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+{"text": "Inappropriate medication use is a major healthcare issue for the elderly population. This study explored the prevalence of potentially inappropriate prescriptions (PIPs) in long-term care in metropolitan Quebec.A cross sectional chart review of 2,633 long-term care older patients of the Quebec City area was performed. An explicit criteria list for PIPs was developed based on the literature and validated by a modified Delphi method. Medication orders were reviewed to describe prescribing patterns and to determine the prevalence of PIPs. A multivariate analysis was performed to identify predictors of PIPs.Almost all residents (94.0%) were receiving one or more prescribed medication; on average patients had 4.8 prescribed medications. A majority (54.7%) of treated patients had a potentially inappropriate prescription (PIP). Most common PIPs were drug interactions (33.9% of treated patients), followed by potentially inappropriate duration (23.6%), potentially inappropriate medication (14.7%) and potentially inappropriate dosage (9.6%). PIPs were most frequent for medications of the central nervous system (10.8% of prescribed medication). The likelihood of PIP increased significantly as the number of drugs prescribed increased  and with the length of stay . On the other hand, the risk of receiving a PIP decreased with age.Potentially inappropriate prescribing is a serious problem in the highly medicated long-term care population in metropolitan Quebec. Use of explicit criteria lists may help identify the most critical issues and prioritize interventions to improve quality of care and patient safety. Inappropriate medication use is a major health care issue for the elderly population -3. OlderPrevalence estimates of PIPs are likely to vary with the criteria that are applied. Some authors have based their assessment on the Beers criteria -7,9-12. In Canada, a list of explicit criteria was developed by a panel of experts in 1997 . The CanThe long-term care elderly population is particularly vulnerable to inappropriate medication use; it is composed of frail older patients who typically have functional disabilities and acute and chronic medical histories that require complex medication regimens ,21. AsseA cross-sectional chart review of long-term care patients aged 65 years and over living in the Quebec City area was performed in the period between April 1995 and December 1996. All long-term care facilities located in the Quebec City area were contacted and the majority (29 out of 33) agreed to participate in the study. Within the 29 participating facilities, there were a total of 71 long-term care units. Numbers of beds in these units averaged 41 (10 to 190). Units were visited once during the study period. Data on drugs currently being prescribed the day of the visit was collected using medication charts. Demographic data included age, gender and length of stay. This study was approved by the ethics committees at Universit\u00e9 Laval, H\u00f4pital Saint-Fran\u00e7ois d'Assise and H\u00f4pital de l'Enfant-J\u00e9sus.For each medication order, the name, dosage, frequency of dosing and nature of prescription (scheduled or given on an as-needed basis) were collected. To capture the fullest possible extent of potentially inappropriate prescribing, it was assumed that all medications prescribed on an as-needed basis were taken. The total daily dose of an as-needed prescription was calculated by multiplying the prescribed unit dose with the indicated daily frequency of administration. Prescriptions for creams, ointments and drops were not included. Each medication was classified using the Anatomical Therapeutic Chemical (ATC) classification system . The maxA list of explicit criteria for PIP in older patients was developed based on a review of the literature ,13,14,22Medication charts were reviewed and compared with the list of explicit criteria. PIPs were categorized as:\u2022 Potentially inappropriate medication;\u2022 Potentially inappropriate duration;\u2022 Potentially inappropriate dosage; and\u2022 Potentially inappropriate drug-drug interaction.t tests were used to compare proportions and means, respectively. Association between age and drug utilization was evaluated by analysis of variance. Factors predicting PIP were identified by logistic regression analyses. Independent variables were age, sex, number of prescribed drugs and length of stay. An initial bivariate analysis allowed calculation of crude odds ratios, identification of variables individually associated with the risk of PIP, and determination of the appropriate scale for each variable. A multivariate analysis with a significance threshold of 0.10 for the inclusion of variables subsequently yielded adjusted odds ratios for the number of prescribed medications, age and length of stay. Data were analyzed for collinearity and overdispersion. Data analyses were performed using SAS version 6.12 .Drug prescribed and PIP data were stratified by age and gender. Chi-square and Student p = .0001).The study population included 2,633 individuals, aged 65 years and older, residing in long-term care facilities for a mean duration of 8.5 years. Mean age was 82 \u00b1 8 years and the majority of individuals were women (74.2%). Women were older than men  had one or more prescribed medications and 48%  of the total population had five or more medications. Residents had on average 4.8 prescribed medications. Proportions of patients by number of prescribed medications were similar for men and women, but varied according to age. The oldest patients, aged 85 years and more, received significantly less medications than their youngest counterparts aged between 65 and 74 years; 43.8% of patients aged over 85 years received five medications or more, compared to 59.4% of those aged 65 to 74 years. Of the 12,707 medications prescribed, 86% were scheduled administrations and 82% were prescribed for more than three months.A majority of patients  had a prescription for medications of the central nervous system (CNS). Cardiovascular medications  and medications of the alimentary tract and metabolism  were the following most frequently prescribed anatomical groups of medications. Most commonly prescribed therapeutic classes included analgesics (48.0%), anxiolytics (41.4%), antipsychotics (35.0%) and loop (high-ceiling) diuretics (18.6%)  had one or more PIPs; 29.5% had one PIP, 12.5% had two PIPs, 7.5% had three PIPs and 5.3% had four or more PIPs.A total of 12,707 drugs were prescribed of which 1807 were given on an as-needed basis. The proportion of PIPs among scheduled and as-needed prescriptions were 9.2% and 11.5%, respectively. If we exclude as-needed prescriptions, 46.4% of all residents had one or more PIPs.The most common type of PIP was drug-drug interaction, affecting 33.9% of patients treated with drugs, followed by potentially inappropriate duration (23.6%), potentially inappropriate medication 14.7%), and potentially inappropriate dosage (9.6%) ; more than half of those PIPs were for the anxiolytic oxazepam Table . A substMultivariate analysis indicated that patients with a length of stay 10 years or over were 1.78 times at greater risk of being prescribed a PIP than those with less than 10 years of stay  . Estimates of PIP prevalence in the literature vary between 4.8%  and 45.61.5%. EstA large number of patients were receiving CNS medication 85%) and the most common PIPs were related to that category of drugs. Thirty-five percent of patients were prescribed antipsychotics and 22.9% had benzodiazepine for potentially inappropriate duration, defined as more than a month . A numbe% and theThe length of stay was positively associated with PIPs, while the prevalence of PIPs decreased with age. Although the association between length of stay and the likelihood of receiving a PIP in nursing homes was studied in the past , to our The results presented here should be viewed in light of potential limitations. As in previous studies , we did This study evaluated prescription patterns rather than the actual consumption of medication. The low prevalence of as-needed medication (14%) and the long-term care setting, in which medication is administered to patients by a health caregiver, suggest that this limitation did not have a significant impact on the results. As-needed prescriptions may have accounted for repeat prescriptions, which may in turn have led to overestimation of the number of drug-drug interactions. However, even after excluding as-needed prescriptions from the analysis, the proportion of residents with a PIP remains high.Predictors of PIPs were assessed using a multivariate analysis. It allowed us to adjust for potential confounding variables. However, we were not able to adjust for facility variables as those were not available.This study is the first to describe and qualify prescribing practices in long-term care facilities in urban Quebec. In particular, it highlights the extent of potentially inappropriate prescribing in elderly long-term care patients, which are among the frailest of society ,21. InapInappropriate prescribing is highly prevalent in the elderly long-term care population in metropolitan Quebec. The use of a explicit criteria list to identify PIPs is a first step towards identifying most critical issues and implementing strategies to improve quality of care and patient safety. Identifying predictors of PIPs may help to target problems and prioritize interventions that are most needed in the rapidly expanding older population.Carol Rancourt and Jean-Pierre Gr\u00e9goire were employed by Merck Frosst Canada at the time of the preparation of this article.CR, in partial fulfillment for the grade of M.Sc., lead the protocol development, expert panel consultation, data analyses, discussion of results, and manuscript preparation. JM contributed to all steps of this research project and manuscript preparation. LB contributed to protocol development, presentation and discussion of results and manuscript preparation and participated in the expert panel to define the explicit criteria. RV is the principal investigator for the initial research project which generated the drug prescription data used for this study. He contributed to protocol development, presentation and discussion of results, manuscript preparation and participated in the expert panel to define the explicit criteria. DL was a co-investigator for the initial research project, which generated the drug prescription data used for this study, and contributed to protocol development, data analyses and manuscript preparation. All authors read and approved the final manuscript. JPG contributed to protocol development presentation and discussion of results, manuscript preparation and participated in the expert panel to define the explicit criteria.The pre-publication history for this paper can be accessed here:Appendix: List of explicit criteria used to assess the quality of prescribing in long-term care for elderly patients provide as additional fileClick here for file"}
+{"text": "Pseudomonas aeruginosa and Acinetobacter baumanniii has led to the re-use of polymyxins. Our objective was to study the toxicity of prolonged intravenous administration of colistin (polymyxin E).The intravenous use of polymyxins has been considered to be associated with considerable nephrotoxicity and neurotoxicity. For this reason, the systemic administration of polymyxins had been abandoned for about 20 years in most areas of the world. However, the problem of infections due to multidrug-resistant (MDR) Gram-negative bacteria such us An observational study of a retrospective cohort at \"Henry Dunant\" Hospital, a 450-bed tertiary care center in Athens, Greece, was undertaken.Patients who received intravenous colistin for more than 4 weeks for the treatment of multidrug resistant Gram-negative infections were included in the study. Serum creatinine, blood urea, liver function tests, symptoms and signs of neurotoxicity were the main outcomes studied.We analyzed data for 19 courses of prolonged intravenous colistin  in 17 patients. The median creatinine value increased by 0.25 mg/dl during the treatment compared to the baseline (p < 0.001) but returned close to the baseline at the end of treatment . No apnea or other evidence of neuromuscular blockade was noted in any of these patients who received prolonged treatment with colistin.No serious toxicity was observed in this group of patients who received prolonged intravenous colistin. Colistin should be considered as a therapeutic option in patients with infections due to multidrug resistant Gram-negative bacteria. Pseudomonas aeruginosa and Acinetobacter baumannii resistant to most classes of antimicrobial agents has made the medical community to rethink of colistin, an old, basically abandoned for the last two decades antibiotic. Intravenous colistin (polymyxin E) has been used in the industrialized countries during the last years mainly in patients with cystic fibrosis infected with Pseudomonas aeruginosa resistant to other available antimicrobial classes , and at least two of the following: new onset of purulent sputum or change in character of sputum, increased respiratory secretions or increased suctioning requirements, new onset or worsening of cough or dyspnea or tachypnea, rales or bronchial breath sounds, or worsening gas exchange . Unresponsiveness, defined as persistence or worsening of presenting symptoms and/or signs of the infection during intravenous colistin administration, was observed in 5 of the 19 episodes of infection (26.3%).All patients had been admitted to the ICU at some time during their hospitalisation. In 12 courses out of the 19 analyzed the whole colistin therapy was administered in the ICU, in 1 course it was administered in a hospital ward, and in 6 courses it was given both in the ICU and a hospital ward.Figure At the initiation of colistin administration the median value of baseline blood urea for the 18 courses was 42 mg/dl. During the 18 prolonged courses of colistin administration there was a slight increase in blood urea values; the median maximum value was 60 mg/dl. However, at the completion of colistin therapy median blood urea value returned to 41 mg/dl.During the 19 courses of colistin administration, other potentially nephrotoxic drugs were given. Specifically, aminoglycosides were co-administered in 12 courses , teicoplanin in 10 , vancomycin in 8 , amphotericin in 4 .Four patients had history of chronic renal dysfunction, with only one of them receiving hemodialysis treatment prior to admission. This patient continued the hemodialysis during his treatment with colistin and died due to sepsis. For the other 3 patients the baseline, the maximum, and the end-of-therapy creatinine values (mg/dl), were 1,8/2,1/2,1 for the first patient, 3,7/3,7/2,4 for the second patient and 2,4/3,8/2,3 for the third patient.Acinetobacter baumannii pneumonia, sensitive only to colistin and gentamicin. Apart from a slight elevation of serum creatinine  on the 5th day of treatment no other sign of nephrotoxicity was observed during a 34-day administration of colistin .Only one patient had acute renal failure prior to colistin use, which was due to severe haemorrhage during surgery. This patient received 13 courses of hemodialysis. One day after the last course, colistin therapy had to be initiated for the management of th day of colistin treatment and became worse on the 27th day. From then on, and although colistin was continued for 11 more days, the symptoms gradually subsided. No confirmatory electromyographic testing was performed. The second patient was transferred from another ICU where he was diagnosed as having myopathy due to sepsis. Fifteen days after his admission, and while his myopathy was improving, therapy with colistin was initiated; the treatment did not affect the gradual improvement. An electromyography performed on his 21st day in our ICU, showed mild axial sensory-motor polyneuropathy and myopathy. He received colistin for a total of 52 days and made full recovery from his neuropathy/myopathy during the course of colistin administration. The third patient developed ICU polyneuropathy one week prior to colistin administration and, again, recovered gradually from it while he was on colistin treatment. The fourth patient was transferred from another ICU, where he had developed ICU polyneuropathy. Only this patient had moderate worsening of the neuropathy while he was receiving colistin. Despite that, he received colistin for a total of 35 days for a respiratory infection with persisting Pseudomonas aeruginosa sensitive only to colistin. His neuropathy improved gradually after the end of colistin treatment. Thus, our assessment regarding these 4 patients who experienced polyneuropathy and/or myopathy is that colistin therapy was probably associated with the development of neurotoxicity only in one of the 4 patients.No apnea or other evidence of neuromuscular blockade was noted in any of these patients who received prolonged treatment with colistin. Four patients were clinically diagnosed to have polyneuropathy and/or myopathy before or during colistin treatment. In the first patient the polyneuropathy symptoms appeared while she was on her 25Data for the possible hepatobiliary toxicity of colistin were collected. Both laboratory and clinical findings were taken into consideration. In subgroup analyses of patients for whom data were available, no substantial changes on liver function tests was found, as described in Figure The main finding of the study is that no significant deterioration of renal function was found in patients with baseline normal serum creatinine value during prolonged administration of intravenous colistin. In addition, in the group of patients with pre-existing chronic renal dysfunction  the administration of the medication for a long duration did not influence further their renal function. The observed slight increase in the serum creatinine values at the end of treatment  cannot be attributed only to the prolonged colistin administration as other factors such as concomitantly administered potential nephrotoxic agents, and sepsis per ce may have had an effect. In addition, apnea or other evidence of neuromuscular blockade was not observed in any of the 17 patients who received prolonged treatment with colistin.Pseudomonas aeruginosa and E. coli strains from the excised specimen. This patient developed acute tubular necrosis (confirmed by autopsy) and apnea requiring intubation and mechanical ventilation. However, in this case the dosage of colistin used was 30 mg/kg of body weight every six hours (120 mg/kg/day) whereas the recommended dose is 2,5 \u2013 5 mg/kg/day. One year later, several reports showed high renal adverse reaction rates after colistin administration [Early experience with colistin revealed a high incidence of toxicity, mainly nephrotoxicity. In 1969, Ryan et al. reported the fatal case of a previously healthy 4-year-old child with persistent fever after appendectomy who died due to cardiac arrest . The chistration . In manyAcinetobacter baumannii, who received either intravenous colistin (21 patients) or imipenem (14 patients), showed an almost double increase in nephrotoxicity rates among the patients in the imipenem group compared to colistin group [However, recent data from published reports do not corroborate this finding . Notablyin group . In addiin group .The conclusion in the majority of the published studies during 1960 \u2013 1970 was that colistin must be used in serious infections where other antimicrobial agents have failed, always with close monitor of renal function, and with precaution in the co-administration of other possible nephrotoxic or neutotoxic agents ,4. SubseOur study is not without limitations. It is a retrospective study with the inherited problems related to this study design, including difficulties in the determination of colistin associated sensory neurotoxicity. In addition, the number of studied patients is relatively small and there is no control group. However, our study offers insight related to interesting experience from a group of patients who received prolonged treatment with colistin for various infections, mainly as a salvage treatment of persisting and serious infections.st century.In conclusion, intravenous colistin seems to be a safe therapeutic intervention for serious infections due to multidrug-resistant Gram-negative bacteria even when it is administered for a prolonged duration (more than four weeks). More studies will be needed to further clarify the correct use of this old antibiotic in the 21The author(s) declare that they have no competing interests.MEF and AM conceived the idea; MR, IAB, KR, and SKK collected the data; all authors contributed in the writing and preparation of the manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Recently completed clinical trials have shown that certain interventions improve the outcome of the critically ill. To facilitate the implementation of these interventions, professional organizations have developed guidelines. Although the impacts of the individual evidence-based interventions have been well described, the overall impact on outcome of introducing multiple evidence-based protocols has not been well studied. The objective of this study was to determine the impact of introducing multiple evidence-based protocols on patient outcome.A retrospective, cohort study of 8,386 patients admitted to the medical intensive care unit (MICU) of an academic, tertiary medical center, from January 2000 through June 2005 was performed. Four evidence-based protocols  were introduced in the MICU between February 2002 and April 2004. We considered the time from January 2000 through January 2002 as the pre-protocol period, from February 2002 through March 2004 as the transition period and from April 2004 through June 2005 as the protocol period. We retrieved data including demographics, severity of illness as measured by the Acute Physiology and Chronic Health Evaluation (APACHE) III, MICU length of stay and hospital mortality. Student's t, Kruskal-Wallis, Mann-Whitney U, chi square and multiple logistic regression analyses were used to compare differences between groups. P-values < 0.05 were considered significant.The predicted mean mortality rates were 20.7%, 21.1% and 21.8%, with the observed mortality rates of 19.3%, 18.0% and 16.9% during the pre-protocol, transition and protocol periods, respectively. Using the pre-protocol period as a reference, the severity-adjusted risk  of dying was 0.777 (0.655 \u2013 0.922) during the protocol period (P = 0.0038). The average 28-day MICU free days improved during the protocol period compared to the pre-protocol period. The benefit was limited to sicker patients and those who stayed in the MICU longer.The introduction of multiple evidence-based protocols is associated with improved outcome in critically ill medical patients. Although the numbers of acute care hospitals and hospital beds have declined over the last two decades, the number of intensive care unit (ICU) beds has risen . DespiteAlthough there is ample evidence supporting the benefit that certain interventions improve the clinical outcome of the critically ill, including length of stay, duration of mechanical ventilation, and mortality, there are barriers to translating the evidence into clinical practice -10. To oAlthough the impacts of the individual evidence-based interventions have been well described, the overall impact on outcome of introducing multiple evidence-based protocols has not been well studied. We have implemented multiple, evidence-based protocols in our medical intensive care unit (MICU) in the last 4 years. We have an Acute Physiology and Chronic Health Evaluation (APACHE) III database that is used for severity adjusted outcome measure including mortality and length of stay . We intrIn this retrospective, cohort study, we reviewed the APACHE III database of patients admitted to the MICU of Mayo Medical Center, Rochester, Minnesota. Mayo Medical Center is a tertiary, teaching institution with two hospitals comprising approximately 1,900 in-patient beds. The study was approved by the Mayo Foundation Institutional Review Board. The MICU was a closed unit throughout the study period. It had a 15-bed capacity at the beginning of the study period. The capacity was increased to 19-bed in August 2002 and 24-bed in December 2002. A critical care service team consisting of attending intensivists, critical care fellows, residents, pharmacists, nurses and respiratory therapists staffed the MICU. The non-physician staffing was consistent throughout the study period. The nurse to patient ratio was 1 to 1 or 1 to 2. Nurses, pharmacists and respiratory therapists participated during the daily rounds. All attending intensivists had internal medicine background and critical care or pulmonary/critical care subspecialty training. Fellows and internal medicine residents provided 24-hour in-house coverage. The attending intensivists did not routinely stay in-house at night but were available by phone and came to the ICU as needed. Clinically important decisions in the ICU were made by, or under the direct supervision of, the attending intensivists.Patients who did not authorize their medical records to be reviewed for research were excluded. Data retrieved included demographics, MICU admission diagnosis; Acute Physiology Score (APS), APACHE III score, and hospital predicted mortality rate based on the first MICU day values; length of MICU stay and hospital mortality. The hospital predicted mortality rates were calculated based on the admission diagnoses, APACHE III score and location prior to MICU admission, using software provided by Cerner Corporation  . SubgrouWe developed and started implementing four evidence-based protocols as follows: lung protective strategy for ALI in February 2002, activated protein C for severe sepsis/septic shock in October 2002, intravenous insulin for hyperglycemia control in September 2003 and a protocol for sedation/analgesia in April 2004. The protocols were developed with the participation of all MICU staff including physicians, nurses, respiratory therapists and pharmacists. The protocol for lung protective strategy was based on providing tidal volume not greater than 6 mL/kg ideal body weight in patients with ALI or Acute Respiratory Distress syndrome (ARDS). The activated protein C protocol was applicable for adults with severe sepsis/septic shock and multiple organ failure with no risk factor for bleeding and who opted for full resuscitation and life support. The hyperglycemia control protocol was activated if patients' glucose was > 150 mg/dL. A continuous intravenous insulin infusion was titrated to maintain blood glucose level between 100 and 119 mg/dL. There was also a protocol for the treatment of hypoglycemia based on symptoms or blood glucose level < 60 mg/dL. The sedation/analgesia protocol had two parts, one for patients anticipated to remain intubated for 48 hours or less and another protocol for those expected to remain intubated longer than 48 hours. The less than 48 hours sedations/analgesia protocol used morphine or fentanyl for analgesia and propofol or midazolam for sedation. The longer than 48-hour protocol used morphine or fentanyl for analgesia and lorazepam for sedation. Both analgesia/sedation protocols used numeric pain scales and Richmond Agitation-Sedation Scale (RASS) for titration and the continuous infusion of the opioids and sedatives was interrupted daily.  and from April 2004 through June 2005 as the protocol period . During the pre-protocol period, the implementation of evidence-based practice was based on the individual physicians' knowledge and discretion. During the development of the protocols, all MICU staff became more familiar with the available evidence. With the activation of the protocols, the elements that constitute evidence-based practice were easily available in order-set forms and were implemented with the active participation of intensivists, fellows, residents, nurses, respiratory therapists and pharmacists.The 28-day ICU free days were calculated by subtracting the actual ICU length of stay in days from 28. The 28-day ICU free day was considered 0 if a patient died before hospital discharge or stayed in the ICU for > 28 days ,22. We uWe summarized data as mean (standard deviation) (SD), median (interquartile range) (IQR) or percentages. Student's t, Kruskal-Wallis, Mann-Whitney U, and chi square tests were used to compare differences between groups. In order to determine the impact of the protocol on severity adjusted patient outcome, we created a multiple logistic regression model consisting of hospital mortality as a dependent variable and the APACHE III predicted hospital mortality rate and the three study periods as independent variables. All independent variables were entered into the model simultaneously. The pre-protocol period was considered as reference in this logistic regression model. When appropriate, the 95% confidence intervals (CI) were calculated. We considered P values < 0.05 as statistically significant. We used StatView version 5.0  and Confidence Interval Analysis version 2.0.5  computer softwares for statistical analysis. We used variable life-adjusted display (VLAD) to show the differences between the cumulative and actual deaths during the three periods of the study ,24.After exclusion of 186 patients (2.2%) who did not authorize their medical records to be reviewed for research, 8,386 patients were included in the study. Of the 8,386 patients, 7,910 (94.3%) were white and 4,346 (51.8%) were male. Their mean (SD) age was 62.3 (19.1) years. The mean APS, APACHE III score and predicted hospital mortality rates were 46.5, 60.1 and 21.1%, respectively. The number of patients included in the study was 2,677 in the pre-protocol, 3,513 in the transition and 2,196 in the protocol periods  advocates use of protocol-based bundles in order to apply the best available science into clinical practice and improve patient outcome . In the In the Acute Respiratory Distress Syndrome Network study, lung protective strategy reduced the mortality of patients with ALI from 39.8% to 31.0% . van denThe implications of reducing ICU days include reducing ICU complications and associated costs. The study by Kress and colleagues had shown reduction of ICU stay by 3.5 days using a protocol with daily interruption of sedative infusions . The 28-Our study has several limitations. Since the study was performed in a single medical center with its own unique characteristics, the findings may not be generalizable. The APACHE III database we used for the study did not include information on the rate of compliance with the protocols or evidence-based practice. We did not have the data to determine the eligibility and contraindications for each protocol. Our data also lacked the identification of the individual patients who received treatment based on the protocols. Since the four protocols were introduced at various times of the study, it is not easy to determine the effects of each protocol individually. We may not have accounted for all confounding variables although we adjusted for the severity of illness. Because of the retrospective design, our study cannot exclude the fact that unmeasured changes in patient care and unrelated to the protocols may have contributed to the improved outcome. The current study was performed over a period of 66 months. The transition period to having all the protocols available in the MICU took 24 months. Because of the long time interval it took to complete the study, we cannot avoid the potential confounding effects of frequently imperceptible changes in practice on outcome measures. Our study did not control for factors such as ICU and hospital patient volume and occupancy that may influence outcome. In the early part of our study, there were changes in the structure and staffing of the MICU. The MICU had expanded from 15 to 19 and then to 24 beds and the intensivist to bed ratio had changed from 1:15 to 1:9.5 and then to 1:15 . HoweverThe current study suggests that the introduction of several evidence-based patient care protocols is associated with improved  severity adjusted mortality in a population of critically ill adult patients admitted to a medical ICU. Using sensitivity analysis, Pronovost and colleagues have extrapolated that 167,819 lives can be saved annually by the consistent and appropriate implementation of evidence-based therapies in the intensive care unit . HoweverALI = Acute lung injury APACHE = Acute Physiology and Chronic Health EvaluationAPS = Acute Physiology ScoreARDS = Acute Respiratory Distress syndromeCI = Confidence intervalsICU = Intensive care unitIQR = Interquartile rangeMICU = Medical intensive care unitRASS = Richmond Agitation-Sedation ScaleSD = Standard deviationThe author(s) declare that they have no competing interests.BA was involved in the conception and design of the study, acquisition, analyzing and interpretation of data, writing of manuscript. OG was involved in the conception and design of the study, acquisition and interpretation of data, critical revision of the manuscript. MTK was involved in the conception and design of the study, interpretation of data, critical revision of the manuscript. EGS was involved in the conception and design of the study, interpretation of data, critical revision of the manuscript. RDH was involved in the conception and design of the study, interpretation of data, critical revision of the manuscript. SGP was involved in the conception and design of the study, interpretation of data, critical revision of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Sedation-analgesia protocol A. Describes the management of pain and agitation in patients anticipated to receive invasive mechanical ventilation for 48 hours or shorterClick here for fileSedation-analgesia protocol B. Describes the management of pain and agitation in patients anticipated to receive invasive mechanical ventilation for longer than 48 hoursClick here for file"}
+{"text": "During the 2003 severe acute respiratory syndrome (SARS) crisis, we proposed and tested a new protocol for cardiac arrest in a patient with SARS. The protocol was rapidly and effectively instituted by teamwork training using high-fidelity simulation.Phase 1 was a curriculum design of a SARS-specific cardiac arrest protocol in three steps: planning the new protocol, repeated simulations of this protocol in a classroom, and a subsequent simulation of a cardiac arrest on a hospital ward. Phase 2 was the training of 275 healthcare workers (HCWs) using the new protocol. Training involved a seminar, practice in wearing the mandatory personal protection system (PPS), and cardiac arrest simulations with subsequent debriefing.Simulation provided insights that had not been considered in earlier phases of development. For example, a single person can don a PPS worn for the SARS patient in 1 1/2 minutes. However, when multiple members of a cardiac arrest team were dressing simultaneously, the time to don the PPS increased to between 3 1/2 and 5 1/2 minutes. Errors in infection control as well as in medical management of advanced cardiac life support (ACLS) were corrected.During the SARS crisis, real-time use of a high-fidelity simulator allowed the training of 275 HCWs in 2 weeks, with debriefing and error management. HCWs were required to manage the SARS cardiac arrest wearing unfamiliar equipment and following a modified ACLS protocol. The insight gained from this experience will be valuable for future infectious disease challenges in critical care. Morbidity and Mortality Weekly Report published a description of the SARS outbreak on 21 March 2003 [Severe acute respiratory syndrome (SARS) is a newly identified atypical pneumonia that can be life threatening. Attention was drawn to the disease in February 2003 when a physician and subsequently 12 other hotel guests staying in a hotel in Hong Kong became ill [\u00ae Instruments, Kalamazoo, MI, USA).SARS created a crisis in healthcare in Toronto. The lack of literature, uncertainty about treatment, and fear of the disease caused great concern among HCWs. In late April 2003, our Critical Care Department was asked to urgently develop and implement a protocol for the management of cardiac arrest in the SARS patients. At the time there were directives from the Ontario provincial government mandating the use of a personal protection system (PPS) during the intubation of SARS patients . A PPS wThe cardiac arrest scenario was of great concern because care had to be delivered immediately. We knew from previous simulation experience that a HCW required 1 1/2 minutes to dress in the Stryker T4 . Hence, We used simulation to perfect the protocol as well as to train the cardiac arrest team. Simulation has been used to improve individual and team performances -9. It haSimulation was used to design a protocol and then to train over a two-week period all HCWs who might be involved in a SARS cardiac arrest.A modified ACLS protocol was designed and referred to as 'Code Blue Special' (CBS). We were aware that there was minimal scientific evidence, and there were no guidelines, for decisions related to having HCWs apply protective equipment that would delay time to definitive ACLS care. The Critical Care Department convened committee meetings involving experts representing the disciplines involved in the treatment of cardiac arrest . The infection control service provided consultants to the committee. An initial protocol was developed by this committee.A group of educators then assessed this protocol in a teaching area by repeated simulations. The infection control service monitored the simulations for breaches of infection control. After these simulations, discussions between educators and infection control personnel resulted in a modified protocol that was accepted by the multidisciplinary committee Figure . During Finally, the group of educators conducted a cardiac arrest simulation with a manikin  placed in a bed in an empty negative-pressure patient room on a ward. In preparation, all necessary equipment to manage a SARS cardiac arrest was placed outside the room and all HCWs that would respond to an actual SARS cardiac arrest  were present. A full arrest scenario was then simulated, including the transport of the resuscitated patient to the intensive care unit (ICU). During this simulation an educator and the director of infection control noted any flaws. Phase 1, the protocol development, took 4 days to complete.The goal of Phase 2 was to train the on-call cardiac arrest teams in CBS. We acquired a dedicated training area in the hospital consisting of five adjoining rooms with computer and Internet access. We obtained call schedules for the arrest teams and began the training with the team members who were on call during the next two days.Using our experience in Phase 1, we decided to train HCWs in groups of eight. We planned to train HCWs in the use of the PPS in groups of two, and because four educators were available daily we decided that the maximum number of HCWs for each session was eight.A two-hour training session proceeded as follows:1. All HCWs attended a PowerPoint presentation highlighting pertinent principles for the care of the SARS cardiac arrest patient. Each received a handout and had time for questions and answers. We stressed all the modifications to the standard ACLS protocol, especially relating to defibrillation, airway management and infection control.2. The group then observed two educators describing and demonstrating the dress-up and the dress-down method for the PPS.3. Next, the group was divided into two sets of four who went into separate rooms. Here individualized practice sessions were done with the trainees donning and then removing the PPS Figure . A 2:1 r4. Last, we simulated cardiac arrest scenarios. Four trainees managed one unknown ACLS scenario . We timed how long it took the first person to don the PPS. The other four trainees observed.5. After the simulation we debriefed the entire group and discussed and reinforced pertinent points. Then the four remaining HCWs managed a different simulation. The groups were chosen to mimic the arrest team, namely an anesthesia resident, ICU nurse, medical resident and respiratory therapist.6. All physicians trained with their peers concurrently on call. There were six anesthesia residents and six medical residents, but many more nurses and respiratory therapists, to be trained. Hence, once we had trained the residents, we modified the simulation for the remaining nurses and respiratory therapists. Subsequent simulations involved only basic cardiopulmonary resuscitation and intubation because hospital policy mandates defibrillation by physicians only.The first protocol required a PPS for everyone entering the patient's room as part of the arrest team. This was based on the assumption that it would take 1 1/2 minutes to don the PPS and this was felt to be an acceptable delay before providing patient care.During Phase 1 we found that dressing took longer. When four members of the arrest team were simultaneously dressing it took between 3 1/2 and 5 1/2 minutes for the fastest team member to dress, even with assistants aiding verbally and physically. This longer time seemed to be due to HCWs and assistants talking at the same time to request equipment, and HCWs reaching across each other for equipment.With the assistance of the logistics department we developed a cart for the PPS. The cart was easily portable and allowed four HCWs to access it simultaneously; it also had numbered equipment labels allowing HCWs to follow the dress-up procedure visually without memorizing the steps.To expedite dressing we put up wall posters demonstrating the dress-up procedure and we used one dressing assistant per two team members. This represented a realistic number of people probably available to help at an actual arrest. It was also an acceptable total number of people (six) that could fit around the equipment cart.Once we discovered that the time to don the PPS in a team situation was at least 3 1/2 minutes, there was concern about the delay to defibrillation. After discussion with infection control and reviewing the available literature, we determined that there was no evidence that the person defibrillating needed to don a PPS. We therefore changed the protocol so that any physician on the ward could defibrillate, even if not part of the arrest team. This physician was required to wear routine protective SARS gear: an N95 respirator, goggles, a gown and two pairs of gloves. The N95 respirator is a face mask that filters 95% of particles greater than 0.3 \u03bcm in diameter. Respirator is the terminology used by the Centers for Disease Control and Prevention (USA) and the National Institute for Occupational Safety and Health.We also proposed having all SARS patients on cardiac telemetry, so that a defibrillator could be brought into the room by the first responder. Available resources did not permit a defibrillator in each room.Although a PPS was not worn for defibrillation, we noted that if instead of applying paddles, multifunction defibrillation electrodes capable of both pacing and defibrillation  were applied to the chest, hands-off defibrillation could be accomplished. The defibrillator machine could be placed about 2 m from the patient when multifunction electrodes were used, and the buttons on the machine could be pressed for defibrillation. There was uncertainty about the mode of transmission of the SARS virus, but 1 m is approximately the distance that organisms travel by droplet spread . We reco2 detector as the initial method to confirm tracheobronchial placement of the endotracheal tube. If the end-tidal CO2 was felt to be unreliable owing to low cardiac output we allowed a second person to place the stethoscope earpieces under direct vision. We did not use an esophageal detector device because of infection concerns with applying negative pressure to the airway of a SARS patient.A problem we encountered previously with the PPS was the risk of dislodging the PPS helmet when a stethoscope was placed in the ears . To miniWe also noted ergonomic limitations when wearing the PPS such as the following: claustrophobia; an inability to balance when removing equipment, which increased the risk of self contamination; the need to perform shorter periods of cardiopulmonary resuscitation to avoid heat fatigue; and the need to have an easy-to-follow poster of degowning placed on the wall to avoid making errors during the degowning process.In situ intravenous access was added to the protocol to expedite drug delivery. No drugs were permitted via the endotracheal tube.Positive-pressure ventilation was permitted only by HCWs wearing the PPS. To minimize the exposure of HCWs to the SARS virus, patients received a neuromuscular blocker before intubation. During Phase 1 we noted that once the arrest team dressed in the PPS entered the room and began resuscitation, the initial HCWs in the room without a PPS were at risk. HCWs without a PPS were therefore instructed to position themselves at least 2 m from the patient during positive-pressure airway manipulation.During the simulations it became apparent that many seemingly simple actions during removal of the PPS were more complicated than expected and had been inadequately described. An example was the difficulty in removing the contaminated outer pair of gloves without contaminating the clean inner pair of gloves. Most instructions merely instruct one to 'remove the gloves'. The education and infection control team simulated every step of the dressing and undressing to ensure safety and clarity, and then scripted and photographed the process.The usual cardiac arrest team at the time had ward nurses assisting the arrest team. This was changed during Phase 1 because it would have required training too many ward nurses in the application of the required PPS. Instead, ward nurses were trained as dressing assistants.When the cardiac arrest was simulated in a negative-pressure room on the ward, we noted that there were items in the room that could not be disinfected, such as cork bulletin boards. Subsequently, we went to every negative-pressure room in the hospital to ensure infection control safety.During the planning for the simulation on the ward, we realized that the CBS protocol lacked a scripted transport policy for moving the resuscitated patient from the ward to the ICU. This policy was immediately developed in conjunction with the infection control, housekeeping and security services.We trained 275 HCWs over a two-week period. Training sessions were held on Monday to Friday. All physicians were successfully trained in teams that mirrored their on-call schedule. The largest group to train was the 225 ICU nurses. It was difficult to free eight nurses for two hours because of concurrent patient care obligations and because we could not run sessions during ICU breaks. We ran three sessions during the day shift (07:30 to 19:30) and one session during the evening shift (20:00 to 22:00).The program was evaluated by the trainees. They were asked to complete an evaluation form at the end of each session. The results are summarized in Table During each session the educators checked both individual and team performances of the trainees in the cardiac arrest protocol, as well as the infection control policy.We noted and corrected common ACLS deficiencies, for example an inability to attach the multifunction pacing/defibrillation electrodes to the defibrillator machine, or an inability to adjust the transcutaneous pacemaker settings such as the pacemaker output.Mistakes in infection control practice by HCWs were noted and corrected. Common errors noted were the inability to remove the contaminated outer pair of gloves without contaminating the clean inner pair of gloves, the inability to remove the gown without contaminating the uniform underneath, the failure to disinfect hands appropriately, and not administering neuromuscular blocking agents before intubation.The theme that needed constant attention was that removing the PPS always posed a great danger of self-contamination. Trainees were required to repeat the PPS removal until no errors in technique were noted. There were no known instances of self-contamination of HCWs in our institution. The effect on bedside practice was difficult to evaluate properly because only one cardiac arrest actually occurred in a patient suspected of having SARS.We describe the use of high-fidelity simulation to design a modified practice of cardiac arrest resuscitation for an 'at risk of contamination' situation and to train caregivers as individuals and as a team. Simulation was used to delineate flaws and omissions in a modified ACLS protocol. We used scenario-based simulation training as an educational tool for different cardiac arrest etiologies. In all, 275 HCWs were trained in this SARS-specific cardiac arrest protocol.One unexpected but crucial result was that the time to don the PPS was prolonged for a group (3 1/2 to 5 1/2 minutes) in comparison with a single HCW (1 1/2 minutes) donning the same equipment. This observation resulted in a major change to the initial protocol, namely not requiring the wearing of a PPS for defibrillation. A PPS was mandatory for any positive-pressure airway manipulation. We designed the protocol to minimize HCW contact with airway secretions.We were concerned with the possibility of human error in this scenario, especially because of the reported transmission of SARS to protected HCWs involved in the intubation of a SARS patient .We had to repeatedly reinforce our observation that although applying the PPS correctly was important, it was the undressing and removal of contaminated clothing that was even more important to prevent self-contamination. Undressing had to be done without the use of the dressing assistant, while wearing multiple layers of protective equipment. When simulation occurred in a negative-pressure patient room instead of the teaching area, we discovered unexpected infection control problems with furniture as well as our lack of a scripted transport protocol to move the patient to the ICU.Some limitations of our approach became apparent and may help in planning for future disasters. Specific logistical challenges noted during our training period included the following:1. The need for educators to have dedicated time freed from their regular duties.2. The need for a high ratio of educators to trainees, to ensure careful observation of newly learned infection control practices.3. The need for night training sessions for staff who work only night shifts.4. The limited time in a crisis situation to simulate multiple scenarios.5. The ongoing need for resources such as a dedicated training area, supplies and assistants.6. The difficulty of quickly freeing up HCWs to train when they also have patient care obligations.Because of the urgent nature of the crisis and time restraints we were unable to make a full evaluation of the effectiveness of our training. We evaluated only satisfaction with the program content, namely level one of the four levels of evaluation according to Kirkpatrick's model . AlthougThe cost of the training program was substantial, although we do not have exact totals. This would include time for the dedicated educators, costs for educational materials and costs of the non-reusable equipment. In addition we needed two assistants. One was responsible for bookings, providing handouts, keeping sign-in records and collating evaluations. A second assistant was required to restock disposable equipment  and to clean the rooms between sessions. Finally this project monopolized the high-fidelity simulator, excluding its use by others.Planning can improve crisis management for future disasters. High-fidelity simulations of infectious disease protocols can be an invaluable asset for staff and patient safety. A written protocol can be developed and simulated, and core groups of people can be trained in the protocol before the crisis occurs. Once the crisis occurs, some HCWs should be immediately transferred from their usual duties to manage the patients. The other previously trained HCWs would immediately begin arranging training sessions in a pre-identified training area.High-fidelity simulation proved to be a crucial tool in the evaluation and implementation of a new, urgently developed SARS-specific cardiac arrest protocol, as well as in the subsequent training of team members in the use of unfamiliar protective equipment. It was used to detect and correct flaws and omissions in a theoretical protocol specific to the SARS patient. We used scenario-based simulation training to prepare our HCWs to manage a cardiac arrest in a SARS patient.\u2022 \tWe found that simulation was a valuable tool for evaluating a new treatment protocol in a novel and rapidly evolving crisis.\u2022 \tWe found that scenario-based simulation training was effective, but resource intense.\u2022 \tWe found that simulation was suited to teamwork training for disaster management.\u2022 \tWe suggest that simulation be used to prepare precise protocols for future serious events.\u2022 \tWe recommend that for uncommon events, simulation be done both in the teaching area and the actual patient environment.ACLS = advanced cardiac life support; CBS = Code Blue Special; HCW = healthcare worker; ICU = intensive care unit; PPS = personal protection system; SARS = severe acute respiratory syndrome.The authors declare that they have no competing interests.SDA and SC were the lead educators in developing the cardiac arrest protocol and in arranging and delivering the simulation-based training. SDA was the lead writer of the article. SC reviewed the article. FB was involved in helping to develop the cardiac arrest protocol and was involved in the proof reading of the article. All authors read and approved the final manuscript."}
+{"text": "AIDS-associated cryptococcal meningitis has a high mortality. Fluconazole was the only systemic antifungal therapy available in our centre. From 1999\u20132001 we used low-dose fluconazole , and did not offer therapy to patients perceived to have poor prognoses. In 2001 donated fluconazole became available, allowing us to use standard doses . Antiretroviral therapy was not available during the study period.Retrospective chart review of adult patients before and after the fluconazole donation.205 patients fulfilled the inclusion criteria, 77 before and 128 after the donation. Following the donation fewer patients received no antifungal treatment , and more patients received standard-dose fluconazole . In-hospital mortality was 25%. Impaired consciousness, no antifungal treatment received and cerebrospinal fluid antigen titre > 1,000 were independent predictors of in-hospital mortality. Concomitant rifampicin did not affect in-hospital survival. Thirteen patients were referred to the tertiary referral hospital and received initial treatment with amphotericin B for a mean of 6 days \u2013 their in-hospital survival was not different from patients who received only fluconazole (p = 0.9). Kaplan-Meier analysis showed no differences in length of survival by initial treatment with standard or low doses of fluconazole (p = 0.27 log rank test); median survival was 76 and 82 days respectively.Outcome of AIDS-associated cryptococcal meningitis is similar with low or standard doses of fluconazole. The early mortality is high. Initial therapy with amphotericin B and other measures may be needed to improve outcome. AIDS-associated cryptococcal meningitis is a severe opportunistic infection with a high mortality, even in developed countries . Early mIn developing countries access to antifungal therapy is limited . In sub-In our centre we had no access to amphotericin B or flucytosine, but had access to fluconazole. Initially, because fluconazole was expensive, we selected patients with fair prognoses and we used low doses: 200 mg daily for initial therapy followed by 100 mg daily for maintenance. There was evidence for this low dose approach: fluconazole 200 mg daily for initial therapy compared to amphotericin B resulted in similar survival and time to negative cultures ; and in \u00ae, Pfizer Inc., was used throughout the study. Amphotericin B (administered at a dose of 0.7 mg/kg/day) was only available at the regional tertiary referral hospital, but following the donation referrals for amphotericin B were no longer accepted. Following discharge most patients were referred to their local primary care clinics for follow up. Antifungal therapy was provided free to patients throughout the study period. During the study period there was no public sector access to antiretroviral therapy.A retrospective chart review was conducted at GF Jooste hospital, a public sector secondary level hospital that serves a population of approximately 1.3 million people with a high HIV prevalence. All HIV-infected patients who presented with a first episode of cryptococcal meningitis between January 1999 and December 2002 were included. Donated fluconazole became available in April 2001. Low dose fluconazole  was generally used prior to the donation. Furthermore, because of the expense of fluconazole, prior to the donation those patients deemed to have a poor prognosis received no antifungal therapy and were referred for palliative care. The South African protocol for treating cryptococcal meningitis with donated fluconazole was 400 mg daily for 8 weeks followed by 200 mg daily for life. Amphotericin B for initial therapy up to two weeks was optional, but was not available in many smaller hospitals such as ours due largely to intense bed pressure, difficulty in supervising infusions and monitoring for toxicity. DiflucanPatients were identified from computerised laboratory records if one or more of the following tests of cerebrospinal fluid (CSF) was positive: India Ink stain, cryptococcal culture and cryptococcal latex agglutination test titre >1:4 . Patients were excluded if their HIV status was unknown or negative, or if they received anti-retroviral therapy. Demographic characteristics, prior AIDS , baseline clinical and laboratory data, antifungal treatment, duration of hospitalisation and outcome were extracted from the medical records. The level of consciousness at the time of admission was classified into three stages using a modified version of the classification used for tuberculous meningitis : stage 1Data were analyzed using Stata 8.0 . Bivariate associations were described using chi-square, Fisher's exact and Wilcoxon rank-sum tests as appropriate. Because we were unable to ascertain date of death for patients referred to palliative care facilities, we analysed survival as the time to the combined endpoint of date of death or transfer to the palliative care facility. Kaplan-Meier survival curves were generated and survival expressed in days from the date of diagnosis to the time of death or transfer to a palliative care facility (failures) or the date of the last visit when the patient was known to be well (censored). Survival curves were compared using the log-rank test for the equality of the survivor function across groups.The study was approved by the Research Ethics Committee, University of Cape Town.A total of 249 patients with cryptococcal meningitis were identified during the study period: 44 were excluded from the study  leaving 205 evaluated patients. Demographic, laboratory, clinical and antifungal treatment data of the patients before and during the donation programme are shown in Table + T-lymphocyte count was 34 cells/\u03bcL (range 3\u2013186), but was only performed in 30 patients.CSF pleocytosis (>3 cells/\u03bcL) was present in 69% of patients, with lymphocytic predominance in 82%. Median CSF leukocyte count was 16 cells/\u03bcL. CSF protein was elevated (>0.4 g/L) in 92% and hypoglycorrhachia (CSF glucose <2.2 mmol/L) was present in 59%. In total 12 (6%) patients had a normal CSF . CSF pressure was not recorded due to the absence of manometers. The median CD4No significant baseline differences were noted between patients who received cryptococcal meningitis treatment prior to or during the fluconazole donation programme Table . HoweverThe median duration of the first hospital stay was 5 days. Fifty-two patients 25%) died during the first hospital admission after a median of 6 days (range 1\u201397 days), and 4 patients (2%) were discharged to a palliative care facility. Of the 149 patients who left the hospital alive and were not discharged for palliative care, 110 (74%) were discharged without disability, 27 (18%) had neurological impairment, and in 12 (8%) the neurological status upon discharge was not recorded. There were no significant differences in length of hospital stay, inpatient mortality or outcome in the pre- compared with the post-donation period  or between patients who received initial amphotericin B and fluconazole at any dose (p = 0.9). Patients who received no antifungal treatment, those with lower level of consciousness, and CSF CLAT titre > 1,000 had a higher risk of the combined endpoint of in-hospital death or palliative care facility transfer in the Cox proportional hazard model Table . PatientMedian follow up for the patients discharged from hospital alive was 36 days. The median survival from time of cryptococcal meningitis diagnosis was 76 days; 10% of patients were known to be alive after six months and 3% after one year. Kaplan-Meier analysis showed no overall differences in length of survival by initial treatment with fluconazole 400 mg versus fluconazole 200 mg (p = 0.27 log rank test)  and standard-dose  fluconazole groups. We have previously reported 7-month median survival in patients surviving until hospital discharge using the low-dose fluconazole strategy in a cohort followed up at hospital-based HIV clinics . HoweverThis study has several limitations. Firstly, it is a retrospective study and comparisons between different treatment groups should be made with caution. Secondly, as noted in the discussion on initial treatment with amphotericin B, some of the groups were too small to draw meaningful conclusions. Thirdly, follow-up after hospital discharge was poor. Our centre was operating largely as an acute care hospital with very limited outpatient facilities during the study period. However, most of the analyses were done on hospital outcomes.The fluconazole donation has allowed us access to standard-dose fluconazole treatment for AIDS-associated cryptococcal meningitis. The outcome appeared no better than when we were selecting patients for low-dose fluconazole therapy, but other benefits of the fluconazole donation were not measured. The early mortality in our study was better than in other African studies. Fluconazole monotherapy may be an option in resource-poor settings with limited access to antifungal therapy.The author(s) declare that they have no competing interests.CFS drafted the manuscript and acquired the data. GAM and FAP helped to draft the manuscript and contributed to study design. CM performed the statistical analyses and helped to draft the manuscript. GM conceived the study, participated in study design, and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Accurate cost estimate and a profit and loss analysis are necessary for health care practice. We performed an actual financial analysis for an intensive care unit (ICU) of a university hospital in Japan, and tried to discuss the health care policy and resource allocation decisions that have an impact on critical intensive care.The costs were estimated by a department level activity based costing method, and the profit and loss analysis was based on a break-even point analysis. The data used included the monthly number of patients, the revenue, and the direct and indirect costs of the ICU in 2003.The results of this analysis showed that the total costs of US$ 2,678,052 of the ICU were mainly incurred due to direct costs of 88.8%. On the other hand, the actual annual total patient days in the ICU were 1,549 which resulted in revenues of US$ 2,295,044. However, it was determined that the ICU required at least 1,986 patient days within one fiscal year based on a break-even point analysis. As a result, an annual deficit of US$ 383,008 has occurred in the ICU.These methods are useful for determining the profits or losses for the ICU practice, and how to evaluate and to improve it. In this study, the results indicate that most ICUs in Japanese hospitals may not be profitable at the present time. As a result, in order to increase the income to make up for this deficit, an increase of 437 patient days in the ICU in one fiscal year is needed, and the number of patients admitted to the ICU should thus be increased without increasing the number of beds or staff members. Increasing the number of patients referred from cooperating hospitals and clinics therefore appears to be the best strategy for achieving these goals. Intensive care units (ICUs) of university hospitals and advanced medical centers are indispensable for providing critical and intensive care for patients who have either undergone major surgery or who have received emergency care. Hospitals can obtain greater revenue from national insurance by a short admission in the ICU than by admission in other hospital departments. For example, the average length of ICU stay for postoperative patients who have undergone coronary artery bypass grafting (CABG) is 3.5 \u00b1 2.5 days (average \u00b1 SD) in our hospital. The medical revenue for CABG is US$ 3,122, which is expressed in purchasing power parity (PPP) by World Health Organization (WHO) US dollars, and the revenue for postoperative admission in the ICU is US$ 888 per day. The latter is much higher than the revenue of US$ 247 per day in the case of postoperative admission in a department other than the ICU. However, the cost of ICU stay for a patient who has undergone CABG at our hospital is estimated to be US$ 1,539 per day. As a result, the patient stay in ICU after CABG is not profitable, but it remains unclear as to whether ICU in Japan is profitable or not. There have so far been no reports on the profit performance of ICUs in Japan despite an abundance of such reports from other countries [The cost estimation and a profit and loss analysis are necessary for health care practice. In this study, we performed a profit and loss analysis of the ICU of a university hospital in Japan based on an estimation of the break-even point (BEP). A BEP analysis is a cost accounting method commonly used to determine how much revenue is necessary to cover the total cost . It has We explain the terminology used in this article as noted below. First, we define \"cost\" as it is used in this paper. The cost is defined as the amount of money that our hospital must pay to perform medical services in the ICU, for example, medical material expenditures, personnel fees, as well as lighting and heating expenses ,2. The cThe Japanese health care system, which consists of the Social Health Insurance Organization and the Governmental Health Insurance Organization, covers 100% of the population. Until 2003, a fee-for-service system was used for all medical services in Japan. Seventy percent of all fees were paid by the insurance organizations and the remaining 30% were paid by the patients. The revenue of university hospitals in Japan consists of two main components: reimbursement for medical services and official support from the government . In addiIn 2003, a new reimbursement system was introduced in Japan. In this new system, the reimbursement per day is predetermined for each of the 1860 combinations of diagnoses in ICD10 and the corresponding procedures named the Diagnosis Procedure Combination (DPC) [The data on the size, utilization and patient population of the ICU at our hospital were obtained from the hospital information system. In this study, we calculated the patient days (bed occupancy days) using the \"day to day\" method. For example, if a patient was admitted at 20:00 on a Monday and discharged at 10:00 on a Wednesday, this would be counted as 2 patient days. If a patient was admitted and discharged on the same day, this would be counted as one patient day -23. The The annual revenue data for the ICU were obtained from the hospital accounting system. The total revenue can be obtained by the summation of the individual medical payments of patients treated in the ICU. The revenues of surgery and surgery-related laboratory tests, medical imaging diagnoses and medications were deducted from the total patient revenue because these revenues belong to the department in which the surgery was performed.The cost estimation for the total cost was divided into the direct cost accounting for the ICU, the indirect cost estimation from the co-medical departments of the ICU, and the hospital overhead costs for the ICU such as the building depreciation cost, lighting, water, cleaning and garbage disposal contracts, and telephone charges.The direct costs were calculated using the data obtained from the hospital accounting system. The hospital's overhead costs were allocated to the ICU corresponding to the percentage of space, the number of employees and the number of patients.Ai, i = 1,...,14, which indirectly provided services to ICU as shown in the first column of Table Xi of Ai beforehand, and set the corresponding cost drivers to each activity as shown in the second and third columns of Table Di, and the sub-volume of Di for the ICU, di, were investigated and also showed in Table Xi, Ci, is obtained by the following equation:The ABC method was used for indirect cost accounting. Using the ABC method, all events or transactions that create costs are recognized as \"activities\" and a specific cost driver, which is an index for allocating indirect costs appropriately to the cost object, corresponds to each activity. In this study, the ICU is the cost object. We identified 14 activities Ci = Xi \u00d7 di / Di.C, allocated to ICU is expressed as:Finally, the total indirect cost, We performed an annual profit and loss analysis for the ICU with a break-even point analysis ,25,26. TNiigata University Medical and Dental Hospital is a local central hospital that has 770 beds and serves the 2,470,000 people who live in Niigata Prefecture. The hospital had more than 240,000 inpatient days in 2003, and the average length of hospital stay was 20.5 days. The ICU in the hospital has six beds and 1,549 inpatient days with 417 new inpatients per year. The ICU staff includes six doctors and 17 nurses. In 2003, the average bed occupancy rate was 71% and the average length of ICU stay was 3.8 days. Table Table The costs that do not vary with the number of patient days were US$ 1,741,380 which was 65% of the total cost. These costs included that 38.1% was labor cost , 22.7% was equipment cost , and 4.2% was the sum of building depreciation, and others expenses costs. The costs that change with the change in the number of patient days were US$ 936,672, which was 35% of the total cost. These costs included that material costs of US$ 735,661 (27.5%), co-medical service cost was US$ 151,871 (5.7%) and other costs (1.8%). The actual number of patient days in the ICU was 1,549. Figure Consequently, the current losses for this fiscal year were US$ 383,008 as estimated from the total revenue and total costs .The results of this study based on a profit and loss analysis indicate that ICUs in Japanese hospitals may not be profitable at the present time and that an increase of 437 patient days per year is needed to make up for the deficit in our hospital.All Japanese national universities, including the attached hospitals, changed over to a new system called the \"national university corporation\" in April 2004 under the National University Corporation Law. The hospitals are required to carry out self-management and self-responsibility, and will no longer receive national support funding within a few years. Actually, the Japanese government has declared the intention to cut annual national support funding to university hospitals by 2% to 3% and with a goal of a complete cut-off of such funding within five years. Therefore, university hospitals must consider practical strategies to increase the number of inpatients and cut costs.Reducing the ICU total cost is difficult because of the difficulty in reducing the costs that do not vary with the number of patient days. Almost 88.3% of the total costs were the labor costs of the ICU staff, the equipment costs and the material costs . National university hospitals are obliged to provide the highest and most advanced medical services to patients who stay in the ICU. To maintain and improve the quality of patient care in the ICU, reducing the number of staff members and equipment in the ICU is not a realistic option. Moreover, the cost for inpatients who stay in the ICU of our hospital is almost the same as that in other countries ,7-9. TheIn 2003, the national university hospitals in Japan had an average bed occupancy rate of 86.4% which thus resulted in many unoccupied beds. To enhance the bed occupancy rate and shorten the length of stay of inpatients, Japanese university hospitals need to strengthen the partnership among university hospital and clinics. In 2003, our hospital had 370,000 outpatients and 240,000 inpatients (patient days), including 1,549 patients in the ICU (patient days). If the total number of patients could increase by more than 10% by increasing the number of patients referred from hospitals and clinics, then an increase of more than 25,000 inpatients (patient days) and more than 170 inpatients (patient days) for the ICU will be possible based on proportional calculations.Another strategy to increase the number of patients admitted to the ICU is to increase the number of emergency patients. At almost all Japanese university hospitals, the ICU and emergency department are managed as a single unit. With an increase in the number of emergency patients, the number of patients transferred from the emergency room to the ICU would be expected to increase. Emergency inpatients accounted for 20.4% of all inpatients who stayed in the ICU in 2003. Since the emergency outpatients increased another 75% in 2004 in comparison to that in 2003, the unoccupied beds in the ICU could therefore be most easily filled with emergency patients.We think that the results and implications of this study can be generalized to other ICUs in Japan. Actually, the ICU in our hospital is located in an average position of ICUs in Japan regarding various aspects, as shown in Table The methods of department level ABC for the cost estimation of the ICU and the profit and loss analysis based on BEP analysis are useful to understand that what is the profit or loss for the ICU practice, and how to evaluate and to improve it. In this study, the results indicate that ICUs in Japanese hospitals may not be profitable at the present time. As a result, in order to increase the income to make up for this deficit, an increase of 437 patient days in the ICU in one fiscal year is needed, and the number of patients admitted to the ICU should thus be increased without increasing the number of beds or staff members. Increasing the number of patients referred from cooperating hospitals and clinics therefore appears to be the best strategy for achieving these goals.This paper received a grant from the Institute for Health Economics and Policy and the Health Care Science Institute, which provided funding for portions of the study.No editorial control was allowed.The article contains nothing that is unlawful, libellous, or which would, if published, constitute a breach of contract or of confidence or of commitment given to secrecy.Author 1 (PC) designed the study, performed the cost and profit and loss analysis, and drafted the manuscript. Author 2 (ST) conceived of and performed the data collection with the medical data, and participated in drafting the manuscript. Author 3 (TA) performed the data collection related to the management data . Author 4 (KA) guided the analysis on hospital management and policy, and participated in designing and drafting the manuscript.All authors have all read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Medication errors in the intensive care unit (ICU) are frequent and lead to attributable patient morbidity and mortality, increased length of ICU stay and substantial extra costs. We investigated if the introduction of a computerized ICU system  reduced the incidence and severity of medication prescription errors (MPEs).A prospective trial was conducted in a paper-based unit (PB-U) versus a computerized unit (C-U) in a 22-bed ICU of a tertiary university hospital. Every medication order and medication prescription error was validated by a clinical pharmacist. The registration of different classes of MPE was done according to the National Coordinating Council for Medication Error Reporting and Prevention guidelines. An independent panel evaluated the severity of MPEs. We identified three groups: minor MPEs ; intercepted MPEs ; and serious MPEs  or ADEs, being MPEs with potential to cause, or actually causing, patient harm).P < 0.001). There were significantly less minor MPEs in the C-U than in the PB-U . Intercepted MPEs were also lower in the C-U , as well as the non-intercepted potential ADEs . There was also a reduction of ADEs . No fatal errors occurred. The most frequent drug classes involved were cardiovascular medication and antibiotics in both groups. Patients with renal failure experienced less dosing errors in the C-U versus the PB-U .The C-U and the PB-U each contained 80 patient-days, and a total of 2,510 medication prescriptions were evaluated. The clinical pharmacist identified 375 MPEs. The incidence of MPEs was significantly lower in the C-U compared with the PB-U (44/1286 (3.4%) versus 331/1224 (27.0%); The ICU computerization, including the medication order entry, resulted in a significant decrease in the occurrence and severity of medication errors in the ICU. In 1999, the Institute Of Medicine reported that 44,000 to 98,000 people annually die in US hospitals as a result of medical errors -6. AccorStudies published by the ADE Prevention Study Group indicate that prevention strategies targeting systems rather than individuals are more effective in reducing errors . ComputeIn one unit of our ICU, we implemented an ICIS with incorporated CPOE and a moderate level of CDSS. The objective of this study was to evaluate and compare the incidence and severity of medication prescribing errors (MPEs) between this CPOE unit and paper-based units.The study was conducted in a tertiary care University Hospital over a five week period . The 22-bed surgical ICU was divided into three adjacent units of 8, 6 and 8 beds.A prospective, controlled cross-sectional trial was conducted in two paper-based units ) versus one computerized unit , 10 months after implementation of the ICIS in the latter unit. Patients were randomly assigned to either of these units by an independent nurse. All units had a similar case mix of patients. Medical staff, consisting of five senior intensivists and three residents, rotated continuously over these units, usually on a one-week basis. One month after the completion of the study, the ICIS was implemented in the two other remaining units. Approval of the ethics committee was obtained; informed consent was waived.A surgical ICU-independent clinical pharmacist with experience in medication errors analyzed every medication order of randomly selected patients during this five week period and recorded every possible MPE. Physicians and nursing staff at the units were completely unaware of the ongoing study. As it was not possible to screen every patient on a daily basis because of lack of time, patients were picked with a minimal pause of one day between selections. All medication and fluid prescriptions were checked for errors in:1. Drug (brand or generic) name .2. Dosing .3. Dosage interval .4. Pharmaceutical form.5. Preparation instructions .6. Adequate drug monitoring .7. Route of administration .8. Infusion rate of continuous medication .9. Double prescriptions.10. Clinically important drug-drug interactions.11. Contra-indications to the prescribed drug.12. Known allergy to the prescribed drug.\u00ae, Waltham, MA, USA) and an interaction data bank  were used. Errors were identified within 24 hours after prescription, and further classified into different types, categories and possible causes, according to the National Coordinating Council for Medication Error Reporting and Prevention (NCC MERP) guidelines, which provide a standard language for reporting medication errors [The appropriateness of drug choice was not considered. Transcription errors in the PB-U were taken into account. The pharmacist retrieved information out of the medical and nursing file and the laboratory data. Renal function was noted for every patient and renal failure was defined as calculated creatinine clearance less than 50 ml/minute. The parameters needed to calculate the creatinine clearance were always available in both the PB-U and the C-U. In addition to the pharmacists' own professional knowledge, clinical guidelines , with full connections to monitors, ventilators, syringe pumps and also connection with the hospital information system for administrative patient data and laboratory results. The CDSS consisted of several different functionalities. There was a possibility for facilitated medication prescription by means of protocols for specific patient groups, for example, liver transplant patients or neurotrauma patients, with separate protocols for subgroups with renal failure or sedation. When choosing a drug, the most commonly used prescription with corresponding drug dose was shown, together with the different dosing schemes for renal insufficient patients  and for patients with severe liver dysfunction. All these prescriptions had a fully preconfigured template. Clinically important interactions of commonly prescribed medication appeared at the time of prescription as pop-ups. Physicians were also notified about a number of important and possibly life-threatening drug-related complications . The allergy status of the patient was shown by means of a differentially colored highlighted icon in the toolbar as well as in the general prescription window. Sophisticated CDSS in the form of real-time alerts notifying the physician to adjust drug dosages to changing organ failure was lacking.The primary outcome measure was the difference in incidence and severity of MPEs in the C-U versus the PB-U. Secondary endpoints were univariate correlations between patient characteristics  and the number of MPEs.U tests. These data are presented as median values (with 25th and 75th percentiles). Nominal data were compared by using chi-square analysis or by Fisher's exact test as appropriate. Correlations between continuous variables were calculated by the Spearman rank correlation test. All reported tests are calculated two-tailed, and P < 0.05 was predetermined to represent statistical significance. All statistical analyses were carried out with SPSS 12.0 .Nonparametric data were analyzed with the Kruskal-Wallis and Mann-Whitney During the five week study period we analyzed 160 patient-days in 90 different patients. Both the C-U and the PB-U group contained 80 patient-days. Patient characteristics are shown in Table P < 0.001). Overall, the ICIS resulted in a relative reduction of 86.7% for all types of errors associated with medication ordering. These results are shown in Table A total of 2,510 medication and fluid prescriptions were evaluated by the clinical pharmacist, comprising 1,286 in the C-U and 1,224 in the PB-U. In the C-U, 44 MPEs occurred versus 331 in the PB-U , and problems with continuous infusion prescriptions . Another example of intercepted MPE involved the wrong prescription of a tenfold overdose of a beta-blocker, where rapid intervention of the clinical pharmacist intercepted the administration of this overdose. The non-intercepted potential ADEs were mainly dosing errors or incompleteness of low molecular weight heparin prescriptions. The two ADEs that occurred in the C-U involved an antibiotic overdose (level 2) and a vasopressin infusion overdose causing cardiac ischemia (level 3.5).In the PB-U, there were many minor MPEs, mainly because of illegible writing, incomplete orders, or abbreviations. The intercepted MPEs were mostly errors of negligence  or transcription errors. The ADEs were mainly dosing errors .P < 0.001) was observed. In the PB-U, 91% of these serious MPEs were due to dosing errors, which is significantly higher than the proportion of dosing errors in the C-U .For patients with renal failure, a three-fold reduction of serious MPEs in the C-U versus the PB-U  are shown in Figure n = 25); C-U, 23% (n = 8)), cardiovascular medication ; C-U, 37% (n = 13)) and sedatives ; C-U 12.5% (n = 4)).The most common drug classes associated with intercepted and serious MPEs were antibiotics  on MPE's simultaneously in a paper-based and an already computerized ICU. Most other articles studying the impact of CPOE on MPEs have a before-after design, which induces an important bias in time ,22,30,31Our study, like others, shows that CPOE has the potential to almost completely eliminate minor MPEs ,32. The The incidence of intercepted MPEs was four times lower in the C-U than in the PB-U. A few of these errors concerned problems with trailed zeros, but most of them were double prescriptions, which were identified by the nurse or the physician. These types of errors did not occur in the PB-U, meaning they were caused by the CPOE system itself. But as these errors did not reach the patient, we choose not to assign a severity level. This is in contrast to the study of Shulman and colleagues , who ratRegarding the intercepted and serious MPEs, we observed a 67% decrease, which is similar to several other studies that reported decreases of 55% to 86% . Many paThe amount of ADEs was significantly reduced by the CPOE. The two ADEs that did occur in the C-U could not have been avoided by our current CPOE and moderate level of CDSS. Comparison between studies remains difficult because there is no consensus for medication error classification. But when we compare our results with those of Shulman and colleagues , we do fWe believe that our estimate of reducing medication errors in the ICU by implementing a CPOE is conservative. First, there could be a bias since the physicians working in the C-U as well as in the PB-U had an opportunity to learn how to prescribe a drug correctly , which can account for a lower incidence and severity of MPEs in the PB-U. Secondly, this study only investigated prescription errors, and not dispensing or administration errors. Administration errors are the second most frequent cause of medication errors, but are rarely studied in the ICU -35. ICISAs in our study, it already has been shown previously that CPOE can create new problems, such as inconsistent or duplicate orders ,36,37. CThe allergy notation was properly filled in 69% of the patients in the C-U, whereas only 2% had an allergy notation in the charts of the PB-U. The only allergy error we encountered was in the C-U in a patient whose allergy status was not noted in the ICIS, although it was clearly notified in the patient charts. The study, however, was conducted four weeks after an upgrade with installation of the allergy notification, and a recent evaluation showed a more adequate registration. Our study, however, has several limitations. First, the study took place at only one tertiary care teaching hospital. The effect of CPOE on the incidence of MPEs depends on the implemented system; therefore, our results may not be generalized to other ICU settings and other ICISs.Secondly, the absolute numbers of ADEs in both our groups (C-U and PB-U) are higher than those reported in previous trials ,38. In tAnother potential bias in this study could be that some patients were at least double screened . However, no patient was screened on two consecutive days. In the C-U, one identical non-intercepted potential ADE occurred in a patient who was screened with an interval of three days. In the PB-U, four patients had at least one completely identical medication error, with a total of eight identical MPEs. Of these errors, there was one intercepted MPE, three non-intercepted potential ADEs, and four minor MPEs. Finally, although rotating physicians and nurses were unaware of the study registration by the clinical pharmacist, we cannot exclude the possibility that some bias may have resulted from some interventions that were made by the clinical pharmacist to prevent a potentially serious or life threatening error to occur.Implementation of CPOE with a moderate level of CDSS showed a significant reduction in incidence and severity of MPEs, and significance was found through all levels of severity. However, CPOE had the highest potential to eliminate MPEs at the lowest level of severity. Furthermore, evaluation of the CPOE enabled us to identify newly introduced problems, and gave us the opportunity to take corrective actions.This study once again underscores the importance of evaluating newly installed systems, even if it is a vendor-built product. To be able to compare different studies, it would be of great benefit to have a more standardized way of error classification and detection. This would substantially simplify the discussion about whether CPOE alone, or with a varying degree of CDSS, is a more or less effective way of improving quality of care.\u2022 This is the first ICU study with a prospective cross-sectional design evaluating MPEs and ADEs in a CPOE unit versus a paper-based unit.\u2022 CPOE had the highest potential to eliminate MPEs at the lowest level of severity but also the amount of ADEs was significantly reduced.\u2022 For patients with renal failure, a three-fold reduction of serious MPEs with CPOE was observed, mainly by reducing dosing errors\u2022 By evaluation of the ICIS we identified newly introduced problems, and were able to take corrective actions.ADE = adverse drug event; CDSS = clinician decision support system; CPOE = computerized physician order entry; C-U = computerized unit; ICIS = intensive care information system; ICU = intensive care unit; MPE = medication prescribing error; NCC MERP = National Coordinating Council for Medication Error Reporting and Prevention; PB-U = paper-based unit.The authors declare that they have no competing interests.All of the authors were involved in designing the study. KC was responsible for conceiving the study, data acquisition, analysis of the data, statistical analysis and drafting of the manuscript. BC was responsible for data acquisition, analysis of the data, and drafting of the manuscript. JD was responsible for conceiving the study, statistical analysis and critical revision of the manuscript. AS, KV and HR were responsible for critical revision of the manuscript. All authors read and approved the final manuscript."}
+{"text": "The clinical and economic consequences of the emergence of multidrug-resistant Gram-negative bacteria in the intensive care unit (ICU) setting, combined with the high mortality rate among patients with nosocomial pneumonia, have stimulated a search for alternative therapeutic options to treat such infections. The use of adjunctive therapy with aerosolized colistin represents one of these. There is extensive experience with use of aerosolized colistin by patients with cystic fibrosis, but there is a lack of data regarding the use of aerosolized colistin in patients without cystic fibrosis.We conducted the present study to assess the safety and effectiveness of aerosolized colistin as an adjunct to intravenous antimicrobial therapy for treatment of Gram-negative nosocomial pneumonia. We retrospectively reviewed the medical records of patients hospitalized in a 450-bed tertiary care hospital during the period from October 2000 to January 2004, and who received aerosolized colistin as adjunctive therapy for multidrug-resistant pneumonia.Acinetobacter baumannii (in seven out of eight cases) and Pseudomonas aeruginosa (in one out of eight cases) strains. Half of the isolated pathogens were sensitive only to colistin. The daily dose of aerosolized colistin ranged from 1.5 to 6 million IU (divided into three or four doses), and the mean duration of administration was 10.5 days. Seven out of eight patients received concomitant intravenous treatment with colistin or other antimicrobial agents. The pneumonia was observed to respond to treatment in seven out of eight patients (four were cured and three improved [they were transferred to another facility]). One patient deteriorated and died from septic shock and multiple organ failure. Aerosolized colistin was well tolerated by all patients; no bronchoconstriction or chest tightness was reported.Eight patients received aerosolized colistin. All patients had been admitted to the ICU, with mean Acute Physiological and Chronic Health Evaluation II scores on the day of ICU admission and on day 1 of aerosolized colistin administration of 14.6 and 17.1, respectively. Six of the eight patients had ventilator-associated pneumonia. The responsible pathogens were Aerosolized colistin may be a beneficial adjunctive treatment in the management of nosocomial pneumonia (ventilator associated or not) due to multidrug-resistant Gram-negative bacteria. Pseudomonas aeruginosa and Acinetobacter baumannii strains, is among the most serious complications that occur in the intensive care unit (ICU) setting. Mortality, morbidity and health care costs are substantially increased by this type of infection  or leucocytosis ), and at least two of the following: new onset of purulent sputum, change in the character of sputum, increased respiratory secretions, or increased requirement for suctioning; new onset or worsening of cough, or dyspnoea or tachypnoea; rales or bronchial breath sounds; or worsening gas exchange. Pneumonia was considered to be ventilator associated (VAP) when its onset occurred 48 hours after the initiation of mechanical ventilation, and was judged not to have been incubating before the initiation of mechanical ventilation  versus 30/45 patients [66.7%], P = 0.67).Of 152 patients who received treatment with intravenous colistin for infections with multidrug-resistant Gram-negative bacteria during the period of study, 55 had received less than 72 hours of intravenous colistin and were excluded from all analyses. Medical records were not available for three patients; in addition, one patient was in the hospital during data collection. Thus, 93 patients were further analyzed. Forty-five of these patients received intravenous colistin for the treatment of nosocomial pneumonia due to Gram-negative bacteria. Survival and clinical cure rates for the infection were better, although not statistically significantly so, in patients with pneumonia who received additional aerosolized colistin than in patients who received only intravenous colistin treatment  in the nebulized form for the management of pneumonia due to Gram-negative bacteria is not new. In 1963, Pino and coworkers  reportedonchitis -23.P aeruginosa strains. Notably, studies found that nebulized colistin reduced the number of relapses of lung infections and subsequently the decline in lung function among patients with cystic fibrosis [There is extensive experience with administration of aerosolized colistin to patients with cystic fibrosis, in whom this type of treatment is used to prevent or treat lung infections with fibrosis -27.The pharmacokinetic properties and dosing strategies of aerosolized colistin are not well defined. Whether the various forms of colistin used for inhalation therapy  or the different types of nebulizing systems influence the effectiveness and safety of colistin remains to be determined -31.2 agonists before the initiation of aerosolized colistin was able to prevent the development of such side effects in the respiratory system. Another significant concern regarding the use of aerosolized colistin for the treatment of nosocomial pneumonia is dissemination of multidrug-resistant bacteria through nebulizer devices [Adverse effects of aerosolized colistin or polymyxin B are a major concern; potential adverse effects include bronchoconstriction, chest tightness and apnoea due to neuromuscular blockade. One study conducted in 58 children with cystic fibrosis who received nebulized colistin for the treatment of lung infections  reported devices ,35. HoweOur study is not without limitations. It is a small case series and is of a retrospective design. In addition, there is no control group of patients receiving treatment with only intravenous antimicrobial agents. Furthermore, some of the patients also received intravenous treatment with other antimicrobial agents, which might have influenced the outcomes.P aeruginosa and A baumannii, resistant to colistin; and an increase of infections due to Gram-positive and Gram-negative pathogens, such as Proteus and Serratia spp., inherently resistant to colistin. Consequently, there is an urgent need to restrict the use of colistin use in order to minimize these risks.Two major risks are arising from the wide use of colistin: the emergence of Gram-negative bacteria, such as P aeruginosa strains is commonly treated with aerosolized colistin alone. Randomized controlled trials studying the possible additional benefits and risks associated with use of nebulized colistin, as an adjunct to intravenous antimicrobial treatment, in patients with pneumonia due to multidrug-resistant Gram-negative bacteria are urgently needed.Inhaled colistin may be beneficial in the treatment of nosocomial pneumonia (ventilator associated or not) due to multidrug-resistant, Gram-negative bacteria. However, the severity of these infections in the ICU setting means that treatment just with aerosolized colistin is unlikely to be sufficient. This is in contrast to therapeutic strategies employed in patients with cystic fibrosis, in which initial lung colonization with \u2022 \tAerosolized administration of colistin is a promising adjunctive therapy for management of patients with pneumonia (whether ventilator associated or not) due to multiresistant Gram-negative bacteria\u2022 \tAerosolized colistin was safe in this group of patients.\u2022 \tThere is an urgent need for randomized controlled trials examining the efficacy and safety of aerosolized colistin for the management of patients with nosocomial pneumonia.ICU = intensive care unit; VAP = ventilator-associated pneumonia.The author(s) declare that they have no competing interests.AM and MEF conceived the study. SKK, ZM, KR and AMK collected data. All authors contributed to the writing and preparation of the manuscript."}
+{"text": "Clinical trials throughout the world must be evaluated by research ethics committees. No one has yet attempted to clearly quantify at the national level the activity of ethics committees and describe the characteristics of the protocols submitted. The objectives of this study were to describe 1) the workload and the activity of Research Ethics Committees in France, and 2) the characteristics of protocols approved on a nation-wide basis.Retrospective cohort of 976 protocols approved by a representative sample of 25/48 of French Research Ethics Committees in 1994. Protocols characteristics , number of revisions requested by the ethics committee before approval, time to approval and number of amendments after approval were collected for each protocol by trained research assistant using the committee's files and archives.Thirty-one percent of protocols were approved with no modifications requested in 16 days (95% CI: 14\u201317). The number of revisions requested by the committee, and amendments submitted by the investigator was on average respectively 39 (95% CI: 25\u201353) and 37 (95% CI: 27\u201346), per committee and per year. When revisions were requested, the main reasons were related to information to the patient (28%) and consent modalities (18%). Drugs were the object of research in 68% of the protocols examined. The majority of the research was national (80%) with a predominance of single-centre studies. Workload per protocol has been estimated at twelve and half hours on average for administrative support and at eleven and half hours for expertise.The estimated workload justifies specific and independent administrative and financial support for Research Ethics Committees. Clinical trials in biomedical research throughout the world must be evaluated by research ethics committees. No one has yet attempted to clearly quantify the activity of ethics committees and describe the characteristics of the protocols submitted, and only five studies-4 have dFrance was one of the first country to affirm these principles through the Huriet-S\u00e9rusclat Act of 1988 , which lA European harmonization was needed since a long time and was decided in 2001 . All EurWe describe one year of activity in a sample comprising half of the French RECs and the full characteristics of the protocols approved in these RECs during this year.All biomedical research protocols approved by a representative random sample of every other French RECs the participating committees between 01 January 1994 and 31 December 1994 were included. This year was chosen in order to allow studies to be completed and so to collect data on all amendments, information and serious adverse effects.The definitions used in this study are drawn from French law on the subject table .Ethics committee activity and protocol characteristics were gathered into four thematic groups. The first two comprised committee activities: (i) the process of approval , (ii) the modifications requested after approval and the information transmitted. The other two comprised protocol characteristics: (iii) the legal and administrative characteristics of protocols  and (iv) the proposed scientific characteristics . Research assistants in charge of data collection were trained in order to ensure homogeneous results. The questionnaires were completed using the committee's archives, and were then sent to the coordinating centre. An identification number was given to each protocol in order to ensure anonymity of the investigator.Each formal step was studied, i.e., the number of protocols approved, revisions requested by the ethics committee and amendments submitted by the investigator were studied. The number of revisions, amendments and total exchanges (defined as the sum of revisions and amendments) were calculated per approved protocol.At the REC level, each protocol is registered and assessed for approval. A list of the different tasks was established by the Lyon B REC members and administrative staff and submitted to a national panel of REC members table . The tim\u00ae. A Kaplan-Meier survival analysis [\u00ae to establish probability of approval curves based on cumulative hazard function in order to study the time between the submission of the protocol and final approval (in days). A log-rank test [As this was mainly a descriptive study, we presented frequency distributions calculated with SAS softwareanalysis  was perfank test  was thenA multiple correspondence analysis  was alsoTo compare number of revisions, amendments and time per protocol between the clusters an ANOVA was performed .A total of 25/48 RECs throughout France participated in this national study. There were 1143 declared protocols approved by these committees during the year 1994. One hundred sixty-seven had to be excluded because the inclusion criteria were not fulfilled  was, respectively, 16 days , 27 days (24\u201330) and 48 days (43\u201352). The comparison between the three groups resulted in a significant log-rank test  and the number of amendments was 37 . Thus the average number of exchanges (revisions and amendments) was 76  per year and per committee, ranging from 13 exchanges for phase I studies on drugs, through 19 for studies not investigating drugs and to 45 for studies on drugs other than phase 1.Revisions were requested for 555 (57%) protocols, but only 181 (19%) required a second revision or more. A revision could contain one or more points to be modified by the investigator. In total 1438 points for modification were cited (Table The main reasons for revisions were information to the patient (28% of responses), consent modalities (18%), inclusion criteria (8%), scientific factors (7%) and legal and administrative requirements (6%). This global ranking of reasons remained the same when considering the sub-group of protocols with only one revision and the sub-group of protocols with two revisions.th position (for protocols with one or two revisions) to the 9th position. However, requests for more specific methodological and statistical information, which were more complex to solve, were more frequently cited, rising from the 11th rank (for protocols with one or two revisions) to 7th rank.The ranking of reasons for protocols with three or more revisions showed that legal and administrative points, which were quite easy to solve, became less important, falling from the 4Over half of the investigators submitted no amendments (57%), although few protocols were not implemented and therefore could not have led to amendments. For the 416 protocols requiring at least one amendment, 875 reasons were mentioned. An average of 1.2 reasons per amendment was cited. A single reason was cited in 52% of cases.The main reasons for which protocols had to be modified after approval were a change in the number of subjects to be included (18%), a modification of inclusion criteria, a modification of the timetable and changes in examinations and treatments Table . RegardiAs information on the progress of research is not mandatory, 81% of investigators did not send any further information regarding their study. When they did it was mainly to warn of the end  of the study (30%), to declare adverse side effects (25%) and to inform the committees of the intermediate or final study results (18%).The administrative staff panel estimated that administrative tasks  required 4.5 hours per initial protocol recording, 4.5 hours per revision and 3.5 hours per amendment table . The expThese figures show, per year and per month respectively, an estimated administrative workload of 480.50/40 hours and an estimated expertise workload of 453.25/38 hours per REC .Table Sponsors were mainly pharmaceutical firms (64%). The research setting was a tertiary teaching hospital in 55% of cases; another 15% of protocols were conducted simultaneously in a tertiary teaching hospital and another type of institution. The item \"other\" included other combinations of these settings and also private offices, etc.Drug evaluation was the object of research in 68% of cases. For the research topic, the item \"other\" was cited in 8% of cases, usually for studies on genetics or vaccines, i.e., other types of clinical trials. It was also pointed out that participating RECs encountered problems in reporting expected duration since 26% of the answers on questionnaires neglected to mention this information.Reasons for revisions varied according to investigators' status  and to place of research  and reasons for amendments according to sponsor , place of research , study phase , study scope  and study duration .the scree test resulted in the selection of two axes explaining 40% of the inertia. The modalities most contributing to the construction of each axis were represented on figure The second axis separated descriptive studies sponsored by the public sector from experimental studies on drugs, mostly sponsored by private pharmaceutical firms.vs. 68% in the global population), with direct individual benefit, and phase II and phase IV studies . Cluster 2 grouped phase I drugs trials (89% vs. 17%), without direct individual benefit (97% vs. 34%), in a single centre (99% vs. 47%), lasting less than 2 months and sponsored by pharmaceutical firms. Cluster 3 also concerned drugs, but protocols were phase III studies (64% vs. 23%), with direct individual benefit, multicentre and international (42% vs. 16%) and with a randomized design (87% vs. 52%). Finally, cluster 4 grouped protocols not evaluating drugs (89% vs. 32%), the studies were descriptive (32% vs. 15%) and the sponsor was in the public sector (58% vs. 27%).This analysis yielded four clusters explaining 75% of the variance. The first cluster included protocols on drug testing (98% in this cluster We have linked the different clusters to the number of revisions and amendments table . The meaThe mean time spent per protocol (workload) was estimated in each cluster, cluster III protocols are the most time-consuming.Time spent by each cluster was obtained by multiplying number of protocols by mean time per protocol. At a national level 31% of committees' time is spent for cluster IV protocols, 28% for cluster III, 27% for cluster I and 13% for cluster II.On average, each REC studied 39 protocols, 39 revisions and 37 amendments per year, representing an annual workload of 934 hours  including neither committee meetings nor training of the members since there is too much variability across committees. Most protocols evaluated drugs (68%), were experimental (85%) and were monocentric (47%).The main advantage of our study is to have collected exhaustive information for half of French RECs over a full year . Moreover, homogeneous data collection was guaranteed by the initial training followed by research assistants. The choice of the year 1994 was based on initial studies conducted by the French Ministry of Health -16 showiMoreover the French law is broader than EC 2001/20 European Directive , since iThe major difficulties encountered concerned definitions. Although the law clearly defines what are direct approvals and revisions, some RECs evocated direct approval with revision and postponed approval without revision. However this issue is not related to our study methods but to a difficulty in understanding and putting into effect the French law. Despite their training, research assistants had difficulties to retrieve information on study design and to categorise it. Some data were often missing in the protocols, such as the expected duration.The four articles previously published collected information on only one or two ethics committees-4. TheseOne of our major results is that 46% of revisions concerned patient information and consent.The above article showed that when studies were conditionally approved, deferred or rejected, most queries also concerned the patient information leaflet (85%). But only protocols with five or more centres were assessed; consequently these protocols were not representative of all biomedical research on human beings.A French report  also gavAlmost all protocols anticipate recruitment in terms of sample size rather than in terms of study duration. We think that the first point shows compliance with good clinical practices , and theThe workload theme in terms of numbers of protocols, number of revisions and number of amendments is very relevant since French RECs' members work for free during or after their workday, and at least administrative and financial support of the REC structure is obviously needed in order to guarantee independence in each country. The results of our study are fully supported by the recommendations of the Ad Hoc advisory group on the operation of NHS Research Ethics Committees , asking Across countries, RECs may not have the same structure , the same remit  and the same functioning since the number of protocols reported in the above article was very heterogeneous in the different countries. When looked at in terms of protocols approved per year and per committee, clear differences appeared: 110 protocols approved in the multicentre REC of London , 180 proIt would be of great importance to launch a similar study in more recent years and in other settings at country level to see if things have changed, and if so in which way. Studies in different European countries will allow to collect information which would be very useful to ease harmonisation. It would also be interesting to launch a prospective recording of all tasks and time needed on a random sample of committees.Up to now, one study described the activity of one REC specialised in multicentre trials and three other studies were carried out on the fate of protocols approved by one committee but nothing was known at a nation level.Our study showed that 976 protocols were approved in one year by half of the French RECs and that median approval time ranges from 16 days (if no modification) to 48 days. Moreover, most protocols were carried out in France only (79%) for drugs evaluation (68%)Revisions before approval relates first and foremost to patient information and consent modalities (46%). For a protocol evaluation , scientific and administrative workload varied on average from 21.3 hours up to 31.7 hours according to protocols' characteristics.The author(s) declare that they have no competing interest.ED coordinated the study, managed the data, performed the statistical analysis and drafted the manuscript.VL participated in the design of the study, coordinated the study and managed the data.FC designed, submitted and coordinated the study, interpreted data, and helped to draft the manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "The medical problem list is an important part of the electronic medical record in development in our institution. To serve the functions it is designed for, the problem list has to be as accurate and timely as possible. However, the current problem list is usually incomplete and inaccurate, and is often totally unused. To alleviate this issue, we are building an environment where the problem list can be easily and effectively maintained.proposed medical problems drive the foreground application designed for management of the problem list. Within this application, the extracted problems are proposed to the physicians for addition to the official problem list.For this project, 80 medical problems were selected for their frequency of use in our future clinical field of evaluation (cardiovascular). We have developed an Automated Problem List system composed of two main components: a background and a foreground application. The background application uses Natural Language Processing (NLP) to harvest potential problem list entries from the list of 80 targeted problems detected in the multiple free-text electronic documents available in our electronic medical record. These The set of 80 targeted medical problems selected for this project covered about 5% of all possible diagnoses coded in ICD-9-CM in our study population (cardiovascular adult inpatients), but about 64% of all instances of these coded diagnoses. The system contains algorithms to detect first document sections, then sentences within these sections, and finally potential problems within the sentences. The initial evaluation of the section and sentence detection algorithms demonstrated a sensitivity and positive predictive value of 100% when detecting sections, and a sensitivity of 89% and a positive predictive value of 94% when detecting sentences.The global aim of our project is to automate the process of creating and maintaining a problem list for hospitalized patients and thereby help to guarantee the timeliness, accuracy and completeness of this information. The problem list is an important piece of the medical record as well as a central component of the problem-oriented electronic medical record in development in our institution . To serve the functions it is designed for, the problem list has to be as accurate and timely as possible. In most of our inpatient settings, we are converting to an electronic problem list from a paper-based tool. However, the current problem list, hand written on a form in the paper chart, is usually incomplete and inaccurate, and is often totally unused. While we hope that the advantages of a universally available problem list will change this behavior, as an addition incentive, we are building an environment where the problem list is easily and effectively maintained.More than three decades ago, Larry Weed proposed the Problem-Oriented Medical Record as a remedy for the complexity of the medical knowledge and clinical data, and for weaknesses in the documentation of medical care ,2. He no\u00ae) [0-9a-z%\"])\\.(?=[ \\n\\r]+[A-Z0-9])This regular expression splits text at periods preceded by one number (0\u20139), lowercase letter (a-z), or period (.), this character being followed by one number, lowercase letter, percent sign (%), or quote (\"). The period must be followed by one to many spaces or carriage returns, the latter being followed by one uppercase letter (A-Z) or number. For all twelve regular expressions used, see .A small pilot evaluation of our section and sentence detection algorithms was executed to determine their effectiveness in the clinical text documents planned for our subsequent studies. For this small evaluation, 20 documents from the test set described above were randomly selected, and sections and sentences were determined by the first author to build the reference standard. Each document was analyzed three times, in different random order, and at an interval of at least 3 days. Sections and sentences determined by the majority of the three reviews were finally used as reference standard. The section and sentence detection algorithms were then applied to these documents and results compared with the reference standard.angina, but hasn't suffered from anginal pain for more than two years\". The first mention of the problem is stated as present, and the second as absent, and it will finally be considered present, since mention of this problem in the problem list with an inactive status is desirable.After the algorithms described above split the document's text into sections and sentences, each sentence is passed to the NLP module with context information including the document type and the section descriptor. The NLP module then analyzes each sentence and extracts potential medical problems, inferring the state of the problem using this contextual information. For example, a problem found in the Family History section will be stated as absent in the patient, if not found in another section of the document. At the sentence level, priority is given to the state present if the same problem is found more than once in the sentence. Thus, if the same problem is found once absent and once present in the same sentence, it is categorized as present at this sentence level. The priority of the state present is justified by examples like \"the patient is known for At the document level, priority is given to the state absent. This means that if the same problem is found present in one sentence and absent in another, it will finally be categorized as absent in the document, unless the absent problem reference was found in the Family History section. Medical problems are often mentioned more than once in a document, and their states usually match, but examples of multiple mentions of the same problem with different states are common in documents like Discharge Summaries. A problem could be mentioned in the patient medical history, and then negated at the end of the document, when explaining that the problem was successfully treated during the hospitalization and is now absent. In this case, no mention of the problem in the problem list is desirable .The NLP module was developed in two different versions. The first version was based on the NLP application developed locally and called SymText. This application is designed to do syntactic and semantic analysis, the latter using Bayesian Networks. It is trainable for different contexts and the semantic part was adapted to accommodate the clinical documents and medical concepts necessary to identify medical problems . Training for this tool applies principally to the semantic model. Its semantics are represented as collections of Bayesian Networks representing the relationship between the words and phrases in a sentence and the concepts that they represent. Once the structure of the network is defined, the relationships among the semantic elements are captured as tables of probabilities. The Bayesian Network used by our application was made of 11 nodes and is displayed in Figure Right Bundle Branch Block was linked to Incomplete Right Bundle Branch Block, Complete Right Bundle Branch Block, and Other or unspecified Right Bundle Branch Block). All phrases corresponding to the 80 concepts and their subconcepts were first retrieved from the MRCONSO table (6928 phrases). We then did some filtering, removing all phrases containing brackets, angled brackets, commas, forward slashes, squared brackets, dashes, and the words NOS or unspecified \", \"Anemia, aplastic\", etc.). After removal of duplicates, the final table contained 4570 phrases.To capture these relationships, training cases are needed. We created those training cases using a semi-automated technique depicted in Figure Once this algorithm had selected target sentences, each sentence and its corresponding medical problem, along with the document type, the section title, and the sentence text were then proposed to a human reviewer (the first author). He proceeded to extract the word or phrase expressing the state of the problem, and added the state of the problem (present or absent) to the case. A medical problem was considered present if mentioned in the text as probable or certain in the present or the past , and considered absent if negated in the text or not mentioned at all . The resulting file contained 4436 training cases. Training cases were prepared in a tab-delimited format as required by for training by Netica , the BayAfter the training, the tables in the NLP application's Bayesian Networks contained a statistical representation of the relationship between the words and phrases in the sentences and the medical problems that we had identified for extraction.The second version of the NLP module was based on MetaMap Transfer (MMTx) , the JavIn addition to detecting problems referenced in medical documents, the system formats the documents according to the CDA standard. After section and sentence detection and NLP analysis, this XML-based CDA version of the source document is created, with each medical problem embedded as an encoded Observation, and with markup of the source sentences to allow the creation of the customized view of the document when a problem list user views the source documents associated with a problem. An example of a CDA document in both its XML form and its customized and rendered HTML (Hypertext Markup Language) form is displayed in Figures To avoid displaying repeated instances of a previously recognized problem, a patient's problems that are already stored in the CDR are analyzed. If the same problem has already been previously stored, and if this problem is not of \"Family History\" type, then the medical problem found by our system will not be stored in the CDR. In all other cases, the problem will be stored with a reference to the CDA version of the source documents. This link is required to allow recovery of this source document for display to users in the problem list management application. Finally, the medical problems recognized by the system as present are stored in the CDR, along with the resulting CDA document. These are given the status of \"proposed\" to distinguish them from problems stored manually in the CDR.The problem list management application is the users' interface to the Automated Problem List system. It is the window through which end users interact with the medical problems proposed by the Automated Problem List system described above. For this project, an earlier problem list management application was rewritten to take advantage of the proposed problems. This new application for managing the problem list includes additional functionality focused on the proposed medical problems. It is designed to prompt the user to consider adding these extracted problems into the problem list.Our clinical information system's user interface, called the Clinical Desktop, offers secured access to clinical data through specialized modules like the \"Patient search\", \"Labs\", \"Medications\", or \"Problems\" module.The \"Problems\" module is our problem list management application. Features already present allow viewing, modifying, and adding medical problems in the problem list. An \"infobutton\" also allows access to medical knowledge relevant to the problem listed . FiltersFunctions added to take advantage of the proposed problems include the capability to list these problems with a new \"proposed\" status, and the provision of a link back to the source document to allow viewing of the document from which the problem was extracted. Human intervention is required to officially add a medical problem to the problem list, but for problems mentioned in the clinical documentation, addition is guided and simplified by this extended interface. To accept a problem, the user simply changes the status of the problem from \"proposed\" to \"active\" or \"inactive\", and to reject it, changes the status to \"error\". A \"source button\" has been added to each proposed problem listed, giving access to a viewer pop-up window displaying the source document with the source sentences of the medical problem highlighted in red Figure . The useObservation elements with the corresponding content elements in CDA documents. This preliminary phase therefore uses some more advanced XML manipulation features provided by a library for working with XML and XSLT on the Java\u2122 platform. It searches the CDA document for Observation elements with codes corresponding to the medical problem's code. If Observation element(s) are found, corresponding content elements in the text element of the same section are searched and their tag name changed from content to bold when found, as described in Figure Preliminary processing of the XML document is required due to some missing features in XSLT, the most striking being an inability to change variables' values after their declaration and instantiation. This latter feature is needed to link the encoded To estimate the proportion of coded diagnoses covered by our set of 80 targeted problems, all unique ICD-9-CM codes assigned to cardiovascular adult inpatients during 2003 in our institution  were retrieved. A total of 1531 different codes had been used. Our set of 80 targeted problems therefore covered only 5.2% of all possible codes. In the data set we found a total of 24160 ICD-9-CM code instances, and 15449 of them corresponded to one of our 80 targeted problems. Our set of problems therefore covered 63.9% of all code instances.A small pilot evaluation of the section and sentence detection algorithms showed good performance. The section detection algorithm reached a sensitivity of 1.00 and a positive predictive value of 1.00. It detected all 146 sections present in the 20 randomly selected documents from our dataset, and detected only those 146 sections. With the sentence detection algorithm, a sensitivity of 0.889  and a positive predictive value of 0.946 (0.907-0.985) were measured. 687 out of 731 sentences present in the test set of 20 documents were detected. Of those 687 sentences, 662 were correctly detected (i.e. true positives), and 25 were false positives.The development and functions of the Automated Problem List system have been reported in this paper, along with results of two preliminary evaluations: The coverage of the set of 80 targeted problems was only about 5% of all possible diagnoses coded in ICD-9-CM, but about 64% of all instances of these coded diagnoses. The section and sentence detection algorithms performed well, with a sensitivity and positive predictive value of 100% when detecting sections, and a sensitivity of 89% and a positive predictive value of 94% when detecting sentences.Some potential issues concerning this system have to be discussed. A key issue is the level of use of the problem list. Our goal is a system that eases the effort of maintaining an accurate, up-to-date problem list. However, our system can only be beneficial if users feel a commitment to maintain a problem list.In the environment represented by the electronic medical record, there are much improved incentives to do so. Electronic implementation helps to mitigate key reasons for a lack of proper problem list maintenance. With the list available in one place, the EMR, rather than independently in each of the sites where the patient receives care, there is a significant incentive to both maintain and consult this document.Another issue could be insufficient accuracy in the NLP portion of our system, with deficient sensitivity, positive predictive value, or speed. A sufficient sensitivity is required to significantly improve the quality of the problem list. We are seeking a sensitivity higher than 80%. A sufficient positive predictive value is also desirable to avoid overloading the collection of proposed problems with false positives. Those incorrectly proposed medical problems could make the use of the APL slower by requiring the user to reject an excessive number of incorrectly proposed problems. We are seeking a positive predictive value higher than 60%.A last issue, which might effect our evaluation of this system, would be speed with which proposed medical problems are returned from the target documents. Our focus here is on the speed of the NLP module. This is clearly the slowest part of our system, requiring heavy computing power to allow deep text analysis. The evaluation of the NLP module will allow us to select the version to use (i.e. based on SymText or on MMTx/NegEx) for subsequent use and evaluation in a clinical setting. We will continue to study the issues of speed, along with scalability and accuracy as we evolve the system to manage problems beyond the cardiovascular domain.In the future, the NLP module will be evaluated first, and, if satisfactory, the system described above will be evaluated in a clinical setting to determine its effectiveness in guaranteeing the accuracy, completeness, and timeliness of the medical problem list. We expect an increased proportion of correct problems, a reduced proportion of incorrect problems, and a reduced time between problem identification and addition to the problem list.Coverage of instances of problem mentions in the free-text medical documents by the set of UMLS-derived phrases described in the Methods section could affect the accuracy of the NLP module, but this will not be evaluated in the future. We will focus on the bottom line question: performance of the NLP module when detecting medical problems.The proposed Automated Problem List will be beneficial for many reasons: A better problem list should improve patient outcomes and reduce costs by reducing omissions and delays, improving the organization of care, and reducing adverse events. It will enhance decision-support for applications that require knowledge of patient medical problems to function optimally. A timely and accurate problem list should improve patient safety, an important and timely issue that has received substantial attention since the 1999 Institute of Medicine report .We have developed an Automated Problem List management system using NLP to extract potential medical problems from free-text documents in a patient's EMR. This system's goal is to improve the problem list's quality by increasing its completeness, accuracy and timeliness. By encouraging the use of a problem list of good quality, this system could potentially improve patient outcomes and security, improve the organization of care, reduce costs, and reduce adverse events.The medical problem list figures prominently in our plans for computerized physician order entry and medical documentation in the new Clinical Information System (HELP2) currently under development at IHC. A well-maintained list will significantly enhance HELP2's applications. We believe that this clinical tool, which has been taught in medical schools and used sporadically in medical practice for decades, will become a key component for managing clinical information in systems that are developed to provide a longitudinal view of the health record.The author(s) declare that they have no competing interests.SM has conceived the Automated Problem List, developed it with the help of some acknowledged programmers, and implemented it. SM also has drafted this manuscript. PJH has proposed the general design and aim of the project, has guided its development and implementation, and has critically revised this manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Sentence detection algorithm regular expressions Text file listing all regular expressions used for sentence detection.Click here for fileXSLT stylesheet Stylesheet used to transform the CDA version of the medical problem source document into its customized HTML version for viewing in the problem list management application.Click here for file"}
+{"text": "The increasing problem of infections due to multidrug-resistant Gram-negative bacteria has led to re-use of polymyxins in several countries. However, there are already clinical isolates of Gram-negative bacteria that are resistant to all available antibiotics, including polymyxins.We present a case series of patients with infections due to pathogens resistant to all antimicrobial agents tested, including polymyxins. An isolate was defined as pandrug-resistant (PDR) if it exhibited resistance to all 7 anti-pseudomonal antimicrobial agents, i.e. antipseudomonal penicillins, cephalosporins, carbapenems, monobactams, quinolones, aminoglycosides, and polymyxins.Pseudomonas aeruginosa or Klebsiella pneumoniae was observed in 4 out of 6 patients with combination of colistin and beta lactam antibiotics.Clinical cure of the infection due to pandrug-resistant (PDR) Gram-negative bacteria, namely Colistin, in combination with beta lactam antibiotics, may be a useful agent for the management of pandrug-resistant Gram-negative bacterial infections. The re-use of polymyxins, an old class of antibiotics, should be done with caution in an attempt to delay the rate of development of pandrug-resistant Gram-negative bacterial infections. Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. Colistin was used for about two decades after its discovery in 1950, but the reported nephrotoxicity and neurotoxicity led to gradual decrease of its use. However, the antibiotic recently regained some popularity in several countries as a salvage antimicrobial agent against bacteria resistant to all other classes of antibiotics. Fortunately, several studies during the last five years did not verify the high rates of toxicity of the medication reported in the old literature , and at least two of the following: new onset of purulent sputum or change in character of sputum, increased respiratory secretions or increased suctioning requirements, new onset or worsening of cough or dyspnea or tachypnea, rales or bronchial breath sounds, or worsening gas exchange [The definition of the site of the infection and of the responsible pathogen were based on the clinical symptoms and signs of individual patients, imaging findings, and on the isolation of a possible pathogen from evaluable clinical specimens. Specifically, diagnosis of pneumonia required two or more serial chest radiographs with at least one of the following: new or progressive and persistent infiltrate, consolidation, cavitation, or pleural effusion. In addition, patients must have had fever >38\u00b0C with no other recognized cause, or abnormal white blood cell count [leukopenia (< 4000 WBC/mmexchange .and at least one of the following: a) common skin contaminant  grown from two or more blood cultures drawn on separate occasions or b) common skin contaminant  grown from at least one blood culture from a patient with an intravascular line and physician- instituted antimicrobial therapy [Bacteremia required either growth of a recognized pathogen from one or more blood specimen cultures or at least one of the following signs or symptoms: fever (>38\u00b0C), chills, or hypotension  therapy .Infections at other body sites or fluids, such as urinary tract infections, meningitis, and central venous catheter-related infections were defined based on guidelines from the Centers for Disease Control and Prevention .Identification of all causative microorganisms was performed by classic microbiologic methods. Susceptibility testing was performed both by the disk diffusion method and according to an automated broth microdilution method . The breakpoints were those defined by the Clinical and Laboratory Standards Institute (CLSI) . The MIC\u00ae, Kent, UK). One milligram of the colistin used is approximately equal to 12,500 IU. Other data related to the hospitalization of these patients are summarized below.Seven patients were identified to have infection due to pandrug-resistant Gram-negative bacteria during the study periods. The isolated organisms were found to be resistant to the following antibiotics; amikacin, aztreonam, cefepime, ceftazidime, ciprofloxacin, gentamicin, imipenem, meropenem, netilmicin, pefloxacin, piperacillin/tazobactam, ticarcillin/clavulanic acid, trimethoprim/sulfamethoxazole, cefpirome, colistin, and isepamicin. The clinical features of patients, including the isolated pandrug-resistant strains, the antimicrobial agents used for the management of the infections, as well as their outcome, are shown in the table. The colistin formulation that was administered to the patients was sodium colistimethate  was isolated from his bronchial secretions. The infection was gradually controlled and, finally, the patient was discharged from the hospital in a good condition.A 47-year-old male was transferred from another ICU due to multiple chest injuries after a car accident, respiratory tract infection, and ICU polyneuropathy. Based on clinical and microbiological evidence, therapy for severe pneumonia was initiated on the 2th day of ICU stay, grew a MDR Pseudomonas aeruginosa strain  and ceftriaxone was substituted with piperacillin/tazobactam and meropenem. Due to deterioration of her clinical condition several combinations of antimicrobial agents were subsequently given, including ciprofloxacin for 19 days, chloramphenicol for 11 days, colistin for 23 days, and gentamicin for 6 days. On the 62nd day of the ICU stay, a PDR Pseudomonas aeruginosa strain was isolated from a cerebrospinal fluid specimen. Until the isolation of the PDR strain, repeated CSF specimen cultures grew MDR Pseudomonas aeruginosa. A gradual change in the Pseudomonas susceptibility was observed from multidrug-resistance to pandrug-resistance. The patient died on the 66th day of hospitalisation due to Pseudomonas meningitis.A 47-year-old female was admitted to the ICU due to possible meningitis. She had been operated for meningioma and had a ventriculo-peritoneal shunt. The shunt was removed at the day of admission with simultaneous insertion of external cerebrospinal fluid drainage. The initial cultures of CSF specimens were negative. The patient was treated with intravenous ceftriaxone and vancomycin. A CSF specimen, taken on the 24nd and 3rd cervical vertebrae and concomitant acute respiratory failure type I. Pneumonia was diagnosed clinically and confirmed microbiologically. Staphylococcus aureus and COS Acinetobacter baumannii were repeatedly isolated from bronchial secretions during the second and the third week of his hospitalization, respectively. Different combinations of antibiotics were provided, due to the persistence of severe pneumonia. The antibiotics were vancomycin, broad-spectrum cephalosporins, clindamycin, piperacillin/tazobactam, aminoglycosides, quinolones, meropenem and colistin . On the 34th day of ICU stay PDR Pseudomonas aeruginosa grew from his bronchial secretions. At that time, he was receiving colistin, meropenem, ofloxacin and gentamicin. He continued to receive the same antimicrobial combination. PDR Pseudomonas aeruginosa was not isolated again from subsequent cultures of bronchial secretions. Instead, Acinetobacter baumannii grew again from bronchial secretions on the 35th day of hospitalization and in subsequent cultures by the 45th day of ICU stay. Meanwhile a gradual improvement of the infection was noted. The patient was transferred to a specialized orthopedic center after 78 days of ICU stay, in good general condition, without evidence of pneumonia or other infection.An 18-year-old male was admitted to the ICU with fractures of the 2Pseudomonas aeruginosa and acute respiratory distress syndrome (ARDS). During the last trimester, she had been hospitalized twice for episodes of UTIs. Treatment with intravenous colistin and meropenem was initiated on ICU admission and 15 days later urine specimen cultures were negative. However, due to persistence of fever the treatment was continued. Vancomycin was added to the regimen after isolation of Enterococcus faecium from cultures of the tip of the central venous catheter (CVC) on the 8th day of ICU stay. On the 33rd day, PDR Klebsiella pneumoniae was isolated from the tip of the CVC. Due to unavailability of antimicrobial agents with in vitro effect against the pathogen, no change in the antimicrobial regimen was performed. Her clinical condition was complicated on the 37th day with lower respiratory tract infection and bacteremia due to COS Pseudomonas aeruginosa, for which a short course of gentamicin was added to the treatment. From the tip of the CVC, MDR Klebsiella pneumoniae resistant to all tested drugs (not tested for colistin) was isolated again on the 48th day, and COS Klebsiella pneumoniae on the 63rd day of ICU stay. Her clinical condition improved gradually, although COS Pseudomonas aeruginosa kept being isolated in cultures of bronchial secretions up to the 78th day of ICU stay. She was transferred in good condition to another hospital for continuation of her care, without fever or other clinical and laboratory evidence of infection, after 85 days of ICU stay.A 72-year-old female was admitted to the ICU with a complicated, severe urinary tract infection (UTI) due to COS th day of ICU stay the patient was re-operated for infection of the thoracotomy with pus collection, which required drainage. On the 52nd day PDR Klebsiella pneumoniae was isolated from bronchial secretions. He had already received 31 days of colistin treatment, combined with ampicillin/sulbactam (for 22 days) and trimethoprim/sulphamethoxazole (for 8 days), all given for treatment of polymicrobial pneumonia caused by MDR strains of Acinetobacter baumannii and Klebsiella pneumoniae and bacteremia by MDR Klebsiella pneumoniae strain. By the time of isolation of the PDR microorganism the patient's condition was steadily deteriorating; eventually irreversible septic shock and multiple organ failure led to his death on the 68th day of ICU stay.A 56-year-old male was admitted to the ICU after elective operation for a thoraco-abdominal aortic aneurysm and intra-operative hemorrhage. On the 24Pseudomonas aeruginosa. The patient had a history of rupture of an aneurysm of the circle of Willis that was surgically managed. During his prolonged hospitalization, episodes of obstructive hydrocephalus and CNS infection complicated his clinical condition, which necessitated admission to the ICU. On the 32nd day of ICU stay, PDR Pseudomonas aeruginosa was isolated from a urine specimen. By that time, he had received several combinations of antimicrobial agents, including 6 days of treatment with colistin, for lower respiratory tract infection with COS Pseudomonas aeruginosa. After isolation of the PDR pathogen he received combination treatment with colistin and ceftazidime leading to clinical cure of the UTI. The clinical condition of the patient gradually improved and he became afebrile. However, the patient's respiratory tract remained constantly colonized with MDR and COS Pseudomonas aeruginosa strains. He was transferred to the neurosurgical ward after 134 days of ICU stay, in improved condition. Thirty days later, pneumonia relapsed and progressed into septic shock and multiple organ failure, and subsequent death.A 35-year-old male was transferred to our hospital from another medical center with infection of the lower respiratory tract and UTI due to COS Pseudomonas aeruginosa strain. Treatment with intravenous piperacillin/tazobactam was initiated. A quick improvement of the cellulitis was noted. The therapeutic regimen was not changed when a result for isolation of a PDR Pseudomonas aeruginosa strain from the blood cultures became available. The patient was discharged to home after 10 days of treatment, with the cellulitis cured. In addition, subsequent blood cultures did not reveal any isolates.An 82-year-old male was admitted in the nephrology clinic due to cellulitis of the right lower limb. The patient had a history of chronic renal failure and a recent episode of bronchiolitis obliterans organizing pneumonia (BOOP). Blood specimen cultures performed on admission and 24 hours later, grew both PDR We present the first case series that focuses on clinical information and outcome of infections due to pandrug-resistant Gram-negative bacteria. Our study shows that the isolation of a pandrug-resistant Gram-negative rod from clinical specimens does not necessarily mean a bad outcome. This may be explained by several reasons. First, the achieved concentration of several antimicrobial agents in some body fluids, including the urine may exceed the minimal inhibitory concentration of the isolated pathogen, even if the in vitro susceptibility result would place the isolate in the resistant category. Second, infections, even severe, sometimes are self-limited without the use of antimicrobial agents, as the pre-antibiotic era taught us. Third, pandrug-resistant bacteria may be colonizers of the respiratory tract in patients receiving several classes of antimicrobial agents for a long period of time, while the real pathogen may not be isolated. Fourth, it has been shown that occasionally MDR organisms may exhibit decreased virulence compared to other more sensitive organisms of the same species.In addition, some of our patients received combinations of colistin with antimicrobial agents such as carbapenems, third generation cephalosporins, and quinolones. This fact represents an important practical observation of our study. Specifically, 4 patients achieved complete cure from the pandrug-resistant Gram-negative bacterial infection, with combinations of colistin with other antimicrobial agents, with which may have a synergistic effect. Previous experimental and clinical studies showed promising results regarding the synergistic effect of colistin with antibiotics from other classes such as \u03b2-lactams , aminoglycosides , sulfonamides (trimethoprim/sulfamethoxazole), rifamycins (rifampicin), and quinolones (ciprofloxacin) against MDR Gram-negative bacteria -17. HowePseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii exhibited a greater than 98% susceptibility to colistin or polymyxin B. However, reports about clinical parameters and outcome of infections due to colistin-resistant Gram-negative bacteria are rare in the literature. A search of PubMed and Current Contents databases revealed only 2 such reports. Both of them referred to cystic fibrosis patients [Pseudomonas aeruginosa chronic bronchial infection developed resistance to colistin. In the second study, colistin resistant Pseudomonas aeruginosa was found in 5 out of 150 children with cystic fibrosis over a 5-year period of follow up. Our study confirms the rarity in the incidence of such infections since in a study period of several years we recorded only seven such infections in two hospitals.Colistin resistance among Gram-negative organisms has been reported up today in some in vitro studies, as well as in surveillance studies for antimicrobial resistance ,8,18. Inpatients ,6. In thPseudomonas aeruginosa strains is that the outer membrane protein OprH blocks the self-promoted uptake pathway of polymyxins by replacing Mg2+ on the LPS molecule. Thus, the overexpression of OprH caused by mutation or as a result of adaptation to an Mg2+-deficient medium can be associated with resistance to polymyxins [Gram-negative bacteria can develop resistance to polymyxins through mutation or adaptation mechanisms . Mutatiolymyxins ,23.Pseudomonas aeruginosa strains have suggested that cell surface changes are related with the development of resistance. These include alterations of the outer membrane of the bacteria cell . Specifically, the absence of 2-hydroxylaurate, the presence of 4-aminoarabinose, and increase in the palmitate content of lipid A has been associated with resistance of Pseudomonas aeruginosa to polymyxins [Salmonella spp. have shown that the two component systems PhoP-PhoQ and PmrA-PmrB regulate polymyxin resistance. The transcription of PmrA-PmrB is activated by the PhoP-PhoQ or by growth in mild acidic conditions (pH = 5.8) and micromolar concentrations of Mg2+ (10 \u03bcM). The products of PmrA-PmrB activated genes are responsible for the modifications on the LPS molecules of Gram-negative bacteria. Consequently, the affinity of polymyxins to the bacterial cell surface is reduced [Studies on polymyxin-resistant lymyxins ,24. In aYersinia spp. demonstrated that an efflux pump/potassium antiporter system may be associated with the mediation of resistance to cationic antimicrobial peptides in general, including polymyxin B [Bacillus polymyxa subsp.colistinus produces colistinase that inactivates colistin [In addition, a recent study in ymyxin B . Althougcolistin .Guidelines from Clinical and Laboratory Standards Institute (CLSI) the about the in vitro determination of the minimum inhibitory concentrations (MICs) of different microorganisms to colistin using broth dilution and agar dilution techniques were established in 1970. However, due to the rare use of colistin in the United States and most countries, the CLSI guidelines were not modified after 1981 and were withdrawn in 2000. The re-use of polymyxins during the last years will make necessary the re-evaluation of the susceptibility breakpoints. A common method for susceptibility testing of colistin has been the disk diffusion method that uses 10 micrograms of colistin sulfate disk . Isolates are considered sensitive if the inhibition zone is \u2265 11 mm. It is important to emphasize that in clinical practice it is colistimethate sodium, not colistin sulfate that is used for intravenous administration. There is generally agreement in the results obtained from agar dilution and broth microdilution methods regarding testing of colistin sulfate . HoweverWe should acknowledge several limitations of this study. First, molecular typing was not performed thus the possibility that PDR isolates derived from the previous isolated microorganisms cannot be determined. Second, MDR or PDR bacteria were markers of the patients' condition and the effect of the infection and of the treatment on the outcome of the patients cannot be adequately evaluated.In an era of alarmingly increasing bacterial resistance, our study adds clinical information about the outcome of infections due to pandrug-resistant Gram-negative bacteria. Further work is needed to elucidate the possible in vitro synergistic effect of combinations of colistin with antibiotics from several classes and their effectiveness in clinical practice.The author(s) declare that they have no competing interests.MEF conceived the idea for the study; IAB, SKK and PA collected the data; MEF drafted the manuscript. All authors contributed in the writing and preparation of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "The frequency of drug prescription errors is high. Excluding errors in decision making, the remaining are mainly due to order ambiguity, non standard nomenclature and writing illegibility. The aim of this study is to analyse, as a part of a continuous quality improvement program, the quality of prescriptions writing for antibiotics, in an Italian University Hospital as a risk factor for prescription errors.The point prevalence survey, carried out in May 26\u201330 2008, involved 41 inpatient Units. Every parenteral or oral antibiotic prescription was analysed for legibility  and completeness . Eight doctors (residents in Hygiene and Preventive Medicine) and two pharmacists performed the survey by reviewing the clinical records of medical, surgical or intensive care section inpatients. The antibiotics drug category was chosen because its use is widespread in the setting considered.Out of 756 inpatients included in the study, 408 antibiotic prescriptions were found in 298 patients . Overall 92.7% (38/41) of the Units had at least one patient with antibiotic prescription. Legibility was in compliance with 78.9% of generic or brand names, 69.4% of doses, 80.1% of frequency of administration, whereas completeness was fulfilled for 95.6% of generic or brand names, 76.7% of doses, 83.6% of frequency of administration, 87% of routes of administration, 43.9% of dates of prescription and 33.3% of physician's signature. Overall 23.9% of prescriptions were illegible and 29.9% of prescriptions were incomplete. Legibility and completeness are higher in unusual drugs prescriptions.The Intensive Care Section performed best as far as quality of prescription writing was concerned when compared with the Medical and Surgical Sections.Nevertheless the overall illegibility and incompleteness (above 20%) are unacceptably high. Values need to be improved by enhancing the safety culture and in particular the awareness of the professionals on the consequences that a bad prescription writing can produce. Adverse drug events (ADEs), usually defined as injuries caused by the use of a drug, are a major health concern for the patient in most clinical settings.It has been estimated that ADEs account for approximately 5% of all hospital admissions, occur during 10\u201320% of hospitalisations and are responsible for 7\u20139% of hospitalisation days.[Some ADEs are caused by errors called medication errors that have similar consequences as well as lowering patient satisfaction. Chart reIf we consider harm caused by any error, medication error is the fourth most frequent category among all sentinel events collected by the Joint Commission between January 1995 and June 2008 after wrong site surgery, suicide and op/post op complication.A medication error can occur at any step of the medication use process: prescribing, transcribing, dispensing and administering. Prescribing and administering errors are the two most frequent types of medication errors, but while 48% of the former can be intercepted, only 2% of the latter are intercepted.. The repA broad definition of prescribing error includes errors in decision making and errors in prescription writing. PrescribThe aim of this study is to analyse, as a part of a continuous quality improvement program, the quality of prescription writing for antibiotics, in an Italian University Hospital. We did not analyse the appropriateness of the molecule choice, but we evaluated the completeness and legibility of information present in the clinical records as risk factors for prescription errors.The study, a point prevalence survey, took place between 26\u201330 May 2008 in a North-Eastern University hospital. All 41 inpatient Units were involved, except for ophthalmology and dermatology since their antibiotic use, when present, is mainly topical, an administration route not considered in this survey.For study purpose Units were grouped together in medical, surgical and intensive care sections, as follows:\u2022 Medical section: cardiology, haematology, infectious diseases, internal medicine, nephrology, neurology, oncology, pediatrics, post acute care, pulmonology, radiotherapy, rheumatology, pain control, nursery.\u2022 Surgical section: general surgery, maxillofacial surgery, plastic surgery, vascular surgery, heart surgery, vertebral (spine) surgery, neurosurgery, urology, gastroenterology, orthopedics and traumatology, obstetrics, gynecology, otorhinolaryngology.\u2022 Intensive Care section: anesthesia and intensive care unit, medical intensive care unit, neonatology, coronary unit.The surveyors looked at the clinical record, both nursing and medical data, of the inpatients present in ward at 8.00 AM on the day of the survey. In the hospital different formats exist for recording drug prescriptions. A healthcare worker (HCW) was in charge of giving information to the surveyors in case the clinical record was not clear enough. All antibiotic prescriptions for parenteral or oral use were included in the survey. Every Unit was surveyed in one single day. Patient's age, gender and number of admitted patients were recorded. For those patients who had any parenteral or oral antibiotic prescriptions we collected further information: antimicrobial agent, dose, date of prescription, prescriber signature, frequency of administration, route of administration, indication for given therapy or target for prophylaxis in medical records and presence of microbiological culture before therapy. When the indication was surgical prophylaxis it was specified whether it was single dose or lasting \u2264 24 h or > 24 h.Each prescription was analysed for: legibility  and completeness . Each item was classified as compliant when it was filled in and legible; if partially compliant it was classified as non compliant. The total completeness and legibility were calculated considering all the specific items.The Anatomical Therapeutic Chemical (ATC) classification system was used to class the antibiotics.The adopted definition of completeness was \"having all necessary parts or components\" while the one adopted for legibility stated \"easily readable by someone who is not familiar with the context examined\".Eight doctors (residents in Hygiene and Preventive Medicine) and 2 pharmacists performed the survey. They gave each patient an identifying code, so that the data collected were anonymous.A meeting was held before starting the survey so that the surveyors could analyse the items on the form and standardize data collection with the aim of reducing the inter operator variability. To this end pairs of surveyors were created to visit each ward, too.This observational study was performed in agreement with the local ethical committee in compliance with the Italian law.Data were processed using the software program SPSS version 12.0. The statistical analysis was performed using the Chi-Square, non parametric k-sample  and 2-sample (Mann Whitney) tests assuming as significant a p value \u2264 0.05.Out of 756 patients included in the study, 408 antibiotic prescriptions were found in 298 patients . Overall, 39.4% of patients used antibiotic and 92.7% (38/41) of the Units had at least one patient with antibiotic prescription. Table Antibiotic prescriptions from the medical section were significantly higher than prescriptions from the surgical section (p = 0.019), whereas the mean number of prescriptions per observed patient proved to be significantly higher in intensive care section (p = 0.008) and medical section (p = 0.04) compared to the surgical section.For 165/408 (40.4%) prescriptions the indication was prophylaxis both medical and preoperatory; the remaining 243/408 (59.6%) had a therapeutic indication.Of the 243 therapeutic prescriptions overall 55.6%(135/243) had a previous written request for a microbiological culture without differences among different areas.Written reasons for prescribing antibiotic were found in 58.1%(237/408) of the prescriptions, more frequently for therapeutic indication 166/237 (70%) than for prophylaxis 71/237 (30%); in the remaining cases it was necessary to request information from the medical or nursing staff.In medical section written reasons for using the antibiotic were present in 65.2%(144/221) of prescriptions, in surgical section in 46.2%(60/130) and in intensive care section in 57.9%(33/57) of the cases.Prescriptions for perioperative prophylaxis in the surgical section were for more than 24 hours in 87.7% (79/90) of the cases.To obtain a global view of legibility and completeness in the hospital antibiotic prescriptions we analysed drug name, dose and frequency and route of administration, prescription date and signature classified by section [see Additional file A more detailed analysis showed that the legibility versus illegibility of the drug's name was higher in the medical section than in the other two sections (p = 0.003). Intensive care completeness was significantly higher than the other two as far as dose (p < 0.001), frequency of administration (p = 0.035) and prescription date (p < 0.001) are concerned.In the medical and intensive care sections the route of administration was completed in a significant greater portion of the prescriptions than in the surgical section (p < 0.001 and p < 0.035 respectively). Overall 23.9% of prescriptions were illegible and 29.9% of prescriptions were incomplete.The distribution of legibility and completeness by antibiotic category is shown in additional file The study highlights the need to pay attention to antibiotic prescription writing: in fact 1 in 4 prescriptions were not fully completed or were illegible. We think this is a field that could be improved, particularly for some items, like dosage legibility prescription, date and physician's signature.We found a widespread use of antibiotics: almost all Units (92.7%) on the day of the survey had at least one patient with an antibiotic prescription.This is not unusual in acute care hospitals as reported in other studies.,12 Such Our study focused attention on formal quality characteristics that is part of the medication error risk. Certainly this risk should be evaluated hospital by hospital and, eventually in each department or Unit, possibly adopting proactive methods like the Health Failure Mode Effect Analysis (HFMEA).Nevertheless, we think that data presented in this paper can serve to increase health care workers' awareness of drug use safety, and specifically of written communication, i.e. how to fill in clinical documentation precisely.Monitoring these aspects is quite simple since based on existing clinical documentation. We performed a hospital wide survey but this is not strictly necessary in a routine program since a few cases per Unit would be enough to show attitudes and behaviours of the professional teams.If any hospital quality and safety oriented team would adopt the same methodology it should involve some professionals to read prescriptions: no specific training is required but the recommendation that professionals are different from those of the observed Units.In fact doctors and nurses have to use this information to decide on further clinical actions: if they are not able to find or read and understand written prescriptions, they can immediately realise the risks related to patient safety.Through this methods we can detect also the use of acronyms that we know are a risk factor when different professionals do not share their meaning, or worse, they are misinterpreted..Our data show that Intensive Care Units have some characteristics we should consider:\u2022 patients with antibiotic prescriptions and mean number of prescriptions per observed patient are higher than in the other sections because of the expected severity and typology of inpatients;\u2022 they show the best performance in terms of completeness in all items, except for the signature of the physician that remains unfulfilled. These data may mirror the fact that the Intensive Care Units are selected settings where the patient safety culture is more widespread because they often have to deal with critical situations like emergencies. The diffusion of risk reduction strategies such as protocols or checklists in these settings have long been appreciated and are more frequently used in these sections.Written reasons for antibiotic prescriptions cover approximately half of the cases (58.1%), more frequently if the indication is therapeutic. Microbiological cultures are requested before starting the therapy in a percentage slightly over the half as well (55.6%). In our opinion both aspects should be improved and they must be part of a wider quality improvement program related to antibiotic use appropriateness.We found that perioperatory prophylaxis lasting > 24 h was high (87.8%) in spite of the international guidelines recommending a short or ultra short first choice prophylaxis, leaving to a limited number of selected patients the possibility to extend it over one day . This fiLegibility and completeness are higher in unusual drugs prescriptions.In general, there are consistent differences in legibility and completeness when different chemical principles are concerned but we cannot compare all these percentages, because, in the case of specific drugs (i.e. Antimycotics for systemic use), we analysed only one hospital and the performance can be influenced by a limited number of Units prescribing that chemical principle.Further important but simple directions that come out as priorities from this survey are the necessity to print prescriptions and the need to record the route, dose and frequency of administration of the drug. The lack of prescription date and doctor's signature were the most critical areas in terms of prescription completeness, both were absent in more than 50% of the prescriptions.We think that a systematic use of feedback together with the adoption of formats where spaces for prescription date, signature of the physician and route of administration are more emphasised would simplify the prescriber's task.It is reported that computerized physician order entry and computerised physician decision support, in fact, significantly reduce prescription errors improving drug safety . NeverthThis study analysed only the legibility and completeness as risk factors for prescription errors but despite this, we think it has an operative relevance and it is essential in improving quality of healthcare and reducing errors.A limit of the study is that only some of the risk factors in prescription writing can be detected through chart review, thus for instance the potential error of omission is an aspect that cannot be detected through this methodology but could be considered for further studies that involve the direct observation of the professionals.The paper addresses an important issue of antibiotic safety that reflects a global problem. In fact this is not only a problem in Italy but also in other hospitals around the world. It would be also interesting to include in a further study numerous hospitals. The magnitude of this problem could be even more severe in the less resourced countries such as Africa and Asia.The findings confirm that there is a problem that needs to be attended to and serve to sensitise stakeholders in health delivery about this issue.The survey confirms the extensive use of antibiotics in an acute care hospital.Written reason for the use of an antibiotic is poor as is the request for microbiological culture in case of therapeutic indication.Overall legibility is good in more than three out of four cases, while completeness is poor mainly concerning the date of prescription and the signature of the physician.The feedback of the objective data to the Units is a great opportunity to improve the awareness of safety and to stress the need for accuracy in prescription writing. As the measurements are objective they can be repeated to monitor trends over time.Since several easily identified risk factors are associated with a large proportion of medication prescribing risk factors, an intervention is needed to enhance the safety culture in all settings by improving clinical documentation and through enhanced the professional awareness of potential medication errors related to bad prescription writing.The authors declare that they have no competing interests.All authors have substantially contributed to the research. Specifically CL, BS conceived the idea for the study, and PA, AL, LC, TMG, participated in its design. All authors were involved in data collection and CL, PA, AL performed the analysis and interpretation of data and the statistical analysis. CL, PA, AL, LC, QR have been involved in drafting the manuscript and BS and TMG critically revised it. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Legibility and completeness of antibiotic prescription by section (n. 408 prescriptions). the data provided represent the legibility and completeness of antibiotic prescription in the medical section, surgical section and intensive care section.Click here for fileDistribution of legibility and completeness of prescribed drugs for antibiotic category (n. 408 prescriptions). the data provided represent the legibility and completeness of antibiotic prescription related to antibiotic type.Click here for file"}
+{"text": "Despite being a critical part of improving healthcare quality, little is known about how best to move important research findings into clinical practice. To address this issue, the Department of Veterans Affairs (VA) developed the Quality Enhancement Research Initiative (QUERI), which provides a framework, a supportive structure, and resources to promote the more rapid implementation of evidence into practice.This paper uses a practical example to demonstrate the use of the six-step QUERI process, which was developed as part of QUERI and provides a systematic approach for moving along the research to practice pipeline. Specifically, we describe a series of projects using the six-step framework to illustrate how this process guided work by the Diabetes Mellitus QUERI (DM-QUERI) Center to assess and improve eye care for veterans with diabetes.Within a relatively short time, DM-QUERI identified a high-priority issue, developed evidence to support a change in the diabetes eye screening performance measure, and identified a gap in quality of care. A prototype scheduling system to address gaps in screening and follow-up also was tested as part of an implementation project. We did not succeed in developing a fully functional pro-active scheduling system. This work did, however, provide important information to help us further understand patients' risk status, gaps in follow-up at participating eye clinics, specific considerations for additional implementation work in the area of proactive scheduling, and contributed to a change in the prevailing diabetes eye care performance measure.Work by DM-QUERI to promote changes in the delivery of eye care services for veterans with diabetes demonstrates the value of the QUERI process in facilitating the more rapid implementation of evidence into practice. However, our experience with using the QUERI process also highlights certain challenges, including those related to the hybrid nature of the research-operations partnership as a mechanism for promoting rapid, system-wide implementation of important research findings. In addition, this paper suggests a number of important considerations for future implementation work, both in the area of pro-active scheduling interventions, as well as for implementation science in general. The need to more rapidly move important research findings into clinical practice is recognized as a critical part of closing the quality chasm ,2. OftenSeries of articles documenting implementation science frameworks and approaches developed by the U.S. Department of Veterans Affairs (VA) Quality Enhancement Research Initiative. QUERI is briefly outlined in Table Series' introductory article [QUERI Series. The Diabetes Mellitis QUERI (DM-QUERI) is one of the current QUERI Centers, and is one of the original eight Centers established in 1998 [This article is one in a  article  highligh in 1998 ,9. Type  in 1998 -13. Amon in 1998 ,15. Redu in 1998 -19In this paper we illustrate the use of the QUERI six-step process Table  as a fraAlthough work by the diabetes QUERI is multi-faceted, preventing diabetes-related visual loss is a specified area of concern. As depicted in the QUERI six-step process, implementation is part of a continuum or pipeline that progresses from identifying high priority conditions/populations to determining evidence-based practices and quality gaps to designing, implementing and evaluating quality improvement programs. In the following sections, we describe a series of projects using the six QUERI steps to illustrate how this process guided work by DM-QUERI to assess and improve eye care for veterans with diabetes. We begin with an overview of the scope of the problem (QUERI Step 1) and then focus on specific projects for Steps 2\u20136, including a brief discussion of the project background, methods, results and implications, as the full results of these projects are published elsewhere ,21. GiveDiabetes is the leading cause of new cases of blindness in adults ages 20\u201374 in the U.S. . In the Evidence suggests that 90% of visual loss due to diabetic retinopathy can be prevented through optimal medical and ophthalmologic care, including early detection and laser therapy -27. TherThis research was conducted using simulation techniques (a Markov model) and a population of patients with diabetes based on data from the Third National Health and Nutrition Examination Survey (NHANES III) . The simThe study showed that risk of blindness varies by both age and a patient's level of glycemic control over the past 2\u20133 months. The patients who benefit most from annual screening and for whom it is cost-effective are those with very poor glycemic control. However, for those patients whose previous exam was normal , routineThe results of this Step 2 project, along with similar findings by other researchers ,29, provEye screening is important, but screening alone does not prevent visual loss or blindness. In fact, since FY2002 retinal screening rates for VA patients with diabetes have been greater than 70% according to performance measurement reports prepared by the VA Office of Quality and Performance. To better understand the circumstances surrounding preventable visual loss among patients with diabetes, a study was undertaken that focused specifically on the timing of retinal photocoagulation  as a key issue in preventing visual loss .Physician reviewers examined medical records from a university ophthalmologic center and two VA Medical Centers for 238 patients who had photocoagulation for proliferative diabetic retinopathy or macular edema. Based on pre-specified criteria , the revThe results of this Step 3 study identified a lack of close follow-up of those with known disease as a potentially important gap in quality of care. Moreover, these findings suggested that the prevailing performance measure, which encouraged an annual exam for all patients with diabetes, could potentially decrease true quality. Trying to screen everyone annually consumes much of the eye care clinics' limited resources, thereby making it more difficult to aggressively monitor and follow veterans at highest risk of blindness .\u00ae) [\u00ae and the VA's quality monitoring system was actively being debated. Efforts directed toward changing the current measurement policies began well before the eye care implementation project and continued throughout much of the study period, as described in more detail in the next section. Second, DM-QUERI received funding through VA's Health Services Research and Development Service's (HSR&D) service-directed project mechanism, which was specifically established for implementation studies, to support an eye care implementation project. The proposed implementation project was a small scale multi-site study (or phase 2 project as described in Table With a high-priority issue identified (QUERI Step 1), evidence to support a change in the diabetes eye screening performance measure (QUERI Step 2), and an identified gap in quality of care (QUERI Step 3), the next step was implementation. Accordingly, DM-QUERI focused on two initiatives: 1) an intensive lobbying effort to revise the existing Health Plan Employer Data and Information Set (HEDIS\u00ae)  and VA pThere are many studies of interventions to improve the management of patients with diabetes ,32. HoweTo help guide the implementation process , we emplA cornerstone of the eye care intervention was a system for automatically tracking patients based on risk status \u2013 \"Progressive Reminder and Scheduling System (PRSS)\" Figure . The PRS\u00ae Access database. Initial development of the PRSS took place at one study site (Site A) with the intent of developing similar but organizationally tailored systems at two other study sites (Sites B and C).Despite the sophistication of VA's health information technology , only apThe database was populated by identifying a cohort of patients with diabetes using encounter and prescription data obtained from national VA databases ,38. NextInformation about risk status and recommended follow-up time from the check-out forms was entered into the Access database. Based on the number of months specified by the provider, a recommended follow-up date was calculated for those patients identified as high risk. Diabetes patients who had a normal exam, and no other condition specified, were automatically assigned a two-year follow-up appointment, while those with mild disease were assigned a one-year follow-up appointment. This information could then be merged with data from the scheduling system to identify patients with high-risk eye conditions who either were not scheduled for an appointment within the recommended time-frame or who were already past the time for their recommended appointment . This information also facilitated the pro-active follow-up, by clinic staff, of those individuals at greatest risk for preventable visual loss.In addition, the PRSS database was used to identify patients with no eye appointment in the past two years. This step was not part of the original study plan but was requested by the service-directed project review committee. After discussions with VA Ophthalmology personnel and the ambulatory care service leadership at Site A, it was decided to send a letter to individuals with no identified appointment in the past two years, signed by the Associate Chief Of Staff for primary care. Along with the letter, veterans received a brief questionnaire asking them to indicate whether they received their eye care outside the VA system or at another VA facility and, if not, whether they would like to be contacted so that a visit could be scheduled.In conjunction with creating a supportive technology infrastructure, efforts to address other factors, as identified in our implementation framework  at each study site) also were underway. More than 80 eye clinic personnel including attending physicians, residents, nurses, technicians, and clerks completed a mailed survey, and approximately 45 participated in semi-structured interviews. Information collected as part of the survey and interviews focused on the perceived adequacy of clinic resources, job satisfaction, clinic goals, functional issues, and suggestions for improvement. Subsequently, this information was used to identify how the PRSS might be tailored to function at each site. It also was used to provide a platform for discussing the potential advantages or disadvantages of the proposed changes relative to the current system with key persons in the organization .\u00ae and the VA's quality monitoring/performance measurement system. Although the VA/Department of Defense (DoD) diabetes guidelines already included a risk-stratified approach for diabetes eye care based on insulin use, level of glycemic control, and risk status, the approach was difficult to implement as a performance measure. The proposed alternative measure simply advocated for every-other-year eye exams for patients at low risk, and continued, annual or more frequent exams for patients at high risk.Creating tension for change and identifying effective alternatives required becoming more actively involved in the policy arena. Not only were researchers involved by producing evidence (as described in Step 2 above), they also served as technical experts while policy discussions about changing the diabetes eye care performance measure were in progress. In particular, DM-QUERI \u2013 through research publications and direct representation on the National Diabetes Quality Improvement Alliance \u2013 strongly advocated revising the diabetes eye care quality measure used in HEDISUnderstanding context is important but not sufficient when implementing changes in a clinical setting . The resThe initial PRSS database at study site A contained a cohort of approximately 5,500 unique veterans with diabetes. From November 2004 through June 2005 more than 780 checkout forms were completed during an eye-care visit with a diabetes patient at study site A. The provider assigned risk status for these visits as shown in Table More than 2300 patients with no identified eye-care visit in the past two years were identified using the site A PRSS database. Approximately 60% (1375) completed the mailed survey that showed that 952 (69%) patients were receiving eye care services elsewhere  the revised performance measure allowing every other year eye exams for patients at low risk  was adopted by HEDISA number of meetings with administrative, clinical, and clerical personnel were conducted at all study sites throughout the study period. Revised check-out forms, which included information about risk status and recommended follow-up for patients identified as high-risk, were developed and used by eye clinic providers at two of the three study sites. The amount of support for enacting the changes needed to implement the PRSS was variable both across sites and over time. In addition, the extent to which the check-out forms were used at the two sites cannot be fully assessed due to incomplete information about the number of patients or patient visits while the forms were in use.The QUERI process provides a systematic approach for moving along the research to practice pipeline. Guided by the six-step QUERI process, DM-QUERI conducted several research projects (QUERI steps 1\u20133) that, in turn, provided the basis for an implementation project. Specifically, a high-priority issue was identified, evidence to support a change in the diabetes eye screening performance measure was developed, and a quality gap was identified. Building on information generated by these studies, we undertook an implementation project to improve eye care and prevent visual loss among VA patients with diabetes. This project involved shifting responsibility for the coordination of diabetes eye care to the eye clinic and using automated tools to facilitate less frequent screening of low-risk patients and more aggressive follow-up of veterans at higher risk. We accomplished and learned much during the course of this implementation project. However, despite devoting substantially more resources to the project than were originally budgeted, we did not succeed in developing a fully functional system. In retrospect, we now appreciate the potential value of the four phases within the QUERI implementation framework .Second, this project highlights the importance of aligning national policy with a planned change in practice, as part of creating tension for change. Our surveys and interviews suggested that many clinic staff were not entirely satisfied with current clinic operations, and key individuals at the sites agreed that the proposed changes might be beneficial for improving the delivery of eye care for patients with diabetes. Neither of these conditions was sufficient, however, to overcome the pressure exerted by the existing demands on the eye clinics, including the current performance measure, which still emphasized annual visits at the time of the project. Specifically, the research team was encouraging changes based on the evidence and an anticipated change to every-other-year exams, but due to the political nature of the negotiation process it took two years longer than expected for this change to be adopted by HEDISThird, as others have found , garneriFourth, this project suggests that in some situations to change one element you may need to change the entire system. We set out to help the eye clinic develop a proactive scheduling system for diabetes-related eye care because of a quality problem with that clinical condition. However, perhaps we should have focused on designing a more efficient, proactive scheduling system that would apply to all patients seen in the clinic, not just for veterans with diabetes. This issue began to emerge during our discussions with clinic staff, but the reasons for pursuing such a strategy are even more apparent after the fact. In particular, most everyone is interested in a more efficient and effective scheduling system, so you develop common ground even with those that may not care about diabetes eye exams, specifically. It also is easier to get people to change the general procedures for every case than it is to get them to use a new system for patients with diabetes, while using the old system for other cases. In fact, as we learned, approximately 20% of eye-care visits for people with diabetes are not related to diabetes eye disease, thus making a system for diabetic eye disease even more confusing.Moving important research findings into clinical practice to ensure the efficient and effective delivery of healthcare services is important for improving healthcare quality. This article provides an example of how the VA QUERI program, specifically the QUERI six-step process and dedicated funding support through VA/HSR&D's service-directed project mechanism, facilitated a fairly rapid progression from developing evidence to inform the eye care screening debate \u2013 to identifying quality gaps related to close follow-up of high-risk patients \u2013 to implementation of a quality improvement intervention that addresses both eye screening and follow-up for patients with diabetes. While it is not possible to know how this series of activities might have progressed without QUERI, it seems doubtful that such an integrated set of projects could have been conducted in an approximately five-year timeframe without the process and structural support of the QUERI program. Moreover, without a specified funding mechanism for implementation work, it is unlikely that our work in this area would have progressed beyond Step 3 or identifying the quality gap.The eye care implementation project was essential for the collection of important data to further characterize the risk status of veterans with diabetes who receive eye care services in the VA, and to better examine the extent to which there may be problems with close follow-up of high-risk patients. In addition, a revised diabetes eye care performance measure was adopted inside and outside VA, and we developed a prototype, proactive, risk-stratified system that can be used to support and inform future ongoing work in this area. Our experience with the eye care implementation project also has provided further insight into the implementation of scheduling interventions including issues related to time, project scope, and the importance of aligning policy with practice.However, even with the support provided by QUERI a feasible and sustainable change in the delivery of eye care services for veterans with diabetes is yet to be accomplished. In addition to the project specific issues just discussed, we would like to highlight a few other issues that affected our work and deserve special consideration for advancing the field of implementation science. While implementation research is an integral part of improving healthcare, there are certain constraints associated with the hybrid nature of this type of research and operations enterprise that need to be addressed. In particular, both funding and timeline requirements for conducting implementation studies must be sufficient and flexible enough to support the scope of the project.Funding and operational issues precluded our use of project funds to pay facility information technology staff for time they devoted to the project. Moreover, project funds were not available until the research requirement of obtaining institutional review board approval was completed, which caused significant delays. Obtaining institutional review board approval also required significant staff time and resources since the project had to be reviewed and approved at each participating site. Moreover, one of the original project sites was eventually excluded from the study and replaced with another site because of difficulties associated with trying to meet the specific human subjects' requirements at the original site. Resource constraints at the facility level also posed a substantial problem in our attempt to facilitate follow-up of patients with no visit in the past two years, which had actually been included in the project at the request of the scientific review board that approved the project for funding. Finally, despite the importance of learning by doing we also must be cognizant of the potential adverse consequences of unsuccessful implementation efforts, which for this project included not meeting the expectations or needs of both providers and patients, as we do not want to jeopardize future initiatives that could lead to significant improvements in patient care and outcomes.In conclusion, we believe that work by DM-QUERI to promote changes in the delivery of eye care services for veterans with diabetes demonstrates the promise of the QUERI process in facilitating the more rapid implementation of evidence into practice. There remain many challenges for those engaged in implementation work; however, by continuing to share our experiences we can overcome many current \"implementation blocks.\" Such active learning is already underway within the QUERI program, as evidenced by the continuing progress of QUERI with rolling out a major regional demonstration project for collaborative care, and also is likely to benefit others as the implementation imperative continues to take hold.The author(s) declare that they have no competing interests.SLK participated in the conduct of the implementation project, data acquisition and analysis, and she wrote the manuscript. SJB participated in the design and conduct of the implementation project and assisted in writing the manuscript. CEF participated in the conduct of the implementation project, data acquisition, and writing the manuscript. FM participated in the conduct of the quality gaps and implementation projects, data acquisition, and writing the manuscript. CLG participated in the conduct of the implementation project and writing the manuscript. BW participated in the conduct of the implementation project and writing the manuscript. SV conducted the cost-utility project and participated in writing the manuscript. RAH conducted the quality gaps project, participated in the conduct and design of the cost-utility and implementation projects, and participated in writing the manuscript. All authors read and approved the final manuscript."}
+{"text": "Acinetobacter baumannii are often treated with colistin, but there are few data comparing its safety and efficacy with other antimicrobials.Nosocomial infections due to multi-drug resistant A. baumannii infections in intensive care units (ICUs) at Groote Schuur hospital. Colistin was used for A. baumannii isolates which were resistant to all other available antimicrobials. In the tobramycin group, 53% of the isolates were only susceptible to tobramycin and colistin. We assessed ICU mortality, nephrotoxicity and time to the first negative culture.A retrospective cohort study of patients treated with colistin or tobramycin for 32 patients, with similar admission APACHE scores and serum creatinine, were treated with each antimicrobial. There were no significant differences between the colistin and tobramycin groups in ICU mortality (p = 0.54), nephrotoxicity (p = 0.67), change in creatinine from baseline to highest subsequent value (p = 0.11) and time to microbiological clearance (p = 0.75). The hazard ratio for total in-hospital survival in patients treated with colistin compared to tobramycin was 0.43 (95% CI 0.19 to 0.99).A. baumanii infections when the organism is resistant to other available antimicrobials.Our study suggests that colistin and tobramycin have similar risks of nephrotoxicity and are equally efficacious. Colistin is an acceptable antibiotic for the treatment of  Colistin (polymyxin E) has been available since 1959. Since the 1970's, other classes of antimicrobials, such as the aminoglycosides or carbapenems were favoured for gram-negative infections ,3.Dosing recommendations for colistin vary considerably , and, unAcinetobacter baumannii infections have become common. Multi-drug resistant organisms are defined as resistant to three or more antimicrobial classes normally used for the treatment of infections [A. baumannii infections but resistance to tobramycin occurs commonly (41% of isolates in 2006). All A. baumannii isolates at Groote Schuur Hospital remain susceptible to colistin, which is prescribed when the organism is resistant to all other available antimicrobials. Tobramycin or colistin are used as monotherapy for Acinetobacter baumannii infections at Groote Schuur Hospital. The aim of our study was to compare the safety and effectiveness of colistin versus tobramycin in ICU patients with A. baumannii infections.In the intensive care units (ICUs) at Groote Schuur Hospital, multi-drug resistant fections . The amiA. baumannii infections in Groote Schuur Hospital ICUs between January 2003 and December 2005 were identified from pharmacy records.A retrospective chart review of patients in the ICUs of Groote Schuur Hospital was conducted. Groote Schuur Hospital is an 867-bed tertiary referral centre in Cape Town, South Africa, with 56 ICU beds . All patients treated with colistin for A. baumannii infections. Two patients had received both colistin and tobramycin during their ICU stay and were excluded from the analysis. A total of 64 medical records were evaluated. Every fourth patient who received tobramycin, in order of date of admission to ICU, was included.Figure The patient's health status on admission as measured by the Acute Physiology and Chronic Health Evaluation II (APACHE) score, clinical course in ICU and other antimicrobials used were captured using a standardised data collection form. Laboratory results were obtained from the Groote Schuur National Health Laboratory Services electronic database.Acinetobacter baumannii was tested for susceptibility to piperacillin-tazobactam, colistin, ceftazidime, cefepime, imipenem, meropenem, gentamicin, amikacin, tobramycin and ofloxacin/ciprofloxacin and colistin. For all antibiotics except colistin, susceptibility testing was performed using the Kirby-Bauer disc diffusion method on Mueller Hinton agar (Oxoid). Minimum inhibitory concentrations for colistin were determined by E-test  according to the manufacturer's instructions. Interpretative criteria according to Clinical Laboratory Standards Institute guidelines were used.A baumannii: positive blood culture for A. baumannii or culture of A. baumannii from other sites associated with clinical signs of infection  documented within 48 hours of commencing the antimicrobial [The following were regarded as criteria to support the use of antimicrobials for \u00ae Aventis, Bellon, France) is administered at a dose of 2 million units 8 hourly in patients with normal renal function. The manufacturers recommend 50 000 IU/kg/day in 2\u20133 divided doses [At Groote Schuur Hospital, colistin  to the highest subsequent recorded value within 10 days of initiation of the antimicrobial. Participants receiving haemodialysis at the time antimicrobial therapy was first administered were excluded from this analysis. The proportion of participants in each group who increased their serum creatinine to greater than 50% above the upper limit of normal of the local reference values  was compared. All other adverse effects reported in the patient clinical records were recorded.The study was approved by the University of Cape Town Health Science Faculty Research Ethics Committee.Data analysis was performed using Intercooled STATA\u2122 version 8.2 . Continuous variables were summarised using means and standard deviations if normally distributed, and medians and ranges if not normally distributed. 95% confidence intervals (CIs) or interquartile ranges (IQRs) were calculated for all summary statistics and parameter estimates. Between-group comparisons of continuous data were performed using a Student's t test if normally distributed, and the Wilcoxon rank sum test if not normally distributed. Proportions were compared using a two sample test of proportions when the observed frequencies were \u2265 5 in each group and the Fisher's exact test if the expected frequencies were < 5. Kaplan-Meier curves were plotted for time to microbiological clearance and time to death while in ICU, and survival curves compared with a log-rank test. Patients were censored when they were discharged from ICU for the analysis of death and for death in the case of microbiological clearance. For all statistical analyses, a p value of less than 0.05 was regarded as sufficient evidence to reject the null hypothesis.A. baumannii  in the colistin group and 22 (68.75%) patients in the tobramycin group met the criteria for sepsis (culture-positivity in conjunction with 2 or more components of the Systemic Inflammatory Response Syndrome) [Either agent at our hospital is commenced after a review of the case by a clinical microbiologist. In addition, all, except one subject in the each group, had at least one feature to support the use of antimicrobial therapy for their yndrome)  at the tAll the isolates in the colistin group were resistant to all antimicrobials tested, except colistin. All the isolates in the tobramycin group were susceptible to tobramycin. In 17 of the tobramycin cases (53%), the organism was only susceptible to tobramycin and colistin. Carbapenem resistance was present in 24 (75%) of the tobramycin group.The median length of ICU stay after initiation of the antimicrobial was 6 days IQR 4 to 21) in the colistin group and 9 days (IQR 4 to 21) in the tobramycin group (p = 0.06). Eleven colistin-treated patients (34.4%) and 7 tobramycin-treated patients (21.9%) died in ICU (p = 0.54). There was no significant difference in time to death in ICU by Kaplan-Meier survival analysis (log rank p = 0.09)  in the colistin group and 27 days (IQR 12 to 56) in the tobramycin group (p = 0.08) Sixteen patients treated with colistin (50%) and 9 patients treated with tobramycin (28.1%) died in hospital. The hazard ratio for total in-hospital survival in patients treated with colistin compared to tobramycin was 0.43 (95% CI 0.19 to 0.99) (logrank p = 0.04) . The median time to clearance of A baumannii was 3 days for colistin and 4 days for tobramycin (p = 0.46). There was no significant difference in time to microbiological clearance by Kaplan-Meier analysis  in those on colistin and 17 \u03bcmol/mL (IQR 6 to 22) in those on tobramycin (Wilcoxon rank sum test p = 0.11). The geometric mean change in creatinine in the colistin group was 42 \u03bcmol/L (95% CI 24.4 to 74.9) and 19.6 \u03bcmol/L (12.1 to 31.6) in the tobramycin group. Only one participant in the cohort required initiation of haemodialysis after antimicrobial therapy was commenced, a male patient with a baseline serum creatinine of 118 \u03bcmol/mL 6 days after colistin initiation. The proportion of patients with normal renal function at baseline who increased their creatinine concentrations to greater than 50% above the upper limit of normal were similar in the two groups: 2 of 23 (8.7%) in the tobramycin group and 4 of 21 (19%) in the colistin group (Fishers exact test p = 0.67). No other adverse effects were documented.Ten patients in each group had bloodstream infections. Colistin was used for a mean of 13 days (IQR 2 to 12.5) and tobramycin 7.5 days (IQR 3.5 to 11.5) for bloodstream infections (p = 0.25). Five patients in the tobramycin group (50%) and 3 patients (30%) in the tobramycin group with bloodstream infections died in ICU (p = 0.58).Six patients with bloodstream infections in each group (60%) were demonstrated to have achieved sustained microbiological clearance.We found no significant differences in ICU survival, time to microbiological clearance and elevations in serum creatinine between the tobramycin and colistin groups. These findings are encouraging, considering the uncertainties regarding the optimal dose of colistin to maximise efficacy while avoiding toxicity. Patients treated with colistin had higher in-hospital mortality; however we feel that in-ICU mortality is the more important measure as the ICU admission was the time period within which patients were exposed to colistin or tobramycin.Two uncontrolled studies reported an incidence of nephrotoxicity associated with colistin usage of 14% ,6. HowevA. baumannii isolates are resistant to the carbapenems. Seventy five percent of isolates in the tobramycin group in our study were carbapenem-resistant. Because of increasing carbapemen resistance, A baumannii infections in our institution are likely to be treated with either the aminoglycoside tobramycin, or colistin. To our knowledge there has been no study which directly compared aminoglycoside monotherapy with colistin. In one comparative study, 48% of patients in the non-colistin group received an aminoglycoside  [Several studies have compared colistin with the carbapenems in ICU patients, and found that the mortality and nephrotoxicity rates were not different in the colistin and carbapenem groups -14. Howeinolone) . These iinolone) .We compared colistin with tobramycin. In a study of once daily administration of gentamicin and tobramycin in ICU patients, 14% of participants were reported to have a rise in creatinine of 45 \u03bcmol/mL or more . AlthougA. baumannii infections from any site. Clinical patient information was gathered retrospectively, resulting in some missing data and we were not able to control for inter-observer variability. The sample size is small, however as colistin is only prescribed when there are no other available antimicrobials and sample sizes in other studies have been similar. The primary outcome was the change in creatinine concentration in those patients who were not receiving dialysis. There was a high degree of variability in the change in creatinine in the colistin group and the 95% confidence intervals for the geometric mean are wide. It is therefore possible that we failed to demonstrate a difference between the colistin and tobramycin groups because of our limited sample size. In the ICU setting, sepsis, hypotension and the use of other nephrotoxic drugs contribute to impairment of renal function. We are not able to exclude the confounding impact of these variables.Our study has several limitations. The study population is heterogenous, as we included all intensive care unit patients with A. baumanii infections when the organism is resistant to other available antimicrobials.Our data suggests that colistin is not significantly different to tobramycin in terms of efficacy or nephrotoxicity and that it is an acceptable treatment of The authors declare that they have no competing interests.RG contributed to the design of the study, the acquisition and analysis of data and preparation of the manuscript. CB contributed to study design, the acquisition of data and preparation of the manuscript. RZS was involved in the study design and the preparation of the manuscript. KC contributed to the analysis of the data and the preparation of the manuscript. GM contributed to the design of the study, data analysis, the preparation of the manuscript, and supervised the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/26/prepub"}
+{"text": "The presence or absence of intestinal metaplasia of the stomach was determined in 272 patients by direct vision biopsy of standardized intragastric sites using fiberoptic gastroscopy. Analysis of ABO blood groupings and Rhesus factor failed to reveal an association with metaplasia."}
+{"text": "Sir,We present an unusual case of acute hepatitis caused by hair dye.Hair dye contains various mutagenic and carcinogenic chemicals and is examined as a risk factor for various malignancies. Hair dye-induced hepatitis is a rare condition. There is only one case of hair dye-induced hepatitis reported in the literature.A 33-year-old healthy young female presented with pruritus, skin lesions and jaundice 2 days after using a new hair dye rather than her usual one. Clinically, she was febrile, icteric, with hepatosplenomegaly but no ascites. Laboratory investigations showed hypereosinophilia of 20%, abnormal liver profile with total bilirubin of 13.5 mg/dL, conjugated 6.8 mg/dL, alanine transaminase 190 IU/L, aspartate transminase 152 IU/L, alkaline phosphatase of 227 IU/L and albumin being 3.7 g/dL. An ultrasound of the abdomen showed features of acute hepatitis and a normal biliary system. The prothrombin time was normal.Work-up for acute hepatitis indicated negative IgM antibody for hepatites A, E, and anti-HBc antibody. Additionally, hepatitis B virus DNA was undetectable. Serology for cytomegalovirus, Herpes simplex virus, Epstein barr virus, leptospira and malaria were negative. Wilson's and autoimmune markers were also negative. Drug-induced hepatitis was considered in view of chronology of events. Because the patient had an allergic reaction, lymphocyte activation test was performed to study if the hair dye in question induced immune-mediated drug reaction leading to acute hepatitis.in vitro lymphocyte activity experiment strongly suggest that hair dye causes immune-mediated drug reaction in hypersensitive individuals [Lymphocytes were isolated using Histopaque, 1077  by centrifugation. Lymphocyividuals .Hair dye is a well-recognized cause of contact dermatitis. Routinely used hair dyes contain multiple components viz Toluene-2, 5-diamine, resorcin, cetearyl alcohol, polyethylene glycol, etc. Chemicals of the hair dye can be absorbed into the body through a wound, damaged skin or by aspiration of the spray during dyeing. The pathogenesis of drug-induced hepatotoxicity usually involves either the parent drug or its metabolite, which affects the hepatocytes directly or elicits an immune response.in vitro lymphocyte reactivity to hair dye supported hair dye-induced immune-mediated hepatitis.[The chronological course of the present patient, allergic skin manifestations and epatitis. Liver biThus, the present case suggests a possible role of hair dye in inducing an immune-mediated drug reaction in hypersensitive individuals and a lymphocyte activation test may be utilized to diagnose the immune-mediated drug-induced hepatotoxicity."}
+{"text": "Generalised spike wave (GSW) discharges are the electroencephalographic (EEG) hallmark of absence seizures, clinically characterised by a transitory interruption of ongoing activities and impaired consciousness, occurring during states of reduced awareness. Several theories have been proposed to explain the pathophysiology of GSW discharges and the role of thalamus and cortex as generators. In this work we extend the existing theories by hypothesizing a role for the precuneus, a brain region neglected in previous works on GSW generation but already known to be linked to consciousness and awareness. We analysed fMRI data using dynamic causal modelling (DCM) to investigate the effective connectivity between precuneus, thalamus and prefrontal cortex in patients with GSW discharges.We analysed fMRI data from seven patients affected by Idiopathic Generalized Epilepsy (IGE) with frequent GSW discharges and significant GSW-correlated haemodynamic signal changes in the thalamus, the prefrontal cortex and the precuneus. Using DCM we assessed their effective connectivity, i.e. which region drives another region. Three dynamic causal models were constructed: GSW was modelled as autonomous input to the thalamus (model A), ventromedial prefrontal cortex (model B), and precuneus (model C). Bayesian model comparison revealed Model C (GSW as autonomous input to precuneus), to be the best in 5 patients while model A prevailed in two cases. At the group level model C dominated and at the population-level the p value of model C was \u223c1.Our results provide strong evidence that activity in the precuneus gates GSW discharges in the thalamo-(fronto) cortical network. This study is the first demonstration of a causal link between haemodynamic changes in the precuneus - an index of awareness - and the occurrence of pathological discharges in epilepsy. The existence of a link between physiological and environmental factors and the occurrence of epileptic seizures is well documented in the literature Such a link is particularly evident in patients affected by Idiopathic Generalized Epilepsy (IGE) in whom, for example, sleep deprivation, alcohol and stress seem to act as activating factors for seizures occurrence The prototypical seizure type in IGE is the absence with its electroencephalographic hallmark, generalised spike and wave (GSW) discharges. Clinically, absences are characterized by a blank stare and impaired consciousness. Activities requiring vigilant attention have been coupled with a lesser likelihood of absences whereas an increased frequency of these seizures during relaxation is well established Recent functional imaging studies have revealed the existence of a set of brain regions which show increased functional and metabolic activity during rest, compared to attention-demanding tasks The DMN shows decreased activity both during attention-demanding tasks and equally during states of reduced vigilance and, especially the posteromedial cortical regions, during altered states of consciousness EEG-correlated functional magnetic resonance imaging (EEG-fMRI) studies have shown a common pattern of blood oxygen level-dependent (BOLD) signal decrease in the precuneus and the other default mode areas, together with a thalamic BOLD signal increase, during ictal and interictal GSW discharges in vivo intracellular recordings in the GAERS rat model (Genetic Absence Epilepsy Rats from Strasbourg), Polack and colleagues The pathophysiological substrate of GSW remains enigmatic and several studies, both in animals and humans, have tried to answer the historical debate regarding the putative role of the thalamus and cortex as generators. Data from invasive recordings and manipulations in well-validated genetic models of absence epilepsy have supported the hypothesis that absence seizures are of cortical origin. Depth electrode recordings from the thalamus and suprasylvian cortex in cats have shown a primary role of the neocortex in producing seizures consisting of spike and wave complexes However, despite the suggestion of the involvement of dorsal cortical regions  in GSW discharges from neuroimaging studies Dynamic Causal Modelling (DCM) offers the possibility of characterising the effective connectivity, defined as \u201cthe influence that one neural system exerts over another\u201d, in other words: it can be used to test which brain region drives which We applied DCM to EEG-correlated fMRI data to understand the dynamic interaction between brain regions known to be involved in the initiation and cessation of GSW discharges and with a brain region known to be related to conscious awareness, the precuneus. We compared a family of models of effective connectivity focusing on a set of cortical regions and the thalamus. We tested and compared the following models in relation to the GSW discharges: when treated as autonomous input GSW activity enters the cortico-thalamic loop: 1. via the thalamus ; 2. via the ventromedial prefrontal cortex (vmPFC) ; or 3. via the precuneus ; see In order to apply DCM analysis, we re-analysed the resting-state EEG-fMRI data of 32 IGE patients studied previously 2, 64\u00d764 matrix) were acquired over a 35-min session on a 1.5 Tesla Horizon EchoSpeed MRI scanner . Patients were asked to rest with their eyes closed and to keep still.The methods pertaining to data acquisition are described elsewhere http://www.fil.ion.ucl.ac.uk/spm/).FMRI data were processed and analysed using SPM8b  was constructed to assess the presence of regional GSW-related BOLD signal changes. GSW events were represented as variable-duration blocks beginning at the onset of GSW as identified on the MR-synchronised EEG by two expert observers (AEV and RT) and ending upon GSW cessation.Motion-related effects were modelled in the GLM by 24 regressors of the 6 scan realignment parameters and a Volterra expansion of these The GSW event blocks were convolved with the canonical hemodynamic response function (HRF), and its temporal and dispersion derivatives, to form regressors testing for GSW-related BOLD signal changes. Significant positive and negative BOLD signal changes correlated with GSW were identified by means of an F-contrast across the three regressors of interest and recorded as activation and deactivation depending on the response shape. The resulting SPMs were thresholded at p<0.001 http://ric.uthscsa.edu/project/talairachdaemon.html); the Talairach coordinates and equivalent Z-scores of the selected regions are listed in The DCM analysis was performed for three ROI: thalamus, vmPFC, precuneus. For all ROI we used spherical volumes with a 5 mm radius. For ROI selection within the thalamus we chose the axial slice that showed the largest cluster and placed the ROI so as to cover the region. In patients with bilateral thalamus involvement, we selected only one ROI, on the side of the largest cluster. For ROI selection within vmPFC, we placed the ROI in the axial slice within the Brodmann Area 10 and the side containing the largest area of signal BOLD change. The precuneus ROI was placed within the medial sagittal slice, rostrally to the middle of the parieto-occipital sulcus. In patients showing bilateral precuneus involvement, we placed the ROI on the side of the largest cluster. The ROI positions were defined using Talairach Daemon, The regional responses were filtered, whitened and the nuisance effects  were subtracted to leave only GSW-related effects. To account for the effect of scan nulling of large motion events  Using the DCM module as implemented in SPM8b three linear models were constructed. Each comprised the three ROI as reciprocally (forward and backward) connected regions and GSW event blocks considered as autonomous input to each of the three regions, one at a time a) GSW as autonomous input on the thalamus (Model A), b) GSW as autonomous input on vmPFC (Model B), c) GSW as autonomous input on the precuneus (Model C). Hence, three models were evaluated per subject - ln p(2y | m). If this difference is greater than about 3 (i.e. the relative likelihood is greater than 20\u22361) then one asserts that there is strong evidence in favour of one of the models. This is commonly calculated based on the F value of each model, which is the negative marginal log-likelihood or negative log-evidence: F\u200a=\u200a\u2212ln p(y | m). For more details about BMC, see After the estimation of parameters of each competing model, they were compared using Bayesian Model Comparison (BMC) where the evidence of each model, computed from estimated parameters distributions, is used to quantify the model plausibility Assuming that data from each subject are conditionally independent, the evidence at the group level is obtained by multiplying the marginal likelihood, or, equivalently, by adding the log-evidences from each subject Good quality EEG was obtained following pulse and gradient artefact subtraction, allowing reliable identification of epileptiform discharges see . EEG disIn accordance with the selection criteria, significant GSW-associated BOLD signal changes were found in the thalamus, in the frontal lobe limited to the vmPFC, and precuneus see . FrontalF values (i.e. the negative log-evidence) in absolute numbers. p value calculated for each model were extremely close to zero for models B and A, and close to 1 for model C, demonstrating the latter to be very likely at the population level.Assuming that all patients in the group are representative of IGE we generalized the results of group analysis to the population level. The We investigated the causal relationship between neuronal activity as reflected by BOLD signals in three brain regions, namely the thalamus, the vmPFC and the precuneus, in relation to the onset and offset of GSW in 7 patients affected by IGE. The thalamus and the frontal cortex are key structures in well established hypotheses on GSW generation Applying DCM to fMRI data simultaneously acquired with EEG we found that, for the models tested, in five out of seven patients studied, electroencephalographic discharges first affected the precuneus. This finding became more evident at the group and population level so that the evidence in favour of model C (GSW as autonomous input to precuneus) was significantly higher than for models A  and B (GSW as autonomous input to vmPFC). In the remaining two cases, BMC showed model A to be the best. The discrepancy between the results of the analysis of the single subjects is not unexpected. We note that in one of the pioneer DCM studies on evoked potentials, Garrido et al Our finding, that GSW onset and offset are more directly linked to the neural activity  in the precuneus than in the other tested regions, implies a dependency of the cortico-thalamic loop on the precuneus and hence its state, i.e. a causal link. A possible interpretation is that changes in the precuneus state , which reflects spontaneous fluctuations in awareness, act on the thalamic- cortical network facilitating the development of GSW. This is in contra-distinction to previous suggestions A similar hypothesis has been already proposed by Archer et al Additionally, an fMRI study showed BOLD signal decrease in the posterior cingulate in IGE subjects following photic stimulation whether or not GSW occurred, while control subjects showed no change in this region According to the current thinking of the pathophysiology of GSW, there are two prerequisites for the occurrence of this pathological activity: 1) the pathological thalamo- cortical interactions and 2) the so-called mild diffuse epileptogenic state of the cortex The precuneus' neuronal state, and hence the level of awareness, may, consequently, reflect a \u201cphysiological initiator\u201d of generalized synchronous discharges. The existence of a transient facilitating state of the brain which increases cerebral susceptibility to GSW generation has been recently demonstrated in patients with absences, by synchronization measures and MEG source imaging methods Previous EEG-fMRI studies showed BOLD signal decrease in the DMN in relation to GSW apparently not correlated with clinical manifestations While there is little evidence of a strict consequentiality between a particular state of vigilance and the occurrence of GSW discharges, there is a notable lack of studies focusing on the possible role of cortical structures (particularly the precuneus) other than the thalamus and frontal cortex in GSW. However, evidence from scalp EEG source reconstruction analysis suggests that the precuneus participates in the generation of fast sleep spindles at 14 Hz In contrast to surface electrophysiological recordings, fMRI studies with concurrent EEG in patients with GSW discharges have shown common significant haemodynamic changes not only in the thalamus and frontal cortex, but also in the precuneus and other brain regions  of the DMN FMRI's relatively homogeneous sensitivity across the brain relative to that of scalp EEG may explain why recent EEG-fMRI studies have been able to reveal precuneal involvement in epilepsies characterized by impaired consciousness and in particular associated with GSW The application of connectivity analysis techniques based on fMRI data may improve our understanding of the interactions between brain regions haemodynamically involved during GSW discharges. David et al., applied DCM analysis to fMRI data acquired in an animal model of absences Brain connectivity based on fMRI data, can be investigated via two different approaches: functional connectivity and effective connectivity analysis. Functional connectivity is data-driven and assesses statistical dependencies between fMRI signals from different brain regions without consideration of the underlying neuronal activity. In contrast, analyses of effective connectivity test hypotheses based on modelling of neuronal activity and a forward model describing how this activity is translated into the fMRI signals There are different approaches for modelling effective connectivity from functional MRI, which include structural equation modelling (SEM), vector-autoregression models and DCM The causal link revealed in our study (i.e. precuneus activity facilitates GSW) is limited to onset and offset of GSW discharges. Therefore, our findings do not preclude a reverse causal relationship in which GSW accompanied by impairment of consciousness leads to (further) deactivation of the precuneus. This could be addressed by studying ictal GSW data using a similar methodological approach.In common with all DCM-based inferences, our conclusions are valid solely with respect to the family of tested models; there may be brain areas which are involved in the GSW generation processes that were overlooked because of their apparent lack of haemodynamic involvement.In this study we have demonstrated an active role in generalised epilepsy for the precuneus, a region previously neglected in electrophysiological studies of GSW. Using Dynamic Causal Modelling based on EEG-fMRI data we showed that the precuneus not only is strongly connected with the frontal cortex and the thalamus but also that the neuronal activity in this area may facilitate epileptic activity within a thalamo-cortical loop, the existence of which is well established. These findings suggest that GSW may arise through the direct influence of the neuronal state of the precuneus associated with spontaneous changes in the level of awareness.Table S1Description of fMRI results for the cases that did not satisfy the selection criteria for the DCM analysis. Summary of results for all cases for which GSW activity was captured during EEG-fMRI but did not meet the selection criteria for the DCM analysis (extracted from (0.06 MB DOC)Click here for additional data file."}
+{"text": "Because variable results of capsaicin challenges may be due to the incomplete solubility of capsaicin, we sought to determine if the use of Tween-80 in solutions of capsaicin improves actual concentrations of freshly prepared and stored solutions.Capsaicin solutions ranging from 0.5\u2013128 \u03bcM were mixed with and without Tween-80. Samples of various concentrations were then stored under 4 environmental conditions: 4\u00b0C, protected from light; room temperature, protected from light; room temperature, exposed to light; -20\u00b0C. All samples were analyzed initially, and at 2 and 4 months.While freshly prepared solutions with Tween-80 had consistently higher concentrations than those prepared without Tween-80 (83% vs. 69%), Tween-80 does not facilitate complete solubility. For solutions stored at 4\u00b0C and protected from light, there was a significant decrease after 2 months in low concentration solutions of both the Tween-80 and non-Tween-80 solutions. Both Tween-80 and non-Tween-80 containing solutions significantly decreased in concentration after 2 months when stored at room temperature and protected from light, room temperature and exposed to light, and -20\u00b0C. Concentrations of solutions made of 4 \u03bcM or higher are stable when stored at 4\u00b0C and protected from light for 4 months.While the inherent difficulty of forcing capsaicin into solution cannot be eliminated, it can be improved with Tween-80. However, the addition of Tween-80 does not prevent the breakdown of stored capsaicin solutions. We recommend preparing and storing capsaicin solutions according to the methods and results of this study. Inhalational challenges using solutions of capsaicin have been used to determine cough thresholds in subjects , and to While Tween-80 has been utilized by others -9 when mTwo stock solutions of 128 \u03bcM concentrations of capsaicin were made. Solution 1 (without Tween-80) was made by dissolving 39 mg of capsaicin powder  in 12.5 mL of 100% ethanol; this was then diluted with 0.9% sodium chloride solution to a total volume of 1 liter. Solution 2 (with Tween-80) was made by dissolving 39 mg of capsaicin powder in 5 mL of 100% ethanol. To this, 5 mL of Tween-80  was then added, and the solution was diluted with 0.9% sodium chloride solution to a total volume of 1 liter.The two stock solutions of 128 \u03bcM concentration of capsaicin were repeatedly diluted 1:1 with 0.9% sodium chloride solution to make 2 sets of solutions of 64, 32, 16, 8, 4, 2, 1, and 0.5 \u03bcM. One set contained Tween-80 (from stock Solution 2) and the other set did not contain Tween-80 (from stock Solution 1). Aliquots of these freshly made solutions were then analyzed for actual concentration.Additional aliquots of each of the freshly prepared solutions, both with Tween-80 and without Tween-80, were separated for storage under several environmental conditions. Samples of all nine of the concentrations of both the Tween-80 and non-Tween-80 solutions were stored at 4\u00b0C and protected from light. Samples of solutions of 4 \u03bcM, 8 \u03bcM, and 16 \u03bcM capsaicin concentrations were stored at room temperature and protected from light, and room temperature exposed to light. Finally, samples of solutions of 32 \u03bcM, 64 \u03bcM, and 128 \u03bcM were stored in a freezer at -20\u00b0C. Samples to be kept in the dark  were placed in amber vials, and the vials were then stored in light protecting boxes. Samples to be stored exposed to light were placed in clear vials and stored on a laboratory shelf. Samples were tested at baseline and then every 2 months for a total of 4 months. During testing, each sample was tested twice. The concentration of each individual sample was determined by calculating the average of the 2 tests. However, if the results of the two tests on any one solution differed by more than 3%, they were discarded and two additional tests were performed to obtain data within the acceptable variations.18, 150 \u00d7 4.6 mm, 5 \u03bcm particles, 100 \u00c5 pores) . The method was isocratic, with a flow rate of 1.5 mL/min and a run time of 12 minutes. Injection volume was 20 \u03bcL. Capsaicin eluted at 5 minutes.The concentrations of the solutions of capsaicin were measured by high performance liquid chromatography (HPLC). Measurements were made both via electrochemical detection and ultraviolet detection. Initial calibration tests demonstrated that electrochemical detection produced non-linear results for concentrations above 16 \u03bcM. Therefore, electrochemical detection was used only on the solutions of 16 \u03bcM or less, and ultraviolet detection was used for the 32 \u03bcM, 64 \u03bcM, and 128 \u03bcM concentrations. The HPLC system consisted of an ESA 540 autosampler , an ESA 580 pump, and a Spectra-Physics SP8490 UV detector  set at 280 nm. Data were acquired and analyzed by PC/Chrom+ software . The column was a Phenomenex Luna . The 0.05 level of significance was used throughout.The initial stock solution containing Tween-80 was clear in color, while the non-Tween containing stock solution had a whitish, cloudy appearance. The subsequent concentrations of the non-Tween containing solutions became less cloudy with further dilution with saline as described in the Methods Section. The Tween-containing solutions remained clear even after further dilution.Predicted and actual initial concentrations of Tween-80 and non-Tween-80 containing solutions are summarized in Table These data are summarized in Table These data are summarized in Table These data are summarized in Table These data are summarized in Table These data are summarized in Table These data are summarized in Table These data are summarized in Table These data are summarized in Table For the solutions stored at 4\u00b0C and protected from light, there was a significant drop in the concentration of the non-Tween-80 containing solutions when compared to the Tween-80 solutions at 2 and 4 months (p = .003) (Table When comparing the lower concentrations (0.5 \u03bcM and 1 \u03bcM) of solutions with and without Tween-80 in Table While there were significant decreases in all solutions stored at room temperature and protected from light, the decrease in the non-Tween-80 containing solutions was significantly greater than the Tween-80 containing solutions at 2 and 4 months (p < .001) Table .Identical results were seen with all solutions stored at room temperature and exposed to light. Whether containing Tween-80 or not, there were significant decreases in all concentrations at 2 months (p = .001) and 4 months (p < .001) when compared to the baseline concentration Table . The decWhile all concentrations of solutions stored at room temperature significantly decreased at 2 and 4 months compared to baseline, the decreases were greater in the solutions exposed to light vs those protected from light (p < .001 at 2 and 4 months).When compared to the baseline concentration, both the Tween-80 containing solutions and those without Tween-80 had a significant decrease in concentration after 4 months when stored at -20\u00b0C and protected from light (p < .001). There was no significant difference between the decrease in concentration of the Tween-80 compared to the decrease in the non-Tween-80 containing solutions after 4 months (p = .136).From our study, two notable findings emerged that have not been previously reported regarding the use of Tween-80 in preparing capsaicin solutions. First, our results demonstrate that the use of Tween-80, an emulsifier and dispersing agent, significantly improved the actual concentration of freshly prepared solutions of capsaicin compared to solutions not containing Tween-80. Second, the use of Tween-80 did not prevent solutions from decreasing in concentration when stored at room temperature and protected from light, room temperature and exposed to light, or frozen at -20\u00b0C after 4 months. This trend is consistent with the results of our first study in which we analyzed only solutions made without Tween-80 .With respect to the use of Tween-80 to prevent the breakdown of capsaicin solutions over time, when stored at various environmental conditions, we found that solutions of 2 \u03bcM and less of both the Tween-80 containing and non-Tween-80 containing solutions did significantly decrease in concentration after 2 months compared to the higher concentration. However, it did appear that the higher concentration of Tween-80 containing solutions were more stable after 4 months when stored at 4\u00b0C and protected from light compared to the non-Tween-80 solutions. The non-Tween-80 data in this study are similar to the results in our initial study of non-Tween-80 containing solutions . AlthougTween-80 is commonly used in pharmaceutical products to improve solubility of water insoluble medications. While Tween-80 is felt to be safe for intravenous and inhaled use, there have been a few reported cases of toxicity and hypersensitivity that have been associated with Tween-80 found in some medication preparations. Liver and kidney damage in infants who received intravenous vitamin E has been attributed to Tween-80 , as has While there have been several reported methods for making capsaicin solutions without Tween-80 ,2,5, we While the addition of Tween-80 in preparing fresh solutions of capsaicin improves the actual concentration of capsaicin with our method of preparation, Tween-80 does not facilitate complete solubility of capsaicin. Also, the addition of Tween-80 does not prevent the breakdown of capsaicin solutions over time. The FDA considers Tween-80 to be safe for use in intravenous and inhaled medications, but we know of no prospective safety studies that demonstrate that there is no increased risk of side effects associated with inhaled capsaicin solutions made with Tween-80. Therefore, we recommend using Tween-80 cautiously until prospective studies are available to confirm its safety.When prepared capsaicin solutions are to be used, we recommend the following: When preparing fresh solutions of capsaicin, Tween-80 should be added because it improves the actual concentration. Nevertheless, even with use of this this emulsifier and dispersing agent, it should be appreciated that the actual concentration will still be less than 100%.If solutions of capsaicin are to be stored for future use, we recommend making a stock solution of 128 \u03bcM with Tween-80, and storing the solution at 4\u00b0C and protected from light, as solutions of 128 \u03bcM made without Tween significantly breakdown when stored at these conditions after 4 months (see table When making solutions to be stored, the addition of Tween-80 does not appear to prevent a decrease in the 0.5 \u03bcM, 1.0 \u03bcM, and 2.0 \u03bcM concentrations after 2 months when stored at 4\u00b0C and protected from light. Tween-80 also does not prevent the decrease in concentration of solutions stored at room temperature or frozen at -20\u00b0C.While the inherent difficulty of forcing capsaicin into solution cannot be eliminated, it can be improved with Tween-80. To decrease the variability of results of capsaicin tussigenic challenges, it is important to standardize all aspects of the methodology including preparing and storing capsaicin solutions. Until further refinements in capsaicin solution preparation are forthcoming that improve upon the present situation, we recommend preparing and storing capsaicin solutions according to the methods and results of this study.The authors declare that they have no competing interests.SK, RI, and RD conceived the study and participated in the design of the study, SK and RI drafted the manuscript, SK performed the statistical analysis, RD, MB, and TM prepared the solutions, oversaw the storage of the solutions, and performed the analysis of all the solutions. All authors read and approved the final manuscript."}
+{"text": "The purpose of this pilot study is to assess the feasibility, acceptability, and safety of a new feeding protocol designed to enhance the delivery of enteral nutrition (EN).In a prospective before and after study, we evaluated a new protocol compared to our standard feeding protocol. Innovative elements of the new protocol included setting daily volume based goals instead of hourly rate targets, initiating motility agents and protein supplements on Day 1, liberalizing the gastric residual volume threshold, and the option to use trophic feeds. Bedside nurses filled out questionnaires to assess the acceptability of the new approach and we assessed patients' nutritional and clinical outcomes.P = 0.33 and 0.13). When the subgroup of patients prescribed to receive full volume feeds in the after group were evaluated (n = 18), they received 83.2% and 89.4% of their energy and protein requirements by EN respectively (P = 0.02 for energy and 0.002 for protein compared to the before group). The rates of vomiting, regurgitation, aspiration, and pneumonia were similar between the two groups.We enrolled 20 mechanically ventilated patients who stayed in the Intensive Care Unit for more than three days in the before group and 30 such patients in the after group. On a scale where 1 = totally unacceptable and 10 = totally acceptable, 30 nurses rated the new protocol as 7.1 (range 1 to 10) and no incidents compromising patient safety were observed. In the before group, on average, patients received 58.8% of their energy and 61.2% of their protein requirements by EN compared to 67.9% and 73.6% in the after group (This new feeding protocol seems to be safe and acceptable to critical care nurses. The adoption of this protocol may be associated with enhanced delivery of EN but further trials are warranted to evaluate its effect on nutritional and clinical endpoints.ClinicalTrials.gov NCT01102348 P = 0.014)) and an increased number of ventilator-free days (VFDs) . Since the main route of providing energy and calories was enteral nutrition, efforts to increase the enteral provision of macronutrients are warranted.Several observational studies have described an association between inadequate feeding and poor clinical outcomes in critically ill patients -3. In a Repeated efforts over the past few years have not significantly improved the amount of calories delivered via the enteral route -7. If wevolume-based goal represents a significant shift in practice from traditional hourly rate goals in which nurses can increase the hourly rate depending on how many hours they have left in the day to ensure that the patient receives the 24-hour volume within the day. 2) For patients who are deemed unsuitable for high volume intragastric feeds, we provide an option to initiate trophic feeds at a low volume of a concentrated feeding solution. By trophic, we mean a minimal volume of EN designed to maintain gastrointestinal structure and function, not designed to meet the patients caloric or protein needs. When deemed suitable, trophic feeds can be advanced to full feeds. 3) Rather than wait for a protein debt to accumulate because of inadequate delivery of EN, protein supplements are prescribed at initiation of EN and can be discontinued if EN is well tolerated. 4) We propose to start motility agents at the same time EN is started with a re-evaluation in the days following to see if it is necessary and we raised our gastric residual volume threshold from 200 to 250 ml. It has been shown in one randomized trial that a feeding protocol that starts a motility agent empirically at the time of initiation of feeds and uses a higher threshold for a critical gastric residual volume (250 ml) improves nutritional adequacy [We propose a new approach that protocolizes an aggressive approach to providing EN and shifts the paradigm from reactionary to proactive followed by de-escalation if nutrition therapy is not needed. The key components of this new protocol are the following: 1) Starting feeds at the target rate based on increasing evidence that some patients tolerate starting nutrition at a higher rate of delivery and that slow start ups are not necessary ,9. For padequacy .Since the bedside nurses initiate and utilize feeding protocols to achieve target goals, we will couple this newer generational feeding protocol with a comprehensive nurse-directed nutritional educational intervention that will focus on its safe and effective implementation. This focus on nursing nutrition education represents a major shift away from traditional education which has focused on dietitians and physicians.Protein-Energy Provision via the Enteral Route in Critically Ill Patients: The PEP uP Protocol.\"The Enhanced We hypothesize that the combination of these components will safely improve the provision of energy and protein compared to usual care. We postulate that this increased provision of calories and protein may translate into improved clinical outcomes, particularly for the patients at the extremes of weight, but the current study is not powered to demonstrate such a difference. The purpose of this pilot study is to assess the feasibility, acceptability, and safety of this new feeding protocol, \"This is a prospective before and after study conducted in a 21-bed medical surgical tertiary care ICU with a usual nurse-to-mechanically-ventilated-patient ratio of one-to-one that admits approximately 1,000 patients per year. All care in the ICU is directed by an attending intensivist. The clinical staff in the ICU also includes a full time registered dietitian. Enteral feeds are guided by a standard feeding protocol specified by pre-printed ICU admission orders. The admitting physician has the option of initiating the enteral feeding protocol or keeping the patient nil per os (NPO). If EN is ordered, the bedside nurse initiates a standard polymeric feeding formula at 25 ml/hr once the position of the feeding tube is confirmed. Gastric residual volumes are monitored every four hours and in the absence of a gastric residual volume above 200 ml, feeds are advanced to their target hourly rate by 25 ml/hr every four hours. In the setting of persistent (two or more consecutive) high gastric residual volumes, feeds are reduced by 25 ml/hr and the nurse is instructed to ask the physician to order gastrointestinal prokinetic agents , if appropriate. The dietitian usually assesses the nutritional needs and prescribes the appropriate solution and hourly target rate within 24 to 48 hours of admission. In the summer of 2008, we evaluated the efficacy of our standard protocol in a cohort of eligible patients.full volume feeds, trophic feeds, or NPO. Beside each tick box, there were indications and contraindications listed for each selection. If an admitting physician selected NPO, they had to fill in a reason or justification. A sample of justifications was provided on the form. To simplify the administration of this protocol, we chose one feeding formula to be used initially. We chose a semi-elemental, concentrated feeding solution that would be useful in both full volume and trophic fed patients . The dietitian could suggest changes to this formula after the protocol was started based on further assessment. There was also a tick box to initiate a motility agent in the absence of contra-indications  and to initiate a protein supplement . Nurses were given instructions on how to set the hourly rate based on the 24-hour volume prescribed. For example, if the total goal for the day was 1,500 ml of a nutritional solution to meet their caloric requirement, then the hourly rate would be 62.5 ml/hr. If feeds were held for several hours while the patient underwent a radiological procedure and now there are nine hours left in the day and the patient has only received 400 ml, the new rate would be  122 ml/hr for the remaining 9 hrs. Beginning the next day, the target would shift back to 62.5 ml/hr. We arbitrarily set a limit of a maximum of 150 ml/hr for PEP uP Protocol patients. We increased the threshold gastric residual volume to 250 ml from 200 ml and since patients were already on metoclopramide as per the protocol, erythromycin was added if the threshold gastric residual volume of 250 ml persisted.Starting in January 2009, we implemented our new protocol combined with our nursing educational intervention. The concepts and the details of the PEP uP Protocol were presented at our multidisciplinary critical care rounds and augmented by small group educational in-servicing for bedside nurses. We operationalized the protocol with a pre-printed order form and a series of bedside algorithms and instructions. On the pre-printed order form, there were tick boxes for the admitting physician to choose refeeding syndrome with the aggressive feeding strategy, the protocol also required frequent assessment of electrolytes, magnesium, phosphorus, and calcium during the first 72 hours of admission of all patients.We encouraged nurses to calculate the nutritional adequacy  daily and report it during daily multidisciplinary rounds. Given the concerns raised about the possibility of precipitating a These protocol documents  and a slide presentation coupled with educational reminders (posters and bedside notices) and practice helps  were made available to all nurses working in our unit, in bedside manuals and on the local intranet. Beginning 2 February 2009, we formally introduced the protocol and evaluated its adoption in the next series of eligible patients. Nurses attending to patients enrolled in the after phase of the study received daily bedside academic detailing (one on one instruction) by the ICU Dietitian.Our study patient population consisted of consecutive mechanically ventilated adult patients (>18 years old) who remained in ICU for more than 72 hours. In the before group, we enrolled 20 patients prospectively. To ensure a larger number available for evaluation of the new protocol, we enrolled 30 patients in the after group.In both groups, we retrospectively collected the following information related to the prospectively enrolled patients from their hospital chart: admission category , primary admission diagnosis, sex, age, weight, height, and Acute Physiology And Chronic Heath Evaluation II (APACHE II) score . We recoThe primary outcome of this pilot study was the feasibility of the new feeding protocol as judged by a nursing questionnaire that evaluates their opinion of its safety and acceptability. We asked about the acceptability of each of the novel parts of the feeding protocol and the overall protocol using a scale where 1 = totally unacceptable and 10 = totally acceptable. This evaluation questionnaire was administered to the bedside nurse involved in caring for a patient on the new protocol (in the after group). Secondary outcomes included nutritional endpoints (adequacy of EN and timeliness of initiation of EN) and safety endpoints . In addition, one of the investigators reviewed the charts to determine if there were any undetected serious adverse events related to the nutritional management of the patient and whether the initial prescription of full volume, trophic feeds, or NPO was appropriate.P-value < 0.05. Institutional ethics approval was obtained from the Health Sciences Research Ethics Board at Queen's University, Kingston, Ontario, Canada. The need for informed patient consent was waived given the nature of this study .No formal sample size calculation was done as the primary purpose of this before and after study was to evaluate the feasibility and safety of the new protocol and not its effect on mortality or length of stay. Categorical variables are reported as counts and percents and compared between cohorts by the Fisher's Exact test. Length of stay variables are described by medians and quartiles and compared by the log-rank test. Other continuous variables are described by their means and standard deviations and compared by the Wilcoxon-Mann-Whitney test To assess nutritional adequacy, the total amount of energy or protein received from either EN or parenteral nutrition (PN), inclusive of propofol, was divided by the amount prescribed as per the baseline assessment and expressed as a percentage. For the purposes of this evaluation of this enteral feeding protocol, we compared adequacy from EN sources between the two groups as the primary outcome over the first seven ICU days. Patients receiving no EN were excluded from this analysis of EN adequacy. We also report a subgroup analysis of the EN adequacy of just those patients indicated to receive full volume feeds, as per the order on the admission pre printed order forms. Statistical analysis was completed using SAS v9.1.3 . All tests were two-sided with statistical significance considered as a In the before group, we screened consecutive admissions and enrolled 20 mechanically ventilated patients who stayed in the ICU more than three days. In the after group, we again screened consecutive patients but this time enrolled 30 patients who remained in the ICU more than three days. The characteristics and outcomes of all enrolled patients are shown in Table On average, across the two time periods, study patients were prescribed 24 to 26 kcal/kg and 1.1 to 1.2 grams/kg of protein by the ICU dietitian. Details of the nutritional assessment and the nutritional prescription of study patients can be seen in Table P = 0.72) (see Table P = 0.22). In the before group, no patients received motility agents or protein supplements on Day 1 of admission whereas in the after group, the number of patients that on Day 1 of admission received protein supplements and motility agents was nine (30.0%) and seven (23.3%) respectively (see Table P = 0.11) and protein supplements  compared to the before group (see Table The average time to starting EN was 16.0 hours in the before group and 17.7 hours in the after group (was nine 0.0% and was nine 0.0% and P = 0.33 and 0.13)  started on Peptamen 1.5. The average duration of use of Peptamen 1.5 was 3.0 days, with standard deviation of 1.8 days. Only one patient in the before group was started on Peptamen 1.5, the majority of the remaining were started on a polymeric solution. In the before group, on average, patients received 58.8% (range 0 to 116%) of their energy and 61.2% (range 0 to 104%) of their protein requirements from EN. In the after group, patients received 67.9% (range 0 to 139%) and 73.6% (range 0 to 119%) of their energy and protein requirements respectively (P = 0.78). There were no documented electrolyte complications in the after group. Morning blood glucose (8.0 \u00b1 0.9 vs. 8.1 \u00b1 1.0 mmol/L) and the proportion of measurements spent above 10 mmol/L  were similar between the two groups.There was no increase in complications related to the PEP uP Protocol. The rates of vomiting, regurgitation, aspiration, and ventilator-associated pneumonia (VAP) were similar between the two groups  indicated that they witnessed an event or incident that, in their opinion, compromised patient safety. These incidents were recorded in open text as the nurse perceptions that the patient was getting too much volume and concerns about new feeding pumps that had nothing to do with the protocol but unfortunately were deployed the same time we evaluated the new protocol. There were no indications from the nurses that patients had regurgitated or aspirated while on this feeding protocol. Nurses rated the acceptability of the protocol and its individual components as follows: the acceptability of the 24-hour volume based target was 7.0 (range 1 to 10); the acceptability of starting at a high hourly rate was 5.9 (range 1 to 10); the acceptability of starting motility agents right away was 7.4 (1 to 10); the acceptability of starting protein supplements right away 7.6 (1 to 10); and the acceptability of the overall protocol was 7.1 (range 1 to 10) on a scale where 1 = totally unacceptable and 10 = totally acceptable.Worldwide, there is considerable controversy around the optimal amount of and route of feeding in critically ill patients. However, our recent observational research shows that the amount of energy and protein received during the early stages of ICU admission impacts on patient mortality . A revieWhat this paper introduces is a new philosophy for delivery of EN, where the clinician starts at near maximal therapy, optimizing as many strategies as possible. This means the clinician starts at goal rate with automatic initiation of prokinetic agents, uses a small peptide formula that would be tolerated by the most number of patients, optimizes protein delivery with early supplementation, minimizes interruptions by setting a higher gastric residual volume threshold, and promotes volume-based feeding so that nurses can make up for lost time due to diagnostic tests and surgical/endoscopic procedures. The goal is to optimize chances for tolerance, prevent ileus, shorten the time frame to achieve goal feeding, and take advantage of the window of opportunity early after admission to the ICU to change outcome with the EN. This philosophy is a radical departure from conventional provision of nutrition where clinicians typically start at a low rate of infusion, ramping up slowly, cautiously evaluating tolerance and adding prokinetic agents only after evidence of ileus is already present.Although the current study is small and utilizes a weak study design, its value is in the new ideas it espouses to maximize EN delivery to critically ill patients. Based on nurse-rated questionnaires and a retrospective chart review, we conclude that the new PEP uP Protocol is safe, feasible, and acceptable to nurses working in our ICU. Moreover, for those patients who were prescribed volume based feeds, they achieved almost 90% of their prescribed protein and energy requirements. These gains were achieved with volume-based feeds despite the fact that they started EN the same time as the before group. Thus it appears that this protocol may enhance the delivery of enteral nutrition; however, this finding requires testing in a prospective randomized fashion. We acknowledge that there is evidence that aggressive, intragastric EN can cause harm . Hence, NPO, 20% of protocolized patients were still ordered NPO at the outset of their ICU admission, some without adequate justification. These deficiencies highlight the need for improved training and education on these points to ensure better compliance with these new ideas. To the extent that we are more successful at their implementation, we will have greater success with EN adequacy than currently reported. Furthermore, we can increase the maximal hourly rate of the 24-hour volume based feeds (from 150 to 200 ml/hr) and/or increase the threshold gastric residual volume from 250 to a higher amount and these changes will likely also increase EN delivery.In implementing this protocol at our site, we observed several challenges which decreased the effectiveness of the protocol. First, although motility agents and protein supplements were meant to start on Day 1 on all PEP uP Protocol patients, in reality, this only happened in about one-third of patients. Also, while we intended for a ready to use, easily absorbed semi-elemental diet to be prescribed at initiation of the feeding protocol, this did not happen in 25% of the patients. Finally, despite our introduction of the concept of trophic feeds, as an alternative to We believe that the best way forward to implement these novel changes to the way we deliver EN is in the context of a feeding protocol. Such nurse-driven bedside feeding protocols have been shown to be effective in reducing interruptions and improving nutritional adequacy . HoweverWhereas significant underfeeding of critically ill patients occurs around the world, this new feeding protocol has the potential to dramatically improve nutrition practice world wide. This current study is valuable in that it introduces some key philosophical and practical changes to how enteral nutrition is prescribed and delivered to critically ill patients. We have shown, in the context of a single-center before and after study, that this new protocol, the PEP uP Protocol, is safe, feasible, and may improve nutritional adequacy. This important single center study justifies the need for a larger multicenter, multinational RCT to further determine the safety and efficacy of this novel approach to feeding the critically ill patient.\u2022 Traditional enteral feeding protocols do not adequately provide sufficient protein and calories to critically ill patients.\u2022 A new feeding protocol that challenges traditional assumption and incorporates innovative ideas on maximizing the benefits and minimizing the risks of EN is needed.\u2022 The PEP uP Protocol uses 24-hour volume-based feeding goals instead of hourly rate targets, initiating motility agents and protein supplements on Day 1, liberalizing the gastric residual volume threshold, and the option to use trophic feeds.\u2022 In this before and after pilot study, we demonstrate that this new feeding protocol seems to be safe and acceptable to critical care nurses.\u2022 The adoption of this protocol may be associated with enhanced delivery of EN but further trials are warranted to evaluate its effect on nutritional and clinical endpoints.APACHE II: acute physiology and chronic health evaluation score; CI: confidence interval; EN: enteral nutrition; ICU: Intensive Care Unit; NPO: nil per os; PN: parenteral nutrition; VAP: ventilator-associated pneumonia; VFD: ventilator-free days.Daren K Heyland has received research grants and a speaking honorarium from Nestle. All other authors have no competing interests to declare.DKH was responsible for the initial design of the study and writing of the manuscript. JM, AA and FA were responsible for data collection. MW and AGD conducted the analysis. JM, JWD, SAMC, RD and NEC provided input on the design and interpretation of the study. All authors have contributed to, seen, and approved this manuscript."}
+{"text": "This information can aid in the application of deoxyArbutin for many future uses.Tyrosinase is the key and rate-limiting enzyme responsible for the conversion of tyrosine into melanin. Competitive inhibition of tyrosinase enzymatic activity results in decreased or absent melanin synthesis by melanocytes in human skin. DeoxyArbutin (4-[(tetrahydro-2H-pyran-2-yl)oxy]phenol), a novel skin whitening agent, was synthesized through the removal of hydroxyl groups from the glucose side-chain of arbutin. DeoxyArbutin not only shows greater inhibition of tyrosinase activity but is also safer than hydroquinone and arbutin. Hence, deoxyArbutin is a potential skin whitening agent for cosmetics and depigmenting drugs; however, stability of this compound under some conditions remains a problem. The lack of stability poses developmental and practical difficulties for the use of deoxyArbutin in cosmetics and medicines. Improving the thermostability of deoxyArbutin is an important issue for its development. In this research, we established an analytical procedure to verify the amount of deoxyArbutin in solutions using a high performance liquid chromatographic (HPLC) method. The results indicate that this novel skin whitening agent is a thermolabile compound in aqueous solutions. Additionally, the rate constant for thermodegradation ( Although human melanin is the skin\u2019s most important protection against the harmful effects of UV light, the dark skin caused by melanin accumulation is not considered cosmetically pleasing to most people \u20133. The iin vivo in several studies . Hy. Hy10].  studies ,13. Deox studies . This co studies ,13. Hencin situ upon absorption into skin, thus, it has potential instability and tends to change color due to oxidation at higher temperature in formulation [k) and half-life (t1/2) of deoxyArbutin were also determined.Arbutin produces the hydroquinone mulation . BecauseThe major purpose of the work was to investigate the thermostability of deoxyArbutin in an aqueous solution. For this reason, the first part of the study examined the solubility and measured the UV spectrum of deoxyArbutin in an aqueous solution. Second, we established the analytical procedure to confirm the quantity of deoxyArbutin in solutions using an HPLC method. Using this method, the kinetics of deoxyArbutin thermodegradation were determined.Because the hydroxyl groups of the glucose side-chain are removed , deoxyArThe United States Food and Drug Administration (FDA) has determined propylene glycol to be a Generally Recognized As Safe (GRAS) ingredient for use in cosmetics, food, and medicines. The World Health Organization (WHO) has also identified it as safe for use . Similar2O) with 10% propylene glycol were placed in a quartz cell and then scanned using a UV-Vis spectrophotometer between wavelengths from 200 to 400 nm. The results are shown in To establish an analytical method for deoxyArbutin, we used a UV-Vis spectrophotometer to collect the UV spectrum of deoxyArbutin aqueous solutions. Both 0.05 and 0.1 mM deoxyArbutin in deionized distilled water  of deoxyArbutin and hydroquinone were injected into an HPLC to establish the analytical method. The chromatogram, shown in In several studies, hydroquinone and other compounds were separated using HPLC with a conditioned mobile phase such as methanol-water at 80:20 (v/v) and acetonitrile-water-formic acid at various ratio \u201321. Thus\u22124 M) solutions in ddH2O (pH 7) and 10% propylene glycol were placed in glass bottles and kept in the dark. The solutions were then exposed to various temperatures in an incubator. The temperatures were 4 \u00b0C (low temperature), 25 \u00b0C (middle temperature) and 45 \u00b0C (high temperature). At each time point the samples were analyzed using the HPLC method, and the resulting deoxyArbutin retention percentages are shown in DeoxyArbutin  of 15.4 days at 20 \u00b0C [90% of about 1.9 days at 25 \u00b0C  and the initial amount of deoxyArbutin in an aqueous solution, respectively. The k) was obtained at various temperatures from 4 to 45 \u00b0C and gave half-life (t1/2) values for deoxyArbutin as shown in k values for deoxyArbutin thermodegradation were 0.0037, 0.0312 and 0.0947 at 4 \u00b0C, 25 \u00b0C and 45 \u00b0C, respectively  in an aqueous solution [1/2 value of deoxyArbutin at 25 \u00b0C in an aqueous solution is only about one week (7.11 days) and is decidedly shorter than the t1/2 value of arbutin at 20 \u00b0C  for solutions and buffers was obtained using a Milli-Q system .DeoxyArbutin was purchased from Denjelly Co., Ltd . Hydroquinone was purchased from Wako Pure Chemical Industries . HPLC-grade methanol was purchased from Merck . Analytical grade propylene glycol and other chemicals were purchased from Sigma-Aldrich . Deionized distilled water . The UV wavelength was set to scan from 200 to 400 nm and the scanning spectral bandwidth was 2 nm with a scanning speed of 200 nm/minute. The resulting UV spectrum was analyzed with the UVWin5 Software (PG Instruments).To overcome the low solubility of deoxyArbutin in pure water, deoxyArbutin was initially dissolved in propylene glycerol then mixed with ddHThe 20-\u03bcL samples of the deoxyArbutin solution were injected into an HPLC . A pre-packed C18 reversed-phase column  was used. The HPLC UV detector wavelength was set to 280 nm in accordance with the UV-Vis Spectrophotometer analysis results . The mob\u22124 M) in ddH2O (pH 7) containing 10% propylene glycol were placed in glass bottles and kept in the dark. In an incubator, these samples were exposed to the following temperatures: 4 \u00b0C (low temperature), 25 \u00b0C (middle temperature) and 45 \u00b0C (high temperature). At select times, the solutions were analyzed using the previously described HPLC method to monitor the thermodegradation progression of the compound. The rate constant for thermodegradation (k) and half-life (t1/2) of deoxyArbutin were calculated according to standard first-order kinetics [Solutions of deoxyArbutin  and half-life (t1/2) data for deoxyArbutin can help us understand the thermodegradation kinetics of deoxyArbutin. In conclusion, we believe that these results may aid in the development of deoxyArbutin\u2019s use for many applications, including cosmetics and medicines.In summary, we established an analytical procedure to verify the amount of deoxyArbutin in solutions using an HPLC method. We also demonstrated that this novel skin whitening agent is a thermolabile compound in an aqueous solution. The rate constant for thermodegradation ("}
+{"text": "Rapid reviews have emerged as a streamlined approach to synthesizing evidence - typically for informing emergent decisions faced by decision makers in health care settings. Although there is growing use of rapid review 'methods', and proliferation of rapid review products, there is a dearth of published literature on rapid review methodology. This paper outlines our experience with rapidly producing, publishing and disseminating evidence summaries in the context of our Knowledge to Action (KTA) research program.The KTA research program is a two-year project designed to develop and assess the impact of a regional knowledge infrastructure that supports evidence-informed decision making by regional managers and stakeholders. As part of this program, we have developed evidence summaries - our form of rapid review - which have come to be a flagship component of this project. Our eight-step approach for producing evidence summaries has been developed iteratively, based on evidence (where available), experience and knowledge user feedback. The aim of our evidence summary approach is to deliver quality evidence that is both timely and user-friendly.From November 2009 to March 2011 we have produced 11 evidence summaries on a diverse range of questions identified by our knowledge users. Topic areas have included questions of clinical effectiveness to questions on health systems and/or health services. Knowledge users have reported evidence summaries to be of high value in informing their decisions and initiatives. We continue to experiment with incorporating more of the established methods of systematic reviews, while maintaining our capacity to deliver a final product in a timely manner.The evolution of the KTA rapid review evidence summaries has been a positive one. We have developed an approach that appears to be addressing a need by knowledge users for timely, user-friendly, and trustworthy evidence and have transparently reported these methods here for the wider rapid review and scientific community. Rapid reviews have emerged as a streamlined approach to synthesizing evidence in a timely manner -typically for the purpose of informing emergent decisions faced by decision makers in health care settings. Although there appears to be a growing use of rapid review 'methods', and a proliferation of rapid review products, there is a dearth of published literature on rapid review methodology. With limited transparency, it is impossible to determine the validity, appropriateness and, ultimately, the utility of these products. To address the gap, this paper outlines our experience with rapidly producing, publishing and disseminating evidence summaries in the context of our Knowledge to Action (KTA) research program to date.The KTA research program is a two-year project designed to develop and assess the impact of a regional knowledge infrastructure that supports evidence-informed decision making by regional managers and stakeholders. It is a collaborative effort between researchers with expertise in knowledge synthesis and knowledge translation based at the Ottawa Hospital Research Institute (OHRI), and knowledge users involved in decision making around health service delivery based at the Champlain Local Health Integration Network (LHIN) - a regional health authority in Ontario, Canada. As part of the knowledge intelligence services that comprise the knowledge infrastructure, we have developed 'evidence summaries', our form of rapid review, which have come to be a flagship component of this project.According to the Cochrane handbook, a traditional systematic review is a review that \"attempts to collate all empirical evidence that fits pre-specified eligibility criteria in order to answer a specific research question. It uses explicit, systematic methods that are selected with a view to minimizing bias, thus providing more reliable findings from which conclusions can be drawn and decisions made\" . While sDespite the increasing production and use of rapid review products, its methodology remains underdeveloped. In fact, there is no universally accepted definition of what constitutes a rapid review. Given their potential deficits in the absence of an approved methodology, many experts have questioned the validity of rapid reviews ,4. GivenThe KTA evidence summary was conceived as an overview of the available evidence addressing a research question or set of research questions related to a single topic (an area of need and priority identified by knowledge users of the Champlain LHIN) produced within a short timeframe (four to five weeks). Because they were developed under the auspices of the KTA research program, and therefore as a means to facilitate collaboration between researchers of the OHRI and knowledge users of the Champlain LHIN, the first several evidence summaries were developed in the absence of a specific methodology; rather, their production was informed by a general intent to consult and synthesize a broad range of quality evidence quickly.Following positive responses to early evidence summaries and the progressive development of a collaborative relationship between the OHRI and Champlain LHIN, considerable effort had been invested toward the advancement of their methods. For example, we have continued to experiment with incorporating more of the established methods of systematic reviews, while maintaining our capacity to deliver a final product in a timely manner. We have also experimented with various user-friendly formats, reporting styles and document lengths to increase usability. Usability has been an oft-neglected component in traditional systematic reviews despite evidence that has shown its capacity to improve the quality and cost effectiveness of health care ,6.Potential uses of an evidence summary may include the following:\u2022 to serve as an informative brief that prepares stakeholders for discussion on a policy issue;\u2022 to support the direction and evidence-base for various health policy initiatives;\u2022 to support the development of clinical interventions and/or health services programs.From November 2009 to March 2011 we have produced 11 evidence summaries. Our eight-step approach for producing evidence summaries has been developed iteratively; it is outlined in Table Our evidence summaries begin with a proposal for an evidence synthesis by a knowledge user about a clinical or health services or system topic. We have defined knowledge users in the context of the Champlain LHIN as policymakers, administrators, stakeholders, managers or decision makers. Probing consultation with knowledge users is recognized as an important initial stage of synthesizing information for the purpose of supporting evidence-informed decision making ; as suchIn particular, we have found that the research team may need to elicit additional information from knowledge users about the specific needs and interests related to their proposed topic or question. This process serves the dual objective of refining the scope such that it is suitable to our proposed methods and ensuring that the final product is meaningful and useful for its intended audience and their objectives. In cases where a proposed topic has been difficult to address in the context of a rapid evidence summary , we have strived to retain the knowledge users' priorities and objectives and, where feasible and appropriate, adapt our methods to address their specific needs. Our methods and evidence summaries have benefited from this dynamic approach.http://www.ohri.ca/kta and have included the following :Generally, knowledge users in our context have not demonstrated a strong capacity for formulating effective research questions; while they are clear about the broad strokes of what they want to ask, they seem less able to provide insight into the critical details that make a research question more precise and, therefore, answerable. To address this problem, we have established a requirement that knowledge users make an initial time investment (around 1 to 2 hours in addition to the needs assessment) to collaboratively develop a clear and effective research question. Using information gleaned from the initial needs assessment , the research team attempts to facilitate the question refinement process by proposing research questions to be vetted by the knowledge user. For questions of effectiveness, we attempt to operationalize the Participants/Populations, Interventions, Comparators, and Outcomes framework as reasonably as possible; when addressing questions related to health systems and/or health services, we modify accordingly. This is an iterative process whereby questions are edited and refined by both the research team and knowledge user until a suitable research question (or series of questions) satisfying both parties (as outlined in the needs assessment section above) has been established. Questions posed by evidence summaries to-date can be accessed on the KTA website 1. Pre-diabetesst century2. Health services for the 213. Electronic health records4. What is known about postpartum interventions for women with a history of gestational diabetes mellitus?5. What is known about the timing of elective repeat Cesarean section?6. What is known about options and approaches to fetal surveillance and intrapartum management of women with gestational diabetes mellitus?7. What evidence exists to describe the effect of interventions that use pedometers to reduce risk for and manage chronic disease?8. What are the drivers of in-hospital formula supplementation in healthy term neonates and what is the effectiveness of hospital-based interventions designed to reduce formula supplementation?9. What is known about third and fourth degree lacerations during vaginal birth?10. What is known about the maternal and newborn risks of non-indicated induction of pregnant women at term?11. What is the evidence of the effectiveness and safety of emergency department short stay units?12. Of note, the first three evidence summaries were produced in the absence of a primary research question but rather using a series of research questions developed by the research team. In part, the lack of clarity and direction supporting these efforts was a primary precipitating factor behind the researchers' subsequent demand for greater involvement of the knowledge users in formulating and establishing precise research questions.In the initial stages of the KTA project, it was not yet clear to the research team which of the program's offerings would be most useful to Champlain LHIN knowledge users. As such, the first few evidence summaries primarily reflected an effort on the part of the research team to identify what may or may not be useful for the knowledge users of the Champlain LHIN. Because of this, a formal proposal was not produced for these first few evidence summaries. However, as enthusiasm among knowledge users grew and additional evidence summaries were requested, it became clear that there was a need for a formal document to succinctly summarize the outcomes of the needs assessment and question refinement stages as well communicate any other particulars relevant to the completion of the report that may or may not have been discussed. In its current form, the proposal aims to include: question background; finalized research question or questions; proposed methods, deliverables and timelines; and knowledge user-research team agreements . In addition to providing a point of reference for the knowledge users and research team  the proposal may also serve to inform extended members of the research team (such as the information scientist) and provide necessary documentation for securing outside funding, when applicable. A brief and concise proposal template has been developed to expedite this step, thus maximizing the time for conducting the evidence summary itself.Depending on the nature of the question, purpose of the report and magnitude of the literature, a variety of types of evidence have been targeted in the searches for evidence summaries. In most cases , emphasis has been placed on locating and summarizing evidence from relevant and high quality systematic reviews. Evidence from systematic reviews is prioritized in order to limit unnecessary duplication, to minimize resources needed to screen and summarize primary level evidence and to minimize the potential bias and/or error which could be incurred by reviewing primary evidence rapidly. In the absence of systematic reviews, however, high-quality and/or recent primary studies may be included, as well as landmark and/or oft-cited studies. Commentaries may also be considered  as a means of providing background and/or context to the body of literature, as well as guidelines, economic analyses and other non-clinical reports. Finally, quasi-experimental and/or observational studies of high quality  may also be considered. The inclusion of evidence from primary studies and other reports was more commonly employed with earlier summaries, when our rigorous approach was still being refined and the questions being addressed were broader and more topic-based. Selection of 'high-quality' studies was determined subjectively, based on methodological expertise, by the research coordinator responsible for the particular evidence summary. Recent summaries have addressed narrower questions and, with the exception of relevant information garnered from guidelines, have almost exclusively drawn from evidence reported in systematic reviews. All literature is considered regardless of publication status.Grey Matters, a seminal guide for searching grey literature produced by the Canadian Agency for Drugs and Technologies in Health [Searches for the first four evidence summaries were conducted by the research coordinator in the absence of an information specialist, any reference management or study selection software, or the use of tools to search for grey literature. As enthusiasm grew among knowledge users, we aimed to improve upon the rigor of our methods by consulting a senior information scientist to perform systematic searches, ensuring appropriate concepts, keywords and subject heading terms, as well as relevant grey literature, were incorporated. For example, to ensure comprehensive coverage of grey literature, the information scientist uses n Health . With th\u00ae; Thomson Reuters, New York, NY) where search strategies, dates, yield and duplicate counts are recorded in a search log. Citations are then uploaded to DistillerSR\u00a9 , an internet-based systematic review software program intended to facilitate study selection by the research team.To manage the records retrieved, search yields are downloaded into a bibliographic database software , enters screening questions within these levels and sets limits and rules regarding the screening process . The screening questions operationalize the eligibility criteria; initially these were informed by the needs assessment, however, subsequent to the addition of the question refinement stage, they were informed by the study question.Once citations have been uploaded to DistillerSRUsually the retrieval of full text documents takes place following one level of title and abstract screening. Occasionally, however, upon first pass of the literature, researchers will discuss and refine eligibility criteria with the knowledge users and, using the revised criteria, a second level of screening title and abstracts is performed. Full text is obtained largely through the journal subscriptions held by the Ottawa Hospital and the University of Ottawa; records not available electronically are pragmatically excluded as timelines do not permit for delays incurred by interlibrary loans. Similarly, due to limited time and resources available for translation, only English reports are included. Although exclusion of languages other than English is undesirable, it is considered to be a reasonable practice given limited time and resources and there is some evidence to suggest that it may not markedly bias review findings . In mostScreening for the first few evidence summaries was undertaken by one reviewer. It is recognized, however, that a single reviewer introduces a level of error that is not desirable , and a sWhile about half of published systematic reviews include a meta-analysis , the KTAIn addition to summarizing the evidence, each study is assigned a level of evidence based on a modified framework established by the Cochrane Musculoskeletal Group . The goaThis step includes developing a concise report that succinctly yet methodically covers all components we set out to address in the proposal. An initial template for the evidence summary report was developed iteratively based on early feedback from the knowledge users. Later formats have incorporated ideas from the structured summaries of systematic reviews developed by the Supporting Policy relevant Reviews and Trials Collaboration  as well Once the content of the report has been inserted, the final phase of aesthetic formatting occurs . We have learned not to underestimate the time needed to perform this critical last step which, in addition to the quality of the content, we believe may facilitate dissemination and implementation of the evidence summaries to its intended audience.Evidence summaries were conceived within the context of a research program that seeks to build relationships between health policymakers and health services researchers. As such, the evidence summaries developed by the research program serve as a basis for researchers to learn more about what health knowledge users need to incorporate evidence into their work. We continue to engage with knowledge users  eliminate them completely. While research remains ongoing, our experience to-date has shown KTA evidence summaries to be effective tools for addressing the evidentiary needs of health services decision makers in the Champlain region and are highly valued by researchers and knowledge users alike.DM is a co-editor-in-chief at Systematic Reviews.SK contributed to the design, coordination and execution of the study and its associated evidence summaries and drafted the manuscript. KK contributed to the design, coordination and execution of the study and its associated evidence summaries and oversaw revisions of the manuscript. RC contributed to the conception, design and execution of the study. JG contributed to the conception, design, coordination and execution of the study. DM contributed to the conception, design, coordination and execution of the study and its associated evidence summaries. All authors participated in the critical revision and read and approved the final manuscript."}
+{"text": "Background. In the last decades, several hospitals have adopted this concept of integrative medicine for the treatment of chronic and acute states of illnesses in in-patient treatment. The aim of this paper was to summarize the current evidence for a possible effectiveness of integrative on-patient treatment in patients' quality of life by means of a meta-analysis. Material and Methods. The databases MEDLINE, EMBASE, AMED, PsycInfo, PsycLit CCMED, and CAMbase were screened to find articles. We also screened publisher databases to find relevant information. Articles were included if patients were treated in a hospital. To guarantee comparability SF-36 was the predefined outcome measure for patients' quality of life. Data of pre/posteffects on the mental and physical scores of the SF-36 were extracted and effect sizes were calculated and entered into a random effect meta-analysis. Results. Eight articles published between 2003 and 2010 were included in the final meta-analysis. Random effect meta-analysis of the eight studies revealed an overall effect size of 0.37  in the physical score and 0.38  in the mental score of the SF-36. I2 statistics indicate a high heterogeneity in the effects in both the physical and mental scores of the SF-36 . Discussion. This meta-analysis might help to rediscover the importance of integrative in-patient treatment for patients, physicians, and stakeholders. Integrative medicine according to the definition of the consortium of the Academic Health Centers for integrative medicine is \u201cthe practice of medicine that reaffirms the importance of the relationship between practitioner and patient, focuses on the whole person, is informed by evidence, and makes use of all appropriate therapeutic approaches, healthcare professionals, and disciplines to achieve optimal health and healing\u201d . It therIn the last decades, several hospitals have adopted this concept of integrative medicine for the treatment of chronic and acute states of illnesses in in-patient treatment , 3. ThisAlready in the very early years of these institutions such whole systems evaluations, that is, with the focus on comparative health economic analysis, demonstrated the therapeutic potential of these approaches . NowadayIn the appraisal of patient's benefits several measures like patient' mood, depression, or pain perception were applied to demonstrate the effects of integrative in-patient treatment. However health related quality of life very early became the main and most important outcome parameter and denotes the least common denominator of such evaluations .Up to now, published data is widespread and no systematic review so far has collected the results of the studies to get a broader picture of the effects of integrative in-patient treatment. The aim of this paper was to summarize the current evidence for a possible effectiveness of integrative in-patient treatment on patients' quality of life by means of a meta-analysis.The following databases were used to find articles: MEDLINE, EMBASE, AMED, PsycInfo, PsycLit CCMED, and CAMbase . We alsoArticles were included if patients were treated in a hospital (no out-patient or day clinic treatment). To guarantee comparability SF-36 was the predefined outcome measure for patients' quality of life. To get a picture the sustainability of the effects, we decided to concentrate on the differences between \u201cbaseline\u201d and \u201cfollowup\u201d with a follow-up duration of three months. Finally the aspect of \u201creal world data\u201d was covered and thus controlled clinical trials of a single drug or treatment were excluded.All articles were fully read and their reference lists were checked for further relevant publications. To guarantee validity of the selection process, all abstracts of excluded papers were double checked. The complete search was performed between March and May 2012. The reporting of the results adhered to the MOOSE and QUOROM guidelines .Details of eligible studies were extracted and summarized using a data extraction sheet including the study indicators year, origin, institution, therapeutic approach, diseases, treatment duration, number of patients, and mental and physical scores of the SF-36 (mean and standard deviations at baseline and followup). Extracted data was cross-checked again.d between the two time points and its standard deviation STD(d) according to the recommendations of Dunlap et al. [m1, s1 and m2, s2 denote the means and standard deviations of the pre- and post-SF-36 scores and r represents Pearsons correlation coefficient between them. In cases where the correlation between pre- and post-measures was not reported, we set r = 0.7, which according to [When a trial was found to be eligible, data of pre/post effects on the mental and physical scores of the SF-36 were converted into effect sizes and their standard deviation using an MS Excel sheet. We used the formulasp et al. , where mrding to  is a suiI2 coefficient measuring the percentage of total variation across studies due to true heterogeneity rather than chance. Results were displayed using a forest plot.To calculate overall estimates of the treatment effect we chose a random effects model according to the recommendations and algorithms given in Borenstein et al.  assumingA total of 364 records were found, of which 36 could be identified as reviews. After screening the abstracts of the remaining 328 records, 268 records were excluded because they did not fit to the inclusion/exclusion criteria. The remaining 60 articles were assessed for eligibility and other 52 were excluded according to the inclusion/exclusion criteria after reading the full text as they provided data on out-patient treatment or did not report on SF-36 quality of life data. Thus eight articles published between 2003 and 2010 were included in the final meta-analysis. A flow chart of the inclusion process is provided in Six of the eight articles described a traditional European medicine in-patient treatment strategy including the five therapeutic elements \u201chydrotherapy,\u201d \u201cphytotherapy\u201d, \u201cexercise therapy,\u201d \u201cnutrition/dietetics,\u201d and \u201clifestyle modification\u201d of classical naturopathy as originally described by Kneipp. One of the studies included \u201ctraditional Chinese medicine\u201d as an additional therapeutic element; another one had a focus on spa therapies. The remaining two articles reported on an integrative mind body approach and on a biopsychosocial treatment strategy. Seven of the eight studies were conducted in German hospitals or hospital departments. Only one study provided data from integrative in-patient treatment from the USA.The mean number of patients enrolled was 897 ranging from 22 to 4253. The treatment duration varied between two and three weeks. The majority of patients were treated because of diseases of the musculoskeletal system and connective tissue (ICD chapter M00\u2013M99) including pain syndroms. The data on the 8 included articles is summarized in I2 statistics indicate a high heterogeneity in the effects in both the physical and mental scores of the SF-36 .Random effect meta-analysis of the eight studies revealed an overall effect size of 0.37  in the physical score and 0.38  in the mental scores of the SF-36. d = 0.16 and d = 0.18 in the studies of Greeson et al. [d = 0.50 and d = 0.51 in the studies of Weidenhammer et al. [In the physical dimension effect sizes were quite heterogenous ranging from small effects of n et al.  and Wiebn et al.  to moderr et al.  and Buchr et al.  . Howevez et al.  .In both dimensions the overall effect is mainly influenced by the huge cohort study of Weidenhammer et al. from 2007 , which iThis is the first systematic review and meta-analysis to cover whole systems evaluations of integrative in-patient treatment. Based on the data of 7180 patients treated with integrative concepts ranging from classical naturopathy to traditional Chinese medicine we were able to calculate moderate total effect size almost three months after discharge from hospital. Quite fortunately all scientific evaluations have used standardized outcome measures and most of them included the SF-36 as a standardized measure for health related quality of life (HrQoL). Although setting parameters and patient characteristics did differ to a certain extent between the included studies, the results of this meta-analysis both from the perspective of sample size and indications and outcome measures can be regarded as a valid indicator of effectiveness for integrative in-patient treatment.The by far most treated conditions in the 8 included studies are musculoskeletal and pain disorders . It is wWithout question, due to its high relevance and burden, effective multimodal interventions are needed and a moderate total effect size almost three months after discharge from hospital proves the value of this special approach especially but not exclusively in these fields of medicine. Further frequent diagnoses for integrative in-patient treatment are chronic cardiovascular, gastrointestinal or pulmonary diseases, or even oncological diseases, but currently, data to evaluate these fields of interest are lacking.Therefore, this meta-analysis only digs a small corridor in the field of evidence. Some of the studies included in our analysis have tried to identify responders and nonresponders to integrative medicine. Although they finally did not succeed in doing so, this might still be an option if data from these studies are aggregated and reanalyzed. Apart from conducting an individual patient data meta-analysis as proposed by Vickers et al. , this apHowever this idea is somehow limited. The fact should not be hidden that there are still several studies on whole systems evaluation of integrative in-patient treatment which have not seen the light of publication. One of the most deplorable examples in this respect is the model project Charlottenstift which aimed at integrating traditional European and traditional Chinese medicine . Thus, this meta-analysis might be seen as an episode one of in-patient evaluation and might help to rediscover the importance of this field for patients, physicians, and stakeholders of the health care system."}
+{"text": "Drug addiction is a term applied across substance use disorders (SUDs) and defined as a chronic, relapsing complex brain disease characterized by compulsive drug seeking, craving, loss of self-control, and impaired decision making  for nicotine  for alcohol and opioids   Enoch  and for \u03b15-\u03b13-\u03b24 nicotinic cholinergic receptor subunit gene cluster on chromosome 15q, which sparked the discovery of several loci related to smoking and its more dire health consequences, such as lung diseases and peripheral artery disease  in addition to the more common \u03b14/\u03b22 nicotinic receptors, which are thought to mediate nicotine reward and dependence  and the interpeduncular nucleus (IPN), brain regions that express high levels of the Changeux , and whiDRD4 gene with individuals showing a greater risk for attention deficit hyperactivity disorder (ADHD) and for SUD. These individuals also show an increased sensitivity to both adverse as well as positive environmental factors resulting in either worse or better outcomes than those without the 7-repeat DRD4 VNTR  for SUD is mediated though their influence on brain development, connectivity, and function, which in turn influences brain responses. This more integrated perspective on the role of genes and gene regulation in SUD provides a better understanding of why there is significant comorbidity between SUD and mental illness, including not only the frequent co-occurrence of both of these disorders but also overlap in brain circuits, in risk alleles and in environmental insults. Particularly prominent are circuits involved with \u201cself regulation and control,\u201d which implicate frontal networks and those involved with saliency circuits, which implicate striatal and limbic regions . CNR1 is densely localized in the hippocampus, the amygdala, and the prefrontal cortex  and obesity risk , orexin, and corticotropin-releasing factor (CRF) systems and they can remain engaged even when the stressor is no longer present Koob . The CRFIt has become increasingly evident that epigenetic factors are instrumental in their ability to affect several genes simultaneously with or without the underlying genetic variation. Epigenomic regulation mediates cellular responses to external stimuli such as environmental toxins, infection, stress, and drugs of abuse. Epigenomic regulation of cocaine responses in brain reward circuitry has been particularly well studied Nester . ExposurIn summary, genetics and epigenetics have expanded our understanding of the neurobiological basis of SUDs and related disorders through an interdisciplinary process of human and molecular genetic research Fig. . The rem"}
+{"text": "Day-and-night rotating shiftwork was negatively associated with NCDs and CMRFs (p < 0.001). Sedentary behavior should be considered a health risk among workers. Hence, to promote a healthy lifestyle and safe workplace, organizations should encourage standing activities during break and physically active commutes, and have workers avoid prolonged sitting.Although prolonged sitting appears as a novel risk factor related to health outcomes for all ages, its association needs to be replicated in occupational conditions. This study explored the associations between sedentary behavior and four noncommunicable diseases (NCDs) as well as two cardiometabolic risk factors (CMRFs) among workers in a petroleum company, Thailand. All workers were invited to complete the online self-report questionnaire. Sedentary behavior was measured as the amount of time sitting at work, during recreation, and while commuting. Out of 3365 workers contacted, 1133 (34%) participated. Prevalence of NCDs and CMRFs was 36% and was positively associated with sedentary behavior. After adjusting for age, BMI, and exercise, the risk of NCDs and CMRFs for sedentary office work was 40% greater compared with more active field work. Those who took a break without sitting more than twice a day and commuted by walking or cycling had less risk of NCDs and CMRFs. The total duration of sedentary behavior was 10 h/day, and two-thirds of that total was workplace sitting. This was significantly associated with NCDs and CMRFs ( Noncommunicable diseases (NCDs) are causes of morbidity and mortality worldwide. Four common NCDs, cardiovascular diseases (CVDs), diabetes mellitus (DM), chronic respiratory diseases (CRDs), and cancers (CAs), account for more than 38 million deaths globally . Social,Four common NCDs caused 71% of deaths among Thai people age 15 years or older in 2014 . No progThe impact of urbanization on human lifestyle discourages participation in PA and motivates sedentary behavior . SedentaThe identification of sedentary behavior influencing overweight or obesity and disease occurrence has been reported in many studies ,15,16,17To examine the association between occupational sitting and health outcomes, van Uffelen and colleagues selected 43 papers . OccupatThe association between sedentary behavior and disease occurrence has been inconclusive, so many researchers have examined potential factors ,15,16,17We conducted an online cross-sectional survey research among petroleum workers between 2013 and 2015. Their names, employee identification numbers, and positions were anonymous for personal information protection and confidentiality. We guaranteed that their participation in this study would have no consequences to their job security. This study was approved by the Mahidol University Institutional Review Board (COA. No.2013/109.3010).http://www.pt.mahidol.ac.th/ergo/index.php. The content validity and reliability test of the OSCWE were acceptable  [An online self-reporting questionnaire on computer work-related exposure (OSCWE) consisted of five domains to survey health and related factors among computer workers . The OSC34\u20130.93) . For datSubjects reported personal factors, health conditions, and sedentary behavior data. Personal factors were as follows: age comprised of four-decade groups , BMI categorized into four groups based on the WHO guideline for the Asian population  , diabetes mellitus (DM), chronic respiratory diseases (CRDs), cancers (CAs)\u2014and two cardiometabolic risk factors (CMRFs)\u2014hypertension (HT) and hyperlipidemia. Subjects who reported at least one NCD, one CMRF, or a combination were defined as \u201chaving diseases\u201d. Those who were healthy or had other conditions such as musculoskeletal disorders, gastrointestinal problems, and others were classified as \u201cnot having diseases\u201d.According to previous studies ,9,10, weRecreation was classified by type of activity performed at home consisting of playing with a computer/tablet/smart phone, exercising, and performing other activities. The duration of recreational activity was collected as hours daily. Commute was defined as the mode and duration (h/day) of commuting between home and workplace. The modes of transportation consisted of walk, bicycle, motorcycle, personal car, public transportation, and employee shuttle bus.This study examined whether personal factors and sedentary behavior was associated with having diseases (4 common NCDs and 2 CMRFs). The risk of having diseases associated with personal factors was determined in order to identify potential confounding factors. We then controlled for these factors to determine the risk of sedentary behavior associated with disease prevalence.The sample size was estimated using a single proportion formula . The Z sp < 0.05.Data were analyzed using SPSS\u00ae . The number and percentage (%) of the total population were calculated for categorical data. Mean and standard deviation was used to report the average amount of time spent working, engaging in recreation, and commuting. Total duration of sedentary behavior represented as hours daily (h/day) was calculated by summarizing time spent sitting for work, sitting and playing with computer/tablet/smart phone for recreation, and using a passive mode of transportation when commuting . The pren = 102), a CMRF (n = 237), or a combination (n = 66) was reported by 36% of subjects. The majority of subjects selected more than one category of health problem. Subjects reported having one (n = 309), two (n = 78), three (n = 17), and four conditions (n = 1). They reported CVDs (n = 11), DM (n = 24), CRDs (n = 129), CAs (n = 11), HT (n = 82), and hyperlipidemia (n = 263). Of 1133, 728 (64%) subjects reported being healthy (n = 308) or having other conditions (n = 420) such as musculoskeletal disorders, gastrointestinal symptoms, thyroid disease, eyes problems, migraine, stress, and injury.Of a total of 3365, 1295 subjects (39%) registered for the online questionnaire and 1133 workers (34%) completed the OSCWE. A NCD . They reported a weight change within two years including a weight gain of 3.26 \u00b1 4.02 kg and a weight loss of 5.28 \u00b1 4.81 kg. Only 15% of workers performed frequent exercise (more than 3 times weekly) such as playing sports , performing yoga, and exercise for physical fitness. Other recreational activities included reading, listening to music, watching TV or movies, knitting, shopping, gardening, and walking their dogs.n = 405/1133). p < 0.05). Workers with high BMI (>27.5) were twice as likely to have diseases compared to those with an acceptable BMI 18.5\u201323.0 . Decreased risk of having diseases was demonstrated in the subjects who reported more frequent exercise in one week.The prevalence of diseases among workers was 36% (n = 357/917) of office workers reported having diseases. The odds of having diseases was 3.06 times  higher in office workers compared to field workers. The daily workplace sitting time ranged from 5 to 12 h. Those with 12 h rotating shift work were less likely to have diseases than those who had regular work. Of the total, 1030 subjects (91%) reported having breaks daily, and the time ranged from 5 to 30 min for each break. Subjects taking fewer breaks were more likely to have diseases than those who had more breaks. Approximately 80% of the subjects reported that, during breaks, they performed a variety of activities including having coffee while standing or walking (n = 695) and sitting or inclining to play with a computer/tablet/smart phone (n = 140). Those who were active during breaks were less likely to have diseases compared to those who were not active during break.More than 39% (The risk of having a disease increased among workers who spent time using a computer/tablet/smart phone at home. Recreation at home was reported by 1124 subjects. There were 535 subjects who reported spending time using a computer/tablet/smart phone and of these 505 subjects spent 1 to 4 h daily. An average of 2 h of sitting for recreation at home was reported by subjects who had and did not have disease .n = 550), public transportation (n = 168), an employee shuttle bus (n = 55), or a motorcycle (n = 20). The time of transportation between home and work was 15 min\u20133 h for using passive modes and 15\u201375 min for walking or bicycling. The subjects who used passive modes to commute were more likely  to have diseases than those who walked or biked.In terms of commute, most subjects 70% used passive modes of transportation including a personal car (p < 0.001)  .p < 0.001) and recreation  was associated with NCDs and CMRFs. We found a positive association between sedentary office and shift work, rest during breaks, and sedentary recreation at home and the occurrence of diseases . Prolonged sitting time at work could be used as a predicting factor to determine whether workers are at a high risk of disease occurrence. Recent findings by Pronk and colleagues [r = 0.35\u20130.47, p < 0.05). They found that stand-up projects during sedentary work could reduce 66 min of time spent sitting and improve neck and back musculoskeletal pain, fatigue, mood, and depression.The average total time of daily sedentary behavior was 10 h/day, and two-thirds of this time occurred in the workplace\u20148 h of time spent sitting per day. Previous studies have reported that the total time for daily sedentary behavior was 7 h for Dutch workers , 10 h folleagues  reportedOne unexpected finding in this study was the lower risk of diseases for shift workers after adjusting for age, BMI, and exercise. Previous studies found a low level of physical activity (PA) and a high level of sedentary behavior among shift workers compared with regular workers ,31. Day p < 0.01). Ekelund and colleagues [The negative health risk effect of sedentary behavior could be alleviated by PA ,33. Bakrlleagues  determinp-value less than 0.05 and 0.001 was demonstrated in many parameters in our study. Third, the self-reporting questionnaire could have resulted in recall or information bias. These problems are inevitable in survey-based research. However, we minimized these problems by using an online self-reporting questionnaire that was user-friendly, took only 20 min to administer, and was appropriate for surveying health risk factors among workers.Some limitations occurred in this study. First, we conducted a cross-sectional survey that might not demonstrate causality between sedentary behavior and disease occurrence among workers. However, we provided strong evidence of a positive association by eliminating the effect of confounding factors. We filled in the gap of controversial relationships and recommendations for workers. Second, the low questionnaire response rate (34%) might not have represented all workers. However, the number of participants was greater than the sample size calculation. Statistical significance at a In conclusion, we found positive associations between sedentary behavior and four common NCDs as well as two CMRFs among workers. Based on our findings, recommendations to prevent health risk were developed. First, workers should reduce time spent sitting at work by arranging short and more frequent active breaks. Second, workers should avoid prolonged sitting during commuting by walking or cycling. Third, workers should be conscious of performing more PA or exercise in their spare time than inactive or sedentary recreation. Finally, an organization should implement strategies to promote PA to compensate for prolonged sedentary behavior while working such as stand-up projects or active work stations."}
+{"text": "Cardiovascular (CV) diseases (CVDs) are the leading cause of mortality worldwide. Globally, there is a growing interest in understanding and addressing modifiable psychosocial risk factors, particularly depression and anxiety, to prevent CVDs and to reduce morbidity and mortality. Despite the high premature mortality rate from CVDs in Latvia, this is the first Latvian study to examine the association of depression and anxiety with CVD morbidity in a primary care population.This cross-sectional study was carried out in 2015 within the framework of the National Research Program BIOMEDICINE at 24 primary care facilities throughout Latvia. Consecutive adult patients during a one-week time period at each facility were invited to join the study. Assessments onsite included a 9-item Patient Health Questionnaire (PHQ-9) and a 7-item Generalized Anxiety Disorder scale (GAD-7) followed by a socio-demographic questionnaire and measurements of height, weight, waist circumference, blood pressure, and total cholesterol. The diagnostic Mini International Neuropsychiatric Interview (MINI) was conducted over the telephone within 2\u00a0weeks after the visit to the general practitioner. A multivariate model was developed using binary logistic regression.p\u2009=\u20090.04) for current depressive symptoms (PHQ-9\u2009\u2265\u200910) and 2.08 (p\u2009=\u20090.002) for lifetime depressive episode (MINI).From the 1565 subjects , CVD was detected in 17.1%. Depression screening was positive (PHQ-9\u2009\u2265\u200910) for 14.7%, and anxiety screening was positive (GAD-7\u2009\u2265\u200910) for 10.1% of the study subjects. According to the MINI, 10.3% had current and 28.1% had lifetime depressive episode, and 16.1% had an anxiety disorder. Depression, not anxiety, was statistically significantly related to CVDs with an odds ratio (OR) of 1.52 (Current depressive symptoms (PHQ-9\u2009\u2265\u200910) and a lifetime depressive episode (according to the MINI) were significantly associated with increased risk of CV morbidity. Therefore, CV patients should be screened and treated for depression to potentially improve the prognosis of CVDs. Enhanced training and integration of mental health treatment in Latvian primary care settings may improve clinical outcomes. Anxiety and major depressive disorder (MDD) are the most common mental disorders in the European Union (EU) affecting 20.9% of the population (99.4 million people every year). MDD is already now the most important single contributor to the total disease burden not only in the EU but also worldwide, as measured by disability-adjusted life years (DALYs) , 2. AnxiDepression and anxiety are highly prevalent in patients with CVDs . ApproxiOver the last 25\u00a0years, a large body of evidence has demonstrated that depression and anxiety are not only more common in CV patients, but that these two psychiatric conditions are also risk factors for increased cardiac morbidity and recurrent CV events and mortality, independent of traditional CV risk factors , 20. TheThe largest cross-sectional surveys on CV risk factors in the EU called European Action on Secondary and Primary Prevention by Intervention to Reduce Events (EUROASPIRE) were conducted to determine whether the Joint European Societies guidelines on CVD prevention are being followed in clinical practice , 33. EURWe therefore aimed to conduct the first study on the association of depression and anxiety with CVDs in a primary care population in Latvia. This study addresses an important gap in the literature by focusing on Latvia. Recent evidence reported major cross-country differences in the determinants of disability among patients with heart disease and supported implementation of country-specific programs to reduce disability among CV patients . TherefoThe cross-sectional study was carried out in 2015 within the framework of the National Research Program BIOMEDICINE 2014\u20132017 to assess the prevalence of the most frequent mental disorders in primary care settings in Latvia. Patients were recruited from 24 primary care facilities all over the country . The survey was conducted in Latvian and Russian (the two most commonly spoken languages in Latvia).This study was approved by the Ethics Committee of the Riga Stradins University, Riga, Latvia (No. 8/ 18.06.2015.). The project was carried out in accordance with the Declaration of Helsinki and its subsequent amendments. All respondents were enrolled only after providing written informed consent.The inclusion criteria were consecutive treatment-seeking patients visiting a general practitioner (GP), patients aged 18 or older, and patients who had provided their informed consent. The exclusion criteria were patients who refused to participate in the study, patients younger than 18\u00a0years of age, and patients with acute medical condition requiring urgent hospitalization.During a one-week period at each primary care facility, all consecutive patients who corresponded to the inclusion criteria were invited to complete a nine-item Patient Health Questionnaire (PHQ-9) and a seven-item Generalized Anxiety Disorder scale (GAD-7) in Latvian or Russian (language as preferred by participant) followed by an interview with a structured socio-demographic questionnaire and measurements of height, weight, waist circumference, blood pressure and total cholesterol on the same study visit. The Mini International Neuropsychiatric Interview (MINI) was conducted over the telephone by specially trained psychiatrists within a period of two weeks after the first contact. Diagnoses of CVD were confirmed using medical records. CVD was defined as an atherosclerotic vascular disease in the heart , brain  and periphery , or a combination of these conditions . AdditioThe PHQ-9 is a self-rating tool that was developed from the Primary Care Evaluation of Mental Disorders (PRIME-MD) questionnaire in the late 1990s to screen and to diagnose patients with depressive disorders in primary care settings . It consThe GAD-7 is a self-reported measure of anxiety , 48. ParThe MINI is a short, structured, diagnostic interview that was developed for DSM-IV and the psychiatric disorders portion of the 10th version of the International Classification of Diseases (ICD-10). It was validated in relation to the SCID-P  and the CIDI  . It consThe structured questionnaire contained questions about demographics , the medical reason for a visit to the general practitioner, CV risk factors, history of CVDs, diabetes mellitus and psychiatric disorders, use of CV and psychotropic medications, and days absent from work during last 3\u00a0months.Family history of premature CVD was defined as a fatal or non-fatal CVD event or/and established diagnosis of CVD in first degree male relatives before 55\u00a0years of age or female relatives before 65\u00a0years of age.The question about leisure-time physical activity was: \u201cHow often do you do physical exercise at leisure lasting at least 30 min. making you at least mildly short of breath or perspire?\u201d It was categorized as follows: 1) Unable to perform; 2) 1 time a week or less; 3) 2\u20133 times a week; 4) 4\u20136 times a week; 5) Every day.The variable describing the prevalence of smoking was based on several questions regarding different aspects of smoking . It was categorized as follows: 1) Ever smoked and 2) Never smoked.Alcohol consumption representing episodes of heavy drinking was measured with the following question: \u201cHave you used 5 or more doses of alcohol at once during the last 12 months?\u201d It was dichotomized as follows: 1) Yes and 2) No. Dose of alcohol was defined as equal to 10\u00a0mL by volume of 8\u00a0g by weight of pure alcohol.The question about the use of fresh vegetables and fruits was: \u201cDo you consume at least 200 g of fresh vegetables or/and fruits per day?\u201d And it was categorized as follows: 1) Yes and 2) No.Consumption of fish was considered as a protective factor if patient responded positively to the following question: \u201cDo you consume fish at least 2 times per week (one of which to be oily fish)?\u201dAssessment of CV risk factors was based on recommendations and criteria of the 2012 Joint European Societies\u2019 guidelines on cardiovascular disease prevention  and quesp\u2009<\u20090.05. Crude and stratified percentages were used for descriptive statistics. To identify factors associated with the presence of CVDs, univariate and multivariate analyses were carried out. The multivariate model was developed by using binary logistic regression. To avoid multi-collinearity, separate regression models were elaborated for two indicators of depression as independent predictors of CVD- lifetime depressive episode (identified by the MINI interview) and current depressive symptoms (detected through the PHQ-9 instrument). For the final model of multivariate analysis, generalized anxiety disorder (according to the MINI) was chosen, as it gave the best-fitting regression model when compared to other anxiety disorders identified by the MINI or to clinically relevant anxiety symptoms detected by the GAD-7 questionnaire.Statistical Package for the Social Sciences (SPSS) version 20.0 (IBM SPSS Corp.) was used for all statistical analyses. Statistical significance was evaluated at the level of n\u2009=\u2009268) of the studied population. The prevalence of CVD was slightly higher among men than women, with values of 18.4% and 16.5%, respectively.The mean study response rate was 91.3% (from 1756 approached patients 152 refused to participate), it varied in the range between 86.3\u201393.7% across 24 primary care facilities all over the country. Those who refused did not significantly differ in the basic socio-demographic characteristics from the rest of the group. In this study, 1604 patients were invited to complete the PHQ-9 and the GAD-7 questionnaires, and 1585 of the approached patients completed both questionnaires. For those who completed both questionnaires, information about the presence of CVD diagnosis was available in 1565 subjects, 489 (31.2%) men and 1076 (68.8%) women, which were included in the final analysis. CVD  was detected in 17.1% (One third 31.2%) of the study sample were males, and one third (28.8%) had a university degree. Slightly more than a half (55.8%) of the respondents had reached or exceeded the age of 55\u00a0years, and half (51.9%) were employed. One fifth (19.9%) of the study subjects were residing in the capital city, Riga  of the study subjects were tobacco smokers, 16.4% had 5 or more drinks of alcohol at least once during the preceding year, one quarter (25.8%) did not include fresh fruits and vegetables in their meals daily, and 60.6% reported that they did not eat fish regularly . Daily moderate physical activity was reported by one fifth (20.5%) of individuals. More than two thirds (69.2%) of the patients were overweight according to BMI, and more than half had increased levels of total cholesterol (60.8%). The prevalence of diabetes among the respondents was 9.1%. Systolic or diastolic hypertension were present in 42.6% and 31.1% of patients, respectively. Thus, at the time of the questionnaire, 49.1% of the individuals were taking antihypertensive medications, and 15.2% were taking cholesterol lowering medicines (Table n\u2009=\u2009228) of the individuals. According to the MINI questionnaire, 10.3% (n\u2009=\u2009149) had current and 28.1% (n\u2009=\u2009407) had lifetime depressive episode. Antidepressants were used by 3.0% of individuals, which could have been for depression or anxiety disorders.As shown in Table In the univariate analyses, the factors statistically significantly associated with the presence of CVD  were older age, lower education, economical inactivity, urban place of residence, smoking status, episodes of heavy drinking, being overweight, the presence of diabetes, systolic hypertension, depression and intake of antihypertensive or cholesterol lowering medications Table\u00a0.Table 2Fp\u2009<\u20090.001). Economic inactivity  doubled the odds of having a CVD (p\u2009<\u20090.001). Residence in the capital city (Riga) increased the risk of CVD approximately nine times (p\u2009<\u20090.001), and living in another urban area increased the odds more than 3 times (p\u2009<\u20090.001) when compared to living in a rural area (Table p\u2009=\u20090.03). Patients who were using antihypertensive or cholesterol lowering medications had three times higher odds of having a CVD (p\u2009<\u20090.001).After adjustment, only 6 out of the mentioned 11 factors remained significant predictors of CVD. Older age had one of the strongest associations with the CVD, and it increased the odds approximately five times (ea Table . Individp\u2009=\u20090.046) and for lifetime depressive episode (detected by the MINI) the OR was 2.11 (p\u2009=\u20090.002) (Table The odds ratio (OR) for current depressive symptoms (according to the PHQ-9) was 1.50 (p\u2009=\u20090.004) higher odds of having CVD in women, but not in men (data not shown).Gender stratified analysis of study population showed that current depressive symptoms (according to the PHQ-9) were associated with 2.04 (p\u2009=\u20090.002) times higher odds of having a CVD, and a lifetime depressive episode according to the MINI was associated with an adjusted OR for CVD of 1.52 .To the best of our knowledge, this is the first study in Latvia that explores the association between CVD and depression and anxiety in the primary care population. The main findings of this cross-sectional study were that individuals with current depressive symptoms (PHQ-9\u2009\u2265\u200910) demonstrated 2.08  published up to April 2014 with a follow-up duration ranging from 2 to 37\u00a0years. They found a more modest association of depression, with CHD and MI reporting pooled RRs of 1.30 (95% CI: 1.22\u20131.40) and 1.30 (95% CI: 1.18\u20131.44), respectively [An overview of 59 prognostic studies from 3 meta-analyses demonstrated that depression was associated with a 1.5- to 2.7-fold increased risk of incident CVD , 54. A mectively . Therefop\u2009=\u20090.001) but found no statistically important correlation between current anxiety symptoms and CVD. On the other hand, a cross-sectional study involving 1094 patients from 23 primary care practices across Estonia that explored which co-morbid diseases are associated with depression  did not confirm higher co-morbidity of CVD in depressed patients when compared to non-depressed individuals [Evidence about the comorbidity of depression and CVD in other Baltic countries is contradictory. A cross-sectional study in Lithuania involving 317 individuals from primary care centers in 20 cities investigated the association between psychosocial stress, manifested as anxiety and depression, and CVD using the Hospital Anxiety and Depression Scale (screening tool for detection of elevated anxiety and depressive symptoms) . Burokieividuals .Specific genetic, behavioral and pathophysiological factors have been established as contributing to the initiation, progression, and clinical manifestation of athero-thrombotic CVD in those suffering from depression and anxiety disorders. Behavioral factors are related to an unhealthy lifestyle  and poor treatment adherence  \u201360. PathIn contrast to depression, which in numerous studies has been linked to the increased morbidity and mortality of CVDs, less is known about the influence of anxiety symptoms and anxiety disorders. The most recent meta-analysis, summarizing a total of 37 studies including 1,565,699 participants, demonstrated a 52% increased risk of CVD onset  for patients with anxiety symptoms and disorders. This finding seemed to be independent of traditional CV risk factors and depression . The mosp\u2009=\u20090.25). This could be explained by the insufficient sample size of our study population. Only 89 individuals corresponded to the MINI diagnostic criteria of GAD. Similarly, a previously mentioned cross-sectional study in the primary care population of Lithuania (n\u2009=\u2009317) by Burokiene et al. also reported no significant correlation between anxiety and CVD [We have not found a statistically significant association of GAD (detected by the MINI) with CVD  for individuals living in the urban area of Riga  compared to those living in rural areas. This result seems to be consistent with the data obtained in the Prospective Urban Rural Epidemiologic cohort study involving more than 150,000 adults in 17 high-, middle-, and low-income countries [Another new finding was the highest adjusted odds ratio of 8.98  and lifetime depressive episode (according to the MINI) with increased risk of CVD morbidity; however, the same association was not found for anxiety disorders or current anxiety symptoms. Living in an urban area, especially in the capital city, has been established as an important associated factor that was rarely used in previous research. The findings of this study suggest that CV patients should be screened and treated for depression to potentially improve the prognosis of CVD. Enhanced training and integration of mental health treatment in Latvian primary care settings may improve clinical outcomes. Further research with a larger sample size should be undertaken to better assess the effect of anxiety on CVD morbidity and to investigate the optimal PHQ-9 threshold for the Latvian CVD population."}
+{"text": "Within-species hybrid incompatibility can arise when combinations of alleles at more than one locus have low fitness but where possession of one of those alleles has little or no fitness consequence for the carriers. Limited dispersal with small numbers of mate potentials alone can lead to the evolution of clusters of reproductively isolated genotypes despite the absence of any geographical barriers or heterogeneous selection. In this paper, we explore how adding heterogeneous natural selection on the genotypes  that are involved in reproductive incompatibility affects the frequency, size and duration of evolution of reproductively isolated clusters. We conducted a simulation experiment that varied landscape heterogeneity, dispersal ability, and strength of selection in a continuously distributed population. In our simulations involving spatially heterogeneous selection, strong patterns of adjacency of mutually incompatible genotypes emerged such that these clusters were truly reproductively isolated from each other, with no reproductively compatible \u201cbridge\u201d individuals in the intervening landscape to allow gene flow between the clusters. This pattern was strong across levels of gene flow and strength of selection, suggesting that even relatively weak selection acting in the context of strong gene flow may produce reproductively isolated clusters that are large and persistent, enabling incipient speciation in a continuous population without geographic isolation. Hybrid incompatibility refers to when hybrids between species exhibit reduced viability, lower fertility, and/or phenotypic abnormalities, and is a form of postzygotic reproductive isolation. A number of researchers have argued that hybrid incompatibility is important to the speciation process  from the two alleles, A and a. Selection is then implemented through differential survival of offspring as a function of the relative fitness of the offspring's genotype at the location on that surface where the dispersing individual settles . All loci experienced a 0.0005 mutation rate per generation  using the K allele model, a commonly used mutation model Balloux, , free reOur simulations combined dispersal in an isolation-by-distance (IBD) framework with heterogeneous natural selection for genotypes involved in reproductive incompatibility. The simulation modeling experiment involved all combinations of three factors  were each favored by selection in one of the two habitat types. We produced 10 replicate landscapes for each H-value to assess stochastic variation among simulated landscapes.The second factor is the pattern of landscape heterogeneity of two habitat types providing differential selection for the genotypes involved in heterogeneous selection. Specifically, we used the neutral landscape model, QRULE Gardner, , to simus) determined by genotypes at the selected loci. Selection strengths included s = 0.02 or \u201c2%,\u201d s = 0.04 or \u201c4%,\u201d s = 0.08 or \u201c8%,\u201d s = 0.16 or \u201c16%,\u201d s = 0.32 or \u201c32%,\u201d and s = 0.64 or \u201c64%\u201d (see Table A and B), two-allele selection model . We assumed that alleles a and B are incompatible and individuals that have these two alleles simultaneously have zero viability. This was implemented through relative fitness surfaces of 0.0 across the landscape for the genotypes AaBB, AaBb, aaBB, and aaBb as in Landguth et al.  if they occurred in \u201caabb\u201d habitat patches. AABb individuals had mortality less than 50% but greater than the favored AABB individuals. Individuals with aabb and Aabb genotypes experienced the opposite selection gradient from those of AABB and AABb, respectively. For example, in the s = 0.02 scenarios there would be a net 2% difference in fitness between aabb and AABB genotypes in the two habitat types, with AABB having 51% survival in its favored habitat type, and 49% survival in its disfavored type, while aabb would have 51% survival in its favored type and 49% survival in its disfavored type. The AAbb genotypes experienced a uniform selection of s = 0.5 or 50% mortality across the entire surface. Table Across these different heterogeneous landscapes and dispersal distances, we tested the third factor: strength of selection, defined as the difference of relative fitness of genotypes involved in hybrid incompatibility in the two habitat types and mediated in the simulations through density-independent  mortality  and (2) a RI event persisting in consecutive generations. To define an RI event, we used the density-based spatial clustering algorithm  within a neighborhood  emerged with the above criteria (RI events) was reported and averaged across the 10 Monte Carlo runs for each dispersal scenario. To assess persistence of RI events, we simply recorded the duration (in generations) of each RI event and reported the average time duration across each replicate and for each dispersal strategy. We also recorded the size of each RI event in terms of the number of individuals in the reproductively isolated cluster.Following Landguth et al. , we defiF-value was more than four times higher for selection and dispersal than for landscape heterogeneity, suggesting larger differences in cluster duration across levels of selection and dispersal than levels of habitat heterogeneity. There were significant interactions between landscape heterogeneity and selection and dispersal, and weaker interaction between landscape heterogeneity and selection.Factorial analysis of variance found highly significant main effects for landscape heterogeneity, strength of environmental selection, and dispersal ability on the mean duration that reproductively isolated clusters of individuals persisted in the simulations Table . The F-vTo explore the main effects and the predominant interaction between landscape heterogeneity and dispersal we produced histograms in a dispersal \u00d7 selection space, across the three levels of landscape heterogeneity , factorial analysis of variance found highly significant main effects for landscape heterogeneity, strength of environmental selection, and dispersal ability on the size of reproductively isolated clusters of individuals Table . The F-vThe histograms displaying size of reproductively isolated clusters across combinations of dispersal ability and strength of environmental selection Figures  size.aviLandguth et al.  found thIn this paper, we show that adding heterogeneous selection for the genotypes involved in reproductive isolation led to dramatic increases in the duration, number, and size of reproductively isolated patches. Landguth et al.  found thWe also found that strength of selection and dispersal ability affect the size, duration, and number of isolated clusters in roughly the same degree, and much more so than does the heterogeneity of the landscape. However, landscape heterogeneity does have substantial effects, such that when there is extremely high heterogeneity reproductively isolated clusters are less likely to emerge since there is a highly mixed pattern of selection that inhibits formation of large, aggregated clusters. This suggests that in evolutionary landscape genetics, as well as neutral differentiation (e.g., Cushman et al., In addition to the much larger total number, size, and duration of reproductively isolated patches when there is environmental selection, the pattern of cluster adjacency changes in critical ways that enable persistence of reproductively isolated clusters and therefore the potential for incipient speciation. Specifically in the Landguth et al.  simulatiIn contrast, when we added environmental selection on the genotypes involved in hybrid incompatibility very strong patterns of adjacency of mutually incompatible genotypes emerged such that these clusters were truly reproductively isolated from each other as there were no other reproductively compatible \u201cbridge\u201d individuals in the intervening landscape to allow gene flow between the clusters. This pattern was very strong across levels of gene flow and strength of selection, suggesting that even relatively weak selection acting in the context of strong gene flow may produce reproductively isolated clusters that are large and persistent, enabling incipient speciation in a continuous population without geographic isolation.There are several lines of future work which should be explored to extend the scope of what was found in this paper. First, this paper used a simple two-locus model of hybrid incompatibility. While this is a model that is widely used in theoretical evolutionary ecology (Dobzhansky, SC and EL designed research. EL ran simulations. SC and EL performed the analyses, interpreted the data, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Terrestrial laser scanning (TLS) has become a powerful data acquisition technique for high-resolution high-accuracy topographic and morphological studies. Conventional static TLS surveys require setting up numerous reflectors (tie points) in the field for point clouds registration and georeferencing. To reduce surveying time and simplify field operational tasks, we have developed a rapid TLS surveying method that requires only one reflector in the field. The method allows direct georeferencing of point clouds from individual scans to an East\u2013North\u2013Height (ENH) coordinate system tied to a stable geodetic reference frame. TLS datasets collected at a segment of the beach\u2013dune\u2013wetland area in Freeport, Texas, USA are used to evaluate the performance of the rapid surveying method by comparing with kinematic GPS measurements. The rapid surveying method uses two GPS units mounted on the scanner and a reflector for calculating the northing angle of the scanner\u2019s own coordinate system (SOCS). The Online Positioning User Service (OPUS) is recommended for GPS data processing. According to this study, OPUS Rapid-Static (OPUS-RS) solutions retain 1\u20132 cm root mean square (RMS) accuracy in the horizontal directions and 2\u20133 cm accuracy in the vertical direction for static observational sessions of approximately 30 min in the coastal region of Texas, USA. The rapid TLS surveys can achieve an elevation accuracy (RMS) of approximately 3\u20135 cm for georeferenced points and 2\u20133 cm for digital elevation models (DEMs). The elevation errors superimposed into the TLS surveying points roughly fit a normal distribution. The proposed TLS surveying method is particularly useful for morphological mapping over time in coastal regions, where strong wind and soft sand prohibit reflectors from remaining strictly stable for a long period. The theories and results presented in this paper are beneficial to researchers who frequently utilize TLS datasets in their research, but do not have opportunities to be involved in field data acquisition. Terrestrial laser scanning (TLS), also known as ground-based light detection and ranging (LiDAR), has become a powerful research tool for tracking ground surface deformation over time and space, such as landslide movement ,2,3,4,5,Laser measurements require line-of-sight (LOS). An object that is not in the sight of a TLS scanner will not be scanned. Since natural topography is usually not flat and smooth, numerous scans are required from different directions to fully scan field objects. For example, for scanning a landslide in Puerto Rico with an approximate area of 150 m by 200 m, 25 individual scans were conducted to fully cover the landslide surface [The theories behind the registration and georeferencing are the same: using a seven-parameter Helmert transformation algorithm to align two three-dimensional (3D) Cartesian coordinate systems. In general, registration focuses on stitching point clouds from individual scans together according to certain common points or topographic features, while georeferencing focuses on aligning point clouds to a regional geodetic coordinate system according to GPS measurements. A minimum of three common points (reflectors) is required to align two 3D Cartesian coordinate systems, but a greater number of common points will increase the numerical stability and reliability of the transformation. In practice, five or more common reflectors are often used for deriving key parameters for each coordinate transformation, and ten or more reflectors are used in a field project to make sure two neighbor scans share at least three common reflectors. As a consequence, conventional TLS surveys require numerous tripods, reflectors, GPS units, and significant manpower in the field for point clouds registration and georeferencing. For our previous TLS survey projects at the Freeport beach area  and the The authors have been monitoring coastal erosion problems in Freeport, Texas, since the summer of 2013. A 7-km long and 0.5-km wide beach\u2013dune\u2013wetland segment has been periodically scanned twice per year, using the rapid surveying method since 2015 and the conventional surveying method in earlier years . A RieglTLS points are initially positioned with respect to a scanner-intrinsic spherical coordinate system consisting of one horizontal rotation angle and one vertical altitude angle of the laser source at the moment the laser pulse is sent out. The spherical coordinates are transformed into a 3D right-handed Cartesian coordinate system (XYZ) with respect to the SOCS . For thehttp://www.riegl.com/products/software-packages/riscan-pro), the companion software package for Riegl\u2019s 3D laser-scanners, was utilized to manage field data acquisition, registration, georeferencing, and the basic process. The primary process included removing outliers, octree filtering, and terrain filtering. The size of the cabin of the octree filtering was 3 cm in the horizontal directions and 1 cm in the vertical direction. The pre-processed coordinates were exported into the American Standard Code for Information Interchange (ASCII) format. The ASCII files were further processed with the Generic Mapping Tools (GMT) software package [In this study, the RiSCAN PRO 2.0  for the case study. The value of the orthometric height was calculated according to the GEOID12B model [The essence of georeferencing is to transform point clouds from XYZ-coordinates with respect to a SOCS or a PROCS to a regional geodetic coordinate system. In surveying and geodesy communities, a geodetic coordinate system is often described by easting (E), northing (N), and height (H). In practice, E and N coordinates are often aligned to the easting and northing coordinates of the Universal Transverse Mercator (UTM). The study area was located in UTM Zone 15R. 2B model . In Nort2B model . A stabl2B model ,28,29. TThere are several classical methods for coordinate transformation between two Cartesian coordinate systems. The Helmert transformation is the one that is frequently used in geodesy to produce distortion-free transformations between two 3D Cartesian coordinate systems. The transformation requires seven parameters: three translations  to replace the onboard single-frequency GPS unit. For the Riegl VZ-2000 scanner a, the prgle (Rz) a,b. The As mentioned earlier, the rapid surveying method utilized seven parameters for georeferencing. Four of these seven parameters were derived from static GPS observations on the scanner and the reflector\u2014munities . OPUS offers two approaches, OPUS Static (OPUS-S) and OPUS Rapid-Static (OPUS-RS), for solving a static position. OPUS-S is used for observations longer than two hours; OPUS-RS is used for observations longer than 5 min but less than 2 h. OPUS-S solves a position using three single baselines from three known Continuously Operating Reference Stations (CORS). OPUS-RS solves positions for shorter sessions (<2 h) by interpolating the troposphere and ionosphere over the user\u2019s point, using up to nine nearby CORS. Detailed information about CORS and OPUS may be consulted in a number of articles included in a monograph edited by Soler , and recAccording to our field experience, it takes approximately 5 min to set up equipment at a scan site, 15 min for the scanner to finish a panorama scan, and 10 min for identifying, rescanning the reflector, and packing up. The GPS unit mounted on the scanner is able to collect about 25 min of static data at a scan site. In practice, the reflector is often set up at a relatively high site and can be used for four or more scans. Thus, the GPS unit on the reflector often collects over two hours of data. GPS data collected on the scanner and reflector can be processed by OPUS-RS and OPUS-S, respectively. The performance of OPUS-S within the greater Houston region has been investigated by Wang and Soler  and WangThe U.S. Geological Survey (USGS) has been conducting repeated coastal surveys in Texas coasts for several decades. A permanent surveying benchmark was installed on a concrete bridge near the Freeport beach . The benhttp://www.rtklib.com), which is an open source package for precise Global Navigation Satellite Systems (GNSS) positioning. The performance of RTKLIB has been investigated by previous studies [In order to assess the elevation-accuracy of the TLS points georeferenced by the rapid surveying method, kinematic GPS surveys were performed in two washover-fans, as shown in  studies ,43,44. IAs aforementioned, the vertical accuracy of the kinematic GPS measurements was below 1 cm. So, the GPS measurements can be regarded as true references to assess the elevation-accuracy of TLS measurements. Bare earth DEMs are often regarded as final products of TLS topographic surveys. By default, the accuracy of TLS surveys is often referred to as the elevation-accuracy of DEMs rather than discrete laser points. The accuracy is often assessed by comparing the elevations of DEM grids with kinematic GPS measurements. As mentioned earlier, the vertical RMS-accuracy (uncertainty) of OPUS-RS solutions for 15 to 25-min sessions is also at a level of 2 to 3 cm d,f. ThusAs aforementioned, the essence of registration and georeferencing is the seven-parameter coordinate transformation, which assumes that the crust block covered by the reference points is rigid. Nevertheless, certain coordinate distortions will be induced during each coordinate transformation because the surveying block is not strictly rigid. The distortion can be significant if the transformation utilizes a few reflectors while covering a large area. The conventional surveying method uses approximately five reflectors with GPS measurements (GPS-reflectors) to transform the PROC coordinates of the whole study area to a regional geodetic reference frame. As a consequence, the conventional surveying method often produces considerable coordinate distortions (errors), particularly in the areas where the distribution of GPS-reflectors is sparse. However, the rapid surveying method introduced in this article directly transforms point clouds from individual scans that cover a small area to a regional geodetic coordinate system. Thus, the rapid method avoids or minimizes coordinate distortions during coordinate transformations. The spatial distribution and numbers of tie points (reflectors) are critically important for aligning two coordinate systems. According to our experiences from landslide projects, georeferenced TLS points, as well as DEMs, do not retain a consistent or homogeneous elevation accuracy in space ,18,45. TA map depicting the differences between two DEMs acquired at different times is often called DDEM. DDEM and topographic profiles are highly effective in depicting topographic changes  over time and space ,46,47. TIt appears that the DDEM map and cross-dune profiles did a great job of quantitatively depicting the coastal topographic changes over space and time. However, TLS data users need to realize that the elevation-accuracy of a DEM is a function of the density and accuracy of survey points, gridding resolution, method of interpolation, and the complexity (or roughness) of terrain features. Data gaps and rough terrains can significantly degrade the accuracy of DEMs ,49. The LiDAR techniques have resulted in a new era of micro-topography analysis, using dense topographic measurements over a large geographic area. This study introduced a rapid TLS surveying method that takes advantage of onboard inclination sensors equipped with model laser scanners. According to this investigation, the overall accuracy of the georeferenced TLS points is dominated by the positional accuracy of the GPS antennas on the scanner and reflector; the onboard inclination sensors contribute a minor fraction of the total error budget. We proposed to use OPUS-S and OPUS-RS for GPS data post-processing. The accuracy of OPUS-RS solutions strongly depends on the length of the occupation period in the field. According to this study, OPUS-RS solutions (15 to 25 min observations) would retain a 1.5-cm horizontal accuracy and a 2 to 3-cm vertical accuracy in the Texas coastal region. For surveyors who want to further improve the positional accuracy of GPS and, in turn, the accuracy of TLS surveys, the advice is quite simple: slow down the scanning process and collect a longer period of GPS data at the scanning site. A 40-min GPS data session will be able to considerably improve the positional accuracy compared to a 20-min data session, particularly in the vertical direction ,38. OPUSTLS and GPS integrated technologies have provided practicing geologists with a new way to map the Earth\u2019s surface at an unprecedented spatial resolution and accuracy over large areas and long-time spans, which is opening new ways of investigating coastal erosion problems. Many key scientific questions require high-resolution, high-accuracy, and repeated topographic datasets. These standards demand creative and well-designed data acquisition and processing procedures. This article introduced a rapid TLS surveying method for high-resolution (handprint) and high-accuracy (<5 cm) topographic mapping. The surveying method utilized one scanner, two GPS units, and only one reflector in the field. A team of two field crews is able to conduct over 20 scans within an 8-h fieldwork time. The proposed rapid surveying method will benefit both researchers and professional surveyors who use TLS for high-accuracy terrain mapping and geomorphological studies. This investigation indicated that OPUS-RS can achieve 1 to 2 cm horizontal accuracy (RMS) and 2 to 3 cm vertical accuracy for static observations of approximately 30 min in the Texas coastal region. The rapid TLS surveys can achieve an elevation-accuracy (RMS) of approximately 3 to 5 cm for georeferenced points and approximately 2 to 3 cm for TLS-derived DEMs. The elevation errors superimposed into the georeferenced TLS points roughly fit a normal distribution.Recently, mini unmanned aerial vehicles (mini-UAVs) bearing LiDAR and photogrammetry techniques have been demonstrated as powerful tools for topographic mapping in coastal regions ,53,54,55"}
+{"text": "United States (US) and European Union (EU) legislation attempts to counterbalance the presumed discrimination in pediatric drug treatment and development.We analyzed the history of drug development, US/EU pediatric laws, and pediatric studies required by US/EU regulatory authorities and reviewed relevant literature.The US and EU definitions of a child are defined administratively  as being aged <17 years and <18 years, respectively. However, children mature physiologically well before their seventeenth or eighteenth birthdays. The semantic blur for these differing definitions may indicate certain conflicts of interest.Pediatric healthcare today is better than ever. Regulatory-related requirements for \u201cpediatric\u201d studies focus on labeling. Most of these studies lack medical usefulness and may even harm \u201cpediatric\u201d patients through administration of placebo and/or substandard treatment, despite the resultant publications, networking, patent extensions, and strengthened regulatory standing. Clinicians, parents, and ethics committees should be aware of these issues. New rules are needed to determine new pharmaceutical dose estimates in prepubescent patients, and when/how to clinically confirm them. Internet-based structures to divulge this information should be established between drug developers, clinicians, and regulatory authorities. A prerequisite for the rational use of pharmaceuticals in children would be to correct the flawed concept that children are discriminated against in drug treatment and development, and to abandon separate \u201cpediatric\u201d drug approval processes. Preconceptions and long-standing traditional treatments can be tenacious and difficult to change. There have been many incorrect opinions related to a number of pediatric conditions, including the idea that depression,Legislation in the USA and the European Union (EU) has sought to counterbalance the presumed discrimination in pediatric drug development and treatment.In 1900, few efficient systemic drugs existed, and industrial production of antibiotics only began during the Second World War. Today, drug efficacy and benefit\u2013risk ratios are not only well documented but also undergo an extensive approval process. Pharmaceuticals have a huge impact on society, ranging from intended medical use to their societal and economic impact: prescribed by physicians, pharmacy sales, public debates, insured versus uninsured costs and reimbursement, healthcare jobs, therapeutic expectations by the public, and more. In addition, the role of regulatory authorities has transitioned from administrative organizations to powerful institutions, and clinical studies now have a major role in the drug approval process. Drugs both make lethal diseases treatable,pre-approval clinical studies, a principle now accepted worldwide.25Originally \u201cdrug labels\u201d (labeling) simply described the packaged medication. Since 1906, drug labeling has evolved to include therapeutic characteristics.Central regulatory authority involvement has changed and shaped society\u2019s relationship with medications. Clinical studies are regarded as the gold standard for drug treatment decisions. Participation in clinical studies and publications have become key factors in a clinicians\u2019 career. Many studies are sponsored by companies that anticipate retrieval of invested money via post-approval sales.Conflicts of interest exist when professional judgment concerning a primary interest, including patient welfare or the validity of research, may be influenced by another interest. Healthcare, approval of effective drugs, and the pharmaceutical industry itself are entangled in a world where conflicts of interest abound. Beyond financial compensation, clinicians profit from participation in international studies by related international meetings, networking, conference presentations, and publications. \u201cEvidence-based medicine\u201d suggests that medical decisions are based on evidence.27This review discusses\u2014and thereby opens a Pandora\u2019s box for\u2014these issues.We reviewed literature related to the history of drug development, the FDA approval process, and how children entered into this equation. We examined the ramifications of international recruitment, EU pediatric legislation, exemplary clinical areas for pediatric studies triggered by regulatory decisions, and how academia reacted and behaved. Regulatory documents were internet-retrieved.The concept of children as \u201ctherapeutic orphans\u201d began in 1962, when the FDA started to control prescription medicine advertising and corporate lawyers started inserting specific pediatric warnings into drug labeling.34The FDA\u2019s concept is discussed and justified in several papers, claiming that: (1) drugs prescribed for children were not sufficiently studied in children; (2) pharmaceutical companies have limited interests to study drugs in \u201cchildren;\u201d and (3) lack of pediatric studies and pediatric labeling leads to additional risks.39(pp91\u201392) These positions would be true if children remained as vulnerable as premature newborns until their seventeenth birthday.The focus of pediatric FDA-requested studies has been regulatory, not clinical. A connection between pediatric labeling, improved clinical use, and avoidance of adverse events is claimed;40Clearly, the needs of very young children versus older children have been confused. The limited awareness of infant vulnerability to drugs in the 1950s/1960s has been translated into a generalized warning of alleged treatment dangers in \u201cchildren,\u201d ignoring physical maturation. The literature maintains this semantic blur for different meanings of \u201cchildren,\u201d i.e. the very young versus the FDA-defined child <17.In 2006, the EU defined \u201cchildren\u201d as being under 18 years of age (<18).The EMA PIP scheme is mechanistic, often requiring placebo-controlled efficacy studies for multiple sclerosis,The confusion engendered by the administrative definitions of \u201cchild\u201d and the bureaucracy involved for pharmaceutical approvals is evident in a number of clinical areas.Use of antidepressants in pediatrics has a confusing history that leaves physicians in a quandary. Contributing to this is the administrative definition of \u201cchildren,\u201d which prevents young patients from receiving effective depression treatment. Suicide in young persons is a higher-ranked cause of death than malignomas.Today, pediatricians and psychiatrists treating depression in minors must either follow regulatory recommendations or administer off-label prescriptions for potentially life-saving medications. The American Psychiatric Association (APA) stated that antidepressants save lives, with \u201cno care at all\u201d being the greatest threat to a depressed child. It expressed concerns that the black-box warning could reduce appropriate prescribing. The American Academy of Child and Adolescent Psychiatry (AACAP) has stated that the black-box warning was inconsistent with research and clinical experience.Pediatric oncology studies triggered by the FDA endorse the semantic blur of bureaucratically defined \u201cchildren.\u201d These studies assumed that, except for CML, the biology of malignancies differs in adults versus children.68The FDA issued 25 written requests for carcinoma drugs; only clofarabine, everolimus, and imatinib received pediatric labels.69The pemetrexed written request asked to investigate \u201crefractory or relapsed pediatric malignancies\u201d in infants >1 month to adolescents.71All but one of the FDA-triggered oncology studies were open-label with one chemotherapeutic agent.75One FDA-program rewarded pediatric melanoma studies with ipilimumab, but 13 PIPs demanded \u201cpediatric\u201d studies in solid tumors, including melanoma. Two PIP-demanded \u201cpediatric\u201d melanoma studies had to be terminated because monotherapy with ipilimumab and vemurafenib, respectively, became sub-standard and recruitment waned.32Fludarabine studies revealed relatively low efficacy.80The FDA-required pediatric oncology studies were not designed to promote survival and quality of life, as did the earlier studies performed by the pediatric oncology researchers,The FDA-requested pediatric oncology studies were mostly performed in heavily pretreated, relapsed or refractory patients,75The FDA-requested pediatric oncology studies were not scientifically motivated, in contradistinction to the pediatric oncology networks studies.Finally, not all malignancies in underage patients are \u201cpediatric\u201d cancers\u2014for example, conventional melanoma,Hypertension, frequent in adults, is rare in younger patients. The FDA rewarded \u201cpediatric\u201d antihypertensive studies,Four oral drugs for type 2 diabetes mellitus (T2DM) have been FDA-evaluated for \u201cpediatric\u201d use: metformin, glimepiride, rosiglitazone, and metformin+ glyburide. Each drug reduced glycemic parameters; three failed to reach the FDA-demanded efficacy threshold, and only metformin was FDA-approved in \u201cchildren.\u201d These studies did not contribute to better diabetes treatment in children. The academic publication described \u201cpediatric drug development\u201d as what it is: regulatory approval/non-approval of drugs whose efficacy in humans is already well proven.89Neonatology has continuously advanced.91Chloramphenicole toxicity in neonates is well known. The toxicities in preterm newborns that triggered industry\u2019s pediatric warnings occurred with antibiotics.Lacking scientific rationale are the FDA and EMA requirements for active-controlled or even placebo-controlled comparisons of anti-inflammatory compounds in multiple sclerosis (MS).Pediatric clinical pharmacology had and has a key role in promoting separate \u201cpediatric\u201d studies.www.accelerate-platform.eu/) discusses pediatric oncology studies. Without acknowledging the flaws of US/EU pediatric legislation, it recommends inclusion of adolescents in promising adult cancer studies,97The multi-stakeholder group \u201cACCELERATE\u201d  epilepsy, efficacy extrapolation is accepted from adults down to 4-year-olds;47As stated above, \u201cpediatric drug development\u201d originated as a response to the US thalidomide catastrophe. New procedures were imposed on the administratively defined pediatric population in an attempt to keep dangerous substances from the market and improve pediatric healthcare; the main proponent were the AAP and the FDA, endorsed by the clinical community. For many practitioners common sense prevailed, as noted already by Shirkey in 1968: most clinicians ignored the pediatric warnings.The key issue is the administrative definition of the \u201cpediatric\u201d population: <17/<18.110The flawed definition of \u201cchildren\u201d\u2014supplemented by FDA assumptions about pediatric cancerMany publications on \u201cpediatric\u201d studies pretend to investigate a scientific question and omit mentioning that the studies were FDA/EMA-required.8Ongoing research is required in neonates. Institutional review boards/ethics committees should re-assess all ongoing pediatric studies. Those found to be questionable should be suspended and newly submitted questionable ones rejected.Based on this review, we believe that (1) treatment of neonates, infants, children, adolescents, and adults is today better than ever in history; (2) the justifications for separate \u201cpediatric drug development\u201d are flawed; (3) these flaws are not a conspiracy, but reflect the complex path toward rational use of drugs, biologics, devices, and technology; (4) the flawed concept of \u201cpediatric drug development\u201d has created many conflicts of interest since becoming US law; (5) this flawed US-born concept was adopted and augmented by the EU; (6) the EU further potentiated conflicts of interest of many parties and institutions that profit from separate \u201cpediatric\u201d studies; (7) the EU exaggerations finally facilitated detection of the fundamental flaws; and (8) academic critical reflections have not pinpointed the flaws of \u201cpediatric drug development.\u201d112It is possible that underlying conflicts of interest will trigger angry responses from individuals, parties, and institutions; the ensuing public debate could rock public trust in science and institutions. Nevertheless, conflicts of interest are not only financial and do not follow traditional boundaries of institutions; hence, protective mechanisms against fraud and professional misconduct are needed and should be reflected in required revisions to US/EU pediatric legislation. Also, the International Committee of Medical Journals Editors (ICMJE) guidelines should be accordingly revised.The current framework allowed and allows pediatric researchers on the one hand to support pediatric legislation and demand more pediatric studies, but on the other hand to omit, in the resulting study publications, the regulatory background and the reason companies had to sponsor the studies.The FDA- and EMA-triggered \u201cpediatric\u201d studies might represent the largest abuse of patients in medical research, dwarfing even the atrocities unveiled by Beecher in 1966 and other projects that led to the Belmont Report.115New guidelines are needed, including when and how drug developers should estimate doses in prepubescent patients and when and how to confirm them in \u201copportunistic\u201d settings.Finally, a prerequisite to moving ahead is requiring the FDA, EMA, AAP and its European counterparts to reject the flawed concept of children as \u201ctherapeutic orphans\u201d and the need for separate drug approval in \u201cchildren.\u201d This flawed concept is now outdated and should be discarded."}
+{"text": "To date, there have not been any workforce-based Japanese cohort studies investigating work sustainability after return to work (RTW). The objective of this study was to investigate the post-RTW cumulative recurrent sick leave rate and cumulative resignation rate among female cancer survivors.Among Japanese employees who were registered in the Japan sickness absence and return to work (J-SAR) study, the subjects were those female employees who returned to work after sick leave due to newly clinically diagnosed cancer , based on a physician\u2019s certificate, between 2000 and 2011. The last day of the follow-up period was December 31, 2012. The recurrent sickness leave rate and resignation rate were calculated using competing risk survival analysis.Of 223 cancer survivors, 61 took further physician-certified sick leave after their RTW. The median duration of the post-RTW work period among all cancer survivors was 10.6\u2009years. The work continuance rates of the female cancer survivors were 83.2 and 60.4% at 1 and 5\u2009years after they returned to work, respectively. There was a steep reduction in the work continuance rate during the first post-RTW year. There were considerable differences in the work continuance rate according to the primary cancer site. Cumulative recurrent sick leave rates of 11.8 and 28.9% were seen at 1 and 5\u2009years after the subjects returned to work. The cumulative resignation rate was 5.0 and 10.7% at 1 and 5\u2009years after the subjects returned to work. Most recurrent sick leave occurred in the first year after the subjects returned to work, followed by the second year.Sixty percent of female cancer survivors were still working at 5\u2009years after returning to work, although the work continuance rates for different types of cancer varied significantly. According to the statistics of the Ministry of Internal Affairs and Communication, Japan 2010), the working age population in Japan includes 81.3 million people; however, it is estimated that it will decrease in size by almost 50% in the next 50\u2009years 0, the wo. In JapaAs cancer survival rates have improved due to recent advances in cancer treatment, more cancer survivors are able to resume their everyday lives and return to work (RTW) during or after treatment , 7, 8. WIn February 2016, the \u201cguidelines for the support of therapy and working life in the Japanese workforce\u201d were published by the Japanese Ministry of Health, Labour and Welfare . As a reWhile returning to work would seem to indicate that a cancer survivor has completely recovered from their illness and the adverse effects of treatment, it actually only demonstrates that their ability to perform their job has recovered to a level where they can be reinstated. After returning to work, cancer survivors often encounter difficulties associated with the balance between the demands of treatment and recovery and the requirements of the workplace \u201314. SomeAlthough a number of studies of cancer survivors have been carried out in Europe and the US, there have not been any workforce-based Japanese cohort studies investigating the frequency of recurrent sick leave or resignations among female cancer survivors that they RTW , 19\u201322. The objective of this study was to investigate the cumulative recurrent sick leave rate and cumulative resignation rate after Japanese female cancer survivors RTW and to analyze predictors of recurrent sick leave, resignation between different cancers. This study is the first cohort study to examine the work sustainability of cancer-affected female employees in Japan.The Japan sickness absence and return to work study (J-SAR study) was a retrospective, observational cohort study conducted in Japan. The J-SAR study analyzed data regarding sick leave that were registered in the health data system of a private occupational health center. This occupational health center had signed occupational health service with the following companies: regional, international telecommunication, big data and mobile services , logistics, energy, and construction etc., as described elsewhere , 23\u201328. In Japan, there is no law insuring sickness absence for employees who are not able to work. To our knowledge, almost all large Japanese companies have their own sickness insurance system for employees. The time limit for sickness absence varies depending on the company. In the Japanese sickness absence system, part-time sickness absence combined with part-time work is not so common. The fact is that many small and medium-sized enterprises in Japan do not have such an established sickness insurance system. We guess that cancer survivors who work at small and medium-sized enterprises have no choice but to quit because of their companies\u2019 economic circumstances, among other factors. On the other hand, this Japanese company group has more improved sickness absence system and RTW system than other companies, as described in our previous studies , 23\u201328.The subject inclusion criteria for this study were as follows: female employees, aged 18 to 60\u2009years, who returned to work after their first period of sick leave due to clinically diagnosed cancer  based on a physician\u2019s certificate between January 1st, 2000, and December 31st, 2011. Two hundred forty-five female employees had sick leave due to cancer newly during the duration, as described in our previous study . Of thesThe work continuance rate was estimated via Kaplan-Meier survival analysis. Numbers of person-days were calculated based on the follow-up period. The day used to measure the beginning of the follow-up period was the first day of the subject\u2019s RTW after their sick leave due to cancer. In this study, the data were censored at the end of the follow-up period  or the day of retirement (March 31 of the year that the subject became 60\u2009years old), whichever came first. The event day for this analysis was the first day of recurrent sick leave due to any physician-certified illness or the day of resignation before retirement. Deaths were analyzed as \u201crecurrent sick leave\u201d. During the follow-up period, 12 female employees died after RTW.Cumulative recurrent sick leave and resignation rates were calculated via competing risk survival analysis . A survin\u2009=\u200990). The second most common type of cancer was female genital malignancy, which included cancer of the uterus (n\u2009=\u200947) and ovarian cancer (n\u2009=\u200917). The third most common type of cancer was gastric cancer (n\u2009=\u200918), followed by lung cancer (n\u2009=\u200916). The other types of cancer (n\u2009=\u200935) seen among the employees included colon cancer (n\u2009=\u20095) and esophageal cancer (n\u2009=\u20093). The subjects\u2019 mean age on the initial day of their sick leave was 48.2\u2009years old. Just after returning to work, 185 cancer survivors (83.0% of RTW employees) had their work schedules reduced based on OP certificates, and 38 cancer survivors were able to work full-time.In total, 223 female employees returned to work after their first period of sick leave due to cancer. The subjects\u2019 characteristics are shown in Table\u00a0After returning to work, 61 subjects (27.3%) experienced further periods of sick leave (recurrent sick leave) due to medical reasons, which were certified by their physicians. Of these 61 employees, 43 experienced recurrent sick leave due to their original cancer, 4 developed new types of cancer (other cancer), 4 suffered from common mental disorders, and 3 suffered fractures. Twenty-two cancer survivors resigned before reaching retirement age (60\u2009years). Among all cancer survivors, the median duration of the period of work after the subjects initially returned to work was 10.6\u2009years. During the follow-up period, 12 female employees died after the day of RTW. Of the 12 deaths, 9 employees died within the period of recurrent sick leave.According to Kaplan-Meier survival estimates, the work continuance rate of the female cancer survivors was 88.2% at 6\u2009months, 83.2% at 1\u2009year, 73.1% at 2\u2009years, 69.6% at 3\u2009years, 64.2% at 4\u2009years, and 60.4% at 5\u2009years after they initially returned to work, as shown in Fig.\u00a0The work continuance rate of the subjects with breast cancer patients was 82.9% at 1\u2009year, 73.8% at 2\u2009years, and 63.4% at 5\u2009years. The work continuance rate of the subjects with lung cancer was 70.5% at 1\u2009year, 62.5% at 2\u2009years, and 31.3% at 5\u2009years, and these values were lower than those of the other types of cancer. As shown in Fig.\u00a0As far as we know, this is the first longitudinal cohort study to investigate the work sustainability of female cancer survivors after they RTW. We found that about 60% of female cancer survivors were still working at 5\u2009years after their RTW, although the work continuance rate differed significantly among different cancer types. In particular, the lung cancer patients exhibited had the lowest work continuance rate. Though there were no statistically significant difference between different cancers, as for recurrent sick leave and resignation, that could be caused by the rather small sample in this study.n\u2009=\u200961), 25.0, 32.4, and 53.6% required such leave within 6\u2009months, 1\u2009year, and 2\u2009years of their RTW, respectively. The competing risk curve for recurrent sick leave plateaus at 5\u2009years after a cancer survivor returns to work, and hence, is very similar in shape to the Kaplan-Meier curve of recurrent sick leave due to depression produced in a previous study [The main work-related problems experienced by female cancer survivors after they RTW are as follows: the first is recurrent sick leave due to a physician-certified medical reason (including death). As shown in Fig. us study .The second problem experienced by female cancer survivors is having to resign before they reach retirement age (60\u2009years old). In the present study, the cumulative resignation rate was unexpectedly low. The survival curve of the cumulative resignation rate remained below 10% until 4\u2009years after the subjects returned to work. Of all the post-RTW resignations, 17.9, 33.1, 49.0, 56.2, 65.6, and 70.9% had occurred within 6\u2009months, 1\u2009year, 2\u2009years, 3\u2009years, 4\u2009years, and 5\u2009years after the subjects returned to work. Short et al. reported that about 10% of cancer survivors resigned from their jobs within a 4-year period because of cancer-related factors . StewartFemale cancer survivors\u2019 work continuance rates (60.4%) at 5\u2009years after the RTW day were higher than male cancer survivors (48.5%) . We specAs for the strengths of this study, it was the first longitudinal study (it covered a period of >\u200910\u2009years) to investigate the frequencies of recurrent sick leave and resignation after female cancer survivors RTW. Secondly, the registered sick leave data analyzed in this study were based on physicians\u2019 certificates, which increases the study\u2019s validity.As for the limitations of this study, the results of this study should be interpreted carefully. First, the patients\u2019 medical files were not available, and so we did not have any information about the treatments the subjects received, the side effects previous treatments induced, or cancer-related symptoms. As a future task, the effects of clinical factors  on work sustainability among female cancer survivors should beconsidered. Especially, CrF is known to be one of the most influential factors for work sustainability, caused by surgery, chemotherapy, radiotherapy, and hormone treatment \u201344. SecoSixty percent of female cancer survivors were still working at 5\u2009years after they returned to work, although the work continuance rate differed significantly among the various types of cancer. Recurrent sick leave occurred most frequently in the first year after the subjects returned to work, followed by the second year."}
+{"text": "This study aimed to clarify the psychological benefits of brief walks through forest areas. In addition, we aimed to examine the associations between psychological responses and trait anxiety levels. Five-hundred-and-eighty-five participants  were instructed to walk predetermined courses through forest (test) and city (control) areas for 15 min. The Profile of Mood State (POMS) questionnaire and State-Trait Anxiety Inventory were used to assess participants\u2019 psychological responses and trait anxiety levels, respectively. The results revealed that walking through forest areas decreased the negative moods of \u201cdepression-dejection\u201d, \u201ctension-anxiety\u201d, \u201canger-hostility\u201d, \u201cfatigue\u201d, and \u201cconfusion\u201d and improved the participants\u2019 positive mood of \u201cvigor\u201d compared with walking through city areas. Furthermore, a significant correlation was found between participants\u2019 trait anxiety levels and their changes in the subscale of \u201cdepression-dejection\u201d of POMS after walking through forest areas. A more effective reduction in the feeling of \u201cdepression-dejection\u201d after walking through forest areas was observed for participants with high trait anxiety levels than for those with normal and low trait anxiety levels. This study showed the psychological benefits of walking through forest areas and identified a significant correlation between psychological responses to walking through forests and trait anxiety levels. As individuals are exposed to several stressors in daily life, they attempt to adopt effective methods for coping with stress and for relaxing. Nature-based experiences have relaxing effects, and the therapeutic effect of nature has received increasing attention.The positive effects of nature on physical and mental health have been recently reported ,2,3,4, aNumerous studies have focused on and demonstrated the effects of forests in mitigating stress states and inducing physiological relaxation ,10,11,12However, these studies were limited to small sample sizes, and individual differences within these effects have been noted. A previous study revealed the individual differences in changes of blood pressure after walking through forests . AlthougRegarding psychological aspects, the restorative effects of natural environment, including forests, that are associated with psychological stressors or mental fatigue, decreased depressive symptoms, and improved mood states have been reported ,31,32,33Individual differences also exist with regard to psychological responses, and this phenomenon requires further clarification. We previously examined individual differences in changes of mood states such as \u201cdepression-dejection\u201d, \u201ctension-anxiety\u201d, \u201canger-hostility\u201d, \u201cfatigue\u201d, \u201cconfusion\u201d, and \u201cvigor\u201d after walking and viewing forests and found significant correlations between them and participants\u2019 initial values . HoweverWith a large sample population, this study aimed to clarify the psychological benefits of brief walks through forest areas. In addition, we assessed the associations among changes in the mood state of \u201cdepression-dejection\u201d after walking through forest areas and trait anxiety levels because mental health problems such as depression and high anxiety are common in modern societies.From 2005 to 2013, we performed experiments in 52 forest and city areas of Japan. Twelve male Japanese university students participated in each experiment , and no student reported a history of physical or psychiatric disorder. Of the 624 participants, data from 585 participants  were analyzed. The demographic parameters of the participants are given in Twelve participants visited the orientation site in each experimental region on the day before (39 areas) or the morning of (13 areas) the experiment. Before initiating the experiments, we explained the aims and procedures of the study to all participants and obtained their written informed consent. Participants were randomly divided into two groups of six persons. On the first day, one group performed the experiment in the forest area, and the other performed the same experiment in the city area. On the second day, participants switched field sites to eliminate order effects. On arrival in the forest or city area, the participants awaited their turn in a waiting room and were eventually taken, one by one, to the experimental site. Each participant took a walk along the given course for approximately 15 min .For evaluated participants\u2019 mood state, the Profile of Mood State (POMS) questionnaire was used. POMS is a well-established, factor analytically-derived measure of psychological distress, and its high reliability and validity levels have been previously documented ,36. POMSIn addition, the State-Trait Anxiety Inventory (STAI) form JYZ was used to assess the participants\u2019 trait anxiety level. STAI is a self-reported tool that measures the presence and severity of current symptoms of anxiety and a generalized propensity to be anxious . Unlike The Wilcoxon signed-rank test was used to compare psychological responses after walking through the forest and city areas.Pearson\u2019s correlation test was used to analyze the correlation between scores of the POMS subscales after walking through forest areas  and those of trait anxiety of STAI.Mann\u2013Whitney U test was used to between participants with high trait anxiety levels and those with normal and low trait anxiety levels.p-values of <0.05 were considered to be statistically significant.Statistical analyses were performed using the Statistical Package for Social Sciences . In all cases, p < 0.01). Similar results were obtained for T-A , A-H , F , and C  subscales, and a decrease in negative mood state was observed after walking through forest areas. In contrast, regarding the positive mood state of V, the score after walking through forest areas was 42.6 \u00b1 10.4, which was significantly higher than 35.1 \u00b1 8.9 after walking through city areas (p < 0.01); thus, an increase in positive mood state was observed after walking through forest areas.Significant differences between walking through forest and city areas were observed for all subscales of D, T-A, A-H, F, C, and V . The scox-axis denotes the changes after following walking through forest areas, the y-axis denotes the trait anxiety scores of STAI, and the z-axis denotes the number of participants. A significant correlation was observed between changes in the D subscale after walking through forest areas  and the participants\u2019 trait anxiety levels .Participants with high trait anxiety levels tended to have a more effective reduction in the feeling of \u201cdepression-dejection\u201d after walking through forest areas compared with those with normal and low trait anxiety levels .Participants with higher trait anxiety levels tended to show greater decreases than those with normal and low trait anxiety levels (participants with high trait anxiety, This study found that walking through forest areas decreased the negative moods of \u201cdepression-dejection\u201d, \u201ctension-anxiety\u201d, \u201canger-hostility\u201d, \u201cfatigue\u201d, and \u201cconfusion\u201d and improved the participants\u2019 positive mood of \u201cvigor\u201d compared with walking through city areas. These results, which demonstrate the psychological benefits of forests, are partly consistent with previous findings of the effect of viewing forest scenery or walking in forests ,11,32. PA significant correlation was found between participants\u2019 trait anxiety levels and their changes in the \u201cdepression-dejection\u201d subscale of POMS after walking through forest areas. Our data revealed that psychological responses can differ depending on a participant\u2019s trait anxiety levels and that those participants with high trait anxiety levels tended to have a more effective reduction in the feeling of \u201cdepression-dejection\u201d after walking through forest areas than participants with normal and low trait anxiety levels. Only the feeling of \u201cdepression-dejection\u201d had a significant correlation, and no significant correlation was found between the other subscales of POMS. In future studies, this point must be considered. Very few studies have assessed individual differences in psychological responses, and therefore, more researches in this area are required.More than half of the global population currently lives in urban environments, and 69% of individuals are expected to live in urban areas by 2050 ,41. AlthThis study had several limitations. Firstly, this study was conducted in representative forests in each region to validate the psychological effect of walking in forest areas. Because the experiments were conducted at 52 different sites, the difference according to region might have affected the result. The effects according to the various characteristics of the forests must be examined in the future. Secondly, to generalize the findings, further studies that include various other demographic groups such as females and individuals with different ages are required. Thirdly, for an overall discussion, verifying the effect of forests using other psychological measurements is necessary to demonstrate the psychological effect of forests. Finally, participants\u2019 prior expectations and experience with forests may influence the results. These limitations should be considered in future research.This study demonstrated the psychological benefits of walking through forest areas and revealed a significant correlation between psychological responses and trait anxiety levels."}
+{"text": "The effects of forest activities on health promotion have received increasing attention. The aim of this study was to evaluate the physiological and psychological effects of brief walks in forests on young women. The experiments were conducted in 6 forests (test) and 6 city areas (control). Overall, 12 participants in each area  were instructed to walk in a forest and a city area for approximately 15 min; simultaneously, their heart rate variability, heart rate, blood pressure, and pulse rate were measured to quantify their physiological responses to walking. The modified semantic differential method, Profile of Mood States (POMS), and the State\u2013Trait Anxiety Inventory (STAI) were used to determine their psychological responses. Walking in a forest was associated with significantly higher parasympathetic nervous activity and lower sympathetic nervous activity and heart rate. In addition, scores for the comfortable, relaxed, and natural parameters and vigor subscale of POMS were significantly higher, whereas scores for negative feelings, such as tension\u2013anxiety, depression\u2013dejection, anger\u2013hostility, fatigue, and confusion, were significantly lower, as were the total mood disturbance of POMS and the anxiety dimension of the STAI. The subjective evaluations were generally in accordance with the physiological responses. A brief walk in a forest resulted in physiological and psychological relaxation effects in young women. People are exposed to many stressors in daily life; in response, they try to find effective methods to cope with stress and to relax. One result of this has been the increased focus on using forest environments as places for promoting health through taking in the forest atmosphere, an approach known as \u201cshinrin-yoku\u201d or \u201cforest bathing\u201d ,2. It haNumerous studies have demonstrated the effects of forest environments in mitigating stress states and inducing physiological relaxation ,11,12,13In addition to many studies targeting healthy young men, studies including large sample sizes ,16 and pHowever, most studies involving forest therapy experiments have reported the various effects on male subjects ,24,25,28The aim of the present study was to clarify the physiological and psychological effects on young women of brief walks through forests. Between 2014 and 2017, we conducted experiments in six forests and six city areas in Japan. In each region, we selected safe, well-maintained forest areas, as well as city areas that were located either downtown or near a Japan Railway station. Twelve Japanese female university students participated in each experiment . None of the participants reported a history of physical or psychiatric disorders. Of the 72 participants, data from 60 participants  were analyzed, because of errors in the data collection . During To eliminate order effects, the 12 participants were randomly divided into two groups of six. One group performed the experiment in the forest area, and the other performed the same experiment in the urban area. On the following day, the groups switched field sites. The details regarding the experiment sites in the forest have been summarized in On arrival in the forest or city area, the participants waited in a waiting room and were taken one at a time to the experimental site. After resting in a chair for 5 min at the site, the participant\u2019s blood pressure and pulse rate were measured to obtain the values before walking. The participant then walked along a given course in the experimental area for approximately 15 min at her normal walking pace . The walThe physiological measurements made in this study were of HRV, heart rate, blood pressure, and pulse rate. HRV was analyzed for the periods between consecutive R waves , measured by a portable electrocardiograph  ,33. ThisSystolic blood pressure (SBP), diastolic blood pressure (DBP), and pulse rate in the right upper arm were measured by an oscillometric method using a digital blood pressure monitor . Each was measured twice; if the two values of SBP differed by >10 mmHg and/or the DBP values differed by >6 mmHg, an additional measurement was taken. The mean of the two or three measurements was used in the analysis.The psychological evaluations used the modified semantic differential (SD) method, the Profile of Mood State (POMS) and State\u2013Trait Anxiety Inventory (STAI) questionnaires. The SD method obtains a subjective assessment from the participant through a questionnaire comprising pairs of opposing adjectives, each evaluated on a 13-point scale . Three pThe POMS is a well-established measure of psychological distress derived from factor analysis, and its high levels of reliability and validity have been documented ,41. It sThe STAI form X was used to assess the participants\u2019 state anxiety level. STAI is a self-reported tool that measures the current presence and severity of symptoms of anxiety and a generalized propensity to be anxious . State ap-value <0.05 was considered statistically significant. One-sided tests were used because it was hypothesized that the participants would be physiologically and psychologically relaxed by walking in forests.The analysis included the HRV and heart rate data of 52 participants and the blood pressure and pulse rate data of 46 participants, because of errors in the data collection. The statistical analyses were performed using SPSS version 20.0 . Paired t-tests were used to compare physiological responses between the forest and city areas. The Wilcoxon signed-rank test was used to compare the psychological responses. For all the analyses, a p > 0.05). However, the participants exhibited statistically significant differences in their physiological and psychological responses to the walks in the forest and city areas. The mean value of ln(HF), an indicator of parasympathetic nervous activity, averaged over the entire walking period, was significantly higher for forest walking than for city walking . In contrast, ln(LF/HF), an indicator of sympathetic nervous activity, was significantly lower during forest walking than during city walking . In addition, the mean heart rate was significantly lower during forest walking than during city walking . There were no significant differences in the blood pressure values before and after walking in either area. The pulse rate after walking differed significantly between the forest and city areas , although there was no significant difference before walking . The pulse rate increased between before and after walking in both areas. There were no significant differences in SBP or DBP between the forest and city areas .Significant differences between the forest and city experiments were observed for all the psychological measures. p < 0.01, except for depression and dejection, p < 0.05). In contrast, the vigor subscale score after walking in the forest was 5.6 \u00b1 3.5, which was significantly higher than 2.5 \u00b1 2.8 after walking in a city area (p < 0.01). The total mood disturbance score was significantly lower after walking in a forest area than after walking in a city area .Significant differences between the forest and city areas were observed for all the POMS subscales rated after walking and for the total mood disturbance score . The subp < 0.01; Finally, the state anxiety score of the STAI was 34.8 \u00b1 7.2 after walking in a forest area, significantly lower than 45.3 \u00b1 7.1 after walking in a city area  higher parasympathetic nervous activity; (2) lower sympathetic nervous activity; (3) lower heart rate; (4) feeling more \u201ccomfortable\u201d, \u201crelaxed\u201d, and \u201cnatural\u201d, as assessed by the modified SD method; (5) more improvement in the mood state, as assessed by the POMS; and (6) lower the anxiety level than those caused by walking in the city area. In conclusion, brief walks in forests induced physiological and psychological relaxation in healthy young women."}
+{"text": "Agt) expression, and attenuates systemic hypertension and renal injury in diabetic Hnrnpf-transgenic (Tg) mice. We thus hypothesized that deletion of Hnrnpf in the renal proximal tubules (RPT) of mice would worsen systemic hypertension and kidney injury, perhaps revealing novel mechanism(s). Tubule-specific Hnrnpf knockout (KO) mice were generated by crossbreeding Pax8-Cre mice with floxed Hnrnpf mice on a C57BL/6 background. Both male and female KO mice exhibited elevated systolic blood pressure, increased urinary albumin/creatinine ratio, tubulo-interstitial fibrosis and glycosuria without changes in blood glucose or glomerular filtration rate compared with control littermates. However, glycosuria disappeared in male KO mice at the age of 12 weeks, while female KO mice had persistent glycosuria. Agt expression was elevated, whereas sodium-glucose co-transporter 2 (Sglt2) expression was down-regulated in RPTs of both male and female KO mice as compared to control littermates. In vitro, KO of HNRNPF in human RPTCs (HK-2) by CRISPR gRNA up-regulated AGT and down-regulated SGLT2 expression. The Sglt2 inhibitor canagliflozin treatment had no effect on Agt and Sglt2 expression in HK-2 and in RPTCs of wild-type mice but induced glycosuria. Our results demonstrate that Hnrnpf plays a role in the development of hypertension and glycosuria through modulation of renal Agt and Sglt2 expression in mice, respectively.We reported previously that overexpression of heterogeneous nuclear ribonucleoprotein F (Hnrnpf) in renal proximal tubular cells (RPTCs) suppresses angiotensinogen ( Over-activation of the intrarenal RAS appears to be involved in various kidney diseases7. We and others have reported that overexpression of angiotensinogen  in RPTCs leads to systemic hypertension and kidney injury in transgenic (Tg) mice10, supporting the notion that enhanced intrarenal Agt expression and RAS activation play an important role in the development of hypertension and kidney injury.The kidney contains all components of the renin-angiotensin system (RAS)Hnrnpf) mediates insulin inhibition of Agt gene transcription through binding to the putative insulin-responsive element (IRE) in the rat Agt promoter12. We recently reported that overexpression of Hnrnpf in RPTCs suppresses Agt expression, and attenuates systemic hypertension and renal injury in male Akita (type 1 diabetic murine model) Hnrnpf-Tg mice13 and db/db (type 2 diabetic murine model) Hnrnpf-Tg mice14. Since sex differences may modulate the development of systolic blood pressure (SBP)16, we investigated whether Hnrnpf would affect intrarenal Agt expression in a sex-dependent manner. We generated tubule-specific Hnrnpf KO mice by employing the Pax8-Cre/lox system17 and monitored the development of phenotype in both male and female mice.Our lab has reported that heterogeneous nuclear ribonucleoprotein F , respectively. Treatment with canagliflozin (an inhibitor of Sglt2) had no effect on Agt and Sglt2 expression in HK-2 and in RPTCs of wild-type mice, whereas it induced glycosuria.Here, we report that tubule-specific (Pax8) Hnrnpf KO mice were generated by using Pax8-Cre/lox recombination strategy  which is localized on chromosome 6. Heterozygous of Hnrnpf-floxed allele mice were generated by cross-breeding male Hnrnpf-floxed mice with female Pax8-Cre mice. These mice were further crossbred to generate homozygous Hnrnpf-floxed allele and carried the Cre allele. PCR analysis of genomic DNA extracted from ear punch tissues to distinguish the genotype of Cre (392\u2009bp), floxed (568\u2009bp) and WT (507\u2009bp) is shown in Fig.\u00a0Hnrnpf mRNA expression in RPTs freshly isolated from male and female Ctrl and KO mice at the age of 8 weeks  were also observed in both male and female KO mice compared to Ctrl at 24 weeks of age with no significant difference between male and female Ctrl as well as between male and female KO mice. Fig.\u00a0. Twenty-Fn1  at the age of 23 weeks in male and female mice  was observed in RPTs from female KO mice. However, no significant difference of Sglt2 expression in RPTs was observed between male and female Ctrl as well as between male and female KO mice. WB of isolated RPTs confirmed these changes at the protein level  mRNA expression in RPTs isolated from both male and female KO mice as compared to Ctrl . Four weeks of canagliflozin treatment had no detectable effects on SBP 1Cgn/J) with our Hnrnpffl/fl mice on a C57BL/6 background to explore the phenotype of global Hnrnpf KO mice and found that like the global Hnrnpk deletion23, global Hnrnpf KO also results in embryonic death -Pax8tm1.1(cre)Mbu/J) mouse line17. Several labs have also successfully employed Pax8-Cre mice to delete genes in renal tubules26. Our homozygous Pax8-Hnrnpf KO mice are viable and fertile without symptoms of body weight loss, physiological imbalance and altered hearing. However, they develop hypertension and elevated ACR with increased Agt expression in RPTCs by 8 weeks of age 30 but differs from that in Sweet Pee mice, which are characterized by elevated urinary excretion of calcium and magnesium and growth retardation31, as well as from that in patients with renal Fanconi syndrome32. Intriguingly, male Hnrnpf KO mice exhibited a transient glycosuria between the ages of 6 and 12 weeks and then returned to levels similar to Ctrl. Furthermore, glycosuria correlates with reduced Sglt2 expression in RPTs of male Hnrnpf KO mice. In contrast, female Hnrnpf KO mice displayed persistent glycosuria throughout 6 to 24 weeks of age with similar inhibition of Sglt2 expression observed at both 8 and 24 weeks of age. These data would indicate that male sex hormones rather than female sex hormones may modulate Sglt2 expression in Hnrnpf KO mice. Indeed, this notion had been suggested by Sabolic\u2019s group34 who implicated androgen, but not estradiol up-regulates Sglt2 expression and activity in mice.An unexpected finding of our present study was that Agt expression, we treated HK-2 cells and WT mice with canagliflozin. Canagliflozin had no detectable effects on SBP and blood glucose levels but enhanced the development of glycosuria in both male and female mice as compared to non-treated mice. Furthermore, canagliflozin treatment had no effect on the expression of Agt and Sglt2 expression in RPTs of mice. Thus, our data would argue against a causal relationship between Sglt2 inhibition and Agt expression in RPTCs; rather our data would indicate that inhibition of Hnrnpf expression modulates both Agt and Sglt2 expression in RPTCs.To explore the impact of Sglt2 inhibition on in vivo observations, we studied a human renal proximal tubular cell line (HK-2)35. By employing CRISPR gRNA technology, we obtained several clones of HK-2 cells with HNRNPF KO. Consistent with our findings in Hnrnpf KO mice, HK-2 with HNRNPF KO displayed significantly higher AGT and lower SGLT2 expression as compared to HK-2 controls. These data lend further support our previous observations that Hnrnpf down-regulates RPT Agt expression and RAS activation, leading to improve tubulo-interstitial fibrosis in the kidney. Moreover, consistent with our in vivo results, canagliflozin treatment had no effect on SGLT2 and AGT expression in HK-2 cells.Finally, to replicate our HNRNPF led to down-regulation of SGLT2 transcription in HK-2 cells are unclear. One possibility might be that HNRNPF affects SGLT2 transcription at the promoter activity level. This is unlikely since transfection of the HNRNPF cDNA did not affect the SGLT2 promoter activity  in HK-2 cells  upstream of 2\u2009kb of the SGLT2 promoter. The second possibility is that HNRNPF deletion might alter the splicing of SGLT2 to yield mutant forms of SGLT2. This is also unlikely since only one species of SGLT2 was detectable in HK-2 cells with HNRNPF KO, which was similar to the size of SGLT2 in HK-2  or factor(s) that might have a greater impact (stimulation) on SGLT2 expression and activity. Clearly, further studies are needed to elucidate the mechanisms underlying HNRNPF down-regulation of SGLT2 expression.At present, the underlying mechanism(s) by which genetic deletion of Agt expression is unknown. One possibility is that Hnrnpf binds to the insulin-responsive element (IRE) in the Agt promoter12 and functions as a negative trans-acting protein to inhibit the binding of other positive trans-acting factor(s) to TATA-binding protein (TBP) and RNA polymerase II, subsequently attenuating Agt transcription. This possibility is supported by the studies of Yoshida et al.22 showing that Hnrnpf is associated with TBP, RNA polymerase II and nuclear cap-binding protein complex. A second possibility is that Hnrnpf is associated with Hnrnpk to form an Hnrnpf/k complex and that the Hnrnpf/k complex is more effective in inhibiting Agt transcription. Indeed, we have previously reported that Hnrnpf co-immunoprecipitated with Hnrnpk and that co-transfection of Hnrnpf with HnrnpK was more effective in inhibiting Agt transcription than either Hnrnpf or HnrnpK alone35. A third possibility is that the Hnrnpf/k complex might recruit unidentified repressor molecules and subsequently repress Agt transcription. This third possibility is supported by the studies of Denisenko et al.36 demonstrating that Hnrnpk could bind the murine repressor Zik1. Clearly, more work is needed to elucidate the precise molecular mechanism of action of Hnrnpf on Agt transcription in RPTCs.The exact mechanism(s) of Hnrnpf regulation of Agt and down-regulation of Sglt2 expression in RPTs, respectively. With the recent development of SGLT2 inhibitors as a novel treatment for diabetic patients41, it would be important to understand the regulation of SGLT2 expression at the molecular level. Our findings raise the possibility that Hnrnpf KO mice may be a useful animal model for advancing studies on SGLT2 regulation and familial renal glycosuria in human.In summary, the present study reveals a novel role for Hnrnpf in the development of hypertension, tubule-interstitial fibrosis and glycosuria in mice via up-regulation of http://www.atcc.org). Human SGLT2 gene promoter  was amplified from HK-2 genomic DNA by PCR with specific primers  were purchased from Sigma-Aldrich  and Janssen Inc. , respectively. Dulbecco Modified Eagle Medium (DMEM) Cat. No. 11966-025), Ham\u2019s F12 medium (Cat. No. 11765-054) and fetal bovine serum (FBS) were bought from Gibco . Oligonucleotides were synthesized by Integrated DNA Technologies, Inc.  and listed in Supplemental Table\u00a01966-025,Hnrnpf KO mice were generated by cross-breeding male Hnrnpf-floxed mice with female Pax8-Cre mice17 . Briefly, the mouse Hnrnpf gene (Gene ID: 98758) is localized on chromosome 6: 117,900,340-117,925,622. Four exons have been identified in Hnrnpf with the ATG start codon and TAG stop codon both located in exon 4. The lox-modified Hnrnpf targeting vector was created by including 5\u2032 and 3\u2032 homology arms as well as two loxP sites flanking the fourth exon region amplified from SV129 BAC genomic DNA and confirmed by sequencing. C57BL/6 ES cells were used for gene targeting . These mice allow the excision of exon 4 of Hnrnpf gene and disruption of the protein expression in the presence of Cre recombinase. By cross-breeding male Hnrnpf-floxed mice with female Pax8-Cre mice, heterozygous Hnrnpf-floxed allele mice were generated . These mice were back-crossbred to generate homozygous Hnrnpf-floxed allele and carrying the Cre allele . The Pax8-Hnrnpf KO mice  and control littermates (Ctrl) (genotype: Hnrnpffl/fl) as well as heterozygous littermates  were used in the present studies. Experimental mice were generated from at least three different breeding couples. Offspring were genotyped by PCR to detect the Cre-recombinase as well as the presence or absence of the 5\u2032 loxP site using specific primers  and control littermates (Ctrl) (genotype: Hnrnpffl/fl) were studied. Animal care and procedures followed the Principles of Laboratory Animal Care  and were approved by the CRCHUM Animal Care Committee.Age- and sex-matched male and female KO . SBP was measured with BP-2000 tail-cuff  at least 2 to 3 times per week per animal in the morning without fasting as previously described43. Urines were also assayed for levels of albumin and creatinine 14 and glucose .At 24 weeks of age, twenty-four h prior to euthanasia, mice were housed individually in metabolic cages. Food, water consumption, and urine output were recorded. Mouse serum and urine samples were extracted with C18 Sep-Pak columns  and assayed for Ang II by specific ELISA  according to the recommended number III protocol43. Aliquots of freshly-isolated RPTs from individual animals were used immediately for total RNA isolation and Western blotting.For tissue studies, mice were euthanized at the age of 8 or 24 weeks. Blood samples were collected by cardiac puncture. The kidneys were isolated, decapsulated and weighed. The left kidneys were processed for histology and immunostaining, and the right kidneys were used for isolation of renal proximal tubules (RPTs) by Percoll gradientSerum and urine sodium, phosphorus and calcium were measured by the Comparative Medicine and Animal Resources Centre, McGill University .http://www.diacomp.org/) with slight modifications14.The glomerular filtration rate (GFR) was estimated with fluorescein isothiocyanate inulin as recommended by the Animal Models of Diabetic Complications Consortium  was performed after 6\u2009h fasting in non-anesthetized mice at the age of 23 weeks, as described previouslyHnrnpf, Agt, Sglt2, fibronectin I (FN1) and ribosomal protein L13A (RPL13A) in isolated RPTs as described previously43 with specific primers  analyses were performed to quantify the relative expression of ctin I FN and ribo43. Details of the sources of antibodies and working dilutions are listed in Supplemental Table\u00a0Western Blotting (WB) was performed in isolated RPTs as described previously43. Masson\u2019s Trichrome staining, Sirius Red staining and immunostaining for Fn1 were performed to assess tubule-interstitial fibrosis. Semi-quantitation of the relative staining was done by NIH Image J software (http://rsb.info.nih.gov/ij/). Mean glomerular tuft and RPTC volumes, and the tubular luminal area were determined by the methods of Weibel45 and Gundersen46, as described previously48.Kidney sections were stained with periodic acid Schiff (PAS) as previously described49) . Image quantification and merge were assessed by ImageJ software (http://rsb.info.nih.gov/ij/). To quantify the amount of Sglt2 expression, the pixel intensity of Sglt2 was divided by LTL intensity. To calculate the average ratio, 6 sections per mouse, 6 mice per group were analyzed.Immunofluorescence (IF) staining was performed on 3-\u03bcm tissue sections from mouse kidney fixed in formalin and embedded in paraffin followed by staining with ALEXA FLUOR-594-labeled secondary antibody (Invitrogen). Proximal tubules were identified by fluorescein-labeled lotus tetragonolobus lectin . Briefly, the day before transfection, HK-2 cells (2.5\u2009\u00d7\u2009105 cells per well) were cultured in a 1:1 mixture of DMEM and Ham\u2019s F12 medium containing 10% of FBS in 6-well plate. OPTI-MEM medium with Lipofectamine Cas9 Plus\u2122 Reagent  and the mixture of 37.5 pmol TruCut\u2122 Cas9 Protein v2  and 37.5 pmol gRNA :tracrRNA ) were transfected to HK-2 and cultured for 2 days at 37\u2009\u00b0C. Single cell clones were then isolated by using limiting dilution cloning in 96-well plates. The positive clones were identified for the absence of HNRNPF by WB of cellular extracts and confirmed by PCR of genomic sequence. The clones with HNRNPF expression were used as controls.Human renal proximal tubular cells (RPTCs) (HK-2) cells are derived from a normal adult male human kidney transfected with the human papilloma virus 16 (HPV-16) E6/E7 geneset al.51. WB and RT-qPCR were used to quantify SGLT2 and AGT protein and mRNA expression, respectively.To test the pharmacologic effect of SGLT2 inhibition on SGLT2 and AGT expression, HK-2 cells were harvested after 24\u2009hours of culture in serum-free normal glucose (5\u2009mM) DMEM in the absence or presence of 0.5\u2009mM canagliflozin as described by Pirklbauer Agt expression in RPTCs in vivo, male and female WT mice were treated with or without canagliflozin (0.2\u2009mg/ml in drinking water) at the age of 4 weeks as described previously52. Body weight, blood and urinary glucose and SBP were monitored weekly. The mice were euthanized at the age of 8 weeks. The left kidneys were processed for histology and immunostaining, and the right kidneys were used for isolation of RPTs and were used immediately for total protein and RNA isolation to quantify protein and mRNA expression of Agt and Sglt2 by WB and RT-qPCR, respectively.To investigate the impact of Sglt2 inhibition and t-test or 1-way ANOVA  and the Bonferroni test as appropriate. p\u2009<\u20090.05 values were considered to be statistically significant.The data are expressed as means\u2009\u00b1\u2009SEM. Statistical significance between the experimental groups was analyzed by Student\u2019s Supplementary Figures And Tables"}
+{"text": "In the present work, S-tert-butyl isothiouronium bromide is successfully applied as an odorless surrogate for tert-butyl thiol. The C-S bond formation is carried out under palladium catalysis with the thiolate formed in situ resulting in high yields of tert-butyl aryl sulfides. The subsequent formation of benzobisdithioles is here achieved with scandium(III)triflate, a less harmful reagent than the usually used Lewis acids, e.g., boron trifluoride or tetrafluoroboric acid. This enables a convenient and environmentally more compliant access to high yields of benzobisdithioles.Benzobisdithioles are important building blocks within a range of functional materials such as fluorescent dyes, conjugated polymers, and stable trityl radicals. Access to these is usually gained via  Fuin vitro ,12, as pin vitro , viscosiin vitro , and as in vitro ,16. Addiin vitro ,18. The material . Precursobenzene  via SNArd sodium  in situ.0.33 ppb . Consideth KMnO4  or thermth KMnO4 . While tNAr-type reactions. However, such an approach should avoid the use of free thiols altogether. Although C-S cross-coupling reactions using appropriate thiol surrogates are known, these usually need to be synthesized from the corresponding thiols, such as alkyl thioacetates bisdithioles and this with improved yields. It should be noted, that S-tert-butyl isothiouronium bromide has already been applied as a thiolate source for the functionalization of electron-poor heterocycles via nucleophilic substitution reactions bisdithiole 1a by use of catalytic amounts of Sc(OTf)3 was possible, which in comparison to previous protocols avoids the use of large amounts of hazardous Lewis acids like HBF4 and BF3.In the present study, the olfactory hazard and lack of conversion was circumvented by synthesis of S-eactions ,33. Furttert-butyl isothiouronium halides to form the corresponding thiolate exploitable for in-situ C-S cross coupling was assumed.Although it is well-known that S-alkyl isothiouronium salts form alkylthiols upon treatment with aqueous hydroxide , their btert-butyl isothiouronium halides were occasionally described in older literature bisdithiole tert-butyl isothiouronium bromide , tris(dibenzylideneacetone)dipalladium(0) (2.5 mol%), phosphine ligand (10 mol%) and base (4.0 eq.) were dissolved in dry and degassed DMF (4 mL) under argon atmosphere. The mixture was stirred for 19 h at the given temperature. Afterwards, the reaction was quenched with water (10 mL) and extracted with DCM (3 \u00d7 3 mL). The unified organic phases were repeatedly washed with 2M-HCl-solution (3 \u00d7 5 mL) and dried over MgSO4. The solvents were removed under reduced pressure regularly giving an oil containing rests of DMF. Due to the volatile nature of the products, drying of the resulting oil in high vacuum was not performed.General procedure for condition screening with 4-bromoanisole. 4-Bromoanisole , 1H-NMR in CDCl3 and independently prepared samples of 4-bromoanisole, 4-bromophenol, and 4-methoxy-tert-butylthiobenzene (vide infra) were used as external reference as shown in 3 containing 1% DMF.The analysis of the crude products was performed by tert-butylisothiouronium bromide (1.20 eq.), tris(dibenzylideneacetone)dipalladium(0) (2.5 mol%), triphenyl phosphine (10 mol%), and potassium tert-butanolate (4.0 eq.) were placed in a Schlenk tube and dissolved in 4 mL dry DMF. The reactions were stirred at the indicated reaction temperature and afterwards quenched by addition of 10 mL water and extracted with DCM (3 \u00d7 3 mL). The unified organic phases were repeatedly washed with 2M-HCl-solution (3 \u00d7 5 mL) and dried over MgSO4. The solvents were removed under reduced pressure regularly giving an oil containing rests of DMF. Due to the volatile nature of the products, drying of the resulting oil in high vacuum was not performed.General procedure for screening with other substrates. The respective educt (100 mg), S-tert-butylisothiouronium bromide is exploited as a surrogate for tert-butyl thiol for C-S cross coupling reactions yielding tert-butyl aryl thioethers. Interestingly, the ligand performance dropped with increasing steric congestions, though sterically demanding ligands are known to accelerate the often rate-determining reductive elimination. Therefore, the nucleophilic displacement step is assumed to be rate-determining in the present case. Accordingly, simple Ph3P yields quantitative conversions, while more sophisticated ligands, including those of the biarylphosphine type, failed to provide sufficient reactivity. While the reaction conditions can be tuned even milder with aryl iodides, the presented procedure cannot be expanded to chloro- and fluoroarenes. With activated nitroarenes, however, thioetherification already occurs at room temperate but via an SNAr reaction instead of a catalytic cross-coupling process. Based on this, efficient access to benzobisdithioles via C-S cross coupling of 1,2,4,5-tetrabromobenzene is established, outperforming the previous route via SNAr-reactions. Additionally, the ease of thioketal formation was enhanced by using catalytic amounts of Sc(OTf)3 instead of stoichiometric amounts of less effective and hazardous Lewis-acids.In this work, odorless S-"}
+{"text": "Breastfeeding is natural and the optimal basis of infant nutrition and development, with many benefits for maternal health. Human milk is a dynamic fluid fulfilling an infant\u2019s specific nutritional requirements and guiding the growth, developmental, and physiological processes of the infant. Human milk is considered unique in composition, and it is influenced by several factors, such as maternal diet and health, body composition, and geographic region. Human milk stands as a model for infant formula providing nutritional solutions for infants not able to receive enough mother\u2019s milk. Infant formulas aim to mimic the composition and functionality of human milk by providing ingredients reflecting those of the latest human milk insights, such as oligosaccharides, bacteria, and bacterial metabolites. The objective of this narrative review is to discuss the most recent developments in infant formula with a special focus on human milk oligosaccharides and postbiotics. Nutrition in early infancy and childhood can significantly impact growth and development as well as immediate and later health . The WorA healthy gut development is of major importance during infancy. It contributes to growth and development by ensuring digestion and absorption of nutrients and fluids. The gut is also key in the development of immunity insofar as it maintains a barrier against infectious agents and interacts directly with the immune system to induce mucosal and systemic tolerance, which prevents of allergy. Furthermore, the gut provides signals to the brain to maintain a healthy state .One of the major factors enabling proper gut function and development is a balanced gut microbiota . SeveralTwo human milk oligosaccharides (HMOs), 2\u2032-fucosyllactose (2\u2032FL) and lacto-N-neotetraose (LNnT), are recent examples of optional ingredients being added to infant formula ,9,10,11.Human milk contains many bioactive compounds such as oligosaccharides, immune cells, and varying levels of bacteria and their metabolites. They play an important role in the development of a healthy gut by supporting a favourable intestinal microbiota and in the development of the infant\u2019s immune system ,16,17,18HMOs are pools of complex carbohydrates, the third most abundant component of human milk, and one example of naturally occurring \u2018prebiotics\u2019 . AlthougThe quantity of HMOs in mature human milk is approximately 12\u201315 g/L as reported elsewhere ,25. TherMost HMOs escape digestion in the small intestine  and progHMOs are synthesised in the mammary gland by the prolongation of lactose with monosaccharides forming non-digestible trisaccharides (DP3)  or tetrasaccharides (DP4). The addition of sialic acids leads to two different sialyllactoses (SL), 3\u2032-SL and 6\u2019-SL (DP3), and the addition of fucose leads to two different fucosyllactoses (FL), 2\u2032-FL and 3-FL (DP3), as well as difucosyllactose (DP4) ,36,37 F. HMOs also include galactosyllactoses (GLs), which appear in the form of several structurally distinct isomers and only differing in the glycosidic linkage of the terminal galactose added to lactose, leading to 3\u2032-GL, 4\u2019-GL, and 6\u2018-GL (DP3) ,39 Figu. Those HLarger HMO structures are derived by adding galactose and N-actetylglucosamine as disaccharide building blocks, which leads to tetrasaccharides (DP4), Lacto-N-tetraose (LNT), and Lacto-N-neotetraose (LNnT). Those tetrasaccharides can be further extended by those disaccharide units  and in addition decorated with further single or multiple fucose residues, leading to large neutral HMOs with a molecular size up to 8 kDa  and/or sSize exclusion chromatography, which was further used to describe the complexity of short- and long-chain HMOs, demonstrated a molecular size distribution of short-chain and long-chain HMOs in a ratio of 9:1 . Long-chBifidobacterium spp. and Lactobacillus spp. [Bifidobacterium dominating the gut microbiota of a vaginally delivered infant [Human milk is also an important source of beneficial bacteria  that help colonise the infant gut and contribute to the composition of a favourable gut microbiota, including lus spp. ,53, withd infant ,55. Bactd infant . In gened infant ,57,58,593 and 106 bacteria per millilitre [The total number of bacteria in human milk significantly differs according to detection methods. It has been estimated that human milk contains median values between 10llilitre ,61,62. Tllilitre .Besides bacteria, their metabolites , peptides, oligosaccharides) may also naturally pass into human milk, and this can be detected through metabolomics research using nuclear magnetic resonance spectroscopy ,64. MostThere is increasing research on nutritional postnatal interventions using probiotics, prebiotics, synbiotics, and the so-called postbiotics to promote the establishment of a beneficial microbiota and to have a positive impact on neonatal health . WhereasAccording to a recent consensus definition, probiotics are live microorganisms that confer a health benefit on the host when administered in adequate amounts , whereasBifidobacterium spp. and/or lactic acid bacteria such as Lactobacillus spp., which are generally regarded as safe for food use in the European Union based on the QPS-list  of bacteria. In the United States, a non-mandatory system of safety evaluation is in place, provided by the U.S. Food & Drug Administration (FDA). On request, it evaluates safety assessment filings of specific probiotic strains to be included in the so-called GRAS  Notice Inventory, which is continuously updated [Most probiotic-containing infant formula comprise  updated . L. rhamnosus GG (LGG) ATCC 53103 (at a daily dose ranging from 1 \u00d7 109 CFU to 6 \u00d7 109 CFU) and the combination of B. infantis Bb-02, B. lactis Bb-12, and Str. thermophilus TH-4 (at a daily dose of 3.0 to 3.5 \u00d7 108 CFU of each strain) as it might reduce necrotizing enterocolitis (NEC) stage 2 or 3 in preterm infants (low certainty of evidence), but mortality and sepsis did not show any clear direction in effect size. However, no recommendation could be made in either direction regarding the use of L. reuteri DSM 17938 and the combination of B. bifidum NCDO 1453 (currently reclassified as B. longum) with L. acidophilus NCDO 1748  in preterm infants to reduce the risk of mortality, NEC stage 2 or 3, or sepsis (very low certainty of evidence) [There are data on specific probiotics in infant formula accompanied by a large diversity in study outcomes . Howevervidence) . For terBecause of their stability, low risk of adverse effects, ease of administration, and potential for influencing the composition and function of the microbiota in the gut and beyond, the clinical applications of prebiotics are expanding . In last decades, different prebiotic mixtures of galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS) have been studied. Globally the most studied prebiotic mixture of oligosaccharides in infant formula consists of short-chain (sc) GOS and long-chain (lc) FOS, scGOS/lcFOS (9:1). The mix is in the range of HMOs close to human milk in quantity (8g/L) and diversity (more than 100 different structures of short- and long-chain types in a ratio of 9:1). Although this prebiotic mixture approaches the molecule size distribution of short- and long-chain oligosaccharides in human milk, it is not structurally similar to HMOs . ClinicaIn addition to prebiotics based on GOS and/or FOS, infant formulas have recently been supplemented with specific technically derived HMOs , which have been anticipated as candidate prebiotics  see Sec.Recent research highlights the variety of HMOs in human milk, underlining the importance of scientific assessment of the role of both minor and major oligosaccharides in promoting infant health before adding it to infant formula. E. coli as a microbial cell factory  has prebiotic effects and may deliver functional benefits in infants. In preclinical trials, 2\u2032-FL promoted the growth of specific bifidobacteria ,99, blocInfant formulas containing 2.4 g total oligosaccharides/L  have been demonstrated to be safe in terms of normal growth and were well-tolerated in a prospective, randomised, an controlled growth and tolerance study with 189 healthy, singleton infants . A furthA recently developed infant formula concept combines a prebiotic mixture of scGOS/lcFOS (9:1) and 2\u2032-FL to further mimic the complex composition of HMO structures and their functional benefits. Data from the first in vivo pre-clinical investigations showed positive effects of this combination in a rotavirus model  and in aBifidobacterium infantis [Streptococcus pneumoniae in children (6 months or older). Although colonisation was not reduced, LNnT proved to be well-tolerated at the tested concentration of 200 mg/L [LNnT has been assessed in vitro for function as a prebiotic inducing growth and metabolic activity in infantis . As an i200 mg/L . Infant formula supplemented with a combination of 2\u2032-FL (1.0 g/L) and LNnT (0.5 g/L) was demonstrated to be safe and well-tolerated and to support age-appropriate growth in a multicentre, randomised, double-blind trial with 175 healthy infants, with weight gain as a primary study outcome. Secondary outcomes were also found including associations of lower parent-reported morbidity (particularly bronchitis) and medication use (antipyretics and antibiotics) in infants fed the supplemented formula compared to the control .HMO 3\u2032-GL is naturally present in human milk and was already isolated from human milk in 1988 . RecentlIn pre-clinical studies, 3\u2032-GL, 4\u2019-GL, and 6\u2019-GL prepared from colostrum individually accounted for specific immunomodulation of polyinosinic:polycytodylic acid-induced interleucin-8 levels in an immature human intestine tested at a concentration of 200 \u00b5g/mL each . AnotherAlthough these first pre-clinical data may indicate that certain galactosyllactoses, such as 3\u2032-GL, have protective and immunomodulatory effects in the gut, more research is needed to further explore their role as HMOs.Since 3\u2032-GL in infant formula is a fermentation by-product, the safety of 3\u2032-GL can be considered assured by the long history of safe use of the fermented formula in France . The cliBeneficial synergistic effects may be expected from a combination of probiotics and prebiotics, which are called synbiotics, using prebiotics to selectively increase abundance of both endogenous beneficial bacteria and beneficial microbes in the infant gut .Bifidobacterium breve M-16V already demonstrated promising results. This synbiotic combination compensated for delayed bifidobacteria colonisation in infants delivered by caesarean section  in an exploratory, randomised, double-blind, controlled study with 153 infants with detection of total faecal bifidobacteria as primary study outcome [Given the evidence for an aberrant gut microbiota in infants with allergy ,115 and  outcome . Seconda outcome , lower f outcome . Further outcome . At the  outcome . However, even if some synbiotic combinations already display promising clinical results in infants, more randomised trials with longer follow-up are needed for the determination of their physiological and metabolic impact on the host .In contrast to probiotics, bacterial viability in postbiotics is not seen as an essential requirement for health benefits, providing a potential opportunity to foods that are not convenient for carrying viable bacteria , e.g., lLactobacillus and Bifidobacterium strains, which are also generally the most used probiotics [In infant formulas, the concept of postbiotics is yet to be defined, although specific fermented infant formulas with postbiotics have been commercially available in Europe for decades. So far, most known postbiotics are derived from obiotics .Recently, a provisional definition has been formulated that stipulates that postbiotics are compounds produced by microorganisms and released from food components or microbial constituents, including non-viable cells that, when administered in adequate amounts, promote health and well-being . Compounds deriving from bacterial metabolism, such as exopolysaccharides, vitamins, lactic acid, bacteriocins, enzymes, surfactants, antioxidants, and SCFAs.Complex molecules released from food compounds , such as peptides and galacto-oligosaccharides, e.g., 3\u2032-GL and 6\u2018-GL.Components released from lysed cells including DNA, RNA, cell walls and, perhaps, other cytoplasmic components, and surface layer proteins.Examples of postbiotics have been summarised as follows : CompounA consensus definition on postbiotics is currently being formulated by ISAPP .For many thousands of years, fermentation of food has been applied as a natural process to generate foods with particular properties, palatability, taste, and health benefits . Most heStreptococcus thermophilus  from lactose fermentation [For cow\u2019s milk-based infant formulas, fermentation processes typically use lactic acid-producing bacteria as a starter culture . In addientation . The ferentation . Bifidobacterium breve C50 and Streptococcus thermophilus 065) and naturally delivering postbiotics [Recently, a specific fermentation process has been developed for infant nutrition, using two specific types of food-grade lactic acid producing microorganisms  stimulated morphological  and functional  gut maturation more similar to the mother-fed situation than infant formula without pre- or postbiotics . Gut perThe first clinical study with a fermented infant formula was already published in 1989, reporting positive effects on gut function . More reBifidobacterium breve C50 and Streptococcus thermophilus 065\u2014with and without prebiotic scGOS/lcFOS (9:1)\u2014demonstrated its capacity to impact immune and gut health benefits in otherwise healthy infants  and beneficial bacteria  and their metabolites . In infant formula, these nutritional concepts are provided by different pre- and probiotic mixtures and a mixture of both (synbiotics) and, additionally, by using partly fermented infant formula with beneficial compounds produced by microorganisms that are released from food components or microbial constituents, including non-viable cells (postbiotics). One of the most current examples of such compounds is 3\u2032-galactosyllactose (3\u2032-GL), which is present in human milk and is a natural derivative of milk fermentation. Although such developments may pave the way for future infant formula alternatives for those infants who are not able to be (fully) breastfed, human milk feeding will always remain the unmatched goal for infant nutrition and development as well as provide many benefits for maternal health."}
+{"text": "Infectious spleen and kidney necrosis virus , nervous necrosis virus , and infestations with ectoparasites during the rainy season in juvenile grouper (Epinephelus spp.) farmed in Aceh, Indonesia. The survey was intended to detect aquatic pathogens present at 10% prevalence with 95% confidence, assuming 100% sensitivity and specificity using a sample size of 30 for each diagnostic test. Eight populations of grouper from seven farms were sampled. Additional targeted sampling was conducted for populations experiencing high mortality. Infection with NNV was detected at all farms with seven of the eight populations being positive. The apparent prevalence for NNV ranged from 0% (95% CI: 0\u201312) to 73% (95% CI: 54\u201388). All of the fish tested from the targeted samples (Populations 9 and 10) were positive for NNV and all had vacuolation of the brain and retina consistent with viral nervous necrosis (VNN). Coinfections with ISKNV were detected in five populations, with the highest apparent prevalence being 13% (95% CI: 4\u201331%). Trichodina sp., Cryptocaryonirritans and Gyrodactylus sp. were detected at three farms, with 66% to 100% of fish being infested. Hybrid grouper sourced from a hatchery were 5.4 and 24.9 times more likely to have a NNV infection and a higher parasite load compared to orange-spotted grouper collected from the wild (p < 0.001). This study found that VNN remains a high-impact disease in grouper nurseries in Aceh, Indonesia.A cross-sectional survey was used to estimate the prevalence of infections with the  Grouper (subfamily Epinephelinae) aquaculture is a well-established industry in Asia with 155,000 tonnes produced annually. Behind China and Taiwan, Indonesia is the third largest producer with 17,000 tonnes per year  ,3,4.Epinephelus spp.) comprises three stages; hatchery, nursery and grow-out, which tend to be concentrated in geographically distinct areas across the island nation [In Indonesia, the production cycle for grouper  with a 1 mm mesh size at a density of 800 fish/m3 [3 and fed a diet of chopped up low value \u201ctrash\u201d fish harvested from the local estuary [Epinephelus coioides), which are captured from the wild, and tiger grouper (Epinephelus fuscoguttatus), which are reared in hatcheries [Epinephelus lanceolatus) [The nursery phase for grouper production can be undertaken either in shore-based tanks or in coastal ponds . In pond fish/m3 ,7. Gener fish/m3 ,7. For t estuary ,7. In Actcheries . Novel seolatus) ,10. HybrIn a survey of pond-based grouper nurseries in Aceh, survival was generally observed to be approximately 75% but varied from 15% to 98% . AnecdotBetanodavirus, nervous necrosis virus (NNV) [Megalocytivirus [Megalocytivirus, specifically red sea bream iridovirus (RSIV) and Infectious spleen and kidney necrosis virus (ISKNV), frequently occur in east and Southeast Asia [Larval and juvenile grouper are susceptible to epidemic mortality outbreaks of disease caused by infection with us (NNV)  and by iytivirus . Fish noytivirus . VNN is ytivirus  and is rytivirus . Diseaseast Asia  with morast Asia . Megalocast Asia  under a ast Asia , United ast Asia , Australast Asia ,21, and ast Asia .Grouper are known to exhibit prolonged sub-clinical infections with NNV and megalocytiviruses with disease manifesting under conditions involving various combinations of host and environmental risk factors ,24. InfeEpinephelus were sampled, including wild-sourced orange-spotted grouper (Epinephelus coioides) and hatchery-produced hybrids known as Cantang (E. fuscoguttatus \u2640 \u00d7 E. lanceolatus \u2642) and Cantik (E. fuscoguttatus \u2640 \u00d7 E. polyphekadion \u2642)  and ISKNV (0.702). There was no significant association between a fish having any observed abnormality or skin/gill abnormality with the parasite load (p = 0.405 and p = 0.707). All populations of fish had gross pathological changes associated with the hematopoietic organs  and abnormalities were frequently observed for the skin and gills (p < 0.001).The necropsy identified that the fish had internal and external abnormalities, such as skin ulcers, changes in skin colour , gill pallor or haemorrhage, emaciation, unilateral or bilateral exophthalmia, cataract, short operculum, fin erosion and/or haemorrhage, ascites, changes in the size and the colour of the spleen, anterior kidney, posterior kidney and liver . The prond gills . The pro3 copies of the genome per mg tissue, in the eight populations sampled in the cross-sectional survey.Infection with NNV was detected at all farms and in seven of the eight populations from the representative sampling . The appn = 15). However, NNV was detected at Farm 2 in the hatchery-sourced Cantang (Population 2) with an apparent prevalence of 40% (95% CI: 23\u201359%)  and no NNV-associated lesions were observed in the histopathological sections of these fish ( 23\u201359%) . These tp < 0.001). In other words, wild grouper had a 14% chance of being infected with NNV compared to hatchery-sourced fish with a 76% chance. NNV infection was not associated with farm factors including number of net-pens (p = 0.094), regional district (p = 0.138), and pond size (p = 0.711). NNV infection was not associated with fish factors including weight (p = 0.416), number of days at the farm (p = 0.831) and being classified as having no gross pathological changes (p = 0.536). Fish length was associated with NNV infection (p = 0.001). Fish less than 5 cm in total length had a 77% chance of infection compared to fish measuring 5\u201310 cm having a 25% chance and fish over 10 cm having a 41% chance.From the representative sample, hybrid grouper sourced from the hatchery were 5.4 times more likely to have NNV infection compared to orange-spotted grouper collected from the wild  were positive for NNV and all fish had viral nervous necrosis disease-specific vacuolation of the brain and retina ; Table 3Five of seven farms and five of eight of the representative populations were infected with ISKNV, with the highest apparent prevalence being 13% (95% CI: 4\u201331) . Charactp = 0.912), regional district (p = 0.429), and pond size (0.561). Nor was ISKNV infection associated with fish factors including hatchery or wild source (p = 0.129), weight (p = 0.225), length (p = 0.992), number of days at the farm (p = 0.663) and being classified as having no observed gross pathological changes (p = 0.522). Both target populations (Population #9 and #10) were infected with ISKNV at a similar apparent prevalence to the representative sample of the same population . The viral quantity for ISKNV was considered moderate to high (up to 3.0 \u00d7 108 copies of the genome per mg tissue) (ISKNV infection was not associated with farm factors including number of net-pens ( tissue) .Gyrodactylus spp. and the ciliated protozoans Trichodina spp. and Cryptocaryon irritans. All three parasites were found at each farm with the exception that C. irritans was not found at Farm 6 . Hatchery fish were 24.9 times more likely to have a higher parasite load than wild-sourced fish (p < 0.001). The predicted mean \u00b1 standard error for hatchery- and wild-sourced grouper was 12.2 \u00b1 1.2 parasites/g and 1.3 \u00b1 0.88 parasites/g, respectively (p < 0.001). Length was not found to be significant in the multivariate model (p = 0.189).Parasite examinations were completed at three farms and two thirds to 100% of the fish had at least one ectoparasite . Based ot Farm 6 . The mosparasite . HatcherDisease has been identified as the primary constraint to the production of aquatic species, impeding both economic and social development in many developing countries ,28,29. TDisease-specific histopathological lesions associated with ISKNV infection were detected at one farm while infection was detected at five of seven farms. The disease in grouper caused by ISKNV infection has a range of presentations from acute disease outbreaks with 60\u2013100% mortality, but also chronic disease with low grade mortality over a period of several months . Whilst Trichodina spp., Gyrodactylus spp. and Cryptocaryon irritans, all of which are known to cause reduced growth and mortality in grouper aquaculture [There was a high prevalence of parasitic infestations and high parasite load at the three farms where parasite examination was possible. The parasite burden and diversity of species for hatchery-sourced grouper was significantly higher compared to wild-sourced fish. The pparasitic taxa identified were aculture ,33. The Both NNV and ISKNV are cosmopolitan viral pathogens that can infect over 40 species of marine fish and are endemic in many parts of the world including grouper in Indonesia ,12. The Megalocytivirus and numerous vaccines have been described for NNV [Our study demonstrated that a high proportion of fish from each farm were showing a wide range of gross pathological changes that were independent of being infected with NNV and ISKNV or infested with ectoparasites. It is postulated that this has arisen from a combination of infectious, environmental and nutritional diseases. An integrated approach to disease management would reduce exposure to these pathogens through biosecurity, addressing risk factors for disease severity and vaccination for preventative measures. Relevant biosecurity measures would include batch culture, sourcing infection-free seed, appropriate disposal of dead and sick fish and reducing incursion of wild and trash fish. Expression of VNN and the impact of disease can vary with stress . The occ for NNV . VaccinaE. lanceolatus \u00d7 E. fuscoguttatus) is encouraged if they can be purchased certified free of NNV and ISKNV, as they have been shown to have improved growth rates and disease resistance to bacterial challenge compared to the their parent species [In agreement with a survey on marine aquaculture in Vietnam , we obse species . FurtherParasitic diseases have a major impact on global finfish aquaculture and in many regions they represent a key constraint to production, sustainability and economic viability . We obseE. fuscoguttatus) compared to those fed commercial pellets, and there was no economic benefit to using pellets [E. coioides) being fed trash fish or commercial pellets [Trichodina) had higher prevalence and abundance in the group-fed trash fish [All of the farms in this study fed a low value \u2018trash\u2019 fish diet and it is considered a common management practice for this stage of grouper production in Indonesia . This pr pellets . However pellets , althoug pellets . It was ash fish . The ecoThis study showed that a major cause of disease in juvenile grouper during the rainy season was VNN caused by infectious with NNV. Additionally, co-infestations with ectoparasites and infections with ISKNV may have contributed to poor growth and survival. Hatchery-sourced fish had a significantly higher chance of being infected with NNV (5.4 times) and ectoparasites (24.9 times) compared to the wild-sourced grouper. In order to prevent disease outbreaks at the grouper nurseries, several regional-specific biosecurity recommendations could be implemented, in particular the requirement to stock fish that were certified disease-free.In July 2015, approximately four months prior to the rainy season, an informal in person discussion in Bahasa Indonesia was conducted with the owners at each of 24 grouper nurseries in Aceh, Indonesia. Farms were located along the coast of the Malacca Strait covering approximately 400 km between Sagoe (5\u00b030\u203258.0\u2033 N 95\u00b047\u203242.1\u2033 E) and Langsa (4\u00b028\u203223.0\u2033 N 97\u00b058\u203232.5\u2033 E). Seven grouper nurseries were selected for inclusion in the cross-sectional disease survey based on the availability of fish in the farm at the time of the study, willingness to participate, personal safety and accessibility to the laboratory within 24 h ; Table 1A sample size of 30 fish per population was determined to provide 95% confidence of detecting a disease with a minimum expected prevalence of 10%, assuming 100% sensitivity and 100% specificity of the test . In ordeAt each farm, a population of fish was defined as individuals of the same species and age. For all farms, each population of grouper was held in one pond but was distributed amongst several net-pens (hapas) . When thTo select the fish, the net-pen was pulled almost entirely out of the pond and tilted to one side to crowd the fish together. Fish were sampled using a scoop net and were collected into a bucket. The fish were selected by convenience without deliberate bias. This was the only method accepted by the farm managers that was suitable to minimize disruption to the health and production of the remaining fish. At two farms, the farm manager reported severe mortality in the population included in the cross-sectional survey. An additional targeted selection of moribund fish (15 fish per diagnostic test) showing clinical signs was completed and fish were dissected and sampled as described below. The clinical signs included abnormal swimming, skin lesions, loss of appetite and skeletal deformity.Necropsy was performed directly on site at the farms as the fish were collected. Fish were euthanized in pond water using 400 to 500 mg/L clove oil . Fish were weighed, measured and examined for gross pathology. In order to avoid cross contamination, the workspace was disinfected using 200 ppm sodium hypochlorite then 70% ethanol, and equipment was disinfected using 200 ppm sodium hypochlorite for 10 min before rinsing in clean fresh water. A new sterile scalpel blade was used to necropsy each fish. Samples for PCR were preserved in 95% ethanol. For histopathology, tissues were fixed in 10% neutral buffered formalin and processed if needed to look for evidence of disease in populations of fish that tested positive for virus.Parasitology was conducted at three nurseries where electricity was available to enable onsite microscopy. Examination for ectoparasites was conducted from a scraping of the mucus layer of the skin, a gill biopsy and a fin clip . Brieflyw/v final dilution after air drying to remove ethanol. A pool of an equal portion of kidney, liver and spleen from one individual fish was prepared for Megalocytivirus tests and a pool of both retinas and brain to test for NNV. The homogenates were clarified by centrifugation for 10 min at 900\u00d7 g and a 200 \u00b5L aliquot of the supernatant was used for nucleic acid purification using the High Pure Viral Nucleic Acid Kit  according to the manufacturer\u2019s directions.Tissues were homogenized using sterile disposable pestle grinders in phosphate buffered saline (PBS) at 1/10 6 to 101 copies of linear plasmid DNA with NNV RNA2 sequence in nuclease-free water was amplified in duplicate on each plate for quantification of positive samples. A standard curve was prepared by plotting the log of the plasmid standard template quantity against the Ct value. This provided a measure of the quantity of NNV RNA, estimated as the number of copies of the NNV capsid protein gene per mg of tissue within the range of the standard curve, where the efficiency was between 90% and 110%.Real-time, reverse transcription PCR (RT-qPCR) was used to detect NNV according to previously described methods ,42. SampMegalocytivirus, including RSIV and the many genotypes of ISKNV. Briefly, the assay was performed in 25 \u00b5L reactions containing: 2.5 \u00b5L of template DNA; 250nM forward and reverse primers; 12.5 \u00b5L of Quantitech SYBR green master mix ; and 7.25 \u00b5L molecular biology-grade nuclease-free water. Each plate included the following control reactions: positive control fish tissue infected with Megalocytivirus; negative control nuclease-free water (NTC 1) and nuclease-free water subjected to the nucleic acid purification process (NTC2 EXT). The PCR assay was performed using a 7500 real-time PCR system (Applied Biosystem) under the following conditions: 1 cycle of initial denaturation at 95 \u00b0C for 15 min; 40 cycles of denaturation at 95 \u00b0C for 30 s, annealing at 62 \u00b0C for 30 s, and extension at 72 \u00b0C for 30 s, with fluorescence acquisition at the end of the annealing step. The threshold cycle (Ct) values were determined with quantitative PCR software. Post amplification, a dissociation curve was determined by: dissociation of reaction products at 95 \u00b0C for 1 min; annealing at 55 \u00b0C for 30 s; heating to 95 \u00b0C at a rate of 1 \u00b0C every 30 s. Continuous collection of SYBR fluorescence signal indicated the temperature at which the reaction products dissociated. A single peak in the dissociation curve with a melting temperature of 85.0 \u00b0C \u00b1 0.5 \u00b0C confirmed the specificity of the amplification product and was required to assign a positive result. A 6-step, serial 10-fold dilution series in nuclease-free water that contained 106 to 101 copies of the ISKNV major capsid protein gene sequence in linear plasmid DNA was amplified in duplicate for quantification of positive samples. This provided a measure of the number of genome copies per mg of tissue in the range of the standard curve that had an efficiency between 90% and 110%. Conventional PCR was used to amplify segments of the major capsid protein and ATPase genes as previously described . Purified amplification products were submitted to the Australian Genome Research Facility for Sanger sequencing. The ISKNV genotype was confirmed for at least one qPCR-positive fish from each population, as the number of bases different to the ISKNV reference sequence (GenBank Accession AF371960) was 1 or less.Quantitative PCR for detection of megalocytiviruses was performed according to the method described by Rimmer, et al. . This asSamples processed for histopathology included all samples from the two populations with disease (#9 and #10) and all fish with a positive qPCR for ISKNV from Populations 1 to 8. A random selection of 15 fish from Population 3 was included for comparison, as it had no positive detections for NNV and ISKNV. All other histopathology samples were held for future study.The retinas, brain, liver, kidney and spleen were cut into 2 to 3 mm blocks and placed in one cassette for each fish. The tissues were processed by dehydrating through an ethanol concentration gradient and cleared using xylene, before embedding in a paraffin block. Sections were cut at 5 \u00b5m thicknesses, mounted onto a slide and stained with hematoxylin and eosin (H&E). The sections were examined by an experienced pathologist (R. Whittington) for up to 10 min per slide at up to 400\u00d7 magnification. The microscopic examination was focused on observing specific lesions, such as vacuolation of the brain and retina consistent with viral nervous necrosis and megalocytic cells or inclusion bodies in each of the targeted organs. The megalocytic cells or inclusion bodies are defined as basophilic hypertrophic cells in the spleen, liver, heart, kidney, gills and other tissues .p value < 0.05 was considered significant. For the apparent prevalence, exact binomial confidence intervals at the 95% level were calculated. For the regression analyses, categorization of continuous variables were as follows: days at farm , number of hapas per pond , and fish weight . Due to variation in population numbers, fish length was categorised differently for testing associations with viral pathogens  and parasitology testing . The gross pathology was categorized with 0 representing normal and changes in pathology coded from 1 to 3 representing changes in appearance  . A  colour) .The association between fish weight, fish length, percent of fish with any gross abnormality, gross abnormality in the hematopoietic organs , gross abnormality in the skin and gills, or population (randomly selected vs. targeted) was assessed using Restricted Maximum Likelihood (REML) models. A Generalised Linear Mixed Model (GLMM) with an underlying binomial distribution was used to determine the effect of regional district, days at farm, pond size, number of net-pens, fish length, fish weight, species, source, hapa size, and gross pathology on presence of either ISKNV or NNV.The parasite load was calculated as the total number of observed parasites divided by the fish body mass. The total number of parasites per fish and parasite load were coded to levels either 0, 1, or 2 corresponding with values 0, 1\u201310 and 10+ for number of parasites and 0, <1 or >2 for parasite load. Ordinal logistic regressions were conducted to determine the effect of fish source and length on categorised total number of parasites and parasite load. The effect of fish source, farm, fish length and weight and days at farm on the total number of parasites present and parasite load was investigated using REML models."}
+{"text": "The AA metabolite pathway showed significant changes in centrifuged CSF pellets and supernatants from before to 24\u00a0h after surgery , from before to 6\u00a0weeks after surgery , and from 24\u00a0h to 6\u00a0weeks after surgery . These findings indicate that AA, DHA, and EPA derived lipids are detectable in human CSF, and the AA metabolite pathway shows postoperative changes in centrifuged CSF pellets and supernatants.Arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) derived lipids play key roles in initiating and resolving inflammation. Neuro-inflammation is thought to play a causal role in perioperative neurocognitive disorders, yet the role of these lipids in the human central nervous system in such disorders is unclear. Here we used liquid chromatography\u2013mass spectrometry to quantify AA, DHA, and EPA derived lipid levels in non-centrifuged cerebrospinal fluid (CSF), centrifuged CSF pellets, and centrifuged CSF supernatants of older adults obtained before, 24\u00a0h and 6\u00a0weeks after surgery. GAGE analysis was used to determine AA, DHA and EPA metabolite pathway changes over time. Lipid mediators derived from AA, DHA and EPA were detected in all sample types. Postoperative lipid mediator changes were not significant in non-centrifuged CSF ( Preclinical models suggest that this neuroinflammatory response to surgery may play a causal role in perioperative neurocognitive disorders (PNDs) such as delirium and postoperative cognitive dysfunction/neurocognitive disorder postoperative6. Indeed, neuroinflammation has detrimental neurocognitive effects in central nervous system (CNS) disorders such as multiple sclerosis, autoimmune encephalitis, and Alzheimer\u2019s disease (AD)9.Both animal and human studies show that peripheral tissue trauma induces a significant inflammatory response within the central nervous system11. SPMs, such as lipoxins are derived from arachidonic acid (AA), whereas resolvins, maresins, and protectins, are synthetized from docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). These SPM sub-families play different, yet well-orchestrated roles in resolving inflammation, including suppression of NF-kB dependent inflammatory gene expression (reviewed in12). SPMs, including aspirin-triggered RvD1 and maresin 1 (MaR1), play key roles in resolving neuroinflammation and reducing memory deficits after surgery in mice14.Recent works have also demonstrated that inflammation is actively regulated by polyunsaturated fatty acid-derived lipids known as specialized pro-resolving molecules (SPMs)17. To this end, we measured CSF lipid mediators derived from AA, DHA, and EPA before and after surgery in older adults who were enrolled in the prospective cohort study MADCO-PC (Markers of Alzheimer\u2019s Disease and Neurocognitive Outcomes after Perioperative Care)18. Further, we performed these measurements on non-centrifuged CSF, centrifuged CSF supernatants, and centrifuged CSF cell pellets to determine the extent to which these mediators are present in the cellular fraction versus aqueous phase of CSF.To date, no studies have thoroughly profiled SPMs in the human central nervous system before and after surgery. This represents a necessary first step toward ultimately determining the role of SPMs in humans following surgical recovery, and may have therapeutic implications for conditions like PNDs and other postoperative neurologic complications such as stroke19. We excluded patients who were correctional facility inmates, receiving anticoagulation therapy that would preclude lumbar puncture, or had a personal or family history of malignant hyperthermia. Patients who received chemotherapy or who experienced a traumatic brain injury between the baseline and 6-week postoperative study visits were also excluded. Patients who agreed to participate were scheduled for baseline study visits within one month prior to their scheduled surgery and signed informed consent prior to study participation.CSF samples were obtained from patients enrolled in the Markers of Alzheimer\u2019s Disease and neurocognitive Outcomes after Perioperative Care (MADCO-PC) study, which was approved by the Duke IRB and registered on clinicaltrials.gov (NCT01993836) in accordance with the Declaration of Helsinki. MADCO-PC enrolled Duke surgical patients who were English-speaking,\u2009\u2265\u200960\u00a0years of age, and scheduled for non-cardiac/non-neurologic surgery under general anesthesia for at least 2\u00a0h with a planned postoperative hospital stay of at least 1 night20 using a 25-gauge pencil point spinal needle. A 10-mL polypropylene syringe was used to aspirate CSF from the spinal needle prior to placing the CSF into a 15-mL centrifuge tube  pre-chilled to 4\u00a0\u00b0C. Since lipid mediators are largely hydrophobic/lipophilic, we centrifuged CSF samples from a second set of patients from the same study in order to separately measure lipid mediators in the cell pellet vs aqueous supernatant. To accomplish this, CSF samples were centrifuged at 800\u00a0g for 10\u00a0min at 4\u00a0\u00b0C. The resulting supernatant was decanted into a separate 15-mL tube that was pre-chilled on ice, and aliquoted into 1.5-mL polypropylene tubes  that were also pre-chilled to 4\u00a0\u00b0C, and then stored at \u2212\u00a080\u00a0\u00b0C. For cryopreservation, the resulting cell pellet was re-suspended in 0.5\u00a0mL 90% Hyclone heat-inactivated FBS  and 10% Hybri-Max DMSO  that was pre-chilled to\u2009~\u20094\u00a0\u00b0C, and placed into a BioCision Cool Cell  prior to freezing at \u2212\u00a080\u00a0\u00b0C. Samples were transferred to long-term storage at \u2212\u00a0140\u00a0\u00b0C after 1\u00a0week at \u2212\u00a080\u00a0\u00b0C.CSF samples were obtained at a baseline study visit\u2009\u2264\u20091\u00a0month prior to surgery, and 24\u00a0h and 6\u00a0weeks after surgery. Lumbar punctures were performed as described21. For each time point, 3-mL CSF aliquots (non-centrifuged CSF or centrifuged CSF supernatant) or cryopreserved centrifuged CSF pellets were sent to Wayne State University  for LC\u2013MS analysis. Samples were assigned anonymous codes; thus, assays were blinded to sample identity. In brief, each sample for each time point was thawed on ice and spiked with internal standards. LC\u2013MS-grade methanol was then added to each sample (15% concentration). Samples were sonicated for 2\u00a0min, and then left on ice in the dark for 1\u00a0h. Samples were applied to a C18 solid phase extraction cartridge  that was pre-conditioned with 2\u00a0mL methanol and 2\u00a0mL water and 15% methanol. Cartridges were washed first with 2\u00a0mL 15% methanol in water and then with 2\u00a0mL hexane, and dried under vacuum. Cartridges were then eluted with 1\u00a0mL methanol with 0.1% formic acid directly into HPLC autosampler vials, and the resulting eluate was evaporated under nitrogen at 25\u00a0\u00b0C. The resulting residue was reconstituted in methanol, flushed with nitrogen, and stored at \u2212\u00a080\u00a0\u00b0C.SPMs derived from AA, DHA, and EPA were measured using liquid chromatography with mass spectrometry (LC\u2013MS) as outlined22.For LC\u2013MS measurements, samples were thawed at room temperature, and an equal volume of 25\u00a0mM aqueous ammonium acetate was added. The mixture was then vortexed and loaded in the autosampler at 15\u00a0\u00b0C. HPLC was performed on a Prominence XR system  using a Luna C18(2), 3 \u00b5, 2.1\u2009\u00d7\u2009150\u00a0mm column (Phenomenex) with a mobile phase gradient between methanol\u2013water-acetonitrile (10:85:5 v/v) (A) and methanol\u2013water acetonitrile (90:5:5 v/v) (B), each containing 0.1% ammonium acetate. The elution gradient with respect to mobile phase B: 0\u20131\u00a0min 50%, 1\u20138\u00a0min 50\u201380%, 8\u201315\u00a0min 80\u201395%, and 15\u201317\u00a0min 95%) and a flow rate of 0.2\u00a0mL/min. The HPLC eluate was then introduced to the electrospray ionization (ESI) source of a QTRAP5500 analyzer  in negative ion mode, with conditions set at: curtain gas, GS1, and GS2: 35 psi, temperature 600\u00a0\u00b0C, ion spray voltage \u2212\u00a02500\u00a0V, collision gas set at low, declustering potential \u2212\u00a060\u00a0V, entrance potential \u2212\u00a07\u00a0V. The eluate was monitored by Multiple Reaction Monitoring (MRM) to detect distinct molecular ion-daughter ion combinations. Optimized collisional energies (18\u201335\u00a0eV) and collision cell exit potentials (7\u201310\u00a0V) were used for each MRM transition. Mass spectra were then recorded for each lipid mediator, and the detected peaks were verified via Enhanced Product Ion spectra. Data were collected through Analyst 1.6.2 (SCIEX), and MRM transition chromatograms were quantitated by MultiQuant software (SCIEX) with internal standard signals for each chromatogram used for normalization for recovery and quantitation of each lipid analyte. The resulting area ratios for non-centrifuged CSF, CSF cell pellets, and CSF supernatants were assessed for validity, and retained for analyses only if the signal-to-noise ratio was greater than 3For any lipid mediator(s) with a concentration value below the lower limit of detection , this lower limit of detection (0.0001\u00a0ng/mL) was imputed. In non-centrifuged CSF and centrifuged CSF supernatant samples, we considered a lipid analyte to be present only if its median value across patients exceeded the lower limit of detection at one or more timepoints .Centrifuged CSF pellets were resuspended in FBS and 10% DMSO (as discussed above) and stored at \u2212\u00a0140\u00a0\u00b0C prior to LC\u2013MS. To address the possibility that FBS with 10% DMSO could contain measurable levels of lipid mediators (which could confound sample measurements), we also measured lipid mediator concentrations in three separate 0.5\u00a0mL samples of FBS with 10% DMSO . Post hoc tests included testing for factor interactions and Tukey\u2019s Honestly Significant Difference (HSD) test. For individual lipid analytes, family-wise error rate was corrected using the Bonferroni correction.23 which uses a two-sample t-test for pathway significance. Since this analysis was performed to examine changes over time within each lipid  derived metabolite pathway, samples were excluded for this analysis from any patients who did not have samples available at all 3 time points . This resulted in the exclusion of samples from 20 of the 92 patients whose non-centrifuged CSF samples were used for lipid measurements. Values were normalized to have zero mean and unit standard deviation within each group. Multiple comparison corrections were performed for GAGE analysis using the false discovery rate .To study changes in the AA, EPA and DHA derived metabolite pathways over time, LC\u2013MS data were analyzed using Generally Applicable Gene-set Enrichment (GAGE) analysis in the computing language R (version 3.6.2) as outlinedBaseline characteristics of the patients whose CSF samples were not centrifuged N\u2009=\u200992) and those whose CSF samples were centrifuged  are presented in Tables  and thos2, PGE2, 5-HETE, 11-HETE, 12-HETE, 15-HETE, and 5(S),12(S)-DiHETE; the DHA mediators PD1, 4-HDoHE, 13-HdoHE, and 14-HdoHE; and the EPA mediators RvE3, 5(S),15(S)-DiHEPE, and 18-HEPE for at least 1 of the observed timepoints ,15(S)-DiHETE, 5(S),12(S)-DiHETE, and 15-epi LXA4, the EPA mediator RvE2, and the DHA mediator 10S,17S-DiHDoHE were detectable at all time points -HHTrE, and 5(S),12(S)-DiHETE, the EPA mediator 18-HEPE, and the DHA mediators PD1, 4-HDoHE, 13-HDoHE, 14-HDoHE, and 17-HDoHE were detected (i.e. above the lower limit of quantitation) for at least 1 of the observed timepoints ,12(S)-DiHETE, 5-HETE, 15-HETE, 12-HETE, 11-HETE, 4-HDoHE, and 13-HDoHE. Median log concentrations of lipid mediators for the centrifuged samples cohort (i.e. centrifuged CSF pellets and supernatants) were plotted over each timepoint . Timepoint  did not have a significant effect on lipid mediator concentration except for 12-HETE. Post-hoc testing for 12-HETE showed a significant increase in its concentration from 24\u00a0h post-op to 6\u00a0weeks post-op (p\u2009=\u20090.0012). There was also a\u2009~\u2009180% increase in 12-HETE level from before to 6\u00a0weeks after surgery (p\u2009=\u20090.0014), though there were no significant differences in 12-HETE levels between the pre-op and 24\u00a0h post-op timepoints (p\u2009>\u20090.05). Interaction between sample type and timepoint was not significant for any analyte (p\u2009>\u20090.05), except for 12-HETE at 6\u00a0weeks post-op (p\u2009=\u20091.10E\u22128).ANOVA was performed on lipid mediators whose median analyte concentration values were greater than 0.1\u00a0ng/mL in the centrifuged cell pellet and supernatant group: PGEp and q-values). In both centrifuged CSF cell pellets and supernatants, significant changes were seen in the AA derived metabolite pathway over all 3 time periods (q\u2009<\u20090.05 for each). In contrast, the DHA and EPA derived metabolite pathways did not show changes over any of the time periods in centrifuged CSF cell pellets or supernatants. Non-centrifuged CSF showed no significant changes in any of the 3 pathways. AA, DHA and EPA derived metabolite changes for non-centrifuged CSF , in each sample type  over each time period. These time-period comparisons were made from before to 24\u00a0h after surgery, before to 6\u00a0weeks after surgery, and 24\u00a0h after surgery to 6\u00a0weeks after surgery  across all time points, consistent with the notion that inflammation is a delicate balance between inflammatory (pro/anti) and pro-resolving factors11. Further, we provide the first evidence that human CSF displays dynamic postoperative lipid mediator changes in the AA pathway, but not in the EPA or DHA pathways.Here, we provide evidence that AA-derived pathway mediators showed dynamic changes from before to after surgery in centrifuged CSF supernatants and in cell pellets. Further, we found that SPMs derived from the EPA, DHA, and AA pathways are detectable in centrifuged CSF supernatants, centrifuged CSF cell pellets, and non-centrifuged CSF samples obtained from older adults before and after surgery. A mix of pro- and anti-inflammatory lipid mediators was detected in each sample type , SPMs tend to be generated at higher concentrations locally at sites of active inflammation, and become diluted when they enter circulation resulting in lower SPM concentrations in plasma24. The observation that centrifuged CSF pellets showed enrichment of lipid mediators also suggests a compartmentalization of these molecules within cells as opposed to the surrounding fluid. That the enzymes, such as 5-LO (reviewed in25) and 15-LOX-126, responsible for the endogenous production of SPMs are present within leukocytes lends further credence to this finding. Indeed, macrophages have been shown to switch from pro-inflammatory molecule generation to SPM production via the action of HMGB1-C1q27; since this happens via conversion of polyunsaturated fatty acids, the substrates for this enzymatic action must be available within cells.To our knowledge, this is the first comparison of lipid mediator levels in centrifuged CSF cell pellets, supernatants, and non-centrifuged CSF samples. Notably, lipid mediators observed in centrifuged CSF cell pellets were higher than those seen in centrifuged CSF supernatants, even after subtraction of baseline lipid mediator levels in FBS\u2009+\u200910% DMSO. This suggests that SPMs are likely present at higher concentrations in the CSF cellular/pellet fraction than in aqueous CSF. This finding is analogous to the results of prior studies that measured plasma SPM levels; while SPMs can be generated and act in situ in blood  have been found in patients with active multiple sclerosis36. Clearly, more studies will be necessary to understand the biology, timing, and function of immunomodulatory lipid level changes in the human CNS in multiple neurocognitive disease states. Our data from 50 lipid mediators detected by LC\u2013MS demonstrate the feasibility of this approach for such studies.Indeed, a detailed characterization of SPMs from the AA, EPA, and DHA pathways in human CSF may contribute to novel biomarkers not only relevant to PNDs but other neurocognitive disorders, especially given the increasing evidence for inflammatory dysregulation in various neurologic disease states16. Second, the non-centrifuged CSF samples and centrifuged CSF samples were obtained from two different patient cohorts. The 10\u201312-mL volume of CSF obtained per patient in both of these cohorts is larger than the CSF volume obtained in many other studies39. Yet, this 10\u201312-mL volume is too small to split into 2 parts, one for centrifugation and one without centrifugation, since it takes a full 10\u201312\u00a0mL of CSF to generate a sufficient cell pellet after centrifugation19. Thus, it would be difficult if not impossible to measure lipid mediators in individual human CSF samples split so that half could be processed with centrifugation, and half without centrifugation. Patient and/or procedural differences between these two cohorts may explain why AA pathway changes were seen in centrifuged CSF pellet and supernatant samples but not in non-centrifuged CSF. Alternatively, since the highest concentrations of these lipids were detected in the cell pellets, the levels in non-centrifuged CSF samples may simply be too dilute to measure biologically active intracellular changes in these lipid levels. Third, the CSF samples studied here were from patients enrolled in the MADCO-PC study18; thus, these data cannot necessarily be generalized beyond this population . Fourth, it is unclear whether the changes in CNS lipid mediator levels observed here are driven by peripheral40 factors that cross the blood\u2013brain barrier, or by factors that originate within the CNS itself15, or both. This is a general question that has been posed about the origin of neuroinflammation across multiple neurologic disease states, and applies not only to the immunomodulatory lipid pathways studied here but also to more \u201ctraditional\u201d neuroinflammatory mediators such as cytokines that have been more extensively studied41.This study has at least four limitations. First, the sample size for centrifuged CSF samples was relatively low (N\u2009=\u200920), although the non-centrifuged CSF sample size (N\u2009=\u200990) was similar in size to prior SPM CSF investigations using LC\u2013MSIn conclusion, we have reported dynamic perioperative changes in the CSF lipidome of older surgery patients. The levels of these mediators and the variance among them provides essential preliminary data for powering future studies to decipher their role in various disease states. Such future studies will be required to clarify the role of these mediators in PNDs, and their therapeutic potential for curtailing neuroinflammation and improving cognition in neurologic conditions.Supplementary Information 1."}
+{"text": "Most pancreatic neuroendocrine tumors (PNETs) are indolent, while pancreatic ductal adenocarcinomas (PDACs) are particularly aggressive. To elucidate the basis for this difference and to establish the biomarkers, by using the deep sequencing, we analyzed somatic variants across coding regions of 409 cancer genes and measured mRNA/miRNA expression in nine PNETs, eight PDACs, and four intestinal neuroendocrine tumors (INETs). There were 153 unique somatic variants considered pathogenic or likely pathogenic, found in 50, 57, and 24 genes in PDACs, PNETs, and INETs, respectively. Ten and 11 genes contained a pathogenic mutation in at least one sample of all tumor types and in PDACs and PNETs, respectively, while 28, 34, and 11 genes were found to be mutated exclusively in PDACs, PNETs, and INETs, respectively. The mRNA and miRNA transcriptomes of PDACs and NETs were distinct: from 54 to 1659 differentially expressed mRNAs and from 117 to 250 differentially expressed miRNAs exhibited high discrimination ability and resulted in models with an area under the receiver operating characteristics curve (AUC-ROC) >0.9 for both miRNA and mRNA. Given the miRNAs high stability, we proposed exploring that class of RNA as new pancreatic tumor biomarkers. Pancreatic neuroendocrine tumors (PNETs) arise from the endocrine portion of the pancreas, and pancreatic ductal adenocarcinomas (PDACs) originate from epithelial cells. Although most are indolent, PNETs are unpredictable, ranging from nearly benign to aggressive, metastasizing neoplasms. Larger tumor size, higher grade, and liver metastases indicate a less favorable prognosis ,2. WhileThe histological diagnosis of neuroendocrine tumors is confirmed by immunohistochemical analysis of chromogranin A and synaptophysin expression and the Ki-67/MIB1 proliferation index . HoweverKRAS, TP53, CDKN2A, and SMAD4, while several other genes are mutated in only a small fraction of tumors; of these, only some can be drivers of pancreatic tumorigenesis [MEN-1, encoding menin, a component of a histone methyltransferase complex; in genes encoding either of the two subunits of a transcription/chromatin remodeling complex consisting of DAXX (death-domain associated protein) and ATRX ; in genes encoding components of the mTOR  signaling pathway [MEN1 and DAXX and focal amplification of MYCN concomitant with loss of APC and TP53, suggesting that different cellular pathways may contribute to PNET progression [Complex genetic and epigenetic changes are involved in the initiation and progression of both PDAC and NETs; the malignant behavior of neoplastic cells is driven by somatic mutations in oncogenes and tumor suppressor genes that affect cellular pathways, including cell growth regulation, cell interaction with the extracellular environment, and DNA repair systems . The mosigenesis ,17,18,19 pathway . Another pathway . The gengression .To elucidate the basis of the distinct clinical courses of PNETs and PDACs, we compared the molecular signatures of mostly well-differentiated NETs and PDACs using multi-omics profiling.To compare potential driver mutations in neuroendocrine tumors and ductal adenocarcinomas of the pancreas, targeted sequencing of 409 genes was performed in 13 NETs (nine PNETs and four INETs) and 8 PDACs; detailed characteristics of the patients are shown in The median mapped read count was 20,820,942. The median percentage of bases with greater than 100\u00d7 coverage was 98.41%. Each read was assigned to its respective amplicon to perform mutation calling and estimate the mutant allele frequency.After quality filtering, we identified 1288 unique variants across 234 genes, ranging from 106 to 206 variants for each tumor sample. Among them, 153 were considered pathogenic or likely pathogenic according to the VAST or CHASM algorithm or had a high impact on protein coding  . Of thesHNF1A, CREBBP, RNF213, KRAS, CASC5, APC, and TP53) were found in 50% or more of PDAC samples. Two genes  and six genes  were found to be mutated in 44% and 33% of PNET samples, respectively. Eleven genes  were found to be mutated in two of four INET samples. Of these, the most frequently mutated genes were CREBBP and CASC5 in all three tumor types, HNF1A and APC in both PDACs and PNETs, while two, four, and eight genes were mutated only in PDACs, PNETS, and INETs, respectively  of these genes revealed visible separation of PDACS from NETs and some, but not so clear, second principal component separation of PNETs from INETs .The pairwise comparison revealed that 2082 (1372 downregulated and 710 upregulated), 2621 (1966 downregulated and 655 upregulated), and only 107 (86 downregulated and 21 upregulated) mRNAs differentiated PDACs and PNETs, PDACs and INETS, and PNETs and INETs, respectively . There wThere were 798, 1659, and 54 differentially expressed mRNAs that exhibited a high ability to discriminate between PDACs and PNETs, between PDACs and INETs, and between PNETS and INETs, respectively, with an area under the receiver operating characteristics curve (AUC \u2212 ROC) > 0.9 ; of thesRNAseq data sets were functionally analyzed by annotation to the Reactome signaling pathway database. Several Reactome pathways were found to be discriminative between PDACs and PNETs. The genes that were significantly downregulated in PNETs compared with PDACs could be classified into 53 functional groups . The firp-values < 0.05) between PDACs and PNETs, PDACs and INETS, and PNETs and INETs, respectively  for both miRNA and mRNA in the second component . There wPNETs are relatively uncommon neoplasms, constituting 1\u20135% of pancreatic tumors ,23,24. THNF1A, CREBBP, RNF213, KRAS, CASC5, APC, and TP53; in the PNET samples, CASC5, APC, HNF1A, CREBBP, KAT6B, MLL2, MLL3, and JAK3; in the INET samples, STK11, CASC5, CREBBP, RNF213, PAX7, NFKB2, FLT4, NOTCH1, FLT3, ZNF521, and EP300. While mutations in HNF1A, CREBB, RNF213, CASC5, and APC were shared by NETs and PDACs, mutated KRAS, TP53, and SMAD4 were found only in PDACs.Our massive parallel sequencing of 13 NETs, 11 of which were well-differentiated, and 8 PDACs identified 1288 unique variants across 234 genes. Of these, proliferating driver mutations were present in a fraction of genes, and variants that were considered pathogenic or likely pathogenic were found in 57, 24, and 50 genes in PNETs, INETs, and PDACs, respectively. The most mutated genes in the PDAC samples were MEN1 and VHL are identified in distinct NET hereditary syndromes, and those involving DAXX, ATRX  [PTEN, TSC2, NF1, PIK3CA) are commonly found in sporadic PNETs [KRAS, TP53, SMAD4, and CDKN2A, and mutations in other oncogenes , tumor suppressor genes , and DNA mismatch repair genes  have been found [KRAS, 89.8%; TP53, 66.1%; SMAD4, 22.5%; CDKN2A, 18.5%; ARID1A, 7.6%; LRP1B, 5.7%; RNF43, 5.5%; KMT2C, 5.5%; KMT2D, 5%. A study using a comprehensive cancer gene panel has found the most frequent somatic mutations in PDACs in KRAS, PIK3CD, TAF1L, MTOR, and TP53 [KRAS and inactivating mutations of RB1 and TP53 are typically absent in well-differentiated PNETs, they are commonly seen in both PDACs and pancreatic neuroendocrine carcinomas [As reported previously, recurrent mutations of n pNETs) , and mTOic PNETs ,31,32. Ien found . As descen found  have revrcinomas ,35,36,37MEN1, DAXX, and ATRX were mutated in 36.7%, 22.4%, and 10.2% of tumors, respectively, and that PTEN, SETD2, ATM, MTOR, NUMA1, TP53, and KMT2C were mutated at a frequency between 7.1 and 3.1% [MEN1 and DAXX were found to be mutated in one-fifth of our PNET samples; other genes, including ATRX, PGFRA, MTOR, ATM, TSC2, and VHL, were mutated in individual samples. In summary, we demonstrated that the mutational burden of NETs did not differ from that of PDACs, and while some of the mutated driver genes were shared between all three tumor types, others differentiated one tumor type from the other two.Whole-genome sequencing of sporadic PNETs, mostly of G1\u2013G2 grade, revealed that and 3.1% . Of thesIn contrast to genetic analysis, deep sequencing of the mRNA and miRNA transcriptomes identified much more visible separation between PDACS and NETs at the molecular level. Of 20,520 mRNAs and 1532 mature miRNAs identified by RNAseq, 2082, 2621, and 107 mRNAs and 258, 297, and 129 significantly dysregulated miRNAs differentiated PDACs and PNETs, PDACs and INETS, and PNETs and INETs, respectively. In addition, 220 correlations were found, mostly negative, between relative levels of mRNAs and miRNAs for variables that contributed to three components in DIABLO analysis.2+ signaling, long-term potentiation, and long-term depression, and that downregulated genes are enriched in pancreatic secretion, protein digestion and absorption, and other metabolic pathways. Interferon-stimulated gene protein 15, somatostatin, and synaptosomal-associated protein 25 kDa are identified as hub proteins [The rarity of PNETs has limited the number of large-scale transcriptomic studies, in contrast to numerous studies previously conducted in pancreatic cancer. Bioinformatics analysis of the two data sets from ICGC and two from the NCBI GEO database has identified the five common differentially expressed genes in PNETs  related to their development and prognosis . A microproteins .When our mRNAseq data sets were annotated according to Reactome signaling, downregulated transcripts in PNETs, when compared with PDACs, were classified into 53 functional groups, including interleukin-10 signaling, immune system, extracellular matrix organization, degradation of the extracellular matrix, and syndecan interactions. Upregulated transcripts fell into 25 groups, including the neuronal system; GABA synthesis, release, reuptake, and degradation; neurotransmitter receptors and postsynaptic signal transmission; GABA receptor activation; regulation of gene expression in beta cells . These fIn fact, NETs originate from the diffuse neuroendocrine system, and functional NETs can secrete different peptides and amines categorized as nonspecific markers  and specific markers . Our functional annotations of differentially expressed genes confirmed significant biological differences between PNETs and PDACs.Each of the approximately 1800 human miRNAs may have regulatory roles in multiple cell signaling pathways, and some mediate and/or their expression correlates with oncogenesis. Oncogenic miRNAs are linked to several pro-tumorigenic processes, such as apoptosis resistance, cell proliferation, chemoresistance, and EMT, depending on the function of their respective target mRNAs . miRNAs miRNAs profiling may have diagnostic and prognostic potential, correlating with cancer clinical outcomes . Our ownwww.cancerstaging.orgajcc@facs.org). All subjects provided written informed consent prior to participation, and the study complied with the Declaration of Helsinki. The study was approved by the ethics committee . Written informed consent was obtained from all participants.A total of 21 patients were enrolled in this study from January 2018 to December 2018, including 9 with PNETs, 8 with PDACs, and 4 patients with intestinal neuroendocrine tumors (INETs); of the latter, one tissue sample was obtained from liver metastasis. All cases had adequate clinical and pathologic information. The patient\u2019s sex, age, tumor site, size, stage, histology, grade, type of surgery performed, additional treatment, and follow-up data were recorded. Tumor grade and stage were evaluated according to the criteria of the 8th edition of the AJCC staging manual (2017) (PNET patients. The median age of PNET patients (8 women and 1 man) was 45.4 years (range 24\u201370). The median tumor size was 2.9 cm (range 1.3\u20133.5); five tumors were located in the pancreatic tail. Two tumors were G1, six were G2, and one was G3, respectively. Six tumors were at stage II, one at stage III, and one at stage IV. In all, but one, patients, distal pancreatic resection or pancreaticoduodenectomy was performed.PDAC patients. The median age of PDAC patients (6 women and 2 men) was 60.3 years (range 38\u201372). The median tumor size was 2.7 cm (range 1.7\u20133.3); six tumors were located in the pancreatic head. All tumors were classified as ductal adenocarcinoma. Five tumors were G2, and six tumors were at stage III and 2 at stage IV. In five patients, potentially curative surgery was performed, and in three others, the only laparotomy.INET patients. The median age of the INET patients (one woman and three men) was 64 years (range 59\u201367). The median tumor size was 2.6 cm (range 2\u20133). Three primary tumors were G1, and one liver metastatic tumor was G3. There was one tumor at stage II, one at stage III, and one at stage IV.All tissue samples for molecular analysis were unfixed postoperative specimens, and all, but one, were primary tumors. The tissue was frozen in liquid nitrogen and stored until use at \u201370 \u00b0C.Total nucleic acids were isolated from tumor tissue specimens using the ALLPrep DNA/RNA Micro Kit , following the manufacturer\u2019s protocol. DNA sample concentrations were measured using a NanoDrop spectrophotometer, following the manufacturer\u2019s instructions, and the DNA was stored at \u201320 \u00b0C. The purity and quantity of total RNA were measured using a Qubit Fluorometer and assessed using an Agilent 2100 Bioanalyzer with an Agilent RNA 6000 Nano Kit . RNA samples were stored at \u201370 \u00b0C.Ion AmpliSeq Comprehensive Cancer Panel  libraries were prepared from DNA samples for the analysis of the coding regions of 409 oncogenes and tumor suppressor genes by sequencing on an Ion Proton sequencer with the Ion PI Hi-Q Chef Kit, loading four samples onto an Ion PI Chip, as described previously .p-value after FDR correction was smaller or equal to 0.1 for a single variant, (ii) the variant was a non-synonymous variant in a known driver gene, or (iii) the variant was pathogenic according to the VEST algorithm.Raw reads were processed using the Torrent Suite analysis pipeline version 5.10 and mapped to human genome assembly hg19 using TMAP . VariantmRNA and miRNA cDNA libraries were prepared with the Ion AmpliSeq Transcriptome Human Gene Expression Panel and Ion Total RNA-Seq Kit v2 for Small RNA Libraries, respectively, and an Ion Xpress\u2122 RNA-Seq Barcode Kit , according to the manufacturer\u2019s protocol. The amplified libraries of ligation products (94\u2013114\u2009bp) were assessed on the Bioanalyzer 2100 using a High Sensitivity DNA Kit . Eight mRNA barcoded library templates at a concentration of 50 pM and up to 16 miRNA libraries at a concentration of 40\u2009pM were loaded onto Ion PI chips and sequenced on an Ion Proton Sequencer  with the Ion PI Hi-Q Chef Kit, according to the manufacturer\u2019s instructions.p-values were adjusted for the testing of multiple hypotheses using the Benjamini\u2013Hochberg procedure to control the false discovery rate. The false discovery rate threshold was set to 0.05, and only probe sets exhibiting a minimum 2-fold change in mean relative expression were included in the functional analysis. Overrepresentation of gene ontology and Reactome pathways with hypergeometric testing was determined using ClueGO version 2.5.4, with false discovery rate p-value correction [Raw reads were processed using the Torrent Suite analysis pipeline and mapped to the AmpliSeqTranscriptome version of the human genome assembly hg19 by use of TMAP. Reads corresponding to each gene were counted using htseq-count  version rrection .Unmapped bam files were converted to fastq files using a bamToFastq script from bedtools version 2.26 . Read mahttp://mixomics.org/mixdiablo/case-study-tcga/.Transcriptome and miRNA data integration (including PLS-DA and correlation computation) were conducted using DIABLO with mixOmics functions  package MEN1 and DAXX, mutations in which were found in one-fifth of PNET samples but not in other tumor types. Among the most-mutated genes, KRAS and TP53 were mutated only in cancers, while five other genes  were mutated with similar frequencies in adenocarcinomas and neuroendocrine tumors. However, most other driving mutations were found only in individual tumor samples or in small percentages of tumors. In contrast to the genetic findings, the clearest differences were transcriptomic. While differentially expressed genes may indicate different sites of origin for adenocarcinomas and neuroendocrine tumors, they cannot be linked to significant differences in the aggressiveness of PNETs and PDACs. Finally, our study revealed that both mRNA and miRNA profiles had a high ability to discriminate between tumor samples. In addition, integrative analysis using the novel algorithm DIABLO allowed us to construct a multi-omic biomarker model, based on quantitative data from miRNA and mRNA. Both profiles showed high discriminatory powers, mainly in the second component, where they reached AUC of 1 for all the groups. Unsurprisingly, the miRNA profile was also highly correlated to mRNA expression. Hence, miRNAs can be considered as a new class of pancreatic tumor biomarkers due to their high stability.To sum up, our study was the first to compare directly and simultaneously mutational and transcriptional profiles of well-differentiated neuroendocrine tumors and ductal adenocarcinomas of the pancreas using massively parallel sequencing. We confirmed the presence of two driver genes,"}
+{"text": "Correction to: Laboratory Investigation10.1038/s41374-019-0353-3https://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.The article Extra-mitochondrial citrate synthase initiates calcium oscillation and suppresses age-dependent sperm dysfunction, written by Woojin Kang, Yuichirou Harada, Kenji Yamatoya, Natsuko Kawano, Seiya Kanai, Yoshitaka Miyamoto, Akihiro Nakamura, Mami Miyado, Yoshiki Hayashi, Yoko Kuroki, Hidekazu Saito, Yasuhiro Iwao, Akihiro Umezawa and Kenji Miyado, was originally published electronically on the publisher\u2019s internet portal on 19th December 2019 without open access. With the authors\u2019 decision to opt for Open Choice the copyright of the article changed on\u00a09th January to \u00a9 The Author(s) 2019 and the article is forthwith distributed under a Creative Commons Attribution 4.0 International License (The original version of this article was revised due to a retrospective Open Access order."}
+{"text": "The Mini-Mental State Examination-Second Edition (MMSE-2) consists of three visions: a brief version (MMSE-2:BV), a standard version (MMSE-2:SV), and an expanded version (MMSE-2: EV). Each version was equipped with alternate forms (blue and red). There was a lack of evidence on the practice effect and test-retest reliability of the three versions of the MMSE-2, limiting its utility in both clinical and research settings. The purpose of this study was to examine the practice effect and test-retest reliability of the MMSE-2 in people with dementia.t-test and Cohen\u2019s d. The test-retest reliability was examined using the intraclass correlation coefficient (ICC).One hundred and twenty participants were enrolled, of which 60 were administered with the blue form twice  and 60 were administered with the blue form first and then the red form . The practice effect was evaluated using a paired p\u2009=\u20090.061\u20131.000), except for the MMSE-2:SV total score (p\u2009=\u20090.029). In the AF group, no statistically significant differences were found for all three versions of the total scores and subtests (p\u2009=\u20090.106\u20131.000), except for the visual-constructional ability subtest (p\u2009=\u20090.010). Cohen\u2019s d of all three versions\u2019 total scores and subtests were 0.00\u20130.20 and 0.00\u20130.26 for SF group and AF group, respectively. For the test-retest reliability, ICC values for all three versions and eight subtests in SF and AF groups were 0.60\u20130.93 and 0.56\u20130.93, respectively.For the practice effects, in the SF group, no statistically significant differences were found for the MMSE-2:BV and MMSE-2: EV total scores and eight subtests . Caution should be taken when interpreting the results of visual-constructional ability, registration, and recall subtests of the MMSE-2.The online version contains supplementary material available at 10.1186/s12877-021-02732-7. It has been estimated that about 46.8 million people are suffering from dementia worldwide and that this figure will increase to 74.7 million by 2030 and to 131.5 million people by 2050 [Four commonly-used cognitive screening tools have been used for the early detection of dementia including the Mini-Mental State Examination (MMSE), the Short Portable Mental Status Questionnaire, the Montreal Cognitive Assessment, and the Saint Louis University Status Examination . Each ofThe MMSE-2 preserves the clinical utility and efficiency of the original MMSE while expanding its application in populations with dementia . The MMSSecondly, each version of the subtests of the MMSE-2 has two alternate forms (blue and red forms) in order to decrease the practice effects that might occur over repeated testing . Almost An assessment would be considered useful if it could produce stable and consistent results with repeated administration . The praA convenience sample of people with dementia was recruited from Department of Psychiatry or Department of Neurology of two teaching hospitals in northern Taiwan between March 2019 and April 2020. The following criteria were used to determine the eligibility of people with dementia to participate in this study: (1) diagnosis of probable dementia and dementia according to the National Institute on Aging and Alzheimer\u2019s Association [Prior to the study, three raters  familiarized themselves with the MMSE-2. The three raters reviewed the user manual of the MMSE-2 and received 4 hours of training from the corresponding author on the administration of the MMSE-2. Then, the three raters performed the MMSE-2 on the corresponding author, and their score results were checked. If there were any discrepancies in the score results, discussions with the corresponding author were required to ensure proper administration procedures and scoring. Finally, the three raters independently administered the MMSE-2 to five people with dementia. The corresponding author observed the assessments and gave MMSE-2 scores simultaneously to confirm that all three raters performed the MMSE-2 correctly in a standardized manner. In addition, when the study began, the raters did not discuss the results of scores with each other to avoid potential bias.The blue form was administered twice in a two-week interval by raters A and B to the participants, who were from hospital A.The alternate forms  were administered by rater C 2 weeks apart to the participants who were from hospital B. The AF group completed the MMSE-2 in a fixed order .All assessments were conducted in a quiet room to avoid interference that might have affected the performance of the participants. Between each assessment, the participants were allowed to break to minimize fatigue. The demographic data of all participants were collected from their medical records.The MMSE-2 was developed to assess cognitive impairment. The MMSE-2: BV (score range: 0\u201316) is composed of three subtests: registration, orientation, and recall. The MMSE-2: SV (score range: 0\u201330) is composed of six subtests: three subtests of the MMSE-2:BV along with attention and calculation, language, and visual-constructional ability. The MMSE-2: EV (score range: 0\u201390) is composed of eight subtests: six subtests of the MMSE-2:SV, as well as story memory and processing speed .Paired  effects . As multrrection  for 11\u2009t2,1) was calculated using a two-way random analysis of variance with absolute agreement. An ICC value of 0.81\u20131.00 indicates excellent reliability, 0.61\u20130.80 indicates good reliability, 0.41\u20130.60 indicates moderate reliability, and\u2009<\u20090.40 indicates poor reliability [95) was calculated on the basis of the standard error of measurement (SEM) to estimate the random measurement error of the MMSE-2 [To examine the test-retest reliability of the MMSE-2, the intraclass correlation coefficient , which was independent of the units of measurement, and used it to determine a relatively true change between two assessments: MDC%\u2009=\u2009\u2009\u00d7\u2009100 . In thist\u2009=\u20091.781, p\u2009=\u20090.080, gender, \u03c72\u2009=\u20090.094, p\u2009=\u20090.759, education, t\u2009=\u200939.992, p\u2009=\u20090.105, or CDR, t\u2009=\u20093.436, p\u2009=\u20090.752. The characteristics of the participants are shown in Table\u00a0A total of 120 participants participated in this study; of these, 60 from hospital A were assigned to the SF group and 60 from hospital B were assigned to the AF group. The participants in the SF group had a mean age of 81.5\u2009\u00b1\u20097.8\u2009years, 55% were female, and 58.3% had an educational level below elementary school. The participants in the AF group had a mean age of 79.1\u2009\u00b1\u20096.8\u2009years, 67.3% were female, and 43.3% had an educational level below elementary school. There were no significant group differences in age, p\u2009=\u20090.061\u20131.000). There was a statistically significant difference in the MMSE-2:SV total score (p\u2009=\u20090.029). The Cohen\u2019s d for all three versions total scores and subtests, except for the visual-constructional ability subtest, ranged from 0.00 to 0.15, indicating no or trivial practice effects. Cohen\u2019s d for the visual-constructional ability subtest was 0.20, indicating a small practice effect , except for the visual-constructional ability subtest (p\u2009=\u20090.010). Cohen\u2019s d for all three versions\u2019 total scores and subtests, except for the visual-constructional ability subtest, ranged from 0.00 to 0.10, indicating no to trivial practice effects. Cohen\u2019s d for the visual-constructional ability subtest was 0.26, indicating a small practice effect , showed good to excellent reliability (ICC\u2009=\u20090.67\u20130.93) Table . At the In the AF group, all three versions and subtests, except for the registration and recall subtests (ICC\u2009=\u20090.56), showed good to excellent reliability (ICC\u2009=\u20090.67\u20130.93)  in the AF group showed moderate test-retest reliability, indicating that these two subtests may not consistently assess specific subtest functions over repeated assessments. One possible reason might be that alternate forms (blue and red forms) were used in the AF group, which may have resulted in more variation compared to using the same form as the SF group. Although the alternate forms used diminished the carryover effect, they increased the variation due to random errors in measurement. Thus, caution is needed when using the alternate forms of registration and recall subtests in people with dementia.The\u00a0MDC values can be used as thresholds to indicate whether an individual\u2019s changed score between two successive assessments is due to real improvement or due to measurement errors . For exaOur results showed that the three versions of the MMSE-2 total scores and all subtests were all below 30% of the highest score of all test data, indicating an acceptable random measurement error. Thus, the MMSE-2 appeared reliable for describing cognitive function in people with dementia. Although the random measurement error was acceptable, we found that the MDC% of the visual construction ability subtest in both groups and the registration subtest in the AF group were higher than those in the other subtests. The large amount of MDC% values indicates that the scores of these two subtests are unstable and thus may obscure the real changes of a person with dementia. Possible reasons for these results might be the nature of practice effects and moderate test-retest reliability. Future studies are needed to investigate the causes of variations in people\u2019s responses to visual-constructional ability and registration subtests between repeated assessments. The results could help users exclude factors that increase random measurement errors and improve the utility of the MMSE-2.p\u00a0=\u20090.003), had non-significant differences at baseline (p\u00a0>\u20090.05) , the results of the AF group with a sample size of 51were not dramatically different when compared with the previous results using a sample size of 60  used in the AF group might have influenced the results of the study. To confirm our findings, future studies that randomized the alternate form order may be needed. Fourth, the inter-rater reliability between rates has not yet been established, which may jeopardize our current validation of the MMSE-2. Future studies are needed to examine the inter-rater reliability of the MMSE-2 in people with dementia. Fifth, we did not collect information regarding whether the patients had been assessed with the MMSE or MMSE-2 before or during our study, in which case the participants might have become familiar with the screening tools. Familiarity with the screening tools might have caused underestimations of the practice effect and test-retest reliability in this study.Overall, the practice effect of the MMSE-2 diminished when the alternate forms were performed. In addition, the MMSE-2 had good to excellent test-retest reliability with an acceptable random measurement error, which supports the use of this measure in both clinical and research settings. However, the visual-constructional ability subtest showed small practice effects, and both the registration and recall subtests were found to have moderate test-retest reliability. Thus, caution should be exercised when interpreting the results of the visual-constructional ability, registration, and recall subtests of the MMSE-2.Additional file 1. Practice effects of the MMSE-2 in the alternate-form group (AF group).Additional file 2 Results of independent sample t-test of the versions and subtests in the MMSE-2 between the SF (n\u2009=\u200960) and AF (n\u2009=\u200960) groups at baseline."}
+{"text": "Among urinary metabolites, lower levels of citrate, ethanolamine, formate, and hypoxanthine were positively associated with all three DR outcomes . Higher levels of serum/plasma 3-hydroxybutyrate and lower levels of urinary 3-hydroxyisobutyrate were associated with VTDR. Comprehensive metabolic profiling in three large Asian cohorts with DR demonstrated alterations in serum/plasma and urinary metabolites mostly related to amino acids, lipoprotein subclasses, kidney function, and glycolysis.Diabetic retinopathy (DR) is a microvascular complication of diabetes mellitus, a metabolic disorder, but understanding of its pathophysiology remains incomplete. Meta-analysis of three population-based cross-sectional studies (2004\u201311) representing three major Asian ethnic groups  was performed. A panel of 228 serum/plasma metabolites and 54 urinary metabolites were quantified using nuclear magnetic resonance (NMR) spectroscopy. Main outcomes were defined as any DR, moderate/above DR, and vision-threatening DR assessed from retinal photographs. The relationship between metabolites and DR outcomes was assessed using multivariate logistic regression models, and metabolites significant after Bonferroni correction were meta-analyzed. Among serum/plasma metabolites, lower levels of tyrosine and cholesterol esters to total lipids ratio in IDL and higher levels of creatinine were positively associated with all three outcomes of DR (all  In 2019, an estimated 463 million adults (20\u201379 years old) globally had diabetes, and this is expected to increase to 700 million by 2045 [Diabetes mellitus is a metabolic disorder associated with insulin resistance, altered lipid metabolism, subclinical inflammation, and oxidative stress ,5,6. MetTwo main techniques of metabolomic profiling are currently used\u2014mass-spectrometry (MS) or nuclear magnetic resonance (NMR). These techniques provide complementary insights, due to their respective strengths and limitations. MS allows for in-depth characterization of metabolic profiles due to high mass resolution and accuracy, as it is able to resolve and identify thousands of small molecules in a sample with high sensitivity. However, it is labor intensive, placing limitations on study sample size. Nuclear magnetic resonance (NMR), on the other hand, is effective for metabolite quantification in large-scale epidemiological studies as it requires minimal sample preparation and processing, offering high throughput capabilities. While NMR is still less sensitive than MS, technological developments have greatly increased the resolution and sensitivity of NMR, making it an overall more accessible and cost-effective tool for metabolic analysis, and it has been broadly applied in large-scale epidemiologic studies . Indeed,Several previous studies have identified metabolites associated with DR, mainly proliferative DR (PDR), in serum, plasma, or vitreous of patients with diabetes using MS ,10,11,12n = 3280), the Singapore Indian Eye Study , and the Singapore Chinese Eye Study . The detailed recruitment and methodology of these studies have been published elsewhere [Data for this study were derived from Chinese, Malay, and Indian participants aged \u226540 years who participated in the baseline visit of three independent population-based studies in Singapore: the Singapore Malay Eye Study  and had their metabolites measured . Of these, after excluding those with ungradable retinal photographs or missing values for key covariates included in the multivariable model, 485 Chinese, 945 Malays, and 1213 Indians were included for the final serum/plasma metabolites analysis. Serum samples were collected from the Chinese and Indian participants, whilst plasma samples were collected for analysis from the Malay participants. The Malay cohort was not included in the analysis of urine metabolites as data on the urinary metabolites were not available for two thirds of the Malay cohort. Hence, for urinary metabolites, 509 Chinese and 1153 Indians with data on urinary metabolites and key covariates were included for the final analysis.For the current analysis, we included only those with diabetes  field 1 which is centered on the optic disc, and field 2 which is centered on the fovea. These photographs were taken using the Canon CR-1 Mark-II Non-mydriatic Digital Retina camera. DR was considered to be present if any characteristic lesions were noted on the retinal photographs, including microaneurysms, hemorrhages, cotton wool spots, intraretinal microvascular abnormalities, hard exudates, venous beading, and/or neovascularization. DR severity grading was conducted by trained retinal graders from the University of Sydney, Australia. Based on the modified Airlie House classification system, retinopathy severity was categorized as minimal (level 20), moderate (level 43\u201347), severe non-proliferative (level 53), and proliferative (level > 60) . Taking Information on age, sex, ethnicity, personal history of diabetes, duration of diabetes, and medication were collected using standardized questionnaires. Blood pressure (BP) measurements were taken using a digital automatic blood pressure monitor after the participant was seated for at least 5 minutes and an average of two measurements were taken as the blood pressure value for that individual. HbA1c and random blood glucose were assessed from blood samples.Blood and urine samples for our study were collected at baseline and stored at \u221280 \u00b0C until time of metabolite analysis between 8 to 15 years after collection. The average time taken to process the samples from the time of collection to the time of storage was 4\u20136 h. The samples were subsequently stored at \u221280 \u00b0C until the time of analysis.A number of studies have assessed the effect of storage of samples at \u221280 \u00b0C over various durations on metabolites ,22,23,24A high-throughput NMR metabolomics platform  was used to quantify 228 metabolic measures from baseline serum/plasma samples and 54 metabolites from baseline urine samples. Serum/plasma metabolites included routine lipids, lipoprotein subclasses with lipid concentrations within 14 subclasses, fatty acid, amino acids, ketone bodies, and glycolysis related metabolites. The 14 lipoprotein subclasses include six subclasses of VLDL , IDL, three subclasses of LDL , and four subclasses of HDL . Lipid concentration within each lipoprotein particle included triacylglycerol, total cholesterol, non-esterified cholesterol and cholesteryl ester levels, and phospholipid concentrations. Urine metabolites included amino acids, dietary, microbial, phenyl alanine, and pyrimidine. Metabolic measures were quantified, either as absolute concentrations of each metabolic measure or as ratios. Details of the NMR metabolomics quantification have been described previously [e (metabolite) transformed. To facilitate comparison of associations independent of the concentration ranges, we standardized each transformed concentration to mean 0, variance 1 for each cohort separately. Standardized values \u22654 or \u2264\u22124 were considered outliers and thus excluded. Next, we implemented logistic regression adjusting for age, sex, duration of diabetes, HbA1c, and systolic blood pressure to estimate the odds ratio (OR) and 95% confidence interval (95% CI) for each metabolite in each cohort separately. Metabolites having p-values < 0.05 in at least one cohort and associations of the same direction in Chinese, Malay, and Indian cohorts for serum/plasma, or Chinese and Indian cohorts for urinary, were carried into meta-analysis, where results from individual cohorts were combined using generic inverse variance-weighted fixed effect model. The presence of heterogeneity was evaluated using p-values was pre-specified as 0.05/n using Bonferroni correction (BC) where n was the number of serum/plasma or urine metabolites passed to the meta-analysis accordingly. After BC, statistical significance for serum/plasma metabolites was considered at p-value < 0.00075, 0.00072, and 0.00161 for outcomes any DR, moderate/above DR, and VTDR respectively, while for urine metabolites it was <0.00714, 0.00417, and 0.005, respectively. Our approach to meta-analysis was similar to the methods adopted by recently published studies that analyzed NMR profiled metabolites associated with diabetes [https://www.R-project.org/, accessed on 8 May 2020).For analyses, metabolic concentrations below the detection limit were imputed by the minimum values detected for the corresponding metabolites. To alleviate the skewness of distributions, all metabolite concentrations were logdiabetes  and impadiabetes . Lastly,Participants with and without DR were not statistically different in age and gender . Those wp < 0.05 were carried to meta-analysis. However, after accounting for multiple testing using BC, only five metabolites were significantly associated with any DR  and creatinine were positively associated with any DR.In total, 13 metabolites were significantly associated with moderate/above DR after BC. Among the lipoprotein subclasses, ratios of cholesterol esters to total lipids in chylomicrons and extremely large VLDL, large VLDL and very large VLDL, and total cholesterol in chylomicrons and extremely large VLDL, were positively associated while ratios of cholesterol esters and total cholesterol to total lipids in IDL, phospholipids in small LDL, and total cholesterol to total lipids ratio in IDL and very large HDL were protectively associated with moderate/above DR . In addiNine metabolites were significantly associated with VTDR after BC. Among the lipoprotein subclasses, ratio of cholesterol esters to total lipids in chylomicrons and extremely large VLDL, ratio of total cholesterol to total lipids in chylomicrons and extremely large VLDL, and triglycerides in IDL were positively associated . Ratio oSeven urinary metabolites were carried to meta-analysis for association with any DR, based on ethnic group analysis, of which five were found to be associated with any DR after BC . Twelve p-values < 0.00714  and correspondingly lower urinary levels of 3-hydroxyisobutyrate  were found to be associated with VTDR.p < 0.001) for any DR; 0.824 (0.797\u20130.852) vs. 0.854 (0.829\u20130.879), p = 0.003 for moderate/above DR; 0.835 (0.803\u20130.867), vs. 0.868 (0.8441\u20130.895), p = 0.004 for VTDR.  of 0.777 (0.756\u20130.798) vs. 0.795 (0.774\u20130.815) for traditional + serum/plasma metabolites , sphingomyelins, and phospholipids in small LDL, cholesterol esters to total lipids ratio in IDL, chylomicrons and large/very large/extremely large VLDL, triglycerides to total lipids in IDL, chylomicrons and extremely large VLDL, and total cholesterol to total lipids ratio in chylomicrons and extremely large VLDL, IDL, and large HDL. The urinary metabolites associated with at least one of the three DR outcomes were citrate, ethanolamine, formate, hypoxanthine, 3-hydroxyisovalerate, 3-hydroxyisobutyrate, alanine, glutamine, uracil, and glycolic acid. We identified three serum/plasma metabolites and four urine metabolites that were significantly associated with all severities of DR: higher levels of tyrosine and higher cholesterol esters to total lipids ratio in IDL were found to be protective for all severities of DR, while higher levels of creatinine were found to be positively associated with all three DR outcomes. For urinary metabolites, higher levels of citrate, ethanolamine, formate, and hypoxanthine were negatively associated with all three DR outcomes. In ROC analyses, addition of significant serum/plasma metabolites modestly improved the prediction accuracy of any DR, moderate and above DR, and VTDR beyond traditional risk factors.DR is a complex chronic metabolic disease, involving an interplay of various metabolic pathways. The pathophysiology of DR remains incomplete. Current understanding can be broadly discussed in three main aspects\u2014hyperglycemia induced damage, inflammation, and retinal neurodegeneration.Similar to the previous studies, our study found that lower serum/plasma levels of the amino acid tyrosine were positively associated with DR. This is in agreement with a previous hospital-based cross sectional study that found that low plasma tyrosine and phenylalanine levels were a risk factor for DR in patients with type 2 diabetes . One aspSerum/plasma creatinine is used clinically as a measurement of kidney function. Both nephropathy and retinopathy are known microvascular complications of diabetes. The prevalence of both of these complications is known to increase proportionally to the duration of type 2 DM ,30. A feSeveral studies have been done to investigate the correlation between elevated serum/plasma cholesterol and lipid levels with DR. In particular, studies focusing on the treatment of hyperlipidemia have highlighted a possible correlation between serum/plasma cholesterol and lipid levels with DR. Our study noted that lower levels of serum/plasma cholesterol esters to total lipids ratio in IDL and higher levels of total cholesterol to total lipids ratio in chylomicrons and extremely large VLDL were significantly associated with moderate/above DR. Furthermore, lower levels of ratio of saturated fatty acids to total fatty were associated with VTDR. Previous studies have found that diabetes affects the synthesis and metabolism of triglyceride-rich lipoprotein particles . HoweverThe association between serum/plasma saturated fatty acids and diabetes has not been conclusive. Most studies done on this subject so far support the role of odd-chain saturated fatty acids being protective and even-chain saturated fatty acids having an adverse effect ,37,38,39Few studies have been done to investigate urinary metabolites. Interestingly, our study identified significantly higher levels of serum/plasma 3-hydroxybutyrate in participants with DR and participants with VTDR, and correspondingly, lower levels of urinary 3-hydroxyisobutyrate in individuals with moderate and above DR, and VTDR. This suggests a correlation between serum/plasma and urinary levels of metabolites involved in ketone metabolism. Understanding of the physiological role of formate remains limited. It acts as an intermediary metabolite in folate-mediated one-carbon metabolism\u2014a network of biochemical reactions essential for the biosynthesis of purines and generation of methyl groups . It is pPast studies have shown that patients with chronic kidney disease have increased acid retention as estimated glomerular filtration rate (eGFR) decreases with disease progression . FurtherThe major strength of the current study is quantifying metabolites in large population-based, well characterized and diverse ethnic cohorts and evaluating associations using a combination of both serum/plasma and urinary metabolites. Although previous metabolomic studies on DR employed mass spectrometry which is more sensitive than NMR profiling, they were limited by the small sample sizes of less than 100 patients and 100 controls, resulting in large variability in findings and difficulties making conclusive associations.Our study has some limitations. Being a cross-sectional study, it does not provide a longitudinal picture, as a prospective study would be able to do. Furthermore, while our study identifies metabolites that are present in significantly different levels in participants with different severities of DR, it does not provide insight into the causal relationship between the metabolites and DR. In addition, we included only Chinese and Indian adults for the urinary metabolite analyses since urine samples were not available in the Malay cohort.In conclusion, we identified sixteen serum metabolites and ten urinary metabolites to be associated with DR. If confirmed in future longitudinal analysis, adding detailed metabolic profiling to traditional risk factors could help in the formulation of disease prevention strategies for reducing the burden of DR."}
+{"text": "Currently available structural variant (SV) detection methods do not span the complete spectrum of disease\u2010causing SVs. Optical genome mapping (OGM), an emerging technology with the potential to resolve diagnostic dilemmas, was performed to investigate clinically\u2010relevant SVs in a 4\u2010year\u2010old male with an epileptic encephalopathy of undiagnosed molecular origin.OGM was utilized to image long, megabase\u2010size DNA molecules, fluorescently labeled at specific sequence motifs throughout the genome with high sensitivity for detection of SVs greater than 500\u00a0bp in size. OGM results were confirmed in a CLIA\u2010certified laboratory via mate\u2010pair sequencing.de novo 90\u00a0kb deletion and inversion on the X chromosome disrupting the CDKL5 gene. Detection of the mosaic deletion, which had been previously undetected by chromosomal microarray, an infantile epilepsy panel including exon\u2010level microarray for CDKL5, exome sequencing as well as genome sequencing, resulted in a diagnosis of X\u2010linked dominant early infantile epileptic encephalopathy\u20102.OGM identified a mosaic, OGM affords an effective technology for the detection of SVs, especially those that are mosaic, since these remain difficult to detect with current NGS technologies and with conventional chromosomal microarrays. Further research in undiagnosed populations with OGM is warranted. CDKL5 gene. Detection of the mosaic deletion, which had been previously undetected by chromosomal microarray, an infantile epilepsy panel including exon\u2010level microarray for CDKL5, exome sequencing as well as genome sequencing, resulted in a diagnosis of X\u2010linked dominant early infantile epileptic encephalopathy\u20102. OGM affords an effective technology for detection of SVs, especially those that are mosaic, since these remain difficult to detect with current NGS technologies and with conventional chromosomal microarrays.In a participant with an epileptic encephalopathy of undiagnosed molecular origin, optical genome mapping (OGM), identified a mosaic, de novo 90\u00a0kb deletion on the X chromosome disrupting the  CNVs arOptical genome mapping (OGM) developed by Bionano Genomics is a novel technology with high sensitivity for the detection of SVs ranging from 500\u00a0bp to aneuploidies , a nationwide research study funded by the National Institutes of Health Common Fund, aims to discover diagnoses for the most difficult\u2010to\u2010diagnose individuals (including many with non\u2010diagnostic CMA and ES) through collaborative science and adoption of novel diagnostic methods  was performed to investigate clinically\u2010relevant SVs in a 4\u2010year\u2010old male with an epileptic encephalopathy of the undiagnosed molecular origin and an exhaustive non\u2010diagnostic evaluation, including both ES and GS. Informed consent had been obtained from the participant's parent to participate in the NIH\u2010UDN protocol (15\u2010HG\u20100130).2.1A 4\u2010year\u2010old male was accepted to the UDN due to refractory epilepsy, severe hypotonia, cortical visual impairment and severe global developmental delay. Seizures began at age one month and consisted of infantile spasms and tonic\u2013clonic seizures. Treatment with ACTH resulted in cessation of seizures for one month, with a relapse once ACTH was discontinued. Since then, the seizures have been refractory to numerous antiepileptic drugs (AEDs), and continue to occur multiple times daily, despite AED polytherapy and vagal nerve stimulator implantation. The family history was non\u2010contributory; there was a healthy older brother. MRI brain at age three years demonstrated diffuse brain parenchymal volume loss and confluent T2 white matter signal abnormality with posterior cerebral predominance. Extensive metabolic and genetic testing prior to the UDN had been non\u2010diagnostic, including chromosome analysis, CMA (Affymetrix Cytoscan HD array), an infantile epilepsy panel in 2013 , mitochondrial genome sequencing and trio ES  in 2014.de novo variant of unknown significance  in ARHGAP45 had been reported on ES by the commercial laboratory. However, ARHGAP45 had no known disease association, the c.838G>C variant was observed in five alleles in gnomAD, and case matching efforts through GeneMatcher were unsuccessful. Research reanalysis of the ES data through the UDN utilizing a phenotypic agnostic pipeline was non\u2010diagnostic and did not reveal additional reportable variants. Trio GS  was completely negative, no variants were reported.At the time of the UDN evaluation, the participant had demonstrated little developmental progress; he was unable to hold his head up without support, could not sit independently or grasp objects with his hands. He was able to make sounds but had no speech. On exam, dysmorphic features including coarse facial features, anteverted nares, and thick, prominent lips with tongue protrusion were noted. The participant also had growth delay (height at the third percentile); weight and head circumference at the 25th percentile; soft, doughy skin; hirsutism; severe, diffuse hypotonia; mild lower extremity hemihypertrophy; and small hands and feet. A 2.2A fresh blood sample in EDTA was collected from the participant and both parents for OGM. Collected blood was processed with the manufacturer's guidelines for the extraction of ultra\u2010high molecular weight (UHMW) DNA (Bionano Genomics). DNA labeling was performed following the manufacturer's protocols (Bionano Genomics). The fluorescently labeled DNA molecules were imaged sequentially across nanochannels on a Saphyr instrument. Effective genome coverage of greater than 80X was achieved for all samples.De novo genome assembly, variant calling and analysis were performed using software solutions provided by Bionano Genomics. SVs were identified based on the alignment profiles between the de novo assembled genome maps and the Human Genome Reference Consortium GRCh38 assembly. If the assembled map did not align contiguously to the reference, but instead was punctuated by internal alignment gaps (outlier) or end alignment gaps (endoutlier), then a putative SV was identified. Targeted rare variant analyses were performed to estimate the percentage of mosaic levels for identified SVs. Briefly, mosaic variant frequencies were determined by a custom workflow. First, the raw molecules were realigned to all consensus genome maps and the reference GRCh38, then the allele frequency of a variant was computed by dividing the variant\u2010allele coverage on the consensus map by the coverage on the reference assembly and all other maps in the vicinity. SVs were annotated with the combination of Variant Annotation Software (Bionano Genomics) and an open\u2010source R package, nanotatoR . The OGM base\u2010pair resolution is limited due to utilization of label patterns and measurement of distances between them. This means that the actual breakpoint could be anywhere between the two labels showing SVs. On average this should be approximately 3\u00a0kb, but could be as large as 10\u00a0kb. Confirmation of the deletion and refinement of the breakpoints in a CLIA\u2010certified laboratory was obtained via mate\u2010pair sequencing at Mayo Clinic Laboratories.2.3DNA was extracted from a peripheral blood specimen using the PureGene protocol (Qiagen), and 1 ug was utilized for mate\u2010pair sequencing (MPseq). Library preparation was performed using the Illumina Nextera Mate Pair library kit (Illumina). Library preparation consisted of cleavage and tagging of DNA, strand displacement to fill any gaps, and overnight circularization (16\u201320\u00a0hours) to produce stabile 2\u20135\u00a0kb DNA fragments. Ampure purification  was performed after the tagmentation and strand displacement steps  to ensure the selection of only the longest fragments. After circularization, non\u2010circularized DNA was digested with exonuclease prior to mechanical shearing of the circularized fragments with a Covaris LE220 System (Covaris). The resulting biotinylated DNA fragments were bound to Dynabeads M\u2010280 Streptavidin (Thermo Fisher Scientific) and subsequently processed through end repair, A\u2010tailing, ligation of 7\u00a0bp Illumina adapters (a component of the TruSeq DNA library prep kit), and PCR using the PCR Primer Cocktail (Illumina) and KAPA HiFi HotStart Ready Mix PCR Kit (KAPA Biosystems). A 0.67X Ampure purification was performed to complete library preparation. MPseq libraries were multiplexed at two samples per lane, and paired\u2010end sequencing (101\u00a0bp) was then performed on the Illumina HiSeq 2500. The average bridged coverage was 47X. Data were aligned to the reference genome (GRCh38) using BIMAv3, and abnormalities were identified and visualized using SVAtools and Ingenium, which are in\u2010house developed tools  in the UDN participant, the details of which are outlined below. Utilizing the UDN diagnosis coding tool, the diagnosis was classified as certain  3.2CDKL5 gene and mosaic inversion at the same location , also known as CDKL5 deficiency disorder (CDD). CDD affects nearly four times as many females as males, with complete loss of function thought to be lethal in males  which removes exons 1\u20133 of the CDKL5, however, mosaic events may not be detected by exon aCGH. Deletions involving three exons are generally amenable to detection by CNV analysis of NGS data . Mosaic intragenic CNVs are difficult to routinely detect on ES or GS. GS in particular has improved sensitivity over ES in detecting most types of constitutional CNVs, but the lower number of reads makes identification of mosaicism challenging. Retrospective manual inspection of the GS data demonstrated evidence of the deletion in the participant in the form of coverage drop , notably with the ability to detect low\u2010level mosaicism starting at 5% alternate allele fraction (Genomics, HB and EV own a limited number of stock options of Bionano Genomics Inc. HB is also employed part\u2010time by Bionano Genomics Inc. All other authors declare no conflict of interest.HC, NW and VS participated in the patient's UDN evaluation and collected the clinical data. HB, SB, YF and EV performed optical genome mapping and analysis. NH and CM performed mate\u2010pair sequencing and analysis. CR and KD performed microarray and analysis. AA performed a reanalysis of ES and GS data. HC drafted the manuscript. HB and NH contributed to the writing and revision of the manuscript. SB, YF, NH, CM, NW, CR, KD, AA, EV and VA edited the manuscript. All authors approved the final manuscript.Fig S1\u2010S2Click here for additional data file."}
+{"text": "The Argonaute (AGO) and the Trinucleotide Repeat Containing 6 (TNRC6) family proteins are the core components of the mammalian microRNA-induced silencing complex (miRISC), the machinery that mediates microRNA function in the cytoplasm. The cytoplasmic miRISC-mediated post-transcriptional gene repression has been established as the canonical mechanism through which AGO and TNRC6 proteins operate. However, growing evidence points towards an additional mechanism through which AGO and TNRC6 regulate gene expression in the nucleus. While several mechanisms through which miRISC components function in the nucleus have been described, in this review we aim to summarize the major findings that have shed light on the role of AGO and TNRC6 in mammalian chromatin biology and on the implications these novel mechanisms may have in our understanding of regulating gene expression. Most of the mammalian genome is transcribed into non-coding RNAs (ncRNAs) that play a variety of structural and regulatory roles in cells. Small-size RNAs (sRNAs), ranging from ~18 to 30 nucleotides, are a well-known class of ncRNAs involved in both the cytoplasmic and nuclear regulation of gene functions. Their primary sequence provides the information necessary to guide repressor or activator complexes to complementary binding sites within a target nucleic acid. The mode of recognition of nucleic acids by sRNAs exploits the pairing combinations provided by the Watson\u2013Crick complementarity. This leads to a higher degree of binding specificity than that allowed by proteins, which instead rely on a limited number of binding domains to recognize DNA and RNA sequences.Mammalian genomes code for three main groups of sRNAs: microRNAs (miRs) , endogenIn contrast, AGOs are expressed ubiquitously and show a remarkable versatility in function. This is supported by the fact that AGOs are embedded in a variety of complexes that range from 100kD to several MD ,8, implyCaenorhabditis elegans (C. elegans) as heterochronic genes [The most studied among AGO functions in mammalian cells is its miR-dependent post-transcriptional gene repression (PTGR) of protein-coding genes . miRs weic genes . Since tic genes .seed\u201d sequence within the loaded miR, and a binding site usually situated within the 3\u2019UTR or the coding region of the cognate target mRNA. Next, the AGO\u2013miR complex associates with one member of metazoan-specific TNRC6 proteins, a molecular docking platform for the recruitment of mRNA decay enzymes, namely decapping factors and deadenylases [During PTGR, AGO is directed onto target mRNAs via base pairing between a 7 to 8nt \u201cenylases   , the effIt has been demonstrated that multiple molecular pathways allow for the controlled shuttling of AGO and TNRC6 between the nucleus and cytoplasm. Nuclear import of AGO is controlled, at least in part, by Importin 8 , althougSimilarly, TNRC6 proteins can shuttle between the cytoplasm and the nucleus, which is supported by the fact that some TNRC6 members contain both a nuclear localization signal (NLS) and nuclear export signal (NES). Accordingly, TNRC6 export has been shown to be mediated by the interaction of its NES with Exportin-1 .As the physical interaction between AGO and TNRC6 seems to be conserved between the nucleus and cytoplasm,  it is noAlthough AGO\u2013TNRC6 complexes can still mediate PTGS on target mRNAs before they reach the cytoplasm , they caSchizosaccharomyces pombe S. pombe). From yeast to humans, heterochromatin constitutes a group of transcriptionally silent chromatin domains essential for the maintenance of chromosome integrity and for the regulation of gene expression [chromodomain. H3K9 methylation spreads along chromatin through sequential cycles of methylation, mediated by H3K9 methyltransferase enzymes, coupled to the oligomerization of HP1 family members [S. pombe. The mechanistic basis of this link began to be elucidated with the isolation of the RNA-induced transcriptional silencing (RITS) nuclear complex, whose core components are Chp1 (a chromodomain-containing protein), Ago1, a Dicer-dependent sRNA, and Tas3 [dicer with the consequent loss of rasiRNAs, induces defects in heterochromatin maintenance. Once bound to the chromatin, the RITS recruits the RNA-dependent RNA polymerase complex (RDRC) formed by Rdp1 polymerase, the helicase Hrr1, and the non-canonical poly(A) polymerase Cid12. The RDRC complex amplifies the silencing process initiated by RITS in a positive feedback loop consisting of the further synthesis of target-specific dsRNA, their processing by Dicer and subsequent production of rasiRNA, and additional recruitment of more RITS to the region [The isolation of the RITS in pression . Heterocpression  through  members . A link and Tas3 , a proteand Tas3 . The RITand Tas3 , are proand Tas3 . Deletioe region   Clr4, the Cullin scaffold protein Cul4, the RING finger protein Pip1, the DNA damage binding protein 1 homolog Rik1, and the DCAF-like protein delocalization of Swi6 1 and 2 (Dos1 and Dos2) ,44,45,46S. pombe. For example, in Arabidopsis thaliana, the DICER-LIKE proteins DCL1 to 4 produce sRNAs that are loaded into 1 of the 10 Argonaute proteins encoded by the genome of this organism. Of note, in plants, sRNA-induced TGS relies principally on DNA methylation at cytosine residues by the RNA-directed DNA methylation pathway (RdDM). RdDM directs de novo DNA methylation while other DNA methyltransferases, such as MET1 or CMT3, mediate DNA methylation maintenance [The mechanism underlying Ago-mediated heterochromatic modifications in plants resembles the mechanism in ntenance . Drosophila, Piwi proteins/piRNA complexes operate in the nucleus where they recognize nascent transposon transcripts and direct the TGS of the corresponding transposon loci [The piRNA pathway. Mechanisms of TGS are also present in animals. TGS in animals is best understood in the piRNA pathway, where the Piwi clade of Argonautes direct the silencing of transposable elements (TE) in gonadal tissues ,49. In Dson loci ,51. Recoson loci . Panoramson loci ,53,54, ison loci ,56,57  the recognition of nascent transcripts by an Argonaute family loaded with sRNA; (2) the recruitment of heterochromatin factors; and (3) silencing of the targeted loci. As described in the next section, similar mechanisms also take place in mammals.EF1A) promoter could repress the expression of an integrated EF1A promoter\u2013reporter transgene, and, although to a lesser extent, the expression of the endogenous EF1A locus [S. pombe, and did not involve RNA-mediated PTGR events [TGS triggered by exogenous sRNAs. Several examples of TGS in mammals have been described . Early s1A locus . In thisR events .progesterone receptor (PR) gene [E-cadherin (CDH1) promoter could promptly induce transcriptional repression, this process could take place even in cells depleted of virtually any capacity to perform DNA methylation [However, it became clear that DNA methylation was not always associated with sRNA-mediated TGS ,68,69. Fhylation .EF1A promoter upon transfection of promoter-directed sRNAs. Interestingly, H3K9 methylation was even induced at sequences hundreds of bps downstream of the EF1A promoter, suggesting that the sRNA-mediated targeting may induce the spreading of chromatin marks to regulate neighboring regions [While the extent to which DNA methylation participates in sRNA-induced TGS is still under debate, more compelling evidence has been provided for sRNA-induced silencing through repressive histone modifications ,70. For Drosophila and C. elegans inspired the search for a potential role of AGO as the physical interface between sRNAs and the chromatin-modifying machinery. Such a role for AGO came to light in chromatin immunoprecipitation (ChIP) experiments in human cells. These studies showed that the intracellular delivery of sRNA could induce up to a 20-fold increase of AGO1 and H3K9me2 presence in proximity of the sequences complementary to the sRNA [The observation that promoter-directed sRNAs could induce chromatin changes in mammalian cells ignited interest towards the factors that could mediate this process. The structural and functional information previously gathered from the RITS and the piRNA pathways in the sRNA . Interesthe sRNA . The recS. pombe. For example, while Clr4 is the only H3K9-specific methyltransferase within the RITS in S. pombe, mammalian genomes encode for several HMTs with H3K9 specificity, any of which could, in principle, function during RNA-induced TGS. So far, among these enzymes a role in TGS has only been documented for SETDB1, as evidenced in a study in which sRNAs directed to the androgen receptor (AR) promoter increased the local recruitment of both AGO2 and SETDB1 around the sRNA binding site [AR promoter required a nascent ncRNA spanning the promoter region. This observation provides further evidence that a nascent RNA transcript may be the preferential recognition platform for sRNA-guided chromatin remodeling of the template DNA. Notably, DNA methylation is not induced following targeting of the AR promoter, consistent with the notion that the induction of repressive histone modifications may play a greater role in mammalian TGS.Recent studies have further elucidated the identity and modus operandi of the enzymatic activities recruited by AGO during TGS, although the high complexity of mammalian genomes has made the search for these enzymes more challenging than the search previously undertaken in ing site . Moreoveing site . These oPOLR3D, and its depletion induced an increase of POLR3D expression at the transcriptional level [POLR3D promoter, consistent with a role of miR-320 in the induction of repressive chromatin at the targeted site. Sense transcripts spanning the POLR3D promoter were also detected, although authors have not investigated their role in AGO1 recruitment and gene repression. Given the central role of POLRD3 in the cell cycle progression [miR-dependent pathways of TGS. The discovery that exogenous sRNAs could induce TGS and chromatin silencing in mammalian cells led to the speculation that endogenous sRNAs with analogous function may exist. miRs were an obvious class of candidates to explore, given their similarity in size with the RITS-associated sRNAs. Early attempts to discover an example of miRs-mediated TGS relied on computational tools that search for sequences within gene promoters with extensive complementarity to endogenous miRs. This approach led to numerous examples of miR-induced TGS in mammals . Authorsal level . Moreovegression , this stprogesterone receptor (PR) and of immunoglobulin superfamily member 1 (IGSF1). Among those, the overexpression of miR-423-5p was shown to decrease Pol II occupancy, and to induce accumulation of H3K9me2 at the promoters of the two mentioned genes, in a DNA-methylation-independent manner. Moreover, TGS required the recruitment of AGO2 onto a ncRNA-encompassing gene promoter [PR promoter have ruled out a role for TNRC6 in the TGS of this locus [Using computational methods to screen for miR target sites within gene promoters, Younger et al. found multiple miRs that were able to target the promoter of the human promoter . Of noteis locus .DNMT3b, whose promoter contains a binding site for miR-133a. These authors were able to directly link the nuclear enrichment of miR-133a with the transcriptional silencing of DNMT3, as the transfection of cells with antisense oligonucleotides against miR-133a binding sites prevented DNMT3b repression. ChIP analysis showed that the inhibition of Wnt signaling was also accompanied by AGO2 localization at the wnt promoter, an increase in the histone repressive mark H3K27me3, and a decrease of H3K4me3 that marks active transcription. Interestingly, DNMT3B was also recruited at the miR-133a binding site and was physically associated with AGO2. As a consequence of the recruitment of DNMT3B, DNA methylation levels at the promoter increased, forming a negative feedback mechanism through which the miR-mediated recruitment of DNMT3B resulted in the repression of its own expression.More recently, Di Mauro et al. have found a link between Wnt signaling and miR-mediated epigenetic regulation in cardiac cells . They hamatrix metalloproteinase 14 (MMP-14). This gene is activated by the transcription factor Yin Yang 1 (YY1), and its overexpression or downregulation promotes or inhibits tumor progression, respectively. Zheng et al. noted that a YY1 binding site is located just 30\u201340 bp downstream of a putative binding site for miR-584-3p within the MMP-14 promoter, suggesting a possible functional interaction between the two elements. Indeed, in gastric cancer cells, the authors found that miR-584-3p binds to the MMP-14 promoter in an AGO2-dependent manner, leading to increased levels of the repressive histone marks H3K27me3 and H3K9me2. The induction of heterochromatin hinders the recruitment of YY1 and consequently suppresses MMP-14 expression. Importantly, treatment with RNase H reversed miR-584-3p-mediated chromatin modification, indicating that miR-584-3p directly interacted with the MMP-14 promoter via an RNA\u2013DNA hybrid. Authors of this study showed that, by impeding the binding of YY1 to the MMP-14 promoter, an AGO2\u2013miR-584-3p complex can suppress the tumorigenesis and aggressiveness of gastric cancer cells, both in cell lines and xenograft tumors [Endogenous miRs have also been involved in the transcriptional repression of the metastasis-promoting gene t tumors .It is unclear how widespread miR-mediated TGS occurs within mammalian gene networks, and to what extent either DNA or RNA acts as a recruitment platform. Nevertheless, the expression of ncRNA-spanning promoter regions seems to be a common feature of several mammalian promoters ,84, whicBeyond their typical role in the repression of gene expression, complexes that include sRNAs, AGO proteins, and, in isolated cases, TNRC6, have also been shown to promote gene expression via a mechanism known as transcriptional gene activation (TGA)  , and VEGF, surprisingly stimulated, instead of inhibited, gene transcription in a sequence-specific manner. This process required AGO2 and was accompanied by the loss of the repressive mark H3K9me2 [CDH1 promoter was susceptible to TGS via the induction of histone methylation [The first evidence of AGO-mediated TGA came from experiments where sRNAs, named small activating RNAs (saRNAs), targeting the promoter region of  H3K9me2 . Notablyhylation . Therefohylation .PR gene increased the expression of the locus. ChIP analyses showed that TGA was accompanied by the reduced acetylation of H3K9 and H3K14, reduced presence of HP1, and increased levels of H3K4me2 and H3K4me3. Importantly, the recruitment of the AGO\u2013saRNA complex required a nascent antisense RNA transcript overlapping the promoter [PR gene transcription via the recognition of DNA, rather than of a nascent transcript [PR promoter and observed no impairment of TGA at this region, thereby arguing against a possible role of nascent transcripts in this instance. As evidenced by the conflicting observations from these studies, the modality of target recognition during TGA is still an object of intense debate. As noted earlier, sRNAs directed to the PR promoter can also repress gene expression [TGA was further confirmed in studies where saRNA that was directed to the promoter of the promoter ,90. Howeanscript . To supppression . AlthougCDH1 and CSDC2 genes, whose promoters contain sequences complementary to miR-373, was upregulated by the overexpression of miR-373. This early study, although it provided evidence for endogenous pathways that mediate TGA, did not investigate the potential involvement of AGO proteins or nascent transcripts in this process [TGA mechanisms act through a promoter and enhancer. The first evidence that endogenous miRs can induce TGA came from a study in which the transcription of the  process .CCNB1 promoter and subsequently enhance CCNB1 expression in mouse cell lines. ChIP analysis revealed that the expression of miR-744 and miR-1186 induced the localization of AGO1 on the CCNB1 promoter, accompanied by increased levels of RNA Pol II and H3K4me3 at the CCNB1 TSS [CCNB1 overexpression promotes tumorigenesis [CCNB1 overexpression, this work indicates that TGA may be a process through which miRs can contribute to cancer development.The mechanistic details on miR-mediated TGA were soon provided by Huang at al. By using a combination of in silico and wet lab approaches, these authors found that miR-744 and miR-1186 can bind to complementary sites within the CNB1 TSS . Since Cigenesis ,102, andIL24 and IL32, enhancing their expression via TGA. This process led to induced apoptosis and cell cycle arrest, and impaired the invasive properties of prostate cancer cells. Accordingly, the overexpression of miR-205 was accompanied by an enrichment of Pol II, H3K4me2, and H3/H4ac in the promoter of both genes, indicating transcriptional activation [Further studies have similarly supported a role of TGA in tumor formation and progression. Majid et al. showed that miR-205 binds to complementary sites within the promoters of the tumor suppressor genes tivation . Howeverheparanase (HPSE) gene, which was accompanied by a corresponding decrease of H3K9me2 and H3K27me3, and an increase of H3K4me3 and RNA Pol II on the HPSE promoter. HPSE gene activation requires the presence of AGO1, but not AGO2, as only a knock-down of AGO1 abolished the miR-558-induced enrichment of active epigenetic markers on the promoter.By using neuroblastoma cells as a model, Qu et al. found that the overexpression of miR-558 induced the expression of the HPSE promoter, suggesting that the AGO1\u2013miR-558 complex relies on DNA\u2013RNA pairing for promoter recognition, such a mechanism has not been thoroughly investigated and no direct evidence has been provided for this model of targeting in this study [Although RNase-H treatment reduced TGA on the is study .FBP1 and FANCC. TALEN deletion of the enhancer blocked the ability of miR-24-1 to activate FBP1 and FANCC. Importantly, TGA on this locus was abolished in a AGO2-null background, implying that miR-24-1-dependent TGA is mediated by AGO2. Moreover, miR-24-1 overexpression induced an enrichment of Pol II and the consequential transcription of RNAs overlapping the enhancer region, concomitant with increased levels of AGO2, p300, H3K27ac, and H3K4me1, as well as a reduced level of H3K9me3. Notably, the overexpression of miR24-1 not only led to an increased H3K27ac within the proximal enhancer, but also at thousands of enhancers with sequences complementary to the miR-24-1 [miRs can mediate TGA by promoting a favorable chromatin status not only at the promoters, but also at the enhancers of target genes. Xiao et al. observed that about 25% of loci involved in the production of nuclear-enriched miRs overlap with markers of active enhancers, namely p300/CBP, H3K4me1, and H3K27ac, as well as Dnase I-sensitive regions. Moreover, they observed that the expression of this subgroup of miRs positively correlated with the expression of surrounding loci. These observations prompted them to test the hypothesis that miRs may target enhancers overlapping their own loci and, in turn, activate both their own expression and the expression of surrounding genes. This hypothesis was confirmed by ectopically overexpressing miR-24-1, whose locus maps within a region with enhancer function. Indeed, the overexpression of miR-24-1 led to the expression of the endogenous miR-24-1, as well as the expression of its neighbor protein-coding genes miR-24-1 .It is also important to note here that, although promoters and enhancers seem to be the preferential target sites for TGA in mammals, the possibility that other genomic regions may also be recognized by AGO\u2013miR complexes to initiate permissive chromatin has yet to be determined.CDKN1A (p21) as a model gene, Portnoy et al. have isolated the RNA-induced transcriptional activation (RITA) complex, which, in addition to the saRNA\u2013AGO2 complex, includes the nuclear DNA helicase II (RHA) and CTR9 [The purification of the RNA-induced transcriptional activation complex. Recently, a complex able to mediate TGA has been characterized. Using the human and CTR9 . RHA is and CTR9 . The preand CTR9 . RITA alThe building blocks of chromatin are the nucleosomes, which are composed of histone proteins wrapped by genomic DNA. Chromatin-remodeling complexes include enzymes that ensure the proper spacing between nucleosomes, which ultimately contribute to controlling the regulation of gene expression . By perfInterestingly, the pool of SWI/SNF-associated AGO2 was associated with a group of DICER-dependent sRNAs that do not belong to any previously characterized sRNA class, and that, instead, preferentially map around SWI/SNF-bound TSSs. This class of sRNAs, named swiRNAs, was found to overlap with more than 4000 TSSs genome-wide, and co-localized with histone markers of open chromatin, such as H3K9ac and H2Az, suggesting widespread roles for swiRNAs within mammalian gene networks. As expected, micrococcal nuclease digestion (which detects open chromatin regions) followed by RNA-seq revealed that in an AGO2-null background, nucleosome occupancy at the +1 position of the targeted TSS is altered, indicating that AGO2 not only physically interacts, but also functionally cooperates, with SWI/SNF to control chromatin remodeling. However, the low expression of swiRNAs (they are about 4 orders of magnitude less expressed than miRs), in addition to the fact that the AGO2 ablation did not result in significant perturbation of expression of swiRNAs-targeted genes, indicates that more studies are needed to establish the precise role of this class of sRNA in vivo.In 3D, chromosomes are organized into active and inactive territories , which aCD44 gene [Mechanisms where AGO regulates alternative splicing via chromatin modification have been also described. All\u00f3 et al. have shown that exogenous sRNAs targeting both intronic and exonic regions of the fibronectin gene were able to regulate its alternative splicing by inducing an increase of H3K9me2 and H3K27me3, histone markers associated with gene silencing. This process was dependent on AGO1 and AGO2, and leads to a decrease of Pol II elongation, an event that ultimately affects splicing . SimilarD44 gene . ImportaD44 gene .As discussed above, the role and contribution of AGO proteins in chromatin modification and organization has been amply demonstrated. In contrast, even though TNRC6 is associated with AGO also in the nucleus, evidence for its role in these nuclear-specific processes are scarce.COX-2 as a model, Matsui et al. provided the first evidence of a role of TNRC6 proteins in nuclear TGA through a mechanism mediated by endogenous miRs [COX-2 promoter, which led them to investigate the possibility that this miR may transcriptionally regulate COX-2 expression. Indeed, anti-sense mediated knock-down of miR-589 or the introduction of miR-589 mimics in mammalian cells led to decreased, or increased, COX-2 expression, respectively. Importantly, the knock-down of a promoter-associated transcript reduced the levels of COX-2 mRNA upon miR-589 overexpression, consistent with a role of promoter-associated RNAs as a docking platform for the recruitment of gene-activating machineries.Nevertheless, TNRC6 proteins have been involved in TGA in some circumstances. Using the pro-inflammatory gene ous miRs . They foCOX-2 induction was paralleled by the accumulation of AGO2 and RNA Pol II at the promoter. Importantly, an accumulation of TNRC6 at the same site was also observed. siRNAs directed against either AGO2 or TNRC6 reduced the ability of miR-589 mimics to induce COX-2 expression, suggesting a functional role of these two proteins in the miR-dependent activation of this locus. Moreover, knocking down the sense strand of the promoter-associated RNA also resulted in the loss of miR-568-dependednt COX-2 activation.Upon the delivery of miR-589 mimics, COX-2 gene.Together with the observation that AGO and TNRC6 physically interreact in the nucleus, these data are consistent with a model according to which AGO2 forms a complex with TNRC6 and miR-589, which, by recognizing nascent promoter-associated RNAs, induces the activation of the COX-2 promoter. Accordingly, WDR5 recruitment was accompanied by the enrichment at the promoter of the activating histone markers H3K4me3 and H4Ac. Knocking down WDR5 resulted in a complete loss of miR-mediated COX-2 activation. These observations are consistent with a role of AGO and TNRC6 proteins as a functional and physical interface between ncRNAs and chromatin-modifying complexes.miR-589 mimics also induced the accumulation of WDR5, a protein scaffold that stimulates HMT activity , on the COX-2 locus. Other groups of TNRC6-associated proteins with potential chromatin-modifying functions were CCR4\u2013NOT, which, in addition to its role in PTGR in the cytoplasm, has been shown to induce activating histone markers, including H3K4 tri-methylation, as well as H3 and H4 acetylation [Building upon these observations, Hicks et al. aimed to isolate additional factors that may be involved in the intercommunication between AGO/TNRC6-containing complexes and the chromatin modifying machinery. To this aim, they performed a mass spectrometry analysis to identify TNRC6 partners in the nucleus . In additylation .COX-2 system represents a clear example of co-participation of AGO\u2013TNRC6 complexes and chromatin-modifying factors, it also provides insights on the possible role of miRs in promoting chromatin 3D organization. This notion stems from the observation that all of the manipulations that affected the miR-589-dependent regulation of COX-2 also perturbed the expression of the pro-inflammation gene PLA2G4A. COX-2 and PLA2G4A loci map within the same chromosome 150kb apart, and are found in a head-to-head organization. Interestingly, an miR-589-dependent increase of the activating histone markers H3K4me3 and H4Ac within the COX-2 promoter was accompanied by the same changes of histone markers spanning the PLA2G4A promoter. Importantly, no RNA was found to overlap both promoters, ruling out the possibility that miR-589 may simultaneously target COX-2 and PLA2G4A through the same mechanism. Instead, chromosome conformation capture (3C) analysis revealed that miR-568 promotes a physical linkage between the two genes by inducing chromatin loops that place the two promoters in proximity to each other. Through this mechanism, the miR-dependent recruitment of AGO\u2013TNRC6, with the consequent recruitment of chromatin-remodeling enzymes, may simultaneously induce histone modification on both the COX-2 and PLA2G4A promoters. However, how prevalent this mechanism is in mammalian gene networks is yet to be determined.Although the miR-589/Studies conducted over the past 15 years have established that in mammals, sRNAs can carry the information necessary to direct chromatin-remodeling machinery on specific genomic regions to regulate gene expression. However, the underlying mechanisms are still largely unknown, and many questions remain open. For example: (1) What are the determinants that drive either TGA or TGS on a given target gene? (2) What is the relative contribution of the AGO1-4 paralogues in the silencing or activating process? (3) Is AGO2\u2019s slicing activity required? (4) Does the sRNA recognize the DNA sequence or a nascent transcript? (5) To what extent is DNA methylation involved in the silencing process? Like most biological processes, it is conceivable that context-specific conditions dictate the modality through which TGS and TGA function at a specific gene locus, which would explain the mechanistic complexity, and at times inconsistency, that emerges from the studies discussed in this review.COX-2 remains, so far, the only instance in which of TNRC6 seems to be involved. However, it is possible that the use of the AGO\u2013TNRC6 dimer in TGA/TGS may be more widespread than currently appreciated. This idea came from the observation that the RITS, which plays a central role in TGS in S. pombe and contains a member of the Ago protein family, shares important structural similarities with mammalian TNRC6. Indeed, mammalian TNRC6 contains both the AGO-hook, a domain of Tas3, and an RNA-recognition motif (RRM), a part of Chp1 [Another important question pertains to the requirement of TNRC6 paralogues in sRNA-mediated transcriptional regulation, as TGA of  of Chp1 . To dateOur groups have recently generated a mouse model that allows the inducible disruption of the interaction between AGO and TNRC6. It is based on the expression of a small protein that competes with TNRC6 for binding to AGO, thereby interrupting PTGR without affecting the expression of miRs ,121. WheThe ability to control gene expression by directing sRNAs to promoters and enhancers has motivated the research of possible approaches to transcriptionally adjust gene expression for therapeutical purposes . For exaIn conclusion, there is compelling evidence of the existence of mammalian mechanisms that utilize the information contained in sRNAs to direct transcriptional gene regulation via chromatin modification and remodeling. This information has been used to create new sRNA-based drugs to adjust gene expression, which, though still in their infancy, have opened a promising new field of therapeutical intervention, although more mechanistic studies are needed to improve the efficacy and safety of this new class of molecules."}
+{"text": "In mental health research, it has proven difficult to find measures of brain function that provide reliable indicators of mental health and well-being, including susceptibility to mental health disorders. Recently, a family of data-driven analyses have provided such reliable measures when applied to large, population-level datasets. In the current pre-registered replication study, we show that the canonical correlation analysis (CCA) methods previously developed using resting-state magnetic resonance imaging functional connectivity and subject measures (SMs) of cognition and behaviour from healthy adults are also effective in measuring well-being (a \u2018positive\u2013negative axis') in an independent developmental dataset. Our replication was successful in two out of three of our pre-registered criteria, such that a primary CCA mode's weights displayed a significant positive relationship and explained a significant amount of variance in both functional connectivity and SMs. The only criterion that was not successful was that compared to other modes the magnitude of variance explained by the primary CCA mode was smaller than predicted, a result that could indicate a developmental trajectory of a primary mode. This replication establishes a signature neurotypical relationship between connectivity and phenotype, opening new avenues of research in neuroscience with clear clinical applications. By mode. 1.2et al. [et al. chose 158 measures that were quantitatively and qualitatively appropriate for analysis.In their landmark 2015 study, Smith et al.  investiget al. . To studr = 0.87, p < 10\u22125) between the primary CCA mode connectome weights and SM weights. From the primary CCA mode, they derived a \u2018positive\u2013negative axis' that quantified the correlation of various SMs to the CCA mode, linking lifestyle, demographic and psychometric SMs to each other and to a specific pattern of brain connectivity [Using canonical correlation analysis (CCA), which can simultaneously consider two variable sets from different modalities to uncover essential hidden and high-dimensional associations between the sets , the autectivity . SMs comA hierarchical clustering analysis of the 200 connectome nodes revealed clusters of nodes in four regions: one cluster in the sensory, motor, insula and dorsal attention regions; and three clusters covering the default mode network (DMN), subcortical and cerebellar regions . The brar = 0.25 between the primary CCA mode connectome and SM weights in the testing dataset, strongly supporting the primary CCA mode finding [Finally, to test the predictive value of their model, the authors conducted a train-test split in which 80% of subject data  were used to train a CCA model, which was then tested on the remaining 20% of the data. This process was repeated 10 times, with statistical significance estimated via permutation testing (1000 permutations) of family structures in each iteration. This train-test process yielded a statistically significant mean correlation of  finding .et al. [The findings from Smith et al.  suggest et al. . EstabliAs a precursor to the current study, using code published by the original authors, we computationally replicated their findings in both the HCP 500 and 1200 subject releases to validate our own analysis pipeline .. 1.3et al.'s positive\u2013negative axis and primary CCA mode findings in novel data from the Adolescent Brain Cognitive Development (ABCD) study. This is not a replication in the sense that we seek to find results identical to those of the original authors . Ratheret al. for multiple reasons. The 22- to 35-year-old subjects in the HCP dataset, which was fairly homogeneous in terms of subject demographics and scan quality [et al. used neuroimaging data of youth (8\u201322 years old) and found positively correlated patterns of functional connectivity and psychiatric symptoms across four dimensions of psychopathology. Each dimension was associated with a distinct pattern of abnormal connectivity while still showing common features (e.g. loss of network segregation) distinguishing them from normal brain function. To account for potential confounds, the authors controlled for age, sex, race and motion and they also found that these findings replicated in an independent dataset [We selected the ABCD dataset to replicate Smith  quality , are vas dataset . The impFinding a primary CCA mode and positive\u2013negative axis in the ABCD data would have implications beyond speaking to the robustness of the original study and the presence of the phenomena in a dataset other than HCP. It would suggest that the high-dimensional relationship between brain connectivity and phenotype may be independent of age, demographics, scanner type, preprocessing methods and even the SMs, which can vary in how they index cognitive constructs. Such observations would be pivotal for the establishment of a signature neurotypical relationship between connectivity and phenotype, which could then be applied to a variety of clinical populations potentially increasing its practical application in medicine. Most importantly, finding the positive\u2013negative axis in the baseline ABCD dataset would enable researchers to study it longitudinally over subsequent ABCD releases for the next 10 years. In doing so, researchers could explore how the positive\u2013negative axis changes with regard to age, behavioural characteristics, mental and physical health outcomes, education, demographics and environment, and possibly even determine which factors form the basis for the positive\u2013negative axis. We aim to design the analysis pipeline such that it can easily be re-run by other researchers who wish to explore how these factors impact the CCA results over time as subsequent ABCD datasets are released. Given these potential implications, we feel that replicating the positive\u2013negative axis in a heterogeneous dataset is important not only to lend validity to the original study but also to open new avenues of research in neuroscience.. 1.3.1et al. to perform a CCA on ABCD subject connectomes and SM data . After applying similar preprocessing steps to the resting-state scan data, we derived a 200-dimension group-ICA functional parcellation. This parcellation was used to calculate subject-level connectomes, which were aggregated to form the connectome matrix used in the CCA. The choice of parcellation can have significant effects on connectivity results [et al., which has been shown to have greater predictive power compared to a priori functional and anatomical parcellations [In this conceptual replication, we followed the same methodology as Smith  results . For repllations ,14.The ABCD study has over 60 000 SMs  measures), which we quantitatively filtered as per the original study. After removing quantitatively inappropriate SMs, we performed a one-to-one matching of ABCD SMs to their counterparts in the HCP dataset. This process identified 89 ABCD SMs, of which 74 were deemed appropriate for the CCA. We regressed out confounds similar to those in the original study prior to conducting the CCA.post hoc analyses from the original study, including generating a positive\u2013negative axis that correlates the SMs  with the primary CCA mode; a hierarchical analysis of the connectomes, and determining which functional connections correlate most strongly with the primary CCA mode; a clustering analysis to identify the major brain regions into which the 200 nodes fall; and a train-test split to evaluate the predictive performance of the CCA model on unseen data.After conducting the CCA, we performed multiple  (1)The primary CCA mode explains a statistically significant amount of variance in the connectomes and SMs relative to the null distribution generated via permutation testing. (2)z-scores for connectomes and SMs are at least a factor of 2 and 3 greater, respectively, than the next largest z-scores for connectomes and SMs (from any of the other modes).The primary CCA mode  (3)p < 0.001).A statistically significant correlation exists between the primary CCA mode SM and connectome weights  were as follows:nt modes . Since t. 2Royal Society Open Science. Following IPA, the accepted Stage 1 version of the manuscript, not including results and discussion, was pre-registered on the OSF (http://doi.org/10.17605/OSF.IO/HMC35). Minor deviations from the protocol are identified in footnotes, and detailed explanations of deviations are provided in \u00a73.3.This article received in-principle acceptance (IPA) at . 2.11The sample composition of the ABCD Release 2.0.1 before and after filtering out subjects is shown in n = 285) or triplets (n = 30) but were missing zygosity data; these subjects were considered non-twin siblings for our study to match the procedure followed by Smith et al. Prior to filtering, there were 738 MZ, 1082 DZ, 1908 non-twin siblings (1593 non-twin siblings plus 315 twins/triplets treated as non-twin siblings) and 8147 single children.The ABCD dataset contained multiple types of sibships (children with the same parents): monozygotic (MZ) and dizygotic (DZ) twins, as well as siblings and single children. As in the original study, the sibship data were used to generate permutations of the ABCD SM data for statistical testing. Twins were designated as MZ or DZ based on genomic data, and, when zygosity was unavailable, twins were simply treated as siblings. There were 315 subjects that were designated as twins (n = 197) or triplets (n = 22), but were missing zygosity data; again, these subjects were considered as non-twin siblings for our study. The final sibship counts were: 562 MZ, 777 DZ, 1245 non-twin siblings (1026 non-twin siblings plus 219 twins/triplets treated as non-twin siblings) and 5226 single children.After filtering, there were 219 subjects that were designated as twins , processed by the Oregon Health & Science University Developmental Cognition And Neuroimaging Lab , which included preprocessed and concatenated resting-state fMRI data.2 Preprocessing steps in the DCAN pipeline were in accordance with the HCP recommended minimal preprocessing [3 Collection 3165 also provides censor files for each subject, which denote time points that exceed 0.3 mm in head motion and only segments of 5 or more time points are preserved. To generate the final time-series files, we first removed time points flagged by the censor files and then truncated the data to include only the first 10 min to ensure that all subjects had the same number of time points. After truncating, we excluded subjects from analysis for having less than 10 min of data. This was performed in order to properly process the scans in the group-ICA pipeline and to mimic the HCP dataset in which all scans were of the same length .The ABCD fMRI data were obtained from the National Institute of Mental Health Data Archive (NDA). We used Collection 3165 . The SMs were provided in a subjects-by-SMs matrix (.Rds file), containing all subjects and their available baseline and follow-up SM data up to the ABCD 2.0.1 release. The data were already processed with qualitative variables factored, summary variables generated and missing data removed .The SM data were obtained from the NDA ABCD RDS release a. Subject must have preprocessed and concatenated resting-state fMRI data.b. Subject must have a motion summary file associated with the resting-state scan data, and motion data and censoring data must be present in the file.Availability of resting-state scan data and motion summary data in the NDA ABCD collection 3165. (2)Subject must have at least 10 min of \u2018good\u2019 resting-state scan time  threshold of 0.3 mm). (3)Subject's mean FD must not be anomalous in the overall distribution . (4)a. At least one T1 anatomical scan which passed both QC and protocol compliance checks, for registration.b. At least two resting-state scans that passed both QC and protocol compliance checks.Subject's scans must meet QC requirements, based on QC data included in the SM data matrix.Subjects that were missing any of the aforementioned data , had less than 10 min of \u2018good\u2019 scan time after motion censoring, had anomalous FD motion values, and/or did not pass the QC thresholds were removed from our sample. The cutoffs for anomalous mean FD motion values were 0.1663 mm for the top 0.25% and 0.0212 mm for the bottom 0.25%To determine which subjects were appropriate for our analysis, we applied the following data inclusion criteria, and included only subjects that met 5 out of the original 10 0386 subjects remained as possible candidates for our analysis after this stage of filtering.After this initial filtering, 434 subjects were dropped due to criteria one, 1743 due to criteria two, 9 due to criteria three and 40 due to criteria four, for a total of 2226 subjects flagged for removal. As a result, 7812. 2.3.2et al., a quantitative filtering was performed in order to remove SM data unsuitable for CCA due to insufficient variance, too much missing data, or potential outliers that could skew the CCA [Following the same procedure as Smith  the CCA ,8. Only all of the following criteria, derived from Smith et al., were met: (1) (a)Defined as at least 50% of subjects having data for a given SM.There was enough data available. (2) (a)Defined as less than 95% of subjects having the same SM value.There was sufficient variation in the SM. (3) (a)max(sY) greater than 100*mean(sY), where sX is a vector of all subjects' values for an SM s, and vector s =Y (s \u2212Xmedian(sX))2.Specifically, a SM contained an extreme outlier if: The SM did not contain an extreme outlier value based on the most extreme value from the median.The ABCD 2.0.1 release included a total of 64 148 SMs. After applying these criteria, 5699 SMs were dropped due to criteria one, 2765 due to criteria two and 41 383 due to criteria three, for a total of 49 847 SMs dropped, resulting in 14 301 out of the original 64 148 SMs passing as quantitatively appropriate for our analysis. For a list of all SMs and why they were dropped according to the quantitative criteria, see electronic supplementary material, file 1.A SM was kept only if https://www.fmrib.ox.ac.uk/datasets/HCP-CCA/). When possible, exact SM matches were selected ; otherwise, SMs were selected to be as similar as possible. Where multiple candidate matching SMs were present, we included all reasonable matches. This one-to-one matching identified 89 ABCD SMs which were deemed to be exact or approximate matches to HCP SMs in the original study.From the remaining 14 301 SMs, we then identified ABCD SMs to include in our study via a one-to-one matching of the remaining ABCD SMs with the 461 HCP SMs published by the original authors ( 1.abcd_site).Scanner site ( 2.mri_info_manufacturer).MRI scanner manufacturer ( 3.remaining_frame_mean_FD).Mean frame displacement .Weight .BMI ( 6.smri_vol_subcort.aseg_wholebrain).Cube-root of total brain volume .Cube-root of total intracranial volume . To be consistent with the original study, they were excluded from the analysis.In the original study, a total of nine confound variables were identified and regressed out of the SM matrix  . However7 At this point, we removed any subjects that were missing more than 50% of the final 74 SMs (n = 2).8 Thus, the final SM matrix had dimensions 7810 by 74 (subjects-by-SMs).9 Although only this selected subset of 74 SMs was used as inputs in the CCA, we examined the following 4 SMs in our post hoc positive\u2013negative axis: BMI, age, household income level and parent education level. These SMs were selected because they were analysed post hoc in the original study and because we deemed the ABCD measures quantitatively appropriate for correlation analysis. BMI and age are continuous variables whose magnitude has interpretable meaning. Household income level and education level were factored such that higher scores correspond to higher income or higher education level. For a list of the final 74 SMs, see appendix A; for the one-to-one ABCD/HCP SM matching, see electronic supplementary material, file 2.After excluding the seven confounds and eight undesirable SMs, 74 SMs remained for use as the input to our analysis.. 2.3.3temporal concatenation option for multi-session fMRI data (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/MELODIC). After calculating the group-ICA, we used FSL's dual regression utility to derive the individual subject parcellated time series. Specifically, dual-regression stage-1 was used to estimate the node-time series, in which the 200-dimension group-ICA map was used as a spatial regressor against the full time-series data for each subject, estimating one parcellated time series per subject. The result is 200 nodes' time series of 750 time points (for 600 s of scan time) for each subject. Finally, we derived the subject-level functional connectivity matrices (netmats) via network modelling using the FSLNets toolbox (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/FSLNets). Netmats were estimated for each of the 7810 subjects10 using partial correlation with L2 regularization (rho = 0.01) via FSLNets\u2019 Ridge Regression netmat option (ridgep). The symmetric netmats had dimensions 200 \u00d7 200, where each entry of the matrix was a number representing the strength of the edge between two nodes (indicated by the row and column indices). The netmat values were converted from Pearson's r values to z-scores via Fisher's transformation, and a group average partial correlation network matrix was estimated by averaging the z-scored netmats across all 7810 subjects. Full correlation netmats were also calculated using FSLNets' full correlation option (corr), and a group average full correlation network was estimated by averaging the z-scored full correlation netmats across all 7810 subjects.Using the preprocessed resting-state scan data, we calculated a 200-dimension group-ICA functional parcellation using FSL's MELODIC tool . We usedet al., the subject-level functional connectivity matrices were combined to create a single subjects-by-edges matrix. This was accomplished by half-vectorizing each subject's symmetric functional connectivity matrix (200 dimensions) to obtain a vector of length n(n \u2212 1)/2  per subject, and concatenating these subject-level vectors to produce the final subjects-by-edges matrix with dimensions 7810 \u00d7 19 900.11 Preparation of this matrix can be accomplished with the code provided in our project repository.As in Smith . 2.4. 2.4.1https://www.fmrib.ox.ac.uk/datasets/HCP-CCA/) and the steps described in the original study [et al. study [The CCA pipeline was based on the code provided by the original authors at their website  out of matrix S2.The 7810 \u00d7 74 subjects-by-SM data matrixS3 (1.73% of data was missing). To do so, we estimated a subjects-by-subjects covariance matrix one SM at a time for matrix S3, where, for any pair of two subjects, SMs missing in either subject resulted in the comparison being ignored. The approximated covariance matrix was then projected onto the nearest valid positive-definite covariance matrix using the nearestSPD toolbox [S4 (dimensions 7810 \u00d7 74), which had no missing data and thus accounted for missing data without needing to impute missing SM values. There was a strong correlation (r = 0.9999) between the before and after covariance matrices.In order to run the pre-CCA PCA reduction ,8 on the toolbox  in MatlaS4 was input to a 70-dimension PCA, generating matrix S5 which was the SM data matrix input to the CCA. Although a 100-dimension PCA  was not possible due to having fewer valid SMs than in the original study, Smith et al. noted that there were no statistically significant differences in the final CCA model when the pre-CCA reduction of SMs and netmats was run with a much smaller number of PCA components .13 was imported into Matlab and stored in N0. From N0, two matrices (N1 and N2) were generated, and then horizontally concatenated to form matrix N3. N1 was formed by first demeaning N0 column-wise, then globally variance normalizing the matrix. N2 was formed by normalizing each column of N0 relative to its mean, followed by dropping any columns whose mean values were too low (less than 0.1), then demeaning the matrix column-wise and finally globally variance normalizing it. The 12 confound variables were then regressed out of N3, forming matrix N4. N4 served as the input to the 70-dimension PCA to reduce dimensionality, estimating the top 70 eigenvectors of the connectomes to form matrix N5, which was the connectome data matrix input to the CCA.The functional connectomes were processed in a similar manner. First, the 7810 \u00d7 19 900 subjects-by-edges data matrix. 2.4.2canoncorr function (from the Statistics and Machine Learning Toolbox), using the 70-eigenvector matrices for the SMs (S5) and connectomes (N5) as inputs. The CCA estimated 70 modes, optimizing de-mixing matrices A and B such that the resulting subject-weight matrices 5*AU = N and 5*BV = S (both had dimensions 7810 \u00d7 7014) were maximally similar to each other. We then determined the correlation between the subject connectome and SM weights for each of the 70 CCA modes by correlating the corresponding column pairs in matrices U and V. The Pearson's r value for each CCA mode indicated the strength for which a mode of covariation exists in both the subject connectomes and SMs.The CCA was run using the Matlab U and V), we performed a 100 000 permutation-based significance test in which the CCA was re-run on each permutation of the SM matrix to generate a null distribution of CCA results. As in the original study, the permutations (preserving family structure) were generated using the package hcp2blocks (https://github.com/andersonwinkler/HCP/blob/master/share/hcp2blocks.m) [15 Sibships were determined based on subject zygosity and their parent/family IDs; all related subjects were matched according to their family IDs. Within these family groupings, MZ twins were permuted, DZ twins were permuted, and non-twin siblings were permuted. After the subject-level permutations were created, entire families that contained the same types of sibships and number of children were permuted. As discussed in \u00a72.1, to match the procedures of the original study, any twins/triplets whose zygosity data were unavailable were treated as non-twin siblings when generating permutations. Since there are many related subjects in the ABCD dataset  the family structure was maintained when permuting so that a valid null distribution could be built.To estimate the significance of the correlation between the weights of each CCA mode (i.e. correlation between the corresponding columns of matrices locks.m) .15 SibshS2) and connectome (N0) data matrices. Connectomes and SMs were analysed separately.The null distributions were used to calculate thresholds for significance in the percent variance explained in the connectomes and SMs by each CCA mode. Specifically, the 5th and 95th percentiles of variance in the null distributions were used to determine which of the first 20 CCA modes explained a significant percentage of the total variance in the original SM (. 2.4.3S7 (dimensions 7810 \u00d7 78 subjects-by-SMs).16 Matrix S8 was then calculated by regressing the 12 confound variables (identified in \u00a72.4.1) out of matrix S7 . We correlated each column of matrix S8 with the primary mode SM weights (i.e. the first column of matrix V) to find Pearson's r values for each of the 78 SMs against the primary mode, and then plotted these correlations on an axis. In addition to the Pearson's r correlation values, we calculated z-score for each SM and the percentage of variance in the primary CCA mode that was explained by each SM. The percent variance was calculated by solving the following expression for each SM:pinv is Matlab's pseudo-inverse function, vector V1 is a demeaned copy of the primary CCA mode weights for SMs (a 7810 \u00d7 1 vector)17 and vector 18 is a demeaned copy of the raw values for the ith SM in matrix 8S.To understand which SMs were most strongly correlated with the primary mode and determine the directionality of the relationship (a positive or negative correlation), we generated a positive\u2013negative axis that quantified the level of variance explained by each SM. This analysis included the final 74 SMs that were input to the pre-CCA reduction, and 4 additional SMs  for a total of 78 SMs whose raw data were stored in a new matrix, et al., we entered the group-averaged full correlation network  into Ward's clustering algorithm implemented in Matlab. The low-dimensional results were then examined and assessed for correspondence with large-scale clusters observed in Smith et al. . To calculate the correlation between the CCA connectome-modulation weights  and the original population mean connectome, we first obtained the relative weights of involvement of the original set of connectome edges by correlating the primary CCA mode connectome weight vector U1 (i.e. the first column of matrix U) against N0, resulting in the \u2018full length\u2019 edge weight vector F1A (a 19 900 element vector), effectively mapping the primary CCA mode onto the original connectome data matrix. F1A was then correlated against the original population mean connectome.To facilitate interpretation of our ICA and connectivity results, we submitted the nodes derived from the group-ICA to a hierarchical clustering algorithm. As in Smith 19 Since the ABCD dataset contains sibships, the training and test subsets were generated without splitting any families across the subsets. To calculate the \u2018training\u2019 CCA, we applied the same steps outlined in \u00a72.4.1 to the training subset of the connectome and SM data to calculate the de-mixing matrices Atrain and Btrain such that Utrain= N5,train*Atrain and Vtrain= S5,train*Btrain.In addition to the 100 000 permutation-based significance test of the main CCA result, a 80\u201320 split train-test cross-validation was performed in which approximately 80% (approx. 6249) of subjects were used in a \u2018training\u2019 CCA, and the remaining 20% (approx. 1562) of subjects were left out as a test validation set.20 subjects; see \u00a72.4.2). The primary mode weight vectors for connectomes and SMs  were correlated against the primary mode weight vectors for connectomes and SMs from the original analysis (vectors U1 and V1).We then evaluated the primary mode of the \u2018training\u2019 CCA to determine how similar it was to the initial CCA result  were then correlated. In order to measure the significance of correlation between the \u2018testing\u2019 CCA primary mode connectome and SM weight vectors Utest,1 and Vtest,1, we performed a 1000-permutation test (respecting family structure). This test-train and significance testing process was repeated 10 times, each time with a randomly selected subset of test and train subjects, and the average correlation of Utest,1 and Vtest,1 from all 10 runs was calculated.The matrices  2.5https://github.com/nih-fmrif/abcd_cca_replication). We have also uploaded a compressed archive of the code in the Github repository to the OSF project (http://doi.org/10.17605/OSF.IO/HMC35) which also contains the full text of the pre-registration. Questions about the code may be posted as issues on the github project or emailed to the corresponding author directly.The code to run this entire pipeline is available on this project's Github repository . Scatterplots in et al., we colour the points by an example SM; here we chose fluid cognition as measured by the NIH Toolbox as it was most similar to the fluid intelligence measure used in fig. 1b from Smith et al. While fluid cognition does not appear to be associated with the relationship between SM and connectome weights in Mode 1, we observed a positive relationship in Mode 2 such that subjects with greater Mode 2 weights for both SMs and connectomes have higher fluid cognition scores. However, the relationship between CCA weights and fluid cognition in post hoc positive\u2013negative axis analysis we quantitatively relate all SMs to the primary CCA mode.The CCA yielded four modes whose correlation between the SMs and connectomes weights were statistically significant by permutation test . For the SM matrix, Modes 2 and 3 explained a significant amount of variance . Figure 3a,b shows the connectome variance and SM variance explained for each of the top twenty modes.We next examined the variance from the full SM and connectome matrices explained by the CCA modes by converting the Pearson's From these results, we determine that Mode 2 is the primary, \u2018strongest\u2019 mode according to our replication criteria (see \u00a71.3.1), since it maximally explains variance in both SM and connectome matrices (criterion 1) and exhibits a statistically significant correlation between SM and connectome weights (criterion 3). The z-score for SM variance explained by Mode 2 was greater than the next highest z-score by more than a factor of 2 (criterion 2).Z = 2.99, Mode 2: Z = 3.16). Although not part of our criteria, we did note that both of these z-scores were a factor of 2 larger than Mode 3 (Z = \u22120.34). With regard to the SM matrix, we expected the primary mode's variance explained z-score to be a factor of 3 larger than the next highest mode. Instead, we observed that the z-score of Mode 2 (8.56) was a factor of 2.35 greater than the next highest mode (Mode 3: Z = 3.64). Thus while our results are numerically similar to our expectations, the magnitude of variance explained by the primary CCA mode did not reach our a priori hypotheses.The only replication criterion not met is criterion 2, which addressed the variance explained by the connectome and SM weights. We expected that the connectome variance explained by the primary CCA mode would be a factor of 2 greater than the next highest mode. Instead, we observed that Modes 1 and 2 explained similar amounts of variance in the connectomes , thus confirming our main CCA results. Further, our 80\u201320 train-test split results are numerically similar to the results from Smith et al. . Notably, while still statistically significant, the mean correlation between the Utest and Vtest (r = 0.22) is smaller than the corresponding correlation from the full CCA (r = 0.44). We attribute this decrease in correlation to the possibility that the CCA model defined on the full dataset may be overfit and a generalizable correlation between canonical variates would likely be smaller in magnitude.To validate the CCA results, which were calculated on the full SM and connectome matrices, we then submitted the data to a 80\u201320 train-test split cross-validation procedure. Here we implemented a CCA on 80% of the sample and then estimated the subject weight vectors for Mode 2 in the left-out 20%. The weight vectors were then correlated and compared against 1000 permutations where we shuffled the SM matrix and performed the same analysis. This procedure was repeated 10 times. The mean correlation between The results presented thus far used data from a preprocessing pipeline that removed structured artefacts using an ABCD-trained ICA-FIX classifier. However, to remain as close to the original study as possible, we also repeated the main CCA and variance analysis on data preprocessed with an ICA-FIX classifier trained using data from the HCP. While there were small numeric differences in the results from these two pipelines, the overall findings are consistent, suggesting that our results are not dependent on which ICA-FIX classifier we use. See electronic supplementary material, S2.3.. 3.2We next sought to determine the relationship between the CCA modes and specific SMs and connectome edges. Here we focus on Mode 2 as this is the primary mode according to our replication criteria (see \u00a71.3.1).et al., we found a positive\u2013negative axis where positively valenced SMs  correlated positively with the CCA mode, whereas negatively valenced SMs  correlated negatively with the CCA mode. Interestingly, no negatively valenced SMs were on the positive end of the spectrum and no positively valenced SMs were found on the negative end. See r = \u00b10.2 and electronic supplementary material, figure S6, for the unthresholded axis.As in Smith et al. we do not observe a clear organization in which all nodes topographically similar to the DMN . Here we calculated correlations between subject-specific connectomes and the connectome weights from the primary CCA mode. . 3.3et al. All of the deviations detailed below were made while preprocessing the data. The final CCA mode calculation, on which successful replication was defined, was not conducted prior to the deviations and did not influence our decisions. The accepted Stage 1 protocol is available on the project's Open Science Foundation page (http://doi.org/10.17605/OSF.IO/HMC35).On closer inspection of the ABCD dataset, we realized that deviations from our pre-registration were necessary to more closely adhere to the analysis methods used in Smith et al. removed structured artefacts using ICA + FIX [et al. We, therefore, decided to use the DCAN's BIDS-formatted input data and their abcd-hcp-pipeline  to do preprocessing in accordance with the HCP recommended minimal processing .In our pre-registration, we planned to use the fully preprocessed and concatenated ABCD resting-state data distributed by DCAN. The DCAN pipeline removes motion and respiratory artefacts through nuisance regression and filtration , whereasCA + FIX ,24. If wIn our stage 1 pre-registered manuscript, we failed to specify which ICA-FIX classifier we would use to classify and remove structured artefacts. Therefore we elected to run two versions of our pipeline: one using an ICA-FIX classifier that we trained using ABCD data and another using the ICA-FIX classifier distributed with the ICA-FIX software which was trained using HCP data. We present the results from the ABCD-trained ICA-FIX pipeline in the main paper and the HCP-trained ICA-FIX pipeline in S2.3 of the electronic supplementary material. See S1.1 in the electronic supplementary material for information regarding ICA-FIX classifier signal and noise component classification performance and methods regarding training our own classifier. 1.Subject must have at least two \u2018good\u2019 runs of resting-state data (see below). 2.Subject must have a total of at least 10 min  of post-censoring resting-state data (see below for time point censoring criteria). 3.Subject must have at least one T1-weighted image that passes QC and protocol compliance metrics provided by ABCD.Using published studies that use the ABCD fMRI data as a guide [ 1.Mean FD for the run is less than 0.3 mm. 2.The pre-censoring run length (i.e. length of the raw scan) is at least 50% of the expected length of 380 time points .Runs that passed the above criteria were then submitted to ICA-FIX to remove structured artefacts. Censor files were then created using an FD threshold of 0.3 mm. To create the censor files, we used head motion parameters generated during pre-processing to flag time points that exceeded an FD of 0.3 mm for removal. In addition, segments of the time series were also flagged for removal if fewer than 5 consecutive time points had an FD displacement below the threshold. After ICA-FIX, runs were censored  and concatenated. Finally, we truncated the time series to 10 min for all subjects.Since we elected to not use the preprocessed, concatenated fMRI time series included in the DCAN ABCD release, it was necessary to examine the quality of the four individual fMRI runs for each subject before passing them to ICA-FIX. Here we noticed many runs had low numbers of time points (i.e. less than the full acquisition protocol) and/or high mean framewise displacement (FD) values, which could lead to spurious results. Thus, we applied the following revised data inclusion criteria. The addition of the following inclusion criterion 1 is the only deviation in inclusion proctol. Inclusion criteria 2 and 3 remained the same as the pre-registered protocol. a guide ,29,30, aAfter the 10 038 subjects in Collection 3165 were filtered according to the new criteria and only those with \u2018good\u2019 time points were retained, 5013 subjects remained as possible candidates for our analysis. This sample is smaller than the 7810 described in our pre-registered protocol because our new analysis excluded individual runs that lacked sufficient time points or exceeded a mean FD of 0.3 mm.resiliency_6b\u2019 was removed for having too much missing data , leaving 73 final SMs. No subjects in the new 5013 subject sample were dropped due to missing 50% or more SM data.The SM filtering protocol was unchanged. However, given the revised sample of 5013 subjects, SM \u2018N = 5013) had the following demographic breakdown: 47.9% male, 52.1% female; age in years : 9.99, 0.625, 9.0\u201310.21; race: 59.6% White, 10.4% Black, 17.5% Hispanic, 1.9% Asian, 10.5% Other; sibships: 360 MZ, 514 DZ, 810 non-twin siblings (687 siblings + 111 twins missing zygosity + 12 triplets), 3329 single.Our final sample  exhibited a positive correlation with the primary CCA mode, and the negatively valenced SMs (e.g. anger) exhibited a negative correlation. The authors also found that connectivity within the DMN was positively correlated with the primary CCA mode.In this study we have shown that a single axis of co-variation spanning \u2018positive\u2019 and \u2018negative\u2019 attributes links diverse participant characteristics with specific patterns of brain connectivity in 9- and 10-year-old children. We set out to replicate the landmark paper from Smith et al. is that the primary mode in the original paper was Mode 1, whereas we observed Mode 2 as primary. CCA modes are ordered according to the magnitude of correlations between the corresponding linear low-rank projections of the left and right input matrices\u2014that is, mode order relies on the strength of the relationship between the canonical variates [relationship between SM and connectome matrices, Mode 2 was more successful at explaining the variance in each matrix separately. As a result, Mode 2 met our criteria for the primary mode. Further, while Smith et al. found a single statistically significant mode, we found four, though only one met our criteria as primary. Subsequent work examining the multidimensional relationship between brain imaging and SMs suggests that multiple interpretable modes may exist and account for a variety of brain-behaviour dimensions [et al. in young adults: a surprising amount of variation in brain connectivity can be explained by a single axis of \u2018positive\u2019 and \u2018negative\u2019 attributes.Our analysis met 2 of the 3 strict numerical replication criteria described in our pre-registration (see \u00a71.3.1). For our first criterion, we found that Mode 2 of our CCA explained a significant amount of variance in both the connectome and SM matrices, according to a permutation test. For our third criterion, the connectome and SM CCA weights for Mode 2 exhibited a statistically significant positive correlation. Only our second criterion was unsuccessful. We expected that the primary CCA mode would explain a majority of the variance relative to the other modes, as indicated by z-scores of a factor of 2 for the connectome matrix and a factor of 3 for the SM matrix greater than the next largest z-score. In hindsight, we would judge the specific numeric magnitude factor criteria as overly conservative and not necessarily a critical finding of the original study. Further, one noteworthy difference between our findings and Smith variates . While omensions ,32. Despet al. replicate so well, since previous work has shown poor reliability when combining data from multiple sites using methods such as intraclass correlation coefficient [et al. , it is possible that the current analysis is not as susceptible to heterogeneity compared to other methods [Further, our analysis has shown that this brain-behaviour relationship is robust to significant heterogeneity in acquisition and analyses. First, the HCP dataset used in the original study was collected at a single site on a single scanner , whereasfficient . While w methods  since th methods .et al. are also robust to the many differences in demographics between the subjects of HCP versus those of ABCD. The ABCD dataset attempts to reflect the race and ethnicity breakdown of the United States in general [The findings from Smith  general , whereas general . Moreoveet al. noted that the primary mode could be evidence for a neural correlate of the general intelligence g factor [As in the original study, we also found evidence for a positive\u2013negative axis linking specific SMs to the primary CCA mode. Here SMs typically regarded as positive qualities  were positively correlated with the primary mode, whereas SMs typically regarded as negative qualities  were negatively correlated with the primary mode. Smith g factor . Interesg factor . They alg factor ,39. SES g factor ,41, whicOn the negative-correlation side of the positive\u2013negative axis, the appearance of clinical measures such as subscales of the Child Behaviour Checklist  for ADHDet al., we found that connectivity within the DMN was positively correlated with the primary CCA mode. The DMN is a network of distributed brain regions implicated in introspective and abstract thought, social cognition and autobiographical memory [et al. found nodes representing a broad swath of the DMN within the edges most strongly related to the CCA mode, we only found that edges between nodes within the posterior midline and temporo-parietal areas were most strongly related to the CCA mode. Further, while the default mode nodes in Smith et al. all clustered together, we observed that these nodes were represented in multiple clusters.Finally, we also found a specific pattern of functional connectivity edges that was related to the primary CCA mode. The nodes whose edges were most strongly correlated with the CCA included nodes topologically similar to sensory areas and the DMN. As in Smith l memory . HoweverThis is consistent with the findings on the development of the DMN and higher-order association cortices in general, such that these regions undergo a protracted development into early adulthood . That iset al. and extends their findings into the more heterogeneous ABCD dataset. We found a primary mode of correlation between brain functional connectivity and SMs meeting two of three pre-registered numerical criteria. We also found evidence of the positive\u2013negative axis first reported by Smith et al. where positive SMs were positively correlated with the primary mode and negative measures were negatively correlated. Finally, like Smith et al., we also found that connectivity within regions of the DMN were positively correlated with the primary mode, although this pattern of results in the ABCD dataset was not as dominated by default mode, and connections involving several other brain regions emerged as significantly linked to the primary mode; this may reflect developmental differences. The current study is situated within more recent efforts to examine the multidimensional relationship between multiple imaging derived phenotypes, physiological measures, genomics and behaviour showing interpretable modes related to phenotypes such as fluid intelligence, handedness and cardiovascular disease [In conclusion, the current study sought to replicate Smith  disease ,30. The Click here for additional data file."}
+{"text": "It is important to understand the effects of the COVID-19 pandemic not only on individuals\u2019 daily lives, but also their close partners. Current literature suggests that the COVID-19 pandemic has impacted older adults\u2019 lives in several ways, including the frequency of social interactions and change in various life habits . Data from 42 middle-aged and older, long-term married or cohabitating dyads were collected as part of an ongoing study of everyday cognition and functioning among couples. Participant age ranged from 40-85+, and couples were partnered for 9-60+ years. During this study, COVID-19 pandemic impact was assessed using six items (1 = No change to 4 = Severe change) examining daily routines, medical and mental health access, social contacts, and pandemic and family-related stress; reports ranged from six to 19. On average, women reported significantly higher COVID-19 pandemic impact compared to men. For both partners, the greatest disruptions reported related to routines and social contacts. Further analysis examined COVID-19 pandemic impact in dyads. For eight dyads, both partners reported relatively lower COVID-19 impact (6-11), whereas for six dyads, both partners reported higher impact scores (14-19). Discussion focuses on within-dyad and between-dyad differences related to perceptions of the pandemic\u2019s impact."}
+{"text": "K\u2009=\u200940 studies were identified, of which k\u2009=\u200920 studies met the eligibility criteria. For BTI paradigms using visuotactile stimulation, most studies found elevated sense of ownership ratings in SCZ-S populations compared to healthy controls (HC). Implicit illusion measures , in turn, did not generally indicate elevated embodiment levels in SCZ-S populations. Likewise, no consistent group differences emerged between SCZ-S populations and HC with respect to BTI paradigms using visuomotor stimulation. Furthermore, BTI vividness was found to correlate significantly with core symptoms of schizophrenia and various subclinical characteristics related to the SCZ-S. In line with the self-disturbance hypothesis, SCZ-S populations appear to be affected by aberrations in bodily self-awareness. Review registration: PROSPERO (identifier: CRD42022287960).Schizophrenia has been viewed as a disorder of the self. Accordingly, the question arises if and how senses of ownership and agency are impaired in schizophrenia. To address this question, several body transfer illusions (BTIs) have been investigated in schizophrenia patients and other schizophrenia spectrum (SCZ-S) populations. The objective of the study was to systematically review the current evidence from BTIs in the SCZ-S. A systematic literature search in PubMed and CENTRAL  was conducted on BTI studies carried out in SCZ-S populations. Studies were included if they were published in English after peer review, reported original research data, related to the SCZ-S, and used a BTI as its study method. Conference papers, study protocols, and reviews were excluded. For each included BTI study, various study characteristics and outcomes were retrieved, and a risk-of-bias score was calculated based on six study quality criteria.  Schizophrenia (SCZ) is a clinical disorder in which one\u2019s own self-awareness may be particularly disturbed2. While modern classification systems do not list self-disturbances as a key symptom, presumably due to their atheoretic approach, SCZ has historically been regarded as a disorder of the self. Emil Kraepelin, for instance, considered a disunity of consciousness (an \u201corchestra without conductor\u201d) to be a central feature of SCZ4, and Kurt Schneider regarded SCZ as \u201ca loss of the very contours of the self\u201d5. Consequently, he added several \u201cego-disorders\u201d (German: \u201cIch-St\u00f6rungen\u201d) to his \u201cfirst-rank symptoms\u201d of SCZ, that are still in use today. Moreover, in line with these traditional views, more recent SCZ theories like the Ipseity-Disturbance Model (IDM)2, have also stressed the importance of self-disturbances in SCZ. Likewise, recent meta-analyses by Burgin et al.6 and Raballo et al.7 have confirmed a high prevalence of self-disturbances in SCZ patients. Specifically, Burgin et al.6 reported a 2.5\u201312 times higher prevalence of self-disturbances in SCZ and associated conditions as compared to both healthy populations and populations with other mental illnesses. Similarly, Raballo et al.7 stated that self-disturbances selectively aggregate in SCZ and associated conditions, have validity as a phenotypic marker of vulnerability to varying degrees of severity of SCZ and, importantly, can be distinguished from a broader proneness to psychosis.Although we usually do not actively reflect on ourselves in everyday life, instead taking it pre-reflexively for granted, this tacit self-awareness can be severely disturbed under various clinical conditions9. Whereas SoO describes an experience of \u201cmineness\u201d toward one\u2019s own feelings, sensations, thoughts, and body parts , SoA refers to the authorship experience of initiating and controlling an action 9. As such, SoO and SoA are considered subcomponents of embodiment10 and several empirical studies suggested that alterations in SoO and SoA are specifically associated with SCZ symptoms13.If SCZ indeed involves a disorder of the self, the question arises if and how bodily self-awareness is also affected. In particular, it is not clear to which extent the sense of ownership (SoO) and sense of agency (SoA) are impaired in SCZ, given their ubiquitous involvement in bodily self-awareness14. That is, participants undergoing a BTI perceive that a presented artificial or virtual limb belongs to their own biological body (SoO) and that they can control its movements and actions (SoA). By now, various kinds of BTIs have been developed and tested . The original and most common BTI is the rubber hand illusion (RHI)15: by simultaneously stroking both an artificial hand placed in an anatomically plausible position in front of a subject and the subject\u2019s (hidden) real biological hand, an illusory SoO toward the artificial hand can often be evoked15. In addition, as an implicit sign of artificial hand embodiment, a so-called proprioceptive drift can frequently be observed, which reflects a shift of the estimated position of the participant\u2019s real hand toward the artificial hand16. Interestingly, however, the RHI decays in healthy populations when the artificial hand is positioned in an anatomically incongruent position 17, or when the tactile and visual stroking is applied asynchronously15. In addition to the RHI, further BTIs have been developed over time, such as the projected hand illusion (PHI)18 , the mirror box (MB)19 , and the full-body illusion (FBI)20 . Likewise, besides the original visuotactile induction method , several further BTI induction methods have been explored, including the visuomotor induction method . The advantage of this latter induction method is that, in addition to studying SoO and artificial hand embodiment, SoA can also be systematically investigated. For example, the moving RHI paradigm of Kalckert and Ehrsson21 examines how the cause of movements of the artificial hand affects SoO and SoA .Body transfer illusion (BTI) paradigms are a promising approach to investigate bodily self-awareness. In brief, BTIs can be defined as perceptual illusions that induce SoO and sometimes also SoA over artificial or virtual limbs22). In short, and following Tsakiris\u2019 taxonomy22, these theories can be arranged on a continuum between bottom\u2013up and top\u2013down approaches. Whereas bottom\u2013up accounts suggest that successful BTI induction mainly depends on multisensory integration  and only marginally on internal body maps, top\u2013down accounts assume stronger involvement of internal body maps .Based on the results revealed by these BTI investigations, various neurocognitive theories have been put forward over the years on how and why BTIs emerge , these BTI studies have not been systematically summarized and synthesized yet. Therefore, the aim of this preregistered systematic review is to summarize, link, and evaluate previous findings on BTIs in individuals with SCZ and related (sub)clinical conditions, as well as to give an overview of open research questions and potential clinical applications. To comprehensively review the entire body of BTI literature available in the schizophrenia spectrum (SCZ-S), the current work also includes studies that examined individuals with schizoaffective disorder, high psychosis- or delusion-proneness, or high levels of schizotypy. Of course, not all of these states are pathological, and the question rightfully arises to what extent firm conclusions about SCZ can be drawn for instance from subclinical SCZ-S states, such as high schizotypy . In the present review, the umbrella term SCZ-S is used whenever statements refer not exclusively to SCZ but also to schizoaffective disorder or any subclinical condition . Wherever applicable, however, the specific population under investigation will be mentioned.Originally, BTIs were primarily tested in healthy populations, but more recently they have also been studied in patients with SCZ as well as in populations with related clinical or subclinical conditions. With the exception of a mini meta-study25 and preregistered in PROSPERO (identifier: CRD42022287960).This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-AnalysesA systematic literature search on titles, abstracts, and keywords was performed in PubMed and CENTRAL . A conjunctive search query was made, whose first operand contained a disjunctive set of keywords  relating to clinical and subclinical SCZ-S phenotypes. Appropriate truncations were hereby used to search for all variations of a word stem. The second operand searched for BTI paradigms and contained a disjunctive set of relevant keywords .To be eligible, a study had to be published in English, to be peer-reviewed, to report original research data, to be SCZ-S-related, and to have used a BTI as its study method. Conference papers, study protocols, and reviews were excluded. No inclusion restrictions were made with respect to sample age, BTI paradigm implemented, study outcomes, or control conditions.Ordering/assignment-control: Was it ensured that the BTI conditions under comparison were (pseudo-)randomized or counterbalanced?Control group: Was there a HC group to which the SCZ-S sample was compared?Control condition: Was there a control condition , to which the actual BTI condition  was compared, in order to detect general suggestibility effects?Control items: Did the BTI questionnaire include a statistical comparison to control questions to detect general suggestibility effects and response biases?Registration: Was the study enrolled in an official study register?Blinding: Were the study staff blinded with regard to the different intervention groups?For each included study, various characteristics were retrieved. Moreover, to assess the study quality, a risk of bias (RoB) score (in %) was calculated, based on the following Yes/No criteria:A RoB of 100% (no criteria fulfilled) was defined to indicate a low study quality and a RoB of 0%  a high study quality.Study eligibility assessments and RoB evaluations were performed by two independent reviewers , and disagreements were resolved by discussion or by consulting a third reviewer (N.B.).k\u2009=\u200940 studies were identified, out of which k\u2009=\u200920 studies were deemed eligible and included in the review , were not related to the SCZ-S (k\u2009=\u20097), duplicates (k\u2009=\u20096), or were excluded for other reasons (k = 5). The main study characteristics and results of included studies are summarized in Table M\u2009=\u200960%) but varied considerably (SD\u2009=\u200918.26%) between studies and paradigms . Regarding SCZ-S populations, studies were performed in patients with SCZ (k\u2009=\u200911) and in mixed groups of patients with SCZ or schizoaffective disorder (k\u2009=\u20094), varying degrees of schizotypy (k\u2009=\u20092), psychosis-proneness (k\u2009=\u20091), psychotic-like experiences (k\u2009=\u20091), or delusion-proneness (k\u2009=\u20091). Concerning the BTI paradigms that were implemented, k\u2009=\u200911 studies relied on the classical RHI, k\u2009=\u20093 on a PHI version, k\u2009=\u20091 on the MB paradigm, k\u2009=\u20091 on the FBI, k\u2009=\u20092 on the moving RHI, and k\u2009=\u20092 on an otherwise modified RHI. Regarding BTI parameters, most studies collected phenomenological data based on standardized BTI questionnaires (k\u2009=\u200919). Implicit embodiment measures such as the proprioceptive drift (k\u2009=\u20098), somatosensory evoked potentials via electroencephalography (EEG) (k\u2009=\u20091), skin temperature (k\u2009=\u20091), the self-localization drift (k\u2009=\u20091), or the forearm bisection task (k\u2009=\u20091) were, in turn, used less often. Except for two studies in which skin temperature and EEG were obtained, no other physiological embodiment measures, such as the electrodermal fear response during artificial limb threatening28, were collected.The excluded 29). If multiple facets of embodiment  were in turn collapsed into one general rating, the term embodiment rating will be used .In terms of content, the studies primarily addressed four research questions, with some addressing more than one at a time: (1) Do populations of the SCZ-S display stronger SoO and overall embodiment levels toward BTIs than HC? (2) Do SCZ-S populations show differences in SoA during BTI inductions? (3) How relevant is multimodal synchronicity for BTI induction in SCZ-S individuals? (4) Are there associations between SCZ-S symptoms and BTI parameters? The results of the included studies are presented following these four main questions. Regarding self-report data, studies differed in their inclusion and analysis of questionnaire items. For example, while some studies specifically analyzed SoO toward the artificial limb, others examined more general facets of BTI embodiment. Therefore, in the following, questionnaire items are only considered as SoO ratings if they exclusively addressed a mineness experience toward the artificial limb or some location shift toward the artificial hand and k\u2009=\u20092 studies did not find any group differences29.Regarding visuotactile BTIs, a majority of studies found that individuals within the SCZ-S show higher SoO ratings during BTI induction than HC. More specifically, out of the 31 reported higher embodiment scores for both synchronous and asynchronous stroking, whereas the RHI studies by Mirucka34 and Peled et al.36 solely implemented a synchronous visuotactile RHI condition and reported higher SoO scores. The RHI study by Zopf et al.30, in turn, not only conducted classical synchronous and asynchronous stroking conditions, but also (a)synchronous conditions applied with an LED  and a tactile stimulator (for tactile feedback) attached to the participants\u2019 index fingers. Here, higher embodiment ratings were found across all conditions in the SCZ group. Finally, Shaqiri et al.20 implemented an FBI and found higher SoO ratings toward a whole virtual body among patients with SCZ compared to HC. However, the authors did not interpret these results as a specific indication for altered SoO of individuals with SCZ, given that additional control items assumed to be unrelated to body ownership were also rated higher in the patient sample and there was no significant three-way interaction between question type , synchrony (synchronous vs. asynchronous condition), and group (SCZ vs. HC)20.Of the studies that found higher SoO or embodiment ratings in SCZ-S populations, Thakkar et al.\u2019s RHI study40 reported lower SoO ratings in individuals within the SCZ-S. However, the RHI induction method was quite different in that study: Instead of actually undergoing hand strokes, participants only observed the experimenter\u2019s hand approaching the artificial hand under anatomically congruent and anatomically incongruent RHI conditions. While in the congruent condition, which yielded the strongest SoO ratings in both groups, the SCZ group reported lower SoO ratings compared to the HC group, no group differences were found in the incongruent RHI conditions40. In the other study, Lev-Ari et al.33 implemented the synchronous RHI condition on three trial days to test for a potential illusion learning effect. On the first trial day, both groups reported similar SoO ratings, while in subsequent trials more individuals of the HC group than the SCZ group showed an SoO learning effect .Among the studies finding opposite effects, Ferri et al.29 applied the classical RHI and did not find a group difference in SoO. Graham et al.18, in turn, applied the PHI to a mixed population of patients with SCZ or schizoaffective disorder and also did not find a significant SoO difference compared to HC.Regarding the aforementioned null findings, Prikken et al.k\u2009=\u20091 study reported a higher proprioceptive drift in individuals within the SCZ-S31, k\u2009=\u20094 studies32 did not find any proprioceptive drift group differences. A potential explanation for this discrepancy between SoO rating and proprioceptive drift results might be that both measures address different aspects of artificial limb embodiment45. For instance, Gallagher et al.43 hypothesized that subjective questionnaires reflect cognitive processes such as the personal body image, while the proprioceptive drift reflects the integration of multisensory stimuli . Following this approach, explicit and implicit illusion measures assess distinct aspects of bodily self-awareness that both are complementary and of individual importance.In contrast to these self-rating results, hardly any evidence for an increased BTI susceptibility of individuals with SCZ-S has been found in implicit embodiment measures. While 38, Graham-Schmidt et al.39 and Laurin et al.37 did not compare individuals within the SCZ-S and HC, the study by Rossetti et al.19 found lower illusion experiences in the implicit measure among individuals within the SCZ-S. More specifically, Rossetti et al.19 applied the MB paradigm, in which the participants performed movements with their hidden hand while observing the experimenter\u2019s synchronous (In-Phase), asynchronous (Anti-Phase), or random hand movements in a mirror. As an implicit measure, the forearm bisection task was used during which the participants were instructed to blindly point at the midpoint of their tested forearm. A successful illusion induction was assumed if a distal shift  was observed. Here, the SCZ group showed a proximal shift in all movement conditions, while the HC group showed a distal shift in the In-Phase condition.Regarding visuomotor BTIs, the few conducted studies revealed no clear indication of greater susceptibility toward BTIs in individuals within the SCZ-S compared to HC. While the studies by Louzolo et al.k\u2009=\u20097 studies also separately surveyed SoA. Of those, k\u2009=\u20093 relied on visuotactile stimulation47 and k\u2009=\u20094 on visuomotor stimulation39.In contrast to the multitude of BTI studies assessing SoO, only K\u2009=\u20093 studies39 found less sensitivity to visuomotor incongruency  in SCZ patients in general and in different SCZ subgroups. Rossetti et al.19 used a visuomotor MB paradigm in which the level of multisensory synchrony was systematically varied across a synchronous, an asynchronous, and a random visuomotor feedback condition. While in the HC group SoA turned out to diminish with declining synchrony, it remained constant across conditions in the SCZ group. Due to data transformation to counteract possible response biases, no conclusion about the actual SoA level experienced in the different study conditions was possible. In line with this, Graham et al.18 found that individuals with current passivity symptoms reported statistically similar degrees of SoA under both a synchronous and an asynchronous visuotactile PHI condition, while HC report decreased SoA after asynchronous stimulation. Graham-Schmidt et al.39 extended these results by applying a PHI but providing visuomotor instead of visuotactile feedback. They found that both patients who currently showed passivity symptoms and those who never experienced passivity symptoms reported similar levels of SoA after synchronous and asynchronous visuomotor induction, while HC and patients with past passivity symptoms showed significantly lower SoA under visuomotor asynchrony.37, in turn, used a moving RHI paradigm to compare SCZ patients with and without first-rank symptoms. The authors thereby referred to the original definition by Schneider5, which specified auditory hallucinations, thought broadcast, insertion, and withdrawal, as well as delusional perceptions as first-rank symptoms. While, on average, SCZ patients with first-rank symptoms reported no SoA over the artificial hand in an asynchronous movement condition, SCZ patients without first-rank symptoms did report SoA in that study.Laurin et al.38, SoA and psychosis-proneness46, and SoA and psychotic-like experiences47. Germine et al.46 applied the classical visuotactile RHI and found a significant correlation between psychosis-proneness and SoA under both visuotactile synchrony and asynchrony. As the authors noted, however, these correlation results for the asynchronous condition could have also derived from a carry-over effect, given that the asynchronous stimulation always followed the synchronous condition. Using a moving RHI, Louzolo et al.38 reported a significant correlation between SoA and delusion-proneness in passive (synchronous and asynchronous) but not in active conditions. Finally, Graham et al.47 found no specific effects of psychotic-like experiences on SoA in their applied visuotactile PDI.The remaining three studies explored potential relationships between SoA and delusion-proneness38 for asynchronous BTI conditions, where SoA usually diminishes. However, given that conclusions are largely based on null findings, (unplanned) subgroup analyses, and self-report data, these findings should be interpreted with caution. Further BTI studies on SoA with more rigorous experimental designs are therefore necessary.In sum, some evidence exists that SCZ patients in general, some specific SCZ subpopulations  as well as some SCZ-S populations  show \u201cover-inclusive agency\u201dk\u2009=\u200910 visuotactile48 and k\u2009=\u20092 visuomotor BTI studies39 also examined the role of multimodal synchronicity in the induction of BTIs in individuals within the SCZ-S.In addition to investigating whether individuals within the SCZ-S show higher susceptibility toward BTIs, k\u2009=\u20097 studies found higher SoO ratings47, k\u2009=\u20093 studies found higher embodiment ratings32 and k\u2009=\u20093 studies found higher SoA ratings under synchronous than asynchronous stroking within individuals of the SCZ-S46. However, in the study by Graham-Schmidt et al.39, this SoA effect was observed only under active but not under passive visuomotor stimulation, and k\u2009=\u20092 other studies19 also found merely similar levels of SoA (in some subgroups) in SCZ individuals after synchronous and asynchronous induction. Also, k\u2009=\u20094 studies found that patients with SCZ appear to give higher SoO29 and embodiment ratings31 than HC under asynchronous stimulation.Concerning explicit illusion measures, k\u2009=\u20099 studies applied implicit measures48, out of which k\u2009=\u20095 studies reported evidence for a greater proprioceptive drift in the synchronous conditions48, while k\u2009=\u20094 studies did not find an effect of synchronicity for their proprioceptive drift measure47 or global self-localization measure20.Regarding implicit illusion measures in the SCZ-S , K\u2009=\u200914 studies examined whether specific SCZ-S-related symptoms are correlated with BTI parameters. Out of those, k\u2009=\u20096 studies assessed subclinical symptoms in healthy individuals49 and k\u2009=\u20098 studies investigated symptoms in patients with SCZ or schizoaffective disorder40.31 found moderate to large positive correlations of positive and negative schizotypy with SoO ratings in synchronous and asynchronous classical RHI conditions. Partly in line with this, Asai et al.\u2019s49 RHI study found a moderate positive correlation between embodiment (composite score based on the proprioceptive drift and SoO ratings) and positive but not negative schizotypy. Similarly, psychoticism (interpreted as vulnerability for schizotypy) was found to correlate strongly with SoO in Kallai et al.\u2019s13 classical RHI setup. Furthermore, Germine et al.46 detected a moderate positive correlation between positive psychosis-proneness and SoO under visuotactile synchrony, as well as between positive psychosis-proneness and SoA under both visuotactile synchrony and asynchrony. Similarly, applying a moving RHI, Louzolo et al.38 reported moderate positive correlations between delusion-proneness and SoO as well as between delusion-proneness and SoA during passive but not active movement. Finally, higher scores in psychotic-like experiences in healthy individuals screened for SCZ-S symptoms were found to be positively associated with an overall PHI rating 47. Notably, however, when analyzing all BTI components separately, this correlation only persisted for disembodiment of the biological hand.Regarding healthy participants, Thakkar et al.k\u2009=\u20093 studies reported moderate positive correlations between hallucination severity and different BTI parameters. Specifically, Peled et al.35 found a correlation with two SoO rating items, Rossetti et al.19 with SoO, SoA, and a hand location subscale, and Thakkar et al.31 for an overall SoO rating. Prikken et al.29, in turn, found a positive correlation between delusion severity and SoO during a synchronous RHI condition, while Ferri et al.40 found a positive correlation between anhedonia and SoO during a congruent condition in a modified RHI which focused on the anticipation of a touch experience. In contrast, k\u2009=\u20093 other studies32 did not find any significant correlations between BTI parameters and positive or negative symptom severity in patients with SCZ or schizoaffective disorder. Overall, the pattern of associations is therefore not clear, especially since the latter studies failed to detect significant associations between SCZ-S symptoms and BTI parameters.Concerning SCZ patients, k\u2009=\u200920 eligible studies. Overall, based on the four research questions addressed here, evidence points toward various alterations of bodily self-awareness in the SCZ-S: First, most studies reported that individuals within the SCZ-S tend to give higher SoO or embodiment ratings in visuotactile BTIs than HC36. Whether this effect also applies to visuomotor BTIs, however, remains unanswered, as no studies have been conducted on this issue so far. Second, individuals within the SCZ-S also appear to present alterations in their SoA. Unlike HC, who typically demonstrate reduced SoA after asynchronous compared to synchronous stimulation, individuals on the SCZ-S appear to report similar SoA levels  under synchronous and asynchronous conditions39. Third, in line with results in HC, many studies found higher SoO47 and embodiment ratings32 after synchronous compared to asynchronous stimulation in individuals on the SCZ-S. However, some studies reported that individuals on the SCZ-S show higher SoO29 and embodiment ratings31 than HC during asynchronous conditions. Finally, the severity of positive SCZ symptoms , as well as higher scores of subclinical SCZ-S expressions  of the SCZ-S, appear to be associated with alterations of SoO and SoA49.This systematic review investigated whether SCZ-S populations show altered bodily self-awareness in BTI paradigms. More specifically, a systematic literature search in two databases was carried out, which identified 20 that the observed results are merely methodological artifacts and do not represent specific changes in bodily self-awareness of SCZ patients. Next, assuming that the results are not merely methodological artifacts, we discuss these findings on a neurocognitive and phenomenological level, whereby both explanation attempts are not considered as competing, but as complementing each other at different levels of analysis.In the following, we first discuss the possibility raised by Shaqiri et al.20 on SoO aberrations during BTI paradigms in individuals with SCZ. Interestingly, and at first glance in contradiction to the present results, Shaqiri et al. reported no evidence that SoO is elevated in individuals with SCZ. Of note, however, Shaqiri et al.20 applied rather strict criteria in their meta-analysis. Specifically, they exclusively focused on a two-way interaction between group (SCZ patients vs. HC) and condition (synchronous vs. asynchronous stimulation), based on the assumption that SoO aberrations in individuals with SCZ can only be demonstrated, if a group difference exclusively emerges under multisensory synchrony and not under multisensory asynchrony. Their underlying assumptions were (1) that multisensory asynchrony usually impairs SoO and (2) that increased SoO ratings could also be due to response bias unrelated to the actual experience. That is, individuals with SCZ, might show a generally increased confirmation bias and therefore report higher self-ratings on BTI paradigms than HC in general20. Given that Shaqiri et al.\u2019s meta-study revealed no statistically significant interaction, they concluded that the hitherto reported \u201cSoO differences\u201d between SCZ patients and HC are more likely due to a general response bias of SCZ individuals rather than a specific SoO aberration.Prior to the present systematic review, one meta-analysis had been carried out by Shaqiri et al.While acknowledging this possibility, we consider it much more likely that at least part of the observed effects in fact reflects specific SoO aberrations in individuals with SCZ, for the following reasons. First, Shaqiri et al.\u2019s meta-study was based on a small number of only four studies. Second, the question arises whether reporting SoO also during multisensory asynchrony actually constitutes sound evidence for a response bias. Just because healthy individuals usually report little SoO under multisensory asynchrony, it cannot be inferred that individuals from the SCZ-S have a similar experience. Instead, as discussed in the next section, individuals on the SCZ-S might for instance have an increased temporal binding window, that would explain the occurrence of SoO and SoA also after multisensory asynchrony.In sum, our review cannot exclude the possibility that response biases had an impact on the observed results. On the other hand, we see no evidence that a response bias alone explains the results sufficiently, and assume that aberrations in SoO and SoA explain the data at least as well.50, various potential neurocognitive mechanisms have been proposed as potential explanations for higher SoO ratings of individuals on the SCZ-S during BTIs. Examples include an over-dominance of visual information, a longer temporal binding window, a stronger reliance on external sensory input than on pre-existing body representations, and a disturbed SoA, which in turn might affect SoO. As concluded by Klaver and Dijkerman50, the most convincing and unifying explanation is probably that patients with SCZ rely more heavily on multisensory information than on their stored body representations. According to this idea, the observed higher SoO ratings in SCZ-S individuals stem from the fact that SCZ-S individuals rely more heavily on their momentary visual input  than on their proprioceptive input or pre-existing body representations. More specifically, during the BTI induction, SCZ-S individuals rely more on visual input  than on contradictory proprioceptive information concerning the position of their biological hand, or the consistency of their internal body representation .As reviewed and critically discussed by Klaver and Dijkerman20 assumed that higher SoO scores after asynchronous stimulation in individuals with SCZ compared to HC are due to response biases, an alternative explanation would be longer temporal binding windows among SCZ-S individuals and a resultant reduced multisensory temporal acuity50. According to this hypothesis, the general and often replicated finding51 that individuals with SCZ show an increased time interval during which temporally separated sensory stimuli are still perceived as occurring simultaneously, also affects the BTI induction. More specifically, due to the enlarged temporal binding window, the multimodal asynchrony during asynchronous BTI conditions is possibly not (as much) encoded as asynchronous by SCZ individuals compared to HC50. Therefore, the influence of asynchrony on SoA and SoO is reduced in these subjects. In addition, regarding SoA, defective motor predictions have been associated with SoA persistence under multimodal asynchrony. According to this idea, SCZ is associated with impaired motor predictions that lead to difficulties in detecting kinematic dissimilarities caused by visuomotor incongruency; and therefore, SoA also persists under multimodal asynchrony19. Finally, the finding that a subclinical population high in delusion-proneness reported similar SoA ratings under active and passive movement has been attributed to a combination of reduced motor predictions and hypersalient processing of external input38. That is, for inferring agency, these individuals would rely less on their own motor predictions and intentions, but more on their sensory input perceived, and as a result experience SoA also during passive movement.These explanations might also partially account for the finding of similar SoA under synchronous and asynchronous stimulation in SCZ-S individuals (see research question 2) and the occasionally persisting SoO under asynchronous stimulation (see research question 3). While Shaqiri et al.52. And third, a disturbed \u201chold\u201d or \u201cgrip\u201d which refers to the loss of stability and salience of the field of awareness4. Based on these three aberrations of self-experience, higher SoO ratings of SCZ-S populations can be explained by a reduced self-demarcation. Accordingly, individuals on the SCZ-S might be more susceptible to perceive external objects  as belonging to their own body due to diminished self-other boundaries.The reported findings can also be addressed from a phenomenological perspective. Referring to the aforementioned IDM, for instance, altered SoO demonstrated by SCZ-S populations during BTI exposure (cf. research question 1) might be a result of a disordered first-person perspective of experience  characterized by three main aberrations: First, hyper-reflexivity. i.e., an excessive self-awareness in which aspects of oneself are experienced as alien and akin to external objects. Second, diminished self-presence, i.e., a reduced sense of being an agent of action and a reduced SoO toward everyday experiencesFurthermore, the IDM also serves to explain the findings addressed in research question 2. Similar SoA ratings for synchronous and asynchronous stimulation in individuals on the SCZ-S hints toward an \u201cover-inclusive\u201d agency, as SoA ratings usually decrease in HC for asynchronous conditions. An \u201cover-inclusive\u201d agency could be the result of a reduced self-demarcation, making it more difficult for individuals on the SCZ-S to distinguish between their self and the external world. In addition, this could also be caused by a hyper-reflexivity  on, in this case, external stimuli.53 offered a parsimonious approach to explain the association between erroneous multisensory integration in BTIs and positive SCZ symptoms (cf. research question 4). According to the authors, conflicting sensory input  may lead to perceptual incoherence that could cause self-disorders as described in the IDM. For example, sensory imbalance or reduced sensory input promote hyperfocused attention. Simultaneously, it could be that unconscious efforts to regain perceptual coherence may result in delusions and hallucinations. Hence, the authors\u2019 idea was that both increased BTI susceptibility and positive symptoms are secondary consequences of perceptual incoherence and therefore correlated.Finally, connecting the neurocognitive and phenomenological perspectives, Postmes et al.M\u2009=\u200960%; SD\u2009=\u200918.26%) of many conducted studies . In fact, none of the k\u2009=\u200920 studies carried out a preregistration of their research project to reduce potentially problematic research practices 54. Likewise, none of the studies applied a form of blinding  to avoid investigator effects. Also, k\u2009=\u200914 studies did not include control items into their questionnaire. The inclusion of such control items, however, appears crucial to control for possible response biases  and to verify the illusion specificity of the experimental manipulations55 . Similarly, k\u2009=\u20095 studies omitted the integration of an adequate control condition , which may help to rule out general responses biases20. Moreover, k\u2009=\u20096 studies did not perform adequate randomization or counterbalancing, implying that sequence effects cannot be excluded.One limitation is the rather low quality , exploring implicit as well as explicit BTI measures appears particularly important, since both measures capture different aspects of artificial limb embodiment44. Moreover, the proprioceptive drift measure is probably less prone to general suggestibility effects and response biases than a BTI questionnaire. Therefore, future BTI research should include different implicit measures to operationalize the illusion. One promising approach is recording the participant\u2019s electrodermal activity while the virtual hand is threatened by a virtual syringe, as implemented in some BTI studies57 and using this physiological marker as another implicit measure of artificial hand embodiment.Another methodological weakness is that many studiesk\u2009=\u20096 studies58 found illusion scores \u22640 on a Likert scale ranging from \u22123 to +3 and still interpreted these scores as indicative of a successful BTI induction. Even if relative SoO differences might still be detectable despite weak embodiment (SoO) levels, the question arises, whether a meaningful manipulation of bodily self-awareness can still be assumed. A promising approach to counteract over-interpretations could be the integration of an absolute illusion threshold  in future research for a successful SoO and/or SoA induction.A further limitation concerns the interpretations of some of the studies, particularly regarding the questionnaire results. For example, among the studies that reported raw means of the questionnaire items, 39, or first-rank symptoms37. Focusing on specific aspects of the SCZ-S therefore appears to be a particularly interesting approach to study bodily self-awareness within the SCZ-S in further detail.Another limitation of the reviewed studies is that some of them reported strong interindividual differences within the SCZ-S, with illusion ratings ranging from strong disconfirmation to strong confirmation. Consequently, the samples acquired might not be sufficiently homogenous to justify a generalizable statement about bodily self-awareness within the whole spectrum of SCZ-related states. Instead, it appears that some BTI experiences are not explained by SCZ per se, but rather by subsets of SCZ-related symptoms. Three of the included studies in fact divided their SCZ samples into multiple subgroups according to symptomatology, such as current, past, and no experience of passivity symptoms59 or the Inventory of Psychotic-Like Anomalous Self-Experiences (IPASE)60. To validate the hypothesis that SCZ is a disorder of the self and that BTI paradigms can contribute experimental evidence to this hypothesis, it is important to conduct thorough assessments of prevalent self-disorders in addition to the application of BTI paradigms.Moreover, concerning the fourth research question on associations between SCZ-S symptoms  and BTI parameters, it has to be noted that none of the reviewed studies directly assessed self-disorders by means of dedicated assessment tools such as the interview-based Examination of Anomalous Self-Experiences (EASE)61 could provide additional insight. If SCZ is indeed primarily caused by a \u201cdisunity of consciousness\u201d , individuals with SCZ-S should be particularly vulnerable to this modified RHI.Another aspect concerns the question of which conclusions can actually be drawn from the BTI results so far. Notably, most reviewed studies merely focused on the extent to which SCZ-S individuals are more prone to \u201cexperientially replace\u201d a biological limb with an artificial one. That is, they all relied on BTI paradigms that expand already experienced SoO toward one\u2019s biological limb to an artificial limb, but that do not instantiate any new limb in the participant\u2019s body matrix or drastically change a limb\u2019s perceptual appearance. Therefore, the question arises as to how much can actually be learned from observing BTI vulnerability differences and to what extent a higher (or lower) susceptibility to BTIs must be seen as pathological. Therefore, it might also be interesting to explore whether more drastic changes in bodily self-awareness can be induced and investigated in future studies. For instance, the effect of a \u201csupernumerary limb illusion\u201d, a modified RHI under which participants perceive more limbs than they physically possessFinally, an interesting application-oriented approach could be to investigate whether BTI paradigms prove useful as a therapeutic or psychoeducational element in patients with SCZ: Experiencing a BTI intuitively demonstrates that our bodily boundaries\u2014which we usually take for granted and constant\u2014can easily be modified within seconds. As a consequence, other SCZ-related symptoms such as shifts in the perception of reality  or reduced demarcation of inner and outer world , might be evaluated differently after experiencing a BTI in the therapeutic context.63 or dexamphetamine64, which can induce psychosis-like experiences. The comparison of these studies with studies of patients on the SCZ-S remains difficult, which is why studies actively inducing psychosis-like symptoms were not included in the present review. Nevertheless, this approach represents a novel and promising way of conducting research in the SCZ-S. Third, to get an overall picture of the results, and because the number of published studies is still quite small, some interpretations refer to the whole SCZ-S and may ignore differences between populations. However, with more data available, a detailed comparison of populations within the SCZ-S with respect to BTI might be possible in the future. Finally, regarding the phenomenological underpinnings of possible alterations of bodily self-awareness presented here, we focus on the framework laid out by the IDM. It has to be noted that other phenomenological theories like the perceptual anomalies approach65 might offer additional insights into the subjective experience of BTIs. While also highlighting the potential over-salience of external stimuli, as well as describing the phenomenon of giving new meaning to what is experienced in SCZ individuals, this approach considers alterations in low-level, perceptual/automatic processing and resulting disruptions of the perceptual field at the core of these experiences65. As a consequence, it relies less on theoretical assumptions about the self  and could be a more parsimonious framework to explain alterations of SoO and SoA.The present systematic review has some limitations itself that must be taken into account. First, our literature search only included two databases, PubMed and CENTRAL, and only considered articles published in English. Therefore, it is possible that some publications were not identified. Second, a number of studies investigated SCZ and psychoses in healthy participants through the administration of substances such as ketamineOur review indicates that during BTI exposure, populations on the SCZ-S demonstrate altered bodily self-awareness. Regarding SoO, especially SCZ patients appear to give stronger SoO ratings under most visuotactile BTI paradigms, whereas under visuomotor BTI paradigms results are less conclusive, presumably due to a lack of studies. Moreover, concerning implicit illusion measures, the majority of studies do not find higher SoO in SCZ-S populations. Regarding SoA, some studies report similar SoA levels after synchronous and asynchronous stimulation in SCZ individuals, while HC typically report significantly lower SoA under visuomotor asynchrony. Also, positive associations emerge between BTI measures and subclinical SCZ-S states  as well as between BTI measures and SCZ symptoms . While on the neurocognitive level, a stronger influence of external stimuli or weaker stored body representations could be responsible for these findings, on the phenomenological level a reduced ego demarcation might be accountable. These findings, however, need to be interpreted with caution, given that many of the studies included in this review lacked sufficient methodological rigor. Therefore, further and more rigorous research is needed to identify possible pathomechanisms of bodily self-awareness in SCZ-S populations.Risk of Bias Evaluation"}
+{"text": "Burkholderia (B.) mallei is a host-adapted equine pathogen that causes glanders, a re-emerging zoonotic disease, which is endemic in Pakistan and other developing countries and seriously impacts the global equine movement. Due to globalization, the geographical restriction of diseases vanishes and the lack of awareness of and experience with eradicated diseases in industrialized countries also promotes the re-introduction of infections in these regions. Owing to the high equine population, the Pakistani province Punjab is a potential hotspot where several glanders outbreaks have been seen over last two decades. For determining the genomic diversity of B. mallei in this and other equine-populated prefectures, the genomes of 19 B. mallei strains isolated between 1999 and 2020 in different locations were sequenced and their genotypes were determined. Particularly, for genetically highly homogenous pathogens like B. mallei genotyping techniques require a high discriminatory power for enabling differentiation on the strain level. Thus, core-genome single nucleotide polymorphism (cgSNP) analysis was applied for distinguishing the highly similar strains. Furthermore, a whole-genome sequence-based core genome multi locus sequence typing (cgMLST) scheme, specific to B. mallei, was developed and additionally applied to the data. It was found that B. mallei genotypes in Pakistan persisted over time and space and genotype clusters preferred connection with a time point rather than the place of isolation, probably due to frequent equine movement, which promotes the spread of glanders. The cgMLST approach proved to work in accord with SNP typing and may help to investigate future glanders outbreaks. Burkholderia mallei that mainly affects equines [B. mallei can also infect other mammals, including humans, making it a zoonotic pathogen. Laboratory workers, veterinarians and animal caretakers are at highest risk [International animal trading poses the risk of global dissemination of pathogens. Even on smaller scales , unrecognised infection carriers can have a fatal impact, causing outbreaks among the native population . Such tr equines ,3. Horse equines . Althougest risk ,6. Thereest risk ,8.B. mallei infection led to the classification of glanders as a re-emerging disease [In the past, glanders received wide attention due to the fatal nature of the disease, but now it has been eradicated in many countries due to strict measurements and culling policy . However disease ,10,11,12B. mallei is sporadically disseminated to other equine populations by asymptomatic animals [One of the earliest documentations of glanders in Pakistan dates back to 1877 and several outbreaks have been reported since the beginning of the present century ,14,15. H animals . Additio animals ,18,19.B. mallei strains on a molecular basis and therefore the tracing of infection sources is complicated, as the global population of this pathogen is genetically highly homogenous [B. mallei are at hand, most of which were originally developed for the closely related B. pseudomallei making them hardly applicable for B. mallei. In a multi locus sequence typing (MLST) scheme most B. mallei belong to a single genotype, as the chosen target genes are highly conserved [The differentiation of mogenous . Severalonserved . Variablonserved  provide onserved . Thus, tB. mallei yet. Girault, et al. [B. mallei population, which was later confirmed by whole genome SNP analysis [B. mallei populations caused outbreaks of glanders in Bahrain in 2010 and 2011 [Acinetobacter baumanii, Bacillus anthracis and Listeria monocytogenes [SNP typing has rarely been applied for the differentiation of , et al.  used 15 analysis . Furtherand 2011 . Allel-band 2011 ,28. In fytogenes ,30,31.B. mallei genotypes circulating in Pakistan based on whole genome sequencing. By developing a B. mallei-specific cgMLST scheme, we add an additional method to the epidemiologist\u2019s toolbox for determining connections between outbreak events and infection chain tracing.In this study we aim at elucidating the diversity of B. mallei strains  isolated from different outbreaks of glanders between 1999 and 2007  per sample with an average length of 255 bp and sufficiently high coverage for further analysis . GenomesIn contrast, by pursuing a hybrid assembly strategy combining Illumina short-read and nanopore long-read sequencing data, a higher level of contiguity could be reached for the genomes of four strains from our strain collection .The investigated strains from Pakistan were compared in a cgSNP analysis  to sevenWithin the Pakistani cluster, the strains formed smaller separate clusters, which were defined by sampling decade rather than by location of isolation, i.e., the strains from 2017 to 2020 did not mix in clusters with the PRL-named strains (1999\u20132007). In a cgSNP analyses merely including the strains sequenced in this study, all in all, 660 core genome SNPs were detected and SNP differences of 0\u201396 SNPs were observed . In the It was striking that the more contemporary strains exhibited fewer clusters, with less than 10 varying SNPs, implying a higher genomic heterogeneity of strains from 2017 to 2020. The strains from Faisalabad  and Islamabad  were the most similar among these strains, as they were separated merely by two SNP, while all other strains exhibited at least 10 SNP differences. They belonged to the most prominent cluster, cluster III, formed by strains from 2017 to 2019 from almost every sampled location. When comparing the two sampling decades, the strains exhibited 10 to 96 SNPs difference.B. mallei genotyping studies were not represented in the set of chosen public database entries. Thus, we additionally sequenced four strains of our strain collection , which was not possible by cgSNP (zero SNP differences). However, the PRL and more recent strains still did not form clusters defined by isolation location or year. The three exemplary Indian strains that were included in the cgMLST analysis did not mix with Pakistani strains.Additionally, in silico multiple-locus variable number tandem repeat analysis (MLVA) was conducted for the Pakistani strains . For somB. mallei genotypes in this region. The present study is the first extensively employing whole genome sequencing for molecular genotyping of B. mallei outbreak strains from Pakistan. Often, typing studies suffer from the availability of only a few strains that can be investigated, which might pose a problem for revealing the true genomic variability within this specie and determining the method that is best suited for differentiation of more distantly related strains as well as highly congruent outbreak isolates. Thus, we investigated 19 strains that cover a sampling period of over two decades and originate from different locations in Pakistan.Although glanders has been endemic in Pakistan since at least the 19th century, little is known about the distribution of different Burkholderia-specific MLST scheme [B. mallei strains while an MLVA scheme [B. pseudomallei, the assumed progenitor of B. mallei, was proposed [B. mallei strains (unpublished data), a B. mallei-specific cgMLST scheme was developed in the present study. The percentage of targets identified as suitable core genome genes included in this scheme was well in range with other studies, although this value is highly specie-dependent. The number of identified target genes, 2838 targets, was lower than the published number of 3456 B. mallei core genome genes [For a reliable differentiation of species and strains, it is necessary to identify unique molecular signatures with a high discriminatory power. Several methods are at hand. However, the T scheme  fails toA scheme  providesproposed . As thisme genes . Howeverme genes ) and it me genes .B. mallei scheme when challenged with sequences from worldwide strains covering the complete diversity of the species, as well as the Pakistani outbreak strains. Likewise, the new scheme was compatible with cgSNP analysis as both revealed the same epidemiological patterns. However, Pakistani strains that were identical in the SNP analysis showed differences in cgMLST allelic profiles. Thus, we believe the cgMLST scheme might help outbreak investigations, in which highly congruent strains have to be differentiated. For other species, thresholds of allelic differences have been determined that define a single outbreak event, e.g., five and twenty alleles in case of Bacillus anthracis and Enterococcus faecium, respectively [B. mallei. Such a threshold is also not known yet for B. pseudomallei cgMLST analyses, as even two alleles separate outbreak strains of a single transmission event from unrelated isolates and epidemiological connections were merely assumed for isolates differing by one allele [B. mallei.In a well-defined cgMLST scheme the retrieval rate of these targets in outbreak strains should constitute at least on average 95% to 97.5% ,31. Thisectively ,30. As ie allele . A similB. mallei strains, the resulting polytomy matched the known patterns and was in accordance with the cgSNP typing results. The analysis of duplicate strains from different sequencing projects deposited in public databases gave allelic patterns that for some strains differed in several targets. However, differences can be attributed to the age of the isolates resulting in different replication cycles in the laboratories, as well as varying sequence qualities. Observed differences in allelic numbers of one and the same strain could be attributed to ambiguities in the assemblies. In particular, the N50 value of the assemblies proved crucial for the success of cgMLST analysis. In fragmented assemblies, genomic elements might be truncated or missing. In order to overcome this lack of high-quality assemblies for the development of the cgMLST scheme, we added genome sequences of four strains to the set of query genomes. The assemblies for those strains were generated by a hybrid assembly approach combining Illumina and nanopore sequencing data, which improved genome completeness and accuracy.When the newly developed cgMLST scheme was employed for the analysis of a diverse set of global B. mallei in the Punjab province [Although glanders is endemic in Pakistan and neighboring countries for at least 150 years ,14,15,18province , to whicB. mallei phylogeny that can be distinguished from closely located Indian strains. Apparently, there is no extensive mixing between B. mallei populations from both countries.The whole genome genotyping approaches employed in this study showed that the Pakistani strains form a group within global B. mallei PRL strains have been investigated before by MLVA [B. mallei in Lahore, which might be the source of infection of a mule, host to PRL44, 1.5 years later. However, the MLVA profiles of the strains differ [The  by MLVA , on the  by MLVA , the hosB. mallei during passage through a host [B. mallei lineages circulate in Pakistan. However, the connection between genomic links and epidemiological links remains difficult given the complex epidemiological situation where horses often move between different cities and regions.When comparing the cgSNP and cgMLST data, strains that differed merely by a couple or by no SNPs, although they were isolated from different places and/or years apart, showed larger differences in cgMLST profiles, as would be expected and this makes sense from an epidemiological point of view, as comparably high numbers of genome alterations occur in h a host . The samh a host . PRL 1 aB. mallei throughout the country. However, the currently circulating strains are distinct from NCTC 3709, isolated at the beginning of the 20th century in Lahore. It is known that glanders was re-introduced in India in the 1960s during the Indo-China war by imported horses and also employed in warfare [B. mallei strains in Pakistan.The analysis is complicated by a gap of 11 years between the sampling periods and by the fact that various undetected glanders outbreak events can be expected in the region . In pref warfare ,18, whicB. mallei strains would not only help to elucidate the origin of Pakistani strains, but also the detection and surveillance of glanders worldwide. Laroucau et al. [fliP-IS407A gene did not give a positive result, probably due to genetic variation in the infecting strains. Solely by applying more extensive methods, like SNP and MLST typing, the serological results could be confirmed. For such cases, in silico analysis based on whole genome sequences might help improving diagnostics, which is also important for countries where glanders is eradicated for decades, as veterinarians do not recognize the symptoms of the disease easily [Thorough genotype characterization of u et al.  reportede easily . The appHere, we could show that the read-dependent cgSNP approach works well together with cgMLST analysis that is based on assemblies, and we believe that future studies could benefit from employing both methods, especially when the analysis must be based on assemblies, when no raw read data is available.B. mallei strains were isolated from blood and puss samples of clinically suspected glanders equids (horses and mules), which were brought to the Veterinary Medical Teaching Hospital (VMTH) of the University of Agriculture (UAF), Pakistan, between 1999 and 2020. Blood cultures were carried out in Oxoid Signal Blood Culture System . For this purpose, approximately 10 mL peripheral venous blood collected from the jugular vein was inoculated and incubated on a shaker at 37 \u00b0C for 56 h. Samples where the indicator devices showed positive culture signals were sub-cultured on 5% sheep blood agar plates and presumptive colonies were biochemically tested and confirmed by conventional PCR targeting the B. mallei bimAma gene as described elsewhere [B. mallei strains were grown in brain-heart infusion broth containing 4% glycerol at 37 \u00b0C for 2d. DNA was extracted using enzymatic digestion and phenol-chloroform extraction, according to standard protocols [For DNA isolation, the Pakistani rotocols .B. mallei, four B. mallei strains from the Friedrich-Loeffler Institutes\u2019 strain collection  were selected, for which no high-quality genome assemblies were deposited in the public databases, yet. The strains were grown on nutrient agar  supplemented with 3% glycerine and 7.5% blood for 48 h at 37 \u00b0C. DNA was isolated using the NucleoBond HMW DNA kit  and subsequently used for Illumina and nanopore sequencing library preparation.For the development of a cgMLST scheme covering a high diversity of Short-read sequencing libraries were prepared from the isolated DNA with the Nextera XT library preparation kit  and subsequently sequenced using v3 chemistry on a MiSeq system (Illumina) in paired-end mode. Four strains  were additionally sequenced by nanopore long-read technology (ONT). For this purpose, libraries were prepared with the Ligation Sequencing Kit SQK-LSK 109  together with the Barcoding Kit EXP-NBD 104  and sequenced on an R9.4.1 flow cell with a MinION Mk1B sequencing device  for 24 h. Sequencing raw data and hybrid assemblies were deposited at the European Nucleotide Archive under project number PRJEB52165.Basecalling and demultiplexing of the ONT data were conducted with Guppy basecaller v5.0.7  applying the \u201csuper-accuracy\u201d model. The read quality was checked by NanoPlot v1.32.1 . Finallyhttps://github.com/tseemann/shovill, accessed on 11 April 2022). Short reads and assemblies were analyzed using the pipeline WGSBAC v2.2  including a check for species identity and contaminations by kraken2 [https://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 11 April 2022) and Quast v5.0.2 [The Illumina reads were assembled using Shovill v1.0.4 , as well as 23-loci MLVA [https://github.com/Papos92/MISTReSS, accessed on 11 April 2022) were conducted utilizing WGSBAC. B. mallei ATCC 23344 (GCF_000011705.1) was used as reference strain for SNP typing. Trees were visualized with FigTree v1.4.3  and figures were made publication-ready using Inkscape v1.1 . For further genotyping by cgMLST using a scheme generated in the framework of this study, Ridom SeqSphere+ v7.7 [Furthermore, core genome SNP typing with Snippy v.4.6.0  and NCBI GenBank were browsed on 20 January 2022 for Illumina read data and assemblies of B. mallei on 11 October 2021. Metadata and function of these sequences are listed in Public databases were searched for genome assemblies of + target definer incorporated in Ridom SeqSphere+ v7.7 with default parameters, as previously described [B. mallei ATCC 23344 (GCF_000011705.1) served as the seed genome.The cgMLST scheme was generated by a genome-wide gene-by-gene comparison using the MLSTescribed , includiB. mallei raw reads from the NCBI SRA database were downloaded  in conjunction with RAxML v8.2.12 [For validation of the scheme, publicly available  v8.2.12  and Pars v8.2.12 , respect v8.2.12 . Bootstr"}
+{"text": "Journal of Clinical Medicine (JCM) . The management of salivary gland (SGs) disorders encompasses a broad array of diseases, both benign and malignant, and the great international interest in this topic has led to the publication of the second part of the Special Issue, thusly entitled \u201cDiseases of the Salivary Glands-Part II\u201d, which reflects the diverse nature of SG dysfunction and is focused on various topics ranging from diagnosis and therapeutic implications to the study of the molecular mechanisms underlying autoimmune and neoplastic diseases of the salivary glands.This Special Issue, \u201cDiseases of Salivary Gland-Part II\u201d, was born as a continuation of the volume \u201cDiseases of the Salivary Gland\u201d, published, with great success, in 2021 in the prestigious The first part of this Special Issue concerns research articles focused on new discoveries in the diagnostic field of SG diseases ,3,4,5,6 Given the scientific importance of the articles collected, I am thrilled to reiterate that this Special Issue aims to provide insights into SGs diseases and summarizes the current knowledge of underlying pathophysiological mechanisms, yielding surprising results."}
+{"text": "In-stent neoatherosclerosis (ISNA) is an important reason for stent failure. High lipoprotein (a) [Lp (a)] level is an independent predictor of in-stent restenosis (ISR). To date, the relationship between the level of serum Lp (a) and the incidence rate and vulnerability of ISNA has never been verified.A total of 119 patients with 125 drug-eluting stent ISR lesions who underwent percutaneous coronary intervention guided by optical coherence tomography were enrolled in this study. According to their Lp (a) level, the patients were divided into two groups . The clinical baseline, angiographic characteristics, and optical coherence tomography data of both groups were recorded and analyzed.P\u2009>\u20090.05). The incidence rate of ISNA in the high Lp (a) group was significantly higher than that in the low Lp (a) group . The incidence rate of thin-cap fibroatheroma in ISR lesions was significantly higher in the high Lp (a) group than in the low Lp (a) group .No significant differences in clinical and angiographic characteristics were found between the two groups (A high Lp (a) level is associated with the high incidence rate and plaque vulnerability of ISNA.The online version contains supplementary material available at 10.1007/s10554-022-02736-3. In-stent neoatherosclerosis (ISNA) is one of the types of in-stent restenosis (ISR) and is an important cause of late stent thrombosis and late ISR. Neoatherosclerosis (NA) is characterized by the accumulation of lipid foamy macrophage within neointima, with or without necrotic core, and the presence of calcium  within tLipoprotein (a) [Lp (a)] is a serum lipoprotein whose structure is composed of apolipoprotein (a) [Apo (a)], apolipoprotein (b) [Apo (b)], and low-density lipoprotein (LDL)-like segment . Lp (a) The correlation between Lp (a) and adverse clinical outcomes, such as ISR after percutaneous coronary intervention (PCI), remains controversial. Most previous studies were conducted on patients with PCI receiving bare metal stent (BMS) . Recent This single center, retrospective observational study enrolled patients hospitalized at the Chinese People\u2019s Liberation Army (PLA) General Hospital from March 2014 to March 2022. From a total of 805 ISR patients with DES implantation, we included 324 patients treated with OCT-guided ISR lesions. We then excluded patients with BMS-ISR (n\u2009=\u200911), without serum Lp (a) values (n\u2009=\u2009182), and with poor OCT image quality (n\u2009=\u200912). Finally, 119 patients with 125 DES-ISR lesions were included in this study  analysis was performed in the core laboratory of the Chinese PLA General Hospital, where QAngio XA software  was used by a blinded trained technician to analyze the angiographic images of all patients. Mehran classification was used for ISR classification : type I,ISR lesions were imaged using OCT catheters  and frequency-domain OCT , and the OCT catheter was introduced into distal ISR lesions with a pullback rate of 18\u00a0mm/s and rotation rate of 100 frames/s . During For quantitative and qualitative analyses, OCT cross-sectional images between proximal and distal reference lumen areas were analyzed at 1-mm intervals. For qualitative indicators, the presence of target features in any section can be considered as the existence of this feature. For quantitative indicators, minimum stent area (MSA) in ISR lesion area was analyzed. The main indicators analyzed were the plaque structural characteristics and multiple luminal area characteristics of ISR. For quantitative analysis, the percentage of maximum neointimal hyperplasia (NIH) was calculated as ([stent\u2013lumen area]/stent area) \u00d7 100%. Proximal and distal reference lumen areas were the slices with the largest lumen area within 5\u00a0mm proximal and distal to the stent edges, respectively. Underexpansion was defined as minimum MSA/the mean of the distal and proximal area\u2009<\u200980% .For qualitative analysis, the accumulation of lipid foamy macrophage within neointima (with or without necrotic core) in the presence of calcium within the culprit stent was defined as NA . The thrIn the OCT images, lipid plaque was defined as a signal-poor diffuse region, and the TCFA in lipid plaques was \u2264\u200965\u00a0\u03bcm . Neointi\u00b1\u2009SD was used for description, and a comparison between the two groups was performed by t test. For data with skewed distribution, the median was used for description, and the rank-sum test was used for comparison between the two groups. Count data were expressed as rates (%), and the \u03c72 test was used to compare the data between the two groups. Univariate and multivariate logistic regression analyses were performed to determine the independent factors for the presence of OCT-ISNA that suggest an association with the presence of ISNA  were observed in the patients with Lp (a)\u2009\u2265\u200930\u00a0mg/dL compared with those with Lp (a)\u2009<\u200930\u00a0mg/dL. Microvasculature  and neointimal macrophages  appeared more frequently in the patients with high Lp (a) than in those with Lp (a)\u2009<\u200930\u00a0mg/dL  and multivariate  analyses indicated that Lp (a) level was independently associated with the presence of ISNA.Data of 125 lesions were used for logistic regression. As shown in Table\u00a0P\u2009<\u20090.001) for the identification of OCT-ISNA and 0.738  for the identification of OCT-TCFA. According to the ROC analysis results of ISNA and TCFA, the best cut-off of Lp (a) value was 26.6\u00a0mg/dL for OCT-ISNA detection  and 30.5\u00a0mg/dL for OCT-TCFA detection .ROC curves were established and analyzed to evaluate the ability of Lp (a) to identify OCT-ISNA Fig.\u00a0\u2013A and OCIn the qualitative OCT assessment, inter- and intra-observer variabilities  were 0.89/0.89 for ISNA and 0.91/0.91 for TCFA.To investigate the relationship between Lp (a) and in-stent NIH characteristics, this study retrospectively analyzed the relationship between serum Lp (a) level and ISR with DES implantation. The main findings on the relationship between Lp (a) and ISNA were as follows: (1) In patients with ISR, the prevalence of ISNA and TCFA was significantly higher in the high Lp (a) group than in the low Lp (a) group; (2) Lp (a) level was independently associated with ISNA plaque vulnerability. Therefore, we infer that a high Lp (a) level is associated with a high ISNA proportion in ISR and increased plaque vulnerability.2, fiber cap thickness (FCT)\u2009<\u200975\u00a0\u03bcm, lipid core Angle\u2009>\u2009180\u00b0 and macrophage infiltration under OCT were associated with increased risk of coronary events [This paper focuses on the relationship between Lp (a) and plaque vulnerability under OCT. Many previous studies have demonstrated that plaque vulnerability is associated with adverse clinical outcome events. The CLIMA study examined the predictive value of high-risk plaques on OCT for coronary events within 12 months, The results showed that the minimum lumen area (MLA)\u2009<\u20093.5mmy events . The greHigh Lp (a) values are associated with plaque vulnerability and a high risk of major adverse cardiovascular events in patients with de-novo atherosclerosis . Dahlen Recently published PACMAN-AMI  randomized trial has shown that aggressive lipid-lowering leads to degeneration of plaque vulnerability features, including macrophage infiltration . Lp (a) The LDL-like segment of Lp (a) can be phagocytosed by macrophages, which then form foam cells that contribute to the formation and development of atherosclerotic plaques . Lp (a) To date, the pathophysiological mechanism of Lp (a) in promoting ISNA increasing plaque vulnerability has not been proven by relevant basic studies. The current work may provide a new way for the exploration of the pathogenesis and development of ISNA and may contribute to the clinical prevention and treatment of ISNA.This study has some limitations. First, this research was a retrospective study conducted in a single center with small sample size. When screening the patients included in this paper, more patients did not have OCT examination or Lp (a) detection, so the included patients had a certain selection bias. In addition, the stents originally implanted at the ISR target lesions may be first- or second-generation DES, which may affect plaque formation to some extent. Thus, the findings of this article cannot be generalized. Second, on the basis of the suggestion of Chinese experts, we set 30\u00a0mg/dL as the critical Lp (a) value in this study. However, this cut-off is not universal, and different results may be produced in regions and countries other than China. Third, we were unable to distinguish whether prior pharmacological therapy had an effect on the incidence and structural characteristics of ISNA and TCFA in patients with ISR. In particular, the inconsistent use of statins and antiplatelet agents may influence the differences in TCFA and other vulnerable plaque characteristics in targeted vessels between the two patient groups. Fourth, OCT has intrinsic limitations in the qualitative analysis of restenotic tissues, as recently reported by Lutter et al. Its image features are not completely consistent with pathological results , thus crIn patients with ISR, the incidence of OCT-ISNA and OCT-TCFA was significantly associated with their Lp (a) level. Therefore, a high Lp (a) level is an important factor in the increased vulnerability of ISNA plaques.Supplementary Fig.\u00a01. Prevalence of ISNA (A) and TCFA (B).According to Lp (a) quartile level, ISR lesion were divided into four groups: (a) Lp (a)\u2009\u2264\u20098.48\u00a0mg/dL; (b) 8.48\u00a0mg/dL\u2009<\u2009Lp (a)\u2009\u2264\u200923.23\u00a0mg/dL; (c) 23.23\u00a0mg/dL\u2009<\u2009Lp (a)\u2009\u2264\u200946.25\u00a0mg/dL; (d) Lp (a)\u2009>\u200946.25\u00a0mg/dL. OCT-ISNA, in-stent neoatherosclerosis on OCT images; OCT-TCFA, thin-cap fibroatheroma on OCT images.Below is the link to the electronic supplementary material.Supplementary Material 1"}
+{"text": "Low-dye taping (LTD) is widely used by athletes and medical practitioners but the research regarding its impacts on athletic performance is lacking. This study investigated the effects of using low-dye taping on plyometric performance and muscle activities in recreational basketball players with overpronated feet.Twelve collegiate males with at least three years basketball training experience and navicular drop (ND) value \u226510 mm performed the navicular drop, drop jump and countermovement jump tests. Surface electromyography of selected lower limb muscles were observed during bilateral free squat. All tests in non-taped (NT) and taped (TAP) conditions were counterbalanced using repeated crossover study design. Paired t-test with an alpha level of 0.05 and non-clinical magnitude-based decision (MBD) with standardized effects were used to analyze data.Contact time and reactive strength index (RSI) in the TAP condition were significantly shorter (p = 0.041) and higher (p<0.01) than the NT condition respectively. No significant difference in CMJ performance between NT and TAP was observed. MBD demonstrated clear effects on both ND (standardized effect: -1.54\u00b10.24), flight time (standardized effect: 0.24\u00b10.30), contact time (standardized effect: -0.27\u00b10.21), RSI (standardized effect: 0.69\u00b10.35) and eccentric activities of inferior gluteus maximus (standardized effect: 0.23\u00b10.35), gluteus medius (standardized effect: 0.26\u00b10.29) and tibialis anterior (standardized effect: 0.22\u00b10.06).LDT is effective in correcting overpronated feet by increasing ND height. Meanwhile, it provides a small increase in RSI and gluteal muscle activity during the eccentric (down) phase of the bilateral squat, and without affecting CMJ performance. Conditioning coaches or therapists may use LDT to enhance gluteal activation for reducing injury occurrence and reactive strength performance in drop jump tasks. Overpronation of the feet (flat feet) is a common foot malalignment issue where the medial longitudinal arch (MLA) is decreased, leading to increased medial plantar pressure . It is rWhen the MLA collapses and lengthens, it directly impacts the kinetic chain of the lower limbs during weight-bearing activities  \u20134. Such A common method and long-term solution to relieve undesirable stress produced by overpronation are using orthotic insoles to provide additional support to the MLA. However, tight-fitting specialized shoes  and unfit orthotic insoles may cause additional discomfort and plantar pressure . An alteRegarding the biomechanics of functional activities such as walking or running, the tightening of the plantar fascia may provide additional support to the MLA, as explained by the windlass mechanism . HoweverAlthough there are proposed benefits of applying LDT before physical activity, further scientific support is needed for the performance change in team sport athletes such as basketball players, as prior research has predominantly focused on foot stability, joint alignment, stance, and gait \u201324. To tThis study investigated how the application of LDT to correct overpronation effect on drop jump (including RSI) and CMJ performance, and lower extremity muscle activity during a bilateral squat. Participants performed all tests after non-taped (NT) and taped (TAP) conditions as a repeated crossover study design. All tests were completed in one day to maximize reliability . ParticiTwelve collegiate male recreational basketball players  participated in this study. The inclusion criteria included: 1) a minimum of three years of basketball training experience (at least twice per week); 2) navicular drop (ND) \u226510 mm regarded as overpronation and; 3) body fat \u226417.5% for optimum sEMG signal. Participants with recent lower limb injury (within 12 months) or health conditions that could affect their performance or completion of this study, ND value \u22649 mm, and body fat >17.5% were not recruited. All participants completed a PAR-Q and informed consent form, and all experimental risks and benefits were disclosed. All participants passed an allergy test by putting adhesive rigid tape on the left ankle for at least 24 hours to ensure no adverse reactions. Participants were required to wear their competitive basketball shoes. This study was approved by the Human Research Ethics Committee.Warm-up. Before each performance test, a standardized dynamic warm-up protocol was used to increase body temperature and readiness of participants Reviewers' comments:Reviewer's Responses to Questions <font color=\"black\"> Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions? </font> The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1:\u00a0YesReviewer #2:\u00a0Partly********** <font color=\"black\"> 2. Has the statistical analysis been performed appropriately and rigorously?  </font> Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified. </font> The Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 4. Is the manuscript presented in an intelligible fashion and written in standard English? </font> PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes********** <font color=\"black\"> 5. Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0The manuscript meets the publication criteria established by the journal. However, there are some issues that concern me.TITLEI would recommend using a more concise and impactful title. Now it's a bit repetitive and longABSTRACTThe objective of the study is not clearly defined in the abstract. \u201cEffects\u201d of DLT on what?LDT abbreviations are not predefined in the abstractMATERIAL AND METHODSWas there a prior familiarization session for the tests?Is there any information on the comfort level of individuals after taping placement? You should Indicate this like a limitation of the study, since the level of comfort could influence the performance achieved.To determine the possible influence of fatigue, it would be interesting to report about the time that the individuals were performing the tests. 1, 2 hours or how many hours? What rest time was there between the treatment conditions (NT test and TAP)?Line 287: superscript what does it refer to? Check and correctRESULTSTable 1. Indicate that the flight time refers to the drop jump, and not to the CMJ. It could confuse the reader.DISCUSSIONGeneral speaking, the discussion regarding the effects on performance in vertical jumps is too brief. It is necessary to provide more information about this topic. Why does DJ performance improve and not the CMJ, in which there is also a concentric phase and an eccentric phase of the movement? Could being overpronated, neutral or supinator influence the results? What has been observed in other studies? The following articles will help you in this task.Struzik A, Stawarz M, Zawadzki J. The effect of low-Dye taping on hopping performance in handball players. Acta Bioeng Biomech. 2020;22(3):3-8. PMID: 33518734.Radford JA, Burns J, Buchbinder R, Landorf KB, Cook C. The effect of low-Dye taping on kinematic, kinetic, and electromyographic variables: a systematic review. J Orthop Sports Phys Ther. 2006 Apr;36(4):232-41. doi: 10.2519/jospt.2006.36.4.232. PMID: 16676873Line 390, 398 If the journal instructions do not prevent it, it is preferable to write the citation number immediately after the authors Line 391-393. It is speculative. Provides scientific reference and justificationReviewer #2:\u00a0Thank you for the opportunity to review this paper. I think that you should better describe the potential practical applicability of your study. Specific remarks are given below.INTRODUCTION: consider shorteningMETHODS: Subjects Exclusion and inclusion criteria must be better described (more information is needed). What were the criteria for overpronation? ND value? If so, please state that in the text and report clear cuttoff for inclusion, so that readers can understand who were the participants in this study.Why you didn\u2019t use contact mat for CMJ as well? And additional don\u2019t you think that force plate would be best choice for both tests? Please elaborate that as potential limitation.Statistics: state clearly which effect size was usedRESULTS: Table 1 and 2 are nice, clear. However, you are introducing MBI without any explanation what that is. Please correct.DISCUSSION.To shorten your findings you have only managed to show that LDT does change ND values in basketball players with overpronation without almost any effect on performance. In light of such findings you should go through your practical application once again to see if you data really support your claims that \u201cOur findings offer conditioning coaches and therapists alternate techniques to reduce lower limb injuries and enhance plyometric and RSI performance. Furthermore, it provides a method for increasing muscle activation of the gluteal group during squat-related exercises without decreasing the CMJ performance.\u201dhttps://pubmed.ncbi.nlm.nih.gov/33518734/ that also showed no effect of LDT on mean jump height, mean ground contact time and mean vertical stiffness.Also there is a study ********** <font color=\"black\"> 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose \u201cno\u201d, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. </font> Reviewer #1:\u00a0NoReviewer #2:\u00a0Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at\u00a0figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,\u00a0 31 Aug 2022Reviewer 1Reviewer: I would recommend using a more concise and impactful title. Now it's a bit repetitive and longResponse: Thank you for the comments and suggestions. Yes we have struggled a bit on deciding the title and were greedy to try including everything inside. Now the title is revised to more precise and appealing as \u201cLow-dye taping may enhance physical performance and muscle activation in basketball players with overpronated feet\u201d (line 1-2)Reviewer: The objective of the study is not clearly defined in the abstract. \u201cEffects\u201d of DLT on what? LDT abbreviations are not predefined in the abstractResponse: Thank you for your suggestions. We have added (LTD) in line 40 and also indicate clearly the effects in line 42-43 as \u201cThis study investigated the effects of using low-dye taping on plyometric performance and muscle activities in recreational basketball players with overpronated feet.\u201dReviewer: Was there a prior familiarization session for the tests?Response: Thank you for your question. We did not have additional familiarization session for subjects adapting the use of low-dye taping. But we have given practice trials for the drop jump test (line 216), CMJ (line 235), squat (line 247).Reviewer: Is there any information on the comfort level of individuals after taping placement? You should Indicate this like a limitation of the study, since the level of comfort could influence the performance achieved.Response: Thank you for pointing out this. We didn\u2019t measure the comfort level. Therefore we have added such limitation in line 463-465.Reviewer: To determine the possible influence of fatigue, it would be interesting to report about the time that the individuals were performing the tests. 1, 2 hours or how many hours? What rest time was there between the treatment conditions (NT test and TAP)?Response: Thank you for pointing this out and your reminder. We have indicated back the resting period (30 min) between conditions in line 152. Yes, we agree there can be carryover fatigue from the testing activities in the former conditions brought to the latter one. Since only a total of 5 drop jump  and 5 CMJ , and another 5 trials of free squat (2 practice + 3 repetitions) without failure were performed, whereas we have given resting period between tests and trials in jumping tasks and MVC tests, we assume no, or very minimum fatigue will be induced and cumulated from the first conditions to the latter one. To tackle this we performed counterbalance order for NT and TAP conditions to offset such potential bias.Reviewer: Line 287: superscript what does it refer to? Check and correct Response: Thank you for pointing this out. This should be the citation but we used the inconsistent format before. Now rectified as shown in line 279Reviewer: Table 1. Indicate that the flight time refers to the drop jump, and not to the CMJ. It could confuse the reader.Response: Thank you for pointing this out. We have indicated \u201cthe drop jump\u201d in table 1 for better clarity.Reviewer: Line 390, 398 If the journal instructions do not prevent it, it is preferable to write the citation number immediately after the authors Response: Thank you for your reminder. We have amended per your suggestions as shown in line 382 and 394 now.Reviewer: General speaking, the discussion regarding the effects on performance in vertical jumps is too brief. It is necessary to provide more information about this topic. Why does DJ performance improve and not the CMJ, in which there is also a concentric phase and an eccentric phase of the movement? Could being overpronated, neutral or supinator influence the results? Whathas been observed in other studies? The following articles will help you in this task.Struzik A, Stawarz M, Zawadzki J. The effect of low-Dye taping on hopping performance in handball players. Acta Bioeng Biomech. 2020;22(3):3-8. PMID: 33518734.Radford JA, Burns J, Buchbinder R, Landorf KB, Cook C. The effect of low-Dye taping on kinematic, kinetic, and electromyographic variables: a systematic review. J Orthop Sports Phys Ther. 2006 Apr;36(4):232-41. doi:10.2519/jospt.2006.36.4.232. PMID: 16676873Response: Thank you raising this out, your resources and suggestions. We have extended the paragraph regarding CMJ performance in line 412-427 using Struzik et al., 2020 and another paper by Ho, Kong, Chong & Lam (2019). We have also extended the content for drop jump part in line 428 to 446 while why DJ and CMJ yieided different results was explained from line 446-451.Line 391-393. It is speculative. Provides scientific reference and justificationResponse: Thank you raising this out. We have amended this part and also give additional justification with reference 38 and 39 to support such speculation (not belief). Line 383-388.Reviewer 2Reviewer: consider shorteningResponse: Thank you for the suggestion. We have reviewed this part again and simplified/cut some sentences without affecting the meaning. The number of lines were reduced from a total of 65 lines to now 59 lines.Reviewer: METHODS: Subjects Exclusion and inclusion criteria must be better described (more information is needed). What were the criteria for overpronation? ND value? If so, please state that in the text and report clear cuttoff for inclusion, so that readers can understand who were the participants in this study.Response: Thank you for point out this. We have revised this section to state clearly about the exclusion and inclusion criteria including the clear cut-off point line (157-162).Reviewer: Why you didn\u2019t use contact mat for CMJ as well? And additional don\u2019t you think that force plate would be best choice for both tests? Please elaborate that as potential limitation.Response: Thank you for pointing out this. We agreed that force-plate is widely used and also kind of accepted as one of the gold-standards in jump measurement. However, recent studies shown variable jump heights using different devices. The contact mat is reliable but can underestimate high performer . Meanwhile a very recent paper by Concei\u00e7\u00e3o et al., 2022 (DOI: 10.3390/app12010511) shown high jump height error/inconsistency when compared with 3D measured displacement in all devices. Our study aims to measuring the true jump height value instead of an estimated value and therefore, we use a relatively novel method. But we agree that we should add potential limitation in the discussion section and we have added this in line 455-457.Reviewer: Statistics: state clearly which effect size was usedResponse: Thank you for point out this. It is Cohen\u2019s d and now added this back to line 299.Reviewer: RESULTS: Table 1 and 2 are nice, clear. However, you are introducing MBI without any explanation what that is. Please correct.Response: Thank you for your suggestion. We just found that Will Hopkins recently changed the name from magnitude-based inference to magnitude-based decision and we have also renamed all of them accordingly. Meanwhile we highlight the full form and abbreviation first in line 296 such that readers will not be confused in the result section.Reviewer: To shorten your findings you have only managed to show that LDT does change ND values in basketball players with overpronation without almost any effect on performance. In light of such findings you should go through your practical application once again to see if you data really support your claims that \u201cOur findings offer conditioning coaches and therapists alternate techniques to reduce lower limb injuries and enhance plyometric and RSI performance. Furthermore, it provides a method for increasing muscle activation of the gluteal group during squat-related exercises without decreasing the CMJ performance.\u201dResponse: We have revised the practical applications to make all the claims more accurate and in line to our data/findings (Line 468-473)https://pubmed.ncbi.nlm.nih.gov/33518734/ that also showed no effect of LDT on mean jump height, mean ground contact time and mean vertical stiffness.Reviewer: Also there is a study Response: Thank you for giving us the recent similar study that we haven\u2019t covered. Since in their study, they didn\u2019t specifically recruit subjects with overpronated feet and also didn\u2019t measure any change on the navicular alignment to check the change of medial arch, we have highlighted such differences between their and our studies in our discussion (line 434-441).  .AttachmentResponse to Reviewers1.docxSubmitted filename: Click here for additional data file. 12 Sep 2022Low-dye taping may enhance physical performance and muscle activation in basketball players with overpronated feetPONE-D-22-03490R1Dear Dr. Luk,We\u2019re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you\u2019ll receive an e-mail detailing the required amendments. When these have been addressed, you\u2019ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they\u2019ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Emiliano C\u00e8Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 2 Oct 2022PONE-D-22-03490R1 Low-dye taping may enhance physical performance and muscle activation in basketball players with overpronated feet Dear Dr. Luk:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofProfessor Emiliano C\u00e8 Academic EditorPLOS ONE"}
+{"text": "The fractionation of diatomaceous earth (DE) using sedimentation made it possible to obtain separate unbroken diatom fractions from broken or agglomerated bodies with a range of particle sizes. The produced filler was used to prepare polylactide (PLA)/diatomaceous earth biocomposite samples containing different particle sizes, which were subjected to mechanical testing , colloidal testing , and thermal testing . Modification of the PLA containing the smallest particle size with diatomaceous earth (Fraction 5) resulted in a higher impact strength compared to both the pure PLA and the PLA/DE composite that contained base diatomaceous earth. Furthermore, the melt flow rate was improved by more than 80 and 60% for the composite modified with fractionated diatomaceous earth (Fraction 4) compared to pure PLA and base diatomaceous earth, respectively. The elasticity of the composite was also improved from 3.3 GPa for pure polylactide to 4.4 GPa for the system containing the smallest diatomaceous earth particles (Fraction 5). For many years, an intense increase in the global production of plastic has been observed. This is due, among other factors, to the easy access to their raw materials, improved living standards, and thus a higher consumption. Domka  identifiPreserving and enhancing natural capital by the control of limited resources and the balancing of renewable resource flows;Optimizing raw material use by keeping products, components, and materials in circulation with their highest utility in both cycles;Developing system efficiency by identifying and removing negative externalities.Degradation of the natural environment, development of climate policies, new pro-environmental regulations, and increasing consumer awareness/expectations are changing how business is conducted. A circular economy linked to biological and technical cycles is guided by the following three main principles: The developed concept of the circular economy is, therefore, an approach in which products and materials remain in circulation as long as possible, resulting in reduced waste at landfills. In response, the search for new materials to replace conventional thermoplastics has begun. A ubiquitous thermoplastic, with many applications, that requires a reduction in its environmental impact is PLA. The latter is a biodegradable polymer derived from renewable resources with very attractive properties  ,4. The mIn this study, a naturally occurring filler  is used. With this in mind, our paper acts as a response to the search for suitable alternatives to conventional polymer composites. Diatomaceous earth is chosen because of its unique properties and wide availability. Diatomite is characterized by its porous structure, developed specific surface area, and high silica content ,18. MoreIn previous papers ,24, we fIn this study, diatomaceous earth is fractionated using a sedimentation process and the fractions that result are then used to modify the polylactide and create composites. Composites that contain four different diatomaceous earth particle sizes  and four different modifier concentrations  are obtained. Next, particle size tests (Dynamic Light Scattering\u2014DLS) are conducted and SEM images of the sediments after fractionation are produced. In addition, mechanical, rheological, surface (contact angle), and ageing tests are performed on the composites.Polylactide (PLA)-type Ingeo 4043D was purchased from NatureWorks . Diatomaceous earth  was derived from diatomite deposits. Synthetic wax WTH-B microbeads were bought from WTH GmbH .Amorphous diatomaceous earth  (28 kg) was placed into Barrel 3 that can hold 180 L, which was then filled with demineralized water to approximately 100 L. The suspension was then mechanically stirred for 3 min (at a speed of 2000 rpm) and left to undergo sedimentation. After 2 h, the supernatant liquid was pumped into Barrel 4 by using a water pump and again left to undergo sedimentation; Barrel 3 was refilled with demineralized water to about 120 L. After 24 h, the supernatant liquid from Barrel 4 was transferred to Barrel 5. The suspension in Barrel 3 was mechanically stirred and, after 2 h, the supernatant liquid was pumped into Barrel 4. The sediment remaining in Barrel 3 was put into Barrel 2, and Barrel 2 was then filled with demineralized water of volume 140 L. As shown in The following diatomaceous earth fractions were selected for further testing: base, Fraction 2, Fraction 4, and Fraction 5. The detailed study of these fractions is given in The ZAMAK MERCATOR WG 150/280 laboratory mill  was used to homogenize polylactide (PLA 4043D) with base and fractionated diatomaceous earth. For this purpose, 500 g of PLA was plasticized at 210 \u00b0C for 15 min with diatomaceous earth added in portions so that the concentrations of 5, 10, 20, and 30% and 1% of WTH-B Microbeads synthetic wax were obtained. Wax-free systems were also prepared for comparison purposes. The systems were then ground using the WANNER C17.26 sv mill  and dried for 24 h at 60 \u00b0C. The prepared masterbatches were diluted 1:1 with PLA using the Engel e-victory 170/80  injection molding machine. g) was determined as the midpoint of the glass transition temperature range. The cold crystallization temperature (Tc) and melting temperature I were taken as the peak temperatures of the cold crystallization and melting, respectively. The measurements for the hiding power were performed by placing samples of the prepared resin systems into the optical path between the light source (LED) and the UV-NIR spectrophotometer, a AvaSpec-Mini2048CL . Dynamic mechanical analysis (DMA) was performed using a Q800 DMA  using a dual cantilever mode that accords to ASTM D4065-01. From each composite, three rectangular specimens, which were 60 mm long and 10 mm wide, were cut and used in the testing. The analysis was conducted from 0 \u00b0C to 130 \u00b0C, with a heating rate of 3 \u00b0C/min at a frequency of 1 Hz and amplitude of 30 \u00b5m. The glass transition temperature (Tg) was determined from the peak value in the storage modulus, the loss modulus, and the damping factor curves.For flexural and tensile strength tests, the materials obtained were printed into type-1B dumbbell specimens, in accordance with EN ISO 527:2012 and EN ISO 178:2006. The tests of the obtained specimens were performed on the INSTRON 5969 universal testing machine  with a maximum load force of 50 kN. The traverse speed for the tensile strength and the flexural strength measurements were set at 2 mm/min. The Charpy impact test (with no notch) was performed on the Instron Ceast 9050 impact-machine  and accorded with ISO 179-1 . The powder morphology was characterized by a scanning electron microscope  operating at an applied voltage of 5 kV. Before observations via the SEM, the samples surfaces were sputtered with a gold-palladium layer for 90 s at an electric current of 10 mA and voltage of 2 kV. The size of the diatoms used to prepare the composites was measured with a Mastersizer 3000 . The measurements were taken for samples in water suspension (Hydro EV attachment). The parameters of the measurements for the wet samples were a stirrer revolution speed of 2330 RPM and an ultrasound power of 70%. Contact angle analyses were performed by use of the sessile drop technique (5 \u03bcL) at room temperature and atmospheric pressure, with the Kr\u00fcss DSA100 goniometer . Ageing tests were performed in the ESPEC ARS-0220 environmental stress chamber using 10 heating and cooling cycles of \u221210 \u00b0C + 50 \u00b0C at an 85% humidity. The melt flow rate (MFR) was measured using the Instron CEAST MF20 melt flow tester , which accords to EN ISO 1133 at 210 \u00b0C for a load of 2.16 kg. The thermal properties of the materials were studied using a Q1000 Differential Scanning Calorimeter . Samples with a weight of 8.0 + 0.2 mg were placed into an aluminum hermetic pan. First, the samples were equilibrated at \u221290 \u00b0C, then heated to 280 \u00b0C with a scan rate of 10 \u00b0C/min, and cooled to \u221290 \u00b0C with a scan rate of 10 \u00b0C/min. Finally, they were again heated to 280 \u00b0C with a scan rate of 10 \u00b0C/min. The process was conducted in a nitrogen atmosphere. Using Universal V4.5A TA software , the glass transition temperature , which is due to the low adhesion of the polymer to the filler particles. Compared to the tensile tests, the synthetic wax does not cause a significant deterioration in the mechanical parameters relating to flexure. However, the particle size of the filler has a significant effect on the flexural strength of the systems. Fraction 2, which has the highest average particle diameter, causes the greatest decrease in the tensile strength parameters for the highest filling. In each case, the flexural modulus is higher for the filled samples compared to the base PLA , with an increasing trend with more filling. Moreover, the fractions with the largest and smallest particles show the highest values of the flexural modulus, especially for the highest concentration (15%). The systems that are modified with the base diatomaceous earth, and Fraction 4, show a similar tendency.The addition of diatomaceous earth to the polylactide, irrespective of the fraction, results in an initial increase and then a decrease in the impact strength at most concentrations . The optDiatomaceous earth is also shown to affect the brittleness of the material: the higher its addition, the lower the impact strength values. This correlation is the most and least visible for Fraction 5  and Fraction 4 , respectively. In the latter case, the addition of 5% or more does not cause any significant changes, so the impact strength remains at a similar, constant level.To assess the influence of atmospheric conditions, i.e., the effects of the differences in humidity and temperature on the mechanical and visual properties , the samConditioning the composites in a climatic chamber for 5 days results in a slight increase in the elongation at the break point. However, further exposure of the samples to harsher climatic conditions (for 20 days) cause clear changes in the structure, which manifest in terms of an increased flexibility and, thus, an increased elongation at the break point. The smallest changes are observed, each time, for the highest proportion of the filler, which relates to the lowest light transmission (which is the factor responsible for the photodegradation) within the samples. Photodegradation, together with the hydrolysis, results in a reduction in the average molecular weight of the polymer, which translates into higher sample tensility. The lack of any significant differences between the samples with and without wax is due to a low proportion of photodegradation for samples with a high proportion of the filler. This also indicates that, at higher filling levels, the tensile strength of the samples is so low that their degradation has little to no further effect on this parameter.Conditioning of the samples in a climate chamber also affects their flexural strength . The fleFor each system, the maximum contact angle is observed for a given proportion of the filler in the composite. This relates to the balance between the increasing proportion of porous filler and an intensifying micro-roughness of the sample, and the impact of the polar character of the filler surface on the polarity of the composite surface. However, the polar effect is observed to a lesser extent on the presence of wax in the composites. As the size of particle diatoms in the systems increases, the hydrophobicity also increases. The lowest values are observed for the systems that are modified with Fraction 5 (69.3\u00b0 on average), while for Fraction 4 and Fraction 5, the results are comparable: 86.7 and 84.8\u00b0 . SystemsThe exposure of the composites to increased humidity, and variable temperature conditions (within the climatic chamber), causes changes in their properties. The system with the smallest diatomaceous earth particles has a higher contact angle for the whole series, regardless of the modifier concentration. Similarly, for systems that are not modified with synthetic wax, the 15% diatom sample for Fraction 2 achieves the highest contact angle result  of 110.05\u00b0. Holding the systems in the climate chamber for a longer period of time results in decreased values for the contact angle for most systems, which can be explained by the degradation of the polylactide; the latter is evidenced by the decrease in the contact angle for pure PLA after 20 days from 83.4\u00b0 to 70.6\u00b0. The initial increase in the contact angle is related to the development of a microporous structure on the polymer surface . FurtherThe electron microscope is also used to provide images of the fractures that are formed after the impact testing of the composites . The comHiding power tests, based on the visible light transmittance of the samples, show the impact of both the diatomaceous earth content and the particle size of the individual fractions on the pigmentation efficiency of the composites. The higher the DE content, irrespective of the modifier type, the higher the hiding power. Once again, the highest results are obtained for fractions that contain the diatomaceous earth with the smallest particle size, i.e., Fraction 5. This occurs due to the excellent dispersion characteristics of the particles with small sizes in the polymer matrix, as confirmed by the SEM images . The comg of neat PLA is less pronounced and observed in the 60\u201363 \u00b0C range. The application of the fractionated diatomite leads to a slight change in the dynamic mechanical properties of the composites. Most importantly, the Tg transition is significantly more distinctive. Moreover, it can be observed that the addition of a different fraction of DE enables an increase in the glass transition temperature (Tg), which is determined from the storage modulus (E\u2019), loss modulus (E\u201d), and the damping factor (tan\u03b4). A sharp decrease in the storage modulus, the loss modulus (E\u201d), and the damping factor (tan\u03b4) of the composites with diatomaceous earth is related to the glass transition of PLA. The glass transition temperature (Tg) increases slightly when the filler particle size decreases. This shows that the presence of a different volume fraction of DE restricts the segmental motion of the polymer chains.Based on the DMTA curves at normal scale, cold crystallization and subsequent melting are observed after the glass transition temperature. In the process of cold crystallization, a new fraction of crystallites is formed due to improved mobility and ordering ability of the polymer chains in the amorphous phase, which consequently results in an increase in modulus, and a significant peak in the tan delta curve is observed, with an onset at ~90 \u00b0C for PLA composites. This phenomenon was discussed in the review by Cristea et al. . As the g) near 55 \u00b0C, (b) a cold crystallization peak (Tcc), and (c) an endothermic melting peak (Tm).A DSC analysis is performed to investigate the thermal behavior of the PLA and PLA/diatoms composites. 1 and Tm2). For the 15BSW and 15Fr2SW materials, there is only one melting peak, and hence, its Tm1 equals Tm2. The values of the glass transition temperature, cold crystallization temperature, specific cold crystallization enthalpy, melting temperature, and the specific melting enthalpy are listed in With regard to cold crystallization, m) is around 150 \u00b0C. While the PLA/GF composites reveal a double melting peak with two melting temperatures (Tm1 and Tm2), the double melting peak could be related to the recrystallization of the generated crystals. For the 15Fr4SW and 15Fr5SW composites that crystallize around 110 \u00b0C, the crystals typically show a less-than-perfect structure and they are much thinner than the crystals generated at the higher temperatures during cold crystallization. However, these thinner crystals can melt and recrystallize to generate a closer to perfect and thicker structure. Due to a temperature increase, melting again occurs and the second melting peak (Tm2) is achieved [With a further increase in the temperature, the crystals that are generated during cold crystallization begin to melt. As a result, a melting peak appears in the DSC thermograms. It can be observed that the unmodified PLA and the 15Fr2SW composite show a single melting peak, and its melting temperature  increases, which varies depending on the diatomaceous earth fraction. It was also determined that the MFR of the unfractionated diatoms has significantly lower values compared to the others. When measuring the tensile strength of the composites, the optimum amount of additive is 5% diatomaceous earth, which produces the highest obtained results; this is regardless of particle size. The highest impact strength is obtained for the composites that are modified with diatomaceous earth that contain the smallest particle size (Fraction 5). The highest hiding degree is achieved for composites that have diatomaceous earth with the smallest particles (Fraction 5). With an increase in the modifier concentration within the system, the hiding degree also increases. The thermal analysis revealed that the particle size of the diatomaceous earth, and its tendency to form agglomerates, both have an effect on the blocking the PLA chain. The addition of synthetic wax provides the PLA composites with diatoms and the resulting anti-ageing properties, which has a positive effect on the mechanical properties after an acceleration of the ageing."}
+{"text": "Lagenaria siceraria (Mol. Standl) genotypes, representing three different geographical origins: South Africa , Asia (Philippines and South Korea), and Chile . The RSA changes were evaluated at four substrate depths (from 0 to 40 cm). Bottle gourd genotypes were grown in 20 L capacity pots under two contrasting levels of irrigation (well-watered and water-deficit conditions). The results showed that the water productivity (WP) had a significant effect on plasticity values, with the Chilean accessions having the highest values. Furthermore, Illapel and Chepica genotypes presented the highest WP, MRD, and RV values under water-deficit conditions, in which MRD and RV were significant in the deeper layers (20\u201330 and 30\u201340 cm). Biplot analysis showed that the Illapel and Chepica genotypes presented a high WP, MRD, and RV, which confirmed that these may be promising drought-tolerant genotypes. Consequently, increased root diameter and volume in bottle gourd may constitute a response to a water deficit. The RSA traits studied here can be used as selection criteria in bottle gourd breeding programs under water-deficit conditions.In many agricultural areas, crop production has decreased due to a lack of water availability, which is having a negative impact on sustainability and putting food security at risk. In plants, the plasticity of the root system architecture (RSA) is considered to be a key trait driving the modification of the growth and structure of roots in response to water deficits. The purpose of this study was to examine the plasticity of the RSA traits  associated with drought tolerance in eight  Drought is a compelling ecological issue that significantly damages plant development and growth ] was one of the earliest plant species domesticated for human utilization  Heiser] and American/African bottle gourd (L. siceraria. subsp. siceraria) , were selected to determine changes in the root system at different substrate depths under drought and well-watered conditions. This information will be useful for the development of bottle gourd varieties that can tolerate drought stress conditions by developing optimal root systems.A recent study conducted with bottle gourd reported that drought-tolerant genotypes showed a reduced length and density of lateral roots, constituting a response to a water deficit  of South Africa, two accessions were from the Genetic Resource Center of Japan, specifically from the National Agriculture and Food Research Organization (NARO), and three were collected from three different regions of Chile. Details about the bottle gourd accessions are shown in The seeds of the bottle gourd accessions were sterilized by immersion in 2% (v/v) sodium hypochlorite in water for 10 min, rinsed 2 times with deionized water for 10 min, and germinated for 5\u20137 days at 20\u201325\u00b0C in 7 \u00d7 7 \u00d7 8 cm (0.23 L) pots with peat and sand substrate in an equal ratio of 1:1. The plants with the first fully expanded true leaf and with an absence of damage or disease were considered as criteria for transplantation to pots.2. The experiment was established in a randomized complete block design (RCBD) with an 8 \u00d7 2 factorial arrangement and three replicates by block. Factors consisted of eight bottle gourd accessions and two water regimes (well-watered and water-deficit regimes) totaling 16 treatments.The experiment was conducted in February 2021 in glasshouse conditions using a shade net cover (Raschel sun-shading net with 50% light transmittance), the average air temperature was 23.8 \u00b1 2.7\u00b0C with relative humidity of 54% and a solar radiation level of 27 Mj/m3). Prior to transplant, each pot was saturated with water, allowed to drain, and covered with plastic bags to avoid evaporation for 24 h according to Opazo et al. (\u00ae Mini 3M 16-8-12(+2) N-P2O5-K2O(+MgO+S) at the initial stage of the experiment, after which no fertilizer was applied during the entire experiment to avoid confusion about the applied stress. The experiment ended when the weight of the pots under WD conditions had no variations and the plants had died.Each pot with 20 L of capacity  was filled with 30 kg of the substrate (1:1 peat/sand v/v)  was estimated from the water-supplied data. To avoid evaporation, the surface of the pot was covered with black bags. For that, three plants per genotype and water treatment were harvested at the end of the experiment. Root and aerial (leaves and shoots) tissue dry matter weights  were measured using the leaves, shoots, and roots of each plant, which were separated and dried in an oven at 60\u00b0C to obtain the dry weights. The root:shoot ratio was determined by dividing the RDW and SDW for each plant. The plant water consumed over the 4-week period was estimated from the sum of the daily water consumption (WU) minus the water drained out of the pots and plant biomass gain. For this, the water drained was estimated through control pots, which included holes for water drainage (without plants), while the plant biomass gain (PBG) was estimated at the end of the experiment and corresponds to PBG = [final fresh weight\u2014initial fresh weight]/[number of days in the experiment]. The WU was determined as follows:i corresponds to each of the n measurements (being i-1 the past measurement), PW and CPW represent the weight of the pots and control pot, respectively. Finally, the WP was determined as follows:where The whole root system of each of the bottle gourd plants was analyzed. The root length (RL), root surface area (SA), mean root diameter (MRD), and root volume (RV) were determined using WinRhizo 2019a software . The roots were cleaned by washing them over a sieve. Each sample was scanned with a flatbed scanner . The roots were partitioned into nine diameter classes in 0.5 mm steps , and the root lengths for each root diameter class were computed. The root images were analyzed at four different substrate depths .The phenotypic plasticity for a trait is related to the difference in this trait between two individuals of the same genotype under different water conditions  was performed after testing the homogeneity of variances and the normality of the residuals using Bartlett and Shapiro\u2013Wilk tests, respectively. A two-way ANOVA was performed for RSA traits, WU and WP traits, while a one-way ANOVA was used for RDPI traits . Fisher's least significant difference (LSD) test and orthogonal contrasts were performed on multiple comparisons of the mean values of the genotypes. Statistical analyses were carried out using R 4.0.5 software  were used to compute the Pearson's linear correlation coefficients using the \u201cchart. Correlation\u201d function of the PerformanceAnalytics package  based on the correlation matrix was performed using the \u201cprincomp\u201d function in R. The PCA-biplot was then generated using the \u201cggbiplot\u201d function of ggbiplot2 package Vu,  in R to p < 0.01) in the RDW among two South African bottle gourd genotypes (BG-78 and GC) and the Osorno, Philippines, and South Korea genotypes in the drought treatment, which was not observed in WW (The root dry weight (RDW) of the plants in the drought treatment ranged from 1.2 (Osorno) to 2.6 g (GC); while for the well-watered (WW) plants, the dry weight varied from 2.0 (Philippines) to 4.6 g (GC). The leaves and shoots dry matter weight (SDW) ranged from 14.4 (Philippines) to 22.4 g (Chepica) for plants under water-deficit (WD) conditions and 68.8 (Philippines) to 119.7 g (GC) for WW . There wed in WW . This ined in WW .Analysis of variance showed a significant effect of the water regime in all traits except for WP . MoreoveIn most of the tested bottle gourd genotypes, the contrasting means in the comparison of the WW and WD water regimes  showed tp < 0.01) correlation with SDW (0.78). Similarly, WU was significantly and positively associated with SDW (0.94) and RL (0.73). Furthermore, RDW showed strong and significant positive correlations with RL (0.88), SA (0.95), and RV (0.67). In particular, WP had a negative correlation with MRD (\u22120.70) . Furthermore, RDW showed a positive correlation with RL (0.80) and SA (0.63)  . These rA (0.63) . These rUnder the WW condition, significant genotypic differences in RL values were detected for most substrate layers (except 0\u201310 cm), while differences in SA and RV only were observed in the deepest layer (30\u201340 cm) . In thisThe RL, SA, and RV root traits were classified into different classes based on root diameter intervals, i.e., 0\u20130.5, 0.5\u20131.0, 1.0\u20131.5, 1.5\u20132.0, 2.0\u20132.5, 2.5\u20133.0, 3.0\u20133.5, 3.5\u20134.0, and 4.0\u20134.5 mm. This was performed to compare the fine root distribution across different diameter intervals under different depths for both water regimes.The highest RL values in the four depths and both water conditions were recorded in the 0.5\u20131.0 mm diameter interval, then the values decrease according to the increase in diameter intervals. Moreover, in the 0.5\u201310 mm interval, significant genotypic differences were detected for the second depth layer (10\u201320 cm) under the WD condition, with the GC and Illapel genotypes having the highest and lowest values, respectively. Similarly, for SA, the highest values were observed in the 1.0\u20131.5 mm interval, and then, as the diameter of the intervals increased, the SA values decreased. It should be noted that under the WD regime, the fourth depth layer (30\u201340 cm) showed stable SA values after a diameter interval of 0.5\u20131.0 mm. Moreover, the RV values increased according to the increase in root diameter intervals independent of water regime and depth layer .A principal component analysis was carried out to discover the most contributing root traits under two contrasting water regimes and for the differentiation of drought-tolerant and sensitive bottle gourd genotypes. Under the WW condition, the two first principal components (PCs) explained 96% of the cumulative variance observed, with 56% at the first and 40% at the second axes, with eigenvalues of 1.67 and 1.40, respectively . The parPlasticity responses to a water deficit were compared for WP, root traits, and biomass production by means of the RDPI. In this sense, the Chilean genotypes had significantly higher plasticity in WP , with OsL. siceraria genotypes and investigated the various root traits at different substrate depths and root diameter intervals under a water-deficit condition. For instance, different root-related traits, such as primary root length, number and length of lateral roots, and average root diameter, have been proposed as important traits that contribute to regulating water uptake, which is critical under drought stress  and very fine roots ( \u22640.5 mm diameter) in the second (10\u201320 cm) and third (20\u201330 cm) layers, while the Illapel and Chepica genotypes presented the highest root length in the thicker roots (>2 mm diameter) in the last layer (30\u201340 cm) . It has \u22121 (and preferably <1 gL\u22121) if want to minimize growth restrictions for harvest. In this context, this study reported ratios of biomass/root volume between 1.06 and 2.58 gL\u22121 for each genotype treatment, which is in agreement with the proposed by Poorter et al. . In the present study, under water-deficit conditions, the Chilean and South African bottle gourds showed different strategies for tolerating drought stress. In particular, water-deficit conditions may lead to an increase in the diameter and volume of roots, which, in turn, may contribute to improving water capture under these conditions.Understanding the associations among the relevant root traits associated with drought adaptation/tolerance is important for improving the selection of superior genotypes for drought tolerance and the breeding of bottle gourd. In this context, PCA is a powerful statistical procedure that reduces the dimensions of the variables and, in our case, helps to discriminate highly correlated traits in order to identify clusters of individuals in response to drought stress  that focus on water capture and nutrient uptake, and which are suited for sustainable production in cucurbit crops  and reduced the MRD and RV. Thus, both groups of bottle gourd used different strategies associated with root trait modifications in response to a water deficit. Notably, the genotypic variation observed for these root traits at different substrate depths, especially under water-deficit conditions, was also associated with drought tolerance in The original contributions presented in the study are included in the article/RC-S, DZ, CM, and FM-P conceived the research plans. CM, FM-P, and RC-S analyzed the phenomic data, wrote the first draft of the manuscript, and reviewed and edited the final version of the manuscript. RC-S, DZ, CM, and LD supervised the field experiments. RC-S and CM performed the data curation. All authors reviewed and approved the paper for publication.This work was supported by FONDECYT (Grant Number 11180278).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "The tumor necrosis factor (TNF) receptor superfamily is a structurally and functionally related group of cell surface receptors that play crucial roles in various cellular processes, including apoptosis, cell survival, and immune regulation. This review paper synthesizes key findings from recent studies, highlighting the importance of clustering in TNF receptor superfamily signaling. We discuss the underlying molecular mechanisms of signaling, the functional consequences of receptor clustering, and potential therapeutic implications of targeting surface structures of receptor complexes. The TNF receptor superfamily (TNFRSF) comprises a diverse group of cell surface receptors involved in regulating immune responses, inflammation, and cell survival . DysreguMembers of the TNFRSF are type I, single pass membrane proteins with their C-terminal end anchored in the membrane. Their elongated ectodomains contain 1-6 cysteine rich domains (CRDs). TNF receptors can be grouped into three distinct groups see . TNFSF lOne potential issue with this model is that the number and size of the extracellular CRD domains of TNFRSFs vary and not all members have been shown to be able to form interactions via their PLAD. This raises the question whether the proposed model can hold for TNFRSF receptors that are unable to form stable dimers on their own. The answer has been provided by the structure of herpesvirus entry mediator  in complex with the regulatory protein B- and T-lymphocyte attenuator (BTLA) (BTLA is the member of the Immunoglobulin (Ig) superfamily (IgSF) that comprises of proteins that play crucial roles in the immune system and other biological processes. These proteins are characterized by the presence of one or more Ig domains, which are structurally conserved regions that contain about 70-110 amino acids arranged in a sandwich-like structure . BTLA fucis configuration BTLA does not interfere with LIGHT or LT-\u03b1 binding, instead it serves to facilitate HVEM oligomerization on the cell surface and to inhibit the ligand independent activation of HVEM. BTLA rather than being a true ligand of HVEM as previously proposed, serves as a regulatory protein modulating HVEM oligomerization and controls receptor activation. There are several TNFRSF receptors with three or fewer CRD domains that could potentially utilize a co-regulatory receptor to aid their oligomerization on the cell surface  and edges (e), where n represents the input signal and e the output signal  can activate TNFR2 very effectively but soluble TNF (sTNF) is a weak activator of TNFR2. We can illustrate how clustering can potentially explain these differences. eceptors . The FasA low amplitude signal may not reach the threshold of activation and may result in not just quantitatively but qualitatively different signaling outcomes. This could explain how activation of the same receptor can result in different outcomes in the TNFRSF. Interestingly a recent paper on DR5 signaling provides an explanation of how the long isoform of the FLICE-like inhibitory protein (FLIP(L)) can act as both an inhibitor and promoter of caspase-8 at the death-inducing signaling complex (DISC) . The outFLIP has three functionally different isoforms. In addition to FLIP(L), two shorter isoforms FLIP(R) and FLIP(S) also exist and together they play a pivotal role in switching between cell survival, apoptosis and necroptosis. While FLIP(L) plays an important role in modulating apoptosis, FLIP(s) is important for assembling the necrosome to induce necroptosis. Necroptosis is the caspase independent regulated inflammatory form of cell death via cell lysis and necrosis. Receptor-interacting serine/threonine protein kinase-1 and 3 (RIPK1/3) and mixed lineage kinase domain\u2013like (MLKL) are central mediators of TNF induced necrosis via TNF receptor 1 (TNFR1). Interestingly, TNFR1 internalization is an important first step in necroptosis . As we d). sTRAIL can also more easily activate DR4 that have a short stem region but not DR5 that has a longer stem  region of the receptor plays an important role as well. In most cases, the stem region is defined by the sequence between the last CRD domain and the transmembrane domain of the receptor. When the stem regions of TNFR1 and TNFR2 are switched, sTNF can readily activate TNFR2 but not TNFR1 . TNFR1 hger stem . LT\u03b2R wiger stem . Table\u00a01ger stem . We beliWe are now going to illustrate how taking into account the natural 3D structure of an antigen on the cell surface can guide the successful development of therapeutics with examples from the TNFRSF.reg expansion. On the receptor side, it has been challenging to create therapeutic antibodies against the TNFRSF. Antibodies against the TNFRSF can either block signaling and function as antagonists or promote signaling to function as agonists. It is only during the last few years that we have begun to understand how antibodies binding to different epitopes and surface structures can achieve these opposing functions  receptors by antibodies that bind on the outside of the receptor, opposite the ligand binding site, may provide the best solution , 38 Fig and alsoThere are conflicting data in the literature on the need for Fc\u03b3 recruitment but it is not an absolute requirement for receptor agonism , 42\u201344. 2 structure is able to successfully block TNF binding. The data supports a mechanism where the best antagonists bind to the antiparallel dimer form of the receptor locking in the non-signaling form of the receptor (Historically, it has been difficult to create effective antagonist antibodies against the TNFRSF. For those working in the field, it is broadly appreciated by trial and error that the natural ligands to the TNFRSF not only have high affinity but also high avidity and can, in most cases, successfully compete against antagonist antibodies in challenge assays. For instance, antagonist antibodies raised against TNFR2 can block TNF binding with different effectiveness . Charactreceptor , 36 (Figreceptor . In thisreceptor .Several groups have mapped the surface of TNF receptors to see how the epitopes influence function. As the above examples show, there is no clear connection between the epitopes position on the CRD and agonism or antagonism. However, the consensus that seems to be emerging is that the best agonists are bivalent or multivalent antibodies that cross-link and stabilize receptor complexes in the hexagonal cluster , 38 and The antibody isotype can also have a huge influence on the function of antibodies both for agonism and antagonism. Several anti-CD40 agonist antibodies have been shown to benefit from isotype switching from the IgG1 to IgG2 isotype , 49. TheFor ligand-based therapeutics targeting the TNFRSF, approaches that mimic the membrane-bound form result in much improved signaling. For a good review on different strategies see De Miguel et\u00a0al. . The minWe have reviewed the current state of our understanding of TNFRSF signaling mechanism. We have shown that TNFRSF signaling can be described by a unified model that orders the receptors and ligands into a honeycomb cluster. The hexagonal lattice of TNF receptors is optimized for signal transduction as it provides the most economical way to build a stable scaffold. Clustering also results in signal amplification that depends on cluster size and geometry in agreement with experimental data showing TNF receptors are arranged in small nanoclusters on the cell surface. We have shown that high occupancy of a receptor cluster by ligands leads to a sharp, high amplitude signal, while random occupancy leads to broad low amplitude signal that is directly proportional to the concentration of RING dimers or caspase-8 generated and could explain differences in signaling outcomes between membrane and soluble TNFSF ligands. Building of more detailed signaling models in the future combined with experiments will further improve our understanding of the intricacies of TNFRSF signaling.Beyond the TNFRSF, there are a growing number of hexagonal biological systems that indicate this may be a common arrangement of signaling networks in general. In addition to TNF receptors and their downstream signaling partners, chemo- or phototaxis receptors also cluster into hexagonal core complexes, consisting of trimers of dimers that further assemble to form large hexagonal arrays \u201371. SignAs the examples have shown a better understanding of receptor conformations on the cell surface can lead to the development of more effective therapeutics. Beyond antibody- and ligand-based therapeutics, the detailed knowledge of cell surface structures could also aid the development of small molecule drugs. Due to the high structural homology among members of the TNFRSF, strategies that work for the targeting of one receptor can be applied to others.EV and DF contributed to the design, writing and revision of the article. EV and DF approved the final content of the article."}
+{"text": "Demineralized dentin matrix (DDM) is an osteoconductive and osteoinductive material that has been successfully used in sinus floor augmentation and alveolar ridge augmentation in clinical applications. It releases bone morphogenetic proteins (BMPs) and other growth factors, making DDM a suitable grafting material. However, the granular particle of DDM makes it difficult to anchor into the bone defect area. The aim of this study was to investigate the biological effects and osteoinductivity of the combination of DDM and Fibrin Glue (FG) at an optimal ratio on bone healing from a critical bone defect in an animal model. The mouse osteoblastic cell line (MC3T3-E1) was co-cultured with various ratios of DDM and FG to examine their effects on osteoblast proliferation and differentiation, as indicated by alkaline phosphatase (ALP) activity, osteocalcin (OC) production and mineralized nodules formation. The optimal ratio was then chosen for further study with a rabbit calvarial defective model, in which they were implanted with DDM or DDM-FG1 (1\u00a0g: 0.1\u00a0ml) and DDM-FG2 (1\u00a0g: 0.5\u00a0ml) compounds, or left blank for 2, 4, 8 and 12\u00a0weeks to investigate soft tissue and new bone regeneration. Micro-CT and histology analysis were used to evaluate the total grafting properties according to the different healing periods. The result from in vitro studies demonstrated that the ratio of 1:0.1 induced more ALP activity and mineralized nodules, while the ratio of 1: 0.5 (DDM-FG combined) induced more osteocalcin (OC) at specific time points. In the animal model, the 3D new bone volume in all DDM-FG treatment groups was significantly greater than that in the blank group at 2, 4, 8 and 12\u00a0weeks. Furthermore, the new bone volume was greater in DDM-FG2 when compared to the other groups during the early weeks of the healing period. In histological analysis, clusters of osteoblasts were formed adjacent to the DDM particles, and newly formed bone was observed in all groups, suggesting an osteoinductive property of DDM. Moreover, the greater new collagen synthesis observed at 4\u00a0weeks suggested that early bone healing was induced in the DDM-FG2 group. This study demonstrated that at an optimal ratio, the DDM-FG compound enhances osteogenic activities and bone regeneration. However, granular bone grafts have the limitations of being easily dispersed and scattered from the implanted site. One solution is to combine them with a barrier membrane to achieve a space-maintain function. Nevertheless, alternative choices are limited in term of high cost and ethical aspects. Therefore, these drawbacks have led to developments of other bone substitutes as alternative grafting materials.In the recent bone augmentation, various types of bone graft materials have been applied. Among these materials, autologous bone demonstrated, which demonstrates conductibility, bone inductivity and osteogenesis performance, is considered the gold standard of bone transplantation3. Although the matrix of dentin and bone have different structures, they have similar chemical compositions and may induce bone formation as consistently as bone matrix4. Both bone and dentin are mineralized tissues composed of approximately 18% collagen (most of them is type I collagen), 2% noncollagenous proteins (NCPs), and 70% hydroxyapatite (HA). DDM can release several growth factors, such as bone morphogenetic proteins (BMPs), vascular endothelial growth factor (VEGF), insulin\u2011like growth factor (IGF), transforming growth factor\u2011\u03b2 (TGF-\u03b2), and basic fibroblast growth factor (FGF)6. BMPs are the most effective bone-inducing growth factors, belonging to the TGF-\u03b2 superfamily, which are multifunctional cytokines. BMPs can significantly induce the different stages of the bone healing process such as the inflammatory phase, angiogenesis, the callus formation, and bone remodeling7. VEGF improves angiogenesis via induce effects in endothelial cells8. TGF-\u03b21 can induce new bone formation in bone defect animal model9, and it cooperatively induces osteogenic differentiation with BMPs10. These identified growth factors play a crucial role in promoting cell migration to the bone defect region, proliferation and differentiation to form bone synthesizing osteoblasts11, in addition to promoting angiogenesis.DDM is a type of dentin that has recently been used as a bone graft material. It is obtained from extracted teeth after being demineralized, and essentially represents demineralized dentin13. Fibrin is composed of linked fibrinogens, they play important roles in coagulation, inflammation, fibrinolysis, tissue remodeling, cellular and matrix interactions, and wound healing. In this study, we combined the DDM granular and the fibrin glue, resulting in the conversion of the granular bone grafting material into gelatinous sticky bone, which aided in the integration of the particle bone grafting material into the alveolar bone and anchored to the bone defect surface. The objective of this study was to investigate the optimal ratio of the DDM and FG in the compound, in order to determine the effect of DDM-FGs on mouse osteoblastic-cell proliferation and differentiation. Additionally, the compounds of DDM and DDM-FG were investigated in an animal model of the rabbits calvarial bone defects which can be evaluated for bone healing capacity and osteogenesis.Fibrin glue (FG), also called fibrin sealant, generally consists of two main components: fibrinogen and thrombin. Fibrin is an activated form of fibrinogen that is activated by the thrombin, and the activated fibrin glue has a certain viscosity, which is conducive to blood coagulation and wound closureThe ground DDM was observed in pale yellow Fig.\u00a0A1. At\u2009\u00d7\u2009After 72\u00a0h decalcification, calcium was found in supernatant at 5.19\u00a0mmol/L, the released calcium from DDM kept increasing after 72\u00a0h. The release of calcium from DDM confirmed that the DDM we used in this present study, was partially decalcified  which is higher than other groups. There are only few mineralized nodules in FG group that is similar to blank control group.After different DDM-FG compounds were co-cultured with MC3T3-E1 cells in transwell system. The results of visible nodules and the staining intensity of each group were quantified in Fig.\u00a0The ALP activity from MC3T3-E1 cultured in osteogenic medium with DDM, DDM-FG1 and DDM-FG2 were significantly higher when compared to the MC3T3-E1 alone at day 3 and day 7 as shown in Fig.\u00a0P\u2009<\u20090.001).The osteocalcin secretion detected at day 14 and 21, were significantly higher in DDM\u3001DDM-FG1 and DDM-FG2 group than control and FG groups  . Later, the bone density of DDM, DDM-FG1 and DDM-FG2 were significantly induced (P\u2009<\u20090.001) at 4\u00a0weeks. However, the density from experimental group began to slightly decrease at 8\u00a0weeks, and the decreases in bone density from all group were more distinct at 12\u00a0weeks. Nevertheless, the bone density from all treated groups was significantly increased compared to that of the blank group at 12\u00a0weeks (P\u2009<\u20090.05). This may be the result of the DDM particles, while yielded the initial density value, while the new bone regeneration started to take over and bone remodeling occurred in later stages.The increase of BV/TV were significantly detected in DDM, DDM-FG1 and DDM-FG2 groups at 4\u00a0week compare to blank control group (05) Fig.\u00a0B. The BV05) Fig.\u00a0C. IntereIn the blank group, there was no evidence of hard tissue formation in the defective area until 4\u00a0weeks, when thin mineral-stained tissue was detected at the margin of the bone defect  . At 8\u00a0weeks, the collagen formation in DDM-FG2 group was significantly higher than that of DDM-FG1 (P\u2009<\u20090.05).Histological findings of Masson-stained sections are similar to the HE stained sections Fig.\u00a0A, and th01) Fig.\u00a0C. The av15. Furthermore, animal experiments have demonstrated that DDM is biocompatible and osteoinductive, comparable to decalcified bone matrix (DBM)16. Fibrin glue (FG) is biopolymeric material introduced as having excellent biocompatibility, controllable degrade rate, and the ability to enhance cell adhesion, improve angiogenesis. The material can be administered as a liquid to irregularly shaped sites, which adds to its surgical usefulness17. Given the biological characteristics of DDM particles and FG, and a need of an alternative suitable grafting material, the present study aimed to investigate the optimal condition of the combining DDM and FG for osteogenesis and bone regeneration in a rabbit calvarial defect model.The utilization of bone graft substitutes and materials in dentistry has significantly increased in recent years due to advancements in dental implant and the rising demand for the recovery of various types of bone defects. Demineralized Dentin Matrix (DDM), obtained from extracted teeth, has been used as a bone graft material in bone augmentation surgeries because of its osteoinductive and osteoconductive properties, as confirmed by several studies18. In this study, we observed dentin tubule openings in our self-made DDM, DDM-FG1 and DDM-FG2 after effective partial demineralization. The biological effect of FG has been reported to improve osteoblast differentiation (MC3T3-E1) and cell proliferation after directly co-culture with fibrin19. Furthermore, in a direct seeding on osteoblasts, the concentration of thrombin dose-dependently changed the fibrin structure, providing a suitable microenvironment for osteoblast attachment and differentiation, as confirmed by level of calcium deposition, ALP activity and level of Runx220. Unlike previous reports, FG alone could not effectively induce osteoblast proliferation and differentiation when compared to DDM or DDM-FGs in the present study. Meanwhile, the combination of 0.1 and 0.5\u00a0ml FG to DDM further improved cell differentiation by inducing higher ALP activity, more calcium nodules, and osteocalcin. It is possible that in our experiment\u2019s transwell system, cells were indirectly exposed with FG and other materials, leading to a different biological response than that observed with direct cellular binding capacity.The morphological differences between the DDM, DDM-FG1, and DDM-FG2 revealed that the physical characteristics of DDM particles clearly improved with increasing FG ratio. The optimal ratio of FG maintained the naturally porous structure of DDM. In a previous study on the implantation of the DDM in a calvaria bone defect over 12\u00a0weeks, ultrastructural investigation of the interface between DDM and the surrounding new bone illustrated a network connection of the cellular processes of the osteocytes in the new bone tissue to the DDM, which extended into the dentinal tubules. This robust evidence indicated that the presence of dentine tubules provides more areas for cell attachment, facilitates substance interchange, and promotes blood vessels grown into the deep material22. The number of vascular sections indicated that: the 1:1 ratio was mostly effective in stimulating an angiogenesis. Thus, the combination of granular material and FG in a ratio of 1:1(g/ml) appeared to demonstrate significant bone regeneration and angiogenesis. However, in contrast to a previous animal study, our study did not find good cellular responses when using a 1:1 ratio of DDM (mg) and FG (ml) while other ratios (1:0.1 and 1:0.5) did result in good cellular responses. Moreover, the additional 0.1 and 0.5\u00a0ml of FG to DDM demonstrated supplemental effects on osteoblast differentiation, and promoted bone regeneration and angiogenesis when implanted into the rabbit calvarial bone defects. It is possible that excessive amounts of FG may not always complement the biological properties of good grafting materials, as they can completely block dentin tubules and cause the loss of the porous structure of DDM, thus interfering with cellular responses and bone regeneration.In an animal model, the combination of calcium phosphate cement (CPC) with FG was implanted into rabbit bone defects with varied ratios. The 3D reconstruction images demonstrated that the 1:1 ratio produced the greatest bone formation23. The optimal particle size is essential in ensuring a proper resorption rate during bone reconstruction while maintaining defect volume for new bone ingrowth24. The particle size of our self-made DDM was 400\u20131000\u00a0\u03bcm, which provided good bone regeneration in the bone defect, similar to other previous studies26. Among other scaffold proteins, FG was proven to be a good carrier for BMP-2 release, and could prolong the BMP-2 releasing time28. Meanwhile, it is known that BMP-2 is produced and slowly secreted from DDM, with the presence of FG, it would create a suitable environment for osteogenesis and bone healing in our DDM-FGs groups than the DDM alone.Stable space-making and maintenance are crucial factors that could determine the success of bone regenerationIn conclusion, both DDM-FG1 and DDM-FG2 demonstrated good biocompatibility and were found to promote bone regeneration by enhancing osteogenic differentiation, improving the osteogenic microenvironment, and increasing capillarization in the bone defect area. These compounds were more practical due to their better physical properties and slower rate of degradation into the host bone. Therefore, based on the optimal ratio of the combined DDM-FGs, it enhanced osteogenic activities and bone regeneration in addition to DDM, as confirmed by the in vitro and in vivo results. This study provides new knowledge about a combination of grafting materials that can potentiate DDM as an effective biomaterial for oral surgery.In this experiment, the source of the dentin prepared for the DDM is from undecayed molar, premolar or impacted tooth from patients who came to have tooth extraction in the oral and maxillofacial surgery department, faculty of dentistry, Mahidol University. Although, the sample collection did not retain any identifying information, informed consent was obtained from all subjects and/or their legal guardian(s). All methods were carried out in accordance with relevant guidelines and regulations approved by ethic committee of the Faculty of Dentistry/ Faculty of Pharmacy, Mahidol University, Institutional Review Board (MU-DT/PY-IRB 2020/029.1007).29. Briefly, cold normal saline (NS) was used to wash the extracted tooth, and a dental high-speed drill was used to entirely removed the soft tissue, cementum and enamel  was used in this experiment Fig.\u00a0C,D. The 2+ release was detected using a calcium detection kit (Nanjing Jiancheng Bioengineering institute) to confirm the extent of decalcification of DDM from 1 to 96\u00a0h.The CaThe DDM particles and the DDM-FG mixed compounds were observed by scanning electron microscope (SEM) (Hitachi SU8220 Japan). After freeze-drying, the specimens were fixed with 2.5% glutaraldehyde for 24\u00a0h. After washing 3 times with PBS, ethanol gradient dehydration and air drying were done. The specimens were sputter-coated with gold spraying and morphologically observed under an SEM at 12\u00a0kV.2 and humidity of 70\u201380%. The medium was replaced every three days. The co-culture system was conducted in transwell (with pore size of 8\u00a0\u03bcm) for the cell proliferation calcium nodule and osteocalcin experiments, as shown in Fig.\u00a0Twelve hours after seeding, MC3T3-E1 were cultured in basal medium: Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% streptomycin solution with a temperature of 37\u00a0\u00b0C, 5% CO4 were grown on the transwell system  at lower chamber of 24 well plate for 4\u00a0h, the different DDM- FGs (DDM-FG1 representing ratio of 1\u00a0g DDM with 0.1\u00a0ml FG and DDM-FG2 representing ratio of 1\u00a0g DDM with 0.5\u00a0ml FG) were placed into the upper chamber. Then, after 1, 3, 5 and 7\u00a0days, the original culture medium was replaced by 500\u00a0\u03bcl DMEM with 10% FBS containing 50\u00a0\u03bcl CCK-8 reagent. After incubating at 37\u00a0\u00b0C for 4\u00a0h, 100\u00a0\u03bcl of the above solution was taken from each sample and added to a 96-well plate. Three parallel replicates were prepared. The absorbance at 450\u00a0nm was determined using a microplate reader . The test was independently repeated three times.A cell count kit-8  was used to quantitatively evaluate MC3T3-E1 cell proliferation. After 500\u00a0\u03bcl cell suspension with a density of 1\u2009\u00d7\u2009104/ml were inoculated to the surface of different DDM-FG compounds directly and cultured in 37\u00a0\u00b0C incubator. At 3 and 7\u00a0days, the cell lysis buffer was centrifuged at 1200\u00a0rpm/min at 4\u00a0\u00b0C for 25\u00a0min, and the supernatant was taken for testing. After 100\u00a0\u03bcl reaction termination solution was added to each well, the absorbance was measured at 405\u00a0nm with a microplate reader . The amount of ALP required for hydrolysis of para-nitrophenyl phosphate to produce 1\u00a0\u03bcmol of p-nitrophenol per minute was defined as an enzyme activity unit, alkaline phosphatase activity was calculated. The test was independently repeated three times.To investigate bone ALP activity, an Alkaline Phosphatase Assay Kit  was used. The osteogenesis was induced by the osteogenic differentiation medium  containing basal medium mixed with 200\u00a0\u03bcM ascorbic acid 10\u00a0mM \u03b2-glycerophosphate, 100\u00a0nM dexamethasone. Different DDM-FG compounds; DDM-FG1 representing ratio of 1\u00a0g DDM with 0.1\u00a0ml FG and DDM-FG2 representing ratio of 1\u00a0g DDM with 0.5\u00a0ml FG, were prepared and placed in 6-well plates until FG was completely gelatinized. MC3T3-E1 cells with a density of 1\u2009\u00d7\u2009104/ml MC3T3-E1 cell was inoculated in transwell lower chamber in a 6-well plate, different DDM-FG compounds  were prepared in transwell upper chamber and cultured in an incubator at 37\u00a0\u00b0C . The intensity of the solution was immediately measured (\u03bb\u2009=\u2009450\u00a0nm).2\u2009\u00d7\u20091030. Rabbits were monitored daily throughout the study period by an accredited veterinarian. During this experiment, the rabbits were fed with food and water ad libitum. After a 4-week acclimation period, the treatment was randomized by a blinded performer who neither performed nor investigated the result, thus being \u201cblind\u201d to the study.The animal study protocol has been approved by the Ethical Committee of Animal Experiments, Kunming Medical University, Kunming, Yunnan Province, China (kmmu2018029). All methods were carried out in accordance with relevant guidelines and regulations, which are reported in accordance with ARRIVE guidelines. For this study, forty male adult Japanese big ear rabbit age 12\u2009\u00b1\u20093\u00a0months, weight of 3.5\u2009\u00b1\u20090.3\u00a0kg were chosen31 . Two experienced surgeons performed all surgical procedures in 30\u00a0min, following the steps in Fig.\u00a0General anesthesia was induced with an intramuscular injection of 0.15\u00a0ml/kg Xylazine Hydrochloride32 at 2, 4, 8 and 12\u00a0weeks. The skulls containing the surgical defect area were cut and subjected to sample preparation for \u00b5CT scan and histological analysis.Each ten rabbits were euthanized by an overdose of ketamineThe bone samples were soaked in 4% neutral formaldehyde, and store at 20\u00a0\u00b0C. Samples were scanned using a Micro-CT scanner  with a scanning resolution of 11.4\u00a0\u03bcm, Voltage (70\u00a0kVp), Current (114\u00a0\u03bcA) at a standard rotation angle of 360\u00b0 for 150\u00a0min. After scanning, data was reconstructed (\u03bcCT Ray V4.2 image processing software) and performed a quantitative analysis of all bone parameters using \u03bcCT Evaluation Program V6.6 software to examine the growth of new bone in different phases.The 4% neutral buffered formalin solution was used to fix the samples and then stored in 70% ethanol at 4\u00a0\u00b0C before histological processing. The rabbits calvaria were soaked in Rapid Cal. Immuno\u2122 , pH\u2009<\u20091, for about 6\u00a0days until they were completely decalcified. Then, the samples were rinsed for 10\u00a0min in running tap water, dehydrated with ascending graded alcohol, cleared in xylene, and embedded in paraffin. Serial sections of 5 \u03bcm thick were cut from the center of the original bone defect. Paraffin sections were stained with hematoxylin and eosin (H&E) and Masson\u2019s-trichrome dyes and then observe under Stereo Microscope  and Light Microscopy  to investigate bone healing and collagen type I deposition, respectively. Images were then captured using NIS-Elements F 4.60.00 64-bit and ZEN 3.0 (blue edition) software. To quantify the collagen area on the Masson sections, the 3 areas of certain squares located in the middle field of each slide were calculated by Image-Pro Plus 6.0 software.p value\u2009<\u20090.05.All the data were presented as mean\u2009\u00b1\u2009standard deviation Animal ethical approval for this study was obtained from the Ethical Committee of Kunming medical University (kmmu2018029). The ethic approval of the human study was granted by the Faculty of Dentistry/ Faculty of Pharmacy, Mahidol University, Institutional Review Board, Thailand (No. MU-DT/PY-IRB 2020/029.1007)."}
+{"text": "The addition of waste glycerol slightly increases the calorific value of the pellet (17.94 MJ/kg for 7.5% additive). A 10-kW domestic biomass boiler has been employed to burn the tested pellets. The consumption of analyzed fuels during boiler operation was determined. The concentration of CO, CO2 and NOx in exhaust gases has also been examined. It was observed that the addition of 7.5% of waste glycerol contributes to the reduction in NOx concentrations by 30 ppm and CO2 by 0.15%. The obtained experimental results were compared with the numerical calculations made with the use of ANSYS Chemkin-Pro. The conducted research indicates the legitimacy of utilizing waste glycerol as an additive to wood pellets. In addition, this type of addition has a positive effect on, among others, the increase in calorific value, as well as lower emissions of combustion products such as NOx and CO2.The shortage of fossil fuels and their rising prices, as well as the global demand for renewable energy and the reduction in greenhouse gas (GHG) emissions, result in an increased interest in the production of alternative biofuels, such as biodiesel or biomass pellets. In this study, the possibility of utilizing waste glycerol, as an addition to pine pellets intended for heating purposes, has been investigated. The usefulness of pellets containing glycerol additions has been compared in terms of applicable quality standards for wood pellets. The highest values of moisture (4.58%), ash (0.5%) and bulk density (650 kg/m This is due to the higher share of hydrogen and oxygen, lower share of carbon and lack of nitrogen in pure glycerin . The produced pellets therefore meet the EN ISO-17225-2:2014 standard  in terms2, CO and NOx is shown in The results of the initial simulations were carried out for pine pellets (PP). Due to the fact that during the experiment the coefficient of excess combustion air oscillated within \u03bb = 2.0 (\u00b1 0.5), numerical simulations were carried out for \u03bb = 2.0 and 2.5. The dependence of the combustion temperature on the concentration of CO2 and CO at lower temperatures, with the largest ones observed for \u03bb = 2.0 and T = 300 \u00b0C and amounting to 0.53% and 0.52%, respectively. In the case of NOx concentration dependence on temperature, the largest discrepancies can be observed for higher temperatures (624 ppm for T = 850 \u00b0C and \u03bb = 2.0). For the lowest temperatures considered, these discrepancies are much lower (average 54 ppm). Due to the existing discrepancies, in order to select the mechanism for further consideration, a comparative analysis with experimental studies was carried out. During the combustion of the analyzed pellets, the temperature was close to 600 \u00b1 50 \u00b0C and the excess air coefficient was \u03bb = 2.0 \u00b1 0.5. Therefore, the results of numerical simulations obtained for T = 600 \u00b0C T = 650 \u00b0C, \u03bb = 2.0 and \u03bb = 2.5 were implemented for the comparative analysis. A summary of the results for both mechanisms and experimental results for the pine pellet (PP) is shown in From the analysis presented in x concentration. The CO2 concentration depends on the coefficient of excess combustion air and shows a similar tendency for both mechanisms. In the case of the CO concentration, there are significant discrepancies between experimental and calculated results. However, in the case of the Glarborg mechanism, these discrepancies are smaller. Such a similarity was also observed in a previous article [2, CO and NOx concentrations on temperature for the four considered pellets are shown in Comparing the results of the numerical simulations with the experimental results, a greater convergence with the calculations carried out using the mechanism developed by Glarborg was found a. In the article . Due to 2 concentration in the entire range of the analyzed temperatures. A similar tendency is noticeable in the case of CO concentration in the lower temperature range, up to about 500 \u00b0C. At higher temperatures, the differences resulting from the different additions of glycerol are not so significant. In the case of the analysis of the NOx concentration, no significant deviations depending on the temperature are observed. To illustrate the exact range of concentration differences, taking into account the previous considerations (3.1) and knowing the conditions of the experiment, the results of the simulation for the temperature of 650 \u00b0C were taken into account for the comparative analysis  only or with a small admixture of waste glycerol (PP-G2.5) does not significantly reduce the NOx concentration, the introduction of 5% and 7.5% glycerol to pellets clearly reduces such a concentration. This favorable phenomenon is due to the lower share of nitrogen in fuels containing glycerol  reactor was used for numerical simulations, which ensured perfect mixing of reagents. Such conditions are not always possible to meet during the experiment. Therefore, the small discrepancies visible between the results of the experiment and the calculations should be explained by the instability of the combustion process.Comparing the results of experimental data and numerical calculations, one can observe convergent trends for the exhaust gas components in question. With the increase in the share of glycerol in the burned pellets, a decrease in the concentration of COglycerol . A simil\u22123) were observed for PP. The addition of waste glycerol slightly increases the calorific value of the pellet (17.94 MJ/kg for PP-G7.5).All four types of analyzed pellets meet the requirements of the EN-ISO-17225-2:2014 standard in terms of calorific value, moisture content, ash, bulk density and dimensions The highest values of moisture (4.58%), ash (0.5%) and bulk density  of the base material (pine sawdust).\u2212x and CO2 concentrations in exhaust gases. The addition of 7.5% glycerin waste contributes to the reduction in NOx concentrations by 30 ppm and CO2 by 0.15%.The addition of waste glycerol contributes to the reduction in nitrogen and carbon content in the fuel, which has a positive effect on the reduction in NO\u2212x between the considered mechanisms).The Glarborg mechanism used in numerical calculations shows a high convergence with the experimental results in comparison to the Creck mechanism (over 400 ppm difference at 650 \u00b0C for NO\u2212Difficulties in burning pellets with a 7.5% and higher content of waste glycerol result from adapting the burner to burning wood fuels.\u2212The obtained test results indicate the legitimacy of further analysis in terms of the efficiency of the combustion of this type of pellet in boilers equipped with an industrial burner.In the era of the energy crisis and the limited resources of fossil fuels, it is extremely important to find cheap, alternative, environmentally friendly solutions, the so-called bio-fuels. The role of this type of fuel is to meet expectations, both in terms of ecology and the economy. This article examines the possibility of using waste glycerol as an additive to pine pellets. This solution allows for the cheap management of post-production waste, and it may also contribute to the improvement of selected physicochemical properties of the fuel and thus lowering the emission of harmful combustion products. The experimental and numerical research carried out allowed the following conclusions to be formulated:"}
+{"text": "Tribolium castaneum is an important pest of stored agricultural products and the first beetle whose genome was sequenced. So far, one high-copy-number and ten moderate-copy-number satellite DNAs (satDNAs) have been described in the assembled part of its genome. In this work, we aimed to catalog the entire collection of T. castaneum satDNAs. We resequenced the genome using Illumina technology and predicted potential satDNAs via graph-based sequence clustering. In this way, we discovered 46 novel satDNAs that occupied a total of 2.1% of the genome and were, therefore, considered low-copy-number satellites. Their repeat units, preferentially 140\u2013180 bp and 300\u2013340 bp long, showed a high A + T composition ranging from 59.2 to 80.1%. In the current assembly, we annotated the majority of the low-copy-number satDNAs on one or a few chromosomes, discovering mainly transposable elements in their vicinity. The current assembly also revealed that many of the in silico predicted satDNAs were organized into short arrays not much longer than five consecutive repeats, and some of them also had numerous repeat units scattered throughout the genome. Although 20% of the unassembled genome sequence masked the genuine state, the predominance of scattered repeats for some low-copy satDNAs raises the question of whether these are essentially interspersed repeats that occur in tandem only sporadically, with the potential to be satDNA \u201cseeds\u201d.The red flour beetle  Eukaryotic genomes are rich in repetitive DNA sequences, among which transposable elements and satellite DNA (satDNA) sequences are the most abundant. Unlike transposable elements, which are scattered throughout the genome, repetitive units of satDNAs are arranged tandemly in arrays, sometimes encompassing megabase-sized regions. Although more than 60 years have passed since their discovery , satDNAsMegaleporinus macrocephalus [Pontastacus leptodactylus [The discovery and experimental study of satDNAs have historically been hampered by their repetitive nature and predominant location in heterochromatin regions. In the pre-genomic era, restriction digestion of genomic DNA followed by gel electrophoresis was one way to discover the most abundant satDNAs in the genome of a species under study, which was also usually considered species-specific (reviewed in ). PCR facephalus  and as mdactylus , as revidactylus . After rdactylus , Ruiz-Rudactylus , but theIn terms of evolution, related species may have the same or similar satDNAs. According to the concept of concerted evolution, when related species share a particular satDNA, monomeric repeats within a species show greater similarity than repeats between species . The libT. castaneum  (Coleoptera: Tenebrionidae) is one of the most important cosmopolitan pests of stored grain products. Its potential for laboratory research was recognized five decades ago [T. castaneum (2n = 20) counts 18 autosomal chromosomes and an Xyp pair of sex chromosomes, but the genome sequence is anchored to 9 autosomes and the X, while the yp chromosome remains unassembled. According to reassociation kinetics, nearly 40% of the T. castaneum genome consists of repetitive DNA [T. castaneum, a major satDNA, TCAST, comprising 17% of the genome was discovered in the pre-genomic era with a restriction digestion approach [The red flour beetle ades ago , and sinades ago ). As a rades ago , and itsades ago . Howevertive DNA , which itive DNA . In T. capproach . TCAST iapproach , also exapproach . In addiapproach . HoweverT. castaneum and, in particular, to determine the low-copy-number satDNAs that presumably exist in the genome but have not been revealed by the approaches used in previous studies. By employing low-coverage Illumina reads, we predicted potential satDNAs via assembly-free graph-based sequence clustering using the TAREAN tool. In addition to previously detected satDNAs, in this work, we detected 46 novel tandem repeats. By characterizing their distribution and organizational patterns using the current T. castaneum assembly, as well as unassembled scaffolds/singletons, we found 29 of them to be present exclusively in a tandem organization, while for 17 of them, the scattered repeats were also annotated. Experimental characterization of the three low-copy-number tandem repeats discovered in silico allowed us to validate them as satDNAs.The aim of this work was to examine the entire satellitome of T. castaneum that was originally obtained from the USDA-ARS  in 2015. The insects were routinely reared in whole wheat flour at 28 \u00b0C and 50% relative humidity in the dark and subcultured every four weeks. Total DNA was extracted from 200 mg of adult beetles  using the DNeasy Blood and Tissue Kit . DNA quantity was measured with a Qubit 4 fluorometer  using a Qubit dsDNA BR Assay Kit , while DNA quality was determined using a NanoDrop Spectrophotometer  and 1% agarose gel electrophoresis.All experiments were performed using a laboratory culture of a highly inbred GA2 (Georgia 2) strain of the red flour beetle T. castaneum genomic DNA library of ~500 bp fragments was prepared with a Kapa HyperPrep Kit  and the whole-genome sequencing (WGS) was performed on an Illumina NextSeq platform by Admera Health . The Illumina sequencing resulted in 8,122,518 paired-end reads (2 \u00d7 101 nt), and the raw sequencing data were deposited in the Sequence Read Archive (SRA) database under BioProject study accession number PRJNA606031. Quality control checks of raw Illumina reads were undertaken with FastQC [https://repeatexplorer-elixir.cerit-sc.cz/galaxy/ accessed on 25 August 2022). The reads were cleaned from adapter sequences, quality-filtered using quality scores \u2265 10 over 95% with no Ns allowed, and trimmed to 89 nt. The interlaced reads were randomly subsampled in order to scale down large datasets to low genome coverage. Seven randomly subsampled sets containing 230,000 to 2,300,000 reads corresponded to genome coverage of 0.1\u20131\u00d7 (T. castaneum satDNAs [T. castaneum reference genome assembly Tcas5.2 [T. castaneum genome. A criterion for declaring a satDNA was an array of at least five consecutive copies with \u226570% similarity to a TAREAN cluster consensus present in the assembled or unassembled genome sequence. The consensus sequences of satDNAs detected in this work were used as the query sequences for BLAST searches against the NCBI nucleotide collection (https://blast.ncbi.nlm.nih.gov/Blast.cgi accessed on 14 September 2022) to check similarity with published sequences. Geneious Prime was used to perform local BLAST searches against the Tribolium RepeatScout library, which contains 4475 repeat families detected in T. castaneum categorized into several repetitive classes: HighA (31 families), HighB (5 families), Mid (304 families), Low (3237 families), 360 bp satellite (1), and transposable elements (897) [A f 0.1\u20131\u00d7 . SatDNA  satDNAs ,30 was i satDNAs . TCAST sts (897) . The conts (897) . The conhttps://github.com/rambaut/figtree/releases accessed on 19 October 2022). The satDNA monomer unit sequences annotated in the Tcas5.2 assembly and the unplaced scaffolds and singletons were extracted and used for further analyses. For satDNAs TCsat12, TCsat13, and TCsat14, monomeric unit sequences from the cloned fragments see . were alT. castaneum satDNAs [The genomic environment of low-copy satDNAs was examined for the presence of genes, transposable elements, and previously detected  satDNAs ,28,30. F satDNAs  by using satDNAs . Only hi satDNAs  on the G satDNAs . For eacT. castaneum genomic DNA by PCR in a 30 -\u00b5L reaction mixture containing 10 ng genomic DNA, 0.2 \u00b5M of each specific primer, 0.2 mM dNTP mix, 2.5 mM MgCl2, 0.25 U GoTaq G2 Flexi DNA polymerase, and 1\u00d7 Colorless GoTaq Flexi Buffer . PCR amplification included a predenaturation step at 94 \u00b0C for 3 min, 35 amplification cycles , and a final extension at 72 \u00b0C for 10 min. The primers\u2019 sequences, the optimal annealing temperatures, and the lengths of the amplified fragments for each satDNA are listed in Based on the consensus sequences, specific primers for satDNAs TCsat12, TCsat13, and TCsat14 were designed using Primer3 software . The fraDNA probes for Southern blot hybridization and fluorescence in situ hybridization (FISH) were generated from cloned satDNA fragments, the nucleotide sequences of which were verified by sequencing. In order to ensure the representation of different variants of the satellite monomeric sequence, a mixture of several different plasmid clones in an equimolar ratio was used in the preparation of each probe. Specifically, TCsat12-cl1/2/16/25/32 clones were used for the TCsat12 probe, TCsat13-cl27/32/40/47/50 clones for the TCsat13 probe, and TCsat14-2/10/31/32/34 clones for the TCsat14 probe (nucleotide sequences for specified clones are presented in 2HPO4 (pH 7.2), 20% SDS, 1 mM EDTA, 0.5% blocking reagent, and 20 ng/mL of the DIG-labeled specific probe with agitation overnight. Hybridization was carried out under high-stringency conditions at 68 \u00b0C, allowing >90% homology. Posthybridization washes were applied at 65 \u00b0C in the buffer containing 20 mM Na2HPO4, 1 mM EDTA, and 1% SDS. Chemiluminescent detection was undertaken using a DIG DNA Labeling and Detection Kit, CDP Star, and X-ray films .For Southern blot analysis, genomic DNA was digested with different restriction endonucleases according to the manufacturer\u2019s instructions . For each restriction digestion, 2 \u00b5g of genomic DNA was digested to completion with 20 U of restriction enzyme overnight at 37 \u00b0C. Digested genomic DNA was electrophoresed in a 1% agarose gel and blotted onto a positively charged nylon membrane . Hybridization was performed in the buffer containing 250 mM NaT. castaneum chromosomes was assessed using FISH. Chromosome spreads were prepared from pupae male gonads with the squash method as described previously [2HPO4, and 10 ng/\u03bcL of the probe. Posthybridization washes were performed in 50% formamide/2\u00d7 SSC at 37 \u00b0C. Biotin-labeled probes were detected with a fluorescein avidin D and biotinylated antiavidin D system  via signal amplification through three layers of fluorophore conjugates and antibodies using the following dilutions: 1:500 fluorescein avidin D, 1:100 biotinylated anti-avidin D, and 1:2000 fluorescein avidin D. Finally, the slides were counterstained in 4\u2032,6-diamidino-2-phenylindole (DAPI) solution for 15 min, air-dried, and embedded in Mowiol 4\u201388 mounting medium . Slide visualization and image acquisition were undertaken using a Leica TCS SP8 X confocal laser scanning microscope  equipped with an HC PL APO CS2 63\u00d7/1.40 oil objective, 405 nm diode laser, and a supercontinuum excitation laser . Images were captured separately for each fluorochrome and processed with ImageJ [Localization of TCsat12, TCsat13, and TCsat14 satDNAs on the eviously . FISH wih ImageJ  and AdobT. castaneum satellitome, we sequenced the whole genome of the flour beetle T. castaneum de novo using Illumina sequencing. Randomly subsampled sets of Illumina reads were used in seven series of TAREAN clustering  over the entire length of the sequence . Using the sequence similarity criterion of \u226570%, we annotated the consensus sequences of the remaining clusters in the current T. castaneum genome assembly Tcas5.2, which consists of ten chromosome/linkage groups [The consensus sequences of clusters annotated as putative satDNAs in T4 and T7 analyses were BLAST-searched against the NCBI GenBank nucleotide database. In this way, we identified the clusters corresponding to the 11 previously described satDNAs ,28,30 ane groups . We alsoTribolium RepeatScout library, sharing similarity with 1 HighB, 5 Mid, and 3 Low repetitive elements, annotated according to [Among the 46 novel satDNAs, 9 showed partial matches to repeat elements recorded in the rding to  , monomer length , or A + T content .The monomers of the 46 satDNAs annotated in the Tcas5.2 assembly and unplaced scaffolds/singletons were extracted  and aligT. castaneum satDNA with the shortest repeat units  was found between the proportion of scattered repeats and the distribution across different chromosomes and unassembled sequences. The proportion of scattered repeats also showed a positive correlation with satDNA abundance , as well as with nucleotide divergence  (p = 0.494).Despite the incompleteness of the current genome assembly, we tried to decipher the organization of low-copy satellites based on available data and annotated monomer copies. First, we wanted to determine the extent to which the low-copy satDNA monomers were tandemly repeated or scattered as individual copies. Using \u22655 consecutively repeated monomers as a criterion for tandem organization, we found 29 satellites that had monomers exclusively repeated in tandem , whereas< 0.001) . No corrNext, we aimed to gain insight into the genomic environment of low-copy satDNAs. The analysis focused on the assembled part of the Tcas5.2 genome because it has well-curated annotations. We searched for the presence of genes, transposable elements, and previously described satDNAs ,28,30 inT. castaneum satellitome, we experimentally characterized three novel satDNAs with clusters that were declared as putative satellites in all TAREAN analyses: TCsat12, TCsat13, and TCsat14  that had only one cutting site in a monomeric sequence . Digestimonomers  explaine profile B. On thevergence . Therefomonomers , revealemonomers C. Howevemonomers , the hybmonomers C. In sumT. castaneum chromosomes, we performed FISH using the cloned PCR fragments as specific probes. On the haploid complement, FISH mapping of TCsat12 revealed its presence on the two chromosomes, with a consistently stronger signal on one of them centromeric satDNA TCAST, which was discovered nearly three decades ago [Tribolium confusum [T. freemani [Tribolium species that followed in subsequent years and were also based on restriction digestions of genomic DNA demonstrated that one or two highly copious and species-specific satDNAs dominate in other Tribolium genomes as well [T. castaneum genome assembly, it has become clear that TCAST is not the only satDNA in T. castaneum, as ten other satDNAs 20\u201385 times less abundant than TCAST have been found [Triatoma delpontei [T. castaneum than the 11 previously characterized, and cataloging its satellitome became the main objective of this research.The genome of the representative coleopteran species ades ago . This ficonfusum  and T. ffreemani , in whic as well ,44,45,46en found ,30. The en found  and kissen found , more then found ,49, and en found , while aelpontei . TherefoT. castaneum satDNA, we identified 46 new satDNAs in the genome of this important model insect. This expands the T. castaneum satellitome to a total of 57 satDNAs, which cumulatively make up 23.8% of the genomic sequence. The most striking difference between the newly discovered satDNAs and those previously characterized is their low proportions in the genome, with 45 satellites each accounting for less than 0.1% and thus being designated as low-copy-number satDNAs. The T. castaneum satellitome, in which 79% of the members are low-copy satDNAs, is in line with many insect satellitomes, in which 70\u201380% of the total numbers of detected satellites belong to satDNAs that individually make up less than 0.1% of the genomes [To address this task, we applied Illumina sequencing and took advantage of satDNA detection without the need for genome assembly. In this work, a quarter century after the discovery of the first  genomes ,48,52.T. castaneum satellites. One of them is the high A + T content of the repeat units, which predominantly ranges between 60 and 80%. High A + T composition is a frequent characteristic of satDNAs in many organisms, and it has been suggested that AA/TT periodicity in satellite monomers may contribute to nucleosome stabilization centromeric regions [T. castaneum satellitome contains satDNAs, which, despite drastic differences in abundance and different chromosomal positions and chromatin environments, incline to high A + T compositions and preferred lengths for repeat units. It could be hypothesized that the low-copy satellites included in the T. castaneum satellitome, having certain structural sequence attributes, might have the potential to be propagated into more abundant satDNAs under certain evolutionary circumstances. Despite the large difference in abundance, the newly described satDNAs share some common features with already known iewed in ). Howeverding to , as also satDNAs ,55. Inte regions ,29 but aT. castaneum satellites is the chromosomal distribution. In contrast to the previously described satDNAs, the copies of which are annotated on most chromosomes, in the current Tcas5.2 assembly, we mainly detected most low-copy satDNAs on one or only a few chromosomes. It is intuitive to expect that low-represented satDNAs will not be distributed over a larger number of chromosomes, and the presence of low-copy satDNA families restricted to one or only a few chromosomes has also been evidenced in other organisms [T. castaneum genome, which abounds in large heterochromatin blocks, the low-copy satellites are nevertheless mainly embedded in euchromatic regions, as was also found for its moderate-copy satDNAs [Hippodamia variegata [Rhynchophorus ferrugineus [T. castaneum, their genomes are dominated by a major, highly abundant satDNA that is primarily, although not exclusively, distributed in the (peri)centromeric heterochromatin of all chromosomes, while low-copy satDNAs mostly have a scattered and sporadic distribution along euchromatic regions. That low-copy satDNAs can successfully exist outside of heterochromatic regions has also been confirmed in heterochromatin-poor organisms, such as the Pacific oyster Crassostrea gigas, in which short satDNA arrays, often incorporated into Helitron/Helentron mobile elements, are widely dispersed throughout the genome [Drosophila virilis and Drosophila americana, the transition zones between densely condensed heterochromatin and lightly packed euchromatin were detected as regions of recently formed, abundant tandem repeats derived from DNA transposons, suggesting that the less compacted environment of the higher recombination rates may favor amplification of satDNA arrays [The feature that clearly distinguishes the low-copy satDNAs discovered in this work from the previously known rganisms ,49,56. Iariegata  and Rhynrugineus  have shoe genome ,58. One A arrays ,60. T. castaneum low-copy satDNAs. Transposable elements are also a type of sequence with which certain T. castaneum low-copy satDNAs share similarities in some segments of their repeat units. The link between satDNAs and transposable elements has been extensively demonstrated and elaborated in the literature [D. virilis, for example, tandem repeats within the Tetris DNA transposon led to the TIR-220 satDNA [Lathyrus sativus showed that 9 of the 11 most abundant satDNAs also had short tandem arrays located within LTR-retrotransposons [T. castaneum, TCAST-like sequences were found to be embedded in a complex unit resembling a DNA transposon, but TCAST-like elements were also detected within the CR1-3_TCa non-LTR retrotransposon [T. castaneum genome [T. castaneum genome is still missing, and perhaps ultra-long sequencing will reveal the genome regions that contain longer TCsat23 arrays. Long-read sequencing, which unravels extensive tandem arrays, and comparison of related genomes are promising tools for deciphering the origin of satellites. Applying such an approach to the plant L. sativus, it was deduced that its three satDNAs evolved from the tandem subrepeats originally contained in the LTR retrotransposon sequence [Transposons dominated in the analyzed genomic environment of terature ,61,62. T0 satDNA . A recennsposons . For T. ansposon . In thism genome , and TCsm genome . Scalvenm genome  suggestesequence .T. castaneum satellitome characterization, we experimentally evaluated three low-copy satDNAs predicted by TAREAN. Despite their low representation in the genome (0.015\u20130.046%), Southern hybridization successfully confirmed their tandem organization and yielded hybridization profiles consistent with the variability calculated from PCR-amplified fragments and monomers annotated in the genome assembly. In addition to the 46 satDNAs cataloged in this work, TAREAN also proposed a certain number of clusters as potential satellites for which we could not annotate tandemly organized copies either in the current genome assembly or among the unplaced scaffolds/singletons, so we did not include them in the satellitome collection. We assume that if these potential sequences have tandemly organized repeats, they are placed in the 20% of the genome sequence that has not yet been assembled. It is expected that future long-read sequencing will disclose whether these are indeed satDNAs, and it is possible that the current satellitome collection will be expanded. The long-read sequencing will also reveal whether some of the low-copy-number satDNAs discovered in this work form longer tandem arrays than those we were able to annotate in the current assembly.Within the frame of T. castaneum satellitome, we resequenced the genomic DNA of the highly inbred GA2 strain used in the T. castaneum genome sequencing project [Triatoma infestans, although they share most of the satDNAs detected in their genomes, show significant differences in the representativeness of certain satDNA families [T. castaneum, the population-specific profiles were found for the three subfamilies of the satDNA TCAST2 [T. castaneum collection of 57 satDNAs presented here is an initial reference, but it is possible that future analyses of other strains/populations will unveil the plasticity of the T. castaneum satellitome in terms of the number of satDNA families and/or their genomic abundance.To define the  project  and on w project . It shoufamilies ,67. In TA TCAST2 . The T. T. castaneum will certainly help to approach these findings.The advent of ultra-long-sequencing technologies and the complete T2T-CHM13 human genome sequence have ushered in the telomere-to-telomere (T2T) genomics era . Althoug"}
+{"text": "Beeswax is a by-product of the bee honey industry obtained from the honeycomb of the honeybee and other bees as the crystalline substance formed from honey sugars. From the harvesting of alfalfa, a large quantity of beeswax is commonly available in Egypt. This study evaluated the effects of the supplementation of beeswax on performance, nutrient digestibility, rumen fermentation, blood metabolites, and sustainability in terms of feed costs in Assaf lambs. Eighteen lambs were allotted to three experimental groups fed with a basal diet supplemented with 0, 2, and 4 g of beeswax/day for 90 days. The results revealed an increased performance in the beeswax-supplemented groups, resulting in a higher efficiency and lower feed cost and thus optimizing farming efficiency and profitability. In addition, beeswax inclusion in the feed formulation enhanced the nutrient digestibility by enhancing rumen fermentation and decreasing the ammonia emissions. In conclusion, the use of 4 g/day of beeswax supplementation in growing Assaf lambs could promote zootechnical performance, nutrient digestibility, rumen fermentation and thus lower the cost of feed formulation and support the sustainability of lamb farming.n = 6 lambs/group). The lambs were fed a basal diet without supplementation (G1) or supplemented with 2 and 4 g beeswax/head/day in G2 and G3 groups, respectively. Zootechnical performance was evaluated over a 90 day period. Feed digestibility was assessed in faeces through the acid insoluble-ash method, and rumen liquor was collected to measure ammonia (NH3-N) and total volatile fatty acid (TVFA) levels. Blood samples were obtained for the titration serum metabolites by colorimetric tests. The findings showed that G3 had an improved performance compared to the other groups (p < 0.01). The lambs in G3 revealed the highest nutrient digestibility and feed use, followed by G2, and G1. G3 recorded the highest economic efficiency followed by G2 and G1 (p < 0.01). The TVFA, acetate, and propionate concentrations were higher and the pH values, NH3-N, and butyrate concentrations were lower in G3 compared to G2 and particularly to G1 (p < 0.01). The concentrations of total protein, globulin, and glucose were significantly higher with 4 g beeswax (p < 0.05). However, albumin, cholesterol, total lipids, urea, creatinine, glutamic oxaloacetic transaminase (GOT), and glutamate pyruvate transaminase (GPT) concentrations as well as the albumin to globulin ratio decreased significantly with both levels of beeswax (p < 0.05). The addition of beeswax at the level of 4 g/head/day for growing Assaf lambs significantly improved the growth performance, digestibility, rumen fermentation, and blood serum parameters in addition to the economic efficiency.The aim of this work was to assess the effects of beeswax supplementation on growth rate, feed intake, nutrient digestion, rumen fermentation, blood parameters, and economic sustainability in Assaf lambs. Eighteen growing Assaf (5 months old) lambs were separated into three experimental groups ( For centuries, bee-derived products such as honey, propolis, and beeswax have been used as natural therapeutics in folk medicine healing due to their properties and their high content of bioactive compounds . Today, Beeswax has been employed as an additive for several industries in products, or during processing. In pharmaceutics, it has been exploited as a thickener, binder, and carrier. Beeswax products have been used since ancient times both for their nutritional value and for a broad spectrum of therapeutic purposes. Beeswax products are deemed to be a potential source of natural antioxidants which can counteract the effects of oxidative stress underlying the pathogenesis of many diseases ,9.The livestock system plays a key role in food security, and the Agriculture Organization of the United Nations (FAO) reported that animal production supplies 34% of the total protein intake globally . It has The role of diet has a great impact on the quality of life, and the use of functional products with high concentrations of bioactive molecules of a natural origin is preferable to synthetic products ,16. AnimThe aim of this study was to determine the effects of beeswax supplementation on growth performance, feed intake, digestibility, rumen fermentation, blood metabolites, and economic efficiency in growing Assaf lambs.The experiments were performed according to the guidelines of a local ethics committee for animal care and welfare (Number 08/2016 EC). Eighteen growing male Assaf lambs  with an average age of 5 \u00b1 0.04 months and a live body weight of 23.47 \u00b1 0.27 kg were allotted to three groups of six animals each for a total duration of 90 days. A beekeeper in Saint Catherine, in the South Sinai Governorate of Egypt, provided the beeswax for this investigation. The beeswax additive  used in the following trial was mainly composed of 57.4% total esters , 15.7% total hydrocarbons , 18% total free fatty acids, and 0.6% total free fatty alcohols . The waxLambs were housed in an open yard with a shed area of about one third of the total area and individually fed (at 8 a.m.) a total mixed diet comprising 50% concentrate feed mixture (CFM), 30% corn silage (CS), and 20% alfalfa hay (AH) to meet the recommended requirements for growing sheep [A total mixed diet was offered every day at 8 a.m. Water was freely available throughout the day. Every two weeks, the amount of total mixed diet was adjusted to account for variations in live body weight.After a 16 h fast, lambs were weighed biweekly before feeding in the morning. Dietary conversions were calculated as kg of DM (dry matter), TDN , CP (crude protein), and DCP (digestible crude protein) per kg of live body weight (LBW). The ratio between the income from daily weight gain and the cost of daily feed consumed was used to assess the economic efficiency. The cost in 2020 for CFM was LE 5000 per ton, LE 600 per ton for CS, LE 3500 per ton for AH, LE 65 per kg for beeswax, and LE 55 per kg for live body weight gain.Three digestion trials were undertaken for three experimental groups to determine the nutrient digestion and feeding values of the tested diets using acid insoluble ash (AIA) as a natural marker . Fecal sTotal digestible nutrients (TDN) and digestible crude protein (DCP) were calculated by standard formula of McDonald et al. . TDN = (CP \u00d7 CPD + CF \u00d7 CFD + EE \u00d7 EED \u00d7 2.25 + NFE \u00d7 NFED)/100DCP = (CP \u00d7 CPD)/100CPD: Crude protein digestibility, CFD: crude fiber digestibility, EED: ether extract digestibility, NFED: nitrogen free extract digestibility.3-N) was determined according to the AOAC method [Rumen liquor samples were collected from three lambs each in group 3 h after feeding using a rubber ruminal probe. Rumen liquor was strained through double layers of cheese cloth and immediately measured for pH using an Orian digital pH meter. Ammonia nitrogen , glucose, cholesterol, total lipids, urea, creatinine, glutamic oxaloacetic transaminase (GOT) and glutamate pyruvate transaminase (GTP) were actually determined calorimetrically by spectrophotometer (Milton Roy company spectronic 20 D) using commercial diagnostic kits .p \u2264 0.05.The data gathered were statistically analyzed using IBM SPSS Statistics 28.0  [p < 0.01). G2 showed a significantly lower performance compared to G3, but increased growth compared to G1 (p < 0.01). The average daily gain of G2 and G3 increased by 16.75% and 35.81% compared to G1, respectively.The results of growth performance are reported in p < 0.05; p < 0.01).Feed conversion efficiency by lambs improved significantly (p < 0.01). On the other hand, G3 showed the highest price for weight gain (p < 0.01), net revenue, and economic efficiency followed by G2, but G1 registered the lowest values. Beeswax supplementation in G2 and G3 increased the net revenue by 25.06% and 54.10% compared to G1, respectively.The economic efficiency of lambs  showed tp < 0.05). The intake of TDN and DCP was the highest in G3 followed by G2, and the lowest values were registered in G1 (p < 0.01)  .p < 0.01). Nutrient digestibility was the highest in G3 for all considered parameters and feeding values, followed by G2, and then G1 which presented the lowest values .Blood serum parameters are presented in This study evaluated the use of beeswax supplementation in feed on performance, nutrient digestibility, rumen fermentation, blood metabolites, and economic sustainability in growing Assaf lambs.Staphylococcus aureus, Salmonella enterica, Candida albicans, and Aspergillus niger [Growth performance revealed that the G3 group showed the highest performance in terms of final body weight, total gain, and ADG. In contrast, the G1 group showed the lowest performance compared to the other groups. Although the detailed composition of the beeswax used in the present study was not analyzed, the results found might be attributed, at least partly, to the presence of bioactive compounds such as remains of pollen, metamorphosis wastes, and other compounds with antimicrobial, antioxidant, and other protective effects that possibly can promote animal health and performance . Indeed,us niger ,30.Additionally, recent studies have found that isorhamnetin derivates, kaempferol derivates, myricetin derivates, quercetin derivates, chlorogenic acid, and tiliroside obtained from beeswax byproducts exert antimicrobial and radical scavenging activity  and, thuFeed conversion efficiency showed the same trend as growth performance, revealing lower values for DM, TDN, CP and DCP per kg of body weight by increasing the beeswax supplementation. The feed conversion ratio is important for meat production when evaluating the feed conversion efficiency, and smaller feed conversion ratio values indicate a higher efficiency of feed use. Beeswax additive improved growth performance and reduced the amount of feed required for each kg weight gain. We have identified a paucity of information on the contribution of key biological processes, including appetite regulation, post-ruminal nutrient absorption, and cellular energetics and metabolism to the efficiency of feed utilization. Increasing the amount of feed consumed after the supplementation of beeswax suggests an improvement in the palatability of the feed, an increase in the animal\u2019s appetite, or a higher growth rate. Also, our study showed that beeswax supplementation at 4 g/head/day reduced the cost of gain as well as increasing the output of gain and all economical parameters. In addition, a previous study obtained an increased return and economic efficiency with bee bread extract supplementation  in Zaraibi goats considering the price of milk yield .The ingestion rate of DM and CP showed higher values in G3 compared to G1, and G2 revealed comparable values to G3 and G1; however, TDN and DCP were different among experimental groups. Similarly, DM intake by lactating goats was higher in a 3% beeswax-supplemented diet followed by 1% beeswax-supplemented diet compared to the control . This ef3-N concentrations, thus further supporting a possible effect on ruminal microflora. In rumen liquor samples of beeswax-supplemented groups compared to the control lambs, TVFAs increased significantly, while NH3-N and pH decreased. Rumen fermentation is a complex process involving animal physiology and microbiota [The rumen liquor analysis revealed a modulation of pH as well as TVFAs and NHcrobiota . Ruminancrobiota . It was crobiota , thus thcrobiota . A totalcrobiota  suggestsIn our study, blood serum metabolites showed a modulation after the supplementation of beeswax for 90 days of the experimental trial. The levels of total protein, globulin, and glucose were significantly higher and the albumin:globulin ratio was lower in G3 compared to the G1 group. In contrast, cholesterol, total lipids, urea, creatinine, GOT, and GTP values were lower in G3 compared to G1. Increased concentrations of total protein, albumin, and their ratio indicate an enhanced metabolism in response to higher feed consumption and nutrient digestibility, particularly regarding the protein component of the diet and an increased microbial protein supply from the rumen . GlucoseDietary supplementation of beeswax at 2 and 4 g/head/day to growing Assaf lambs increased growth performance by increasing the feed intake and digestibility of nutrients and modulated rumen fermentation and serum metabolites, thus resulting in a higher economic efficiency. Our results highlight the use of 4 g/head/day of beeswax as functional feed additive for growing Assaf Lambs aimed at enhancing animal performance, efficiency, and farming sustainability. Further studies will be necessary to fully characterize the beeswax and understand the role and interaction of different bioactive compounds contained in this valuable honeybee\u2019s byproduct."
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