diff --git "a/deduped/dedup_0182.jsonl" "b/deduped/dedup_0182.jsonl" new file mode 100644--- /dev/null +++ "b/deduped/dedup_0182.jsonl" @@ -0,0 +1,44 @@ +{"text": "Rivastigmine, a butyl- and acetylcholinesterase inhibitor, is approved for symptomatic treatment of Alzheimer's disease (AD). Data supporting the safety and efficacy of second-generation cholinesterase inhibitors, such as rivastigmine, are available for treatment up to 1 year, with limited data up to 2 1/2 years. The purpose of this report is to present safety and effectiveness data for rivastigmine therapy in patients with mild to moderately severe AD receiving treatment for up to 5 years.An observational approach was used to study 37 patients with originally mild to moderate AD receiving rivastigmine as a therapy for AD in an open-label extension .The initial trial demonstrated rivastigmine was well-tolerated and effective in terms of cognition, global functioning and activities of daily living. In this open label extension, high-dose rivastigmine therapy was safe and well tolerated over a 5-year period. Two thirds of the participants still enrolled at week 234 were in the original high-dose rivastigmine group during the double-blind phase, suggesting that early therapy may confer some benefit in delaying long-term progression of symptoms.Long-term cholinesterase inhibition therapy with rivastigmine was well tolerated, with no dropouts due to adverse effects past the initial titration period. Early initiation of treatment, with titration to high-dose therapy, may have an advantage in delaying progression of the illness. Alzheimer's disease (AD) is the most common form of dementia affecting elderly people in the United States. Prevalence is 1% to 2% at age 65 years, but increases markedly to 35% or greater by age 85. Because of a demographic shift toward a more aged population, the percentage of affected individuals is rapidly increasing. This trend is expected to continue for the foreseeable future. Therefore, accurate and timely diagnosis and effective treatments are critical to optimal outcomes over the 8- to 10-year course of the illness Figures When evaluating qualitative data, it is important to examine particular patients in terms of the data collected and treatment response at end of study. Data for the youngest man and the elderly woman, considered side-by-side, seem counterintuitive. Part of the explanation, however, may rest with the caregivers' experiences with and beliefs about the patients. This information can be accessed by reviewing the \"symptoms most troubling\" item from the CIBIC-Plus. This item asks, \"With respect to the above symptoms, which are the biggest problems for you and/or other caregivers?\" The spouse of the 57-year-old man reported that the \"symptoms most troubling\" for her were the patient's \"selective difficulties and his attitude problem\" and that the patient \"doesn't try\" . These comments suggest a lack of acceptance of the patient's diagnosis and a lack of belief regarding the patient's documented decline that could explain continued clinic visits until, at week 234, the patient's MMSE score was 0. In contrast, the caregiver for the 76-year-old woman cited the fact that the patient \"doesn't want to leave the house\" (week 12) and the patient's anxiety about coming to clinic appointments (week 26) as the \"most troubling\" symptoms. These comments suggest that the caregiver was encountering patient resistance and observing patient distress, which were exacerbated by efforts to participate in the study. Therefore, further clinic visits were declined. Retrospectively, it is impossible to determine how the caregiver's beliefs and experience with the patient affect perceptions about disease status, treatment response, and decisions to continue or terminate treatment. These are interesting hypothesis-generating observations that should be explored in future investigations.Limited data are available on the tolerability and effectiveness of cholinesterase inhibitor therapy for periods up to 3 years -7, but nOf note, approximately two thirds of the participants still enrolled at week 234 were in the original high-dose rivastigmine group during the double-blind phase; this finding suggests that early therapy with rivastigmine may confer some benefit in delaying long-term progression of symptoms, as has been previously suggested by analysis of the combined 26 weeks of double-blind and first 26 weeks of open-label data from the B352 US trial . ThroughIn summary, long-term therapy with the ChE inhibitor rivastigmine was well tolerated with no dropouts due to adverse effects past the first 9 months of the open-label extension. In contrast to the generally reported community experience of relatively brief duration of therapy with ChE inhibitors in AD, 25% of patients in this study were still taking rivastigmine by the end of 5 years. Of interest is the multifactorial nature of reasons for treatment discontinuation. Only 1 of these patients withdrew from the study because of perceived treatment failure over the 5-year period. Disease progression accounted for the withdrawal of 4 patients.These results are informative both for the duration of treatment, and because the majority of the patients who continued treatment during this 5 year period were from the group originally titrated up to a high dose during the initial double-blind phase. Given that the sample size prohibits significance testing, these data suggest that rivastigmine therapy may be sustained over long periods of time in a significant percentage of patients with AD, and that high-dose therapy, particularly when begun early, may have an advantage in delaying the progression of the illness.MRF has received grant support and has served as consultant and received honoraria from Novartis Pharmaceuticals Corporation. MLL has no financial interest to declare.MF carried out the original clinical trial, conceived of the design for the observational study, and participated in the drafting and revisions of the manuscript. ML participated in the design of the observational study, performed statistical analysis, and participated in the drafting and revisions of the manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"} +{"text": "Following our previous publication we have received critical comments to our conclusions as well as new data that are strengthening our findings.With the new data, 11 suicide attempts among patients on paroxetine against 1 among patients on placebo, we found with a Bayesian technique that the posterior probability that medication with paroxetine is associated with an increased intensity per year of a suicide attempt is from 0.98 to 0.99, depending on the prior.We found that the comment to our article by GSK representatives contained errors, misunderstanding and unwillingness to accept Bayesian principles in the analysis of clinical trials.We were in our previous publication, with preliminary data and a Bayesian approach, able to raise a concern that suicide attempts might be connected with the use of paroxetine. This suspicion has now been confirmed. Last year we wrote a paper \"Suicide attempts in clinical trials with paroxetine randomised against placebo\" that hitWe analyzed the data GSK presented in their latest report by the same Bayesian approach used by us in our article. We included only the double blind, parallel design studies with patients randomized to either paroxetine or placebo, as recognized by GSK in the Briefing Document. These 19 studies contained 3455 and 1978 patients to the treatment and placebo groups, respectively. They resulted in 11 and 1 suicide attempts, respectively, as compared to 7 and 1 in our study. The studies lasted 6 \u2013 12 weeks, and we obtained 601 and 333 patient years in the treatment and placebo groups, respectively. Since we did not have access to the points of time of censoring, we were not able to correct for this when computing the patient years. We took the same model approach as in our article, and refer to it for details regarding the model. We let \u03b8p and \u03b8d be the mean number, or intensity, per year of a suicide attempt for a random patient in the 19 studies in the placebo and treatment group, respectively. We performed simulations by making 80000 random draws of \u03b8p and \u03b8d, from their independent posterior distributions. We computed the logarithms of the ratios \u03b8d/\u03b8p, and constructed posterior probability density plots by applying a standard density estimation technique to these 80000 numbers. (The logarithm was introduced to avoid an unwelcome feature of the density estimation method). Note that the logarithm of the ratio \u03b8d/\u03b8p is greater than zero whenever \u03b8d is greater than \u03b8p. Hence, we calculated the probabilities that medication with paroxetine is associated with an increased intensity of a suicide attempt per year as the proportions of logarithmic ratios greater than zero in the samples. We applied three different prior assumptions based on earlier work by others, as described in the article. We chose to express the pessimistic view as equivalent to observing 0.86 events with paroxetine during 50 patient years and 0.43 with placebo during 50 patient years, adding up to 1.29 attempts per 100 patient years, which is similar to the observed average value for paroxetine and placebo taken together. We based the calculations on a total of only 100 patient years in the prior, compared to 934 patient years in the real data, in order to increase the importance of the real data over the prior information. For the slightly optimistic and slightly pessimistic priors we assigned the numbers of suicidal patients on paroxetine and placebo per 50 patient years to be respectively 0.58 and 0.71 and vice versa. We found that the posterior probability that medication with paroxetine is associated with an increased intensity per year of a suicide attempt is 0.99 with the pessimistic prior, 0.98 with the slightly optimistic prior and 0.99 with the slightly pessimistic prior. Hence, we can be at least 98 % sure that paroxetine increases suicide attempts. This is stronger evidence than the p value equal to 0.058 given in the Briefing Document [In addition to calling into question the statistical method applied, the authors of the GSK comment denied our findings by disputing the selection of data. They state that \"analysis was based solely on early data submitted to regulatory authorities in 1989. The studies analyzed included only 916 paroxetine-treated patients. Today we have data from more than 13,000 adult patients treated with paroxetine in well-controlled clinical trials.\" Interestingly, GSK has now in the Briefing Document reduced the corresponding number to 3455 and also admitted that they previously had several patients with a history of suicide attempts from an earlier trial placed in the treatment and placebo groups. With such a case history these patients were more likely to attempt suicide again. They should obviously not have been included since they blurred the results. This can be described as 'data-drowning', and is at least poor methodology.One item in the comment by GSK concerns two cases that they believe we had misplaced. One patient (no 1.12.037) was claimed to show only suicidal ideation but has by GSK in the Briefing Document been re-classified to represent a suicide attempt. The other patient (no 04.02.056) was from a cross over continuation study. We do not agree that this patient should have been excluded.GlaxoSmithKline state in their comment to our article that the risk may be closely related to the point of time observed in a study, and further that they would expect to have 6 attempts in the treatment group out of a total events of 8. The number is more precisely 5.79. Hence, our number 7 represents a 20.9 % increase compared to what is expected. They apply a Fishers Exact Test, which in our opinion is a very coarse method. Both in their earlier report in which they applied the Mantel-Haenszel test, and in their new analysis in the Briefing Document [In the comment to our article it was claimed that we should have compared a hypothesis of an increased number of suicide attempts to one in which the rate is identical for the treatment group and placebo group. This hypothesis is meaningless. No one believes that the rates for these two groups are exactly equal. Secondly, we do not in any way attempt to do any form of hypothesis testing. We are merely computing the probability that medication with paroxetine is associated with an increased intensity per year of suicide attempts. GSK questions our statement that '...the data strongly suggest that the use of SSRIs is connected with an increased intensity of suicide attempts per year'. We feel that this is all about understanding and accepting the Bayesian approach as a way of analysing data. Recently FDA issued a drafted guidance for using Bayesian statistics in clinical trials . They wrWe conclude that irrespective of the various prior assumptions, we now see clearer that the data strongly suggest that the use of antidepressant drugs is connected with an increased intensity of suicide attempts per year, not only among young people, but also among adults. Moreover, information from studies on antidepressants and other drugs must be made open for researchers and presented in the most informative way possible. We believe that this would create an open minded, trusting dialogue between consumers, manufacturers and scientists working towards the same goal: finding the best and safest treatment for the patients.The author(s) declare that they have no competing interests.IA collected the data, presented the problem and drafted the manuscript along with BN, who suggested the statistical solution based on earlier work by the present authors ; IFT didThe pre-publication history for this paper can be accessed here:"} +{"text": "Inclusion of unpublished data on the effects of antidepressants on children has suggested unfavourable risk-benefit profiles for some of the drugs. Recent meta-analyses of studies on adults have indicated similar effects. We obtained unpublished data for paroxetine that have so far not been included in these analyses.The documentation for drug registration contained 16 studies in which paroxetine had been randomised against placebo. We registered the number of suicides, suicide attempts and ideation. We corrected for duration of medication and placebo treatment and used a standard Bayesian statistical approach with varying priors.There were 7 suicide attempts in patients on the drug and 1 in a patient on placebo. We found that the probability of increased intensity of suicide attempts per year in adults taking paroxetine was 0.90 with a \"pessimistic\" prior, and somewhat less with two more neutral priors.Our findings support the results of recent meta-analyses. Patients and doctors should be warned that the increased suicidal activity observed in children and adolescents taking certain antidepressant drugs may also be present in adults. The debate about whether the use of antidepressant drugs increases suicidal activity has recently been sharpened after more than 10 years of turmoil . Inclusith BMJ editorial [In a February 19ditorial accompanditorial ,5, the aditorial containeWe included only double blind, parallel design studies with patients randomised to either paroxetine or placebo. Altogether 16 studies met these criteria (references 79 to 93 and 95 in the Expert Report), containing respectively 916 and 550 paroxetine and placebo treated patients. The study period was in most instances 6 weeks. One important exception was a study (reference 91) with a preponderance of paroxetine use over placebo and lasting for 17 weeks. Patients were excluded from the studies after a suicide-related event. Taking this censoring into account, paroxetine treatment made up 190.7 patient years altogether and placebo 73.3 patient years. Suicide-related events could be found in tables in the Expert Report, in the adverse reactions section in the individual study reports, and in the individual patient descriptions.p be the intensity per year of a suicide attempt in the placebo group and \u03b8d the intensity per year in the drug group, for a random patient in the 16 studies; correspondingly, Xp and Xd represent the total numbers of suicide attempts. We can have at most one suicide attempt for each patient. Taking this censoring into account, we denoted the corresponding patient years in the 16 studies combined by mp and md. In addition, patients in both the placebo and drug groups are supposed to behave in a similar manner. It then follows that the likelihood of the experiment corresponds to Xp and Xd having Poisson distributions respectively with parameters (mp\u03b8p) and (md \u03b8d). In addition, we assume that the two variables were conditionally independent given the parameters. The corresponding observed data are and , and the prior information is denoted by and .We let \u03b8p and \u03b8d . This does not mean that these parameters are to be interpreted as random variables, but our knowledge of the parameters is uncertain and we describe this uncertainty with the help of probability distributions. Probability distributions describing our initial uncertainty are called prior distributions . When the real data are taken into account, the prior distributions are updated by Bayes' formula to posterior distributions. An excellent introduction to Bayesian methods in medicine is given by Spiegelhalter et al. [The Bayesian approach is based on the construction of probability distributions for \u03b8r et al. .p is gamma, with parameters xop and mop, while correspondingly \u03b8d has the parameters xod and mod and is assumed to be independent of the prior distribution for \u03b8p. Hence, standard Bayesian theory gives the posterior distribution of \u03b8p as gamma, with parameters xop + xp and mop + mp, while \u03b8d will have the parameters xod + xd and mod + md. We performed simulations by making 80000 random draws of \u03b8d and \u03b8p from their independent gamma posterior distributions, computed the logarithms of the ratios \u03b8d/\u03b8p, and constructed diagrams by applying a standard density estimation technique to these logarithms. (The logarithm was introduced to avoid an unwelcome feature of the density estimation method.) Note that the logarithm of the ratio \u03b8d/\u03b8p is greater than zero whenever \u03b8d is greater than \u03b8p. Hence, we calculated the probabilities that medication with paroxetine is associated with an increased intensity of a suicide attempt per year as the proportions of logarithmic ratios greater than zero in the samples. This corresponds to areas below the densities to the right of zero in the diagrams.We assume that the prior distribution for \u03b8od) events with paroxetine during 50 (mod) patient years and one (xop) with placebo during 50 (mop) patient years, adding up to 3 attempts per 100 patient years, which is similar to our observed average value for paroxetine and placebo taken together. We based the calculations on a total of only 100 (mod + mop) patient years in the prior, compared to 264 (md + mp) patient years in the real data, in order to increase the importance of the real data over the prior information. The slightly optimistic and slightly pessimistic priors represent respectively a paper by Lapierre [vice versa.The grounds for a pessimistic prior have been given by Healy and Whitaker who, relLapierre with thWe believe that the chosen studies are similar enough to be pooled for analysis. This view is supported by the similarities of the protocols for the various studies, although the populations that were studied differed considerably. We also believe it is best to count patient years rather than patients, although claims have been made for the contrary . At leasp and the ratio \u03b8d/\u03b8p, with a common, relatively weak prior for \u03b8p in all three formulations. This was our approach in a previous publication using basically the same method [Another statistical approach would have been to express the prior distributions in terms of independent priors for \u03b8e method . To makeAlthough we report a small data set, by taking various priors into account the data strongly suggest that the use of SSRIs is connected with an increased intensity of suicide attempts per year. The two meta-analyses and our contribution taken together make a strong case for the conclusion, at least with a short time perspective, that adults taking antidepressants have an increased risk of suicide attempts. We also conclude that the recommendation of restrictions on the use of paroxetine for children and adolescents recently conveyed by regulatory agencies should bThe author(s) declare that they have no competing interests.IA collected the data, presented the problem and drafted the manuscript along with BN, who suggested the statistical solution based on earlier work by the present authors ; IFT didThe pre-publication history for this paper can be accessed here:"} +{"text": "This study seeks to determine whether perception of weight status among the overweight has changed with the increasing overweight/obesity prevalence.2) from NHANES III (1988\u20131994) and overweight participants from NHANES 1999\u20132004. Perception of weight status was assessed by asking participants to classify their weight as about the right weight, underweight or overweight. Comparisons were made across age groups, genders, race/ethnicities and various income levels.The perception of weight status was compared between overweight participants (BMI between 25.0\u201329.9 kg/mFewer overweight people during the NHANES 1999\u20132004 survey perceived themselves as overweight when compared to overweight people during the NHANES III survey. The change in distortion between the survey periods was greatest among persons with lower income, males and African-Americans.The increase in overweight/obesity between the survey years (NHANES III and NHANES 1999\u20132004 has been accompanied with fewer overweight people perceiving themselves as overweight. The Surgeon General's Call to Action to Prevent and Decrease Overweight and Obesity was issued in response to the increasing population prevalence of overweight and obesity and the resulting public health threat [2 \u2013 29.9 kg/m2) and obesity (BMI \u2265 30 kg/m2) among adults was 34% and 30.5%, respectively, based on data from the National Health and Nutrition Examination Survey (NHANES) 1999\u20132000 [In 2001, ty BMI \u2265 0 kg/m2 aDistorted weight perception is not a new phenomenon and is actually very well documented -16. Stud2 [Caucasians are more likely to perceive themselves as overweight when compared to African Americans and Mexican Americans. Overweight people of higher socioeconomic status (SES) are more likely to perceive themselves overweight when compared to those of lower SES. Individuals with BMIs greater than 30 are more likely to perceive themselves overweight when compared to those with BMIs between 25.0\u201329.9 kg/m2 . This obThe overweight public's failure to accurately recognize their own overweight status prior to becoming obese may prevent them from changing behaviors that might contribute to additional weight gain. Therefore, it is important to understand the magnitude of weight status distortion within persons with BMI scores within the overweight range.The intent of the present study was to determine if there is a trend for more individuals observed in the \"overweight\" BMI range to perceive themselves to be of \"normal\" or healthy weight by comparing NHANES (1999\u20132004) to NHANES III (1988\u20131994). This study also assessed whether income, race and gender moderated this effect.This study used data from NHANES III (1988\u20131994) and NHANES 1999\u20132004. NHANES is a multi-stage, stratified probability sample of non-institutionalized U.S. civilians, ages 2 months and older. The survey is conducted by the National Center for Health Statistics (NCHS), Centers for Disease Control and Prevention (CDC) and includes administration of questionnaires, a physical exam, and a battery of laboratory tests. Details of the questionnaires, the exam, and the tests have been described elsewhere -20.2 , were included in our sample. Respondents were excluded if they were pregnant or under the age of 20 (children/adolescents). Pregnancy status was determined using self-reported information as well as results of the urine test administered during the medical examination.Adult participants (\u2265 20 years of age) classified as overweight, identified by a measured BMI: 25.0\u201329.9 kg/mHeight and weight were measured by trained examiners during the extensive medical evaluation at the Mobile Examination Center (MEC). Participants were weighed using a scale with a digital display readout, and height was measured using a stadiometer.Body Mass Index (BMI) was calculated based on these weight and height measurements and calculated as weight (kg)/height (meters) squared. Participants were then classified as obese (\u2265 30.0 kg/m2), overweight (25.0\u201329.9 kg/m2), or normal weight (18.5 \u2013 24.9 kg/m2), which is consistent with the NIH Clinical Guidelines on the Identification, Evaluation and Treatment of Overweight and Obesity in Adults \u2013 The Evidence Report [ 18.5 \u2013 2.9 kg/m2,Weight perception was ascertained asking subjects during the face-to-face interview if they considered themselves to be overweight, underweight, or about the right weight. Less than 0.01% of respondents were missing weight status data, and these individuals were excluded from analyses examining weight distortion. At the time respondents were asked to report their weight status, they were also told that their weight would be measured at a follow-up session.Respondents were asked to select the category of income that most accurately reflected their total combined family income over the last 12 months. Reported income was then used to develop the poverty income ratio (PIR), which is the ratio of income to the family's appropriate poverty threshold. Using the PIR allows income data to be compared across various survey years. Income was categorized, as suggested by NCHS, using the PIR eligibility cut points for the United States Department of Agriculture (USDA) food assistance programs, into the following categories: low (0.000\u20131.850), middle (1.851\u20133.500), and high (3.501+) incomes . IndividNon-age specific estimates were age-standardized to the 2000 Census using the standard population structure as suggested by NCHS. For specific estimates examining age, the following categories were used: 20\u201334, 35\u201349, 50\u201364, and 65+. These categories enable exploration of how weight perception differs across the age spectrum while providing a sufficient number of subjects in each group.NHANES participants during both survey periods were asked to select the race and ethnicity that they most readily identified with. Responses were then systematically collapsed into the following categories of Non-Hispanic (NH) Whites, Non-Hispanic (NH) Blacks, Mexican-Americans and Other ,22. OnlyAnalysis of the NHANES data was conducted using SAS and SUDAAN statistical software packages. To account for unequal probabilities of selection and non-response, all our analyses incorporated sample weights in order to provide national estimates of population proportions. Standard errors were calculated in SUDAAN based on the Taylor Series Linearization method . Analyse2 and < 30 kg/m2) respondents in NHANES 1999\u20132004 identified themselves as overweight when asked whether they considered themselves underweight, overweight or just the right weight compared to 68% who were asked the same question in NHANES III (1988\u20131994). In both surveys, the percentage of overweight females who accurately identified their weight status as overweight was greater than the percentage of overweight males who accurately identified themselves as overweight. This was consistent across all racial/ethnic and age groups. However, those in the youngest age group (ages 20\u201334) in both genders experienced statistically significantly more alteration in perception of weight status than the older groups between the survey years with 13% fewer males and females ages 20\u201334 accurately identifying themselves as overweight in the 1999\u20132004 survey compared with the NHANES III (1988\u20131994) survey. See Table Sixty-two percent of measured overweight 59.7% of NH blacks had an accurate perception of their weight status whereas 82.1% of NH white females had an accurate perception. This represents a shift from the NHANES III (1988\u20131994) data, which when compared to the NHANES 1999\u20132004 data shows that fewer Non-Hispanic Blacks , fewer Non-Hispanic Whites and fewer Mexican American males accurately perceive their weight status in the latter survey. It is also important to note that the weight perception of Mexican Americans of both genders and white males did not change significantly over the survey years. In addition among all ethnic groups, NH blacks experienced the greatest change in weight status perception between the survey periods. See Table As PIR increased, the trend was for more people to correctly perceive themselves as overweight. This trend was consistent across all racial ethnic groups. However, whites with lower and middle PIR and blacks with lower PIR realized the greatest growth in distortion between surveys with 8%, 9% and 18% (respectively) fewer persons accurately identifying themselves as overweight (p < 0.05). Mexican Americans with lower PIR were more likely than their NH black counterparts and less likely than NH whites of the same income level to perceive themselves overweight. However, there was an insignificant difference across survey years for the low income Mexican Americans. See Table The intent of the present study was to determine if the increasing population prevalence of overweight and obesity has been accompanied by a trend for fewer individuals categorized in the \"overweight\" BMI range to perceive themselves to be overweight when comparing NHANES (1999\u20132004) to NHANES III (1988\u20131994). The results of the study clearly show that fewer overweight people with BMIs within the overweight range identified themselves as overweight in the 1999\u20132004 NHANES Survey than did in the NHANES III survey. In order to gain a better understanding of the degree of distortion along the BMI continuum, an ad hoc analysis was conducted to examine the shift in weight perception among males and females with a BMI of 25.0\u2013<27.5 compared to those with a BMI of 27.5\u2013<30 between NHANES (1988\u20131994) III and NHANES 1999\u20132004. In both males and females, there was a statistically significant decrease in the number of overweight adults with an accurate weight perception among those with a BMI of 25.0\u2013<27.5 (p = 0.004 and p < 001 respectively). While individuals in the higher BMI category (BMI 27.5\u2013<30) experienced a decrease in accurate weight perception, this difference was not statistically significant. Among the individuals with a BMI greater than 30, there was no change in perception across survey periods. The majority of males and females with a BMI above 30 are aware that they are overweight (87.0% and 95.5% respectively). The shift at lower BMI levels suggests the perception of a healthy weight is expanding to include those who are mildly overweight.The findings of our study are consistent with those found in a 2006 Pew research survey where 9 in 10 Americans acknowledge that there is a weight problem in this country and 7 in 10 acknowledge that their family and friends have a problem, yet only 4 in 10 say they themselves are overweight. Within the same survey 51% of respondents whose reports of their own height and weight would result in them being classified as overweight perceived themselves to be just right . The finThe results of this study speak to the need to increase the awareness of the public's perception of weight status. As the weight status perception becomes more distorted it lessens the likelihood that individuals will make the recommended behavioral changes necessary to realize the health benefits associated with even small weight loss.The author(s) declare that they have no competing interests.WJT conceived of the study, participated in the design of the study, the analysis, drafted the manuscript and incorporated other author comments. RF participated in the design of the study, the analysis, assisted with the draft of the manuscript. VH participated in the design of the design of the study. PSR and PE assisted with the draft of the manuscript. All authors read and approved the final manuscript."} +{"text": "There is a typo in the first sentence of the abstract. Please read the correct sentence below:Omega-3 long-chain (\u2265C20) polyunsaturated fatty acids (\u03c93 LC-PUFA) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are critical for human health and development."} +{"text": "The following information was missing from the funding section: National High Technology and Development Program of China (grant no. 2008AA10Z152).It should also be noted that the correct listing of the corresponding authors should be:whwei@oilcrops.cn (WW); cmlu@oilcrops.cn (CL)"} +{"text": "Background. The orofaciodigital syndromes (OFDS) are a heterogeneous group of syndromes that affect the face, oral cavity, and the digits. OFDS type IV (OMIM %258860) is rare and characterized by broad nasal root and tip, orbital hypertelorism or telecanthus, micrognathia, hypoplastic mandible, and low-set ears. Oral symptoms may include cleft lip, cleft or highly arched palate, bifid uvula, cleft or hypoplastic maxillary and mandibular alveolar ridge, oral frenula, lingual hamartoma, and absent or hypoplastic epiglottis. Dental anomalies are common and generally include disturbances in the number of teeth. Case Report. This report presents a six-year-old girl, referred with the chief complaint of missing teeth. She was diagnosed as having OFDS type IV based on clinical findings. Her parents reported three deceased children and two fetuses that had the same phenotype. She was the seventh child of consanguineous parents who were first cousins. Conclusion. This is a very rare syndrome. Many reported OFDS type IV cases have consanguineous parents, consistent with an autosomal recessive trait. Manifestation of cleft palate in the healthy sibling may be mild expression of the disorder or an unrelated isolated cleft. The oral-facial-digital syndromes (OFDS) are a heterozygous group of conditions that affect the face, oral cavity, and the digits. Eleven different types of orofaciodigital syndromes have been reported (OMIM and OFD1\u201311) . ClinicaThis report presents the craniodentofacial manifestations of a case of OFDS type IV with five deceased siblings with the same phenotype and one sibling with cleft palate only .A six-year-old girl was referred to Istanbul University, Faculty of Dentistry, Department of Maxillofacial Prosthodontics with a chief complaint of missing teeth. She was diagnosed as having OFDS type IV at the age of 25 days . She wasAs the proband's birth certificate indicates, she was born with a birth weight of 2450\u2009gr, length 45\u2009cm, and her head circumference was 32.5\u2009cm . At age of 25 days, she was reported to have hypertelorism, depressed nasal bridge, microrethrognathia, median pseudocleft of the upper lip, small and lobulated tongue, pseudocleft of the posterior palate, and bifid epiglottis. She had mesomelic shortness of the limbs with club feet, duplication of great toes, and polysyndactyly. Her radiographs revealed an extra phalanx between the fourth and fifth fingers, bifid thumb, and postaxial digit in the right hand. The left hand radiography showed a forked fifth metacarpal with an extra postaxial digit and her tibias were short and broad. The hemogram report and the urine analysis were normal. The chest radiograph, ECG, abdominal ultrasound, and cranial MR revealed no abnormalities. Hearing loss was detected in her left ear with auditory brain responses. Chromosome analysis using G-banding technique revealed a 46,XX karyotype. At that time, she was diagnosed as having OFDS type IV .The necropsy report of the third child (IV-3) who had died at 1 day of age reported that she had a lobulated, tethered tongue and bifid epiglottis. Her right hand revealed six fingers and thumb was wide and flat. Syndactyly was noted between fourth and fifth fingers. Eight fingers were present on the left hand and the thumb was broad; syndactyly was seen between fourth and fifth, fifth and sixth, and seventh and eighth fingers. Both hands were slightly flexed forward. Mesomelic shortness of the limbs was noticed. She had megaloureter and lobar pneumonia which was reported as the probable cause of death.The proband had difficulty walking. She had a broad nasal bridge, root, and tip, protruding ears, and bifid frenula . She hadIntraoral examination revealed cleft palate and a pseudo-cleft lip, sublingual hamartoma, and lobulated tongue that had been operated on bilaterally because of tumor-like lesions . Dental The OFDS are rare and demonstrate considerable clinical heterogeneity, making definitive diagnosis problematic. This case presented with findings of both OFDS types II and IV. The lobulated tongue with hypertrophied frenula, median pseudocleft of the upper lip, and hearing loss are findings characteristic of OFDS type II whereas the tibial dysplasia and club foot deformities are characteristic of OFDS type IV. Both conditions are reported to show overlapping oral findings including high arch palate, cleft palate, and lobulated tongue with nodules. Temtamy and McKusick first reIn their review of OFD syndromes, Toriello classified the syndrome into 11 groups as types I through IX, Mohr-Majewski syndrome, and Egger-Joubert syndrome . Mohr syMost of the reported clinical findings for OFDS type IV cases come from necropsy reports of fetuses. The proband reported here is six-year old and has all characteristic features of the syndrome. Dental findings of oligodontia and significant caries were also noted. Conductive hearing loss which is also a characteristic of OFDS type IV was present in this case. As the parents are consanguineous, transmission of the syndrome in this family is also consistent with an autosomal recessive trait. The presence of six and possibly seven affected offspring in the same family provides a unique opportunity to see the phenotypic variability of this condition. While family reports support autosomal recessive transmission of the condition, it is unknown if OFDS type IV results from mutation of a single gene locus or if locus heterogeneity contributes to the observed clinical variability.There are only sixteen patients reported in the literature with this syndrome, and few report dental findings and report longitudinal followup describing developmental progression of OFDS type IV. The palatal pseudocleft reported in infancy has progressed to a frank palatal cleft. Most OFDS type IV cases report consanguineous parents. In the current family, both affected males and females were observed, consistent with autosomal recessive inheritance of the trait. The affected individuals also demonstrate the striking heterogeneity reported for the condition, ranging from neonatal lethality to infant lethality to a fully affected OFDS type IV sibling. The male sibling with cleft palate may represent a mildly affected spectrum of the OFDs Type IV phenotype.Of the eleven OFDS types reported, OFDS type IV cases are very rare, and the range of clinical heterogeneity is unclear. This case reports longitudinal followup of an OFDS case first reported in early infancy. The palatal pseudocleft has progressed to a frank palatal cleft, and additional dental findings are reported. The proband had one healthy sibling with a cleft palate. It is thus concluded that the inheritance of this syndrome is most consistent with an autosomal recessive trait and the cleft palate of the healthy sibling may reflect a mild expression of the syndrome. Although this syndrome is referred to as Baraitser-Burn syndrome in the OMIM database, consideration of all clinical reports is most parsimonious with the OFDS type IV Mohr-Majewski syndrome."} +{"text": "Castor oil bean cement (COB) is a new material that has been used as an endodonticsealer, and is a candidate material for direct pulp capping.The purpose of this study was to evaluate the biocompatibility of a newformulation of COB compared to calcium hydroxide cement (CH) and a control groupwithout any material, in the subcutaneous tissue of rats.The materials were prepared, packed into polyethylene tubes, and implanted in therat dorsal subcutaneous tissue. Animals were sacrificed at the 7th and 50th daysafter implantation. A quantitative analysis of inflammatory cells was performedand data were subjected to ANOVA and Tukey's tests at 5% significance level.Comparing the mean number of inflammatory cells between the two experimentalgroups (COB and CH) and the control group, statistically significant difference(p=0.0001) was observed at 7 and 50 days. There were no significant differences(p=0.111) between tissue reaction to CH (382 inflammatory cells) and COB (330inflammatory cells) after 7 days. After 50 days, significantly more inflammatorycells (p=0.02) were observed in the CH group (404 inflammatory cells) than in theCOB group (177 inflammatory cells).These results demonstrate that the COB cement induces less inflammatory responsewithin long periods. Conservative endodontic techniquesfacilitate the maintenance of teeth with pulpal alterations, minimizing the unwantedsequelae of their unplanned extraction9. Biocompatibility is as important as the physical and chemicalproperties when selecting a material for endodontic therapy because of the directcontact with the vital tissue. However, some currently used pulp capping materials havea tissue-irritating potential17.In direct pulp capping procedures, a biocompatible or bio-inductive material is placedonto the exposed pulp tissue, preserving its vitality, stimulating the repair process,and promoting the formation of hard tissue barrier29 and antibacterial action25. CH has been thematerial of choice for direct pulp capping because it seems to stimulate a rapiddifferentiation of odontoblast-like cells that form a hard tissue barrier in thepulp25. On the other hand, this action is not exclusive of CH and thismaterials suffers mechanical wear and solubility for long periods9.Calcium hydroxide (CH) cement presents properties such as low cytotoxicity, highpHRicinus communis) is polyester formed by anamino radical, which confers bactericidal effect and has biocompatibility with livingtissues3. It has great potentialto facilitate tissue healing, excellent structural properties, low cost and does elicittoxic effects. COB14 has been testedin rabbits as a matrix for bone and joint replacement. After 40 days of surgery, thehistological examination showed absence of late inflammatory reaction and no signs ofsystemic toxic effects.The castor oil bean (COB) (6 (1997) analyzedhistometrically the alveolar bone healing around castor oil bean implanted immediately aftertooth extraction. The material was biologically compatible, as it was progressivelyintegrated into alveolar bone in the healing process.Carvalho, et al.1 (2003) investigatedin vivo the biocompatibility of Ricinus communispolyurethane with three different chemical compositions. Modification of the polymer\u2019schemical composition by the addition of calcium carbonate or calcium phosphate promotedmatrix mineralization, these materials being more biocompatible than pure resin.Mastrantonio and Ramalho22 (2003)evaluated the subcutaneous tissue reaction in mice, after the implantation of castor oilbean with or without calcium carbonate and showed that both materials arebiocompatible.Barros, et al.28.In endodontics, COB has been used in retrograde filling materials in paraendodonticsurgeries, irrigating agents and endodontic sealers15. The development of newer biocompatible, bactericidal, inductivematerials that promote tissue repair and present adequate sealing can result inlongevity of pulp capping procedures10. In addition, material selection is important for the success orfailure of these treatments15.The use of a certain material must be based on experimental and laboratory studies thatprove its biocompatibility and other properties26 , although it is known that some reactions observed inthis test cannot be considered identical to those occurring in living dentaltissues.With this objective, some methods have been developed to evaluate the irritatingpotential of dental materials. The implantation of materials in the subcutaneousconnective tissues of small experimental animals is considered an adequate methodologyto determine the biocompatibility of endodontic materials12. For this reason, thereis an interest to increase the knowledge of the biocompatibility of COB because thismaterial can be a candidate for direct pulp capping. The purpose of this study was toevaluate the biocompatibility of a new formulation of COB compared to CH and a controlgroup without any material, in the subcutaneous tissue of rats.It is believed that the pulp reaction can vary with the use of different availableproducts, depending on their biocompatibility, which could cause severe damage to thistissueThis study was performed in accordance to the ethical Principles of Animalexperimentation and was approvedby the local Research ethics Committee (process no. 003/2006-PA/CeP).Rattus norvegicus) aged 90 days andweighing 350 to 400 g were used. The animals were maintained with food and waterad libitum.Forty-two male Wistar rats and a CH cement . The COB cement was prepared according to themanufacturers\u2019 instructions, mixing liquid polyol (5 mL), liquid prepolymer (5 mL), andcalcium carbonate (5 g) until homogenization was obtained. CH cement was hand-mixedaccording to the manufacturer\u2019s directions.Polyethylene tubes (10-mm long x 1.5 mm inner diameter) were washed with 70% alcohol anddistilled water, autoclaved, and filled with the experimental materials using a lentulospiral at low speed. All carriers and glass platesused in the study were previously sterilized.The sample comprised three experimental groups of 14 animals each, half of which werekilled after 7 and half at 50 days. each animal received a polyethylene tube containingCOB cement or CH cement in the dorsum. In the control group, the animals received anempty polyethylene tube in the dorsum. For the surgical procedures, the animals were anesthetized by intramuscularadministration of 38.5 mg/kg of ketamine HCL , and 14.2 mg/kg of xylazine . The back of the animal was shaved and cleaned with 1% iodine inethanol. Incisions were made on the dorsum, and one subcutaneous pocket was carefullyprepared by blunt dissection. The base of the pocket was located at 10 mm from theincision line. A tube containing cement was then placed into each pocket and theincision was closed with surgical gut sutures.The animals were killed after periods of 7 and 50 days, and the tubes were removed alongwith the surrounding tissue and immersed in 10% buffered formalin.per rat.After fixing for 48 h, the tissue was processed for paraffin embedding. The tubes wereremoved during this procedure. A paraffin block was oriented in such a way that it wasparallel to the long axis of the tube, and serial sections were cut to a 5-\u00b5mthickness. The sections were stained with hematoxylin and eosin. Histologicalqualitative and quantitative analyses of the inflammatory response were performed onlight microscope. Color images of stained sections were acquired with a high-resolutioncamera at 200x originalmagnification for histomorphometric analysis. Only 1 view standardized total area analyzed. It was positioned in the exact center of end of tubein each one of the 5 semi-serial slides A single investigator blinded to the groups examined all specimens. Inflammatory cellswere counted with an automated image analysis software, a public domain image processingand analysis program , using the point tool. The criteria ofhistological quantitative evaluation were based on microscopic aspects. Lymphocytespresented small, round, very darkly staining nuclei and little surrounding cytoplasm.Macrophages presented larger, paler, oval or bean shaped nuclei and a somewhat largeramount of cytoplasm. Neutrophils were easily identified because of their polymorphicshape nucleus. Other inflammatory cells were not obvious in this study. Immaturefibroblasts displaying large oval nuclei and mature fibroblasts with fusiform nucleiwere observed but were not quantified. A mean number of inflammatory cells were obtainedfor each animal. Data were subjected to descriptive and inferential analysis. The meansof inflammatory cells were tested by two-way ANOVA. The control, CH cement and COBcement groups were compared considering the following factors as variables: material andperiod of sacrifice (7 or 50 days after implantation). When the ANOVA showedstatistically significant difference, the Tukey\u2019s multiple-comparison test was used. Thelevel of significance was 5% for both tests.The groups were compared qualitatively and no difference was observed between the CHand COB cement groups. In the sections obtained at the 7th day, a moderate to severe chronic inflammatoryprocess was observed, except for one specimen that presented mild intensity.Inflammatory infiltrate composed of mononuclear cells, mainly lymphocytes, waspresent near the material. In some specimens, there were also plasma cells,neutrophils and eosinophils in the inflammatory infiltrate. Only one case exhibitedextravasation of COB granules, which were surrounded by numerous inflammatory cells.The connective tissue in contact with the material showed mild inflammatory reactionand a zone of necrosis was detected in few cases. The presence of multinuclear giantcells was not prominent in this experimental group. These cells appeared in a smallnumber close to the material and presented three to five nuclei.In the sections obtained at the 50th day, a mild to moderate inflammatory cellinfiltrate was observed, mainly composed of lymphocytes and plasma cells. Thepresence of extravasated material in the subcutaneous tissue was associated with anincreased inflammatory process. In most cases, the tissue was organized in a capsulararrangement with parallel collagens fibers, interspersed with fibroblasts and matureblood vessels. Multinuclear giant cells with three to five nuclei were found in mostspecimens, although they appeared in a small number and were distributed near thematerial.In the sections obtained at the 7th day, moderate to severe inflammatory cellinfiltrate was observed. It was increased around the extravasated CH granules in thesubcutaneous tissue. Except for one specimen that presented numerouspolymorphonuclear leukocytes, mainly neutrophils, the tissue reactions showed aninflammatory infiltrate composed of lymphocytes and macrophages. Few plasma cellswere observed. The macrophages were distributed around the overflow granules ordiffusely in the granulation tissue. Sometimes, the cytoplasm of these macrophagespresented some material particles. In some specimens, multinuclear giant cells withdifferent amounts of nuclei and irregular cytoplasmic outline were found. The nucleiwere distributed at random, characterizing a foreign body reaction. The connectivetissue in contact with the material showed different degrees of reaction and a zoneof necrosis was detected in many cases.In sections obtained at the 50th day, moderate fibroblast and angioblasticproliferation was observed. The fibrous area presented a capsular arrangement withmoderate amount of collagen fibers. Moderate to severe inflammatory infiltrate wasverified, except in some specimens in which it was of mild intensity. Theinflammatory cells were distributed around the extravasated CH granules. In thisgroup, there was predominance of mononuclear cells, mainly lymphocytes and plasmacells, except for two cases exhibiting numerous neutrophils and eosinophils.Multinuclear giant cells and necrosis were not evident in this period.Seven days after implantation, granulation tissue was observed in contact with thetube, presenting fibroblasts and new blood vessels. Moderate to mild inflammatoryinfiltrate composed of mononuclear cells, mainly lymphocytes and plasma cells, wasobserved. On day 50, a dense collagenous tissue was observed with scarce inflammatorycells, characterizing a fibrous capsule.The analysis of the morphometric results shows that in the COB group, the number ofinflammatory cells decreased from days 7 to 50.Comparison of the mean number of inflammatory cells between the two experimentalgroups (CH and COB) and the control group by two-way ANOVA revealed significantdifference (p=0.0001). There were no significant differences between tissue reactionto CH (382 \u00b1 123.8 inflammatory cells) and COB (330 \u00b1 106.9inflammatory cells) after 7 days. However, after 50 days, the CH group presented alarger mean number of inflammatory cells than the COB group (404 \u00b1 118.8versus 176 \u00b1 60.2 inflammatory cells). The mean number of inflammatory cellsin the control group at 7 and 50 days was 69.0 \u00b1 35.1 and 50.0 \u00b1 33.5,respectively.19 because it is believed that, if made bycalibrated examiners, it can determine reliable results. However, histomorphometricanalysis based on counting of the number of inflammatory cells presents more reliableresults than qualitative analysis with scores, for example23.Histomorphometric and/or quantitative analyses can be used to verify the inflammatoryand repair phenomena and other reactions of dental materials in the subcutaneous tissueof rats. Some studies have used quantitative analysis16. There are many ways to applycomputerized tools in morphometry. Semi automatic counting of inflammatory cells wasused in this study to verify the intensity of the reaction caused by the implantedmaterial. According to the obtained results, the simple qualitative analysis ofspecimens did not demonstrate any inflammatory differences between CH and COB implants.On day 50, the histomorphometric analysis showed that the COB cement presented asignificantly smaller number of inflammatory cells than CH, and also showed nodifference from the control group. On the other hand, qualitative analysis showed aninflammatory reaction similar to both cements, with mild to moderate intensity in thesame evaluation period. Therefore, quantitative analysis was very important to showdifferences in tissue reaction to the tested cement.The Image J software is widely used in quantitative experiments15. Fourteen daysafter implantation, the volume of tissue reaction was measured histomorphometrically.The highest flow values were obtained with CH cements, but the flow did not correlatewith the degree of inflammatory response.CH cement produced minimum inflammatory reactions in a previous study that determinedthe flow characteristics and subcutaneous tissue reactions to CH and zinc oxide-eugenolendodontic sealers19 (1998) evaluatedthe in vivo biocompatibility of CH sealer in root canals. The intensityof reaction, initially severe, decreased on the 60th day, and this reduction continuedprogressively up to the 120th day.Kolokouris, et al.\u00ae CH cement exhibited the highest water sorption and solubility values after an evaluation of itsmechanical properties11.In the present study, after 50 days, it was verified that the CH cement still showed aninflammatory process of moderate to severe intensity, mainly close to overflow CHgranules. The presence of this material outside the tube increased not only theextension, but also the intensity of the inflammatory reaction. Therefore, beforeselecting a material for pulp capping procedures, it is important to know its mechanicalproperties. The ideal material should present low flow and should not be too muchfriable. Hydro C29 , in the present study the CH cement hasless biocompatible than the COB cement. However, if we had used longer periods ofevaluation, CH would be similar to COB cement. A large number of dental materialspresent cytotoxic effects when applied close or directly to the pulp, and the onlymaterial that seems to stimulate early pulp repair and dentin hard tissue barrierformation is CH. CH products are the best choice for conservative pulp treatments due totheir therapeutic and biological potential, and the property of stimulating theformation of sclerotic and reparative dentin with a consequent protection of the pulpagainst thermal stimuli24.Although CH capacity of inducing the formation of a hard tissue bridge is an importantproperty of pulp capping materials12.The COB cement has been used in Medicine in the reconstruction, substitution or fillingof bone defects presenting good results21.Studies have persisted in the search for materials with high biocompatibility and goodphysicochemical properties, since the materials used in endodontic procedures can causedifferent reactions on pulp tissue3 (2001)and Carvalho, et al.6 (1997) implanteda COB-derivative natural resin in the extraction wounds in rats to add information aboutthe biocompatibility of this material. Their results were similar to those of thepresent study because after 6 weeks there was no persistence of inflammatory reaction,yet a small number of giant cells were observed in the tissue in contact with thematerial. Also, there was no foreign-body reaction or persistence of the inflammatoryreaction in the study of Carvalho, et al.7 (1997). They evaluated histometrically the bone healing aroundpolyurethane resin implants derived from castor bean and verified progressiveosteogenesis in conjunction with a decrease in the fibrous capsule thickness.In Dentistry, Calixto, et al.Ricinus communitis polyurethane followed by synthetic body fluidincubation could be a useful alternative to improve the biological properties, as boneformation, of this polyurethane2.Other authors have demonstrated that the incorporation of alkaline phosphatase to the8 (1997), the COB cement presented acceptable biocompatibility undermicroscopic analysis when tested in subcutaneous implants in rats. The biocompatibilityof COB cement has also described by Perassi, et al.28 (2004), who evaluated the tissue response of subcutaneousimplants filled with COB cement and other endodontic sealers. The COB cement showed lesstissue response than any other sealer in both experimental periods (7 and 50 days),which can be explained by the structure of this material with high pureness, lack ofsolvent, debris, stabilizers and degradation products present in other polymers, whichwould lead to adverse organic responses28.According to Costa, Marcantonio and Hebling22 (2003)evaluated the subcutaneous tissue reaction in rats after the implantation of COB cementwith or without calcium carbonate and showed that both materials induced mildinflammatory response after 7 days. Barros, et al.1 (2003) verified that the addition of calcium phosphate or calciumcarbonate to the Ricinus communis polyurethane improved itsbiocompatibility implanted in rabbit femurs.Mastrantonio and RamalhoThese results are according with those of the present study, which showed lessinflammatory response and acceptable biocompatibility after 50 days of observation.in vitrostudy4 using primary humanpulp-derived cells. However, new complementary studies are necessary to evaluating thismaterial over the pulp tissue.In summary, the findings of the present study indicate that the COB cement is apromising material. Furthermore, extracts of COB slightly induced cell proliferation anddid not present genotoxicity without formation of micronuclei in V79 cells, ormodification of the normal cell cycle in a previous These results demonstrate that the castor oil bean cement (COB) induces lessinflammatory response within long periods."} +{"text": "P < .05). Moreover, remarkably up\u2010regulated ciRS\u20107 and RELA expressions, as along with down\u2010regulated miR\u20107 expressions, were found within NSCLC tissues and cells in comparison with normal ones (P < .05). Besides, overexpressed ciRS\u20107 and underexpressed miR\u20107 were correlated with increased proliferation, migration and invasion, yet reduced apoptosis rate of NSCLC cells (P < .05). More than that, ciRS\u20107 specifically targeted miR\u20107 to reduce its expressions (P < .05). Ultimately, the NSCLC cells within miR\u20107 + RELA group were observed with superior proliferative, migratory and invasive capabilities than those within miR\u20107 group (P < .05), and RELA expression was also significantly modified by both ciRS\u20107 and miR\u20107 (P < .05). In conclusion, the ciRS\u20107/miR\u20107/NF\u2010kB axis could exert pronounced impacts on the proliferation, migration, invasion and apoptosis of NSCLC cells.The purpose of this study was to figure out the effect of ciRS\u20107/miR\u20107/NF\u2010\u03baB axis on the development of non\u2010small cell lung cancer (NSCLC). In response, the expressions of ciRS\u20107, miR\u20107 and NF\u2010\u03baB subunit (ie RELA) within NSCLC tissues and cell lines were determined with real\u2010time polymerase chain reaction (RT\u2010PCR) and Western blot. Moreover, the NSCLC cells were transfected with pcDNA3\u2010ciRS\u20107\u2010ir, pcDNA3\u2010ciRS\u20107, miR\u2010NC and miR\u20107 mimic. Furthermore, the targeted relationships between ciRS\u20107 and miR\u20107, as well as between miR\u20107 and RELA, were confirmed by luciferase reporter assay. The proliferation, migration and apoptosis of NSCLC cells were, successively, measured using CCK\u20108 assay, wound\u2010healing assay and flow cytometry test. Consequently, ciRS\u20107, miR\u20107, histopathological grade, lymph node metastasis and histopathological stage could independently predict the prognosis of patients with NSCLC (all The fresh specimens were cryopreserved at \u221280\u00b0C within 30 minutes after exairesis. All cases were pathologically confirmed by >2 senior professionals, and none of them had received chemotherapy or radiation before surgery. The histopathological grading of NSCLC was evaluated in light of World Health Organization (WHO),2.22 and saturated humidity at 37\u00b0C.Human NSCLC cell lines and the normal human embryonic lung fibroblast cell line (ie MRC5) were purchased from Shanghai Institute of Biochemistry and Cell biology, Chinese Academy of Sciences. All the cell strains were inoculated within cell medium that contained 10% bovine foetal serum, 2 \u03bcmol/L glutamine, 100 IU/mL penicillin and 100 \u03bcg/mL streptomycin sulphate. Subsequently, the cells were managed to grow in 5% CO2.3\u2212\u25b5\u25b5Ct method was used to calculate relative quantification (RQ) values, and each experiment was repeated for at least 3 times.Total RNA was extracted by consulting the operational manual of routine TR1201 reagent , and the extracted RNAs were preserved at \u221280\u00b0C. Then, the purity and concentration of RNA were measured by applying spectrophotometry. Moreover, the expressions of miR\u20107 and ciRS\u20107 were measured through qRT\u2010PCR (Invitrogen Corporation), and their primers were designed and composed by Sangon Biotech (Shanghai) Corporation, China .2.5The cells at the exponential phase were digested with pancreatins, and they were processed into cell suspension after low\u2010speed centrifugation. Subsequently, after being mixed with the trypan blue saline solution (percentage: 0.4%), they were dropped upon the blood counting chamber, and cell counting was completed within 3 minutes. Finally, under the microscopic observation, the cells dyed to blue were determined as dead, while the colourless and transparent ones were judged as live.2.63 cells, and the cells were cultured in 5% CO2 at 37\u00b0C. After each hole was added with 50 \u03bcg MTT, the cells were again cultured for another 4 hours. Then, the supernatants were sucked out before the addition of 200 \u03bcl DMSO. Ultimately, the optical densities of each hole at the wavelength of 570 nm were measured with utilization of microplate reader at the 24th, 48th, 72nd and 96th hours, respectively. The inhibition rate (IR) of cell proliferation was calculated according to the following formula: Each hole of 96\u2010well culture plates was added with 100 \u03bcL cell suspension that contained 2 \u00d7 102.7Transwell chambers were placed within 24\u2010well plates, and the bottom membranes were coated with the diluted Matrigel. Serum\u2010free DEME nutrient solution was used to dilute cell sediments, and the concentration was adjusted to 5 \u00d7 10\u00b3/mL. The upper and lower Transwell chambers were, respectively, added with 200 \u03bcL cell suspension and 500 \u03bcL DEME solution that included 10% foetal bovine serum. After being cultured for another 12 hours, cells were washed with pre\u2010cooled PBS for twice. Methyl alcohol was prepared to immobilize membranes and cells for 15 minutes, which were then dyed with 1% crystal violet for 30 minutes. Under the high\u2010power microscope, the invasive cells below the chamber membrane were quantified, and 5 views were randomly observed to get the average results.Besides, the migration experiment was conducted mostly in accordance with the above procedures, except that Matrigel glue was hardly applied.2.85/mL. Flow cytometry was employed to detect the cell apoptotic conditions, following the instructions of Annexin V\u2010 fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit. The resultant scatter diagram was explained in the following way: (1) the left\u2010lower quadrant indicated live cells marked with FITC\u2010/PI\u2212; (2) the left\u2010upper quadrant indicated live cells marked with FITC\u2010/PI+; (3) the right\u2010upper quadrant indicated dead cells marked with FITC+/PI+; and (4) the right\u2010lower quadrant indicated the early apoptotic cells marked with FITC+/PI\u2212.The cells at the logarithmic phase were seeded within the culture vessels at the density of 1.25 \u00d7 102.9PCR was performed to amplify the miR\u20107 fragment that embraced the binding site for ciRS\u20107. Subsequently, the products were cloned into the pmirGLO Dual\u2010luciferase miRNA Target Expression Vector, deriving wild\u2010type miR\u20107 (ie miR\u20107\u2010wt). Besides, the same putative binding site was mutated, and miR\u20107\u2010Mut was drawn. With assistance of Lipofectamine 2000 , the cells were, respectively, transfected with pcDNA\u2010ciRS\u20107 and pcDNA\u2010ciRS\u20107\u2010ir and pRL\u2010TK reporter gene carrier. As for the targeted relationship between miR\u20107 and RELA, the RELA fragment that covered the binding site of miR\u20107 was also amplified by PCR before being cloned into the pmirGLO Dual\u2010luciferase Expression Vector , in which way the reporter vector RELA\u2010wild\u2010type (ie RELA\u2010wt) was constructed. Similarly, the RELA with the mutated binding site of miR\u20107 was called RELA\u2010mutated\u2010type (ie RELA\u2010Mut). Subsequently, the cells were, respectively, transfected with miR\u20107 mimics and miR\u2010NC, and each group was then transfected with RELA\u2010Wt 3\u2032UTR\u2010PGL3 plasmid/RELA\u2010Mut 3\u2032UTR\u2010PGL3 plasmid and pRL\u2010TK reporter gene carrier, respectively. The activities of Firefly and Renilla luciferase were evaluated successively by applying dual\u2010luciferase reporter assay system (Promega) in 48 hours after transfection. All the assays were repeated for at least 3 times.2.10By means of the 10% SDS\u2010PAGE electrophoresis, the extracted total protein was transferred onto nitrocellulose membranes for 60 minutes. Then, PVDF membranes were sealed at room temperature for 2 hours, and rabbit anti\u2010human RELA primary antibody was incubated overnight. Subsequently, the rat anti\u2010rabbit second antibody was added and incubated for another 2 hours at room temperature. Then, enhanced chemiluminescence (ECL) was applied for exposure and development, and gel image analysis system was employed to analyse the electrophoresis strips. The \u03b2\u2010actin was regarded as the control of ciRS\u20107 regarding calculation of the intensity values. All the assays were repeated for at least 3 times.2.112 test, while measurement data (mean \u00b1 SD) were contrasted using t\u2010test. The method of Kaplan\u2010Meier was applied to fit overall survival (OS) curves of the included subjects. It would be considered statistically significant when P value was <.05.All the statistical analyses were conducted with SPSS 20.0 software. The enumeration data were compared with \u03c733.1P < .05), whereas the miR\u20107 expression followed an inverse trend (P < .05). The different trends also rendered a significantly negative correlation between ciRS\u20107 and miR\u20107 expressions within NSCLC tissues (P < .05) Figure B. Analog) Figure C.3.2P < .05). Moreover, Kaplan\u2010Meier analysis also manifested that NSCLC subjects with highly expressed ciRS\u20107 or lowly expressed miR\u20107 possessed inferior OS to ones with lowly expressed ciRS\u20107 or highly expressed miR\u20107 (P < .05) (Table The incorporated patients with NSCLC were divided into higher ciRS\u20107 expression (>average ciRS\u20107 expression) group and lower ciRS\u20107 expression (99%. Moreover, this plasmid displayed high sequence similarity to the previously reported epidemic IncX3 blaNDM-5-carrying plasmids, with dynamic changes observed only in blaNDM-5-surrounding elements. Interestingly, the IncX3 blaNDM-5-carrying plasmids showed strong stability in clinical isolates when cultured in antibiotic-free medium. However, after the conjugation inhibitor linoleic acid was added, a gradual increase in the level of IncX3 plasmid loss could be observed. Clinical isolates displayed 10% to 15% blaNDM-5-carrying plasmid loss after coculture with linoleic acid for 5\u2009days. These results showed that the IncX3 plasmid facilitated the dissemination of blaNDM-5 among multiclonal K. pneumoniae strains in children and that conjugal transfer contributed significantly to IncX3 plasmid stability within K. pneumoniae.NDM-5 carbapenemase was mainly identified in IMPORTANCE The emergence and spread of New Delhi metallo-\u03b2-lactamase (NDM)-producing Enterobacteriaceae have been a serious challenge to public health, and NDM-5 shows increased resistance to carbapenems compared with other variants. NDM-5 has been identified mostly in E. coli but has rarely been described in K. pneumoniae and other Enterobacteriaceae isolates. Here, we present the dissemination of highly similar 46-kb IncX3 blaNDM-5-carrying plasmids among multiclonal K. pneumoniae strains in children, highlighting the horizontal gene transfer of blaNDM-5 among K. pneumoniae strains via the IncX3 plasmid. Moreover, the IncX3 blaNDM-5-carrying plasmids displayed strong stability in clinical strains when cultured in antibiotic-free medium, and the plasmid maintenance was attributed partly to conjugal transfer. Plasmid conjugation is mediated by the type IV secretion system (T4SS), and T4SS is conserved among all epidemic IncX3 blaNDM-5-carrying plasmids. Therefore, combining conjugation inhibition and promotion of plasmid loss would be an effective strategy to limit the conjugation-assisted persistence of IncX3 blaNDM-5-carrying plasmids. Escherichia coli carbapenemase is an important type of carbapenemase with the ability to hydrolyze almost all \u03b2-lactams, and 24 NDM variants (NDM-1 to NDM-24) have been identified to date were recovered from blood, urine, and normally sterile body fluids (SBF) of patients in Shanghai Children\u2019s Medical Center from January 2016 to December 2018. All isolates were analyzed by PCR for blaKPC, as well as blaNDM,blaIMP,blaOXA-48, and blaVIM was performed for the selected NDM-5-positive isolates (K. pneumoniae strains belonged to 9 different sequence types (STs) . In acco10.1128/mSphere.00917-20.1FIG\u00a0S1K. pneumoniae strains. Numbers represent the strain codes. M, DNA marker. Download FIG\u00a0S1, JPG file, 0.3 MB.PFGE analysis of the representative NDM-5-producing Copyright \u00a9 2020 Zhu et al.2020Zhu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the K. pneumoniae isolates possessed 2 to 3 plasmids and that the blaNDM-5 genes were all located on plasmids with similar sizes (\u223c46\u2009kb) . The blan typing . The gen10.1128/mSphere.00917-20.2FIG\u00a0S2K. pneumoniae strains. Bands indicated with arrows show positive signals in Southern blot hybridization with the blaNDM-5 probe. M, DNA marker. Download FIG\u00a0S2, JPG file, 0.4 MB.S1-digested plasmid DNA and Southern blot hybridization for representative NDM-5-prducing Copyright \u00a9 2020 Zhu et al.2020Zhu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00917-20.3TABLE\u00a0S1blaNDM-5-carrying plasmids. Download Table\u00a0S1, DOCX file, 0.02 MB.Primers used for screening the backbone of Copyright \u00a9 2020 Zhu et al.2020Zhu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00917-20.4TABLE\u00a0S2blaNDM-5 transconjugants. Download Table\u00a0S2, DOCX file, 0.02 MB.Antimicrobial susceptibility testing of Copyright \u00a9 2020 Zhu et al.2020Zhu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the K. pneumoniae strain K2-7 was randomly selected, and plasmid DNA (pSCK27-NDM5) from the corresponding E. coli J53 transconjugant was extracted using a Qiagen Plasmid midi kit and was sequenced with an Illumina MiSeq system . The reads were assembled de novo into contigs using SPAdes 3.9.0, and gaps were closed through PCR and Sanger sequencing. Comparative analysis of the representative fully sequenced IncX3 blaNDM-5-carrying plasmids was performed to assess the genetic context of the blaNDM-5 gene. The functional genes were identical across IncX3 blaNDM-5-carrying plasmids, with all plasmids carrying genes for replication (repB), partitioning (parA and parB), and conjugative transfer . StructublaNDM-5-carrying plasmid within K. pneumoniae was investigated, and three NDM-5-producing strains were randomly selected. The proportion of the bacterial population that retained the blaNDM-5-carrying plasmid was determined over a period of 5\u2009days (blaNDM-5-carrying plasmid, the conjugation inhibitor linoleic acid was added to LB broth at final concentrations of 2.5 and 5\u2009mM. The culture was diluted each day, and each dilution was plated on LB agar and incubated overnight at 37\u00b0C. A total of 100 colonies were randomly collected from all dilutions and spotted on LB plates in the presence and absence of meropenem. Plasmid retention was calculated by comparing the number of colonies on the LB agar plate containing meropenem with that on pure LB agar.The stability of the IncX3 f 5\u2009days . BacteriblaNDM-5-carrying plasmids showed strong stability in clinical isolates, without apparent plasmid loss after serial subculture for 5\u2009days (blaNDM-5-carrying plasmid loss could be observed in all three strains (blaNDM-5-plasmid conjugation efficiency but did not exert any tremendous effect on bacterial growth (blaNDM-5-carrying plasmid loss after coculture with linoleic acid for 5\u2009days, indicating that conjugal transfer contributed significantly to the persistence of IncX3 blaNDM-5-carrying plasmid. Plasmid conjugation is mediated by T4SS, and T4SS is conserved among all epidemic IncX3 blaNDM-5-carrying plasmids (blaNDM-5-carrying plasmid.The IncX3 r 5\u2009days . It seem strains . Linolei strains , 13. Linl growth . These splasmids . TherefoK. pneumoniae to ameliorate the associated fitness costs of plasmid carriage (K. pneumoniae for relatively long periods would definitely lead to an increased level of plasmid loss in bacterial populations. In addition, 3% to 5% plasmid loss was still observed in clinical strains after 1\u00a0day of culture with 5\u2009mM linoleic acid, suggesting that inhibition of conjugal transfer is likely to promote IncX3 blaNDM-5-carrying plasmid loss from K. pneumoniae.It is commonly believed that a plasmid-free bacterial host can compete successfully with bacterial cells harboring plasmids, due to the fitness costs of plasmid carriage . Howevercarriage . Though blaNDM-5-carrying plasmids among multiclonal K. pneumoniae strains in children, highlighting the horizontal gene transfer of blaNDM-5 among K. pneumoniae via the IncX3 plasmid. Moreover, the IncX3 blaNDM-5-carrying plasmids displayed strong stability in clinical strains when cultured in antibiotic-free medium, and conjugal transfer contributed significantly to plasmid maintenance within K. pneumoniae.In summary, this study presented the dissemination of highly similar 46-kb IncX3 All procedures in this study that involved human participants were performed in accordance with the ethical standards of the Institutional Review Board Ethics Committee of Shanghai Children's Medical Center. For this type of retrospective study, formal consent is not required.MT663954.The complete sequence of plasmid pSCK27-NDM5 was submitted to the GenBank database under accession number 10.1128/mSphere.00917-20.5DATA SET\u00a0S1Data Set S1, TXT file, 0.04 MB.The complete plasmid sequence of pSCK27-NDM5. Download Copyright \u00a9 2020 Zhu et al.2020Zhu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the"} +{"text": "In China, bacteria carrying these plasmids are increasingly being detected from diverse samples, including hospitals, communities, livestock and poultry, and the environment, suggesting that IncX3 plasmids are becoming a vital vehicle for blaNDM dissemination. To elucidate the fitness cost of these plasmids on the bacterial host, we collected blaNDM-negative strains from different sources and tested their ability to acquire the blaNDM-5-harboring p3R-IncX3 plasmid. We then measured changes in antimicrobial susceptibility, growth kinetics, and biofilm formation following plasmid acquisition. Overall, 70.7% (29/41) of our Enterobacteriaceae recipients successfully acquired the blaNDM-5-harboring p3R-IncX3 plasmid. Contrary to previous plasmid burden theory, 75.9% (22/29) of the transconjugates showed little fitness cost as a result of plasmid acquisition, with 6.9% (2/29) of strains exhibiting enhanced growth compared with their respective wild-type strains. Following plasmid acquisition, all transconjugates demonstrated resistance to most \u03b2-lactams, while several strains showed enhanced biofilm formation, further complicating treatment and prevention measures. Moreover, the highly virulent Escherichia coli sequence type 131 strain that already harbored mcr-1 also demonstrated the ability to acquire the blaNDM-5-carrying p3R-IncX3 plasmid, resulting in further limited therapeutic options. This low fitness cost may partly explain the rapid global dissemination of blaNDM-5-harboring IncX3 plasmids. Our study highlights the growing threat of IncX3 plasmids in spreading blaNDM-5.The wide dissemination of New Delhi metallo-\u03b2-lactamase genes ( Klebsiella pneumoniae isolate in 2008 [blaNDM is often associated with other resistance genes, which complicates treatment and results in higher mortality rates [The World Health Organization has identified the global spread of antimicrobial resistance as one of the three greatest threats to human health ,2. A New in 2008 . Since t in 2008 ,5,6,7. Nty rates .blaNDM-carrying plasmids, IncX3 appears to be the most common type (117/355 = 32.96%) [blaNDM-carrying IncX3 plasmids have been recovered from China, indicating an epidemic clustering of these plasmids. However, blaNDM-carrying IncX3 plasmid have also been reported in Asia, Europe, and North America [blaNDM-1, blaNDM-4, blaNDM-5, blaNDM-6, blaNDM-7, blaNDM-13, blaNDM-17, blaNDM-19, blaNDM-20, and blaNDM-21, have been identified on IncX3 plasmids, indicating that IncX3 plasmids provide an efficient platform for the spread and evolution of blaNDM genes and a likely vehicle for the spread of \u03b2-lactam resistance [Enterobacteriaceae species and encode a type IV secretion system enabling conjugative transfer, providing accessory functions such as resistance gene acquisition and biofilm formation to their host strains [blaNDM-5-carrying IncX3 plasmids have been isolated from clinical settings, companion animals, agriculture, and the environment, indicating that IncX3-type plasmids have played a crucial role in disseminating blaNDM-5 amongst Enterobacteriaceae [Plasmids are key elements in the dissemination of antibiotic resistance via horizontal transfer. Among the reported 32.96%) . Interes America . To datesistance . IncX3 p strains . Strainseriaceae ,15.blaNDM-5-carring IncX3 plasmids need to be further explored. A previous study described the physiology and pathogenicity of hyper-virulent K. pneumoniae following acquisition of the p24835-NDM5 plasmid [Escherichia coli J53 and K. pneumoniae PRZ [Enterobacteriaceae isolates from different sources, including the environment, chickens, meat, flies, humans, swine, and companion animals, to assess the success rate of conjugative transfer of a blaNDM-5-harboring p3R-IncX3 plasmid. We then evaluated the fitness costs associated with plasmid acquisition. Our study indicated that p3R-IncX3 plasmids are highly stable across multiple generations and can slightly improve membrane formation in different wild-type recipient hosts. The low fitness cost of this plasmid in Enterobacteriaceae may contribute to the spread of antibiotic resistance.However, although plasmids can help their host to adapt to diverse environmental conditions, they also have a fitness cost in the absence of selection for plasmid-encoded traits . General plasmid . A recenniae PRZ ; howeverblaNDM-5-harboring p3R-IncX3 plasmid used in our study was 46,149 bp in length and had a G+C content of 46.64%. The plasmid had a similar genetic background to that of IncX3 plasmid pNDM_MGR194, originally recovered from a K. pneumoniae isolate from India (GenBank accession number KF220657) [E. coli 3R (ST156) harboring plasmid p3R-IncX3, was isolated from a chicken cloaca sample from a farm in Qingdao in 2015. PacBio whole-genome sequencing of strain 3R identified four plasmids with different Inc types . The transformants were first selected using meropenem, the PCR-based screening using the four specific primers was then conducted to identify the transformants harboring the p3R-IncX3 only was conducted to select blaNDM-negative strains from our laboratory culture collection for use as wild-type recipients. Briefly, laboratory-stored strains of various sources were given serial numbers, then certain numbers were randomly selected by a computer. During random sampling, each member of a population has an equal chance of being included in the sample. The recipient strains were originally isolated from various sources, including humans, chickens, swine, companion animals, flies, meat, and environment samples. We aimed to select three E. coli and three K. pneumoniae isolates from each of the different origins; however, if this was not possible, other Enterobacteriaceae strains were used as alternatives. Detailed information on the wild-type recipients is available in Simple random sampling (http://www.genomicepidemiology.org/). Minimum spanning trees were constructed for E. coli and K. pneumoniae using BioNumerics version 7.0. The main phylogroups of the E. coli isolates were identified using Clermontyping [https://itol.embl.de/).Whole-genome sequencing of all recipient strains, the DH5\u03b1 donor strain, and their transconjugants was performed using the Illumina HiSeq 2500 sequencing platform . Genomic DNA was extracted using a TIANamp Bacteria DNA Kit . The draft genomes were assembled using SPAdes version 3.9.0 . Resistaontyping . The STshttp://www.eucast.org). Reference strain E. coli ATCC 25,922 was used as the quality control strain.The MICs of a range of antibiotics against all of the bacterial isolates were detected using the agar dilution method as per the Clinical and Laboratory Standards Institute recommendations and the blaNDM-5 and the plasmid replicon of p3R-IncX3 to verify the presence of the plasmid. The stability assays were performed in triplicate.Plasmid stability experiments were performed as described previously . Briefly600) of 0.5. Samples were then diluted 100-fold in LB broth medium in 96-well microtiter plates and incubated at 37 \u00b0C for 24 h. OD600 measurements were taken hourly to construct a growth curve. Growth kinetics assays were performed in triplicate.Growth curves for the recipients and transconjugants were performed in 96-well flat-bottom plates as described previously . BrieflyBiofilm formation assays were conducted as described previously . BrieflyblaNDM-5-harboring p3R-IncX3 plasmid, 41 NDM-negative wild-type recipient strains from different sources, including environmental samples, chickens, meat, flies, humans, swine, and companion animals, were randomly selected (E. coli 3R (ST156), isolated in our previous study [blaNDM-5-harboring p3R-IncX3 plasmid. In the current study, we used strain 3R as a donor to generate a blaNDM-5-harboring p3R-IncX3 plasmid-carrying DH5\u03b1 strain, which was confirmed by PCR-based screening was higher than rates for K. pneumoniae (63.6%) and other species (57.1%) . Of note (57.1%) . In comp (57.1%) . The higE. coli and K. pneumoniae strains were constructed from core-genome single-nucleotide polymorphism data, which also included information on the collected strains, including STs, phylogroups, sources, Inc types, and antimicrobial resistance gene profiles belonged to non-pathogenic phylogroup A. Phylogroup B strains composed the second-largest group, which included donor strain 3R. Among the phylogroup B strains, E. coli strain No. 28 (ST131), originally isolated from a human, belonged to highly virulent phylogenetic group B2 [mcr-1, a plasmid-mediated colistin resistance gene that can breach the efficiency of colistin as a \u201clast-line\u201d resort for treatment of MDR bacterial infections [mcr-1 and blaNDM-5 in E. coli strains, especially the highly virulent ST131 strain, is particularly concerning for public health. Plasmid Inc type analysis showed that two E. coli strains both contain an IncX1 plasmid belonging to the same family as the IncX3 plasmid; however, the presence of this plasmid did not affect the ability of these two strains to capture the IncX3 plasmid. The coexistence of different Inc-families in a single strain increases the antimicrobial resistance spectrum of the recipient strains.To investigate the phylogenetic relationships among the recipient strains and to verify the transconjugants, all 41 wild-type recipient strains and 29 transconjugants were subjected to whole-genome sequencing. Phylogenetic trees for the profiles . The mai plasmid , indicatgroup B2 , and cougroup B2 . SignifiblaNDM-carrying p3R-IncX3 plasmid, the MIC of meropenem against DH5\u03b1 increased from 0.032 to 16 mg/L. For some wild-type recipient strains, the meropenem MICs increased from 0.032 to \u2265 128 mg/L 2017 guidelines: S, \u2264 2 mg/L; R \u2265 8 mg/L). Strains No. 4 and No. 13 were the exception, with meropenem MIC values of 4 mg/L for both strains. However, all 29 strains that successfully acquired the p3R-IncX3 plasmid demonstrated high-level resistance to meropenem following plasmid acquisition. For example, after acquiring the 128 mg/L . These d128 mg/L . The expK. pneumoniae rapidly lost a blaNDM-5-harboring p3R-IncX3 plasmid after monoculture for 100 generations without antibiotics [To evaluate the stability of the p3R-IncX3 plasmid, we selected nine transconjugants and passaged them daily for 15 days in the absence of antibiotic selection. Unlike a previous study showing that hyper-virulent ibiotics , our resblaNDM-5-harboring p3R-IncX3 plasmid, growth kinetics assays were performed for the strains with and without the p3R-IncX3 plasmid. Interestingly, results vary for the different strains. The growth of DH5\u03b1 carrying the p3R-IncX3 plasmid was almost indistinguishable from that of wild-type DH5\u03b1 (E. coli No. 11 (p < 0.01) and K. pneumoniae No. 14 (p < 0.01) (C. freundii strain No. 21 (p < 0.01) (E. coli strain No. C45 (p < 0.01) (E. coli strain No. 0970 (p < 0.01) (E.coli strain No. 10 (p < 0.01) C, chicke < 0.01) F, swine- < 0.01) J, and co < 0.01) K, indica < 0.01) D and com < 0.01) L showed < 0.01) M. Thus, blaNDM-5-harboring p3R-IncX3 plasmid should impose a fitness cost on the recipients in the absence of antibiotic pressure as was observed in a previous study wherein a 140-kb blaNDM-1-carrying IncA/C-type plasmid imposed a high fitness cost in E. coli J53 and K. pneumoniae PRZ [blaNDM-5-harboring p3R-IncX3 plasmid used in the current study carries H-NS-like genes, which may explain the low fitness cost of this plasmid. Similarly, two carbapenemase-encoding plasmids, pG12-KPC-2 and pG06-VIM-1, had low biological costs when they transferred from K. pneumoniae to E. coli [blaNDM-5-harboring p3R-IncX3 plasmid used in this study had different fitness costs in different wild-type recipient strains. However, this is consistent with a previous study showing that the fitness costs of newly acquired mobile genetic elements depend on the genetic makeup of the recipient and/or environmental factors [It is widely believed that in the absence of antibiotic pressure, plasmids containing the corresponding resistance genes will impose certain fitness costs on their hosts . We did niae PRZ . Howeverniae PRZ . For exaniae PRZ . Furtherniae PRZ . The bla E. coli . Interes factors .E. coli DH5\u03b1 demonstrated only weak biofilm formation, but it increased significantly after getting the p3R-IncX3 plasmid from chicken manure compost and K. pneumoniae strain No. 14 (p < 0.01) from a chicken cloaca swab , meat , flies , humans , and swine C\u2013G. Howe < 0.01) I.Enterobacteriaceae [blaNDM-carrying p3R-IncX3 plasmids have the capacity to expand their host range among E. coli with different MLST profiles [mcr-1, also demonstrated enhanced biofilm formation following acquisition of the p3R-IncX3 plasmid. In addition, we previously showed that biofilm formation by Staphylococcus aureus accelerated the spread of plasmid-borne antibiotic resistance genes via conjugation or mobilization [blaNDM genes, but also increase the possibility of treatment failure rates.Biofilm formation requires the cooperation of different bacterial strains and species, allowing them to prosper and protect each other. Biofilms can enhance the tolerance of bacteria to harsh environmental conditions, and can increase antibiotic resistance ,35. In oeriaceae , a growiprofiles ,12. Givelization . Thus, tblaNDM-5-harboring p3R-IncX3 plasmid among a wide range of wild-type Enterobacteriaceae. The increased resistance to \u03b2-lactam antibiotics and enhanced biofilm formation ability as a result of plasmid acquisition may reduce drug efficacy. Further, transfer of this plasmid to highly virulent strains will increase their pathogenicity and multidrug resistance, with the likely consequence of increased treatment failure rates. The low fitness cost of the plasmid further indicated that p3R-IncX3 plasmids may adapt to an expanding range of hosts, thereby disseminating blaNDM genes among a large number of strains. These findings highlight the need to recognize the importance of IncX3 plasmids and take measures to control their spread.This is the first study to confirm high conjugative transfer rates of a"} +{"text": "Panopea generosa, to repeated exposures of elevated pCO2 in a commercial hatchery setting followed by a period in ambient common garden. Respiration rate and shell length were measured for juvenile geoduck periodically throughout short-term repeated reciprocal exposure periods in ambient (~550\u00a0\u03bcatm) or elevated (~2400\u00a0\u03bcatm) pCO2 treatments and in common, ambient conditions, 5\u00a0months after exposure. Short-term exposure periods comprised an initial 10-day exposure followed by 14\u00a0days in ambient before a secondary 6-day reciprocal exposure. The initial exposure to elevated pCO2 significantly reduced respiration rate by 25% relative to ambient conditions, but no effect on shell growth was detected. Following 14\u00a0days in common garden, ambient conditions, reciprocal exposure to elevated or ambient pCO2 did not alter juvenile respiration rates, indicating ability for metabolic recovery under subsequent conditions. Shell growth was negatively affected during the reciprocal treatment in both exposure histories; however, clams exposed to the initial elevated pCO2 showed compensatory growth with 5.8% greater shell length (on average between the two secondary exposures) after 5\u00a0months in ambient conditions. Additionally, clams exposed to the secondary elevated pCO2 showed 52.4% increase in respiration rate after 5\u00a0months in ambient conditions. Early exposure to low pH appears to trigger carryover effects suggesting bioenergetic re-allocation facilitates growth compensation. Life stage-specific exposures to stress can determine when it may be especially detrimental, or advantageous, to apply stress conditioning for commercial production of this long-lived burrowing clam.While acute stressors can be detrimental, environmental stress conditioning can improve performance. To test the hypothesis that physiological status is altered by stress conditioning, we subjected juvenile Pacific geoduck, During typical hatchery practice, geoduck are reared from \u2018setters\u2019 to \u2018seed\u2019 in either downwellers or stacked trays; juveniles are then planted in situ to grow for several years until market size. Following aquaculture practice, trays were filled with a 5-mm depth of rinsed sand (35\u201345\u00a0\u03bcm grain size) that allowed juvenile geoduck to burrow and siphons could clearly be seen extended above the sediment throughout the experiments. To enable measurements of metabolic activity and shell growth, 30 geoduck were placed in an open circular dish (6.5\u00a0cm diameter and 3\u00a0cm height) with equal mesh size and sand depth submerged in each tray, the remaining 50 geoduck in each tray burrowed in the surrounding sediment. Seawater at the Jamestown Point Whitney Shellfish Hatchery was pumped from offshore (100\u00a0m) in Quilcene Bay , bag-filtered (5\u00a0\u03bcm) and UV sterilized before fed to 250-L conical tanks at rate of 1\u00a0L\u00a0min\u22121. Four conical tanks were used as replicates for two treatments: elevated pCO2 level of ~\u20092300\u20132500\u00a0\u03bcatm and ~\u20097.3 pH and ambient hatchery conditions of ~\u2009500\u2013600\u00a0\u03bcatm and ~\u20097.8\u20137.9 pH . The elevated pCO2 level was set with a pH-stat system . The experiment began with an initial exposure period of 10\u00a0days under elevated pCO2 (2345\u00a0\u03bcatm) and ambient treatments before secondary exposure for 6\u00a0days to reciprocal treatments of elevated pCO2 (2552\u00a0\u03bcatm) and ambient treatments throughout the 30-day experiment with a programmable dosing pump (Jebao DP-4 auto dosing pump). Large algae batch cultures were counted daily via bright-field image-based analysis \u00a0\u00f7\u00a0(7\u2009\u00d7\u2009W\u2009\u00d7\u2009C); this equation accounts for a feed ration of 0.4\u00a0mg dried algae mg live animal weight\u22121\u00a0week\u22121, the live animal weight (mg) of spat and cell concentration of the culture (cells\u00a0\u03bcl\u22121) to calculate the total volume (V) of each species in a mixed-algae diet. Tray flow rates and food delivery were measured and adjusted daily.Juvenile geoduck were fed semi-continuously with a mixed algae diet (30% \u22121). Stacked trays, commonly used for incubation of finfish, present a promising innovation for geoduck aquaculture; the experiment stack occurred alongside prototype stacked growing trays stocked by Jamestown Point Whitney Shellfish. The juveniles were fed cultured algae ad libitum daily for 157\u00a0days before shell length and respiration rates were measured.All geoduck survived the exposure periods. Half of the remaining juveniles burrowed in each tray were maintained at the hatchery, positioned in the same replicate trays and stacked for continuous and high flow of ambient seawater , each filled with 0.2\u00a0\u03bcm filtered seawater from corresponding trays. Three blank vials per tray, filled only with 0.2\u00a0\u03bcm filtered seawater, were used to account for potential microbial oxygen consumption. Respiratory runs occurred within an incubator at 15\u00b0C, with the vials and sensor placed on a rotator for mixing. Each set of measurements lasted ~\u200930\u00a0min, and trials ceased when oxygen concentration declined ~\u200970\u201380% saturation to avoid hypoxic stress and isolate the effect of pCO2 treatment on respiration rate. Siphons were observed pre and post-respirometry and were fully extended (~1\u20132 times shell length). Geoduck were subsequently photographed, and shell length was measured using ImageJ with a size standard (1\u00a0mm stage micrometer).Juvenile geoduck were measured on Days 2, 5, 8 and 10 of initial exposure, Days 0, 2, 4 and 6 of secondary exposure and 157\u00a0days after the exposure period (cumulatively as Day 187) to assess rates of oxygen consumption normalized to shell length. Calibrated optical sensor vials were used to measure oxygen consumption in 4\u00a0ml vials on a 24-well plate sensor system (Presens SDR SensorDish). Juveniles in each treatment dish were divided into three sensor vials and observations was run individually for each respirometry measurement over the full 30-min record as a \u2018reference\u2019 dataset. These are considered to be the most robust parameters as suggested by the R package authors . To determine the optimal set of parameters, respiration data was calculated using three alpha values and data truncations and each was compared to the initial reference dataset with two curve fitting steps to calculate unbiased and reproducible rates of oxygen consumption similar to the reference . Final metabolic rates of juvenile geoduck were corrected for vial volume, rates of oxygen change in the blank vials, and standardized by mean shell length (\u03bcg O2\u00a0h\u22121\u00a0mm\u22121).Rates of respiration (oxygen consumption) were calculated from repeated local linear regressions using the R package LoLinR (\u22121 seawater) water samples were collected from trays once daily during treatment periods, in combination with measurements of pH by handheld probe , salinity and temperature . Seawater chemistry was measured for three consecutive days during the 14\u00a0days of ambient common garden between initial and secondary treatment periods. Quality control for pH data was assessed daily with Tris standard (Dickson Lab Tris Standard Batch T27) and handheld conductivity probes used for discrete measurements were calibrated every 3\u00a0days. TA was measured using an open cell titration and TA measurements identified <\u20091% error when compared against certified reference materials (Dickson Lab CO2 CRM Batches 137 and 168). Seawater chemistry was completed following Guide to Best Practices and time\u00d7pCO2 interaction for respiration and shell length during initial exposure. A t test was used to test the effect of initial pCO2 treatment on respiration rate and shell length prior to the secondary exposure . For the secondary exposure period, a three-way ANOVA was used to test the effects of time (fixed), initial pCO2 treatment (fixed), secondary pCO2 treatment (fixed) and their interactions on respiration rate and shell length. No significant differences in seawater chemistry were detected between trays of the same treatment ; thus, tray effects were assumed negligible. Significant model effects were followed with pairwise comparisons with a Tukey\u2019s a posteriori HSD. We used a two-way ANOVA to analyze the effects of initial (fixed) and secondary (fixed) pCO2 treatments on respiration and shell length after 157\u00a0days in ambient conditions. In all cases, model residuals were tested for normality assumptions with visual inspection of diagnostic plots was used to analyze the effect of time (fixed), 2\u00a0h\u22121\u00a0mm\u22121 (mean\u2009\u00b1\u2009SD). Elevated pCO2 had a significant effect on respiration rate over the initial 10-day exposure with a 25% reduction in respiration rate in elevated pCO2 treatment relative to ambient with a 3.6% increase in shell length between Days 2 and 10 prior to exposure was 0.29\u2009\u00b1\u20090.16\u00a0\u03bcg O ambient . Juvenil2 and 10 , but thel length . Signifiexposure . In contn garden .pCO2 treatments nor between treatments and time on respiration rate or shell length with a 31% increase in average respiration rate between Days 2 and 6. Initial pCO2 treatment had a significant effect on shell length, with on average a ~\u20094% reduction in shell size under high pCO2 relative to ambient initial exposure . This same trend was present under the secondary high pCO2 exposure, with 3.20% smaller shells for individuals exposed to elevated pCO2 treatments. There were pairwise differences in shell size between animals only exposed to ambient and animals repeatedly exposed to elevated pCO2 .There was no interaction between initial and secondary l length . There wpCO2 treatments on respiration rate or shell length , where average shell lengths were 5.8% larger in juveniles exposed to initial elevated pCO2. Secondary 6-day exposure had a significant effect on respiration rates after 157\u00a0days in ambient common garden with an average of 52.4% greater respiration rates in juveniles secondarily exposed to elevated pCO2. Visual examination during screening indicated low mortality (1\u20134\u00a0tray\u22121) over the ~\u20095-month grow-out period. Shell lengths of dead animals (as empty shells) were similar to the size of juvenile geoduck during the 30-day exposure period suggesting low mortality occurred at the start of the grow-out period possibly due to handling stress.There was no interaction between initial and secondary l length . The iniP. generosa present a novel application of hormetic framework for resilience of a mollusc to acidification. To date, within-generation carryover effects remain poorly understood for marine molluscs are planned to determine the threshold between low-dose stimulation and high-dose inhibition from stress conditioning.Juvenile geoduck repeatedly exposed to elevated generosa and prompCO2 , overlaid with the standard timeline for geoduck industry, does not present additional expenses. Further related tests on stress conditioning and production of resilient strains (i.e. phenotypes and/or epigenotypes) must account for distinct life-stages and species-specific attributes in aquaculture practice.Our findings infer both positive and negative implications for aquaculture. Although advantageous to elicit carryover effects exhibited by stress-conditioned animals, results imply greater feed (ingestion rate) to sustain enhanced aerobic metabolism and compensatory shell growth; this can heighten labour and financial costs for industry, likely not incentivized by a marginal 5.8% increase in shell size. However, typical protocols for geoduck aquaculture yield 5-month-old juvenile clams in the hatchery before grown in situ. As conditions in coastal bays report deteriorating water quality optimizes establishment of resilient phenotypes and genotypes during hatchery-rearing? (iii) does stress history under elevated pCO2 affect the stability and longevity of carryover effects later in life? Answers to these challenges will result in effective implementation of conditioning to both reduce pressure on wild stocks and sustain food security under environmental change.Shellfish farming has adapted in recent years to implement \u2018climate-proofing\u2019 technology to maintain production and combat both coastal and climate-related stressors , pre-burrowing time (time elapsed to anchor into substrate and obtain upright position), and burrowing depth are directly related to growth and survival with physiological responses throughout reproductive and offspring development under environmental stress (Data in this present study provides evidence of capacity to cope with short-term acidification for an understudied infaunal clam of high economic importance. Survival of all individuals over the 30-day experiment demonstrates the resilience of this species to low pH and reduced carbonate saturation. Juvenile geoduck exposed to low pH for 10\u00a0days recovered from metabolic depression under subsequent stress exposure and conditioned animals showed a significant increase in both shell length and metabolic rate compared to controls after 5\u00a0months under ambient conditions, suggesting stress \u2018memory\u2019 and compensatory growth as possible indicators of enhanced performance from intragenerational stress conditioning. Our focus on industry enhancement must expand to test developmental morphology, physiology, and genetic and non-genetic markers over larval and juvenile stages in a multi-generational experiment to generate a more holistic assessment of stress hardening and the effects of exposure on cellular stress response for advaS.J.G., B.V., S.B.R. and H.M.P. designed the experiments, S.J.G. conducted the experiments, S.J.G., B.V., S.B.R. and H.M.P. drafted, revised, read and approved the final version of the manuscript.This work was funded in part through a grant from the Foundation for Food and Agriculture research; Grant ID: 554012, Development of Environmental Conditioning Practices to Decrease Impacts of Climate Change on Shellfish Aquaculture. The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of the Foundation for Food and Agriculture Research.Supplementary_Figure_1_coaa024Click here for additional data file."} +{"text": "Hangover resistance may be linked to an increased risk of continuing harmful drinking behaviours as well as involvement in potentially dangerous daily activities such as driving while hungover, mainly due to the absence of negative consequences the day after alcohol consumption. The aim of this study was to examine the occurrence of claimed alcohol hangover resistance relative to estimated blood alcohol concentration (eBAC). A total of 1198 participants completed an online survey by answering questions regarding their demographics, alcohol consumption and occurrence of hangover. Two methods were used to calculate eBAC, one based on the modified Widmark Equation (N = 955) and the other from an equation averaging the total body water (TBW) estimates of Forrest, Watson, Seidl, Widmark and Ulrich (N = 942). The percentage of participants who claimed to be hangover resistant decreased rapidly with increasing eBAC and only a small number of hangover resistant drinkers remained at higher eBACs. Comparisons of the eBACs calculated by the two methods revealed significantly higher BACs when using the modified Widmark equation. These findings suggest that additional research for eBAC calculations is needed to improve accuracy and comprehensiveness of these equations for future alcohol hangover research. Alcohol hangovers are often the result of a night of heavy drinking and are a familiar phenomenon worldwide. The alcohol hangover refers to the combination of negative mental and physical symptoms, which can be experienced after a single episode of alcohol consumption, starting when blood alcohol concentration (BAC) approaches zero . HistoriA hangover can result in impairment in both cognitive and psychomotor functioning . This poBecause of the substantial impact of alcohol hangovers on health, economy, and society, it is important to determine the causes of alcohol hangover as well as factors that contribute to symptom severity. Recent research showed that hangover-resistant drinkers may be at increased risk of continuing harmful drinking behaviours as well as involvement in potentially dangerous daily activities, such as driving, mainly because of absence of negative consequences the day after drinking ,17. LoweIdentifying how hangover-resistant drinkers differ from non-hangover resistant drinkers may help to unfold the pathology of alcohol hangovers. Thus far, several theories exist as to why hangovers occur and what factors might influence severity, but this phenomenon remains largely unclear . PreviouQuantity of alcohol consumed directly impacts the severity of hangovers and associated impairments . As suchThe most widely used eBAC calculation was developed by Widmark ,27. ThisThis equation has been modified as research has shown that the original Widmark formula had a tendency to overestimate BAC . SpecifiHowever, the use of eBAC has limitations when applied to real world drinking experiences. It is likely to introduce more variability of BAC and the accuracy of eBAC equations in naturalistic settings is relatively unknown . AdditioApproximately 20\u201325% of alcohol consumers are classed as \u2018hangover resistant\u2019, in that they report no hangover symptoms after a night of heavy drinking . PreviouAnother factor that needs to be considered when investigating hangover resistance is the definition that is being used. A Canadian study used two definitions to describe hangover resistance. The first definition stated \u2018Never having had a hangover during one\u2019s lifetime\u2019 and the second definition stated \u2018Never having had a hangover during a certain time period \u2019 . A recenDue to the frequent use of eBAC in alcohol hangover research, it is vital to explore accuracy, comprehensiveness, and possible improvements for currently used formulas. This study aimed to assess whether the previously discussed calculations differ and explores the use of an additional and more comprehensive eBAC evaluation when assessing hangover resistance to explore similarities and differences in eBAC outcomes within an Australian population when compared with a previously used calculation in international research. This additional and more comprehensive method calculates eBAC by averaging the total body water (TBW) estimates of Forrest , Watson in hours .In line with previous research, it was predicted that around 50% of the participants would have relatively low eBACs . It was The study was approved by the Swinburne University Human Research Ethics Committee (Reference 2012/045) and was conducted in accordance with the Declaration of Helsinki.This was an online survey assessing alcohol consumption behaviours and hangover resistance occurrence amongst an Australian population with the use of a commonly used method to calculate eBAC as well as an additional and more comprehensive calculation for eBAC.Overall, 1748 respondents opened the online anonymous questionnaire. Data provided by non-drinkers were removed from the dataset. In total, 1198 participants completed the online survey , mostly consisting of students (65.3%). The mean age of the participants was 23.10 years old .Participants answered questions regarding demographics , use of medications, tobacco, and illicit drugs.Participants were questioned on their alcohol intake . Alcohol consumption was defined using standardized Australian alcohol units . The consumption questions assessed frequency and quantity of alcohol consumed across various timescales for each type of drink . Specifically, participants were asked the number of standard drinks and the number of hours that they had spent drinking.Responses to alcohol consumption questions regarding the heaviest drinking session within the previous 30 days were used to calculate eBAC with two different methods. Both methods computed the eBAC separately for males and females. Method 1 is the most frequently used method in previous research and is a modified Widmark equation . The modTwo groups were created, depending on the answer to the question: \u201cI have had a hangover the morning after I had been drinking\u201d over the past year. If the answer was \u2018yes\u2019, the participant was assigned to the hangover sensitive group. If the answer was \u2018no\u2019, the participant was assigned to the hangover resistant group.Participants were recruited via word of mouth, flyers, and advertisements on social media. Participation was voluntary and anonymous. Participants provided informed consent by agreeing to the survey terms online, after which they completed the online questionnaire. On completion of the survey, participants could choose to go into the draw to win one of two iPads by entering their email address.p < 0.05.Data were collected online using SurveyMonkey and analysed using the Statistical Package for the Social Sciences version 25 . Initially, the data were screened for any participants who did not meet the criteria, i.e., surveys completed between 2:00 a.m. and 7:00 a.m., and surveys submitted from the same IP address. Finally, any participant who answered \u2018no\u2019 to the final question, whether they had honestly and correctly answered all questions, were excluded. The mean, standard deviation and frequency distributions were calculated for the hangover sensitive group and the hangover resistant group. Comparisons between the two methods to calculate eBAC were made using nonparametric tests, since the eBAC data were skewed. Pearson\u2019s Chi square test was used to measure associations between the different eBAC levels of the two methods and hangover resistance. Effects were regarded as statistically significant if Two methods were used to calculate eBAC. First, the most commonly used method in previous alcohol hangover research was used and described, Method 1 ,27. SecoParticipants aged 36 years and older were excluded, since this question was assessed in a multiple-choice format and the exact age was needed in the eBAC calculation. This resulted in the removal of 100 participants, leaving 1098 participants. Participants with an estimated eBAC of 0.00% were excluded in both methods used because this suggests either no or extremely low alcohol consumption. Participants with an estimated eBAC of 0.40% and higher were also excluded in both methods, as this is an unreliably high estimation which suggests overestimation of the alcohol consumption and thus eBAC. This resulted in 20.3% of participant exclusion in Method 1 (leaving 955/1198) and 21.4% in Method 2 (leaving 942/1198). A general description of the typical drinking pattern is provided in Of N = 955 remaining participants, 196 (20.5%) reported no hangover the day after drinking while 759 (79.5%) reported experiencing a hangover.Consistent with previous research , particiThe data show that 39.9 of drinkers fell within an eBAC below 0.08%. When considering only the subset of drinkers with eBACs above 0.08% the proportion of this subset who claimed hangover resistance was 11.5% (65/567). When the smaller subset of drinkers with eBACs above 0.20% was considered, the prevalence of hangover resistance was 12.5% (21/168), respectively.Of N = 942 remaining participants, 19.9% reported no hangover symptoms the day after drinking (N = 187) and 755 (80.1%) reported experiencing hangover symptoms. The participants were divided into seven groups to examine the occurrence of hangover resistance the day after drinking within different eBAC ranges, which are shown in The data shows that 49.4% of drinkers fell within an eBAC below 0.08%. When considering only the subset of drinkers with eBACs above 0.08% the proportion of this subset who claimed no next day adverse effects after drinking was 10.7% (51/476). When the smaller subset of drinkers with eBACs above 0.20% was considered, the prevalence of no adverse effects the day after drinking was 14.5% (16/110), respectively.z = \u221211.07, p < 0.001.eBAC data was skewed for both eBAC methods, since most people are distributed along the lower eBAC ranges. Therefore, a Wilcoxon signed-rank test, which evaluated the difference between medians for eBAC Method 1 and eBAC Method 2 was conducted. The results indicated that eBAC levels of Method 1 were statistically significantly higher than eBAC levels of Method 2, A chi-square test of independence showed that there were no significant differences in reported hangover resistance in the two eBAC methods and across the 7 BAC groups . ResultsThe current study assessed the occurrence of hangover resistance amongst an Australian population and compared two eBAC equations. Several of our hypotheses were supported. Both eBAC methods showed that almost half of the drinkers were distributed at eBAC levels below 0.08% (Method 1: 39.9% and Method 2: 49.4%), which supports the findings of previous research ,38. PrevThe present study confirms that the percentage of people who claim to be hangover resistant decreases with increasing eBACs, independent of eBAC method. However, contrary to previous research, a slight increase of hangover resistance claims occurred above 0.20% in the present study for both eBAC methods. This is likely a chance fluctuation due to smaller numbers of participants claiming hangover resistance at higher eBACs ,37,40.When comparing the two methods to calculate eBAC, several similarities were found. Both methods were able to show a decrease in hangover resistance prevalence with higher eBACs. Both methods showed that between 22\u201325% of the drinkers reported no hangover, however almost half of these drinkers were distributed at eBAC levels below 0.08%, which is a threshold to determine impaired driving in several countries .The differences between the two methods are found in the mean eBACs, with a significantly higher mean eBAC for Method 1 than for Method 2. Interestingly, hangover resistance prevalence in several eBAC increments was observed to be slightly, but not significantly, lower with the use of Method 2. An explanation for this could possibly be found in the notion that previous research found that Method 1 (based on a modified Widmark formula) ,27 mightDifferences found between the two eBAC methods used in this study suggest that additional research exploring the accuracy and comprehensiveness of eBAC equations is needed for future hangover research. As illustrated earlier, previous alcohol hangover research most commonly uses the modified Widmark calculations for BAC ,31,32,33Limitations of this study include relying on self-reported alcohol intake and eBAC calculations to estimate BAC obtained during the drinking episode. Heavy drinking often results in memory impairment, which may lead to less accuracy and recall-bias . This liObjective measures of ethanol assessments are absent in this study, which could pose the question whether the calculations for both BAC methods are accurate. It is unclear whether similar results would be found in controlled settings based on these study findings. However, the main purpose of this study was to assess whether the two methods to calculate eBAC differ rather than assess the accuracy of the two methods. An objective measure of ethanol assessment, such as blood and saliva, should be utilized for future research purposes. A controlled setting would allow equal quantity of alcohol consumption in both groups of drinkers. This ensures valid comparison of hangover-resistant and non-hangover-resistant drinkers in a controlled setting, which could then be translated to naturalistic designs. Including measures on hangover frequency and severity in future research will enhance our understanding of differences between hangover-resistant and non-hangover resistant drinkers.The findings presented here show that almost half of drinkers had an eBAC below 0.08%, irrespective of applying Method 1 or Method 2 to calculate estimated BAC. The present results support previous findings and show that the percentage of people who claim to be hangover resistant decreases rapidly with increasing eBACs. However, a small number of hangover resistant drinkers persists, independently of higher eBACs. Differences between the two eBAC methods are found in the mean eBACs, with a significantly higher mean eBAC for Method 1. Future research should assess influences of eBAC on hangover resistance prevalence with the use of an objective and more extensive assessment of hangover (severity) and should further explore research on eBAC calculations to improve their accuracy and comprehensiveness for future research. Notwithstanding the absence of significant findings, this study highlights the importance of continuing assessment of the methodology used to assess hangovers to increase knowledge of alcohol hangover resistance and pathology."} +{"text": "ESM1, GUCA2A, and VWA2 for males and CLDN1 and FUT1 for females.Colorectal cancer (CRC) is the third leading cause of cancer deaths. Advances within bioinformatics, such as machine learning, can improve biomarker discovery and ultimately improve CRC survival rates. There are clear sex differences in CRC characteristics, but the impact of sex has not been considered with regards to CRC biomarkers. Our aim here was to investigate sex differences in the transcriptome of a normal colon and CRC, and between paired normal and tumor tissue. Next, we attempted to identify CRC diagnostic and prognostic biomarkers and investigate if they are sex-specific. We collected paired normal and tumor tissue, performed RNA-seq, and applied feature selection in combination with machine learning to identify the top CRC diagnostic biomarkers. We used The Cancer Genome Atlas (TCGA) data to identify sex-specific CRC diagnostic biomarkers and performed an overall survival analysis to identify sex-specific prognostic biomarkers. We found transcriptomic sex differences in both the normal colon tissue and in CRC. Forty-four of the top-ranked biomarkers were sex-specific and 20 biomarkers showed a sex-specific prognostic value. Our data show the importance of sex in the discovery of CRC biomarkers. We propose 20 sex-specific CRC prognostic biomarkers, including Colorectal cancer (CRC) is the third leading cause of cancer deaths among both women and men in the US . In SwedIdentification of biomarkers, which can improve the diagnosis and disease monitoring, could significantly improve the survival rates. The advances in bioinformatics tools provide opportunities to speed up biomarker discovery and have been integrated for several cancers, including CRC ,7. TransBRAF) mutations and a higher microsatellite instability (MSI) status compared to men, whereas men have a higher number of NRAS proto-oncogene, GTPase (NRAS) mutations [Sex-specific CRC recurrence and survival rates have been reported . The incutations . Recentlutations . Some ofutations ,14,15.ESM1, guanylate cyclase activator 2A/GUCA2A, claudin 1/CLDN1) and novel ones (fucosyltransferase 1/FUT1 and von Willebrand factor A domain containing 2/VWA2).Despite the sex differences seen in CRC, most research is done without considering sex in study designs or interpretations. Sex-specific strategies for screening, prevention, and treatment should be considered in order to reduce CRC mortality. In the present study, we evaluated sex differences in the transcriptome of both non-tumor colon epithelium and CRC. Additionally, we studied sex differences in relation to diagnostic and prognostic biomarkers. Our study highlights sex differences in the normal colon, related to bile acid secretion, vitamin digestion and absorption, and in the tumor, especially related to immune response. Moreover, our study shows the importance of sex in the discovery of prognostic biomarkers. We identified 20 sex-specific prognostic biomarkers, including previously proposed biomarkers > 1), respectively (CPS1), remained differentially expressed between the sexes in both conditions and prognostic biomarker cell migration inducing hyaluronidase 1 (CEMIP) ,17. We v by qPCR . Next, w by qPCR . Next, wectively A. The maectively B. Interenditions A. Biolognditions C. The senditions C. Thus, Since it is well known that the sexes present differences in tumor location and characteristics ,11,12, wNext, we compared alterations between the normal colon and CRC transcriptomes for each patient using pairwise comparisons and investigated if the sexes showed different profiles. In females, 7156 genes were differentially expressed between the paired normal colon and tumor, whereas 2611 genes were differentially expressed in males A. Nearlyn = 18 for females and n = 6 for males). In order to exclude the effect of the imbalanced data we performed differential expression analysis on six randomly selected female tumor samples (from subtype 3 and 4) and matched normal samples in three individual runs overlapped with the female-specific tumor expression in the unbalanced data . With the selected cutoff, 54, 46, and 19 of the features that passed the feature selection were differentially expressed for the female, male, and Swedish mixed cohorts, respectively , minimum redundancy\u2014maximum relevance (MRMR), and Boruta algorithm A. Due toectively . The PCAectively C. The biectively D. In addectively D.CDH3) and ESM1 were ranked as the top biomarkers and were both upregulated in the tumors (THBS2)) of the upregulated Swedish mixed cohort from the previous analysis remained. Eighteen biomarkers were found in all three cohorts , matrix metallopeptidase 7 (MMP7), and collagen type XI alpha 1 chain (COL11A1) were identified as biomarkers in all cohorts, and are all secreted. Furthermore, cystatin SN (CST1) detected in female TCGA data, transcobalamin 1 (TCN1) detected in the Swedish mixed cohort, and palmitoleoyl-protein carboxylesterase (NOTUM) detected in female and male TCGA data are also secreted and thus of potential interest as screening biomarkers.Our data demonstrate that there are both common and sex-specific biomarkers. In order to evaluate if the best biomarkers are common or sex-specific, we performed machine-learning techniques to rank the features according to importance A. Randome tumors C,D. In ae tumors A. In orde tumors A. Reassu cohorts A. Twenty cohorts B,D and s cohorts C,D. FurtESM1, an early biomarker and strong top candidate in all cohorts, showed a prognostic value when combining the sexes and had a clear unfavorable prognostic value specifically in males and kinesin family member 26B (KIF26B) were also significant when the sexes were combined, and showed a non-significant trend in the other sex , FUT1, and four-jointed box kinase 1 (FJX1) presented a sex-specific prognostic value, they presented a significant prognostic value when the sexes were combined and a non-significant trend in the other sex (data not shown). Overall, our data show that females and males indeed presented a number of sex-specific top biomarkers. Even more striking is that the prognostic value of the biomarkers was highly dependent on sex, with 20 biomarkers showing a sex-specific prognostic value. This suggests that some of the diagnostic biomarkers can have a profound impact on predicting CRC prognosis when sex is taken into account, and our results indicate that sex is an important factor when evaluating CRC biomarkers.Interestingly, although some of the top biomarkers were common in both sexes, the prognostic value of these could be sex-specific, and vice versa. We performed OS analysis with Kaplan\u2013Meier plots and found that in males . CLDN1, ifically . Furtherifically . Worth nther sex . Additioc values . AlthougOur objective with this study was to evaluate if there are sex differences in the gene expression of a normal colon and CRC, and whether separating the sexes can improve the diagnostic and prognostic CRC biomarkers. Several studies have shown that there are sex differences in CRC, regarding incidence and mortality, tumor location, and mutation status ,10,11,12Our findings revealed significant sex differences, which, if incorporated into biomarker discovery and the clinic, could impact CRC patient outcome. First, we demonstrated sex differences in the normal colon, especially among pathways related to gluconeogenesis, bile secretion, and carbohydrate, vitamin, and lipid metabolism, all known to be dysregulated in CRC. The sex differences in the normal colon might shape the tumor characteristics and microenvironment. This can help explain the differences in male and female incidences of CRC. Estrogen menopausal hormone therapy has indeed been shown to correlate to a lower CRC incidence ,22,23. ACLDN1, CEMIP, keratin 80/KRT80, CDH3, and ESM1) were ranked as top features in our paired cohort. This further validates the results in a study published by Long et al., who found CLDN1, CEMIP, and CDH3 amongst the most important features and potential diagnostic biomarkers [CLDN1 has potential as an unfavorable prognostic biomarker specifically in females. CLDN1 has previously been proposed both as a marker for CRC prognosis and as a therapeutic target [ESM1 showed an unfavorable prognostic value in males. ESM1 regulates CRC cell growth and metastasis by activation of NF\u03baB and has been shown to be of prognostic value for disease recurrence, and to correlate with a worse survival outcome [FTSJ1, CST1, and glutamate ionotropic receptor NMDA type subunit 2D/GRIN2D) and male-specific , and cyclin P/CCNP/CNTD2) top-ranked features, based on the TCGA data. Of note, CST1 and NOTUM are secreted and can be potential sex-specific diagnostic markers.The sex-independent potential diagnostic biomarkers , presented an unfavorable prognostic value specifically in males. FJX1 has also been shown to be involved in angiogenesis and associated with an unfavorable prognosis of CRC [GUCA2A was downregulated in CRC in both sexes and showed a favorable prognostic value in males. GUCA2A mRNA and protein loss is among the most common gene losses in CRC, occurring in more than 85% of tumors [S100A2) was associated with an unfavorable prognostic value specifically in males. S100A2 has been shown to reprogram glycolysis and induce proliferation in CRC, and suggested as a therapeutic target [Moreover, s of CRC . The comf tumors , and hasf tumors . GUCA2A f tumors . Ligand f tumors . Such inc target . High exc target .CLDN1, CEMIP, and CDH3 and propose new potential biomarkers. Interestingly, we did not find a single significant biomarker showing a prognostic value independent of sex, while we identified 20 diagnostic features with a sex-specific prognostic value, in particular, ESM1, GUCA2A, FJX1, and S100A2 for males and CLDN1 for females. Importantly, our study highlights the need to take sex into account in CRC research, which may improve CRC mortality.Overall, in this study, we identified sex differences in the normal transcriptome, which may explain the sex differences in CRC susceptibility. Furthermore, we validated the previously proposed sex-independent diagnostic biomarkers n = 24, 18 women and 6 men) undergoing surgery in Stockholm after informed consent. The study was approved by the regional ethical review board in Stockholm (2016/957-31 and 2017/742-32). In addition, gene expression for 641 (299 women and 342 men) colorectal cancer (CRC) and 51 (28 women and 23 men) noncancerous mucosal tissues were downloaded from TCGA. The COAD and READ data were combined, the data were downloaded on 31st of January 2019, and the bioconductor package from R (Rversion 3.6.1) via the NCI Genomic Data Commons (GDC) data portal was used (TCGAbiolinks version 3.8). The molecular subtypes were determined on the Swedish cohort based on the status of the MSI, BRAF-, and KRAS mutations. The MSI status was determined using MSIsensor [BRAF- and KRAS mutation status was analyzed using the integrative genomics viewer [Clinical samples were collected from patients (SIsensor and the n 2.5.2) . A detai"} +{"text": "We noticed an error in figure 5 in our published manuscript"} +{"text": "We noticed an error in Figure"} +{"text": "The primary outcome was the comparison of the \u201cPI ratio 10,\u201d which was defined as the ratio of the PI 10 to the baseline. Receiver operating characteristic (ROC) curves were constructed to determine the accuracy of the PI in predicting the block success at each time interval. The PI ratio 10 was 2.7 (1.9\u20134.0) in non-epinephrine group and 3.3 (2.2\u20134.4) in epinephrine group . The ROC curves compared without group identification were not significantly different over time. The cut-off value for the PI and PI ratio at 5\u00a0min (PI ratio 5) were 7.7 and 1.6 , respectively. The perineural epinephrine did not affect the PI following a SCBPB. The PI ratio 5\u2009>\u20091.6 might be considered as a relatively accurate predictor of a successful SCBPB.The perfusion index (PI) is an objective tool used to assess a successful nerve block. Epinephrine is a widely used adjuvant to local anesthetics, and it may affect PI values because of the vasoconstrictive property. The aim of this study was to investigate the influence of epinephrine on PI as an indicator of a successful block in ultrasound-guided supraclavicular brachial plexus block (SCBPB). In this randomized controlled trial, 82 adult patients underwent upper limb surgery under SCBPB were recruited between July 2018 and March 2019 in a single tertiary care center. Participants were randomly assigned to one of two groups: non-epinephrine group (https://cris.nih.go.kr. CriS No. KCT0003006).Trial registration: This study was registered at the Clinical Trial Registry of Korea ( Among these, the perfusion index (PI) is free of many factors associated with subjective interpretation and can be easily applied to most patients.The evaluation of peripheral nerve block success is typically assessed using sensory and motor blocks. Recently, several objective methods for the evaluation of block success have been developed to replace conventional methods with inherent limitations 7. It is affected by changes in intravascular volume, elasticity, and intravascular pulse pressure. Therefore, it corresponds to changes in blood volume and varies in values depending on the distensibility of vascular walls and pulse pressure7. The blockade of sympathetic nerve fibers after successful nerve block results in increased local blood flow and vasodilation, eventually increasing the PI8. Several studies have investigated the utility of the PI for assessing regional anesthesia success and reported that the PI might be used to determine the success of peripheral nerve blocks9. However, there is a lack of research to evaluate the usefulness of the PI depending on whether some adjuvants are added to the local anesthetics (LAs). Since the PI is affected by the blood flow in the blood vessels, its value can vary depending on the adjuvants of LAs that affect the condition of blood vessels.The PI is a noninvasive tool for assessing the ratio between pulsatile and non-pulsatile blood flow using a pulse oximeter, and it can also measure sympathetic stimulation or peripheral perfusion with a sensor attached to a finger10. Therefore, epinephrine could lead to vasoconstriction and alter PI values depending on the extent of vasodilation11. As a result, the aim of this study was to assess the effect of epinephrine as an adjuvant to local anesthetics on the PI values and also as an indicator of successful supraclavicular brachial plexus block (SCBPB).Epinephrine is a widely used adjuvant to LAs to detect unintended anesthetic injections, prolong the duration of sensory blockade in regional anesthesia, and delay the absorption of LAs by inducing vasoconstriction at the injection site\u22121 epinephrine in the epinephrine group. Participants were enrolled by one of the authors (D.K.) and all study drugs were prepared by one of the authors (J.S.J.) who was not involved in either the SCBPB or the outcome assessment. The unilateral upper limb orthopedic surgery was performed by one surgeon (M.J.P.) who was blinded to the allocation groups and clinical patient data were masked until completion of this study.A statistician who was unrelated to this study generated a random allocation sequence. The participants were randomly assigned to one of two groups using a computer-generated randomization sequence and a 1:1 ratio with sealed, opaque envelope techniques: a non-epinephrine group and an epinephrine group. Study drugs were mixed with 12.5\u00a0ml of 2% lidocaine, 12.5\u00a0ml of 0.75% ropivacaine, and 0.1\u00a0ml of normal saline in the non-epinephrine group or 12.5\u00a0ml of 2% lidocaine, 12.5\u00a0ml of 0.75% ropivacaine, and 5\u00a0mcg\u00a0ml\u00ae, Geisingen, Germany) was inserted following a lateral to medial direction using the in-plane technique. The needle was advanced to the intersection of the first rib and the subclavian artery . After confirming that the aspiration test was negative, 10\u00a0mg of the study drug were injected. Then the needle was repositioned in the neural cluster; once a threshold current less than 0.5\u00a0mA14 was confirmed, the remaining 15.1\u00a0ml of the study drug were injected.On arrival in the block room, standard monitors including electrocardiogram, noninvasive blood pressure (NIBP), heart rate, and pulse oximetry were applied. To avoid influencing any change in blood flow, the NIBP was measured at the ankle; the temperature in the block room was kept constant (25\u00a0\u00b0C) to minimize the impact from the environment. SCBPB was performed by one expert anesthesiologist with extensive prior experience performing regional anesthesia (J.S.K.). Before the SCBPB, 1.0\u20131.5\u00a0mg of midazolam was administered for anxiolysis. Patients were placed in a semi-fowler\u2019s position with the head turned 45\u00b0 to the opposite side of the surgical area. Two percent chlorhexidine and 70% isopropyl alcohol were used for skin sterilization. After skin infiltration with 2\u20133\u00a0ml of 1% lidocaine, a high-frequency linear transducer was used to identify the brachial plexus in the supraclavicular fossa. A 50-mm, 22-gauge insulated nerve-stimulating needle , median (thumb opposition), ulnar (thumb adduction), and musculocutaneous (elbow flexion) with grading on a three-point scale . The block onset time was defined as the time from the completion of the injection of the study drug until a sensory score of 0 was reported, which indicated the complete loss of sensory function in the radial, median, ulnar, and musculocutaneous nerves. Block success was defined as the complete loss of sensory function in the aforementioned nerve distribution within 30\u00a0min after completion of the SCBPB.After the completion of the SCBPB, the degrees of sensory and motor blocks were assessed every 5\u00a0min (min) for a total duration of 30\u00a0min. The sensory block was evaluated in the distributions of the radial, median, ulnar, and musculocutaneous nerve dermatomes with the pinprick test using a blunt-tip needle with a three-point scale \u00ae; Masimo Corporation, Irvine, CA, USA), a type of attaching tape, was used to monitor the PI. Two separate machines were applied to the middle fingers of each hand to measure the PI (in both the blocked and unblocked arms). The PI was recorded at the following time-points: before the SCBPB (baseline) and every 5\u00a0min for 30\u00a0min .A pulse oximeter using a skin thermometer . The same surrounding environment and supine positions were maintained until all parameter measurements were completed to prevent changes in the PI.The primary outcome was the comparison of the \u201cPI ratio 10,\u201d which was defined as the ratio of the PI 10 to the baseline. To correct the high skewness of the baseline PI, we used the PI ratio instead of the PI as the primary outcome.Secondary outcomes were to evaluate the relationship between the complete sensory block and the time of the peak PI, and between the skin temperature of the arm and the PI over time, and to determine the best cutoff value for the PI and PI ratio in the detection of a successful block. The accuracy of the PI to predict the block success was measured at each time interval.n\u2009=\u20095) and 5.5\u2009\u00b1\u20093 in the non-epinephrine group (n\u2009=\u20095). At a power of 0.80 and an alpha error of 0.05, we calculated that 37 patients per group were needed to detect this degree of difference between the two groups. Considering a 10% dropout rate, 82 patients (41 participants for each group) were required to participate in this study.In our preliminary (unpublished) study, the mean\u2009\u00b1\u2009standard deviation (SD) of the PI ratio 10 was 4.0\u2009\u00b1\u20091.1 in the epinephrine group , as appropriate, and categorical variables are presented as numbers with percentages. The continuous variables were explored for normality using the Shapiro\u2013Wilk test and were compared using the Student\u2019s t-test or the Mann\u2013Whitney U test where appropriate. The categorical variables were compared using a chi-square test or Fisher\u2019s exact test as appropriate. Generalized estimating equations were used to compare the PI between the two groups or between the blocked and unblocked arm with post hoc pairwise comparisons using the Bonferroni test. Because there were no failed blocks, a receiver operating characteristic (ROC) and area under the ROC (AUROC) curve using data of the blocked and unblocked arm was constructed to determine the extent of the PI change and the ability of the PI ratio to predict block success at each time interval. Concurrently, the sensitivity, specificity, positive predictive value, and negative predictive value were calculated. A paired t-test was also used to compare the blocked and unblocked arms at each time point. The relationship between the block onset time and the time of the highest value of the PI, and between the temperature of the blocked arm and the PI in each group were analyzed using Spearman\u2019s correlation. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS), version 22.0 . https://cris.nih.go.kr/cris/, KCT0003006, Principal investigator: Justin Sangwook Ko, Date of registration: July, 20th, 2018). Written informed consent was obtained from all participants before enrollment in this study. We enrolled adult patients between 19 and 76\u00a0years of age with American Society of Anesthesiologists Physical Status Classifications from I to III who were scheduled for elective unilateral upper limb orthopedic surgery between July 2018 and March 2019. Exclusion criteria included pregnancy, a body mass index \u2265\u200930\u00a0kg\u00a0m\u22122, preexisting neurological deficits or neuropathy, those taking \u03b1 or \u03b2 blocking agents which can affect vasoconstriction and concentration of epinephrine13, contraindications to peripheral nerve block, coexisting cardiac and pulmonary problems, diabetes mellitus, and complex regional pain syndrome. All methods were performed in accordance with the relevant guidelines and regulations.This single-center, prospective, randomized control trial was approved by the Samsung medical center's Institutional Review Board (IRB # SMC 2018-03-060) and written informed consent was obtained from all subjects participating in the trial. The trial was registered prior to patient enrollment at the Clinical Trial Registry of Korea . There were no differences in the patient characteristics . The median (IQR) of the PI ratio 10 was 2.7 (1.9\u20134.0) in the non-epinephrine group and 3.3 (2.2\u20134.4) in the epinephrine group .There were no differences in the PI and PI ratios between the two groups according to the time interval (ly) Fig.\u00a0. The PI Because the PI and PI ratios were not significantly different between the two groups, the ROC curves were compared without group identification, and they showed no significant differences over time Fig.\u00a0. SubsequP\u2009=\u20090.947; epinephrine group: correlation coefficient\u2009=\u20090.177, P\u2009=\u20090.269, respectively). In addition, the skin temperature and PI were not significantly correlated . The times to reach peak PI, block onset, and peak skin temperature were not statistically difference between the two groups . Regardless of the group, the times to reach peak PI, block onset, and peak skin temperature were 13.8\u2009\u00b1\u20097.7\u00a0min, 14.6\u2009\u00b1\u20095.8\u00a0min, and 16.7\u2009\u00b1\u20097.9\u00a0min, respectively.There was no significant correlation between the time to reach the highest PI value and the time of block onset Table . No partIn this study, perineurally injected epinephrine combined with ropivacaine did not have any influence on either the PI or the PI ratio after the SCBPB in patients undergoing forearm surgeries. However, the PI and PI ratio 5 showed a good ability to predict a successful SCBPB. There was no significant correlation between the times to reach peak PI and complete sensory block, or between the skin temperature and the PI values of the blocked arm.5. However, it is speculated that the use of adjuvants with local anesthetics can change the degree of vasodilation and theoretically affect the PI values. Epinephrine is one of the most popular adjuvants in peripheral nerve blocks and is known to produce vasoconstriction and subsequently alter PI values. The peripheral nervous system receives dual blood supply from blood vessels in the endoneurium and also in the epineurial space16. Of these, the extrinsic blood supply in response to adrenergic stimulation is the action site of epinephrine16 and it results in a marked decrease in blood flow17. However, in our study, the PI and PI ratio showed no difference over time regardless of the administration of epinephrine. Perineurally injected epinephrine is slowly absorbed to alter heart rate and blood pressure, but has no effect on the mean arterial pressure17. Since the mean arterial pressure is important when assessing the relationship between systemic resistance, blood flow, and pressure, this might be one of the reasons for the unaffected PI and PI ratio when epinephrine is injected perineurally.Previous studies have shown that the PI is a useful indicator to predict successful peripheral nerve blocks18. For example, Abdelnasser et al. determined the PI ratio 10 as an indicator of a successful block, and suggested that the cut-off values of the PI and PI ratio 10 were 3.3 and 1.4, respectively4. However, in our study, the PI ratio 5 was determined as an indicator of a successful block, and the cut-off values for the PI and PI ratio 5 were 7.7 and 1.6, respectively. A previous study measured the PI values at relatively wide intervals (10\u00a0min), but we used shorter intervals (5\u00a0min). Although there were no mentions of the PI ratio, previous studies have compared the baseline PI with the PI value after 5\u00a0min and reported its statistical significance21. In addition, it was confirmed that the PI at 5\u00a0min after the SCBPB was the fastest reference point for the change in the PI as a result of AUROC analysis, and the AUROCs were not significantly different across time intervals. Therefore, we recommend the PI ratio 5 as an indicator of successful blocks. PI values may vary depending on the instrument used to measure the PI. However, the PI ratio can be expressed as a constant value because it shows the ratio of the baseline value and each time interval despite the aforementioned difference. In practice, although the cuff-off value of PI was more than two times higher, the cut-off value of the PI ratio was similar to that found in a previous study. Therefore, a PI ratio 5\u2009>\u20091.6 in our study can be useful predictors of the block success of an SCBPB. In addition, future study is warranted to approve the predictive value of PI ratio 5.Because of the high variability of the PI values, the PI ratio might be more accurate than the absolute value of PI8, laser doppler perfusion imager22, skin electrical resistance23, and the PI4. The skin temperature and PI can be measured relatively easily compared to other methods. In our study, the time to reach the highest PI (14\u00a0min) was not correlated with the time to reach the highest skin temperature (17\u00a0min). Although the assessment of skin temperature is regarded as a reliable and objective method to evaluate block success, the time required to reach the peak value was also longer than that of the PI. For example, in epidural-induced sympathectomy, the PI was shown to be a faster and more sensitive indicator of block success than the skin temperature8. This outcome may be due to a discrepancy in skin blood flow control during the changes in peripheral vasodilation24.Previous methods used to predict block success included skin temperature25 and produce dose-dependent relaxation26, and may increase PI and overestimate the value. Lastly, we added 5\u00a0mcg\u00a0ml\u22121 of epinephrine to the ropivacaine. Although this dosage is frequently used in clinical practice, it is expected that its extent of influence on the degree of vasoconstriction might vary with the varying dose of epinephrine. Future research is needed to determine the dose of epinephrine that can be used to maintain the reliability of the PI.There are several limitations to this study. First, for the ROC curve analysis, data from both the blocked and unblocked arm of the same patient were used. In previous studies, data from successful and failed blocks were used. However, in our study, all SCBPBs were successful, so the data of the unblocked arms were regarded as the failed block data. Second, in the current study, we could not suggest the validated PI as an objective indicator of the successful regional anesthesia. However, the PI ratio 5 was demonstrated as a surrogate endpoint and may be a useful indicator in clinical situations. Third, we administered midazolam to ease patient\u2019s anxiety before the SCBPB. Midazolam, a short-acting benzodiazepine, can lead to peripheral vasodilationIn conclusion, the use of epinephrine as an adjuvant to local anesthetics did not affect the PI and PI ratio. The PI ratio is a useful indicator for evaluating a successful SCBPB regardless of the presence of epinephrine. A PI ratio at 5\u00a0min\u2009>\u20091.6 is suggested as a relatively accurate predictor of a successful SCBPB."} +{"text": "Diet based on cereal, vegetables, oleaginous and dried fish are providing essential metallic elements. It can be also a source of exposure to toxic metallic elements. The aims of this study were to evaluate the contents on nine metallic trace elements in some major raw foodstuffs including rice, maize, peanut, tomato and dried fish in Burkina Faso and assess the health risk of these elements. Two hundred twenty-two samples were collected and analyzed by atomic absorption spectrometry. The health risk assessment was based on the United States Environment Protection Agency (USEPA) model. Iron and Zinc were the elements with the highest concentrations in the investigated foodstuffs. The iron highest median value (68.80\u00a0mg/kg) was observed in dried fish followed by maize (43.09\u00a0mg/kg) and peanuts (28.92\u00a0mg/kg). Rates of 77.95%, 66.66% and 32.5% obtained respectively fro tomato, maize and rice samples were above the maximum limit of lead set by Codex Alimentarius while 47.6%, 71.16% and 0% of maize, tomato and rice samples respectively have shown concentration above the maximum limit of cadmium. Chromium had shown higher contribution rate to the maximum daily intake of 167.11%, 34%, 2% and 8.53% for rice, maize and peanut respectively. A non-cancer risk situation has been observed on rice, maize and peanut consumption. None of the index risk values was above the threshold set by USEPA. Therefore, food safety is a major public health concern and its demand by consumers worldwide has stimulated research regarding the risk associated with consumption of foodstuffs contaminated by various contaminantsAmong the chemical contaminants recognized as potential food threats, metallic trace elements (MTE) are of great concern for human health.2.The term Trace elements is used for elements existing in natural and perturbed environments in small amounts, whose excessive bioavailability has a toxic effect on the living organism3. They are present in trace quantities; however, high intake of these elements could also cause health damage4. Whereas, heavy metals like cadmium (Cd), chromium (Cr), mercury (Hg), lead (Pb) arsenic (As) generally refer to metals having densities greater than 5\u00a0g.cm-3.5 have no established biological functions, and are considered as non-essential metals6.There are two classes of trace elements, essential and non-essential metals. Elements such as copper (Cu), manganese (Mn), selenium (Se) and zinc (Zn) also called micronutrients are essential for living organism and play important roles in the functioning of the critical enzyme system7. Foodstuffs can also be contaminated during their process, storage and marketing. The food sold in the open market can easily accumulate a high level of heavy metals8.Trace elements can occur as residues in foodstuffs through their presence in the environment as results of human activities such as industry, farming and car exhaust9.Due to their non-biodegradability and toxicity at low concentration, MTE intake through the food chain is a problem receiving increasing attention10.The USEPA and the International Agency for Research on Cancer (IARC) have classified some elements as either \u201cknown\u201d or \u201cprobable\u201d human carcinogens based on epidemiological and experimental studies showing an association between exposure and cancer incidence in humans and animals11. According to the high frequency consumption of these food groups12, cereal (maize and rice), legume (tomato), oleaginous (peanut) and dried fish have been selected as major consumed food items. Although these foodstuffs are an important source of a wide range of essential trace elements for humans. They can also carry toxic metals. Number of studies have been reported on the MTE contents in cereal, oleaginous, legumes and fish17 in over the world. However, there are few data on MTE contamination in these food items in Burkina Faso. Furthermore, the risk assessment of dietary exposure remains unknown. Therefore, the objectives of this study were to (1) evaluate the contents of Iron, Zinc, Cadmium, Cobalt, Cupper, Lead, Manganese, Nickel, Silver and Chromium in maize, rice, peanut and tomato, (2) estimate daily MTE intake through consumption of these food and (3) determine the cancer and non-cancer risks associated with the MTE intake using determinist risk assessment approach.The diet in Burkina Faso is based on staple foods, mainly cereals, legumes, roots, and tubersThe sampling was carried out across the following locations: Ouagadougou, Bobo-Dioulasso, Niangoloko, Dakola and Cinkanse. The samples were collected from different open markets located in these cities. A total of 222 samples including 40 samples of rice, 19 samples of maize, 59 samples of peanut, 59 samples of tomato and 45 samples of dried fish were collected from 2 November to 3 October 2020. The dried samples were kept at room temperature while tomato sample were frozen prior for analyses.-1 purchased from MERK .The de-ionized water was obtained from the water purification system Lab Tower Aft . Nitric and chlorhydric acids were provided by Hiperpur, Panreac AppliChem, Darmstadt, Germany and the individual stock standard solutions containing 1000\u00a0mg.LRassMill from Romerlab (Austria) was used for grinding cereal samples and the stainless-steel blender for peanut and tomato samples. The sample digestion was carried out with the Techne heat block . The elementary analyses were performed by an atomic absorption spectrometer VARIAN 240FS .18. Briefly 0.5\u00a0g of sample was digested in a test tube by 5\u00a0ml of the acid mixture at 150\u00a0\u00b0C for 2\u00bd hours. The volume of the digestate was completed to 100\u00a0ml with de-ionized water and filtered. The analysis was done with atomic absorption spectrometry on flame mode with external calibration curve.HNO3/HCl (3:1) mixture was used for the sample digestion as described by Demirel et al.19.The quality control of analytical performance was apply as described by Bazi\u00e9 et al.9. The average daily dose (ADD) (mg/kg/day) of metals was determined as follow:22The USEPA human health risk assessment model was applied21.24.The ratio of the average daily dose and the reference dose (RfD) of the MTE expressed as the hazard quotient (HQ) was used25:To assess multiple MTE exposure, the non-carcinogenic risk is expressed as the hazard index (HI), which is the sum of the HQ21The carcinogenic risk was determined according to the following equation:The SPSS package version 23.0.0.0. was used for statistical analysis. The data were check for normal distribution. The non-parametric tests have been performed.The design and conduct of the survey were approved by the Institutional Ethics Committee Board of the Ministry of Health of Burkina Faso since the experimental research on plants complied with the relevant institutional, national, and international guidelines and legislation.Since the data failed to show a normal distribution, the metallic trace elements contents are presented in Tables 26.The order of the median level of the top four metallic trace elements in the samples was found to be Fe\u2009>\u2009Zn\u2009>\u2009Mn\u2009>\u2009Cu, Fe\u2009>\u2009Zn\u2009>\u2009Cr\u2009>\u2009Pb, Fe\u2009>\u2009Zn\u2009>\u2009Ni\u2009>\u2009Co and Zn\u2009>\u2009Fe\u2009>\u2009Mn\u2009>\u2009Cu respectively in cereals, tomato, dried fish and peanuts. Similar results were reported by Ertugrul et al.,2008 in mushrooms from Black Sea region in Turkey29. However, when their intake is excessively elevated, essential metals can produce toxic effects30. Zinc is known to be involved in most metabolic pathways in humans; thus, zinc deficiency can lead to loss of appetite, growth retardation, skin changes, and immunological abnormalities. In the investigated foodstuffs, peanuts have the most abundant zinc content (31.59\u00a0mg/kg) which was 2 times higher than the concentrations found in cereals. The lowest median concentration was recorded in the tomato samples (4.39\u00a0mg/kg) The mean Zn concentration (13.178\u00a0mg/kg) in rice reported in Bangladesh15 is similar to the median value of zinc content in rice in this study. The content of Zn and Fe in tomato reported by Gebeyehu et al.31 were 24.50\u00a0mg/kg and 85.10\u00a0mg/kg, respectively which were too lower than the median values found in the current study.Iron and Zinc were the elements with the highest concentration in the investigated foodstuffs. The iron highest median value (68.80\u00a0mg/kg) was observed in dried fish followed by maize (43.09\u00a0mg/kg) and peanuts (28.92\u00a0mg/kg). Kolmogorov\u2013Smirnov test stated that there is a significant difference on iron content in the investigated food item. It is know that adequate iron in a diet is very important for decreasing the incidence of anemia31 in Ethiopia reported mean concentration of chromium in tomato (1.49\u00a0mg/kg) higher than the median concentration of tomato samples. Chromium in the diet is of a great importance because it is an essential trace element. It plays a key role on insulin function and lipid metabolism33. However, high concentrations may cause adverse health effects34.Chromium was detected in 72.22% of maize samples, 96.92% of tomato samples while 100% of peanuts, dried fish and rice contained Chromium. The highest concentration (5.40\u00a0mg/kg) was found in peanut sample collected in Dakola. Maize has the lowest median concentration (0.08\u00a0mg/kg) followed by tomato (0.95\u00a0mg/kg). Gebeyehu et al.31 in their study found in the tomato samples a mean concentration of 0.63\u00a0mg/kg which is similar to the median content obtained in this study. Cobalt is essential for human health since it is a part of vitamin B12. Cobalt is also used in the treatment of anemia in pregnant women for red blood cells stimulation. However, exposure to high concentrations of cobalt could lead to lung adverse effects, such as asthma and pneumonia35. The IARC has listed cobalt and cobalt compounds within group 2B as possibly humans carcinogenic agents35.Hundred percent of all the samples contained Cobalt. The cereal, rice (0.88\u00a0mg/kg), maize (0.40\u00a0mg/kg) and tomato (0.64\u00a0mg/kg) have shown less Cobalt content than dried fish (2.45\u00a0mg/kg) and peanuts (3.12\u00a0mg/kg). Gebeyehu et al.14 which is comparable to the current study. Ahmed and Shaheen15 and Rahman et al.36 reported 0.213 and 0.01\u00a0mg/kg respectively the mean concentration of nickel in rice in their study. These concentrations are largely lower than the median concentration of nickel in rice found in this study. Gebeyehu et al.31 reported 1.86\u00a0mg/kg in tomato which is comparable to the median value found in this study. Nickel is essential in small quantities, but it can endanger human health at high concentrations.Nickel was found in 94.7% of maize, while 100% of other foodstuffs contained Nickel. Peanuts and dried fish have shown the higher contents of 5.31\u00a0mg/kg and 4.17\u00a0mg/kg. rice (1.5\u00a0mg/kg) and tomato (1.09\u00a0mg/kg) had similar content and the lowest concentration was observed in maize samples. A mean concentration of Ni at 5.20\u00a0mg/kg in fish samples in Bangladesh has been reported by Islam et al.14 reported Cu content in fish ranging from 2.02 to 7.37\u00a0mg/kg which is higher than the values found in this study.Copper concentrations in samples are ranged from 1.05 to 5.00\u00a0mg/kg (rice), from 1.57 to 3.93\u00a0mg/kg (maize), from 0.09 to 1.24\u00a0mg/kg (tomato), from 0.15 to 0.67\u00a0mg/kg (dried fish) and from 0.26 to 16.26\u00a0mg/kg (peanuts). The highest median value (9.12\u00a0mg/kg) was recorded in peanut sample while the lowest (0.18\u00a0mg/kg) was found in dried fish samples. Islam et al.35. Copper can also pose public health hazards at high concentrations.Copper is known to be both vital and toxic for many biological systems. It plays a role in activation of more than 30 enzymes of which some are involved in the synthesis of the main component of connective tissues called collagen37. It is quite toxic at high doses. Its toxicity to humans manifest through a psychological and neurological disorder, termed as manganism that closely resembles Parkinson\u2019s disease38.The highest concentration (34.04\u00a0mg/kg) of manganese was recorded in peanuts sample collected in Dakola and two samples of maize from Bobo-Dioulasso did not show the presence of manganese. Tomato shown the lowest median concentration (1.50\u00a0mg/kg) followed by maize (2.85\u00a0mg/kg) and rice (4.48\u00a0mg/kg). Manganese has considerable biological significance, it is associated as an enzymatic cofactor in mitochondria, participates in the regulation of cell metabolism, in receptor binding, and signal transduction pathways39. The Inorganic lead compounds are classified as possibly carcinogenic for human40. Lead was found in 82.5% of rice sample, 72.22% of maize sample, 74.4% of dried fish samples, 78.69% of tomato sample and 86.45% in peanut samples. The highest concentration (5.80\u00a0mg/kg) was recorded in peanut sample from Dakola. Rates of 77.95%, 66.66% and 32.5% of respectively samples of tomato, maize and rice above the maximum limit set by Codex Alimentarius. The mean concentration of lead (3.63\u00a0mg/kg) in tomato reported by Gebeyehu et al.31 in Ethiopia was higher than the median content found in the tomato sample considered in this study.Lead is a toxic metal that has no known vital or beneficial effect on organisms, and is bio-accumulative which can cause serious injury to the brain, nervous system, red blood cells, and kidneys to animals and humans15 was similar to the median value of the Cadmium content in the current study, while the median content of Cadmium in tomato was lower than the value reported by Gebeyehu et al.31 (0.56\u00a0mg/kg).Cadmium was detected in 92, 5% in rice samples, 44.44% in maize sample, 91.81% in tomato sample, in 86.45% peanut sample and 76.75% in dried fish sample. The highest concentration (9.00\u00a0mg/kg ) was recorded in tomato sample collected in Cinkanse. Peanut has shown the lowest median value (0.24\u00a0mg/kg). Codex Alimentarius has set the maximum limit of cadmium in rice (0.4\u00a0mg/kg), in other cereal (0.1\u00a0mg/kg) and vegetables (0.05\u00a0mg/kg). 47.6% and 71.16%, 0% of sample of maize, tomato and rice respectively shown concentration above the maximum limit. The mean concentration (0.088\u00a0mg/kg) of Cd in rice reported by Ahmed et al.41.Cadmium is a non-essential and toxic element for human: kidney and bones are the critical target organs of Cadmium contamination. IARC has classified Cadmium as a carcinogen42. These elements can most likely be attributed to rapid development, increased traffic emissions into the atmosphere, industrial activities, and to the lack of sophisticated management of wastes43, since the foodstuff are collected through the various agricultural zones, stored and sold in different open markets located in the main cities of the country.The large variability observed on the metallic trace elements contents in the different samples may be related to the variability of environmental both field production and selling conditions. Indeed, metal ions in the environment are a big problem for human beings44 while tomato (0.6\u00a0g/day), peanuts (27\u00a0g/day), and dried fish (0.06\u00a0g/day) consumption data were collected from FAO database45.The estimated chronic daily intake (CDI) of the nine investigated trace metals were evaluated according to the average concentration of each metal in each food group and the consumption data of these food items Table . The con50.Due to the higher consumption rate of both rice and maize, these two cereals have shown the biggest contribution of EDIs. The essential metallic trace elements such as iron (2032.35\u00a0\u03bcg/day\u20131240.06\u00a0\u03bcg/day) and zinc (2349.69\u00a0\u03bcg/day\u20132899.52\u00a0\u03bcg/day) had the higher EDIs followed by manganese (607.62\u00a0\u03bcg/day\u2013756.22\u00a0\u03bcg/day). However Chromium had shown higher contribution rate to the maximum daily intake of 167.11%, 34%, 2% and 8.53% for rice, maize and peanut respectively. This higher contribution was followed by Nickel with rice (84.40%), maize (49.04%) and peanut (47.79%). The contribution of rice (0.84%) tomato (0.02%) dried fish (0.00%) and peanut (0.54%) to the MTDI of Lead were less than 1%. According to a total diet study made by the Catalan Food Safety Agency, fish and shellfish account for 12.4% of total Pb intake for the Catalonia population50.Among the food group, rice (2.70%) provided more Cadmium followed by peanut (1.30%) while maize, tomato and dried fish were contributed poorly. It has been reported by the Spanish Food Safety and Nutrition Agency that fish and shellfish only represent a fraction of the total Cadmium intake in the diet, which has been estimated to be between 17.3 and 33.9%The non-cancer risks were expressed as the cumulative hazard index (HI) which was the sum of the individual metal hazard quotient (HQ) Fig.\u00a0.Figure 1The HI values recorded on tomato and dried fish consumption of both men and women varied from 0.009 to 0.23. These values were below the reference value (HI\u2009=\u20091). Consequently, there was no non-cancer risk recorded at this level of consumption of these food items.8.However, non-cancer risk situations have been observed on rice, maize and peanuts consumption. Rice was the food item with highest HI value 9.66 and 8.74, respectively recorded for women and men. This was followed by maize (6.14\u20135.55) and peanut (5.16\u20134.67). In their study, Nuapia et al. 2017, have reported that in Johannesburg and Kinshasa, the combined HI values were greater than 1 for all the food samples including cabbage, bean, beef and fish for both men and women. These results indicate high potential risk to the local consumers in Kinshasa and Johannesburg via consumption of the food sold in the open markets\u22121\u00a0day\u22121. Fe and Zn have shown the highest daily intakes, but their contributions to the risk indices were very low. This is due to their higher RfD because of their essential status.Cobalt was the main contributor to the risk index. The HQ of Cobalt contributed for about for 85% of Hi. This high contribution is related to its concentrations in the samples but especially to its risk oral reference dose (RfD) of 0.0003\u00a0mg\u00a0kg\u22126 to 8\u2009\u00d7\u200910\u22129. None of the index risk values was above the threshold set by US-EPA (IR\u2009>\u200910\u2009\u2212\u20094); consequently, the consumption of these food groups in Burkina Faso was free of cancer risk from lead contamination.The results obtained Fig.\u00a0 show theThis study was consisted to evaluate metallic trace elements contents in some major raw foodstuffs in Burkina Faso and assess the health risk. Metallic trace elements including Fe, Zn, Mn, Co, Cd, Pb, Cu, Ni, Cr were investigated in maize, rice, tomato, peanuts and dried fish. The obtained results showed that the investigated foodstuffs contained an appreciable among of essential trace elements since these food staples are higher contributing to the daily intake. Iron and zinc had the higher EDIs followed by manganese through the consumption of cereal. The non-essential traces elements were found in large portion with more than half of the samples having lead and cadmium concentrations above the limits set by the Codex Alimentarius. The estimation of the daily intake revealed that the population of Burkina Faso are exposed to a non-cancer risk linked to metallic trace elements associated to rice maize and peanut consumption. However, the cancer risk was not a concern."} +{"text": "Modern perioperative medicine has dramatically altered the care for patients undergoing major surgery. Anaesthetic and surgical practice has been directed at mitigating the surgical stress response and reducing physiological insult. The development of standardised enhanced recovery programmes combined with minimally invasive surgical techniques has lead to reduction in length of stay, morbidity, costs, and improved outcomes. The enhanced recovery after surgery (ERAS) society and its national chapters provide a means for sharing best practice in this field and developing evidence based guidelines. Research has highlighted persisting challenges with compliance as well as ensuring the effectiveness and sustainability of ERAS. There is also a growing need for increasingly personalised care programmes as well as complex geriatric assessment of frailer patients. Continuous collection of outcome and process data combined with machine learning, offers a potentially powerful solution to delivering bespoke care pathways and optimising individual management. Long-term data from ERAS programmes remain scarce and further evaluation of functional recovery and quality of life is required. The field of perioperative medicine has undergone radical change in the last 30 years witnessing major advances in anaesthetic and surgical technique. Along with this, traditional models of care have been disbanded in favour of enhanced recovery programmes in almost every surgical specialty. In this review, we have presented the evolution of modern perioperative care; we have discussed current practice, areas of contention, and future directions for advancing the field.Historically, patients knew very little of what to expect following a major surgery. Perioperative care was often characterised by prolonged fasting, aggressive bowel preparation, nasogastric decompression, bed-rest, and prolonged convalescence. A paradigm shift came during the 1990s with the work of Henrik Kehlet on the physiological stress response and organ dysfunction following surgery. He hypothesised this was a key factor in postoperative morbidity and that combined approaches to inhibit this response would improve clinical outcomes .Kehlet and others pioneered modern recovery \u2013 advocating multimodal analgesia and regional anaesthetic techniques combined\u00ae) study group. The group sought to address the variability . H. H51]. HOther areas of controversy include intraoperative fluid therapy, which has been an important component of ERAS from its inception. Balancing fluid therapy to achieve adequate splanchnic perfusion whilst avoiding oedema, paralytic ileus, and fluid overload remains challenging. This is particularly true amongst high-risk patients such as those with limited physiologic reserve, severe cardiopulmonary disease, renal impairment, or patients undergoing extensive surgery . Early tERAS challenged all the dogmas of conventional perioperative care, but it needs to continue to evolve, or else risk becoming dogmatic itself. ERAS must be responsive to the latest research evidence as well as novel surgical approaches and technologies. Enhanced recovery programmes have already begun to influence the care of emergency general surgery , 61] an an61] anResearch within ERAS is also evolving with on-going work to investigate ways of enhancing the success of ERAS. Studies to predict which patients are likely to deviate from the expected perioperative course has been of interest as it may allow remedial action to be taken to avert this . Recent Deconstructing ERAS protocols by analysing the effectiveness of specific items may seem counterintuitive given the evidence base is required to justify each component. Efforts to simplify and streamline protocols are primarily borne from a desire to improve compliance, but may also represent a response to criticism that ERAS has become overcomplicated and unwieldy . Debate What is increasingly clear in perioperative medicine is that one size does not fit all. The individual stress response to surgery remains highly variable without a means to measure or predict this currently. The increasing complexity of patients\u2019 medical needs combined with heterogeneity in service infrastructure, operative, and patient factors are driving a need for more personalised care programs. This is particularly true in the context of an aging global population and the rising number of elderly and comorbid patients undergoing surgery. The increasing prevalence of frailty and geriatric syndromes amongst this patient group places them at increased risk of adverse outcome following surgery including medical complications, prolonged hospitalisation, institutionalisation, and readmission as well as short and long-term mortality .Although series have demonstrated that ERAS is safe and beneficial in caring for elderly surgical patients , a numbeThe growing complexity of perioperative care and the need for increasingly personalised and bespoke pathways has stimulated interest in digital technological solutions and automated care processes. Digital technology is likely to have a major role in shaping the future of perioperative care and a number of advances relevant to enhanced recovery programmes have been investigated. These include apps directed at lifestyle modification and preoperative optimisation, objective nociceptive measurements, portable non-invasive sensors calibrated to recognise postoperative cardiopulmonary complications, and activity trackers to monitor postoperative ambulatory recovery .An exciting frontier in medical technology is in combining big data analytics with artificial intelligence in order to guide patient management. Artificial neural networks and machine learning programmes have exhibited superior performance to conventional prediction models in diagnosing acute appendicitis , selectiThis may well revolutionise care of the surgical patient within the next 20 years. We envisage a system whereby data from electronic health records combined with metrics prospectively measured throughout the perioperative period are harvested by machine-learning programmes. This continuously updates the optimum care pathway with targeted adaptations or adjustments for an individual patient as well as the local population in an automated fashion .There is cause for great optimism about the future of perioperative medicine. There has been greater consensus and collaboration on issues such as nutrition , anaemiaStudies examining patient satisfaction and health related quality of life with ERAS have so far found no significant difference when compared to conventional care. Some evidence supports a reduction in postoperative fatigue and earlA limited number of reports have now been published examining medium to long-term survival, which reveals both an overall and cancer-specific survival advantage with ERAS , 96], , . It is lCrucially these studies have reasserted the importance of adherence to ERAS protocols which was strongly associated with survival independent of cancer stage and postoperative complications , 96], , .From a global perspective there is still considerable progress required in perioperative care. In 2015 the Lancet commission on global surgery highlighted the alarming deficit in essential, life-saving surgical and anaesthesia care in low and middle income countries . The repClick here for additional data file."} +{"text": "Castleman\u2019s disease (CD), otherwise known as angiofollicular lymph node hyperplasia, is a rare, poorly understood disorder, which often occurs in a mediastinum. Involvement of parotidgland is a considerably infrequent event. We present a 15-year-old boy patient with a swelling in the left parotid gland that has been diagnosed with CD. The clinical features,radiographic findings, and treatment plan are discussed. Furthermore, a thorough literature review demonstrated 57 published cases of CD in salivary gland with their summarized features. Castleman\u2019s disease (CD) is a rare non\u2010neoplastic lymphoproliferative disorder, first known in 1956 by Dr. Benjamin Castleman who described a 40-year-old male with a mediastinal lymphnode mass characterized histologically by lymph node hyperplasia and follicles with hyalinized centers . BecausThe CD diagnosis is basically confirmed by biopsy. The characteristic histopathological criteria included angiofollicular lymph node hyperplasia in a lymph node region . Due toA 15-year-old boy patient referred to Plastic Surgery Department at Imam Khomeini Cancer Institute with chief complaint of left facial swelling for past 4 yearsFigure and b. HFollowing the CTs, ultrasonography (US) examination was administrated for the patient and in the US findings; an oval and bean-shaped mass measuring approximately 30*6.5 mm accompanied bycentripedal vascularity in the buccal space was reported. In addition, presence of multiple giant lymph nodes with increased wall thickness in the both sides of jugular space and submandibularspace was noted. The patient underwent a left superficial parotidectomy with preservation of the facial nerve under general anesthesia. On gross examination tangray salivary gland was identified,which showed central area of well-demarcated gray nodule with firm consistency. Microscopic examination of referred specimen revealed salivary gland tissue composed of mostly serous aciniand related ducts with preserved architecture and intra parotid lymph node with marked vascular proliferation and hyalinization of germinal center with appearance corresponded to transformgerminal centers regressively. Concentric aggregation of lymphocytes at the mantle zone was noted. Interfollicular stroma showed multiple vessels admixed with few plasma cells.Immuno histo chemistry (IHC) study for CD-21 showed strong positive in follicular dendritic cells within germinal center, which confirmed diagnosis. Postoperatively, the patient did well, and he experienced no major complications, that the authors obtained written and signed informed consent\u00a0from patient's guardians for publishing this\u00a0case report.CD is a rare pathological disorder that can rarely affect the parotid gland .Based oIn the present case, the lesion was located in buccal side of left parotid gland mimicking a salivary gland tumor. et al. [ et al. [ Several theories have been proposed for the un known etiology of this disease. The strongest hypotheses indicate robust relation between virus or chronicinflammation and lymphoproliferation . In 197et al. identif Clinically, CD is sub classified into unicentric (UCD) and multicentric (MCD) types. The UCD form usually presents as an asymptomatic painless palpable enlarged lymph node mass which reflectsits benign feature. On the other hand, MCD is more aggressive and presents with systemic symptoms such as fever, loss of weight, and splenomegaly and can be associated with some malignancies,such as Kaposi\u2019s sarcoma, non-Hodgkin\u2019s lymphoma, Hodgkin\u2019s disease, and POEMS syndrome . In ourAccording to histopathological pattern, the HV is the most common subtype, accounting for 80-90 % of the cases .This vTotal excision is the golden standard treatment for UCD in the head and neck area. However, the non-operable patients need radiotherapy. Because of aggressive policy of MCD form,it is only cured by palliative treatment . AccordIt is crucial for the physicians to be aware of this entity for early diagnosis and to provide timely and adequate treatment. Concerning the possibility of malignant transformation, long follow-ups should be regarded."} +{"text": "Parkellus (Parkellus zschokkei (Parkellus. Parkellus hagiangensis sp. nov. is characterized by a medium-sized buccal cavity, posterior position of the dorsal tooth located below the beginning of the pharynx, males having the ventromedian cuticular pores above and below the excretory pore, short spicules with conical proximal part; females with very faint pars refringens vaginae and small teardrop-shaped pieces, short pars distalis vaginae, the presence of small ventromedian vulval papillae. Parkellus tuyenquangensis sp. nov. is characterized by a medium-sized buccal cavity, posterior position of the dorsal tooth located above the beginning of the pharynx, males having the ventromedian cuticular pores above and below the excretory pore, medium-sized spicules with a cylindrical proximal part, very short lateral guiding pieces, females with very strongly sclerotized pars refringens vaginae, medium size teardrop-shaped pieces, short pars distalis vaginae thickened at the junction with pars refringens vaginae. The newly described species are morphologically most similar to P. parkus and P. zschokkei. An identification key to Parkellus species is presented.Two new species of arkellus from Vie Predatory nematodes play a substantial role in the soil food-web as regulators of other trophic groups: bacterial, fungal, algae, and herbivorous feeders, they are also suitable bioindicators of soil condition . UnfortuParkellus are provided. Parkellus for the new species P. parkus collected in Korea from forest soil surrounding the roots of Euonymus oxyphyllus Miq. The main feature that distinguished the newly described genus Parkellus from the morphologically very similar Coomansus is the location of dorsal tooth in the posterior half of the buccal cavity. Coomansus to the genus Parkellus. Parkellus from the genus Coomansus because the distinguishing characters such as posteriorly situated dorsal tooth apex and a complex gubernaculum can be observed also in some species of Coomansus and regarded the genus Parkellus as a synonym of the genus Coomansus. However, Parkellus from the genus Coomansus. He has separation of several genera based on the position of dorsal tooth apex on dorsal metarhabdion: the new genus Supronchus from the genus Iotonchulus, Megaiotonchus from the genus Iotonchus (Iotonchidae), the new genus Pentonchella (Anatonchidae) .Parkellus species described so far, nine are found in Asia, three in Europe, and one in North America or in a DESS mixture for mol, and mou-Well Thermal Cycler, Applied Biosystems, Foster City, CA, USA) at 65\u00b0C for 3\u2009hr followed by a 5\u2009min incubation at 100\u00b0C. The obtained single nematode lysate (crude DNA extract) was used either immediately as a DNA template for a PCR reaction or stored at \u2012 20\u00b0C for future use.After conducting the microscopic studies and preparing the photographic documentation, all nematodes subjected to molecular analyses were taken out from the temporary slides. Each retrieved nematode was transferred to a separate dish filled with PBS solution. The dishes with nematodes were placed on a laboratory shaker. The nematodes were initially washed in PBS for 3\u2009\u2009hr. Subsequently, the nematodes were transferred into dishes containing fresh PBS and were washed on a shaker overnight. In the final washing stage, the nematodes were transferred into dishes containing sterile milliQ water and were washed with it for 4\u2009hr. After this procedure, the nematode individuals were transferred to separate 0.2\u2009ml polymerase chain reaction (PCR) tubes containing 25\u2009\u03bcl sterile water. An equal volume of the lysis buffer, as described in Parkellus individuals in two overlapping fragments, using the following primer combinations: 988F combined with 1912R and 1813F combined with 2646R as follows: an initial denaturation step at 94\u00b0C for 3\u2009min, followed by 40 cycles at 94\u00b0C for 30\u2009sec, 54\u00b0C (58\u00b0C or 62\u00b0C in case of D2A-D3B primer combination) for 30\u2009sec and 72\u00b0C for 60\u201370\u2009sec with a final incubation for 7\u2009min at 72\u00b0C. Amplicons were visualized under UV illumination after Simply Safe gel staining and gel electrophoresis. Excess dNTPs and unincorporated primers were removed from the PCR product using the enzymatic mixture of Fast Polar-BAP \u2013 Thermosensitive Alkaline Phosphatase and Exonuclease I \u2013 .All PCR reactions contained 12.5\u2009\u03bcl Color Perpetual OptiTaq PCR Master Mix (2x) , 1\u2009\u03bcl of the forward and reverse primer (5\u2009\u03bcM each), the 3\u2009\u03bcl DNA template and sterile Milli-Q water to 25\u2009\u03bcl of the total volume. All PCR reactions were performed in Veriti 96Parkellus rDNA sequence fragments, as well as ribosomal DNA fragments of Coomansus parvus used in this study as a close reference species, were deposited in GenBank under the following accession numbers: MT799665\u2013MT799669 (18S rDNA) and MT799670\u2013MT799673 (28S rDNA).After sequencing the obtained Parkellus, newly acquired Coomansus parvus and available sequences of Mononchida representatives from GenBank. Representatives of Dorylaimida, Mermithida, and Trichinellida were used as an outgroup. The final multiple-sequence alignments consisted of 1,688 overlapping characters in case of 18S rDNA and 1,047 characters in case of 28S rDNA. Genetic distances were estimated with the DNADist tool implemented in the BioEdit, using the default options (Kimura 2-parameter model of nucleotide substitution).The newly obtained rDNA sequences were analyzed using the BioEdit program : v.7.2.56 generations in case of the 18S rDNA data set and for 1\u2009\u00d7\u2009106 generations in case of the 28S rDNA data set. Sample frequency in both cases was 100 generations. The sampled trees from each run were combined in a single 50% majority-rule tree. Stabilization of the likelihood and parameters was checked with the program Tracer Suborder Mononchina Superfamily Mononchoidea Family Mononchidae Parkellus Coomansus acuticaudatus Coomansus cobbi Coomansus monticola Coomansus mucronatus Coomansus paraamphigonicus Coomansus silvius Parkellus simmenensis Iotonchus simmenensis Coomansus simmenensis Iotonchus zschokkei Coomansus zschokkei Coomansus scopulosus Parkellus hagiangensissp. nov.Holotype female and two paratype males, in permanent mounts in glycerine, deposited in the nematode collection at the Department of Nematology, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology. One paratype female and one paratype male deposited in the nematode collection of the Museum and Institute of Zoology, PAS, Warsaw, Poland.Soil samples from pristine forest containing specimens originated from Du Gia natural conservation area at Minh Ngoc Commune, Bac Me District, Ha Giang Province .The name of the species refers to its geographical origin from Ha Giang Province in Vietnam.Measurements see Relaxed specimens arcuate, more curved ventrally at posterior end. Body tapering slightly anterior to base of pharynx but more sharply toward posterior end. Maximum body width at the level of vulval region. Cuticle smooth, 6.4 (5.3\u20137.5) \u03bcm thick at the base of pharynx. Sublateral, subdorsal, and subventral body pores distinct along the entire body excluding tail.Lip region rounded, 3.4 (3.1\u20133.7) times as wide as high, slightly offset by a depression, its diameter almost the same as the adjacent body. Labial papillae small, conical, slightly protruding. Cephalic papillae comparatively more prominent, broadly rounded, slightly protruding beyond the body outline. Amphidial fovea cup-shaped, its aperture 5.4 (5.1\u20135.8) \u00b5m wide, located 20.8 (18.6\u201322.8) \u00b5m from the anterior body end. Buccal cavity spacious, oval-shaped, 1.8 (1.7\u20132.1) times as long as wide, with funnel-shaped base, its wall strongly sclerotized. Dorsal tooth medium-sized, located at the base of the vertical dorsal plate of the buccal cavity, below the beginning of the pharynx. Apex of dorsal tooth located 41.1 (38.1\u201344.6) \u00b5m or 71\u201376% of buccal cavity length from its anterior end, opposite to it lies a thin, subventral longitudinal ridge. Nerve ring encircling cylindrical, muscular pharynx at about 26.5 (23.8\u201329.2%) of its length. Excretory pore distinct, males have there are three well developed ventral pores, first one at 40.7\u201346.6\u2009\u00b5m above and the next two at 24.7\u201329.7 and 74.3\u201382.6\u2009\u00b5m behind the excretory pore. Pharyngo-intestinal junction non-tuberculate. The intestine content nematodes, diatom shells, earthworm setae and detritus are visible. Rectum wide, arcuate, 0.8 times the anal body diameter long. Tail conical, ventrally bent, regularly tapering, with rounded tip. Hyaline part well developed, 17.0 (10.9\u201323.8)\u2009\u2009\u00b5m long. Caudal glands and terminal opening absent.pars dilatata with distinct lumen, it is connected with the uterus by a muscular sphincter, uterus, relatively long, contains numerous sperm cells mainly in its distal part. Vagina extending inwards for 27.8\u201328.3\u2009\u00b5m or 35\u201343% of body diameter, pars proximalis vaginae longer than wide, pitcher-shaped, surrounded by strong circular muscles, pars refringens vaginae with a very faint, small (6.7\u2009x\u20094.7\u2009\u00b5m), teardrop-shaped pieces, pars distalis vaginae short, vulva a transverse slit. Small, ventromedian vulval papillae, one or two on each side of the vulva are visible. Female tail with four pairs of sublateral caudal pores.Genital system didelphic-amphidelphic, the sexual branches almost equally developed, anterior 225\u2013238\u2009\u00b5m long or 11\u201313% of body length, posterior 140\u2013187\u2009\u00b5m long or 8\u20139% of body length. Ovaries on alternate sides of the intestine, well developed, reflexed with numerous oocytes, not reaching the uterus-oviduct junction, oviduct takes the form of a slender tube, which then expands to form a well-developed \u2009\u00b5m long. Ventromedian supplements 19\u201323 in number, conical and regularly arranged, occupies 12.9\u201313.3% of the body length, the anterior most situated at 307\u2013326\u2009\u00b5m from cloacal aperture, distance between the first and last supplement 287\u2013307\u2009\u00b5m. Above the series of supplements, 1\u20132 cuticular pores are visible. Male tail with two pairs of sublateral, two pairs of subdorsal caudal pores and two pairs of small subventral papillae.Genital system diorchic, testes opposed. Sperm cells elongate spindle shaped. Spicules total length along arc 1.2 times that at chord, 1.4\u20131.5 times longer than body diameter at cloacal aperture, relatively thin, 9.1\u20139.4 times longer than wide ventrally curved, curvature 114\u2013119\u00b0 median piece marked between hump and lateral guiding pieces, occupying 39.9\u201341.3% of spicule total length. Dorsal contour arcuate, ventral side with moderately expressed hump and hollow, the former located at 27.3\u201328.7% of spicule total length from its anterior end, head oval-shape, offset by a shallow depression, proximal part of spicules conical, posterior end bifurcate, 4.4\u20135.0\u2009\u00b5m broad. Lateral guiding pieces with arched edges and bifurcate terminus, this furcation is symmetrical and poorly marked. Gubernaculum well developed, 31.9\u201334.7Parkellus hagiangensis sp. nov. is characterized by its large sized body ; males having the ventromedian cuticular pores above and below of the excretory pore; position of amphidial aperture (18.6\u201322.8\u2009\u00b5m from the anterior body end); 50.8\u201360.2\u2009\u00b5m long buccal cavity; dorsal tooth apex at 38.1\u201344.6\u2009\u00b5m from anterior margin of buccal cavity or 71\u201376% of buccal cavity length, located at the base of the vertical dorsal plate of the buccal cavity, below the beginning of the pharynx; pars refringens vaginae with very faint and small (6.7\u2009x\u20094.7\u2009\u00b5m) teardrop-shaped pieces; short pars distalis vaginae; small ventromedian vulval papillae and 82.3\u201397.3\u2009\u00b5m long spicules with rounded head and conical proximal part. P. hagiangensis differs from all so far known Parkellus species by having more posterior dorsal tooth apex from anterior margin of buccal cavity.Parkellus hagiangensis sp. nov. is similar to P. parkus , and P. tuyenquangensis sp. nov. From P. parkus, it is distinguished by having lower position of dorsal tooth apex (71\u201376% vs 61\u201365%), longer female tail (116\u2013128 vs 85\u201390\u2009\u00b5m) and males having the ventromedian cuticular pores above and below of the excretory pore. From P. zschokkei it differs by lower position of dorsal tooth apex (71\u201376% vs 47\u201360%), lower position of amphidial aperture (18.6\u201322.8 vs 12.0\u201316.0\u2009\u00b5m) from anterior body end, males having the ventromedian cuticular pores above and below of the excretory pore and spicule curvature (114\u2013119\u00b0 vs 104\u2013109\u00b0). From P. tuyenquangensis sp. nov. it is distinguished by having lower position of dorsal tooth apex (71.0\u201376.0% vs 60.0\u201370.0%), appearance of very faint and small pars refringens vaginae pieces vs strong sclerotized and medium size, structure of pars distalis vaginae (not thickened vs thickened at the junction with pars refringens vaginae), presence of small vulval papillae (vs absence), structure of proximal part of spicule .In general appearance . parkus , P. zschP. hagiangensis sp. nov. (MT799665) while identical, 1\u2009kb long, 28S rDNA sequences were obtained from three individuals (MT799670). The results of the phylogenetic analyses using these molecular data are presented in Nearly full length , identical 18S rDNA sequences were acquired from two individuals belonging to Parkellus tuyenquangensissp. nov.Holotype female, three paratype females, and three paratype males from Cham Chu and Na Hang population, in permanent mounts in glycerin, deposited in the nematode collection at the Department of Nematology, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology. Nine paratype females and nine paratype males from Cham Chu and Na Hang population deposited in the nematode collection of the Museum and Institute of Zoology, PAS, Warsaw, Poland. Two paratype females and three paratype males from Na Hang population deposited in State Museum of Natural History NASU, Lviv, Ukraine.Cham Chu population: soil samples from pristine forest containing specimens originated from Cham Chu natural conservation area at Yen Thuan Commune, Ham Yen District, Tuyen Quang Province .Na Hang population: soil samples from pristine forest containing specimens originated from Na Hang natural conservation area at Na Hang District, Tuyen Quang Province .The name of the species refers to its geographical origin from Tuyen Quang Province in Vietnam.Measurements, see Relaxed specimens arcuate, more curved ventrally at posterior end. Body tapering slightly anterior to base of the pharynx but more sharply toward the posterior end. Maximum body width at the level of vulva. Cuticle smooth, 7.1 (6.2\u20138.5) \u03bcm thick at the base of the pharynx. Sublateral, subdorsal, and subventral body pores visible along the entire body excluding tail.Lip region rounded, 3.4 (3.0\u20133.7) times as wide as high, slightly offset by a depression, its diameter slightly wider than an outline of the adjacent body. Labial papillae small, conical, slightly protruding. Cephalic papillae bigger, broadly rounded, slightly protruding beyond the body outline. Amphidial fovea cup-shaped, its aperture 5.2 (4.2\u20136.4)\u2009\u2009\u00b5m wide, located 19.5 (17.7\u201322.0)\u2009\u2009\u00b5m from the anterior body end. Buccal cavity spacious, oval-shaped, 2.0 (1.9\u20132.1) times as long as wide, with funnel-shaped base. Dorsal tooth small, located at the vertical dorsal plate of the buccal cavity, above the beginning of the pharynx. Apex of dorsal tooth located 35.7 (31.6\u201337.7) \u00b5m from anterior margin of the buccal cavity or 60\u201370% of the buccal cavity length from its anterior end, opposite to it lies a thin, subventral, longitudinal ridge. Nerve ring encircling cylindrical, muscular pharynx at about 25.6 (22.0\u201328.6%) of its length. Excretory pore, ampulla and renette cells well visible. Males with three or four very well-developed ventral pores near the excretory pore, first one located at 75.4 (71.9\u201383.8) \u00b5m and the second one (in three specimens only) at 22.0\u201352.4\u2009\u00b5m above the excretory pore, the next two at 39.0 (15.6\u201347.4) and 83.4 (72.0\u201389.5) \u00b5m behind the excretory pore. Pharyngo-intestinal junction non-tuberculate. In the intestine content visible nematodes, diatom shells, earthworm setae and detritus. Rectum wide and arcuate, 0.8\u20130.9 times the anal body diameter long. Tail conical, ventrally bent, regularly tapering, with rounded tip. Hyaline part well developed, 16.8 (14.9\u201321.7) \u00b5m long. Caudal glands and terminal opening absent.pars dilatata with distinct lumen, it is connected with the uterus by a muscular sphincter, inner part of this sphincter heavily sclerotized, uterus, relatively long, contains numerous sperm cells mainly in its distal part, sometimes spermatozoids are also visible in sphincter and proximal part of pars dilatata. Vagina extending inwards for 29.1 (26.2\u201333.4) \u00b5m or 29.8 (24.8\u201335.7%) of body diameter, pars proximalis vaginae longer than wide, pitcher-shaped, surrounded by strong circular muscles, pars refringens vaginae medium size (9.0\u201312.9\u2009\u00d7\u20094.8\u20138.0\u2009\u00b5m), very strongly sclerotized, teardrop-shaped pieces, pars distalis vaginae very short, thickened at the junction with pars refringens vaginae, vulva transverse slit, sometimes the vulval lips extend beyond the body contour. No papillary structures before and behind the vulva, sometimes only single cuticular pores are visible, one on each side of vulva. Female tail with four pairs sublateral caudal pores.Genital system didelphic-amphidelphic, the sexual branches almost equally developed, anterior 364 (291\u2013430)\u2009\u2009\u00b5m long or 12.9 (11.2\u201315.0%) of body length, posterior 322 (223\u2013384)\u2009\u2009\u00b5m long or 11.4 (8.2\u201313.4%) of body length. Ovaries on alternate sides of intestine, well developed, reflexed with numerous oocytes, not reaching the uterus-oviduct junction, oviduct takes the form of a slender tube, which then expands to form a well-developed Genital system diorchic, with opposed testes. Sperm cells elongate spindle-shaped. Ejaculatory glands in tandem, generally distinct. Spicules total length along arc 1.2\u20131.3 times that at chord, 1.5 (1.4\u20131.9) times longer than body diameter at cloacal aperture, relatively thin, 9.2 (7.8\u201310.1) times longer than wide, ventrally curved, curvature 115 (112\u2013119\u00ba), median piece marked between hump and lateral guiding pieces, occupying 36.7% (31.0\u201340.5%) of spicule total length. Dorsal contour arcuate, ventral side with moderately expressed hump and hollow, the former located at 24.4% (18.4\u201329.6%) of spicule total length from its anterior end, head oval-shape, proximal part of spicule almost cylindrical, posterior end bifurcate, 5.4 (4.6\u20136.0)\u2009\u2009\u00b5m broad. Lateral guiding pieces with arched edges and bifurcate terminus, this furcation is symmetrical and poorly marked. Gubernaculum well developed, 33.9 (30.1\u201340.6)\u2009\u00b5m long. Ventromedian supplements 16\u201320 in number, conical, regularly arranged, occupies 12.9% (12.1\u201314.8%) of body length, the anterior most situated at 19.1 (15.7\u201329.0)\u2009\u2009\u00b5m from cloacal aperture, distance between the first and last supplement 334 (240\u2013408) \u00b5m. Above the supplements visible two cuticular pores. Male tail with two pairs of sublateral and two pairs of subdorsal caudal pores and two pairs of small subventral papillae.Parkellus tuyenquangensis sp. nov. is characterized by its large sized body ; males having the ventromedian cuticular pores above and below of the excretory pore; 52.5\u201360.7\u2009\u00b5m long buccal cavity; dorsal tooth apex at 31.6\u201337.7\u2009\u00b5m from anterior margin of buccal cavity or 60\u201370% of buccal cavity length); pars refringens vaginae with a very strongly sclerotized, medium size (9.0\u201312.9\u2009\u00d7\u20094.8\u20138.0\u2009\u00b5m) teardrop-shaped pieces; pars distalis vaginae very short and thickened at the junction with pars refringens vaginae; and spicules 95\u2013110\u2009\u00b5m long with rounded head and cylindrical proximal part and lateral guiding pieces 13\u201318\u2009\u00b5m long.Parkellus tuyenquangensis sp. nov. is similar to P. parkus , and Parkellus hagiangensis sp. nov. From P. parkus it is distinguished by males having the ventromedian cuticular pores above and below of the excretory pore , longer spicules (95\u2013110 vs 82\u201392\u2009\u00b5m), length and shape of lateral guiding pieces (13\u201318 vs 22\u201325\u2009\u00b5m), distal part barrel-shaped vs cylindrical) and longer female tail (120\u2013169 vs 85\u201390\u2009\u00b5m). From P. zschokkei it differs by the lower position of the dorsal tooth apex (60\u201370% vs 47\u201360%), lower position of amphidial aperture (17.7\u201322.0 vs 12.0\u201316.0\u2009\u00b5m) from anterior body end lack of ventromedian vulval papillae, males having the ventromedian cuticular pores above and below of the excretory pore spicule curvature (112\u2013119\u00b0 vs 104\u2013109\u00b0). The differences between Parkellus tuyenquangensis sp. nov. and Parkellus hagiangensis sp. nov. were described above in the diagnosis in the description of Parkellus hagiangensis sp. nov.In general appearance . parkus , P. zschP. tuyenquangensis sp. nov. we were able to obtain only the second part of the 18S rDNA gene from one of the two molecularly-analyzed nematode individuals. Identical, 1\u2009kb long, 28S rDNA sequences were successfully obtained from both of these nematodes (MT799671). The results of the phylogenetic analyses using these molecular data are presented in For the Parkellus zschokkei Parkellus zschokkei from three Ukrainian populations: (I) Uzhhorod, Zakarpattia region, Ukraine, oak forest ; (II) Uzhhorod, Zakarpattia region, Ukraine, oak forest ; (III) Uzhhorod, Zakarpattia region, Ukraine, oak forest N 48\u00b035\u203247.88\u2033; E 22\u00b022\u203241.51\u2033, 170 m a.s.l.). In total, 13 females and 11 males (population I) fixed with 4% TAF, mounted in glycerine, on permanent microscope slides and 16 females and 6 males fixed with DESS mixture and submitted for molecular study .Specimens of Measurements see Relaxed specimens arcuate, more curved ventrally at posterior end. Body tapering slightly anterior to base of pharynx but more sharply toward posterior end. Maximum body width at the level of vulva. Cuticle smooth, 4.3 (3.1\u20135.1) \u03bcm thick at the base of pharynx, subcuticle with faint transverse striae. Sublateral, subdorsal, and subventral body pores distinct along the entire body excluding tail. Tail with four pairs of sublateral caudal pores.Lip region rounded, 3.4 (3.0\u20134.0) times as wide as high, offset by a depression, wider than outline of the adjacent body. Labial and cephalic papillae conical, protruding beyond of the body contour, cephalic papillae comparatively more prominent. Amphidial fovea cup-shaped, its aperture relatively large, 7.3 (6.4\u20139.5) \u00b5m wide, located 13.5 (12.0\u201316.0) \u00b5m from the anterior body end. Buccal cavity spacious, oval-shape, 2.0 (1.9\u20132.2) times as long as wide, with funnel-shaped base. Dorsal tooth small, at the vertical dorsal plate of the buccal cavity, above the beginning of the pharynx. Apex of dorsal tooth located 28.1 (24.7\u201330.3) \u00b5m from anterior margin of buccal cavity or 47\u201360% its length from anterior end, opposite to it lies a thin, subventral longitudinal ridge. Nerve ring encircling cylindrical, muscular pharynx at about 29.4% (27.7\u201331.5%) of its length. Excretory pore and the ampulla well marked. Pharyngo-intestinal junction non-tuberculate. In the intestine content visible nematodes, diatom shells, earthworm setae, and detritus. Rectum wide and arcuate, 0.8 (0.7\u20131.0) times the anal body diameter long. Tail conical, ventrally bent, regularly tapering, with rounded tip. Hyaline part well developed, 12.9 (9.4\u201318.6) \u00b5m long. Caudal glands and the terminal opening absent.pars dilatata with a distinct lumen, it is connected with the uterus by a muscular sphincter having a heavily sclerotized inner part, uterus, relatively long, contains numerous spermatozoids mainly in its distal part, sometimes sperm cells are also visible in sphincter and proximal part of pars dilatata. Vagina extending inwards for 37.4 (34.3\u201341.0) \u00b5m or 43.2% (37.3\u201354.4%) of the body diameter, pars proximalis vaginae longer than wide, pitcher-shaped, surrounded by strong circular muscles, pars refringens vaginae with strong sclerotized, teardrop-shaped pieces, 13.8 (11.2\u201315.6)\u2009\u00d7\u20094.8 (4.1\u20136.0) \u00b5m, pars distalis vaginae very short, thickened at the junction with pars refringens vaginae, Vulva as a transverse slit, sometimes the vulval lips extend beyond the body contour. Small, ventromedian vulval papillae, one to three on each side of vulva are visible.Genital system didelphic-amphidelphic, the sexual branches almost equally developed, anterior 348 (256\u2013423) \u00b5m long or 12.1% (9.3\u201314.6%) of body length, posterior 349 (297\u2013423) \u00b5m long or 12.1% (10.5\u201316.3%) of body length. Ovaries on alternate sides of intestine, reflexed and well developed with numerous oocytes, not reaching the uterus-oviduct junction, oviduct takes the form of a slender tube, which then expands to form a well-developed Genital system diorchic, with opposed testes. Sperm cells elongate spindle shaped. Ejaculatory glands in tandem, generally distinct. Spicules total length along arc 1.3\u20131.4 times that at chord, 1.6 (1.4\u20131.8) times longer than body diameter at cloacal aperture; relatively thin, 8.4 (8.1\u20138.6) times longer than wide, ventrally curved, curvature 105.6\u00b0(104\u2013109.5\u00b0), median piece marked between hump and lateral guiding pieces, occupying 26.3% (22.0\u201330.1%) of spicule total length. Dorsal contour arcuate, ventral side with moderately expressed hump and hollow, the former located at 30.2% (26.4\u201332.9%) of spicule total length from its anterior end, head narrow, on a dorsal side offset by a shallow depression, proximal part of spicules almost cylindrical, posterior end bifurcate, 4.9 (4.2\u20135.3)\u2009\u2009\u00b5m broad. Lateral guiding pieces with arched edges and a bifurcate terminus, this furcation is symmetrical and well-marked. Gubernaculum well developed, 41.6 (36.2\u201346.3)\u2009\u2009\u00b5m long. Ventromedian supplements 21\u201324 in number, conical, regularly arranged, occupies 13.1% (11.8\u201313.9%) of body length. Above the supplements two cuticular pores are visible, the posterior most situated at 15.6 (13.8\u201316.6) \u00b5m from cloacal aperture, distance between the first and last supplement 360 (317\u2013399) \u00b5m. Parallel to the supplements, on both sides of them, five to six subventral papillae of varying sizes are visible, largest one lies at the level of the anus. Almost at half of the tail length, on its ventral side a large medioventral caudal papilla is situated, below it lies one pair of subventral and two pairs of subdorsal papillae.P. zschokkei. Three of the 18S rDNA sequences differed from the other eight in 1 extra nucleotide. All the acquired 28 rDNA sequences were identical. 18S rDNA: MT799667\u2013MT799668 (approx. 1.6\u2009kb); 28S rDNA: MT799672 (1\u2009kb).The phylogenetic relationships of the acquired sequences are presented in Both, the 18S and 28S rDNA \u2013 derived sequences were acquired from 11 independent nematode individuals belonging to Parkellus have been recorded. The following key is modified from Currently, 12 species of the genus 1.\u2013 Length of buccal cavity less than 61\u2009\u00b5m............\u20092\u2013 Length of buccal cavity more than 65\u2009\u00b5m.........\u20099P. mucronatus2.\u2013 Tail tip mucronate; gubernaculum thick, hooked proximally.......................................\u2009\u2013 Tail tip without mucro; gubernaculum not hooked proximally..........................................................\u200933.\u2013 Tail terminus slender, acute orsubacute.............\u20094\u2013 Tail terminus conspicuously rounded.................\u20095c\u2032\u2009=\u20093.5\u20134.0.................................................\u2009P. cobbi4.\u2013 Position of dorsal tooth apex 60\u201361% of buccal cavity length from its anterior end; c\u2032\u2009=\u20092.5\u20133.4................................................\u2009P. silvius\u2013 Position of dorsal tooth apex 50\u201353% of buccal cavity length from its anterior end; a is close to 40).............................................\u2009P. simmenensis5.\u2013 Tail in posterior third cylindroid with broadly rounded terminus; body slender (ratio a is close to 30).....\u20096\u2013 Tail uniformly narrowing with finely rounded terminus; body not so slender ; dorsal tooth rounded in contour.......... \u2013 Tail strongly curved (90\u00b0 or more); dorsal tooth more or less pointed..............................................\u200911P. acuticaudatus11. \u2013 Buccal cavity shorter (66\u2009\u00b5m); tail tip acute. .....................................................\u2009P. monticola\u2013 Buccal cavity longer (70\u201375\u2009\u00b5m); tail tip rounded......................................................\u2009Parkellus speciesAnalysis of interspecific sequence variation and phylogenetic relationships of the investigated Parkellus species were analyzed in this study: P. zschokkei from Ukraine, P. hagiangensis sp. nov. and P. tuyenquangensis sp. nov from Vietnam. These are the first molecular data available for the genus Parkellus. The interspecific nucleotide variation within the acquired 18S rDNA sequences was: P. zschokkei vs P. tuyenquangensis sp. nov.\u2009=\u20090.04, P. zschokkei vs P. hagiangensis sp. nov.\u2009=\u20090.02 and P. tuyenquangensis sp. nov. vs P. hagiangensis sp. nov.\u2009=\u20090.01. The interspecific 28S rDNA variation was: P. zschokkei vs P. tuyenquangensis sp. nov.\u2009=\u20090.05, P. zschokkei vs P. hagiangensis sp. nov.\u2009=\u20090.04 and P. tuyenquangensis sp. nov. vs P. hagiangensis sp. nov.\u2009=\u20090.02.Molecular sequences of three Parkellus species clustered together in a monophyletic group and were positioned within the M3 clade , the DNA distance between Coomansus and Parkellus are at the same level (3\u20135%) as the one between Prionchulus and Clarkus. Moreover, the Coomansus gerlachei species, currently represented only by the sequences deriving from the work of zschokkei-group\u2019 was already mentioned by Coomansus and Parkellus should be investigated in more detail in further research on Mononchida.Our research delivers so far the only molecular data that are available for the genus ecularly . That isParkellus from Vietnam, Parkellus hagiangensis sp. nov. and Parkellus tuyenquangensis sp. nov. are discovered based on the morphological and molecular characteristics. Their phylogenetic position in between Mononchida is fitted well within the M3 clade.Two new species of the genus"} +{"text": "Iotonchus lotilabiatus n. sp. from Bat Xat Nature reserve in Lao Cai Province, Vietnam is described and the molecular data (18S and 28S rDNA) are given. Females of the new species are characterized by large body size (L\u2009=\u20093.8-5.2 mm); barrel shaped buccal cavity of large size (41\u201354\u00d770\u201389 \u00b5m) with dorsal tooth apex located 22 to 23.5% of buccal cavity length from its base; reproductive system didelphic-amphidelphic, vagina long, corresponding almost half of body width at the vulva with distinct parrefringens vaginae, sclerotized and triangulated pieces in optical section; tail long filiform, ventrally arcuate, with three small caudal glands in tandem and prominent terminal spinneret. Males with spicules 119 to 148 \u00b5m in length, and slender, slightly curved gubernaculum, 39 to 44 \u00b5m long and with 13 to 14 ventromedian supplements. The new species Iotonchus lotilabiatus n. sp. is closest to I. miamaensis , but differs by having larger size of body length and buccal cavity, lower position of dorsal tooth apex; longer female tail with lower c but higher c\u2032 ratios and the presence of advulval cuticular structures.The new species Iotonchus was proposed by Mononchus into five subgenera Mononchus, Prionchulus, Mylonchulus, Iotonchus, Anatonchus, and ultimate raised in rank to genus level. The known species became the type species of the genus as I. gymnolaimus , I. montanus , I. spinicaudatus , Vietnam. Observations of morphological diagnostic features and light microscope were taken with a Nikon digital camera mounted on a Nikon Eclipse Ni microscope. Illustrations were drawn using a Nikon Eclipse Ni microscope equipped with a Nikon Y-IDT drawing tube. Photographs and illustrations were edited by using Adobe Photoshop CC 2018.They were heat killed, fixed in 4% formaldehyde or in a DESS mixture for mol, and mouIontonchus lotilabiatus n. sp. rDNA sequence fragments were deposited in GenBank under the following accession numbers: MW218936-MW218937 (18S rDNA) and MW218934-MW228378 (28S rDNA).Nematode DNA was extracted from a single individual as described by Iotonchus lotilabiatus sp. nov. and available sequences of Mononchida representatives from GenBank. The prepared multiple alignments of 18S and 28S rDNA generated by the ClustalW algorithm were routinely manually edited in order to eliminate improper phylogenetic signals. The phylogenies were constructed with the program MEGA 7 version 7.0. Maximum likelihood with T92+G substitution model for both, 18S and 28S data sets was used.For phylogenetic relationships, analyses were based on 18S and 28S rDNA. The newly obtained rDNA sequences were analysed using the BioEdit sequences avalaible in GenBank using the ClustelW aligment tool implemented in the MEGA 7 version 7.0 . The finIotonchus lotilabiatus n. sp. No. 1; four paratype femaleson slides Iotonchus lotilabiatus n. sp. No. 2-5, and six paratype males on slides Iotonchus lotilabiatus n. sp. No. 6-11 in permanent mounts in glycerin. All slides have been deposited in the nematode collection at the Department of Nematology, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology (VAST), Vietnam.Holotype female, four paratype females and six paratype males in good condition of preservation. Holotype female on slide Soil around the roots of random forest trees in the Bat Xat Nature reserve , Bat Xat District, Lao Cai Province, Vietnam.The specific epithet refers to the morphology of the labial region that characterizes this species.Measurements: see Large size of nematodes. Body ventrally arcuate after fixation. Body tapering slightly anterior to vulva position but more sharply toward posterior end. Maximum body width at the level of vulva. Cuticle smooth, 4.7 (3.4-5) \u00b5m thick at the base of pharynx, sub-cuticle distinctly striated. Labial region offset by a depression from the body contour, 2.8 to 3 times as wide as high. Labial region slightly separated six lips in the shape of lotus-petals with prominent labial papillae. Anterior sensilla arranged in two circles: an anterior one of six inner labial papillae, posterior crown with six outer labial papillae and four cephalic papillae protruding beyond the body outline. Buccal cavity begins at 19-22 \u03bcm from the anterior end of the body, large size, 1.6-1.8 as long as wide, barrel shaped, its wall strongly sclerotized, vertical plates parallel, basal oblique plates flattened. Amphidial fovea small, cup-shaped, with oval aperture 4-5 \u03bcm wide, located around at the beginning of buccal cavity or slightly lower. Dorsal tooth small size, located at the base of vertical plates, apex pointed forward, at 22.5 (21.6-23.4) % its length from the base of buccal cavity. Two small but prominent foramina present at the base of the buccal cavity. About the posterior fifth of buccal cavity embedded in pharyngeal tissues.Body diameter length at the pharynx base about 1.2 to 1.4 times head width. Pharynx cylindrical, surrounded by the nerve ring, located at 26 (24-27) % of its length, measured from the anterior body end, respectively. Secretory-excretory (SE) pore conspicuous, situated just posterior to nerve ring at 29 (28-30) % of its length from anterior body end. Pharyngo-intestinal junction tuberculate; cardia with conoid projection into wide intestinal lumen. Distance between pharynx base and vulva 1.7 to 2.0 times of the pharynx length. Rectum slightly curved, as long as anal body width. Tail long filiform, 0.7 to 1 mm long, ventrally curved, correspondence 16 to 22% of body length or 12.2 to 19.0 anal body diameters long; three small caudal glands in tandem and opening on terminal tail.pars dilatata, connected to the uterus with a muscular sphincter. Vulva a transverse slit, situated on the posterior half of the body. Zero to one pre- and one to two conspicuous post advulval papillae. Vulva-anus distance 1.4 to 1.7 times its tail length. Uterus in one paratype female with two large eggs (177 \u03bcm long\u2009\u00d7\u200963.4 \u03bcm wide in anterior branch and 200.4 \u03bcm long\u2009\u00d7\u200961 \u03bcm wide in posterior branch). Vagina length corresponds to almost half of body width. Pars proximalisvaginae as long as wide, gradually expanded in distal part, surrounded by strong circular muscles. Pars refringensvaginae appearing as two prominent, triangular sclerotized pieces (6.7\u00d77 \u00b5m) in longitudinal optical section. Pars distalisvaginae short. Vulva transverse in ventral view.Genital system typical for the genus, didelphic-amphidelphic, both branches about equally developed: anterior branch occupies 9.4 (8.9-10.4) % of body length and posterior branch occupies 9.0 (8.5-10.2) % of body length. Ovaries reflexed, 100 to 145\u03bcm long in anterior branch and 85 to 120 \u03bcm long in posterior branch; oocytes arranged in a row except at its tips. The uterus is relatively long, 170 to 240 \u03bcm long in each branch; sphincter present at the oviduct-uterus junction, valve sclerotized. Oviduct length 133 to 203 \u03bcm in anterior branch and 123 to 200 \u03bcm in posterior branch, with well-marked Males are similar to females in general morphology, but more strongly curved to coiled in the posterior region. Buccal cavity smaller than females, 36\u201341\u00d761.5\u201367 \u03bcm. Genital system diorchic, testes pared, outstretched and opposed, 1.2 to 1.7 mm long. Spicules slender, ventrally arcuated with bifurcate terminus, 2.1 to 2.4 times longer than body diameter at cloacal aperture and 8 to 11 \u03bcm long at the widest part. The head of spicule round-shape, 13 to 19 \u03bcm long and 7.5 to 9.5 \u03bcm wide; offset by shallow depression. The median piece is 104 to 115 \u03bcm long and 3.5 to 4.5 \u03bcm wide; round blade part. Lateral guiding pieces straight with bifurcate terminus, 18 to 24 \u03bcm long. This furcation is symmetrical and well-marked. Gubernaculum is well developed, 39 to 44 \u00b5m long (following Iotonchus lotilabiatus n. sp. is characterized by large adult body size (3.8-5.2 mm); long length of buccal cavity (69.5-88.5 \u00b5m); posterior position of dorsal tooth apex (21.6-23.4)% of buccal cavity length from its base; didelphic-amphidelphic with both branches equally developed, pars refringens vaginae sclerotized (6.7\u00d77 \u00b5m), intriangulated shape; the presence of small ventromedian papillary structures on the vulval region; the presence of males with long spicules (119-148 \u00b5m) with rounded head and blade part, gubernaculum (39-44 \u00b5m) and lateral guiding piece (17-24 \u00b5m) well developed; 13 to 14 ventromedian supplements.Iotonchus lotilabiatus n. sp. is similar to I. apapillatus (I. brachylaimus (I. miamaensis (L>3 mm), long length of buccal cavity (>65 \u00b5m), didelphic-amphidelphic, caudal glands opening on terminal tail and the presence of males. From I. apapillatus, it differs by having larger body length (3.8-5.2 vs 2.7-3.5 mm), slenderer nematodes (a\u2009=\u200949-59 vs 35-39); larger buccal cavity (41-54\u00d770\u201389 \u03bcm vs 40-44\u00d768-70 \u03bcm); lower c value (4.5-6.3 vs 10-13) but higher c\u2032 value (12.2-19 vs 4.3-5.8); vulval more anterior (V\u2009=\u200954.2-59.4% vs V\u2009=\u200962-74%), the presence of advulval cuticular structures and the number of ventromedian supplements in males (13-14 vs 17-19). The new species Iotonchus lotilabiatus n. sp. differs from I. brachylaimus by having larger body length (3.8-5.2 vs 3.2 mm), slenderer nematodes (a\u2009=\u200949-59 vs 33); longer length of buccal cavity (70-89 \u03bcm vs 64 \u03bcm); lower c value (4.5-6.3 vs 17) but higher c\u2032 value (12.2-19 vs 3.7); the presence of advulval cuticular structures; longer spicule length (130-141 vs 95 \u03bcm) and the number of ventromedian supplements (13-14 vs 16).In general appearance pillatus , I. brachylaimus , and I. amaensis based onI. miamaensis, it is distinguished by having larger size of body length (3.8-5.2 vs 3.2-3.5 mm); larger size of buccal cavity (41-54\u00d770-89 \u03bcm vs 42-45\u00d764-72 \u03bcm), more posterior dorsal tooth apex (21.6-23.4% vs 24-27%); longer female tail (0.57-0.97 vs 0.4-0.43 mm), lower c (4.5-6.3 vs 7.6-8.4) but higher c\u2032 (12.2-19.0 vs 7.5-8.3) ratios and the presence of advulval cuticular structures.From Iotonchus species morphometrics for comparative purposes ; I. longisaccatus ; I pharoensis , and I. terminus Buccal cavity length >50 \u00b5m (53-55\u00d730-31) \u2026\u2026\u2026\u2026.\u2026 Buccal cavity length <50 \u00b5m \u2026\u2026\u2026\u2026\u2026\u2026\u2026 12acuticaudatus ; \u2642: unknown \u2026\u2026..\u2026\u2026..\u2026.. ilvallus barqrii ; \u2642: unknown \u2026\u2026...\u2026\u2026. barqrii recessus V<66 \u2026\u2026.............................................................. 17L\u2009=\u20092 mm; V\u2009=\u200963-64 .\u2026.........................................................................\u2026 lacuplanarum ; \u2642: unknown ............ ...................................... nsimilis gymnolaimus ; \u2642: unknown \u2026.........................................\u2026\u2026\u2026. nolaimus , 1916Post-uterine sac completely absent \u2026\u2026\u2026\u2026 22Post-uterine sac present \u2026\u2026.......................... 26L\u22641.3 mm \u2026\u2026\u2026\u2026...\u2026 23Shorter body length: L>1.3 mm \u2026.........................................................\u2026\u2026\u2026. trichurus \u2026\u2026\u2026\u2026......................................................... burensis Buccal cavity length >30 \u00b5m \u2026\u2026\u2026\u2026\u2026\u2026. 24V\u2009=\u200968-72; larger buccal cavity\u2009=\u200936-40\u00d721-29 \u00b5m; \u2642: unknown ............................\u2026\u2026\u2026\u2026.\u2026 \u2026...\u2026.................... nepotum , labial papillae weakly developed; buccal cavity flattened at base \u2026\u2026\u2026\u2026... ongensis damsanensis (Lip region higher (21-22 \u00b5m); labial papillae prominent, protruded; buccal cavity narrowing posteriorly \u2026\u2026\u2026....................................................................\u2026 sanensis L\u2009=\u20092.2-2.5 mm; slenderer: a\u2009=\u200936-43; c\u2009=\u200934-41; buccal cavity\u2009=\u200944-48\u00d728-33 \u00b5m \u2026\u2026\u2026\u2026... aequabilis Caudal spinneret subterminal \u2026\u2026\u2026\u2026..... 51Caudal spinneret terminal \u2026........................\u2026 58Caudal spinneret subdorsal .................\u2026\u2026\u2026. 52Caudal spinneret subventral \u2026\u2026\u2026.\u2026\u2026...\u2026 54mboticus Tail>14 anal body diameter long \u2026......................................................\u2026..\u2026 Tail<14 anal body diameter long \u2026\u2026\u2026.....\u2026 53c\u2032\u2009=\u20097-8; V\u2009=\u200957; buccal cavity\u2009=\u200946\u00d722 \u00b5m; \u2642: unknown \u2026\u2026.. rayongensis Vulva anteriorly: a\u2009=\u200950-57; c\u2032\u2009=\u200917-18; tail terminus conoid-rounded; caudal pores absent \u2026\u2026\u2026...\u2026\u2026\u2026..\u2026 kilumicus V\u2009=\u200960-65; buccal cavity\u2009=\u200958-60\u00d738-40 \u00b5m \u2026\u2026\u2026\u2026\u2026.............................................\u2026. sagaensis , GenBank accession No. MW218936, MW218937 and two 747 bp length D2D3 expansion of rRNA (28S), GenBank accession No. MW218934 and MW228378 were obtained for phylogenetic analyses. This is the first sequences of both 18S and 28S rDNA are provided for a representative of the genus Iotonchus and submitted to GenBank. There are only a few sequences of super family Anatonchoidea presented on the Genbank.Molecular sequences of two individuals of Iotonchus lotilabiatus n. sp. revealed 99% similarity to unidentified species Miconchus sp. 1JH\u20132014 (accession No. KJ636436) and 98% similarity to Anatonchus tridentatus . Based on the 28S rDNA sequences, the new species Iotonchus lotilabiatus n. sp. only identically 89 to 94% to sequences of Anatonchus tridentatus .A BLAST search for matches to the partial 18S rDNA sequence of new species Iotonchus lotilabiatus n. sp. were positioned within the M4 clade (following the nomenclature of Anatonchusand Miconchus (Anatonchoidea super family) and 6. TIotonchus lotilabiatus n. sp. is supported by its morphological and molecular characterization. An updated key to species of the genus Iotonchus (During this study, the new species otonchus based on"} +{"text": "Prionchulus jonkershoekensis n. sp. is described from South Africa and illustrated using morphological, morphometric, and molecular techniques. This species is characterized by its body length (1.78\u20132.14\u2009mm); the size of buccal cavity (38\u201344\u2009\u00d7\u200924\u201331\u2009\u00b5m), lower dorsal tooth position in relation to buccal cavity base, the position of amphidial aperture just above dorsal tooth apex, pars proximalis vaginae with almost straight walls, and tail 144\u2013158\u2009\u00b5m long with sickle shaped posterior third part. Phylogenetic analyses based on 18\u2009S rDNA and 28\u2009S rDNA of P. jonkershoekensis n. sp. revealed close relationships of the new species with Prionchulus punctatus and Prionchulus muscorum. This is also an additional geographical record for the genus from South Africa. PrionchulusPrionchulus have been revised and updated by different scientists in the last decade Species belonging to the genus The genus has a cosmopolitan distribution, with Europe being particularly rich in species . To datePrionchulus was collected. A thorough investigation revealed that this was an undescribed species, and is described herein as Prionchulus jonkershoekensis n. sp. This species was characterized using both morphological and molecular techniques, its phylogenetic relationships are also discussed based on 18\u2009S and 28\u2009S rDNA genes.During a survey of the Jonkershoek Mountains in Stellenbosch, South Africa, samples were collected from seven localities to study nematodes associated with leaf litter. Among the various nematodes extracted, one population of Prionchulus jonkershoekensis n. sp. specimens were extracted from leaf-litter samples collected from one locality in the Jonkershoek Mountains by using the Baermann tray method (y method . NematodPrionchulus specimens were made with the aid of a Nikon 80i microscope equipped with a drawing tube. Micrographs were taken with an automatic camera system mounted (Axiocam ICc 5) on Zeiss Axiophot microscope. Using the drawings made with a drawing tube as a template, digital drawings were constructed in CorelDRAW X5.Measurements and drawings of mounted Mounted specimens were removed carefully from slides, hydrated in distilled water, dehydrated in a graded ethanol series, critical point dried, and coated with gold , and obswww.vacutec.co.za). Two nuclear loci were amplified and sequenced: partial 18\u2009S rDNA (SSU) using primers SSU F04 (5\u2019\u2013GCTTGTCTCAAAGATTAAGCC\u20133\u2019), and SSU R26 (CATTCTTGGCAAATGCTTTCG) (www.inqaba-southafrica.co.za).Genomic DNA was extracted from a single female specimen using the modified Chelex method as described by GCTTTCG) ; partialGCTTTCG) . The PCRwww.geneious.com) and consensus sequences were extracted and used for further analyses. The newly obtained consensus sequences of 18\u2009S and 28\u2009S rDNA genes were compared to those available for other species in Genbank using BLAST search. Two data sets were prepared for 18\u2009S and 28\u2009S genes using available mononchid sequences in Genbank; newly obtained sequences were also included. Both 18\u2009S and 28\u2009S rDNA datasets were aligned using MUSCLE (www.geneious.com). The General Time Reversible model with a Gamma distribution (GTR\u2009+\u2009G) was selected as the most appropriate nucleotide substitution model for both data sets according to jModelTest 2.1.10 analyses were conducted using MrBayes v3.1.2 implemenPrionchulus jonkershoekensis n. sp. nematodes, 1.78\u20132.14\u2009mm long. Body cylindrical, tapering toward tail terminus. Habitus ventrally curved at posterior end upon fixation, J-shaped. Cuticle moderately thick, with two layers, outer layer thinner than inner layer, with fine transverse striations. Cuticle appears more thickened at vulva and caudal region (2\u20134\u2009\u00b5m). Lateral chord 27\u201338% of midbody diameter. Body pores obscure. Lip region rounded, offset by weak depression but of similar width with adjacent body, about 3\u20134 times as wide as high. Lips amalgamated, rounded to angular. Labial papillae slightly more conical and protruding than cephalic papillae.Amphidial fovea cup-shaped, opening at level of anterior denticle above dorsal tooth apex, 3\u20136\u2009\u00b5m in diameter and occupying 1/5\u20131/6 of lip region width. Stoma consists of a vestibulum and a buccal cavity 1.4\u20131.7 times as long as wide, with 2\u20134\u2009\u00b5m thick walls and tapering at its base, funnel shaped. Dorsal tooth of medium size situated in lower anterior half of buccal cavity, its apex sharply pointed, located 30\u201334\u2009\u00b5m or 75\u201381% of the buccal cavity length from its base, its upper edge slightly curved, directed forward. Subventral denticles present, 11\u201314 in number, relatively large, orientated anteriorly, separated, arranged irregularly in some specimens. Two small foramina observed. Pharynx muscular, cylindrical, about 8\u201310 times as long as wide, surrounding basal part of stoma. Pharyngeal gland nuclei and outlets indistinct. Nerve ring located at 29\u201333% of the neck length. Secretory-excretory pore visible, situated 30\u201338% of the neck length. Cardia oval to rounded, smooth, 0.3\u20130.5 times as long as wide, 0.2 times as long as body diameter at neck base, non-tuberculate. Intestine uniformly granulated; inner surface lined by short rod-like structures (bacillary layer).pars dilatata, 40\u201362\u2009\u00b5m in width. Uterus and oviduct separated by a moderate sphincter constriction. Uterus thick-walled, bipartite, 0.5\u20131.0 times as long as body diameter. Sperm absent. Vagina extending over 33\u201347% of body diameter. Pars distalis vaginae short 4\u20136\u2009\u00b5m long with rounded walls. Pars refringens vaginae with well-developed teardrop-shaped sclerotized pieces, with combined width of 10\u201314\u2009\u00b5m, extent along the lumen of 6\u20138\u2009\u00b5m. Pars proximalis vaginae 14\u201322\u2009\u00b5m long, longer than wide, with almost straight contours proximally but curved outwards distally, surrounded by circular muscles. Vulva slit transverse, with lips slightly protruding. Eggs ovoid, smooth egg shell. Rectum 36\u201348\u2009\u00b5m long, mostly shorter than anal body diameter. Three unicellular rectum glands present.Genital system didelphic-amphidelphic. Genital branches almost symmetrical; anterior branch 300\u2013390\u2009\u00b5m and posterior branch 252\u2013362\u2009\u00b5m long. Ovaries reflexed, well developed, similar; anterior ovary 124\u2013218\u2009\u00b5m and posterior ovary 124\u2013186\u2009\u00b5m long; not reaching oviduct-uterus junction. Oviduct with well-marked Tail conical, 144\u2013158\u2009\u00b5m long, bent ventrally, distal 1/3 more curved ventrad, sickle shaped, tapering to finely pointed terminus. Hyaline portion 7\u2009\u00b5m long, 5% of tail length. Caudal glands absent. Three pairs of caudal pores present.Males: Not found.The type material was collected from leaf litter and moss near the First Waterfall hiking trail in the Jonkershoek mountains, Stellenbosch, South Africa .Holotype female (slide 51180), and one female and three juvenile paratypes (slide 51181) deposited in the National Collection of Nematodes (NCN), Agricultural Research Council-Plant Health and Protection (ARC-PHP), Pretoria, South Africa; 23 female paratypes deposited in the nematode collection of the University of the Free State (UFS), Bloemfontein, South Africa.The specific epithet refers to the geographical origin of the new species in the Jonkershoek Mountains in South Africa.Prionchulus jonkershoekensis n. sp. is characterized by females having medium body length (1.78\u20132.14\u2009mm); labial papillae conical, slightly larger than cephalic papillae; buccal cavity length 38.0\u201344.0\u2009\u00b5m, buccal cavity less than 1.7 times as wide as high; pars proximalis vaginae with almost straight walls; pars refringens vaginae with teardrop-shaped sclerotized pieces with smooth surface; tail 144\u2013158\u2009\u00b5m long, with terminal part ventrally curved, sickle shaped. Moreover, the novelty of the species was confirmed based on sequence of both 18\u2009S and 28\u2009S rDNA genes that revealed enough differences to the other closely related species and by 16\u201318\u2009bp differences from P. punctatus in the 18\u2009S rDNA gene . The new species also differs from P. punctatus by 60\u2009bp of the 28\u2009S rDNA gene (accession number MG994945). Due to lack of 28\u2009S rDNA sequences of P. muscorum in public sequence databases, this species was not included in the phylogenetic analysis of that gene.unctatus Andr\u00e1ssyP. jonkershoekensis n. sp. from other species of the genus. Prionchulus jonkershoekensis n. sp. resembles P. muscorum, P. hygrophilusP. punctatus in size, lip region rounded and offset by slight depression, position of the vulva (V\u2009=\u200959\u201367%), and an a-value (a\u2009=\u200923\u201329). However, it differs from P. punctatus in the number of subventral denticles (11\u201314 vs. 16), b-value (b\u2009=\u20093.0\u20133.5 vs. 3.6\u20133.9), neck length (580\u2013610 vs. 360\u2013475\u2009\u00b5m), teardrop shaped pars refringens vaginae vs. triangular, tail sickle shaped strongly curved ventrad with finely pointed terminus vs. plump tail with broadly rounded tip, and egg surface smooth vs. echinulate; from P. hygrophilus it differs in buccal cavity length (38\u201344 vs. 48\u201349\u2009\u00b5m), buccal cavity length to width ratio 1.4\u20131.7 vs. 1.8\u20132.0, vulval lips protruding vs. not protruding, c-value (c\u2009=\u200912\u201314 vs. 10\u201312), c\u2019-value (c\u2019\u2009=\u20092.9\u20133.5 vs. 4.2\u20135.0), and shorter tail (144\u2013158 vs. 173\u2013207\u2009\u00b5m); from P. muscorum it differs by smaller body size [1.78\u20132.14 vs. 2.50\u20133.50\u2009mm and 98% similarity to Coomanus parvus Jairajpuri & Khan, 1977 (accession number AY284767). Based on 28\u2009S rDNA sequence, the new species only showed 92% similarity to an unidentified isolate of Prionchulus (accession number KY750805) and 91% similarity to P. punctatus (accession number MG994945).During the survey, two sequences for each gene were generated. However, since they were identical only one sequence of 18\u2009S rDNA and one sequence of 28\u2009S rDNA were selected for phylogenetic analyses. A BLASTn search for matches to the partial 18\u2009S rDNA sequence of The 18\u2009S alignment was prepared using 47 sequences (739 nt long) in which 223 nt were variable, while the 28\u2009S alignment contained 23 sequences (760 nt long), of which 494 nt were variable.P. jonkershoekensis n. sp. closely with populations of P. punctatus and P. muscorum (P. punctatus (Prionchulus and Clarkus Jairajpuri, 1970 are closely related genera.Bayesian inference analyses using partial 18\u2009S rDNA sequences clustered muscorum . The 28\u2009unctatus . InteresPrionchulus representing a new species was found in South Africa and described herein as P. jonkershoekensis n. sp. using morphological, morphometrical, and molecular approaches. This also represents an additional geographical record for the genus Prionchulus. Two notable traits of the new species are its smaller buccal cavity dimensions and lower position of dorsal tooth. The phylogenetic position of P. jonkershoekensis n. sp. was resolved using sequences of partial 18\u2009S and 28\u2009S rDNA genes, however the monophyly of the genus Prionchulus was not established based on either of these genes. Although the new species shares some similarities with P. muscorum, phylogenetic analyses showed that this species and P. jonkershoekensis n. sp. are clearly distinct (During this study, a population of distinct ."} +{"text": "A 24-week non-blinded, randomized pilot study to assess the efficacy of subcutaneous exenatide 2.0\u00a0mg once weekly plus oral dapagliflozin 10\u00a0mg once daily (Group A) compared to a control group (Group B) in 56 patients with type 2 diabetes awaiting bariatric surgery was conducted . Both groups received an energy-deficit low-fat diet. The primary endpoint was the proportion of patients running off the criteria for bariatric surgery at the end of the follow-up period (BMI\u2009\u2264\u200935.0\u00a0kg/m2 or a BMI\u2009\u2264\u200940.0\u00a0kg/m2 plus an HbA1c\u2009\u2264\u20096.0%). Changes in the BMI were also of interest. The proportion of patients who ran off the criteria for bariatric surgery was larger in Group A than in the control group . Participants in Group A exhibited an absolute decrease in body weight and BMI of 8.1\u00a0kg (95%IC: \u2212\u00a011.0 to \u2212\u00a05.2) and 3.3\u00a0kg/m2 (95%IC: \u2212\u00a04.5 to \u2212\u00a02.2), respectively (p\u2009<\u20090.001 for both in comparison with Group B). A higher percentage of participants in Group A reached a BMI\u2009<\u200935\u00a0kg/m2 (45.8 vs 12.0%) and lost\u2009>\u200910% of their initial body weight (20.8 vs 0%) compared to Group B. The combination of exenatide plus dapagliflozin appears as a strategic option to reduce the waiting list for bariatric surgery, especially in those patients with type 2 diabetes.The glucagon-like peptide-1 receptor agonist family together with the renal sodium/glucose cotransporter-2 inhibitors have garnered interest as potential therapeutic agents for subjects with type 2 diabetes and obesity. In these patients, bariatric surgery is indicated based in a BMI\u2009\u2265\u200935\u00a0kg/m Obesity increases the risk of a variety of comorbid conditions, including hypertension, dyslipidaemia, obstructive sleep apnoea and type 2 diabetes2. Since type 2 diabetes is associated with obesity, and obesity is the main etiological cause of type 2 diabetes, the term \u2018diabesity\u2019 has been proposed3. Unfortunately, few safe and effective drugs are currently available for the treatment of obesity, leading to a marked increase in use of surgery to treat it in Western countries4. However, less than 2% of patients with severe obesity potentially eligible for bariatric surgery eventually undergo these procedures5. Therefore, a massive residual pool of patients with diabetes that meet medical guidelines to undergo bariatric surgery is still waiting for a solution.The prevalence of obesity has increased dramatically in recent decades and is more than 30% in some European countries7. In the human pancreas, actions of GLP-1 include the enhancement of glucose-dependent insulin synthesis and secretion, and stimulation of beta-cell proliferation7. In addition, GLP-1 also plays a role in regulating satiety, feeding behaviour, and body weight6. As compared with healthy controls, patients with type 2 diabetes and obesity have an attenuated secretion of GLP-1 in response to either an oral carbohydrated load or a mixed meal9. In this setting, the GLP-1 receptor agonist (GLP-1ra) family has garnered intense interest as a potential therapeutic agent for subjects with diabesity10.In recent years, there has been an increased understanding of the role of gastrointestinal hormones, such as the glucagon-like peptide-1 (GLP-1), in the control of glucose metabolism and body weight12. The SGLT2, located mainly in the first two segments of the proximal tubular system, mediates most of the renal reabsorption of glucose from the glomerular filtrate. Therefore, its therapeutic inhibition induces glucose excretion with urine, reaching two beneficial effects for patients with type 2 diabetes: glycaemic control and weight loss14.More recently, the emergence of inhibitors of the renal sodium-linked glucose transporter type 2 (SGLT2i) has provided more hope to patients with diabesityOn this basis, our purpose was to evaluate the percentage of patients with obesity and type 2 diabetes who no longer met the criteria for bariatric procedures after a combined therapy with both GLP-1ra plus SGLT2i added to an hypocaloric diet. Therefore, we designed a non-blinded, randomized, pilot study in patients with type 2 diabetes awaiting for bariatric surgery to assess the efficacy of subcutaneous exenatide 2.0\u00a0mg once weekly plus oral dapagliflozin 10\u00a0mg once daily, together with a low-fat, energy-deficient diet. The control group only received an energy-deficit low-fat diet.No differences at baseline were observed between both groups regarding the main anthropometry, clinical and metabolic parameters, nor in the measurement of quality of life. Table .Table 1Avs. 12.0%, p\u2009=\u20090.010) in comparison with the control group (Group B) (45.8% 2 (95%IC: \u2212 4.5 to \u2212 2.2), respectively, in comparison with participants in Group B (p\u2009<\u20090.001 for both) and 3.3\u00a0kg/m2 in comparison with the 12.0% of participants in Group B . Similarly, the percentage of participants who lost both\u2009>\u20095% or\u2009>\u200910% of their initial body weight were higher in Group A than in Group B. Similar results were detected for changes in anthropometric measurements when men and women were assessed separately.Waist circumference also experienced a significant decrease among participants receiving dapaglifloxin plus exenatide in comparison with the control group Table . Regardip B Fig.\u00a0. On the p\u2009=\u20090.097). However, the composite objective of\u2009>\u20095.0% decrease in body weight plus a\u2009>\u20091.0% decrease in HbA1c from baseline to 24-weeks of therapy was achieved in the 50% of patients under exenatide plus dapagliflozine in comparison with the 16.0% in Group B (p\u2009=\u20090.012). There was not a significant effect treatment in lipid profile, in blood pressure or in the IWQOL-Lite , respectively. Seven of the 29 AEs were related to gastrointestinal disorders (15.7% in Group A vs. 30.0% in Group B). In Group A, 4 AEs related to administration site conditions (21.0%) and 3 related to urinary tract infections (15.7%) were reported, with no cases in Group B. No participants in either treatment group experienced confirmed hypoglycaemia or hypotension, and neither AE caused treatment discontinuation. One patient in Group B experienced twice the same serious adverse event .16.To the best of our knowledge, the DEXBASU study is the first to provide clinical evidence that the combination of exenatide plus dapagliflozin exhibits a positive impact on patients with type 2 diabetes awaiting for bariatric surgery. This combination achieves a reduction by almost half the number of patients who meet the criteria for bariatric surgery through its double effect on weight and hyperglycemia. Considering that both severe obesity and type 2 diabetes will increase their prevalence in the coming years, and when bariatric surgery is accessible to a very limited number of patients, combinations of pharmacological treatments emerge as a valid option for both patients as for the National Health Systems4. In fact, bariatric surgery is an effective therapy for obesity with an acceptable safety profile and provides appropriate treatment for people who do not achieve the recommended treatment goals with medical therapies, especially when there are other important comorbidities such as type 2 diabetes 5. The International Federation for the Surgery of Obesity and Metabolic Diseases reported that almost 580,000 metabolic procedures were performed worldwide in 201417. Similarly, the American Society for Metabolic and Bariatric Surgery estimated that in the United States the initially 158.000 metabolic procedures performed in 2011 raised to 252.000 in 201818. However, although this exponential increase in obesity-related surgical procedures less than 2% of eligible patients are treated annually5. Determining factors to explain these data include lack of awareness of the surgical option in some communities and negative attitudes towards obesity, but also misunderstanding about optimal management of patients with obesity and type 2 diabetes and the role of metabolic surgery procedures in its treatment scheme. According to the 1991 National Institutes of Health guidelines patients with type 2 diabetes appears as candidates for metabolic surgery if their BMI is higher or equal to 35\u00a0kg/m219. This group of patients with type 2 diabetes mellitus and candidates for surgery have been the study population in the DEXBASU study.The disappointing medium and long-term results in body weight provided by dietary and behavioral treatment, as well as the dearth of drug treatment, have led to an exponential increase in use of surgery to treat obesity in Western countries21. Treatment-associated weight loss can be significant, with a low risk of hypoglycemia, and some molecules provide proven cardiovascular benefits, which differentiates these classes of drugs from most other antidiabetic agents22. Therefore, the combined treatment of GLP-1ra plus SGLT2i could be an attractive option for patients with type 2 diabetes and obesity awaiting bariatric surgery. However, no previous data was previously available in this particular setting.Both GLP-1ra and SGLT2i, with complementary mechanisms of action, have been shown to be very useful options in the treatment of type 2 diabetes, particularly in patients with obesity223. For 52\u00a0weeks the combination of exenatide once weekly plus dapagliflozin once daily showed greater average body weight loss than exenatide and dapagliflozin alone, with 37.7% of participants achieving an HbA1c\u2009<\u20097.0% and a 30.7% of participants who achieved weight loss\u2009\u2265\u20095.0% at the end of the follow-up period. These benefits had already been achieved by reaching week 28 and extended to 104\u00a0weeks after randomization25. In combination therapy participants, reductions in body weight were greater in women than in men, in patients with basal HbA1c\u2009\u2265\u20098.0% to\u2009<\u20099.0%, and as BMI increased26. In the DEXBASU study, in which all participants had a BMI\u2009\u2265\u200935\u00a0kg/m2, the percentage of subjects who managed to lose\u2009>\u20095% of their baseline body weight increased to 58.3%. In addition, no differences in the evolution of anthropometric measurements between men and women were observed in our study. Weight reduction with this drug combination is associated with a decresase in total volume, abdominal visceral and subcutaneous adipose tissue assessed by magnetic resonance imaging of body composition, and also with improved markers of liver steatosis such as fatty liver index and FIB-4 scores29. Combination therapy with exenatide and dapagliflozin may also have synergistic effects on markers of renal function compared with therapy alone or with placebo in obese patients with type 2 diabetes30. In an attempt to explain the possible beneficial effects of the combination of exenatide and dapagliflozin in poorly controlled type 2 diabetes, Ferrannini et al.suggested that the combination abolished the dapagliflozin-induced rise in the \u03b2-hydroxybutyrate, reduced the exenatide induced increase in fasting insulin-to-glucagon molar ratio, maintained glycosuria, and increased haematocrit, while mitigating the risk of ketoacidosis31.The DURATION-8 study recruited 695 patients (47.9% of men) with type 2 diabetes poorly controlled with metformin, with a mean BMI of 32.7\u00a0kg/m32. Unfortunately, we do not have data regarding the body composition of participants in the DEXBASU study.In patients with obesity without type 2 diabetes, increased body weight loss after 24\u00a0weeks with exenatide plus dapagliflozin was associated with lower basal adiposity and lower basal insulin secretion, as well as with the single-nucleotide polymorphisms rs10010131, a variant associated with the GLP-1 pathway33. Again, the result was a significant and clinically relevant improvement in glycaemic control and weight loss with dulaglutide 1.5\u00a0mg once weekly compared to placebo after a 24-week follow-up period. Similarly, in the SUSTAIN-9 trial, when 1.0\u00a0mg subcutaneous semaglutide was added once weekly to SGLT2i therapies for 30\u00a0weeks, HbA1c [\u2212 1.42% (95% CI: \u2212 1.61 to \u2212 1.24)] and body weight [\u2212 3.81\u00a0kg (\u2212 4.70 to \u2212 2.93)] were further reduced versus those randomised to volume-matched placebo34. Lastly, the efficacy and safety of insulin degludec/liraglutide (IDegLira) was evaluated when added-on to SGLT2i therapy in patients with inadequately controlled type 2 diabetes. IDegLira did not show inferiority with respect to insulin glargine 100 units/mL regarding to the average reduction in HbA1c, but superiority with body weight [\u2212 1.92\u00a0kg (\u2212 2.64 to \u2212 1.19)]35. In two recent meta-analysis, the GLP-1ra/SGLT2i combination was associated with an improved HbA1c control, along with enhanced weight reduction and control of blood pressure and lipid profile37. In the DEXBASU study, both groups achieved a similar reduction in HbA1c, which must be attributed to the intensification of glycemic control in both groups. The fact that the percentage of patients treated with pioglitzone (a peroxisome proliferator-activated gamma receptor agonist that is highly effective in the treatment of insulin resistance but is associated with adverse events such as weight gain and retention of liquids) in group B increased from 8.0% at the beginning to 60.0% at the end of the follow-up could help to better understand the differences in the evolution of weight.Different combinations, beyond exenatide and dapagliflozin, have been evaluated in randomized clinical trials. In the AWARD-10 dulaglutide was assesed as adjunctive therapy to SGLT2i in patients with inadequately controled type 2 diabetes238. After 6\u00a0months of follow-up, a significant reduction in HbA1c levels (\u2212 1.1%), BMI (\u2212 2.1\u00a0kg/m2) and systolic blood pressure (\u2212 13\u00a0mmHg) was observed. In addition, the greatest reduction in HbA1c levels and weight was observed in patients who started both drugs simultaneously.Although age is a limitation for bariatric surgery, Carretero G\u00f3mez et al.evaluated the real-world efficacy and safety of SGLT2i and GLP-1ra combination therapy in 113 patients with type 2 diabetes over 65\u00a0years with an average BMI of 36.5\u00a0kg/mThere are a some potential limitations that need to be considered when contemplating the results of our study. First, we evaluated a pretty minor number of patients with type 2 diabetes and obesity who were inclined to participate in a clinical trial, meaning that no irrefutable clinical consequences can be inferred to general population. Second, the treatment period was limited to 24\u00a0weeks, when we know that obesity is a chronic and recurrent condition. Therefore, longer-term studies are needed in the same population. Third, it was a pilot study with a control group lacking a placebo, which may weaken the power of our findings. Fourth, we cannot rule out that changes in the treatment of comorbidities, including diabetes, may have influenced part of the results.In conclusion, given the opportunity to achieve remarkable reductions in body weight and ameliorate glycemic control, without further risk of hypoglycemia, our data strongly support the GLP-1ra/SGLT2i combination as a strategic option in the management of patients with type 2 diabetes awaiting bariatric surgery. By providing a rational basis for designing new strategies for the treatment of patients with obesity and type 2 diabetes, the information obtained from the DEXBASU study may be useful in reducing the healthcare costs associated with diabesity and will contribute towards improving the quality of life of these patients.Spanish Agency for Medicines and Health Products as well as for the human ethics committee of the Arnau de Vilanova University Hospital (CEIC 2017/0801). All potential participants provisioned a written informed consent to join the trial, which was conducted according to the Helsinki Declaration and the Good Clinical Practice Guidelines. The DEXBASU trial has been registered in the European Clinical Trial Database . The reporting has been done following the CONSORT guideline for randomized trials39.The DEXBASU study was approved by the Centers of Obesity Management under the umbrella of the European Association for the Study of Obesity (COM-EASO). The study was open for recruitment from November 2017 to May 2020, when the target sample size was obtained. The study flow chart is displayed in Fig.\u00a0The DEXBASU study is a pilot, phase II, randomized, non-blinded study to assess the efficacy of subcutaneous exenatide 2.0\u00a0mg once weekly plus dapagliflozin 10\u00a0mg once daily in a 24\u00a0weeks duration trial in patients with type 2 diabetes awaiting bariatric surgery. The study was performed in the Arnau de Vilanova University Hospital of Lleida. This center is one of the 2 that were awaiting bariatric surgery. This implies a time of evolution of severe obesity greater than 5\u00a0years. The cut-off of 42.5\u00a0kg/m2 was an arbitrary set point. Other inclusion criteria comprised stable body weight (<\u20095% reported change during the previous 3\u00a0months) and HbA1c between 7.0 and 10.0%.Eligible participants included men and women aged 18\u201360\u00a0years, with type 2 diabetes and a body mass index (BMI) between 35.0 to 42.5\u00a0kg/m2, serum creatinine\u2009>\u20091.7\u00a0mg/dl, abnormal liver function test , pregnancy, breast-feeding and females of childbearing potential who were not using adequate contraceptive methods. Patients were also excluded from the trial if there was a history of acute or chronic pancreatitis, cholelithiasis, personal or familial history of medullary thyroid carcinoma or multiple endocrine neoplasia type 2, cardiovascular disease, heart failure and/or stroke, or recurrent urinary tract or genital infections. Patients enrolled in the study who initiated SGLT2i, GLP-1ar or oral corticosteroids were also excluded from the final analysis.Exclusion criteria included previous surgical obesity procedures, use of approved weight lowering pharmacotherapy, previous or active treatment with GLP-1ra, SGLT2i or warfarin, current drug or alcohol abuse, uncontrolled psychiatric illness, an estimated Glomerular Filtration Rate\u2009<\u200960\u00a0ml/min/1.73mNational Institutes of Health criteria for bariatric surgery comparing with placebo at the end of the 24-week follow-up period19. Therefore, the inclusion of 46 patients achieved a 80% of statistical power (\u03b2\u2009=\u20090.2), with a significant level of 5% (\u03b1\u2009=\u20090.05) to detect this significant increase. However, sample size was rised to a total of 56 patients according to an expected drop-out rate of 20% in relation to adverse drug effects.This was a pilot study in which we assumed that a GLP-1ra plus SGLT2i based therapy will lead to an absolute 30% risk increase of the rate of patients with type 2 diabetes and obesity running off the 2). Participants assigned to Group A received: (i) 2-week initial period with dapagliflozin 10\u00a0mg once daily, along with an energy-deficit low-fat diet, followed by (ii) 22-week of dapagliflozin 10\u00a0mg once daily plus subcutaneous exenatide 2.0\u00a0mg once weekly, along with a reduction in their daily energy intake to 500\u00a0kcal below their individual requirements. Participants assigned to Group B underwent a 24-week treatment period with a low-fat diet with about 500\u00a0kcal per day deficit below their individualized requirements, that were assessed using prediction equations. Qualified professionals prescribed the calorie reduction diet in person on day 0, and the advice was repeated at each visit throughout the study. No specific recommendations on increasing physical activity were given to the participants. All participants started the assigned treatment within 2\u00a0weeks of randomization. Twenty four participants from Group A and 25 participants from Group B completed the treatment period of 24\u00a0weeks , sulphonylureas (25.0 and 20.0%), dipeptidyl peptidase-4 inhibitors (50.0 and 44.0%), pioglitazone (0.0 and 8.0%), basal insulin (33.3 and 32.0%), and fast-acting insulin (4.1 and 0.0%) in Groups A and B, respectively. None patient was treated with diet alone.All subjects underwent treatment intensification to improve glycemic control according to our routine medical practice, except for the exclusion of SGLT2i and GLP-1ar. At the end of the study, the proportion of patients receiving metformin (91.6 and 92.0%), and fast-acting insulin (4.1 and 4.0%) in Groups A and B was almost the same. However, modifications were observed in those treated with sulphonylureas (16.0 and 36.0%), dipeptidyl peptidase-4 inhibitors (0.0 and 80.0%), pioglitazone (0.0 and 60.0%), and basal insulin (16.6 and 36.0%), respectively.National Institutes of Health criteria for bariatric surgery after the 24-week follow-up period 16. Mainly, proportion of patients who achieved a BMI\u2009\u2264\u200935.0\u00a0kg/m2 or a BMI\u2009\u2264\u200940.0\u00a0kg/m2 plus an HbA1c\u2009\u2264\u20096.0%.The primary endpoint was to assess the proportion of patients with type 2 diabetes awaiting bariatric surgery running off the (i) body weight, classification of weight category by BMI and waist circumference; (ii) percentage of patients who achieve both a 5% and 10% decrease in body weight; (iii) percentage of patients who achieve a decrease of 1% in their HbA1c level; (iv) proportion of patients who achieve the composite objective of both a\u2009\u2265\u20095% decrease in body weight plus a\u2009\u2265\u20091.0% decrease in HbA1c levels; (v) changes in blood pressure and lipid profile; and (vi) changes in quality of life and and proportion of patients that refuse the surgery option at the end of the follow-uop period.As secondary endpoints the study assessed the effects of treatment from baseline to 24-week on: 40. Waist circumference was measured with a non-stretchable tape with an accuracy of 0.1\u00a0cm, midway between the lowest rib and the iliac crest, on the horizontal plane with the subject in a standing position41. All measurements were performed by one experienced nurse, using the same device to avoid inter-observer and inter-device variability. The Impact of Weight on Quality of Life-Lite (IWQOL-Lite) was administered to quantitatively assess the individual\u2019s perception of how weight affects day-to-day life42.\u00a0It is comprised of 31 items grouped into five dimensions: physical functioning, self-esteem, sexual life, public distress, and work. The measure provides scores for each separate dimension and a total score.Weight and height were measured without shoes and light clothing with and standard equipment, to the nearest 0.5\u00a0kg and 1.0\u00a0cm, respectively. BMI was calculated as the ratio of body weight (kg) to the height (m) squared t test for continuous variables were used to assess differences between groups. All endpoints were analyzed longitudinally from randomization to 24\u00a0weeks and cross-sectional at 24\u00a0weeks. A chi-squared or exact Fisher test was done to compare the proportion of patients who don\u2019t accomplish the National Institutes of Health criteria for bariatric surgery at the end of the study. All the contrasts were bilateral with a threshold for statistical significance threshold set at 0.05 (p\u2009<\u20090.05). The data was analyzed with the Statistical Package for the Social Sciences software .A normal distribution of the variables was established using the Kolmogorov\u2013Smirnov test, and data are expressed as the mean\u2009\u00b1\u2009standard deviation, median (interquartile range), or as a frequency (percentage). A homogeneity analysis was undergone to compare patients in both groups with regards to basal clinical and anthropometric variables. Mann\u2013Whitney test for categorical variables and Student\u2019s Baseline description of the study population was made in the intention to treat population . Efficacy analyzes were performed on the per protocol population (restricted to participants who completed the study without major protocol deviations), whereas safety analyzes were carried out on the safety population (participants who received at least one dose of any study product)."} +{"text": "The smart engineering of novel semiconductor devices relies on the development of optimized functional materials suitable for the design of improved systems with advanced capabilities aside from better efficiencies. Thereby, the characterization of these materials at the highest level attainable is crucial for leading a proper understanding of their working principle. Due to the striking effect of atomic features on the behavior of semiconductor quantum- and nanostructures, scanning transmission electron microscopy (STEM) tools have been broadly employed for their characterization. Indeed, STEM provides a manifold characterization tool achieving insights on, not only the atomic structure and chemical composition of the analyzed materials, but also probing internal electric fields, plasmonic oscillations, light emission, band gap determination, electric field measurements, and many other properties. The emergence of new detectors and novel instrumental designs allowing the simultaneous collection of several signals render the perfect playground for the development of highly customized experiments specifically designed for the required analyses. This paper presents some of the most useful STEM techniques and several strategies and methodologies applied to address the specific analysis on semiconductors. STEM imaging, spectroscopies, 4D-STEM (in particular DPC), and in situ STEM are summarized, showing their potential use for the characterization of semiconductor nanostructured materials through recent reported studies. Advances in the development of novel and improved functional materials require deep characterization analyses to fully understand and exploit their physical properties. Within this context, transmission electron microscopy (TEM) is a powerful tool providing a broad variety of techniques, which allow in-depth analyses of the materials\u2019 micro-/nano-/atomic structure and composition, as well as addressing some related physical properties.TEM techniques may be first catalogued as those performed illuminating the region of interest using a parallel electron beam (TEM), and those employing an electron beam probe scanning the area of interest . Conventional and high-resolution (HR-) TEM imaging relies on diffraction and phase-contrast imaging of the transmitted electrons, providing a comprehensive picture of the microstructure of the materials. STEM-related techniques take advantage of different emerging signals from the electron\u2013matter interaction while scanning the electron probe over the sample, including chemical or opto-electronic analyses, among others.The emergence of novel electron sources, with increased brightness and stability, along with the appearance of aberration correctors and electron monochromators, have driven the development of advanced methodologies, particularly in STEM mode. The lat-est advances in terms of faster and better detectors, enabling detection down to a single direct electron with high speed read-outs , and theAnother primary distinction among imaging techniques distinguishes between bright field (BF) and dark field (DF) techniques, depending on whether the collected electrons are on-axis or off-axis, respectively, referred to the electron microscope optical axis. Interestingly, the image formation in HRTEM and BF-STEM relies on the same electron pathway with opposite trajectories, which is known as the principle of reciprocity . NowadayImportantly, different electron\u2013matter related signals can be monitored while work-ing in STEM. Therefore, different spectroscopic techniques can be implemented to address both, composition and opto-electronic properties. On one hand, measuring the energy lost by inelastically scattered electrons is an out standing technique with chemical capabilities, attaining information on bond-ing/coordination/oxidation states while it is also suitable to address opto-electronic properties and even vibrational modes . On the Moreover, either in situ sample holders, equipped with chips for electrical bias-ing/heating, etc., or environmental microscopes with sample chambers operating at high-er pressures than that of the TEM column, impose additional milestones for the study of nanoscale dynamic processes see .It is worth mentioning the relevant role of computational schemes to handle and un-derstand the obtained results. Software improvements and updates oriented to enhance acquisition and analysis skills are continuously growing. Particularly, machine learning approaches, due to their prediction ability, are a powerful tool within this context, with applications from structural analysis to data denoising, among many others see .Thereby, the unique capabilities of STEM along with the many operational modes render an ideal tool to face countless correlative studies, thanks to the struc-ture\u2013properties relationships attainable. In the case of semiconductor materials, there are many studies focusing on STEM characterizations to cover their structural properties, in-cluding structural defects, atomic ordering, polarity, and related phenomena such as growth mechanisms, strain relaxation, and quantum structures, as well as those ad-dressing the electronic and opto-electronic properties, as reviewed elsewhere for the The present review shows the potential of STEM techniques applied to semiconductor materials, ranging from atomic resolution imaging to electric field measurements, covering a variety of spectroscopies and in situ analyses to fully address the structure, composition, and functionality of the materials. The referred STEM techniques are briefly explained, and some of the last instrumental advancements and deriThe imaging capabilities of STEM techniques have been widely exploited over recent years in material science, providing valuable insights into subtle structural features, which influence the system properties. As already mentioned, the emergence of electron beam probe correctors and brighter electron sources enable atomic-resolution imaging in STEM mode under a variety of techniques. Probe correctors compensate for the aberrations of the electromagnetic lenses driving the electrons from the source towards the sample, providing sub-angstrom electron probes to scan the area of interest, which results in atomic resolution imaging . These tModern electron microscopes are usually equipped with at least two annular detectors with adjustable detection ranges, leading to the simultaneous performance of different techniques. The most commonly employed STEM imaging technique is high-angle annular dark field, HAADF, also referred to as Z-contrast imaging since the image contrast scales with the atomic number, Z, of the sample constituents as follows:Although \u03b1 values range from 1.2 to 1.8 as a function of the actual collection angle , and theNotably, there are many methodologies based on the analysis of atomic resolution images, which cannot be covered in detail here. For instance, atomic resolution images contain information on the lattice strain and relaxation mechanisms known to impact the performance of semiconductors, which can be analyzed by different approaches, as geometric phase analysis (GPA) or peak The poor imaging ability of HAADF to visualize light atoms due to their weak scattering power, especially when combined with heavier elements, drove the popularity of annular bright field (ABF) few years ago, proving its capability of imaging even the lightest element . In thisThere are other useful angular collection ranges other than HAADF and ABF, such as low-angle annular dark-field, LAADF. Under LAADF experimental conditions, involving DF of partially coherent scattered electrons . The characterization under ABF, HAADF and LAADF reveals the formation of Cu2ZnSnSe4\u2212xOx owing to O placed at Se sites near the grain boundary, leading to the observed strain-contrast arising from compositional differences (O content).The simultaneous acquisition of atomic-resolution images at different collection angles entails huge advantages for the design of specific methodologies. For instance, III-V semiconductors have been widely studied over recent years under HAADF-ABF atomic-resolution conditions to address the relative position of the atomic species within the crystal lattice of binary compound nanostructures . The ext2 (Mo), at the hexagon center of the MoS2 structure and on top of a S site (TS), classified as ground state (TMo) and metastable states according to their relative DFT-calculated energies. The most likely pathway for Mo diffusion within MoS2 is through the metastable H state.Excitingly, atomic-resolution STEM imaging, supported by ab initio calculations, can be applied to the study of dynamic processes such as atomic diffusion. Tracking the migration of atomic species or vacancies by means of sequential imaging (time-lapsed) allows for unrevealing the atomic pathways of diffusion phenomena, as in the case of a single Mo atom in MoS2 b 6]. Th. Th2 of the convergent beam electron diffraction (CBED) pattern, being linearly proportional to the (projected) electric field. Therefore, the recorded DPC signal corresponds to the COM movement, containing phase information. The 2D integration of the two components of the DPC image, leads to phase retrieval , and it ectively :(3)\u22072\u03a6, TMDs, offer an ideal playground for implementing DPC-STEM. For example, sub-angstrom resolution studies have proven electrostatic field fluctuations related to the location of crystal defects in TMD monolayers, where in-line vacancies lead to electron-rich areas of 1D conduction channels . Remarkably, EELS analyses may reach a sub-angstrom spatial resolution, along with a high energy resolution, achieving up to a few millielectronvolts if using monochromated electron probes. On one hand, modern instruments equipped with sophisticated electron guns and aberration correctors focus the scanning electrons into sub-angstrom bright probes. On the other hand, the implementation of monochromators at the microscope column, cutting out deviant wavelengths, improves the energy resolution down to <10 meV . AdditioThe exact amount of energy lost by the electrons irradiating the sample is characteristic of the atomic species constituting the material, providing an accurate analytical tool. Indeed, EELS capabilities are far beyond compositional analyses, as the energy onset and fine structure of the ionization edges contain information on the oxidation state, local coordination , or bondEELS measurements allow simultaneous ADF acquisition, and both spectroscopy and imaging may be performed with a sub-angstrom resolution, which is highly important for the study of heterostructures and interfaces. For instance, these sorts of analyses are useful for understanding electronic properties at heterojunctions, whose band alignments are affected by possible diffusion phenomena. The abruptness of interfaces at complex nanostructures is intimately related to the growth mode employed, and the coexistence of abrupt and graded interfaces within individual nanostructures is possible . For insIn addition to providing chemical identification and quantification, the spectral shape of the ionization edges at the core loss contains information on the bonding and coordination configurations, appealing for the study of atomic arrangements and crucial to fully understanding either intended or undesired doping and related consequences. The techniques applied to the study of single dopants (atomic species) have revealed different bonding configurations of individual atom impurities . As EELSThe lower energy range of an EEL spectrum reflects opto-electronic features arising from the interaction of the microscope electron probe with the outer electron shells of the atomic species composing the analyzed material. Low-loss EELS (LL-EELS) is therefore also referred to as Valence-EELS (VEELS) and is particularly interesting for the characterization of photonic materials, in order to assess their functional properties. In addition to interband transitions and band gap determinations, LL-EELS proves collective electron oscillations and as the latest state-of-the-art it has achieved vibrational spectroscopy, resolving spectral features within the IR range.2-II-VI-IV4 compounds the varying electrostatic potential induced in non-stoichiometric combinations has been reported to detrimentally shifting the band gap offset . T. T2 growters see .Remarkably interesting is the fact that accessing the materials structure at different experimental conditions brings the chance to explore metastable phases or phase-transition phenomena. As an example, recently published in situ STEM measurements address the structural origin of the metal-to-semiconductor transition observed in PbTe .Annealing processes can also be investigated dealing with heterostructured systems subject to solid-state reactions, such as those reported, for instance, for different Ge-based combinations. In situ experiments have shown thermally induced diffusion phenomena driving by the formation of Ge axial discs thinner than 10 nm within Al NWs, which are thermally induced In the case of the Ge-Cu material system, the annealing process via Joule heating (Ge NWs with Cu contacts) causes both germanium and copper diffusion in opposite directions, resulting in intermetallic phases (\u201cgermanidation\u201d) .The implementation of machine learning algorithms has enormous potential regarding the abovementioned techniques, resulting in an invaluable complement to experimental methodologies. Currently, different methods achieve successful structural phase identification, either based on supervised or unsupervised learning approaches , which aMachine learning algorithms also find applications facing spectral data, which is useful, for example, to distinguish the signal sub-space from the noise by dimensionality reduction of EDX data, which in turn improves the identification of light elements , thus ofx clusters to a triangular MoS2 clusters enables relating the extracted experimental patterns to individual atom sites and sublattices, successfully achieving single-dopant recognition, providing an efficient computational tool for image interpretation . MoreoveThis review evidences the usefulness of STEM-related techniques for the smart engineering of advanced semiconductor systems. The unique capabilities of STEM, in terms of spatial and spectral resolutions, along with the many meaningful signals arising from the electron\u2013matter interactions, result in a broad range of techniques, finding applications in countless methodologies. Nowadays, technological advances and faster computer-processing capabilities continue pushing the development of improved STEM instruments, driving some of the latest breakthroughs. Furthermore, this review highlights the valuable insights attainable by STEM methodologies applied to semiconductor materials, summarized through recent publications. The gathered examples illustrate the wide scope of characterization provided by STEM, allowing the performance of correlative studies strongly benefiting semiconductor materials research.The provided overview aims to encourage future in-depth semiconductor STEM characterizations, broadening the understanding of their behavior, alongside the smart engineering of better and novel functional systems."} +{"text": "Breast cancer is the most frequent cancer among women, and metastases in distant organs are the leading cause of the cancer\u2010related deaths. While survival of early\u2010stage breast cancer patients has increased dramatically, the 5\u2010year survival rate of metastatic patients has barely improved in the last 20\u2009years. Metastases can arise up to decades after primary tumor resection, hinting at microenvironmental factors influencing the sudden outgrowth of disseminated tumor cells (DTCs). This review summarizes how the environment of the most common metastatic sites is influenced by the primary tumor and by the varying dormancy of DTCs, with a special focus on how established metastases persist and grow in distant organs due to feed\u2010forward loops (FFLs). We discuss in detail the importance of FFL of cancer cells with their microenvironment including the secretome, interaction with specialized tissue\u2010specific cells, nutrients/metabolites, and that novel therapies should target not only the cancer cells but also the tumor microenvironment, which are thick as thieves. Metastases can arise decades after primary breast tumor resection. This review by M. Bentires\u2010Alj and colleagues describes how the environment of the most common metastatic sites is influenced by the primary tumor and the varying dormancy of disseminated tumor cells, with a focus on feed\u2010forward loops. Cancer dormancyCellular dormancy describes a reversible non\u2010proliferative state of a cancer cell that can last for several years. Tumor mass dormancy represents the offset of cancer cell proliferation by cell death, resulting in net\u2010constant cell numbers.Two forms of dormancy have been observed: ColonizationGrowth of micrometastases into macrometastases.Disseminated tumor cells (DTCs)Cancer cells that infiltrate and survive in distant sites. DTCs may succumb, remain dormant, or colonize the tissue.Extracellular Matrix (ECM)Three\u2010dimensional network surrounding cells, consisting of macromolecules and minerals, including collagens, glycoproteins, and cell adhesion proteins. The ECM provides essential structural support and serves diverse biochemical activities. Components of the ECM are produced intracellularly by resident cells and subsequently secreted into the extracellular space. The composition thus varies widely between organs and can be transiently remodeled upon physiological injury or chronic stimuli, including cancer/metastases.Extracellular Vesicles (EV)EV is a collective term covering a variety of cell\u2010derived membranous structures that encapsulate and transport cellular materials, and nearly, all cell types can produce them. EV cargo include proteins, lipids, microRNAs (miRs), mRNA, and noncoding RNAs. One example of EVs are exosomes. In the context of cancer, EVs released from primary tumors can establish a pre\u2010metastatic niche in distant organs.Inter\u2010site heterogeneityHeterogeneity between lesions in different sites. Inter\u2010site heterogeneity is generally used to describe cancer cells; however, it also applies to other cell types that are part of the tumor microenvironment.Intra\u2010site heterogeneityHeterogeneity within the same primary tumor or the same metastasis. Commonly, intra\u2010site heterogeneity is used for cancer cells only; however, it also applies to other cell types that are part of the tumor microenvironment.MetastasisMetastasis is a multi\u2010step process in which cancer cells invade surrounding tissues at the primary site, intravasate, and survive in the circulation as circulating tumor cells (CTCs) that extravasate at distant organs . Metastases are responsible for the majority of breast cancer\u2010related deaths.Minimal residual disease (MRD)This disease stage is when a patient is in remission, and only a small number of cancer cells have persisted therapy. Minimal residual disease can endure for several months or, in some cases, up to decades and represents a significant challenge for long\u2010term remission because it is a reservoir of cancer cells that can regrow anytime. Additionally, these cells are often of a more aggressive type because they are treatment\u2010resistant and will therefore be more challenging to eradicate.OrganotropismProcess of cancer cells spreading to and surviving in distant organs in a non\u2010arbitrary way. Broadly categorized, it depends on cancer cell intrinsic factors and non\u2010cancer cell autonomous features . These factors allow seeding to specific distant organs and enable survival in this foreign microenvironment.Pre\u2010metastatic Niche (PMN)Primary tumors secrete soluble factors and EVs that reprogram distant sites and facilitate future cancer cell homing and survival. Numerous factors influence PMN formation, including vascular changes, stromal cell activation, immune cell recruitment, ECM remodeling, and metabolic reprogramming.Tumor Microenvironment (TME)The TME can include endothelial, immune, tissue\u2010resident cells, nerve cells, adipocytes, a stroma composed of extracellular matrix, cancer\u2010associated fibroblasts, mesenchymal cells, and numerous soluble factors. Breast cancer cells and the TME co\u2010evolve dynamically through reciprocal interactions that corrupt homeostatic networks and contribute actively to disease progression.et al, et al, et al, et al, et al, et al, Metastases are the leading cause of cancer\u2010related deaths in breast cancer patients . Finally, we discuss the concept that novel therapies should target not only the cancer cells but also the tumor microenvironment.et al, et al, An elaborate interplay of the primary tumor\u2019s secretome with immune and tissue\u2010resident cells results in a microenvironment in secondary organs (the pre\u2010metastatic niche\u2014PMN) that favors subsequent cancer cell homing. PMN alterations can be broadly categorized into (i) vascular changes including vascular leakiness, expression of adhesion molecules, clot formation, (ii) activation of stromal components and extracellular matrix (ECM) reorganization, for example, by distant secretion of matrix metalloproteinases, (iii) immune cell recruitment, (iv) changes in resident cells, including metabolic adaptions preferentially home to specific secondary organs (\u201csoil\u201d) are a common occurrence in multiple solid cancers. Clinically, breast cancer often spreads to several distant organs and different subtypes have been associated with differential patterns of metastasis. The most common site of metastasis shared between all subtypes is the bone, with the highest percentage in the estrogen receptor (ER)\u2010positive subtype. In ER\u2010positive/HER2\u2010negative patients, there is a pattern of bone only disease that persists several years to decades without clinical detection is referred to as \u201cdormancy\u201d. Two forms of dormancy have been proposed: cellular dormancy and tumor mass dormancy. Cellular dormancy is characterized by three traits: dormant DTCs persist in foreign organs (\u201csoil\u201d), they are\u2014for the time being\u2014arrested in G Ghajar, . In tumoet al, et al, et al, et al, et al, et al, et al, et al, Cancer cells may enter or exit cellular dormancy through cancer cell\u2010intrinsic mechanisms is the last and most fatal stage of breast cancer and the most difficult to treat due to tumor heterogeneity, metabolic flexibility, and complex interactions of cancer cells with the tumor microenvironment. It is important to note that no single therapeutic agent has been approved that specifically targets breast cancer metastases. That clinically detectable metastases can develop years after resection of the primary tumor highlights the fact that colonization is the most complex and rate\u2010limiting phase of the metastatic cascade Fig\u2009 Massagu. The siget al, et al, et al, et al, One example is CCL2 secretion from mammary tumors, which recruits C\u2010C motif receptor 2 (CCR2)\u2010expressing inflammatory monocytes to the metastatic site can also result in detrimental self\u2010enhancing loops that increase the survival and proliferation of metastatic tumor cells. One example is that mammary tumor\u2010initiating cells exhibit elevated G\u2010CSF production due to increased mTOR signaling, which then leads to accumulation of myeloid\u2010derived suppressor cells (MDSC). Notch activation by MDSCs subsequently increase tumor\u2010cell initiating frequency , to secondary organs, where they are potent orchestrators of PMN formation through immune\u2010modulation and immune\u2010suppression cells or VEGF\u2010A, TGF\u03b2, and TNF\u03b1\u2010evoked induction of serum amyloid A proteins that recruit MDSCs , including exosomes released from the primary tumor, can dictate organotropic behavior of metastatic tumor cells , small non\u2010coding RNAs that are crucial master regulators of gene expression in several cancer\u2010related signaling pathways identified EV markers from tumor and plasma that might improve early cancer detection and characterization from lung\u2010resident fibroblasts is inhibitory for metastatic cancer cells Fig\u2009. Howeveret al, et al, The ECM protein tenascin C (TNC) secreted from breast cancer cells arriving in the lung initiates the formation of a metastatic micro\u2010niche Fig\u2009 signals Fig\u2009. Initialet al, + T cells and polarization of TAMs into anti\u2010inflammatory M2 macrophages in a STAT3\u2010dependent manner increases the secretion of exosomes carrying miR503 Fig\u2009. Exosomaet al, et al, et al, et al, et al, et al, et al, et al, et al, The liver is one of the organs most affected by breast cancer metastasis, which often results in fatal disease progression is a gatekeeper in this YAP\u2010mediated process and the loss of CCM3 in CAFs enhances tissue remodeling while exacerbating matrix stiffness. The result is reciprocal YAP/TAZ activation in neighboring tumor cells and dissemination to distant organs from de novo lipogenesis and exogenous uptake are linked to cancer progression is the other main pathway of FA generation and this was shown recently to be involved in breast cancer brain metastasis can be produced by host cells but are also available for cancer cells through dietary uptake. Proline catabolism is higher in metastases than in primary tumors of mice and human patients , will provide powerful tools to unravel the metabolic states of metastatic cancer cells type, TME composition, and the metastatic organ need to be monitored with biopsies before and during therapy (Dey Additionally, it should be noted that preclinical studies are often suboptimal compared to the clinical situation of patients. In\u2010depth preclinical research and clinical validation that focus on controlling dormant cells at the different metastatic sites are needed. The tumor dormancy window offers a means to prevent incipient metastases from reawakening. This has the advantage of intervening pharmacologically and/or by modifying risk factors at a stage when the number and heterogeneity of cancer cells are still relatively low. Moreover, technologies that identify harbingers of the switch from dormancy to metastatic outgrowth will allow therapeutic intervention during the early stages of metastases. These may include secreted cytokines and chemokines, as well as circulating cells, nucleotides, metabolites, and lipids. Nevertheless, given current detection methods, some patients present to the clinic only at later stages of disease when metastases are detectable, fully established and have already a rewired microenvironment; thus, research on late\u2010stage disease is also needed (Ganesh & Massagu\u00e9, Zora Baumann: Conceptualization; Investigation; Visualization; Methodology; Writing\u2014original draft; Writing\u2014review and editing. Priska Auf der Maur: Conceptualization; Investigation; Visualization; Methodology; Writing\u2014original draft; Writing\u2014review and editing. Mohamed Bentires\u2010Alj: Supervision; Funding acquisition; Writing\u2014original draft; Writing\u2014review and editing.CRediT author contributions listed above, the contributions in detail are:In addition to the ZB, PADM, and MB\u2010A wrote and reviewed the manuscript. ZB and PADM designed the figures. ZB and PADM contributed equally, and order was chosen by flipping a coin. Both authors are allowed to refer to this article with their name in the first position.ZB, PADM do not have any disclosures. MBA is a member of EMBO. MBA owns equities in, and receives laboratory support and compensation from Novartis, and serves as consultant for Basilea.Development of more sensitive biomarkers for premetastatic niches and minimal residual disease in patients.Therapies aiming at normalizing pre\u2010metastatic niches back to tumor suppressive homeostatic conditions.Design of drugs interrupting feed\u2010forward loops to halt disease progression efficiently.Development of treatments targeting specific metastatic sites.Investigating and defining points of no return in breast cancer disease progression to understand if they can be prevented.Improvement of translatability of preclinical to clinical studies.Creating pan\u2010cancer clinical trials targeting specific metastatic sites."} +{"text": "Adenium obesum commonly known as \u201cdesert rose\u201d belongs to the family Apopcynaceae and has previously been reported for its anti-influenza, antimicrobial, and cytotoxic efficacies and well-known for their ethno-medicinal applications. In the present study, ethanolic extracts of A. obesum (AOE) were analyzed by gas chromatography-mass spectrometry (GC\u2013MS) to identify the important phytochemical compounds. The GC\u2013MS analysis of AOE detected the presence of 26 phytochemical compounds. This plant is traditionally used for the treatment of various diseases. In this report, the antioxidant, anti-inflammatory, and anticancer activities of ethanolic leaf extract from A. obesum (AOE) were studied. The antioxidant potential of ethanolic extract of AOE was examined by different antioxidant assays, such as antioxidant capacity by the DPPH, ABTS, superoxide, hydroxyl radical scavenging, and lipid peroxidation inhibition assays. The antioxidant activities of various reaction mixtures of AOE were compared with a reference or standard antioxidant (ascorbic acid). In addition, we also evaluated the anticancer activity of AOE, and it was observed that AOE was found to be cytotoxic against A549 lung cancer cells. It was found that AOE inhibited the viability of A549 lung cancer cells by inducing nuclear condensation and fragmentation. Furthermore, ethanolic AOE demonstrated the anti-inflammatory potential of AOE in murine alveolar macrophages (J774A.1) as an in vitro model system. AOE showed its potential in reducing the levels of inflammatory mediators including the proinflammatory cytokines and TNF-\u03b1. The results obtained in the present investigation established the antioxidant, anticancer, and anti-inflammatory potency of AOE, which may account for subsequent studies in the formulation of herbal-based medicine. Since ancient times, many natural compounds isolated from different medicinal plants have been extensively used for the treatment of numerous chronic diseases. There are various secondary metabolites such as flavonoids, phenolic acids, lignans, quinones, coumarins, and alkaloids, which showed substantial antioxidant and other activities and have played an important role in the treatment of cancer . NaturalOxidative stress is recognized as an imbalance between ROS generation and their elimination by protective mechanisms, which can lead to chronic inflammation. Reports have demonstrated that oxidative stress plays a pathogenic role in chronic inflammatory diseases. Moreover, ROS have been highlighted as one of the main causes of several inflammatory diseases such as cardiovascular diseases (CVD), type II diabetes, and cancer .Adenium obesum (Forssk) Roem and Schult is commonly called \u201cdesert rose\u201d and belongs to the family Apopcynaceae and has earlier been reported for its anti-influenza and 2,2_-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) were purchased from Sigma\u2013Aldrich . Ascorbic acid (vitamin C), gallic acid, rutin, nitro blue tetrazolium (NBT), and butylated hydroxytoluene (BHT) were purchased from Hi-Media, India. The remaining chemicals and solvents used were of standard analytical grade and HPLC-grade. 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA) and Hoechst 33342 dye were obtained from from Sigma . DMEM-high glucose medium, fetal bovine serums (FBS), and antibiotic\u2013antimycotic solution were procured from Himedia India, Ltd., Mumbai, India, whereas a colorimetric kit specific for caspase-8, -9, and -3 was provided by BioVision, CA, United States . The DyNAmoColorFlash SYBR Green qPCR Kit (F415L) along with the Verso cDNA synthesis kit were procured from Thermo-Scientific, United States . The primers used in the present work were synthesized and purchased from IDT, United States .A. obesum were initially washed with running water and shed-dried for about 6\u20137\u00a0days and then cut and crushed separately in a grinder. The powdered form is obtained and stored in dry tubes. Thereafter, the powdered samples were packed in a Soxhlet apparatus and extracted with 150\u00a0ml of 70% ethanol and 30% water as it showed the best extraction yield (A. obesum (AOE) was stored at 4\u00b0C. The extraction yield (%) was calculated as follows cells and J774A.1 murine macrophages were procured from the repository of the National Centre for Cell Sciences (NCCS), Pune, India. Both the cells were maintained in DMEM-high glucose completed with 10% FBS and 1% antibiotic\u2013antimycotic solution in a controlled humidified atmosphere at 37\u00b0C with at least 5% CO0 is the absorbance of pure DPPH or control and A1 is the absorbance of DPPH in the presence of various extracts or ascorbic acid.1,1-diphenyl 1-2-picryl-hydrazyl (DPPH) assay was used to determine the free radical scavenging activity . In orde2,2\u2032-azino-bis (3-ethylbenzothiazoline- 6-sulfonic acid) (ABTS) cation decolorization assay is similar to the DPPH assay, and it was also performed for the measurement of antioxidant potential of the plant extracts by using standard protocols described earlier with slight modifications . 1\u00a0ml alvia the oxidation of NADH and estimated through the reduction of NBT. In the present experiment, the superoxide radicals were produced in 3\u00a0ml of sodium phosphate buffer containing 1\u00a0ml of NBT (150\u00a0mM) solution, 1\u00a0ml of NADH (468\u00a0mM) solution, and different doses of the AOE in water. This is followed by the addition of 1\u00a0ml PMS solution (60\u00a0mM) to the mixture, and the reaction mixtures were incubated for 5\u00a0min at room temperature. The absorbance was calculated against the corresponding blank solution. Ascorbic acid was used as the reference standard. The decline in the extent of NBT reduction, as evaluated by the absorbance of the reaction mixture, correlates with the superoxide radical scavenging activity of the AOE extract. The percentage of superoxide radical scavenging was measured by using the aforementioned equation.The superoxide radical scavenging activity was evaluated as described previously . Superox2, 90\u00a0ml of 1\u00a0mM 1,10-phenanthroline, 2.4\u00a0ml of 0.2\u00a0M phosphate buffer (pH 7.8), 150\u00a0ml of 0.17\u00a0M H2O2, and 1.5\u00a0ml of AOE extract at different concentrations. Addition of H2O2 initiated the reaction. Thereafter, reaction mixtures were incubated for 5\u00a0min, and the absorbance of the mixture was measured at 560\u00a0nm with a spectrophotometer. The scavenging of hydroxyl radicals was calculated by the aforementioned equation.The scavenging activity for hydroxyl radicals was evaluated with the Fenton reaction as described previously by Yu et al. . The rea3+/ascorbic acid\u2013mediated lipid peroxidation in bovine brain extract was performed as described previously in the presence and/or absence of AOE or the reference compound. The final volume of the reaction was 330\u00a0\u00b5l, and the reaction was briefly incubated for 1\u00a0h at 37\u00b0C. The generation of hydroxyl radicals provided the impetus for lipid peroxidation, which further augmented the synthesis of malondialdehyde (MDA). MDA was subsequently quantified with the TBA reaction through ELISA reader at 532\u00a0nm. Ascorbic acid during the study served as a positive control. The results were expressed as percentage inhibition activity calculated as.Non-enzymatic Feeviously with subControl was the absorbance of the control and ATest was the absorbance of the sample.3 A549 cells/well were seeded in a 96-well plate and allowed to adhere in a humidified atmosphere. A549 cells were treated with different concentrations of AOE and further incubated for 24\u00a0h under standard conditions. MTT dye was added to all the wells and further incubated for 4\u00a0h (37 \u00b0C). Finally, the formazan or purple-colored crystals were solubilized by supplementing 100\u00a0\u03bcl DMSO. Thereafter, each well was assessed for absorbance of formazan crystals at 490\u00a0nm using a microplate reader , and the cell viability was calculated as percent (%) cell viability in comparison with the untreated control.In order to study the cytotoxic potential of AOE, an MTT assay was performed as per the earlier described protocol with slight modifications . BrieflyPhase-contrast microscopy was performed to study the morphological alterations within the AOE-treated A549 lung cancer cells. Cells were incubated overnight in a 96-well plate approximately and cultured with different concentrations of AOE for 24\u00a0h. Morphological alterations within the AOE-treated A549 cells along with the control group were visualized and captured using the relief phase channel of the FLoid imaging station at \u00d720 magnification .3\u00a0cells/well) were seeded and allowed to adhere and thereafter exposed to AOE treatment (24\u00a0h) at different concentrations as mentioned earlier and incubated under standard conditions. After that, the media were decanted, and cells were further stained with Hoechst 33342 dye under standard conditions. Last, fluorescent nuclei of treated cells were visualized, and photomicrographs were captured using a blue fluorescence channel (Excitation: 390/40\u00a0nm\u2013Emission: 446/33\u00a0nm) of FLoid imaging station .Hoechst 33342 dye assay was used to study the nuclear condensation in AOE-treated A549 lung cancer cells . A549 ceROS generation in AOE-treated A549 lung cancer cells was qualitatively analyzed by using DCHF-DA staining as described earlier . The celA colorimetric caspase assay kit specific for caspase-9 and -3 was used as per the manufacturer\u2019s instructions. The results were interpreted as a percentage increase in the activity of specific caspases in comparison with the control cells.Approximately 1 million A549 cells were exposed to AOE treatment for 24\u00a0h followed by total RNA extraction using a commercial grade kit according to the manufacturer\u2019s protocol. 2 \u03bcg of extracted RNA was further used for cDNA synthesis by using the Verso cDNA synthesis kit as per the protocol. The primer sequences (0.5\u00a0\u00b5M each of forward and reverse primers) used in this study are listed below as described previously . Further6 J774A.1 cells/well were transferred to a 6-well plate and stimulated with or without 100\u00a0ng/ml LPS for 24\u00a0h. Post simulation, the cells were exposed to varying concentrations of A. obesum ethanolic leaf extract for an additional 24\u00a0h under standard culture conditions. Subsequently, the levels of inflammatory mediators, namely, IL-1\u03b2, TNF-\u03b1, IL-10, and PEG2 were quantified through commercially available ELISA kits in accordance with the manufacturer\u2019s protocol. Cells with and without LPS stimulation served to be positive and negative control, respectively.Nearly 10p < 0.05, \u2217\u2217p < 0.01, and \u2217\u2217\u2217p < 0.001.All reported data are expressed as the mean \u00b1 SEM of three individual experiments performed thrice through GraphPad Prism (Ver. 5). Statistical analysis among different treatment groups was determined using one-way ANOVA followed by Dunnett\u2019s post-test. Significance: \u2217A. obesum , whereas ascorbic acid also considerably inhibited the lipid peroxidation by 82.84%. The AOE-mediated inhibitory effect on lipid peroxidation was found to be dependent on the concentration of AOE.Lipid peroxidation was colorimetrically assessed by quantifying TBA at 532\u00a0nm. As shown in 50 of AOE against A549 lung cancer cells was found to be IC50 \u00b1 256.75\u00a0\u03bcg/ml. AOE treatment significantly decreased the cell viability of A549 cells as shown in In order to study the anticancer or cytotoxic effects of AOE on A549 lung cancer cells, an MTT assay was performed. A549 cells were treated with different concentrations of AOE for 24\u00a0h. The ICMorphological analysis of AOE-treated lung cancer cells was undertaken using a phase contrast microscope. A dose-dependent morphological alteration was observed in AOE-treated A549 cells. Lung cancer cells undergo several morphological alterations including round morphology with slight shrinkage and nuclear condensation in the presence of different concentrations of AOE . These morphological changes within lung cancer cells were more obvious with the increase in the dose of AOE, whereas control cells exhibited flattened morphology .Nuclear condensation and fragmentation are the two peculiar hallmarks of apoptosis. Hoechst 33342 staining was performed to qualitatively analyze whether the AOE-induced cytotoxicity in lung cancer cells was due to apoptosis induction. As observed from the fluorescent micrographs, treatment of AOE induces nuclear condensation and fragmentation in A549 cells in a dose-dependent manner as indicated by the white arrows, while the control cells exhibited unaltered morphology .In order to study the effect of AOE on ROS, we perform DCFH-DA staining. A549 cells were stained with DCFH-DA to detect the changes in the levels of intracellular ROS after 24\u00a0h treatment of various concentrations of AOE. The fluorescent micrographs exhibited that treatment with different doses of AOE resulted in stronger DCF-fluorescence intensity in lung cancer cells, which indicated an enhanced intracellular ROS generation caused by AOE .Bcl-2 family members and caspases are primarily responsible for regulating apoptosis or programmed cell death in multicellular organisms. It is subdivided into two types: extrinsic (death receptor) and intrinsic pathways. As a result, we performed caspase assay on AOE-treated A549 cells, and it was observed that AOE (100\u2013400\u00a0\u03bcM) treatment significantly increased the caspase-9 and -3 activity by 36.31 \u00b1 2.31%, 58.39 \u00b1 4.02%, and 82.25 \u00b1 4.29% and 49.03 \u00b1 3.96%, 74.46 \u00b1 5.47%, and 109.20 \u00b1 5.56% respectively .We studied the mRNA levels of Bax, Bad (proapoptotic protein), and Bcl-2 (antiapoptotic protein) which are chiefly involved in mediating the mitochondria-dependent apoptotic pathway. The mRNA levels of Bax, Bad, and Bcl-2 in AOE-treated A549 cells were measured through qRT-PCR analysis. The mRNA levels of Bax and Bad enhanced to 1.88 \u00b1 0.07, 2.30 \u00b1 0.13, and 2.92 \u00b1 0.24 folds and 1.52 \u00b1 0.24, 1.88 \u00b1 0.13, and 2.40 \u00b1 0.06 folds respectively, comparatively with the untreated cells . HoweverMitochondria play an important role in inducing apoptosis by mitochondrial or intrinsic apoptotic pathways. As shown in A. obesum substantially reduced the level of TNF-\u03b1, IL-6, and IL-1\u03b2 in a dose-dependent manner . Similar effects were observed on PGE2 levels within J774A.1 cells treated with ethanolic leaf extract of A. obesum again in a dose-dependent manner and human prostate cancer LnCap and 22 RV1 cells . This is followed by the addition of AOE to this pre-formed radical cation which was converted to ABTS in a concentration-dependent manner. The result is analogous to the earlier mentioned DPPH assay, suggesting that AOE acts as a potent antioxidant.Initially, we performed a DPPH assay to investigate the antioxidant potential of AOE, and we observed dose-dependent scavenging of the DPPH radical. In addition, the reaction between ABTS and potassium persulfate leads to production of a blue-colored chromophore and decreased the expression of anti-apoptotic proteins (Bcl-2) in lung cancer.Earlier published data on the effect of AOE extracts on cancerous cells are limited, and the mechanisms are not yet fully deciphered. In our present study, apoptosis induction was the central motif and from the results, we observed changes in the morphology of the cells upon treatment with AOE extracts. The lung cancer cells (A549) exhibited cell shrinkage, spikes, and other attributes resembling DNA fragmentation; these characteristics were similar to those reported in the past as evidence that cells are undergoing apoptosis 11\u201315. Our results suggested that AOE induced cell death in A549 cancer cells m after treatment with AOE. We found that the AOE enhances the level of ROS, significantly mediating apoptosis.Caspases are the crucial regulators of apoptosis and are family members of the cysteine proteases . Our res2O2), singlet oxygen, and hydroxyl radicals. Moreover, increasing evidence suggests that escalated production of intracellular ROS may directly or indirectly induce damage to nucleic acids, proteins, and lipids, which eventually leads to induction of apoptosis (Oxidative stress (OS) in the cells arises due to an imbalance between the formation and elimination of oxidant species . Reactivpoptosis .in vitro screening system. The cells were stimulated with LPS which has been established for its potential to instigate the TLR-4-NF-\u03baB\u2013mediated inflammatory axis within these cells (The acute inflammatory response is a prerequisite for homeostatic functioning of the innate immune response and bridging it with the adaptive arm of immunity. Indeed, chronic activation of inflammatory response results in onset of diseases such as arthritis and neurodegenerative disorders . Chronicse cells . AOE shoin vitro, suggesting that AOE could be a reservoir of natural antioxidants. Our findings open up the possibility in the future to identify the potential therapeutic agents from AOE for the development of herbal-based medicine.In conclusion, our present investigation has shown that AOE exhibited antioxidant, anticancer, and anti-inflammatory properties. AOE exhibited powerful antioxidant activity evaluated"} +{"text": "A third of people with juvenile myoclonic epilepsy (JME) are drug-resistant. Three-quarters have a seizure relapse when attempting to withdraw anti-seizure medication (ASM) after achieving seizure-freedom. It is currently impossible to predict who is likely to become drug-resistant and safely withdraw treatment. We aimed to identify predictors of drug resistance and seizure recurrence to allow for individualised prediction of treatment outcomes in people with JME.https://osf.io/b9zjc/).We performed an individual participant data (IPD) meta-analysis based on a systematic search in EMBASE and PubMed \u2013 last updated on March 11, 2021 \u2013 including prospective and retrospective observational studies reporting on treatment outcomes of people diagnosed with JME and available seizure outcome data after a minimum one-year follow-up. We invited authors to share standardised IPD to identify predictors of drug resistance using multivariable logistic regression. We excluded pseudo-resistant individuals. A subset who attempted to withdraw ASM was included in a multivariable proportional hazards analysis on seizure recurrence after ASM withdrawal. The study was registered at the Open Science Framework . Recurrence of seizures after ASM withdrawal (n\u00a0=\u00a0368) was predicted by an earlier age at the start of withdrawal, shorter seizure-free interval and more currently used ASMs, resulting in an average internal-external cross-validation concordance-statistic of 0\u00b770 (95%CI 0\u00b768\u20130\u00b773).We were able to predict and validate clinically relevant personalised treatment outcomes for people with JME. Individualised predictions are accessible as nomograms and web-based tools.MING fonds. A third of people with JME are drug-resistant, and three-quarters of individuals with JME who attempted to withdraw treatment after becoming seizure-free experienced a seizure recurrence. We last performed a systematic literature search in PubMed and Embase on 11 March 2021 by searching for \u2018juvenile myoclonic epilepsy\u2019 and \u2018treatment outcome\u2019 and various synonyms. We found no sufficiently powered multivariable analyses that assessed potential predictors of drug-resistant JME or predictors of seizure recurrence after ASM withdrawal.This individual participant data (IPD) meta-analysis (n\u00a0=\u00a02518) identified nine independent predictors of drug-resistant JME, seven previously reported, and two novel: ethnicity and family history of epilepsy. We found three predictors of seizure recurrence after treatment withdrawal. The strongest predictor for post-withdrawal seizure recurrence in JME \u2013 earlier age at the start of withdrawal \u2013 had an inverse direction of effect compared to other epilepsy types. We used these variables to create prediction models of drug-resistant JME and of seizure recurrence. Internal-external cross-validation showed robust predictive performance of our models.We created and validated prediction models, available as nomograms and web-based tools, to improve and personalise JME treatment. We expect that the models will aid in improving and personalising the treatment and counselling of people with JME.,,Juvenile myoclonic epilepsy (JME) is the most common idiopathic and presumed genetic generalised epilepsy syndrome, affecting 5\u201310% of all people with epilepsy.,,Predicting who is likely to become drug-resistant and who could safely withdraw ASM treatment after a certain period of sustained seizure-freedom, has clinical benefits. Drug withdrawal improves quality of life by avoiding the adverse effects of potentially unnecessary treatment.We aimed to identify independent predictors of\u00a0drug resistance and post-withdrawal relapse risk based on individual participant data (IPD) from previously published study cohorts. We developed and validated predictive tools to calculate these risks.https://osf.io/b9zjc/). The methods and reporting are consistent with the PRISMA-IPDWe performed a meta-analysis of individual participant data according to a pre-registered protocol website, other publications by the same authors and performed a manual internet search for alternative contact details.Authors who agreed to collaborate were asked to provide treatment outcome data and potential predictors by filling in a standardised data entry sheet containing 41 variables . AlternaAs in our previous meta-analysis, we used the Newcastle\u2013Ottawa quality assessment scale for cohort studies to assess the methodological quality of all the included studies.Our study was a meta-analysis of de-identified individual data and did not require ethical approval or specific informed consent. Local research ethics committees or other entities overseeing personal data had approved the original studies. Where applicable under local regulations, data sharing agreements were signed before receiving individual data.We used a combination of outcome measures to define drug resistance and seizure recurrence after ASM withdrawal. For the primary analysis, we used the definition of drug-resistant epilepsy formulated by the ILAE, taking each seizure type into account.We assessed seizure recurrence in a subset of people who attempted to withdraw treatment after a period of seizure freedom. Seizure recurrence was evaluated at two and five years after initiation of ASM withdrawal. Our primary analysis comprised recurrence of any seizure after start of ASM withdrawal. We also specifically assessed GTCS recurrence.We selected candidate predictors of drug resistance and seizure recurrence based on our previous meta-analysis on refractory JME,2 test, which is defined as the percentage of total variation across studies that is due to heterogeneity rather than chance.The supplementary methods provide a detailed overview of all analyses and statistical methods. In brief, the proportion of drug-resistant JME was assessed with random-effects meta-analysis and a meta-regression of drug resistance by publication year. Between-study heterogeneity was assessed using the I,Cox proportional hazards analyses were performed to assess the time to seizure recurrence after start of ASM withdrawal. Univariable predictors at p\u00a0<\u00a00\u00b72 were used for multivariable analyses, after which backward selection was performed to remove the least contributing predictors. We performed an internal-external validation by splitting the 18 cohorts with data on post-withdrawal seizure recurrence into three datasets of 6 cohorts, balanced on sample size. We trained the prediction model on 12/18 cohorts and assessed the external predictive value of this model on the left-out 6 cohorts, quantified with the concordance-statistic (C-statistic).AUC and C-statistic values range between 0 and 1, where a value of 0.5 represents no better prediction than chance, and 1 represents perfect predictive performance. A value\u00a0<\u00a00.7 is generally considered poor, \u22650.7 is deemed acceptable, and \u22650.8 is considered excellent.All statistical analyses were performed in RStudio Version 1.3.1093, using the packages: MICE, metafor, glmer, rms, coxme, rsample, purrr, survminer, tidyverse, ggplot, and survAUC.To aid use in clinical practice, we converted our multivariable models to nomograms and web-based tools. The nomograms are visual representations of the mixed-effects logistic regression analysis on drug resistance and the Cox proportional hazards model on seizure recurrence within 2 and 5 years. They come with instructions to manually estimate clinical outcomes for an individual. Similarly, we translated the models into web-based tools where a user can fill in predictors to obtain the associated probability of a clinical outcome for an individual.The funder had no role in the study design, data collection, data analysis, data interpretation, drafting of the report or the decision to submit. RS, DA-T, WMO, BPCK, FEJ, KPJB had full access to all the data. All authors interpreted results, reviewed and critically revised the article, and approved the final version for submission.We screened 1334 articles and identified 53 eligible studies . The autIn total, 2518 individuals from 18 countries and various ethnicities were included in the predictive analyses of drug resistance. Missing data before imputation ranged between 0% and 38% per variable . Among variables included in the drug resistance analysis, three variables had missing data between 25% and 40%; eight variables were missing between 10% and 25%, five variables between 1% and 10%, and data was complete for seven variables . Among vFollow-up duration ranged from one to 73 years . Information on current ASM treatment was available for 2365 people, of which 805 (34%) were on multiple ASMs, and 1560 (66%) were on monotherapy . Among t2\u00a0=\u00a088%, p\u00a0<\u00a00\u00b70001). Funnel plots of people with JME were drug-resistant , with siel plots and EggeUnivariable mixed-effects logistic regression analysis identified 18 predictors of drug resistance at p\u00a0<\u00a00\u00b72 , some oWe performed internal-external cross-validation to assess the external predictive value of the multivariable model, which showed an AUC of 0\u00b770 (95%CI 0\u00b767\u20130\u00b772). The AUC varied between 0\u00b756 and 0\u00b784 per left-out cohort , with smaller cohorts on both ends of the distribution . A plot As further sensitivity analyses, we assessed how well we could predict freedom of any seizure and freedom of GTCS in the last one, two and five years of follow-up. We used the same predictors . These individuals were older and included more people of Asian ethnicity compared to people who did not try to withdraw treatment but did not differ in other predictors of drug resistance . Five yeUnivariable analyses showed ten predictors of seizure recurrence at p\u00a0<\u00a00\u00b72 . SubsequWe collected IPD from a large group of people, enabling the creation of prediction models for individual assessment of drug resistance (n\u00a0=\u00a02518) and seizure recurrence risk after withdrawal of ASM treatment (n\u00a0=\u00a0368) in JME. We validated the prediction models and found good calibration. Three-quarters of people who attempted to withdraw ASMs experienced a seizure recurrence within five years, for which we found three predictors. A third of people with JME were drug-resistant, for which we found nine independent predictors.,,,We confirmed multiple previously found risk factors of drug resistance.,,We identified two predictors not previously associated with drug-resistant JME: family history of epilepsy as a risk factor, and Asian ethnicity was protective compared to Caucasians. The most likely explanation for this association is that both predictors are proxies of the presumed population-specific genetic basis of JME.We found three predictors of seizure recurrence after ASM withdrawal. There are several predictors of seizure recurrence in the broader epilepsy population,,Our results showed a higher AUC for drug resistance prediction than seizure freedom prediction in the last one, two, and five years. The higher AUC suggests that the formal definition of drug resistance as defined by the ILAE,We found a higher AUC for predicting seizure freedom and recurrence of seizures after ASM withdrawal when assessing any seizure type compared to the analyses confined to GTCS. Analyses on GTCS may lack statistical power. Alternatively, freedom of GTCS might be inherently more challenging to predict since GTCS often occur less frequently than myoclonic or absence seizures. For example, assessing GTCS freedom in the last year of follow-up might be an unreliable measure for someone only having GTCS every other year. Hence, we would advocate using the ILAE definition of drug resistance, which considers all seizure types and pre-treatment seizure intervals.,Our study has limitations. The included cohorts were primarily obtained from tertiary care centres, potentially limiting the generalisability of our prediction model to primary and secondary care. Potential selection bias and selective loss to follow-up of drug-responsive people could further reduce the representativeness of our dataset. We provided a standardised data entry sheet, but significant intra- and inter-observer variability likely remain. We found considerable heterogeneity between cohorts. Potential sources of heterogeneity include differences in demography, study ascertainment, country-specific healthcare organisation and accessibility. In particular, differences in ethnicity between studies could explain part of the heterogeneity in the proportion of drug resistance, and age differences might explain heterogeneity in seizure recurrence rate after withdrawal. We mitigated the influence of between-study heterogeneity by using random-effect statistical models, although heterogeneity might have still limited the predictive performance of our models. As most studies were several years old, collecting all potential predictors for each individuals was impossible. We mitigated this by performing multiple imputations of missing data, reducing bias and increasing precision.http://epilepsypredictiontools.info/). We expect that the models will aid in improving and personalising the treatment and counselling of people with JME.In conclusion, we assessed whether we could predict the likelihood that an individual with JME will become drug-resistant or has a seizure recurrence after ASM withdrawal. After validating these predictions, we created nomograms and developed publicly accessible web-based tools to help estimate individualised risks (RS, FEJ, HJL, JWS, BPCK, WMO and KPJB contributed to study conceptualisation and design. RS and DA-T analysed the data and created the figures. WMO and HJL supervised the statistical analyses. RS and DA-T verified the integrity of the full dataset. RS, DA-T, WMO, BPCK, FEJ, KPJB have full access to all the data. RS wrote the first draft of the report, with input from DA-T, WMO, FEJ, BPCK and KPJ. Data were obtained by AAA, MF, GC, SJ, AP, \u00c7\u00d6, SA, JG, CPB, LJS, MJB, GU, AR, JH, ET, RK, ECI, CDB, JPS, LEH-V, MLM-A,YZ, DZ, NP, NS, GJ, SB, MJ, PK, MS, KKS, BJV, MH, LGV, SS, BB, EAA, FvP, JS, US, AV-A, IK, WD, JWS. All authors interpreted results, reviewed and critically revised the article, and approved the final version for submission.http://epilepsypredictiontools.info/. The main analyses\u2019 study protocol and R scripts are available on https://osf.io/b9zjc/. The signed data-sharing agreements between the different cohorts participating in this study do not allow the de-identified individual participant dataset to be publicly released. An exception can be made to replicate the results in this manuscript by an academic third party, after signing data sharing agreements with all collaborating centres.The individualised prediction models for drug resistance and recurrence of seizures after ASM withdrawal in JME are available as nomograms in this manuscript. They will be made available upon publication as a web-based tool at 10.13039/501100012155National Institute for Medical Research Development, royalties for a book publication from 10.13039/501100007723Oxford University Press, and speaker fees from Cobel Daruo, Tekaje, and Raymand Rad. CPB received research grants and honoraria from 10.13039/100011110UCB and 10.13039/501100003769Eisai, support for attending meetings by UCB, and served in the advisory board of Arvelle. CDB received consulting fees and honoraria from 10.13039/100014114GW Pharmaceuticals, 10.13039/100015661UCB Pharma, 10.13039/501100003769Eisai, 10.13039/501100006546Angelini Pharma and Bial. JPS received grants from the 10.13039/100000002National Institutes of Health, 10.13039/100000005Department of Defense, and the 10.13039/100000001National Science Foundation; and consulting fees from 10.13039/100015661UCB Pharma, AdCel Biopharma, LLC, iFovea, SK Life Sciences, and 10.13039/100013410LivaNova; and has stock options for iFovea and AdCel Biopharma. LEH-V participates in the Young Epilepsy Society, received speaker honorario from Armstrong, and was supported by Abbott pharmaceuticals to attend the Mexican Congress of Neurology. NP received honoraria from Zogenix and Ethos for Angelini Pharma. NS received honoraria from Biomarin, Livanova, GW Pharma, Zogenix and Marinus; and support to attend meetings from Livanova, GW Pharma and Zogenix; and participated on a data safety monitoring board for Marinus. SB received speaker fees from Eisai. PK received lecture honorarium from UCB Pharma and Eisai, consulting fees from 10.13039/501100003769Eisai and 10.13039/100013410LivaNova and his institution received research grants from 10.13039/100015661UCB Pharma and 10.13039/501100003769Eisai. MS received speaker honoraria from UCB Pharma and Eisai. KKS received research grants from the 10.13039/501100005416Norwegian Research Council, the DAMFoundation and the Norwegian National Advisory Unit on Rare diseases; and a networking grant from the 10.13039/501100004785NordForsk Foundation; and she acted as a paid PhD defense opponent at the University of Bergen, and attended a meeting for Nordic clinicians organised by Eisai. BJV received grants from the 10.13039/501100007870German Society for Epileptology and the 10.13039/501100001659Deutsche Forschungsgemeinschaft; and honoraria from University Medical Center Schleswig\u2013Holstein and Cornelsen Verlag. MH received consulting fees and honoraria from Arvelle, Bial, Desitin, Eisai, GW Pharmaceuticals, UCB Pharma, and Zogenix. FvP has received speaker honoraria from Bial, Eisai, GW Pharmaceutical companies, Angelinipharma, Zogenix and UCB Pharma; and scientific advisory board honoraria from GW Pharmaceutical companies, UCB Pharma, and Angelinipharma. WD's salary is part-funded by The University of Melbourne; he has received travel, investigator-initiated, scientific advisory board and speaker honoraria from UCB Pharma Australia and Global; investigator-initiated, scientific advisory board, travel and speaker honoraria from Eisai Australia and Global; advisory board honoraria from Liva Nova and Tilray; educational grants from Novartis Pharmaceuticals, 10.13039/100014476Pfizer Pharmaceuticals and Sanofi-Synthelabo; educational; travel and fellowship grants from GSK Neurology Australia, and honoraria from SciGen Pharmaceuticals; and he has an equity interest in the device company EpiMinder. CPB received honaries and research support from EISAI, UCB and Arvelle. ET received speaker's honoraria from Arvelle, Abbott, Angelini Pharma, UCB, Biogen, Gerot-Lannacher, Bial, Eisai, Epilog, Takeda, Newbridge, Hikma, GW Pharmaceuticals, Sunovion Pharmaceuticals Inc., LivaNova and Novartis; consultancy funds from 10.13039/501100006546Angelini Pharma, Argenix, Arvelle, Epilog, 10.13039/100011110UCB, 10.13039/100005614Biogen, Gerot-Lannach, Bial, 10.13039/501100003769Eisai, 10.13039/100007723Takeda, Newbridge, 10.13039/100014114GW Pharmaceuticals, 10.13039/100009655Sunovion Pharmaceuticals Inc., Marinus, and 10.13039/100004336Novartis; directorship funds from Neuroconsult GmbH. ET's Institution received grants from 10.13039/100005614Biogen, Red Bull, 10.13039/100004334Merck, 10.13039/100011110UCB, 10.13039/501100000780European Union, FWF \u00d6sterreichischer Fond zur Wissenschaftsf\u00f6rderung, and 10.13039/501100007148Bundesministerium f\u00fcr Wissenschaft und Forschung. JWS reports personal fees from Arvelle, personal fees from 10.13039/100011110UCB, grants from UCB, grants from 10.13039/100003997NEF, grants from UCB, personal fees from 10.13039/100012739Zogenix, grants from GW Phama, outside the submitted work; and his current position is endowed by the Epilepsy Society, he is a member of the Editorial Board of the Lancet Neurology, and receives research support from the Marvin Weil Epilepsy Research Fund. All other authors declare no potential competing interests. None of the above mentioned declarations represent a conflict of interest directly related to the present publication.AAA received a grant from the"} +{"text": "Robertson-Schr\u00f6dinger uncertainty function, which is always non-negative for quantum systems, but not necessarily so for classical systems. Here, quantum refers to noncommutativity of the canonical operator pairs. From the nonequilibrium free energy, we succeeded in deriving several inequalities between certain thermodynamic quantities. They assume the same forms as those in conventional thermodynamics, but these are nonequilibrium in nature and they hold for all times and at strong coupling. In addition we show that a fluctuation-dissipation inequality exists at all times in the nonequilibrium dynamics of the system. For nonequilibrium systems which relax to an equilibrium state at late times, this fluctuation-dissipation inequality leads to the Robertson-Schr\u00f6dinger uncertainty principle with the help of the Cauchy-Schwarz inequality. This work provides the microscopic quantum basis to certain important thermodynamic properties of macroscopic nonequilibrium systems.Thermodynamic uncertainty principles make up one of the few rare anchors in the largely uncharted waters of nonequilibrium systems, the fluctuation theorems being the more familiar. In this work we aim to trace the uncertainties of thermodynamic quantities in nonequilibrium systems to their quantum origins, namely, to the quantum uncertainty principles. Our results enable us to make this categorical statement: For Gaussian systems, thermodynamic functions are functionals of the Uncertainty in simultaneous measurements of canonical variables is one of the building blocks of modern quantum field theories. Already in the early days of quantum mechanics, they found their formalization in, e.g., the Heisenberg Uncertainty principle for zeroWhile it might be no surprise that uncertainty relations will leave their marks on macroscopic (thermodynamic) parameters of the system, general statements in nonequilibrium systems are scarce. Importantly, we should mention entropic uncertainty relations , fluctuaIn our work, we intend to approach this issue in two stages. In this first paper, we do not adhere to any specific formulation or particular interpretation of thermodynamic uncertainties such as offered in the above references, but explore and strengthen their theoretical foundations using the conceptual framework of open quantum systems (OQS) ,23,24 anIn future work , we shalafter this equilibrium condition is met can one explore whether such relations exist or not. In the recent works by two of us , would With this condition we then compare the result of the internal energy with two different, but equally plausible definitions of internal energies in the nonequilibrium setting at strong coupling. On one hand, a customary example is the expectation value of the system Hamiltonian equality in the zero-temperature limit at late times. However, it turns out that, in general, the fluctuation-dissipation inequality cannot by itself resolve into the fluctuation-dissipation equality at late times, but this is the case only for the absolute zero-temperature quantum bound. Only with additional information on the bath spectral density can one deduce the famous spectral Returning to the equilibration process of the system, we find that the fluctuation-dissipation inequality, i.e., a nonequilibrium inequality on the magnitude of the respective fluctuation and dissipation kernels in the system, can lead to a fluctuation-dissipation Lastly, looking at the big picture, we comment on two aspects extending what we have done here: (1) TURs in terms of currents: Many thermodynamic uncertainty relations are expressed in terms of thermodynamic quantities such as energy flux and entropy production in multiple-bath scenarios. In our next paper on this topic , the cen"} +{"text": "Ten DRD, 14 cervical dystonia patients and 12 controls were included. Univariate- and network-analysis did not show differences in binding between DRD patients compared to controls. Sleep disturbances were correlated with binding in the dorsal raphe nucleus and participants with a psychiatric disorder had a lower binding in the hippocampus . Post-hoc analysis with correction for psychiatric co-morbidity showed a significant difference in binding in the hippocampus between DRD patients and controls (p\u2009=\u20090.00). This suggests that psychiatric symptoms might mask the altered serotonergic metabolism in DRD patients, but definite conclusions are difficult as psychiatry is considered part of the phenotype. We hypothesize that an imbalance between different neurotransmitter systems is responsible for the non-motor symptoms, and further research investigating multiple neurotransmitters and psychiatry in DRD is necessary.GTP-cyclohydrolase deficiency in dopa-responsive dystonia (DRD) patients impairs the biosynthesis of dopamine, but also of serotonin. The high prevalence of non-motor symptoms suggests involvement of the serotonergic pathway. Our study aimed to investigate the serotonergic system in vivo in the brain of`DRD patients and correlate this to (non-)motor symptoms. Dynamic [ The most common type of dopa-responsive dystonia is caused by an autosomal dominant inherited mutation in the GCH1 gene (OMIM#600225) affecting the enzyme GTP cyclohydrolase 12. This enzyme is the first and rate-limiting step in the biosynthesis of tetrahydrobiopterin (BH4), which is an essential co-factor involved in the biosynthesis of mono-amine neurotransmitters, such as dopamine and serotonin.Dopa-responsive dystonia (DRD) is a rare inherited metabolic disorder in which patients present with dystonia characterized by diurnal fluctuations and an excellent response to levodopa treatment5. The pathophysiology of non-motor symptoms in dystonia is not known, however, serotonin is suggested to play an important role6. As already indicated, the biosynthesis of serotonin, identical to that of dopamine, is impaired due to the GCH1 mutation and subsequent deficiency of BH4. In line with this, decreased levels of 5-Hydroxyindoleacetic acid (5-HIAA), the final breakdown product of serotonin, in cerebral spinal fluid of DRD patients has been described7. Furthermore, the serotonergic system is important in the regulation of mood and sleep, and is implicated in the pathophysiology of many psychiatric disorders.The biochemical changes in dopamine are considered to be responsible for the motor phenotype of dystonia and Parkinsonism in DRD patients and hence the excellent response to levodopa therapy. Besides these motor manifestations, attention has been drawn to non-motor symptoms in DRD with an increased prevalence of psychiatric and sleep disorders compared to controls11C]-3-amino-4-(2-dimethylaminomethyl-phenylsulfanyl)benzonitrile ([11C]DASB) is a well-validated and selective radio-ligand that binds to the serotonin reuptake transporter (SERT) and qualifies as an excellent biomarker to study the serotonergic system in vivo8.To investigate the potential role of the serotonergic system in the brain of DRD patients, positron emission tomography (PET) can be used. The radiotracer DASB PET scans was detected using this method in Parkinson\u2019s disease11.Dystonia, including DRD, is considered to be a network disorder, with multiple brain regions involved in its pathophysiology11C]DASB PET scans. Therefore, this study aimed to examine group differences in serotonergic system integrity between controls and DRD patients using both univariate- and network-based analysis. The second aim is to investigate the relationship between (non-)motor symptoms and the serotonergic system in the brain of DRD patients.To the best of our knowledge, there are no published studies investigating SERT status in vivo in DRD patients using DASB (mean dose 382\u2009\u00b1\u200941\u00a0MBq)16. PET images were reconstructed from list-mode data into 23 frames . Head movement was minimized with a head-restraining band. DRD patients were allowed to continue using their levodopa treatment during the day of the scan.Individual axial 3D T1-weighted gradient-echo images of the brain were acquired from all participants. PET imaging of DRD patients was performed using the same protocol as previously published for CD patients and controls with either a Biograph 40-mCT or 64-mCT . The protocol consisted of a 60-min dynamic acquisition scan starting simultaneously with an intravenous bolus of [11C]DASB images was performed using the 3D OSEM algorithm (3 iterations and 24 subsets), point spread function correction, and time-of-flight, resulting in a matrix of 400\u2009\u00d7\u2009400\u2009\u00d7\u2009111 of isotropic 2-mm voxels, smoothed with 2-mm filter at full width at half maximum (FWHM).Reconstruction of the dynamic DASB PET scans revealed high BPND in the dorsal midbrain, thalamus, and striatum in all groups and the AUC of the ROC curve was 0.48, indicating that the pattern did not effectively discriminate between the two groups and BPND in the sDRN , indicating a higher BPND in participants with a worse quality of sleep. This positive association was also present in the DRD patients, this almost reached statistical significance after correction for multiple comparisons was also found, indicating a lower BPND in patients with more fatigue. This finding was not present in the whole group.In all participants a significant association was found between quality of sleep and BPND in the hippocampus . In DRD patients this finding was also present, but did not reach statistical significance .Participants who were at least once in their life diagnosed with a psychiatric disorder had a lower BPND in the hippocampus between DRD patients and respectively CD patients and healthy controls in the globus pallidus.In the DRD group no correlations between dosage of levodopa and 11C]DASB PET scans. Both the univariate- as well as the network analysis did not show any differences in SERT binding between DRD patients compared to CD patients or healthy controls. However, the highly prevalent non-motor symptoms did have an association with SERT binding; sleep disturbances and psychiatric co-morbidity were correlated with respectively the BPND in the raphe nuclei and hippocampus.This is the first in vivo study assessing the serotonergic brain metabolism in patients with GTP cyclohydrolase deficient DRD using DASB PET scans we measured SERT, an important regulator of the synaptic concentration of serotonin, but we did not directly measure serotonin concentrations.The deficiency of the enzyme GTP-cyclohydrolase 1 in patients with DRD leads to an impaired synthesis of BH4, resulting in a decreased synthesis of dopamine known to cause motor symptoms, while a decreased synthesis of serotonin hypothetically can be linked to the non-motor symptoms. However, we did not observe any differences in brain SERT binding in patients with DRD, suggesting that the number of SERT or their binding capacity was not different. Our finding is in line with a post-mortem case report that did not show altered levels of serotonin markers, including SERT protein in the striatum of a DRD patient25 and are likely involved in non-motor symptoms in dystonia as well. Further studies assessing not only the serotonergic system, but combined with other neurotransmitter systems are necessary.Another explanation for our negative findings could be that alterations in the serotonergic metabolism in DRD are too subtle to be detected with a single PET scan assessing only the binding to SERT. As we know that other neurotransmitters, such as dopamine and acetylcholine, are also involved in dystonia, it is very likely that the non-motor symptoms, in a similar way, can be the result of an imbalance between multiple neurotransmitters, rather than a disruption in the pathway of only serotonin. Other neurotransmitters such as dopamine, noradrenaline and acetylcholine are extensively studied with regard to psychiatric disorders and sleep disordersAssessing all participants we did, however, find an association between psychiatric co-morbidity and SERT binding in the hippocampus, indicating that the serotonergic system is involved in psychiatric disorders. A subsequently performed post-hoc analysis to correct for psychiatry in the VOI-based analysis revealed a significant difference in SERT binding in the hippocampus between DRD patients and the two control groups. This suggests that there might be an altered serotonergic system related to the motor symptoms in DRD patients, overshadowed by the high prevalence of psychiatric disorders and corresponding disturbances in the serotonergic system. This is a difficult concept as the psychiatric symptoms are currently considered part of the phenotype in DRD patients. Further studies in a larger group of DRD patients is required to further study the relationship of DRD patients with and without psychiatric symptoms with the serotonergic system.26. Serotonin has a dual function in sleep and waking and this is now believed to depend, among other things, not only on the brain region and serotonin receptor type involved, but also on the interaction with other neurotransmitter systems27. This might explain the contrasting finding of an association between fatigue and a lower BPND in the median raphe nucleus in DRD patients. Again, the correlation was not statistically significant after correction for multiple comparisons (p\u2009=\u20090.05), probably due to the small sample size. Comparable results were found in two [11C]DASB studies in Parkinson\u2019s disease, where patients with sleep dysfunction or fatigue had a lower BPND in several brain areas including the raphe nuclei as compared to patients without sleep problems29. Further studies assessing the role of serotonin in sleep and fatigue in dystonia patients are warranted to validate the findings of our study. Our results support that the serotonergic system is associated with both sleep and psychiatric disorders, but that this is not specific for DRD.Furthermore, the correlation analysis in all participants showed that a worse quality of sleep was associated with higher SERT binding in the DRN. This was also observed in the DRD group, though the correlation did not reach statistical significance after correction for multiple comparisons. In the past, several studies showed evidence of the complex interaction between the raphe nuclei and sleep30, so one would expect to find more differences in SERT binding. Furthermore, we did not find a correlation between dosage of levodopa and BPND in any of the VOIs in the DRD group. None of the participants were taking serotonergic medication during the study, however, one DRD patient used paroxetine 7\u00a0years prior to the scan, this might have had some influence on our results. Next, the number of participants was relatively small which is an inevitable reality when one investigates a rare disorder such as GTP cyclohydrolase deficient DRD, but this may account for our negative findings.This study has several limitations that should be taken into consideration. The levodopa therapy that our patients were taking might have influenced our results. However, it was shown that levodopa therapy is able to reduce levels of serotonin and 5-HIAA in the brain even more11C]DASB PET scans alone. Associations between sleep problems, psychiatric diagnosis, and SERT binding were found in the whole group and were not specific for DRD. However, when corrected for psychiatric co-morbidity a significant difference in SERT binding in the hippocampus was found between the DRD patients and controls. This suggests an altered serotonergic metabolism in DRD, masked by the high prevalence of psychiatric disorders. Further research investigating psychiatric co-morbidity and multiple neurotransmitters, like dopamine, norepinephrine or acetylcholine together with serotonin, are necessary to shed light on the pathophysiology of the highly present non-motor symptoms in DRD.To conclude, no differences in SERT binding in DRD patients were found, indicating that either there is no altered serotonergic metabolism, or differences are too subtle to be detected with [Supplementary Information."} +{"text": "The Hatter Cardiovascular Institute biennial workshop, originally scheduled for April 2020 but postponed for 2\u00a0years due to the Covid pandemic, was organised to debate and discuss the future of Remote Ischaemic Conditioning (RIC). This evolved from the large multicentre CONDI-2\u2013ERIC\u2013PPCI outcome study which demonstrated no additional benefit when using RIC in the setting of ST-elevation myocardial infarction (STEMI). The workshop discussed how conditioning has led to a significant and fundamental understanding of the mechanisms preventing cell death following ischaemia and reperfusion, and the key target cyto-protective pathways recruited by protective interventions, such as RIC. However, the obvious need to translate this protection to the clinical setting has not materialised largely due to the disconnect between preclinical and clinical studies. Discussion points included how to adapt preclinical animal studies to mirror the patient presenting with an acute myocardial infarction, as well as how to refine patient selection in clinical studies to account for co-morbidities and ongoing therapy. These latter scenarios can modify cytoprotective signalling and need to be taken into account to allow for a more robust outcome when powered appropriately. The workshop also discussed the potential for RIC in other disease settings including ischaemic stroke, cardio-oncology and COVID-19. The workshop, therefore, put forward specific classifications which could help identify so-called responders vs. non-responders in both the preclinical and clinical settings. Remote Ischaemic Conditioning (RIC) has been shown consistently to be an effective experimental intervention for the reduction of ischaemia\u2013reperfusion injury in all organ systems and animal species that have been studied to date. The phenomenon is not new. Przyklenk\u2019s group first described in the heart that non-injurious ischaemia in one coronary artery territory would lead to protection in an adjacent coronary vascular bed almost 30\u00a0years ago . MacAlliHowever, attempts at clinical translation thus far have not realised the uniformly effective intervention that had been either anticipated or hoped for. In recent years, large patient outcome trials looking at the efficacy of RIC in the context of primary percutaneous intervention and cardiac surgery have proven predominantly neutral . The samWhile gaps in knowledge belying RIC signalling were not felt to represent a significant obstacle to clinical translation, recent neutral clinical outcome trials will force the field to redress these omissions.In the\u2009~\u200930\u00a0years since the first description of RIC, the field has expanded significantly, leading to a far greater understanding of both the mechanisms preventing cell death following injurious ischaemia and subsequent reperfusion, and the key target cyto-protective pathways recruited by protective interventions, such as RIC. However, there remain significant gaps in our scientific knowledge as to how RIC recruits signalling from one tissue bed, such as a limb, to result in protection in remote organs. This knowledge gap in how RIC functions could be critical, since it could have led to vulnerabilities of this pathway being overlooked. For example, the potential for the interaction of co-morbidities and coincident medical interventions to interfere with the effective recruitment of cytoprotective signalling , 56. AnoThe current model of RIC signalling from the limb to a remote internal organ suggest that this is via a neuro-humoral pathway Fig.\u00a0 3, 57]., 57.3, 5Perhaps surprisingly, the duration of limb ischaemia, the number of cycles required and even the pressure of the cuff used to occlude arterial blood supply to the limb have received limited attention , 81, 93.Perhaps the closest we can currently come to addressing this question is to look at the data that have already been published in the setting of both acute myocardial infarction and ischaemic stroke. Several groups have undertaken a meta-analysis of remote conditioning protocols in basic and clinical settings, with the aim of ascertaining their ability to protect and whether any observation can be made with respect to the ischaemic stimulus itself in these two disease settings , 62, 94.Yellon\u2019s group showed that in some cases, co-morbidities can be overcome through increasing the number of cycles of RIC (effectively increasing the \u201cdose\u201d of the conditioning stimulus) to overcome a weakening of fundamental cyto-protective signalling cascade response to standard RIC protocols in animal models, but multiple co-morbidities or indeed co-medications are rarely, if ever, fully investigated in animal models. In reality, the true model that we need to consider here is the human \u201cmodel\u201d, more specifically the patient who commonly presents with an acute ischaemic syndrome, with all the attendant co-morbidities, such as atherosclerosis, age, diabetes etc., that these patients inevitably present with. In retrospective analyses of the limited existing clinical data, there is less solid clinical evidence for an effect of co-morbidities and co-medications on cardioprotection. Evidence for an influence of co-medications comes from studies using antiplatelet agents which can, in themselves, elicit cardioprotection thus limiting the potential for further protection , 98. In In terms of optimising RIC \u201cdose\u201d in humans, currently the only way a dose\u2013response curve can currently be constructed is through assessment of a clinical endpoint, such as infarct size as the biomarker of RIC efficacy. This is a difficult and expensive way of optimising a RIC regimen that may require many iterations in many different populations of patients with differing co-morbidities to identify the ideal RIC protocol. It is possible that flow-mediated dilatation or heart-rate response could be used as a biomarker of an effective RIC application , but bedIt would be very helpful to be able to identify the reasons for failure to protect against injurious ischaemia by RIC, and thereby identify likely responders and non-responders. As alluded to previously, patients are frequently multi-morbid and are often on therapy that can modify cytoprotective signalling.As discussed above, the timing and dose of the RIC intervention is important for obtaining maximal cardioprotection. As such, inappropriate timing or insufficient dose may result in failure of RIC ) are frequently found themselves to be cardioprotective. We have previously discussed the so called \u201csuccess hypothesis\u201d 3481777) , and buiOther organ systems could be similarly targeted, for which ischaemic emergencies are encountered or transient ischaemia can be anticipated (for example in surgery and particularly in the handling of donor organs)\u2014and it could be argued that it may be worth revisiting certain studies such as the REPAIR Study in the lWhile the focus of large clinical trials to date has appropriately been on ischaemia and reperfusion injury, there are other emerging fields in which RIC may potentially find a role. Discussion in this workshop covered a range of novel targets that may be amenable to RIC intervention. In this regard, Redington\u2019s group recently demonstrated the ability of RIC to improve cardiac output and organ function in the context of lipopolysaccharide-induced sepsis . OptimisSepsis is associated with an increase in cytokines similar to that observed in patients diagnosed with COVID-19 . This paAnother area discussed, which is becoming increasingly relevant to cardiologists and oncologists alike, is the impact of cancer therapy upon cardiac injury and the risk for the late emergence of heart failure and iatrogenic cardiomyopathy. It is certainly clear that repeated cycles of anthracyclines lead to an accumulative injury to the heart, often realised by the release of troponin . Of note, in some cases anthracycline cardiotoxicity results in cardiac contractile impairment without death of cardiomyocytes. These cases might not be picked up by hs-Tn evaluation, and more sensitive modalities, such as cardiac magnetic resonance imaging or strain echocardiography should also be considered. In fact, in some ways, anthracycline cardiotoxicity may be a more suitable target for RIC than STEMI, since the timing for the intervention of RIC is more convenient , there is no issue regarding the area at risk or coronary collateralization extent. This has inspired studies of RIC in the setting of anthracycline cardiotoxicity , 20, 29,However, the mechanism of anthracycline-mediated myocardial injury remains unclear, and how chemotherapy may potentially impact the generation of the RIC signal has also not been described , 89. MorThe workshop participants concluded that, although some aspects of RIC remain an enigma it is recognised as a powerful cytoprotective intervention in experimental models. Furthermore, it has led to remarkable advances in our understanding of the mechanisms and progression of cell death following lethal ischaemia and reperfusion and has provided novel insights into neuro-humoral signalling that appears to link different organ systems under conditions of stress. However, thus far at least, it has failed to translate into an effective clinical therapy.Applying RIC in modern clinical practice has proven considerably more difficult than expected\u2014the reasons for its failure have been widely discussed. One conclusion of those attending this workshop was the importance to return to basics: to fully define the whole RIC cyto-protective signalling arc and to understand how co-morbidities and concomitant medical therapies may interfere with RIC signalling and the downstream cytoprotective signalling in the organ itself. On that basis, we propose a simple scheme for breaking down and understanding how RIC protection signalling may be inhibited, and based on this understanding, ensure that future clinical trials are cognisant of these potential issues and patient inclusion/exclusion criteria are developed to ensure that trials can be appropriately powered to provide a definitive answers to the question: is RIC a potentially useful clinical intervention, or should it remain an incredibly useful research tool in which to expand our knowledge of ischaemia and reperfusion injury.It is also clear that we need a fuller understanding of the neuro-humoral signalling pathway and need for biomarkers to measure activation of the RIC signalling pathway, so that conditioning protocols can be optimised and potentially identify patients who are RIC responders, and those who are not.In pre-clinical studies, there are already excellent guidelines for the design and reporting of fundamental science studies, such as those advised in neuroprotection: the Stroke Treatment Academic Industry Roundtable (STAIR) guidelines , the 20/The workshop participants debated whether pre-clinical studies should be made more complex so as to more accurately reflect the clinical scenario when patients present with an acute ischaemic syndrome. There is certainly a place for such studies, but there is also an argument that increasing complexity of basic studies moves away from discovery into something else entirely, and as part of the replacement, reduction and refinement to minimise the use of animals, then the only model that really counts is the patient model\u2014and here, through study design refinement, it should be possible to construct a study with the best chance of revealing a positive result when powered appropriately."} +{"text": "Cellulose is known to interact well with water, but isinsolublein it. Many polysaccharides such as cellulose are known to have significanthydrogen bond networks joining the molecular chains, and yet theyare recalcitrant to aqueous solvents. This review charts the interactionof cellulose with water but with emphasis on the formation of bothnatural and synthetic fiber composites. Covering studies concerningthe interaction of water with wood, the biosynthesis of cellulosein the cell wall, to its dispersion in aqueous suspensions and ultimatelyin water filtration and fiber-based composite materials this reviewexplores water\u2013cellulose interactions and how they can be exploitedfor synthetic and natural composites. The suggestion that celluloseis amphiphilic is critically reviewed, with relevance to its processing.Building on this, progress made in using various charged and modifiedforms of nanocellulose to stabilize oil\u2013water emulsions isaddressed. The role of water in the aqueous formation of chiral nematicliquid crystals, and subsequently when dried into composite filmsis covered. The review will also address the use of cellulose as anaid to water filtration as one area where interactions can be usedeffectively to prosper human life. To dismiss hydrogen bonding completely seemscounter to what might occur in the assembly, whereby these nonbondedinteractions give rise to favorable conformations of the chains tofacilitate other interactions, e.g., hydrophobic. Much research hasbeen carried out into the self-assembly of synthetic amphiphilic blockcopolymers, and their use for a number of applications including thesynthesis of drugs and gene delivery,10 ordered polymer matrices,11 and for arange of products including rheological modifiers, stabilizers forlatexes, etc.12 Similar studies have beenconducted on incorporating polysaccharides into what are called glycopolymers,where synthetic polymers are decorated with pendant sugars, or polysaccharidesare modified with synthetic polymers.13 Some merging of these disciplines is however required, and a deeperunderstanding of the interplay of side chain modification in the solubilityand then perhaps switching to an insoluble state needed. The definitionof what constitutes a glycopolymer is also somewhat confused13 and perhaps should only refer to a syntheticpolymer with pendant carbohydrates or sugars.14 Probably most pertinent to this review will be what are called amphiphilicglycopolymers (AGPs) where hydrophilic polysaccharides, or sugars,are modified with hydrophobic groups.13 It may be possible that such \u201cglycopolymers\u201d formin the cell wall, during synthesis, but this remains a topic for futurework.Indeed, much has in recent times been made aboutthe importanceof hydrophobic interactions in cellulose, viewing the molecule as\u201camphiphilic\u201d, with the Lindman hypothesis stating thatthis is a major reason why cellulose is recalcitrant to most solvents,including water itself.15 So it is quite possible that water interrupts the ability of certainsolvents to act on cellulose and can be used as a quench for the dissolutionor as a coagulating agent, for instance in the ionic liquid spinningof cellulose fibers.16Cellulose is known to interact well with water, giventhe largenumber of hydroxyl groups along its molecular chains. Cellulosic materialsswell and will disperse in water. These interactions have been usedas a processing and activation step in the dissolution in other solvents,and the medium provides the means for the dispersion of fibers toproduce paper. However, it has been shown that water acts as an antisolvent,and much better dissolution is obtained in its absence for difficultto dissolve celluloses.21 These early observations showed that there was a hysteresis betweenthe water adsorbed and desorbed from the structure, which was thenunderstood on the basis that wood behaved like a swelling gel.21 Gels themselves are materials that have receiveda lot of attention in recent times, especially where polysaccharideslike cellulose are concerned. The use of nanocelluloses for the productionof gels has recently been reviewed.22 However,it is probably not a good direct comparison to compare wood and gelsbecause orders of magnitude differences exist between the modulusand strengths of these two materials.The swellingof cellulose in water is thought to take place dueto the presence of water molecules \u201cpacking\u201d into thedisordered regions of the semicrystalline structure. Understandingof the interactions of cellulose with water began with observationsof materials such as wood.23 Also, plant fibers haveplayed a significant role in traditional applications of biomass,including for the construction of ropes, sails, and also paper. Inthe 1930s and 1940s, both in the U.S. and Europe, dwindling suppliesof traditional materials led pioneers such as Henry Ford, George WashingtonCarver,24 and Norman de Bruyne25 to incorporate natural fibers in automotiveand aerospace applications. It is known that natural fibers are susceptibleto moisture, showing typical sorption/desorption hysteresis behavior,26 which can limit their use in certain applications.As such, there have been moves to use more highly crystalline cellulosematerials, with the premise that a reduction in the so-called \u201camorphousfraction\u201d of the materials might result in less susceptibilityto these effects.Cellulose based naturalfibers are numerous as sources of biomasson the planet. They come in a variety of forms, depending on the plantsource, e.g., flax, hemp, jute, ramie, sisal, kapok, cotton, bamboo,and miscanthus. Geographically, certain plant fibers are enormouslyimportant to the economy of specific countries. For instance, jutein India and Bangladesh accounts for the vast majority of the world\u2019sproduction and has in the past significantly contributed to theirown economies.31 CNFs are typically produced through the mechanical and/or chemical/enzymaticbreakdown of plant matter, using processes such as homogenization,grinding, and microfluidisation.32 Similar nanofibrilsof cellulose can also be made using bacteria, so-called bacterialcellulose (BC).33 All of these forms ofCNMs have been used to make composite materials, where the high stiffnessof CNFs and CNCs, due to the intrinsically high modulus of crystallinecellulose (130\u2013150 GPa),34 enablesreinforcement of polymer matrices.35 Inaddition, given the switchable interactions (\u201con/off\u201d)between cellulose nanomaterials by the introduction and extractionof water, adaptable properties actuating stiffness upon drying, andflexibility on wetting, can be achieved.37 Indeed, nature has synthesized a complex composite system with highstrength and flexibility in wood and plants, which can be changedby the addition of water. Moreover, the actual synthesis of plantcell walls requires the presence of water. This review will startby exploring the biosynthetic process of cellulose and its interactionwith other components of the plant cell walls, emphasizing the roleof water, and then explore extracted nanomaterials and their interactionswith water in composite systems and assemblies.To address these issues, among others, cellulosenanomaterials(CNMs), in particular cellulose nanofibrils (CNFs) and cellulose nanocrystals(CNCs), have been extensively researched and reported on extensivelyover the past decade.22.140 The cell walls of plants are divided into the primary cell wall(PCW) and secondary cell walls (SCW). The components of the PCW, whichinclude cellulose, hemicellulose, and pectin function in a cohesivemanner to enable the enlargement of plants while maintaining theirstructural integrity.43 The SCW isdeposited after cell growth has stopped and contains a greater portionof cellulose alongside hemicellulose, lignin, and a smaller amountof pectin.45 The plant cell wall and the major components of the PCW and SCWare represented in The growth of plants occurs via enlargement and differentiationof cells enclosed within polysaccharide-based walls.46 The biosynthesis ofcellulose occurs at the plasma membrane of plant cells by a largemobile enzyme complex known as the cellulose synthase complex or rosette.39Cellulose biosynthesis in the cell wall of plantsis a highly complexand concerted sequence of natural processes. However, advances ingene sequencing have paved the way for a better understating of cellulosesynthesis in the cell walls.Arabidopsis thaliana possesses 10 differentCESA proteins, and three different CESA proteins are needed to makea functional cellulose synthase complex. AtCESA1,3, and 6 are involved in cellulose biosynthesis in the PCW, while AtCESA4, 7, and 8 are responsible for the synthesis of cellulosein the SCW. Each CESA protein uses uridine diphosphate (UDP)-glucoseas substrate to catalyze the addition of \u03b2-d-glucopyranoseto the growing cellulose chain via a (1\u21924) glycosidic bond.47 While early studies have suggestedthe basic cellulose microfibril is composed of 36 chains,38 more recent work using wide-angle X-ray scatteringand solid-state NMR49 all suggests an 18\u201324glucan chain cellulose microfibril50 An 18-glucan chain model51 was further supported by several studies on CESA protein structureand organization, including a recent study using cryoelectron microscopylooking at poplar CESA8, part of the cellulose synthase enzymes inthe SCW of poplar.52 The structure demonstratedthat PttCESA8 formed a homotrimer that would be consistent with arosette synthesizing an 18 chain microfibril and being composed of6 trimers occur via hydrogen bonding,van der Waals forces, and electrostatic and hydrophobic interactions.38 Hemicellulose is a branched polymer of different sugarunits that binds well to cellulose in the cell wall.59 Pectin, a complex polysaccharide, supports cell growthby forming swollen gels that cause microfibrils to glide past eachother during growth or lock them after growth.60 There is evidence that some types of hemicellulose canbind to cellulose in a manner that they are able to alter the packingand the crystal structure of cellulose microfibrils.62 Cultures of Acetobacter xylinum prepared in acetylglucomannan61 and xylan62 hemicellulose media resulted in loosening of the microfibrilpacking and a reduction in the cellulose 1\u03b1 content becauseof the binding of these hemicelluloses. It is worth noting that onthe contrary, cellulose microfibrils prepared in pectin media didnot affect cellulose packing or crystalline structure.In plant cells, the matrixpolysaccharides, hemicellulose, and pectin, are synthesized in theGolgi apparatus and transported to the cell wall.38 The coating of cellulose microfibrils with matrixpolysaccharides functions as a \u201cglue\u201d for the nearbymicrofibril bundles. In a recent study,63 the interaction of cellulose with five hemicelluloses found in PCW and SCW were studied using molecular dynamics simulationsin hydrated and nonhydrated systems and in isolation of other cellwall components. This study showed that 4-O-methylglucuronoarabinoxylanhas the highest binding energy to the hydrophilic plane of cellulose,while fucogalactoxyloglucan showed the least binding energy. A bindingassay study of cellulose\u2013xyloglucan interaction however showedthat the binding capacity of xyloglucan depends on the molecular weight,as lower molecular weight xyloglucan resulted in higher binding capacitythan those of higher molecular weight.64 This study also postulated that the interaction of xyloglucan withhydrated cellulose involves the formation and breakage of hydrogenbonds.64 Nevertheless, the binding capacityof either xyloglucan or xylan depends on the source of cellulose,whether of bacterial or plant origin and the differences in packingwithin the cell wall.65Somepostulations of cellulose\u2013hemicellulose interactionsin the PCW may involve a spontaneous binding of neighboring microfibrilbundles by hemicellulose or the entrapment of hemicellulose duringmicrofibril formation and subsequent coating by the same.66 a well-definedxylan interaction with the hydrophilic plane of cellulose within thesecondary cell wall is possible if the xylan chains conformed to a2-fold helical screw, such as seen for cellulose, where only one sideof the xylan chain is bearing well distributed acetyl or [4-O-methyl] glucuronic acid functional groups. However, irregularbinding of xylan to the hydrophobic planes of cellulose was postulatedto occur. The authors also asserted that this concerted covering ofthe hydrophilic plane of cellulose by xylan exposes the hydrophobicplanes of cellulose to interaction with lignin as represented in 66Accordingto Busse-Wicher et al.66 was later confirmed to facilitate xylaninteraction with cellulose, as evidenced by solid-state nuclear magneticresonance (ssNMR) studies using whole/untreated plant stems.67 However, this study shows that xylan acts asa bridge between the cellulose and lignin and that the cellulose\u2013lignininteraction is limited. Also, it was indicated that the xylan\u2013lignininteraction occurs via intermolecular hydrogen bonding between theoxygen of the lignin methoxy groups and the hydrogen of the hydroxylgroups of xylan68 and very minimally by a hydrophobicinteraction.67 Molecular dynamics simulationof the interaction of lignin with cellulose, however, showed higherlignin binding affinity on the hydrophobic plane of cellulose.69 It is therefore likely that lignin interactswith cellulose and other cell wall components via both electrostaticand hydrophobic interactions, which is something that could be potentiallymimicked in synthetic systems. There is clearly much more work todo, however, to ascertain which interaction is dominant and the mechanismby which cellulose interacts with xylan and lignin in the cell wall.The 2-fold helical screw conformation postulatedfor xylan by Busse-Wicheret al.68 In an attempt to mimic the cell wall of wood, honey-combed cellulosefilms were fabricated and adsorbed with hemicellulose and/or lignin.71 The films with adsorbed lignin showed improved mechanical strengtheven at high moisture content in comparison with films without ligninat the same conditions.Indeed, lignin is a hydrophobic aromatic polymer which is predominantin the secondary cell wall of plants and woody materials, and contributesto the mechanical and waterproofing properties of these materials.72 Water uptakesupports the growth of plants by increasing the volume and relaxingthe stress generated in the cell wall from the synthesis of cell wallpolymers.74 Water is therefore an essential componentof the complex cell wall composite that interacts with the other cellwall components at different rates.75Water is actually a major cell wallcomponent that regulates thegrowth of plants but is less talked about relative to the solid andpolymeric structural constituents.76 Another moleculardynamics simulation of cellulose\u2013hemicellulose interactionshowed that water molecules were not adsorbed within the crystallineregions but adsorbed on hemicellulose and the interphase between thetwo polymers, leading to increased spacing and a reduction in modulus.77 Simulations that did not include hemicellulose,but only cellulose and water, showed that there is an increase inthe volume of the crystalline region of cellulose on interaction withwater.57 This model, however, may not berepresentative of the cell wall as it is devoid of other cell wallcomponents.In simulated cellulose\u2013hemicellulose interactions, waterwas implicated to cause a reduction in the amount of force neededto shear the cellulose\u2013hemicellulose system by acting as lubricantand plasticizer within the interface.78 cellulose, the most abundant material on earth.While this lack of consensus can appear challenging with many beggingquestions yet to be answered, there lies a great opportunity to harnessthe manifold knowledge and understanding of cellulose formation andinteractions to create sustainable materials for specific applications.There have been many attempts to harness the self-assembling natureof cellulose, and this review now turns to a few examples of thosetrying to understand the role that water plays in these processes.One stark observation is the disparities in theliterature on theunderstanding of cellulose formation in the cell wall and the interactionsof cell wall components. This lack of agreement stems from the greatdiversity and heterogeneity of the material in question,33.179 Cellulose itselfforms into many chiral shapes, including at the fibril level structuresfor a variety of biopolymers have been known about since the early1800s and has been observed in aqueous solutions of DNA,90 polypeptides,93 cellulose,95 virus suspensions,97 helical filaments,98 and amyloid fibrils.99 We know from all of these studies that LC phasesare often formed due to the presence of charged rod-like particlesin an aqueous phase.100 Indeed, althoughan aqueous environment is often used, this effect will occur in othersolvents. A combination of and a balance between chiral steric interactionsand Coulombic charge repulsions, often CNCs that form LC phases arenegatively charged with sulfate half ester groups, is key to the formationof the lyotropic phases.100 CNCs in a diluteaqueous suspension are known to form isotropic fluids, but at a veryspecific concentration they will form LC phases.101 The specific nature of these LC phases are chiral nematics,wherein CNCs are ordered nematically, but a continuous twist is observed phasesoccur, to a value below that which causes purely geometry driven kineticarrest (or the formation of a gel).102The exact physical reason why CNCs form this phaseis unknown,nor is it known what role water plays in this transition to a chiralphase. What is known though is that colloidal stability, often forCNCs controlled by the presence of sulfate monoester groups on theirsurface, is extremely important.103 The approach has recently been a subject of renewed interestdue to the ability then to take such structures and covert them tosilica, and glass-like materials,104 butto also scale the production to make colored films and iridescentglitters.105 The formation of the filmsinvolves controlled drying, which initially focused on the dryingof pinned droplets to a substrate,106 althoughsuch approaches have recently demonstrated that coffee ring effectscan give rise to nonuniformity in both thickness and structural color.107 The coffee ring effect has been known aboutfor some time for other aqueous suspensions of nanomaterials.108 What is most interesting here though in a dryingfilm of CNCs is the movement or flow of the water and how that canbe controlled to enable the better formation of the resultant films.Later, the drying kinetics of the film are also important. It is knownthat Marangoni and capillary flows are in competition with each otherin a drying droplet of aqueous CNC suspensions.109 The Marangoni effect has long been known and is the masstransfer along the interface between two fluids due to a gradientin the surface tension, and it is known that it has to be suppressedin a drying droplet for coffee rings to form.110 The manipulation of this flow has been studied for CNCbased aqueous droplets and shown to be insignificant compared to thecapillary flow.109 If an infusion of ethanolinto the droplet is however allowed, then Marangoni flows can be usedto ensure the production of uniform films of structural color.109 Height profiles of the drying droplets havebeen shown to be more uniform do notform chiral nematic structures due to rapid gelation,113 and this is due to the hydrophobic interactionsbetween the CNCs. However, little has been reported on any such effectsin unmodified CNC systems or indeed whether such effects do occur.Some understanding of the shape of confined water around CNCs, withhydrophilic and hydrophobic moieties present, has recently been described.114 The drying rates of CNC films has been controlledby the presence of hydrophobic groups (phosphonium modification).114 This work showed that the dielectric constantof absorbed water is significantly lower than that of the bulk, andthe shape of the confined layer is more spherical for hydrophobicallymodified CNCs and platelet-like for hydrophilic materials.114 The shape of the confined layer of water thenhas an effect on the pitch of the cholesteric twist, and thereby thecolor, with hydrophilic CNCs giving rise to a tighter twist, and ared-shift in color.114 Although the morehydrophobic forms of CNCs and other nanocelluloses do not seem toform chiral structures, they do however aid self-assembly and offerinteresting routes to structured materials. The next section dealswith this topic, highlighting again the role of water in this process.This control of the flow of water is just one aspectof the formationof the entire film of a deposited cellulosic structure. There havebeen very few studies on the role of water in the interstices betweenthe CNCs in a forming chiral nematic structure. It is known that becauseCNCs are themselves parallelepiped structures, with twists of theirown, that they pack in a chiral structure to maintain efficiency.3.3115 The adsorption of chemicalspecies to the surface of cellulose, unmodified or not, is best understoodin terms of thermodynamics.115 It has beenshown that for a wide range of materials that they demonstrate an invariance in the binding basedon thermodynamic considerations, with a constant change in the GibbsFree Energy (\u0394G).115 Linear relationships between the enthalpy change and entropy changewere plotted, showing a constant slope equal to the reference temperature, for a wide range of different adsorbants.115 The mechanistic differences were that the bindingof proteins was found to be enthalpy and entropy driven, whereas chargedmolecules and ions were found to be driven by entropy, wherein watermolecules are displaced but there is no heat exchange.115 These considerations are important when itcomes to discussions about so-called hydrophobic interactions becausethere are many recent publications that claim that cellulose has hydrophobicand hydrophilic properties, i.e., it is amphiphilic.117Amphiphilicproperties of charged forms of cellulose, such as sulfated cellulosenanocrystals and nanofibrils, are well-known, although sometimes overstated.It is understood that the surface of cellulose itself is relativelyinactive and requires some modification to increase its chemical activity.Nevertheless, adsorption of many different polymeric and other chemicalspecies to its surface is possible, driven by both enthalpic and entropicprocesses.118 The hydrophobic effect,or interaction, as it should be called (it is not a bond) has beenstudied extensively and is probably best understood from the simplepartitioning of oil\u2013water emulsions.119 It has been shown that the hydrophobic effect, even in these simplesystems, has subtleties in the balance of entropic and enthalpic effectsas a function of temperature.119 Southallet al. showed that there is a big penalty for the opening of a cavityin a solvent.119 These \u201ccavities\u201dare often viewed in aqueous systems as a structuring of the watermolecules around the solute. The role of the size of the solute hasbeen recently shown to be critical in this structuring of the solvent,and the solvent also dictates the solute identity.120 It is known that cellulose binds to water through hydrogenbonding and also to itself by the same interaction. But there is nodifference in the strength of either bond ,and in that sense cellulose will just as readily bind to water asit will do to itself. Indeed, a recent overview of the role of hydrogenbonding and the exaggeration of its importance has recently been presentedand has shown that their strength and relevance in the cohesion ofcellulose is relatively small compared to dispersion and hydrophobiceffects.5In simulation studiesof cellulose\u2013graphene interfaces, it has been shown that certainfaces of the cellulose molecule in idealized structures will adhereto graphene via a hydrophobic effect, wherein water is displaced betweenthe surfaces.121 The presence of a hydrophobic \u201cface\u201dto cellulose is somewhat fraught with difficulty because, for fibrillarstructures, it is not completely known what their shape is and whetherthis varies between materials made from different starting materials.Lahiji et al.122 have summarized severalidealized cross sections for cellulose nanocrystals of the films considerably, whereas when dry the filmswere found to be much stiffer and stronger.133 The ability for cellulose to \u201cswitch\u201d from interactionswith itself, to interactions with water, is grounded in the fact thata hydrogen bond within the structure of cellulose is no stronger,nor more energetically favorable, than with water.6 However, it is also known that there is a certain amountof \u201cfree water\u201d at the surface of cellulose even atvery low moisture contents,134 and thatas moisture content increases this also increases, relying on thepresence of bound water also at the surface. Quite what the role waterplays in mediating the interactions between cellulose fibers, particularlyat the nanoscale, is unknown. Additional to this, plasticization,which is often talked about in the literature, where the hydrogenbonding is switched from cellulose\u2013cellulose interactions tocellulose\u2013water interactions is commensurate with a dramaticincrease in the free water.134 It has beenshown that it is the bound water that indeed breaks the hydrogen bondingbetween cellulose,135 which begs the questionas to whether it is just hydrogen bonding at play in switchable nanocellulose-basedcomposites?Building on the work presented in the previous section on chiralnematic films and liquid crystalline states of CNCs, switchable interfacesin chiral-based epoxy\u2013CNC composites have been also achievedby combining cholesteric phase liquid crystalline based films withepoxy resin; a water responsive chiral nematic composite.4.1136 Furthermore, this effect was demonstrated tobe wholly due to the disruption of the internetwork bonding (assumedhydrogen bonding) between CNCs inside the composite, and not matrixplasticization.136 This was demonstratedby swelling the materials in 2-propanol (IPA), which is known to havea similar effect to water; no decrease in the mechanical propertieswas however observed. This unique interplay between water and cellulosefurther enabled switchable interfaces in other nanocomposites, includingwith polyurethane (PU), demonstrating shape-memory deformation.6 The shape-memory effect relied on the abilityto disengage the interactions between a network of CNCs within thePU matrix, enabling an orientation of the rod-like particles 37 Upon drying, the CNCs remained in this oriented state,only relaxing again upon further addition of water whereupon the networkreforms its shape and connectivity.37 Similarresults were also obtained by another group, again showing shape-memoryeffects.137One example of a highly impactful study on stimuli-responsive nanocompositesbased on cellulose was by the group of Christoph Weder, and others,where they showed that the addition of water as a \u201cchemicalregulator\u201d to an ethylene oxide\u2013epichlorohydrin/CNCnanocomposite resulted in a significant reduction in the stiffnessof the material (from 800 to 20 MPa on immersion in deionized water).4.2138 has demonstrated that diffusioninto a polymer matrix\u2013CNC composite is increased with the additionof CNCs into a PU/CNC composite and that the dry and wet state mechanicalproperties can be modeled using percolation and Harpin\u2013Kardosmodels, respectively. The percolation model is governed by the equation140E\u2032 is the storagemodulus of the composite, Es\u2032 and Er\u2032 are the experimentally determinedmoduli of the matrix and the reinforcing phase, respectively, Xr is the volume fraction of the reinforcingphase, Xc is the critical percolationvolume fraction, and \u03c8 is the volume fraction of the reinforcingphase that take part in load transfer. It is worth going back to theoriginal papers on these models, specifically the Takayanagi model,139 and the further developments by Ouali et al.140 for multiphase polymers, although their equationis largely based on the former. The models are phenomenological andbased on viscoelastic constructions and variations of series-parallelmodels. They really comprise three phases, not the two (reinforcementand matrix) that might be assumed on first sight, with a series parallelarrangement as depicted in dispersed stiff phase in a more compliant one. In fact, the later model byHalpin and Kardos142 were critical of theuse of a parallel phase are boundby the upper and lower ,with S-shape laws of reinforcement for all composite structures placedin between these bounds of a composite, asE1 and E2 are the moduli of the matrix and the reinforcingphase, respectively.One of the major issues with the use of these modelsis that beingphenomenological, they have limited physical meaning beyond what hasbeen already described. Their use has been proposed to describe thewater interactions between CNCs within a network, but little has beendone to fully interrogate the models and adapt them for this purpose.It is assumed that the models developed by Takayanagi et al. are bestused for networks of CNCs, whereas Halpin\u2013Kardos for disengagednetworks when wet. However, given the 3-phase nature of the modelssee 13, itouUsing this equation to plot a range ofcurves for different valuesof \u03b1 15, it is144 and originally proposed by McCullough et al.145 This approach often gives reinforcement curvesthat sit between the upper and lower bounds to assist with printing and curing. Shape changing biomorphicstructures from the surrounding media interact with the adsorbent surface. Theinteractions are governed by \u03c0\u2013\u03c0 interactions,forces that produce physical bonds between contaminants and the adsorbentsurface, e.g., van der Waals forces, hydrogen bonding, as well aschemical bonds, e.g., ion exchange and complexation.162 Surface modification by acid hydrolysis often results in the modificationof C-6 OH groups, as these are most accessible, although modificationof C-3 and C-4 OH moieties have also been reported, e.g., followingphosphoric acid hydrolysis.163 Finally,a less explored approach to surface modification involves reducingend modification160 which offers the possibilityof imparting dual functionalities on a single anhydroglucose unitthrough the C6 OH group, thereby increasing adsorption efficiency.The applicationof nanocellulose as the active adsorbent is based on the presenceof appropriate surfaces which functionalities can be imparted, throughapproaches broadly classified as (i) direct modification of the surface,e.g., through acid hydrolysis, TEMPO oxidation, periodate oxidation,or (ii) grafting of polymers with the desired functionalities.\u2013), phosphates (PO43\u2013), and thiol (\u2212SH)groups. The capture of anionic contaminants, on the other hand, ispromoted by positively charged surface groups which can be impartedthrough amination and quaternisation reactions.Surface charges play a significant role in the interactions betweenadsorbates and adsorbent surfaces. As such, it is important, in thedesign of adsorbents, to consider the nature of the target contaminant.Positively charged contaminants, e.g., metal ions and cationic dyes,are efficiently captured using adsorbents with negatively chargedsurface groups, e.g., carboxylates (\u2212COOPearson\u2019shard soft acid base (HSAB) concept also offersmeaningful guidance in selecting surface modifications to impart tonanocellulose surfaces. Soft acids form more stable covalent bondswith soft bases, hence improving the removal of Hg(II), Ag(I), andAu(I) ions by thiolate and thiocyanate containing adsorbents. Whilehard acids such as Cr(III), Cr(VI), Mg(II), and Fe(III) ions are betteradsorbed by hydroxides, carboxylates, and ammonia groups (hard bases).Borderline elements, such as Fe(II), Co(II), and Pb(II), are bestremoved using adsorbents with pyridine, aniline, nitrate, and sulfatemoieties.5.1.1164 ammonium persulfate (APS), hydrogen peroxide,165 and periodate\u2013chlorite combination,166 anhydrides of succinic, phthalic, and maleicacids,167 as well as organic acids, e.g.,oxalic acid169 and citric acid.170 Organic acid derived materials seem to havehigher aspect ratios compared to CNFs generated using sulfuric acid,170 i.e., aspect ratios of 144 versus 83, whichis beneficial for aerogel formation by physical bonding, precludingthe use of chemical cross-linkers. Dye and metal(Cu2+) removal by the physically cross-linked aerogelswas 132.98 and 45.05 mg g\u20131, respectively. Similarremoval efficiencies: 110 and 51.1 mg g\u20131 for MBand Cu2+, respectively, were reported for CNCs with a carboxylicacid content of 2.2 mmol g \u20131, that had been preparedby hydrogen peroxide oxidation.165 Carboxylationby this approach is driven by H+ ions and OH\u2022 generatedfrom Fe2+ catalyzed degradation of H2O2. H+ ions protonate the \u03b2(1\u21924) glycosidicbonds resulting in its disintegration, while the free radicals attackthe OH groups to produce carboxylic acid groups. Adsorption of dyesis then driven by hydrophobic ring\u2013ring interactions with thecellulose molecules, as well as electrostatic interactions betweencationic centers on the MB ring and the carboxylate groups on CNCs.This may explain the higher adsorption of MB relative to copper, asonly a single mechanism is involved in uptake of the latter.Carboxylation isone of the most studied surface modifications of nanocellulose materials.Carboxylated CNCs and CNFs can be prepared using a variety of oxidantsincluding 2,2,6,6-tetramethylpiperidine-1-oxygen (TEMPO),171 examined the use ofcyclic anhydrides as oxidizing agents in the preparation of CNFs fromwheat fibers. CNF prepared with succinic anhydride (S-CNF) generatedthe highest concentration of COO\u2013 groups (3.8 mmol g\u20131), much higher than those of CNFs prepared using maleic anhydride(0.9 mmol g\u20131) and phthalic anhydride (1 mmol g\u20131). When added to paper filters at a 5 wt % concentration,S-CNF led to an increase in Pb2+ removal from 50 to 96.5%.Sehaqui et al.173 It is considereda greener approach because, besides precluding the traditional pretreatmentsteps of bleaching and delignification, waste products of the processcan be converted to fertilizer, thereby reducing chemical and waterconsumption. The process involves reacting nitric acid and sodiumnitrate in the presence of excess acid to generate the oxidizing agent:nitroxonium ions (NO+), which then attack the primary hydroxylgroup of cellulose to generate carboxylate groups. Chen et al.174 used this approach to isolate CNFs from Moringaplants before applying them for Hg2+ adsorption. Nanofibers10\u201312 nm in width and 250\u2013300 nm in length, with 0.97mmol g\u20131 carboxylate content, were obtained. Themaximum Hg2+ adsorption capacity for the CNFs was 257.07mg g\u20131. Drying the CNFs reduced Hg2+ captureslightly, from 81.6 to 74.3%, suggesting that drying for economicallyviable transportation does not substantially reduce performance ofthe adsorbent. Higher capacities were, however, reported when adsorbentsprepared by this approach were applied for removal of UO22+ and Cd2+ions, i.e., 1470 and 2550 mg g\u20131, respectively.173The production of CNF by nitro-oxidation was popularized by Hsiao\u2019sgroup at Stony Brook University.175 Cu2+ ions adsorbedonto CNFs were reduced to Cu0 and CuO nanoparticles polypropyleneglycol-block-polyethylene glycol-block-polypropylene glycol) and used for the adsorption of acetaminophen,sulfamethoxazole, and N,N-diethyl-meta-toluamide (DEET).176 Thebinding of Jeffamine ED 600 to CNFs decreased the zeta potential ofthe composite. As such, at low pH where unbound carboxylate groupsof CNF were protonated, the adsorbent was uncharged, and aggregated,leading to low adsorption. At higher pH, deprotonation and chargedsurfaces allowed for distribution of the adsorbent in solution. Similarfindings were reported for the adsorption of salbutamol (a bronchodilator)by succinylated CNFs.177 As salbutamolexisted largely as protonated or zwitterionic ions below pH 11, itsadsorption was dependent on changes in the surface charge of CNFs.Only when the pH increased above 7 and they became deprotonated wasadsorption of the positively charged salbutamol species possible.An important note for covalent bonding in adsorbent synthesis: whileit ensures more stable cross-linking, it also uses up the reactivecarboxylate groups required for adsorption. As such, a robust adsorbentwith poor efficiency may be created.Finally, carboxylated nanocellulose can be usedfor adsorptionof organic contaminants including pharmaceutical drugs and pesticides.TEMPO\u2013CNF were covalently attached to Jeffamine ED 600 -trimethoxysilane (MPTMS),2+ and Pb2+. Geng et al.178 showedthat the adsorption of Hg2+ was twice as fast with aerogelsmade from MPTMS-functionalized CNFs than with TEMPO oxidized CNFs,i.e., 5 h versus 10 h. Importantly, CNFs without MPTMS had a removalefficiency of 23%, demonstrating that carboxylate groups are alsoinvolved in Hg2+ capture, a finding confirmed also by XPSanalysis. The calculated maximum adsorption capacity of the thiol-modifiedTEMPO CNF of 729.9 mg g\u20131 was close to the experimentalvalue of 718.5 mg g\u20131. Li et al.182 reported an even higher adsorption efficiency (923 mg g\u20131) using l-cysteine-modified CNCs. In theirapproach, CNCs generated from sulfuric acid hydrolysis were oxidizedusing sodium periodate to generate dialdehyde CNCs. Aldehyde groupswere then reduced by using sodium cyanoborohydride (NaBH3CN) before reaction with l-cysteine removal. Both materials were functionalizedwith l-cysteine by Schiff base coupling after the periodateoxidation, resulting in attachment of thiol moieties at C-2 and C-3,but this difference was lower than would be expected considering thatTEMPO CNFs (\u223c5 nm in diameter) are 6000 times smaller in diameterthan typical microfibers (\u223c30 \u03bcm in diameter). Thus,theoretically, the surface area for functionalization per gram ofadsorbent is far greater for CNFs. However, cysteine content was 648mg g\u20131 in the nanofibers and 497 mg g\u20131 for the microfibers.183 Further, As(III)removal by the two materials also did not vary much: 344.82 versus357.14 mg g\u20131 for the micro- and nanoscale materials,respectively. Neither did the temperature at which thermal degradationbegan: 101 \u00b0C for CNF and 115 \u00b0C for microfibers. Together,these data suggest that nanofibers may not always present considerablyhigher adsorption capacities, and it may be worthwhile consideringmicroscale materials whose preparation involves fewer steps and reagents.Periodate oxidation may lead to partial defibrillation in microfibersresulting in a material with large inner surface areas for functionalization.Some findings with relevance to the design of adsorbentsfor watertreatment were reported by Chen et al.184 Silylated CNFs were modified using MPTMS.185 The materials had a turbidity removal efficiency of 90\u201399%at a concentration of 4000\u20138000 ppm and showed high selectivityfor chalcopyrite and pyrite as removal of quartz particulates wasonly 30%. Silylated CNFs could therefore potentially be useful inthe treatment of surface water around mine dumps that are often contaminatedby fine particulates deposited by wind or by rainfall driven attritionof waste heaps.Thiol-modified nanocellulose has also been investigated for selectiveflocculation of chalcopyrite and pyrite minerals.5.1.3186 pharmaceutical drugs,187 pesticides,188 chromates,189 and other anions .190 Different methods and a varietyof cationization agents have been reported, with varying degrees ofsuccess. It is worth noting here that while addition of amino functionalities,e.g., using polyethylenimine (PEI) also leads to cationic surfaces,the surface charge is pH-dependent, and so amine functionalized materialsare only positively charged at low pH.191 Tertiary amine groups of PEI (2 \u2264 pKa \u2265 3) are protonated at a pH of 2.192 Below this pH, therefore, CNF surfaces are positively chargedand would repel rather than adsorb cations. In contrast, quaternaryammonium compounds (QACs)193 are positivelycharged over a wide pH range, e.g., 3\u20138, and are, therefore,more attractive for cationic functionalization. QACs reported in theliterature for functionalization of nanocellulose include Girard\u2019sreagent T ((2-hydrazinyl-2- oxoethyl)-trimethylazanium chloride),195 epoxy-propyltrimethylammonium chloride (EPTMAC),197 glycidyltrimethylammonium chloride,198 imidazolium,199 aminoguanidine,201 pyridinium,202 and a deep eutectic solvents made from aminoguanidinehydrochloride and glycerol203 or boricacid and glycidyl trimethylammonium chloride.204Modifying nanocellulosesurfaces with cationic groups allows for the adsorption of a broadrange of negatively charged contaminants including anionic dyes,205 This was possible because the adsorbents lost their surfacecharges once saturated with the contaminant, leading them to settleout of solution. Similarly, the formation of flocs with lateral dimensionsof several millimeters when cationic cellulose was used for the flocculationof kaolin has also been reported.195 Asthe flocculation performance of cationic cellulose has already beenshown to be better than that of commercial polyacrylamides,204 it is plausible that if produced via economicallyviable methods, it could replace polymeric flocculants in conventionalwater treatment.In water treatment applications, cationic nanocelluloseis attractive because it offers ease of adsorbent recovery after adsorption.In a recent study, cationized cellulose was recovered after the adsorptionof Cr(VI) ions by decanting or light centrifugation.198 A second approach reported by Zaman et al.198 involved mixing powdered CNCs with NaOH before the addition of wateror a water/dimethyl sulfoxide (DMSO) mixture. Glycidyltrimethylammoniumchloride (GTMAC) was then added dropwise to the mixture before heatingat 60 \u00b0C for 4 h. They found that CNCs made using the DMSO processhad much higher surface charge density (2.05 mmol g\u20131) than CNCs cationized in water (0.35 mmol g\u20131).The maximum fraction of water in the water/DMSO mixture for optimalcationization was determined to be 36 wt %. Together, these studiesshow substitution of a fraction of the water with organic solventscan improve cationization efficiency.One of the main challenges faced in aqueouscationisation approaches,however, is how to control the amount of water to achieve a high degreeof substitution. Of course, one of the other issues of adding cationicgroups is that you often immediately get adsorption of anionic polyelectrolytes,which can effectively neutralize the surface. The process is usuallycarried out in the presence of water and NaOH, with the former actingas a nucleophile to activate cellulose OH groups toward etherification.However, water promotes the formation of side reactions, reducingthe efficiency of etherification and, subsequently, the degree ofsubstitution (DS). Odabas et al. attempted to address this challengeby substituting some of the water with 2-propanol or THF and foundthat this increased the DS. Replacing up to 90% of the water withTHF increased the DS from 0.05 to 0.35.\u20131 were synthesized aftertreatment at 80 \u00b0C for 10 min.203 Thissurface charge was more than two times greater than when the samecationizing agent was used in a heterogeneous system, i.e., 1.07\u20131.70mmol g\u20131.201 The DEScould also be reused five times before replenishing of aminoguanidinewas required. Deep eutectic solvents, therefore, provide a more efficientand sustainable approach to cationisation.A third and more environmentallyfriendly solution is presentedby the use of deep eutectic solvents (DES). Using an aminoguanidinehydrochloride and a glycerol DES system, CNFs and CNCs with a chargedensity of 2.48 mmol g 5.1.4212 Phosphorylation is also known to improve thermal resistance213 and may therefore be used to improve the thermalstability of membranes intended for high temperature applications.Although relativelyless explored in comparison to other functionalization approaches,addition of phosphate functionalities has significant benefits forwater treatment, ranging from uptake of hard metal ions, e.g., lanthanide,actinide, and transition metal ions.163 showed byusing NMR studies that hydroxyl groups at C-2 and C-6 may also bephosphorylated. Various phosphorylating agents have been reported,including phosphorous acid (H3PO3),214 orthophosphoric acid (H3PO4),216 phosphoruspentoxide (P2O5),217 phosphorus oxychloride (POCl3),218 and sodium or ammonium phosphates, e.g., NaH2PO4,219 NH4H2PO4,220 (NH4)2HPO4,223 or other agents, including cyclotriphosphate(Na3P3O9)209 and hexachlorocyclotriphosphazene (P3N3Cl6).218 Shi et al.224 also reported functionalization with phosphate esters preparedby the reaction of 1-octanol or 1-octadecanol with P2O5.Phosphorylation of native nanocellulose is an esterification reactionthat involves mostly the C-6 hydroxyl group due to its greater nucleophilicity.Nevertheless, Lemke et al.,225 Heterogenous reactions involve solvents such as water,225 pyridine, hexanol, dimethylformamide (DMF), and tetrahydrofuran.226 To improve the degree of substitution, some workers have includeda swelling pretreatment step using NaOH,228 but urea,210 DMF, and hexanol have alsobeen reported, although the latter results in degraded end products.216Phosphorylation reactions may be classified as homogeneousor heterogeneous.The former, excluding water and phosphorylating agents, e.g., metaphosphoricacid are simply dissolved in molten urea.229 It seems to play multiple roles, including as a swelling agent,a solvent, as well as protecting fibers against excessive degradation.230 Granja et al.,216 for example, found that while high phosphorylation efficiencieswere possible in the absence of urea, the yield was low due to dissolutionof the cellulose fibers. Finally, recent work has suggested that ureadoes not simply act to create basic conditions in phosphorylationreactions. Blilid et al.218 used K2CO3 in the phosphorylation of nanocellulose withphosphorus oxychloride (POCl3) and found that the presenceof this base hardly improved the success of the phosphorylation, i.e.,0.29\u20130.31%. Urea, therefore, seems to exert additional advantagesbesides a basic environment. Akin to NaOH, it likely breaks down vander Waals and hydrogen bonds in the fibrils, thus facilitating exposureto phosphorylating agents. Together, these data suggest that ureaplays a key role in both homogeneous and heterogeneous phosphorylationreactions.Urea plays a key role in the nanocellulosephosphorylation reaction;some studies have reported little or no phosphorylation when ureawas left out of reactions.\u20131, respectively) because of the lower numberof N\u2013H groups available for hydrogen bonding with cellulose.In contrast, using urea resulted in phosphate concentrations as highas 3300 \u00b1 160 mmol g\u20131 and a degree of substitutionof 26 \u00b1 1%.217Not all urea containing molecules facilitate phosphorylation.Stericallyhindered derivatives, e.g., 2-imidazolidone and tetramethyl urea,has resulted in far lower phosphorylation . Higher temperature also leads to higherDS. Increasing the reaction temperature from 85 to 105 \u00b0C increasedthe DS from 0.6 to 2.5, and the reduction of reaction time from 6to 2 h.59Heterogeneousreactions tend to have lower phosphorylation efficienciesthan homogeneous reactions, which has been linked to the presenceof water.22+,211 Ho3+, Sm3+, La3+, Cu2+, Cd2+, Co2+, Zn2+, Cd2+, Ni2+,206 Fe3+, Fe2+, Bi3+, and Ag+.209 Despite the high adsorption capacities of theadsorbent prepared by Inoue et al., e.g., up to 5727 mg g\u20131 for Fe2+ ions, the long reaction times required for itspreparation (14 days at 50 \u00b0C) make the approach rather impractical.Nevertheless, the ion affinity was found to be in the order: Bi3+ > Fe3+ > Fe2+ > Ag+. Whatseemed to be contradicting findings with respect to Ag+ and Fe3+ ions were later reported by Liu et al.,209 i.e., that selectivity was higher for Ag+ than Fe3+ in both single ions and mixed ion solutionsand that Ag+ adsorption attained equilibrium in half thetime it took Fe3+ ions, i.e., 6 h versus 12 h, respectively.At face value, it seems paradoxical that a soft acid (Ag+) was adsorbed to a hard base (phosphate) and faster than a hardacid (Fe3+). The answer, however, may lie in the fact thatat the 62.5 mg L\u20131 concentration used for both ions,far more Ag+ than Fe3+ ions were present insolution. In fact, based on their molar masses, the concentrationof ferric ions was about half that of silver ions. Silver ions, therefore,benefitted from a greater drive to adsorption sites than the ferricions, leading to a greater adsorption for Ag+ in both singleion and mixed ion solutions. Where the authors used molar concentrationsand concentrations were comparable, i.e., 55 mol Fe3+ and63 mol Cu2+ ions, adsorption followed the HSAB theory.Adsorption of cupric ions by phosphate functionalized CNFs was somewhatsubdued (19.6 mg g\u20131) and ferric ions were preferentiallyadsorbed. Uptake of uranyl ion, a hard metal, by phosphate-modifiedCNFs was also high as 1550 mg g\u20131.Phosphorylated cellulosehas high adsorption capacities for a rangeof ions, including UO231 As such, themore stable phosphonates, despite being less efficient in adsorption,may be worth considering. Phosphonated nanocellulose can be synthesizedby reacting dialdehyde cellulose with sodium alendronate,232 and an adsorption capacity of 1.98 mmol g\u20131 for vanadium ions has been reported for this approach.208Finally,despite being good chelators, phosphate groups are relativelyunstable to hydrolysis.5.1.5Polymer graftingallows for the covalent attachment of polymer chains to CNCs and CNF.Grafting approaches are broadly classified as being either grafting\u201cto\u201d, where a preformed polymer is attached to the nanocellulosesurface, or grafting \u201cfrom\u201d, where polymerization isinitiated at the surface of the CNC or CNF. Each approach has advantagesand disadvantages. While grafting to allows for aqueous synthesisand thorough characterization of the polymer before grafting, sterichindrance from long or entangled polymer chains often limits the extentof polymer attachment in this approach, resulting in lower graftingdensity. Grafting from approaches, on the other hand, result in highergrafting density, but involves multiple synthetic steps, and poseschallenges with characterization of the attached polymers, e.g., length.The approach also involves side reactions that produce free/ungraftedhomopolymers that are difficult to separate from modified materials,leading to impure products.233 copolymers,234 and polyurethane.235 Grafting from approaches fall under three broadcategories: (i) ring-opening polymerization, which is often used forgrafting hydrophobic polymers, (ii) free radical polymerization, whoseconvenience lies in the fact that it can be performed in water anddoes not require prior attachment of initiators, and (iii) controlledradical polymerization (CRP), which is further divided into atom transferradical polymerization (ATRP), reversible addition\u2013fragmentationchain-transfer polymerization (RAFT), and nitroxide-mediated polymerization(NMP).159 Despite requiring prior attachmentof an initiator and tedious postattachment purification steps to removethe catalyst, CRP techniques, particularly ATRP, are attractive becausethey enable control of the polymer length and grafting density. Wepresent selected instances where grafting to and grafting from approachesfor modification of nanocellulose have been used or present opportunitiesfor water treatment applications.Grafting to can be by (i) carbodiimidecoupling, where carboxylate groups on nanocellulose react with aminegroups of polymers being grafted, (ii) epoxy ring opening, where deprotonatedsurface hydroxyl groups react with the epoxy ring of the polymer,or (iii) isocyanate mediated, which is used to couple CNCs with hydrophobicpolymers, e.g., caprolactone homopolymers,236 prepared pH-responsiveflocculants by surface-initiated graft polymerization of 4-vinylpyridine,using ceric(IV) ammonium nitrate as an initiator. Because 4-vinylpyridineis hydrophilic in its protonated state (pH < 5) and hydrophobicin the deprotonated/uncharged state, grafted CNCs produced by thisone-pot synthetic approach showed reversible flocculation and precipitationwith changes in pH (PAA) and elastin-like polypeptides(ELPs). The resulting CNC\u2013polymer complexes had both pH andtemperature responsiveness, the former because of PAA and the latterdue to ELPs. The carboxy groups of the PAA were protonated and positivelycharged at pH 3.0 but deprotonated and negatively charged at pH 8.4.Kan et al.es in pH 19a. It iSimilarly, the functionalized CNCs were agglomeratedabove 20 \u00b0Cbut formed a homogeneous dispersion below 20 \u00b0C 19b. Such238 synthesized a nanocellulose aerogel for theremoval of Pb2+, Cd2+, Zn2+, andNi2+ ions by modifying CNFs with a copolymer, methacrylicacid-co-maleic acid, via radical polymerization (\u20131), and the maximumadsorption capacities for the modified aerogel were 165, 135, 138,and 117 mg g\u20131 for Pb2+, Cd2+, Zn2+, and Ni2+, respectively. Similar findingswere reported by Jin et al.,241 who showedthat grafting of poly(ethylenimine) (PEI) increased Cu and Pb removalby bacterial cellulose from 90.91 to 111.11 mg g\u20131 and 100 to 125 mg g\u20131, respectively.Polymer modified nanocellulose has also been investigated for thetreatment of metals and dye contaminated water. Maatar and Boufirization 19c. The 241 andPEI243 to CNFs. PVA-modified CNF aerogels showedhigh removal capacities for Congo Red (869.1 mg g\u20131), Acid Red (1469.7 mg g\u20131), and Reactive LightYellow (1250 mg g\u20131).241Amine-graftedcellulose has also been applied for dye removal bygrafting of poly(vinylamine) (PVA)244 The concentration of grafted thiolranged from 0.4 \u00b1 0.05 mmol g\u20131 when the ratioof carboxylate to cysteine was 1:2, to 0.9 \u00b1 0.1 mmol g\u20131 when the ratio was 1:8. Thus, increasing the cysteine concentrationby a factor of 4 led to a doubling of the thiol concentration. Depositionof these modified CNF onto polyacrylonitrile electrospun fibers resultedin increased adsorption of Cr(VI) and Pb(II) ions (\u20131) was reported for amine-modified PEI-grafted CNCs.245Sulfhydryl moieties can be grafted into cellulose by carbodiimidecoupling of amine groups of cysteine and carboxylate groups of TEMPO-oxidizedCNF.II) ions 19d. Howe\u20131 versus 0.955\u20132.646 mmol g\u20131, respectively,160 carboxyl,247 thiol,248 and amino249 groups can be grafted to these reducing ends to createadsorbents. Zoppe et al.249 attached cationic(N-isopropylacrylamide and [2-(methacryloyloxy)ethyl]trimethylammonium chloride) and an anionic polymer (sodium 4-vinyl-benzenesulfonate) to CNCs in aqueous media, demonstrating a protocolfor the use of reducing end-modified CNCs that could be applicablein the design of adsorbents for metal ions and dyes.249 Thiol and amino-modified CNCs were synthesized by Li etal.240 by grafting triazole moieties toend-modified CNCs. While these were used to improve the mechanicalproperties of natural rubber, the presence of thiol and amino moietieson the CNCs makes the material suitable for complexation of both softand hard base metal ion pollutants. Zhong et al.250 have also recently demonstrated the antimicrobial propertiesof CNCs modified with thiol groups on their reducing ends, which allowedfor templated nucleation of silver nanoparticles.Although aldehyde groups can be 50\u2013150 timesfewer thanhydroxyl groups on the cellulose surface, i.e., 0.018\u20130.0738mmol g249Grafting ofpolymers on surface OH moieties and reducing ends ofcellulose therefore both present opportunities for increased contaminantremoval and water treatment efficacy. We note, however, that the latterpresents a challenge with adsorption efficiency due to the lower numberof sites available for grafting. Challenges have also been found withcharacterization, e.g., low signal intensities for NMR, due to thelow number of attached groups.5.2255 Nanocellulose has also been used to control aggregation256 as well as to tune the porosity of aerogels.257 Conducting polymers such as poly(pyrrole),polythiophene, and polyaniline are attractive for water treatmentdue to their high adsorption capacities for metal ions and the easeof synthesis and functionalization.259 However,they tend to aggregate during synthesis, reducing sorptive surfaceareas. On its own, poly(pyrrole) has a specific surface area of only12.21 m2\u00b7g\u20131 and an adsorption capacityfor Cr(VI) of \u223c16 mg\u00b7g\u20131 at pH 2.260 However, when complexed with bacterial cellulose,a specific surface area of 95.9 m2 g\u20131 has been reported and the adsorption capacity at the same pH increasedto 555.6 mg g\u20131.261 Complexationwith nanocellulose was also shown to facilitate the use and recoveryof polyaniline, which is an excellent adsorbent but whose recoveryfrom treated wastewater is impeded by high buoyancy.262This section looks at the application of cellulose as a scaffolding/supportmaterial in adsorbents rather than as the active material in processof adsorption itself. The premise for the application of nanocellulosein this manner is simply to take advantage of properties inherentto the material itself. This means that the materials lack the activeadsorbent and therefore develop composites with superior porosity,mechanical strength, or increased surface charges.263 as reductants,264 or in the control of aggregation.266 Finally, during adsorption, nanocelluloses can also enhance performanceand longevity of adsorbents by controlling attrition/loss of nanoparticlesfrom the adsorbent surface.270 In the following sections, we survey indetail, nanocellulose-containing composites and their water treatmentapplications.In composites, nanocellulose surfaces also serveas nucleation and growth sites for metal organic frameworks (MOFs)and nanoparticles,5.2.1271 They are characterizedby an infinite network structure, high specific surface area, andthermal stability, in addition to chemical properties unique to theorganic and inorganic components.273 As such,it is possible to tailor MOFs for wide-ranging applications includingin water treatment, where they have been used for the adsorption ofcations, dyes, and other organic contaminants. However, MOFs comein powders that compact during water flow-though, which not only diminishesflux through the adsorbent column but also limits accessibility ofthe MOF\u2019s sorption sites, thereby limiting contaminant capture,desorption, and reuse.Metal organic frameworks (MOFs) are porous crystalline materialsmade by the interaction of inorganic metal ions or metal clustersand organic ligands.270 found that inaddition to acting as scaffolds for ZIF-8 MOFs, TEMPO-oxidized CNFsalso provide nucleation and growth sites for the MOFs. Ionic interactionsbetween Zn2+ ions and CNF carboxylic groups led to nucleationand growth of the ZIF-8 crystal. In addition, depletion of MOFs fromthe aerogel was impeded by hydrogen bonding and ionic interactionsbetween MOFs and CNFs. The rhodamine B adsorption rate (0.036 g mg\u20131 h\u20131) and maximum adsorption capacity(83.3 mg g\u20131) of the aerogels was also much higherthan that of plain ZIF-8 MOFs . Clearly, therefore,the formation of the CNF\u2013MOF complex significantly improvedboth the performance and durability of the adsorbent.Various properties of nanocellulose,particularly the scaffolding ability of CNFs, can address this shortcoming.Furthermore, Zhu et al.4\u2022\u2212), to degradeorganic pollutants. Sulfate radical based AOPs are preferred due totheir higher redox potential (2.5\u20133.1 V) and their applicabilityover a wider pH range (2\u20138), hence the increased use of peroxymonosulfate(PMS) as a sulfate radical source. A catalyst is required for thegeneration of the free radicals and transition metals have traditionallybeen used for activation of PMS. However, leaching of such elementsas Co2+ into treated water is an undesirable secondaryeffect of the process. MOFs can be used to create heterogeneous catalysts,thereby addressing this challenge. Ren et al.267 showed that the degradation of p-nitrophenolby cobalt containing MOFs was high and rapid (90% of p-nitrophenol in 1 h) and that Co 2+ loss was reduced.When a zinc-based MOF (ZIF-8) was incorporated into TEMPO-oxidizedcellulose nanofibers, good flux (84 L m\u20132 h\u20131 bar\u20131 for 24 h) in a dead-end filtrationsetup, and high removal of Janus Green B and Methylene Blue dyes was reported.274 Together, these data demonstrate that combining nanocellulose withaerogels can reduce catalyst loss and improve flux by reducing compaction.Nanocellulosehas also been key in the improvement of advancedoxidation processes (AOPs) for water treatment, which involve theuse of powerful oxidizing agents, particularly hydroxyl radicals (\u2022OH)and sulfate radicals , remained above 90% after 6 cycles. Themembrane was equally effective at removal of the endocrine disruptors,bisphenol A and bisphenol S, 1-naphthyl amine (carcinogen), and propranolol(an antihypertension drug).There are increasing reports on the use of wood as substrates forMOFs. Wood offers linear channels useful for confinement of MOFs,thus minimizing loss and maximizing remediation efficiency by increasingadsorbent\u2013contaminant contact. Guo et al.basswood 20. Zirco2), Liu et al. attained a 5.81 mg g\u20131 (71.7 wt %)uranyl ion capture from seawater.269 Further,selectivity for the ions was maintained even after 30 days in seawater.Wood\u2013MOF composites have also been usedfor the captureof metal ions from water. In a strategy that used polydopamine toattain a high MOF loading of zirconium MOFs reduction capacity was preserved above 80% even after10 runs.276 A note for magnetic adsorbents:the choice of iron precursor can influence the nature of the adsorbent,especially when bacterial nanocellulose is used. While compositesmade with Fe(II) sulfate and Fe(III) chloride result in higher magnetizationthan those made with Fe(II) acetate and Fe(III) chloride, the latterproduce more porous materials, making them preferable for synthesisof adsorbents.277 Together, these studiesshow that when paired with MOFs, nanocellulose can act as a adsorbentsupport as well as a template for nucleation and growth of MOFs.A Zn(II)-based MOF magnetic aerogel, however, showed better metaladsorption efficiency, i.e., 558.66 mg g5.2.2278 However, just aswith MOFs, their powder state substantially hinders applications inwater treatment applications. Synergistic effects have, however, beenobserved for nanocellulose-clay composites, suggesting that in additionto acting as a scaffold, CNF hydroxyl and carboxylate moieties contributeto contaminant sorption. The adsorption of methylene blue, for example,was highest for CNC-montmorillonite composites (183.8 mg g\u20131), than when the clay or CNCs were applied separately i.e., 140.6mg g\u20131 versus 96.8 mg g\u20131, respectively.279As a result of their high specific surface areas, excellent physicaland chemical stability, high cation exchange capacity, and low cost,clays hold great potential as adsorbents of dyes, metal ions, pesticides,and organic matter.2+ adsorptioncapacity of CNF\u2013attapulgite complexes decreased from 87.1 to56.5 mg g\u20131 when the CNF to clay ratio rose from4 to 12 due to aggregation of the clays and poor pore accessibilityat higher clay concentrations.280One of the main challenges facing nanocellulose\u2013claycomposites is achieving high clay loadings while maintaining theirdispersion. Increasing clay concentrations in composites may not alwaysresult in higher adsorption capacities. The Cu281 reported a technique to enhance moisture resistance in CNF\u2013montmorillonitecomposites that could also be useful for adsorbents in water treatmentapplications. In their work, dopamine functionalized CNFs were conjugatedto montmorillonite (MTM) platelets via catechol/metal ion chelationand hydrogen bonding to generate layered nanocomposite films and an increased waterflux (78.9 to 106.9 L\u00b7m\u20132 h\u20131) without nanoparticle loss.287 The membraneswere also resistant to chlorine degradation even after a 4 h exposureperiod to a 6000 ppm chlorine solution. Good chlorine resistance andflux enhancement were also reported for membranes with TEMPO-oxidizedCNFs.288Nanofiltration membranes (1\u201310nm pore diameters) are attractive in water treatment because of theirhigher flux and lower operating pressure and energy consumption comparedto reverse osmosis membranes. Thin film composite membranes, whichare commonly used in nanofiltration, are usually fabricated by theinterfacial polymerization of piperazine and trimesoyl chloride onan ultrafiltration substrate. However, as with other polymeric membranes,thin film composite membranes are challenged by chorine degradation,biofouling, and flux reduction, etc. While nanomaterials includingzeolites, graphene oxides, and carbon nanotubes have been used toaddress these challenges, poor compatibility with these inorganicmaterials and the polyamide skin layer often results in their detachmentor dissolution from the polyamide layer.289 In mixedmatrix membranes, nanocelluloses also increase hydrophilicity, waterflux (92\u2013195 L\u00b7m\u20132 h\u20131), and bacterial resistance.155 They canbe incorporated into membranes during, or after, synthesis. Postsyntheticmodification by carbodiimide conjugation of thiol-modified CNFs toan electrospun polyacrylonitrile membrane resulted in a high fluxmicrofiltration membrane with Cr(VI) and Pb(II) adsorption capacityof 87.5 and 137.7 mg g\u20131, respectively.244 The synthetic approach, however, is lengthy.Nevertheless, these membranes had a higher sorption efficiency forPb(II) than that of thiol-modified membranes prepared by deacetylationof electrospun cellulose acetate (22 mg g\u20131).290A separate approach involvesincorporation of nanocellulose asinterlayers between the hydrophobic ultrafiltration membrane and thefunctional thin film layer. Besides improving permeability, theseinterlayers improve the coating efficiency of the substrate withoutsacrificing salt rejection.291 Manipulation of the humidity conditions can allow for the productionof porous fibers,292 which could exposethe enclosed CNCs and modify the surface and adsorption propertiesof the electrospun membrane.To prepare nanocellulose-modified electrospunmembranes in a single-stepprocess, water-in-oil emulsions are used to stabilize nanocellulosesuspensions in the polymer dope. During spinning, the solvent in theregion close to the surface evaporates faster, resulting in higherviscosity in the outer layer and inward movement of emulsion dropletsto the center, where they are simultaneously condensed and stretchedunder the force of a high voltage field.5.4293 There is therefore a needfor absorbents for treatment of oil-contaminated water. Nanocelluloseis attractive as a material for the design of absorbents for oil/waterseparation because of its ability to form light aerogels with highsurface areas that can be hydrophobically modified to increase oleophilicityand hydrophobicity. Although these have been mentioned a number oftimes already, aerogels from nanocellulose may be synthesized viachemical vapor deposition,296 click chemistry,299 and atomic layer deposition.300Besides large-scale marine oil spills from shippingand oil exploration, many industries including metal/steel industriesand mining, produce oily wastewater. A typical mining operation, forexample, loses over one-fifth of its total oil purchases through spills,leaks, and overflows, producing 140000 L of oil-contaminated watereach day.Ochroma pyramidale), an attractivetemplate for the design of adsorbents. The process involves delignificationto produce sponges with a honeycomb295 ora lamella structure.301 The latter seemsto have superior compression recovery than the former. Lamella woodsponges can bear compressive strain as high as 60% and fully recoverto their original height after the stress is removed: a height retentionof 93% after 100 compression cycles. Honeycomb-structured sponges,in contrast, only recovered to 60% of their original height. Thissuggests that the additional treatment step, namely using NaOH tobreak down the honeycomb structure , and larger surface areas, while those withlower CNF content had compact structures with smaller pores sizes(\u223c50 \u03bcm). CNFs also acted as anchors for the NR latexparticles responsible for hydrophobicity.Despite lower compressivemoduli, high absorption capacities havebeen reported for nanocellulose\u2013aerogel composites. A CNF/naturalrubber latex composite was found to have a 92% porosity and a surfacearea of 226 m302 The high absorption capacity of the aerogels 373 g g\u20131 was attributed to the interconnected pore structure and the highsurface area provided by the fibrous cellular walls , Pb(II), Cu(II),and Ag(I)), implying that they maintained surface charges enough forelectrostatic adsorption of the ions, even after the hydrophobic treatment.Polyvinyl acetate (PVA)\u2013cellulose aerogels, on theotherhand were found to be compressible for 50 cycles without any significantdecrease in mechanical strength and with an absorption capacity upto 32.7 times of their weight when absorbing chloroform from water.5.5304 The low pH they exert has negativeeffects on protein activity, membrane permeability, and nutrient absorption.305 However, an acid-dependent strategy would belimited in water treatment applications due to dilution and neutralization.Uncharged nanocellulose has no intrinsic antimicrobial properties.However, oxidized variants, e.g., by 2,3-dialdehyde nanocellulose,have potent and often immediate antimicrobial action in vivo and invitro.308 or (ii) complexation with nanomaterials having antimicrobial propertiesincluding zeolites,309 graphene oxide,154 organic agents,310 and metal/metal oxide nanoparticles, e.g., Ag,311 ZnO,312 and TiO2.313 In this latter approach, nanocelluloses mayact as nanoparticle nucleation sites to decrease nanoparticle agglomeration,thereby increasing sorptive surface areas and antimicrobial action,265 or they may act as reductants.311 Taking advantage of the reductive propertiesof TEMPO-oxidized CNF, Zhang et al. prepared Ag-doped nanofibers byhydrothermal synthesis.264 The resultingcomposite had antimicrobial action toward both Gram negative (Escherichia coli) and Gram positive (Staphylococcusaureus) bacteria, as well as a strain of fungus . Nath et al.266 also reported antimicrobial action by a polyaniline supported ZnO/CeO2/nanocellulose composite against E. coli and Bacillus subtilis, in addition to a high adsorption efficiencyfor arsenic ions. In their work, the presence of nanocellulose inhibitedthe aggregation of the ZnO/CeO2 nanoparticles, therebypromoting an even incorporation in the polyaniline matrix.Notwithstanding, several nanocellulose antimicrobial materialssuitable for water treatment have been synthesized by (i) surfacemodification with polymers having antimicrobial properties, e.g.,quaternary ammonium compounds,314Theother strategy for generating nanocellulose with antimicrobialproperties involves grafting of polymers, e.g., quaternary ammoniumcompounds (QACs), which possess these properties. Positive centersin QACs bind to the negatively charged phosphate groups in peptidoglycansof Gram-positive bacteria or phospholipids of Gram-negative bacteria.The bacterial cell membranes are gradually disrupted, leading to membranerupture and cell death.306 compared cationizationof CNFs by etherification with epoxypropyl trimethylammonium chloride(EPTMAC) and by redox-initiated graft copolymerization using [2-(methacryloyloxy)ethyl]trimethylammonium chloride ,318 TiO2319 have been used as solar absorbersatop bacterial or plant cellulose (\u20132, the CNT-coated aerogel absorbed 97.5% ofincident light from 300 to 1200 nm, resulting in an evaporation rateof 1 L m\u20132 h\u20131. A similar waterproduction rate: 1.26 L m\u20132 h\u20131 was reported for a black titania/bacterial cellulose solar absorber.319Cellulose nanofibers areparticularly amenable to fabrication of solar desalinators due totheir hydrophilicity, which favors water transport, high aspect ratio,which allows for channel formation upon directional freezing, andgood mechanical properties.ellulose 24a. When320 attemptedto improve thesolar absorption efficiency of magnetite by increasing adsorptivesurface areas. By magnetically stirring cellulose\u2013magnetitemixtures, the absorbents were transformed from 2-dimensional planarstructures with 76.9% solar absorption to 3-dimensional structures(\u20132 h\u20131. However, salt deposition on the 3D cellulose film slightly decreasedperformance after 6 h.Gan et al.ructures24b with317 attempted to address thischallenge of salt deposition by coating the cellulose aerogels withpolydopamine. The resulting material had, in addition to resistanceto salt deposition, greater antifouling capacity due to increasedsurface roughness, high hydrophilicity, and dye sorption capacityfor methylene blue and rhodamine B. Furthermore, surface temperaturescould rise from 22 to 40 \u00b0C in 10 min, with 1 sun radiation inboth seawater and oil-contaminated water. The superhydrophilic PDA-coatedcellulose aerogels repelled the oil contaminants and delivered waterto the surface of the aerogel, resulting in an evaporation rate of1.36 L m\u20132 h\u20131. No salt depositswere found on the aerogel after 10 days, and the evaporation ratewas not diminished, confirming the durability of the aerogel.Zou et al.316 A water evaporation efficiency of 95.9% and an evaporationrate of 1.83 L m\u20132 h\u20131 was reportedwhen these aerogels were covered with a layer of soot. When used togenerate clean water from an oil\u2013water mixture, the absorptioncapacity decreased only slightly, from 34 to 29 g g\u20131, even after 29 adsorption\u2013removal cycles and a stable evaporationrate of 1.75\u20131.83 L m\u20132 h\u20131 while irradiated continually for 10 days, suggesting it could withstandlong-term deployment. Nevertheless, as with all treatment technologies,water production rate, cost, and durability will be key for determiningmarket entry for solar desalination devices. It is, nevertheless,cheaper than bottled water but more expensive than tap water, andas there are still locations around the world where tap water is unavailable,solar desalinators may be worth considering in such situations.Resistance to disintegration in the harsh conditions of salinewater poses a significant challenge for aerogel use in thermal solardesalination. Silane functionalization seems to hold some potentialin addressing this challenge by imbuing aerogels with superhydrophilicityand interconnected radial-aligned channels that allow for high watertransfer rates from solution to the aerogel surface.6Cellulose has a uniqueinteraction with water. This interactionis something which needs to be harnessed in the drive toward greenand sustainable chemistries for a range of applications, where thereplacement of oil-based materials is of paramount importance andneed. In the cell wall, cellulose intimately contacts with other materials,and water plays a key role in mediating those interactions in whatperhaps could be considered the most green of manufacturing processes.When extracted from the plant cell wall, cellulose continues to interactwith water, something which can be exploited in the self-assemblyof higher-order structures. This review has covered those, includingchiral nematic liquid crystals and amphiphilic cellulose derivatives.More research is needed in these areas to better understand how watermediates these self-assembly processes. This will be key in our movementtoward a green materials revolution, with cellulose at the centerof this to produce new technologies. As human beings we also facegrave difficulties in accessing clean water, particularly as climatechange takes effect. In water treatment applications, nanocellulosepresents benefits, ranging from strengthening and increasing fluxand antifouling efficiency of membranes to applications in solar desalination,and as hosts of reactive agents including catalysts, clays, and othernanoparticles. While many studies have investigated the optimizationof contaminant capture by these materials, including applicationsto real samples, consideration needs to be given to other water components,particularly organic matter, to obtain accurate efficiencies of adsorbentmaterials.For low-cost treatment solutions, nanocellulose isan expensiveoption largely due to the production methods. However, as shown bysome of the studies reviewed, the performance of less processed microfibrillatedcellulose can sometimes be comparable to that of nanofibrillated alternatives.More studies comparing the performance of these two sets of materialsare needed to determine instances when nanoscale or micrometer-scaleare most appropriate.Large-scale application of nanocellulosein water treatment alsocalls for synthetic approaches that are greener and leaner, economically.Deep eutectic solvents are one route through which materials can besynthesized using reagents that are relatively cheap, biodegradable,and environmentally friendly. Mechanical treatment in the absenceof heat and solvents may be another alternative."} +{"text": "Cellulose is the most abundant natural polymer and deserves the special attention of the scientific community because it represents a sustainable source of carbon and plays an important role as a sustainable energent for replacing crude oil, coal, and natural gas in the future. Intense research and studies over the past few decades on cellulose structures have mainly focused on cellulose as a biomass for exploitation as an alternative energent or as a reinforcing material in polymer matrices. However, studies on cellulose structures have revealed more diverse potential applications by exploiting the functionalities of cellulose such as biomedical materials, biomimetic optical materials, bio-inspired mechanically adaptive materials, selective nanostructured membranes, and as a growth template for inorganic nanostructures. This article comprehensively reviews the potential of cellulose structures as a support, biotemplate, and growing vector in the formation of various complex hybrid hierarchical inorganic nanostructures with a wide scope of applications. We focus on the preparation of inorganic nanostructures by exploiting the unique properties and performances of cellulose structures. The advantages, physicochemical properties, and chemical modifications of the cellulose structures are comparatively discussed from the aspect of materials development and processing. Finally, the perspective and potential applications of cellulose-based bioinspired hierarchical functional nanomaterials in the future are outlined. T[BMIm]Cl .Cellulose hydrogels are physically or chemically cross-linked three dimensional hydrophilic cellulose networks capable of absorbing large amounts of water and swelling. The hydrogel stability is ensured either by physical interactions or chemical cross-links . Aqueous\u22123), high porosity as well as high specific surface area, low thermal conductivity, excellent shock absorption, and low dielectric permittivity -cycloadditions of organic azides with alkynes (Click reaction) and for adsorbing/eliminating of the residual copper species from the reaction solution. The cellulose paper was functionalized with thiol (-SH) groups by grafting the cellulose with thioglycolic acid. The -SH grafted cellulose (Cel\u2013SH) had a dual function, that is, promoting the Cu-catalyzed Huisgen synthesis of 1,4-disubstituted triazoles through the reduction of CuSO4\u00b75H2O to Cu2O as well as adsorbing and eliminating copper byproducts from the reaction medium. The thiol-modified cellulose paper reduced CuII to catalytically active CuI and acted as an effective adsorbent for copper, thereby simplifying the synthetic process and resulting in the reaction mixture being almost free of copper residues after a single filtration , lightweight, and they were actuated by a small magnet to absorb water and release it during the compression. Due to their high porosity, large surface area, and flexibility, these aerogels are promising materials for electronic actuators and microfluidic device applications . A. A253]. 3+) doped, chiral nematic, mesoporous luminescent films of ZrO2 (EDCNMZrO2) with tunable optical properties were prepared by a hard template process using silica templated by cellulose nanocrystals (CNCs). Chiral nematic SiO2 films were formed by replicating the chiral nematic structure of CNC using an evaporation-induced self-assembly (EISA) approach, followed by calcination at 600 \u00b0C for 6 h, and subsequent silica etching. The obtained EDCNMZrO2 showed selective suppression of the spontaneous emission of Eu3+ ions as well as the possibility of modulating their luminescence lifetime. This study is a showcase of how the capability of CNC to organize into the liquid crystal nematic phase can be exploited as an indirect template to prepare the inorganic (ZrO2) photonic material, which has potential application in novel smart optical nanodevices [Europium have been prepared through this process. The reported approach to create hollow fibers was demonstrated to be scaled-up easily and the application of elaborated processes as well as expensive starting materials was avoided. The potential applications of the prepared hollow inorganic fibrous materials are in microfluidics, as microreactors, and in catalysis [A special case represents the carbon nanofibers formed from various cellulose structures by pyrolysis in an inert atmosphere. Such structures are thermally stable and electrically conductive. When bacterial nanocellulose is used, templated carbon networks are obtained. Depending on the raw cellulose material, a whole variety of porous carbon nanostructures can be formed. For example, when wood was first delignified, the resulting cellulose hierarchical structure was exposed to cellulose enzymes to partially degrade its structure to form micro- and nanopores, followed by carbonation at 1000 \u00b0C for 3 h in a Ntability . Porous tability ,257. Vartability . Carboniatalysis .2 where the bacterial nanocellulose (BNC) was applied as the precursor and template. The obtained carbon networks functioned as conductive networks for the integration of active electrode materials providing the fast electron transfer and as a support for the preparation of high capacitance electrode materials. MnO2 was formed on the surface of the activated carbon network by the redox reaction using KMnO4 as a precursor. The prepared asymmetric supercapacitors, based on activated carbon/carbon-MnO2, showed a maximum power density of 227 kW kg\u22121 and a high energy density of 63 W h kg\u22121 in an aqueous solution of 1.0 M Na2SO4. Their energy density was higher than most values reported for the activated carbon//MnO2 asymmetric supercapacitors. Additionally, the resulting supercapacitor also showed a formidable cycling performance with a 92% specific capacitance retention after 5000 cycles. The reported approach represents an environmentally-friendly and low-cost preparation process of electrode materials with potential application in high-performance energy storage devices [Nitrogen-doped carbon networks were formed by carbonizing the polyaniline (PANI) coated BNC at 850 \u00b0C for 2 h in a flow of N devices .\u2212 ORR). NiCl2 was first deposited on BC by its immersion in an aqueous solution of the precursor (NiCl2-conc. = 0.1 mol L\u22121) for 24 h and subsequently freeze-dried. The resulting material was pyrolyzed first at 360 \u00b0C for 2 h and then at 700 \u00b0C for an additional 2h both in an Ar atmosphere. After cooling down to RT, the solid product was ground in a mortar, washed several times with deionized water and ethanol, and dried for further use. In 0.1 mol L\u22121 KOH, the Ni-NPs/BCCF-20.7 sample showed a high electrocatalytic performance toward 2e\u2212 ORR, exhibiting a H2O2 selectivity of ~90% and an onset potential of 0.75 V vs. the reversible hydrogen electrode (RHE). By applying this catalyst, a high Faradaic efficiency (93.9% \u00b1 4.2% at 0.5 V vs. RHE in bulk ORR electrolysis) and a high H2O2 yield rate (162.7 \u00b1 13.7 mmol gcat\u22121 h\u22121 at 0.2 V vs. RHE) were achieved. This work provides new insights into the design and development of high efficiency 2e\u2212 ORR electrocatalysts for H2O2 production [The thermal reduction of metallic nanoparticles in the presence of cellulose is questionable due to its rather low thermal stability, but the thermal reduction of metals in an inert atmosphere accompanied with carbonization of the cellulose is possible. For example, metallic Ni nanoparticles deposited on bacterial cellulose-derived carbon fibers (Ni-NPs/BCCF) were synthesized using a simple impregnation-pyrolysis method as efficient electrocatalysts for a two electron oxygen reduction reaction were homogeneously distributed and tightly adsorbed onto the cellulose structure by the strong intermolecular interaction between the CdS and cellulose fibers, thus preventing their aggregation. The CdS/cellulose composite films showed a substantially enhanced photocatalytic activity due to the porous structure of the cellulose template and the presence of the CdS nanoparticles. Additionally, for the promotion of H2 evolution, Pt was applied as a cocatalyst. This was formed in situ on the surface of the CdS\u2013cellulose films by photo deposition using H2PtCl6 as a precursor. The cellulose fiber supported CdS nanoparticles showed a high efficiency in the photocatalytic evolution of hydrogen, and as a portable photocatalyst, they were easily recovered, avoiding environmental pollution [By using the porous structure of the regenerated cellulose, the CdS nanostructures were formed from the CdClollution . 2 was prepared. The cross linking between Mo4+ cations and carboxyl (\u2013COOH) as well as hydroxyl (\u2013OH) groups on the cellulose surface caused intense interaction between the MoS2 nanosheets and the cellulose fibers, thus enabling the formation of an aerogel with a density of 0.00473 g cm\u22123 and a 97.36% porosity. The resulting material showed outstanding self-extinguishing capabilities and fire resistant properties as characterized by cone calorimetry as well as by the vertical burning test. The MoS2 structure in the aerogel remained unchanged after the vertical burning test, as shown by Raman spectroscopy. This study represents a new approach toward the preparation of fire resistant and lightweight aerogel materials for application in the construction of fire resistant and energy efficient buildings as well as aerospace applications [An innovative nanocomposite aerogel composed of TEMPO oxidized microfibrillated cellulose embedded in a layer of previously synthesized ultrathin, fire retardant nanosheets of MoSications .2, Pb(NO3)2, and CdCl2 with H2S gas. In the controlled growth of defined semiconductor nanoparticles, the application of a cetyltrimethylammonium bromide (CTAB) cationic surfactant was the crucial factor. The pH of the salt solution and precursor concentration were varied to control the packing density and average particle size of the sulfide semiconductor nanostructures on the CNC surface. This procedure could serve as an innovative universal platform for engineering a variety of nanoscale functional materials for optical and electronic nanodevices [Various semiconducting sulfide nanostructures such as ZnS, PbS, and CdS have been prepared by using cellulose nanocrystals (CNCs) as supports combined with cationic surfactants as stabilizers. Their synthesis on the CNC surface was carried out by reacting precursor salts such as ZnClodevices .2+, Cd2+, PO43\u2212, and NO3\u2212 ions from the aqueous solution. The MFC was initially treated with NaOH and CHA was subsequently deposited on its surface by the reaction of CaCl2 and NaH2PO4. The obtained results revealed that the removal of all of the studied ions was not dependent on the pH and was highly effective for Ni2+, Cd2+, and PO43\u2212. Namely, the adsorption efficiency for NO3\u2212 removal from an aqueous solution reached more than 50% while the adsorption efficiencies for the other three ions were higher than 90%. The highest observed removal capacities of the studied CHA/MFC adsorbent for the NO3\u2212, PO43\u2212, Cd2+, and Ni2+ ions were 0.209, 0.843, 1.224, and 2.021 mmol g\u22121, respectively. The adsorption kinetics was very fast for all of the studied ions. This study demonstrated that CHA/MFC was an environmentally-friendly and practical absorbent, exhibiting high removal efficiencies for both the metallic and phosphate ions while the removal efficiency for the nitrate ions was only moderate [Carbonated hydroxyapatite (CHA) modified microfibrillated cellulose (MFC) was studied as an adsorption substrate for Nimoderate . A biomimetic approach was applied for the development of hydroxyapatite/bacterial cellulose nanocomposites with bone healing properties. The nucleation of calcium-deficient hydroxyapatite (cdHAp) was initiated by the adsorption of carboxymethyl cellulose (CMC) on two forms of bacterial cellulose (BC) (pellicles and tubes). The dynamic simulated body fluid (SBF) was used in the formation of the cdHAp in vitro over a one-week period. The atomic content of phosphorus and calcium varied from 0.3 to 11.2 atomic % and 0.4 to 7.7 atomic %, respectively, while the Ca/P overall ratio changed from 1.2 to 1.9 and the crystal size of cdHAp decreased with the decrease in the density of bacterial nanocellulose fibrils. The differentiation and morphology of the osteoprogenitor cells were studied through alkaline phosphatase gene expression and fluorescence microscopy to determine the viability of the scaffolds in vitro. The BC surface coated with cdHAp crystals showed an enhanced cell attachment, indicating that the BC-cdHAp scaffolds were a feasible and promising bone scaffolding material .2 was added and stirred at 90 \u00b0C until complete dissolution. Subsequently, the Na2HPO4 solution (Ca/P = 1.67) was admixed into the prepared solution with the pH adjusted to 10 using a NaOH solution. After this, the solution was stirred for 2 h at 90 \u00b0C followed by aging at RT for 24 h. At the end, the resulting precipitate was freeze-dried and calcined at 800 \u00b0C to prepare the n-HA. By using uniaxial compression, the composite aerogel material with a density close to that of natural bone was prepared with a high mechanical strength and toughness. When the ratio of the cellulose, n-HA, and SF was 8:1:1, the composite aerogel showed the same mechanical strength range as the cancellous bone. HEK-293T cells, cultured on these composite aerogels, exhibited a high ability of adhesion, differentiation and proliferation. The studied biodegradable composite aerogel has high potential to be applied as a bone tissue substitute in bone-tissue engineering [Cellulose (BC) is a high-strength fibrous and extensively used biomaterial while hydroxyapatite (HAp) has outstanding osteoconductivity and bioactivity. Composite materials composed of cellulose matrices and inorganic nano-hydroxyapatite (n-HA) fillers have received considerable attention in bone engineering, although the brittleness and low strengths limit their application. Therefore, the improvements in the toughness and mechanical strength of aerogel-based biomaterials are necessary. A urea alkali system was applied to disperse and gelate cellulose mixed with silk fibroin (SF) to prepare the composite aerosol. n-HA was prepared by swelling PAA with water for 20 h and then CaClineering .The one-step electrophoretic deposition process (EPD) was used for the preparation of an innovative bioglass\u2014a cellulose nanocrystal (BG-CNC) bioactive hybrid material\u2014to be applied for the surface coating of orthopedic implants with the purpose to enhance their contact with the bone tissue. The characterization of the prepared materials revealed that the network of CNCs functioned as an in situ template tailoring the mineralization rate and morphology of the formed hydroxyapatite (HA) crystal phase . The BG N-methylmorpholine-N-oxide (NMMO) using a dry\u2013wet method and by subsequent surface modification of the fibers by the deposition of the Gd4O3F6:Eu3+ doped with 5 mol % of the Eu3+. During its dissolution process in NMMO, the photoluminescent nanostructures were admixed as a powder into the cellulose matrix. Photoluminescence spectroscopy was used to study the relationships between the emission intensity and the excitation energy as well as the concentration of Gd4O3F6:Eu3+ nanoparticles in the final composite products. About 80% of inorganic particles had a diameter below 300 nm when the samples contained up to 1% of the modifier, and with an increase in the modifier concentration, the agglomeration of the particles was intensified. The obtained cellulose fibers with UV activity have potential application as a security marking for documents, textile, and paper products [Cellulose fibers with UV activity have been prepared by spinning of 8 wt.% \u03b1\u2212cellulose solution in products .2O4) nanostructures were formed on the surface of the cellulose structures from CoCl2 and FeSO4 as precursors in the presence of KNO3 and NaOH. These inorganic nanoparticles were applied as labels for fine cellulose fibers, representing an approach that allows for the detection of micro- and nanostructured celluloses in paper sheets in combination with various imaging technologies. The addition of nanostructured CoFe2O4 as a labeling material during the paper manufacturing process enables imaging in scanning electron microscopy/energy-dispersive X-ray spectroscopy experiments and provides contrast in X-ray microtomography. By combining these two techniques, the fines in the paper sheets were located, evaluated, and quantified. It was demonstrated that the water retention value, the tensile indices, and the air permeability of the handsheets were not changed by the addition of the CoFe2O4 labeled fines compared to the sheets where the nonlabeled fines were added. The knowledge about the distribution of nano- and microscale cellulosic structures in a cellulose fiber matrix is important to improve the properties of the existing paper products and in the future, the design of new materials in the paper industry [Cobalt ferrite materials based on the cellulose aerogel for super capacitive application. Supercapacitors are a highly propulsive area of research and have already found commercial application [3O4, cationic polymer, SiO2, poly(vinylpyrrolidone), and cyclodextrine was studied and its potential application in the separation of pharmaceutical compounds was demonstrated. Similarly, the CNC/Fe3O4/Au (CNFeAu) nanocomposite was used as a support matrix for the immobilization and separation of papain from the reaction solution with a high enzyme loading; the conjugated biomolecules retained their high enzyme activity and exhibited high stability and reusability. Inorganic nanostructures can be synthesized in situ on the surface of the cellulose fibrillar structure or they can be admixed to the cellulose. In this way, ZnO nanoparticles were formed in the presence of various saccharides, and such saccharide capped ZnO nanostructures were further deposited on the pine cellulose fibers that were subsequently incorporated into the alginic acid sheets, producing paper materials with a high degree of immobilization of biomolecules for the detection of blood type. Instead of chemical methods, physical methods of nanostructure formation can also be applied. For example, cellulose\u2013Cu and Al2O3 sandwich nanostructures were prepared by magnetron sputtering and RF reactive sputtering, producing promising materials for EMI shielding applications. Recently, cellulose paper substrates have attracted tremendous interest from both academia and industry as a platform for biosensing applications. Cellulose fibers have an interesting combination of features that makes this substrate advantageous or even ideal for conducting chemical and biological analyses comprising the passive transport of fluids through their capillary wicking, excellent biocompatibility and biodegradability, large surface-to-volume ratio, porous structure, high abundance, and most importantly, their low-cost [Aside from metallic, metal oxide, or other inorganic nanoparticles, various complex hybrid nanostructures have also been formed on the cellulose surface. The specific fibrous structure and high number of hydrogen bonds give the cellulose sufficient porosity and high specific surface area, which can serve as a matrix to support other functionalized materials and as a template to form various inorganic hierarchical nanostructures. New materials with interesting combinations of properties and functions have been prepared by combining different inorganic nanostructures and polymer chains with various cellulose structures. Other than cellulose supported or templated inorganic compounds, more complex hybrid modifications or decorations of the cellulose structures have also been studied and reported. Complex hybrid cellulose materials have a wide variety of potential applications lication . A compllow-cost . Since mA sensitive paper-based electrochemiluminescence origami device (PECLOD) for the detection of the human immunoglobulin G (H-IgG) antigen based on the rolling circle amplification (RCA) using the Au-paper working electrode (Au-PWE) was developed . The Au-4TEA) and with gold nanoparticles that provided durability. This impregnated paper-based sensor platform was applied for the development of the fluorescence turn-on cyanide (CN) assay in aqueous media. The detection mechanism was based on two processes: (a) Ligand exchange of TEA with the CN\u2212 ion stimulating the enhancement of cyanide specific fluorescence, and (b) the dissolution of gold nanoparticles, causing the recovery of fluorescence. The described CN assay demonstrated a visible transition of color as a function of CN\u2212 exposure in the concentration range from 10\u22122 to 10\u221212 M. Additionally, an algorithm of image processing was developed that enabled the color change calibration and quantification of the CN\u2212 concentration. The reported algorithm was applicable to an Android smart phone, thus transforming it into a quantitative detector of CN\u2212 ions. The reported approach enabled sensitive, fast, and simple instrument free CN\u2212 ion detection in water as well as the possibility of remote water monitoring and controlling in the laboratory [Ordinary filter paper with a 6 \u03bcm pore size was applied as a solid support that facilitated the impregnation with europium tetrakis dibenzoylmethide triethylammonium (EuDboratory .2) films with chiral nematic ordering were prepared and further applied as a hard template for the formation of silver nanoparticles (AgNPs) from AgNO3 by reduction with NaBH4. The resulting SiO2 films deposited with the Ag NPs showed optical activities in the vicinity of the surface plasmon resonance (SPR) of the Ag NPs, as shown by the circular dichroism (CD) measurements. By conducting the experiments with three different SiO2 hard templates loaded with AgNPs dried and soaked with water, it was shown that the optical activities only originated from the long-range organization of the Ag NPs in the nematic chiral silica template. Since the SPR of metallic NPs is important in biosensing, the application of the response based on the chiral organization of metallic AgNPs embedded in a chiral mesoporous host will lead to the development of new biosensors. This is a showcase of the indirect application of CNC nematic liquid crystals in the formation of optically active inorganic structures [By using cellulose nanocrystals (CNC) as a template, mesoporous silica have been used for the preparation of large surface area bioactive hybrid scaffolds by exploiting the self-assembly and adsorption of the previously formed oxidized nickel (Ni/NiO) nanoparticles (NPs). The Ni/NiO NPs enabled the oriented binding of functional His-tagged protein G to the cellulose surface as it was confirmed by the subsequent binding of fluorescent antibodies. It was shown that the interaction between the polyhistidine-tagged protein and the nickel substrate was chemically inert and highly resilient. The targeted conjugation of His-tagged protein G enabled the oriented binding of anti-Salmonella antibodies, enabling the attachment of Salmonella bacteria to the substrate . This si3)2 and AgNO3 as precursors and hexamethylenetetramine as the precipitating and reducing agent. Before synthesis, the surface of cellulose was activated by H2O2 with a relatively mild effect on the contact angle with H2O. The formed ZnO/Ag nanostructure was a nanodispersed semiconductor/metal hybrid with an exceptional collective plasmonic structure never previously observed. The experiments with a single precursor solution enabled the separate study of the interaction of Zn2+ and Ag+ ions with the cellulose surface and in this way, the reaction mechanism in the mixed precursor solution was explained. A specific interaction between the Zn2+ and the cellulose substrate was confirmed while no specific non-thermal effects of microwave heating were detected [\u03b1-Cellulose was applied as a support in the formation of hybrid zinc oxide/silver (ZnO/Ag) nanostructures on its surface by the microwave solvothermal synthesis using Zn(OOCCHdetected .S. aureus and E. coli) as well as strong resistance toward cellulase producing fungus G. trabeum [For the antibody immobilization, pine cellulose fiber sheets decorated with saccharide capped-ZnO nanoparticles have been applied as a bioactive substrate. Initially, ZnO nanoparticles were formed in the presence of starch and alginic acid as well as sucrose and glucose. The fibers of pine cellulose were subsequently surface modified by the prepared saccharide capped ZnO nanoparticles that were finally incorporated into sheets of alginic acid. The immobilization of antibodies on the surface of the fibers was significantly enhanced by the adsorbed ZnO nanoparticles. The observed retention of antibodies was about 95% after rewetting the ZnO\u2013alginic acid modified sheets with the saline solution. For the possible application of bioactive- or biosensing-papers for the detection of blood types, a high degree of the biomolecule immobilization is an important characteristic. The possibility of detecting blood types was successfully demonstrated by using A-, B-, and D-antibodies. Additionally, the ZnO nanoparticles introduced a strong activity against bacteria 2 and hexamethylenetetramine and thermal treatment at 75 \u00b0C for 6 h produced cellulose fibers decorated with CuO NSs. The resulting CuO NSs/paper was coated with ZnO seeds and subsequently transformed into ZnO NRAs through hydrothermal synthesis at 85\u201390 \u00b0C for 3 h. The prepared device exhibited the I\u2013V characteristics with rectifying behavior at 0.93 V of the turn-on voltage. This simple process for the ZnO NRA formation on the CuO NS-modified paper proved to be a useful strategy toward the formation of paper-based nanostructures for applications in electronic devices [ZnO nanorod arrays (NRAs) were formed on the CuO nanospindle (CuO NS)/paper by using a hydrothermal process to prepare the cellulose supported p\u2013n junction device. Soaking with an aqueous solution of Cu modified with fluoroalkyl silane (FAS) under moderate reaction conditions to prepare the magnetic flexible hybrid membranes with an amphiphobic surface. The surface chemical properties of the BC nanofibers (ether and hydroxyl groups) and the ultrafine network architecture both functioned as effective nanoreactor cavities for the formation of magnetite nanostructures. The FS10 sample with homogeneously dispersed magnetite nanoparticles on the BC showed the maximal amphiphobicity with an oil contact angle of 112\u00b0 and a water contact angle of 130\u00b0. It also showed the excellent superparamagnetic properties (sM of 8.03 emu g\u22121) as well as the highest tensile strength (186 MPa) among the fluorinated magnetic membranes, and magnetic actuation was also demonstrated. This is an effective process in which to prepare self-cleaning, magnetic, and flexible BC membranes that can find potential application in EMI shielding, information storage, magnetographic printing, or as electronic actuators [Fectuators .2 nanoparticles, polypyrrole-nanofibers, and polypyrrole coated carbon nanotubes were synthesized separately and subsequently deposited into the 3D cellulose aerogel structure in one step during the aerogel assembly, providing a huge accessible surface area of the capacitive material, thus enhancing the charge storage nanocomposite was developed as a support for the adsorption and separation of papain, having a maximum loading of 186 mg protein g\u22121 on the CNFeAu from the reaction solution was formed by dipping the previously prepared CNC photonic films, prepared by an evaporation-induced self-assembly (EISA) method, into the TiO2 sol. CNC photonic films acted as a chiral biotemplate, which was removed by calcination at 500 \u00b0C for 4 h and mesoporous chiral TiO2 film was formed. The stopband of the TiO2 photonic films was controlled by the quantity of D-glucose added to the system. Finally, mesoporous TiO2 was decorated with Au nanoparticles using HAuCl4 as a precursor and NaBH4 as a reducing agent to increase its photocatalytic activity. The prepared mesoporous chiral TiO2/Au hybrid films were tested as catalysts in the photocatalytic splitting of water for the production of H2. It was demonstrated that the photo generation of H2 was more than eight times higher compared to the conventional mesoporous TiO2 loaded with Au nanoparticles [Mesoporous TiOarticles .4, H2PtCl6, AgNO3, or H2PdCl4 while the most frequently used reducing agents are NaBH4, Na citrate, or ascorbic acid. The most frequently used process for the growth of Au nanorods is the one reported by Busbee et al. [The number of publications on cellulose supported or templated hybrid inorganic nanostructures is extensive due to the large number of hybrid combinations and potential applications. These are summarized in e et al. . The mos12 tons, which is comparable to the reserves of the main fossil and mineral resources on our planet. Cellulose, with its fibrillary structure, contributes greatly to the diversity in the field of the synthesis and application of hierarchical inorganic nanostructures with additional alternatives. Based on the large number of hydroxyl groups on its surface, it influences the formation of inorganic nanomaterials. It serves as a support or template, it can control crystallization or act as a nanoreactor for in situ precipitation, and it can also prevent their aggregation and agglomeration to a large extent. Cellulose in any form enables the formation of hierarchically organized organic/inorganic hybrid nanostructures that cannot be obtained otherwise.By intensifying the global climate changes and shortages in natural resources, increased attention has been devoted in recent years to sustainable resources and environmentally-friendly solutions. Cellulose is the most abundant natural polymer on the Earth, with an annual production of ~1.5.10There are two types of cellulose/inorganic composite structures: (i) Cellulose supported inorganic composite materials and (ii) pure inorganic materials with cellulose templated porosities. In the first type, cellulose is present in the final composite material while in the second case, it serves as a sacrificial template and is removed by calcination, leaving the pure inorganic material. The inorganic component can be synthesized or formed in situ on the surface of the cellulose or it can be synthesized ex situ, and subsequently deposited on the cellulose. In the first case, cellulose serves as a template and support, while in the second case, it only serves as a support. When the inorganic (nano)structures are formed or grown on the surface of the cellulose, its surface characteristics affect and control their formation. On the other hand, when the cellulose is removed by calcination and a porous inorganic material is obtained, then the dimensions of the cellulose structures become important since they directly or indirectly define the dimensions of the pores in the inorganic matrix. The cellulose aerogel, as an example, acting as a support, produces lightweight, highly flexible, porous, mechanically resistant, and environmentally-friendly structures accompanied with interesting catalytic, electronic, optical, magnetic, and antibacterial properties. The potential areas of applications are very broad .The most widely studied area of application is medicine, where hydrophilic cellulose aerogels are combined with metallic Ag or/and ZnO nanoparticles to achieve the antibacterial effect. There are numerous publications reporting on the enhanced inhibiting effect on the bacterial or fungal growth. In many cases, the cellulose supported nanoparticles showed higher antibacterial activities compared to the neat nanoparticles, thus presenting a highly promising alternative for antimicrobial wound dressings or antimicrobial packaging materials. Highly promising biomaterials are cellulose/hydroxyapatite hybrid materials used as bone-tissue scaffolds for bone healing.Heterogeneous catalysis is another application area of cellulose-templated inorganic nanostructures. Metallic or metal oxide nanoparticles immobilized on the cellulose structures show superior catalytic activities compared to conventional catalytic systems while the removal of a catalyst is not the issue, since nanoparticles are attached to cellulose structures. Cellulose templated pure inorganic materials also showed enhanced activities in heterogeneous catalysis due to their porosity and high specific surface area. The highly important growing area comprises catalytic systems in alternative energy sources such as fuel cells and high capacity batteries.Supercapacitors based on nanocellulose supports with high charge storage, excellent capacitance retention, and very low internal resistance represent another fast growing application field of cellulose supported inorganic or hybrid nanostructures.5+, Pb2+, Cd2+, Cr6+, Mn2+, etc. from the aqueous medium gives the cellulose supported inorganic structures a high potential for use as a water decontaminant, and also in the purification of water or air from various organic chemicals. The selective and efficient adsorption of toxic metallic ions such as AsAnother highly promising area of application includes fast response humidity, chemical, and biomedical sensors, where the porous structure of cellulose-based paper with a high specific surface area certainly has advantages over conventional materials. The rapidly growing subfield of microfluidic cellulose fiber (paper)-based analytical devices is a highly promising approach toward simple, portable, disposable, and low-cost devices for molecular analysis, environmental detection, and health monitoring.A comprehensive review on the application of cellulose in all of its forms as a support or template for various inorganic materials demonstrates the role of cellulose as a highly versatile support and template for the formation of inorganic nanostructures as well as for bioinspired hierarchical structures and assemblies with interesting combinations of properties. Cellulose, as a fibrillar and functional biomaterial support or template, offers new approaches and pathways to the nucleation and controlled growth of inorganic nanoparticles with a defined shape and porosity as well as to hierarchically organized nanostructures that are difficult or even impossible to prepare through conventional processes. Cellulose, and especially cellulose nanostructures with abundant hydroxyl functionalities on their surface, can intensively interact with various metallic ions. Cellulose thus influences the formation mechanism of metallic and metal oxide nanoparticles and serves as a templating agent, which enables the preparation of (nano)structures that cannot be obtained by conventional methods. Due to the strong interactions of hydroxyl groups with inorganic ions, cellulose prevents the aggregation and agglomeration of growing inorganic nanostructures, indicating a higher interaction energy of these structures with the cellulose surface compared to the driving energy of particle aggregation and Ostvald ripening. Since the cellulose-supported nanostructures do not agglomerate and grow into larger structures, they retain their nanodimensional size and extremely large specific area, which is highly important in the heterogeneous catalysis of various chemical reactions. However, the formation and growth of inorganic nanostructures should be performed in a more sustainable way to reduce the use of toxic compounds and waste generation as well as by applying low temperature aqueous solution synthetic protocols, thereby reducing the production costs and enhancing manufacturing safety. Additionally, the implementation of such structures may cause some environmental issues associated with their application. The environmental impact should be assessed from many different aspects including their potential toxicity when released into ecosystems or the human body. The application of cellulose in any form offers answers to the majority of the previously mentioned issues. The review of publications up to the present shows that research in this area is heading away from the simple deposition of one type of inorganic nanostructure onto the (nano)cellulose structure toward the deposition of hybrid structures combining metallic, metal oxide, or sulfide with carbon nanotubes or graphene oxide or conducting polymers. These cellulose-supported hybrid inorganic materials form functional hierarchical (nano)structures with targeted applications in (photo)catalysis, Li-batteries, bone tissue engineering, supercapacitors, wound healing, waste water cleaning, colorimetric analysis systems, in sensing, or in other areas of electronics. Recent advances in the preparation of cellulose-based inorganic nanostructures suggest that the cellulose application will facilitate overcoming the major drawbacks of traditional synthetic methodologies. This review provides an insight into a rather specific area of cellulose-based inorganic (nano)structures and the trends of their exploitation in specific applications, demonstrating their huge potential to become as important as other well-known cellulose applications such as a bioreplacement for fossil fuels, organic chemicals, and/or organic materials based on fossil fuels. A large number (~500) of publications on this topic in the last ten years, mostly in the high impact scientific literature, strongly supports the growing importance of cellulose-based inorganic (nano)structures in materials, technologies, and applications of the future.Biomaterials based on cellulose are emerging as highly promising solutions to a number of technological challenges. Cellulose and cellulose-based materials are abundant and biocompatible but also possess structures for transformative device performance. A special case is that of cellulose\u2013inorganic hierarchical nanostructures, since this scientific field is a combination of nanotechnology and environmental science. Currently, a substantial number of promising devices and applications of hybrid cellulose\u2013inorganic structures have been demonstrated, but there are still important challenges in the basic scientific research and understanding to bring cellulose-based hybrid materials to a commercial reality in many innovative applications such as: energy saving and transformation applications, sustainable catalytical systems, green electronics, innovative low-cost sensors or detectors, and biological or biocompatible devices. These challenges comprise (i) low-cost solutions in the extraction of cellulose structures from wood or other cellulose resources and in the production of either components or devices; (ii) high performance cellulose\u2013inorganic hierarchical nanomaterials and devices based on these materials with suitable lifetime and durability; and (iii) the integration of systems composed of multiple devices.Innovative developments using cellulose\u2013inorganic hybrid materials elaborated in this review, especially those based on nanocellulose, will generate new solutions influencing our everyday life in the future. To cope with the emerging global challenges, environmentally-friendly and forward-thinking strategies are expected to sustain the research initiatives, and cellulose and cellulose\u2013inorganic hierarchical materials will certainly play an important and decisive role in this demanding task."} +{"text": "Inspired by ideas from NMR, we have developed Infrared Diffusion\u2010Ordered Spectroscopy (IR\u2010DOSY), which simultaneously characterizes molecular structure and size. We rely on the fact that the diffusion coefficient of a molecule is determined by its size through the Stokes\u2013Einstein relation, and achieve sensitivity to the diffusion coefficient by creating a concentration gradient and tracking its equilibration in an IR\u2010frequency resolved manner. Analogous to NMR\u2010DOSY, a two\u2010dimensional IR\u2010DOSY spectrum has IR frequency along one axis and diffusion coefficient along the other, so the chemical structure and the size of a compound are characterized simultaneously. In an IR\u2010DOSY spectrum of a mixture, molecules with different sizes are nicely separated into distinct sets of IR peaks. Extending this idea to higher dimensions, we also perform 3D\u2010IR\u2010DOSY, in which we combine the conformation sensitivity of femtosecond multi\u2010dimensional IR spectroscopy with size sensitivity. Infrared diffusion\u2010ordered spectroscopy is a simple and cost\u2010effective way to simultaneously characterize size and chemical structure of molecules, molecular aggregates/complexes, or small particles. With the increased importance of self\u2010assembly in chemistry and biochemistry, size has become an increasingly important molecular parameter: besides polymers, one can think of supramolecular complexes, self\u2010assembling protein nanoparticles, and amyloids. With infrared (IR) spectroscopy, such complexes and aggregates are often difficult to distinguish from their monomeric building blocks: this is because IR spectroscopy is sensitive to the functional groups and conformation of a molecule,We took our inspiration from NMR Diffusion Ordered Spectroscopy,11, 12Just like NMR\u2010DOSY, IR\u2010DOSY also relies on the fact that the diffusion coefficient of a molecule (or particle) is determined completely by its size, but the diffusion\u2010coefficient selectivity is obtained in a different manner: instead of using a pulsed field gradient, we create a spatially inhomogeneous distribution of solute molecules using a flow method. The resulting two\u2010dimensional IR\u2010DOSY spectrum is similar to an NMR\u2010DOSY spectrum, but has IR frequency along the horizontal axis instead of the chemical shift. In an IR\u2010DOSY spectrum, one obtains the IR spectrum and the diffusion coefficient of a compound at the same time; and mixtures of compounds with different sizes are nicely separated into distinct sets of peaks. We think IR\u2010DOSY can be a useful complement to NMR\u2010DOSY, for instance for paramagnetic compounds, compounds with strongly overlapping NMR spectra, or when the molecular tumbling is too slow for solution\u2010phase NMR.Figure\u20052O. In the initial experiments we added 2\u2009% poly\u2010ethylene glycol (PEG) to ensure optimal flow, but we found that we also obtain stable flow in absence of PEG. In pure D2O, we obtain identical results, apart from slightly different diffusion coefficients, as expected from the Stokes\u2013Einstein relation (see Supporting Information for the measurements in pure D2O). The IR spectrum while pumping the sample solution and solvent through the channel. The liquid\u2010liquid contact is a straight line at the center of the rectangular channel, as expected since the flow\u2010injection rates of the two liquids are the same. The bottom panel of Figure\u2005t=0. The top row shows the absorption at the IR\u2010frequency of acetone, the bottom row at the frequency of dialanine (1595\u2005cm\u22121). The absorption is proportional to the species concentration, and we can clearly observe that acetone becomes evenly distributed on a faster time scale than dialanine, due to its higher diffusion coefficient.To investigate if the device works as predicted, we first use IR microscopy to image an experiment on a test sample in real time (in absence of the slit). As a sample, we use a solution of acetone and dialanine in D\u22121 frequency range. At each IR frequency, the time\u2010dependent absorption profile is the sum of that of acetone and dialanine, with weight factors determined by the absorption of each species at that IR frequency. We keep the value of the two diffusion coefficients of the two compounds fixed to the values obtained at their peak\u2010absorption frequencies, and treat the amplitudes of acetone and dialanine at each frequency as fit parameters. Similar to NMR\u2010DOSY,Figure\u2005\u22121 is generally highly congested because of the overlap of many side\u2010chain modes, such as the CH\u2010bending modes. In the IR\u2010DOSY spectrum, we observe the spectral bands separated into two rows centered at D\u22482\u00d710\u22126\u2005cm2\u2009s\u22121 and D\u22483.5\u00d710\u22126\u2005cm2\u2009s\u22121. In the top row, the spectral bands are centered at 1695, 1450 and 1360\u2005cm\u22121, which correspond to the absorption frequencies of acetone . In the bottom row, we observe spectral bands at 1665, 1600, 1490, 1400 and 1360\u2005cm\u22121, which correspond to the absorption frequencies of dialanine . The IR\u2010DOSY spectrum neatly resolves the congested FTIR spectrum , which is very sensitive to molecular structure, but in the case of samples containing more than one compound tends to be congested when using conventional IR spectroscopy. Comparing the conventional and IR\u2010DOSY spectra of the cell, we use a slit to ensure spatial selectivity Figure\u2005A. The sl\u2212 stretch) appear first, while the BSA amide\u2010I band appears much later. Figure\u2005\u22126, 2.7\u00b10.6\u00d710\u22127 and 4.5\u00b10.5\u00d710\u22126\u2005cm2\u2009s\u22121). In the bottom row, we observe two signatures at 1580\u2005cm\u22121 and 1650\u2005cm\u22121, which are due to the carboxylate and amide groups of BSA . It is interesting to note that the carboxylate peak at 1580\u2005cm\u22121 is difficult to distinguish in the conventional infrared spectrum of the mixture spectroscopy due to depletion of the v=0 state by the IR pump pulse, and the positive signal at lower probe frequency (red contours) is due to the induced absorption of the v=1\u21922 transition. In this particular sample there are no cross peaks, since acetone and BSA both have only one vibrational mode in this frequency region. With increasing time, first the 2D\u2010IR peaks of acetone appear (vpump=1700\u2005cm\u22121), and at later times the 2D\u2010IR peaks of BSA (vpump=1660\u2005cm\u22121). We fit the absorption profiles taken at the bleach position of the two pair of diagonal peaks to obtain the diffusion coefficients, and using these values, we globally fit the time\u2010dependent 2DIR spectra, obtaining a three\u2010dimensional IR\u2010DOSY spectrum (see Supporting Information for details). Similarly to the IR\u2010DOSY plots, we observe 3D\u2010spectral signatures at two different diffusion coefficients, 3.4\u00b10.5\u00d710\u22126 and 3.5\u00b10.7\u00d710\u22127\u2005cm2\u2009s\u22121. The 3D\u2010spectral signature at the lower diffusion coefficient consists of the 2D\u2010IR peaks of the BSA, while the one at the higher diffusion coefficient consists of the 2D\u2010IR peaks of the acetone. Comparing the tilt of the 3D peaks with respect to the pump\u2010frequency axes for BSA and acetone, it can immediately be seen that the former has not only larger size but also more structural heterogeneity than the latter.To combine the enhanced structure sensitivity of 2D\u2010IR spectroscopy with molecular\u2010size selectivity, we perform 2D\u2010IR spectroscopy with the DOSY cell to obtain 3D\u2010IR\u2010DOSY spectra, somewhat similar to higher\u2010dimensional NMR\u2010DOSY.27, 283D\u2010IR\u2010DOSY is a powerful analytical tool, opening up novel applications of 2D\u2010IR to study supramolecular complexes, self\u2010assembly protein fibers, and amyloids. Since recent work has demonstrated the potential of 2D\u2010IR as a biomedical and diagnostic tool,30, 31Infrared diffusion\u2010ordered spectroscopy is a simple and cost\u2010effective way to simultaneously characterize size and chemical structure of molecules, molecular aggregates or complexes, or small particles. The two\u2010dimensional spectra are analogous to NMR\u2010DOSY spectra, and we hope that IR\u2010DOSY will become a useful addition to NMR\u2010DOSY. The separating power is comparable to that of NMR\u2010DOSY, and less than in typical chromatographic methods, but compared to chromatography it has the advantage that no prior knowledge of the chemical structure of the compounds in the sample is required. The separating power might be increased by adding elecrophoresis to separate the species in the sample solution. IR Spectroscopy generally provides less specific structural information than NMR, but there may be samples for which NMR is difficult to use, or practical reasons to prefer using an FTIR rather than an NMR spectrometer. Furthermore, 3D\u2010IR\u2010DOSY can provide conformational and ultrafast dynamical information about the size\u2010selected species that may be difficult to obtain using other methods.In the experiments reported in this article, the quantitative analysis of the time\u2010 and frequency dependent data could be done in simple manner. However, for more complicated samples more advanced analysis methods might be necessary, and we think that the extensive framework of sophisticated data\u2010analysis methods already developed in NMR research33, 34One can think of a number of interesting applications of IR\u2010DOSY (patent pending). Supramolecular complexes often contain carbonyl or other functional groups with intense IR bands, that can be valuable markers for complexation. Mixtures of such complexes with their monomeric building blocks or small oligomers may be difficult to characterize with conventional IR spectroscopy, but with IR\u2010DOSY the monomers and different complexes can be investigated separately. Another interesting application are protein aggregates and fibrils, where the (2D)IR spectrum provides conformation\u2010sensitivity36, 37The authors declare no conflict of interest.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re\u2010organized for online delivery, but are not copy\u2010edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file."} +{"text": "HCC is one of the most common malignant tumors worldwide. Although traditional treatment methods have been improved in recent years, the survival rate of HCC patients has not been significantly improved. Immunotherapy has shown extremely high clinical value in a variety of tumors. In this study, we found that TUG1 could regulate the expression of PD-L1 through JAK2/STAT3 to mediate immunosuppression. Here, The expression of TUG1 and PD-L1 in HCC tissues was evaluated through analysis of databases and verified in HCC tissue and HCC cancer cells by qRT-PCR. The effect of TUG1 on tumor immune escape was detected by coculture, and cell viability was detected with a CCK8 assay. The results demonstrated that TUG1 was closely associated with anticancer immunity. TUG1 and PD-L1 were highly expressed in HCC tissues and HCC cancer cells, and high expression of TUG1 and PD-L1 was related to the poor prognosis of HCC patients. In addition, knocking down TUG1 expression could reduce PD-L1 expression and enhance the cancer cell-killing capability of T cells. Downregulating TUG1 expression could also decrease the mRNA and protein expression of JAK2 and STAT3. To sum up, TUG1 and PD-L1 are overexpressed in patients with liver cancer and are related to the poor prognosis of these patients. Silencing TUG1 expression reduced the mRNA and protein expression of PD-L1 by affecting the JAK2/STAT3 pathway. Currently, therapies of HCC include hepatectomy, liver transplantation and tumor ablation, as well as other palliative treatments such as transarterial chemoembolization (TACE) including conventional TACE and Drug-eluting bead (DEB)-TACE and sorafenib3. Although some progress has been made in the treatment of liver cancer, the prognosis of HCC is not ideal because HCC has the characteristics of metastasis and high recurrence1,4. These observations indicate the urgent need to find new diagnostic and treatment options.Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide and one of the tumors with the highest mortality rate6, are widely expressed on the surface of a variety of cells and are considered costimulatory molecules related to negative immune regulation8. It has been reported that PD-1/PD-L1 is very likely involved in malignant tumor progression and lymph node metastasis11. Fortunately, in recent clinical trials, it has also been shown that PD-1 inhibitors have significant therapeutic effects on adult patients with advanced and/or refractory solid tumors13. Therefore, furthering the understanding of the regulatory mechanism of PD-L1 in tumors could help improve immunotherapy.Tumor immune escape plays an extremely important role in the progression of tumors. Programmed cell death receptor-1 (PD-1) and programmed cell death ligand (PD-L1) have been shown to play irreplaceable roles in tumor immune escape14. The role of LncTUG1 has been reported in various cancers, such as triple-negative breast cancer15, osteosarcoma16, acute myeloid leukemia17, and hepatocellular carcinoma18. These studies have mainly focused on the correlation of LncTUG1 with tumor progression, but whether lncTUG1 affects antitumor immunity has been reported in only a limited capacity. Tumor immune microenvironment (TIME) plays an important role in tumorigenesis and therapeutic response19. Tumor immune microenvironment is also an important factor in regulating immune escape. The infiltration of immune cells in the tumor microenvironment plays an important and complex role in the formation and development of tumors. Interestingly, research shows that the expression of TUG1 was positively correlated with the infiltration levels of immune cells in liver cancer tissues20. TUG1 has an important effect on tumor immune microenvironment of HCC. Therefore, we examined the mechanism by which TUG1 interacts with PD-L1 in HCC and evaluated its contribution to tumor immunology. Therefore, we may provide a beneficial direction to enhance the effect of PD-1/PD-L1-targeting treatments.Taurine upregulated gene (TUG1) is identified as a transcript whose expression is upregulated by taurine treatmentHepatocellular carcinoma and adjacent normal tissue samples were collected from 36 diagnosed patients who received surgical treatment at the Second Affiliated Hospital of Nanchang University during 2018 and 2020. None of the patients received any medications for treatment before sample collection. All tissue samples were then immersed in liquid nitrogen and stored at \u2212\u200980\u00a0\u00b0C for later use. All studies in this project were approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University. Prior to the collection of any samples, all participants signed an informed consent form, which complied with the Declaration of Helsinki.Human normal liver cells (HL-7702) and four HCC cell lines were purchased from the Shanghai Institute of Cell Biology . All cell lines were cultured in high-glucose DMEM supplemented with 10% FBS , 100\u00a0\u00b5g/mL streptomycin and 100 U/mL penicillin at 37\u00a0\u00b0C and incubated in 5% carbon dioxide. To treat HCC lines, JAK2 inhibitor , STAT3 inhibitor , and recombinant human IFN-\u03b3 were added to the medium for 24\u00a0h.21(http://timer.comp-genomics.org/). TIMER provides immune infiltrate abundances estimated by multiple immune deconvolution methods and allows users to dynamically generate high-quality Figs to comprehensively explore immunological, clinical and genomic features of tumors. We evaluated LncTUG1 as an anticancer immunity-related lncRNA with ImmLnc22. ImmLnc is a web-based resource for investigating the immune-related functions of lncRNAs across cancer types. In this resource, users can query lncRNA pathways, lncRNA-immune cell type correlations, and cancer-related lncRNAs across 33 cancer types. The prognosis of patients with TUG1 expression was confirmed by UALCAN analysis . UALCAN now provides long noncoding RNA (lncRNA) expression and patient survival information across cancer types using level 3 RNA-seq (hg38) TCGA datasets25. The correlation of TUG1 and PD-L1 expression was also confirmed by Gene Expression Profiling Interactive Analysis (GEPIA2) (http://gepia2.cancer-pku.cn/). Patient prognosis was related to TUG1 expression with The Cancer Proteome Atlas (TCPA) .We estimated TUG1 expression in various cancers, and TUG1 expression was correlated with patient prognosis and immune checkpoint genes in various cancers by TIMERFor stable silencing of TUG1 expression in HCC cell lines, nonsense and TUG1 shRNA plasmids were manufactured by Jikai Gene Biology Co., Ltd. . The sequences of shTUG1:GCTTGGCTTCTATTCTGAATCCT. JAK2 (NCBI Ref Seq: NM_004972.3) and STAT3 (NCBI Ref Seq:NM_139276.2) overexpression plasmids were manufactured by Sino Biological. Lipofectamine 3000 and P3000 were used to transfect cells.26. Briefly, tissue and cell proteins were extracted with RIPA buffer with 1% phenylmethanesulfonyl (PMSF); the extracted protein was separated by 10% SDS/PAGE and transferred to polyvinylidene fluoride membranes . Then, the membranes were incubated with a primary antibody overnight at 4\u00a0\u00b0C. The membranes were washed three times with 1\u2009\u00d7\u2009TBST for 10\u00a0min each time. Subsequently, they were incubated with secondary antibodies at room temperature for 1\u00a0h. Finally, the blots were detected and analyzed with software . Protein detection was performed by using the following primary antibodies: anti-PD-L1 , anti-GAPDH , anti-JAK2 , anti-pJAK2 Tyr1007/1008 ,anti-STAT3 , anti-pSTAT3 Tyr705 . Western blot experiments in the manuscript were performed by multiple authors. The C, D and E in Fig.\u00a0Standard Western blotting procedures were used to detect protein expression in tissues and cells, as described previously\u2013\u0394\u0394Ct method. Glyceraldehyde-3 phosphate dehydrogenase (GAPDH) was used as an endogenous control. The 2-\u2206\u2206CT method was employed to determine the relative mRNA expression in Human normal liver cells and four HCC cell lines cells, while the relative mRNA expression in hepatocellular carcinoma and adjacent normal tissue samples was calculated using 2-\u2206CT.Total RNA was extracted from HCC cells or clinical tissues using TRIzol according to the manufacturer\u2019s instructions. Reverse transcription was performed to synthesize complementary DNA followed the instructions of a reverse transcription kit . Next, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed using an ABI PRISM 7500 system according to the instruction kit of SYBR Premix Ex TaqTM II . Primers were synthesized and provided by the Beijing Institute of Genomics (China) and activated with an anti-CD3/CD28 T cell activator . After 3\u00a0days of directly coculturing tumor cells and T cells in 24-well plates, the wells were washed with PBS three times to remove the T cells, and tumor cell viability was measured with a CCK-8 assay.24; P\u2009<\u20090.05 was considered significant.Statistical analyses were performed using GraphPad Software . Data for continuous variables are expressed as the mean\u2009\u00b1\u2009standard deviation (SD) and were analyzed using Student\u2019s t-testWe first analyzed TUG1 expression in various cancers and found that TUG1 was especially highly expressed in CHOL, CESC, HNSC, LIHC, LUAD, LUSC, and STAD tumor tissues and A12232 (STAT3 inhibitor). As expected, after 24\u00a0h, AZ960 and A12232 treatment of HCC cells caused significant decreases in PD-L1 mRNA and protein expression compared with no treatment , neutrophils (TANs), and dendritic cells mainly exert the role of promoting tumor progression. Research confirmed that high TUG1 expression and high levels of infiltration of pro-tumor immunocytes in liver cancer tissue leading to the malignant progression20. Study also found that lnc-TUG1 was significantly correlated with quiescent NK cells, suggesting that lnc-TUG1 had the potential to become a tumor immunotherapy target19. We identified that the expression of TUG1 was positively correlated with PD-L1 expression in HCC. Knockdown of TUG1 expression could reduce the mRNA and protein expression of PD-L1. Furthermore, our results showed that silencing TUG1 expression could partially improve effector T cell-induced killing of cancer cells. These results indicate that TUG1 can decrease PD-L1 expression in tumor cells, which subsequently affects anticancer immunity.With in-depth study of the tumor microenvironment, researchers have discovered that the tumor microenvironment can help tumors escape attack by the human immune system44. Moreover, there is mounting evidence that JAK2/STAT3 signaling regulates PD-L1 in many cell types46. Guru et al.47 found that the V617F mutation in JAK2 is accompanied by increased PD-L1 expression and that this PD-L1 overexpression is mediated by JAK2 (V617F) mainly through STAT3. miR-375 overexpression suppresses PD-L1 expression in gastric cancer via the JAK2/STAT3 signaling pathway48. In this study, we observed that silencing TUG1 decreased JAK2/STAT3 expression and that inhibition of JAK2/STAT3 signaling reduced the expression of PD-L1. Thus, our results indicate that TUG1 positively regulates PD-L1 via the JAK2/STAT3 signaling pathway and can be considered a promising therapeutic target in HCC.Current studies indicate that activation of the JAK/STAT pathway promotes the occurrence and development of various diseases, including various inflammatory diseases, lymphoma, leukemia, and solid tumorsIn summary, our study demonstrates that TUG1 and PD-L1 are highly expressed in liver cancer tissues and have potential as biomarkers and therapeutic targets. Importantly, we discovered that silencing TUG1 expression could improve the effector T cell killing capacity by suppressing the JAK2/STAT3/PD-L1 signaling pathway in vitro, which suggests that LncTUG1 may be developed to improve PD-1/PD-L1-based therapeutics for HCC. There are limitations to this study that need to be acknowledged. Firstly, additional hepatocellular carcinoma and adjacent normal tissue samples are needed to validate the results obtained at protein level and RNA level and to analyze clinicopathological relationships. Secondly, more comprehensive in vivo and in vitro studies are necessary to elucidate the mechanisms of TUG1 promotes hepatocellular carcinoma immune evasion via upregulating the JAK2/STAT3/PD-L1 signaling pathway. Lastly, clinical translation needs to be investigated to optimize therapeutic application. These work are the focus of our later research.Supplementary Legends.Supplementary Figure 1.Supplementary Figure 2.Supplementary Table 1."} +{"text": "In China, adolescents account for about a quarter of those treated for mental disorders each year, and adolescent mental health issues have become a social hotspot. Although several epidemiological surveys of mental disorders have been conducted in China, no study has yet focused on the prevalence of mental disorders among adolescents in a certain region of Zhejiang.In the first stage, 8219 middle school students aged 12\u201318\u00a0years in a city of Zhejiang Province (Shaoxing) were screened with the mental health screening checklist. In the second stage, participants who screened positive were tested with the Mini International Neuropsychiatric Interview for Children and Adolescents (MINI-KID) and the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). Then, the prevalence of mental disorders were calculated.The overall prevalence in this population was 12.4%, with prevalence rates exceeding 20% in both the 17- and 18-year-old age groups. The most common mental disorders were obsessive\u2013compulsive disorder (OCD) (9.1%) and major depressive disorder (MDD) (8.9%).Mental disorders are common among middle school students, and girls are at higher risk than boys. As the most prevalent mental disorders, OCD and MDD should receive timely attention, especially for upper grade students. Mental disorders refer to a diverse range of conditions that can impact an individual's thinking, feeling, behavior, and overall well-being, which can arise from a confluence of genetic, environmental and psychological factors . Some ofGlobally, epidemiological studies estimate the prevalence of any mental disorder to be between 3.5% and 38.3% , 2. Not In China, mental and psychiatric disorders rank first in the country's total burden of disease, with adolescents accounting for about a quarter of those treated for mental disorders each year , 8. The What is more, the imbalance between the psychological development and physical development of adolescents makes them more prone to difficulties in psychological integration, leading to negative states of mind, such as worry, anxiety, depression, loneliness and low self-esteem, and thus mental health problems. In recent years, the number of children and adolescents experiencing mental problems worldwide has been on the rise and is of increasing concern to families, schools and society. Although there are more research studies on the mental health status of adolescents, the findings are not consistent across different geographical areas and different levels of economic development , 8.The aim of this study was to investigate the prevalence and distribution of common mental disorders in Shaoxing, China. A two-stage epidemiological survey was conducted. The mental health status of a total of 8219 students in eight middle schools was screened using a questionnaire, and then the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) criteria were used to determine the diagnosis to clarify the current status of mental disorders among these students. This study will contribute to the future provision of medical resources, health policies and prevention strategies.Shaoxing, a city located in Zhejiang Province, was chosen for this study. Eight secondary schools were randomly selected and a total of 8219 participants were included. Of these, 205 refused to participate, 291 did not complete the questionnaire, 75 of those who completed the questionnaire had missing data (more than 20% of the questionnaire was incomplete), and 131 were out of age. Ultimately, a total of 7493 eligible questionnaires (91.2%) were obtained for this study.The Mental Health Screening Checklist completed by the students was used, which consists of five scales: symptom inventory 90 (SCL-90), Self-rating depression scale (SDS), Self-rating anxiety scale (SAS), Yale-Brown obsessive compulsive scale (Y-BOCS) and Pittsburgh sleep quality index (PSQI). The SCL-90 is a self-reported mental health instrument proposed by Derogatis and is widely used to detect clinical psychiatric symptoms and mental health conditions . It can Two types of interviews were conducted sequentially based on the Mini International Neuropsychiatric Interview for Children and Adolescents (MINI-KID) and DSM-IV criteria , 17. TheThe project was approved by the Ethics Review Committee of the Zhejiang Rehabilitation Medical Center. This study consisted of two steps, with the first step using MHSC as the investigative tool. The survey was conducted by a team of psychiatrists, qualified doctors and medical students. The survey data were initially processed by a statistician and after excluding items with incomplete or confusing responses, all individuals with complete MHSC records were analyzed. Of the 7723 participants who completed the questionnaire, 7493 were eligible, with 28.6% of the students scoring positive, i.e., scoring positive on at least one of the five scales included in the MHSC. Then, the second step included all participants with MHSC positive, as well as 1000 randomly selected participants with a negative result, who were chosen to assess the false-negative rate of MHSC. The second step consisted of two interviews. The psychiatrist first conducted a semi-structured interview based on the MINI-KID, followed by a DSM-IV interview to confirm the diagnosis, and finally a total of participants were identified as having at least one psychiatric disorder. The procedure is illustrated in Fig.\u00a0T tests and Chi-square tests. All statistical tests were two-tailed with a significance level of 0.05. Logistic regression analysis was used to study the age group, gender, region, prevalence and the interaction among them.Data analysis was performed using SPSS 25.0 software. Prevalence, frequencies and 95% confidence intervals (CIs) were estimated for mental disorders and comorbidities. The gender, age and regional distribution of the sample, as well as the prevalence of different mental disorders in different age groups, were assessed and subsamples were compared using \u03c72\u2009=\u200941.736, p\u2009<\u20090.01; Table \u03c72\u2009=\u200982.768, p\u2009<\u20090.01).A total of 8,219 students participated in the MHSC questionnaire and 7493 qualified questionnaires eventually returned, with a gender distribution of 3702 males and 3791 females. Among them, 2140 students were found to have psychological problems, with a detection rate of 28.6% (95% CI 27.5\u201329.6). The prevalence rate of girls was significantly higher than that of boys had the highest prevalence , followed by major depressive disorder , anxiety disorders , attention\u2013deficit hyperactivity disorder , oppositional defiant disorder and sleep disorder (Table p\u2009>\u20090.05).As shown in Table It is noteworthy that the prevalence of overall mental disorders increases with age, as shown in Table Comorbidities of the several common mental disorders are shown in Table The mental health of adolescents has become a major public health concern in recent years. Mental disorders are highly prevalent among adolescents and can significantly affect their academic, social, and emotional functioning. Therefore, understanding the prevalence and distribution of mental disorders among them is essential for developing effective prevention and intervention strategies. This study aimed to discuss the findings on the prevalence and distribution of four mental disorders among a large sample of middle school students and examine the gender and age differences in their prevalence rates.In this study, 2,140 students with mental problems were identified from valid data of 7493 middle school students (detection rate: 28.6%), indicating that more than one in four students were affected by one or more psychiatric disorders. The prevalence rate was generally consistent with data from national and international studies , 19, butp\u2009<\u20090.01), contrary to the results of some studies [What is more, following the MINI-KID interview and DSM criteria for diagnosis, four types of psychiatric disorders were diagnosed in a total of 930 students out of 7943 participants, for an overall point rate of 12.4%. This result is consistent with those reported in epidemiological studies of mental disorders based on a two-stage approach conducted in other countries 7.0\u201319.8%) , 20, 21. studies , 20, 21..8% , 20,According to the results of two-stage epidemiological survey, there is a general trend of increasing prevalence among students aged 12\u201318, which may be attributed to the fact that they are in transition from childhood to adulthood, not only going through physical and psychological changes, but also facing other adverse factors that lead to mental health problems, such as academic pressure and family relationships. The 17\u201318 age group has the highest prevalence rate , which may be related to the transition to university, as they face more difficult courses, and university entrance exams (considered the most important exam of a student\u2019s career in China), with increasing pressure from teachers and parents. On the other hand, we found that the most common comorbidity was MDD rather than ADHD, which is inconsistent with previous findings (children aged 6\u201316\u00a0years) . This maThus, early identification and treatment of psychiatric disorders are essential for preventing negative outcomes, so schools and healthcare providers should screen adolescents for psychiatric disorders and provide appropriate interventions for those who screen positive. Gender and age differences in the prevalence of psychiatric disorders suggest that prevention and intervention strategies should be tailored to specific subgroups of adolescents. For example, targeting girls or older adolescents for prevention and intervention programs may be particularly effective in reducing the prevalence. In addition, the high prevalence of OCD and MDD among adolescents highlights the need for targeted prevention and intervention strategies for these disorders. The findings suggest that prevention and intervention strategies should be tailored to specific subgroups of adolescents and targeted at reducing risk factors and increasing protective factors for specific disorders.In conclusion, our study showed that nearly one in ten middle school students in Shaoxing suffer from a mental disorder, with OCD and MDD being the most common disorders in the sample, and the prevalence increases almost with age. The study provides valuable insights into the prevalence of psychological disorders among students and the gender and age differences in their prevalence rates. In addition, our findings highlighted the need for early screening of middle school students for mental disorders, which facilitates the allocation of public mental health resources and policy development. Further research is needed to explore the underlying factors contributing to these gender and age differences and develop effective interventions to improve mental health outcomes in students.There are several limitations to this study that should be considered when interpreting the findings. First, academic stress and family relationships may be important factors influencing prevalence, but the lack of relevant data collection prevented a correlation analysis in this study. Second, the study did not assess the duration of these mental disorders, which may be important factors in determining the need for intervention. Finally, the study was conducted in a specific geographic region, which may limit the generalizability of the findings to other populations."} +{"text": "BDAI), has been studied on Au(111). Intermolecular hydrogenbonding determines the formation of highly organized linear structureswhere surface chirality, resulting from the 2D confinement of thecentrosymmetric molecules, is observed. Moreover, the structural featuresof the BDAI molecule lead to the formation of two differentiatedarrangements with extended brick-wall and herringbone packing. A comprehensiveexperimental study that combines scanning tunneling microscopy, high-resolutionX-ray photoelectron spectroscopy, near-edge X-ray absorption finestructure spectroscopy, and density functional theory theoreticalcalculations has been performed to fully characterize the 2D hydrogen-bondeddomains and the on-surface thermal stability of the physisorbed material.The control of molecular structures at the nanoscaleplays a criticalrole in the development of materials and applications. The adsorptionof a polyheteroaromatic molecule with hydrogen bond donor and acceptorsites integrated in the conjugated structure itself, namely, benzodi-7-azaindole( Quinacridone has been comprehensively studiedon different surfaces such as highly oriented pyrolytic graphite (HOPG),Ag(111), Ag(100), and Cu(111).33 Although different degrees ofstrength in the adsorption of quinacridone have been observed dependingon the molecule\u2013substrate interactions, in all cases, homochirallinear structures were observed for each of the expected surface enantiomers.This arrangement results from the complementary hydrogen bonds setbetween the carbonyl and the NH groups present in the structure ofquinacridone. Similar results were reported for the surface self-assemblyof indigo on Cu(111), where enantiopure one-dimensional chains wereobserved.34 Diketopyrrolopyrrole (DPP)is another building block frequently used in the synthesis of organicsemiconductors. Its structure also contains two carbonyl groups andtwo NHs representing the same hydrogen bonding motif as quinacridoneand indigo. The short conjugation length of DPP can be easily enlargedby attaching aromatic substituents. These derivatives form hydrogen-bondedlinear structures intercalated with solvent molecules when depositedfrom long alkanoic acid solutions on HOPG.36 The N-heteroacene dihydrotetraazapentacene constitutesanother conjugated molecule whose ability to self-assemble on Au(111),Cu(110), and c-plane sapphire surfaces has been studied.39 In this case, the N\u2013H\u00b7\u00b7\u00b7N interactions betweentetrahydropyrazine and pyrazine-like nitrogens induce the formationof well-ordered molecular rows that grow in homochiral domains.Focusing our attentionon conjugated molecules, which have gainedmuch relevance due to their use as semiconductors in different electronicapplications , their charge transportproperties are determined by their intermolecular interactions andtheir disposition at the substrate interface.40 The self-resolution resulting from the formation of energeticallyfavored hydrogen-bonded hexamers led to a two-dimensional framework.The reciprocal hydrogen bonding set through strategically located7-azaindole units has revealed its suitability as a building blockfor controlling the molecular disposition of conjugated systems aswe have demonstrated in different electronic applications.42 Accordingly, herein we report the integration of this building blockinto a structurally related molecule, namely, benzodi-7-azaindole(BDAI), resulting from the condensation of two 7-azaindoleunits to a central benzene core (pyridine\u00b7\u00b7\u00b7H-Npyrrole) and differs from most ofthe previously reported molecules based on C=O\u00b7\u00b7\u00b7HNinteractions. Moreover, the fused 7-azaindole building block leadsto fully conjugated systems that contrast the cross-conjugation commonlyobserved in most of the abovementioned compounds.Within this context, we have recently reported a supramolecularapproach based on the surface self-assembly of a conjugated tripodalsystem that formed expanded enantiopure domains.ene core 1. This sBDAI moleculeson Au(111), in ultrahigh vacuum (UHV), as well as their thermal stability.The chosen substrate Au(111) provides a weak substrate\u2013moleculeinteraction and allows the observation of molecular structures closeto a free-standing situation.The surface chirality emerging from the adsorptionof this moleculewith a centrosymmetric structure can produce two surface enantiomerswhen deposited on Au(111). Ideally, the molecules can form extendedunidirectional hydrogen-bonded structures. Besides, the lateral packingof these structures leads to expanded domains with either brick-wallor herringbone arrangement depending on the homochiral or racemiccomposition, respectively. In this work, we use several surface-scienceexperimental techniques, including STM, high resolution X-ray photoelectronspectroscopy (HR-XPS), and near-edge X-ray absorption fine structurespectroscopy (NEXAFS), combined with density functional theory (DFT)calculations, to get detailed characterization of the geometry andbonding of self-assembled 2D monolayers of 2b:4,5-b\u2019]di(7-azaindole), BDAI, has been reportedelsewhere.43 The preparation and characterizationof physisorbed layers have been performed in two different UHV systems:a home-laboratory with STM, low energy electron diffraction (LEED),quadrupole mass spectrometry (QMS), and Auger electron spectroscopy(AES) techniques, and a synchrotron laboratory for HR-XPS and NEXAFS.The synthesisof 6,12-dihydrobenzo directions.An increase in coverage produces a further packing of the homochiralrows, with hydrogen bonding between adjacent molecules, extendinglaterally the assembly. The formation of enantiopure domains has beentypically observed for other centrosymmetric molecules whose hydrogenbonding on the surface has been characterized.38 Interestingly, the particularities of the BDAI structure,with a pseudolinear skeleton and the hydrogen bonding sites locatedat both ends of the polyheteroaromatic system, lead to a self-assembledstructure with a noticeable intermolecular shift within the supramolecularrows. As a consequence, these rows can adopt two different moleculararrangements distinguishable in the STM images: the brick-wall packing nm2 with an angle of 106\u00b0. In the latter packing, some rowsof molecules are narrower, probably due to electronic effects. Inboth domains, intermolecular stabilization of the adlayers is mainlydriven by complementary donor\u2013acceptor N\u2013H\u00b7\u00b7\u00b7Nhydrogen bonds between adjacent molecules in the rows . Additionally, differentedge-to-edge intermolecular interactions (with distances ranging between2.2 and 2.6 \u00c5) contribute to the lateral packing of homochiralor enantiomer rows producing densely packed domains in both phases. \u2013). The interaction with the substrate in this physisorption regimeis intense enough to anchor the molecules to the surface but not sufficientto distort the gas-phase molecular morphology, which has its reflectionin that the molecules of both phases are essentially flat on the surface(vide infra for NEXAFS discussion) at perpendiculardistances of 3.19 and 3.32 \u00c5 and binding energies per moleculeof \u22121.12 and \u22120.93 eV for the brick-wall and herringbonemolecular arrangements, respectively. Binding energies have been obtainedas the difference between the computed total energy of the whole interfacialsystems and the sum of the total energies of the corresponding surfacesand molecular adlayers separately. This slight difference of 0.19eV in the adsorption energies between both phases seems to arise froma more efficient packing of the brick-wall arrangement, which alsomanifests in the lower adsorption energy and a slightly lower adsorptionmolecular distance. The less robust packing of the herringbone adlayerfavors a slightly lower decoupling degree from the substrate withrespect to the brick-wall phase, thus decreasing the binding energywith the Au surface. Experimentally, in a rough analysis of the STMimages, we also observe a preference for the brick-wall phase witharound 67% of the covered areas, even though this homochiral structurerequires segregation of the enantiomers.The unit cell obtained for the brick-wall arrangementis (1.06\u00d7 2.09) nmWe have also simulatedtheoretical STM images based on the Keldish\u2013Greenformalism for the optimized interfacial models obtained under theconstant-current regime as in the experiments. Excellent match betweenthe experimental and simulated images has been obtained for both structures:brick-wall 3a and he3.2BDAI fitted with three componentsat binding energies of 398.35, 399.13, and 399.85 eV was carried out by HR-XPS probing both C 1s and N 1s levels4. The N 99.85 eV 4a; detai53The small component located at 399.85 eV can beattributed to (\u2212NH)that does not form H bonds or hydrogenated pyridine.Concerning the C 1s core level for the 0.8 ML onAu(111), the deconvolutionof the spectra fitted well with three components located at 284.06,284.74, and 285.70 eV with 52, 35, and 13% of relative weight, respectively4b. The mBDAI/Au brick-wall and CLSfinal(Na2), behaving, as expected, as donor and acceptor sites, respectively.These values lead to a difference [CLSfinal(Na2) \u2013 CLSfinal(Na1)] of +0.63 eV withthe pyrrolic N atom showing higher binding energy than the pyridinicone for the brick-wall domain. As far as the calculations for theherringbone domain are concerned, values of \u22120.39 and +0.32eV were obtained for CLSfinal(Nb1) and CLSfinal(Nb2), behaving again as donor and acceptorsites, respectively. These values lead to a difference [CLSfinal(Nb2) \u2013 CLSfinal(Nb1)] of+0.71 eV, with the pyrrolic N having a higher binding energy. Thecomputed values of +0.63 and +0.71 eV agree with the experimentalXPS result of +0.78 eV for the difference between N atoms in the pyrroleand pyridine rings.These calculations led to values of \u22120.42and +0.21 eV forCLS3.3BDAI film areshown in 57 and azaindole.58 Fromcomparison with the literature on pyrrole,59 azaindole,58 and imidazole,60 we may associate the broad and asymmetric resonancepeak at 402.05 eV (peak C) with the lowest energy contribution fromthe pyrrolic nitrogen atom, corresponding to a N 1s \u2192 \u03c0*(N\u2013H) transition. The assignment of the small absorption featureat 400.36 eV (peak B) is less straightforward. We can exclude an interfacialorigin because it is alsoobserved in multilayers (grown at low temperatures) with similar weight. A relatively weaker shoulder of themain peak at 1.4 eV higher energy was reported in the case of pyridine.57 Former NEXAFS measurements on imidazole alsoreported a clear peak in between the two main C=N-C and N\u2013H\u03c0* resonances, as due to the transition of N 1s electrons (C=N-C)to the 2\u03c0* orbital.60 We may speculatethat the sharper appearance of resonance B in the BDAI spectra with respect to azaindole may be associated with the overallincrease in the spatial spread of the molecular orbitals due to theconjugation of the benzene ring.We performed linearlypolarized C and N K-edge NEXAFS measurements to elucidate the chemicaland geometrical properties of the molecules on Au(111). The p- ands-polarized NEXAFS spectra of the 0.8 ML BDAI film are shown in Polarized C and N K-edge NEXAFS measurements wereperformed toelucidate the chemical and geometrical properties of the moleculeson Au(111). The p-polarized and s-polarized NEXAFS spectra of the0.8 ML BDAI molecule, and they correspond to the C 1stransitions into the LUMO. The components at 286.8 eV (peak F) andat 287.3 eV (peak G) correspond to the C 1s transitions into the LUMO+1states. The LUMO+1 is quite similar in shape and intensity to theLUMO. Finally, the broad peak located at 290.8 eV is assigned to a\u03c3* symmetry resonance.Regarding the p-polarized C K-edgespectrum 6b,the fBDAI NEXAFS C K-edge showsthat the average tilt angle of the molecules with respect the surfaceplane is 3.2\u00b0 \u00b1 1.5\u00b0 for almost one monolayer. We alsomeasured the NEXAFS for less than one monolayer (0.8 \u00c5), andthe average tilt of the molecules is 3.6\u00b0 \u00b1 1.7\u00b0. Theselow average tilt angles are indicative of molecules lying essentiallyflat on the substrate surface and show good agreement with the DFTcalculations.The relatively sharp C and N K-edgepeaks are evidence of the weakinteraction between molecules and the substrate as it is expectedfor the Au(111) surface. When the polarization dependence of the NEXAFSspectra is analyzed, a strong dichroism is observed. The \u03c0*transitions show the maximum intensity when the electric field isalmost normal to the surface, p-polarization, and their intensitydecreases considerably when the electric field is parallel to thesurface, s-polarization. The reverse behavior is observed for the\u03c3* symmetry resonance. Therefore, we can determine the averageorientation of the molecules relative to the surface through the refs , 62. The3.4BDAI HR-XPSspectrum at different coverages, monolayer and multilayer, has beenstudied at different temperatures. Regarding the core level N 1s measurementsfor a BDAI multilayer, coverage of 11.7 \u00c5, depositedat \u221280 \u00b0C, the spectrum shows one single broad componentat 399.20 eV . Two types of 2D chiral domains, namely, brick-wall and herringbone,stabilized by complementary N\u2013H\u00b7\u00b7\u00b7N hydrogenbonding have been observed for BDAI molecules. NEXAFSand DFT experiments have revealed that the molecules lie essentiallyflat on the surface. The spectroscopic characterization of the flatmonolayer by HR-XPS has confirmed the composition of the 2D self-assembledsupramolecular structure and more importantly has revealed the effectof hydrogen bonding on the absolute binding energies shifts measuredfor N atoms in the pyridine and pyrrole rings. Finally, the thermaldesorption studied at different coverages has proved the high stabilityof hydrogen-bonded monolayers formed by BDAI. These resultsreinforce the utility of hydrogen bonding as a tool for controllingthe molecular organization at the nanoscale and improving the robustnessof conjugated materials.In summary, we have combinedfirst-principles calculations withHR-XPS, NEXAFS, and STM experimental data to investigate the hydrogenbond-directed self-assembly of"} +{"text": "Introduction: The aim of this study was to evaluate the results of a 75 g oral glucose tolerance test (OGTT) performed in the second trimester of pregnancy, the prevalence of gestational diabetes mellitus (GDM), and perinatal outcomes in pregnant women with a history of coronavirus disease 2019 (COVID-19) infection\u00a0and to examine the effect of COVID-19 infection history on OGTT results and GDM prevalence.Methods: We retrospectively analyzed the data of 463 patients who visited the Gynecology and Obstetrics Department of Kayseri City Hospital between March 2020 and January 2023 and were administered a 75-g OGTT in the second trimester of their pregnancy. Accordingly, we traced COVID-19 history, OGTT results, GDM prevalence, and newborn outcomes among the patients.Results: OGTT glucose values were higher in the study group with a history of COVID-19 infection, but there was no significant difference between the groups. GDM developed in 13 (23.2%)\u00a0pregnant women in the group with a history of COVID-19 infection and 88 (21.6%) pregnant women in the control group without a history of COVID-19 (p: 0.348). In addition, in pregnant women diagnosed with GDM, insulin requirement was 8.9% in the COVID-19 (+) group and 5.1% in the COVID-19 (-) group, and the results were not statistically significant (p: 0.178).\u00a0There was no significant difference in neonatal outcomes between the groups.Conclusions: In our study, we found that glucose values were higher and the prevalence of GDM was higher in pregnant women with a history of COVID-19 infection before the OGTT. It is necessary to be more careful about issues such as blood glucose regulation and GDM risk in pregnancy follow-up after infections such as COVID-19, which may have widespread systemic inflammatory effects, and patients should be informed in detail for pregnancy follow-up. Coronaviruses are renowned for being prominent human and animal pathogens. Exponentially spreading pneumonia cases in Wuhan, China toward the end of 2019 were discovered to be caused by a novel coronavirus. The spread of the virus was unstoppable, causing an outbreak across China, followed by an unprecedented number of pneumonia cases worldwide. In February 2020, the World Health Organization (WHO) identified the cases as coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [The virus can be transmitted by droplets from infected individuals through coughing, sneezing, and contact with contaminated surfaces and the patient's hand to the mucous membranes of the mouth, eyes,\u00a0and nose . TransmiCOVID-19 appears to precipitate severe diabetes symptoms, including diabetic ketoacidosis, hyperosmolar hyperglycemic state, and severe insulin resistance . The preA follow-up study investigating the pathogenesis of pancreatic lesions in 2010 found that pancreatic islets were strongly immune-positive for ACE2, while exocrine tissues were only weakly positive. DM developed in 20 of 39 patients who did not receive corticosteroids during SARS illness, and these patients required early insulin treatment, possibly due to the destruction of pancreatic beta cells or increased insulin resistance due to multiple mechanisms. Six of them were diagnosed with DM while being discharged . It shouGlucose intolerance resulting in hyperglycemia that is first diagnosed during pregnancy is often called gestational diabetes mellitus (GDM) -12, and Pregnancy is a process that makes women vulnerable to viral infections and causes partial suppression of the immune system. Even in seasonal influenza seen especially in winter months, morbidity rates increase during pregnancy. There are studies showing that COVID-19 infection during pregnancy is risky ,17. In tIn our literature review, we found that there are very few studies on the possible relationship between COVID-19 and GDM. The aim of our study was to evaluate the results of the 75 g OGTT performed in the second trimester of pregnancy, GDM prevalence, and perinatal outcomes in pregnant women with a history of COVID-19 infection and to examine the effect of COVID-19 infection history on OGTT results and GDM prevalence.We carried out this study with the ethical approval of the Ethics Committee of Kayseri City Hospital (No.: 393 dated 04.29.2021) and in accordance with the principles in the Declaration of Helsinki. In this retrospective, single-center study, we considered the data of patients who visited the Gynecology and Obstetrics Department of Kayseri City Hospital between March 2020 and January 2023 and were administered a 75-g OGTT in the second trimester of their pregnancy. While the COVID-19 positive (+) group consisted of pregnant women who had a positive result of nasopharyngeal swab and antibody tests before the OGTT, those without a history of COVID-19 infection and with negative test results were included in the control group. We then noted down their demographic characteristics, OGTT results, GDM status, mode of delivery, pre-pregnancy body mass index (BMI), and newborn outcomes.Nevertheless, we excluded those with pre-pregnancy diabetes , pre-pregnancy endocrine disorder , a history of bariatric surgery, malabsorption diseases , and multiple pregnancies. We also excluded those not receiving the diagnostic OGTT within the recommended time interval (24+0 to 27+6 weeks of gestation) or prior to the COVID-19 infection. Therefore, we analyzed the data of 463 pregnant women, 56 in the patient group and 407 in the control group.GDM is typically diagnosed based on the 75-g OGTT results, using the criteria suggested by the International Association of Diabetes and Pregnancy Working Groups (IADPSG). These criteria involve glucose levels exceeding the following thresholds: 92 mg/dL when fasting, 180 mg/dL during the first hour, and 153 mg/dL during the second hour (10). Fasting glucose exceeding 126 mg/dL or glucose values exceeding 200 mg/dL twice are considered pre-existing diabetes ,26.Statistical analysisStatistical analysis was performed using IBM\u00a0SPSS\u00a0Statistics for Windows, Version 20 . Data are presented as median or number (percentage) with 95% confidence interval (CI). In addition, chi-square tests were performed for categorical variables and t-tests were performed to compare the means of two groups. All analyses were performed using a two-sided model, taking into account normal distribution as appropriate. Multiple logistic regression was performed to identify factors associated with the incidence of the primary outcome of interest. A p-value less than 0.05 was considered statistically significant.In our study, 463 pregnant women who underwent 75 g OGTT screening between 24 and 28 weeks of pregnancy between 03/2020 and 03/2022 in our clinic were included. It was determined that 56 of the pregnant women 12%) had COVID-19 infection before the OGTT test, while the remaining 407 pregnant women (88%) did not have COVID-19 in their history and tests performed. Demographic characteristics and neonatal outcomes of both groups are presented in Table 2% had COFasting glucose, glucose at 60 minutes, and glucose at 120 minutes were higher in the COVID-19 (+) group than in the COVID-19 (-) group, but no statistically significant difference was observed between the groups . OGTT results of the groups are presented in Table GDM developed in 13 (23.2%) pregnant women in the COVID-19 (+) group and 88 (21.6%) pregnant women in the COVID-19 (-) group, and there was no significant difference in the rate of GDM between the groups (p: 0.348). In addition, in pregnant women diagnosed with GDM, insulin requirement was 8.9% in the COVID-19 (+) group and 5.1% in the COVID-19 (-) group, and the results were not statistically significant (p: 0.178). GDM and insulin requirement rates of the groups are presented in Table The effect of BMI on the frequency of GDM diagnosis is presented in Table The literature hosts a plethora of studies addressing the link between COVID-19 infection and type 2 diabetes ,21. NeveAlthough the exact pathophysiological bidirectional relationship of SARS-CoV-2 infection with diabetes has not yet been fully understood, previous research reported an increased incidence of new-onset diabetes during the COVID-19 disease ,23. It iCOVID-19 or other viral infections may impair beta cell function, leading to type 2 diabetes and even triggering the onset of type 1 diabetes ,25. ThusThe incidence of GDM was found to be higher in women with COVID-19 infection . SimilarIn our study, we found that glucose values were higher and the prevalence of GDM was higher in pregnant women with a history of COVID-19 infection before the OGTT. We concluded that COVID-19 infection may cause glucose intolerance in pregnancy, so caution should be exercised. While the need for insulin is normally limited in women with GDM, it should be kept in mind that the need for insulin increases with a history of COVID-19, and dysglycemia treatment in GDM patients with a history of COVID-19 may require enhanced care in terms of blood glucose regulation." \ No newline at end of file