is not only deposited in the endodermis of plant roots but also in the bundle sheath of plant leaves, the vascular tissues in roots and leaves are thus surrounded by suberized cells. As a transcellular barrier, suberin has a similar function both in root and leaves, which affects the fluxes of solutes and pathogen penetration. Since the vascular tissues are continuous in the whole plant, it is necessary to analyse the function of suberin as a barrier in leaves and roots at the same time. Thereby, better understanding of the function of suberin as a barrier can be obtained. Finally, the biotic and abiotic (nutrient deficiency or toxic) stress can exist at the same time in the field condition. It is necessary to analysis the effect of suberization on pathogen penetration under abiotic condition.AC drafted and revised the manuscript. TL, ZW and XC revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the China Agriculture Research System (CARS-02), the natural Science Foundation of Chongqing (4312100205), China Postdoctoral Science Foundation (2020M673105) and Innovation and Entrepreneurship Program for College Students .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": "Roanoke is addressing problems that confront many small and medium sized cities in the U.S., especially disparities in health and life expectancy between neighborhoods. These disparities are often legacies of decades of racial and economic segregation, resulting in low-income or disinvested communities. Typically, such neighborhoods have fewer parks, higher vacancy rates and less stable affordable housing stock, inadequate public transit systems, too few clinics, too many fast food restaurants and insufficient access to high quality schools. In Roanoke these are the northwest and southeast quadrants, both federally designated Medically Underserved Areas, and characterized by a large proportion of the city\u2019s low-income individuals and families who may be uninsured, underinsured and/or Medicaid recipients. Journal of Appalachian Health by Bob Cowell Jr.,An article in this issue of the Together, Bob\u2019s two pieces describe collective public health activities in this mid-sized city that are remarkable in their depth, breadth and ambition. I have been researching the topic for over 5 years and am not aware of a more comprehensive program in the U.S. The constellation of actors working together includes city government, healthcare providers, schools, libraries, universities, colleges and a school of medicine, police and fire departments, free clinics, Habitat for Humanity, emergency shelters, local nonprofits, garden associations, countless volunteers, and more.Cowell cites the 2012 Community Health Needs Assessment as the catalyst for a collective response, and the genesis of the \u201cbackbone\u201d organization, Healthy Roanoke Valley, which brought together over 50 organizations and agencies. In his words, its mission was to \u201cmobilize community resources to improve access to care, coordination of services, and promote a culture of wellness.\u201dThe Roanoke Valley HOPE Initiative (RVHI) is illustrative. Offering treatment and recovery resources for individuals suffering from substance use disorders, RVHI is a collaborative effort among law enforcement, healthcare systems, treatment, recovery agencies, and safety-net organizations throughout the community. The coalition\u2014today there are fourteen partner agencies\u2014was formed in 2016 by the Roanoke Police Department, based on their acknowledgment that \u201cwe couldn\u2019t arrest our way out of this crisis.\u201d The HOPE Initiative has informed the community about the severity of the opioid (and now hepatitis C virus infection) epidemic, and successfully utilized peer recovery specialists and \u201cAngel\u201d volunteers.Another critical player is the Center for Community Health Innovation (CCHI) at Roanoke College. Founded and directed by Dr. Liz Ackley, its primary functions include helping residents and partner organizations collaborate on equitable community development projects, and to identify challenges and develop innovative strategies to enhance healthy living in the Roanoke Valley. The Center is working with the City of Roanoke on its 2040 Comprehensive Plan, helping to craft policies that promote health and equity. The Center directs the Roanoke Valley Community Healthy Living Index, an annual assessment that monitors health outcomes, engagement in healthy behaviors, and barriers to healthy living among the City\u2019s youth.The Healthy Living Index is a unique experiential opportunity for students\u2014service-driven data collection, analysis, and dissemination of materials that support local projects. \u201cThrough engagement with the Center, students gain real-world exposure to the processes involved in equity-driven, data-informed community development. This work substantiates the true value of a liberal arts education, as it requires students to develop the soft skills and technical skills necessary to work with partners in a variety of sectors, including residents, police officers, city officials, nonprofit leaders, and private developers,\u201d says Ackley.Roanoke is duly recognized for its progress. As Cowell points out, Roanoke has received seven All-America City Awards from the National Civic League and is the first city placed in its Hall of Fame. In addition, Roanoke was recently one of 12 cities chosen to join the National League of Cities pilot \u201cCities of Opportunity\u201d initiative. The program, which receives support from the Robert Wood Johnson Foundation, focuses on three major factors that affect health: economic opportunity, housing, and city planning and design.And I was pleased to learn recently that Roanoke College was the only academic institution among 23 grantees to receive financial and technical assistance awards through the Healthy Food Financing Initiative\u2019s (HFFI) inaugural grants program.So despite Cowell\u2019s understandable impatience at the slow progress in poverty alleviation, achieving gains in the social/structural determinants of health\u2014racism, poverty, transportation, housing, education, and diet\u2014will take time and perseverance.Antwyne Calloway, the Manager of the Healthy Roanoke Valley Pathways HUB says it best: We are finally having fruitful conversations about racism and segregation in our neighborhoods. People are listening to each other and not just reacting. Real progress is happening, but it takes time. We\u2019ll get there."}
+{"text": "A 75\u2010year\u2010old man with no known past medical history presented with acute\u2010onset binocular vision loss and anterograde amnesia. Several hours prior to their presentation to the hospital, he was found in his car after being seen driving erratically and having driven off the road into the grass. On presentation to the emergency department, the patient remained disoriented. During the interview, the patient reported that he was unable to see any of his surroundings in either eye. He could not tell when he had been moved from a darker room to a room with bright lights. Examination showed atrial fibrillation, absent blink\u2010to\u2010threat bilaterally, disorientation, bilateral intact pupillary light responses, and largely intact motor and sensory functions. Laboratory data revealed hypercholesteremia and pre\u2010diabetes. CT angiography and subsequent conventional angiogram revealed bilateral, proximal posterior cerebral artery (PCA) occlusions.The patient proceeded emergently to the interventional angiography suite and underwent successful catheter\u2010based retrieval of both P1 clots resulting in TICI\u20103 reperfusion. Subsequent brain MRI showed acute multifocal infarcts involving medial temporal and occipital lobes, in bilateral PCA territories. His intact pupillary light reflex in the setting of persistent vision loss suggested cortical blindness. After the thrombectomy, he had episodes of confabulation about regaining sight, consistent with Anton syndrome.Bilateral Proximal PCA Territory Ischemic Stroke.Acute stroke management requires rapid clinical reasoning and decision making.Posterior circulation syndromes are underrepresented on standardized stroke evaluations but must remain on an examiner's differential of acute neurologic syndromes.Anton Syndrome consists of cortical blindness and visual anosognosia, often evidenced by confabulation about retaining sight."}
+{"text": "Melanoma is a form of skin cancer that has increased in incidence in the last few decades. The main environmental risk factor is exposure to ultraviolet radiation (UVR). It is the fifth most common cancer in the UK, with a 17% mortality rate. There are new melanoma therapies that show improvement in patient survival; however, there is a significant proportion of patients who do not respond to approved treatments, for whom there are no second line therapies. Developing safe new therapies without significant side effects for patients is a pressing clinical challenge; 3D skin equivalents allow for disease modelling and systematic and safe drug testing for skin cancer. This paper reviews recent advances in creating 3D skin and cancer models for effective drug screening for melanoma.Melanoma is a potentially fatal cancer with rising incidence over the last 50 years, associated with enhanced sun exposure and ultraviolet radiation. Its incidence is highest in people of European descent and the ageing population. There are multiple clinical and epidemiological variables affecting melanoma incidence and mortality, such as sex, ethnicity, UV exposure, anatomic site, and age. Although survival has improved in recent years due to advances in targeted and immunotherapies, new understanding of melanoma biology and disease progression is vital to improving clinical outcomes. Efforts to develop three-dimensional human skin equivalent models using biofabrication techniques, such as bioprinting, promise to deliver a better understanding of the complexity of melanoma and associated risk factors. These 3D skin models can be used as a platform for patient specific models and testing therapeutics. The human skin is the outermost covering of the body and the largest organ of the integumentary system, protecting underlying tissues and organs . As a baAmong the different skin cancers that affect the human body, melanoma is one of the most aggressive and has a poor prognosis when it invades the dermis . The priThis paper aims to provide an overview of the role of bioprinting technologies to fabricate biomimetic 3D models of melanoma to enable advances in understanding melanoma, screening of new drugs and precision medicine. First, we highlight the mechanisms underlying malignant melanoma onset and progression, providing the fundamental knowledge to understand the heterogeneity and complexity of the melanoma tumour microenvironment. Subsequently, the most used approaches for in vitro melanoma modelling are reviewed regarding their ability in reproducing key features of melanoma. Lastly, recent advances in bioprinting of melanoma models are discussed, along with the challenges and future opportunities in the field.Malignant melanoma arises from melanocytes, which are pigment-producing cells that are primarily located in the epidermis, but can also be found in the dermis, and occasionally at rare visceral sites . Thus, iThe cutaneous subtype is the most frequent clinical subtype in the UK, mainly affecting individuals of European descent, and once it metastasises, the prognosis is poor . TherefoMC1R (the red hair gene) . S. S112]. CD271 decreases when the tumour progressed from primary to metastatic melanoma in the same patient. When implanted into a collagen I matrix, CD271 was highly expressed in those cells which were confined to the body of the sphere. However, CD271 was almost absent in the cells that were detached from the spheroid . A. A77]. At manner .Organoids are complex 3D culture systems that recapitulate key the structural and functional characteristics of an organ. The establishment of organoids relies on cell differentiation and self-organisation of either tissue-resident adult stem cells obtained from biopsy samples or pluripotent stem cells (PSCs) . MelanomOrgan-on-chip and tumour-on-a-chip models are being widely developed to reconstruct the complex microenvironment in vitro and to overcome some limitations in organ type cultures, such as inefficient tissue perfusion ,139,140.For the development of effective anti-cancer drugs, it is vital to understand the interactions between cancer cells, neighbouring healthy cells, the ECM, and surrounding tissue and components . Tissue Reconstructed in vitro 3D skin models allow for a sophisticated recapitulation of key aspects of the native tissue, assuming a pivotal role as versatile platforms for the study of melanoma behaviour in human skin. These models are typically created as a top layer of stratified, differentiated keratinocytes, a middle layer of the basement membrane, and a bottom layer of fibroblasts embedded within collagen type I. Patient-derived melanoma cells can be incorporated into such 3D skin models, allowing the recapitulation of the crosstalk between melanoma cells, keratinocytes, and fibroblasts, including paracrine signalling, cell\u2013matrix and cell\u2013cell interactions. It has also been demonstrated that patient-derived melanoma cells cultured within bilayer 3D skin models C recapitTumour cells, such as melanoma cells, could be incorporated into HSE, providing an excellent model for studying tumour growth and invasion as well as a platform for pharmacological analyses, saving time and money typically associated with animal experiments. In vitro generation of melanoma skin equivalent (MSE) has been described using a variety of methods. Initially, these protocols were created to generate healthy HSE, and melanoma cells were seeded with keratinocytes on the reconstructed dermis.One method is to use human de-epidermised dermis (DED). DED uses a skin biopsy of a donor, which is then deprived of the epidermal layer by overnight incubation in sodium chloride and finally digested by Dispase II to remove the basement membrane ,76. Prim\u00ae has been developed by Hill et al. [Recently, a novel humanised HSE using Alvetexl et al. . Most skl et al. . To solvl et al.  developel et al. . Additiol et al. .The interest in using bioprinting to fabricate artificial skin has significantly increased in the last decade. This has been mainly motivated by the unprecedented ability of these technologies in depositing cells, biomaterials, and bioactive molecules in predefined 3D locations with high precision and reproducibility ,146,147.A major advantage of bioprinting relies on the ability to closely mimic the architecture of the native skin through the deposition of bioinks comprised of skin cells and bioinstructive materials into a 3D construct with a tissue-specific organisation. It has been demonstrated that increasing the complexity and biomimicry of bioprinted skin translates to improved biological function, predictive value, and healing ability ,116. TheThe effective fabrication of a multicellular and structurally complex bioprinted HSE requires the use of a suitable bioink. A bioink is a cell formulation that can be processed using biofabrication technologies and that can optionally contain biomaterials  and bioactive molecules  . In contA range of bioink design parameters must be considered during the development of a suitable bioink for skin bioprinting . BioinksSubsequently, a range of approaches to modify specific bioink attributes have been explored to improve printability and biocompatibility ,175,179.However, single material bioinks can struggle to meet the requirements of both printability and biocompatibility, hence the development of multicomponent bioinks ,175,179.Three-dimensional bioprinting encompasses a range of additive manufacturing technologies that can process bioinks into structurally complex and multi-material tissue constructs with high reproducibility. Bioprinting systems can comprise multiple printheads and technologies within a single machine working in tandem to allow multi-material printing. As the skin is a complex organ with a multicellular, multi-layered, and hierarchical organisation; the bioprinting technology selected should be able to replicate this architecture and be suitable for the specific bioink rheological properties. For skin bioprinting, the most relevant techniques are extrusion, material jetting (inkjet), laser assisted, and lithographic bioprinting A.Extrusion-based bioprinting comprises systems that use pneumatic (compressed air), mechanical (screw/piston), or solenoid driven printheads to deposit bioinks onto a platform ,182,183.6 cells mL\u22121), with the additional problem of nozzle clogging at high viscosities. Furthermore, acoustic and thermal actuators can impact cell viability due to the frequencies and temperatures used.Material jetting, continuous inkjet (CI) or drop on demand (DOD), is a bioprinting technique that deposits discrete bioink droplets in a layer-by-layer process onto a platform ,189,190.Laser-assisted bioprinting (LAB) consists of three main elements: a pulsed laser source, a donor slide (ribbon), and a collector ,194,195.8 cells mL\u22121) have been utilised. However, cytocompatibility issues arising from the choice of photoinitiator, wavelength, and light intensity must be considered. Visible light and compatible photoinitiators are becoming an attractive alternative to traditional UV bioprinting due to the lower associated cytotoxicity [Lithographic bioprinting processes, such as stereolithography (SLA), digital light processing (DLP), and two photon polymerisation (2PP) use specific wavelengths and intensities of light to selectively polymerise a bioresin into a 3D structure ,168,196.A new approach in lithographic bioprinting termed volumetric printing  can overcome some of the limitations of conventional bioprinting techniques ,198,199.Three-dimensional bioprinting has allowed the development of complex biomimetic models that begin to recapitulate the structure and composition of native skin. This is important as an advanced melanoma skin model should include epidermal, dermal, and hypodermal layers (full-thickness); appendages ; pigmentation (melanocytes), and a functioning vasculature. The incorporation of all these into a single disease model is a major challenge. However, the complexity of understanding the melanoma TME, interactions with surrounding tissues, and disease progression require a complex and tuneable model. Nevertheless, despite the challenges significant progress has been made in the bioprinting of advanced skin models, which can be translated to cutting-edge models of melanoma ,146,147.The bioprinting of skin appendages, such as hair follicles and sweat glands is an important direction in the development of biomimetic skin models due to their role in thermoregulation, homeostatic maintenance, and wound healing. Abaci et al.  developeSkin pigmentation is a major aspect of developing a viable bioprinted melanoma model. Therefore, studies on bioprinting melanocytes and the evaluation of melanin production are crucial. For example, Ng et al.  exhibiteVascularisation of skin constructs is important as the maximum gas and nutrient diffusion distance that can maintain cell survival is ~100\u2013200 \u00b5m, hence, the long-term viability of the bioprinted skin construct relies on the creation of a functional vasculature ,205. AddAs bioprinting technologies and bioink design have progressed, the ability to bioprint complex full-thickness HSE models has become possible ,210,211.Despite existing skin and melanoma models having been proved to be useful for understanding disease pathology, performing drug screening, and even providing scientific support to clinical trials, a major challenge remains in the development of a melanoma model capable of better reproducing the different features of the tumour microenvironment underlying the in vivo resistance and differential responses in human patients. This is an ambitious and complex task as melanoma cells are characterised by high genetic instability and plasticity, and the ability to dedifferentiate into a variety of states and secret factors that promote melanoma cell viability in an autocrine way and reprogram adjacent stroma cells that influence the tumour microenvironment in a paracrine manner .The potential of bioprinting to create biomimetic melanoma models has been demonstrated in a few studies focused on the bioprinting of melanoma cells and evaluation of cell response. In a recent study, the influence of bioink properties and composition on the proliferation and morphology of metastasis-derived melanoma cell lines (Mel Im and MV3dc) was investigated . ExtrusiSchmid et al.  developeSkin is a complex multi-functional tissue with anisotropic material, structural, and cellular properties. For example, the composition, density, and organisation of cells, appendages, and the ECM varies significantly between skin layers and within the different regions of the layers. Recreating this heterogeneity and architectural features, such as the wavy epidermal\u2013dermal junction is a significant challenge in skin bioprinting.This necessitates the development of enhanced software tools for designing and fabricating such complex constructs; advanced bioprinting systems for fabricating multi-material, hierarchical, and functionally graded skin constructs with high resolution and printing speed.The selection of a bioink for a melanoma model will differ from a bioprinted skin graft. The specific requirement of each application means that the properties of the bioink will be distinct to provide specific microenvironmental cues to both normal and cancer cells. Skin grafts may favour long-term construct stability and promotion of host vasculature in-growth, but the bioink itself may not need to fully recapitulate the native ECM to fulfil its purpose of being a successful skin graft. Whilst a melanoma bioink should from the beginning as closely as possible resemble the native ECM to promote appropriate cell\u2013ECM interactions and maintain markers of melanoma expression, which are crucial in understanding melanoma physiology and progression. Unmodified native skin ECM biomaterials, retaining all instructive and binding sequences, such as collagen may be the most desirable for this objective. Furthermore, with new bioprinting strategies, the difficulty of bioprinting collagen and other native polymers is no longer as much of a challenge ,217,218.The long-term viability of a bioprinted tissue requires the integration of a perfusable vascular network . This isMalignant melanoma is an aggressive form of skin cancer that can be fatal. It is, therefore, essential to understand the genetic drivers and triggers underlying melanoma onset and progression, as well as the dynamics of the tumour microenvironment. While existing 2D models have provided us with core concepts of cancer progression, they are not fully viable for cancer studies as they are conceived under ideal and non-relevant physiological conditions. To overcome these issues, 3D models, such as spheroids and enabling technologies, for example 3D bioprinting have been used to create advanced models that closely recreate the microenvironmental conditions of native tissue as well as the cell\u2013cell and cell\u2013ECM interactions. As a result, a repertoire of melanoma models is available, comprising 2D and 3D tissue cultures, spheroids, organoids, and animal models, such as genetically engineered mice and patient-derived xenografts.Bioprinting is assuming a central position in the biofabrication of 3D melanoma models with improved biomimicry. This has been made possible by translating the understanding gained through the steady development of advanced bioprinted HSE models. Indeed, by developing suitable bioinks and bioprinting strategies it is now possible to create 3D heterogeneous cancer models recapitulating key features of tumour microenvironment through bioprinting. In the context of melanoma, significant progress has recently been made regarding the design of bioinks for the bioprinting of 3D melanoma models containing tumour-associated cells. Efforts have also been made in promoting the vascularisation of melanoma models, though the progress in this field has been more modest. Furthermore, there is still a gap in full thickness skin models to study melanoma as skin is a complex tissue that differs according to race (pigmentation which affects the amount of UVR absorbed), location of the tissue on different parts of the body , and age (elasticity and ECM degradation). In addition, further investigation is also warranted regarding the integration of immune cells and other key cells of the tumour microenvironment to better reproduce the native cellular composition. Bioprinting can also provide a platform for personalised melanoma models utilising patient-derived cells to develop patient specific treatment regimes.Furthermore, advancements in bioprinting also open opportunities for developing models and furthering our understanding of rare non-cutaneous melanoma subtypes, uveal and mucosal. For example, bioprinting of the cornea is rapidly developing and future studies may begin to include the uveal layer ,220,221.Overall, we can conclude that novel developments in improved melanoma models that capture fundamental cell\u2013cell and cell\u2013ECM interactions are required to perform fundamental studies towards identifying new therapeutic targets and perform screening of the efficacy and safety of new therapeutic modalities."}
+{"text": "Finding a safe space for learning and exploration. Participants used peer support to access key information and skills and as a space to safely explore life with HIV. Participants perceived successful peer support as having the right peer while also receiving support at the right location. Study recommendations include further research on how a peer is defined within the U = U era, further research on the peer support needs of young adults, and further research on the accessibility of peer support.In Sweden men account for most new HIV cases, and little is known about the peer support needs of people living with HIV in Sweden. This qualitative study explored how recently diagnosed men perceive and experience peer support in Sweden. Purposively sampled from HIV patient organizations and infectious disease clinics throughout Sweden, data was collected through in-depth individual interviews with 10 men living with HIV, who have experience participating in peer support. Latent and manifest qualitative content analysis produced the overarching theme of  Peer support and its integration within health services is a form of social support that has been explored by many working in illness prevention and management for a variety of health conditions such as HIV, mental illness, and diabetes , 2. For As a resource, peers provide support through exemplifying their experiences and giving advice that is rooted in previous actions , 7, 8. APeers can be defined in many ways and are usually defined by demographic group membership , behaviors , or membership within an illness community  . HoweverSince the 1980s there have been many developments in the field of antiretroviral therapy (ART), with approximately ten years having elapsed since the establishment of convincing evidence on infectiousness and the inability to sexually transmit HIV when virally suppressed \u201312. WithIn Sweden, it is estimated that approximately 8,000 people are living with HIV, with approximately 400\u2013500 new cases each year . In 2019how do recently diagnosed men living with HIV in Sweden perceive and experience peer support? This study is situated within what we have called \u201cthe U = U era\u201d, where participants have experienced adjusting to life with HIV during a time when scientific evidence concerning the effectiveness of ART became increasingly known. Additionally, we defined peers similarly to that of other studies on peer support for PLHIV, where peers are defined as individuals who share the same health condition of living with HIV but not necessarily the same demographic or behavioral identifiers  my luggage was gone with my medication in it for two weeks, and I was horrified because I thought I was going to die and I didn\u2019t know anything and [my family] didn\u2019t know anything, and I was so upset the whole trip (P7).Because of this, peer support played a critical role in providing participants with information about the infection, and opportunities to workshop the skills needed to live successfully with it.When engaging in peer support, many used it as a space to learn essential hard skills. Some of these skills included learning where to seek practical information about HIV, successful strategies for status disclosure, and understanding the mechanics of the infection. Moreover, participants also attended peer support to gain key soft skills. Some of these skills included learning how to get to know other PLHIV and learning the strategies that others have used to emotionally adapt to life with HIV. When learning these new skill sets, participants perceived peer support as facilitating their ability to talk about topics that intersect with life with HIV\u2014such as dating, coping strategies, and opportunities to meet other PLHIV. Participants acquired these skill sets and information through participating in a variety of peer support formats. Some participants perceived peer support as something that was only provided within a formal context at a patient organization. Yet despite this perception, participants experienced both giving and receiving peer support in a variety of ways. Some of these included formal avenues such as activities and support groups organized by organizations, and informal avenues such as engaging with partners, friends, and acquaintances.Yeah, I think it can happen in any kind of way, in any kind of formation. It can be friends over coffee, over lunch, during a walk, it can be more \u2026 I don\u2019t know professional\u2026 it can be any way I think (P7).Participants (N = 6) who provided peer support in a formal capacity did so while holding positions (paid and unpaid) within HIV organizations. The transference of skills and information was bi-directional, where peer support providers also acquired new skills as well. Providers experienced providing peer support as something that gave them confidence. They also experienced it as a pleasurable experience that gave them a sense of fulfillment and gratification.It\u2019s very gratifying and it doesn\u2019t have to do with ego gratification you know what I mean? [\u2026] So something happens that I stop being me a little bit. It\u2019s not about me when I\u2019m there\u2026 you know? Which is quite interesting because you know there\u2019s of course a part of me that likes to be there and I like to give help, and I know I\u2019m doing a good thing (P6).For some, this gratification stemmed from being able to help others in a way that they were once helped.Participants gave peer support for a variety of reasons. Some were motivated to provide peer support out of a desire to change how PLHIV lived with HIV. Others did so because they saw how a lack of access to information about HIV resulted in negative health outcomes among those they knew.I just knew that probably that something\u2026 this should be better [\u2026] Like living with HIV, or with how you deal with it [\u2026]. And there has to be something I can do, and [providing peer support] was probably the only way to like do something more actively [\u2026] (P1).My motivation of talking to people is back in Africa. I\u2019ve seen people out of negligence and stupidity and ignorance pass away. I know a couple of people. Lots of people pass away because they don\u2019t want to be seen in the clinic, they don\u2019t want to be taking pills (P10).Others provided support with the goal of instilling confidence and the ability to do difficult things. During this process, some participants tried to push recipients to achieve key skill sets and milestones which they themselves might not have accomplished yet. This two-way exchange of benefits contributed to a continued learning of skills for peer support providers.If I have given peer support, I will give something that strengthens you in any case. I will try to understand you, I will let you vent, but at the end of the day I will want you to go out of here feeling a little bit taller (P1).As described by the participants, peer support served as an opportunity to explore what it is like to live with HIV via engaging with the lived experiences of others. In doing so, participants were able to learn from both positive and negative experiences without having to go through these experiences themselves. Participants explored experiences such as stigma management and others\u2019 regret of not having accessed peer support while adjusting to life with HIV. For participants, having a space to safely discuss their fears, to share negative experiences, and to see the resilience of other PLHIV was important. It resulted in them gaining a positive outlook on life, and forming friendships that eventually progressed beyond providing HIV related support.When interacting with people not living with HIV (PNLHIV), many participants perceived them as not understanding what coping entails for PLHIV. Moreover, PNLHIV were viewed as not recognizing that having HIV was stigmatized by society at large.There\u2019s still like a great ignorance in the society, so like some people would even think [\u2026] that they are going to die within the next weeks or months or so\u2026 [HIV] is still like a chronic disease that you will be living with for the rest of your life so that in itself is also something that could be very difficult to cope with (P3).And now we think that we\u2019ve normalized it so now it\u2019s not an issue at all and you don\u2019t have to deal with telling people and you don\u2019t have to deal with stigma within the society or people still being scared of you [\u2026].We don\u2019t talk about people\u2019s feelings or narrative, or how it impacts people\u2019s lives, or how you feel or how you react to it, or how you cope with it (P2).Additionally, some participants experienced the support from other PLHIV as carrying more weight and validity than support from PNLHIV sources. This was attributed to PLHIV having the experience of living with HIV and helping participants to humanize the infection.There is a thing with authority, not that [a specific patient organization]is an authority, but it\u2019s more like an authority. When they say something, if they say the exact same thing [that] my dad tells me, everything is fine blah blah blah, it weighs more (P9).Among participants who did not seek peer support, some expressed receiving support from PNLHIV sources such as friends, family, and partners. These participants voiced using PNLHIV sources as spaces to safely explore their questions and anxieties.So, then I started to get to the psychologist and stuff. And then I never sought peer support for HIV because I was offered support for my PTSD and stuff, so I thought it was almost involved in this kind of support I already have (P7).I had a big support from my family, from my parents, and from friends, so I didn\u2019t feel the need to reach out to anyone (P9).Participants expressed a fear of being stigmatized, especially after disclosing their HIV status to others. As a result, participants sought peer support because they perceived it as something that would give them the strategies needed to deal with stigmatization. They found stigma management to be an especially challenging task.I think it\u2019s extremely [needed]. Peer support, because it has nothing to do with you cannot transmit the virus or not. You need peer support because of stigma in the society. That is why you need peer support. Because people are afraid of HIV, and people are afraid of people living with HIV because the knowledge level is so low [\u2026] We still need peer support because of the stigma in the society (P4).When reflecting on their experiences of learning to live with HIV, participants voiced that having an early introduction to peer support would have been beneficial. They spoke of how early access to peer support promotes adopting positive behaviors, while also shortening the journey from a moment of crisis to a state of adaptation.Peer support quickens the healing process [\u2026] I would have found my way here by my own, but it shortened the leap from getting the diagnosis to today. So, it quickens the way when you have someone that has lived through something a bit life changing (P5).Those who did not participate in formal peer support early expressed regret and felt that participating in peer support would have made the adaptation process easier for them. Specifically, they viewed peer support\u2019s ability to provide a non-judgmental and risk-free space to ask questions and learn from others as something that would have helped them to better navigate the adaptation process.[Looking back] I\u2019d tell myself to take every support I could take. Because it would make it easier to go through things together with people who might go through the same thing, although we haven\u2019t the same experience in life. But I think yeah, I would tell myself to take more support. Take because it\u2019s free and there for me, and no one will judge you. It\u2019s stupid not to take it \u2026 not stupid, but yeah (P7).Furthermore, all participants expressed the view that peer support is a service that is still needed for recently diagnosed individuals and should be offered to everyone. They also perceived that peer support should be voluntary.It should be optional to get peer support and it should be something that should be provided to the people that want it. I think that it could be potentially harmful to provide it as something that\u2019s obligatory [\u2026]. But it\u2019s something that is good to be able to provide for people who desire it (P3).Participants described how talking to peers was instrumental in shaping their outlook on life. For many, the first person they contacted after diagnosis was another PLHIV. This was because speaking to peers helped participants to comprehend and conceptualize a life with HIV. They found that the words and experiences of those providing peer support helped them to change inaccurate and negative perceptions.I went to the meetings and [\u2026] and that was the first people I met, and it changed my way\u2026 how I looked upon it totally. [HIV] could be anyone [\u2026] we could look anyway, we could be black, we could be white, we could be heterosexual, homosexual, male, female, whatever; old, young, they were everybody. And so I learned that we\u2019re just people (P4).I think the most positive thing is maybe that you don\u2019t feel alone. And I enjoy the diversity of the people sharing this thing. Because when you think about [HIV], you don\u2019t think about [PLHIV] as being a diverse group with different personalities (P9).Additionally, participation in formal forms of peer support for many participants led to the development of friendships with other PLHIV.I also talked to my friends at [the patient organization]. How they were dealing with relationships, and comparing myself to them, how did they\u2026 some of them had relationships, some of them didn\u2019t (P4).For some, friendships with other PLHIV constituted their most central form of peer support and developed to a point of relating to each other beyond the basis of HIV.He was also the one telling me I gave him peer support in the way that I was open and so you know, didn\u2019t really care about [having] my HIV status out. He had an issue with that, and he had an issue with a lot of other things. But he could also correct me in many ways, and I could correct him [too]. I would teach him or help him in other ways so that was, and we\u2019re still very good friends [\u2026]. So, he continued to be that solid rock and this special friend. That is now not the same, we don\u2019t talk about HIV at all when we meet today [\u2026] (P2).Participants experienced positive peer support experiences as consisting of two components: 1) engaging with the right peer, and 2) receiving support at the right location.Participants described finding the \u201cright\u201d peer as a key component of a successful peer support experience. This was because peers are responsible for providing not only information, but also emotional support and a space to share personal experiences. According to participants, the person whom they regarded as a peer could vary widely. A peer could be a role model, could be any age, and could share similar characteristics or experiences in addition to having HIV. For those who had a partner also living with HIV, they found comfort in having a partner that was also a peer. This comfort arose from having someone with an implicit understanding of what it is like to live with HIV and having someone who exemplifies that it is possible to live well while also having HIV.I remember my boyfriend telling me [after receiving my diagnosis] \u201cIt\u2019s fine. It\u2019s gonna be fine. It\u2019s gonna be super fine\u201d. Like very you know, like no deep talk about anything, but I just knew that it was (P6).[\u2026] I would never choose someone with HIV much more than someone without HIV\u2026 I don\u2019t care about it. It just happened. I just found it actually easier when he actually has it (P7).Both before and while attending their first peer support meeting, participants described feeling a wide array of emotions. These emotions ranged from feeling curious, exciteted, and welcome; to feeling nervous and fearful. The root of these feelings was diverse in nature. For participants with an immigrant background, these feelings sometimes came from previous experiences of judgement and stigmatization that had occurred in their home countries.The first time I was scared [there] would be this hard African mentality, the African feelings that people are going to look at me, and say things, and put me in a group, or say something negative about me\u2026. see all my flaws\u2026and probably people who are [in the building of the patient organization], people around there will stigmatize me (P10).I still remember the first time we met, shook hands, and I kind of just looked at him and said like so this is what a person with HIV looks like [\u2026]. But it was a nice relief (P1).Interestingly, some participants also viewed PNLHIV who had a close proximity to HIV and chronic disease as peers. Such individuals could be patient organization staff, family, friends, and medical staff. This was because these individuals were seen as being able to empathize and understand what PLHIV were going through.[\u2026] he smoked a lot and drank wine and had chronic pain \u2026 so he knew about pain, and living, and enduring it (P5).But he\u2019s told me about his, like he has this depression and all that, so I figured yeah, I\u2019ll tell him about this (P8).Moreover, having the right peer was seen as important because participants experienced the first meeting with another PLHIV as a significant and important moment. In a similar vein, participants felt that finding the right peer included being provided with support that could be tailored to address identity-specific needs if needed. They expressed that an individual\u2019s peer support needs might differ depending on demographic factors such as their geographic location, race, ethnicity, age, sexual orientation, and nationality.[\u2026] but I also think that it is minority stress [\u2026], so that can impact more people than just people with HIV. For example, [among] gay people we know that mental health is not as good as in general the population, and then add on more [like] being a black person, or an immigrant, or and the add on HIV, like it\u2019s always adding on [\u2026] (P2).As a concrete example, some participants wanted more peer support opportunities that would target young people in particular.There were some young people at [the patient organization]. But most of them were older. I was invited to some kind of event for younger people where they kind of got together in [a major city in Sweden]. But I was in that meeting and I liked that because it was a group of younger people and that I miss. That, I would have changed. (P4)It is important to note that participants also mentioned that peer support should be constantly evolving to meet the evolving needs of PLHIV.Lastly, participants perceived having the right location as another key facet of a successful peer support experience. Among participants, formal forms of peer support were provided through patient organizations. They also perceived infectious disease clinics as a safe space. Some even viewed them as a suitable location for peer support to be provided because they are a place that all PLHIV in Sweden frequent.I never imagined that it would have been the NGOs [providing peer support]. I thought that was up to the clinics to do that. I thought that was the service of the clinics, like that would have been the clinics for me (P2).Some participants perceived and experienced online platforms as a safe location to begin speaking with PLHIV before meeting with others in real life. They mentioned using a variety of online platforms such as dating applications, websites, web forums specifically for PLHIV, and mainstream social media applications. Through these platforms, participants were able to have varying degrees of contact with other PLHIV. Some chose to participate through only reading what others had written on the platforms, while others engaged through publicly posting on group pages, individual pages, and web forum threads. Others engaged in one-on-one contact with other PLHIV through private messaging and emailing. One reason why participants chose to engage in peer support via online platforms was because it allowed them to practice forming connections with other PLHIV while also still maintaining some form of anonymity. Additionally, participants also used online platforms because it allowed them to connect with PLHIV around the world.I started to go into these dating sites online, and talking to people online, and in other countries. People in Russia, some people in Belgium, some people in Denmark, Norway. Maybe it was a little bit to like to protect myself, and to just like protect myself like through doing these actions [\u2026] (P4).This was especially the case with PLHIV residing in rural areas, where there were very few other PLHIV in the nearby vicinity.It\u2019s super quiet, and you don\u2019t meet people. It\u2019s a very quiet town, and no one to talk to. So, I usually do my conversation online. I go to dating apps [and] when I meet people, that\u2019s where I try to ask [about their thoughts on HIV] quickly \u2026 and if I know the mentality\u2019s right, I [talk to that person more] (P10).It is important to note that how participants found their way peer support varied. After diagnosis, some participants were referred to peer support services at patient organizations by a clinic professional, while others were left to seek both informal and formal support services on their own. For participants referred to patient organizations, this referral was done at the discretion of clinic professionals.Our findings showed that participants both perceived and experienced peer support as a necessity for recently diagnosed individuals. To our knowledge, this study is the first of its kind within a Swedish context.From our findings it can be inferred that formal referral processes for newly diagnosed PLHIV to peer support services are limited. Participants experienced either being referred to peer support services by a clinic professional or were left to navigate this process on their own. In Sweden, peer support could be housed within, or provided in partnership with infectious disease clinics. This informal referral pathway within infectious disease clinics is noteworthy and suggests a system-wide gap in services to meet the quality of life needs of PLHIV. As such, further research is needed to understand how the accessibility of peer support services may differ among PLHIV in Sweden. It is important to note that not having standardized referral pathways to peer support services for recently diagnosed PLHIV is not unique to Sweden. This practice has also been similarly observed in Australian clinical settings as well , 33.Moreover, participants perceived peer support as something that should be tailored to meet a person\u2019s unique demographic needs. Among participants, peer support was experienced as not solely focusing on acquiring knowledge about HIV, but also on how HIV intersected with other areas of life such as dating, employment, and identity. Those who acquired HIV in their late teens and early twenties experienced peer support organizations consisting of primarily older men and desired spaces and activities specifically for youth. The importance of providing tailored peer support has been observed in other studies , 34\u201337. Furthermore, the results of our study add to existing literature by showing how peer support within Sweden helps recently diagnosed men navigate challenges such as stigma management. According to various quality of life studies, PLHIV encounter stigma, financial stress, mental health issues, and loneliness at higher rates than PNLHIV , 19, 22.Finally, our findings provide new insights about the nature of peer support relationships. In their study, Peterson et al.  found thOur study\u2019s definition of a peer and peer support mirrored the definitions used by others who have previously studied the peer support needs of PLHIV. However, our results indicate that more research is needed to understand the criteria for being a peer during the U = U era. Our findings suggest that existing conceptualizations of what constitutes peer support for PLHIV might be too narrow, because a key criterion for being a peer is having an HIV positive status , 8, 9. TIn our study, a few limitations are present. Because all participants are under 50 years of age, study results may not be applicable to men 50 years of age and older. Moreover, because this study focuses on the experiences of recently diagnosed men, study results may not be applicable to the needs and experiences of recently diagnosed women. Additionally, because we defined recently diagnosed as living with HIV for no more than ten years, there was a risk for recall bias among respondents. Attempts to minimize this were made by having a clearly defined study aim, clearly formulated research questions, and developing an interview guide that facilitated rich conversation. Lastly, even though sufficient information power had been reached via rich descriptions of what it was like receiving and providing peer support, we cannot exclude that additional participants would have contributed additional perspectives.This study has many strengths as well. The analytical process allowed all members of the research team to assess analytical decisions, increasing the confirmability of study results. Additionally, engaging in conversations with a partner patient organization for PLHIV during the study design process enriched and informed the research team\u2019s approach to this study\u2019s design. Moreover, by situating this study within U = U era, our results provide insight into the current peer support needs of men adjusting to life with HIV within a low prevalence, high ART adherence setting. As a result, these results may be transferable to other low HIV prevalence, high ART adherence contexts where similarities are present.In the U = U era, men adjusting to life with HIV in Sweden perceived peer support as assisting with acquiring the skills needed to live successfully with the virus and managing the challenges of HIV related stigma and status disclosure. Further research is needed on the differences in support needs between younger men and older men living with HIV in Sweden. Additionally, further research is needed to assess the accessibility of peer support services in Sweden. Moreover, study results suggest that conceptualizations of peer support for PLHIV within current literature may be too narrow, indicating that further research is needed on how the criteria for being a peer may have changed within this era."}
+{"text": "An increasing number of psychological interventions are shifting to online modes of delivery. One such intervention is peer-to-peer support, which in this context may provide internet users living with mental health disorders an opportunity to connect with and support others living with similar conditions. This paper presents a call for further research into how platforms such as Facebook could be used as channels for peer support and the mechanisms that may underlie their effectiveness. We discuss the background of peer support, how it has transitioned online, and consider theories and models that may have relevance. We also consider the importance of moderation within online peer support and the development of specific social network\u2013based online interventions. We conclude that for social network sites to be used as peer-to-peer support interventions, more research is needed to understand their effectiveness, the role of moderation in these communities, and the mechanisms that produce the benefits experienced by users. Mental illness can be described as \u201chealth conditions that involve changes in emotions, thinking or behavior (or a combination of these)\u201d . Mental Mental illnesses have a significant impact on quality of life, social functioning, work, and family activities. Many of those living with mental illness have educational difficulties, being less likely to finish school or enter college . This isAs the demands for mental health services exceed physical resources, it is more important now than ever that individuals are able to access free and widely available support. The use of social media is widespread across the world, with use in developing countries exceeding that in developed countries . The groThis viewpoint paper considers the background to peer support, how it is currently being used online, and the scope for Facebook to be used in this area, as well as suggesting some models and theories that may help to explain the mechanisms underlying effective online peer support. We suggest next steps in research to enable the development and rollout of online peer-to-peer support interventions for the benefit of those living with mental illness.Peer support within mental health can be defined as support or services provided to individuals experiencing mental health problems by others who have experienced similar problems . Peer suThe roots of peer support services lie in long-established groups, such as Alcoholics Anonymous , designeThe effectiveness of peer support has been examined in relation to multiple outcome measures. A recent meta-analysis of 28 randomized controlled trials investigating the effectiveness of peer support for individuals with a range of mental illnesses demonstrated significant improvements in clinical recovery  and personal recovery  but not functional recovery  . FunctioDespite evidence supporting their effectiveness, these formal services are usually only available through mental health services, which are facing pressures due to cuts in funding  and demaRecent figures suggest that 99% of people aged 16 to 44 years in the United Kingdom are internet users, with 89% of UK adults reporting using the internet daily . There iPeer support can be provided informally online. This support is more informal in that it is not tied to services and can be accessed by anyone on the internet. Asynchronous platforms, such as forums, discussion groups, and bulletin boards, allow users to post topics and engage in discussions, using these platforms to exchange knowledge, ask for help, discuss problems, and share stories . SynchroA systematic review indicated that internet support groups (ISGs) are most often used for emotional and informational support , which creates a sense of social companionship . This reA review of online peer support for individuals with depression suggested several benefits, including increased empowerment and self-efficacy, enhanced coping strategies, and reduced social isolation . HoweverQualitatively, many advantages of online synchronous peer support platforms have been identified, including emotional support, the availability of advice, enablement of positive personal changes, engagement in valuable social interactions, and the opportunity to disclose and express feelings and views ,36. FurtHowever, users have also identified disadvantages, including the potential for others\u2019 experiences to cause personal distress, restrictive rules and moderation, and unhelpful interactions with others . A surveInterventions for mental health, such as online peer-to-peer support, have the potential to exploit the increasing use of social media ,41. SociThe most popular social media platform in the United Kingdom is Facebook , with 23Currently, there is little research into the use of Facebook groups for mental health peer support. In a content and thematic analysis of public Facebook groups for mental health peer-to-peer support, Prescott and colleagues  found thAs social media has developed as a platform for delivering peer support, some researchers have taken this further to develop a novel form of digital intervention called social network site (SNS)-based interventions. These interventions have an SNS format, combining individually tailored therapy with clinical and peer moderation within a peer-to-peer social support network ,49. WhilMany online peer support groups and SNS-based interventions involve moderation, which is important for building a safe and positive community . ModeratPrevious studies of moderators of various health-related online support groups for both mental and physical health conditions have found that most moderators are individuals living with the condition themselves. Some moderators described how moderating online communities fulfilled their own support needs and reported that it was empowering to be able to help others. Other motivations for setting up groups included addressing a lack of good existing support groups, reducing isolation, and having a place to exchange support and information. Moderators described various roles they took within these groups, such as circulating health information; moderating messages; providing support, encouragement, and advice; making announcements; and performing administrative tasks, such as responding to requests, banning harmful users, and organizing discussion threads -56.In a study specifically examining moderation within mental health communities on Reddit , moderatWhile moderators have many critical roles within these groups, there may be some inherent risks given they are often vulnerable individuals themselves. Moderation may become overwhelming, taking up much time and energy, and moderators must set boundaries to focus on their own health. Moderators are required to make decisions on censoring potentially harmful content, to which posters may react angrily ,37. It iSeveral models and theories have been proposed for the mechanisms underlying offline peer support. Gillard and colleagues  proposedIt is as yet unclear whether models like these transfer to online peer support, and there is a gap in research in this area ,59,60. HAside from these models, various theories from across the discipline of psychology have been suggested to underlie peer support, including social learning theory, social comparison theory, and helper therapy . Social Social comparison theory suggests that people seek out others they perceive as having things in common with, such as a similar mental illness, to establish a sense of normalcy and identity . InteracHelper therapy proposes that individuals can benefit themselves through helping others . SuggestIn a study examining the mechanisms of peer support alongside a web-based psychoeducational program, Proudfoot and colleagues  found evWhen considering how these models and theories may be applied to online peer support, it will also be important to consider the extent to which users engage with and contribute to online groups. A common measure is frequency of posts, which has been suggested to broadly fit the 90-9-1 principle, stating that a majority of content will be posted by a small proportion of users (1%), while the vast majority of users (90%) choose to observe rather than participate actively . HoweverAs we emerge into the aftermath of the coronavirus pandemic, it is important now to understand and act on the toll that the pandemic has taken on mental health. There are indications that COVID-19 can have a direct impact on mental well-being through mental and neurological manifestations, with many of those hospitalized with COVID-19 experiencing anxiety or depression . The panResearch shows promise for the use of online SNSs, including Facebook, to provide forums where mental health peer support can take place, with a variety of theories that could plausibly underpin their use. Novel online peer support platforms making use of social network functions may therefore offer a useful way to support people with mental health difficulties. However, it is currently difficult to use or promote online peer-to-peer support as an intervention when we do not have a quantification of the effectiveness of peer support provided in this way or a clear understanding of the benefits its users experience. We argue that in order for existing social network platforms like Facebook to be recommended as peer-to-peer support interventions and for separate peer support interventions to be developed based on SNSs, more research is needed to understand their effectiveness, making use of both quantitative and qualitative methods.We suggest that the effectiveness of novel social network\u2013based peer support interventions should be evaluated using similar experimental designs as applied in prior research into offline peer support ,32-34 wiIt is important also to be mindful of the limitations and potential harms of online peer support. The use of peer support may result in adverse events, such as behavioral contagion, corumination, or unpleasant or negative interactions with other users ,37,81-83Finally, exploring the mechanisms that underlie effective online peer support is crucial to understanding why it is helpful, and how its utility can be maximized. Particularly, in order to develop new interventions that are based on sites like Facebook, that is, SNS-based interventions, it will be important to understand whether theories underpinning offline peer support also apply to online peer-to-peer support in this context. It is also crucial to understand the role of moderation within these communities, and the benefits that moderators have for the workings of online peer support, including safeguarding, to clearly demonstrate their role in future social network\u2013based peer support interventions.Deriving knowledge from existing online peer support groups  will be beneficial for the design of new online peer support interventions. Research in this area has the potential to provide a better understanding of why and how these networks help, and how they can best be organized and managed for maximum effect while reducing risk of harms."}
+{"text": "Research supports that public safety personnel (PSP) are regularly exposed to potentially psychologically traumatic events and occupational stress, which can compromise their well-being. To help address PSP well-being and mental health, peer support is increasingly being adopted (and developed) in PSP organizations. Peer support apps have been developed to connect the peer and peer supporter anonymously and confidentially, but little is known about their effectiveness, utility, and uptake.We designed this study to evaluate the functionality and use of the PeerConnect app, which is a vehicle for receiving and administering peer support. The app connects peers but also provides information  to users; thus, we wanted to understand why PSP adopted or did not adopt the app and the app\u2019s perceived utility. Our intention was to determine if the app served the purpose of connectivity for PSP organizations implementing peer support.A sample of PSP (N=23) participated in an interview about why they used or did not use the app. We first surveyed participants across PSP organizations in Ontario, Canada, and at the end of the survey invited participants to participate in a follow-up interview. Of the 23 PSP interviewed, 16 were PeerConnect users and 7 were nonusers. After transcribing all audio recordings of the interviews, we used an emergent theme approach to analyze themes within and across responses.PSP largely viewed PeerConnect positively, with the Connect feature being most popular (this feature facilitated peer support), followed by the Newsfeed and Resources. App users appreciated the convenience of the app and felt the app helped reduce the stigma around peer support use and pressure on peer supporters while raising awareness of wellness. PSP who did not use the app attributed their nonuse to disinterest or uncertainty about the need for a peer support app and the web-based nature of the app. To increase app adoption, participants recommended increased communication and promotion of the app by the services and continued efforts to combat mental health stigma.We provide contextual information about a peer support app\u2019s functionality and use. Our findings demonstrate that PSP are open to the use of mental health and peer support apps, but more education is required to reduce mental health stigma. Future research should continue to evaluate peer support apps for PSP to inform their design and ensure they are fulfilling their purpose. Public safety personnel (PSP), such as police officers, correctional workers, and firefighters, have a prevalence of mental health disorders 4 times higher than the general population ,2. The hResearch examining 6 types of PSP  reveals that they screen positive for at least 1 mental health disorder, such as posttraumatic stress disorder (PTSD), major depressive disorder, or general anxiety disorder, with a prevalence of 44.5% . I like knowing it doesn\u2019t bother the whole peer support team, and it\u2019s easy for them because I reach out more quickly because I know it\u2019s not this huge process for them.P52 explained how Connect streamlines peer support provision and reduces its administrative load, thus revealing a key utility of the app.In addition to convenience, 5 (31.3%) users discussed how Connect also helped facilitate mental health awareness. P47 appreciated the Connect feature for encouraging everyone to seek support \u201cwithout being in anybody\u2019s face.\u201d They described how users can seek support anonymously and still be followed up supportively to make sure they are doing well.When discussing the Newsfeed feature, 2 users liked how the feature facilitates mental health awareness. The feature was informative for learning about mental health and access to mental health resources, as well as promoting a sense of collectiveness and informing users about events. P8 said they read the resources shared through Newsfeed to \u201clearn about more mental health.\u201d P45 made a similar comment:I like the different articles that are posted in the newsfeed with some mental health issues.One user, P19, deemed Newsfeed convenient, saying that the feature is \u201cfairly easy to work with.\u201d Another user, P14, saw the feature as an effective communication tool:I mean, it functions almost like a social media page, but it\u2019s not bombarded with\u2013not everybody is allowed to post on it. It\u2019s just getting relevant information out to our members specifically. That\u2019s what I like about it.In addition, P14 described how Newsfeed enhances a sense of belonging:The newsfeed is where we posted things. Like I said, we had a family day last week, a family demonstration day, so people could bring their families and the cool units out there, the dogs, and all that kind of stuff. To me, that\u2019s just where I\u2019d put information. But really, we haven\u2019t had any events going on in the last couple of yearsdue to COVID-19Newsfeed is more than just a tool to share news; the feature is also a place to share information that can create a supportive community and allow PSP to share their work life with their family.When discussing Resources, 4 users described the feature as convenient, serving as an information hub where reliable information can be quickly found. P48 valued how the Resources feature has \u201cvetted information\u201d that does not require having \u201cto go searching on my own for it,\u201d saying:I know I can find it easily, and I can access it and look at it whenever I feel that I need to.In addition to convenience and information being vetted, one user, P22, discussed how Resources were shared by most public safety organizations, promoting a sense of occupational belonging:Because it\u2019s shared across first responder organizations. It\u2019s nice to draw from other services.Self-Assessment and Events were both associated with convenience, each being mentioned once. P14 appreciated how:Self-Assessment is built right into the app, and it\u2019s quick, and you don\u2019t have to go searching, you don\u2019t have to go do an internet search to find something, it\u2019s right there.P22 appreciated how the Events feature made connections easier:It\u2019s just another avenue to kind of create that connection to promoting events. Makes it very easy.Thus, the features appeared to provide an opportunity for screening and helped build community by advertising events.Participants were asked what their favorite feature was and what they liked about it. Connect, Newsfeed, and Resources were the favorite features among interview participants. Participants appreciated their convenience and ease of use and how each helped draw awareness to mental health in the workplace and offered occupation-specific and informed mental health support.When asked why Connect was their favorite feature, P14 said that \u201cit streamlines things for us immensely. It really cuts down the leg work that we have to deal with,\u201d making Connect convenient. P22, also talking about the Connect feature, appreciated how Connect helps promote mental health awareness in their service and requires peer supporters to keep up with their peer support skills:It creates organizational coverage where members are getting that face-to-face peer support every year, and it just creates opportunities for people to come forward who need help, and it legitimizes and keeps our peer support team busy and fresh. I would say it keeps them honing their peer support skills.Connect was additionally discussed by P48 as their favorite feature, who reflected on how Connect provided occupation-informed mental health support that would not be available otherwise. They described how Connect allows for \u201cthe ability to talk to somebody that is gonna understand, probably been through a similar situation or at least understanding of the career and how it might be like.\u201d Whereas if they had to reach out to a \u201cpublicly available helpline,\u201d they would \u201chave to explain all the nuances\u201d of their job, potentially hindering the effectiveness of the support.Newsfeed was discussed as a favorite feature by 2 participants. P8 liked that Newsfeed \u201chas a whole bunch of different information on there,\u201d making finding relevant news simple. P19 considered Newsfeed to be \u201cthe best\u201d feature \u201cbecause it has new information and there might be articles there that are of interest to look at,\u201d helping to promote mental health.Resources was the favorite feature of 1 participant, P30, because of convenience:It\u2019s super convenient, one place to get a bunch of different resources. Whether you\u2019re needing like support or you\u2019re just wanting to get some information about something, having it all in one location is very convenient. You don\u2019t have to go searching for it, and if you\u2019re trying to help somebody else, you don\u2019t have to go wasting time searching for it. You can be more effective in how you\u2019re helping other people as well, so it\u2019s just the most convenient thing.Here, P30 describes the value of having mental health and related resources directly accessible in the app, which they describe as convenient and helpful when providing support.Overall, participants, especially peer support providers, felt that having access to the app was essential to support the well-being of employees. Of the 16 app users, 9 said the app improves their well-being at work, while 6 respondents said the app does not. Based on participants\u2019 input, we determined that the app raises awareness of mental health, encouraging well-being at work, and provides a vehicle to connect when support is desired, doing so through an anonymous alternative method, reducing the pressure on peer supporters.According to participants, PeerConnect advanced a culture of care within the services by encouraging employees to mind their well-being and the well-being of their colleagues. As put by P47, \u201cit shows them that people are listening and caring and following up.\u201d They said even workers who feel they do not currently need support appreciate the app as it \u201cgoes full circle even whether they need the peer support or not, it\u2019s there.\u201d Mirroring P47, P33 said a quick text to check up on someone \u201cgoes a long way\u201d to show others how \u201csomebody cares, somebody\u2019s aware of what they went through.\u201dPeerConnect also eased pressure on mental health and wellness coordinators and peer supporters by transforming peer support into an easy and convenient process and making it easier to reach out for help when needed. P40 spoke of how PeerConnect eased some of their stress as a peer support member by \u201csimply knowing that people have it and it\u2019s easy and accessible way to reach out, and they\u2019re not getting overlooked\u2026 [an] email may be overlooked, whereas the app the notification is there, it\u2019s right there.\u201d In essence, the app reduced the administrative burden of peer support provision while allowing for greater anonymity. Complementing P40, P46 said, \u201cit gives me some peace of mind knowing that it\u2019s there\u201d and that \u201cit\u2019s a benefit\u201d for the peer support team as they know that they \u201chave something available for the employees to use as a tool.\u201d PeerConnect was also described as having a positive impact on the services overall. For instance, P52 said that \u201coverall, it has had such a good impact on the entire service\u201d as reaching out is \u201ceasy and close by\u201d and that coworkers \u201care really looking out for each other, and I think it gives people a way to feel they can help without just being helpless.\u201dNonuse of the app was associated with 3 major themes: (1) participants\u2019 lack of interest and commitment to using the app; (2) the app\u2019s lack of innovation, including uncertainty around the need for the app; and (3) the app\u2019s web-based nature.Several factors underpinned the lack of interest and commitment to the app. Some participants were not interested enough in PeerConnect to check out and download the app. For instance, P34 said they had not \u201cchecked out the app yet\u201d and had no idea \u201cwhat\u2019s in it, what\u2019s available or anything.\u201d Others, such as P54, felt as though people did not want to download the app as the ministry\u2019s intentions  were insincere:I think a lot of people are frustrated with the peer support app because it feels like it\u2019s another effort for the ministry to just check a box as opposed to implement real change.Thus, the app was viewed as a tool to distract from a lack of focus on employee wellness\u2014as a band-aid solution\u2014for the Ministry. One nonuser, P37, said that they were familiar with the app, having looked at the Newsfeed a couple of times, but did not continue to use it, giving the reason:I wasn\u2019t sure if I was at a point where I was going to need it right away or at work.Two participants viewed the app as an overlapping resource  that had little to offer to users. To illustrate, P9 had previously used the app, indicating they were \u201chopeful\u201d that it was something they would want to use, but \u201cthat\u2019s not really how it worked out.\u201d They found the app was \u201cvery generic,\u201d and there were not \u201cany actual tools on it to troubleshoot.\u201d Instead, P9 uses a different mental health app with tools like \u201cdifferent yoga practices, like soothing music, deep breathing exercises.\u201d P51 gave a different reason for not using the app, stating they were \u201cnot really sure as to what it would offer other than [connection].\u201d They continued, \u201cif I think that somebody would benefit from check-in, I would reach out to one of the members of the peer group that I know and tell them this happened; you might want to check in on them\u201d rather than going through the app.Additionally, 2 participants called the app\u2019s web-based nature into question, arguing that peers needing support were better off with in-person support than web-based support. P72 argued that the app, due to its web-based nature, could exacerbate feelings of isolation that typically mark compromised well-being:Because half the problem with mental health is the feeling of isolation of being alone and stuff like this and like by enabling more and more online through computer, through virtual, especially through text, instead of through video, it\u2019s almost; it allows someone in a downward spiral, mentally to stay home alone and only interact via text and it almost allows them to stay in the slump instead of getting us out of the slump and yeah.In agreement with the opinion of P72, P9 believed that having an app on your phone is not necessarily helpful because \u201ca lot of people aren\u2019t going to reach out even though they would benefit from talking to somebody.\u201d They went on to say that the app would be more beneficial to have people come to their workplace to \u201chave little in-services or something over people\u2019s lunch hours or even go down to the unit and speak with people about what they offer and kind of putting like a face to a name\u201d to show they are making an \u201cextra effort\u201d to support them.Of the possible strategies to encourage app uptake, users described the value of more communication and promotion about peer support and the app\u2019s existence within the services and combatting mental health stigma.Although there has been some communication and promotion of PeerConnect within the services, participants felt more could be done. For example, P20 said that the app is not \u201cwidely promoted\u201d within their service, suggesting the peer support team and management should use different methods of promoting the app, such as \u201cput a poster on a bulletin board or send us an email once in a while to say just a reminder that this app is available to us and to make use of it.\u201d P22 believed that \u201cthe biggest thing is there has to be more education\u201d and \u201cmore promotion\u201d within the services as \u201cthere\u2019s a lot of paranoia and distrust.\u201d Comparatively, P30 suggested promoting the app as a part of \u201can onboarding\u201d and \u201cpitch it to people as a preventative thing or an \u2018in case of\u2019 sort of thing.\u201d They believe that employees should download the app even if they do not currently need it as \u201cit\u2019s going to be too difficult to like register when you\u2019re in the moment of all of that.\u201d P52 brought up the idea of telling family members about peer support events and PeerConnect because if their families \u201cknew that all of this stuff was available, probably their spouse is going to be the one that talks them into using it if they haven\u2019t used it before.\u201dTwo participants voiced concerns tied to mental health stigma. For instance, P48 believed \u201cthe biggest thing\u201d limiting the usage of PeerConnect is mental health stigma. They said, \u201cI know that a lot of my peers would benefit from having those supports,\u201d but also that \u201cthey wouldn\u2019t necessarily be inclined to reach out\u201d due to the stigma surrounding mental health. Also discussing stigma, P47 said they were unsure if it is \u201cgenerational\u201d as \u201csomebody old school\u201d may be less inclined to use the app. They went on to say that \u201cit becomes an individual thing they got to want to do it themselves\u201d but that employees may not want to due to lack of education around mental health.Like app users, nonusers were asked if their employers could do anything to encourage app uptake. Except for 1 participant who thought the employer should encourage the usage by improving app communication, others believed there was nothing else that employers could do.Participants were asked whether their employers should continue providing the PeerConnect app and were invited to explain their opinions. All users interviewed believed their employers should keep the app for two main reasons: (1) the app facilitates managing peer support services, and (2) it offers tangible gains, such as breaking barriers to mental health access and allowing employees to refer their colleagues for help. Although nonusers were more critical and less engaged with the app than users, 4 out of 7 nonusers believed employers should continue supporting the app, adding that the app represents a source of occupation-informed mental health support. Only 2 nonusers believed no further support should be warranted. One person was unclear about it.Managing peer support services involved several tasks that were facilitated by the app. P40 appreciated the app\u2019s ability to easily track \u201cnumbers,\u201d \u201cresults,\u201d and \u201cdata\u201d to show the \u201chigher ups\u201d within the services that peer support is effective. Referring to PeerConnect as a \u201cvery handy tool,\u201d P63 appreciated how the app \u201cstreamlines that whole process [of referral], so you\u2019re getting contacted by somebody that you already pre-selected but in a safe and discreet way.\u201d Similarly, P20 valued how peer support users \u201ccan personalize who they are comfortable speaking to from their support team or not\u201d instead of other forms of organizing peer support programs. P52 spoke of how PeerConnect \u201ccuts down on so much time for the peer support team\u201d while being \u201cconfidential\u201d and an \u201ceasy\u201d tool to use.Users also believed that the app offers concrete gains to employee well-being, such as breaking barriers to mental health access and allowing employees to refer their colleagues for help. P14 spoke of how PeerConnect \u201cpromotes wellness\u201d and how they \u201cthink it\u2019s helping to break down some barriers for some people, reduce stigma to an extent.\u201d The belief originates in how peers have contacted them directly as they are offered \u201csupports and products and tangible things they can see.\u201d PeerConnect was recognized as \u201ca benefit for staff as a whole\u201d by P47. They said that \u201cthere\u2019s been a lot of positive feedback\u201d and that they have \u201cseen results already\u201d from people using the app, concluding that \u201cif you help one worker, it\u2019s worth the money.\u201dTwo participants spoke of how PeerConnect should continue to be made available by the services because the app offers occupation-informed mental health care. Simply put by P24:It\u2019s good access to someone who can relate to your problems directly.P37 had a similar view, explaining how \u201conce people start using it and realize it\u2019s for their own benefit and other people sharing with other people that \u2018hey, it\u2019s okay to use that.\u2019\u201d They continued, saying, \u201cI think some people are afraid to say anything in fear or reprisal of anything, so I think it\u2019s great that it\u2019s associated with first responders\u201d rather than just people within their service.One participant, P72, was ambivalent about the app\u2019s future. On the one hand, P72 reported that the app was versatile, allowing for \u201cflexibility\u201d by offering help to employees where they were in a \u201cmultitude of ways.\u201d On the other hand, P72 believed the app did not work well, saying that to continue supporting such a tool would equate to paying \u201clip service\u201d to a technology that was \u201cineffective and allows the problem to affect [people] longer.\u201d P72 feared employers would use the app to excuse their responsibility toward employees with compromised health conditions.Participants were asked if they believed that peer support programs in general were effective in promoting their well-being. Answers varied according to whether they were users or nonusers of PeerConnect. However, users and nonusers appreciated how peer support programs offer help informed by occupational knowledge.App users reported that peer support programs effectively improved well-being in the workplace because the programs advanced a culture of care. As put by P3:We want to promote resilience between our members.They said the \u201cgoal is to provide hope and encourage relationships between people within our workplace who really understand the trials and tribulations of being a first responder\u201d that people in other professions would not understand. P30 voiced a similar opinion, saying that peer support can help to \u201ceducate people and take better care of themselves,\u201d which \u201creduces toxicity in the workplace when everybody\u2019s doing better or even if they\u2019re not doing okay, they know they\u2019re not alone in it.\u201d P22 believed peer support \u201ccreates a framework of normality that mental health, it\u2019s experienced by all of us, and we all have challenges\u201d and \u201ccreates just a system of trust and support within an organization.\u201d Another participant, P52, asserted that \u201cpeer support is more effective than everything else,\u201d having more impact than other methods as \u201cthat casual contact, I think that\u2019s kind of the magic of it.\u201dParticipants stated that peer support, unlike other resources, offered occupation-informed mental health support. P45 thought peer support was \u201cextremely useful\u201d as \u201cit gives people a chance to reach out to their fellow coworkers without having to go to see a psychiatrist yet, or they can reach out to someone who understands what they\u2019re going through.\u201d Echoing P45, P48 said that it is beneficial to have \u201cthe ability to reach out and somebody to talk to somebody who you don\u2019t have to explain the nuances to, and just having somebody to talk to about what\u2019s going on and having them understand and probably having been through if not a similar experience.\u201dSimply put by P8:Everybody needs a mentor or just someone to lean on, especially in our type of work. It\u2019s nice to know that there are other people out there that understand and have your back.Overall, nonusers were not enthusiasts of peer support programs. Nonusers questioned peer supporters\u2019 abilities to offer effective help, given that peer supporters are not trained psychologists. P51 did not think \u201cit\u2019s fair to ask someone not trained\u201d as a psychologist, feeling that peer supports are \u201cnot trained enough professionals to help me off the ledge\u201d in a crisis. P37 was rather blunt in their opinion against peer support programs, saying, \u201cI tried to talk to somebody, it just made the situation worse, and made it worse for me.\u201d P72 was not opposed to the use of peer support programs, suggesting that peer support cannot be the only source of help as some employees may not feel comfortable talking to their peers:Because I need supports doesn\u2019t mean that I want to talk to them [peer supporters] about my problems.One participant, P54, questioned the appropriateness of peer support as a source of help for employees, especially in those cases \u201cwhere the person is actually feeling that their mental health is suffering as a result of their employer,\u201d which a participant defined as \u201cmoral injury.\u201d However, they do believe that \u201cpeer support works in certain situations\u201d and that they are \u201ccomfortable\u201d that their employer has \u201cother avenues for people as well other than peer support.\u201dDespite being nonusers, some participants appreciated the ability to talk with people who understand their experiences on the job\u2014people with subject matter expertise. P24 appreciated how peer support provides an avenue to \u201cvent\u201d with someone who \u201cunderstands\u201d and can \u201csympathize,\u201d that is, someone who knows the occupational role. Likewise, P34 said that although there would not be \u201cenough downtime for me during a shift, probably any first responders to make that connection during the shift\u2026 helpful and beneficial [would be] to be able to use the peer connect\u201d after finishing their shift.Both users and nonusers offered suggestions to improve the app and peer support in general. Most suggestions evolved around creating a culture of care within public safety organizations. To demonstrate, P34 said:It would probably be helpful to make some connections with other first responders who are maybe feeling burnt out or anxious.Without these connections, employees do not feel they have \u201cco-workers to be able to vent with.\u201d Mirroring P34, P47 spoke of how \u201cyou got to make the connection a little more personal\u201d for peer support to work, saying that \u201cit\u2019s a work in progress\u201d for their service. Similarly, P33 noted, \u201cthere\u2019s a change in shift from managers and supervisors 20 years ago where they\u2019re maybe not as compassionate\u201d but views compassion as part of their role. They advocated for the app, saying they would be \u201cvery disappointed\u201d if they took away PeerConnect as \u201cit\u2019s definitely in the right direction.\u201dOne participant, P3, suggested customizing the app now that the \u201ctrial pilot is over.\u201d They believe the app is a \u201cgreat way for people to reach resources while they\u2019re at home and not in the workplace.\u201d Rather \u201cthan not knowing where to start and where to look, the app will bring those resources to one easy to use and less overwhelming place to start searching\u201d and being able to \u201ccustomize it and tailor it\u201d to their workplace to help \u201cmake it kind of a less cynical field.\u201d They continue, saying they \u201chope that people can overcome the fear of using another piece of technology\u201d because in more rural workplaces, \u201cthose services can be a little bit harder to come by, especially when there\u2019s a critical incident at work and you need somebody so crucially within those first 48 hours of that event.\u201dWe interviewed 23 participants, 16 of whom were users and 7 of whom were nonusers of the PeerConnect app. In total, 11 users were peer support providers\u2014evidencing the practicality of the app for assisting with peer support administration and tracking\u2014and the remaining 5 self-identified as receiving peer support. Overall, the participants, especially app users, had positive views of PeerConnect. App users appreciated PeerConnect for the convenience the app yielded to peer support services, reducing pressure on peer supporters, and the ability to raise awareness of wellness and reduce stigma.Connect was the most popular of the 5 features, being used by all app users, and represents the primary purpose of PeerConnect. Newsfeed was the next most popular feature, followed by Resources. Overall, when describing what they appreciated about each feature and the qualities of their favorite features, users described all app features as convenient. In particular, peer support providers appreciated the app\u2019s ability to facilitate peer support services, which is a reason why peer support providers in previous research had expressed interest in peer support apps . SimilarAlthough users appreciated PeerConnect, nonusers had their reasons for not using the app. First, nonusers indicated they were not interested in using the app and thus did not use it. Nonusers also indicated the perception of the app as unnecessary, preferring other mental health resources available on the web and in-person. One participant indicated using alternative mental health apps, which largely made PeerConnect redundant, despite the Connect feature. Another participant mentioned how peer support can be conducted in person and how using an app to facilitate peer support seemed unnecessary. Moreover, nonusers did not like how the app was web-based, suggesting a great benefit would arise if people needing support connected in person because the anonymity of the app may exacerbate feelings of isolation. However, we expect nonusers to be unaware of the behind-the-scenes aspects of organizing a peer support program and the app\u2019s benefits for peer support providers; not to say that a completely web-based peer support program is essential, but that such a program, with future research, may serve to help facilitate in-person interactions. Thus, more knowledge of how the functionality of a peer support app can invigorate an existing in-person peer support program is required.An essential goal of PeerConnect is addressing PSP well-being at work, as with any occupationally specific mental health app. As expected, peer support users appreciated the app. PeerConnect was seen as having a positive impact because PSP were aware that their peers were concerned for their well-being, and merely knowing that supports are available, whether or not they were needed at present, provided PSP with a sense of security. Peer support providers also felt a lot less pressure placed on themselves, which provided space to better support their peers. Indeed, a fundamental value of the app was how it contributed to the administration (and reduced the burden) of providing peer support.When asked whether they believed peer support programs in general were effective in promoting well-being, users and nonusers had different views. Users argued that peer support programs advance a culture of care by promoting resilience and providing occupational-informed mental health education and support. Some nonusers also believed peer support programs can promote well-being and appreciated how peer support allows them to vent with other PSP who understand what they face. However, other nonusers did not believe peers were adequately trained to support each other, making them reluctant to use the app. This is a fair point given the lack of regulations surrounding peer support implementation . In the Participants, both users and nonusers, believed that PeerConnect should continue to be offered by the services. Users believed the app should continue to be offered as it helps facilitate peer support while breaking the barriers PSP face regarding mental health support. Despite not using the app, nonusers appreciated the availability of the occupationally informed mental health support that the app offers. Such findings support PeerConnect, suggesting nonusers may use the app in the future if they need support or resources. App users suggested the services continue to further communicate and promote the app to employees and combat mental health stigma. When asked about possible strategies the services could use to encourage the use of PeerConnect, only 1 nonuser offered a suggestion, which was to further communicate and promote the app.Moreover, although efforts have been made to reduce mental health stigma among PSP, the issue remains, making PSP less likely to seek mental health and peer support; this has been documented in previous research ,50,51. RTo improve PeerConnect, services must continue to make efforts toward reducing stigma. Participants were more concerned with stigma than privacy and anonymity, which has been the central issue in previous research. For instance, a study examining user reviews of publicly available mental health apps found that app users appreciated connecting with people with similar concerns for peer support but preferred to do so anonymously as they felt they could open up more . DespiteThere are 4 main limitations to this study. First, 69% (n=11) of participants were support providers; therefore, the data is largely shaped by people who find the administrative benefit of PeerConnect notable. Thus, the findings could only partially capture the opinions of employees using the app to access peer support services, the population for which the app was created and adopted. However, the perspectives of users are instrumental for understanding app uptake. Second, several factors limited research participation, including a lack of support for the app at the service level; uncertainty about future funding to support the app; research fatigue; workload; delays in the app deployment, which prevented all services piloting the app from participating in the interview; and a lack of time to participate in the interview due to a short time frame. Future research should review PeerConnect once it is updated and more services have had the opportunity to use the app. Third, while data saturation was noticed for users, this was not the case for nonusers. The limited number of nonusers (7 participants) had restricted insights on why nonusers do not use the app. However, this limitation was somewhat expected within the research, as nonusers of any product or service tend to be less likely to participate in research about said product or service. Finally, there was limited information on specific features. For instance, the ability to assess the Events feature was compromised by the COVID-19 pandemic, as public gatherings were few. Due to their role as peer support providers, interview participants focused on Connect, offering limited insights into the other app features. Therefore, future research should ask more direct questions about each of the app features to gain deeper insight into the individual features. Knowledge will help overcome the above limitations as this study provides a starting point to inform implementation.This study explored PeerConnect qualitatively. PSP were open to and optimistic about using mental health and peer support apps. However, PSP services need to continue mental health education efforts to reduce stigma, which in turn would increase PeerConnect and general peer support program usage. Future research should continue to evaluate mental health and peer support apps, especially for PSP, as there is currently limited knowledge on their use and effectiveness. If the findings of our study are any indication, then peer support apps should continue to be developed and offered to PSP to support their well-being."}
+{"text": "Digital peer support enhances engagement in mental and physical health services despite barriers such as location, transportation, and other accessibility constraints. Digital peer support involves live or automated peer support services delivered through technology media such as peer-to-peer networks, smartphone apps, and asynchronous and synchronous technologies. Supervision standards for digital peer support can determine important administrative, educative, and supportive guidelines for supervisors to maintain the practice of competent digital peer support, develop knowledgeable and skilled digital peer support specialists, clarify the role and responsibility of digital peer support specialists, and support specialists in both an emotional and developmental capacity.Although digital peer support has expanded recently, there are no formal digital supervision standards. The aim of this study is to inform the development of supervision standards for digital peer support and introduce guidelines that supervisors can use to support, guide, and develop competencies in digital peer support specialists.Peer support specialists that currently offer digital peer support services were recruited via an international email listserv of 1500 peer support specialists. Four 1-hour focus groups, with a total of 59 participants, took place in October 2020. Researchers used Rapid and Rigorous Qualitative Data Analysis methods. Researchers presented data transcripts to focus group participants for feedback and to determine if the researcher\u2019s interpretation of the data match their intended meanings.We identified 51 codes and 11 themes related to the development of supervision standards for digital peer support. Themes included (1) education on technology competency , (2) education on privacy, security, and confidentiality in digital devices and platforms , (3) education on peer support competencies and how they relate to digital peer support , (4) administrative guidelines , (5) education on the digital delivery of peer support , (6) education on technology access , (7) supervisor support of work-life balance , (8) emotional support , (9) administrative documentation , (10) education on suicide and crisis intervention , and (11) feedback .Currently, supervision standards from the Substance Abuse and Mental Health Services Administration (SAMHSA) for in-person peer support include administrative, educative, and supportive functions. However, digital peer support has necessitated supervision standard subthemes such as education on technology and privacy, support of work-life balance, and emotional support. Lack of digital supervision standards may lead to a breach in ethics and confidentiality, workforce stress, loss of productivity, loss of boundaries, and ineffectively serving users who participate in digital peer support services. Digital peer support specialists require specific knowledge and skills to communicate with service users and deliver peer support effectively, while supervisors require new knowledge and skills to effectively develop, support, and manage the digital peer support role. Digital peer support may show longevity past the COVID-19 era. In April 2020, a survey of 180 peer support specialists from 23 states found a 95% increase in peer support specialists offering digital peer support and a 90% increase in peer support specialists\u2019 confidence in digital peer support .Digital peer support expands the reach and practices of in-person peer support and enhances service users\u2019 abilities to engage in mental and physical health services despite barriers such as location, transportation, and other accessibility constraints. Digital peer support sessions have no geographic or time limitations, promote high levels of engagement when developed with peer support specialists as partners, engage service users in mental health services outside of clinical settings, and have access to harder-to-reach groups such as rural residents, older adults, and people experiencing homelessness . Like inAlthough digital peer support has gained traction globally, at present, no formal digital supervision standards have been put in place. Supervision is considered critical for the development of competent mental health workers . SuperviSupervision of peer support specialists can help to enhance problem-solving skills, improve clarity in the decision-making process, empower workers, increase satisfaction, and help peer support specialists to deliver better services due to opportunities for reflection and discussion of work and work-related issues . EffectiThe transition to, and maintenance of, digital peer support offers challenges for both peer support specialists and service users. For example, a recent study about combining web-based and offline peer support discusses challenges related to the transition to digital peer support, such as protecting confidentiality of service users . Similarwhat essential knowledge does a peer support specialist need to offer digital peer support?\u201d \u201cwhat are the essential abilities peer support specialists need to offer digital peer support?\u201d \u201chow do these essential skills vary by lived experience ?\u201dPeer support specialists that currently offer digital peer support services were recruited via email by an international listserv of 1500 peer support specialists, using a convenience sampling method. Participants were eligible if they were (1) 18 years of age or older and (2) a peer support specialist. Participants were asked to complete a web-based presurvey with questions on demographic information  to ensure variation in focus group participants and participated in a 1-hour web-based focus group via a Health Insurance Portability and Accountability Act (HIPAA)\u2013compliant videoconferencing platform in October 2020. The questions presented in the focus groups were coproduced with 4 peer and nonpeer academic scientists and 4 peer support specialists in a community-engaged research approach described as Peer and Academic partnership to help determine important administrative, educative, and supportive guidelines supervisors could use to support the practice of competent digital peer support and support digital peer support specialists . The intThe collaborative development of supervision standards is important within social-environmental historical contexts . To reprTranscripts were formatted into an all-inclusive Excel sheet that included column headings such as question, participant number, and response. Team members assigned codes to each response. After the all-inclusive Excel sheet was produced, the data table was reduced to include only content relevant to the interview questions. The remaining text and codes were then organized into themes. In accordance with the RADar methodology, themes were determined by the incidence of a code aligned with an overarching theme (see Results). The process of member checking was used to ensure the codes were interpreted correctly and correctly organized into themes. Member checking is a qualitative method used to validate findings, resolve conflicting results, and assess the trustworthiness of qualitative results . The perThe Committee for the Protection of Human Subjects at the Dartmouth Health institutional review board approved the project (STUDY02000620). Participants were told in the consent form that they may voluntarily participate in the 1-hour web-based focus group via a HIPAA-compliant videoconferencing platform, and transcripts would be stored via HIPAA-compliant software accessible only to the research team.A total of 59 peer support specialists participated in the 4 focus groups. Participants' characteristics were reported prior to the interview. The majority of participants were female , and the majority of participants had a minimum of high school education . Participants were from 11 states and 3 countries.We identified 51 codes and a set of 11 themes related to the development of supervision standards for digital peer support. Themes covered administrative, educational, and supportive functions that participants believed were integral to the supervision of digital peer support specialists. The eleven themes in order of most frequent to least frequent included: (1) education on technology competency; (2) education on privacy, security, and confidentiality in digital devices and platforms; (3) education on peer support competencies and how they relate to digital peer support; (4) administrative guidelines; (5) education on the digital delivery of peer support; (6) education on technology access; (7) supervisor support of work-life balance; (8) emotional support (emerging); (9) administrative documentation (emerging); (10) education on suicide and crisis intervention (emerging); and (11) feedback (emerging).The most prevalent theme was education on technology competency. This core theme constituted 21.8% (43/197) of the themes discussed in the focus groups. Peer support specialists believed it would be important for supervisors and agencies to help digital peer support specialists to develop knowledge on different technological platforms and devices. One peer support specialist mentioned:A peer support specialist needs to have a comfort with whatever virtual platform they are using to interact with the service user. \u2026I would imagine that, you know, the process of engagement would look very different depending on what kind of technology is being used as the shared platform.Multiple participants added that digital peer support specialists should not only have basic knowledge of technology but should also be able to teach others how to use technology. One participant said:They [digital peer support specialists] need some basic knowledge of computers and a virtual format, like accessing applications like zoom, not only to be able to navigate it, but to teach others how to access it and to navigate it on different platforms, like a computer and a mobile phone.Access to training on digital technology and platforms was encouraged, and some peer support specialists suggested that agencies should offer videos and support in the acquisition of technology knowledge. One peer support specialist mentioned:We offer different training videos and whatnot for different peer support levels\u2026I think that's something that would be interesting to see rolled out to other organizations as well. Having those sort of videos on how to use the technology exactly how to incorporate your thoughts and empathy into words and how to convey exactly what you're hiding.The second theme, education on privacy, security, and confidentiality in digital devices and platforms, constituted 16.8% (33/197) of the themes discussed in the focus groups. Peer support specialists recommended that agencies and supervisors help to clarify and educate peers on certain topics related to privacy, security, and confidentiality. For example, a few participants suggested education on privacy policies, mandatory reporting, and data collection. One participant questioned:There's data, so how is data being collected, if it's being shared? Are there third parties? Who's being shared with? Is it shared\u00a0with treatment teams? Is there data that need is like how does mandated reporting work for certain in certain circumstances with different agencies?...Is there a privacy policy?Another participant recommended the clarification of definitions of privacy, security, and confidentiality and how they relate to the agency and to the service users in which peer support is being offered. One peer specialist said:I think definitely, definitely just what the definition of confidentiality means and how it can be defined differently to different individuals\u2026And I think having that mutuality between you what does confidentiality mean to you, what does it mean to me?Developing knowledge in the evaluation of the security and privacy of digital platforms was not only important to peer support specialists but also having transparency around how privacy, security, and confidentiality guidelines may change depending on the form of peer support. One participant mentioned:Differentiating between a warm line support call versus a crisis call\u2026let people know this is a warm line. And we can talk about certain things. But as soon as you start to talk about harming yourself or others, now, this is turned into a crisis call. It's no longer a warm line call. And what I've been told by emergency services workers in Virginia, one in particular, is that once there is a crisis, confidentiality, HIPAA no longer applies.The third theme, education on peer support competencies and how they relate to digital peer support, constituted 12.7% (25/197) of the themes discussed in the focus groups. Many peer support specialists emphasized the importance of offering training on general peer support and maintaining the important values and principles of peer support during transitions to digital settings. One participant suggested:Peer support requires many different skills. What essential knowledge do peer support specialists need to offer digital peer support? They need to know peer support cold. They need to be aware of how that translates virtually.Other participants discussed the skills they would look for in peer support specialists when hiring them for peer support. They believed that a competent delivery of services would include general peer support competencies. One peer support specialist said:I would want them to be top-notch peer supporter[s] before we even go into digital peer support. I would want someone that knows peers support, [and] that had the certified peer support specialist or a recovery coach so [he/she/one] knows how to deal with substance use, knows good peer support, and has a solid foundation. We\u2019re not hiring people to do digital stuff; we\u2019re hiring people to do peer support and the digital platform is just one way to deliver the peer support.While digital peer support requires new knowledge, skills, and supervisor support, it also requires solidification of the basic principles and values of peer support within the digital environment. One participant mentioned:Peer support is peer support. I\u2019ve been doing this for 23 years, I\u2019ve talked to doctors, nurses, graduate students, etc and they all think that we are the saviors, and we\u2019re going to be able to do the work that they can\u2019t do. I\u2019m just gonna sit down with them, I\u2019m going to shut up, I\u2019m going to listen, and I\u2019m not going to fix them, I\u2019m not going to judge them. So really, peer support, whether it\u2019s digital or face to face, is the same basic principles.The fourth theme was administrative guidelines. The theme of administrative guidelines constituted 10.7% (21/197) of the themes discussed in the focus groups. Peer support specialists recommended the development and transparency of administrative guidelines surrounding topics such as technology security, work-life boundaries, and suicide prevention. One participant said:I think that's essential knowledge for the peer specialist to know what they are, what boundaries they have, for what roles they may be having, and that they're not all the same.For example, participants suggested the creation of agency guidelines around communication methods used in suicide prevention on the internet. One participant mentioned, \u201chaving a real good fine communication flow chart. So that if an event does happen, that, you know, the peer support will know you know what to say, and also who to reach out to, so that they can get the extra support that they need through this as well\u201d around confidentiality and what information peers need to share with supervisors. One peer support specialist said, \u201cthere are certain things that if we discussed and talk about I have to share with my supervisor\u201d and around digital platforms and technologies that are acceptable for digital peer support. For example, one participant observed, \u201cthe state mandates what [digital platforms and technology] you can and can\u2019t use when you\u2019re providing services to their peers or to your peers.\u201dThe fifth theme, education on the digital delivery of peer support, constituted 9.1% (18/197) of the themes discussed in the focus groups. With the transition from in-person peer support to digital peer support, peer support specialists have requested for agencies and supervisors to acknowledge the difference between providing services in-person opposed to digital. Many peer support specialists have appealed for training surrounding digital technologies and the digital delivery of peer support. For example, one participant mentioned:I would want them to attend the digital peer support training. I think some training in digital support, the separate platforms and just talking about software and hardware and how they work together.This includes training and skill development to engage service users on the internet and display empathy in a digital setting. One peer support specialist suggested:Some sort of technical training on empathetic listening or some other ways of being able to convey emotion without necessarily being able to be connect. A personal conversation training maybe.Participants also recommend that supervisors provide peer support specialists with the opportunity to decide whether digital peer support versus in-person peer support would be the best option for them, based on knowledge of both roles. One participant said:Make sure any new hiring you\u2019re having do digital support want to be a part of that. Not just \u2018okay, we\u2019ve hired you as a peer and you\u2019re going to do this also\u2019 because for some people it would be uncomfortable for them to be providing those services digitally.The sixth theme, education on technology access, constituted 8.6% (17/197) of the themes discussed in the focus groups. Access to technology and internet service is an important aspect of digital peer support. However, there are many populations that have difficulties accessing the devices and infrastructure they need to effectively use digital peer support. In order to expand the reach of digital peer support, peer support specialists believe peers need to be aware of the resources and tools available for the support of underserved populations. For example, one participant said:it takes a really, really strong commitment and awareness to really open the doors of the service system much, much wider. Essentially, now that you know, there's so much construction to the pandemic right.Supervisors could potentially help digital peer support specialists to gain knowledge on resources and tools necessary to meet the needs of individuals, such as those experiencing homelessness or those living in rural settings. On participant mentioned:There are a lot of individuals who may be on it, whether they're experiencing homelessness, or don't have consistent access to, for example, like charging or maybe have like, very, very basic phone, like flip phones. And so I would think to having kind of resourcefulness maybe being\u2026having tools to be able to, I think, be proactive and kind of have insight and awareness to meet the needs of various populations in terms of location and in access.The seventh theme, supervisor support of work-life balance, constituted 8.6% (17/197) of the themes discussed in the focus groups. Supervisor support is essential to the maintenance of a healthy work-life balance. Many peer support specialists believe supervisors can help to set time limits and boundaries in service user\u2019s access to telehealth. One participant recommended:Incorporate, you know, some boundaries around your personal time, and to make it a main priority. And again, I think with the accessibility of the, of telehealth, it just is, you know, it just makes it just that much more important. \u2026 the supervisor is going to have to help out with this.Supervisors should be mindful of digital fatigue and help peer support specialists to schedule adequate breaks and time-limits. For example, one peer support specialist recommended:\u2026setting reasonable limits as to the amount of time that is expected of people. So giving people adequate breaks and stuff, so they don't have to do too much. \u2026just being mindful of when you are starting to get that fatigue.Open communication and collaboration between supervisors and peer support specialists can help to address and resolve issues with work-life balance. One participant said:\u2026be upfront and open with those that you're supporting of your availability, because it's also important for you to protect yourself and your boundaries and maintain your health and self-care and all those things.The eighth emerging theme, emotional support, constituted 4.6% (9/197) of the themes discussed in the focus groups. Peer support specialists believe the additional challenges that come with digital peer support require additional support. Many participants believed reaching out for help from a supervisor or other peer can not only help with self-care but can help the peer support specialists to grow in their role as a digital peer support specialist. One participant said:I think an essential ability is to that when you are having those tough moments to make sure that people reach out for help, so that they don't feel like they're struggling on their own, and that they do get feedback on some of those maybe tougher cases\u2026 don't be afraid to reach out for help, because it, it helps you grow in your role. But it also helps with the self-care.Many participants agreed that the transition to digital peer support was often stressful and overwhelming. For example, 1 peer support specialist said:Whether it's a digital or, you know, traditional, but I think more so digital, because a lot of us peer supports are new at this to, like, you know, it was hard enough for us to document now, and now we're trying to like, enter, you know, and do all this stuff on a phone or a computer, that is just a more stressful and I think it just makes me think of it, there's peers that have kind of, you know, resigned because of that, because it's like, Ah, you know.As a result, many believed emotional supervisor support could help peer support specialists to address feelings such as stress and fatigue and other experiences such as retraumatization. One participant mentioned:With peer support, you know, we talk about the trauma informed, because it's a part of our lived experience to share those pieces where it builds connection, we might get re traumatized all over again, and not even realize it until we're trying to sleep that night that something doesn't feel right. You know, and so to be able to talk about it to reach out to your supervisor or another coach or peer, I think is really important and that's foundational.The ninth theme, administrative documentation, was an emerging theme and constituted 3% (6/197) of the themes discussed in the focus groups. Participants agreed that documentation of peer support would differentiate between in-person and web-based support. One participant mentioned, \u201cI think documentation is going to be a little different for what you have to do on telehealth versus what you\u2019re doing in person.\u201d Knowledge of the changes in documentation requirements and regulations is important to the supervisor role. For example, 1 participant suggested knowledge in \u201ccertain regulations\u2026or documentation\u201d and \u201cknowing kind of what with your state are certain requirements or different kind of policies at the state level.\u201d Participants suggested that digital peer support and the use of digital devices and platforms may offer challenges to the process of documentation for supervisors and others. One peer support specialist said, \u201cIt was hard enough for us to document and now we\u2019re trying\u2026to do all this stuff on a phone or computer.\u201dThe tenth emerging theme, education on suicide and crisis intervention, constituted 2.5% (5/197) of the themes discussed in the focus groups. Many participants emphasized the difference between suicide and crisis intervention in-person opposed to digitally. For example, 1 participant said, \u201cwe're all very skilled in in-person crisis response and that completely changes when you're digital.\u201d As a result, peer support specialists believe supervisors should offer trainings on digital suicide and crisis intervention. One participant recommended:A training [or] even just a conversation about how to respond to someone that is in crisis, virtually. When you can't be there to control what's in their environment or their actions, how can you respond to keep them safe while you get them in person assistance? I think that's an important discussion that needs to be had and maybe training that needs to be developed.Digital peer support specialists should know when to contact supervisors in an emergency. For example, 1 peer support specialist suggested the importance of \u201cmaking sure that somebody is sufficient in those competencies such as suicide prevention, how to contact supervisors in a[n] emergency, how to diffuse the situation, [and] talk somebody down\u201d and have access to skill development and trainings in crisis intervention. One participant said:One of the things that you would need to know is when to activate and do an active rescue versus just an imminent risk. If you\u2019re going to take on that role, there\u2019d have to be some additional training of knowing when it is [a] crisis, [or] that they\u2019re just reaching out for help and support.The last emerging theme, feedback, constituted 1.5% (3/197) of the themes discussed in the focus groups. Many peer support specialists believe receiving feedback from supervisors is important to building competency in digital peer support specialists. One participant suggested supervisor feedback is helpful in \u201cpracticing how to do the things that you talk about doing. And making sure that somebody is sufficient in those competencies such as suicide prevention, how to contact supervisors in case there is a need for emergency, how to defuse the situation, how to talk somebody down although that's the same thing with diffusing situation or what I'm saying and yeah that's about it.\u201dFeedback also helps to uncover skills or knowledge supervisors themselves may need to improve upon. For example, 1 peer support specialist suggested that feedback \u201chelps you as a supervisor to know what skills you need to work on both in supervision and you know for trainings.\u201d Peer support specialists recommended feedback methods such as having a supervisor sit in on a meeting or call, or practicing role play in which the supervisor pretends to be in a crisis and then discusses the ways in which the specialist could have improved their digital support. One participant recommended having \u201cthe supervisor sit in on one or two or three or four of the peer support groups or calls\u201d and another recommended having \u201ca fake call or fake message conversation where for an hour the supervisor pretends to be in crisis and reaches out and we have to provide ample support to them and then they critique us on everything that we said after the hours up and they tell us if we are allowed to go on and do peer support provider.\u201dThe following themes emerged from the four focus groups (N=59): (1) education on technology competency; (2) education on privacy, security, and confidentiality in digital devices and platforms; (3) education on peer support competencies and how they relate to digital peer support; (4) administrative guidelines; (5) education on the digital delivery of peer support; (6) education on technology access; (7) supervisor support of work-life balance; (8) emotional support (emerging); (9) administrative documentation (emerging); (10) education on suicide and crisis intervention (emerging); and (11) feedback (emerging). Established supervision standards may help to promote the competent delivery of digital peer support and help to encourage skill development, knowledge, support, and guidelines for the digital peer support role. These supervision recommendations may act to enhance the established supervision standards endorsed by SAMHSA.The purpose of this study was to inform the development of supervision standards for digital peer support and introduce guidelines that supervisors can use to support, guide, and develop competencies in digital peer support specialists. Currently, supervision standards from SAMHSA for in-person peer support include the categories of administrative, educative, and supportive functions. However, the spread of digital peer support during the COVID-19 pandemic requires the expansion of supervision standards to include subthemes such as education on technology and privacy, support of work-life balance, and emotional support. Without digital supervision standards, there are potential risks of a breach in ethics and confidentiality, workforce stress, loss of productivity, loss of boundaries, and ineffectively serving users who participate in digital peer support services.Digital peer support is quickly expanding across the globe. However, the transition to digital peer support brings new challenges and the necessary acquisition of new guidelines and skills. While SAMHSA has developed supervision guidelines for in-person peer support, digital peer support requires the expansion of supervision standards and the significance of administrative, educative, and supportive supervision. Digital peer support specialists require specific knowledge and skills to communicate with service users and deliver peer support effectively. Therefore, supervisors also require new knowledge and skills to effectively develop, support, and manage the digital peer support role.This study is not without limitations. First, there are potentially other supervision standards and guidelines that have not been identified. Second, the sample lacked diversity based on racial and ethnic background. Future studies should consider the inclusion of disadvantaged populations such as Hispanic, and LGBTQIA populations and demographics such as homeless individuals. Third, as technology changes and digital peer support expands, supervision standards will need to be updated. Fourth, the findings cannot be generalized to all digital peer support specialists due to the small sample size. However, we reached saturation when additional data did not provide more information across international boundaries, and the themes identified could be used to promote consistency in the practice of digital peer support. Fifth, the data could not be stratified by volunteer-run services versus paid professional services or the role of the participant . Sixth, Prior work has shown that digital support can be just as effective as in-person support in patient-clinician engagement . There aIntroducing supervision standards for digital peer support is a first step in helping to guide the delivery of digital peer support and the development of digital peer support specialists. As defined by SAMHSA, supervision is important to competency building and skill development in peer support. Supervision has the potential to improve performance, empower workers, and promote knowledge of the peer role. The shift to digital peer support has expanded the reach of in-person support and has shifted how peer support is both delivered and managed. The identification of supervision guidelines for digital peer support has the potential to facilitate the transition from in-person to digital peer support and promote best practices in both digital peer support delivery and the supervision of digital peer support specialists."}
+{"text": "Diabetes and cardiovascular disease (CVD) contribute to significant morbidity and mortality in low-resource settings. Living with diabetes can be overwhelming, isolating, and exhausting, even in settings of resource availability and health care access, while the psychosocial burden of living with diabetes and CVD can be exacerbated by an increased burden of social determinants of health in low-resource settings. Diabetes and CVD care heavily rely on self-management, and health care professionals are now recognizing the importance of peer support in supporting healthy behaviors, psychosocial well-being, and improved clinical outcomes. However, there is currently a lack of consistency in how peer support programs are defined, implemented, and evaluated. Lancet Commission on Diabetes report published in 2020 reveals that people in LMICs face a disproportionate burden of the disease, with 80% of cases occurring in these regions [Noncommunicable diseases (NCDs) are increasingly being viewed as a significant health concern globally, accounting for 74% of global deaths, 86% of which occur in low- and middle-income countries (LMICs) . Cardiom regions . The lin regions , yet mos regions .Diabetes requires daily complex medical decisions related to adjusting food intake, physical activity, and medications to maintain blood glucose levels in a desired target range. The psychosocial burden of living with diabetes is well defined in high-resource settings , and the7810Roadmap on the Prevention of Cardiovascular Disease among People Living with Diabetes [151617The World Heart Foundation\u2019s Diabetes  defines Diabetes  and effeDiabetes 15161718.Journal of the American College of Cardiology, Sherifali et al. report results from a scoping review of peer support interventions for PLWD in LMICs. Inclusion criteria included participants with type 2 diabetes, another CVD, or risk factors for developing CVD and a quantitative study design. Peer support could be delivered in any format  and any setting , as long as it was delivered by a layperson, not a medical professional. Publications were compared with respect to Evan\u2019s definition of peer support, which defines five core functions of peer support, including \u2018being there\u2019, \u2018assistance in daily management\u2019, \u2018social and emotional support\u2019, \u2018linkages to clinical care and community resources\u2019, and \u2018ongoing support\u2019 [In this issue of the support\u2019 . Of the support\u2019 , more reSeveral studies evaluated in the scoping review utilized community health workers rather than PLWD or CVDs to implement peer support. As the authors note, the implication and value of the provision of peer support by a member of the community not living with a similar NCD is unclear. Further research may be necessary to differentiate between the roles of a community health worker or peer supporter and the relative value of both. An additional group who may be valuable in the peer support process includes PLWD or CVD who are also health care providers. Though small, this group has the potential to be extremely impactful in their community through integration of medical knowledge in their existing peer community. Peer support programs may also provide valuable space for health care providers not living with an NCD to learn about the lived experience, challenges and practical tools and tricks from daily experiences. Utilization of lay community workers not living with an NCD may blur the lines between peer and community/social support, while exclusion of health care providers from the peer support process may lead to missed learning opportunities.Use of a broad definition of peer support adds to the strength of this study, given that the needs and structure of peer support programs can vary widely depending on cultural context. Sherifali et al. found that peer support was generally associated with improvement in clinical outcomes such as HbA1c. However, the results highlight a disconnect between the stated goals of peer support programs and how outcomes are currently measured\u2014only 61% of publications reviewed included behavioral or emotional outcomes, while only one looked at the \u2018being there\u2019 aspect of peer support. The concept of \u2018being there\u2019 along with behavioral and emotional support are arguably the most critical aspects of peer support, yet they were rarely measured in study outcomes. While these outcomes may have been addressed in qualitative studies not evaluated in this review, there is also a need for more structured and standardized formats to quantitatively evaluate behavioral and emotional outcomes related to diabetes and CVD management.The importance of inclusion of the voices of those with different NCDs in all levels of care is increasingly being acknowledged in global guidelines, and expanded availability of peer support will be critical to this process . To dateAnother significant area of peer support not yet addressed in the literature is the use of social media and online communities. Today, more and more people are turning to these communities for support in making informed decisions about their health. However, the impact of social media and online diabetes communities on health outcomes is still not fully understood. Potential benefits that come with the ease of connecting virtually with the diabetes community need to be weighed against known risks associated with social media and internet use, including the spread of medical misinformation and stigma.While research on peer support in high-resource settings has largely focused on PLWD, Sherifali et al.\u2019s scoping review highlights the value of a more integrated approach to peer support that acknowledges the dual burden of diabetes and CVD and is inclusive of CVD in addition to diabetes. From a health systems perspective, integrated approaches to NCD care have been shown to improve outcomes in LMICs. Promising models such as the Lancet NCDI Poverty Commission\u2019s PEN-Plus program, which has been adopted by WHO/AFRO to improve access to care for PLWD and CVD in rural SSA, has great potential in strengthening multidisease peer support efforts for severe chronic NCDs at intermediate care facilities in LMICs 23.While broad population-level campaigns are necessary to address the social risk factors that contribute to the development of diabetes and CVD, the estimated projection of 1.3 billion PLWD by 2050 also need improved support. Future studies should focus on defining evidence-based guidelines and programmatic monitoring and evaluation of peer support that is inclusive of emotional and behavioral outcomes. Further exploration of practical ways to enhance peer support for PLWD should be done in collaboration with those living with diabetes and CVD and should focus on cultural perceptions, increasing representation, and prioritizing the diverse needs of those in LMICs ."}
+{"text": "Artificial intelligence (AI) and data sharing go hand in hand. In order to develop powerful AI models for medical and health applications, data need to be collected and brought together over multiple centers. However, due to various reasons, including data privacy, not all data can be made publicly available or shared with other parties. Federated and swarm learning can help in these scenarios. However, in the private sector, such as between companies, the incentive is limited, as the resulting AI models would be available for all partners irrespective of their individual contribution, including the amount of data provided by each party. Here, we explore a potential solution to this challenge as a viewpoint, aiming to establish a fairer approach that encourages companies to engage in collaborative data analysis and AI modeling. Within the proposed approach, each individual participant could gain a model commensurate with their respective data contribution, ultimately leading to better diagnostic tools for all participants in a fair manner. Due to its impressive ability to process large amounts of data quickly and identify patterns and trends that may not be immediately apparent to humans, artificial intelligence (AI) has the potential to greatly assist society in a wide range of sectors, including health care ,2. In meDeciding what to do with your data is always context specific. Your goals determine the most appropriate way to use your data and whether data can be shared with third parties. This involves balancing the benefits and potential harm in light of your context. If you, for example, operate within a public administration, your organization might aim at creating value primarily for all current member inhabitants, with or without a wider perspective on other parts of the world and on future challenges. Now, if you operate in a for-profit company, the primary goal might be centered around achieving economic success, whereas in nongovernmental organizations, the organization may prioritize a political, societal, or environmental agenda. Every setting has its own goals, some explicitly stated and some silently present, including ethical and other generic considerations, all with the power to guide decisions. As long as the data are retained within the organization's boundaries, the organization is in control of what its data are actually used for. Conflicts about opposing goals can be resolved internally, maximizing the potential to do good in a context-specific meaning.As we aim to maximize the potential of using and reusing data by sharing it with others, we find ourselves traversing complex terrain. Sharing data can be approached in various ways along a continuum, ranging from the most constricted, such as running analyses on the data and only sharing condensed bits of information , to the least constricted, such as providing full access as open data, for anyone to use at any time and for any application. Every organization may judge, based on their goals and permissions, which part of their own data to share with whom and in what way. For example, achieving public administration\u2019s goals usually includes sharing data as openly as possible for most data entities in their primary ownership, by that, maximizing the potential for use or reuse of that data for the benefit of all . On the Sharing data can help us gather a significant amount of data to train robust and highly predictive AI models, which could have a profound impact on society, such as improving medical diagnoses for patients. In health care applications, the implementation of health information exchange solutions has facilitated the efficient sharing of health data across different organizations . Health In FL, a central system governs the learning process, while in SL, the parties communicate directly with each other without a central coordinator  Figure . By usinIn specific circumstances, collaborative efforts and sharing data can be advantageous for all parties, even when they are competing companies. This practice of collaborating with competitors, known as co-opetition, is especially helpful for firms seeking to innovate as they allow for pooling resources to reach a goal that an individual firm may not be able to reach on its own. Additionally, co-opetition can lead to greater efficiency and economies of scale . HoweverIn this section, we discuss a case scenario to examine our offered approach.Imagine 3 companies offering the same products or services, with different data collection and management processes, and they aim to develop AI systems, for example, recommender systems, to enhance their revenue. Suppose, for instance, the first company has a basic process for data management with low investments and therefore a lower revenue from their AI system. The second company has optimized its data management with more investments, thus gaining a higher revenue. Finally, suppose the third company has invested the most in data management, obtaining the highest revenue among the 3. If the 3 companies decide to use FL or SL to maximize their revenue, the first company would benefit the most with the highest gain-to-invest ratio, and the third company would benefit the least with the lowest gain-to-invest ratio. This would not be fair from an economic point of view. Based on the economic notion of fairness, we identify an FL or SL system as fair if nodes receive models commensurate with their contributions to the learning process. This cannot be accomplished with a typical FL or SL system in which all nodes ultimately receive the same model. Instead, an alternative framework is necessary to allow nodes to obtain personalized models based on their contributions.In order to investigate this problem, we simulated an SL network in which nodes collaboratively train personalized machine learning models with performance levels proportional to their contributions. In this simulation, we define a node\u2019s contribution as the number of data points they provide for the learning process. Since having more data typically lead to better models, it is reasonable to assume that nodes contributing a larger amount of data play a more significant role in the training process. In this example, we assume that only informative data are used and do not take into account any fraudulent approaches where useless data are provided by a client. The detection of fraudulent behavior in collaborative work through computational methods is a subject for further investigation and is outside the scope of this study.For this simulation, we considered 3 nodes, each with a distinct number of data samples. The nodes use an SL system based on random forest , which iBased on the results, we argue that within such a fair SL framework, first, organizations with more resources are more likely to cooperate with other parties for a collaborative learning task since the payoff will be fair. Second, organizations providing fewer resources still benefit from cooperation with other parties and are still likely to take part in the task. In conclusion, using fair and diverse data sets for training AI is essential for achieving efficient and effective decision-making from an economic perspective. Ensuring that the training data are representative of the population for which it will be used, balancing the costs and benefits of data, and complying with regulations and guidelines can help to promote the responsible and ethical use of AI in the economic sphere."}
+{"text": "In the age of information, new platforms are consulted by patients to acquire consciousness about medical treatments. The aim of this study was to assess the level of understanding and feasibility of video consensus (VC) administration in patients scheduled for radical prostatectomy (RP), comparing it with standard informed consensus (SIC). According to the European Association of Urology Patient Information, we set up a video content for RP that was translated in Italian and implemented with information about possible perioperative and postoperative complications, days of hospitalization etc. From 2021 to 2022, all patients undergoing RP at our institution were prospectively included in this study. Patients received an SIC and after that, a VC about RP. After two consensuses were administered, patients received a preformed Likert 10 scale and STAI questionnaires. On the RP dataset, 276 patients were selected and 552 questionnaires for both SIC and VC were evaluated. Out of these, the median age was 62 years (IQR 60\u201365). Patients reported a higher overall satisfaction for VC (8.8/10) compared to the traditional informed consent (6.9/10). Therefore, VC may play a role in the future of surgery, improving the consciousness and satisfaction of patients and reducing preoperative anxiety. In the current technological era, especially following the COVID-19 pandemic where virtual strategies and \u201csmart learning\u201d activities are being used to continue education ,3; patiehttps://www.urologyhealth.org; accessed on 28 April 2023) and the European Association of Urology (EAU)  have created educational contents based on the AUA Clinical Guidelines [In this setting, the Official Foundation of the American Urological Association (AUA)  may represent a barrier for patients for a full comprehension on the proposed procedure. Thus, the quest for the standardization of an all-inclusive consent with a detailed text and figures, safe graphics or video content, represents a new challenge in the medical scenario.In this panorama, prostate cancer (PCa), the second most common cancer in men , is the Therefore, in our study we attempted to assess the level of understanding and feasibility of VC administration in patients scheduled for radical prostatectomy (RP), comparing it with standard written informed consensus and their overall satisfaction and preoperative anxiety.n = 25), incomplete questionnaires (n = 17) and blind patients (n = 1) were the exclusion criteria.The study was based on the administration of a video content and a satisfaction questionnaire in addition to SIC after the admission to the Urology department of our institution. An ethical board approval was not mandatory. All patients enrolled were requested to be involved, on a voluntary basis, in the study, signing a willing document of inclusion. A prospective analysis of the RP database was performed. From January 2021 to December 2022, the baseline, demographic clinical and perioperative data from 319 patients who underwent laparoscopic RP at our tertiary-referral center were collected. We selected patients with a clear PCa histology after prostate biopsy and with locally confined disease based on preoperative imaging (CT scan and bone scan). All surgeries were performed by two experienced surgeons . Lack of demographic and baseline data  from an Italian Urologist, a certified English speaker with a GMC license to practice and implemented with several information sections at the end of it, such as possible perioperative and postoperative complications, days of hospitalization and catheterization length. The video content lasted about 3 min and 30 s. -Firstly, patients received a print-based traditional consensus of RP administered from a physician in charge of explaining the surgical operation and possible complications.-After the written traditional consensus, video content about RP with several added explanations was shown by a physician to the patient through an iPad.-ComprehensionSatisfactionSimplicityAfter written and video consent, patients filled in a preformed Likert 10 scale questionnaire  with a sOur study was conceived as a four-step procedure:-Finally, patients performed a State-Trait Anxiety Inventory (STAI) questionnaire  , in the Specifically, the STAI questionnaire is a self-assessment questionnaire that consists of a total of 40 questions, composed of 20 items assessing trait anxiety (Y2)  and other 20 items assessing state anxiety (Y1) . In detail, state anxiety is related to how much the person feels anxious \u201cright at that moment\u201d and expresses a subjective feeling of tension and worrying, relational behavior of avoidance and an increase in the activity of the autonomic nervous system  relative to a stimulus situation, therefore transient and of variable intensity; while trait anxiety refers to how the subject usually feels, to a more enduring and stable condition of personality that characterizes the individual on an ongoing basis, regardless of a particular situation . AccordiContinuous variables were presented as median and interquartile ranges (IQRs) and were compared using the Mann\u2013Whitney U test, one-way based on their non-normal distribution. The descriptive and variance analysis was performed through the Statistical Package for Social Sciences (SPSS) software v.28.0 , with an alpha value of significance set at <0.05, comparing the different types of consent.Overall, 43 patients were excluded from the analysis. Consequently, a total of 276 patients were included in our study. We received and evaluated 552 questionnaires for both written and video-based consent and 276 STAI questionnaires. Concerning the clinical and operative features of patients, 91% (251 patients) were aged 50\u201370 years, 9% (25 patients) were over 70 years. The median patient age was 62 years (IQR 60\u201365). After the histological report, out of 276 patients, 53 patients (19.2%) had ISUP 1, 90 patients (32.6%) had ISUP 2, 86 patients (31.1%) had ISUP 3, 35 patients (12.7%) had ISUP 4 and 12 patients (4.4%) had ISUP 5.We evaluated the mean score \u00b1 standard deviation (SD) for each domain of the Likert scale questionnaire between two informed consents. More in detail, the mean comprehension score was 6.87 \u00b1 (0.33) in the written consent group versus 8.47 \u00b1 (0.50) in the video consent group. The mean satisfaction score \u00b1 (SD) was 7.26 \u00b1 (0.44) in the paper-based consensus group versus 9.23 \u00b1 (1.31) in the VC group. The mean simplicity score \u00b1 (SD) was 6.56 \u00b1 (0.50) in the standard consensus group versus 8.74 \u00b1 (0.43) in the VC group .p = 0.000). Afterwards, we also described a higher overall score reported by patients for video consent (8.8/10) compared to traditional informed consent (6.9/10).Additionally, the median and interquartile range were assessed for the same domains and were compared between two informed consents. Concerning the STAI questionnaire, the administration of the RP video content led to a decrease in preoperative patients\u2019 anxiety, from a mean comprehension score \u00b1 (SD) of 66.4 \u00b1 (11.54) to a mean comprehension score \u00b1 (SD) of 52.1 \u00b1 (14.9), while the median and interquartile range were from 67 (IQR 63\u201378) to 53 (38\u201365), respectively. p = 0.004; thumbs up/views p = 0.015). Finally, 77% of these reviewed videos attracted more public engagement (>6 million viewers), despite included latently misinformative and/or biased data within the video or comments section.The rise of patients\u2019 awareness of PCa is related to a growing desire to know more about a disease that affects, potentially, not only overall survival, but also social and sexual life. Despite the invalidating sequelae on erectile function and urinary continence, RP remains the standard of care for the management of clinically significant PCa . Indeed,Social media analysis may help to understand how to reach our public more effectively and how use them in a positive manner , choosinWritten informed consent administration is a fundamental step before surgery, to let patients understand the scheduled procedure, possible complications and extinguish any doubts about it. Three substantial criteria are needed for an adequate informed consent: the patient must be knowledgeable, adequately informed and not obliged .Moreover, before obtaining written informed consent for a surgical procedure, physicians need to let the patient know about the type of the surgery, the expected outcomes, material risks and adverse events, alternative surgical or non-surgical treatments, if available, and the consequences of them. As regards material risks, there are for each procedure specific risks and common risks for all surgeries, such as anesthesiologic troubles, blood loss, potential blood transfusions, infections etc. Otherwise, only in emergency scenarios can surgery be performed without informed consent, when the patient is not comprehensive and there is not available a substitute decision-maker .Despite the doctors\u2019 meticulousness during the consent administration, the lack of a full comprehension of the risk\u2013benefit ratio by patients may represent an unsolved issue. Vikas et al.  assessedp = 0.000). Our results are in line with those reported in the literature by previous studies concerning other fields of surgery, such as bariatric and trauma surgery [In this background, we attempted to investigate the overall satisfaction and preoperative anxiety of patients undergoing RP at our institution, prior and after the administration and explanation of video content. Our evaluation showed interesting findings. First of all, the evaluation of the questionnaires on patients\u2019 satisfaction allowed us to understand how much the patients\u2019 awareness had significantly improved with the administration of video informed consensus compared to the SIC  of men for video consent compared to traditional informed consent (6.9/10). In the age of information, video consent represents a simple and comprehensive tool for patients, which improves their awareness and satisfaction and reduces the preoperative anxiety for the treatment chosen. Our study marks a new era of informative consensus through a shared scientific information supported by video content."}
+{"text": "To design a class of full cover trajectories that satisfy curvature limitations and enhance the buckling load of constructed laminates, a variable stiffness laminate is proposed by applying the cubic Ferguson curve. First, the traditional explicit form of the cubic Ferguson curve is redefined as polar coordinates, two connected Ferguson curve segments with three extra parameters are applied to describe full cover trajectories, and the effects on trajectories introduced by these modifications are discussed. Then, the finite element method is used to introduce parameters for analyzing the buckling load of the designed variable stiffness laminates. Numerical experiments show that automatic fiber placement (AFP) trajectories described by the cubic Ferguson curve can automatically reach  Compared with traditional materials, fiber-reinforced composites have many advantages, including higher strength-to-weight and stiffness-to-weight ratios, resistance to corrosion, and ease of shaping and tailoring their structural configurations. Therefore, they are widely applied in advanced high-technology fields, such as aeronautics, transportation, and electrical power, in areas including aircraft structural components, artificial satellites, and high-speed rail cars. At present, automatic placement technology has become the main processing method for high-performance composite materials and is widely applied in modern aircraft manufacturing enterprises. The technology comprises filament winding, tape placement, and automatic fiber placement (AFP). The AFP can independently operate each prepreg tow, and the width of the prepreg assembled can be adjusted based on the surface features and manufacturing requirements. This approach has a broader applicability than filament winding, and tape placement can be placed in a geodesic manner. Therefore, this technology is particularly suitable for the automatic formation of large curvatures and complex composite components, such as S-shaped inlets and load bearings.As an automatic as well as the most-advanced manufacturing technique for producing fiber-reinforced composite components, efficient and reasonable trajectory generation tactics are key factors for determining the quality and efficiency of fiber placement. The existing approaches to trajectory generation for AFP can be classified into constant stiffness and variable stiffness methods. If the ply structures of these components are the same in all positions, then the stiffness is the same, and they are named constant stiffness components. Several studies have been conducted on this topic: ,4,5,6,7.\u00e9zier curve to define the AFP trajectories and showed that this method can avoid the problem of caps and overlaps [Wang defined three manufacturing constraints and optimized the fiber directions . Based ooverlaps . For moroverlaps ,31,32,33In this study, our main contribution is that we provide a novel scheme using the cubic Ferguson curve to design variable stiffness laminates. The proposed method adjusts the shape of the generated trajectories, reduces their curvature, and enhances the buckling load by locally modifying the introduced parameters. Compared with traditional constant stiffness laminates, the proposed variable stiffness laminate performs better in terms of the buckling load, and the proposed method has several advantages, such as simple expression, d) over the mold surface. Therefore, there must be an initial trajectory and controlling its angle allows a class of variable stiffness trajectories to be obtained that can fully cover the mold surface.For AFP technology, the basis of assumption relative to full cover trajectories is that given an initial trajectory, the next must offset along a special direction up to a given width \u0398<\u03b81(https://www.3ds.com/products-services/simulia/products/abaqus/). The essential boundary and Neiman boundary conditions are illustrated in q is 1. Moreover, the thickness and width of the applied materials are To demonstrate the efficiency of the proposed scheme, we numerically simulated 8-layer square laminates with a width of First, we performed buckle analyses of the constant and variable stiffness. In this case, some optional settings of the fiber directions of the traditional constant stiffness are as follows: <It is not difficult to observe that the optimal fiber direction sequences on constant stiffness laminates are <To simplify the discussion, a symmetric initial trajectory is used in this study, that is, two kinds of parameters In this study, we proposed a novel scheme by applying two piecewise cubic Ferguson segments to design variable AFP trajectories, illustrated the effects of parameter settings on variable stiffness laminates, and provided the results of numerous numerical experiments on trajectory design utilizing finite element analysis. The proposed method can modify the buckling load by adjusting the introducing parameters"}
+{"text": "This study sought to synthesize and reinterpret findings from primary qualitativestudies on the experience of health professionals in caring for people withanorexia nervosa and bulimia nervosa. We conducted a systematic review of theliterature with the SPIDER search strategy assessing six databases. Ameta-synthesis was performed with data from qualitative studies. Two independentreviewers screened and assessed the articles, extracted data from the articlesand elaborated thematic synthesis. Nineteen articles met the inclusion/exclusioncriteria. The meta-synthesis revealed three descriptive themes: Going outsidethe comfort zone: hard relational experiences of health professionals inproviding care for people with anorexia nervosa and bulimia nervosa; Reflectingon treatment: relevance of discussion, communication, and flexibility in healthprofessionals\u2019 work with anorexia nervosa and bulimia nervosa; and Dealing withambivalences: experiences of health professionals with family members of peoplewith anorexia nervosa and bulimia nervosa. We elaborated two analytical themes:Making work with eating disorders palatable: malleability necessary for healthprofessionals in bonding with people with anorexia and bulimia nervosa and theirfamilies; and Leaving the professional comfort zone: transition from multi tointerdisciplinary. Thus, mental health professionals who work with peoplediagnosed with anorexia and bulimia nervosa cope with hard emotional experiencesthat makes them feel out of their comfort zone, requiring flexibility to benefita good therapeutic alliance, but there are still difficulties in promotinginterdisciplinarity. People diagnosed with eatingdisorders present significant alterations in their eating behavior pattern ,Due to the severity and persistence of symptoms, the specialized literaturerecommends that people with eating disorders should receive intense and continuoustreatment conducted by professionals such as doctors, psychologists, nutritional,psychiatrists, among other health professionals. Thus, it is recommended that suchtherapeutics should be carried out by a multidisciplinary team withinterdisciplinary practices ,,,,,Despite extensive research in the area, high rates of non-compliance and difficultiesin adhering to specialized treatment still prevail ,,Research shows that therapeutic alliance is one of the most important variablesrelated to treatment outcome, especially in anorexia nervosa cases ,,,Working with anorexia nervosa/bulimia nervosa people can aggravate stressfulsituations, which make health professionals a vulnerable population regarding mentalhealth ,,,Therefore, knowing that the scientific evidence is about how health professionalsperceive and experience care for people with eating disorders can provide importantclues for understanding the potentials and difficulties encountered whenestablishing a therapeutic bond. This knowledge can provide subsidies to refinetreatment planning, as well as strengthen the mental health and well-being ofprofessionals. Metasyntheses have already been published about the experience ofpatients in treatment In view of the above, this study aims to synthesize and reinterpret the results fromprimary qualitative studies about the experience of caring for people with anorexianervosa/bulimia nervosa from the perspective of health professionals.https://www.rayyan.ai/); (6) Calculation of the kappa index ofinter-rater agreement; (7) Reading the selected articles in full and finalselection of the analysis corpus; (8) Qualitative analysis of the methodologicalprocedures of the studies studied based on the Critical Appraisal Skills Program(CASP) https://provalisresearch.com/); (10) Description and analysis ofthe material.The systematic research trajectory followed 10 steps: (1) Elaboration of theresearch question guided by the SPIDER strategy; (2) Definition of selection andexclusion criteria, and choice of appropriate databases for the research area;(3) Elaboration of the search strategy based on specific descriptors for eachdatabase; (4) Searching the databases, with validation by another researcher whoindependently evaluated the information; (5) Screening and selection from titlesand abstracts, also comparing the results with the second independent reviewerand using the Rayyan tool  was chosen because it is considered anadequate tool for reviewing studies with qualitative methods, providing greaterrigor to the research Eligibility criteria were defined as follows: Inclusion criteria: (a) primaryqualitative studies; (b) studies consistent with the research question developedwith the SPIDER strategy; (c) articles that include the perception of healthprofessionals, trainees, or workers in the Results section as a category orsubcategory. Exclusion criteria: (a) qualitative, mixed, secondary, literaturereview, or theoretical-reflective studies; (b) gray literature, such as theses,dissertations, monographs, books, or chapters; (c) letter to the editor,editorial, commentary, opinion articles, and abstracts; (d) studies that do notinclude health professionals (only patients and/or their families); (e) studiespublished in languages \u200b\u200bother than Portuguese, English, Spanish, or French; (f)studies regarding eating disorders that do not meet the diagnostic criteria foranorexia or bulimia.https://cadernos.ensp.fiocruz.br/static//arquivo/suppl-1-e00223122_2723.pdf).After defining the databases regarding their relevance to the \u200b\u200bknowledge areaand seeking to encompass the national and international research scenario, thesearch terms were selected, which meant choosing the appropriate descriptors foreach database. The SPIDER search strategy was defined by combining thedescriptors of each acronym, suitable for each database. No date restriction wasapplied. The search used the Boolean operators OR between the descriptors of thesame acronym and AND between each one of them, as follows: (S1 OR S2 OR Sn...)AND (Pi1 OR Pi2 OR Pin...) AND (D1 OR D2 OR Dn...) AND (E1 OR E2 OR En...) AND(R1 OR R2 OR Rn...). The \u201cadvanced search\u201d tool was used in the databases. Atrack of descriptors assembled from DeCS/MeSH is shown in Supplementary Material1 .Therefore, the final corpus of this metasynthesis consisted of 19 articles.The kappa index calculation was performed to ensure a sufficiently good agreementrate between reviewers The selection and exclusion process of articles is described in the flowchart developeThe data from the 19 studies were described based on extracting the followingvariables: first author, year of publication, country where the study wasconducted, the study objective, design, data collection and analysis forms, aswell as the number and characteristics of participants .CASP Qualitative Checklist was used to qualitativelyevaluate the methodological properties of the studies. It is a checklist whichconsiders the description and relevance of the objectives, adequacy of themethodological choice and study design, the description of the data collectionand analysis strategy, if the ethical procedures were well described and if thedata were presented in a sufficiently satisfactory manner The Note (questions): 1 - Was there a clear statement of the aims of the research?; 2- Is a qualitative methodology appropriate?; 3 - Was the research designappropriate to address the aims of the research?; 4 - Was the recruitmentstrategy appropriate to the aims of the research?; 5 - Were the data collectedin a way that addressed the research issue?; 6 - Has the relationship betweenresearcher and participants been adequately considered?; 7 - Have ethical issuesbeen taken into consideration?; 8 - Was the data analysis sufficientlyrigorous?; 9 - Is there a clear statement of findings?; 10 - How valuable is theresearch?Thematic analysis was performed according to the following methodology ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,The 19 articles analyzed ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,Regarding data collection, we observed that most studies were conducted throughindividual interviews characterized by in-depth, semi-structured, and open questions(n = 17) A total of 235 participants composed the total sample of this metasynthesis,including the 19 studies. The samples included several specializations in the healtharea, such as: clinical service managers, psychiatrists, consultant doctors, generalpractitioners, endocrinologists, occupational therapists, nurses, dieticians,counseling psychologists, clinical psychologists, social workers, and familytherapists. These participants had anywhere from 28 years of experience to almostnone .The thematic synthesis process revealed 42 codes referring to the results of the 19studies. These were synthesized and described in three descriptive themes: (1) Goingoutside the comfort zone: hard relational experiences of health professionals inproviding care for people with anorexia nervosa/bulimia nervosa; (2) Reflecting ontreatment: relevance of discussion, communication and flexibility in healthprofessionals\u2019 work with anorexia nervosa/bulimia nervosa; (3) Dealing withambivalences: experiences of health professionals with family members of people withanorexia nervosa/bulimia nervosa in the therapeutic context. From this, twoanalytical themes were developed: (1) Making work with eating disorders palatable:malleability necessary for health professionals in bonding with people with anorexianervosa/bulimia nervosa and their families; (2) Leaving the professional comfortzone: transition from multi to interdisciplinary .,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,This theme highlights how the therapeutic relationship between professionals andpatients is experienced, as well as the emotional experiences of theprofessionals. Despite being valued, the therapeutic alliance proves to bedifficult for the participants of the retrieved studies. These obstacles arerelated to the complex and multifactorial characteristics of eating disorders,,,,,,,,arelational space that allows for the development of a positivebond\u201d warm [therapeutic] relationship\u201d Despite the difficulties in establishing a therapeutic alliance, these representsan important aspect for professionals in caring for people with eating disorder.The retrieved studies highlight ,,,,,,,,,,,,,,,outside their comfort zone\u201d However, many emphasize that there are several obstacles to forming andmaintaining a good interpersonal relationship with patients you\u2019re making these kids do something thatabsolutely horrifies them. I saw this as very... very difficult andupsetting\u201d ,,difficult to persuade patients to accept specialistcare\u201d The impasses in establishing a therapeutic relationship with the person withanorexia nervosa/bulimia nervosa in some studies are linked to the need toconvince them to do something that goes against their own will, as is evident inthe following report: \u201c,,,,,,,,It can bechallenging with the patients who don\u2019t have the insight into their illness,because they don\u2019t necessarily want your help\u201d ,,,,,,,,,I found ithard...\u201d Thus, from the perspective of the professionals participating in the recoveredstudies, the attitude of patients in the services which makes the therapeuticrelationship most difficult is the denial of eating disorder as a disease ,,,,,,,She speaks coldly and doesn\u2019t even lookat me\u201d ,,God, what if Isay the wrong thing to the service user?\u201d I used to be quite nervous about saying anything\u201d The selected studies also suggest that the difficulty in communicating withpeople with eating disorders may be a factor that hinders establishing atherapeutic alliance. Participants state that there is little emotionalopenness, distrust, and little ability to express feelings ,,,,,,,,,,Am I doing the right thing?\u201d ,,,,,Others also reported that professionals have doubts when making decisions abouttreatment ,,,,,,,,but if the person is far away andthen, suddenly, they deteriorate severely, then it would be verydifficult\u201d It could just take the littlestthing for you to say to completely regress in their recovery\u201d There\u2019s andanxiety there about holding that risky responsibility\u201d In addition, some of the retrieved studies revealed that professionals feltworried and anxious at times of relapse and worsening of the patient\u2019s condition,,,,,,,How much can I help them? They\u2019ve hadthis disease for months, if not years, and they\u2019re going to have this forthe rest of their lives\u201d Trying to sustain hope and remembering that recovery ispossible... (...) can be quite challenging\u201d Another prevalent feeling in these moments concerns frustration ,,,,,,,,,,,,,,,treatment needsto be tailored accordingly, to meet individual needs through a personaltreatment plan that is focused on the person rather than thediagnosis\u201d The complexity and severity involved in the cases of the analyzed studies, aswell as the difficulty in forming and maintaining a good therapeuticrelationship with the patients are added to the various variables involved indealing with any individual, such as: culture This topic is about the experiences of professionals in health services,especially regarding their work experiences with a specialized multidisciplinaryhealth team, as well as in the daily practices of care in the services based onthe protocols and available resources. The indispensability of working in amultidisciplinary team is highlighted when mentioning the difficulties of thecare scenario. On the other hand, the difficulty of communication betweenprofessionals of the same team or between different professionals of the healthsystem seems to be an obstacle to a good service to service users.,,,,,,,,diverse team with diverse skills\u201d It\u2019s the division of the responsibilities (...)where I feel confident to be able to talk with families andexplain\u201d we decide together\u201d Many studies ,talking to my other consultant colleagues, especially withregard to approaching family and conflicts\u201d ,sometimes I didn\u2019t feel thatcomfortable in asking questions\u201d Other publications also point out that working in a team can help to overcome thedifficulties found in working with eating disorder patients with tips,supervision, and observing the more experienced ,,,... other times you don\u2019t hear back from people, and that can bereally difficult\u201d Communication obstacles are also present between different health centers:between general practitioners/pediatricians and specialized services or in thecommunication between two different specialized services in moments oftransition ,,,,I think one of the problems is when the funding runs out\u201dMore funding for more staff would be the best to beable to provide more care for more patients\u201d Other impasses in the daily practice of health services are related to thestructures of the places where the services are located: hospitals, clinics, andspecialized outpatient clinics. For example, the lack of financial resources,the great demand for services, reducing the time dedicated to each patient, andthe challenges to find an adequate setting or lack of specialized professionals,,,I don\u2019t know ifits linking gaps in my knowledge or if it\u2019s the time to think that might bemore of what\u2019s missing sometimes\u201d The overload of the services, and consequently of the professionals, also leadsthe multidisciplinary team to lack time for study, dedication, and reflectionabout the work carried out ,,,,It\u2019s definitely a disease process thatneeds consistency and regular planning\u201d It might be helpfulto have some kind of training program or something that would mean everyonewas going out to the families and doing the same thing\u201d Some healthcare institutions have stricter diagnostic guidelines, protocols, andimprovement criteria. In some studies, workers experienced the protocols orguides as support in facing doubts, thus facilitating care practice ,,,,,,The actual clinical work wanted diversity. They want to usedifferent models\u201d my fear is that there is a guide, but people areindividuals\u201d On the other hand, others experienced the protocols and guides as instrumentswith parameters that are too rigid, which makes the practice inflexible andlimits the possibilities of adjusting to the needs of each one ,,,patients\u2019 weightbehavior and family circumstances are presented as uncertain evidence ofdiagnosis\u201d uneasy and unsure about theimplications of using a fixed set of criteria recovery\u201d Differential diagnosis was also mentioned in the studies retrieved, and doubtswere reported in formulating diagnostic criteria, especially when there arecomorbidities or in the case of atypical anorexia nervosa ,,,I feel more confident knowing the physical, medicalside\u201d ,,,,,,,,,,Personal resources that help in the daily practice of health services were alsomentioned, such as previous knowledge and experience in the area, making themconfident in providing interventions This theme deals with the experience of professionals with family and companionsof patients treated at the health service and the importance of this supportnetwork for the treatment. Despite recognizing the importance of family membersof people with anorexia nervosa/bulimia nervosa, the participants in theanalyzed studies also felt that they can contribute to maintaining symptoms,hindering, above all, the accountability and autonomy of patients. Therefore,they demonstrate ambivalent feelings about working with family members.,,,,,,,,,,,they play such a key role\u201d They\u2019re going to be the onesto keep an eye out for warning signs. So, it\u2019s important to involve them inthe care as well\u201d Many professionals who participated in the recovered studies reinforced theimportance of the family actively participating in the treatment, seeing it as\u201ccrucial\u201d and even \u201ccentral\u201d ,,,,,,,,With one patient, I felt thefamily was contributing to the ED [eating disorder]\u201d This has been abig issue where parents have gotten together and said even though they are18 we can still make decisions\u201d On the other hand, the family can be seen by these workers as an obstacle totreatment due to their controlling attitudes and often symbiotic patterns ofattachment, making it difficult for patients to take responsibility and developautonomy, and often contribute to the maintenance of symptoms we are bringing the family closer together as well and,kind of, empowering them to support the patient\u201d I think that as a service we haveto support the families because their lives really do change\u201d The professionals participating in the studies therefore recognize that thefamily is essential in the treatment, but can be a factor which contributes tomaintain the chronicity of symptoms. This reinforces that eating disorders careservices must include the family in the therapeutic plan. In the words of aprofessional: \u201cIn view of the results explained in the previous descriptive themes, twoanalytical themes were developed.According to the analyzed studies, working with patients with anorexianervosa/bulimia nervosa is a challenging experience and unpalatable from theprofessionals\u2019 reports. In leaving their comfort zone, workers are faced withthe rigidity of these patients and the tenacity with which they cling to thesymptoms, not recognizing the eating disorder as a problem, not seeking help,and not collaborating with communication. This scenario leads to feelings offrustration, insufficiency, fear, and ambivalence, among others. In thisanalytical theme, we emphasize that the difficult feelings that these patientsand their families place on professionals require flexibility to deal with thedifficulties imposed by the clinical encounter and seek hope, regardless of theobstacles.The professionals showed that flexibility was necessary because of communicationdifficulties, seeking the best way to talk to patients, as well as dealing withtheir own feelings when in the contact with the users of the health services.Malleability can be an adaptive way for professionals to deal with the typicalrigidity of these patients, promoting greater openness to the therapeuticalliance. From these studies, we can see that a lot of flexibility in clinicalmanagement is also needed when dealing with the many variables of each uniquepatient and with family difficulties. Considering the impasses within the carescenario, professionals may have protocols and guidelines, but they had to adaptthem to each case, work as a team to think about the complexity of each personassisted/treated, with flexibility to work with other professionals or evenother services. However, this malleability presupposes that professionals areguaranteed minimum working conditions, such as human and technologicalresources, time for study, discussion, and adequate care and training.Caring for people with anorexia nervosa/bulimia nervosa requires professionals toleave their known world and be flexible when dealing with patients and theirfamilies. However, they still cling to the comfort zone of staying within theirown specialization. In the experience of the professionals participating in thestudies, it is possible to perceive that there is an intense difficulty indialogue because there is no integration between the disciplines, there is noopenness to the various new skills necessary when working with eatingdisorders.Therefore, although the professionals of the analyzed studies emphasize theimportance of a multidisciplinary team, in practice they do not function asinterdisciplinary. This brings obstacles to creating knowledge in practice,which takes place during care in a different way from what they learned before.This lack of interdisciplinarity may be another factor that has implications forthe fact that working with eating disorders is considered difficult and\u201cunpalatable\u201d, and potentially for the difficulty of seeing family members as atarget population of care.This review allowed us to synthesize qualitative evidence on the experience of healthprofessionals in caring for people with anorexia and bulimia nervosa. Threedescriptive themes were developed considering the intense and difficult experienceof working with this type of demand which requires great flexibility fromprofessionals, leaving their comfort zone, both in terms of caring for the patientand their families, as well as in facing a care scenario within the health serviceswhich does not always favor the professional\u2019s work.,,,The specialized literature in the area of \u200b\u200beating disorders ,,,,,,,,,,,,,,,,,,,,,The justifications offered by the professionals of the studies analyzed hereininclude: the complex and multifactorial characteristics of the eating disorders;non-recognition of the need for help on the part of the patient; the difficulty ofcommunication ,,This first analytical theme brings us clues to its answer. People with anorexianervosa/bulimia nervosa have very rigid personality characteristics, with littlecontrol over impulsivity and high intensity of emotional reactions. Which may alsobe the reason for some of their sociability difficulties ,,,,,,,,Participants in the analyzed studies found themselves forced to leave their comfortzone and deal with this rigidity in a malleable way. According to the investigations,,With this careful and open attitude towards the other - going beyond the adjustmentof a protocol or seeking a \u201csuccess\u201d and \u201cimprovement\u201d factor in the treatment -emotional frankness and availability to be with patients has the potential to be afactor that facilitates the bond ,,,,,,,,,,,In the same sense, professionals in the analyzed studies ,,,,,,,,,,,,,,Therefore, this view favors appreciating comprehensiveness in the care provided tothe patient, taking into account a notion of bio-psycho-social health ,,,,,,It is important to point out that this necessary malleability in dealing withpatients and their families assumes that health workers are guaranteed minimumworking conditions, such as human and technological resources, study time,discussions, and adequate care and training. Many of the studies analyzed ,,,,,,,Although the professionals in the studies that composed the corpus of this analysisreported \u201cgetting out of their comfort zone\u201d when in contact with patients, this waslittle observed in regard to openness to communication and practices with otherprofessionals, which can be an obstacle to an interdisciplinary approach, the secondanalytical theme of this study. Professionals recognize that they need tocollaborate with the various knowledge areas involved in the care of people withanorexia nervosa/bulimia nervosa, as recommended by the specialized literature ,As we can see in this study, other studies reaffirm the obstacles faced by physiciansin implementing interdisciplinarity ,,,,,,,,,,The attitude of the healthcare team, from an interdisciplinary perspective, can alsofavor opening the professional to the perception of the family as a target of care,not only making them responsible for treating the member diagnosed with anorexianervosa/bulimia nervosa This study illuminates aspects within clinical practice regarding treatment of eatingdisorders, which can bring relevant implications for such practices in the followingsenses: health professionals must be trained to favor flexibility and affectiveopenness to bond with patients with eating disorders, understanding that theprofessional\u2019s rigidity can intensify the austerity of the patients treated. Thetraining of health workers/professionals must also raise awareness on the importanceof working in an interdisciplinary way, encouraging the professional to being opento listening, transforming, and building new care actions together with otherprofessionals, leaving the comfort zone of the discipline itself and of their ownknowledge.The comprehensive and interdisciplinary look also leads to another way ofunderstanding the families of affected people. From this, we include a thirdimplication of this study for health practices: the relevance of including thefamily in the therapeutic scenario as a care recipient, and not only in the role ofcaregiver. For all of this to be possible, it would be extremely important thathealth services are properly equipped and provided with spaces for discussion,training for the obstacles placed in the care scenario, guaranteeing minimumconditions for health workers.Despite the rigorous methodological procedure, this study has some limitations. Thecorpus analysis did not include Latin American publications. From the point of viewof the methodological quality of the analyzed studies, it was observed that thereare flaws in the reflexivity criterion, meaning that several studies do not explainthe position or influence on the researcher in the context of performing theinvestigations. Another limitation of this review regards the exclusion of greyliterature, such as dissertations and theses. These aspects can illuminate the needfor future studies, especially focusing on the national perspective."
\ No newline at end of file