{"text": "The aim of this analysis was, however, to identify the influencing factors on high catecholamine levels Various potential risk factors that could impact the humoral stress response before induction of anaesthesia were recorded in 84 males undergoing coronary aortic bypass surgery, and were entered into a stepwise linear regression analysis. The plasma level of norepinephrine measured immediately after radial artery canulation was chosen as a surrogate marker for the humoral stress response, and it was used as the dependent variable in the regression model. Accordingly, the mean arterial blood pressure, heart rate and the calculated pressure-rate product were taken as parameters of the hemodynamic situation.-1) on the morning of surgery was the only significant predictor (p = 0.004) of the high variation in preoperative norepinephrine plasma levels. This intervention decreased norepinephrine levels by more than 40% compared to no clonidine administration, from 1.26 to 0.75 nmol·l-1. There was no evidence for dose-responsiveness of clonidine. All other potential predictors were removed from the model as insignificant (p > 0.05). The use of beta-blocker, ace-inhibitors, ejection fraction, and body mass index were significant determinants for the hemodynamic situation  of the patient during the pre-induction period.Stepwise regression analysis revealed that the oral administration of low-dose clonidine (mean dose 1.75 μg·kgThe oral administration of clonidine is the only significant predictor for the observed variation of norepinephrine levels during the preoperative period. Lack of significant dose responsiveness suggests that even a low dose of the drug can attenuate the preoperative stress response and thus is recommended in cardiovascular high risk patients. Even ve drugs ,6-19.However, little attention has been paid to the stress response of cardiac high risk patients when entering the operating area, during initiation of routine monitoring, and finally during awake venous and arterial canulation. Especially the latter procedure can cause significant discomfort for patients even when performed under local anaesthesia . In seveAfter Ethics Committee approval was obtained, patients gave their written and informed consent. Eighty-four consecutive male patients undergoing CABG-surgery were enrolled into this observational study. The only exclusion criterion was emergency operation.-1 and the same-day coefficient of variation for norepinephrine measurements determined by repeated measures of a standardized probe was 3%.Due to the observational character of the study, drugs administered preoperatively (including clonidine) were given at the discretion of the anaesthetist performing the preoperative examination. Each patient received an oral premedication with clorazepate 20 mg in the evening before surgery and in the morning of surgery. In about half of the patients, benzodiazepine premedication was combined with clonidine 75–300 μg. Patients were maintained on their regular cardiac and antihypertensive medication up to the day of surgery but all inhibitors of platelet aggregation were discontinued 3–7 day preoperatively. After arrival at the operating theatre an i.v. line was initiated and 500 ml hydroxyethyl-starch  was infused. A 12-lead channel ECG with an automatic ST-segment analysis, oxygen saturation and invasive blood pressure monitoring were connected to the patients. A radial artery catheter was then inserted after local anaesthesia with 1 ml mepivacaine 1%. Heart rate (HR) and mean arterial blood pressure (MAP) were recorded every 15 seconds online using a Laptop computer connected to a Solar 9500 monitor . HR and MAP were multiplied to receive the pressure-rate product (PRP). These variables were used as a measure of the hemodynamic stress response. To determine humoral stress response, an arterial blood sample for the measurement of norepinephrine plasma level was taken immediately after placing the radial artery catheter (this measurement was the main outcome of the study) and 5, 15, and 60 minutes after endotracheal intubation was performed  and sufentanil (10 μg.kg-1.h-1). After loss of consciousness propofol was reduced to 3 mg·kg-1.h-1 and sufentanil to 1.5 μg·kg-1.h-1. Endotracheal intubation was performed after administration of pancuronium bromide 0.1 mg·kg-1.General anaesthesia was standardized. After administration of midazolam 0.05 mg·kgTo allow comprehensive analysis of potential factors associated with a reduced stress response, the following data were recorded prospectively:• Age• Bodyweight• Height• Body mass index (BMI)• Clorazepate dose per kilogram bodyweight• Clonidine (yes – no)• Clonidine per kilogram bodyweight• Time from morning premedication until observational period• Inhibitors of the angiotensine converting enzyme system (ace-inhibitors)• Beta-blocking drugs• Calcium antagonists• Angiotensin-2 receptor inhibitors• Left ventricular ejection fraction (EF)• Number of affected vessels2 of 0.3 and higher, attributed to 14 independent variables using an F-test with a significance level of 0.05. All potential relevant factors were subjected to a stepwise linear regression analysis using a backward technique. In each step the least significant factor was eliminated when F was lower than 3.96. The quality of the regression model was judged using the Durbin-Watson statistic , and by checking if the standardized residuals are normally distributed. All calculations were performed using SPSS 11.0 for Windows. All continuous data are presented as mean and standard deviation when normally distributed and as median (25th–75th percentile) when normal distribution had to be rejected using the Kolmogorov-Smirnov-test.A power analysis had revealed that 80 patients provide a power of more than 95% to detect an R-1) on the morning of surgery was the only significant predictor (p = 0.004) of the high variation in preoperative norepinephrine plasma levels. This intervention decreased norepinephrine levels by 40% compared to no clonidine administration (from 1.26 to 0.75 nmol·l-1). In this analysis, the dichotomous variable (clonidine administration: yes-no) was a better predictor for the norepinephrine levels than variables including the clonidine dose (absolute dose or dose per body weight), indicating that our data provide no evidence for a strong dose-responsiveness of clonidine in this context.Stepwise regression analysis revealed that the single administration of low-dose clonidine (mean dose 1.75 μg·kgAll other of the investigated factors (see methods) were removed from the regression model as not significant. The two factors that were eliminated with a p < 0.1 during the last but one and during the final step were body mass index (removed in step 11 with a p = 0.064) and age (removed in step 12 with a p = 0.076). Both factors were associated with increased norepinephrine levels.The overall quality of the regression model was excellent. The Durbin-Watson coefficient was 2.04 (very near to the optimum of 2.0) and the standardized residuals were normally distributed.For mean arterial blood pressure, heart rate and the calculated pressure rate product, however, preoperative clonidine administration was not an influencing factor.For the mean arterial pressure (MAP), a higher ejection fraction (EF) was a statistically significant predictor (p = 0.024). Each 10% increase of EF was associated with a 2.7 mmHg higher MAP. Administration of an ace-inhibitor was the second predictor in the final model of MAP (p = 0.03). These patients had a 7.5 mmHg lower MAP than patients without ace-inhibitors.-2 (p = 0.001).For heart rate (HR) there were three significant predictors that remained in the model. Administration of beta-blockers and ace-inhibitors were both associated with a decreased HR (p = 0.004). Each of them decreased HR between 6–7 beats per minute (bpm). Additionally, a higher BMI was associated with a 1.3 bpm higher HR per kg·mSince the PRP is the product of HR and MAP, it is not surprising that similar variables contributed to its prediction. These were the administration of beta-blockers (p = 0.017) and ace-inhibitors (p = 0.004), each of them reducing the PRP, whereas the EF was associated with an increase in PRP (p = 0.014).No patient had signs of cardiac ischemia on arrival at the operating theatre until induction of anaesthesia (defined as ST-T change > 0.1 mV in any ECG lead). There were no major adverse events during the entire induction period and surgery.2-adrenergic receptor agonist clonidine acts by decreasing central sympathetic nervous system activity in all hyperadrenergic situations. In addition to its sedative, anxiolytic, analgesic and antihypertensive properties [The αoperties ,22 it haoperties ,24 and toperties ,26.In many investigations attention has been drawn to the stressful stimulus of endotracheal intubation ,4,6-19, Thus, it was the major aim of this observational study to identify factors that might contribute either to increased humoral stress or that might help to attenuate this response.Our results show, that a single application of low dose oral clonidine was the only factor that was associated with significantly decreased norepinephrine levels on arrival at the pre-induction area. The question that arises from this observation is, if this is simply an association  or if clonidine premedication is the cause for lower norepinephrine levels. In our opinion the latter is the case. Firstly, there were no differences considering any other variables between those patients who had received clonidine and those who had not (see table -1) was low compared to all other trials. Data concerning the appropriate dose of clonidine to attenuate the stress response to intubation vary considerably between 0.625 and 10 μg·kg-1. For example, one trial demonstrated that clonidine 0.625 and 1.25 μg·kg-1 i.v. were sufficient to attenuate pressure response to laryngoscopy and intubation [-1 clonidine i.v. was equally effective as placebo, and only 4 and 6 μg·kg-1 significantly attenuated hemodynamic and adrenergic reactions in an equal manner. It could also be shown that 4 or 6 μg·kg-1 were necessary to reduce norepinephrine levels before induction of anaesthesia, however 2 μg·kg-1 where not sufficient in this setting [However, it is interesting to notice that the mean dose administered to our patients . Furthermore, a post-hoc comparison between the patients receiving either 75 or 150μg clonidine did not show relevant differences (p = 0.91 using the Mann-Whitney U-test).Higher age and higher body mass index showed a non-significant tendency to increase the catecholamine concentration. No other of the investigated factors  had statistically significant impact on norepinephrine levels.An explorative post-hoc analysis of the impact of clonidine premedication (none versus any dose) and clonidine dose on norepinephrine levels during the entire induction period proves the results of the main analysis. There was a pronounced reduction of norepinephrine plasma levels after induction of general anaesthesia with lower values in the clonidine-group. However, a statistically significant interaction term p = 0.012) suggests that the fall of norepinephrine levels are more marked in the untreated group and thus mainly caused by induction of general anaesthesia rather than effects of clonidine (figure 2 suggestThis observational trial demonstrates that patients undergoing coronary artery bypass graft surgery have a great variation of norepinephrine levels when entering the operating theatre. We could identify oral clonidine premedication as the only predictor for increased humoral stress response. There was no strong evidence for a dose dependency, indicating that even small doses, like 75–150 μg attenuate the humoral stress response before coronary artery bypass graft surgery. Clonidine did not have a negative impact on hemodynamic parameters.None declared.AMM processed the data and wrote the manuscript.GG conceived the study, collected the clinical data and participated in its design.US collected the clinical data.MK designed the study and collected the clinical data.HAA performed the laboratory investigations.HW participated in the conception of the study.LHJE designed the study, performed the statistical analysis and extensively revised the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
{"text": "The optimal ScvO2 cutoff value for morbidity prediction was 64.4%. In the first hour after surgery, significant reductions in ScvO2 were observed, but there were no significant changes in CI or oxygen delivery index during the same period.Data from 118 patients was analysed; 123 morbidity episodes occurred in 64 these patients. There were 12 deaths (10.2%). The mean ± SD age was 66.8 ± 11.4 years. Twenty patients (17%) underwent emergency surgery and 77 patients (66%) were male. The mean ± SD P-POSSUM  score was 38.6 ± 7.7, with a predicted mortality of 16.7 ± 17.6%. After multivariate analysis, the lowest cardiac index value (odds ratio (OR) 0.58 ; 2 occur in the immediate post-operative period. These fluctuations are not always associated with changes in oxygen delivery, suggesting that oxygen consumption is also an important determinant of ScvO2. Reductions in ScvO2 are independently associated with post-operative complications.Significant fluctuations in ScvO Readings may be taken intermittently by blood sampling and co-oximetry, or continuously with a spectrophotometric catheter. Experimental studies have shown that changes in ScvO2 closely reflect circulatory disturbances during periods of hypoxia, haemorrhage and subsequent resuscitation [2), although absolute values differ [2 in various groups [2 reductions to below 65% has been demonstrated in trauma [2 conducted so far used a goal of 70% [The successful use of central venous oxygen saturation (ScvOpatients . ScvO2 mcitation . Fluctuas differ . Observas groups . In partn trauma  and cardn trauma . Howeverl of 70% .2I) and related parameters and outcome after major surgery has been well described [2 values in the peri-operative period [2 disturbances is complex. The value of ScvO2 is determined by changes in oxygen delivery and consumption, both of which are subject to considerable variation during the peri-operative period [2 will be similar to those of other patient groups. If ScvO2 is to be used in the haemodynamic assessment of surgical patients, more detailed information is required describing fluctuations during the peri-operative period. The aim of this study was to describe changes in ScvO2 after major general surgery and their relationship to outcome.Although the association between cardiac index (CI), oxygen delivery index  [ScvOpy (GDT) . All patAll patients had arterial and central venous catheters placed before the commencement of surgery. The central venous catheter was positioned with the tip within the superior vena cava immediately above the right atrium. This position was verified by chest radiograph and adjusted if necessary. The following parameters were monitored continuously from arrival in the ICU immediately after surgery and for the next 8 hours: electrocardiograph, pulse oximetry, invasive arterial pressure, central venous pressure and cardiac output. Arterial and central venous blood gas analyses were performed by intermittent blood sampling and co-oximetry  at baseline and hourly during the 8 hours after surgery. This equipment was calibrated each hour, and routine quality control checks were performed. Cardiac output was measured by lithium indicator dilution and pulse power analysis . P-POSSUM  and APACHE II  scores were calculated at admission to the ICU ,18. Comp2I of 600 ml min-1 m-2 with fluid alone (GDT group). Once the 8-hour study period was complete, all patients received standard care for the remainder of their ICU and hospital stay. ScvO2 data were not used to guide clinical management at any stage.Protocols for cardiovascular management during the immediate post-operative period are provided in detail elsewhere . Fluid ct test where normally distributed and the Mann–Whitney U test where not normally distributed. Trends in physiological parameters over time in the two groups were compared with repeated-measures analysis of variance with Tukey's correction for multiple comparisons.Data are presented as means ± SD where normally distributed, as medians (interquartile range) where not normally distributed or, for categorical variables, as a percentage of the group from which they were derived. Normality was tested with the Kolmogorov–Smirnov test. Categorical data were tested with Fisher's exact test. Continuous data were tested with the p < 0.05 was set as the limit for inclusion of new terms. Results of logistic regression are reported as adjusted odds ratios (ORs) with 95% confidence intervals. Receiver operator characteristic curves were constructed to identify optimal cutoff values for association with outcome. The optimum cutoff was defined as the value associated with the highest sum of sensitivity and specificity. Analysis was performed with GraphPad Prism version 4.0 for Windows  and significance was set at p < 0.05.Univariate analysis was performed to test association with complications and death. For data recorded hourly during the study period, the baseline values, lowest values and the mean over the 8-hour study period were tested. A multiple logistic regression model was used to identify independent risk factors for post-operative complications. A stepwise approach was used to enter new terms into the logistic regression model, where 2 data were collected with a spectrophotometric catheter. Sixty-four patients developed 123 complications in all. There were 12 deaths (10.2%). The mean ± SD age was 66.8 ± 11.4 years. Twenty patients (17%) underwent emergency surgery and 77 patients (66%) were male. The APACHE II score was 9.5 ± 4.1, with a predicted mortality of 10.3 ± 9.0%. The P-POSSUM score was 38.6 ± 7.7, with a predicted mortality of 16.7 ± 17.6%. Fifty-seven (49%) patients were extubated within 1 hour of surgery and a further 29 (25%) were extubated before the end of the 8-hour study period.Data was collected from 117 patients between November 2002 and August 2004. Five patients were excluded from the analysis because ScvO2I and ScvO2 were frequently observed, other parameters remained within the normal range or were only slightly abnormal. Univariate analysis identified five variables associated with post-operative complications. These were the lowest ScvO2 value, the lowest DO2I value, the lowest CI value, the P-POSSUM score and the use of GDT. After multivariate analysis, the lowest CI value ; p = 0.018), the lowest ScvO2 value (OR 0.94 (0.89 to 0.98); p = 0.007) and P-POSSUM score (OR 1.09 (1.02 to 1.15); p = 0.008) were independently associated with post-operative complications. The lowest DO2I value and use of GDT were not independent predictors of outcome. The optimal value of ScvO2 to discriminate between patients who did or did not develop complications was 64.4% . Univariate analysis identified no associations with mortality.Commonly measured physiological, biochemical and demographic variables are presented in Tables 2. Those in whom the lowest ScvO2 value was 64.4% or below were defined as the low ScvO2 group and those in whom the lowest value was above 64.4% were defined as the high ScvO2 group  and the low ScvO2 group . DO2I and CI values did not change significantly during this time.Patients were divided into two groups by using the optimal cutoff value for ScvO2 after major general surgery that have prognostic significance. Multivariate analysis identified the lowest ScvO2 value, lowest CI value and P-POSSUM score as independent predictors of complications. This observation supports the hypothesis that the association between reductions in ScvO2 and outcome is similar to that observed previously for CI and DO2I [2I and the use of GDT suggests that the level of DO2I achieved by individual patients is more important than the approach to haemodynamic management.The major finding of this study is the occurrence of considerable fluctuations in ScvOand DO2I -13. It iand DO2I ,18. As m2 for prediction of complications was 64.4%. This is very similar to the value (65%) identified in other patient groups [2 occur during the peri-operative period. Values of ScvO2 decreased significantly during the first hour after surgery, while CI and DO2I remained unchanged. A significant increase in oxygen consumption therefore occurred during this period despite the fact that fewer than half of the patients were extubated within 1 hour of surgery. This finding is consistent with previous findings in cardiac surgical patients [2 cannot therefore be assumed to relate solely to DO2I.The optimal cutoff value of ScvOe value 6% identift groups . Large fpatients , as wellpatients . Post-op2 was 75.0% in patients who did not develop post-operative complications. This value was comparable to previous measurements in healthy conscious patients [2I and ScvO2 were observed in the absence of similar disturbances in other commonly measured biochemical and physiological variables. This was despite the high rates of morbidity and mortality in the study population. It is possible that disturbances in ScvO2, CI and DO2I might indicate the presence of occult tissue hypoperfusion before disturbances in other parameters.The 8-hour mean of ScvOpatients ,20, but patients  and in pThe use of observational data from an interventional trial has both advantages and disadvantages. In this study, goals for arterial oxygen saturation, haemoglobin, heart rate, mean arterial pressure, serum lactate and urine output were the same in all patients. All clinical management and data collection were closely supervised by a member of the research team in accordance with a carefully defined treatment protocol. The benefit of such rigorous study design must be offset against the fact that, in some patients, intravenous fluid administration was guided by central venous pressure, whereas in others fluid management was guided by stroke volume and supplemented with low-dose dopexamine. It is an inherent problem with studies of this type that the predictive nature of certain variables may relate both to the initial cardiovascular disturbance and subsequent attempts to correct it. The large number of statistical comparisons performed in the univariate analyses may seem speculative. This is not the case; comparisons made were of variables in which an association with outcome had previously been suggested ,17,18,212 are common after major surgery and are associated with an increased rate of post-operative complications. Peri-operative changes in ScvO2 relate to both oxygen consumption and delivery. Further evaluation of peri-operative trends in ScvO2 should be performed before this variable is used as a haemodynamic goal in surgical patients.Reductions in ScvO• \tThe successful use of central venous saturation in the management of severe sepsis has led to interest in the use of this variable in surgical patients.• \tThis analysis suggests that central venous saturation may have prognostic significance following major surgery.• \tFurther evaluation of peri-operative trends in central venous saturation is required.2I = oxygen delivery index; GDT = goal-directed therapy; ICU = intensive care unit; OR = odds ratio; P-POSSUM = Portsmouth Physiologic and Operative Severity Score for the enUmeration of Mortality and morbidity; ScvO2 = central venous saturation.APACHE = Acute Physiology and Chronic Health Evaluation; CI = cardiac index; DORP received a travel grant from LiDCO Ltd. to present data at an international meeting. JF has previously performed consultancy work for LiDCO Ltd. DB currently performs consultancy work for LiDCO Ltd. and has previously performed consultancy work for Deltex Ltd. No other competing interests are declared.RP, AR and DB were responsible for study design. RP, DD and JF were responsible for data collection. All authors were involved in data analysis and drafting the manuscript and approved the final version. All authors had full access to data and take responsibility for the integrity of the data and the accuracy of the analysis."}
{"text": "Endogenous airway acidification evaluated as pH in exhaled breath condensate (EBC) has been described in patients with chronic cough. Proton pump inhibitors improve gastro-oesophageal reflux (GOR)-associated cough.We examined pH levels in EBC and capsaicin cough response in 13 patients with chronic cough  associated with GOR before and after omeprazole treatment (40 mg/day for 14 days) and its relationship with clinical response.Omeprazole abolished symptoms associated with GOR. Patients with chronic cough had an EBC pH of 8.28 (SD 0.13) prior to treatment but this did not change with omeprazole treatment. There was a significant improvement in the Leicester Cough Questionnaire symptom scores from 80.8 points (SD 13.2) to 95.1 (SD 17) (p = 0.02) and in a 6-point scale of cough scores, but there was no change in capsaicin cough response.An improvement in GOR-associated cough was not associated with changes in EBC pH or capsaicin cough response. These parameters are not useful markers of therapeutic response. Chronic cough, conventionally defined as a cough persisting for more than 8 weeks, is a common respiratory problem and, at times, presents as a difficult management issue. Asthma, rhino-sinusitis and gastro-oesophageal reflux (GOR) have been identified as the most common diagnoses associated with chronic cough . GOR alo+ ions and this could contribute to the sensitised cough reflex measured with capsaicin since an acid environment has been shown to activate Aδ and C fibres in the airways of rodents [Exhaled breath condensate (EBC) is a simple non-invasive technique for the monitoring of airway inflammation, since it may be representative of the epithelial lining fluid. Endogenous airway acidification, as assessed by the pH of exhaled breath condensate, has been reported in patients with non-asthmatic chronic cough, including GOR . The fal rodents ,9.In order to examine further the significance of acid pH in the pathogenesis of GOR-associated cough, we measured pH of exhaled breath condensate in patients with chronic cough associated with abnormal lower oesophageal pH. We determined whether the improvement in cough associated with treatment with proton pump inhibitors was associated with changes in capsaicin responsiveness and in EBC pH.1  and FVC (103 ± 8.0%) were within the normal range. The chest radiograph was normal and histamine responsiveness measured as PC20  as greater than 16 mg/ml. Skin prick to common allergens were negative and they had no nasal symptoms. Eight of 13 patients reported symptoms of heartburn, regurgitation or dyspepsia; the rest were asymptomatic. All participants were non-smokers. All subjects gave informed consent to participate in the study which was approved by the Royal Brompton and Harefield NHS Trust Ethics Committee.We recruited 13 patients with chronic cough  defined as a cough persisting for more than 2 months, associated solely with GOR as defined by an abnormal 24-hour oesophageal pH measurement from our Cough Clinic. In these patients, we had excluded the presence of asthma and rhino-sinusitis. FEVAn ambulatory 24 hour pH study was performed with the Synectics Digitrapper Mk III . An Antimony pH electrode was placed just above the upper border of the lower oesophageal sphincter. An acid reflux episode was defined as a drop in pH below 4.0. Significant reflux was defined as the total duration of reflux episodes exceeding 3.4% of the total study time.Cough severity was assessed using the Leicester Cough Questionnaire . This coCapsaicin  obtained from Sigma-Aldrich, Gillingham, UK, was dispensed from a nebuliser chamber attached to a breath-activated dosimeter  set at driving pressure of 22 lbs/sq inch and a dosing period of 1 second. As described previously by Lalloo , the proExhaled breath condensate (EBC) was obtained non-invasively by using a condenser  that collected the nongaseous components of the expiratory air. Subjects breathed tidally through a mouthpiece and a two-way non-rebreathing valve, which also served as a saliva trap. They were asked to breathe at a normal frequency and tidal volume, wearing a nose clip, for a period of 10 min. If subjects felt saliva in their mouth, they were instructed to swallow it. The condensate (at least 1 ml) was collected on ice at -20°C, and was transferred to 15 ml Corning tubes. Measurement of pH was performed following de-aeration with argon (350 ml/min for 10 min), using a pH meter .EBC collection, spirometry and capsaicin challenge were performed on the same day in this order. These measurements were performed before and after treatment with omeprazole (40 mg/day for 14 days)10C5. All reported p values are two-tailed. A p value of less than 0.05 was considered statistically significant.Data were analysed using Graph-Prism version 3.0 . Data are expressed as the mean ± SD. Differences between groups were determined using the Mann-Whitney U test. Capsaicin C5 values were analysed as logThe 8 patients with symptoms of gastro-oesophageal reflux reported disappearance of these symptoms. Using the Leicester cough questionnaire, in which the patients assessed their cough and related symptoms on a scale from 19 to 133 points, the patients reported a partial but significant symptomatic improvement after two weeks of omeprazole treatment (80.8 ± 13.2 vs. 95.1 ± 17 points, p = 0.02; Figure After 2 weeks' treatment with omeprazole, we found a partial but significant clinical improvement in cough severity as assessed using the validated Leicester cough questionnaire. This was not accompanied by changes in capsaicin cough response or by changes in pH of the exhaled breath condensate. We conclude that these measurements do not reflect the clinical response. Additionally, omeprazole does not change the pH of exhaled breath condensate, most likely a reflection of the lack of change in pH of the epithelial lining fluid. This may also indicate that direct reflux of gastric acid into the upper airway is an unlikely explanation of GOR-associated cough.GOR is a common associated cause of chronic cough and treatment with gastric acid suppressing proton pump inhibitors is often effective in controlling cough ,5,6. We The baseline EBC pH value in our patients was not lower than that previously published for healthy controls ,14. HoweThe pathophysiological mechanisms underlying GOR-associated cough are not fully understood. Micro-aspiration of oesophageal contents into the larynx and tracheobronchial tree is one of the possible explanations . Our stuWe found that cough sensitivity to capsaicin was increased when compared to a group of historical non-coughing normal volunteers ,18. HoweIn conclusion, our results indicate that EBC pH measurement is not a good tool in the follow-up of GOR-associated chronic cough during treatment with a proton pump inhibitors. In GOR, episodes of micro-aspiration are short and do not produce a persisting level of airway acidification, which may be the reason why changes in EBC pH are not detected."}
{"text": "Chronic cough is a common problem, frequently caused or exacerbated by acid reflux. Diagnosis of acid reflux cough is haphazard currently, often relying on long therapeutic trials of expensive medications. We tested the hypothesis that the most relevant mechanistic component of acid reflux in chronic cough is when it rises to the level of the airway where acid can potentially be aspirated. We further wished to determine if multi-sample exhaled breath condensate (EBC) pH profiles can identify chronic cough patients likely to respond to proton pump inhibitor therapy.59 subjects were recruited for this study. Initially we examined EBC pH (gas-standardized with Argon) in the setting of 15 experimental pharyngeal acid challenges to determine duration of EBC acidification. Subsequently, we enrolled 22 healthy subjects to determine a normal multi-sample exhaled breath condensate pH profile over 1–3 days. We additionally obtained multi-sample EBC pH profiles in 22 patients with chronic cough. These samples were timed to occur after coughing episodes. Exhaled breath condensate pH was measured after gas standardization.We found that exhaled breath condensate pH is substantially reduced for approximately 15 minutes after pharyngeal acid load. Healthy subjects rarely have any low EBC pH values . Patients with chronic cough who subsequently responded well to proton pump inhibition (n = 8) invariably had one or more cough episodes associated with EBC acidification. No patient who had normal EBC pH with each of their cough episodes reported a clinically relevant response to proton-pump inhibition.Patients whose cough responds to proton pump inhibition have transient exhaled breath condensate acidification with coughing episodes, supporting the role of airway acidification in reflux-triggered cough. Multi-sample EBC pH profiles, involving samples collected immediately subsequent to a coughing episode, may be useful appropriately to direct therapy to those patients with cough who have relevant acid reflux. Cough is a leading reason patients consult respiratory physicians. Gastric acid reflux up the esophagus is a well-recognized cause of chronic cough both in the presence and absence of underlying lung or airway diseases. Two mechanisms of this cough have been demonstrated: 1) reflux high into the laryngeal/hypopharyngeal region with laryngeal acid contact with or without aspiration into the airway; 2) esophageal acid contact. Both of these sites of acid exposure lead to cough through vagal-mediated reflex pathways and neurogenic inflammation, but importantly the first also leads to the diverse pathologies resulting from the direct acid injury to the airway[Data are mixed about the utility of proton pump inhibition (PPI) for the treatment of suspected acid reflux cough,3, and iOne explanation is that studies have enrolled the wrong patients. Most studies of acid reflux cough were designed to recruit subjects with respiratory symptoms who also had symptomatic or esophageal pH probe evidence of gastro-esophageal reflux disease (GERD). However, the amount of acid in the airway necessary to trigger airway symptoms such as cough is substantially lower than the amount of acid reflux necessary to trigger esophageal symptoms. Whereas 4% of more esophageal acid contact time may be abnormal from an esophageal standpoint, any acid contact time in the airway, even for moments, is likely capable of causing pronounced symptoms. Esophageal symptoms are commonly not present in patients with acid reflux cough. For theWe hypothesized that acidification of the hypopharynx, such as occurs when gastric acid refluxes above the upper esophageal sphincter into the hypopharynx, should cause exhaled breath condensate(EBC) to be acidic(after gas-standardization). We examined this hypothesis by means of pharyngeal acid challenges. We then tested for spontaneous hypopharyngeal gastric acid reflux by performing EBC pH testing in patients suspected of having acid reflux cough based on history and physical examination. Over a period of one or more days, we tested for acidic breath multiple times per subject, within several minutes of coughing episodes. We compared the EBC pH profiles thus obtained with responsiveness of the chronic cough to a 1 month treatment course with twice daily PPI therapy. This comparison allowed an examination of the ability of the EBC pH profile to predict responsiveness to PPI therapy, which functions as a diagnostic gold standard of sorts. This study design also provided evidence of effectiveness of PPI therapy in the selected population.56 subjects were recruited from the region of the University of Virginia during 2005. Subjects consisted of patients with chronic cough derived primarily from the adult and pediatric pulmonology, allergy and otolaryngology clinics, as well as controls obtained by convenience within the University. The principal enrollment criterion for chronic cough patients was the intention of their doctor to initiate a therapeutic trial of proton pump inhibition as an effort to make a diagnosis of possible acid reflux cough. Chronic cough needed to have been present daily for at least 6 weeks. Minimum age was set as 5 years. To assure real-world utility of the study, subjects with chronic cough were included without regard to the presence or suspicion of other diagnoses, but solely on the basis of the physician's planned therapeutic trial. Exclusion criteria included use of PPI or H2 antagonists within the past 7 days, or a previous attempt to treat the cough with acid blockade. Additionally, if other medication regimen changes were made concurrently, the patients were excluded. No patients had undergone esophageal pH probe testing. The studies were approved by the Human Investigation Committee at the University of Virginia and all subjects provided informed consent.Individual EBC samples were collected without nose clips during 5 minutes of relaxed breathing through a single-use disposable RTube EBC collector , with initial temperature of between -4 and -17°C. The RTube device consists of a polypropylene condensing surface kept chilled with a reusable aluminum cover. Two one-way valves serve to direct exhaled air appropriately through the condenser.To determine how long EBC pH might stay abnormal after a pharyngeal acid challenge, 15 subjects performed EBC collection in the laboratory followed by rapid ingestion of 50 milliliters of an acidic beverage  in a reverse model of acid reflux. They then collected 6 consecutive EBC samples (5 minutes each) for 30 minutes. For comparison, 5 subjects performed the same set of EBC collections, but after ingesting 50 milliliters of tap water (pH 7.8).Subjects were provided a collection kit consisting of 8 disposable RTube EBC collectors and a pre-addressed express mailing box . This sampling procedure was developed specifically to create a multi-sample EBC pH profile for the subjects, which is a clear distinction from all previous published EBC studies. The subjects were asked to collect the 8 EBC samples in their home or work over a period of 1 to 4 days, to include at least 2 samples when they had not been coughing for the previous 1 hour. The remaining samples were requested to be collected specifically when the subject had experienced a coughing episode within the previous 10 minutes. Sample collection duration for each collection was requested to be 5 minutes, and no nose clips were worn. Temperature of collection was determined by the home freezer temperature , which was used to chill the aluminum that provides the cool temperature for condensation in the RTube collection system. Subjects were asked to not collect any samples within one hour of any liquid or food ingestion. After collection of each sample, the subject wrote the date and time of collection on the RTube label along with checking a box to discriminate whether this was a \"cough\" or \"no cough/well\" sample. Each sample was stored in their home freezer until 4 days had passed or all 8 collections were completed, whichever came first. At that point, the subjects placed the RTube EBC samples into the return mailing container for shipment (2-day) to the investigators' laboratory. During this shipping, samples thawed and were not temperature controlled . Healthy subjects provided 8 EBC samples in similar fashion as delineated above, but because they had no cough, the samples were collected at conveniently spaced times during the course of 1–4 days.After providing the multiple EBC samples as described above, chronic cough patients began taking a proton pump inhibitor as prescribed by their physician. The therapeutic regimen was determined by their doctor, and no specific medication or dosing was mandated by the study although all patients were prescribed the medication as a twice daily regimen. Assessment of response to PPI was based on a subjective scoring scale performed 1 month after starting the PPI. Subjects were asked if their cough was 0, 25, 50, 75 or 100 percent improved. 75 % or better was determined in advance of the study to be considered a \"PPI responder.\" An improvement of 0 or 25% was considered a \"non-responder.\" 50% improvement was considered an equivocal response.Upon receipt into the laboratory, data were recorded from the labels of the RTube EBC collectors and the samples removed by plunging the condensers with the internal syringe plunger. A 250 microliter aliquot of EBC was gas-standardized by bubbling with Argon for 8 minutes at 350 ml/min prior to pH measurement, which was performed with an Orion pH glass combo microelectrode attached to an Orion 520 A meter as previously reported[A positive EBC pH profile prospectively was defined as one in which one or more or the patient's coughs occurred with a concurrent low EBC pH value , while The effect on EBC pH of pharyngeal acid challenge was examined graphically and EBC pH at each time period compared by ANOVA on Ranks followed by Dunn's test. Comparison of individual EBC pH values was accomplished by Mann-Whitney Rank Sum, and the number of low EBC pH values for each group compared by Chi-squared analysis. The ability of the system of EBC collections and resulting EBC pH profile in each patient to predict cough responsiveness to PPI therapy was analyzed by Fisher exact test.Ingestion of acidic beverage (as an effort to temporarily acidify the hypopharynx) caused a rapid and pronounced and significant EBC pH decline that persisted for 10–20 minutes , the median (25–75% range) pH of all EBC samples was 8.1 (8.0–8.2)  which was essentially identical to the Paget-Brown normative database of 404 individual collections from healthy subjects. In rega22 chronic cough subjects performed the EBC pH profile (age 28 ± 23 years). These patients were likely to have various causes of their cough. In these subjects, the median EBC pH of all the individual samples collected was 7.9 , which was only minimally, but statistically significantly, lower than controls (p < 0.001). 29/166 samples revealed a low EBC pH value (p < 0.001 compared to this proportion in controls).Of the 22 patients prescribed PPI therapy, 17 patients filled the PPI prescription and initiated therapy as directed by the physician. The other 5 patients never filled the prescription, however follow-up information was available from all subjects. EBC pH profiles were compared to responsiveness to PPI therapeutic challenge or to untreated outcome. After 1 month of therapy with PPI, 8 subjects reported a positive response (75% or more improvement in cough symptoms) and 9 subjects were classified as non-responders (0 or 25% improvement). No subject reported a 50%  improvement. Of the 5 patients who did not start the PPI, all reported substantial resolution (75–100%) of symptoms spontaneously.Subjects whose cough responded to PPI therapy were significantly more likely to have one or more of their coughing episodes occurring in the setting of a low EBC pH than those who did not respond to acid blockade (p = 0.001). 14 out of 32 coughing episodes in these 8 PPI responders occurred in association with a low EBC pH value, and an additional 4 had equivocally low pH value (pH = 7.4). Of the coughing episodes in the 9 PPI non-responders, only 1 out of 47 occurred in association with a low EBC pH, and none had equivocal values . These individual assay data points are graphically presented in Figure profiles derived from all samples collected from each individual subject along with the recorded concurrent symptoms. This was possible because of the availability of a kit designed for multiple collections of EBC samples in patients' homes. To demonstrate this multi-sample EBC pH assay system more clearly, a typical positive EBC pH profile from this study is shown in Table One key purpose of this study was to move away from reliance on analysis of single EBC values, and instead to investigate EBC pH Interpreting the data in this context of a multi-sample profile drew a much sharper contrast between PPI responders and non-responders than did the use of individual collections. In this study, the predictive value of a positive EBC pH profile , in terms of responsiveness to PPI, was 89%. The predictive value of a negative EBC pH profile was 100% Figure .Of the 5 subjects who did not begin taking their PPI medication as prescribed, all showed spontaneous resolution of the cough when contacted at one month. 4 of these 5 subjects had a negative EBC pH profile.Three subjects provided a repeated series of EBC collections in their homes after treatment with PPI and cough resolution. Although few in number, these EBC pH profiles all normalized, with only 1 low EBC pH being found in 24 samples (8 samples each) with that sample having been obtained in the absence of preceding cough).The EBC pH profile developed for this project is novel methodology that distinguishes this approach from previous investigative efforts in which only individual data points were evaluated.In this study, the presence of one or more episodes of cough with a concurrent low EBC pH value in any subject's EBC pH profile strongly predicted responsiveness of the cough to proton-pump inhibition. In the absence of any coughing episodes occurring in association with a low EBC pH value, the patient's responsiveness to proton pump inhibition was minimal or non-existent. The patients were recruited into this study based on the intention of their respiratory medicine physician to initiate a trial of proton pump inhibition for suspected acid-reflux cough. Despite being enrolled from a subspecialty clinic, half of such patients did not respond to a one-month trial of twice daily PPI therapy, and these PPI therapeutic failures could be well identified by a multi-sample EBC pH profile revealing a series of coughs associated with normal EBC pH values.Trials of proton pump inhibition have become common for diagnosis of respiratory symptoms associated with acid reflux. This stEsophageal pH probes are not sufficiently helpful for diagnosis of acid reflux cough. They are expensive, uncomfortable, and neither particularly sensitive nor specific. In clinOur findings regarding EBC pH and responsiveness to proton pump inhibition differ from those published recently, but theA final, and critical, difference is that we collected multiple samples from each subject to develop an EBC pH profile, and this profile allows for much greater sensitivity of the procedure by enhancing the likelihood of finding a correlation of cough with acidity if it is indeed present in a given patient.Coughing may trigger a pharyngeal reflux event, although studies specifically examining concurrent acid reflux and cough find that it is far more common for the reflux to precede the cough. AlthougEBC pH has been reported to be low in acute asthma, stable moderate-to-severe asthma, COPD, and the common cold. We beliDistinguishing acid reflux-induced airway acidification from primary lower airway acidification possibly may be accomplished by seeing normal EBC pH values close in time to low EBC pH values. Reflux leads to rapid airway acidification . Rate of neutralization of this acid insult likely varies, but seems to be rapid in general. A persistently low EBC pH value over the course of hours may be more suggestive of an acidification process other than reflux.Our study would have benefited from a more objective cough score as opposed to a subjective scoring system that has not undergone extensive validation. However, we believe this is overcome in this study because the determination as a PPI responder or a non-responder (in terms of subjective cough score) was in no case equivocal. The number of subjects enrolled was sufficient to identify a highly statistically significant association between a low EBC pH (with cough) and responsiveness of cough to proton pump inhibition.Although there was a trend for different sex distributions between the control groups and the coughing subjects, there were no statistically significant differences in the sexes (by Fisher Exact Test). Additionally, there were no statistically significant differences in the ages of the groups (by ANOVA). However, as a general comment on the likelihood that a patient will respond to PPI therapy, there was a trend for the PPI responsive group to be younger than the PPI non-responders.In conclusion, we have tested the ability of serial (multi-sample) collections of exhaled breath condensate with gas-standardized pH measurement to identify – with high positive and negative predictive values – the likelihood of a patient having a positive response to proton pump inhibition prescribed for their chronic cough. Our data suggest that airway acidification occurs in PPI responders, supporting that hypopharyngeal acidification and probably microaspiration are important contributors to PPI responsive acid reflux cough.Multi-sample measurements of EBC pH in potential study volunteers may be able to decrease the confounding influence of acid reflux cough in future studies designed to test the efficacy of new therapies aimed at the non-acid components of COPD, asthma, and other respiratory diseases. This method of serial EBC pH testing allows for earlier non-invasive diagnosis of acid reflux as a cause of a patient's cough. It should also help more rapidly and efficiently direct the use of PPI medications to the patients likely to respond. Although not yet studied, it is reasonable to expect that this non-invasive tool will lead to more efficient use of medical resources, for example limiting the need for pH probes. This testing has recently started to be used in clinical practice and represents the maturation of the EBC research technique into a rational clinical diagnostic.EBC Exhaled Breath CondensatePPI Proton Pump InhibitorGER Gastroesophageal RefluxGERD Gastroesophageal Reflux DiseaseJH and BG are cofounders of Respiratory Research, Inc., a company that manufactures the exhaled breath condensate collection equipment used in this study. They are both inventors and intellectual property holders of EBC pH assay methodology.JH – first and senior author; YY – planned experiments and data collection, assisted with manuscript preparation; JB – patient recruitment, study design and manuscript preparation; BG – assisted with study design, scientific development, subject recruitment; LN – study design, data collection; DB – clinical research coordinator, patient recruitment, follow up, and interactions with Human Investigation Committee; BW – study design, manuscript assistance, patient enrollment; AS – initial study preparations, assay development, manuscript assistance; SH – patient recruitments, documentation development, manuscript preparation."}
{"text": "Chronic cough is a common symptom which causes significant levels of morbidity. It is becoming increasingly well characterised by research taking place in specialist cough clinics, where successful treatment rates are high. However, there is a paucity of data regarding the symptom complex of chronic cough in the community. This report details the results of a postal questionnaire survey sent to individuals requesting further information on chronic cough.856 chronic cough questionnaires were sent out to members of the public who requested an information sheet following a national UK radio broadcast. Information regarding demography, history of cough, previous treatment and physical, psychological and social effects of the cough was elicited.373 completed questionnaires were returned. Mean age was 65.3 years . 73% were female and 2% current smokers. Median duration of cough was 6.5 years. 66% had no other coexisting respiratory diagnosis, whilst 24% reported asthma. Of those who responded, 91% had consulted a general practitioner regarding the cough and of them, 85% had been prescribed some sort of treatment. 61% had seen at least one hospital specialist. Commonly reported associated physical symptoms included breathlessness (55%), wheeze (37%), fatigue (72%) and disturbed sleep (70%). Incontinence occurred in 55% of women. Similarly, the majority reported psychological effects such as anger or frustration (83%), anxiety (69%) and depression (55%). 64% felt that the cough interfered with their social life.Chronic cough causes a high level of morbidity in the community, which results in a correspondingly high rate of healthcare utilisation. Demography and symptomatology seems to be similar to that reported from specialist centres, but successful treatment of the cough was uncommon, despite a high number of medical consultations in both primary and secondary care. If understanding of this debilitating but eminently treatable condition is enhanced, management of chronic cough will improve and many patients will be helped. Cough is the commonest symptom for which medical advice is sought ,2, and sMost reports of the aetiology and management of chronic cough originate from specialist cough clinics and therefore reflect the experience of chronic cough in secondary care. Indeed, good data on the prevalence and aetiology of cough in the general population are hard to find. For example, the European Community Respiratory Health Survey targeted a large, unselected group from the general population  and included questions on cough . HoweverThis report details the results of a postal questionnaire survey sent to people requesting further information on chronic cough. It provides further information about the demographic and symptomatic profile in a population who consider their cough to be significant.In September 2002 a national UK BBC Radio 4 broadcast took place on chronic cough. This was part of the series \"Check Up\", which offers medical advice on a different health-related topic each week and is broadcast at 3 pm on a Thursday afternoon. Radio Joint Audience Research Limited (RAJAR) published audience figures for Radio 4 of 9.9 million listeners per week  for the third quarter of 2002. The BBC estimated that approximately 700,000 people will have listened to this broadcast. Unfortunately, the authors do not have access to specific demographic data on this program's audience.Of this population, 856 members of the public wrote in with stamped addressed envelopes for an information sheet about chronic cough. The information pack they were sent included a cough questionnaire , with 73% of them being female. 152 (41%) were ex smokers and 8 (2%) were current smokers with a median of 8.0 pack years in these 2 groups (range 0.2–135).Duration of cough ranged from 2.5 weeks to 73 years with a skewed distribution. The median duration was 6.5 years, but 40% of respondents had experienced cough for less than 5 years  respondents, but only 95 respondents (25%) had one or more first-degree relatives with asthma.Only 34 (9%) of the 373 respondents had not consulted their general practitioner about their cough. Of those who had seen the general practitioner 288 (85%) had been prescribed some sort of treatment for the cough. 226 respondents (61% of the whole sample) had seen one or more hospital specialists regarding cough, with 2 people having seen 5 specialists. Of those who had consulted a specialist, 155 (69%) had seen a respiratory physician.A wide range of medications were reported as having been prescribed for the cough with inhaled steroids and beta 2 agonists being the most common. However, despite the high rates of prescribing, 60% said that their symptoms had not been improved by any treatment. Treatments that were perceived to have helped the cough included inhaled steroids, cough syrup, lozenges and water (see figure Cough was commonly associated with other symptoms such as breathlessness (55%), wheeze (37%) and feeling tired or drained (72%). In addition, cough resulted in disturbed sleep in 70%, sore throat in 45% and caused incontinence in 55% of women and 5% of men. Whilst cough syncope was reported by 37 (10%) of subjects, the relatively minor complaint of dizziness on coughing was described by 95 (26%). Most respondents (77%) did not have chest pain as a result of coughing.62% of respondents complained of sputum production but only 6 (1%) expectorated more than 1 cup of sputum per day with 28 (8%) experiencing haemoptysis at some time. 158 (42%) respondents also had heartburn and 250 (67%) complained of post-nasal drip. In addition, cough affected the voice of 67% of respondents. The majority of respondents (63%) were unable to suppress their cough and activities commonly affected by cough included shopping (33%), housework (34%), climbing stairs (24%) and mealtimes (55%).Psychological effects of the cough were common. 83% of subjects felt anger or frustration as a result of cough and 76% felt out of control of their body. In addition, cough made 69% of responders worry about their health, 55% feel depressed, 80% upset and 76% worried about what others might think. However, only 55 (15%) felt that their cough made them dependent on others with 40% of respondents saying that the cough seldom or never significantly altered their lives.64% of respondents felt that cough affected their social life. Many described altering their behaviour such as how often they go to the cinema/bingo (39%) or restaurants (34%) and avoiding things that trigger the cough (60%). For example, 71 respondents (19%) said their cough affected how often they visited friends or relatives. Other areas affected by cough included phone calls (81%) and hobbies (45%).Although only 169 (45%) of the respondents were in employment, 53% of them felt it was affected by the cough. 5 of the 20 smokers (25%) said that cough affected how many cigarettes they smoked.In the past it has been difficult to provide accurate data on the epidemiology of chronic cough. A number of questionnaire surveys have tried to evaluate the prevalence of respiratory symptoms ,6, but tUntil recently understanding of the effects of chronic cough on health status has been limited, although work on cough specific quality of life tools is now starting to provide us with measurable health outcome data ,10. Howeall subjects in the community with cough, due to the usual types of selection bias associated with this type of study. Self selection of questionnaire respondents and factors such as time of day, mode and network of the broadcast mean that the demographics of listeners to the radio broadcast cannot be expected to be wholly representative of the general population. However, the large estimated audience of 700,000 suggests that they are drawn from a wider group than the population usually seen in a specialist cough clinic and the fact that they have responded to an unsolicited questionnaire suggests that these data represent a profile of a clinically relevant group suffering from a troublesome chronic cough.This study population evidently can not be said to represent Notable similarities exist between the demography of our study population and that described in previous reports from secondary care. For example, the high proportion of females (73%) is similar to that reported in the recent literature, with published series from various specialist clinics consisting of between 55% and 78% females . In clinPast experience reveals that although smoking is known to be associated with a dose related increase in reported cough , in pracThis survey confirms that chronic cough is poorly treated in the studied population. Despite a high rate of medical consultations and of prescribing the median duration of cough was still 6.5 years. 24% of respondents claimed to have a pre-existing diagnosis of asthma and 32% had been prescribed either oral or inhaled corticosteroids at some point, but only 9% of respondents reported that these treatments had helped at all. This may be due, at least in part, to the self selected nature of the population as individuals who had gained good effect from prescribed medications might be less likely to respond to the questionnaire; however other explanations are also possible. For example, the accuracy of the diagnoses of asthma cannot be confirmed as we have no information regarding who the diagnosis was made by, or the grounds on which it was made. Even if a correct diagnosis of asthma has been made, this does not rule out the presence of some other additional cause of cough such as reflux disease, which would not improve with steroid treatment. In addition, we have no information regarding the dose or duration of treatment which, if inadequate, might contribute to the likelihood of treatment failure.Cough syrups, lozenges and water, however, ranked highly as treatments that were alleged to help the cough, outranking many prescribed treatments such as beta-2 agonists and nasal steroids. Only 10% of respondents reported that cough syrups and sweets had been prescribed but, when asked which treatments (prescribed or self-medicated) had helped the cough, approximately 10% of respondents stated that syrups or sweets had helped and 12% gave answers such as cold water, chewing gum, alcohol, etc, which were grouped in the \"Other\" category in this report. This may simply reflect the fact that these remedies are much more freely available to the public than prescription medications, but it is interesting to note their perceived efficacy especially since most over the counter cough remedies rely on similar demulcent and non-pharmacological strategies which may have previously been ascribed to \"placebo effect\" . Their rThe impact of chronic cough on health status is varied, ranging from minimal in some patients to a disabling symptom in others. However, the reasons which lead patients to seek advice are complex and poorly understood . Work deAlthough this questionnaire was not designed to be a diagnostic tool, there were several questions which may give clues as to the possible underlying causes of the cough. Previous work suggests that gastroesophageal disease, asthma and rhinitis are the most common causes of chronic cough . In thisThe presumption that chronic cough represents a significant burden on NHS resources and especially on primary care services, is supported by the observation that 91% of respondents to this survey had consulted a general practitioner about the cough and 60% had seen at least one hospital specialist. However, the fact that only 40% of respondents had found a treatment that helped indicates that it is sub-optimally managed in this population, since several series of systematic management show treatment success rates in excess of 90% . AlthougIn conclusion, we have shown that chronic cough causes a high level of morbidity in affected individuals, which results in a correspondingly high rate of healthcare utilisation by these individuals. In the authors' opinion, chronic cough is currently poorly diagnosed and managed outside of specialist cough clinics, mainly due to a widespread lack of knowledge of the aetiology of this debilitating, but eminently treatable symptom. If understanding is enhanced, management of chronic cough may improve and many patients will be helped.CFE collated and analysed data from the returned questionnaires and drafted the manuscript. JAK and RHT both participated in design of the study and of the study questionnaire. RHT also collected and collated data from the questionnaires. AHM conceived of the study, participated in its design and coordination, took part in the initial radio broadcast and helped to draft the manuscript. All the authors read and approved the final manuscript.Chronic cough questionnaire. Blank template of the postal questionnaire survey which was sent to people requesting further information on chronic cough, following the Radio 4 broadcast.Click here for file"}
{"text": "Despite extensive diagnostic evaluation and numerous treatment trials, a number of patients remain troubled by a chronic and uncontrollable cough. Eosinophilic bronchitis, atopic cough and non-acid reflux have been recently added to the diagnostic spectrum for chronic cough. In some cases, failure to consider these conditions may explain treatment failure. However, a subset of patients with persisting symptoms may be regarded as having an idiopathic cough. These individuals are most commonly female, of postmenopausal age and frequently report viral upper respiratory tract infections as an initiating event. This paper seeks to explore the validity of idiopathic cough as a distinct clinical entity. Despite considerable advance in the understanding of cough, the effective management of patients with a chronic cough can be difficult. For the patient, a cough which persists can be associated with considerable distress and impaired quality of life . For theidiopatheia and is defined in the Oxford English Dictionary as a 'disease not preceded or occasioned by another, or by any known cause' [The term 'idiopathic' comes from the Greek word n cause' . In the n cause' . Using an cause' . The subn cause' ,8 and a n cause' ,10 has rn cause' .Although the systematic evaluation of both extrapulmonary and pulmonary causes for cough is widely held to be effective, doubt has been cast on the perception that the diagnostic triad of CVA, PNDS and GORD accounts for the almost all causes of chronic cough ,13. DespThere are a number of possible explanations for the impressive treatment response described by Irwin and others. Firstly, it is probable that the original referral populations included patients with cough following a viral upper respiratory infection. It is now recognised that cough following an upper respiratory tract infection may persist beyond three weeks and only resolve spontaneously some weeks or months later. Therefore some of the 'treatment success' may merely have reflected the natural resolution of a prolonged post-viral cough. Secondly, many patients were prescribed older generation antihistamines, which have an imprecise pharmacological action but presumably exert most of their antitussive effect by crossing the blood-brain barrier and acting directly on the cough control centre within the brain. Crucially, response to such therapy tells us little about the aetiology of the cough. Finally, these original studies reported on short-term treatment outcomes and provided little information on the long-term treatment response. Initial treatment benefit may well diminish over time and the timing of patient follow-up may explain some of the variance in outcome described by different centres .Current guidelines have recommended a combination of diagnostic testing and empirical trials in the management chronic cough . Some auAlternatively, some individuals on relatively high doses of acid suppression may exhibit relative proton pump therapy resistance. This is particularly the case with attempts to suppress proximal and laryngophayngeal reflux where despite single and higher dose treatment regimes, 44% of patients demonstrated abnormal levels of acid exposure on simultaneous oesophagel and laryngeal pH testing . FinallyGiven the extent of associated literature, it is barely conceivable that any respiratory physician is unaware of the most common associations with chronic cough, namely asthma, GORD and rhinosinusitis, more recently termed upper airway cough syndrome. In the last decade, a series of important observations have led to the appreciation of new diagnostic possibilities. Most importantly, the application of induced sputum in the evaluation of cough has led to the identification of eosinophilic airway syndromes . These cThe concept of 'Non-acid reflux' has recently gained attention. Irwin and colleagues  reportedFinally an association between cough, GORD and a familial sensory neuropathy has recently been reported . The locThe common and less common associations with cough must be rigorously excluded before a diagnosis of idiopathic cough can be assigned. None-the-less, this author firmly believes such a condition exists and it will be addressed in some detail in the following section.The accumulation of experience and information regarding idiopathic cough suggests a fairly well defined population of patients. The over-representation of women in the specialist cough clinic referral population is widely acknowledged, and the preponderance of females among idiopathic coughers is particularly striking. Some centers have reported female prevalence rates of more than 80% -16,30-33The human cough reflex consists of an afferent arm comprising cough receptors, afferent pathways, central processing and an efferent pathway. The cough reflex can be modified at any point along this reflex and unraveling the mechanisms responsible is key to a more complete understanding of cough pathophysiology and its successful treatment. Afferent sensory nerves are not static entities and the term 'plasticity' has been used to describe changes in function contributing to the sensitization that occurs in response to various stimuli, in particular those associated with airway inflammatory processes . AlthougOnly a few studies have specifically commented on findings in the airways of patients with idiopathic cough. Birring et al. observed a mild chronic lymphocytic airway inflammation in a predominately female population of idiopathic coughers and highlighted the striking association with organ specific autoimmune disease in particular hypothyroidism . They suGender and sex hormones may have important effects on neuro-immune events within the airway. A number of studies have demonstrated a heightened cough reflex sensitivity in females compared to males both in healthy individuals ,43 and cAlthough inadequate management will continue to explain a significant number of patients with a chronic and uncontrollable cough, an attempt has been made in this article to highlight idiopathic cough as a distinct clinical entity. Although without firm evidence, idiopathic cough may arise as a consequence of the persisting effects of viral infection or other noxious aggravants in susceptible individuals. The excess of middle-aged females with idiopathic cough raises the possibility of some sex hormonal influence. Precision in this area will be greatly hampered unless further research is undertaken."}
{"text": "For many years, the term 'specific cough' has been used as a clinical cough descriptor in children to signify the likelihood of an underlying disease causing the cough. In this case study, we describe a child with specific cough caused by a rare carcinoma, a mucoepidermoid carcinoma of the bronchus. The cough only totally resolved after the primary cause was successfully treated. This report highlights the importance of following up children with cough, especially those with specific cough. An 8-year-old girl from a remote Aboriginal community approximately 2500 km from Brisbane was transferred to our hospital for management of a bronchial lesion. She had received 7-days of intravenous amoxicillin prior to transfer. She had a 4-year history of daily wet and sometimes productive cough, which was worse on exertion. There was no history of exertional dyspnoea, haemoptysis or weight loss. She also had a history of recurrent admissions for pneumonia at the local hospital (3 in the past 6 months). In the child's community, two adults were recently diagnosed with active pulmonary tuberculosis.th percentile, height 25th), appeared well and had a wet cough, reduced air entry over the right side and inspiratory crepitations. Spirometry values were invalid as she could not adequately perform maximum expiratory manoeuvres. Chest x-ray (CXR) showed right upper lobe (RUL) collapse, tram-tracks signs and increased peribronchial and interstitial markings of the right lower lobe. These CXR changes were documented at least 4-months ago  were negative, sweat test and immunological workup were normal. Flexible bronchoscopy revealed a large lesion at the carina (Figure On arrival, the child was thin (weight 5 figures  and 2. C figures  and 4. SWe have described a child with several features of chronic specific cough caused by suppurative lung disease secondary to a rare life threatening lesion, a mucoepidermoid carcinoma obstructing a major bronchus. The child's cough only totally resolved upon removal of the tumour; i.e. after the primary cause was successfully treated. This report illustrates the importance of following-up children with chronic cough. Cough was this child's only symptom that was consistently present between the child's recurrent hospitalisations.Paediatric cough, unlike cough in adults, is generally classified for practical purposes into cough descriptors of 'non-specific' and 'specific' cough ,2. In chLung carcinoma remains the most common cancer in adults but is very rare in children . PulmonaPresentation of patients with MEC is unusual until some obstruction of the involved airway occurs -9. Commo"}
{"text": "Gastro-oesophageal reflux (GOR) is one of the most common causes of chronic cough in Western countries, responsible for 10 to 40% of cases. In Japan, however, GOR-associated chronic cough (GOR-CC) has been rarely reported and its clinical manifestation including frequency of concomitant reflux laryngitis is poorly known.We have analyzed prevalence and clinical characteristics of patients who were diagnosed as having GOR-CC among adult patients with chronic cough (≥ 8 weeks) who visited our asthma and cough clinic over a period of 19 months. Diagnosis of GOR-CC was based on the response of coughing to a proton-pump inhibitor (lansoprazole™) and/or positive results of 24 h ambulatory esophageal pH monitoring. Laryngeal involvement was based on symptoms or objective diagnosis by specialists.2) and high incidence of concomitant reflux laryngitis (5 of 8) in the present 8 patients. Among 4 patients who could stop treatment with temporal resolution of cough, cough recurred in 3 patients, 1 week to 8 months after the discontinuation.GOR-associated chronic cough was diagnosed in 7.1% (8 of 112) of chronic cough patients. In addition to the demographic data which were consistent with the characteristics of patients with GOR-CC in the Western populations, including gender , age , duration of cough (9.9 ± 3.3 months), lack of gastrointestinal symptoms (3 of 8) and complication with other causes of cough (5 of 8), we found the standard range of body mass index (23.9 ± 1.5 kg/mIn conclusion, GOR-CC is a less frequent cause of chronic cough in Japan than in Western countries. Signs or symptoms of laryngitis may be important as clues to suspicion of GOR-CC. Despite the established evidence that gastro-oesophageal reflux (GOR) causes 10 to 40% of chronic cough , this co2 agonists, sustained release theophylline or both, no past history of asthma, or upper respiratory tract infection within the past 8 weeks [We studied 112 consecutive adult patients with chronic cough ≥ 8 weeks) who newly visited the asthma and cough clinic of Kyoto University Hospital from June 2002 to December 2003. Diagnostic investigations included questionnaire, physical examination, blood tests, chest and sinus radiographs, pulmonary function, airway responsiveness and cough sensitivity tests, and sputum induction. Diagnosis of GOR-CC was on the basis of response to 8 week-course of a proton-pump inhibitor  and/or positive results of 24 h ambulatory esophageal pH monitoring  [ 8 weeks . When pa 8 weeks ,8. Diagn weeks wh 8 weeks . If examCauses of chronic cough of the 112 patients were as follows; 38 CVA, 24 probable CVA (12 patients did not undergo airway responsiveness test and 12 presented normo-responsive results but responded to bronchodilator therapy), 17 atopic cough, 9 sinobronchial syndrome, 8 GOR-CC, 7 postinfectious chronic cough, 2 other miscellaneous conditions, 4 unexplained cough. Twelve patients were lost before diagnosis was made. Nine patients had multiple conditions. There were 15 ex-smokers, and 2 current smokers. No patients had ACE inhibitor associated chronic cough. The 8 patients (7.1%) with GOR-CC were diagnosed on the basis of response to the PPI (n = 7) and/or positive results of 24 h ambulatory esophageal pH monitoring (n = 4). One patient who complained of chronic cough and heartburn but did not respond to the PPI showed esophageal pH more than 7 in 66.9% of the 24 h monitoring period, and was diagnosed as having GOR-CC due to alkaline regurgitation.2, which was not significantly different from that of the general population in the present study . Frequent association of reflux laryngitis (5 of 8) was observed. Two patients complained of temporal association of coughing and heartburn. Five patients were complicated with other causes of chronic cough; 3 with CVA/probable CVA on inhaled corticosteroids or an anti-leukotriene receptor antagonist, 1 with sinobronchial syndrome on low dose of macrolide, 1 with atopic cough on an anti-histamine receptor antagonist. Airway responsiveness was tested in 4 patients among whom 2 with complication of CVA showed hyperresponsiveness. Cough sensitivity was examined in 2 patients; one complicated with atopic cough had hypersensitivity, another with CVA did not. All were non-smokers and produced minimal amount of sputum or none.Demographics of the 8 patients were presented in Table In 7 patients, their coughing with or without laryngeal symptoms was alleviated and ceased within a few days after initiation of PPI. However, in 3 of 4 patients who had stopped treatment with resolution of cough, coughing recurred 1 week to 8 months later. Coughing, laryngitis and heart burn observed in one patient with alkaline regurgitation subsided without any treatment.2) [In the present study, GOR-CC was a less frequent cause of chronic cough than in the studies of the Western populations ,2, altho2)  and endo2) ,12. This2)  and 21 t2) .In one patient who complained of cough and heartburn, and presented laryngitis and frequent alkaline regurgitation, the cough had ceased spontaneously as well as heartburn and laryngitis. Although there are no diagnostic criteria for alkaline regurgitation, the patient's cough was clinically considered caused by GOR-CC due to alkaline regurgitation.2 in the present study. Except the frequent incidence of concomitant reflux laryngitis and standard range of BMI, characteristics of the present patients including complication with other causes of chronic cough were consistent with those in the previous studies of GOR-CC in the Western populations [per se can be a trigger of GOR possibly through increased transdiaphragmatic pressure and transient lower oesophageal sphincter relaxation [The present study has shown frequent association of reflux laryngitis and GOR-CC. To date, frequency of this association has not been clarified . In a stulations ,4,15,16.laxation , GOR-CC We conclude that GOR-CC is a less frequent cause of chronic cough in Japan than in Western countries. The presence of reflux laryngitis may be an important clue to suspicion of GOR-CC.BMI = body mass indexCVA = cough variant asthmaGOR = gastro-oesophageal refluxGOR-CC = GOR-associated chronic coughThe ethics committee of our institution approved the study protocol and a written informed consent was obtained from each participant.This study was supported by Takeda Pharmaceutical Company ltd.The author(s) declare that they have no competing interests.H Matsumoto conceived of the study, participated in its design, acquisition, and interpretation of data, and drafted the manuscript.AN conceived of the study, participated in its design, contributed to data interpretation.MT participated in acquisition of dataTU participated in acquisition of dataMY participated in acquisition of dataHM participated in acquisition of dataMJ participated in acquisition of dataKC contributed to data interpretationMM contributed to data interpretation"}
{"text": "Inflammatory mediators are involved in the pathogenesis of airway inflammation, but the role of prostaglandin I2 (PGI2) remains obscure. This study was designed to investigate the role of PGI2 in cough reflex sensitivity of the asthmatic airway, which is characterized by chronic eosinophilic airway inflammation. The effect of beraprost, a chemically and biologically stable analogue of PGI2, on cough response to inhaled capsaicin was examined in 21 patients with stable asthma in a randomized, placebo-controlled cross over study. Capsaicin cough threshold, defined as the lowest concentration of capsaicin eliciting five or more coughs, was measured as an index of airway cough reflex sensitivity. The cough threshold was significantly (p < 0.05) decreased after two weeks of treatment with beraprost [17.8 (GSEM 1.20) μM] compared with placebo [30.3 (GSEM 1.21) μM]. PGI2 increases cough reflex sensitivity of the asthmatic airway, suggesting that inhibition of PGI2 may be a novel therapeutic option for patients with asthma, especially cough predominant asthma. Chronic cough is one of the commonest respiratory symptoms. Cough has been considered to be a defense mechanism of the airway to remove irritant particles or excess mucus, whereas non-productive cough, which is not associated with the clearance of the tracheobronchial mucus, may occur via increased cough reflex sensitivity. Inflammatory mediators such as prostaglandins may adjust the cough reflex sensitivity. However, little is known about how cough reflex sensitivity is influenced by airway inflammatory processes. Although our previous study has clearly shown that arachidonate cyclooxygenase products can modulate airway cough reflex sensitivity to inhaled capsaicin , the effIt has been recognized that prostaglandin I2  is the most abundant prostanoid generated on IgE-dependent challenge of human lung tissue in vitro . Others Twenty-one patients with bronchial asthma  with a mean age of 73.2 ± 1.5 (± SEM) (range 54–83) yrs participated in this study. All patients were lifetime nonsmokers or ex-smokers with no history of viral infection for at least 4 weeks prior to the study. Characteristics of individual patients are shown in Table Each asthmatic patient satisfied the American Thoracic Society definition of asthma, with symptoms of episodic wheezing, cough, and shortness of breath responding to bronchodilators, and reversible airflow obstruction documented on at least one previous pulmonary function study . Reversi-2 M, which was stored at -20°C. This solution was diluted with physiological saline to make solutions starting at a concentration of 0.49 μM and increasing it by doubling concentrations up to 1000 μM. Each subject inhaled a control solution of physiological saline followed by progressively increasing concentrations of the capsaicin solution. Solutions were inhaled for 15 s every 60 s, by tidal mouth-breathing wearing a noseclip from a Bennett Twin nebulizer . Increasing concentrations were inhaled until five or more coughs were elicited. The nebulizer output was 0.21 mL/min. The number of capsaicin-induced coughs was counted by a blinded medical technician in our pulmonary function laboratory. The cough threshold was defined as the lowest concentration of capsaicin that elicited five or more coughs.Cough reflex sensitivity was assessed by capsaicin provocation test . CapsaicThe medication was stopped at 9.00 p.m. on the previous day to allow a washout time of 12 h or more before the measurement of cough threshold to inhaled capsaicin at 10.00 a.m. on each test day to reduce the diurnal variability of the cough response.Each patient attended 4 times separated by 2 weeks, at the same time each day. Control measurement of capsaicin cough threshold was carried out 2 weeks before initiation of the first treatment (run-in). Two weeks treatment with beraprost sodium or placebo was performed separated by a two-week washout period in a randomized, cross-over fashion. Two beraprost sodium tablets (40 μg) and their placebo were taken orally three times a day for 14 days and at 8.00 a.m. on the test day. FEV1 was measured on a dry wedge spirometer  before capsaicin challenge to assess the bronchoactive effect of the treatment regimens. Serum total IgE levels and the number of peripheral eosinophils were measured to assess anti-allergic effect of the test drugs.Capsaicin cough threshold values were expressed as geometric mean with geometric standard error of the mean (GSEM). Forced vital capacity (FVC), FEV1 and maximal mid expiratory flow (MMF) were shown as arithmetic mean values ± SEM. The FVC values, the FEV1 values and the MMF values were compared between each pair of the four groups  by the Wilcoxon signed-ranks test. A p-value of 0.05 or less was taken as significant.Cough threshold to inhaled capsaicin before each treatment  and after treatment with beraprost and placebo are shown in Figure Figure The present study showed that two-week treatment with a stable PGI2 analogue, beraprost, decreased the cough threshold to inhaled capsaicin in asthmatic patients. No difference could be found in the baseline pulmonary function, IgE production or peripheral eosinophil count between beraprost and placebo treatments. These findings suggest that PGI2 enhances the cough reflex sensitivity in the asthmatic airway.Cough is one of the main symptoms of bronchial asthma which can profoundly and adversely affect the quality of patient's lives and social activities, whereas the mechanisms underlying the cough remain obscure. Previous researchers  indicateIt has been revealed that inflammatory mediators such as arachidonate metabolites play major roles in the pathogenesis of bronchial asthma, however, the relationship between inflammatory mediators and airway cough reflex sensitivity remains obscure. Some studies indicated that some inflammatory mediators might modulate the sensitivity of cough reflex ,17. We sIt has been known that PGI2 is the most abundant prostanoid generated on IgE-dependent challenge of human lung tissue in vitro . Others Overall, our data support the conclusion that inhibition of PGI2 formation or action may be a novel treatment for chronic non-productive cough in asthmatic airway, especially in cough variant asthma or cough predominant asthma with normal baseline pulmonary function. This is the first report demonstrating the role of PGI2 in cough reflex sensitivity in the asthmatic airway. Further studies may be required to elucidate the role of PGI2 in other eosinophilic bronchial disorders presenting with non-productive cough with normal baseline pulmonary functions -31.cLT = cysteinyl leukotriene; CVA = cough variant asthma; FEV1 = forced expiratory volume in one second; FVC = forced vital capacity; GSEM = geometric standard error of the mean; PGE2 = prostaglandin E2; PGF2α = prostaglandin F2α; PGI2 = prostaglandin I2; MMF = maximal mid expiratory flow; TxA2 = thromboxane A2."}
{"text": "Manual cough counting is time-consuming and laborious; however it is the standard to which automated cough monitoring devices must be compared. We have compared manual cough counting from video recordings with manual cough counting from digital audio recordings.We studied 8 patients with chronic cough, overnight in laboratory conditions . Coughs were recorded simultaneously using a video camera with infrared lighting and digital sound recording.The numbers of coughs in each 8 hour recording were counted manually, by a trained observer, in real time from the video recordings and using audio-editing software from the digital sound recordings.The median cough frequency was 17.8 (IQR 5.9–28.7) cough sounds per hour in the video recordings and 17.7 (6.0–29.4) coughs per hour in the digital sound recordings. There was excellent agreement between the video and digital audio cough rates; mean difference of -0.3 coughs per hour (SD ± 0.6), 95% limits of agreement -1.5 to +0.9 coughs per hour. Video recordings had poorer sound quality even in controlled conditions and can only be analysed in real time (8 hours per recording). Digital sound recordings required 2–4 hours of analysis per recording.Manual counting of cough sounds from digital audio recordings has excellent agreement with simultaneous video recordings in laboratory conditions. We suggest that ambulatory digital audio recording is therefore ideal for validating future cough monitoring devices, as this as this can be performed in the patients own environment. For more than 40 years there has been an interest in making objective measurements of cough frequency. The original published systems consisted of reel-to-reel tape recorders with patients confined to a single room containing a microphone -3. CoughIn the 1990s ambulatory devices using analogue sound recordings combined with EMG were devised; coughs were identified manually from the visualisation of the waveforms ,5. ValidIn order to make cough monitoring applicable to clinical practice, it is necessary to develop accurate automatic detection and counting of coughs. Automated devices would make large studies feasible and may allow endpoints other than cough counts to be measured e.g. amplitude, temporal pattern and cough sound quality.Although an acceptable automated cough monitoring system is not yet available this area continues to progress -11. WithPrevious studies have used video recordings with real-time manual counting of cough as the gold standard ,13. The The aim of this study was to establish whether video in addition to audio recording was necessary to accurately manually count coughs and hence provide a gold standard for validation of novel cough monitoring systems. We performed simultaneous overnight video and digital audio recordings, in patients complaining of chronic cough and compared the manual cough counts from each media.Eight patients with chronic cough were recruited from the out-patients department of the North West Lung Centre. Simultaneous overnight cough recordings using digital audio and video were made in laboratory conditions. Approval was obtained from the local research ethics committee and all subjects gave written consent.the characteristic sound we recognise as cough. In a peel of coughs, each explosive phase was counted as one cough.Cough sounds were manually counted by a single trained observer. The order in which the recordings were counted for each individual  was randomly allocated. Coughs were quantified by counting the number of explosive cough phases  and digital audio player/recorder . A lapel microphone  was attached to the patient's night clothes and the signal was amplified using a pre-amplifier . The amplified audio signal was channelled through an oscilloscope to allow real time monitoring of the signal and then to the digital recorder and the to video recorder audio input  and a monochrome security camera . The recordings length was limited to 8 hours by the maximum length of the video tape, (4 hour video tape recorded on using long play mode). A continuous display of the time was placed above the patient's bed. The video recordings were played back in real time (i.e. over 8 hours) and explosive cough sounds counted as described above. The position in time of each cough sound was also noted so that any discrepancies between the cough counts from each device could be easily identified.The digital audio recordings were made at a sampling rate of 16 kHz, at 16 bit resolution (preset) and in wav format; this is an uncompressed sound format in common use (unlike mp3). A single 8 hour overnight recording produces a set of files that total 1.8 GB of data and can be archived on compact discs.Explosive coughs sounds were manually counted using CoolEdit2000™ . All sounds present on the digital recordings were listened to. The observer did not just listen to waveforms with the appearance of a typical cough; cough waveforms vary enormously and this would have underestimated the true number of coughs in each recording. The total number and position in time of each cough sound was noted. Using this method manual counting took 2–4 hours per overnight recording, depending on the number of coughs and other extraneous noises.Eight subjects were studied; mean age 55 years SD ± 11.7), 3 men, diagnoses asthma (5), rhinitis (1), gastro-oesophageal reflux disease (1) and idiopathic (1). The median cough frequency was 17.8 (IQR 5.9–28.7) cough sounds per hour in the video recordings and 17.7 (6.0–29.4) coughs per hour in the digital sound recordings  .Manual cough counting is extremely time-consuming and laborious, particularly from video recordings which must be reviewed in real time. It is therefore not applicable to clinical practice. Digital audio recording devices have several advantages over video. Firstly, long ambulatory recordings can be made allowing cough monitoring with unrestricted patient movement, and in their home or work environment. The performance of a cough monitor may be completely different in a subjects own environment with more background noise and movement. Secondly, counting of cough sounds is much quicker and less laborious from a digital sound recorder using audio editing software than from video. Finally, the sound quality is superior and more cough sounds can be correctly identified.Manual counting of explosive cough sounds from digital audio recordings has excellent agreement with simultaneous video recordings in laboratory conditions. As digital sound recorders have significant advantages over video recorders, ambulatory digital audio recording should now provides the gold standard for ambulatory validation of automated cough monitoring devicesThe author(s) declare that they have no competing interests.JAS recruited the subjects, performed the study and the manual counting of the video and digital sound recordings and wrote the manuscript. AW and JEE reviewed the final manuscript."}
{"text": "Chronic cough is a common reason for presentation to both general practice and respiratory clinics. In up to 25% of cases, the cause remains unclear after extensive investigations. We report 4 patients presenting with an isolated chronic cough who were subsequently found to have obstructive sleep apnoea. The cough improved rapidly with nocturnal continuous positive airway pressure therapy. Further studies are required to investigate the prevalence of coexistence of these common conditions. Chronic cough is one of the commonest reasons for presentation to respiratory clinics. Investigations are usually aimed at identifying the three most common causes of chronic cough: cough variant asthma, gastro-oesophageal reflux and upper airway cough syndrome.  In up toA 52-year-old financial advisor was referred by his general practitioner with a 3-month history of productive cough. He described a severe barking cough that occurred both in the day and night time and was exacerbated by lying flat, strong odours and smoky atmospheres. He was prescribed multiple courses of antibiotics, which were unhelpful. He also complained of mild dyspnoea on climbing hills but no wheezing. He had longstanding left nasal congestion without post-nasal drip following a broken nose in childhood and mild symptoms of gastro-oesophageal reflux. He had been diagnosed with hypertension and hypercholesterolaemia two years previously for which he was prescribed Bendroflumethiazide, Valsartan, Doxazoscin and Cervistatin. He was an ex-smoker and accumulated a 15 pack-year smoking history. His clinical examination and physical findings were normal. Initial spirometry and chest radiograph were within normal limits  and a significant improvement in cough specific quality of life  declare that they have no competing interests.SSB conceived of the study, and participated in its design and coordination and wrote the manuscript. AJI, KC and GC participated in subject recruitment and clinical characterisation, and application of cough monitoring techniques to this study. SM participated in the cough monitoring of patients. MDLM and IDP participated in subject recruitment, clinical characterisation, analysis and writing up of the manuscript. All authors read and approved the final manuscript.none"}
{"text": "Although chronic cough is a common problem in clinical practice, data on the prevalence and characteristics of cough in the general population are scarce. Our aim was to determine the prevalence of chronic cough that is not associated with diagnosed respiratory conditions and examine the impact on health status and psychological health, in a representative adult population cohortNorth West Adelaide Health Study  is a representative population adult cohort. Clinical assessment included spirometry, anthropometry and skin tests. Questionnaires assessed demographics, lifestyle risk factors, quality of life, mental health and respiratory symptoms, doctor diagnosed conditions and medication use.Of the 3355 people without identified lung disease at baseline, 18.2% reported chronic cough. In multiple logistic regression models, at follow-up, dry chronic cough without sputum production was significantly more common in males , current smokers , obesity , use of ACE inhibitors , severe mental health disturbance  and older age . Among non-smokers only, all cough was significantly more common in men, those with severe mental health disturbance and obesity.Chronic cough is a major cause of morbidity. Attention to cough is indicated in patients with obesity, psychological symptoms or smokers. Inquiring about cough in those with mental health problems may identify reversible morbidity. Cough is the commonest symptom seen in primary care -3, and cChronic cough is associated with adverse effects on health-related quality of life -17. SuccOur aim was to determine the prevalence of chronic cough in a representative adult population cohort, particularly cough that is not associated with diagnosed respiratory conditions, and examine the impact on health status and psychological health.The North West Adelaide Health Study (NWAHS) is a representative biomedical longitudinal population cohort study of people aged eighteen years or older, randomly selected from the electronic white pages telephone directory and living in the north western suburbs of Adelaide, South Australia . NWAHS initially recruited between 2000 and 2002 with follow-up in 2004-05. The methods of the North West Adelaide Health Study  and the At stage 1, 4060 adults underwent biomedical examination, representing 69% of those who completed the telephone interview. Overall, at Stage 2 follow-up  survey data was obtained on 88% (n = 3574) and clinic data on 79% (n = 3206) of the Stage 1 NWAHS population using the same methodology and questions. One hundred subjects were deceased, 226 persons were unable to be contacted, and 160 refused further participation in the study.2: Underweight: ≤ 18.49; Normal: 18.5-24.9; Overweight: 25.0-29.9; Obesity: ≥ 30.0. Medication use was identified when participants were also asked to bring all current medicines (including complementary medicines) into the clinic at their appointment.Telephone interviews investigated self-reported health status (including asthma and COPD), smoking status and demographic variables. A self-completed questionnaire comprised items on demographic information, risk factors , quality of life, mental health and respiratory symptoms. Smoking was categorized into self-reported current, former or never smoker. Clinic assessment by trained technicians included spirometry according to American Thoracic Society criteria , skin pr1 in the absence of a doctor diagnosis of asthma [1/FVC ratio less than 0.70) [Asthma was defined as current self-reported physician-diagnosed asthma or demonstration of a significant bronchodilator response (SBR) of at least 12% of baseline FEVf asthma ,27. Partan 0.70)  were idean 0.70) ,30. ThisHealth-related quality of life was assessed using the Medical Outcomes Study Short Form 36 Health Survey (SF-36) Physical Health Component Summary (PCS) and Mental Health Component Summary (MCS) scores . The PCSSPSS Version 15.0, SPSS Inc, Chicago, IL). Multivariable logistic regression analyses were conducted to assess the association of GHQ disturbance with chronic cough  after adjustment for sex, age, smoking, BMI and ACE inhibitor use. The models were also adjusted for reflux medication use as a proxy for GERD. An additional model assessing the association of all cough and GHQ disturbance was conducted in the population of never/former smokers.Data were weighted to the 1999 Estimated Residential Population for South Australia  and CensApproval for the NWAHS study was obtained from institutional ethics committees of the North West Adelaide Health Service, and all subjects gave written informed consent.The socio-demographic characteristics and health status of the study subjects at Stage 1 baseline have been described in detail previously ,23. Of tThe prevalence of chronic cough at baseline within various demographic and clinical groups is shown in Table In multiple regression analysis Table , chronicParticipants reporting cough at any time were significantly more likely to have psychological disturbance on the GHQ-12 and report significantly lower quality of life compared to those without cough at any time Table . CompareIn a representative population sample we have shown that chronic, dry cough is common among people without known respiratory disease, with a prevalence of nearly 9% among adults. Cough productive of sputum occurs in around a further 4% of those without known lung disease. People with chronic cough report significant impairments in quality of life and psychological health, compared to those without cough. Across the population, chronic cough was significantly associated with obesity and severe depression, and was more common in men and in people aged over 60 years. Although cough was more common in people who currently smoke, when only non-smokers were analyzed, the significant associations seen with depression, obesity, men and age persisted. The prevalence of cough was not significantly different between former smokers and those who had never smoked.The frequency of chronic cough independent of other lung disease, with its strong associations with impaired mental health, particularly depression, and significantly reduced quality of life, indicates cough is a major contributor to morbidity in the community. The reduction in quality of life in general physical health is similar to that previously reported in Australian populations for asthma , diabeteOur data demonstrates that careful attention should be given to assessment and management of psychological morbidity in the large number of patients with chronic cough in the community, as well as those seen in referral centers. This may be especially the case in people in whom coughing persists in the absence of an identifiable cause and despite extended trials of empirical therapy . ChronicWe found obesity to be significantly associated with dry cough and cough in never/ex-smokers. Janson et al have reported cough was significantly associated with obesity. However, the study population of 20-48 year olds included people with asthma and other respiratory diseases . As obesComparison with previous studies examining the prevalence and associations of chronic cough are difficult due to differences in sampling and other methodological questions. We used a validated symptom score of chronic lung disease to identify cough frequency over the previous 3 months. The use of this tool differs from prior studies and makes direct comparison of prevalence between studies difficult. Studies using selected age groups have either excluded adults aged > 50  or > 60 Similar to other reports we found chronic cough is associated with adverse effects on health-related quality of life -17 and pThe direction of causality regarding cough and psychological problems is difficult to determine. We found that in terms of disturbance on the GHQ-28 that the group with cough a follow-up only was not significantly different from those with cough at both time points, suggesting there may be little effect of chronicity over our follow-up period of 4 to 5 years. However, we do not know if people had cough for all the follow-up period or recurrent cough only in the 3 months prior to each clinic assessment. Although those people with cough at baseline but who were no longer coughing had significantly higher physical health quality of life and were less likely to report disturbance on the GHQ. This can be interpreted as indicating chronic cough has both immediate and longer-term consequences for psychological health that may stem from the significant impact on general health experienced with cough. Alternatively, this may suggest chronic cough is more likely to be seen in those with underlying anxiety or depression, and this may influence an individual's awareness of symptoms. However, anxiety about underlying serious illness has been identified as a concern for most patients with chronic cough . McGarveContrary to anecdotal observations, and consequent to the representativeness of our sample we found cough to be more common in men and in people aged over 60 years, two groups where there is evidence to suggest there is a tendency to under-report symptoms to clinicians . Older pOur study is limited by a lack of specific information regarding some of the common causes of chronic cough, such as upper airways syndrome or gastro-esophageal reflux disease ,5. HowevIn summary, chronic cough is a common problem that is significantly associated with reductions in physical and mental health. Investigation and management of chronic cough is therefore an important medical need. Patients with a history of smoking, obesity, allergy, or use of ACE inhibitors should be questioned regarding cough and active clinical care pursued. Careful attention to symptoms of psychological disturbance, including somatic symptoms, and their management may help identify depression and reduce the burden of this problem. Conversely, specifically inquiring about cough in patients with mental health problems may identify reversible physical and psychological morbidity in this group.SA, DW and AT have no competing interests to declare. RA and RR have received honoraria and speakers fees from Glaxo-Smith Kline over the last 5 years.RA took main responsibility for conceiving the analysis and for drafting of the manuscript. SA undertook the analysis and contributed to revision of the manuscript. DW, AT and RR contributed to the conception and conduct of the study, to analytical approaches and methods, and to revising of the manuscript. All authors have read and approved the final version of the manuscript."}
{"text": "The down-regulation of the cough reflex in patients with aspiration pneumonia can involve both cortical facilitatory pathways for cough and medullary reflex pathways. In order to study the possible involvement of the supramedullary system in the down-regulation of cough reflex, we evaluated the urge-to-cough in patients with aspiration pneumonia.2) and five or more coughs (C5). The urge-to-cough scores at the concentration of C2 and C5, and at the concentration of two times dilution of C2 (C2/2) and C5 (C5/2) were estimated for each subject.Cough reflex sensitivity and the urge-to-cough to inhaled citric acid were evaluated in patients with at least a history of aspiration pneumonia and age-matched healthy elderly people. The cough reflex sensitivities were defined as the lowest concentration of citric acid that elicited two or more coughs (C2 and C5 in the control subjects were significantly greater than those for patients with aspiration pneumonia. There were no significant differences in the urge-to-cough at C2 and C5 between control subjects and patients with aspiration pneumonia. However, the urge-to-cough scores at both C2/2 and C5/2 in patients with aspiration pneumonia were significantly lower than those in control subjects. The number of coughs at C5/2 was significantly greater in the control subjects than those in the patients with aspiration pneumonia whereas the number of coughs at C2/2 did not show a significant difference between the control subjects and the patients with aspiration pneumonia.Both CThe study suggests the involvement of supramedullary dysfunction in the etiology of aspiration pneumonia in the elderly. Therefore, restoration of the cough motivation system could be a new strategy to prevent aspiration pneumonia in the elderly. Morbidity and mortality from aspiration pneumonia continues to be a major health problem in the elderly. A marked depression of cough reflex sensitivity is reported in elderly patients with aspiration pneumonia who show cerebral atrophy and lacunar infarction in the brain . The risCough is usually referred to as a reflex defense mechanism mediated at the brainstem level, where sensory information arising from airway sensory receptors in response to an appropriate stimulus is processed by the medullary respiratory network to produce the motor pattern of cough. However, there is accumulating evidence indicating that human cough is under voluntary control and that higher centers such as the cerebral cortex or subcortical regions have an important role in both initiating and inhibiting reflexive cough ,5. AlthoCough is typically preceded by an awareness of an irritating stimulus and is perceived as a need to cough, termed the urge-to-cough . In a caCough reflex sensitivity and the urge-to-cough to inhaled citric acid were evaluated in patients with at least one history of aspiration pneumonia and age-matched healthy elderly people.Patients were prospectively and consecutively recruited from those referred and admitted to the Geriatric Unit, Tohoku University Hospital for treatment of pneumonia from May 2007 to April 2008. Pneumonia was diagnosed by the presence of pulmonary infiltration on chest radiograph and computed tomography (CT) and according to systemic inflammation as determined according to white blood cell (WBC) count and C-reactive protein (CRP). The criteria for pneumonia were established according to the pneumonia guidelines of the Japan Respiratory Society . In the During the entry period, 41 patients with pneumonia without an apparent past- and present-history of stroke were admitted to our 20 bed geriatric unit, and 34 patients (83%) were diagnosed as aspiration pneumonia. We performed simple chest X-ray in all of them. Among 34 patients, we performed chest CT scan in 30 patients. All 34 patients showed characteristic images of infiltrates compatible with aspiration pneumonia in the posterior segment of any of the lobes and/or lower lobe by simple chest X-ray and/or CT scan. Of 34 patients, 2 patients died and 3 patients eternally tracheostomized. Of 29 recovered patients, due to the difficulty of urge-to-cough estimation, we excluded patients with dementia using the mini-Mental State Examination (MMSE). Of 29 patients who recovered from aspiration pneumonia, 18 subjects with a MMSE score < 24 were excluded. Three patients with apparent paralysis were excluded. Finally, 8 patients (3 men) with aspiration pneumonia (70–88 years old) were enrolled for this study. From 6 patients among 8, we obtained brain images with non-contrast CT scan. The CT scan revealed that 2 patients had infarct in the deep region of middle cerebral artery territory, 2 patients in the superficial region  of middle cerebral artery territory, and 1 patient in both the deep and superficial region of middle cerebral artery territory. One patient had infarct in the superficial region of the posterior cerebral artery territory. The diameters of all infarcts were within 1 cm.Eleven age and sex-matched healthy elderly people (72–84 years old) as control subjects were recruited from the community by advertisement. None of the subjects were demented (MMSE scores > 23). All control subjects were never-smokers, and had no previous history of pneumonia and other respiratory diseases. None of the patients or controls were taking medication which might affect cough sensitivity such as antitussives, narcotics, or ACE inhibitors. A CT scan was obtained from only one control subject.Cough reflex and urge-to-cough was examined more than 3 months after negative conversion of C reactive protein after pneumonia had responded to antibiotics treatment . At the time of evaluation, the subjects were in a stable state until at least 3 months before. Simple standard instructions were given to each subject.2) and the lowest concentration of citric acid that elicited five or more coughs (C5).We evaluated the cough reflex sensitivities using citric acid because we had previously used this method to observe depressed cough in the elderly ,3. CoughImmediately after the completion of each nebulizer application, the subject made an estimate of the urge-to-cough. The modified Borg scale was used to allow subjects to estimate the urge-to-cough . The scaU test or the chi-square test were used to compare patients with controls. A p value of < 0.05 was considered significant.The study protocol was approved by the local ethics committee and informed consent was obtained from all subjects. Data are expressed as mean (SD) except where specified otherwise. The Mann-Whitney All 19 subjects completed the experiments without any difficulty or side effects. Among the 8 patients with aspiration pneumonia, 3 patients had a history of recurrent pneumonia (2–3 episodes). All subjects were leading an independent life. The characteristics of subjects are summarized in Table 2, in patients with aspiration pneumonia (1.5 ± 0.6 g/l) was significantly higher than those of control . The urge-to-cough scores at the concentration of C2 and at the concentration of two times dilution of C2 (C2/2) were estimated for each subject. There were no significant differences in the urge-to-cough at C2 between control subjects (3.0 ± 1.8 points) and patients with aspiration pneumonia (3.3 ± 3.0 points)  were significantly lower than those in control subjects (1.2 ± 0.8 points)  than in patients with aspiration pneumonia (0.0 ± 0.0 times). At C2/2, only one control subject coughed among all subjects.As shown in Figure ) Figure . However) Figure . There w5, in patients with aspiration pneumonia (1.6 ± 0.5 g/l) was significantly higher than those of control . The urge-to-cough scores at the concentration of C5 and at the concentration of two times dilution of C5 (C5/2) were estimated for each subject. There were no significant differences in the urge-to-cough at C5 between control subjects (7.5 ± 1.8 points) and patients with aspiration pneumonia (5.3 ± 3.4 points)  were significantly lower than those in control subjects (3.0 ± 1.9 points)  than in patients with aspiration pneumonia . Actually, 6 patients (75.0%) with aspiration pneumonia did not cough at all at C5/2 whereas 2 control subjects (18.2%) did not.As shown in Figure ) Figure . However) Figure . The num2 and C5 are same value in 1 subject in control group and 5 subjects in the patients with aspiration pneumonia.In the present study, CThis study shows, for the first time to our knowledge, that the urge-to-cough is significantly attenuated in elderly patients with aspiration pneumonia. It has been suggested that the aspiration pneumonia is, at least in part, a consequence of cough reflex impairment. Sekizawa and coworkers demonstrated a marked depression of the cough reflex in elderly patients with aspiration pneumonia . Nakajoh2 and C5, this might be due to too small sample number in this preliminary study. However, as the urge-to-cough precedes the actual cough [2 was not significant between groups. Moreover, the actual cough has possibility to affect the urge-to-cough. In the study, all patients with aspiration pneumonia did not cough at C2/2, and 6 of 8 did not at C5/2. If the actual cough has ameliorating effect on the depressed urge-to-cough in the patients with aspiration pneumonia, the urge-to-cough scores at C2 and C5 became not different between groups. Well-designed and larger sample studies are warranted to clarify this.Although we did not observe significant difference in the urge-to-cough at Cal cough , the dif2 and C5. C5 is considered as a clinically superior value based on better reproducibility compared to C2 [2/2 in order to avoid the effect of actual cough on the result [5/2 whereas there is no significant difference in the number of cough between controls and patients with aspiration pneumonia at C2/2. Therefore, the urge-to-cough at C2/2 may more purely reflect the supramedually involvement of urge-to-cough.In the present study, we estimated the cough reflex sensitivity using Ced to C2 . Howevere result . In the Due to a lack of flow monitoring, we could not accurately distinguish between cough reflex and expiration reflex, both of which are defensive reflexes to remove foreign substances from the airway by producing the expiratory airflow. However, the latency from stimuli to induce expiration reflex was much shorter than that of cough reflex, suggesting that cortical involvement is unlikely in the expiration reflex . TherefoIn stroke patients, an impaired cough capacity is now regarded as one of the main factors accounting for the increased prevalence of aspiration pneumonia -18. The Since it has been proposed that initiation of a reflex cough response requires the urge-to-cough to facilitate it , the depThis study suggests the involvement of supramedullary dysfunction, at least in a part, in the etiology of aspiration pneumonia in the elderly. Therefore, the restoration of the cough motivation system could be a new strategy to prevent aspiration pneumonia in the elderly.2: the lowest concentration of citric acid that elicited five or more coughs; C2/2: The urge-to-cough scores at the concentration of C2 and at the concentration of two times dilution of C2; C5: the lowest concentration of citric acid that elicited five or more coughs; C5/2: Urge-to-cough scores at the concentration of C5 and at the concentration of two times dilution of C5.MMSE: mini-Mental State Examination; CThe authors declare that they have no competing interests.SY, SE and TE participated in the design of the study, collected and analyzed data, and drafted the manuscript. MY, TA, MA and KU participated in the design of the study and collected the data. HA participated in design of the study and helped to draft the manuscript. All the authors read and approved the final manuscript."}
{"text": "The experimental data providing evidence for the above roles of tryptase are summarized in the present review, as well as the effects of tryptase inhibition in animal asthma models. The potential strategies for the development of anti-asthmatic agents based on the inhibition of tryptase are discussed.Recent physiological and pharmacological studies have indicated the potential importance of tryptase, the major protein component in mast cells, in inflammatory diseases . Being released at inflammatory sites after the activation of mast cells, tryptase is capable of causing bronchohyperresponsiveness and infiltration of eosinophils, neutrophils, etc. in animal airways. The mechanisms by which tryptase causes bronchoconstriction involve probably the potentiation of other chemical mediators such as histamine, production of bradykinin via the hydrolysis of kininogen, and cleavage of the bronchodilating peptides VIP  and PHM (peptide histidine-methionine). Tryptase has also been found to be a potent mitogen"}
{"text": "Because 24-h esophageal pH monitoring is quite invasive, the diagnosis of gastroesophageal reflux disease (GERD)-associated cough has usually been made based merely on the clinical efficacy of treatment with proton pump inhibitor (PPI).We recently encountered two patients with PPI-responsive chronic non-productive cough for whom switching from bronchodilators and glucocorticosteroids to PPI resulted in improvement of cough. The cough returned nearly to pre-administration level a few weeks after discontinuation of PPI. Though GERD-associated cough was suspected, 24-h esophageal pH monitoring revealed that the cough rarely involved gastric acid reflux. Following re-initiation of PPI, the cough disappeared again.PPI may improve cough unrelated to gastric acid reflux. Gastroesophageal reflux disease (GERD)-associated cough is a well-known type of chronic, non-productive cough . Asthma,In our institutions, chronic cough lasting more than 8 weeks without history of wheezing was assessed as described in Figure We recently encountered two patients with PPI-responsive chronic non-productive cough in whom 24-h esophageal pH monitoring showed cough rarely associated with a reflux episode. These cases show that PPI may improve cough unrelated to an acid reflux episode.The patient was a 60-year-old man who had been suffering from isolated chronic non-productive cough for about 1 year. He discontinued smoking 9 months before the first visit following development of this cough and had never taken an ACE-I. Although he did not complain of heartburn and other symptoms suggestive of GERD, endoscopic assessment of the esophagus revealed reflux esophagitis (Los Angeles classification Grade B). He had had no respiratory infections during the 8-week period preceding the first visit. No abnormal shadows were noted on chest or paranasal sinus X-rays and chest CT scan. Cutoff points in testing of bronchial hyperresponsiveness and cough reflex hypersensitivity were set at below 10000 μg/ml  and 3.9 The patient was a 34-year-old man who had been suffering from isolated chronic non-productive cough since 31 years of age. He had stopped smoking at 32 years of age and had never taken an ACE-I. Although he did not complain of heartburn or other symptoms suggestive of GERD, endoscopic assessment of the esophagus revealed reflux esophagitis (Los Angeles classification Grade M). He had had no respiratory infections within the 8-week period preceding the first visit. No abnormal shadows were noted on chest or paranasal sinus X-rays and chest CT scan. Airway reversibility to inhaled β2 agonist was 13%, and testing of bronchial responsiveness to methacholine and cough reflex sensitivity revealed hyperresponsiveness (1208 μg/ml) without hypersensitivity (31.2 μM). Cell fractionation of bronchoalveolar lavage fluid revealed percentages of macrophages, lymphocytes, neutrophils, and eosinophils of 92%, 5%, 3%, and 0%, respectively. Neither bronchodilator therapy nor anti-inflammatory therapy improved the cough. PPI was given after discontinuation of bronchodilator and anti-inflammatory therapy. The cough was markedly improved 1 week after initiation of PPI (cough level 1), but had returned nearly to pre-administration level by 3 weeks after discontinuation of PPI (cough level 8). On 24-h esophageal pH monitoring performed prior to re-initiation of PPI, some cough and acid reflux were observed, but little cough-related acid reflux was noted  declare that they have no competing interests.All authors have read and approved the final manuscript. KN had primary responsibility for drafting and submitting the manuscript. HA was involved in the patients' clinical assessment and treatment."}
{"text": "Several studies have suggested that elevated serum alanine aminotransferase (ALT) and asparte aminotransferase (AST) may be markers of hepatitis E virus (HEV) infection. Thus, individuals with elevated ALT and AST may have ongoing subclinical infection of HEV. We estimated the prevalence of anti-HEV antibodies and serum ALT and AST levels among persons who work with pigs in Accra, Ghana.Three hundred and fifty- persons who work with pigs provided blood samples for unlinked anonymous testing for the presence of antibodies to HEV, ALT and AST levels. The median age of participants was 32.85 ± 11.38 years (range 15-70 years). HEV seroprevelance was 34.84%. Anti-HEV IgG was detected in 19.26% while anti-HEV IgM was detected in 15.58% of the persons who tested positive. On multivariate analysis, the independent determinants of HEV infection were, being employed on the farm for less than six months , having piped water in the household and/or on the farm  and consumption of alcohol (OR 4.91: 95% CI 2.65-9.10). Levels >3× the expected maximum were found for both ALT and AST among individuals who tested positive for anti-HEV IgG  and anti-HEV IgM .Consistent with similar studies worldwide, the results of our studies revealed a high prevalence of HEV infection, ALT and AST values in pig handlers. Hepatitis E virus (HEV) infection is one of the major cause of human viral disease with clinical and pathological features of acute hepatitis. The infection represents an important public health concern in many developing countries, where it is primarily transmitted through the faecal oral route due to contaminated water and food , and is The first animal strain of HEV was characterised in pigs in the United States of America ,5 and siGrowing evidence suggests that individuals who work in contact with swine such as pig farmers, veterinarians and slaughterhouse workers are at increased risk of acquiring HEV infection -12. We rSeveral studies -17 suggeA cross-sectional study was carried out between the months of January and October 2008 among workers in 6 commercial pig farms in the Greater Accra Region of Ghana. The pig rearing facilities used for the study ranged from small family-run piggeries (~200 pigs) to large-scale pig farming operations (~4000 pigs) where animal housing conditions, sanitation and overall management were generally of a lower standard. All the farms are situated within the communities in high population density areas. Two of the farms are close to each other while the rest of the farms are about 95 km from the other two farms. The study was approved by the Ethical and Protocol Review Committee of the University of Ghana Medical School, Accra, Ghana.Subjects for the study were male workers of the Farms. The study population was of similar socio-economic and cultural backgrounds. In general, participants had been residing in their respective communities for most part of their lives. Farming is the major source of income; most farmers rear pigs, and other domestic animals such as goats, sheep, cows and poultry for their own consumption and for sale to supplement the family incomes. After an explanation of the purpose of the study, all the workers were invited to participate. They were informed that the study was confidential and that information provided by them would not affect their employment status. A total of 353 persons joined in the research. Written and informed consent was obtained from each participant, and the information regarding the protocol and informed consent was presented at the appropriate literacy level. The study was conducted in a confidential manner and random unique study generated numbers were employed to identify the participants.All the 353 consenting participants completed a structured questionnaire assessing socio-demographic characteristics, and risk factor profile for the infection under investigation.Blood (10 ml) samples were collected from each of the consenting participants in plain tubes. Samples were centrifuged and the sera kept frozen at -20°C until analysed. Sera were tested at the Public Health Reference Laboratories, Korle-Bu, Accra, Ghana, for the presence of IgM and IgG antibodies  to HEV, in accordance with the manufacturer's instructions. The results were scored as positive or negative according to the standard procedures recommended by the manufacturer. Positive and negative controls were included in all the ELISA microplates assayed. The International Immuno-Diagnostics assays were chosen for this investigation because our validation exercises (data not shown) had demonstrated these assays to be sensitive and specific than some other commercially available assays. Liver function tests, particularly ALT  and AST  in serum were determined at the Chemical Pathology Department, Korle Bu Teaching Hospital Central Laboratory, Accra, Ghana, using the Thermo Spectronic spectrophotometer .The Statistical Analysis Software  version 9.1 was used to complete all data analyses. For each generally accepted risk factor for HEV infection, the odds ratio (OR) and the 95% confidence interval (95% CI) were calculated to assess associations with sociodemographic and behavioural variables in univariate analysis. A P value of < 0.05 was considered significant. Independent determinants of anti-HEV reactivity were evaluated by calculating the adjusted OR by univariate and multivariate analysis for the risk factor variables found to be significant and to estimate their joint influence.To examine whether persons who work with pigs are at high risk for HEV infection, 353 subjects , who had been handling pigs for more than 3 months in 6 pig farms in Accra, were recruited to participate in the study between January and October, 2008. The serum samples were assayed for the presence of anti-HEV IgG, anti-HEV IgM, ALT and AST. All the subjects were occupationally exposed to the pigs . Of the subjects occupationally exposed to the pigs, 39.94% (141 out of 353) had been working in the same farm setting for less than 6 months whilst 60.06% (212 out of 353) had been working in the same farm setting for more than 6 months.The overall prevalence of anti-HEV was 34.84% (123 out of 353). Of the persons who tested positive, anti-HEV IgG was detected in 19.26% (68 out of 123) while anti-IgM was detected in 15.58% (55 out of 123). There was a significant difference between them, P < 0.05.Table However, the overall prevalence of antibodies to HEV-IgG was highest 35.71%) among persons 51-55 years of age, followed by 23.08% in 46-50 year group, then 21.82% in 21-25 year group  and anti-HEV IgM . Compared to the different age groups, a higher number of pig handlers, 26-30 years of age, who tested positive for either anti-HEV IgM or anti-HEV IgG had ALT  and a 6.97 fold (95% CI 3.19-15.24) higher risk of elevated ALT and AST levels, respectively. Similarly, pig handlers 26-30 years of age who tested positive to antibodies to HEV IgM had a 41.71 fold (95% CI 18.80-92.56) and a 3.73 fold (95% CI 1.79-7.75) higher risk of increased ALT and AST levels, respectively.Potential risk factors for infection were examined, to determine whether there were associations with anti-HEV prevalence.As shown in Table A similar pattern was observed among persons who work with pigs and had water piped into their homes and/or on the farms and clean barns, assist sows at birth, butcher pigs at the farm, and eat pork (undercooked pork). As shown in Table Of the pig handlers who tested positive to antibodies to HEV, alcohol was consumed by 88.61% (109 out of 123). Our analysis indicated association with alcohol consumption. In univariate analysis, persons who work with pigs and consume alcohol, were at an increased risk (OR 4.91: 95% CI 2.65-9.10) of HEV infection. Pig handlers who consume alcohol above the recommended weekly intake (defined as a maximum weekly intake of 21 units) had a 3.48 fold (95% CI 1.96-6.17) higher risk of HEV infection. In a multivariate analysis, HEV infection was independently associated with length of time a person has been working on the farm, having piped water into their homes and/or the farms and consumption of alcohol.This is the first study in Ghana reporting ALT, AST values and risk factors associated with HEV infection in persons who work with pigs, and demonstrates the high prevalence of and the considerable potential for the transmission of HEV infection in pig farms in Ghana. Although there is no report from the Ministry of Health, Accra, Ghana indicating that Ghana is an endemic area for hepatitis E, this study found very high overall prevalence rates (34.84%) of HEV antibody. In addition, the study found significantly high ALT and AST levels >3× the expected maximum among persons occupationally exposed to pigs, suggesting the possibility of subclinical infections in the country. Further studies with a larger number of pig handlers will be necessary to draw a definitive conclusion. Moreover, additional studies need to be done to isolate both the pig and human HEV strains in Ghana and determine the genomic relationship between them if both the human and pig HEV are isolated.The risk of HEV infection correlated with length of time employed, close contact with animals or animal waste, such as cleaning barns or assisting sows at birth, butchering pigs, consumption of alcohol, and having piped water in homes and/or on the farms. The finding of higher HEV antibody prevalence in persons who work with pigs in Ghana is consistent with literature, and is widely attributable to work-related behaviours practised on the farm settings, although transmission of HEV has also been documented among individuals outside the farm setting and persons who are not occupationally exposed to pigs -22. The Another finding of interest reported herein in our study is that anti-HEV prevalence was significantly higher (P < 0.001) among persons who had been working with pigs in the same farm setting for less than 6 months compared to those who had been working in the same farm setting for more than 6 months . The reason(s) for this disparity cannot be discerned from our study. However, it was observed that newly recruited individuals spend more time cleaning barns and assisting sows at birth, and this perhaps may have accounted for the high seroprevalence rate of HEV infection. Further studies need to be done to define the high prevalence of anti-HEV antibodies in such population.Of particular note, pig handlers studied who tested positive for antibodies to IgG anti-HEV or IgM anti-HEV had ALT  levels >3× the expected maximum. A similar pattern was noted in serum AST levels among pig handlers. Levels 3× the expected maximum were found in pig handlers studied who tested positive for antibodies to IgG anti-HEV  or IgM anti-HEV . Interestingly, among the different age groups, pig handlers, 26-30 years of age, had higher levels of ALT and AST (data not shown).Growing evidence suggest that elevated ALT and AST levels are associated with recent acute HEV infection -17,23. SAnother finding of interest reported herein in our study is that anti-HEV prevalence was associated with consumption of alcohol (OR 4.91: 95% CI 2.65-9.10) among persons who had been working with pigs. Although excess alcohol consumption could compromise hepatic function and predispose pig handlers to HEV infection as suggested by our study, consumption of alcohol may not be probably linked to exposure to HEV infection. However, from our studies it is also possible that excess consumption of alcohol may have resulted in lack of self control therefore leading to higher risk behaviour of HEV infection. Further studies need to be conducted to define the link between alcohol consumption and HEV infection among persons who work with pigs.The small sample size and our inability to test for anti-HEV reactivity in pigs, may be the limitations of this study. However, the detection and prevalence of HEV infection coupled with significantly high values of ALT and AST in persons who work with pigs in Ghana may reflect the prevalence of past and recent HEV infections among pig handlers in the country. Further studies need to be done to define clearly the natural history of HEV infection and transmission in Ghana in order to effectively control and prevent HEV zoonosis.The results reported herein have significant implications for veterinarians, public health officials, persons who work with pigs and farm managers, and suggest urgent need for the introduction of policies to prevent the transmission of HEV on the farms and the general population. These policy strategies must include increasing education of persons who work with pigs about the need for HEV testing and prevention in infected pig handlers. The implementation of a HEV infection prevention programme in pig farms in Ghana should be seen as an opportunity to improve the health status of the infected persons who work with pigs and to prevent further transmission of HEV, within and without the farm settings. The argument for HEV testing among persons who work with pigs in Ghana is compelling, because of the precarious sanitary conditions in most urban and rural areas, increased incidence of acute viral hepatitis without a defined aetiology , and the high infant and maternal mortality. Our findings re-emphasize the suggestion that targeting high-risk pig handlers or universal testing in high prevalence areas, which includes Ghana, could identify most pig handlers, pregnant women, and blood donors infected with HEV at a relatively low cost ,23.This is the first study in Ghana reporting a high prevalence of IgG, IgM anti-HEV antibodies and elevated ALT and AST levels in persons who are occupationally exposed to pigs. The high prevalence of HEV infection coupled with the elevated ALT and AST values suggest that HEV infection should be treated as an occupational illness in persons who work with pigs in Ghana and therefore suggest the urgent need for the introduction of some of the range of effective preventive strategies employed in pig farm settings elsewhere.Fully informed consent was obtained from each study subject. When study subjects were younger than 18 years, informed consent was obtained from their parents.The authors declare that they have no competing interests.AAA, YA, JTA, CAG, JAA, ETN conceived of the study, participated in the design and coordination. All the authors read and approved the final manuscript."}
{"text": "Cough reflex can be induced by the pepper extract capsaicin and by low pH in guinea-pig airways. Transient receptor potential vanniloid-1 (TPRV-1) is expressed in the sensory and afferent nerve fibres in airways.We hypothesized that a novel pyridazinylpiperazine analog TPRV-1 inhibitor can effectively reduce cough reflex stimulated by citric acid and capsaicin.-4M) aerosols, in succession separately by 2 hours.Guinea pigs were injected with specific TPRV-1 inhibitor, V112220, a pyridazinylpiperazine analog of N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1(2H)-carbox-amide (BCTC) (3 mg/kg) intra-peritoneally. One hour before cough response assessment. Coughs were recorded using a recorder system that identified cough sound and accompanying expiratory flows, distinct from sneezes. Guinea-pigs exposed to citric acid (0.4 M) and to capsaicin  and capsaicin  compared to vehicle control.A novel pyridazinylpiperazine analog TPRV-1 inhibitor can inhibit the cough reflex, induced by both low pH and capsaicin, suggesting that it could be clinically beneficial in treatment of cough. Capsaicin is a potent tussive agent in most species including humans. It activates a capsaicin receptor, transient receptor potential vanilloid-1 (TRPV-1), which is a polymodal ion channel  and endoSeveral antagonists of TRPV-1 have now been described . CapsazeThe protocols were approved by the Imperial College BioSciences Group and performed under a Project License from the British Home Office, UK, under the Animals (Scientific Procedures) Act 1986.Pathogen free Male Hartley guinea pigs (600 – 700 g) were used for the study. Animals were screened one week before the in vivo cough examination.Materials used in the study including: V112220, a selective TRPV1 antagonist ; vehicle, 20% hydroxypropyl-β cyclodextrin ; Procaterol hydrochloride ; Citric Acid  and Capsaicin .Conscious guinea pigs were pre-screened to assess their cough response to 0.4 M citric acid one week before the cough study with V112220 or diluent. Low responders (number of coughs < 3) and high responders (number of coughs > 20) were excluded from the study. After pre-screening, animals were allocated into 3 different groups, the control group (n = 4) and two treatment groups .Conscious animals were placed in a 4 L plethysmograph which was equipped with an internal microphone and a pressure transducer, and were connected to a Amplifier Interface Unit series pre-amplifier . Aerosols were generated with an ultrasonic nebuliser  which was connected to a Basic Flow Supplier AIR 200 . Airflow was set at 8 L/min. Coughs were detected in three ways: via the microphone, via the pressure transducer and by observing the guinea-pig behaviour which was also captured with an external camera. Data acquisition was performed with the eDacq  acquisition software.-4 M Capsaicin for 10 min and the cough response was assessed.One week following screening, guinea pigs in the treatment groups were injected with either 1 ml of vehicle or 3 mg/kg V112220 intra-peritoneally (i.p.) 1 hour before cough response assessment. Each guinea pig received 0.1 mg/kg procaterol hydrochloride i.p. injection 10 minutes prior to each cough assessment in order to minimise bronchoconstriction. For cough assessment, animals were exposed to 0.4 M citric acid for 10 min and a 10 min cough response was recorded. Two hours following citric acid inhalation, the same animal was exposed to 10Data were recorded as number of coughs per 10 min assessment. Cough numbers of individual animal were compared among pre-screening, following citric acid inhalation and following capsaicin inhalation. Data from the treatment groups were compared with the control group. Mean values were statistically analyzed by one-way analysis of variance (ANOVA) to evaluate significant differences between groups. Values are expressed as means and 95%CI, with p < 0.05 being considered significant.Guinea-pigs (n = 24) were pre-screened regarding their cough response with citric acid. Ten guinea-pigs (8 low and 2 high cough responders) were excluded and subsequently, fourteen guinea-pigs were divided into 3 groups. No significant difference in baseline cough response was noted among the 3 groups of guinea pigs. Figure Figure Our study demonstrated that blockade of the TRPV-1 receptors with a selective inhibitor, V112220, which is a pyridazinylpiperazine derivative, effectively decreased by 70% coughs evoked by citric acid or capsaicin aerosol exposure in the guinea pig. This is in agreement with a previous study using the earlier TRPV-1 antagonist, capsazepine, which inhibited coughs induced by citric acid or capsaicin but not coughs induced by 7% hypertonic saline solution,15.Recently, 4-(2-pyridyl)piperazine-1-carboxamide analogues as potent TRPV-1 antagonists have been developed . N-(4-teTRPV1 is sensitive to vanilloid molecules, including capsaicin. It can be activated by low extracellular pH ,6,20,21,Chronic persistent cough is a clinical problem, since antitussives available to control cough are often not effective . More poSYL carried out the drugs administration and cough measurements, and performed the statistical analysis. AN helped in the design of the study. ASW & PN participated in animal maintenance. F-XB & QTD assisted in cough measurement, and drugs preparation. KFC conceived of the study, and participated in its coordination. All authors read and approved the final manuscript."}
{"text": "Stimulation of phosphoinositide hydrolysis in myocardium from autoimmune myocarditis mice by ThEA and histamine was assayed. Myocardium from autoimmune heart, but not the normal forms, specifically increased phosphoinositide turnover in the presence of histaminergic agonists. This increment was blocked by a specific H1 antagonist mepyramine and to the same extent by the phospholipase C inhibitor NCDC. By using a binding assay H1 histaminergic receptors were detected in autoimmune heart membrane preparations, but this was not observed in normal heart. These data suggest that autoimmune myocardium expressed a functional H1 receptor that could involve a distinctive mechanism operating in the disease."}
{"text": "Hepatitis E virus (HEV) is a zoonotic pathogen of which several species of animal were reported as reservoirs. Swine stands out as the major reservoir for HEV infection in humans, as suggested by the close genetic relationship of swine and human virus. Since 2000, Genotype 4 HEV has become the dominant cause of hepatitis E disease in China. Recent reports showed that genotype 4 HEV is freely transmitted between humans and swine in eastern and southern China. However, the infection status of HEV in human and swine populations in central China is still unclear. This study was conducted in a rural area of central China, where there are many commercial swine farms. A total of 1476 serum and 554 fecal specimens were collected from the general human and swine populations in this area, respectively. The seroepidemiological study was conducted by enzyme-linked immunosorbent assay. Conserved genomic sequences of open reading frame 2 were detected using reverse transcription-PCR. The results indicated that the overall viral burden of the general human subjects was 0.95% (14/1476), while 7.0% (39/554) of the swine excreted HEV in stool. The positive rate of anti-HEV IgG and IgM in the serum samples was 7.9% (117/1476) and 1.6% (24/1476), respectively. Phylogenetic analysis based on the 150 nt partial sequence of the capsid protein gene showed that the 53 swine and human HEV isolates in the current study all belonged to genotype 4, clustering into three major groups. However, the HEV isolates prevalent in the human and swine populations were classified into known distinct subgenotypes, which suggested that no cross-species transmission between swine and humans had taken place in this area. This result was confirmed by cloning and phylogenetic analysis of the complete capsid protein gene sequence of three representative HEV strains in the three major groups. The cross reactivity between anti-HEV IgG from human sera and the two representative strains from swine in central China was confirmed by Dot-blot assay. In conclusion, although all the HEV strains prevalent in central China belonged to genotype 4, there is no evidence of cross-species transmission between human and swine in this area. Hepatitis E virus (HEV), a member of the genus Hepevirus, is a non-enveloped virus with a positive-stranded RNA genome approximately 7.2 kb in length HEV and antibodies to HEV have been found in a wide variety of animals HEV isolates were divided into four distinct genotypes according to sequence and phylogenetic analyses. Genotype 1 was previously believed to only infect humans, but this has been disputed recently by a report of this subtype in a pig from Cambodia P = 0.462). Our questionnaire showed that 176 individuals had a history of frequent travel to other regions, 9.1% (16/176) of them tested positive for IgG, which was not significantly different from those who did not report frequent travel to other regions (7.8% or 101/1300 P = 0.271). Of the 98 individuals who were positive for anti-HEV IgG, only two have ever been admitted to a hospital because of hepatitis E.The 150-nt PCR-amplified products from 39 swine and 14 human HEV isolates were sequenced. The results identified 28 swine and 14 human HEV isolates with distinct nucleotide sequences. The sequences were deposited in GenBank, isolate names were from Ch-MD-hu1 to Ch-MD-hu14 and from Ch-MD-sw1 to Ch-MD-sw28, and the corresponding accession numbers were FJ641988-FJ641993 and FJ641994-FJ642021, respectively. A BLAST search using these 42 sequences showed that these isolates all belonged to genotype 4 HEV, and some of the matching sequences from GenBank are included in From the three major groups, three representative strains, Ch-MD-sw1, Ch-MD-sw23 and Ch-MD-hu1, were selected and their complete ORF2 sequences were determined. Sequence analysis based on the complete ORF2 sequence indicated that the two swine HEV strains, Ch-MD-sw23 and Ch-MD-sw1, shared 91.7% sequence homology with each other, and 89.7% and 89.3% with the human isolate Ch-MD-hu1, respectively. Their putative amino acid sequences shared 98.5–99.4% sequence identity. Phylogenetic analysis was performed based on the three representative isolates in the present study and 17 referenced, and well characterized, genotype 4 HEV isolates Combining these results, it suggests that there are two different subgenotypes of genotype 4 HEV strains prevalent in swine populations in central China, and they are genetically distinct from the strains which are prevalent in general human populations in this area.The reactivity of the two representative swine HEV strains (Ch-MD-sw1 and Ch-MD-sw23) with the 29 serum samples which were positive for anti-HEV IgG was determined by Dot-blot assay. There is gathering evidence that HEV is enzootic and pigs are considered one of the major reservoirs of human infection with this virus. The zoonotic transmission of HEV from pigs to humans has been suggested previously, particularly for cases in non-endemic areas. This hypothesis was mainly based on phylogenetic analysis, which showed that swine and human HEV strains from same geographic regions share a high genomic similarity Based on the phylogenetic analysis of the 150 nt partial ORF2 HEV sequences from both humans and pigs, Zheng et al reported that genotype 4 HEV transmitted freely between swine and humans in eastern China From the sequencing results, some of the 39 swine HEV strains showed non-unique sequences over the 150nt region. Among these were four strains which had identical sequences but came from two different farms. Phylogenetic analysis also indicated that some of swine or human strains were very closely related  that is being shed enterically from humans and animals may lead to large outbreaks of Hepatitis E Over the last few years there have been a flurry of case reports, case series and descriptions of small scale outbreaks relating to hepatitis E from Japan In the present study, our research was conducted in central China, which is an area of plains with few rivers. Most of the local rural residents consume deeply drilled borehole water. Moreover, the residents in this area are not known to have a penchant for eating raw meat. All these factors make the circulation of HEV in swine or humans independent in central China and thus significantly reduce the chance of HEV cross-species transmission from animals to human. Whether there are other factors which hinder human HEV strains from infecting pigs or vice versa will need to be elucidated by further research.In the present study, all the human serum samples were collected from the general population. None of the 24 persons who were positive for anti-HEV IgM showed evident clinical symptoms of hepatitis, and only two of the 98 persons who were only positive for anti-HEV IgG had a history of being diagnosed with hepatitis E. This suggests that HEV infection in central China is commonly sub-clinical. Thus, HEV infection may persist in disease-endemic populations by serial transmission among susceptible persons in the form of subclinical and asymptomatic infection. These subclinical cases, which go unnoticed, may act as one of the reservoirs for persistence of this infection in the population and as a source of sporadic acute hepatitis E. A similar continual transmission of subclinical infection was the main reservoir of poliovirus in areas in which the disease was endemic. Subclinical HEV infection in laboratory infected cynomolgus macaques was observed by Aggarwal, who reported that subclinical HEV infection in some animals was associated with failure of the development of an immune response when compared with animals with clinical HEV infection Most of the evidence supporting animal–human transmission of HEV is from regions where HEV infection is not endemic. In India, an endemic area for HEV, some reports indicate that zoonotic transmission of HEV from pigs to humans does not exist, and the strains prevalent in human populations all belong to genotype 1 HEV while those prevalent in pigs belong to genotype 4 In the present study, although HEV strains prevalent in human and swine groups in central China all belonged to genotype 4, and the complete ORF2 sequence of representative strains shared nucleotide and amino acid identity of more than 89% and 98%, respectively, they clustered separately into genetically distinct clusters. This suggested that the swine and human HEV isolates in central China are distinct from each other, this phenomenon is not attributed to virus genotype but non-viral factors, such as human living habits, living environments, and local climate.Phylogenetic analysis based on the full or partial ORF2 sequences showed that the human or swine isolates in the present study cross clustered with the swine and human isolates from other regions of China and even Japan  and 2, wSo far, genotype 1 and 4 HEV viruses have been found to be prevalent in human populations in China where genotype 4 HEV has become the dominant cause of hepatitis E disease since 2000 In the present study, we used the primers which had been optimized for genotypes 1 and 4 virus RNA detection In the present study, the age group 20–39 years had the highest positive rate of IgM, suggesting that people in this age group have the highest clinical attack rates in central China. This is consistent with the current opinion that the peak incidence in sporadic cases of hepatitis E in endemic regions occurs in 15-35-year-olds HEV has been isolated and fully sequenced. Sequence analysis of the various genotypes of HEV isolates shows significant similarity of the genomes and many cross-reactive epitopes between these different genotypes. It has been shown that antibodies to one strain are cross-reactive with the other strains, indicating cross-protection between strains. The anti-HEV assays used in the current study used a recombinant peptide of a genotype 1 HEV structural protein that occurs naturally as a homodimer In conclusion, the present study investigated the HEV infection status in the general human population and swine groups in central China. The positive rate of anti-HEV IgG and IgM of the studied subjects was 7.9% (117/1476) and 1.6% (24/1476), respectively. HEV RNA was detected in 0.95% (14/1476) of the general human subjects and 7.0% (39/554) of the swine stool samples. Phylogenetic analysis indicated that although all the HEV strains prevalent in central China belonged to genotype 4, the strains prevalent in swine were genetically distinct from those prevalent in the human populations, which suggested that cross-species transmission of HEV between human and swine might not exist in this area.From September in 2007 to September in 2008, a total of 1476 serum specimens were collected from general populations  of rural communities in central China, including Fuyang ( lies on the boundary of Anhui and Henan Province), Huaibei (lies on the boundary of Anhui and Shandong Province ), and Suzhou (lies on the boundary of Anhui and Jiangsu Province). Most of the participants in the present study were farmers who mainly working on crops growing, and agricultural products processing. All participants ≥16 years of age gave written consent after receiving a full explanation of the study, and written consent was also obtained from the parents of the participants <16 years of age. The Ethics Committee of Shanghai Research Center for Biomodel Organism approved the study. A questionnaire was used to record individual's history of ever being admitted to a hospital because of hepatitis E and frequently tripping to other regions. A total of 554 fecal samples were obtained from pigs aged from 4 to 26 weeks old from March to August in 2008. The swine fecal samples were obtained from 11 swine farms in Fuyang, Huaibei, and Suzhou. Fuyang lies about 200 km from Suzhou and Huaibei. Huaibei lies about 100 km from Suzhou. All the swine farms are formalized and have good sanitation status.Anti-HEV IgG and IgM levels were determined using commercial ELISAs . The ELISAs were performed with a recombinant peptide corresponding to aa 394–606 of the major structural protein specified by open reading frame (ORF) 2 of the HEV genome. Testing was performed in accordance with the manufacturer's instructions.5′-CTGTTTAAYCTTGCTGACAC- 3′) and E5 (5′-WGARAGCCAAAGCACATC-3′) for the first round of PCR and primers E2 (5′-GACAGAATTGATTTCGTCG- 3′) and E4 (5′-TGYTGGTTRTCRTAATCCTG-3′) for the second round. PCR cycling conditions for both rounds consisted of 35 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 53°C, and extension for 40 s at 72°C.Total RNA was extracted from 100 uL of serum sample or feacal suspension, using Trizol . RT-PCR was performed as described elsewhere The complete sequences of ORF2 of the three representative strains of the three major clusters in the phylogenetic analysis were determined using RT-PCR method with the primers designed according to the related sequences available in GenBank.Specific bands for HEV were purified from 1% agarose gel using the QIAquick Gel Extraction kit . The purified PCR products were ligated into pMD18-T Vector . For each product, three positive colonies were selected and sequenced. A consensus sequence of each fragment was saved and used for phylogenetic analysis or assemble of the full ORF2 sequence.www.megasoftware.net/). Prototype HEV strains used as references in the analysis and their GenBank accession numbers and source of origin are marked in the phylogenetic tree.The viral nucleotide or putative amino-acid sequences were aligned using ClustaX1.8 or DNAstar software. Phylogenetic trees were constructed using the neighbor-joining method and evaluated using the interior branch test method with MEGA software  suspensions in PBS, and clarified by centrifugation at 10000 g for 10 min. Then about 30 ul suspension were spotted onto PVDF membrane strips. Blots were blocked by shaking the at 37°C for one hour with TBS-T  containing 5% skim milk powder. Then, blots were washed three times with TBS-T and incubated with each serum diluted 1∶200 in TBS-T (1 h at room temperature with constant shaking). A human serum negative for anti-HEV IgG were included as negative control. After incubation, blots were washed four times with TBS-T, and incubated with a horseradish peroxidaseconjugated goat anti-human IgG  at a 1∶10,000 dilution. After 1 h of incubation at room temperature, blots were washed four times with TBS-T and color development was carried out using a chemiluminescence detection reagent ."}
{"text": "Hidradenoma papilliferum is an uncommon, benign, cystic, papillary tumor that occurs almost exclusively in the female anogenital region. Bartholin's abscess is also an anogenital cystic lesion caused by obstruction of Bartholin's duct with an overlying infection. Concomitant presentation of Bartholin's abscess and Hidradenoma papilliferum is unique.A 43-year-old African American woman presented with a painful cystic mass on the left labia majora. A preoperative diagnosis of Bartholin's abscess was made. During excision and draining, an additional tan-brown dermal nodule was removed which demonstrated histological features of Hidradenoma papilliferum.We present what we believe to be the first case of Bartholin's abscess arising in hidradenoma papilliferum and its clinical significance. Hidradenoma papilliferum (HP) is an uncommon, benign, cystic, papillary tumor that occurs primarily in the vulva of Caucasian women, thought to arise from anogenital glands, and exhibits both eccrine and apocrine differentiation, with the latter being more common . BartholA 43-year-old African American woman presented with a painful cystic mass on the left labia majora. Preoperatively, the lesion was diagnosed as Bartholin's abscess. During the excision and drainage, an additional 2.0 × 0.8 × 0.8 cm tan-brown dermal nodule was identified and submitted for histological analysis. Microscopic examination revealed multiple pieces of fibro-necrotic tissues Fig.  associatPrevious literature has described instances of pathology occurring concurrently with Bartholin's abscess or cyst. Literature describes a case of cellular angiofibroma for which the preoperative diagnosis was Bartholin's glandular cyst . AnotherBartholin's glands are bilaterally located at the base of the labia minora and drain through 2- to 2.5-cm-long ducts that empty into the vestibule at the 4 o'clock and 8 o'clock positions . HidradeDue to hidradenoma papilliferum's mixed eccrine and apocrine features, cyst or abscess formation secondary to the disruption of ductal drainage should be considered . HidradeDiagnostically, hidradenoma papilliferum tends to occur exclusively in post-pubescent white women between the ages of 30 and 70, occurring most commonly in the fourth decade ,9. InterThough it has been suggested surgical excision of Bartholin's abscess is unnecessary due to the low risk of Bartholin's gland cancer , our finWhile hidradenoma papilliferum is a benign tumor, malignant transformations to adenocarcinoma or adenosquamous carcinoma have been reported, although rare -16. TherWe concluded the diagnosed Bartholin's abscess was most likely caused by obstruction of the duct of Bartholin's gland by hidradenoma papilliferum. A diagnosis of Bartholin's abscess should not signal a cessation in further investigation for other pathologies. The clinical presentation of hidradenoma papilliferum itself becomes increasingly indistinct when simultaneously occurring with other cystic lesions of the vulva. As noted previously, though rare, hidradenoma papilliferum does have a potential for malignant conversion. Thus, one should remain diligent in finding a cause of a Bartholin's abscess.Written informed consent could not be obtained. The patient was brought into the emergency room and treated as needed. However, consent was not obtained as the patient left the hospital quickly following treatment. The patient was lost to follow-up as she cannot be contacted due to her unique legal situation. Furthermore, no family contact information was made available. We, the authors, do not believe the patient would object to the writing of our report or the information provided. Lastly, the patient's confidentiality has been protected as no identifiers are given in the report except for age and gender.The authors declare that they have no competing interests.DE was involved in the intellectual analysis of the data and contributed to the writing of the manuscript. WS was involved in the intellectual analysis, carrying out of experimental work on the project, and editing. SD was involved in the intellectual analysis and the major contributor to the writing of this case report. All authors read and approved the final manuscript."}
{"text": "Speech language pathology is an effective management intervention for chronic cough that persists despite medical treatment. The mechanism behind the improvement has not been determined but may include active cough suppression, reduced cough sensitivity or increased cough threshold from reduced laryngeal irritation. Objective measures such as cough reflex sensitivity and cough frequency could be used to determine whether the treatment response was due to reduced underlying cough sensitivity or to more deliberate control exerted by individual patients. The number of treatments required to effect a response was also assessed.The aim of this study was to investigate subjective and objective measures of cough before, during and after speech language pathology treatment for refractory chronic cough and the mechanism underlying the improvement.Adults with chronic cough (n = 17) were assessed before, during and after speech language pathology intervention for refractory chronic cough. The primary outcome measures were capsaicin cough reflex sensitivity, automated cough frequency detection and cough-related quality of life.Following treatment there was a significant improvement in cough related quality of life (Median (IQR) at baseline: 13.5 (6.3) vs. post treatment: 16.9 (4.9), p = 0.002), objective cough frequency , and cough reflex sensitivity .This is the first study to show that speech language pathology management is an effective intervention for refractory chronic cough and that the mechanism behind the improvement is due to reduced laryngeal irritation which results in decreased cough sensitivity, decreased urge to cough and an increased cough threshold. Speech language pathology may be a useful and sustained treatment for refractory chronic cough.Australian New Zealand Clinical Trials Register, ACTRN12608000284369. Chronic cough that persists despite medical treatment (termed refractory cough) is a difficult problem frequently associated with increased cough reflex sensitivity -3. ManagThis study sought to investigate capsaicin cough reflex sensitivity and automated cough frequency monitoring in patients with refractory chronic cough undergoing speech language pathology intervention. Cough reflex sensitivity testing and cough frequency monitoring are two objective measures allowing standardized assessment as well as providing an understanding of possible mechanisms of effect. Capsaicin is an extract of hot peppers and is commonly used as a tussive agent in clinical research because it induces cough in a safe, dose-dependent and reproducible manner -10. Our It was hypothesised that speech language pathology intervention for chronic cough would result in decreased cough reflex sensitivity, reduced cough frequency, improvement in clinical outcome and improvement in cough and laryngeal subjective measures. We also sought to determine how many treatment sessions a patient required to show an improvement and if these benefits were maintained post intervention.A previous pilot study compared 2 behavioral approaches (isolated cough suppression techniques and supportive counselling) for refractory chronic cough (CC) to a CC control group and showed that there was no change in cough reflex sensitivity (CRS) measured as C5 after 1 hour of intervention. These were used to establish the current study in the following ways;1) C5 does not respond to isolated behavioural approaches,2) C5 does not change after 1 × 1 hour session of an isolated behavioural approach, and, 3) CRS testing measured as C5 is a highly reproducible test.\"For detailed description of the participants, procedures, and analysis, see additional file Participant details and results.\"Adult non-smokers n = 17) with chronic persistent cough that was refractory to medical assessment and treatment ,12 and w7 with chnormal to severe. A clinical research officer then administered several questionnaires, [Participants attended for a maximum of 6 visits  over a period of 14 to18 weeks. At visit 1, there was a voice assessment by a qualified speech language pathologist. This involved a clinical case history, symptom frequency and severity rating , auditornnaires, -20 and cnnaires,  and cougnnaires,  during tThe speech pathology programme for chronic cough has been described previously  and consCapsaicin CRS was performed as previously reported with the addition of a participant urge-to-cough score  where thThe LCM is a digital ambulatory cough monitor and external free-field microphone . This waAll analyses were performed using statistical and data analysis software STATA . Comparisons of log cough sensitivity (measured as C5 and cough threshold) between baseline and each visit was undertaken using a generalised linear mixed model (GLMM) with a random intercept term which takes into account the repeated observations on individuals. Standard errors were estimated using bootstrapping  and resuFigures were produced using GraphPad Prism 4 .Seventeen participants  with a chronic persistent cough participated in the study. The participants had a median (IQR) cough duration of 60 147) months and age of 61 (20) years with normal spirometry [Table 47 monthsParticipants responded to the treatment with a significant improvement in cough-related quality of life , laryngeal dysfunction symptom questionnaire score , cough score, p= 0.04 and total symptoms score, p = 0.002 Table . There wThere was also a significant decrease in cough frequency with the speech language pathology treatment for chronic persistent cough. The cough count at baseline was reduced after treatment: Mean ± SD cough frequency, 72.5 ± 55.8 vs. 25 ± 27.9 coughs/hr, p = 0.009 [Individual cough frequency data (baseline v post treatment) represented in Figure Cough threshold at baseline was Mean ± SD log CT 0.47 ± 0.38 and was significantly altered during treatment: treatment visit 1, cough threshold (T1) log CT 0.72 ± 0.60, p = 0.024, treatment visit 2 (T2) log CT 0.80 ± 0.60, p = 0.025, treatment visit 3 (T3) log CT 0.69 ± 0.23, p = 0.002, until maximum effect had been achieved with no significant change at treatment visit 4 (T4) log CT 0.66 ± 0.65, p = 0.122. After completion of therapy, cough threshold improved significantly: log CT 1.14 ± 0.76, p = 0.001 [Individual cough threshold data (baseline v post treatment) represented in Figure There was a significant decrease in urge-to-cough with the speech language pathology treatment for chronic persistent cough. The urge-to-cough at baseline was reduced after treatment: Median (IQR), 5 (1) vs. 1 (4), p = 0.01 [Individual urge to cough data (baseline v post treatment) represented in Figure This is the first study to objectively assess response to a speech language pathology programme for refractory chronic cough using measures of cough sensitivity and cough frequency. We have shown that patients with refractory chronic cough have significantly decreased cough sensitivity and cough frequency together with an improvement in clinical outcome and cough and laryngeal symptoms following the speech language pathology intervention. Participants had an early symptom response to the speech language pathology program that was further improved upon throughout subsequent treatment sessions. Generally, a patient needed 3 to 4 treatment sessions and the response was maintained after the intervention ceased. it was found that isolated components such as specific cough suppression techniques or counselling were not enough for a patient to achieve a clinical response. In a previous study [The speech language pathology program for refractory CC includes several components, and from a previously conducted pilot study us study , we showus study  on coughWithin the large population of patients with CC, there is a small subgroup that does not respond to usual medical treatment ,28. In tThe study does suggest that the effective speech language pathology programme components reduce cough reflex sensitivity. This effect could occur by improvement in vocal hygiene leading to reduce sensory nerve stimulation, and is supported by the improvements in C5 and urge to cough during the programme. It is also possible that the reduction in cough frequency subsequently reduces cough-related airway trauma, and this explains the delayed improvement in cough threshold.In this study we used an open design with objective measures to assess outcome. Although a nonrandomized design is a limitation, our primary purpose was to treat refractory cough patients and determine their response to a therapy outside normal chronic cough treatment. We achieved this aim by using objective measures and presenting novel data showing that cough frequency and cough reflex hypersensitivity significantly improve after speech language pathology treatment. It is possible that a placebo effect such as cough suppression -33 may hWe did not find a heightened cough reflex sensitivity in CC females compared to CC males (power 90%) and this was consistent with our previous research ,27 (for In conclusion, this is the first study to show that speech language pathology management is an effective intervention for refractory chronic cough and that the mechanism behind the improvement is due to reduced laryngeal irritation which results in decreased cough sensitivity, decreased urge to cough and an increased cough threshold. This is accompanied with an improvement in cough symptoms, associated laryngeal symptoms, and cough quality of life. Speech language pathology may be a useful therapy for refractory chronic cough.CC: Chronic Cough; CRS: Cough Reflex Sensitivity; C5: concentration of capsaicin required to elicit 5 or more coughs within 30 secs after dose administration; LCM: Leicester Cough Monitor; LCQ: Leicester Cough Questionnaire; GLMM: Generalized linear mixed model; LDQ: Laryngeal Dysfunction Questionnaire; PVFM: Paradoxical Vocal Fold Movement; IQR: InterQuartile Range; T1-T4: Treatment No.; GORD: Gastro Oesophageal Reflux Disease; Snot-20: 20-item sino-nasal outcome test; HADS: Hospital Anxiety and Depression Scale.The authors declare that they have no competing interests.NR, AV and PG planned the study. AV, SB recruited the subjects, NR performed the objective cough tests. NR, AV and SB performed questionnaires, collected data and calculated scores. NR analysed the data. AV, SB performed speech pathology treatment. AV participated in the manuscript drafting. PG participated in the data interpretation, manuscript drafting and coordination of the manuscript. All authors read and approved the final manuscript.Participant details, supplemental methods and results.Click here for file"}
{"text": "Streptococcus (GBS) genome we found a novel putative surface exposed protein with pullulanase activity. We named such a protein SAP. The sap gene is highly conserved among GBS strains and homologous genes, such as PulA and SpuA, have been described in other pathogenic streptococci. The SAP protein contains two N-terminal carbohydrate-binding motifs, followed by a catalytic domain and a C-terminal LPXTG cell wall-anchoring domain. In vitro analysis revealed that the recombinant form of SAP is able to degrade α-glucan polysaccharides, such as pullulan, glycogen and starch. Moreover, NMR analysis showed that SAP acts as a type I pullulanase. Studies performed on whole bacteria indicated that the presence of α-glucan polysaccharides in culture medium up-regulated the expression of SAP on bacterial surface as confirmed by FACS analysis and confocal imaging. Deletion of the sap gene resulted in a reduced capacity of bacteria to grow in medium containing pullulan or glycogen, but not glucose or maltose, confirming the pivotal role of SAP in GBS metabolism of α-glucans. As reported for other streptococcal pullulanases, we found specific anti-SAP antibodies in human sera from healthy volunteers. Investigation of the functional role of anti-SAP antibodies revealed that incubation of GBS in the presence of sera from animals immunized with SAP reduced the capacity of the bacterium to degrade pullulan. Of interest, anti-SAP sera, although to a lower extent, also inhibited Group A Streptococcus pullulanase activity. These data open new perspectives on the possibility to use SAP as a potential vaccine component inducing functional cross-reacting antibodies interfering with streptococcal infections.Streptococcal pullulanases have been recently proposed as key components of the metabolic machinery involved in bacterial adaptation to host niches. By sequence analysis of the Group B  For example, the malto-oligosaccharide/maltodextrin–binding component of the Group A streptococcus malto-oligosaccharide ABC transporter has been shown to be directly involved in virulence in a mouse model of oropharynx infection et al. demonstrated that in human saliva the transcript levels of several GAS carbohydrate utilization proteins other than glucose are highly expressed S. pneumoniae (SPN) highlighted that a number of α-glucan–active enzymes seems to be virulence factors in a mouse model of lung infection The use of carbon sources is essential to the ability of bacteria to colonize the host and potentially cause disease in humans. In particular, highly polymerized α-glucan polysaccharides, such as starch and glycogen, are most likely to be found in environmental niches. Indeed, it is known that dietary-derived starches are very abundant in the human colon Streptococcus pneumoniae (SPN) pullulanases, named PulA and SpuA respectively, have been recently described Thermotoga maritime and it shares a high specificity for α-glucans. Of interest, PulA has been described to have multifunctional activities as the capability to hydrolyze pullulan, a linear polysaccharide of maltotriosyl repeating units linked by α- glycosidic linkage S. pyogenes gene expression Because of the complex structures of highly polymerized α-glucans, bacteria require an appropriate combination of enzymes for de-polymerization to oligo- and monosaccharides. Among these enzymes are ascribed pullulanases. Pullulanases have a glycosidic hydrolase activity towards α-glucan polysaccharides and are considered key extracellular components in bacterial metabolism. GAS and streptococcus (GBS) is an extracellular mucosal pathogen causing neonatal meningitis and invasive diseases in non-pregnant adults. GBS colonizes the lower gastrointestinal and genital tracts of healthy adults, as approximately 20–30% of healthy women are colonized rectovaginally with GBS Group B streptococcus (GAS) infections In silico analysis of recently sequenced GBS genomes Streptococcus agalactiae pullulanase (SAP). The sap gene is present in all sequenced GBS strains and the protein is highly conserved, with an amino acid identity ranges between 98 and 100%. SAP repeated CBMs bore ∼20% amino acid sequence identity to family 41 CBM found in a secreted Termotoga maritime pullulanase. CBM41 binds tightly to α-glucan polysaccharide comprising primarily α- glycosidic linkages Streptococcus pneumoniae (SPN)  pullulanases respectively  expression vector. As shown in E. coli. This is in agreement with previous data reported in the literature et al., unpublished observations) indicating the same protein pattern for recombinant PulA. On the basis of N-terminal sequencing analysis of the low MW form of SAP, which revealed the MKVQPNDYVF motif, we predicted a second putative GTG translational start codon within the COH1 sap ORF at position +1036 and a possible Shine-Dalgarno region (5′-AGGAGA-3′) 4 bp upstream of this point. The resulting translation product obtained from this start site yield a smaller protein lacking both CBMs. A mixture of the high and low molecular weight forms of SAP (H+L) was purified by affinity chromatography. Moreover, by anionic exchange chromatography, we were also able to separate SAP (L) from SAP (H+L). Both SAP recombinant preparations were used to demonstrate that a pullulanase enzymatic activity was associated to the protein.The The capacity of recombinant SAP to catalyze the degradation of α-glucan polysaccharides was tested by 3,5-dinitrosalicylic (DNS) acid assay . As shown in Of interest, comparison of SAP(H+L) versus SAP(L) preparations, showed that they were both active on pullulan, starch and amylopectin, while only SAP(H+L) was able to degrade glycogen. This finding suggests that the CBM contributes to the specific interaction with glycogen, in agreement with previous reports In addition, we compared the enzymatic activity of SAP versus GAS pullulanase (PulA) using different carbohydrates as substrates. As shown in 1H-1H 2D NMR scalar chemical shift correlation spectroscopy, which gave results in agreement with the assignments reported in the literature 1H NMR spectrum of pullulan after the addition of SAP(H+L)  Maltotriose+H1β(C) Maltotriose) and H1α1-4 (A+B) Maltotriose is 1∶2, we can conclude that the SAP cleaves α- glycosidic linkages between the units A and C generating maltotriose units In order to confirm the classification of SAP as a type I pullulanase we evaluated the modifications of the structure of pullulan after incubation with SAP by NMR spectroscopy. SAP(H+L)  containsGlycogen molecular size distribution before and after the addition of SAP(H+L) was instead determined by size exclusion chromatography. As reported in sap deletion mutant. As expected, the mutant strain was negative for SAP expression as confirmed by RT-PCR . Since bacterial pullulanases are known to be regulated by specific carbon sources y RT-PCR , WB analy RT-PCR  and FACSy RT-PCR .sap strain for the ability to grow in CM supplemented with different carbohydrates. As expected no growth differences were observed among these strains when glucose or maltose, that are not pullulanase substrates, were added to the CM  or anti-SAP antibodies purified from human sera was unsuccessful (data not shown). We hypothesize that the quantity and quality of anti-SAP antibodies derived from adult healthy volunteers that have been in contact with GBS, is not sufficient for our in vitro assay. Unfortunately, no sera from convalescent patients or with GBS invasive disease are at the moment available in our laboratory.In order to test whether the immunoglobulin-mediated response towards SAP impaired bacterial metabolic activity versus α-glucans, we tested by DNS acid assay the capacity of mouse and rabbit anti-SAP sera to prevent GBS pullulanase activity. We performed dose-dependent experiments incubating SAP-expressing bacteria with different sera dilutions in a range between 0.1–2%. As shown in Since SAP CBM appears to be a very immunogenic domain  and thatin vivo during the establishment of vaginal colonization and confirms the specificity of CBMs for glycogen in silico analysis of GBS available genomes revealed that SAP is the only surface associated protein containing glycosidic domains  and since SAP appears to be the only enzyme expressed by GBS capable to catabolize α-glucans, we suggest that this enzyme may be vital for GBS permanence in environmental niches poor in glucose. We are currently testing this hypothesis, and preliminary data have shown that deletion of the sap gene reduces the capacity of GBS to colonize the vagina in a mouse model of infection .The increasing need of new vaccine-based preventive strategies replacing the antibiotic prophylaxis used for eradicating GBS colonization of the genital tract of pregnant women, has recently led to identification of antigens conferring a broad protection in mice in vivo during GBS infections. However, to our knowledge the functionality of streptococcal pullulanase-induced antibody-mediated immune response has not been yet addressed. The fact that incubation of bacteria with an anti-SAP serum reduces GBS pullulanase activity, led us to hypothesize that in vivo antibody-mediated immune response towards SAP might affect GBS adaptive metabolism resulting in a decreased capacity to survive in the host.The importance of α-glucans metabolizing enzymes in streptococcal adaptation to the host is further highlighted by recent reports revealing they are immunogenic. Indeed, 81% of convalescent-phase sera from patients with invasive GAS infections had antibodies reactive with PulA Emerging theories on human-microbe mutualism suggest that the mechanisms that underlie microbial community structure and host–symbiont relationships should be considered for planning prevention strategies for human health. Indeed, as recently proposed by David A. Relman In conclusion, the evidence reported in this paper may draw up the basis for preventing streptococcal infections by using immunogenic metabolic enzymes as target molecules for vaccine development. The fact that, at least for pathogenic streptococci, such enzymes are well conserved opens new perspectives in the development of strategies preventing infections from multiple species.sap gene from 2603 V/R (TIGR Accession SAG_1216), 515 Ia (SAL_1339), NEM316 (gbs1288), H36B (SAI_1308), CJB111 (SAM_1238), A909 (SAK_1302) and COH1 (SAN_1346) strain as well as SpuA (SP_0268) and PulA (Spy_1972) was performed using ClustalW The alignment of SAP protein encoded by S. agalactiae strains COH1 serotype III was used in this study. Escherichia coli DH5α and DH10BT1 were used for cloning purposes and E. coli BL21 (λDE3) for expression of SAP fusion protein. S. agalactiae was cultivated at 37°C in Todd-Hewitt broth (THB) up to desired OD600. E. coli was grown in Luria-Bertani broth (LB); E. coli clones carrying the plasmids pJRS233 or pET21(b)+ and derivates were grown in the presence erythromycin (400 µg/mL) or ampicillin (100 µg/mL), respectively. The complex medium  was used for GBS growth with defined carbon sources. The sugar concentrations were 1% final. To evaluate growth in CM, GBS was initially grown to log phase (OD600 0.3) in THB. The cells were harvested by centrifugation, washed twice in an equivalent volume of phosphate-buffered saline (PBS) and diluted 1 to 50 in CM. Growth was monitored spectrophotometrically at a wavelength of 600 nm.sap gene from S. agalactiae COH1 serotype III was used as a template. The construct was amplified by PCR using specific primers GBS5F and GBS5R introducing NdeI and XhoI restriction enzyme sites  cells. BL21 (DE3) cells were grown in LB-Amp (100 µg/ml ampicillin) and induced with IPTG at a final concentration of 1 mM for 3 hours. The resulting biomass was suspended in 0.3 M NaCl, 50 mM Na-PO4 buffer, pH 8.0 and cells were lysed by enzymatic digestion. The sample was then loaded onto a His-Trap Ni-Activated Chelating Sepharose FF column  at a flow rate of 5 ml/min. Bound proteins were then eluted from the column by running a gradient from 0 to 50% of 500 mM Imidazole, 0.3 M NaCl, 50 mM Na phosphate buffer, pH 8.0 in 12 CV. The IMAC eluted material was collected in 2.5-ml fractions and those ones containing the SAP-His protein pooled. An anionic exchange chromatography was used to separate the two forms of SAP. The pooled fractions from Ni-IMAC were dialyzed against 30mM TRIS, pH 8.0 and then loaded on to a HiTrap Q HP 5 ml column (GE) to further purify the two forms of recombinant SAP. The purification was achieved by running a gradient from 0 to 50% 1M NaCl in 30mM TRIS, pH 8.0 in 16 CV at 5 ml/min. The collected fractions were analyzed by SDS-PAGE  and pooled according to apparent MW. The final preparation of the protein was obtained in PBS, pH 7.4 after dialysis.The PCR products were cloned into the pET21(b)+ vector and the plasmid transformed in sap gene was deleted in GBS strain COH1, according to the procedure previously described XhoI restriction enzyme cleavage sites were incorporated at the 5′-end of the primer to clone the fragment into the XhoI-digested pJRS233 plasmid. After cloning the in frame deletion fragment in pJRS233, the plasmid pJRS233Δsap was obtained.The sap was then transformed into the COH1 strain by electroporation and transformants were selected after growth at 30°C on agar plates containing 1 µg/ml erythromycin. Transformants were then grown at 37°C with erythromycin selection as previously described sap, resulting in the sap deletion on the chromosome. Dilutions of the serially passaged cultures were plated onto agar plates, and single colonies were tested for erythromycin sensitivity to confirm the excision of pJRS233Δsap. The resulting strain was named COH1Δsap.The plasmid pJRS233Δ600 0.4 in CM plus sugars, washed in PBS and incubated for 1 h at 37°C in 500 µl of Tris-HCl 50 mM (pH6.8) containing protease inhibitors and 400 U/ml of mutanolysin . The bacterial suspension was then pelleted and the supernatants containing peptidoglycan associated proteins used for western blotting analysis of SAP. In order to prepare GBS extracts relative to the secreted protein fraction, supernatant of bacteria cultures grown to OD600 0.4 were collected. Proteins in 1 ml of supernatant were precipitated with 10% of trichloroaceticacid (TCA) for 1 hr at 4°C. Protein were then pelletted, washed with cold acetone and resuspended in Tris-HCl pH 6,8.GBS protein extracts were prepared by growing bacteria up to OD600 0.4. Total GBS RNA was isolated using the Rneasy mini kit (Qiagen) according to manufacturer's instructions, except that bacteria were lysed with 100 µl of lysozyme (30 mg/ml) in Tris-EDTA buffer and 2,000 U of mutanolysin, and the mixture was incubated for 15 min at 37°C. Quantification of the transcripts was completed by reverse transcription and semi-quantitative RT-PCR using ImPromII RT (Promega) following manufacturer's instructions. Briefly 2 µg of sample and 0.5 µg of random hexamers were added to a final volume of 5 µl. Samples were incubated in a thermocycler (Biometra) at 70°C for 5 min followed by a quick chill at 4°C. The mixture was used in a 20-µl  cDNA synthesis reaction mixture comprising 4 µl of Improm-II 5× reaction buffer (Promega), 2.4 µl MgCl2 at 25 mM, 2 µl of dNTP mix (each dNTP at 2.5 mM), 0.25 µl of Rnasin RNase inhibitor (Promega) and 1 µl of Improm-II reverse transcriptase. The reaction was performed at 42°C for 60 min. In the negative controls, the reverse transcriptase was substituted with water. 2 µl of cDNA were then added to the PCR reaction consisting of 1× reaction buffer, 200 µM dNTP's, 0.2 µM primer pair, 1 U PlatinumTaq dna polymerase (Invitrogen). GBS Gyrase A (GyrA) was used as an internal housekeeping control. PCR reactions consisted of a 7-min denaturation step 94°C, followed by a variable number of cycles. PCR products were electrophoresed through 2% agarose gels and images were acquired by laser densitometry (Gel-Doc Imaging System).COH1 was grown in CM medium plus sugars up to OD2O, 99.9% atom D – Aldrich was used) to a uniform concentration. Samples were therefore transferred to 5-mm NMR tubes (Wilmad Glass. Co.). 70 µL of SAP were therefore added to the pullulan and glycogen samples in the NMR tubes. For every sample, two NMR spectra were recorded, the first on the native polysaccharide and the second 1 hour later (incubation at 25°C) after the addition of enzyme. Samples of maltose (Sigma) and maltotriose were also prepared by dissolving 10 mg of powder in 0.7 mL of deuterated PBS buffer at pH 7.2. 1H NMR experiments were recorded at 25°C on Bruker Avance 600 MHz spectrometer and using 5-mm probe (Bruker). For data acquisition and processing XWINNMR software package (Bruker) was used. 1-D proton NMR spectra were recorded using a standard one-pulse experiment. 64 scans were collected and averaged, giving a total acquisition time of ca. 10 min. The transmitter was set at the HDO frequency, collecting 32 k data points over a spectral window of 6,000 Hz. 1H NMR spectra were obtained in quantitative matter using a total recycle time to ensure a full recovery of each signal . Spectra were Fourier Transformed to 32 k data points after applying a 0.2 Hz line broadening function and referenced relative to the HDO resonance at 4.79 ppm.Samples of pullulan (Sigma) and glycogen (Sigma) were prepared by dissolving polysaccaride powder (10 mg) in 0.7 mL of deuterated PBS buffer at pH 7.2 (DSamples of glycogen were prepared by dissolving polysaccaride powder (1 mg) in 0.1 mL of PBS at pH 7.2 to a uniform concentration. Samples were therefore transferred to 1 mL vials (Waters). 10 µL of SAP were therefore added to the glycogen sample. Two chromatograms were recorded, the first on the native polysaccharide and the second 1 hour later (incubation at 25°C) after the addition of enzyme.5 Da was used. Samples were loaded onto the gel filtration column and eluted isocratically in 100 mM sodium phosphate + 100 mM NaCl buffer pH 7.2 at a flow rate of 0.5 ml min−1 for 50 min. The elution was monitored with a Ultimate 3000 Photodiode Array detector (Dionex) coupled with the Ultimate 3000 HPLC system (Dionex). For data acquisition and processing Chromeleon software package (Dionex) was used.A TSK G4000PW (TosoHaas) gel filtration analytical column (7.5 mm×30.0 cm) with a fractionation range of Mw PEG/PEO 2,000–3×10600 0.4 in CM with 1% sugar, fixed with 1% PFA for 20 min at RT and incubated with mouse anti-SAP serum or mouse anti-PBS serum (negative control) in 0.1% BSA plus 20% of Normal Calf Serum (NCS) for 1 h at 4°C. Bacteria were then washed in PBS containing 0.1% BSA and incubated with the phicoerytrin (PE) conjugated secondary antibodies  for 45 min at 4°C. After washing bacteria were resuspended in 200 µl of PBS and analyzed by a FACSscan flow cytometer (Becton Dickinson) by using FlowJo software program.In order to quantify the exposure of SAP on the bacterial surface, GBS was grown up to OD600 0.3  in CM plus 1% sugars. Bacteria were then centrifuged for 10 min at 3000 rpm (RT), washed and resuspended in 1 ml of PBS. Formvar-carbon-coated nickel grids were floated on drops of GBS suspensions for 5 min. The grids were then fixed in 2% PFA for 5 min, and placed in blocking solution  for 30 min. The grids were then floated on drops of primary antiserum against the SAP protein diluted 1∶20 in blocking solution for 30 min at RT, washed with six drops of blocking solution, and floated on secondary antibody conjugated to 10-nm gold particles diluted 1∶20 in 1% BSA for 30 min. The grids were examined using a TEM GEOL 1200EX II transmission electron microscope.GBS strains COH1 were grown at 37°C up to OD600 0.4 and washed in PBS. Bacterial pellet were fixed in 2% PFA for 20 min at RT and spotted on POLYSINE™ slides (Menzel-Glaser). The slides were then blocked with 3% BSA for 1 h and incubated with a mix of rabbit anti-serotype III capsule and mouse anti-SAP antibodies diluted in 1% BSA for 1 h at RT. Bacteria were then stained with goat anti-mouse and anti-rabbit Alexa Fluor conjugated antibodies  (Molecular Probes) for 20 min at RT. Slow Fade reagent kit (Molecular Probes) was then used to mount cover slips. The slides were analysed with a Bio-Rad confocal scanning microscope.In order to visualize SAP on bacterial surface, COH1 was grown in CM plus sugars up to ODPullulanase activity was determined by measuring the enzymatic release of reducing groups from α-glucans by the DNS colorimetric method 600 0.6) in THB and THY, respectively. Bacteria were then re-inoculated in CM containing 1% pullulan and grown to log phase (OD600 0.4) to allow the expression of pullulanases on bacterial surface. The cells were then harvested by centrifugation, washed twice with PBS and resuspended in PBS. Bacteria (∼5×108 CFU) were pre-incubated with sera dilutions at 37°C for 15 min, then pullulan was added  and the incubation prolonged for other 2 hours. Samples were centrifuged and supernatants were used for the determination of reducing sugars by DNS acid assay.GBS and GAS were grown at 37°C up to mid-late exponential phase (OD"}
{"text": "Cough is one of the main symptoms of advanced lung disease. However, the efficacy of currently available treatment remains unsatisfactory. Research into the new antitussives requires an objective assessment of cough.The aim of the study was to test the feasibility of a new automatic portable cough analyser and assess the correlation between subjective and objective evaluations of cough in 13 patients with chronic cough. The patients' individual histories, a cough symptom score and a numeric cough scale (1-10) were used as a subjective evaluation of cough and a computerized audio-timed recorder was used to measure the frequency of coughing.The pre-clinical validation has shown that an automated cough analyser is an accurate and reliable tool for the ambulatory assessment of chronic cough. In the clinical part of the experiment for the daytime, subjective cough scoring correlated with the number of all cough incidents recorded by the cough analyser  and the number of cough incidents per hour . However, there was no relation between cough score and the time spent coughing per hour . As assessed for the night-time period, no correlation was found between subjective cough scoring and the number of incidents per hour  or time spent coughing .An automated cough analyser seems to be a feasible tool for the ambulatory monitoring of cough. There is a moderate correlation between subjective and objective assessments of cough during the daytime, whereas the discrepancy in the evaluation of night-time coughing might suggest that subjective analysis is unreliable. Cough is one of the main symptoms of advanced lung disease. However, the efficacy of currently available treatment remains unsatisfactory. Growing knowledge of the pathophysiology of cough has resulted in the implementation of new therapeutic options and research on future potential treatment modalities -3. ReseaIn this study we first tested the feasibility of a new portable automatic cough analyser (CA) which could be applied to 24-hour monitoring in an ambulatory setting Figure . We thenSixteen patients who had been treated at the Department of Pneumonology and Allergology at the Medical University of Gdansk, Poland, were included in the study Table .The inclusion criteria were as follows: chronic cough defined according to the ERS , being oThe exclusion criterion was asthma exacerbation. The study was approved by the Local Research Ethics Committee in Gdansk and patients' individual histories, a cough symptom score and a numeric cough scale (1-10) were used for a subjective evaluation of cough. Portable cough recording was commenced following lung function assessment by spirometry. In the case of ambulatory patients, these were allowed to go home and encouraged to wear the cough-recording device for 24 hours. Moreover, they were asked to self-report their cough during the day and night.The cough analyser (CA) designed for the purpose of the study  was supplied with batteries (2 × AAA 2.4 V) and worn by patients in a special pouch around the thorax Figure . A microThe CA was able to measure acoustic vibrations continuously and the signals being transferred to a recording of sound amplitude. The hardware for the CA allows for a 0.1-second resolution time in registering signals with a cut-off value of 20 dB, whereas the frequency range is 8-1200 Hz.The process of the MES CA validation consisted of three steps.h-m) was assessed according to the following mathematical formula:First - 6 healthy individuals voluntarily produced cough. A comparison of the agreement of manual counting by software using the MES Cough Analyser against counting by the hearing of cough incidents in real time was checked. The counting error  was evaluated for different parameters of automatic analysis, such as time (0.1 s or 1 s) and amplitude of discrimination .Six healthy volunteers coughed in their normal office environments at work  Table . The coua-m was assessed according to the following mathematical formula:ECm - number of incidents counted manually by the software MES Cough AnalyserNa - number of incidents automatically counted by the cough analyserNThe results of the calibration tests showed that the optimal parameters of automatic analysis are time discrimination = 1 second and amplitude discrimination = 10%. This means that the recognition of a signal begins if the amplitude reaches 10% as well as disappearing at the level of 10%.a-m was equal to 2.93% (range 2.27-3.57), thus ECa-m was < 5%.Using these parameters of automatic analysis means that ECThird - automatic counting verified against that achieved via manual counting by the software MES Cough Analyser during prolonged monitoring Table .The thirteen measurements in healthy volunteers lasted a total of 172 hours.The parameters of digital measurement accepted for signal detection were based on the results of the previous step of calibration: discrimination time = 1 second; minimal amplitude of incident = 10%. Thus, the software enabled registration of a 1-second incident as a cough . A 1-second interval was required to discriminate between incidents. The software presented each incident as a percentage of the maximum amplitude achieved by an individual patient. The recognition of a signal began if the amplitude reached 10% and also disappeared at the level of 10%.The results of the validation process enabled the calculation of sensitivity greater than 95% , a specificity greater than 99% , and a median false positive rate = 0.0036 (range 0.0019-0.0054)  during the clinical part, we withdrew signals with an amplitude lower than 50% of the maximum amplitude obtained during cough. We quantified cough in terms of the number of incidents and the time spent coughing in seconds per hour; we analysed day and night periods separately.Patients were asked to self-assess their cough simultaneously with the CA monitoring. The evaluation included the intensity and duration of the symptom based on a Numerical Rating Scale (NRS: 1 = no cough to 10 = worst cough) and a cough scoring system recommended by the ERS ,6.The validation of the CA revealed a sensitivity for discriminating between coughs equal to 95%. Based on clinical experience, we had assumed the sensitivity of the cough score to be 55%. We calculated that to obtain a power of 70% (p = 0.05) for the study we would need a sample size involving 16 patients .The time for monitoring was arbitrarily divided into night (N) and day (D) . The steps for the calculation included: (1) counting the number of cough incidents during D and N separately; and (2) calculating the mean number of incidents per hour for D and N. These mean values were subsequently used for further analysis of correlations between particular features. The same was calculated for time spent coughing. Statistical analysis was conducted using a licensed version of statistical software: STATISTICA PL 5.0 for Windows. The distribution of variables using the Shapiro-Wilk test was abnormal, therefore non-parametric statistical tests were chosen. A Wilcoxon rank test was performed to assess the differences between particular parameters. Spearman correlations were used to examine the relationship between subjective and objective measures of cough.Sixteen patients with chronic cough were included in the CA assessment   who removed it early due to discomfort during the night. Eight other patients decided for themselves to shorten the time of monitoring due to occupational obligations.Among the 16 patients included in the analysis, the median duration time of objective cough monitoring (the entire study period) was 21 hours and 45 minutes (range 15-24 hours) (Table The median time spent coughing for the study group was 16.5 seconds per hour with a wide range between patients (0-52). Time spent coughing was higher during the day than at night. The median time spent coughing during the day was 20 seconds per hour (range 0-78), whereas at night this was 3 seconds per hour . There was a moderate correlation between time spent coughing per hour during D and N .Thirteen patients completed the subjective evaluation form Table . The medFor the daytime, subjective cough scoring correlated moderately with the number of all cough incidents  and the number of cough incidents per hour  (Table In the pre-clinical assessment our new portable automatic cough analyser seemed to be a simple and practical but accurate and reliable method for the objective prolonged monitoring of cough in an ambulatory setting. Our pilot clinical study compared subjective and objective evaluations of cough in patients with chronic cough. The results showed a moderate correlation between cough scoring and the number of cough incidents measured with the CA during the day, whereas there was a lack of correlation between these parameters at night.Until now the relationships between different subjective and objective measures of cough have not been clarified and have been controversial in different trials -11. In cRecently, the need for integrated recording systems for cough assessment has been stressed, particularly in the clinical evaluation of drug efficacy ,5. ThereIt is worth mentioning that a group from Leicester, UK, introduced an automated system for the 24-hour monitoring of cough frequency. Their studies have proved to be significant, with repeatable differences seen in cough frequency between patients with chronic cough and healthy controls ; they haHowever, it is likely that automated cough monitoring is both device and method specific; therefore, the findings of previous studies cannot be extrapolated to new devices.The unsatisfactory effectiveness of currently available antitussives, particularly in patients with advanced lung disease, has provoked increased interest in the development of some novel approaches. One of these is to reach opioid receptors in the trachea-bronchial tract using nebulised opioids  or otherClinical trials on new antitussive drugs would require the thorough objective assessment of the intensity and frequency of cough, which means that introducing portable devices such as a CA might be of clinical relevance.We conclude that appropriate night-time cough evaluation requires both subjective and objective monitoring. The severity of cough defined by a patient may not represent its frequency and duration when measured objectively. The use of ambulatory cough analysers should not be limited to research but might be helpful in clinical practice, particularly when symptomatic treatment is the main or only purpose of the approach.The authors declare that they have no competing interests.MK and IDK were involved in the design of the study protocol, in the patients enrolment and examination, in the analysis and interpretation of data and writing the paper. IDK performed a statistical analysis. LG took a part in the patients enrolment to the study, in a data collection and analysis, and revised the manuscript critically. EJ made substantial contribution to conception and design of the study protocol, analysis and interpretation of data, coordination of the trial and helped to draft the manuscript. All authors read and approved the final manuscript."}
{"text": "This free radical further reacts with oxygen to form the peroxymonosulfate anion radical (−O3SOO•) and sulfate anion radical (SO4• −).Sulfur dioxide, formed during the combustion of fossil fuels, is a major air pollutant near large cities. Its two ionized forms in aqueous solution, sulfite and (bi)sulfite, are widely used as preservatives and antioxidants to prevent food and beverage spoilage. (Bi)sulfite can be oxidized by peroxidases to form the very reactive sulfur trioxide anion radical  to initiate this radical chain reaction, using human serum albumin (HSA) as a model target.We used electron paramagnetic resonance, optical spectroscopy, oxygen uptake, and immuno-spin trapping to study the protein oxidations driven by sulfite-derived radicals.•SO3− was produced, with the concomitant reduction of SOD-Cu(II) to SOD-Cu(I). Further, we demonstrated that sulfite oxidation mediated by Cu,Zn-SOD induced the formation of radical-derived 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin-trapped HSA radicals.We found that when Cu,Zn-SOD reacted with (bi)sulfite, The present study suggests that protein oxidative damage resulting from (bi)sulfite oxidation promoted by Cu,Zn-SOD could be involved in oxidative damage and tissue injury in (bi)sulfite-exacerbated allergic reactions. At high pH, Cu,Zn-SOD exhibits peroxidase activity, with the initial step of the peroxidase cycle being a reduction of SOD-Cu(II) by H2O2 or its deprotonated form, HO2−, to SOD-Cu(I) (3−) buffer sulfite (HSO3−) and (bi)selenite (HSeO3−), may also stimulate the peroxidase activity of Cu,Zn-SOD in the presence of millimolar H2O2 , a metalloenzyme that catalyzes the dismutation of the superoxide anion into OOD-Cu(I) . At neut) buffer . It has lar H2O2 .2SO3) was a one-electron donor substrate for Cu,Zn-SOD, leading to the generation of reactive sulfur radicals via Equations 2–4. We also applied immuno-spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to investigate oxidation of target proteins  to protein radicals sulfite oxidation and found that, under our experimental conditions, SOD1-Cu(II) is slowly reduced to SOD1-Cu(I) by (bi)sulfite. We used optical spectroscopy, electron spin resonance (ESR), and oxygen uptake experiments to demonstrate that (bi)sulfite  were from Sigma Chemical Co. . We determined the H2O2 concentration from its absorbance at 240 nm (ɛ = 39.4 M −1cm −1). DMPO  from Alexis Biochemicals  was sublimed twice under vacuum at room temperature and stored under an argon atmosphere at −80°C before use. Chelex-100 resin was from Bio-Rad Laboratories .We purchased bovine kidney superoxide dismutase (SOD) from Calzyme Laboratories Inc. . HSA (99.99% purity), diethylenetriaminepentaacetic acid (DTPA), sodium sulfite, thiocyanate, azide, cyanide, and HWe obtained ESR spin-trapping data at room temperature using a Bruker EMX spectrometer with 100 kHz modulation frequency and equipped with an ER 4122 SHQ cavity . We placed samples in a 10-mm flat cell  and initiated recording of the spectra within 1 min of the start of the reaction.We recorded low-temperature ESR data at 77 K after the indicated incubation times. Initially, we mixed SOD with (bi)sulfite at room temperature; after incubation, we transferred the reaction mixtures into 1-mL polyethylene syringes and froze them in liquid nitrogen. We added glycerol (10%) to the samples before freezing to prevent cracking of the frozen texture.For oxygen uptake measurements, we added 500 μL sodium sulfite to a chamber equipped with a Clark electrode and a stirrer. We initiated the reaction (1.8 mL) by SOD, and the oxygen uptake curves were obtained at room temperature with an oxygen monitor .2SO3, and 50 μM Cu,Zn-SOD in the presence or absence of 5 mM DMPO in 100 mM phosphate buffer (Chelex-treated with 25 μM DTPA) at pH 7.4 in a total volume of 30 μL. After 1 hr of incubation at 37°C, we stopped reactions with 5 mM reduced glutathione and then diluted the samples with deionized H2O for electrophoresis and immuno-spin trapping analyses.Typically, we carried out reactions of 600 μM HSA, 500 μM NaWe electrophoresed the reaction mixtures under reducing conditions through duplicate 4–12% BisTris NuPage acylamide gels . We performed Western blotting and ELISA analysis as previously described  with min−1cm−1 in the bovine enzyme), which results from the d-d transitions of the Cu atom  using a 500 μM quartz cuvette. We determined Cu,Zn-SOD concentration from the broad band at 680 nm (ɛ = 300 M Cu atom . We carr|| = 135 G) remained unchanged during the incubation time, indicating that (bi)sulfite does not bind directly to the active site Cu(II) (When we added a 20-fold excess of (bi)sulfite to 1 mM Cu,Zn-SOD, the absorption band at 680 nm characteristic of the active site of SOD1-Cu(II) decreased slowly, then completely disappeared as the wild-type protein was reduced to Cu(I) . We recoe Cu(II) .•SO3− adduct of DMPO, DMPO/•SO3− (aHβ= 16.0 G and aN = 14.7 G) (•SO3− is formed when the protein is treated with 1 mM H2O2 in the presence of 20 mM (bi)sulfite sulfite by H2O2, which is known from the literature (2O2 via the following reaction:To determine whether (bi)sulfite is oxidized by Cu(II) in Cu,Zn-SOD, we also performed room-temperature ESR spin-trapping experiments. When we mixed Cu,Zn-SOD (50 μM) with (bi)sulfite (500 μM) in the presence of the spin trap DMPO (100 mM), it generated an intense ESR signal ( 14.7 G) , 1982b. )sulfite . Accorditerature  to proce2O2 (100 μM) and to confirm that •SO3− is generated because of the enzymatic oxidation of (bi)sulfite, we performed control experiments in the presence and absence of H2O2. Contrary to expectation  or Cu,Zn-SOD resulted in no radical adduct formation (2O2 is not involved (data not shown).To determine the effect of low and nontoxic concentration of Hectation , additio•SO3− by the active Cu(II) site of Cu,Zn-SOD proceeds in a one-electron reduction reaction of Cu(II) by (bi)sulfite, similar to the oxidation of (bi)sulfite by HRP and prostaglandin H synthase  almost completely prevented oxygen uptake (−O3SOO• and SO4• −.The proposed mechanism of enzymatic oxidation of (bi)sulfite to synthase . The resreported . To confntration . Addition uptake , that is•SO3−, we mixed 500 μM Na2SO3 with selected inhibitors and initiated the reactions by 50 μM Cu,Zn-SOD in the presence of 100 mM DMPO sulfite to sulfite-derived radicals is possible.To characterize the importance of Cu redox cycling at the enzyme-active site upon the generation of  mM DMPO . The ESR•SO3−, which will initiate the radical chain reaction with formation of −O3SOO• and SO4• − via Equations 2–4. To characterize the ability of these radicals to oxidize amino acid(s) in target proteins, we incubated HSA with the enzyme and (bi)sulfite in the presence of DMPO and analyzed the reaction products by Western blotting using an anti-DMPO polyclonal antibody sulfite is oxidized by Cu,Zn-SOD to antibody . We chos 120 kDa . We alsoby ELISA . This reHSA-derived nitrone adducts also depended on the (bi)sulfite concentration . Omissio2SO3, and 50 μM Cu,Zn-SOD, Western blotting showed that DMPO-HSA radical-derived nitrone adduct production increased with reaction time, reaching saturation at about 1 hr. ELISA data paralleled those from Western blotting —can attack target proteins   sulfite by Cu,Zn-SOD proceeds via a radical mechanism as demonstrated using optical spectroscopy, oxygen uptake, and ESR experiments. Similar results have been reported for some peroxidases  , 1982b. d SO4• − , which—a plasma)   . Previouor •SO3− . In fact(100 mM) , and a dd SO4• − . The vere SO4• − .(Bi)sulfite is one of the few sulfating agents approved by the Food and Drug Administration as a food preservative and antioxidant to prevent or reduce spoilage . However42−), and it is noteworthy that in cases of sulfite oxidase deficiency, the concentration of sulfite in plasma is abnormal (> 1 mM) (2 [(bi)sulfite] forms oxidation products other than sulfate, such as S-sulfonates sulfite is a deficiency of sulfite oxidase, the molybdenum-containing enzyme that oxidizes sulfite to sulfate (SO(> 1 mM) . The cap(> 1 mM) . Howeverlfonates ; this inlfonates  demonstr− (bi)sulfite as a source for generation of oxidants (−O3SOO• and SO4• −) that are diffusible and radicals themselves to show their capability to oxidize the most abundant plasma protein  to protein radicals sulfite provides an enzymatic pathway to generate the reactive intermediates −O3SOO• and SO4• −, which oxidize HSA residues to protein radicals. We also propose that Cu,Zn-SOD may contribute to oxidative damage and tissue injury in (bi)sulfite (sulfur dioxide)–exacerbated allergic reactions. Our results suggest that SOD-dependent, sulfite-mediated oxidation of albumin residues is likely to occur in vivo, particularly at sites where Cu,Zn-SOD concentration is higher. Further studies are necessary to clarify whether alterations in Cu,Zn-SOD activity affect (bi)sulfite toxicity.In summary, our study showed that Cu,Zn-SOD"}
{"text": "Chronic persistent cough can be associated with laryngeal dysfunction that leads to symptoms such as dysphonia, sensory hyperresponsiveness to capsaicin, and motor dysfunction with paradoxical vocal fold movement and variable extrathoracic airflow obstruction (reduced inspiratory airflow). Successful therapy of chronic persistent cough improves symptoms and sensory hyperresponsiveness. The effects of treatment for chronic cough on laryngeal dysfunction are not known.The aim of this study was to investigate effects of therapy for chronic cough and paradoxical vocal fold movement.Adults with chronic cough (n = 24) were assessed before and after treatment for chronic persistent cough by measuring quality of life, extrathoracic airway hyperresponsiveness to hypertonic saline provocation, capsaicin cough reflex hypersensitivity and fibreoptic laryngoscopy to observe paradoxical vocal fold movement. Subjects with chronic cough were classified into those with (n = 14) or without (n = 10) paradoxical vocal fold movement based on direct observation at laryngoscopy.Following treatment there was a significant improvement in cough related quality of life and cough reflex sensitivity in both groups. Subjects with chronic cough and paradoxical vocal fold movement also had additional improvements in extrathoracic airway hyperresponsiveness and paradoxical vocal fold movement. The degree of improvement in cough reflex sensitivity correlated with the improvement in extrathoracic airway hyperresponsiveness.Laryngeal dysfunction is common in chronic persistent cough, where it is manifest as paradoxical vocal fold movement and extrathoracic airway hyperresponsiveness. Successful treatment for chronic persistent cough leads to improvements in these features of laryngeal dysfunction. Chronic persistent cough is responsible for a significant illness burden in the community . LaryngeSubjects with chronic persistent cough (n = 24) were recruited from the Respiratory Ambulatory Care Service at John Hunter Hospital in Newcastle, New South Wales, Australia. Subjects were aged between 18 and 80 years with a persistent cough of more than eight weeks. They were non-smokers or ex-smokers with less than ten pack years, had no other active respiratory or cardiac disease, and were required to have a normal chest radiograph. They were classified into 2 groups based on the presence  or absence  of PVFM observed at fibreoptic laryngoscopy. All subjects provided written informed consent for this study, which was approved by the University of Newcastle's Human Research Ethics Committee and the Hunter New England Human Research Ethics Committee.Subjects attended a total of 5 visits over a period of 18 weeks. At visit 1, clinical history, current respiratory symptoms, medication use, passive smoking history and an in-house rhinitis symptoms score were recorded. A number of questionnaires were also administered and these included a cough specific quality of life questionnaire ) , a gastrAll subjects were non-smokers or ex-smokers with less than 10 pack years and not exposed to current passive smoking and this was confirmed by exhaled carbon monoxide measurement ,15. FracA probability based diagnostic assessment approach was used  with theForced Expired Nitric Oxide (FENO) was measured using an on-line chemiluminescence analyser  according to published European Respiratory Society/American Thoracic Society guidelines . Subject1 fell by more than 15%, 200 μg of salbutamol was administered via a valved holding chamber .Prior to HSC, subjects withheld bronchodilators for their duration of action and antihistamines for 48 hours. Subjects were instructed in the correct performance of inspiratory and expiratory Flow Volume Loops (FVL). The manoeuvre consisted of tidal breathing, deep inspiration to total lung capacity, forced expiration to residual volume followed by deep inspiration to total lung capacity. Hypertonic saline (4.5%) was inhaled for doubling time periods and a inspiratory-expiratory FVL was measured, in duplicate, 60 seconds after each saline dose using a KoKo K323200 Spirometer . Forced expiratory time was held constant at subsequent manoeuvres in order to ensure consistency. If the FEVSolutions of capsaicin  concentrations ranging from 0.98 to 500 μM were prepared daily. Subjects inhaled single breaths  to total lung capacity (TLC)) of capsaicin aerosol from a compressed air-driven nebulizer  controlled by a dosimeter . The inspiratory flow was standardized at 0.5 L/s with an inspiratory flow regulator valve. Cough counting was done for 30 s after exposure to each dose, and the investigation ended when the subject coughed five or more times in response to one dose, or received a dose of the highest concentration.Flexible fibreoptic laryngoscopy  was performed at baseline and immediately after a hypertonic saline challenge ,21. PrioThe gold standard used for the diagnosis of PVFM during the study was a positive laryngoscopy demonstrating paradoxical vocal fold motion at baseline and/or post-HSC while symptomatic.All analyses were performed using statistical and data analysis software STATA . Non parametric quantitative data were compared using the Wilcoxon rank sum test and for parametric data, ttest for matched pair data was used. Significance for 2 group comparison was set at p < 0.05.Twenty-four subjects with a chronic persistent cough participated in the study. The subjects had a median (IQR) cough duration of 24 13–84) months and were predominantly female [Table 3–84 montTen of the 14 subjects with PVFM attended speech language therapy. After treatment, PVFM had resolved in 8 of these 10 subjects (p = 0.039 by McNemar's chi square test). Four of the Cough+PVFM subjects did not attend speech language therapy before returning for their post-treatment visits. PVFM did not resolve in 3 of these 4 subjects but did resolve in 1 subject. Interestingly this subject was the only male in this group of four and had the shortest cough duration (12 months) and youngest age (22 years).In the Cough alone (CC) group, extrathoracic airway responsiveness was not increased and with therapy remained unchanged from baseline Figure . BaselinThis study has identified that paradoxical vocal fold movement and extrathoracic airway hyperresponsiveness are improved by specific treatment for chronic persistent cough, and that this improvement occurs alongside improvements in cough specific quality of life and cough reflex sensitivity. The data provides objective evidence of laryngeal dysfunction in some patients with chronic cough, and shows that it responds to therapy for chronic persistent cough. These results are consistent with Vertigan et al  who founLaryngeal dysfunction is increasingly recognized in chronic persistent cough. Symptoms such as voice hoarseness, dyspnoea, wheeze and cough may all occur as a result of laryngeal dysfunction . Prudon In this study we used an open design with objective measures to assess outcome. Although a nonrandomized design is a limitation, our primary purpose was to determine if the measures of laryngeal dysfunction that occur in chronic persistent cough are responsive to effective therapy. The study achieved these aims by using objective measures and has provided novel data on how PVFM and EAHR improve with therapy of chronic persistent cough. The results extend what is known about how successful therapy works in chronic persistent cough, and provide data that supports the favourable responses reported for symptoms, cough frequency, and measures of cough reflex sensitivity. We now show that laryngeal dysfunction also improves with treatment of chronic persistent cough in those patients with cough and PVFM. Future studies could provide further evidence of efficacy by using a randomized design, and potentially assessing any incremental benefits of speech language treatment.We studied subjects who were representative of those with chronic persistent cough. They were primarily middle-aged females (80%) with a significant cough duration and similar prevalence of the medical conditions that have been associated with persistent cough ,27,28. WThere was a moderately significant  correlation in the Cough+PVFM Group for treatment related changes in extrathoracic airway hyperresponsiveness dose response slope and CRS-C5 Figure . This deIn conclusion, this study identifies that the laryngeal dysfunction that occurs in some patients with chronic persistent cough is responsive to therapy.CC: Chronic Cough; CRS: Cough Reflex Sensitivity; EAHR: Extrathoracic Airway HyperResponsiveness; eCO: exhaled Carbon Monoxide; FENO: Forced Expired Nitric Oxide; FOL: Fibre Optic Laryngoscopy; GORD: GastroOesophageal Reflux Disease; HSC: Hypertonic Saline Challenge; IQR: InterQuartile Range; LCQ: Leicester Cough Questionnaire; LDQ: Laryngeal Dysfunction Questionnaire; PVFM: Paradoxical Vocal Fold MovementThe authors declare that they have no competing interests.NR and PG planned the study. NR recruited the subjects and performed the objective cough and EAHR methods, questionnaires, assisted with fibreoptic laryngoscopy, collected and reviewed data, participated in the design and drafted the manuscript. PG performed patient assessment, physical examinations and fibreoptic laryngoscopy and prescribed medication. AV performed speech pathology treatment and reviewed the manuscript. PG also participated in the manuscript drafting and coordination of the manuscript. All authors read and approved the final manuscript.Nicole M Ryan holds a PhD scholarship from the NHMRC CCRE in Respiratory and Sleep Medicine, Australia.Anne Vertigan holds a post-doctoral fellowship from the NHMRC CCRE in Respiratory and Sleep Medicine, AustraliaProfessor Peter Gibson is an NHMRC Practitioner Fellow."}
{"text": "Nigersaurus taqueti, document for the first time the cranial anatomy of a rebbachisaurid sauropod. Its extreme adaptations for herbivory at ground-level challenge current hypotheses regarding feeding function and feeding strategy among diplodocoids, the larger clade of sauropods that includes Nigersaurus. We used high resolution computed tomography, stereolithography, and standard molding and casting techniques to reassemble the extremely fragile skull. Computed tomography also allowed us to render the first endocast for a sauropod preserving portions of the olfactory bulbs, cerebrum and inner ear, the latter permitting us to establish habitual head posture. To elucidate evidence of tooth wear and tooth replacement rate, we used photographic-casting techniques and crown thin sections, respectively. To reconstruct its 9-meter postcranial skeleton, we combined and size-adjusted multiple partial skeletons. Finally, we used maximum parsimony algorithms on character data to obtain the best estimate of phylogenetic relationships among diplodocoid sauropods. Nigersaurus taqueti shows extreme adaptations for a dinosaurian herbivore including a skull of extremely light construction, tooth batteries located at the distal end of the jaws, tooth replacement as fast as one per month, an expanded muzzle that faces directly toward the ground, and hollow presacral vertebral centra with more air sac space than bone by volume. A cranial endocast provides the first reasonably complete view of a sauropod brain including its small olfactory bulbs and cerebrum. Skeletal and dental evidence suggests that Nigersaurus was a ground-level herbivore that gathered and sliced relatively soft vegetation, the culmination of a low-browsing feeding strategy first established among diplodocoids during the Jurassic.Fossils of the Early Cretaceous dinosaur,  Rebbachisaurus, came to light in Upper Cretaceous rocks in Morocco Some 50 years ago the first bones of an unusual sauropod, dubbed Nigersaurus taqueti  scans of the bones of a single adult individual to recompose the extremely lightweight skull . The del2 . These dNigersaurus have never been reported in any other sauropodomorph dinosaur including closure of the supratemporal opening, five additional fenestrae on the lateral aspect of the skull, and teeth packed into terminal dental batteries that extend laterally beyond the side of the skull Several cranial features of Nigersaurus, however, that fenestra is closed and the line of action to the lower jaw blocked by a sharp bend in the quadrate. As the quadrate shows no sign of a synovial surface or trochlea to bend this line of action (as occurs in some turtles), the origin of the adductor musculature must have migrated onto the quadrate , likewise, appear to have been relatively weak. The elongate basipterygoid processes that buttress the palate against the pull of pterygoideus musculature taper distally to rods no more than 2 mm in diameter.DiplodocusNigersaurus  on the lingual  surface of the upper teeth, which is similar to that in advanced titanosaurs . Light mDicraeosaurusDiplodocusA second high-angle wear facet (ca. 40° from the long axis of the crown) forms on the labial  surface of the upper teeth. This facet is D-shaped, rather than circular, owing to its intersection with the lingual  facet . The labNigersaurus as compared to an average hadrosaurid  composed of only 13 vertebrae, and an adult body length of only approximately 9 m  and modest body length of 10 meters or less characterizes nearly all rebbachisaurids Pneumatic invasion of the centra of the presacral vertebrae has reduced the vertebral body to a hollow shell divided by a thin median septum , left, mNigersaurus forces a consideration of the biomechanical limits of skull and axial design for a large-bodied, land-based herbivore and impacts our understanding of several aspects of sauropod paleobiology and evolution. No other herbivorous tetrapod has evolved a skull of comparable size with as little bone that is able to withstand sustained impact from tooth-to-tooth shearing. The shearing action of its jaws, furthermore, ground down crowns at a faster rate than ever recorded among dinosaurian herbivores and accomplished this dental degradation in the most challenging location from a biomechanical perspective—at the distal end of its jaws, as far as possible from the jaw joint and adductor musculature  when Nigersaurus lived. Previous reference to “head down” feeding by “ventriflexion” Here we distinguish Nigersaurus in particular suggests increasing adaptation to ground-level browsing. Broadening of the muzzle Nigersaurus and the squared muzzles in other diplodocoids would enhance gathering and cropping near a relatively flat surface.Squaring of the muzzle in diplodocoids in general and Nigersaurus  aspect of the dentition in ersaurus  are alsoNigersaurus, in particular, the snout could not have been directed anteriorly to strip leaves or generate an external wear facet without disarticulation of the skull from the neck. Estimating ‘feeding envelopes’ based on cervical vertebrae is revealing The spectrum of previous explanations for tooth form and wear in diplodocoids include molluscivory or piscivory Nigersaurus, this interpretation is open to question. Although the dentition in Diplodocus and some diplodocoids is less compact and more prognathous than Nigersaurus, there are several fundamental similarities, including the distal position of most of the tooth row in a squared jaws, slender crown shape, and increased rate of tooth replacement (judging from the number of crowns in the replacement series). Like Diplodocus and Dicraeosaurus, Nigersaurus has a high-angle labial  wear facet, the product of abrasion against plant or other materials  facet in Nigersaurus and possibly other diplodocoids, thus, as evidence of tooth-to-plant abrasion that occurred as vegetation was gathered near the ground prior to orthal shearing.Finally, jaw movement in dicraeosaurids and diplodocids has long been interpreted as propalinal (fore-aft), either with aterials . Nigersay crowns  that proDiplodocus and Nigersaurus and constraints on forward rotation of the skull seem to favor browsing near the ground, which would have lowered and diversified the ‘browsing profile’ of faunas with several sauropods In sum, although the paradigm for feeding among diplodocoids has shifted from high browsing Nigersaurus and other diplodocoids is speculative due to the paucity of evidence from stomach or fecal contents, the true composition of local flora, and the poor correlation between tooth form/wear and plant type. Nevertheless, the weak bite force and fine scale of scratches on the wear facets in Nigersaurus favor soft understory vegetation, possibly immature ferns or horsetails Nigersaurus and the iguanodontian LurdusaurusThe identity of specific plant groups as food resources for Nigersaurus and other diplodocoids due to their height, resistant structure, absence of a suitable local habitat, and late appearance in the fossil record, respectively. Aquatic vegetation is difficult to imagine as a primary resource in the varied habitats that have yielded diplodocoid remains. Cross-bedded sandstones, rather than sediments indicative of still water, comprise nearly all of the Elrhaz Formation, where the bones of Nigersaurus are found  abrasion facet on its teeth may have been generated during ground-level browsing. The low-angle, planar facet on in the inside of the maxillary crowns indicates that Nigersaurus many cranial and dental features including a squared end to the lower jaw and a labial abrasion facet, they may have shared aspects of their feeding strategies. It seems plausible, at the very least, that other diplodocoids had orthal, rather than a propalinal (fore-aft), jaw movement.Because other diplodocoids share with Nigersaurus are conditions that characterize many diplodocoids and now represent an alternative basal condition for the clade. Nigersaurus is the culmination of a low-browsing feeding strategy among diplodocoids that originated in the mid Jurassic and may have had an ecologically significant impact on surface vegetation on several land areas during the Cretaceous.Modest body length, a proportionately short neck, and downward deflection of the muzzle in Figure S1Nigersaurus taqueti (MNN GAD517) discovered during the 2000 Expedition to Niger. Expedition member G. Lyon is seated inside the curve of the proximal caudal vertebrae of a skeleton planed flat by wind-blown sand at a site in Gadoufaoua, Ténéré Desert, Niger (photo by M. Hettwer).Partial skeleton of (0.34 MB TIF)Click here for additional data file.Figure S2Nigersaurus taqueti were found.Location of outcrops of the Elrhaz Formation where fossils of (0.13 MB TIF)Click here for additional data file.Figure S3Nigersaurus taqueti built from prototyped skull bones (tooth batteries and reconstructed teeth in red) with unknown bones in green modeling clay (photo by T. Keillor).Assembled semi-translucent skull model of (3.47 MB TIF)Click here for additional data file.Figure S4Nigersaurus taqueti (MNN GAD512) derived from surface renderings of µCT scan data. (A)-left lateral view. (B)-left anteroventrolateral view. -ventral view. -dorsal view. -anterior view. -posterior view. Color scheme: cyan blue, cranial endocast; pink, endosseous labyrinth; yellow, nerve canals ; red, arterial canals; dark blue, smaller venous canals. Abbreviations: car, cerebral carotid artery canal; cer, cerebral hemisphere; cvcm, caudal middle cerebral vein; de, dural expansion; fl, flocculus ( = cerebellar auricle); lab, endosseous labyrinth; ob, olfactory bulb; ocv, orbitocerebral vein; opt?, possible optic tectum ( = lobe); pfo, pituitary  fossa; II, optic nerve canal; III, oculomotor nerve canal; IV, trochlear nerve canal; V, trigeminal nerve canal; VI, abducens nerve canal; VII, facial nerve canal; VIII, canal for vestibular branch of vestibulocochlear nerve; IX-XI, shared canal for glossopharyngeal, vagus, and accessory nerves and accompanying vessels; XII, hypoglossal canal.Cranial endocast, endosseous labyrinth, and some endocranial vascular structures in (7.88 MB TIF)Click here for additional data file.Figure S5Nigersaurus taqueti (MNN GAD512), (D-E) the diplodocid Diplodocus longus (CM 11161), and (F-G) the basal neosauropod Camarasaurus lentus (CM 11338). -left lateral view. -dorsal view. (C)-posterior view. Abbreviations: c, cochlea; csc, caudal  semicircular canal; fc, fenestra cochlea ( = round window); fv, fenestra vestibuli ; lsc, lateral  semicircular canal; rsc, rostral  semicircular canal; ve, vestibule of inner ear.Endosseous labyrinths of the left inner ear of  the rebbachisaurid (2.41 MB TIF)Click here for additional data file.Figure S6Nigersaurus taqueti. Colors highlight the partitions used for digital assessment of endocast volumes.Partitioned endocast with transparent osseous labyrinth from (1.27 MB TIF)Click here for additional data file.Text S1Geologic setting.(0.07 MB PDF)Click here for additional data file.Text S2The prototype skull.(0.08 MB PDF)Click here for additional data file.Text S3Endocast and labyrinth.(0.15 MB PDF)Click here for additional data file.Text S4Microwear and incremental lines of von Ebner.(0.13 MB PDF)Click here for additional data file.Text S5Phylogenetic analysis.(0.16 MB PDF)Click here for additional data file."}
{"text": "Outpatient surgery accounts for a significant percentage of anaesthetics delivered in the United States and may be appropriate in infants for certain procedures. The most concerning adverse event after general anaesthesia in an infant is postoperative apnea.There is little specific evidence of the risk of apnea in full term infants. There are facilities that feel comfortable performing outpatient surgeries if the infant is born at greater than 37 wk postconceptual age. However, other ambulatory centers prefer to wait untill the infant is 2-4 wk of age to ensure decreased physiologic jaundice, decreased pulmonary vascular resistance and to give time for the ductus arteriosus to close. As far as sudden infant death syndrome(SIDS) there is no evidence anaesthesia increases the risk−1). In the nonanemicchild with a gestational age of 32 wk and a postconceptual age of 56 wk, the probability of apnea was <1%. With a gestational age of 35 wk, a postconceptual age of 54 wk was the cutoff for apnea to be present in <1% of patients.In the premature infant, apnea is more likely to occur as well as other airway complications such as atelectasis, aspiration pneumonia, stridor, and coughing with desaturation in infants undergoing inguinal herniorrhaphy−1 or placebo, the caffeine group had no postoperative bradycardia, prolonged apnea, periodic breathing or postoperative oxygen saturation <90%, whereas 81% of the patients in the control group had prolonged apnea at 4-6 h postoperativelyCaffeine has been shown to decrease the risk of apnea in preterm infants undergoing general anaesthesia. In 32 preterm infants  who received caffeine 10 mg.kgSpinal anaesthesia alone has been shown to have a lower incidence of postoperative apnea and bradycardia in former premature infants when compared to spinal plus sedation or general anaesthesia9Patients <60wk postconceptual age for hernia repair had shorter times to extubation with no postoperative apnea after thiopental or halothane induction with desflurane maintenance than either halothane or sevoflurane for the entire anaestheticThe cutoff for outpatient surgery in infants born before 37 wk may be 50-52 wk postconceptual age as long as there is no anemia, prior apnea or coexisting disease. However, looking at the evidence based literature to decrease the risk of apneas to <1% patients should be greater than 54 wk postconceptual age without anemia, ongoing apnea or other significant medical problems. Postoperative monitoring ranges from 12-24h, including oxygen saturation, heart rate and impedance pneumography and whether the infants are apnea-free before discharge.Caffeine or spinal anaesthesia may decrease the risk of apnea, but patients should not be discharged if they are not eligible for anaesthesia on an outpatient basis. Full-term infants are acceptable for outpatient procedures provided that they are otherwise healthy and the procedure is not likely to result in significant physiologic changes or postoperative pain requiring opioid medication and the anaesthetic is uneventful. Even in term infants some facilities will not allow outpatient surgery untill they are of 44-46 wk post conceptual age or require longer observation if younger, e.g., 4 h."}
{"text": "Hepatitis E, Central African Republic To the Editor: Outbreaks of hepatitis E virus (HEV) have been documented in many geographic regions and nonindustrialized countries  and Médecins sans Frontières (MSF) teams working in the Begoua Commune Health Center, north of CAR’s capital Bangui, reported an increased number of patients from the Yembi I neighborhood who were showing signs of jaundice and extreme fatigue.Patients suspected of having hepatitis E were defined as those with clinical jaundice (yellow discoloration of the sclera) and symptoms of malaise, anorexia, abdominal pain, arthralgia, and fever. Confirmed cases were those in which patients’ serum samples were positive for HEV immunoglobulin (Ig) M or IgG.Initially, 16 pairs of serum and stool samples were collected from jaundiced patients. Fecal samples were stored at –20°C and sent to the National Reference Center of Enterically Transmitted Hepatitis, Hospital Val de Grâce  for HEV marker testing; serum samples were tested at the Bangui Pasteur Institute for yellow fewer (YF) IgM by MAC-ELISA.,The HEV epidemic was confirmed by the detection of HEV markers: HEV IgG , HEV IgM (Abbott Laboratories), amplification of RNA  had relatives with suspected HEV, and 163 (73.5%) had drunk untreated water from their own wells.These epidemiologic findings suggest the water-borne nature of this outbreak. Environmental testing of water from 2 wells (before chlorination was implemented) showed the water to be unsafe to drink  (The outbreak was not surprising because a 1995 survey in Bangui showed anti-HEV antibodies in 24% of patients tested (,>30% in pregnant women (The observed CFR was similar to that in other reported HEV outbreaks, in which CFR varied from 1% to 4% (We recommended application of preventive measures, including water disinfection, safe disposal of excreta, community health education, and the strengthening of case management and disease surveillance. For the CAR, free access to a safe water supply and drugs was the only way to achieve these goals.The number of HEV cases in the Yembi I neighborhood declined after the crisis team implemented hygienic and chlorination measures in the district, although the number of cases remained constant in other neighborhoods of the commune . DefinitOur results agree with international data on HEV outbreaks in other nonindustrialized countries. However, studies to improve our understanding of this epidemic and to identify the main risk factors involved would be beneficial."}
{"text": "Complex and unusual root canal morphology is an often occurring phenomenon. Understanding the unusual root canal morphology contributes to success in endodontic treatment. One such variant root canal morphology is the ‘S’ shaped or bayonet shaped root canal. This case report discusses endodontic treatment of a maxillary second premolar with an ‘S’ shaped root canal. Variations in root canal system occur so often that it is considered a normally occurring phenomenon. Endodontic therapy will be successful only when a thorough disinfection of the entire root canal system is achieved. Understanding these unusual root canal morphology will contribute to success in endodontics.According to Vertucci,–3 maxillThis report discusses the endodontic management of an ‘S’ shaped root canal.A 34-year-old female patient was referred to the Department of Conservative Dentistry and Endodontics, for the management of a right maxillary second premolar. On clinical examination, tooth 15 had a large carious lesion on the disto-proximal aspect. The patient gave a history of severe pain for the past three days. Radiographic examination of the tooth revealed radiolucency in the disto-proximal aspect, very close to the pulp space. The roots were doubly curved (Bayonet or ‘S’ shaped). From the clinical and radiographic findings, a diagnosis of irreversible pulpitis was made in relation to 15. Hence, endodontic treatment was initiated in 15.The right upper maxillary premolar was anaesthetized with 2% lignocaine and access was opened with a No: 2 round bur, under rubber dam isolation . The comFor determining the patency of the root canals, No:10 SS K-files were used . The bucUnlike the buccal root, the palatal root was negotiable only up to the middle third of the root canal. There was resistance to the movement of the patency file. The palatal root canal was enlarged with protaper rotary instruments (Sx) in a crown down motion, right up to the straight portion of the canal. Once this was done, the patency file slid in easily, to the full length. The working length was established using an apex locator  and confirmed using a radiograph . The roord of the canal (at the expense of the tooth structure) to reduce the angle of curvature. Once this is done, it is easy to negotiate the remainder of the root canal.The ‘S’ shaped canal has two curves, with the apical curve being very difficult to negotiate. The chances of strip perforation are very high in these root canals.  Guttman suggesteIn this case, both the canals were not S-shaped. The buccal canal had only a mild distal curvature; hence this canal was prepared in a crown-down technique using protaper rotary instrument.As the palatal canal was doubly curved, a double flare technique was used to enlarge this canal. The access cavity was flared in the coronal-third, in order to reduce the angle of curvature to the first curve. Reducing the angle of curvature by flaring the access (at the expense of the tooth structure) will make the approach to the second curve much easier. PreflariUnderstanding the complex root canal morphology and choosing a canal preparation technique more suited for such morphology, will contribute to successful endodontic treatment."}
{"text": "Cough is a common and distressing symptom in lung cancer patients. The clinical management of cough in lung cancer patients is suboptimal with limited high quality research evidence available. The aim of the present paper is to present a clinical guideline developed in the UK through scrutiny of the literature and expert opinion, in order to aid decision making in clinicians and highlight good practice.Two systematic reviews, one focusing on the management of cough in respiratory illness and one Cochrane review specifically on cancer, were conducted. Also, data from reviews, phase II trials and case studies were synthesized. A panel of experts in the field was also convened in an expert consensus meeting to make sense of the data and make clinical propositions.A pyramid of cough management was developed, starting with the treatment of reversible causes of cough/specific pathology. Initial cough management should focus on peripherally acting and intermittent treatment; more resistant symptoms require the addition of (or replacement by) centrally acting and continuous treatment. The pyramid for the symptomatic management starts from the simpler and most practical regimens  to weak opioids to morphine and methadone before considering less well-researched and experimental approaches.The clinical guidelines presented aim to provide a sensible clinical approach to the management of cough in lung cancer. High quality research in this field is urgently required to provide more evidence-based recommendations. Cough is a common symptom in about 23-37% of general cancer patients and 47-86% of lung cancer patients . The firWith currently available information, clinicians have difficulty in making appropriate treatment choices, often treating patients on a trial-and-error basis. This has been highlighted through discussions with many clinicians. Our own work with lung cancer patient interviews over time  has showhttp://www.ceco.org.uk) is a collaborative of five UK universities and their clinical partners, funded in 2005 by the National Cancer Research Institute to develop critical mass, capacity and high quality research in supportive and palliative care. Besides its research objectives, the Collaborative also has a remit to have the maximum positive impact on policy and practice. The APM  is a professional society aiming to provide a national and regional network for palliative medicine doctors with a strong focus on developing and implementing educational material, and has strong links with the European Association for Palliative Care.The Task Group was led by the Cancer Experiences Research Collaborative (CECo) with invited experts from the Association for Palliative Medicine (APM). CECo  field. The group met twice, once to agree on the methodology to be used and review the available evidence and once to make sense of this data and develop recommendations.A literature review preceded the meetings over the previous year, with two systematic reviews being undertaken, one with a focus on cough and respiratory illnesses (other than cancer)  and a seAn assessment of the appropriateness, usability and clarity of the guidelines was carried out by international experts N = 15), including 9 consultants in palliative medicine, 4 in medical oncology and 2 specialist palliative care nurses (independent prescribers). This was done through clinicians reading the report and recommendations, and completing a feedback form. A number of items structured in a Likert-type format of 1 = strongly disagree to 5 = strongly agree were included. The areas explored were: the rationale and need for the guidelines ; the need for the development of the guidelines (= 4.8); completeness of literature search (= 4.3); the description of evidence (= 4.6); the methods used to summarise and interpret the evidence (= 4.1); interpretation of the evidence presented (= 4); clarity of the recommendations (= 3.7); agreement with the recommendations made (= 4.1), and feeling comfortable having these guidelines applied in their hospital (= 4). The likelihood of using these guidelines in the reviewers' own practice was explored with a single-item scale ranging from 1 = 'not at all likely to use' to 10 = 'very likely to use', achieving a score of 8.1. After the feedback, the guidelines were modified primarily in relation to the dosages in some drugs, and the clarity and levels of drugs proposed in the cough pyramid  and Aδ fibres (mechanoreceptors) in the airways, carried by the vagus nerve. In disease states, excessive coughing can occur by excessive noxious stimulation of these afferent fibres and/or as a result of sensitization of neurons involved in the cough reflex. In patients with lung cancer, for example, tumour tissue in the central airways may cause mechanoreceptor stimulation directly or indirectly via obstruction and sputum accumulation. The inflammatory mediators associated with infection distal to such an obstruction or mediators released by tumour tissue may further induce coughing by sensitizing peripheral nerves.Patients with lung cancer can experience cough as a result of non-cancer related underlying pathology or cancer itself. The cancer-related causes of cough can include a direct effect of the tumour mass (ie. obstruction), pleural or pericardial effusion, atelectasis, infections, oesophagorespiratory fistulas, lympangitic carcinomatosis, superior vena cava syndrome and treatment-induced cough as a result of radiotherapy or more rarely chemotherapy .Positive and negative trials from our systematic review of cough management in respiratory and non-respiratory diseases are summarized in Table Research into cancer-related cough was even more disappointing, and our Cochrane systematic review  identifiWe also assessed other trials that were not included in the above reviews, phase II trials and experimental case studies. A randomized double blind crossover trial of 16 patients with chronic stable cough was included, receiving Dextromethorphan or Codeine 20 mg) . Results mg . ResThere are, however, some issues with some propositions in the literature, particularly in relation to the effects of opioids. For example Homsi et al  mentioneA thorough history is fundamental to identify the cause of cough and should be taken for each patient with lung cancer in order to identify the causes of cough. This assessment should include the type of cough (productive/non-productive), trigger factors, whether cough is nocturnal or day-time cough, its effects on quality of life or any specific concerns patients may have (e.g. fear of choking during a cough). This assessment should include a careful medication review. Use of a cough scale should be useful in identifying not only the frequency of cough but also its severity and distress. A visual analogue scale is recommended, until validated scales specific for cancer patients are available, which also could be used to assess the effectiveness and responsiveness of initiated interventions. Many of the lung cancer patients have comorbidities with other respiratory diseases (e.g. chronic obstructive pulmonary disease-COPD) and cough may be the result of the underlying respiratory pathology rather than the cancer. The timing of the start of cough is important; any change in cough since the diagnosis with cancer or any new cough is likely to be related with the cancer whereas more chronic cough may be related to underlying respiratory comorbidity. Iatrogenic causes of cough should also be considered, such as radiotherapy and certain medication . Lung cancer patients may not need new investigations, as they may already have x-rays or CT scans that can provide adequate information regarding the cancer-related causes of cough. It may, however, be necessary to carry out such investigations if the causes of cancer-related cough are not apparent from a detailed history.The therapeutic overall goal should first be to treat reversible causes or specific pathology. If the patient has an identified underlying pathology potentially causing cough, he/she should be treated as per available guidelines (ie. BTS guidelines). In cough due to airway diseases such as COPD or asthma, treatments directed at the underlying condition should be used. This could be achieved with the use of inhaled bronchodilators and corticosteroids; for example, salbutamol has little effect in non-asthmatic cough but may be useful when bronchoconstriction is present. The same principle applies to anticholinergics and theophylline. Anticholinergics (e.g. Hyoscine) may be particularly important in the end of life, where the aim is to suppress secretions and subsequent cough. Oral corticosteroids (e.g. Prednisolone 30 mg once daily for 14 days) may also provide rapid relief of cough due to airway inflammation either as a direct result of tumour involvement or associated asthmatic/eosinophilic inflammation. With cough thought to be originating from gastro-oesophageal reflux, PPIs and H2-receptor antagonists may be tried. However, increasing evidence suggests that non-acid reflux pH 4-7) may be associated with chronic coughing in some individuals. Metoclopramide and Domperidone are frequently used to promote GI motility and they should be considered in selected patients where non-acid reflux is suspected. The BTS guidelines . Rap. Rap30]]Symptomatic treatment should start with demulcents such as Glycerol-based ones  and Initial cough management should focus on peripherally acting and intermittent treatment (i.e demulcents); more resistant symptoms require the addition of (or replacement by) centrally acting and continuous treatment. It is noted, however, that peripheral mechanisms may be different in cancer-ulcerated mucosa in localized cancer may result in more or less sensitivity to peripheral agents such as lidocaine or steroids. Furthermore, many patients with advanced cancer will be already receiving strong opioids for pain management which may mean that centrally acting approaches are maximized and less well established treatments should be explored. Behavioural interventions, such as speech pathology training  and vocaA combination of cancer-specific treatments and symptomatic control approaches is likely to be necessary in all patients. The specific balance of these treatments should be determined by the individual patient's needs and a logical stepwise approach to seeking reversible causes and introducing treatments.With the exception of chemoradiotherapy and some treatments for respiratory diseases, the recommendations in the cough management pyramid of Figure Cough is a symptom that has received little attention in cancer supportive care research and it is frequently undertreated in practice as there is a dearth of good quality evidence on which clinicians can base their treatment decisions. These guidelines provide an attempt to rationalize the available evidence and offer a sensible and practical way of managing cough in lung cancer patients. There is an urgent need for more high quality research in the management of cough. Also, as traditional antitussives and other cough suppressants have variable effectiveness and significant side effects, more novel cough treatments need to be developed in the future based on improved understanding of pathophysiological causes of cough in patients with cancer.The authors declare that they have no competing interests.Conception of study, set up of Task Group and coordination: AM.Literature review: AM, JAS, MIB.Task Group participation: All authors.Development of guidelines/recommendations: All authors.Drafting paper: AM.All authors read and approved the final manuscript."}
{"text": "Chronic cough has negative effects on quality of life. However, the changes in health-related quality of life and clinical implications remain unclear in Chinese patients with chronic cough.A standard Chinese version of Leicester cough questionnaire (LCQ) was developed by an established translation procedure and its repeatability was assessed in a preliminary study involving 20 untreated patients with stable chronic cough. The quality of life was measured with the Short form-36 health survey and compared between 110 patients with chronic cough and 90 healthy volunteers. The changes in health-related quality of life were evaluated in the patients with chronic cough with the LCQ just before the specific treatment was initiated and a week after the cough had resolved completely. Cough threshold with inhaled capsaicin, expressed as the lowest concentration of capsaicin required for the induction of ≥5 coughs, was also measured.P < 0.0001). There was a significant correlation between the total score of the LCQ and physical  or mental  component summary of the Short form-36 health survey but not between the LCQ and capsaicin cough threshold.The repeatability of the Chinese version of the LCQ was validated at a four day interval with the intraclass correlation coefficients of 0.89-0.94 for total and domain score (n = 20). The scores of the Short form-36 health survey were significantly lower in patients with chronic cough than those in healthy volunteers. In general, there was no significant difference in overall quality of life between different causes of chronic cough or genders although embarrassment, frustration and sleep disturbance were more common in female patients, as indicated by the LCQ. However, the successful treatment of cough obviously increased the total scores of the LCQ from 14.2 ± 2.7 to 19.5 ± 1.9 (t = 13.7, The quality of life is significantly impaired in Chinese patients with chronic cough. The Chinese version of the LCQ is a valid measure of cough related quality of life and is repeatable and responsive. Chronic cough is a common symptom that involves 20%-38% of the patients seeking medical advice in the respiratory clinic ,2, and aCough symptom score, generic instruments such as the Short form-36 health survey (SF-36), Sickness impact profile and the other respiratory health questionnaires were common tools for the evaluation of HRQOL in chronic cough in early years . Cough sIt has been demonstrated that chronic cough has negative effects on HRQOL of patients in many aspects including physical, psychological and social domains, among which the adverse impact on the psychosocial domain is the most notable and possibly related to gender . Success1) > 80% of predicted and a ratio with forced vital capacity (FEV1/FVC) >70%. Current smokers and ex-smokers of < 2 years were excluded.110 patients with chronic cough referred consecutively to the Department of Respiratory Medicine in Tongji hospital were selected for the study. They were eligible for inclusion in case of the presence of an isolated persistent cough of > 8 weeks, the absence of adventitious lung sounds on physical examination, normal findings on plain chest radiography, a forced expiratory volume in 1 s  of the Chinese version of LCQ over time. The reliability was assessed by administering the LCQ to 20 untreated patients with stable chronic cough at baseline and four day later when the patients did not feel that their cough had changed.cough sensitivity was measured according to the method described by Fujimura  with minThe etiologies of chronic cough were identified and treated by our established protocol . Just bes except for duration of cough and cough threshold C5 which were presented as median (range). Comparisons of variables between the patients with chronic cough and healthy volunteers were made using unpaired t-tests while the difference of gender distribution was examined using X2 analysis. Changes in HRQOL before and after treatment were analyzed by paired t tests. Comparisons between the different etiologies of chronic cough were performed with one way analysis of variance followed by q-test. The repeatability of Chinese version of LCQ was analyzed by calculating intraclass correlation coefficients of LCQ total and domain scores between the two evaluations. The relationships between log transformed cough threshold C5 (Log C5) and LCQ or SF-36 score were calculated using Spearman rank order correlation coefficient. SPSS 10.0 software  was used for statistical calculations. A P-value < 0.05 was considered significant.The data was expressed as P = 0.000) for total score, 0.89  for the physical domain score, 0.93  for the psychological domain score and 0.92  for the social domain score respectively.The intraclass correlation coefficient of LCQ repeatability was 0.94  or MCS  of SF-36 respectively. The median of cough threshold C5 was 3.9 μmol/L . No significant correlation was found between Log C5 and LCQ , PCS  or MCS  of SF-36.There was a weak but significant correlation between the total score of LCQ and PCS (r = 0.39, The quality of life is an important outcome parameter in the study of chronic cough. To investigate HRQOL of Chinese patients with chronic cough, we compared the scores of items in SF-36 between patients with chronic cough and healthy volunteers. The reasons for selection of SF-36 were that SF-36 is one of the most common instruments in general health survey, its reliability and validity have been established with extensive application . MoreoveThe results showed that the quality of life in patients with chronic cough deteriorated significantly when compared with healthy volunteers. The scores of patients with chronic cough were lower in 7 testing multi-item scales of SF-36 except for physical pain than those of healthy volunteers. Among the affected domains, the decrease in the scores of RE, GH and RP related to emotional problems was more obvious. These observations were in accordance with previous reports on HRQOL in patients with chronic cough -8,19.There was comparable HRQOL among the patients in spite of causes of chronic cough, which confirmed the findings of French and Canonica who had observed that HRQOL in the chronic coughers was unrelated to the causes of cough ,20. The Gender is one possible factor determining the quality of life of patients with chronic cough. Most studies have demonstrated that women accounted for the majority of patients with chronic cough seeking medical care, with the ratio of 1:1.2-3.6 between males and females ,25. It sAnother possibility is that the quality of life in the patients with chronic cough visiting hospital was affected in a similar level regardless of gender. Women with chronic cough were more likely to see a doctor than men because of embarrassment and the other psychosocial issues provoked by cough-related urinary incontinence . HoweverWhen cough resolved with successful treatment specific for the cause, the quality of life in the patients could be significantly improved as judged by total score and domain scores in LCQ, which is in accordance with the previous study . TherefoBirring and Kalpaklioglu have found that there was a negative correlation between LCQ and the other tools such as cough symptom score and visual analogue scale respectively ,27. ReceThere was no obvious correlation between LCQ and capsaicin cough threshold, which was similar to the results reported by Birring  and ChanIn conclusion, HRQOL is adversely affected by chronic cough but improves when cough resolves. In general, the changes in HRQOL are not related to the causes of cough and gender differences although some negative emotions are more obvious in female Chinese patients. The Chinese version of LCQ is useful tool for evaluation of the health status in Chinese patients with chronic cough.The authors declare that they have no competing interests.WM was in charge of collection of cases and writing the manuscript, LY was in charge of collection, process, and statistical analysis of data and took part in review of the manuscript, YW, XL and HL took part in the collection of cases and review of the manuscript, ZQ was in charge of design and coordination of the program, review and correction of the manuscript. All authors read and approved the final manuscript.LCQ in Chinese. The Chinese version of Leicester Cough Questionnaire provided is the translation of the original one.Click here for file"}
{"text": "Many tussive agents are components of foods, but little is known about the relationship between cough reflex and oral chemesthesis sensitivities. We investigated the relationships between cough reflex and oral chemesthesis in individuals using two transient receptor potential vanilloid 1 (TRPV1) agonists with different potencies: capsaicin and capsiate.Twenty-eight healthy never-smokers were allocated to evaluate cough and oral chemesthesis of capsinoids. Cough reflex sensitivities are estimated by the lowest concentrations generating five coughs by each TRPV1 agonist inhalation. Oral chemesthesis sensitivities are estimated by the lowest concentrations which generate a hot sensation when filter paper loaded with each TRPV1 agonist is placed on the tongue.There were strong correlations between capsaicin- and capsiate-induced cough reflex sensitivities, and between capsaicin- and capsiate-induced oral chemesthesis sensitivities. However, there were no significant correlations between cough reflex and oral chemesthesis sensitivities induced by both capsaicin and capsiate. The cough reflex sensitivities are significantly greater in females than in males whereas there were no gender differences in oral chemesthesis.The results showed that the sensitivities of sensory afferents were different between cough reflex and oral chemesthesis, suggesting that TRPV1 sensitivities differ between organs within healthy individuals. Capsiate could be a tussigen for the cough challenge test. Although many tussive agents, such as capsaicin, citric acid, and acetic acid, are components of foods, it is unknown whether these chemical stimuli equally stimulate sensory nerves in bronchial airways and the oral cavity. The inhalation of tussive agents as a cough challenge test is a useful method to quantify cough in a clinical setting and to assess the antitussive effects of specific therapies in a laboratory setting . The inhThe physiological effects of capsaicin on cough may be modulated by oral sensory stimuli. Activation of capsaicin-sensitive afferents in the tongue and palate evoke local release of neuropeptides such as substance P and calcitonin gene-related peptides, which are contained in the nerve terminal of the sensory neurons ,4. The nMoreover, in the brain, the gustatory fibers and the sensory fibers that initiate cough may interact with each other because of the close anatomical relationship . In ordeCapsium fruits are used worldwide in foods for their pungency. The pungency felt when eating Capsium fruits is mainly attributed to the activation of oral TRPV1 [Capsaicin acts mainly on the afferent neurons of the non-myelinated C-fibers by the opening of a non-selective cation channel of capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1) . Capsiatal TRPV1 .TRPV1 receptors found on sensory airway nerves are important in the cough reflex . IsolateCapsaicin-induced cough may not solely be mediated through the nerves expressing TRPV1 receptors. Capsaicin inhalation elicits cough through the activation of rapidly adapting receptors (PAR) ,18. The In order to investigate the possible relationship between the perception of sensations mediated by TRPV1, whether directly or indirectly, in different organs, e.g. lung and tongue within individuals, we compared cough reflex and oral chemesthesis sensitivities using two TRPV1 agonists with differential potencies, capsaicin and capsiate. In addition, we evaluated the possibility of the use of capsiate as a cough challenge test.Twenty-eight healthy never-smokers  were allocated to evaluate cough and oral chemesthesis of capsinoids. All were originally recruited via public postings in and around the Tohoku University School of Medicine campus. The mean age was 36.4 ± 2.3 (SE) years. The study was approved by the Institutional Review Boards of Tohoku University School of Medicine. Subjects were without history of pulmonary disease, recent (within 4 weeks) suggestive symptoms, respiratory tract infection and seasonal allergies. Subjects did not take any regular medication.Subjects underwent the sensitivity tests on four successive days at 10:00 am. Each of the four days was assigned to the capsaicin cough sensitivity test, the capsaicin oral chemethesis test, the capsiate cough sensitivity test, or the capsiate oral chemesthesis test. The order of the four tests was randomly decided using a computer program. The day before the start of the test and during the four days, subjects were prohibited from taking any capsinoids in meals or beverages. In order to ensure subjects avoid consumption of capsinoids during meals, various foods and dishes that contain them were explained to the subjects.Cough reflex sensitivities to capsaicin and capsiate were measured on different days using the modification of the method by Fujimura and colleagues . 30.5 mgCapsiate was extracted from CH-19 sweet . Compared with capsaicin, capsiate has an ester bond instead of the amide bond between the vanillyl moiety and fatty acid chain . Increasing concentrations were inhaled until five or more coughs were elicited. The nebulizer output was 0.21 ml/min. The cough reflex sensitivities to capsaicin and capsiate were defined as the lowest concentration of capsaicin or capsiate that elicited five or more coughs (C5). In our preliminary experiments, it was confirmed that the Tween 80 and/or caprylic acid dilutions at any concentration used in saline without capsinoids did not induce cough for 15 s inhalation.Chemesthesis to capsaicin and capsiate was measured with a modification of the semi-quantitative clinical gustometry using a filter-paper disc, which is routinely used for the evaluation of dysgeusia in a clinical setting . Again, In our preliminary experiments, it was confirmed that the Tween 80 and/or caprylic acid dilutions at any comparable concentrations in distilled water without capsinoids did not induce oral chenesthesis, and it was certified that there was no tachyphylaxis of responses to capsinoids with 24-hour intervals for both cough reflex sensitivities oral chemesthesis.Results are expressexd as mean ± SE. Comparisons between each threshold concentration in differential stimuli were performed by a paired t-test. Comparisons between the sensitivities in males and females were performed by the Mann-Whitney test. The correlations between each threshold concentration in differential stimuli were estimated by Pearson's correlation coefficient. A value of p < 0.05 was considered statistically significant.5 value) was significantly greater in capsiate (2.55 ± 0.09 log μM) than in capsaicin (1.20 ± 0.09 log μM) (p < 0.0001). The mean threshold concentration to induce oral chemesthesis by capsiate (2.22 ± 0.10 log μM) was significantly greater than that by capsaicin (1.55 ± 0.11 log μM) (p < 0.0001). The mean threshold concentration for capsaicin application was significantly greater in cough reflex sensitivity than that in oral chemesthesis (p < 0.03).Both cough reflex sensitivities and oral chemesthesis tests were performed without any unpleasant feelings or side effects after the tests for all subjects. The mean threshold concentration to induce cough .As shown in Figure However, there was no significant correlation between cough reflex and pungent taste sensitivities induced by capsaicin . Similarly, there was no significant correlation between cough reflex and pungent taste sensitivities induced by capsiate . These results suggest that the same TRPV1 stimulation induce differential strength of sensation according to the organs within individuals.Table In this study, no significant relationship between cough reflex sensitivity and oral chemesthesis to capsinoids within individuals was found. The cough reflex to TRPV1 stimulations are less sensitive in males than in females whereas there was no significant gender difference in the oral chemesthesis to capsinoids. Here we showed that the usefulness of capsinoids with respect to both their action as a tussigen and the capability to evoke oral chemesthesis.A strong correlation between the threshold concentrations between capsaicin- and capsiate-induced cough was found. Similarly, the threshold concentrations between capsaicin- and capsiate-induced oral chemesthesis significantly correlated. In both sensations, capsiate required a much higher concentration than capsaicin. The intragastric administration of capsiate increases adrenalin secretion and oxygen consumption in mice ,22. In aIndividual variations in cough reflex sensitivities were shown in the cough challenge test even in healthy subjects. The variation exists regardless of methods of cough challenge and tussive stimulants. Cough reflex is reportedly less sensitive in men than women ,9. AlthoThe explanation for an increase in cough reflex sensitivity in healthy females is unknown. One hypothesis is an endocrine influence on the cough reflex. Recently, prolactin was reported to enhance TRPV1 response in the presence of estrogen in rat sensory neurons . HoweverBoth the peripheral and central explanations for why oral chemesthesis are not correlated to cough reflex sensitivity are postulated. The lack of relationship between oral chemesthesis and cough reflex sensitivity within individuals may suggest a differential expression of TRPV1 according to the organs within individuals. In patients with chronic cough, increased expression of TRPV1 in airway nerves was reported . InflammMore importantly, the differential sensitivities to capsinoids between cough reflex and oral chemesthesis could be reflected in the differential contribution of indirect activation of afferent neurons. In cough response, capsaicin is known to activate not only C-fibers that have TRPV1 but also rapidly adapting airway mechanoreceptors (PAR) that do not have TRPV1 ,18. PAR Besides the peripheral factors, central factors may be involved in the differential sensitivities of TRPV1 stimulation between cough reflex and oral chemesthesis within individuals. In contrast to oral chemesthesis, which was finally integrated by cortical processing, cough reflex is essentially a brainstem reflex. Therefore, there is a possibility that the gain of a cortical neural process is involved in the differences in oral chemesthesis, but not in cough reflex. Evidence of gustatory brainstem taste nuclei and cortical connections, which potentially modulate these processes, provide a plausible neural basis for a central gain mechanism ,36. ReceThe lack of relationship between oral chemesthesis and cough reflex sensitivity within individuals might suggest the low possibility of a modulatory effect of capsinoids which were deposited in the oral cavity during the cough challenge test. Although the concentration to induce oral chemesthesis to capsinoids is relatively smaller than that of cough reflex, oral chemesthesis did not trigger cough responses in the present healthy subjects. The lack of gender difference in oral chemesthesis also supports the no modulation hypothesis.In the present study, we found that the capsiate does not induce the sustained irritant airway feeling that is frequently observed in the case of the capsaicin cough challenge test. This might be attributed to the lipohilicity and instability of capsiate. Although this biophysical feature of capsiate is a disadvantage for the preparation procedure, this could be a benefit for the subject to avoid uncomfortable feelings after the cough challenge test .In conclusion, the results showed that the sensitivities of sensory afferents were different between cough reflex and oral chemesthesis, suggesting that TRPV1 sensitivities differ among organs within healthy individuals. The results also suggest that capsiate could be a useful tussigen for the cough challenge test.The author(s) declare that they have no competing interests.MY, SE and TE participated the design of the study, collected and analyzed data, and drafted the manuscript. SF, SY, AM and MY participated in the design of the study and collected the data. HA participated in design of the study and helped to draft the manuscript. All the authors read and approved the final manuscript."}
{"text": "The microcapsules were discrete, large, almost spherical and free flowing with encapsulation efficiency in the range of 75% to 89%, drug loading 75% to 86% and size 440 μm to 500 μm. Rifampicin release from these microcapsules was slow and extended over longer periods of time depending on the polymer coat. Drug release was diffusion controlled and followed first order kinetics. The formulation MC1 with a coating ratio of 1:1  was found to be suitable for oral controlled release.Rifampicin biodegradable microcapsules were prepared by feasible emulsification-ionic gelation method for a novel controlled release product. Sodium alginate and Carbopol 974P were used as coating polymers in different ratios 1:1, 1:2, 1:3 and 1:4 to obtain elegant microcapsules. The formulations were characterized for encapsulation efficiency, drug loading, sieve analysis, scanning electron microscopy and  Controlled release dosage forms are becoming increasingly important, either to achieve the desired level of therapeutic activity required for a new drug entity or to extend life cycle of an existing drug through improved performance or patient compliance. Microsphere technology is one of the established techniques for controlled delivery used to deliver several different types of drugs, such as steroids, peptide4Rifampicin, a semi synthetic hydrazine derivative of rifampicin B was chosen for the study because it is one of the established first-line drugs used to treat tuberculosis and its biological half-life varies from 1.5 to 5 h.Carbopol 974P is a synthetic high molecular weight cross-linked polymer of acrylic acid. It is a safe and nontoxic. It is an excellent thickening, emulsifying, suspending and gelling agent. Because2+, giving both strength and flexibility. Such a cross-linking process stiffens and roughens the polymer and reduces the swelling in solvents. The soluble sodium alginate was cross-linked with calcium chloride, resulting in the formation of the insoluble calcium alginate. This strong thermo stable gel has properties that largely depend on the characteristics of the polymer and the preparation method[Alginates are a family of polysaccharides composed of α-L-glucoronic acid (G) and β-D-mannuronic acid (M) residues, arranged in homopolymeric blocks of each type  and in heteropolymeric blocks of each composition. Alginates form strong gels with divalent cations like Caon method–9.Rifampicin was obtained as gift sample from On Top Pharmaceuticals . Carbopol 974P was obtained from Astra Zeneca Labs. . Sodium alginate was procured from Rolex Laboratories . Tween-80, ascorbic acid LR and calcium chloride were purchased from S. D. Fine Chemicals . Toluene and petroleum ether were purchased from Nice Chemicals , while castor oil was procured from Standard Labs. . All other reagents used were of analytical grade.Microcapsules containing rifampicin were prepared employing carbopol 974P in combination with sodium alginate as coat materials. No methods are reported for micro encapsulation by these polymers. The ionic gelation process11, whichSize of the rifampicin microcapsules was determined by sieve analysis. The microcapsules were separated in to different size fractions by sieving using standard sieves. The particle size distributions of the microcapsules were determined to know the mean particle size of microcapsules. All readings are average of three trials ±SD.The shape and surface morphology of the rifampicin microcapsules were investigated using a Hitachi-S5-20, scanning electron microscope at room temperature using the required magnification. The sample was deposited on alluminium stubs and coated with gold in HUS-5GB vacuum evaporator, to render it electrically conductive. Encapsulation efficiency was calculated using the formula, Encapsulation efficiency= ×100.Rifampicin content in the microcapsules was estimated by using UV spectrophotometer method. A quantity of microcapsules equivalent to 100 mg were powdered and transferred into a 100 ml volumetric flask, sufficient amount of methanol was added to produce 100 ml, shaken for 20 min and filtered. Two milliliters of the filtrate was diluted to 100 ml with phosphate buffer (pH 7.4) containing ascorbic acid (200 μg/ml) and the drug content was analyzed at 475 nm. Three dDissolution studies were carried out using USP XXIV rotating basket method. The stirring rate was 75 rpm. 900 ml of pH 7.4 phosphate buffer having ascorbic acid (200 μg/ml) was used as dissolution medium and maintained at 37±1°. Samples of 5 ml each were withdrawn at regular time intervals, filtered, diluted suitably, analyzed using double beam UV spectrophotometer at 475 nm and an equal volume of fresh medium was immediately added to maintain the dissolution volume. Dissolution studies were carried out up to 12 h. The drug release experiments were conducted in triplicate.t/Mα = Ktn, where Mt/Mα is the fractional solvent absorbed or drug release at time ‘t’; K denotes a constant incorporating the properties of the macromolecular polymeric system and the drug and ‘n’ is a kinetic constant which depends on time and is used to characterize the transport mechanism. While n= 0.45 for case I or Fickian diffusion which is characterized by a square root time dependence in both the amount diffused and the penetrating diffusion front position; n= 0.89 for case II transport, which is completely governed by the rate of polymer relaxation, exhibits a linear time dependence in both the amount diffused and the penetrating swelling front position; n= 0.45<n<0.89 for anomalous behavior or non-Fickian diffusion and polymer relaxation are comparable[The results obtained were fitted according to the following exponential equation, Momparable.IR studies (not shown) indicated that the qualitative composition of the drug formulation and verified the identity of each of the components. No drug interaction or complexation occurred during the manufacturing process.et al, have prepared carbopol microspheres by using modified U-shaped beaker, a two blade paddle stirrer and maintaining a temperature of 60° for a period of 24 h to promote the evaporation of water[Kockisch  of water15. Micro of water17. HenceRifampicin release from the microcapsules was studied in phosphate buffer pH 7.4 containing ascorbic acid (200 μ/ml) in dissolution test apparatus USP XXIV using rotating basket. Rifampicin release from the microcapsules was slow and spread over a period of 12 h and depended on coating ratio  and size of the microcapsules. As the proportion of carbopol 974P was increased, rifampicin release rate was decreased from 91.2, 81.7, and 80.5 to 69.5% respectively.t/Mα) versus Log t . If the polymer does not hydrate quickly the surface barrier or gel is not formed immediately, it results in a large portion of drug to be released during the initial phase of release profile (MC1) . Sodium Spherical microcapsules with a coat consisting of alginate and carbopol could be prepared by emulsification-ionic gelation process. Microencapsulation efficiency was found to be in the range 76-90%. Rifampicin release from the polymer coated microcapsules was slow and extended over longer periods of time and depended on core: coat ratio and size of microcapsules. Drug release from the polymer coated microcapsules was by fickian diffusion and followed first order kinetics. Thus carbopol 974P and sodium alginate coated rifampicin microcapsules were found suitable for oral controlled release products."}
{"text": "A 58-year-old nonsmoker female was referred for evaluation of chronic cough of 13 months duration. After an initial work-up, the patient was diagnosed to have chronic cough due to eosinophilic bronchitis. The diagnostic work-up for eosinophilic bronchitis and bronchial biopsy is discussed. Eosinophilic bronchitis is differentiated from asthma. In addition, the patient developed dyspnea, flushing, and wheezing after the administration of adenosine during a cardiac stress test in spite of a negative methacholine challenge. This indirect stimulus of airway hyperresponsiveness suggests the possible involvement of mast cells in eosinophilic bronchitis. Eosinophilic bronchitis is an uncommon cause of chronic cough in the general practice; however, this entity has been found to be present in 10–20% of patients referred for an investigation by a specialist  and has A 58-year-old post menopausal nonsmoker Caucasian female was evaluated for chronic cough of 13 months duration. The patient has a past medical history significant for hyperlipidemia, and obstructive sleep apnea. The cough was described as a dry cough and was severe enough to cause her to gag and vomit. She reported frequent nighttime awakenings due to cough. Initial work-up at another facility was reported as normal pulmonary function tests, negative methacholine challenge test, normal chest radiogram, normal chest and sinus CT scans, and a normal inspection of vocal cords, trachea, and bronchi by flexible bronchoscopy. A bronchial biopsy was performed during the bronchoscopy and results are reviewed below. She was prescribed an empiric one-week trial of prednisone which resulted in near resolution of her cough. The patient was then started on inhaled fluticasone and tiotropium without a clear diagnosis given. As a consequence, she was uncertain about the use of the inhalers and was noncompliant. The cough came back prompting another evaluation. The cough was not associated with rhinorrhea, sneezing, wheezing, dyspnea, postnasal drip, heartburn, chest pain, fever, sputum production, hemoptysis, weight loss, or night sweats. She denied ever having had exposure to immigrants or any travel outside her home state. No history of ACE inhibitors intake was noted. The patient worked with Christmas trees helping to shear, bale, and make wreaths. She has a dog at home but no other pets. She has no prior history of allergies or allergy testing. The patient did not have a history of childhood asthma, sinusitis, GERD, hayfever or tuberculosis, and no history of indoor hot tub. In addition, the patient complained of bilateral sharp chest discomfort for about 10 months, associated with the cough episodes, nonradiating and not associated with exercise, nausea, or diaphoresis.9/L , and differential evidenced 60% Neutrophils, 1% eosinophils, 35% lymphocytes, and 4% monocytes. Electrolytes including sodium, potassium, chloride, calcium, magnesium, and phosphorus were within normal limits. Serum creatinine and BUN were 0.8 mg/dL , and 12 mg/dL  respectively. Serology for Bordetella pertussis and Respiratory Syncytial virus (RSV) were negative. Her oral exhaled nitric oxide was elevated to 158 parts/billion . The biopsy of the left main bronchus was reviewed and revealed peribronchiolar chronic inflammation with eosinophils and thickened basement membrane . Chest CT showed no infiltrates or pleural effusions, and no abnormal hilar or mediastinal lymphadenopathy. CT scan of the sinuses showed normal mucosal thickening and no air-fluid levels. A 24-hour esophageal pH probe of proton pump inhibitor excluded gastroesophageal reflux disease. CBC showed hemoglobin of 13.6 g/dL , the WBC was 9.2 × 10membrane . Sputum The diagnosis of eosinophilic bronchitis is usually made in patients with chronic cough and airway eosinophilia in the absence of dyspnea, wheezing, airflow limitation, and lack of airway hyperresponsiveness measured by the methacholine challenge test. When facing a patient with chronic cough, it is always very important to exclude other common causes of chronic cough such as gastroesophageal reflux disease, asthma, or upper airway cough syndrome. Asthma, cough variant asthma, and EB share many features including chronic cough and airway eosinophilia . However, the hyperresponsiveness provocative test with methacholine is positive with asthma or cough variant asthma; whereas it is negative with EB and helps to differentiate these entities .Erlich described the eosinophil more than 100 years ago and its presence in the sputum was initially recognized as an important marker in asthma . EosinopThe histological changes described in the biopsy of EB are seen also in asthma and cough variant asthma, and a similar Th2 cytokine driven airway inflammation has been proposed in both entities , 9; howeThe presence of elevated oral exhaled nitric oxide (eNO) has been correlated with predominantly eosinophilic airway inflammation and can be seen in both asthma and EB , 5. TreaOur patient developed symptoms and signs suggestive of airway hyperresponsiveness after the administration of intravenous adenosine for the cardiac stress test that was ordered to evaluate her atypical chest pain. Adenosine type 2 receptors are found in the mast cells and when stimulated can potentiate the histamine release from these cells and therefore precipitate bronchoconstriction . InteresEosinophilic bronchitis is a common cause of chronic cough in referral centers. It shares similarities with asthma such as increased eosinophils in the airways, increased IL-4 and IL-5, and improvement of symptoms with inhaled corticosteroids; however, eosinophilic bronchitis differs from asthma because it presents with normal pulmonary function and normal airway responsiveness. We described the first case of airway hyperresponsiveness after systemic administration of adenosine in a patient with eosinophilic bronchitis presenting as chronic cough."}
{"text": "There is a need for more objective outcome measures for chronic cough. In this pilot study we sought to investigate the utility of the mannitol challenge as a cough-provocation test in non-asthmatic chronic cough. We studied 16 healthy controls and 13 subjects with chronic cough. We assessed cough severity using a visual analogue score, capsaicin cough sensitivity, health status using the Leicester Cough Questionnaire and the dose of mannitol to cause 2 (C2) or 5 (C5) coughs. In all of the subjects with chronic cough and 6 of the controls we assessed the 1-week repeatability of the mannitol challenge. We found that in those subjects with chronic cough the geometric mean (logSEM) mannitol C2 and C5 was heightened compared to controls (C2: 4 (0.2) versus 16 (0.1); p = 0.04 and C5: 63 (0.1) versus 251 (0.1); p = 0.04). Cough visual analogue score, capsacin-induced cough sensitivity and health status were also altered in chronic cough compared to healthy controls, but in those subjects with chronic cough none of these outcomes was correlated with the mannitol C2 or C5. The repeatability of the mannitol challenge assessed by intraclass correlation was C2 = 0.53 and C5 = 0.59. A cut-off in the dose of mannitol of 62 mg/ml for C2 and 550 mg/ml for C5 had a sensitivity of 69 and 62% and specificity of 69 and 81% respectively to distinguish chronic coughers from healthy controls. In conclusion, the mannitol challenge my have potential as a novel cough challenge test and further work is required to extend our findings and to assess whether it has utility in different causes of chronic cough. Chronic cough is the most common presenting symptom in primary care; is a significant cause of morbidity and a considerable health economic burden . The nee1 was measured between each dose of mannitol to measure a drop in lung function. An empty capsule was administered prior to the 5 mg dose, as a placebo. The numbers of coughs within the first 30 seconds following mannitol administration were counted. The challenge was terminated if the subject's FEV1 dropped by 15% (PD15) or more or when the highest dose of mannitol had been attained.Subjects were recruited from hospital staff or from the respiratory clinics at Glenfield Hospital, Leicester, UK. Chronic cough was defined as a cough > 8 weeks as per American College of Chest Physician Guideline (ACCP) . HealthyAll statistical tests were performed using Prism version 4. The concentration of capsaicin or mannitol that caused two coughs (C2) and five coughs (C5) were calculated by the log-dose-response curves. Comparisons between groups were made using t-tests or Mann-Whitney-U test for parametric and non-parametric data respectively. Repeatability was assessed by intra-class correlation and presented as Bland-Altman plots. Receiver-operator curves were generated for mannitol C2 and C5. Correlations were made between the mannitol challenge and other cough outcome measures using spearman rank correlations. A p-value of < 0.05 was considered to be statistically significant.Clinical characteristics for subjects are as shown in table In this pilot study, we report for the first time that mannitol-provoked cough is a repeatable test and is increased in non-asthmatic chronic cough. The advantages of the mannitol challenge compared to more established cough challenge tests is that administration is simple, whereas other tests often require preparation of solutions needing laboratory support, and the mannitol challenge provides additional information on AHR. Our findings support our hypothesis that mannitol-provoked cough is increased in non-asthmatic chronic cough as has been reported in asthma . HoweverThe cause of mannitol-provoked cough is unknown. It is likely that it mediates its effect indirectly via activation of mast cells in the superficial airway to release mediators, which in turn activate local cough receptors. An earlier study in asthma demonstrated that the degree of bronchoconstriction in response to mannitol and the cough response are independent . This suThere a number of potential shortcomings of our study. This was a pilot study of a small number of subjects with non-asthmatic chronic cough. We are therefore unable to determine whether mannitol-provoked cough has a strong association with different causes of cough. Its utility as a cough outcome may therefore differ dependent upon the aetiology of the cough. We have only assessed the short-term repeatability. To fully address the clinical utility of mannitol in the assessment of cough future work will need to determine repeatability over a longer period and investigate responsiveness to therapy in more subjects.In conclusion, the mannitol challenge may have potential as a novel cough challenge test. However, its repeatability and ability to discriminate between subjects with non-asthmatic chronic cough in this pilot study was not yet sufficient to recommend its routine use. Our findings need to be replicated in a larger series of chronic coughers and in particular it may be informative to examine response in chronic coughers with different aetiologies.The authors declare that they have no competing interests.AS and SM undertook the clinical assessments. AS and CB prepared the manuscript. All authors have read and approved the manuscript. CB supervised the study."}
{"text": "We read the recent article by  with keen interest and would like to congratulate them for a job well done. However, we would like to raise a few points relating to the limitations of the study. Antimalarial use in pregnancy is an important determinant of the level of malaria parasitaemia at delivery but Mokuolu et al. , like thThe authors claimed that they took specific note of those patients who used preventive antimalarial drugs, yet they did not provide the proportion of such patients. These patients are likely to procure the drug from the community pharmacies and are at risk of using counterfeit and adulterated antimalarials which are very abundant in Nigeria . In the The inclusion criteria used in the study appear not to be clearly defined by the authors. We are not sure if pregnant women with HIV infection and unbooked patients were part of the cohorts they studied. Perhaps, it would have been proper to exclude these groups of patients because of the possible bias their inclusion may introduce into the study and this may be better avoided by recruiting patients from antenatal clinics and followed to the time of delivery rather than recruiting at the point of delivery. HIV and malaria coinfection has more adverse effects on pregnancy than malaria alone . The preMokuolu et al. found a significant association between malaria parasitaemia at delivery and maternal age less than twenty years. It may be true that pregnancy associated with acquired immunity is lower in younger women than older ones who have obtained adequate immunity from repeated exposures to malaria infections. Teenage pregnancies are likely to be associated with poverty, lack of good education, and poor nutrition. The necessary proteins needed in the synthesis of antibodies against malaria parasites may therefore be reduced in young pregnant women and may indirectly contribute to the high prevalence of malaria parasitaemia observed by the authors in young parturients.Given that the authors are from various disciplines, a future study that will involve a multidisciplinary approach is therefore suggested. The ability of the contributing authors, who are clinical pharmacologists, to assay blood levels of antimalarial drugs in pregnancy will add novelty to the future study."}
{"text": "Acropora palmata, a foundation-species of reefs in the Caribbean and western Atlantic, will advance our understanding of the genetic basis of ecologically important traits in corals and comes at a time when sequencing efforts in other cnidarians allow for multi-species comparisons.Cnidarians, including corals and anemones, offer unique insights into metazoan evolution because they harbor genetic similarities with vertebrates beyond that found in model invertebrates and retain genes known only from non-metazoans. Cataloging genes expressed in A. palmata transcripts were tagged, and provided a rough estimate of the total number genes expressed in our samples . The coral annotation data contained many of the same molecular components as in the Bilateria, particularly in pathways associated with oxidative stress and DNA damage repair, and provided evidence that homologs of p53, a key player in DNA repair pathways, has experienced selection along the branch separating Cnidaria and Bilateria. Transcriptome wide screens of paralog groups and transition/transversion ratios highlighted genes including: green fluorescent proteins, carbonic anhydrase, and oxidative stress proteins; and functional groups involved in protein and nucleic acid metabolism, and the formation of structural molecules. These results provide a starting point for study of adaptive evolution in corals.A cDNA library from a sample enriched for symbiont free larval tissue was sequenced on the 454 GS-FLX platform. Over 960,000 reads were obtained and assembled into 42,630 contigs. Annotation data was acquired for 57% of the assembled sequences. Analysis of the assembled sequences indicated that 83–100% of all Currently available transcriptome data now make comparative studies of the mechanisms underlying coral's evolutionary success possible. Here we identified candidate genes that enable corals to maintain genomic integrity despite considerable exposure to genotoxic stress over long life spans, and showed conservation of important physiological pathways between corals and bilaterians. Hydra magnipapillata and Nematostella vectensis) have revealed that basal metazoans retain genes known only from non-metazoans Cnidarians are valuable for understanding the evolution of metazoan genomes because they comprise a sister group to the Bilateria. From a developmental perspective, comparisons among these groups have yielded much information regarding the evolution of body plans in complex multicellular animals A. palmata are short by comparison  that are responsible for the formation of reefs worldwide de novo assembly of whole transcriptomes from non-model organisms Progress in sequencing technology, chemistry, and bioinformatics now enable the sequencing and Acropora palmata) remains an ecologically important community member A. palmata is one of only a few Caribbean species for which population genetic data is available A. palmata's range Despite a population reduction of >80% throughout its range in recent decades Acropora millepora, Acropora hyacinthus, Montastraea faveolata, Pocillopora damicornis, and Porites astreoidesNematostella vectensis and Hydra magnipapillata were recently completed Transcriptome and EST data are now available from several coral species including A. palmata we present here its transcriptome sequenced with 454 GS-FLX technology, and survey the assembled and annotated sequences for genetic markers, and functional enrichment of important biological processes (with a particular focus on stress response and DNA repair pathways). The genetic resources presented here are a major advance for studies of this species and will promote the study of adaptive trait variation in wild populations of A. palmata as well as comparative genomics among basal metazoans.To develop genome scale tools for 2 treatments, and sampled over the course of development from fertilization to the planula stage. A small amount of adult tissue was also included to represent adult transcripts. We thus expected to find developmental and stress related genes derived from the larval temperature and CO2 treatments, as well as genes related to symbiosis and calcification from the adult tissue.RNA was acquired from genetically diverse larvae collected over a broad geographic range previously identified as comprising two divergent populations A. palmata sample following the library preparation methods of Meyer et al.One plate of a normalized cDNA library generated from a pooled A. palmata ESTs . Assembly yielded 42,630 contigs averaging 1,030 bp long (σ = 623). Contig sizes ranged from 132 to 9,066 bp. The mean depth of coverage was 5.6 sequences  to all 119 genes. Comparison of the distributions of coverage depth between all A. palmata transcripts and those with hits to the 119 single copy orthologs showed that the mean depth of coverage for contigs with hits to these 119 genes (9.7) is greater than that for contigs in general (5.6). This analysis indicated that depth of coverage was high even for single copy genes and that the set of 119 orthologs can serve as a representative sample of the A. palmata transcriptome.Estimation of transcriptome completeness based on two different methods suggest that our single sequencing run tagged between 83–100% of the genes expressed in N. vectensis transcriptome we found hits to 83% of all N. vectensis transcripts. The lengths of the A. palmata sequences were generally greater than those of N. vectensis, suggesting that full length transcripts were obtained for the majority of the A. palmata genes  reflected the longer average lengths of the A. palmata transcripts, and taken together with the high proportion of hits to the N. vectensis transcriptome data indicated very high coverage of the A. palmata transcriptome.Secondly, by comparing our assembled data to the published ta genes . The disAll contigs and singletons  were used to search against the UniProt protein database. Searches of both Swiss-Prot and TrEMBL resulted in hits for 50,118 (57%) of the queried sequences, of which 32,114 (36%) represented unique subject names. 31,888 of these hits corresponded to Gene Ontology (GO) A. palmata dataset (derived from UniProt). A clear match was found for 88% of the 119 annotation descriptions. An additional 9% of matching A. palmata transcripts were described with uninformative species specific codes from UniProt, but functional matches were easily found by looking up the codes in the NCBI UniGene database. Only three of the 119 genes did not match with the A. palmata annotation data, and in all three cases the A. palmata annotation was derived from blast hits to the Florida lancelet or the green puffer, species with limited annotation data. Overall this suggests high (97%) annotation efficiency.To estimate annotation efficiency, the description terms associated with each of the 119 single copy genes were compared to those in the blast results from the 119 orthologs were used to examine the average number of hits to each putatively singe copy gene. Results of this test showed that 63% of the 119 single copy genes had multiple sequence matches , the number of assembled sequences matching to known single copy genes can give an estimate of the level of residual gene fragmentation (either from sequencing gaps or splice variants), and/or assembly difficulty. A. palmata sample. The mean value of 2.2 contigs per transcript was used as an estimate of the redundancy in our dataset. This yielded an estimate of ∼19,377 genes  expressed in our A. palmata sample. Finally, based on the blast results with N. vectensis we found that 43,036 of the A. palmtata sequences matched to a non-redundant set of 17,988 proteins from N. vectensis. Thus we predict that the number of transcripts expressed in A. palmata is in the range of ∼18,000–20,000 genes.The degree of fragmentation in our assembly was also used to generate a rough estimate of the total number of transcripts expressed in the The majority of taxonomic associations of the annotated transcripts matched to metazoan invertebrates and vertebrates (44% and 48% respectively). A small proportion of sequences matched to bacteria (3%), protists (2%), plants and algae (2%). All other groups including fungi, archaea and viruses accounted for 1% or less of matches. Stony coral (Scleractinian) sequences accounted for only 1% of the annotated sequences primarily due to a paucity of annotated coral sequences present in current protein databases.blast annotation associates a given sequence with its closest match in the database, results are limited to previously identified genes. Thus, the vast majority of annotated transcripts in this dataset showed matches to the Starlet Sea Anemone, Nematostella vectensis , the most closely related organism with a sequenced genome blast hits reflected the sequencing efforts on the Florida Lancet, Branchiostoma floridaeblast hits in our annotation database when the best sequence match lacked any functionally relevant data. Viewed this way the number of A. palmata sequences with best matches to N. vectensis was reduced from 36% to 12%. Thus, despite slightly higher sequence similarity of hits to N. vectensis, the search for useful functional annotations was greatly improved by exploring more distantly related hits.Because A. palmata transcripts, we placed A. palmata homologs in the context of known biological pathways using ipa software . Information in the ipa database reflects the level of interest certain pathways have received. Thus, comparison of A. palmata transcripts to the IPA database identified well annotated metazoan pathways for which a high proportion of pathway components were present in A. palmata. These pathways were then used as guides for further analysis. Note that this approach did not make inferences about expression levels of transcripts. Among the most highly represented pathways were many that are expected to be important to heat stressed embryos and who are thus of interest to the current study including protein ubiquitination, cell cycle control, NRF2 mediated oxidative stress response, p53 signaling and DNA double stranded break repair , were chosen to test for positive selection with the program pamlNext, we placed genes involved in the response to DNA damage and oxidative (and irradiant) stress in a phylogenetic context to test for signatures of natural selection. 498 bp of the conserved DNA binding domain from homologs of two A. palmata transcriptome. These included 68 transmembrane ion transporters, including 10 sodium driven bicarbonate exchangers which may play an important role in supplying bicarbonate ions to the reaction site of calcification. Another enzyme, carbonic anhydrase found in the coral calicodermis  Stylophora pistillataCalcification is crucial for coral growth and survival, and numerous genes involved in coral calcification were found in the N. vectensis and A. palmata, 7,754 ortholog pairs were detected, with 9,604 in-paralogs in N. vectensis and 9,365 in-paralogs in A. palmata. The size distribution of paralog groups showed that the number of groups detected declines with increasing group size, and groups of 5 or more were rare  were found, with a large majority consisting of trinucleotide repeats n = 170; . Within A total of 72,605 candidate SNPs were identified from 13,803 contigs spanning 19.8 Mb of sequence. The overall SNP frequency was 1 per 272 bp, with 71% transitions (Ts) and 29% transversions (Tv). Frequencies of different transitions types were similar, as were frequencies of different transversion types . The traTo date, 18 SNPs from 6 contigs have been targeted for amplification using PCR, and the products were sequenced on an ABI3700 fragment analyzer. Of these 18 SNPs, 8 were observed in the sample population (success rate 44%), which consisted of 11 individuals from Florida, 8 from Curacao, and 7 from Puerto Rico. The SNPs detected were in genes for NFkB, melanopsin, Cyano Fluorescent Protein, NRF2, galaxin, and LITAF . OngoingAcropora palmata transcriptome presented here joins transcriptomes and EST data available for several other scleractinian corals, including that of the Pacific congener A. millepora (Matz and Traylor-Knowles pers. comm.) A. palmata  was comparable to that of N. vectensis , and the estimated number of genes expressed in this species  was within the range estimated for A. millepora  and N. vectensis .Coral species are the foundation of reefs because of their association with intra-cellular photosymbionts and ability to calcify. Comparative functional genomic analyses are now possible to elucidate the mechanisms underlying coral's evolutionary success. The A. palmata possesses many of the molecular components present in the more complex Bilateria (particularly in pathways associated with oxidative stress and DNA damage repair) and homologs of p53, a key player in several DNA repair pathways, showed evidence of natural selection along the lineage separating Cnidaria and Bilateria.The high quality and comprehensiveness of the assembled data enabled functional analysis of pathways shared among metazoans as well as those of particular importance to the Cnidaria. A. palmata. Indeed, approximately half of the sequences with blast hits in the ‘protists’ category (555) were associated with the superphylum which includes symbiotic zooxanthellae.Xenobiotic sequences from intracellular parasites are commonly found in transcriptome data of wild species blast with reasonable cutoff parameters.Hits to sequences from scleractinians were rare because of the small number of coral genes in current databases . As such, the genes identified are likely to be highly conserved among the scleractinians, with remaining unannotated transcripts  representing a combination of coral specific genes A. palmata transcriptome  A. palmata.p53 proteins play a central role in the response to DNA damage in vertebrates by initiating pathways leading to apoptosis, or cell cycle arrest and DNA repair N. vectensis and A. palmata are potentially very long lived, as genets can reproduce asexually more or less indefinitely. Thus, minimizing the impact of DNA damage to somatic and germ line cells is critical to survival and fitness. Indeed, our analysis suggests that p53 family proteins have experienced positive selection along the lineage separating Cnidaria from Bilateria  and the Scleractinia  is approximately 225 mya and divergence between the Cnidaria and Bilateria is estimated at 650–1000 mya ilateria . Additio palmata .A. palmata transcriptome. We found similar patterns in the group size distributions of paralogs in both the N. vectensis genome and the A. palmata transcriptome. While it is possible that additional paralogous copies of some genes were not sequenced, such missing transcripts add additional conservatism to this analysis.Paralog group analyses have proven informative in identifying gene and genome duplication events and how they relate to speciation in a variety of organisms A. palmata consisted of immunoglobulin superfamily proteins in the IgLON family involved in the organization of neuronal connections in the developing nervous system in other metazoans N. vectensis and are expressed in developing larvae and planulae Given the rarity of large paralog groups, members of such groups must serve important functions to justify the maintenance of multiple copies. The largest paralog group in A. palmata, including TRAF 2, 3, 6 and a lipopolysaccharide induced TNF alpha factor. In mammals TRAF proteins are important signal transducers mediating innate immune response, apoptosis, bone metabolism and response to stress (including DNA damage) The second most abundant paralog group consisted of homologs of the TRAF proteins. Several TRAF homologs showed multiple paralogs in Drosophila and Caenorhabditis (n = 3 & 1 respectively) These annotations were determined solely by bioinformatic means, thus further verification is required, but it is of interest to note that the six TRAF proteins found in vertebrates are thought to be the result of an evolutionarily recent diversification because few TRAF homologs have been detected in A. palmata transcriptome numerous microsatellite repeats were observed (n = 333), with tri and hexanucleotide motifs representing the most common types (A. millepora (pers. obs.) Microsatellite repeats commonly occur in portions of transcribed mRNA including 5′ and 3′ untranslated regions on types . This isAmong trinucleotide repeats, motifs with high AT content were most frequent in both acroporid transcriptomes . SpecifiA. palmata was 1 per 272 bp . The frequency in A. millepora was slightly higher (1 per 207 bp) A. palmata samples. Only a small set of SNPs were chosen for preliminary validation here, but their wide distribution and the presence of frequency differences in even a small sample size is encouraging  were obtained from the local authorities  for all samples used in this study.Acropora palmata adults contain intracellular symbionts known as zooxanthellae. Like many other corals, symbionts are taken up after settlement and metamorphosis of the planula larvae et al.2 levels.To obtain larval tissue, gametes were collected from adult colonies as they were released during annual spawning events in 2008. Larvae were acquired from colonies at multiple locations in the upper Florida Keys, Puerto Rico, and Curacao to minimize ascertainment bias Low, mean, and elevated temperatures  were used to stimulate the expression of thermal stress response genes. Larval batches were housed in 1 L plastic containers with mesh sides, suspended in four separate 45 L plastic bins filled with filtered sea water at each treatment temperature. Water was circulated with an aquarium pump and changed daily with filtered sea water preheated to the target temperature. Temperatures were maintained within ±1°C with aquarium heaters and chillers, and were monitored with HOBO data loggers .Larval samples were collected throughout development, from immediately following fertilization until day five, to include a full range of developmental genes in the sequencing results. Approximately 100 larvae from each container at each time-point were incubated in RNA later  then frozen in liquid nitrogen following manufacturer's recommendations. Samples were stored at −80°C prior to extraction. Additional samples from adult colonies were also collected to include a component of post metamorphosis transcripts.2 treatments, larvae from Florida were raised in the Climate Change Laboratory at the Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL. Water in the treatment aquaria was held at 28°C and CO2 concentration was manipulated by bubbling with CO2-enriched air to maintain control and high CO2 treatment conditions (400 and 800 µatm respectively) as described in Albright et al. For the COPurification of RNA from all samples was performed using a modified Trizol extraction protocol. Samples were removed from the preservative and immediately submerged in 1 ml of Trizol for 5 minutes. Following the addition of 0.2 ml of Chloroform and incubation for 3 minutes, the samples were centrifuged at 12,000 rpm for 15 minutes at 4°C to separate the phases. The upper aqueous phase was collected and mixed with an equal volume of 70% ethanol, then applied to a Qiagen RNeasy mini spin column  and purified following manufacturer's instructions. Sample concentration was determined using a Nanodrop ND-1000 spectrophotometer  and RNA quality was assessed with an Agilent Bioanalyzer .2 treatment samples, and 4 adult samples from Puerto Rico and Curacao were pooled into a single batch sample such that the contribution of genetic material from each geographic location was equivalent (∼17 ug each). An aliquot of the resulting pool was submitted to the Indiana University Center for Genomics and Bioinformatics  for cDNA preparation, normalization and sequencing on the 454-GS FLX using Titanium chemistry following previously published methods A total of 71 samples, including 1 to 5 replicates each of 13 larval developmental time-points from all 3 locations, 10 COperl pipeline (pipemeta) following the methods of Vera et al.smartscreener script in the pipemeta package (SMART “CAP” primer 5′-AAGCAGTGGTATCAACGCAGAGT-3′), then entered into seqmanprov8 (DNAstar) for additional quality filtering and assembly. Default quality filtering parameters for 454 data were used, and assembly was performed in seqman using parameters suggested by the manufacturer for short read data. Prior to assembly an additional 36,239 high quality EST Sanger reads from a pool of A. palmata samples from different life stages and stress conditions were included to promote the assembly of long contigs. Sanger sequences are available from the SymBioSys database (sequoia.ucmerced.edu/SymBioSys/). 454 sequences and quality scores generated in this study are available from the NCBI short read archive , and the assembled annotated sequences can be downloaded from http://main.g2.bx.psu.edu/u/nickpolato/h/apalmataassembly.The resulting sequencing reads were preprocessed and annotated using a custom blastx to the complete Uniprot database . Acceptable hits were determined by a bitscore of >45 and a corresponding e-value of <1−5. Because two of the most closely related genomes in publicly available databases (Nematostella vectensis and Branchiostoma floridae) are largely unannotated, a large proportion of the highest ranking blast hits were uninformative. To maximize the information content, results were augmented with lower ranking (but more informative) annotations using a perl script and a list of keywords to avoid (i.e. ‘uncharacterized protein’ and ‘predicted protein’). Contig and singleton sequences were also submitted to the KEGG automated annotation server (www.genome.ad.jp/tools/kaas/) for further functional annotation.The resulting contigs and remaining singletons were aligned with blast search was performed comparing all A. palmata transcriptome data against a subset of transcripts from the N. vectensis genome project (“transcripts.Nemve1FilteredModels1” downloaded from genome.jgi-psf.org). The N. vectensis sequences were first compared to themselves to eliminate multiple copies of sequences with >90% similarity (i.e. multi copy genes). Only blast hits between A. palmata and N. vectensis sequences with a bitscore ≥45 were considered. The length of the A. palmata sequence was then divided by the length of the N. vectensis transcript to obtain a percent coverage estimate for each unigene  was downloaded from OrthoDB (cegg.unige.ch/orthodb4) blast was used to identify homologs of these genes in the A. palmata data.A  unigene , using oA. palmata dataset to compare with functional pathway relationships known in other model organisms. IPA was then used to test whether some pathways were more highly represented then others considering the number of sequences from A. palmata that mapped to a given pathway and the pathways' size. This application of IPA did not depend on expression level (i.e. the number of sequencing reads observed per gene). This is appropriate here because the A. palmata transcriptome was generated from a normalized library. IPA bases its statistical analyses on a Fisher's Exact Test (corrected for multiple testing) which is comparable to other well-known enrichment analysis methods Pathway analysis was performed in the Ingenuity Pathway Analysis (IPA) software. IPA is a web-based application that performs functional enrichment analyses to determine the probability that a given gene set is associated with pre-defined reference pathways beyond what would be expected by random chance. Pathway data in IPA is based on information in the Ingenuity Knowledge Base which is a manually reviewed database of pathways and relationships taken directly from the primary literature and public sources including GO, KEGG and EntrezGene. While this software is capable of other functions that consider gene expression levels based on microarray or RNAseq data, our analyses used only presence or absence of pathway components in the A. palmata dataset. Our analyses compared successfully mapped A. palmata transcripts to the IPA reference set consisting of all molecules present in the Ingenuity Knowledge Base. Significance of the association between the A. palmata data set and a reference pathway was measured in two ways: 1) A ratio was calculated by dividing the number of A. palmata transcripts that map to a given reference pathway by the total number of molecules associated with that pathway in the Ingenuity Knowledge Base, and 2) A Fisher's exact test determined the probability that there was a significant enrichment of A. palmata transcripts mapping to a given pathway beyond what was expected by chance alone given the total number of genes involved in that reference pathway. Because some pathways are better characterized than others these results should be interpreted as a guide for selecting conserved and well annotated functional metazoan pathways present in the A. palmata transcriptome.A canonical pathway analysis identified those pathways in the Ingenuity Knowledge Base that were the most well represented in the codeml program in pamlA. palmata homologs of N. vectensis genes (pVS53a and p63) were aligned with 17 other sequences representing a variety of taxa from anemones to humans . jmodeltestgarli (www.molecularevolution.org/software/phylogenetics/garli) with the TIM2+G model. One hundred bootstrap replicates were performed and bootstrap values were matched to the maximum likelihood tree using sumtreespamlh0−lnLh1)) of the difference between the log likelihood of the null model  relative to that of the alternative model , that incorporated positive selection along the branch leading to the Cnidaria by allowing the dN/dS ratio to be >1 along that branch, was performed and the result compared to a χ2 distribution to assess statistical significance.Tests of natural selection in p53 family genes were performed with the A. palmata and N. vectensis was assessed using inparanoidv4.1 N. vectensis (downloaded from genome.jgi-psf.org), and 43,081 ORFs from the A. palmata transcriptome were used in the analysis. Acceptable hits were determined by a bitscore of >40 using the BLOSUM62 scoring matrix.The identification of in-paralog groups in snphunter script in pipemetamysql database and will be made available to the public via the Dryad data archive (http://datadryad.org/) along with sequences containing potential microsatellite markers.Potential SNPs were detected in contigs with sufficient depth of coverage using the snphunter SNPs were validated using PCR and the amplified products were sequenced on an ABI3700. Primers were designed using primer3 , 2 mM of MgCl2, 0.2 mM of dNTPs, and 2 U of Biolase polymerase . PCR was carried out in an Eppendorf Mastercyler Gradient with an initial denaturation step of 95°C for 5 min followed by 35 cycles of 95°C for 20 s; annealing at 56°C for 20 s; and 72°C for 30 s, and a final extension of 30 min at 72°C.To confirm the results obtained by snphunter for each contig. Enrichment analysis of GO terms associated with transcripts showing the top and bottom 5% of all Ts/Tv ratios (<1 or >5) was carried out using wego (http://wego.genomics.org.cn/cgi-bin/wego/index.pl) Transition and transversion ratios were determined based on the number of SNPs in each class identified by http://tandem.bu.edu/cgi-bin/trdb/trdb.exe) tandem repeats finder v4.04 A. millepora transcriptome (www.bio.utexas.edu/research/matz_lab/matzlab/Data.html) To detect repeat elements, the full set of sequences was uploaded to the Tandem Repeats Database Click here for additional data file.Information S2codeml.Results of tests for natural selection using the program (XLSX)Click here for additional data file.Information S3Top and bottom 5% of Ts/Tv ratios.(XLS)Click here for additional data file.Information S4SNP validation summary and primer sequences.(XLSX)Click here for additional data file."}
{"text": "We expand the anecdotic report by Johansson that back-and-forth linear harmonic motions appear uniform. Six experiments explore the role of shape and spatial orientation of the trajectory of a point-light target in the perceptual judgment of uniform motion. In Experiment 1, the target oscillated back-and-forth along a circular arc around an invisible pivot. The imaginary segment from the pivot to the midpoint of the trajectory could be oriented vertically downward (consistent with an upright pendulum), horizontally leftward, or vertically upward (upside-down). In Experiments 2 to 5, the target moved uni-directionally. The effect of suppressing the alternation of movement directions was tested with curvilinear (Experiment 2 and 3) or rectilinear (Experiment 4 and 5) paths.  Humans make striking perceptual mistakes in judging even the simplest kinematics of visual stimuli. Thus, a spot moving at constant velocity along a rectilinear path is perceived as moving fast upon entering the visual field and then decelerating to a constant velocity Runeson The idea that the expected dynamics of natural stimuli biases visual motion perception has been extensively tested in the case of biological movements The motions of masses under force fields afford another rich array of natural events that can bias perception. Gibson A more stringent test of Runeson's hypothesis is possible by considering stimuli compatible with more than one ecological model. The present study focuses on the perception of a very common visual template, the oscillation of an object around a pivot. Many real-world events, including biological movements, are described approximately by such a template that arises whenever a mass is subjected to elastic and/or gravitational forces. In particular, the perceived motion of the pendulum has received considerable attention. When friction is negligible, the instantaneous velocity of a suspended mass under Earth gravity depends only on its initial position and on the length of the pendulum rod. Moreover, in the small-angle approximation, the oscillation is harmonic with a period that depends only on the length of the rod.The earliest evidence that the motion of the simple pendulum is perceptually salient was provided by measuring the accuracy with which one can point to the bob Under specific circumstances, gravity and elastic forces induce similar harmonic oscillations. In general, however, the two forces are very different. Unlike elastic tension, gravity is ubiquitous, almost constant at Earth surface, and acts invariably in the downward direction. In fact, the expected constraints imposed by gravity on object motion can affect visual perception more generally than in the simple oscillatory case We designed six experiments to test these predictions and to investigate the perceptual consequences of modifying some of the features that are normally associated with the motion of a gravitational pendulum or a mass-spring system. We presented a spot moving along curvilinear or linear trajectories with different velocity profiles including both harmonic and constant velocity motion, and asked the observers to choose the profile that appeared most uniform. By varying the law of motion, the shape of the trajectory, and its orientation relative to gravity, we explored a significant range of possible departures from the canonical motion of a simple pendulum. In Experiment 1, the target oscillated back-and-forth along a circular arc. The effects of target kinematics and of the orientation of the trajectory relative to gravity were manipulated independently. In Experiments 2 to 5, the target moved uni-directionally. The effect of suppressing the alternation of movement directions that characterizes the motion of a pendulum was tested with curvilinear (Experiment 2 and 3) or rectilinear (Experiment 4 and 5) paths. In Experiments 1 to 5, no instruction was given concerning eye movements and observers were free to eye-track the target. The anecdotal report by Johansson First, we presented stimuli moving along curvilinear trajectories with constant radius, which are always compatible with the motion of a pendulum. Second, rather than asking a qualitative judgment on just one velocity profile, we asked observers to select the most uniform motion from a full range of alternative profiles, which also included the uniform one. The range of velocity modulations was wide enough to exclude that perceptual misjudgments of the kind reported by Johansson If perception relies heavily on statistical prior models of physics, and if “only natural motions look constant”, the harmonic motion should be chosen as prototype of uniform motion for the three stimulus orientations, which are all consistent with the assumption that oscillations are driven by elastic forces. However, if a prior related to the direction of gravity also affected perception, one would expect that the harmonic motion should be chosen with greater accuracy, precision and inter-individual consistency in the canonical upright orientation of a pendulum than in the other two orientations incongruent with gravity.Note that, although the stimuli could be construed also as a schematic description of a voluntary biological movement (such as the back and forth swing of a limb), they were point-to-point motions with a constant curvature throughout and a reversal of velocity direction at the endpoints of the trajectory. One cannot expect the responses to comply with the 2/3 Power Law, which does not apply to such movements.Twelve participants  volunteered for the experiment. They were all right-handed , had normal or corrected-to-normal vision, and no past history of psychiatric or neurological diseases. All participants in this and the following experiments gave written informed consent to procedures approved by the Institutional Review Board of Santa Lucia Foundation, in conformity with the Declaration of Helsinki on the use of human subjects in research, but were otherwise unaware of the purpose of the experiments.1g) of an ideal simple pendulum under Earth gravity (g = 9.81 m s−2) obeys the differential equation:θ(t) is the angular displacement from the equilibrium position (θ = 0), and L is the rod length. With the initial conditions:0 = π/4. Thus, T = 1.68 s. In the following, A = 2θ0 = π/2 denotes the amplitude of the oscillation. Angular velocity (V1g = dθ(t)/dt) was computed by solving 1g and the law of motion computed in the small-angle approximation (harmonic function) was 0.7% and 1.3% for angular position and velocity, respectively. We use the term Velocity to indicate the unsigned time derivative of the angular displacement.The motion  with the same parameters , but different velocity profiles −V1g (T/4) = V1g (T/4)−V0g (T/4), and V3g (T/4)−V2g (T/4) = V2g (T/4)−V1g (T/4), respectively. The labels 2 g and 3 g denote conditions with a maximum velocity twice and three times as large as 1 g, but do not describe a pendulum motion under a gravity level twice or three times as large as Earth's gravity. In fact the corresponding velocity profiles depart significantly from harmonic functions , and then under-sampling the sequence to match the vertical refresh rate of the display.ions see . FinallyTask and procedures). Stimuli were programmed in C++ and rendered using Autodesk Maya 2009 (Autodesk Inc) with a PNY NVIDIA Quadro FX5600 graphics card.Participants sat 0.58 m in front of a display  in a dimly illuminated room. The height of the chair was adjusted so that participant's eyes were ∼10° above the display midpoint. Responses were entered by pressing one of 4 keys (button-box Empirisoft Corp.) labeled  “Start”, “Forward”, “Backward”, and “OK” (see 2), moving against a uniformly bluish-purple background . Luminance and chromaticity were measured with a Tektronix J17 LumaColor photometer. In each trial, the target oscillated back-and-forth along the arc of a circle  around an invisible pivot  and 275% (K20). For all kinematic profiles except K5 (constant velocity), the ratio exceeded the detection threshold of 25% reported for stimuli with comparable duration and modulation frequency Human sensitivity to visual accelerations is based on the comparison of velocity estimates averaged over short, successive time windows 2, K6, K10, K14, K18,], with the constraint that successive trials could not have the same initial kinematics. There were 5  × 10 (repetitions)  = 50 trials in each session.Participants were tested in a counterbalanced order in three experimental sessions about 15 days apart. In each session, we presented one of the three orientations defined above (block design). In all trials, the target initial position was at π/4 clockwise relative to the trajectory midpoint. The target oscillated continuously with the same law of motion until the participant intervened either to modify the target kinematics (see below), or to end the trial with a response. The initial kinematics for a trial was selected pseudo-randomly from the set . If Ki+1 or Ki−1 fell outside the preset range of conditions, the computer replaced it randomly by one of the three conditions . Participants were informed about neither the number of steps in the sequence nor the condition they were currently exploring. No feedback about response accuracy was provided. No instruction was given concerning eye movements so that participants were free to eye-track the target. Responses were stored together with the preceding sequence of changes. On average, reaching a final decision required 12 adjustments. The typical duration of a session was 40 min. None of the participants reported any sensation of motion-in-depth.The effect of pressing the buttons was to move one step backward  Wnij, respectively. Moreover, in order to treat the stimuli as an ordinal random variable, we define the 3-vector I =  indexing the corresponding kinematics. The results were analyzed at both population and individual level. At population level , the effect of orientation was estimated in several different ways. First, by computing the median (M) and the interquartile range (IQR) of the index of the kinematics judged as uniform. Second, global estimates of the central tendency index and of the variability were derived from a Probit analysis of W: −1 is the probit link function, 0β the intercept and 1β the slope of the linear regression. Goodness of fit was assessed by testing that the deviance was not significantly different from 0 In the following, we use a vector notation (boldface) to denote the performance for the three orientations of the trajectory. Let the 3-vectors I of experimental kinematics) and random-effects predictors (to account for the variability among participants). For participant j the complete model, which also includes the interactions between fixed and random factors, is:Wj is the CDF of participant j, 90D and 180D are dummy variables coding for the orientation (with respect to the 0° orientation reference), 90I·D and I·180D are the interaction terms, 0β, …, 5β are the fixed-effects coefficients (independent of participants), and j0u, …, j5u are the random-effects coefficients for participant j. In particular, 1β estimates the precision of the response in the reference (0°) orientation , 2β (3β) tests whether the intercept is significantly different between the reference and the 90° (180°) orientation, and 4β (5β) tests the same difference for the slope. The model was applied twice, by taking as reference first the 0° orientation (as detailed above), and then the 90° orientation (with the appropriate dummy variables D). The model was fitted to the CDF's using the R package ‘lme4’ As a further analysis at population level, we applied to the data the Generalized Linear Mixed Model GLMM, . The GLMwww.mixedpsychophysics.wordpress.com).Median and JND for the population were again estimated from GLMM using the R package “MERpsychophysics” . There was a significant (P<.001) attractive effect of the initial kinematics on the median. However, the size of the effect was very small, the difference between maximum and minimum median values, computed separately for each starting condition, being equal to 1. Therefore, in the subsequent analyses the results were pooled across repetitions and starting conditions.10 which simulated the effects of a virtual gravity (1 g) acting downwards (0° orientation), leftwards (90° orientation), or upwards (180° orientation). However, the specific distribution of the responses differed as a function of orientation. In particular, the variability was smaller for the 0° orientation (IQR = 1) than for both the 90° (IQR = 3) and the 180° orientation (IQR = 3). Moreover, the proportion of trials in which velocity profiles close to K5 (constant absolute velocity) were judged as uniform was lower for the 0° orientation than for the other two orientations.These results were confirmed by a probit analysis of the population CDFs of the response variable. There was a highly significant difference  between the CDF for orientation 0° and those for either 90° or 180° orientation. Both the median and the slope (response precision) were higher for 0° orientation than for the other two orientations. Differences remained significant  even when the CDFs were computed after equalizing the individual medians. In contrast, the CDFs for 90° and 180° orientation appeared to have the same shape , the same variance , but different median value .Data analysis and modeling). With the only exception of 5β, all parameters of the model were significantly different from zero . Clearly, the responses were much more consistent among participants for 0° orientation than for the other two orientations. The difference across orientations is dramatized in 10 (1 g), but was significantly higher than the median for the other two orientations (90° and 180°). The medians for all stimulus orientations were significantly closer to K10 than to K5 (constant velocity). The slope of the CDF for orientation 0° was significantly higher than the slope for 90° orientation , whereas the slope was not significantly different between 0° and 180° orientations  and between 90° and 180° orientations .The effects of orientation were investigated further by modeling the individual CDFs of the response variable. 0–K9], which includes the uniform motion case (K5), the kinematic profiles presented a discontinuity, because velocity does not go to zero at the endpoints of the trajectory  for the 0° orientation , and the maximum angular velocity of K10 was 32.3° s−1  and were less variable across trials and participants than for the other two orientations (90° and 180°). In other words, the bias was stronger for a trajectory orientation consistent with the interpretation of the stimulus as a canonical upright pendulum accelerated by physical gravity. In the general discussion we elaborate the implications of these findings vis à vis the general notion that perception is influenced by the interpretation of the stimuli in terms of physical events. Two issues must be addressed. 1) Both gravitational and elastic oscillations are characterized by the inversion of movement direction at both ends of the trajectory and by the curvilinear trajectory of the target. The role of these features on velocity perception is investigated in the next 4 experiments. 2) The possible role of eye movements. The last experiment tests whether eliminating the possibility to track the target with eye movements affects velocity judgments.In sum, we found that a large misperception of target kinematics exists for all three orientations of the trajectory. Observers perceived as uniform a quasi-harmonic velocity profile that was strongly non-uniform, velocity changes being greater than 150% within each oscillation. The perceptual bias was significantly modulated by trajectory orientation. Responses for the 0° orientation clustered closer to the 1g-condition  volunteered for the experiment. None of them had served in Experiment 1.Apparatus, task, and general experimental procedures were the same as in Experiment 1. The target moved along the same trajectory and with the same 21 velocity profiles as in the 0° orientation of that experiment. However, motion was unidirectional, from left to right. Its duration was the same (840 ms) as that of a single sweep of the pendulum-like motion in Experiment 1. Upon reaching the rightmost position along the path, the target disappeared for 140 ms; then it reappeared in the leftmost position and immediately started a new sweep. The inter-sweep interval was chosen to permit the eyes to re-foveate the target with a saccade . However, the JND was considerably higher than in the previous experiment. Taken together, the results of Experiment 1 and 2 show that a target moving with quasi-harmonic kinematics is perceived as uniform irrespective of whether full back and forth oscillation cycles or just repeated unidirectional half cycles are displayed. However, the responses tend to be considerably more variable in the latter than in the former case.There was no significant effect of repetition on the response median , and a significant (P<.0001) effect of the starting condition in the sequence . In this experiment we asked whether the perceptual bias toward a quasi-harmonic velocity profile disappears when the trajectory is incompatible with any simple physical model capable of sustaining an oscillatory behavior. The velocity of the targets was modulated as in the previous experiments, but with a different trajectory. Here, the target moved along a circle without ever changing direction and was visible only in the bottom and top quadrants. These two segments of trajectory shown sequentially in time can be perceived as a partially occluded circular motion. Crucially, no physical system is compatible with a harmonic motion along a circular trajectory.Eleven participants  volunteered for the experiment. None of them had served in the previous experiments.Apparatus, task, and general experimental procedures were the same as in Experiment 1. The target moved uni-directionally, anticlockwise, along a circular trajectory centered on the display midpoint . The rad6 (IQR = 3) , and a small but significant effect of the initial kinematics . = 3) see . ParticiIn summary, the change in target configuration abolished almost completely the perceptual bias in favor of the harmonically modulated velocity profiles. Although the trajectory was partly occluded, the stimulus may have evoked the motion of a rotating system. As argued above, no simple such system exhibits harmonic oscillations. By contrast, a mass rotating under the effect of a central force - arguably the simplest physical model for such motion - does indeed move at constant velocity. Therefore, the results are still compatible with the general hypothesis that perception is permeable to pre-conceptions about the possible physical interpretation of the stimuli.This and the next experiment investigate the role of trajectory curvature. In Experiment 2 we showed that even repeated unidirectional sweeps along a circular arc were sufficient to make participants perceive the quasi-harmonic motion of the target as uniform. Here we tested whether the same bias is present also when the target follows a horizontal rectilinear path.Twelve participants  volunteered for the experiment. Ten of them had participated also in Experiment 3. There were about 20 days between Experiment 3 and 4.The target moved on a linear, horizontal path from left to right, encompassing a visual angle of 17.85°. The path length was the same as that of the circular segments in Experiments 1 to 3. The midpoint coincided with the center of the display. Motion duration was the same (840 ms) as that of a single sweep of the pendulum-like motion in Experiment 1. As in Experiment 2, upon reaching the rightmost position along the path, the target disappeared for 140 ms and reappeared in the leftmost position to start a new sweep. Target velocity at any time along the linear trajectory was equal to the tangential velocity at the same time along the curvilinear path of Experiment 1. No instruction was given concerning eye movements. 6 (IQR = 2). 6 or the median of the previous experiment. Finally, the response in this and in the previous experiment had indistinguishable distributions . Therefore, the single difference with respect to Experiment 2 (rectilinear versus curvilinear trajectory) was responsible for eliminating almost completely the perceptual bias toward quasi-harmonic motion. As in the case of Experiment 3, this suggests that the bias disappeared because the stimuli no longer evoked an oscillating physical system.There was no significant effect of repetition on the median of the responses , and a small but significant (P<.005) effect of the initial kinematics (difference between maximum and minimum median  = 1). We tested whether the results of the previous experiment are confirmed when the rectilinear unidirectional motion is vertical instead of horizontal.Twelve participants  volunteered for the experiment. Five of them had participated also in Experiment 1. One of them had participated one year before also in Experiments 2 and 3.The target moved downward along a linear, vertical path of the same length as in Experiment 4 (17.85°). The midpoint coincided with the center of the display. Motion duration was the same (840 ms) as that of a single sweep of the pendulum-like motion in Experiment 1. Upon reaching the bottom position, the target disappeared for 140 ms and reappeared in the top position to start a new sweep. Target kinematics was the same as in Experiment 4. No instruction was given concerning eye movements.5 as the constant velocity profile. 6 (IQR = 5). 6. Taken together, the results of the last 3 experiments show that the perceptual bias towards quasi-harmonic motion is not inevitable, but requires that some cues in the stimuli evoke a context of an oscillatory physical system.There was no significant effect of repetition on the median of the responses , and a small but significant (P<.005) effect of the initial kinematics . Observers were often able to correctly identify KIn all previous experiments, no instructions were given concerning eye movements to participants, who were free to track the target. It has been reported that motions that can be construed as natural events  are easier to track with eye movements than motions deviating from such natural models Six participants  volunteered for the experiment. Two of them had participated also in Experiment 1 about 30 days before.−1), the retinal eccentricity of the target (10.74°) relative to the fovea does not alter velocity estimates substantially The apparatus and general experimental procedures were the same as in Experiment 1. The target oscillated back and forth along the same trajectory and with the same 21 velocity profiles as in the 0° orientation condition. However, throughout each trial, participants were asked to fixate a white cross-hair (0.37°) located at the pivot of rotation of the target, 10° above the display midpoint. The head of the participants was stabilized with the help of a forehead-chin rest. The height of the chair and of the forehead-chin rest was adjusted individually so that the eyes were level with the fixation point. It is known that, in the range of values tested here (average angular velocity: 19.96° s10 (IQR = 3). 10 or from the corresponding median in the 0°orientation condition of Experiment 1. The probit fit to the population CDF and the corresponding CDF in Experiment 1 had the same shape , variance , and median . In conclusion, this control confirmed fully the results of Experiment 1. Preventing the possibility to track the target with eye movements did not suppress the perceptual bias present when target tracking was possible.All participants correctly reported the words presented at the fixation point. Although the target moved in the periphery of the visual field, performance in the main task was very similar to that in Experiment 1. The median response depended on neither the initial kinematics , nor repetition . 10, here denoted as 1g-kinematics), that is, the kind of motion naturally associated with these two physical systems. The bias towards quasi-harmonic motion was most consistent in the upright 0° orientation of Experiment 1, where both the gravitational pendulum and the rotational mass-spring system afford a plausible physical model with almost identical kinematics. When the predictions of the two dynamic models were mutually incompatible (90° and 180° orientation), the bias tended to diminish and to be less consistent across participants. Finally, in the experiments in which the trajectory was incompatible with both a simple pendulum and a rotational mass-spring system  was still compatible with a mass-spring system, but became incompatible with the action of physical gravity. In fact, for trajectories at 180° orientation, gravity would decelerate the target toward the trajectory midpoint and accelerate it away from the midpoint, whereas at 90° orientation gravity would accelerate the target on the way down and decelerate it on the way up. We suggest that, in these conditions, the mass-spring model is the dominant one and is a sufficiently strong prior to attract the perceptual responses much closer to 1g-kinematics than to 0g-kinematics even at these orientations. However, we also argue that the powerful pendulum model is still somehow evoked. The shift of the perceptual bias slightly away from the 1g-kinematics and towards the 0g-kinematics, and the larger variability across trials and participants with respect to the 0° orientation see  is then 6), they were often able to correctly identify K5 as the constant velocity profile (The comparison of the results of Experiment 1 and Experiment 2 (0° and 0°ss  profile  and 7. A6), but they were often able to correctly identify K5 as the constant velocity profile  with a prior related to the statistical distribution of velocities in the natural world File S1Contains: Movies S1–S21. Down-sampled (50 Hz) version of the stimuli presented in the 0° orientation condition of Experiment 1, , Movie S6 to K5 (0 g), Movie S11 to K10 (1 g), Movie S16 to K15 (2 g), and Movie S21 to K20 (3 g). Because each movie shows only one stimulus period, to see the stimuli repeatedly the media player must be set in reply (continuous) mode.(ZIP)Click here for additional data file.File S2Contains: Movies S22–S42. Stimuli presented in the 180° orientation condition of Experiment 1 (0 (−1 g), and Movie S42 to K20 (3 g).riment 1 . Movie S(ZIP)Click here for additional data file.File S3Contains: Movies S43–S63. Stimuli presented in Experiment 4. Movie S43 corresponds to K0 (−1 g), and Movie S63 to K20 (3 g).(ZIP)Click here for additional data file."}
{"text": "Background. Mycosis fungoides (MF) is the most common subtype of cutaneous T-cell lymphoma. Extensive studies on Iranian MF patients are absent. The present study aimed to produce updated clinical information on Iranian MF patients. Methods. This was a retrospective, descriptive, single-center study, including all cases of MF seen in the Department of Dermatology, University Hospital of Isfahan, Iran, between 2003 and 2013. Data systematically recorded for each patient included clinical, biological, histological, and molecular findings. Results. Eighty-six patients with clinical and histologic diagnosis of MF were included in the study. Thirty-nine patients (45.3%) were male. Female predominance was observed in patients . Patients were between 7 and 84 years of age (median: 41). The interval from disease onset to diagnosis ranged from 0 to 55 years (median: 1 year). Eighteen cases (20.9%) had unusual variants of MF. The most common types included hypopigmented and poikilodermatous MF. Childhood cases of MF constituted 5.8% (5/86) of all patients. The early stages were seen in 82 cases (95.34%). Conclusion. The major differences in epidemiologic characteristics of MF in Iran are the lack of male predominance and the lower age of patients at the time of diagnosis. Mycosis fungoides (MF) is the most common subtype of cutaneous T-cell lymphoma (CTCL). MF differs from other primary CTCLs by virtue of unique clinical features and histopathology , 2. PatiThe natural history of MF is characterized by an indolent progression through four stages: patch, plaque, tumor, and visceral involvement, but this progression is not necessarily seen in all patients . Many vaThe peak age at presentation is in excess of 55 to 60 years, with a 2 : 1 male : female ratio , 8.Extensive studies on Iranian MF patients are absent. The present study thus aimed to produce updated clinical information on Iranian MF patients, including epidemiology, patient characteristics, clinicopathologic features, and treatment.Between 2003 and 2013, patients with MF from single-center resident training hospitals had been retrospectively enrolled into the study.A diagnosis of MF was confirmed according to International Society for Cutaneous Lymphoma (ISCL)/EORTC criteria , 9 and, The analyzed clinical data included the age of the patients at the time of diagnosis, the age of the patients at the time of initiation, the cutaneous lesion, sex, and the status of disease at the last follow-up examination. Hematoxylin and eosin-stained slides, immunohistochemical stains for basic B- and T-cell markers (CD20 and CD3 and/or CD45RO), and additional immunostaining, including CD4, CD8, CD30, and CD5, were histologically evaluated by an expert dermatopathologist. Polymerase chain reaction analyses of T-cell receptors (TCR) b or c and immunoglobulin heavy chain gene rearrangement were reviewed.In all the patients, the presence of extracutaneous disease at the time of diagnosis had been excluded by standard staging procedures.Stage workup  and overall survival were obtained by reviewing the clinical records.To determine the stage of MF, the tumor-node-metastasis (TNM) system was used . The cliAfter data collection, statistical analysis was performed by the SPSS software version 19.Eighty-six patients with clinical and histologic diagnosis of MF were included in the study.The interval from disease onset to diagnosis ranged from 0 to 55 years (median: 1 year).From 68 patients with classic MF, 42 patients presented with the patch, 21 with plaque, 4 with a tumor, and only one with erythroderma . From 86Only one patient had folliculotropic MF . One patChildhood cases of MF (younger than 18 years) constituted 5.8% (5/86) of all the MF patients. There were two male and three female patients .There was no familial clustering. None of the patients had HIV or other forms of immunosuppression.According to the TNM classification, among 86 cases of MF, the early stage (stage I + IIA) included 82 cases (95.34%) and the advanced stage (stage IIB + III + IV) included four cases (4.66%). One female patient had a large cell transformation during the course of disease. Progression to the late stages of MF is not seen in childhood/juvenile-onset MF patients.Immunophenotypic analysis demonstrated that 77.6% of the patients with MF were CD4 positive. CD20 was negative in all patients. In our patients, the conventional T-helper phenotype (CD4+/CD8−) was the most common one (63% of patients) .A clonal T-cell receptor rearrangement was detected in the skin biopsy in 9 patients (36%) from a total of 25 patients in whom T-cell receptor analysis was possible.Antibodies to HTLV-1 were not detected in the sera of any patients with MF by ELISA. Seven patients (8.1%) had abnormal lactate dehydrogenase (LDH). None of the patients died during the study period in the early stages.The major differences in the epidemiologic characteristics of MF in Isfahan, Iran, are lack of male predominance and the lower age of patients at the time of diagnosis in those reported from the West. It was reported that the incidence rate of MF in Isfahan is the same as that in other parts of Iran, which is largely similar to the data of other countries .MF is generally rare among Asians and it has a higher incidence rate in blacks , 14; it There is a male predominance in almost all studies on CTCLs and MF, with a male : female ratio of 1.3 : 1 to 2 : 1 –17.The proportion of our patients complaining on the onset of their disease before 21 years was much higher, similar to the epidemiological study in Kuwait . The occSome studies have found an increased incidence of CTCL among workers in chemical science, transportation, and manufacturing industries, whereas other studies have not .Hydrocarbons and petrochemical exposure have been reported to carry an increased MF risk , 21. WheIn the Iran-Iraq War (1980–1988), it is not known whether chemical weapons by Iraq contained Sulfur mustard (SM), a DNA alkylation, and a well-known carcinogenic agent . This hyThe prevalence of childhood MF among different studies has varied between 2.7% and 16.6% , 24. In Among the atypical forms of MF, hypopigmentation is one of the most prevalent forms in Asians . HypopigOur data is compatible with the study of Quaglino et al.  in the fThe median time from symptom onset to diagnosis in retrospective studies is 3-4 years, but it may exceed four decades , 32, 33.The current study differs from that of Quaglino et al. in the fact that the median time from the onset of cutaneous lesions to diagnosis was 2 years .In the present study, the mean time from symptom onset to definite diagnosis was about 5–10 years, which is significantly different in male and female patients.Several studies have shown that nearly all patients with MF are HTLV-1 seronegative . Our finUsually, MF is characterized by an infiltrate of T-helper memory lymphocytes  . HoweverAlthough the group of this study is really not very large and also the number of the patients with more advanced disease is small, it may be the starting point for a larger multicenter study in Iran. Whether the occurrence of MF in younger patients in Iran is related to some ethnic or genetic variations or to environmental factors needs to be determined. A prospective multicenter study on a larger population of patients with a longer follow-up might be useful to confirm the possible causes of these epidemiological differences."}
{"text": "Objectives: Schizophrenia patients often experience relapses once and even more with no limit on number of relapses. The time among relapses are rarely considered in studies. The aim of this study was to identify some risk factors of time to elapses in schizophrenic patients with recurrent events model in survival analysis.Methods: In a retrospective longitudinal study, the medical records of 159 schizophrenic patients who referred to Razi hospital in Tehran from 2003 to 2005 were conveniently sampled, investigated and followed until the end of 2009. The time to recurrent relapses were considered in weeks. The patients with at least one relapse in this duration were included in the study. Event-dependent frailty model, using Bayesian approach, was applied to fit the data and identify the risk factors of time to relapses.Results: In this recurrent failure time model, the effects of age of onset ), gender ), marital status ) and family history ) were significant on the hazard time to relapses. According to the credible interval of frailty variance, elapsed time to relapse is dependent on patients’ characteristics ). Subsequent relapses are likely to be influenced by the occurrence of the first relapse, too ), with decreasing hazard of time to relapse.Conclusions: Subsequent relapses are likely dependent on the first and previous relapses. Age of onset, gender, marital status and family history are important risk factors influencing hazard of time to relapses. More studies are required to clear out the effect of other covariates with this model. Mental disorders have profound social, cultural and economic effects on communities worldwide . Burden The illness tends to develop between the ages of 16 and 30, and mostly persists in the course of patient’s lifetime. Although the causes of schizophrenia are unknown, evidences suggest that genetic factors, early environmental influences , and social factors  play an important role .The inciParticipants and ProcedureA retrospective longitudinal study was carried out on 162 conveniently sampled schizophrenic patients with at least one relapse, who were hospitalized at least once at Razi psychiatric hospital in Tehran between 2003 and 2005, and who were followed up until the end of 2009; data were collected from their medical records. Three patients who had missing values on all covariates in their records were excluded from the total sample. For the rest of the patients, the onset of schizophrenia was considered as the time of their follow up, and they were followed in terms of their relapses. Relapse was defined as observing schizophrenia symptoms in the first re-hospitalization after discharge and following re-hospitalizations. We assessed time to relapses and the number of weeks patients remained to be re-hospitalized. Some patients right censored at their last time period to re-hospitalize. Statistical Analysis Recurrent time data are commonly encountered in longitudinal studies when failure events can occur repeatedly over time for each study subject . Time toThe participants for the studied sample were 134 (84.28%) males and 25 (15.72%) females. The mean age of the patients at the onset of the disease was 21.52 ± 6.84 years (range 10-43). The relative frequency of married patients was 19.59% (31), and 85 (53.46%) patients experienced sudden onset of disease while 76 (47.8% ) patients had a history of head injury. Only 10 (6.29%) patients had schizophrenic patients in their family (grades one to three kinship). In common survival models, positive signs of regression coefficients increase the risk of event occurrence. Distribution of the number of relapses in schizophrenic patients shows that 45.3% of males who did not have a family history of schizophrenia had experienced 3 relapses during the study period . With reChronic diseases such as schizophrenia are, roughly speaking, lifelong transitions between the states of relapse and recovery. The long-term pattern of recurrent times-to-relapse can be investigated with routine register data on hospital admissions. The relapses indicate the time for readmissions to hospital and the time spent at the hospital are considered gaps between subsequent times-at-risk . In thisSubsequent relapses are likely dependent on the first and previous relapses. Age of onset, gender, marital status and family history are important risk factors influencing hazard of time to relapses. More studies are required to clear out the effect of other covariates such as place of residence , education level, economic status and season of birth of patients on time to relapses with this model."}
{"text": "Transformed mycosis fungoides (T-MF) is a rare variant of MF with an aggressive course. In this study, we aimed to describe characteristics of MF/Sezary syndrome (SS) patients with transformation.Patients diagnosed with T-MF among MF/SS patients between 2000 and 2014 in a tertiary single center were evaluated retrospectively. Demographic data, clinical data, laboratory data, immunophenotype features, response to treatment, survival, and histopathologic features were analyzed.Among 254 MF patients, 25 patients with T-MF were identified (10.2%) and included in the study. The male-to-female ratio was 2.6/1. The median time between MF diagnosis and transformation was 32 months (range: 0-192). Nine (36%) patients were diagnosed initially with T-MF. Advanced disease stage and high serum lactate dehydrogenase (LDH) levels were indicators of poor prognosis and treatment response. Five of the 18 patients with progressive disease had undergone allogeneic hematopoietic stem cell transplantation . Allo-HSCT resulted in complete remission in three (60%) patients. Ten (40%) patients died as a result of disease progression. Mean survival time was 25.2±14.9 (2-56) months after transformation.Advanced stage, high serum LDH levels, and loss of CD26 and CD7 expression in the peripheral blood are poor rognostic factors in T-MF. Treatment-resistant tumors and nodules should be cautionary for T-MF. Patients with T-MF have a shortened survival. Some patients may respond to first-line treatments. However, the majority of patients who do not respond to first-line therapies also are unresponsive to second or third-line therapies. Allo-HSCT may be an alternative option in patients with T-MF. Mycosis fungoides (MF) is the most common subtype of cutaneous T-cell lymphoma (CTCL). Generally, MF has an indolent course with slow progression from patch/plaque-stage disease to cutaneous tumors . HoweverDiagnosis of transformed MF (T-MF) is based on the presence of large cells CD30 +/-) exceeding 25% of the infiltrate throughout the lesion or forming microscopic nodules of large cells0 +/- exc.Advanced stage of MF at the time of transformation and folliculotropism are suggested as the most important factors affecting survival. AdditioRisk factors associated with an aggressive course of T-MF are not well described in the literature due to the low incidence of MF/SS and thus T-MF. In different series, the incidence of T-MF has been reported to range between 8% and 55%among MF patients ,10,11. TWe retrospectively evaluated all MF/SS patient records in a single reference center in Ankara, Turkey, from 2000 to 2014. Among all MF/SS patients, T-MF patients with at least one histopathologically confirmed biopsy were included in the study.For each case, clinical features were evaluated by three dermatologists and histopathological findings were reviewed by one pathologist who was an expert in this area.All patients were classified according to the International Society for Cutaneous Lymphomas and European Organisation of Research and Treatment of Cancer revised criteria of 2007 . StagingThe accompanying prognostic factors were also analyzed: age, sex, age at diagnosis of T-MF, presence of folliculotropism in skin lesions, CD30 expression in more than 75% of cutaneous neoplastic T cells, serum lactate dehydrogenase (LDH) levels, serum b2-microglobulin levels, and eosinophilia. The time interval between MF and T-MF, clinical stage at the time of T-MF, and survival were analyzed.Therapies were classified as first-, second-, and third-line treatments according to the 2014 National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology ,14. AlloResponse to treatment was evaluated as follows: complete response (CR), complete resolution of the disease; partial response (PR), at least 50% improvement compared with baseline; stable disease (SD), some improvement (25% to 50% improvement in lesions) plus reduction in the size of axillary and inguinal lymph nodes in the absence of significant evidence of disease; or progressive disease (PD), more than 25% increase in the number or size of clinically abnormal lymph nodes, or development of novel tumors or pathologically positive nodes or visceral disease .The data obtained from patients were analyzed with SPSS 16.0. The Mann-Whitney U test, chi-square test, Spearman’s test, and Mantel-Cox analysis were used to compare variables. The Kaplan-Meier method was used to determine overall survival.The disease stage and exact TNMB stages of patients, initial treatments, and follow-up data of each patient are summarized in 1 , stage IVA2 , and stage IVB ]. Most patients had transformation only at a skin site (96%); in one patient skin and lymph, node transformations were detected simultaneously (4%). Furthermore, 32% of T-MF patients presented with a new or enlarging tumor accompanied by long-standing plaque lesions. Dermatological examination at the time of T-MF diagnosis for the rest of the patients revealed the following: two (8%) patients had long-standing enlarging tumors, four (16%) patients had a new tumor accompanied with erythroderma (one bullous), one (4%) patient demonstrated ichthyosiform erythroderma, three (12%) patients had an enlarging plaque with erythroderma, three (12%) patients had long-standing plaques, two (8%) patients had newly scattered papules distinct from MF plaques, one (4%) patient showed an abrupt onset of multiple pink scattered nodules, and another patient (4%) had follicular papules associated with hair loss within the involved area (Two of 25 patients with T-MF (8%) had early patch and plaque MF . Twenty-three patients had advanced-stage disease [stage IIB , stage III , stage IVAved area .Histopathological examinations of the transformation site showed tumoral lesions in 18 (72%) cases and plaque lesions in seven (28%) cases. Lesion subtype (plaque or tumor) was not significantly correlated with survival (p=0.678). Less prominent or focal epidermotropism was present in 15 (60%) of the 25 patients in our study, and only 2 (8%) patients had Pautrier microabscesses. Folliculotropism was observed in ten cases (40%) with LCT. In eight (80%) of them, there was progression under treatment, while in one (10%) patient PR to treatment and in 1 (10%) patient CR was observed. Folliculotropism was not correlated statistically with survival (p=0.568).+CD4+CD8− T-cell phenotype and one (4%) patient had a double CD4+CD8+ T-cell aberrant phenotype. In most cases (88%), there was partial loss of one or more pan-T-cell antigens. Loss of CD7 expression was seen in 22 (88%) patients. Immunophenotype analysis of the skin biopsies showed that 24 (96%) patients had a CD3CD30 positivity in more than 75% of all the large T cells was present in skin biopsies of five patients (20%). In the remaining 20 (80%) cases, CD30 staining was either completely negative or expressed by only a very few (<5%) large T cells. There was no statistically significant difference either in disease stage or treatment response among CD30-positive and CD30-negative patients . Twelve patients with early  and concurrent  transformation were also evaluated separately for survival (p=0.582).Advanced disease stage at the time of transformation correlated with poor survival (p=0.003). In our study, among the deceased patients, 80% had stage IV disease, whereas only 20% of patients had stage IV disease among the surviving patients (p=0.002). During follow-up, 10 patients died of disease-related events (32%). Three (30%) of 10 patients who died in our study had SS, and the other patients’ stages were as follows: stage IIB , stage III , and stage IVA . The survival curve of the patients is presented in Flow cytometry of peripheral blood showed an increased ratio of CD4/CD8 (>2) in 13 (52%) patients. The ratio was between 2 and 10 in ten (40%) patients and higher than 10 in three (12%) patients. The patients’ disease stages and CD4/CD8 levels showed a statistically significant positive correlation (p=0.038). Increased CD4+/CD26 cell ratio was significantly correlated with poor survival (p=0.017). Loss of CD7 expression (more than 40%) was significantly related to poor survival (p=0.001).Laboratory findings are summarized in All patients received first-line therapy as a combination treatment of two or more of the following: psoralen plus ultraviolet, interferon-alpha, extracorporeal photopheresis, vorinostat, bexarotene, retinoid, low-dose methotrexate, local radiotherapy, or total skin electron beam radiotherapy. Of the 18 (72%) patients showing PD with first-line treatment modalities, 12 (48%) patients received either second- or third-line treatments. Of these 12 patients, six (48%) received second-line treatments either for the induction of remission or in an attempt to decrease tumor burden before allo-HSCT. Second-line therapy included single-agent chemotherapy of either gemcitabine or pralatrexate in 5 (20%) patients to decrease the tumor burden before allo-HSCT. One patient had a lymph node biopsy consistent with concomitant natural killer cell lymphoma and received an Aurora A kinase inhibitor as second-line therapy following five cycles of multiagent chemotherapy. All of the patients’ treatment responses with second-line treatment were evaluated as PD.Seven (28%) patients received third-line therapy due to PD. Additionally, three (12%) patients had received multiagent chemotherapy before first- and second-line treatments before admission to our center. In all patients, the treatment responses of the third-line treatments were evaluated as PD.Five (20%) patients with PD underwent allo-HSCT and CR was achieved in 3 (60%) of them after the procedure. Two patients’ disease recurred 2 and three months after allo-HSCT, and these two patients died 9 and 11 months following transplantation, respectively. One patient in follow-up with complete remission died 24 months after allo-HSCT due to sepsis. Autologous stem cell transplantation was performed in one patient in 2000, and the patient died four months after the procedure due to disease progression. The outcome of patients with HSCT is given in LCT of MF can occur at any stage of MF, and it has been associated with disease progression and poor outcome. Unfavorable prognostic factors for T-MF have been reported previously as advanced stage, presentation of MF with transformation, generalized skin tumors, increased LDH level, and use of combination chemotherapy ,15. CD30LCT at initial diagnosis of MF or within two years has been associated with worse prognosis in several studies ,16,17,18In previous studies, LCT of MF has been reported mainly in advanced disease. In a series with 22 T-MF patients, Arulogun et al.  reportedTransformed folliculotropic MF patients were previously found to have shorter survival time ,21,22. IAccording to a recent study, several clinical characteristics such as a new solitary nodule on MF plaques or rapidly presenting scattered papules may be indicators of the development of LCT for dermatologists . We woulThe treatment strategy is challenging in T-MF. It is important to note that, among our patients with advanced stages of T-MF, none had a CR to treatment under first-line therapies. Among patients receiving first-line therapy, 20% had either SD or PR with these therapies. Notably, all the patients who received second- and third-line therapies had PD. This finding highlights the refractory nature of T-MF. In fact, aggressive treatment strategies and multiple chemotherapies for MF result in a short period of CR, followed by an aggressive relapse . Allo-HSA limitation of the present study was the small number of patients with T-MF, highlighting the rarity of MF/SS. A second limitation was the retrospective design of the study, which may have restricted retrieval of the data from patient archives.Unfavorable prognostic factors in T-MF include advanced stage, high serum LDH levels, and loss of CD7 and CD26 expression in T helper cells. In patients with treatment-refractory tumors and unusual lesions, a biopsy is warranted to exclude T-MF. Patients with T-MF have a short life expectancy. Patients may have CR, PR, or SD with first-line treatments, which underlines the value of less aggressive therapies. However, nonresponders usually do not respond to second- or third-line therapies. Allo-HSCT may be an alternative option for patients with T-MF."}
{"text": "Interferon regulatory factors (IRFs) are a group of closely related proteins collectively referred to as the IRF family. Members of this family were originally recognized for their roles in inflammatory responses; however, recent research has suggested that they are also involved in tumor biology. This review focusses on current knowledge of the roles of IRF-1 and IRF-2 in human cancer, with particular attention paid to the impact of IRF-1 inactivation. The different mechanisms underlying IRF-1 inactivation and their implications for human cancers and the potential importance of IRF-1 in immunotherapy are also summarized. IRF-1, a member of the interferon regulatory factor (IRF) family. IRF-1 was first identified in 1988 as a transcription factor able to induce expression of the gene interferon β (IFN-B) [In human cancers, the accumulation of genetic aberrations is known to affect the normal functions of several genes that control cell proliferation and survival. Amongst these genes is  (IFN-B) . Current (IFN-B) . IRF-1 t (IFN-B) .All members of the IRF family exhibit significant homology in their N-terminal region, which contains a DNA-binding domain (DBD) that includes a cluster of five tryptophan residues. This DBD forms a helix-turn-helix motif and recognizes the interferon-stimulated response element (ISRE) in the promoters of genes targetted by IRFs ,6. The CIRFs were originally recognized for their roles in innate and adaptive immunity, especially in the regulation of interferon-inducible genes in the interferon system . HoweverIRF family members are playing a pivotal role in immune response. Amongst those, IRF-3 and IRF-7 whose antiviral role is well established. In addition, the hematopoietic factors, IRF-4 and 8. IRF-3 is expressed in all cell types and its expression is not triggered by viral infection or downstream to interferon .Expression of IRF-3 is up-regulated upon recognition of viral dsRNA by cellular receptor and, as a consequence of toll-like receptor-3 signaling, IRF-3 is phosphorylated and activated as a result of this post-translational modification . This leUbiquitination of IRF-3 targets its degradation by proteasome enzyme system, a process that can be triggered by propyl isomerase (Pin1) . In contIRF-4 is expressed in B lymphocytes and dendritic cells and is thought to be required for B- and T-lymphocytes’ maturation and differentiation . In thisIRF-5 is another immunomodulatory factor, which has been recognized for its role as a regulator for type I interferon gene expression in response to viral infections. It has a role in regulation and development of host immune response and autoimmune responses; hence it has been recognized as a susceptibility gene for autoimmune disorders . The funIRF-6 gene have been observed in patients with cleft lip and/or palate. In addition, aberrations in IRF-6 predisposes for squamous cell carcinoma and defective development of mammary gland [IRF-6 is a unique member of IRF family being the only family member to be essential for embryogenesis . IRF-6 iry gland . Adding ry gland .IRF-7 gene can be induced by type I interferon and tumor necrosis factor α (TNF-α). On the other hand, regulation of type I interferon gene expression by IRF-7 has been reported, hence the relation between IRF-7 and type I interferon could be described as mutual [Human s mutual ,28. Thiss mutual . Activats mutual .IRF-8, also known as ICSBP is expressed solely in lymphoid and myeloid progenitors . The funIRF-9, p48, or ISGF3-γ contributes to the antiviral response of interferon α, β, and γ. This role is achieved primarily by the binding of IRF-9 to interferon stimulated gene factor3, which interacts with ISRE and regulates ISGs ,36.This review discusses the functions of IRF-1 and IRF-2 in human cancers, with a focus on the potential contribution of IRF-1 inactivation to human carcinogenesis and the future of IRF-1 as a therapeutic target.ras oncogene was seen to result in transformation of IRF-1−/− but not wild-type mouse embryonic fibroblasts (MEFs) [N-ras oncogene in some myeloid cell lines has been shown to suppress proliferation and up-regulate the cyclin-dependent kinase (CDK) inhibitor p21WAF1/CIP1. This suppression was found to be associated with up-regulation of IRF-1, further reinforcing the notion that this IRF exerts an antioncogenic effect [ras oncogene overexpression [The role of the IRF family in oncogenesis was first noted in 1993, when overexpression of IRF-2 was found to transform NIH 3T3 cells and enhance their tumorigenicity in nude mice, a phenotype that was shown to be reversed by IRF-1 overexpression . An antis (MEFs) . Surprisc effect . Moreovec effect . In contpression .PKR) and signal transducer and activator of transcription (STAT) 1 (STAT1) in the Janus kinase (JAK)-STAT pathway [TRAIL), and lysyl oxidase (LOX), the latter of which has also been identified as a tumor suppressor [PUMA) promoter, resulting in up-regulation of the intrinsic apoptosis pathway [in vitro activation of this IRF decreases cyclin D1 expression and CDK 4 (CDK4) activity [The antiproliferative effect of IRF-1 has chiefly been attributed to its induction of the expression of certain target genes that down-regulate cell growth. These genes include protein kinase R  and mediated by binding of the promoter region of p21WAF1/CIP1, which contains binding sites for both IRF-1 and p53. It has also been reported that activation of IRF-1 results in the expression of genes directly involved in various other cellular processes, including regulation of the T cell-mediated immune response to viral infection. Deletion or mutation of IRF-1 and exon skipping (a form of RNA splicing to skip faulty exons) in the corresponding mRNA are also associated with the development of various hematopoietic malignancies and syndromes [The concept that ll cycle . Such chyndromes .IRF-2 gene has been shown to prevent N-ras-induced growth suppression, confirming its pro-oncogenic role [In contrast with IRF-1, IRF-2 exerts a pro-oncogenic effect. IRF-2 has been reported to be up-regulated in pancreatic cancer, in which it is associated with tumor size and differentiation, tumor node metastasis stage, and survival . Moreovenic role . One stunic role . In addinic role ,63. A funic role . Target In conclusion, IRF-1 operates as a tumor suppressor whose loss, in combination with other genetic alterations, may significantly increase risk of malignancy. In the following sections, we summarize its role in different human cancers and the range of mechanisms by which this tumor suppressor loses its function (de novo acute myelogenous leukemia (AML) cases, 50% of cases of secondary AML arising from MDS, and 2% of de novo acute lymphocytic leukemia cases [IRF-1 to band 31.1 of chromosome 5 using fluorescence in situ hybridization with a 19-kb IRF-1 probe. This probe only hybridized to sequences on chromosome 5q and was precisely mapped to 5q31.1 by computer-assisted fluorescence microscopy. Once the IRF-1 locus had been determined, a full-length IRF-1 cDNA probe was used to perform Southern blotting of DNA from patients with different types of leukemia and MDS associated with del(5q) to confirm IRF-1 inactivation in clinical samples. The results of the present study indicated an unusual instability in the 5q region, as deletion of one IRF-1 allele was accompanied by rearrangement or deletion of the second allele in some cases. This led to the conclusion that deletions or rearrangements are more frequent than point mutations at this locus in human leukemia and MDS, and loss of IRF-1 may be critical in the development of AML and MDS [Human leukemia and pre-leukemic myelodysplastic syndrome (MDS) are characterized by a remarkable cytogenetic abnormality, namely, the loss of chromosome 5 or a deletion within its long arm (del(5q) or 5q−) . This abia cases ,72. Willia cases  proposed and MDS .IRF-1 loss is key in 5q− syndrome development, finding that 85.7 % of the patients included in their study exhibited loss of one allele of the IRF-1 gene, with no evidence of homozygous loss [IL-3), IL-4, IL-5, and CSF2 [IRF-1 maps closely [Another group has also investigated the proposal that ous loss . This fiand CSF2 , which I closely .IRF-1 mRNA transcripts obtained from the bone marrow and peripheral mononuclear cells of patients with MDS or leukemia secondary to MDS lacks exon 2 (containing the initiation codon) and exon 3 as a result of accelerated exon skipping. The resulting IRF-1 protein lacks the ability to bind DNA and its tumor-suppressing activity is consequently lost. Thus, accelerated exon skipping comprises a second mechanism, in addition to IRF-1 loss as a result of DNA damage, by which IRF-1 is inactivated. This process may explain the development of hematopoietic malignancies in some 5q− syndrome cases in which both copies of the IRF-1 gene are retained. To validate this proposal, Green and colleagues [IRF-1 allele deletion. Their results showed that accelerated exon skipping is common in patients with a 5q deletion and one deleted IRF-1 allele, and occurs in most APL cases (in which IRF-1 protein expression was found to be absent). Such exon skipping thus leads to loss of IRF-1 function and increases risk of malignancy [In the same context, Harada and colleagues  reportedlleagues  developelignancy .IRF-1 mRNA and elevated presence of abnormal splice variants in chronic myeloid leukemia (CML) patients [To evaluate the extent to which exon skipping-induced IRF-1 inactivation is involved in oncogenesis, investigation of the association between exon-skipped IRF-1 transcripts and other forms of leukemia was also necessary. Mutational analysis and studies of IRF-1 expression patterns have revealed a four-fold reduction in levels of full-length patients . This lepatients . Loss ofpatients .IRF-1/IRF-2 gene expression ratio in AML and normal marrow, concluding that the balance between these factors, rather than the expression level of either in isolation, ultimately determines phenotype. Their study revealed that this ratio was significantly lower in AML patients as a result of low IRF-1 and high IRF-2 transcript levels, and indicated that IRF-1-responsive genes reduce AML risk. In contrast, malignant transformation related to stimulation of IRF-2 gene appears to be relatively common in leukemogenesis [Examining leukemogenesis from a different perspective, Preisler and colleagues  comparedogenesis .in situ (DCIS) and lymph node-positive invasive ductal cancer [Following the discovery of a role for IRF-1 in human leukemia and pre-leukemic myelodysplasia, researchers began to question whether it and other IRF family members might be similarly implicated in other cancers, particularly solid tumors. In one retrospective study, IRF-1 but not IRF-2 was found to be expressed in normal breast tissue, whereas levels of the former were shown to be lower and those of the latter higher in high-grade ductal carcinoma l cancer . This fiIn the development of previous work, Connett and colleagues  used immIRF-1 polymorphism (A4396G) has been identified in breast cancer cell lines, and has been found to be more frequent amongst African Americans [IRF-1 in patients with breast carcinoma and other tumors.At the genetic level, there have been no reports of point mutations that cause IRF-1 inactivation in breast cancer; however, an mericans . It is umericans . It has mericans ,87. The BRCA1 mutation-positive breast tumors. Given that somatic loss of IRF-1 may be a critical event in breast oncogenesis, in 2010, Cavalli and colleagues [IRF-1 locus was found in 32% of cases, providing evidence of a tumor-suppressive effect of IRF-1 in breast cancer [A lack of sufficient evidence concerning the exact contribution of IRF-1 to breast carcinogenesis, together with the well-documented role of this protein in hematopoietic malignancies, triggered a number of comparative genomic hybridization studies. As a result, loss of the 5q31.1 region, to which IRF-1 has been mapped, was found to be common in breast tumors . In addilleagues  investigt cancer .The establishment of a tumor-suppressing role for IRF-1 in breast cancer and leukemia laid the foundations for further investigations exploring its function in other cancers. One such study revealed that 50% of gastric tumors exhibit LOH at the 5q region implying a critical contribution of IRF-1 to the development of stomach carcinoma . In anotIRF-1 to confirm the role of this gene in esophageal carcinoma and stomach adenocarcinoma [IRF-1 gene in gastric adenocarcinoma tissues confirmed LOH at this locus, and led to the identification of a loss-of-function point mutation resulting in a methionine-to-leucine substitution at codon 8 [IRF-1 and consequently, its tumor-suppressing capability is lost. Although it is not clear how this mutation brings about this effect, it has been proposed that it may enhance the interaction of IRF-1 with other factors, impairing the function of this protein as a regulator of transcription [For the correct interpretation of these findings, it was important to test the so-called ‘two-hit hypothesis’ in relation to arcinoma . This hy codon 8 . This mucription . Wang ancription  have furIRF-1 mRNA lacking particular combinations of exons 7, 8, and 9. This alternative splicing results in the absence of the IRF-1 functional domain or the generation of a truncated protein with aberrant transcriptional activity that interferes with that of wild-type IRF-1. Thus, alternative splicing affecting exons 7, 8, and 9 may be another critical mechanism negatively regulating IRF-1 in cervical cancer.A separate group has reported a correlation between IRF-1 expression in human melanoma tissue specimens and less advanced disease, although they were not able to demonstrate a clear relationship between IRF-2 expression and this malignancy in this work . In an eIRF-1 expression is reduced in pancreatic cancer specimens compared with adjacent normal tissues, IRF-2 gene expression is up-regulated. In the same work, it was also found that up-regulation of IRF-1, but not IRF-2, leads to better tumor differentiation, enhanced lymphocyte infiltration, smaller tumor mass, and longer survival [in vitro model of pancreatic cancer has confirmed these clinical observations and thus, the tumor-suppressing and -promoting potentials of IRF-1 and IRF-2, respectively [Recently, it has also been reported that high IRF-1 expression in hepatocellular carcinoma (HCC) is associated with better outcome in terms of frequency of recurrence following surgical resection. In contrast, overexpression of IRF-2 is associated with increased probability of recurrence . In addisurvival . Subsequectively .IRF-2 may be classified as an oncogene, these results are consistent with IRF-1 having tumor-suppressing potential. Kuroboshi and colleagues  have expSeveral mechanisms responsible for attenuated IRF-1 transcriptional activity in human malignancies have been reported. The following section describes the different routes by which the function of IRF-1 and, thereby, its tumor-suppressing role may be lost in human cancers.IRF-1 gene have been reported in both hematologic malignancies and solid tumors. For instance, inactivating rearrangements or deletions in IRF-1 have been reported in AML [IRF-1 has been identified in stomach cancer. This alteration was accompanied by loss of IRF-1 transcriptional and, consequently, tumor-suppressing activity [IRF-1 have also been documented in breast cancer [IRF-1 allele deletion in AML and MDS patients.Alterations in the d in AML , and LOHd in AML . In addiactivity . We prevt cancer . Green at cancer  have alsIRF-1 mRNA is subject to several alterations that ultimately lead to loss of function. For example, Lee and colleagues [IRF-1 mRNA missing combinations of exons 7, 8, and 9. These variants are highly expressed in cervical cancer tissue and associated with attenuated IRF-1 transcriptional activity. miR-23a, which binds to the 3′-UTR of IRF-1 mRNA, is overexpressed in gastric adenocarcinoma, resulting in IRF-1 down-regulation and loss of transcriptional activity, in turn enhancing pro-proliferative and anti-apoptotic conditions [IRF-1 is associated with an absent DBD and loss of the tumor-suppressing action of the encoded protein [IRF-1 mRNA can result from accelerated exon skipping, with affected transcripts lacking a translation initiation site. This constitutes one of the mechanisms leading to IRF-1 inactivation in hematopoietic cancers [lleagues  have idenditions . Skipped protein . Moreove cancers .SUMOylation is a post-translational modification in which lysine residues are modified by attachment of a SUMO group . SUMOylaViruses have evolved various strategies to survive immune responses. Human papillomavirus (HPV), which is associated with increased risk of cervical cancer , counterNPM gene is associated with mislocalization of this protein to the cytoplasm, which has been implicated in loss of the function and antioncogenic effects of IRF-1. Cytosolic NPM has been observed in clinical AML specimens [Nucleophosmin (NPM) predominantly acts as a nuclear shuttling protein; however, translocation of the pecimens . Moreovepecimens . Anti-appecimens .IRF-1 is induced downstream to IFN-γ-type I interferon receptor signal transduction cascade (IRF-1 genes is the major histocompatibility class I (MHCI) gene. Induction of MHCI contributes to the long-known antitumor potential of IRF-1 enhancing tumor antigen presentation that improves the efficacy of the cell-mediated antitumor immune response. IFN-γ also triggers clonal expansion and activation of T cells and production of MHCII [Cancer models have provided evidence on the antitumor potential of interferon and importance of signaling through type I interferon receptor in mediation of anticancer immune response . Importa cascade . In turnof MHCII . Therefoof MHCII .That IRF-1 is a potential target for new therapies has been highlighted by correlations between its inactivation and several types of human cancers. However, the exact IRF-1-related mechanisms that might be targetted to benefit therapy of human malignancies are not yet fully clear. It has been reported that an abnormally low IRF-1/IRF-2 ratio due to defective transcription is a basic characteristic of leukemogenesis, but that administration of certain cytokines (e.g. IL-4) can return this ratio to normal levels . SimilarA recent study  has implUp-regulation of IRF-1 in cancerous lesions is regarded as an approach that could improve prognosis and alleviate resistance to immunotherapy ,122. In"}
{"text": "Approximately 11,000 people die in Scotland each year as a result of smoking-related causes. Quitting smoking is relatively easy; maintaining a quit attempt is a very difficult task with success rates for unaided quit attempts stubbornly remaining in the single digits. Pharmaceutical treatment can improve these rates by lowering the overall reward factor of nicotine. However, these and related nicotine replacement therapies do not operate on, or address, the spatial and contextual aspects of smoking behaviour. With the ubiquity of smartphones that can log spatial, quantitative and qualitative data related to smoking behaviour, there exists a person-centred clinical opportunity to support smokers attempting to quit by first understanding their smoking behaviour and subsequently sending them dynamic messages to encourage health behaviour change within a situational context.We have built a smartphone app—MapMySmoke—that works on Android and iOS platforms. The deployment of this app within a clinical National Health Service (NHS) setting has two distinct phases: (1) a 2-week logging phase where pre-quit patients log all of their smoking and craving events; and (2) a post-quit phase where users receive dynamic support messages and can continue to log craving events, and should they occur, relapse events. Following the initial logging phase, patients consult with their general practitioner (GP) or healthcare provider to review their smoking patterns and to outline a precise, individualised quit attempt plan. Our feasibility study consists of assessment of an initial app version during and after use by eight patients recruited from an NHS Fife GP practice. In addition to evaluation of the app as a potential smoking cessation aid, we have assessed the user experience, technological requirements and security of the data flow.In an initial feasibility study, we have deployed the app for a small number of patients within one GP practice in NHS Fife. We recruited eight patients within one surgery, four of whom actively logged information about their smoking behaviour. Initial feedback was very positive, and users indicated a willingness to log their craving and smoking events. In addition, two out of three patients who completed follow-up interviews noted that the app helped them reduce the number of cigarettes they smoked per day, while the third indicated that it had helped them quit. The study highlighted the use of pushed notifications as a potential technology for maintaining quit attempts, and the security of collection of data was audited. These initial results influenced the design of a planned second larger study, comprised of 100 patients, the primary objectives of which are to use statistical modelling to identify times and places of probable switches into smoking states, and to target these times with dynamic health behaviour messaging.While the health benefits of quitting smoking are unequivocal, such behaviour change is very difficult to achieve. Many factors are likely to contribute to maintaining smoking behaviour, yet the precise role of cues derived from the spatial environment remains unclear. The rise of smartphones, therefore, allows clinicians the opportunity to better understand the spatial aspects of smoking behaviour and affords them the opportunity to push targeted individualised health support messages at vulnerable times and places.NCT02932917.ClinicalTrial.gov,  Despite many therapies that have been introduced over the years, maintaining a quit attempt remains a monumentally difficult task, and most people require several quit attempts before they successfully maintain cessation at 1 year. Unaided quit attempts are successful only about 3–6% of the time [Approximately 11,000 people die each year in Scotland as a result of smoking-related causes . In this paper, we report on the successful completion of the first of these and the use of the initial results to aid the design of the second feasibility study.The design and setting for the initial feasibility study was a maximum of 10 patients within one general practice surgery in Fife, UK; patients were recruited and monitored by JM. We chose this small number of patients from this practice so that we could quickly get the app into the hands of actual smokers, and therefore more quickly get feedback on the app. To recruit these first 10 patients, we created and displayed a poster in the practice. Also, JM reached out to individual patients to notify them of the study and ask them if they would be interested in participating. Patients who expressed interest in participating were subsequently consented with a different PI from JM.The second study will involve up to 100 patients recruited primarily through the NHS Fife Smoking Cessation Services. Target patients for both studies are active smokers interested in quitting, and in possession of a smartphone with a data plan. By active, we mean patients who regularly have one or more cigarettes per week, and specifically that the patients inhale smoke from burning tobacco encased in cigarettes, pipes or cigars. We exclude patients with serious mental illness, and those without a smartphone. The flow of patients through the study is shown in Fig. Live or work in Fife, Scotland, UKAge 18 or greaterActive smoker interested in quittingPossess an Android or iOS (at least iOS 7.0) smartphone with a data planGive informed consentSerious mental illnessLearning difficultyThe primary objective of the first small feasibility study was to see if it is possible to deploy the MapMySmoke app within a National Health Service (NHS) setting; that is, we sought to take the app out of the development lab and into the hands of actual patients. In so doing, we wanted to test the underlying technologies and development infrastructure, and to ensure that data is collected, transmitted, stored and retrieved at suitable levels of security of patient identifiable information. Second, we sought feedback on whether patients find it an acceptable aid. Third, we sought feedback on ways to improve the app ahead of a second, larger, deployment.Each craving or smoking event logged by the user is sent to a secure server stored within the University of St Andrews School of Medicine. The data are transferred from the phone to the server using Transport Layer Security protocol and industry standard for e-commerce. The data that are collected by the MapMySmoke app at each craving or smoking event are stored on the server after two layers of encryption. We use a double key (private and public) system to store the data in a database, with the outer layer protected by a 2048-bit private key held by the project PI (GH). The data transfer is one-way, i.e. from the phone to the server. Once the encrypted data are on the server, there are two further restrictions regarding use by co-principal investigators: (1) only anonymised data are available; and (2) access to these anonymised data is limited to secure access computer terminals within the School of Medicine at the University of St Andrews.We consented eight patients in a surgery in Fife and installed the first version of the MapMySmoke app on their phones Fig. . Of thesIn terms of the original objectives of the study—determine if we could deploy the app with actual patients in a clinical setting, enable secure logging and transmission of private health information and obtain feedback from patients—we were successful on all counts. While we only had a small number of patients log for an extended period of time, their data were all captured and stored securely; they reported positive feedback after using the app Table  and suggThe overarching goals of the MapMySmoke app study, i.e. both the small feasibility study reported here, as well as the larger pilot study, are myriad; the first two are patient focused, while the second two are research focused. First, we wish to facilitate a better understanding of an individual patient’s smoking profile. Similar to a food diary, the goal is to enable users to precisely document where and when they had cigarettes and craving events, and to easily tag each event with quantitative and qualitative metadata. Through the accumulation of data over an initial 2-week logging phase, the app will generate a detailed snapshot of the individual patient’s smoking behaviour.Highlight smoking patterns in time and across spaceIdentify critical cigarettesIdentify critical times for cravingPair smoking behaviour with tailored, evidence-based support messagesThe second goal is to share these data with the GP or healthcare provider, e.g. medical social worker, to generate an individualised plan to support each patient after they start their quit attempt. The ways in which the app will attempt to do that are as follows:The third goal—a research goal—is to see if we can identify optimal times and places to send the user dynamic support messages. Ideally, these messages would be sent to an individual’s phone ahead of a likely craving event. As a patient’s craving increases, they are more likely to want to smoke. This shift in desire could be classified as an underlying change in motivational state. That is, some combination of physiological status and psychological triggers could lead to the individual wanting to smoke . One way to quantify the changing probability of shifting into such a smoking state is to fit hidden Markov models to the data logged during the first 2 weeks. If we can quantitatively identify times and places of these shifts, then we can better time the delivery of in-app support messages to the patient. That way, the user can receive a support message when it is most needed by him/her, as opposed to a generic message delivered at the same time each day for every patient. For example, based on their recorded smoking pattern, patient A might be vulnerable to relapse at 3 pm while they are at work, while patient B might be vulnerable to relapse at 1 pm following lunch, etc. If each patient can receive a tailored message in advance of their unique craving event, then we would expect a priori that the intervention would be more relevant. Research has shown that prospective patients are more interested in receiving tailored messages .The fourth goal is to capture the spatial aspects of relapse. This is accomplished simply by tying the logging of a smoking event to the individual’s quit date. The quit date is set at the patient-GP meeting and stored within the app. Should a user log a smoking event after that date, then that first event is stored as a relapse event. We aim to understand the links between the spatial environment and relapse behaviour through investigation of the data associated with post-quit smoking events. In addition to capturing spatial information associated with the relapse, the app will send different messages to the user after a relapse event. Essentially, the structure of the app remains unchanged during this phase, but the context of a smoking event entered post-quit will automatically be tagged as a relapse event. Because this is a different type of event, the next version of the MapMySmoke app will provide the patient with dynamic messages designed to support the user and encourage them to return to the quit attempt. In summary, this goal is both not only research related—in the sense that we will record the spatial aspects of relapse—but also patient focused through the relapse-related messaging.The results from the feasibility study clearly show that this app can be successfully deployed. Though we caution that it was a very small study limited to one GP practice, we found that patients were comfortable using the app, were able to better understand their smoking behaviour via logging and reported liking the visual feedback from the app. This means that we can use these results and patient-derived feedback Table  to improWe will use the corpus of smoking data in at least four unique ways. First, we will use data obtained during the logging phase in an effort to quantify the probability of a user switching into a smoking state. This is of interest from a clinical standpoint, as it helps uncover the myriad determinants of smoking behaviour. Our intention is to use these state-switching probabilities in order to better target support messages to individual patients. To do this, we will use latent-state models in an effort to understand the temporal distribution of smoking behaviour . In lateAs the project has progressed, we have considered several ways to improve the app. In the apps’ current form, the data encryption and storage scheme does not allow communication from the server back to the phone or to the healthcare provider. Therefore, summary statistics and maps of spatial smoking and craving behaviour are limited to in-app display Fig. . HoweverIn addition, the app could be structured to deliver support to the users at vulnerable times and places. These times and places are unique to each individual, and based on their profiles generated during the logging phase. Prior to implementing this feature, the sampling and communication protocol within the app would be subject to ethical approval within the NHS. While more app-patient interaction and communication is likely to be helpful , this muAnother feature we would like to address in the future is the inclusion of social support with peers. Cheung et al.  showed tIn early pre-release versions of the app, we implemented a random forest algorithm  to delivFinally, in the first feasibility study, we encountered several candidate patients with older phones that did not support the current version of the MapMySmoke app. This is because we are developing and deploying at the leading edge of mobile technology. However, the remit of the NHS Tobacco Support Services is aimed at populations with higher indices of deprivation. We propose to address this issue of device incompatibility in future versions by developing more backwards compatible versions. While this comes with some added development cost, it opens up the study to more potential users and builds-in equity of health promotion.Since more and more humans have and use smartphones, there exists a clear opportunity to harness these tools in the delivery of targeted, specific medical information and support. In particular, we feel there is an opportunity to (a) learn about individual smoking patterns across space and through time and (b) use that information to provide better, more tailored support messages that aid and support a quit attempt. To address this, we have built and successfully deployed a mobile app designed to log individual smoker’s behaviour, provide graphical and statistical summaries of smoking behaviour and support a quit attempt with the delivery of in-app messages designed to provide specific and time-relevant information to the smoker. Initial feedback from users in the first feasibility study was very positive. Thus, the MapMySmoke app could provide practitioners with a new effective tool that supports patients through the difficult process of quitting smoking and maintaining abstinence from smoking."}
{"text": "Generation of attosecond bunches of energetic electrons offers significant potential from ultrafast physics to novel radiation sources. However, it is still a great challenge to stably produce such electron beams with lasers, since the typical subfemtosecond electron bunches from laser-plasma interactions either carry low beam charge, or propagate for only several tens of femtoseconds. Here we propose an all-optical scheme for generating dense attosecond electron bunches via the interaction of an intense Laguerre-Gaussian (LG) laser pulse with a nanofiber. The dense bunch train results from the unique field structure of a circularly polarized LG laser pulse, enabling each bunch to be phase-locked and accelerated forward with low divergence, high beam charge and large beam-angular-momentum. This paves the way for wide applications in various fields, e.g., ultrabrilliant attosecond x/γ-ray emission. The short energetic electron beam has significant potential for applications such as electron diffraction and microscopy4, electron 4D imaging5, and electron injection into a free electron laser (FEL). Especially, this leads to the production of ultrashort x-ray radiation sources with duration down to the attosecond level6, where femtosecond and even attosecond relativistic electron bunches with a narrow energy spread, small divergence angle and large flux are of crucial importance7. For this purpose, significant efforts have been dedicated to acquiring high-quality ultrashort electron bunches using the normal Gaussian laser pulses, for example, by laser irradiating high density plasma boundaries of a channel, hollow cone, wire or droplet target15, underdense plasmas17 or nanofilms19, by the inverse FEL process20, direct laser acceleration in vacuum21, or by a relativistic electron beam scattering off interfering laser waves22. In spite of these different concepts and schemes, it is still very challenging to produce high-quality attosecond electron bunches for potential applications. Even with pC charges, the space-charge repulsion of electrons within the bunch severely limits the bunch lifetime to several tens of femtoseconds with either a low beam charge or large divergence angle. A new solution to suppress the space charge effect in the relativistic regime is required for dense electron bunch generation and acceleration.The generation of ultrashort intense laser pulses pushes the laser-matter interaction to the relativistic regime where the relativistic electron dynamics in the laser fields become dominant. Optical processes driven by relativistic particle motion in plasmas, so-called “relativistic plasma optics”, enable investigation of ultrafast physics phenomena and the development of compact radiation sources23 and charged particles26, including the generation and acceleration of ultrashort electron bunches. For example, a Laguerre-Gaussian (LG) laser pulse is characterized by a hollow intensity distribution, a spiral equiphase surface, and orbital-angular-momentum (OAM)27. The OAM transfer from the laser pulse to relativistic electrons is a topic of fundamental importance on relativistic optics28. It is shown theoretically and experimentally that a relativistic intense LG laser pulse can be generated using a spiral-shaped foil30, via stimulated Raman scattering31 or plasma holograms32. This may offer a new approach to generate and manipulate electron bunches in micron scale by relativistic LG-mode laser-plasma interaction.A proper laser field structure in time and space potentially provides effective manipulation of atomsIn this paper, we report an innovative scheme to generate a dense attosecond electron bunch train efficiently from a laser-driven nanofiber target. In the scheme, a circularly polarized LG laser pulse with OAM but without total AM is employed. The attosecond electron bunches are first dragged out by the radial laser electric fields and are quickly phase-locked and accelerated forward by the longitudinal electric fields. The dense attosecond bunch train results from the unique field structure of the circularly polarized LG laser pulse, enabling each bunch to converge into a dense disk and propagate with a narrow energy spread, high beam charge and large beam-angular-momentum (BAM). Full three-dimensional (3D) particle-in-cell (PIC) simulations indicate that the stable attosecond structure remains intact for several hundreds of femtoseconds, paving the way for many potential applications in various fields.ne = 20nc is used. Here, nc = 1.12 × 1021 cm−3 is the critical density for a λ0 = 1 µm laser. The target could be solid-hydrogen, hydrocarbon or, carbon and silicon materials in experiments33. The fiber radius and length are R0 = 0.5 µm and L0 = 15 µm, respectively. A left-hand circularly-polarized LG laser pulse with mode (x-axis. Here the integer p + 1) represents the number of radial nodes. The laser electric field is given byEL0 = me0cω0a0/qe is the peak amplitude of the laser electric field with me0 the electron rest mass, c the speed of light in vacuum, ω0 the laser angular frequency, qe the charge unit, and a0 the laser dimensionless parameter, r = (y2 + z2)1/2, σ = σ0(1 + x2/f2)1/2 the focal spot radius with σ0 = 3λ0 the beam waist radius, and −cτ ≤ x − ct ≤ cτ with the duration τ = 5T0 and T0 = λ0/c, and k the wave number, R = x + f2/x the curvature radius of the wave-front, φ the azimuthal angle. We use the  mode of the LG laser, which implies an azimuthal phase term eiφ and a spiral wave-front of the laser pulse. Figure a0 = 30. At t = 10T0, a series of short electron bunches with a 1 λ0 spacing are generated along the nanofiber. Each bunch is isolated and doughnut-like, which is markedly different from the sawtooth-like or helical structure in a plasma channel, a hollow cone, a wire, or in the case of micro-droplets using normal Gaussian laser pulses15. These electron bunches move almost at the speed of light with a longitudinal thickness corresponding to a fraction of the laser wavelength (t = 10T0). The inner ring diameter is only 1.0λ0, with the maximal density of 30nc. When the annular electron bunches leave the nanofiber, they are further accelerated and each bunch converges into a dense disk with beam charge up to 0.3 nC, instead of being dispersed in space as in the case with normal Gaussian laser pulses15. As time progresses, the thickness of the disk decreases to, ~200 as, while its peak density is still up to 2nc at t = 50T0. The attosecond structure is very stable and remains intact for a duration >300 fs ] + ω0t − 2 arctan (x/f) is the transformed phase term. One can see that both the electric field Er and Eφ are circularly-symmetric in the transverse cross-section and have nothing to do with the azimuthal angle φ. It is distinct from the normal radially polarized laser pulses36, where only the radial component exists for the electric field. This unique optical field structure would benefit the dense electron bunch acceleration and propagation greatly. When Er is positive, the electrons in the skin layer of the fiber are compressed, forming a rippled structure on the fiber surface; as it becomes negative, the electrons are subject to a radial outward force and are dragged out of the nanofiber, so that an attosecond bunch forms. Since Er is also periodic along the x direction with a period of 1T0, the spacing of the attosecond electron bunches is exactly 1λ0, as seen in Fig. The formation of annular attosecond electron bunches in our scheme results from the unique field structure of the circularly polarized LG laser pulse. Here we considered a left-hand circularly-polarized LG-mode laser pulse with an OAM per photon but without total AM, which is characterized by simultaneously cylindrically-symmetric radially-polarized and azimuthally-polarized components. For convenience, we first convert the laser transverse electric fields into radial and azimuthal equivalents in the cylindrical coordinate system, owing to Ex = −(i/k)(∂Ey/∂y + ∂Ez/∂z):Once the attosecond electron bunches are dragged out, they experience acceleration by the longitudinal electric fields, which exist for a tightly focused circularly-polarized LG laser pulse. Here one can obtain longitudinal electric field from the Poisson equation, i.e., x = 0, r = σ0; For r < σ0, Ex < 0, which is able to accelerate electrons. For each single electron dragged out from the fiber, the electron relativistic equations of motion in the LG laser fields together with the energy equation can be written as γme0c(dβ/dt) = qe[β(β ⋅ E) − (E + cβ × B)]. Here β is the normalized electron velocity by the speed of light, γ is the electron relativistic factor, E and B are the laser electromagnetic fields. In cylindrical coordinates When γ(t) = γ(0) + ηx + η⊥. As expected, the contribution of the transverse electric fields is smaller and most electrons are located within the region cosψ′ < 0 (note kr2/R → 0) and sinψ′ > 0. As seen in Fig. Ex. These bunches are relativistic and co-move with the laser pulse, so that they are quickly phase-locked longitudinally37. Figure Er experienced by an attosecond bunch as marked in Fig. Er, making the doughnut bunches close up after leaving the nanofiber. Figure r < σ0, |Ex| is much stronger than for Er, these electrons are subject to a strong focusing force and are confined transversely. By contrast, as r > σ0, |Ex| gets much smaller so that the electron acceleration is weak and the phase-slip occurs, resulting in an increasing energy spread, even though the electrons are initially in the acceleration phase of the laser pulse. Thus, the appropriate phase for dense attosecond electron bunch formation satisfies ψ′ ∈  in one laser cycle.Dense attosecond electron bunch formation requires simultaneous longitudinal acceleration and transverse focusing, i.e., vx < c), the electron velocity should be high enough. Here the acceleration length approximates to the Rayleigh length, La(c − vx)/c ≤ λ0/4 should be satisfied to keep electrons in phase, which requiresWhen the energetic electrons are well confined near the laser axis, namely γth ≥ 7.5 in order to keep the electrons phase-locked in the LG laser fields. For relativistic LG laser pulses, this is naturally satisfied, because such a laser pulse is capable of accelerating electrons to relativistic energies in less than one laser cycle. Once locked by the LG laser pulse, an electron acquires a velocity of vt = qEx/me × T0 in the first laser cycle, which should be such that a0 = 6. Note that the LG laser pulses with such intensity are already available in several laboratories around the world38.This implies an energy threshold of entclass1pt{minimat = 35T0, which illustrates clearly that most attosecond electrons originate from the left tip of the nanofiber. Since the laser electric field components Er, Eφ, and Ex are circularly-symmetric in the yz plane, these electrons with the same r experience similar acceleration. All these factors contribute to the quasi-monoenergetic electron acceleration as indicated in Fig. t = 60T0. The energy peak gradually increases in width, while the density is still above the critical density at t = 120T0 (400 fs). The final electron energy can be estimated as t = 60T0. The energetic bunches obtained are distinctive with a small divergence angle of <2°, a peak energy of ~400 MeV and nC charges, which have diverse applications in various fields. The corresponding transverse beam root-mean-square emittance εy and εz are 1.4 × 10−2 mm ⋅ mrad and 1.5 × 10−2 mm ⋅ mrad, respectively  makes a charged particle spin with respect to its own axis, while its OAM is able to drive the particle rotate with respect to the laser axis. Under appropriate conditions, both can be transferred to particles, which is a fundamental question from a relativistic plasma optics perspectiveε0 is the permittivity. Considering the relativistic cancellation of the magnetic and electric forces, the net transverse repulsion force approximates to Ne,max < (σ0/re)[p⊥/(me0c)]2, according to the impulse theorem 21. Here, re = (4πε0)−1e2/(mec2) is the classic electron radius. As electrons are dragged out, 10 in our simulations, about ~61.3% of which originate from the left tip. This implies that we may replace the nanofibre with micro-droplets to facilitate experiments. Note that it is easier to produce micro-droplets in large numbers and consistent with high repetition rate lasers41. One can thus test the set-up in experiments with the current and forthcoming LG-mode lasers in the near future.The space charge effect plays an important role in determining the final electron number of each bunch. Here the transverse Coulomb field can be estimated as t = 10T0. Since abundant electrons are dragged out of the nanofiber, strong charge separation fields also form in the nanofiber, resulting in a strong return current to satisfy the plasma neutralization condition42. The return current tends to pump electrons to the left tip, some of which can be distinguished from the trajectories above. During the process, quasi-static electromagnetic fields around the nanofiber are also induced, as seen in Fig. Figure Ex decreases and the phase-slip occurs more quickly, so that the electron energy decreases. Correspondingly, the radial electric field Er increases and more surface electrons are dragged out, and finally the total beam charge increases. In experiments, the pre-pulse may expand the front end of the nanofiber so that the real diameter increases. However, once the diameter becomes larger than a critical value (rth = σ0), the longitudinal electric field Ex in Eq. . The stable process is also valid for a much lower laser intensity and an example simulation with a0 = 10 is given in Supplement. On the other hand, using a larger laser focal spot, the acceleration length of the beam increases and much higher electron energies are achieved. Other experimental factors on attosecond bunch formation are also discussed  is employed, which allows direct fully electromagnetic simulations of relativistic laser plasma interactions. The particle number per cell (PPC) is 27 for both electrons and ions. Absorbing boundary conditions for the fields and particles are employed in the x direction (the laser propagation direction), while periodic ones for the fields and absorbing ones for the particles are used in the y and z directions. The initial electron and ion temperatures are assumed to be 1 keV and 0.01 keV, respectively.We carry out a series of 3D simulations to investigate the physics of the LG-mode laser interaction with a nanofiber target. The PIC code Virtual Laser Plasma Lab (VLPLData associated with research published in this paper can be accessed at http://dx.doi.org/10.15129/f9d7cc35-8f3c-461c-9bbe-ed6a9c8a667b.Supplementary information"}
{"text": "Laser-plasma wakefield accelerators have seen tremendous progress, now capable of producing quasi-monoenergetic electron beams in the GeV energy range with few-femtoseconds bunch duration. Scaling these accelerators to the nanocoulomb range would yield hundreds of kiloamperes peak current and stimulate the next generation of radiation sources covering high-field THz, high-brightness X-ray and γ-ray sources, compact free-electron lasers and laboratory-size beam-driven plasma accelerators. However, accelerators generating such currents operate in the beam loading regime where the accelerating field is strongly modified by the self-fields of the injected bunch, potentially deteriorating key beam parameters. Here we demonstrate that, if appropriately controlled, the beam loading effect can be employed to improve the accelerator’s performance. Self-truncated ionization injection enables loading of unprecedented charges of ∼0.5 nC within a mono-energetic peak. As the energy balance is reached, we show that the accelerator operates at the theoretically predicted optimal loading condition and the final energy spread is minimized. Higher beam quality and stability are desired in laser-plasma accelerators for their applications in compact light sources. Here the authors demonstrate in laser plasma wakefield electron acceleration that the beam loading effect can be employed to improve beam quality by controlling the beam charge. Large-scale facilities such as synchrotrons and free-electron lasers (FELs) provide extremely bright and short wavelength radiation, enabling microscopy at atomic resolution with femtosecond to attosecond time scales. However, conventional radio-frequency electron accelerators currently used to drive such sources are limited to only a few-kiloampere peak current. In contrast, the ultra-high accelerating field gradient in laser-plasma wakefield accelerators can sustain higher bunch charges within a few-femtosecond pulses enabling peak currents up to two orders of magnitude larger than found in conventional accelerators. Knowledge to operate laser-plasma accelerators in the high-current regime would make them ideal drivers for next-generation compact light sources covering high-field THz14. For a sufficiently high driver laser intensity, plasma electrons are expelled from the laser vicinity, thereby creating a co-propagating near-spherical ion cavity15. In this so-called bubble or blow-out regime the accelerating wakefield can exceed several hundreds of GV m−1. In contrast to conventional accelerators, electrons from the plasma background can be self-injected into the wakefield, and therefore no external electron source is required. Electrons that are injected into the right phase of this wakefield will be accelerated to high energies within a quasi-monoenergetic peak. Even though electron beams reaching into the multi-GeV energy range and few-femtosecond bunch durations have already been demonstrated19, bunch charge has been limited to only a few tens of picocoulomb. Scaling the charge to the nanocoulomb range following original predictions15 would yield hundreds of kiloamperes peak-current beams, the key component for next-generation compact light sources. During the submission of this manuscript, Li et al.20 demonstrated generation of electron beams up to 20 kA from laser wakefield acceleration yet of a continuous spectrum extending up to 0.6 GeV. Laser-plasma accelerators generating such high currents accumulate enough charge such that the self-fields of the bunch will superimpose on the wakefield23. As a consequence the plasma cavity structure will be reshaped and the effective accelerating field along the bunch length will be modified affecting the final beam parameters, i.e., maximum energy and energy spread. This phenomenon is generally known as beam loading.In laser-plasma wakefield acceleration, an ultrashort laser pulse propagating through an optically transparent plasma excites a plasma wake by the laser ponderomotive force22 estimated the number of particles that can be loaded into a three-dimensional (3D) nonlinear wake to scale with the normalized volume of the plasma bubble or the square root of the laser power. The same scaling was also found by Gordienko and Pukhov21 with a different coefficient derived from simulations. The linear theory for beam loading in plasma accelerators was developed by Katsouleas et al.24 In this one-dimensional approach it was found that an appropriate bunch shaping can minimize the energy spread typically associated with plasma accelerators. In recent work by Tzoufras et al.25 this theory was expanded for the 3D nonlinear case and an optimum trapezoidal bunch shape was investigated in order to efficiently convert the energy available in the wake into kinetic energy of electrons. In this ideal case, the accelerating field of the wakefield Ez becomes constant (Es) along the bunch and scales as:c is the speed of light, Qs is the total loaded charge, me and e are the mass and charge of an electron and np and kp. For a matched laser condition22, kpRb amounts to approximately Rb and the laser normalized vector potential a0. Eq. 29 process is used (see Methods). This scheme relies on ionization injection, where the helium gas based acceleration medium is doped with a small fraction of nitrogen gas whose K-shell electrons will be ionized and subsequently trapped only near the intensity peak of the laser pulse31. In the STII scheme laser and wakefield evolution restrict the time of electron injection into the wakefield, thus limiting the energy spread. The number of injected electrons can be tuned by varying the nitrogen doping concentration without changing the driver laser and plasma density parameters. Thus, in contrary to the work in ref. 26, the initial position and radius for trapping can be kept equal for various loaded charges. Loading-dependent injection length contributes only weakly to the energy spread  duration. The mean peak energy of the electron beam is 250 MeV with 9% shot-to-shot s.d. The mean absolute energy spread (FWHM) is 36 MeV yielding a relative energy spread of 14%. The accelerator delivers an unprecedented charge within the peak (FWHM) of ∼220 pC ± 40 pC shot-to-shot s.d. with a divergence of 7 mrad. The excellent shot-to-shot reproducibility gives the ability to perform statistical analysis over multiple shots for each data set belonging to a specific experimental parameter.The typical performance of the accelerator is presented in Fig. Emax) is observed. Since the injection dynamics do not change significantly, this energy reduction indicates accelerating field suppression due to the load.The influence of both injected charge and plasma density is clearly seen in the maximum attainable energy as shown in Fig. vertical line in Figs. Beam loading perturbs the wakefield such that the accelerating field strength experienced by the trailing electrons is reduced. In our measurements we have observed a smaller decrease on the mean energy of electrons compared to the maximum attained energy discussed above . Multiple simulations were performed taking realistic experimental parameters in order to study the beam loading effect under realistic laser-plasma dynamics, increasing only the nitrogen doping concentration in order to inject more charge into the wakefield. Although the injected charge in simulations does not reach the optimal loading condition, qualitatively the beam loading effects can be observed until this point. Figure Ez due to beam loading was observed as illustrated in Fig. Ez by ∼50 GV m−1 occurs together with an easing of the accelerating field slope along the bunch compared to the weakly loaded case where a 60 pC bunch is injected. Trailing electrons being affected strongest, this field change subsequently results in a compression of the electron momentum phase-space distribution, resulting in both a reduction in Emax and a reduction of energy spread. This effect is further illustrated in Fig. To support our results, we performed 3D Particle-In-Cell (PIC) simulations using the PIConGPU code34 we could roughly determine a FWHM bunch duration of 5 fs. This is in the range of the typical measured laser wakefield bunch duration which is 4 to 6 fs19. Thus, taking a conservative bunch length value, we estimate that our accelerator delivers a FWHM peak current of ∼50 kA while operating at optimal loading condition with ∼15% relative energy spread.The results presented here will have a strong impact on the parameter design of future laser-plasma accelerators. Minimization of the energy spread by means of the presented beam loading method is a crucial step toward high peak-current beams. At our laser parameters, the optimal loading condition was reached at a charge of ∼300 pC within the FWHM. Although we have no direct measurement of the bunch length, using an indirect duration estimate employing a two-particle model as presented by Rechatin et al.15 with boosting the energy to the tens of GeV range, supported by the tremendous advancements of laser technology pushing peak power above the petawatt level.Working toward driving secondary light sources, future laser-plasma wakefield accelerators will combine even higher peak-current beams by operation in the nanocoulomb range as already predicted in 2002 by Pukhov and Meyer-ter Vehn22 and Gordienko and Pukhov21. The intersection at zero charge for a laser power of 33 TW corresponds to the injection threshold for our experimental parameters. Expanding this scaling and operating in the optimum loading condition we expect that driving a laser-plasma wakefield accelerator with a petawatt laser beam will result in high-quality electron beams with peak currents of over 150 kA. This is two to three orders of magnitude larger than can be achieved in state-of-the art large-scale accelerator facilities.In Fig. Many challenges in the field of laser-plasma acceleration still remain, besides above-mentioned scalings also transverse beam emittance. To correctly measure and further optimize beam emittance for laser wakefield acceleration in the beam loading regime is a nontrivial task and also named as one of big challenges in the field which will be the subject of further studies in this regime. Laser-plasma accelerator-driven FELs are one example of next-generation radiation sources which require low-emittance electron beams. Other next-generation radiation sources, e.g., laboratory-size beam-driven plasma accelerators, THz-radiation sources, betatron sources and inverse Compton X-ray sources, put less stringent demands on beam emittance.23. We have shown that in order to generate high charge beams with a small energy spread, laser-plasma accelerators have to be operated at the optimal loading condition.The work presented here is a significant step toward the application of laser-plasma accelerators as drivers for secondary radiation sources as well as a substantial contribution on advanced accelerator concepts research. It represents an extensive systematic experimental study of beam loading by a quasi-monoenergetic bunch in the bubble regime performed with a nanocoulomb-class laser wakefield accelerator, thereby confirming the scaling theory developed by Tzoufras et al.a0=2.6. Approximately 76% of the laser energy is within the beam waist (1/e2 of intensity), corresponding to an estimated Strehl ratio of 0.9. This energy fraction drops to 62% at 1.5 mm away from the focus position while maintaining a Gaussian laser beam profile. Adjusting the tip tilt and gratings groove orientation of the compressor, the angular chirp of the beam was minimized to less than 0.1 μrad per nm characterized with a spectrally resolved inverted field interferometer. The spectral phase was measured with a spectral-phase interferometry for direct electric-feld reconstruction (SPIDER-A.P.E.) in parallel with a self-referenced spectral interferometry (WIZZLER-Fastlite) and optimized in a closed loop using an acousto-optic programmable dispersive filter (DAZZLER-Fastlite). Before data acquisition, the accelerator performance was further optimized by the phase correction on the second order (group velocity dispersion) and third order (third-order dispersion) at the DAZZLER. Online diagnostics situated at the experimental area were used to monitor the laser’s near field and far field and its temporal stability at each shot.The experiment was performed with the DRACO Ti:Sa chirped pulse amplification laser system operated by Helmholtz-Zentrum Dresden-Rossendorf delivering 30 fs (FWHM) pulses with an energy of 2.5 J on target. The setup allows laser diagnostics on target while the laser system is running at full power. Extra effort has been made to improve the mid field and far field of the laser beam profile as well as the correction for the angular chirp and the spectral phase. An off-axis parabolic mirror (f/20) is used to focus the laser beam onto the gas target, ∼1.5 mm above the nozzle. A wavefront sensor (PHASICS SID4) in closed loop with a deformable mirror provides wavefront optimization which results to a vacuum focal spot size of 20 μm FWHM, yielding a normalized vector potential of 2 was provided by a 3 mm supersonic de-Laval nozzle (Mach 10.4)35 mounted on a fast gas valve (Parker 9-series). The gas-jet profile was characterized with an interferometric method36. It measures a density profile with a flat top region of 1.6 mm with density ramps of ∼0.6 mm on both sides of the jet along the laser propagation axis. By adjusting the gas pressure at which the nozzle is operated, the gas density of the flat top profile can be varied from 1018 cm−3 up to 5 × 1018 cm−3. Different nitrogen doping concentrations from 0.2% to 1.5% were achieved by using premixed bottles with a doping concentration with less than 2% relative error.As plasma medium, a gas jet consisting of mixed He-N28. Operation in the ionization injection regime was confirmed, see Supplementary Note The laser-plasma accelerator is operated in a tailored scheme of the STII process. STII is a combination of ionization injection with conditions such that injection only occurs over a short distance (self-truncation). Laser focus evolution and wake deformation caused by an unmatched laser spot size constrains the electron injection time leading to quasi-monoenergetic energy spectra. A more detailed description of STII can be found in ref. 37 resulting in injection conditions being fulfilled at roughly the center of the gas target. Subsequent laser spot and wakefield evolution restricts the time of electron injection. The remaining acceleration length available after the point of injection is limited by the gas-jet length and thus acceleration is interrupted before the dephasing length is reached.In our experiment the STII scheme was tailored by positioning the laser vacuum focus point 1.5 mm behind the gas-jet exit. Although the vacuum focal spot of 20 μm FWHM is close to the bubble matched spot size condition , the unmatched condition required for the STII process is fulfilled by starting laser-plasma interaction in the laser intermediate field ∼4.5 mm before the laser focus is reached. Focusing of the laser spot given by the geometry is enhanced by the nonlinear self-focusing effectThis tailored scheme highly requires a high laser beam quality with a large fraction of the laser energy located within the beam waist (Strehl ratio >0.9). As the interaction in this tailored scheme starts far before the focus, great care was taken to optimize the laser profile in the intermediate field around the focus together with careful spectral-phase correction .38. Above 200 MeV the spectrometer has a readout error dominated by electron pointing. For 6 mrad pointing error this results to a readout uncertainty of (+1.6/−1.2)% at electron energies of 300 MeV and (+3.1/−2.5)% at 400 MeV. Lower pointing errors lead to a lower readout uncertainty. The spectrometer resolution and detection uncertainty is well below energy and energy spread difference found between data sets. The maximum detection energy of the spectrometer is 550 MeV. The scintillator screens were calibrated for charge at the ELBE accelerator, cross-calibrated to an Integrating Current Transformer  analog to the method described in ref. 39. In order to eliminate optical imaging setup and camera efficiency effects, the cross-calibration against CLS method as described in section III B in ref. 39 was used. Cylindrical glass capsules filled with tritium and covered with scintillating material  were used as constant light sources (CLS). CLS cross-calibration between calibration setup and electron spectrometer was performed within 1 month in order to rule out tritium decay and degeneration of the scintillating material. Charge calibration was performed on scintillating screens from the same type and batch as used in the electron spectrometer.After acceleration, electron beams were dispersed by a 400 mm-long permanent magnetic dipole. The field strength of the dipole was experimentally mapped using a Hall probe (Lakeshore MMTB-6J04-VG). Electron trajectories were simulated using the General Particle Tracer code employing the measured field map. Scintillating LANEX screens (Konika Minolta OG 400) imaged onto CCD cameras (Basler acA1300-30gm) were positioned such that energy resolution is optimized with point-to-point imaging for energies up to 200 MeV33 using a 0.2.0 pre-release40. The simulation box used consists of 704 × 704 × 2,352 cells with a transversal resolution of Δx = Δy = 177 nm and longitudinal resolution of Δz = 44.3 nm, thus resulting in a temporal resolution of Δt = 0.1393 fs. The electric and magnetic field evolution is computed via the field solver by Yee41 while macroparticles are propagated using the particle pusher by Vay et al.42. The current is calculated using the Esirkepov current deposition scheme43 with a triangular-shaped density cloud interpolation44. In order to incorporate ionization into the PIC cycle, similar simulations were performed using the BSI45 and ADK46 ionization methods. The results of the both ionization methods show good agreement for our setup, and thus BSI was selected for performance reasons. By artificially increasing the doping concentration in simulations, we were able to study beam loading effects up to 168 pC of injected charge in the peak. The plasma density was modeled according to the experimentally determined density profile of the gas target used. For the simulation presented in this paper, the electron density was set to reach ne=2.62 × 1018 cm−3 after ionization, independent of the doping used. The laser with wavelength λ = 800 nm was modeled using a Gaussian envelope both transversally and temporally and reached a vacuum peak intensity of a0 = 2.8 in focus. The pulse duration was set to τ=30 fs and the spot size to w0=19 μm (both FWHM of intensity).PIC simulations were performed with PIConGPUThe data that support the plots within this article and other findings of this study are available from the corresponding authors on reasonable request.Supplementary InformationPeer Review File"}
{"text": "Signatures of stochastic effects in the radiation of a relativistic electron beam interacting with a counterpropagating superstrong short focused laser pulse are investigated in a quantum regime when the electron’s radiation dominates its dynamics. We consider the electron-laser interaction at near-reflection conditions when pronounced high-energy gamma-ray bursts arise in the backward-emission direction with respect to the initial motion of the electrons. The quantum stochastic nature of the gamma-photon emission is exhibited in the angular distributions of the radiation and explained in an intuitive picture. Although, the visibility of the stochasticity signatures depends on the laser and electron beam parameters, the signatures are of a qualitative nature and robust. The stochasticity, a fundamental quantum property of photon emission, should thus be measurable rather straightforwardly with laser technology available in near future. In ultrastrong laser fields the quantum properties of electron radiation, the discrete and probabilistic character of photon emission, can be conspicuous. While the discreteness of the radiation photon energy is known to be observed straightforwardly, e.g., in the Compton scattering as a shift of the emission frequency12, the signatures of the stochastic character of photon emission are more subtle and elaborate for observation. The latter has impact on the radiation back-action to the electron dynamics and should be more apparent in the so-called radiation dominated regime (RDR) of interaction14, when multiple emission of photons by an electron becomes probable. One of the conceptual consequences of stochasticity effects (SE) in photon emissions, i.e., the probabilistic nature of photon emission, is the broadening of the energy spread of an electron beam in a plane laser field16, while similar effect can cause electron stochastic heating in a standing laser field17. However, in an experiment in a focused laser beam, competing effects may arise, e.g., an additional energy spreading of the electron beam due to the difference of radiative losses of electrons in the electron-beam cross section. Another SE signature is the so-called electron straggling effect during radiation in strong fields21, when the electrons propagate a long distance without radiation due to SE, resulting in the increase of the yield of high-energy photons. However, we will show below that the straggling effect is rather weak in the considered regime. Different signatures of quantum radiation reaction have been discussed35 which include all quantum effects, such as photon recoil, stochasticity, and interferences35. However, they do not isolate information on the specific role of stochasticity. Unfortunately, so far unequivocal SE have not been observed in an experiment, which would also be important as a clear test for the underlying theories. We underline that in this paper SE refers to the quantum probabilistic nature of photon emission, but not to the chaotic classical dynamics as, e.g., in ref. The next generation petawatt laser systems38, where Fμν is the field tensor, pν =  the incoming electron 4-momentum, and e and m are the electron charge and mass, respectively (Planck units ħ = c = 1 are used throughout). The RDR, when the radiation losses during a laser period are comparable with the electron initial energy, is characterized by the parameter 8, where α is the fine structure constant, ξ ≡ |e|E0/(mω0) the invariant laser field parameter, while E0 and ω0 are the laser field amplitude and frequency, respectively. In the quantum RDR the stochastic nature of photon emission is a fundamental quantum property and has to be taken into account during the high-energy photon radiation42, that significantly affects the electron dynamics and the high-energy photon emission.The quantum effects in strong laser fields are determined by the invariant parameter 14, where the front electrons are reflected and emit gamma-rays in the near-backward direction29: due to the combined effects of the laser focusing and radiation reaction, the front electrons of the electron beam are reflected and emit ultrashort gamma-rays in the near-backward direction with respect to the initial electron motion29. In the considered case with In this paper, we investigate signatures of the stochastic nature of photon emission in the nonlinear Compton scattering in the quantum RDR during the interaction of a superstrong short focused laser pulse with a counterpropagating relativistic electron beam. We consider the interaction in the electron near-reflection regimeγ is the Lorenz factor of the electron, and β the electron velocity scaled by the light speed in vacuum. The two conditions above demand 23–1024 W/cm2 anticipated in next generation facilities2.The considered quantum RDR requires the invariant parameters 42. In superstrong laser fields 43. Then, the photon emission probability is determined by the local electron trajectory, consequently, by the local value of the parameter χ44. The radiation in the quantum regime ignoring the SE are calculated by employing the Sokolov equation24, when the radiation is emitted continuously along the electron trajectory and modifies accordingly the classical equations of motion. In this work we consider the regime when the quantum invariant field parameter χ is not small, The calculation of the radiation is based on Monte-Carlo simulations employing QED theory for the electron radiation and classical equations of motion for the propagation of electrons between photon emissionsz-direction and polarizes in x-direction. The spatial distribution of the electromagnetic fields takes into account up to the ε3-order of the nonparaxial solution47, where ε = w0/zr, w0 is the laser focal radius, k0 = 2π/λ0, and λ0 the laser wavelength. The expressions of the laser fields are represented in the section “Methods”.We employ a linearly polarized tightly focused laser pulse with a Gaussian temporal profile, which propagates along +ϕ = 0° and ±180° correspond to the positive and negative directions of the laser polarization, respectively. The peak intensity of the 6-cycle (FWHM) laser pulse is I ≈ 4.9 × 1023 W/cm2 (ξ = 600), λ0 = 1 μm, and w0 = 2 μm. The electron beam, with radius we = λ0, length Le = 6λ0, and density ne ≈ 1015 cm−3, initially counterpropagates with the laser pulse, i.e. ε0 = 180 MeV  ≈ 0 at θ ≈ 0°.Figures θ with and without SE are essentially different in Fig. θ = 180° and at small angles, without SE it shows a band structure corresponding to the radiation emerging from different laser cycles. A similar behaviour is seen in RAD for photon numbers .The radiation distributions with respect to the polar angle ϕ ≤ +15°, analysing the radiation energy dθ sin(θ)] = ϕ dεR/dΩ and the photon number dθ sin(θ)] = ϕ dNR/dΩ in this domain. The stochastic nature of photon emission is clearly discernible in RAD: a single broad high-intensity gamma-photon peak is formed in the near-reflection direction when SE is included, while in the case without SE multiple radiation peaks emerge corresponding to the emission from different laser cycles. Therefore, the main detectable difference of RADs with and without SE is the peak number of the radiation: the previous has only one radiation peak, and the latter has several peaks corresponding to the laser-cycle structure.A quantitative comparison between RADs including and excluding SE is represented in Fig. z = Le/2 and x = y = 0, is considered). We follow the mean longitudinal momentum We proceed discussing the role of SE in shaping RAD. The dynamics and radiation of a sample electron is analysed in Fig. ξ).In the case without SE, the single-electron radiation angle is well defined at each moment during interaction. In contrast, in the case with SE one observes spreading of the average longitudinal momenta and the emission angles at a certain laser phase χ after the reflection is larger for the first trajectory with respect to the second, yielding significantly more radiative loss for the first trajectory than for the second one, and correspondingly, smaller energy (larger emissison angle) for the first trajectory than for the second one after the reflection . Thus, different probabilistic dynamics of the photon emission, i.e. SE, induces spreading of the photon emission angle at each laser phase.The dynamics of the photon emission in the SE case described above is illustrated in Fig. The angle-resolved radiation intensity and radiation energy are shown in Fig. The RAD with SE for a single electron is shown in Fig. π-region of the azimuthal angle. The straggling effect is observed as an enhancement of the emission of high-energy photons when the SE are included, see Fig. The electron straggling effect in our setup is estimated in Fig. w0 = 2λ0 is optimal, being the same as in Figs w0 = λ0 = we, the near-reflection radiation is rather weak, and there is no laser-cycle structure in the “without SE” case. For w0 = 3λ0, the main (highest) gamma-photon peaks in the region of w0 = 2λ0, since all electrons are much closer to the laser-intensity center, which also weakens the shift of the main peaks in polar angle when the SE are excluded. Consequently, higher angular resolution is required to distinguish the “without SE” case from the one with SE. In the extreme case of a plane-wave laser pulse the structure of multiple gamma-photon peaks in the “without SE” case cannot be observed any more. Therefore, a tightly focused laser pulse with a focal radius of roughly 2 times of the electron-beam radius applies well for the observation of SE.We investigate the optimization of the laser  and electron (energy) parameters for the best identification of SE. The influence of the laser focusing effect on RAD is studied in Fig. λ0 to 10λ0, the main (highest) radiation peak in the region of LL = 10λ0, the laser-field gradient is smaller than for shorter laser pulses, consequently, the ratio between the peaks and the plateaus becomes smaller, and the visibility of the signatures becomes worse. Then, a short laser pulse of LL ≈ 6λ0 is optimal: it has a more-cycle structure than for ultrashort pulses, and the visibility of the laser-cycle structure is better than longer pulses.The dependence of RAD on the laser-pulse length is shown in Fig. ε0 increases from 120 MeV to 240 MeV, the radiation energy εR ∝ χ ∝ ξγ increases as well since ξ being constant is assumed. The peaks in RAD shift right to a larger polar angle since the emission angle after reflection satisfies The role of the initial kinetic energy of the electron beam on the radiation angular distribution is analysed in Fig. Finally, the role of the colliding angle between the laser pulse and the electron beam is investigated, as shown in Fig. We have revealed signatures of the stochastic nature of gamma-photon emission during nonlinear Compton scattering of a superstrong short focused laser pulse by a counterpropagating electron beam in the quantum radiation-dominated regime. The signatures are manifested in the qualitative features of the angular distribution of the radiation in the near-backward direction with respect to the initial electron motion, which arises when the electron energy is at the reflection condition. When the stochasticity effects are included, a single broad gamma-photon peak is formed in the near-reflection radiation angular distribution, while several gamma-photon peaks would arise if there were no stochasticity in the photon emission. The signatures are enhanced with tightly focused and short laser pulses. They are robust with respect to variation of the laser and electron parameters and can be observed in near-future laser facilities, such as ELI and XCELS.z direction and polarized in x direction. The expressions of the electromagnetic fields are presented in the following refs E0 is the amplitude of the laser fields with normalization factor Fn = i to keep η = ω0t − k0z, and ϕCEP the carrier-envelop phase. Note that ϕCEP = 0 is employed throughout.In this work, we employ a linearly polarized focused Gaussian laser pulse propagating along +W is determined by the local electron trajectory, consequently, by the local value of the parameter χ44:k0, k and p are the four-vectors of the driving laser photon, the emitted photon and the electron, respectively, and ωr = 2ω/3(1 − χω). The applicability of the constant crossed-field approximation for the emission probability in Eq. Δη ≥ Nr is fulfilled, where Nr is a uniformly distributed random number in η is inversely proportional to the invariant laser field parameter ξ, i.e., ωmin is the driving laser photon energy, and ωmax equals the electron instantaneously kinetic energy. In addition, the emitted photon energy ωR is determined by the relation:γ for an ultrarelativistic electron, the photon emission is assumed to be along the electron velocity38. The photon emission induces the electron momentum change pf ≈ (1 − ωR/cp)pi, where pi,f are the electron momentum before and after the emission, respectively. In this simulation, the interference effects between emissions in adjacent coherent lengths are negligible since the laser fields employed are superstrong, i.e., Nr until the photon emission happens.In superstrong laser fields 24:τc ≡ 2e2/(3m), and ηi to the final phase ηf read:εR above an arbitrary energy εarb at each step yields:ωarb = εarb.As the stochastic effects are not taken into account, the rate of the electron radiation loss Nstep = 2.7 × 104, and the red-dash curves show the radiation with a smaller simulation step size, i.e., larger step number of Nstep = 5.4 × 104. The simulations clearly show that the behaviour of the photon emission angular distribution does not change with decreasing the simulation step size, i.e., increasing the simulation step number, which indicates the convergence of our results. Besides, the simulations based on the “Sokolov” equation have been compared with those based on a modified “Landau-Lifshitz” equation 48, and they demonstrate good coincidences .To prove explicitly the convergence of our Monte-Carlo simulations, we re-simulate the radiation of Fig. Supplemental materials"}
{"text": "The peak power of this attosecond light source reaches 2 terawatt. The proposed method relaxes the single-cycle requirement on the driving pulse for isolated attosecond pulse generation and significantly boosts the peak power, thus it may open up the route to new experiments tracking the nonlinear response of inner-shell electrons as well as nonlinear attosecond phenomena investigation.Extreme-ultravoilet (XUV) attosecond pulses with durations of a few tens of attosecond have been successfully applied for exploring ultrafast electron dynamics at the atomic scale. But their weak intensities limit the further application in demonstrating nonlinear responses of inner-shell electrons. Optical attosecond pulses will provide sufficient photon flux to initiate strong-field processes. Here we proposed a novel method to generate an ultra-intense isolated optical attosecond pulse through relativistic multi-cycle laser pulse interacting with a designed gas-foil target. The underdense gas target sharpens the multi-cycle laser pulse, producing a dense layer of relativistic electrons with a thickness of a few hundred nanometers. When the dense electron layer passes through an oblique foil, it emits single ultra-intense half-cycle attosecond pulse in the visible and ultraviolet spectral range. The emitted pulse has a peak intensity exceeding 10 They offer an ideal tool with unprecedented time resolution to observe and control the microscopic motion of electrons in atoms, molecules, and nanostructures, and thus can assist the understanding of the fundamental processes in matter5. In general, ultrafast ‘pump-probe’ experiments take advantage of the extremely short temporal resolution of the attosecond pulse. If the photon flux of the attosecond light source, especially the isolated attosecond pulse, significantly increases, it will greatly extend its application for nonlinear ultrafast process investigations8.Attosecond pulses can reveal electron dynamics at the atomic scale, and the attosecond techniques has undergone remarkable development in the past decade11, a loose-focusing geometry has been used to enhance the conversion efficiency, leading to microjoule-energy trains of extreme-ultravoilet (XUV) attosecond pulses. In the laser-solid interaction scenario, multi-hundred terawatts (TWs) laser systems are available to generate relativistic multi-cycle femtosecond pulses, which drive bright XUV/X-ray attosecond pulses in the sub-millijoule energy scale from the solid interface15.Significant amounts of studies have been focused on increasing the peak power of attosecond pulses. In the traditional high-order harmonic generation from a gas target18 W/cm2, which is several orders of magnitude more intense than that from a light-field synthesizer . The intense optical attosecond pulse is emitted from a dense relativistic electron layer with a thickness of a few hundred nanometers as shown in Fig. x-axis in the underdense gas target  of laser pulse has already been reflected by the oblique foil while the down-part (y < 1.7 μm) is still approaching the foil. At the front surface of the foil, the transverse momentum When the dense electron layer forms, it is positioned in the laser front and propagates along with the laser pulse. These electrons inside the layer are directly accelerated by the driving laser pulse, where the electron motion follows the single-electron picture in the local laser electric field24. The current is however screened inside the foil target. When it appears at the rear side of the foil, this transverse current of the electron layer emerges again and emits half-cycle pulse radiation15. The emitted pulse has only half cycle along the + y axis, as same as the electron transverse momentum x axis. Figure y direction, and thus does not require extra phase stabilizing processes in the potential applications1.When the dense electron layer finally reaches the foil, these electrons inside the layer have large longitudinal momenta and a transverse momenta of 16. Such an intense attosecond pulse will make it possible to study the ultrafast electronic quantum dynamics on atomic scale and other attosecond high-field physics.The generated half-cycle attosecond pulse has a broad spectrum, plotted in Fig. 25. The simulation also confirms that there is no visible radiation signal at the case of p-polarized incident laser case while it is constant for the s-polarized laser case . Employing the p-polarized incident laser will enhance the radiation intensity. Additionally, the p-polarized laser could produce a smaller pulse duration of the attosecond pulse owing to the higher longitudinal momenta of the electrons inside the layer. We also learned from the simulation that the intense half-cycle attosecond pulse is generated with well-structure shape and short pulse duration at the case of foil thickness increasing to 15 μm. Therefore the foil target can be very thick in our proposed mechanism, where the foil is strong enough to handle the large gas pressure in experiments. Moreover, such a thick foil will not allow to transmit the initial laser pulse and the radiation of laser-foil interaction is not able to pass through the thick foil.The radiation intensity of optical attosecond pulse strongly depends on the foil angle t = 70 μm/c with a peak field of 4.4 × 1012 V/m and a FWHM transverse size of 8.6 µm. After a propagation distance of 140 µm, the transverse size slowly increases to 10.6 µm, as plotted in Fig. The emitted half-cycle pulse gradually separates from the electron layer when they exits the foil as Coulomb expansion of the electron layer will cause a fast decline of the electron density. The half-cycle attosecond maintains its temporal structure in vacuum for a long distance. A remarkable feature of the half-cycle pulse in our method is that the optical half-cycle pulse is highly-collimated with a large transverse size. The generated half-cycle pulse has a transverse size of around 8.6 µm since the electron layer keeps its electron density and narrow thickness in a large transverse range of 10 µm. Figure y direction, and therefore the emitted energetic electrons will have a certain angle with the attosecond pulse propagation direction (x-axis). The energetic electrons will pass through the thin gas cell wall and be detected, therefore the half-cycle structure of optical attosecond pulse will be verified through the emitted direction measurement of accelerated electrons. We have two gas plasmas of different densities . This specific emission angle relies exclusively on the field structure of the half-cycle attosecond pulse, thus hinting at a new method to experimentally verify the proposed mechanism. The emitted angle The strong electric field of the ultra-intense attosecond pulse can immediately ionize light atoms  and accelerate electrons to relativistic energies. The second gas cell, as shown in Fig. mentclass2pt{minim27, and the relativistic multi-cycle laser pulse is currently available. Here we have verified the physical scheme by taking one laser-target example through the simulation work and the next step is the experimental demonstration. The gas-foil target in proposed method can be realized based on the specially-designed gas cells and replaceable foils in future experiments. Moreover, a simple way is present to measure the produced half-cycle pulse structure. The high-intensity optical attosecond pulse with a small divergence of 0.95° will provide sufficient photon flux on target to ionize heavy atoms and initiate strong field process. Therefore, this intense optical attosecond pulse generation method offers the ability to investigate nonlinear attosecond optics such as electron correlation as well as ultrafast dynamics inside the atom and molecules28.In summary, a novel method is proposed to generate an isolated ultra-intense half-cycle attosecond pulse with a pulse duration of 200 as based on a relativistic multi-cycle laser pulse irradiating a gas-foil target. The generated attosecond pulse in the visible and near-visible spectral ranges reaches a peak power of 2.1 TW. The method fundamentally releases the strict requirement for the high-contrast laser pulse in the laser-solid interactionSupplementary information"}
{"text": "Air quality around the globe is declining and public health is seriously threatened by indoor air pollution. Typically, indoor air pollutants are composed of a series of volatile organic compounds (VOCs) that are generally harmful to the human body, especially VOCs with low molecular weights (less than 100 Da). Moreover, in some situations, more than one type of VOC is present; thus, a device that can detect one or more VOCs simultaneously would be most beneficial. Here, we synthesized a sensor array with 4 units to detect 4 VOCs: acetone (unit 1), benzene (unit 2), methanol (unit 3) and formaldehyde (unit 4) simultaneously. All units were simultaneously exposed to 2.5 ppm of all four VOCs. The sensitivity of unit 1 was 14.67 for acetone and less than 2.54 for the other VOCs. The sensitivities of units 2, 3 and 4 to benzene, methanol and formaldehyde were 2 18.64, 20.98 and 17.26, respectively, and less than 4.01 for the other VOCs. These results indicated that the sensor array exhibited good selectivity and could be used for the real-time monitoring of indoor air quality. Thus, this device will be useful in situations requiring the simultaneous detection of multiple VOCs. Indoor environmental pollution caused by VOCs has become an important issue. For example, acetone, which is a widely used solvent in industry and laboratories, can volatilize easily and affect human health when its concentration exceeds 173 ppm4. Additionally, benzene and its homologs, such as toluene and xylene, are known to be toxic to the hematopoietic system (hematotoxicity) and to cause leukemia5. Methanol possesses strong toxicity, especially to the blood and nervous system. Furthermore, previous studies have shown that methanol is potentially harmful to human optic nerves and retinas7. Formaldehyde has been classified as a mutagen and possible human carcinogen by both the US Environmental Protection Agency and the World Health Organization because of its toxicity, anaphylactic potential and harmful accumulation in the environment9. Effort has been expended to detect these harmful VOCs, and some of the relevant works are summarized in Table 3 (ALFO) and can thus be fabricated on the same substrate, which greatly contributes to the significance and convenience of the application of the device.It is an indisputable fact that volatile organic compounds (VOCs), which are common air pollutants, are harmful to the human body3 (LFO) is a common perovskite-type oxide that exhibits semiconducting behavior10 and is a promising material with an abundance of functionalities, especially in the field of gas sensing. LFO possesses great potential for detecting pollutant gases because of its specific chemical and physical characteristics, including its large surface area, rich active oxygen lattice, good thermostability11, controllable structure12, and strong reducibility17. Thus, LFO is a more attractive gas-sensing material than other metal oxides. In addition, when LFO is doped with Ag (ALFO), some Ag is present that can act as a catalyst in the matrix. Indeed, some of the Ag fills areas between the grains of the matrix and works to decrease the contact potential barrier and enhance the interfacial effects, leading to a lower resistance, and thus, a lower operating temperature18. Therefore, ALFO was selected as the material for the units developed in this study.In contrast to some single-metal oxide semiconductors, LaFeO22. In this approach, the shape and functionality of a template can be transcribed onto microporous materials. The configuration of the functional groups in the template can be memorized within the host polymers. Today, this field is dominated by the recognition and separation of organic macromolecules, such as proteins21 (molecular weight: 40–220 kDa) and enzymes22 (130–140 kDa). However, for small organic molecules, such as VOCs (molecular weight <100 Da), no relevant research has yet been reported.Most importantly, the selectivity of ALFO toward acetone, benzene, methanol and formaldehyde can be modulated via the molecular imprinting technique (MIT). The MIT results in a predesigned molecular recognition capability that can be used to build robust sensorsBased on the MIT, using acetone as a template and choosing an appropriate acetone-related functional monomer, ALFO was prepared as an acetone gas-sensing material (hereafter abbreviated as Mater.a). Similarly, using benzene, methanol and formaldehyde as templates and choosing appropriate functional monomers, ALFO can be prepared as benzene (Mater.b), methanol (Mater.m) and formaldehyde (Mater.f) gas-sensing material. Subsequently, the four materials were brush-coated onto a sensor array, as shown in Fig. 23, as a result, template, functional monomer and initiator could not be detected either. The microtopographies of Mater.a, Mater.b, Mater.m and Mater.f were spherical and uniform in size, with particle sizes in the range of 20–70 nm  spectra of ALFO, acetone, benzene, methanol and formaldehyde and the related functional monomers N,N’-methylenebisacrylamide (MBA), formaldehyde (HCHO), methacrylic acid (MAA) and acrylamide (AM) were obtained  to that in air (Ra). After the ALFO was polymerized with MBA, the gas-sensing mechanism was similar to that of LFO because LFO was the only phase in Mater.a 3·6H2O, 10.0 mmol of Fe(NO3)3·9H2O and 10.0 mmol of citrate were dissolved in 100 mL of distilled water to form solution A. AgNO3 (0.1 mmol) was dissolved in 10 mL of distilled water and added dropwise (12 drops/min) to solution A. Subsequently, polyethylene glycol was added. The final mixed solution was stirred at 80 °C for 8 h and then placed in a microwave chemical device  at 75 °C for 2 h. Thus, the ALFO sol was formed and used as a crosslinker in the molecular imprinting process.Preparation of the crosslinker: All the chemicals were analytical-grade reagents and were used as received from Tianjin Kermel Chemical Reagents Development Center. To prepare the crosslinker, 9.9 mmol of La. The final mixture was treated with ultrasonication for 30 min, stirred at 50 °C for 12 h under a nitrogen atmosphere under reflux and then dried. Finally, the xerogel was heated at 800 °C for 2 h, yielding Mater.a.Preparation of Mater.a: Acetone was used as the template, MBA was used as the functional monomer, azodiisobutyronitrile (AIBN) was used as the initiator and ALFO sol was used as the crosslinker. A schematic diagram of the hydrogen bonding between acetone and MBA is shown in Fig. b. Then, 1.0 mmol of AIBN was dissolved in 20 mL of benzene and mixed with solution Bb and ALFO sol (10.0 mmol). The final mixture was treated by ultrasonication for 30 min, stirred at 50 °C for 12 h under a nitrogen atmosphere under reflux and then dried. Finally, the xerogel was heated at 800 °C for 2 h, yielding Mater.b.Preparation of Mater.b: Benzene was used as the template, FA was used as the functional monomer, AIBN was used as the initiator and ALFO sol was used as the crosslinker. A schematic diagram of the hydrogen bonding between benzene and FA is shown in Fig. m. Then, 1.0 mmol of AIBN was dissolved in 20 mL of methanol and mixed with solution Bm and ALFO sol (10.0 mmol). The final mixture was treated with ultrasonication for 30 min, stirred at 50 °C for 12 h under a nitrogen atmosphere under reflux and then dried. Finally, the xerogel was heated at 800 °C for 2 h, yielding Mater.m.Preparation of Mater.m: Methanol was used as the template, MAA was used as the functional monomer, AIBN was used as the initiator and ALFO sol was used as the crosslinker. A schematic diagram of the hydrogen bonding between methanol and MAA is shown in Fig. f. Then, 1.0 mmol of AIBN was dissolved in 20 mL of formaldehyde and mixed with solution Bf and ALFO sol (10.0 mmol). The final mixture was treated with ultrasonication for 30 min, stirred at 50 °C for 12 h under a nitrogen atmosphere under reflux and then dried. Finally, the xerogel was heated at 800 °C for 2 h, yielding Mater.f.Preparation of Mater.f: Formaldehyde was used as the template, AM was used as the functional monomer, AIBN was used as the initiator and solution B was used as the crosslinker. A schematic diagram of the hydrogen bonding between formaldehyde and AM is shown in Fig. Preparation of the ALFO-based Mater.a, Mater.b, Mater.m, and Mater.f:The prepared materials were further mixed with distilled water and ground to form a paste, which was subsequently printed onto an alumina tube. Two Au electrodes were placed at the ends of the tube. The length of the alumina tube was 4 mm, and the diameter was 1.2 mm. To improve their stability and repeatability, the gas sensors were aged at 150 °C for 170 h in air. The gas-sensing properties were tested using a WS-30A gas senor tester. The relative humidity of the air chest was adjusted by injecting different amounts of water onto a hot plate. As the water was vaporized, the relative humidity changed and was monitored using a hygrometer.−1 to 400 cm−1. The particle morphology of each sample was determined by TEM (JEM-2100). The surface morphology and fracture surface were characterized by scanning electron microscopy . The pore size and Brunauer-Emmett-Teller (BET) surface area were analyzed using Quantachrome QuadRASORB-evo equipment.The XRD patterns were obtained for phase identification with a D/max23 diffractometer using Cu Kα1 radiation (λ = 1.54056 Å); the diffracted X-ray intensities were recorded as a function of 2θ. The accelerating voltage was 35 kV, the applied current was 25 mA, and the sample was scanned from 20° to 80° (2θ) in 0.02° steps. The functional groups were identified by FTIR (FTS-40), and each sample was embedded in a KBr pellet and scanned from 4000 cmSupporting Information"}
{"text": "There was a significant improvement in THI and VAS scores after surgery . The decrease in THI scores in the low-frequency group was significantly larger than that of the mid- and high-frequency groups after surgery . The loudness of tinnitus decreased significantly after surgery (p = 0.031). Tinnitus in patients with VS improved after TLM. Patients with mid-/high-frequency tinnitus and louder tinnitus preoperatively seemed to have a worse prognosis than those with low-frequency and quieter tinnitus.We designed a prospective study to evaluate changes in tinnitus after vestibular schwannoma (VS) surgery. Subjects included 41 patients who were diagnosed with a VS and underwent translabyrinthine microsurgery (TLM) between January 2015 and May 2016. All patients underwent related examinations and were asked to answer the Tinnitus Handicap Inventory (THI) scale and a visual analog scale (VAS) of tinnitus severity both pre- and postoperatively. Of the 41 patients, 31 (75.6%) suffered from tinnitus before surgery. Microsurgery was associated with an overall decrease in tinnitus ( It is a frequent symptom of vestibular schwannoma (VS), occurring in more than half of all patients3. In the past, most surgeons aimed to completely remove the tumor while preserving facial nerve function and hearing; however, recently, more attention has been paid to the patient’s quality of life2. Because tinnitus can reduce the quality of life in these patients6, further evaluation of tinnitus in patients with VS should be considered.Tinnitus is the perception of sound in the absence of an external sound and usually results from a disorder of the somatosensory system or the auditory system7. These findings indicate that tinnitus originates in peripheral organs such as the cochlea or cochlear nerve9. However, some patients still suffer from tinnitus even after tumor removal and vestibulocochlear nerve section, which supports the theory that tinnitus is likely a symptom of central origin14. The objective of this study was to evaluate changes in tinnitus after VS microsurgery.Recently, several retrospective studies have been published focusing on changes in tinnitus after microsurgery; however, the results of these papers have varied. Most of these papers showed that tinnitus improved after surgery regardless of whether translabyrinthine microsurgery (TLM), a retrosigmoid approach, or a middle cranial fossa approach was used16 (see Supplementary Table). Patients without tinnitus have a handicap score of 0 on the THI, and higher THI scores correspond to worse tinnitus18. As in pain research19, visual analog scale (VAS) scales are increasingly being used to assess treatment-induced changes to the extent of annoyance caused by tinnitus20. With a VAS scale, patients self-grade the severity of tinnitus with possible scores ranging from 0 to 10 points; a score of 0 indicates no symptoms and a score of 10 indicates extremely loud tinnitus that seriously affects a patient’s daily life19.The Tinnitus Handicap Inventory (THI) is a reliable and valid measure of tinnitus-related handicap and is a questionnaire that is internationally acknowledgedThe study protocol was approved by the Institutional Review Board of the Affiliated Sixth People’s Hospital of Shanghai Jiao Tong University, and all methods were performed in accordance with the relevant guidelines and regulations. Informed consent for study participation was obtained from all participants.In total, 41 patients diagnosed with unilateral VS were enrolled in this prospective study between January 2015 and May 2016 in the Department of ENT Head and Neck Surgery at the Sixth People’s Hospital affiliated with Shanghai Jiao Tong University. These patients were treated with TLM because their preoperative hearing was already unserviceable, or the size of tumor was too large for hearing to be preserved. Patients who did not undergo surgery but were kept for observation and patients who underwent retrosigmoid or middle fossa approaches were excluded for this study. Microsurgery was performed by the same senior surgeon, and all patients underwent complete excision.21, tumor size, symptom duration, and the frequency and loudness of pre- and postoperative tinnitus were analyzed.All patients underwent pure-tone audiometry, acoustic immittance measurements, otoacoustic emissions measurements, tinnitogram, temporal bone computed tomography (CT), and internal auditory canal enhanced magnetic resonance imaging (MRI). Factors including preoperative pure-tone audiometry, postoperative facial function (House-Brackmann)22.Pure-tone averages (PTAs) were obtained by calculating the average thresholds at 500, 1000, and 2000 Hz23. All measurements were assessed by a senior radiologist and were checked by a senior otolaryngologist. For the tumor size criteria, we referred to the 2012 Acoustic Neuroma Association membership survey and divided the tumors into three groups by size: ≤1.5 cm, 1.6–2.5 cm and ≥2.6 cm24.All internal auditory canal enhanced MRI examinations were performed on an Achieva 3.0 T MRI system . In general, the cerebellopontine angle (CPA) along the long axis of the tumor was used as the maximum diameter, which was considered as the tumor size25. The acoustic signal was selected based on the tinnitus reported by the patient, and the types of acoustic signals were a pure-tone, narrow-band noise, pulse, warble tone and white noise. Then, the appropriate type was chosen according to the patient’s description. The matching process usually adopted the tonal debugging technique26 to adjust the initial test sound to the volume of the tinnitus. According to the pure-tone audiometry and experience of the technician, the participants were presented with a starting stimulus and asked if their tinnitus pitch was higher or lower. After the participants provided their answers, the frequency of the stimulus was adjusted. If the patient answered high, the frequency was increased by 50%; if the patient answered low, the frequency was reduced by 50%. If the tinnitus pitch was equal to the test sound, the test was stopped, and the last frequency was recorded. The test was repeated three times, and the average was calculated. The single ear loudness balance test was usually used to match loudness. Using the selected frequency of tinnitus, we matched the loudness to the individual’s tinnitus loudness in 1 dB steps by asking the participant whether the tinnitus was softer or louder27. For patients with hearing loss, we used the healthy ear to match. In the matching test, tinnitus was classified by frequency: ≤250 Hz, low-frequency; 500–2000 Hz, mid-frequency; and ≥4000 Hz, high-frequency22.Tinnitus frequency matching and tinnitus loudness matching were measured on tinnitograms17.The severity of tinnitus was evaluated using the THI and VAS scales. All enrolled patients were assessed via the THI and VAS scales pre- and postoperatively. The analysis of the THI scores used the total score rather than the subscalesData are presented as the mean ± standard deviation or the median (interquartile range).A postoperative follow-up was performed approximately one year after surgery; this included an interview regarding the patient’s postoperative state and changes in tinnitus and an internal auditory canal enhanced MRI. The follow-up rate was 100%.t-test, Spearman’s correlation analysis, Chi-square test, Fisher’s exact test and analysis of variance (ANOVA). For all analyses, p < 0.05 was considered to indicate statistical significance.Data were analyzed using SPSS . Data from survey responses were analyzed using Student’s p < 0.001).There were 41 patients included in our study, and no patients had lesions in both ears   (Table p = 0.011). We also found a strong correlation between the postoperative THI score and postoperative VAS score  , with a range of 6–82 in the preoperative tinnitus group. The average THI score was 14 (26) (median/interquartile range), with a range of 0–92, postoperatively. This indicates a general improvement between the pre- and postoperative THI scores (p < 0.001).In assessing the THI 20- point threshold as a significant change, we found that of these 31 patients with preoperative tinnitus, symptoms improved in 18 (58.1%), unchanged in 8 (25.8%), and worsened in 5 (16.1%) after surgery; all of these results were clinically significant (p = 0.785)   (Table p = 0.374)  , mid-frequency in 13 (41.9%), and high-frequency in 12 (38.7%). We found no significant correlation between the frequency of the preoperative tinnitus and preoperative THI  (Table p = 0.575)     dBHL, ranging from 20 to 80 dBHL. The postoperative tinnitus loudness ranged from 0 to 100 dBHL, with an average of 20 (74) dBHL. Thus, the loudness of tinnitus decreased significantly after surgery compared with the preoperative value ; 1.6–2.5 cm ; and ≥2.6 cm . Tumor size was correlated with the preoperative THI score and preoperative VAS score     cm. The patients were divided into three groups according to tumor size: ≤1.5 cm    . Age was not correlated with preoperative THI score or preoperative VAS score      cases and grade III in 5 (16.1%) cases; no correlation was observed between facial nerve function and the postoperative THI score or postoperative VAS score     months. The average symptom duration was 6 (60) months for patients with low-frequency tinnitus, 4 (119) months for patients with mid-frequency, and 12 (161) months for patients with high-frequency tinnitus. No significant differences in symptom duration were observed between the low-/mid-frequency and high-frequency groups  or improved (32.2%) for most patients after surgery, indicating a significant overall decrease in tinnitus via microsurgery. Patients with low-frequency and quieter tinnitus preoperatively seemed to have a better postoperative prognosis than those with mid-/high-frequency or louder tinnitus before the surgery.et al.17 reported that tinnitus was neither exacerbated nor relieved after TLM, while many others found that microsurgery improved tinnitus30, consistent with our observations. The underlying mechanisms are still unclear; however, several hypotheses have been proposed. First, the TLM surgical approach sacrifices inner ear and cochlear nerve function3, disrupting the central auditory conduction path. Second, cochlear or auditory nerve lesions can cause increases in the discharge rates of inner hair cells and nerve fibers, and the central auditory system cannot distinguish between pathological and normal physiological excitation and produces errors in hearing, resulting in tinnitus11. From this point of view, surgery may relieve the mechanical stimulation of the auditory nerve by the tumor, thus preventing the abnormal impulses and, consequently, eliminating tinnitus.Previous studies have demonstrated mixed surgical results regarding its effect on tinnitus. Baguley et al.3 reported that tinnitus disappeared or improved in most cases after the retrosigmoid approach and showed no difference in tinnitus incidence whether the vestibulocochlear nerve was resected or not. Park et al.2 suggested that cochlear nerve section may be beneficial for the improvement of postoperative tinnitus by comparing changes in tinnitus after TLM and gamma knife radiosurgery. In this study, the chosen surgery was the translabyrinthine approach because hearing preservation was not intended. During surgery, the cochlea nerve section was definite in all cases31 as suggested by Park et al.2, which may be the reason for the high rates of improvement in tinnitus. Although sectioning of the cochlear nerve creates a condition unsuitable for cochlear implantation32, either simultaneously or prospectively, a BAHA implant is a hearing solution for these patients33.Currently, no consensus exists on whether tinnitus relief is related to cochlear nerve dissection. Kameda 34. The author suggested that high-frequency tinnitus may be related to a longer duration and more severe injury and is therefore associated with greater difficulty in recovery. However, we found no significant relationship between the duration of tinnitus and the different frequencies.We found that the postoperative prognosis was better in patients with low-frequency tinnitus than in those with mid-/high-frequency tinnitus. However, the specific reasons and mechanisms for this finding remain unclear. Interestingly, a previous study found that among patients with sudden deafness accompanied by tinnitus, the rate of recovery was better for those with low-frequency tinnitus than those with high-frequency tinnitus19.The preoperative tinnitus loudness was significantly correlated with the postoperative tinnitus loudness in our cohort, i.e., greater loudness was correlated with greater difficulty in recovery. In theory, louder tinnitus has a greater impact on patients and therefore results in higher scores on the THI and VASet al.35, a 20- point or larger change is considered clinically significant at the 5% confidence level17. In this study, we found statistically significant changes in the THI score, considering any change in the THI to represent a change in tinnitus. Moreover, when we reassessed our results only considering a 20- point or larger change in the THI score, these changes were also statistically significant. Therefore, VS removal caused clinically relevant changes in our cohort.Based on the report by Newman 37. In this study, we found no correlation between them. Kohno et al.31 found that tinnitus appeared in one-fifth of the patients without preoperative tinnitus. We evaluated patients without preoperative tinnitus and found no new-onset tinnitus postoperatively.Whether the severity of tinnitus is associated with tumor size, age, preoperative pure-tone audiometry or postoperative facial nerve function remains controversial17. However, our study was a prospective study and focused exclusively on the translabyrinthine route. Baguley et al.17 and Alvarez et al.16 also focused on the translabyrinthine approach. They demonstrated a change in tinnitus only by the THI scale, whereas our study is the first clinical study to reveal correlations between the preoperative THI score, VAS score, loudness and changes in tinnitus postoperatively. These tests are not interchangeable. In addition, because this study had a relatively limited sample size of 41 patients, it remains possible that unperceived differences existed between the groups38.Some published findings are either retrospective studies or demonstrate that tinnitus improved after microsurgery regardless of which approach was usedTinnitus in patients with VS decreased after TLM surgery, as measured and cross-validated by three separate measures: THI, VAS, and tinnitus loudness matching. Patients with low-frequency and quieter tinnitus preoperatively seemed to have a better postoperative prognosis than those with mid-/high-frequency or louder tinnitus before the surgery.Supplementary Table"}
{"text": "Early prediction of the potential for neurological recovery after resuscitation from cardiac arrest is difficult but important. Currently, no clinical finding or combination of findings are sufficient to accurately predict or preclude favorable recovery of comatose patients in the first 24 to 48 hours after resuscitation. Thus, life-sustaining therapy is often continued for several days in patients whose irrecoverable injury is not yet recognized. Conversely, early withdrawal of life-sustaining therapy increases mortality among patients who otherwise might have gone on to recover. In this work, we present Canonical Autocorrelation Analysis (CAA) and Canonical Autocorrelation Embeddings (CAE), novel methods suitable for identifying complex patterns in high-resolution multivariate data often collected in highly monitored clinical environments such as intensive care units. CAE embeds sets of datapoints onto a space that characterizes their latent correlation structures and allows direct comparison of these structures through the use of a distance metric. The methodology may be particularly suitable when the unit of analysis is not just an individual datapoint but a dataset, as for instance in patients for whom physiological measures are recorded over time, and where changes of correlation patterns in these datasets are informative for the task at hand.We present a proof of concept to illustrate the potential utility of CAE by applying it to characterize electroencephalographic recordings from 80 comatose survivors of cardiac arrest, aiming to identify patients who will survive to hospital discharge with favorable functional recovery. Our results show that with very low probability of making a Type 1 error, we are able to identify 32.5% of patients who are likely to have a good neurological outcome, some of whom have otherwise unfavorable clinical characteristics. Importantly, some of these had 5% predicted chance of favorable recovery based on initial illness severity measures alone. Providing this information to support clinical decision-making could motivate the continuation of life-sustaining therapies for these patients. Cardiac arrest is the most common cause of death in high-income nations . In the Unfortunately, accurate neurological prognostication after cardiac arrest is challenging, particularly in the first 3 to 5 days after resuscitation , 7, 8. AMultiple modalities which might inform early prognostication have been explored , 11, 12.We propose Canonical Autocorrelation Analysis (CAA) as a method for automated discovery of multiple-to-multiple correlation structures within a set of features. Through the introduction of a distance metric between CAA correlation structures, we are able to define Canonical Autocorrelation Embeddings (CAE), a feature space embedding in which each individual/object is represented by the set of its multivariate correlation structures. In this feature space embedding, traditional machine learning algorithms that rely on distance metrics, such as nonparametric clustering and k-nearest neighbors (k-nn), can be applied to compare correlation structures.This methodology is particularly fitting to tasks where multiple potentially correlated data points are recorded over space or time for each individual or unit of study. For example, in clinical medicine several vital signs or other physiological measures may be repeatedly sampled for each patient being monitored. Because physiological processes are interdependent and interact, analyzing the correlation structure between several such processes may reveal otherwise unrecognized patterns that may characterize the current state of the patient , 18. In Because of the difficulty identifying patients with potential for recovery and desire to limit futile care described above, patients may be at risk for withdrawal of life-sustaining therapy when their potential to recover goes unrecognized . To reduIn the remainder of this paper, Section 1 presents a brief review of related work. Section 2.1 discusses the task and data in more detail. In Section 2.1, CAA and CAE are introduced, as well as the use of a k-nn algorithm in the resulting embedded space. Section 3 contains the experimental results, Section 4 discusses our findings and Section 5 summarizes the conclusions and future work.ℓ1 variant of CCA, was proposed by [X and Y are two disjoint matrix representations for one set of objects, so that each matrix is using a strictly different set of variables to describe them. Assuming X and Y have been standardized, the constrained optimization problem is shown in c1 and c2 are small, solutions will be sparse and thus only a few features are correlated.Canonical Correlation Analysis (CCA) is a statistical method first introduced by , useful posed by , 24. ThiThe extension of Sparse CCA for discovery of multivariate correlations within a single set of features to study brain imaging has been previously explored in , 21. UsiCanonical Autocorrelation Analysis (CAA), the methodology we propose, is a generalized approach to discovering multiple-to-multiple correlations within a set of features. variance of data in one-dimensional projections, while CAA finds two-dimensional projections where correlation across two subsets of features is maximized. CAA specifically seeks projections composed by pairs of strongly correlated linear combinations of features, enabling discovery of hidden characteristic correlations in data, which cannot be easily found with other methods such as Sparse PCA. Other methods for finding sparse representations of data comprised in a single matrix include the well-known Sparse Principal Component Analysis (Sparse PCA). While CAA resembles Sparse PCA in the sense that it finds sparse representations of data contained in one matrix, Sparse PCA maximizes retained Extraction of informative projections has been tackled in the past , 26. OurThe comparison of correlation structures and principal components has been explored in the literature for decades. Most prominently,  discusseLearning to defer has been studied in the literature as a means to effectively combine algorithmic and human decision-making , 31. WheThis study was approved by the University of Pittsburgh Institutional Review Board with a waiver of informed consent. The data used in this case study are derived from 451 comatose survivors of cardiac arrest treated at a single academic medical center between 2010 and 2015 . For eacAlso available for each patient are time-invariant clinical characteristics and outcomes, including survival to hospital discharge. For those who lived, the quality of their functional recovery at discharge was measured using two standard outcome scales: Cerebral Performance Category and modified Rankin Scale. We considered “favorable recovery” to be either a Cerebral Performance Category of 1 or 2 or a modified Rankin Scale score of 0-2 at hospital discharge. For those who died, the proximate cause of death is known. X and Y. Applying Sparse CCA when X = Y results in solutions u = v, corresponding to Sparse PCA solutions for X [The goal of CAA is to find multivariate sparse correlations within a single set of variables. In the Sparse CCA framework, this could be understood as having identical matrices ns for X . We overL1 penalty when either u or v are fixed. Replacing the equality constraint by an inequality constraint gives a biconvex problem, while resulting in the same solution. Therefore, we can solve it through alternate convex search [This can be understood as a new generalization of the Penalized Matrix Decomposition . Note thx search , as showAlgorithm 1: CAA via alternate convex search1 Initialize v s.t. ||v||2 = 1;2repeat3  4  s.t. u||1 ≤ c1, 5  6  s.t. v||1 ≤ c1, 7untilu, vconverge;8d ← uTXTXv;u without loss of generality. For a detailed derivation see At each iteration, the resulting convex problem can be solved through the Karush-Kuhn-Tucker (KKT) conditions. The pseudo-code for solving the convex problems at each iteration of the alternate convex search is provided in Algorithm 2, where we solve for Algorithm 2: CAA alternate convex search iteration via KKT conditions12ifthen3  return4else5  Binary search to find λ2 s.t. 6  return7endXTX with a matrix from which the already found correlations are removed, as shown in d = uTXTXv.To find multiple pairs of CAA canonical vectors, Algorithm 1 can be repeated iteratively, replacing xi has a subset Si of associated indices of other features that should not be included as correlates of xi, the resulting optimization problem follows In order to enable the discovery of non-linear correlations by extending the feature space with subsequent powers of the original features , we modi1 penalty at each iteration of the biconvex optimization algorithm. Hence, the problem can still be solved as discussed above, with the only difference that the parameters of the soft-thresholding operator will change.The new constraint for disjoint support can still be understood as a weighted-Lu, v a CAA canonical space, and each CAA model may consist of one or more of such canonical spaces.CAA allows us to find bi-dimensional projections where the data closely follows a linear distribution. Each axis of these projections corresponds to a linear combination of the original features, and their respective coefficients are represented in a pair of vectors u||2 = ||v||2 = 1 ∀i, therefore, the distance between two CAA canonical spaces C1 and C2 can be defined as shown in Since the correlations discovered by CAA are defined by pairs of vectors in It is easy to show that this metric satisfies the necessary conditions for a well-defined distance, see Even though we believe that Having formulated a distance metric between pairs of CAA canonical spaces enables us to employ a range of distance-based machine learning algorithms, such as k-means, hierarchical clustering, or k-nn, to leverage similarities among correlation structures present in data. One additional complexity in our case is that each subset of data being compared may be represented by more than one CAA canonical space, and therefore more than one point in the embedding.k nearest neighbors, and then aggregating over all correlations associated to an object using log-odds, as shown in np,i,j denotes the class label of the jth neighbor of the ith correlation of patient p.This setting can be incorporated into the k-nn framework by calculating the class probability for each correlation structure through the votes of their t, so that log-odds are only calculated over those correlation structures with a class probability that is discriminative enough, as shown in k, indicating the number of neighbors, and t can be tuned through cross-validation.However, it is likely that some type of correlation structures will be common to both classes, while others are discriminative. To reduce noise and allow for those discriminative correlations to lead the decision, we incorporate a threshold Our principal goal is to help improve care given to comatose survivors of cardiac arrest through a decision support system that can boost the accuracy and timeliness of prognostication. To do so, we propose a new way to characterize patients using their latent multivariate correlation structures, and use the resulting featurization of data to build predictive models. The results presented in this section leverage data collected over a five year period at an academic medical center to provide a proof of concept of the proposed methodology.As seen in For each patient, their entire qEEG record is available, with lengths varying from less than an hour to more than a week. We aim to predict recovery as early as possible, but the earlier we attempt prediction, the more challenging it is. For the purposes of this experiment, we target prediction after 36 hours of monitoring. We use CAA to characterize a two hour epoch between hours 34 and 36. We choose 36 hours because we are interested in a period where the EEG is relatively static so that we do not need to account for temporal trends within the analyzed epoch. Clinically, patients are cooled down for 24 hours then allowed to rewarm at about 0.25-0.5C/hr. Both temperature and medications used to suppress shivering can alter the EEG . At 36 hR2 > 0.25. Moreover, to ensure that only reasonably close neighbors are used for matching, we prune connections by only considering distances smaller than k and t in an internal 10-fold cross-validation loop within each training fold, are presented in Figs In order to avoid spurious results, we only consider CAA canonical projections that yield correlations with logistic regression on sets and k-nn on sets, respectively. To emphasize the importance of considering a window of time rather than a snapshot, we also compare against the same two algorithms taking as input the last data point after 36 hours of monitoring, that is, the recording at one time step. We refer to this approach as logistic regression on points and k-nn on points, respectively. The parameters are chosen through 10-fold cross-validation. The results are included in Figs For baseline comparison, we use a popular approach: extract features based on metrics calculated over windows of time and apply standard classifiers to the resulting featurization , 36, 37.Finally, we apply the resulting system to those patients who were withdrawn from life-sustaining therapies. Amongst 31 patients who received life-sustaining therapies for at least 36 hours before withdrawal of life-sustaining therapies, five patients would have been marked by our classifier as very likely to recover at a threshold of FPR equal to 0.025.Recall that the proposed decision-support system is one that only makes recommendations when it has strong indications that the patient is likely to have a positive neurological recovery, and defers in all other cases. We evaluate the performance of the algorithm at the thresholds at which it would make recommendations, which we characterize through low false positive rates (FPR). The true positive rate (TPR) at a given FPR indicates what portion of positives–TPR—would be retrieved while assuring that no more than a given rate of negatives–FPR—will be incorrectly labeled as positive.Due to the gravity of errors in this scenario, the tolerance for false positives should be extremely low. Receiver Operator Curves (ROC) shown in Figs The results presented in Even though consensus guidelines advocate maintaining life-sustaining therapies for at least 72 hours after cardiac arrest , the buri), in a mild to moderate brain injury with good heart and lung function (category ii), in a mild to moderate brain injury but poor heart and/or lung activity (category iii), or severe brain injury with loss of some brainstem reflexes (category iv). While patients in category i have an associated probability of survival of 80%, and 60% probability of having a positive neurological recovery, patients in category iv have probabilities of 10%, and 5%, respectively. At a FPR lower than 0.025, the proposed methodology correctly identified a category iv patient who later went on to have a positive recovery. This constitutes a preliminary indication that the patterns of correlations in neurological activity measured with EEG constitute novel findings and have the potential to improve reliability of prognostication.To appropriately estimate the potential impact of such a decision support system in terms of lives saved, it is useful to compare against physicians’ assessments to validate if the predictions made with the proposed approach are non-redundant to what doctors already know. Each patient in our dataset is classified by Pittsburgh Cardiac Arrest Category, a 4-level, validated prognostic indicator assigned in the first six hours of their stay . This cliv. The remaining three received an initial Pittsburgh Cardiac Arrest Category of ii. While we do not have ground truth regarding counterfactuals of what would have happened if life-sustaining therapies had been continued for these patients, these results provide further indication that CAE is not simply leveraging patterns that are already being used by physicians.As discussed in Section 3, amongst those patients whose cause of death is withdrawal of life-sustaining therapy for perceived neurological prognosis, 5 out of 31 patients who received life-sustaining therapies for at least 36 hours would have been marked by our system as likely to have a positive recovery. Two of these patients had received a Pittsburgh Cardiac Arrest Category of bad outcome label. Meanwhile, the positive recovery label is sure to indicate positive neurological recovery (as well as positive recovery in other areas).While it would also be desirable to identify patients who have a very small probability of neurological recovery, we note that neither of the models would be able to provide confident recommendations to withdraw life-sustaining therapies while guaranteeing low false negative rates (FNR). A research direction that could further improve the performance of CAE is correlation trajectory modeling. While our model captures correlations observed within an interval of time, and in that sense it goes beyond a purely stationary approach, leveraging the sequential structures in data and using all data collected during a patients’ stay is desirable. Methodologically, this calls for the development of models for trajectory modeling of multivariate correlation structures. This could also encompass further exploration of additional distance metrics that could incorporate other types of information. By leveraging more information, such an approach would have the potential of providing earlier and more specific predictions.An additional direction for performance enhancement comes from the fact that our characterization of brain activity with CAA is motivated by the importance clinicians place on correlations. However, the correlations they know to be informative are across raw EEG channel measurements, and it is likely that at the current level of data aggregation, a big portion of the information may be to some extent obfuscated. This does not constitute a risk in terms of the validity of the results presented in this paper, but it means that further promising results may be expected from characterizing correlations in raw EEG signals. Such models could also lead to biological insights that may not be easily derived with the current approach.An important challenge that arises in this setting is the selective labels problem . SelectiCardiac arrest is a leading cause of death around the world, coma after cardiac arrest is common, and good neurological recovery is rare. Everyday, clinicians are tasked with making a prediction that determines whether they will continue life-sustaining therapies for their patients in coma or not. Motivated by the emphasis the clinicians place on potential informativeness of the correlation structures in EEG data, we have proposed a way to characterize and compare patients based on the latent structures of multivariate correlations, and use such information to predict positive neurological recovery. To do so, we have proposed a new formulation of Canonical Autocorrelation Analysis (CAA), a method that automatically finds subsets of features of data that form strong multiple-to-multiple correlations. We have also introduced Canonical Autocorrelation Embeddings (CAE) to enable the comparison of discovered correlation structures. CAE makes powerful and well established machine learning methodologies that rely on the use of distance metrics applicable to the task at hand.The results presented in this paper constitute a proof of concept. Future work involves collecting more data to train and validate the model. It is reasonable to believe that there may be a substantial heterogeneity across patients, hence experiments using more data of more subjects are a necessary next step. Applying CAE to the raw EEG channels rather than to the aggregated featurizations of data would also be interesting to explore once that data becomes available.In addition, we are developing machine learning methodology to tackle the selective labeling problem by incorporating clinicians’ domain knowledge while not reproducing their mistakes. Developing proper evaluation metrics to assess performance under selective labels, and finding ways to tackle the blindness that may result from this problem, is an important ingredient needed to successfully use machine learning to save lives in clinical settings.S1 TableComplete list of qEEG features available and used in this study.(PDF)Click here for additional data file.S1 FileSolution of CAA optimization problem via KKT conditions.(PDF)Click here for additional data file.S2 FileDiscussion of the relationship and differences between CAA and Sparse PCA.(PDF)Click here for additional data file.S3 FileProof of CAA well-defined distance metric.(PDF)Click here for additional data file.S4 FileDiscussion of why principal angles are not a well suited distance for CAA canonical spaces.(PDF)Click here for additional data file."}
{"text": "Aname and Teyl occurring in these local urban remnants. Local predator guilds were significantly influenced by leaf-litter cover (%) and proportion of surrounding parkland. Preference for spider vs. control models was consistent across all predator types , but specialist spider wasps (Pompilidae) only attacked spider models. Generalist predators  were more opportunistic. Lizards and rodents exhibit similar predation behaviour, indicating there may be some inter-specific competition. Invasive generalists (e. g. rodents) or urban adapters (e. g. corvids) are more likely to represent an increased threat to spiders than are co-evolved specialists (e.g. spider wasps).Predator-prey interactions may be altered under human-induced rapid environmental change, such as urbanisation. Extensive clearing in urban areas may leave short-range endemic species, such as mygalomorph spiders, more vulnerable to local extinction through predation in remaining remnants. Predation rates on Australian mygalomorph spiders were assessed using clay models of two size classes , during two time periods in 2016 . Size and phenology of models resembled the mygalomorph genera  Human-induced rapid environmental change, such as urban development, can place severe selective pressures on species to adapt to these changes, move away or persist in fragments or refugia within altered landscapes6.Since 1960 the global human population has dramatically increased and consolidated in urban centres7, some taxa, known as ‘urban exploiters’ e.g. rock pigeons (Columbia livia) and some rodents (e.g. Mus musculus and Rattus spp.) are found mainly in urban landscapes, where they subsist on anthropogenic resources. Other species, e.g. red foxes (Vulpes vulpes) globally8 or ravens (Corvus coronoides) and butcherbirds (Cracticus spp.)9 in Australia can be termed ‘urban adapters’ and benefit from resources such as anthropogenic food or shelter in urban areas but are not limited to urbanised areas. Urban adapters may have profound impact on their native prey species that still persist in patches of urban bushland. While urban adapters may thrive in various landscapes surrounding urban bushland, many native species are restricted to such patches. Negative impacts on native taxa may come from either novel threats (invasive species) or changes in predation behaviour of other native taxa through urbanisation.Response to urban environments varies amongst taxa. Although presence of many species is positively correlated with distance from urban environments10, and may be referred to as ‘urban engulfed’. These traits are present in many species of millipedes, snails, cicadas and mygalomorph spiders11. Such species are known as short-range endemics (SRE)12 and are of high conservation priority in Australia14.Taxa with comparatively low mobility, low-fecundity, poor dispersal and small geographic range may persist in very small natural habitat remnants in urban areas if the quality of the patch is maintained18. There is evidence that invasive rodents can have a severe effect on invertebrates on islands19, which may be analogous to predation in urban bushland fragments, as many native species may be confined to a single fragment. Reptiles, and lizards in particular, are predators of spiders and other invertebrates20. Reptile response in an urban environment seems to vary markedly between species, with some skinks being urban exploiters or adapters21. In Australia, predator-prey interactions involving invertebrates is less studied than vertebrates, probably due to taxonomic impediment and bias towards more charismatic subject species22. However, the predation and parasitisation of spiders by wasps , has been long known23. Exploring predator-prey interactions between urban wildlife will assist in understanding complex interactions, and how these may vary under HIREC.SRE species may face extinction as a result of the additional pressure of predation in small, fragmented populations. Differential predation of taxa across urban areas has not been well documented. Research addressing the threat associated with invasive rodents in Australia has often focused on various impacts on native taxa24. Subsequently, adult males emerge and roam to mate. This venture is a highly seasonal occurrence and appears to vary between species , roams during the summer months. A. mainae is the largest mygalomorph species of the region, reaching up to 5 cm in length. Males of most other species measure up to 2–3 cm in total body length24. These male dispersal events are likely to be the most dangerous times for mygalomorphs as they are exposed to a suite of predators to which they would not be exposed when in burrows. We aimed to test if there were selective or increased seasonal predation of male mygalomorphs while they are exposed on the surface during the mating season. Mygalomorph spider species have specialised microhabitats requirements. Hence, factors such as leaf litter, mid-storey and canopy cover may vary between species in their exposure to predators. In conjunction with other increased pressures present in an urban context, predation may decrease the chances of ongoing persistence of SREs.For most species in Perth, south-western Australia, male roaming occurs during periods of high humidity in winter, possibly due to physiological constraints27. As SRE endemic taxa are of high conservation priority and predation marks can be confidently identified on clay models, we decided this was the most effective and least harmful approach to test our hypotheses27. Our study is the first to use clay models to measure predation types, predation size and frequency on mygalomorph spiders. The questions we asked were:What are the local predator guilds?Is there evidence of competition between identified predator types?Is surrounding land-use correlated with guilds?(2)Is there a significant difference between predator preferences in terms of spider vs. control, size of model, and season?(3)Is predation frequency by identified predator types influenced by microhabitat variables such as leaf litter, mid-storey and canopy cover?(4)Does identified predator type vary in terms of number of attacks or attack location on the body of the spider?We used clay models to explore the effects of habitat patch size, microhabitat and seasonal predation on spiders in urban areas. Clay models have been successfully used to assess predation on small taxa such as lizards, snakes, mice, invertebrates and bird eggs28 as birds (33%), lizards (25%), rodents (29%) and wasps (13%).Of the 2400 models used, 663 (28%) were attacked. From marks on the models predators were identifiedLocal predator guilds were defined based on similar predation behaviour across ninety-six model-patch-season units. Six distinct groups were recognised Fig. : group 1Three extrinsic factors fitted using principal component correlation (PCC) were significant using Monte-Carlo attributes in an ordination (MCAO): leaf litter cover and proportion of parkland surrounding the patch within a 150 m and 250 m zone . Overall model accuracy were moderate  with significant variables reported in Table Wasp attacks were found only on spider models (b). Wasps were the only predator that showed significant p < 0.0001) seasonal differences, predating significantly more in summer than in winter  than once by a single identified predator type. However, attack number varied significantly among identified predator types Table . Birds, Bird attack rates were similar for both middle and edges of models Table . Wasps a29.A clear preference for non spider-mimicking controls by all identified predator types indicated that predators were selecting prey based on visual cues, and not only due to curiosity towards foreign objects. The use of controls is surprisingly rare in experiments involving clay models, but is encouraged30 and such guilds may also interact with each other, influencing their impact on prey31. Prey species that are vulnerable to disturbance10 may be further impacted by changes in local predator guilds. Disturbance can also change local predator guilds by disrupting landscape structure and resources that were previously stable32. Generalist predators may be less affected by disturbance than specialists as their broad prey spectrum does not restrict their activities in any particular area33. Amount of parkland surrounding patches significantly influence predator guilds, increasing towards guilds of predominantly rodents and lizards  back to its lair. Records of pompilids collected in Perth  correlate with Aname males roaming phenology (October to February), though they also occur through to April. Wasps may not be targeting specific spider species, but a specific size of prey which may depend on size of the wasp35. For example, C. bicolor may have been attacking large models mistaking them for sparrassids of a similar size, rather than targeting mygalomorphs. Both Aname and some sparrassids, such as Dingosa36, have open burrows for which wasps may display similar searching behaviour. However, spider response varies – Dingosa avoid wasps by running out of their burrow (pers. obs.), whereas mygalomorphs are more likely to defend burrows through phragmosis i.e. defend the burrow using their body. Predation rates on models are limited in this way as they do not reflect actual outcomes of encounters between wasps and prey.Pompilidae, the family of wasps that prey on spiders, is thought to be highly specific to the species or size of spider preyMissulena which has bright red jaws), predation by rodents may be relatively common. Although bats are known to predate on spiders37, their contribution to predation rates is not known and no bat bite impressions were identified in this study. Predators may use senses other than visual cues to locate their prey39. We suggest future predation experiments either test these factors or be mindful when interpreting results.Microhabitat variables and location of predation events indicate that while rodents and lizards predate at similar rates in the same patches, they occupy different microhabitats. Lizards preferred to forage in areas with high leaf litter and canopy cover, while rodents preferred to forage primarily under high understory cover. Location of lizard and rodent bite marks on the edge of control models or on the legs of model spiders probably reflects the approach of these predators at ground-level. Most lizards are active during the day, but rodents are nocturnal. As mygalomorph spiders are thought to roam primarily at night  since Perth was established makes it impossible to know if predation rates are higher, similar or lower than before urbanisation. It is possible that rodents living in native bushland remnants have a similar deleterious effect as they do on islands19. The adaptation of natural predators such as ravens and magpies in urban areas41 may also have a profound impact on spider populations, but potentially only on spiders active during the day (Missulena). High predator mobility, in conjunction with a higher abundance, may be especially detrimental to species restricted to smaller patches14. We found no significant difference in predation type or rate with size of patch, which is worrying, as it suggests that populations in smaller patches may have a higher extinction debt43.As a novel threat, predation by invasive rodents is of high conservation concernTiliqua rugosa), may encounter only a few models during the week, whereas a fast-moving territorial lizard, such as a scrubland skink (Morethia obscura), may encounter many models and multiple times. Site characteristics such as size and microhabitat variables may be accurate indicators for adequate territory ranges for different species. For example, rodents may require a much lower territory range to satisfy their diets than would corvids because of the vast differences in size and mobility capabilities. For future studies, it is advisable that predator range, if known, be taken into account when interpreting results in regards to size and connectivity of patches.It is possible that predation rates by different taxa are not indicative of an individual attacking one model. For example, a raven may learn quickly that models are not suitable food sources after the first instance. Conversely, a wasp may not learn and continue to attack multiple models in the same area indefinitely. As wasps are highly mobile, attacks on multiple spider models over one quadrat may actually indicate low levels of learning, rather than a high density of wasps. Similarly, a slow-moving lizard, such as a bobtail , located in the South-west Australian (SWA) Global Biodiversity hotspoties Fig. .Figure 3To test any effect of season, sampling was conducted during two different time periods in 2016 . Size and phenology replicate real mygalomorph species that occur in the urban remnant vegetation of Perth  , size (mm), location on the model and number of attacks were identified from distinctive marks left by predators on the clay models see Fig. .Figure 5Leaf litter, mid-storey vegetation and canopy vegetation were recorded. We assumed this was related to the visibility of models to predators. Surrounding land-use area was measured by generating shapefile layers of buildings, roads, parkland and other remnant vegetation within 250 m, 150 m and 50 m buffers. Buffers were measured for both patch and quadrats in the open source program Quantum Geographic Information System (QGIS 2014). The Nearmap plugin was used to determine the proportion of different land-use within each buffer area.45, using PATN3.1146. All 2400 models were included in the cluster analysis (predated and non-predated) and converted into a proportion within each unit. Predator types were used as intrinsic factors and combined patch, model type and season were used as separate units (25 models in each unit). The Gower metric was used to determine the degree of similarity between different identified predator types, followed by hierarchical polythetic agglomerative clustering using flexible UPGMA. We used the Two-Step association measure to determine influence of variable groups on identified predator types. A two-way table is a visual representation of the association between different units (guilds) and the influence of identified predator types to form groups. Darker cells show greater association than lighter cells. MDS ordination portrays the spatial relationship between objects in a way that best preserves relative positions. ‘Stress’ measures the level of distortion from reducing axes (to two in this case), with low stress values (i.e. <0.20) indicating better representation of positions than high stress. The Minimal Spanning Tree (MST) is a form of network analysis that connects each object to its nearest neighbours. If there is a high congruence between cluster analysis, ordination and network analysis, then resulting categories are supported. This allows ‘local guilds’32 of predators to be determined and analysis of the influence site variables. Competition within local predator guilds can then also be assessed. During analysis, we found that attack marks from bird species on the clay measuring ≤2 mm and >2 mm birds diverged. Extrinsic factors were fitted to the ordination using PCC and significance determined using MCAO. The strong clustering formed by using patch, model type and season as units indicated that these factors may be useful in predicting identified predator type behaviour through multinomial logistic regression.As this study examines predator behaviour we used predation as intrinsic factors, and determined whether site variables correlated with the resulting multidimensional scaling ordination axes in numerical taxonomic analysis47. Model coefficients (logit) represent the change in log-odds of a variables influence relative to the reference category (in this case “birds”). A positive logit indicates the effect of the predictor on predation (relative to birds) is positive. We have used “b” to represent the logit in results. Predator type  was the response and site, site size, season, model size and type, attack number, attack location and litter, understory and canopy cover the predictors. Data was split into a train and test set  and the packages ‘caret’ and ‘e1071’ used to assess classification accuracy. The package stargazer48 was used to plot significance levels. Predator size categories were reduced for analysis to: 1 (A), 2 (B) and >2 (C). The location of predation was standardised between model type: ‘Body’ of spider models were subsumed into ‘middle’, and spider model ‘legs’ into ‘edge’. The number of attacks was categorised into: 1–4 (A), 5–8 (B) and >8 (C). A multinomial logistic regression was preferred over other GLM’s as several predictors were categorical and the assumptions of normality and homoscedasticity could not be met. Additionally, as model size and type were recorded at the within plot scale and were to be included in the analysis, data was not averaged to predation rate per plot.A MLR was fitted  using predated data only (n = 510). MLR require a nominal dependant variable with more than two levels and can handle categorical variables"}
{"text": "Cholangiocarcinoma is a malignant tumor originating from bile duct epithelium. Currently, the treatment strategy is very limited and the prognosis is poor. Recent studies reported celastrol exhibits antigrowth and antimetastasis properties in many tumors. Our study aimed to assess the anti‐CCA effects of cholangiocarcinoma (CCA) and the mechanisms involved in it.In this study, the long‐term and short‐term antiproliferation effects was determined using colony formation and Cell Counting Kit‐8 (CCK‐8) assays, respectively. Flow cytometry was performed to quantify apoptosis. Furthermore, wound healing and transwell assays were performed to determine the cell migration and invasion capabilities, respectively. To further find the mechanism involved in the celastrol‐induced biological functions, LY204002, a PI3K/Akt signaling inhibitor, and an Akt‐1 overexpression plasmid were employed to find whether PI3K/Akt pathway was involved in the celastrol‐induced CCA cell inhibition. Additionally, short interfering RNA (siRNA) was also used to investigate the mechanism involved in the celastrol‐induced PI3K/Akt signaling inhibition. Western blotting and immunofluorescence assays were also performed to detect the degree of relative proteins. Moreover, we validated the antiproliferation and antimetastasis effects of celastrol in vivo by constructing subcutaneous and lung metastasis nude mice models.We discovered that celastrol effectively induced apoptotic cell death and inhibited the capacity of migration and invasion in CCA cells. Further mechanistic study identified that celastrol regulated the PI3K/Akt signaling pathway, and the antitumor efficacy was likely due to the upregulation of PTEN, a negative regulator of PI3K/Akt. Blockage of PTEN abolished the celastrol‐induced PI3K/Akt signaling inhibition. Additionally, in vivo experiments conformed celastrol inhibited the tumor growth and lung metastasis with no serious side effects.Overall, our study elucidated a mechanistic framework for the anti‐CCA effects of celastrol via PTEN/PI3K/Akt pathway. Our research provided mechanistic insight into the anti‐CCA effect of celastrol by the regulation of the PTEN expression, and provide a novel therapeutic strategy for CCA. Mechanically, the blockage of PI3K/Akt ‐mediated signaling by upregulating PTEN is responsible for the changes. Our discoveries provided novel insights into celastrol as a promising anti‐CCA agent.22.1Human cholangiocarcinoma TFK‐1 and HuCCT‐1 cell lines were maintained in RPMI‐1640 medium containing 10% FBS and 1% penicillin‐streptomycin solution  in an incubator with 5% carbon dioxide at 37°C.2.2Celastrol was purchased from Sigma, which was dissolved in dimethyl sulfoxide (DMSO) at the concentration of 100 mmol/L before use. LY294002 was purchased from Abcam Corporation . The cytotoxic activity of celastrol on CCA cells was analyzed using CCK‐8 . Primary antibodies were as followed : cleaved caspase‐3, 9, Bax, Bcl‐2, MMP‐2, 9, vimentin, E‐cadherin, PTEN, PI3K, Akt, p‐Akt, mTOR, p‐mTOR, Ki‐67, NF‐κB. In addition, β‐actin  was used as an internal control.2.3CCK‐8 assay was used to determine the cell viability according to the manufacturer's instructions. Briefly, after cells were seeded in a 96‐well plate and incubated with celastrol for indicated hours. The optical density (OD) values were used as the index of cell viability at the absorbance of 450 nm. Five replicate wells were set up in each group.2.4A total of 700/well cells were firstly seeded in 35‐mm dishes until most of them were attached to the bottom. Then, different concentrations of celastrol was used for each dish. After two weeks, the cells were fixed and stained. Colonies containing ≥ 20 cells were counted.2.5TM C6 flow cytometer (Becton‐Dickinson). Each experiment was conducted for three times.Apoptotic degree of cells was measured by Annexin V‐FITC kit (BD Pharmingen). Cells in each group were firstly digested by trypsin without EDTA. Subsequently, the collected cells were resuspended with binding buffer (500 µL) and annexin V–FITC (5 µL) for 30 minutes. After that, PI (10 µL) was added in dark for 10 minutes at room temperature. Finally, the percentage of apoptotic cells were determined using BD Accuri2.64 cells was initially seeded in each well of a 6‐well plate for 24 hours. Cell layer was scratched by a pipette tip (200 µL). Subsequently, cells were then continued cultured with indicated concentrations of celastrol. Representative wound was imaged by an optical microscope. Each experiment was conducted for three times.A total of 5 × 102.74) were initially suspended with or without celastrol into the upper chamber, and the rest of the steps are the same as our previous reports.The experiment was conducted using 24‐well transwell chambers with 50 µL of Matrigel , and the cell invasive ability is assessed by the number of cells passing through the chambers. Cells  for 20 minutes at room temperature. To increase cell membrane permeability, cells were also incubated with goat serum (10%) and Triton (1%). Subsequently, cells were incubated with antibodies for p‐Akt (Thr308) (1:200), p‐Akt (Ser437) (1:200) or PTEN (1:200) antibodies overnight at 4°C. After that, cells were incubated with an Alexa Fluor‐conjugated IgG secondary antibody  for 1 hour at room temperature. Finally, DAPI was used to locate the nuclei. Each experiment was conducted for three times.2.9Cells were transfected with siRNA‐PTN or scrambled siRNA using Lipofectamine 2000 (Invitrogen). The siRNA sequences were listed as follows: siRNA‐PTEN , and scrambled siRNA . PTEN expression was detected by Western blotting. Akt‐1 plasmid was obtained from GeneCopoeia, Inc. These plasmids and siRNA were subsequently transfected into the cells using the Lipofectamine 2000 reagent (Invitrogen).2.10Proteins expression were evaluated by western blotting as our previous studies described.2.117/0.2 mL PBS) subcutaneously into the right flank for xenograft model. After 2 weeks modeling, mice were randomly divided into 2 groups: celastrol (2 mg/kg) or PBS every 2 days (i.p.). Tumor size was measured every 7 days using calipers. (length × width2)/2 was used to analyzed the volume.BALB/c nude mice  were obtained from Beijing Vital River Laboratory Animal Technology Co, Ltd. All the care and use of animals followed the applicable international guidelines and were approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University. For antiproliferation studies: mice were injected with TFK‐1 cells (1 × 106/0.2 mL PBS) via trail vein. After 2 weeks modeling, mice were randomly divided into two groups: celastrol (2 mg/kg) or PBS every two days (i.p.). All the mice were sacrificed and the lung tissue was harvested after four weeks. All the lung nodules were counted before paraffin embedded.For antimetastasis studies: Mice were injected with TFK‐1 cells , vimentin (1:200), cleaved caspase 3,9 (1:100), PTEN (1:100), and p‐AKT (1:50) were incubated overnight at 4°C. The corresponding secondary antibody was incubated for 1h. Finally, DAB (Invitrogen) was used for color development, and the slides were estimated by three independent pathologists individually.2.14t test or one‐way ANOVA were used for the two groups or more than two groups comparison, respectively. P < .05 was considered statistically significant, and P < .001 was highly considered significant.Statistical analysis was used to elevate all the data. Student's 33.1We initially examined the inhibitory effects of celastrol Figure B,C. To f3.2To examine whether the antiproliferative effect was resulted from apoptosis induction, we performed apoptosis assay. After incubated with celastrol , TFK‐1 and HuCCT‐1 cell lines were analyzed by flow cytometry (FCM) analysis using Annexin V/PI assay kit. Figure Furthermore, western blotting was performed to assessed the important signaling proteins involved in celastrol‐induced cell apoptosis. As shown in Figure 3.3To determine whether celastrol could inhibit migration and invasion of CCA cells, wound healing was initially performed after cultivation with celastrol for 24 hours (a low concentration that did not induce cell apoptosis). According to the data in Figure Epithelial‐to‐mesenchymal transition (EMT) act as a primordial role in cell migration and invasion of cancer cells.3.4Aberrant expression of PI3K/Akt signaling promotes the development of CCA oncogenesis, proliferation, and metastasis.To further elucidate the role of PI3K/Akt pathway in celastrol inhibiting CCA development, we initially pretreated TFK‐1 cells with celastrol at different concentrations , then activated or inactivated the pathway by Akt1 transfection and LY294002, respectively. Cell viability was elevated by CCK‐8 assays. Data showed that overexpression of Akt reverse the celastrol‐induced TFK‐1 cells viability inhibition. Nevertheless, LY294002 treatment resulted in a more inhibition compared to the control groups.  mutations occur in CCA patients, and PTEN acts as a negative regulator of PI3K/AKT signaling pathway in CCA cells. We hypothesized that celastrol elevated PTEN expression, and thus inhibited PI3K/Akt activation. As expected, the PTEN expression was observed significantly upregulated in TFK‐1 cells by western blot, following 24 hours of celastrol treatment Figure A. Additi3.63 followed by treatment of vehicle control or celastrol (2 mg/kg) every 2 days. Figure We initially determined the antiproliferation efficiency of celastrol using a subcutaneous xenograft mice model. 12 mice were initially injected subcutaneously with TFK‐1 cells for 2 weeks, and the tumor volume had reached to a mean size of 115 mmWe subsequently determined the antimetastasis efficiency of celastrol using a lung metastatic mice model. 12 mice were injected with TFK‐1 cells via the trail vein for 2 weeks followed by celastrol treatment. After 4 weeks, the number of nodules and incident rate were drastically decreased with celastrol treatment Figure D compareFurthermore, PI3K/PTEN/Akt signaling‐related proteins expression was analyzed in the excised tumor tissues using Western Blot. As expected, the p‐AKT and vimentin expression were decreased, whereas the cleaved caspase‐9 and PTEN expression were increased according to the data. Figure G A immun4Limited clinical option for treatment remains an aggressive challenge for the patients with CCA, especially in advanced stage. Increasing studies suggest that natural compounds may be a new option for the development of potent cancer treatments.Proapoptosis activity acts as one of the most common antitumor mechanisms to control tumor proliferation, and numerous studies proved that mitochondrial apoptosis plays a vital role in human cholangiocarcinoma.EMT is an important molecular mechanism of invasion and metastasis of malignant tumors.The PI3K/Akt pathway acts as a vital role in tumor oncogenesis and development, including cell viability, autophagy, migration, metastasis, angiogenesis, and drug resistance.PTEN (phosphatase and tensin homologue) is known as a tumor suppressor, which was frequently lost or mutated in various human malignances.5In summary, we report a novel component that exhibits a significant effect on CCA cells growth and metastasis in vivo and in vitro. Further mechanism studies demonstrated that the inhibitory effects are attributed to the induction of apoptosis, and the blockade of migration and invasion might be regulated by perturbations in the PI3K/Akt signaling. Furthermore, and these perturbations were regulated by the drug induce‐PTEN upregulation Figure . TherefoThe authors declare no conflict of interest.Biqiang Zhu designed and performed the experiments. Yunwei Wei analyzed the data. Biqiang Zhu wrote the manuscript."}
{"text": "Lupinus angustifolius L.) as a grain legume crop, exploiting discovered alleles conferring low-alkaloid content (iucundus), vernalization independence (Ku and Julius), and reduced pod shattering (lentus and tardus). In this study, a L. angustifolius mapping population was subjected to massive analysis of cDNA ends (MACE). The MACE yielded 4185 single nucleotide polymorphism (SNP) markers for linkage map improvement and 30,595 transcriptomic profiles for expression quantitative trait loci (eQTL) mapping. The eQTL highlighted a high number of cis- and trans-regulated alkaloid biosynthesis genes with gene expression orchestrated by a regulatory agent localized at iucundus locus, supporting the concept that ETHYLENE RESPONSIVE TRANSCRIPTION FACTOR RAP2-7 may control low-alkaloid phenotype. The analysis of Ku shed light on the vernalization response via FLOWERING LOCUS T and FD regulon in L. angustifolius, providing transcriptomic evidence for the contribution of several genes acting in C-repeat binding factor (CBF) cold responsiveness and in UDP-glycosyltransferases pathways. Research on lentus selected a DUF1218 domain protein as a candidate gene controlling the orientation of the sclerified endocarp and a homolog of DETOXIFICATION14 for purplish hue of young pods. An ABCG transporter was identified as a hypothetical contributor to sclerenchyma fortification underlying tardus phenotype.The last century has witnessed rapid domestication of the narrow-leafed lupin ( Lupinus angustifolius L., is a grain legume crop, appreciated as an organic fertilizer that improves soil structure and productivity, as well as providing a source of protein for human and animals. This species has witnessed rapid domestication during the last century. Several important agronomic traits have been identified and transferred into improved germplasm [Ku and Julius), low-alkaloid content (iucundus), reduced pod shattering (tardus and lentus), soft seededness (mollis), white flower color (leucospermus) and anthracnose resistance (Lanr1).The narrow-leafed lupin, ermplasm . These tKu or Julius loci diminished the need of vernalization and enabled temperature-independent sowing of L. angustifolius [Vernalization responsiveness is the natural adaptation to climatic conditions, based on the requirement of a prolonged low temperature period during germination to induce flowering ,3. Naturtifolius .L. angustifolius, and one of them, iucundus, was extensively implemented in breeding [A high level of quinolizidine alkaloids is a typical feature of primitive populations in many lupin species, as these chemical compounds protect plants from pests and fungi , howeverbreeding ,9. Some breeding ,11.L. angustifolius, namely tardus, affecting sclerenchyma strips of the dorsal and ventral pod seams, and lentus, modifying the orientation of the sclerified endocarp of the pod [Shattering of dry pods is natural process of seed dispersal, however, it is a very undesired trait in modern agriculture because it dramatically decreases harvested yield. Two unlinked recessive alleles contribute to reduced pod shattering in  the pod ,13.L. angustifolius is natively adapted to the Mediterranean climate which has hot dry summers, because of one of its survival strategies which is impermeability of seed coat to water. Hard-seeded germplasm has a long dormancy period and irregular germination. Recessive soft-seediness allele mollis confers water permeability and efficient seed germination [L. angustifolius allele for breeding because the desired phenotype is maternally determined [mination . It is ttermined . L. angustifolius has been reduced by high susceptibility to anthracnose, caused by the pathogenic fungus, Colletotrichum lupini (Bondar) Nirenberg, Feiler and Hagedorn [L. angustifolius was revealed to be controlled by several single dominant genes that were discovered in different germplasm resources, namely, Lanr1 in cultivar Tanjil, AnMan in cv. Mandelup, and LanrBo in the breeding line Bo7212 [The agronomic potential of Hagedorn . The rese Bo7212 ,18,19.L. angustifolius seed and flower color. The most widely exploited is the recessive allele leucospermus, affecting anthocyanin synthesis and resulting in bright seeds and white flowers [Several genes contribute to  flowers . iucundus (marker iucLi) [Ku (KuHM1) [mollis (MoLi) [lentus  [tardus  [To generate numerous molecular markers for agronomic trait selection in narrow-leafed lupin breeding programs, microsatellite-anchored fragment length polymorphisms (MFLP) fingerprinting has been exploited ,21. Trair iucLi) , Ku (KuH (KuHM1) , mollis s (MoLi) , lentus nd LeLi) ,24, and nd TaM2) ,26. Narrnd TaM2) ,28,29,30nd TaM2) ,32, and nd TaM2) ,34.L. angustifolius. These new data were exploited for construction of a linkage map and for determination of expression quantitative trait loci (eQTLs) related to selected domestication traits.Recently, a new method of transcriptome-based genotyping-by-sequencing, called massive analysis of cDNA ends (MACE), has been developed . The MACL. angustifolius mapping population (83A:476 × P27255), yielding 11,864 markers. A total of 9304 markers were localized within gene sequences whereas 2560 markers were found in loci lacking any annotation. There were 4185 MACE markers retained after application of total missing data threshold (counting heterozygotes and no data scores), followed by inference of consensus segregation for genes represented by several single nucleotide polymorphism, SNPs. There were 3532 genes represented by single markers, four genes were represented by pairs of markers with heterogeneous segregation patterns, and 645 markers were localized in unannotated loci. The annotation of markers is provided in the The MACE protocol was applied for 89 RILs and for parental lines of L. angustifolius genome physical and linkage mapping. Because the L. angustifolius karyotype carries numerous small and very uniform chromosomes, the BAC-derived markers have been frequently used as chromosome-specific landmarks to validate physical linkage of particular genome regions, as well as to facilitate assignment of particular chromosomes to linkage groups [Ku locus, as well as for comparative mapping of genes from isoflavonoid and fatty acid synthesis pathways [The MACE is a method providing sequences anchored in the 3′-ends of mRNA and can be used to develop sequence-defined markers, as well as to quantify gene expression ,36. In te groups ,45,46,47pathways ,49,50,51Ku, tardus, lentus, mollis, leucospermus, iucundus and Lanr1) [2p-values of distortion from expected 1:1 segregation are provided in Markers used for linkage map development included 4,185 MACE and 10 BAC-end PCR markers developed in this study as well as previously published data including seven trait loci (d Lanr1) , eight td Lanr1) ,25,52,53d Lanr1) ,50,51,54L. angustifolius draft genome sequence [The segregation pattern of 59.7% of the markers was redundant, and therefore the map contains 1735 loci, namely from 60 to 120 loci per linkage group. The lengths of linkage groups vary from 78.5 to 156.38 cM, reaching 2163.63 cM in total. The results of linkage mapping are provided in sequence  and thisL. angustifolius linkage maps [L. luteus [L. albus [L. angustifolius genome mapping study, namely 87 lines with only 78 lines overlapping with previous mapping studies, was too low to provide the high resolution required for significant improvement of genome assembly, and resulted in high marker redundancy, reaching 89.9% [Mapping data from the most recent age maps ,34 were . luteus , as wellL. albus . Neverthng 89.9% . iucundus, 50 for Ku, 35 for leucospermus, 29 for lentus, 17 for tardus, 11 for Lanr1 and five for mollis. The values of the t-Student test association between domestication trait segregation and gene expression patterns, including FDR correction and statistical significance analysis, are provided in iucundus trait segregation highlighted lysine biosynthesis and lysine metabolism, as well as cofactor binding and coenzyme binding, as the most overrepresented processes and functions, respectively  are provided in ectively . This wamollis) to 61 (iucundus) genes had eQTL peaks localized  was cis-regulated, whereas the vast majority (74%) was trans-regulated. Furthermore, from 34 genes for iucundus that had association values between their expression patterns and trait segregation above 0.5 or below −0.5, as many as 30 revealed a positive association with wild, high alkaloid phenotype. Moreover, as many as 16 genes highly associated with iucundus revealed to have their major eQTL locus explaining more than 50% of their observed expression variance localized directly at iucundus, or very close to it , for example, affects nicotine metabolism, as well as regulates the ETHYLENE RESPONSE FACTOR 189, a key transcription factor in nicotine biosynthesis pathway [L. angustifolius quinolizidine alkaloid biosynthetic gene because it revealed similar expression pattern to the LDC gene [MAJOR LATEX PROTEIN 423 , which is hypothesized to be involved in stress responsive activation of biosynthetic pathway of coumestrol, a coumestan isoflavone in soybean [L. angustifolius due to highly elevated expression in bitter P27255 accession [ETHYLENE RESPONSIVE TRANSCRIPTION FACTOR RAP2-7, a candidate locus for iucundus, evidenced by a gene expression study involving transcriptome sequencing of four accessions and quantitative RT-PCR profiling of 14 accessions differing in alkaloid content, as well as by molecular marker development and linkage mapping [RAP2.7 at iucundus locus, another cis-regulated component, Fe SUPEROXIDE DISMUTASE 2 , revealed similarly high LOD and explained eQTL variance values, but opposite direction of association. Other iucundus-associated genes have included Lup005321 (OIW13431.1) and Lup005322 (OIW13432.1) encoding homologs of DIHYDROFLAVONOL 4-REDUCTASE which is one of the key genes from anthocyanin biosynthesis pathway [iucundus region also includes a Lup021586 (OIV95196.1) gene encoding HXXXD-type ACYL-TRANSFERASE . The expression profile of LaAT has been highly associated with alkaloid content in L. angustifolius [pauper locus in L. albus, as evidenced by linkage mapping and validation survey in a set of 127 bitter and 23 sweet accessions [Among the genes with expression positively associated with high alkaloid phenotypes in the RIL population, the highest LOD values of eQTLs were revealed for Lup009726 , Lup028431 , Lup015923 , and Lup007628  a. The Lu studies ,59,60. L pathway ,62. IndeLDC gene . Lup0159 soybean . Lup0159ccession . Lup0076tifolius . Moreovecessions .cis-regulated genes revealed eQTLs localized at iucundus locus, including three hypothetical components of alkaloid biosynthesis pathways, Lup007706 (OIW07664.1), Lup017658 (OIW07643.1), and Lup032669 (OIW21355.1). Lup007706 encodes a representative of a S-adenosyl-l-methionine-dependent methyltransferases superfamily protein. N-methylation of quinolizidine alkaloids was confirmed to occur in crude protein extracts from Laburnum anagyroides carrying S-adenosyl-l-methionine: cytisine N-methyltransferase [S-adenosyl-l-methionine-dependent N methyltransferase catalyzes a nitrogen methylation involved in vindoline alkaloid biosynthesis in Madagascar periwinkle (Catharanthus roseus) [4-HYDROXY-TETRAHYDRODIPICOLINATE SYNTHASE gene which is generally involved in biosynthesis of l-lysine, a precursor of quinolizidine alkaloids. This gene revealed considerably elevated expression in bitter accessions of L. angustifolius, indicating its hypothetical involvement in alkaloid biosynthesis pathway [CARBOXYLESTERASE 1 gene. CARBOXYLESTERASE 1 was evidenced to be involved in one of the final three steps of noscapine alkaloid biosynthesis [iucundus locus. This study provided novel evidences supporting the concept that RAP2.7 may control low-alkaloid iucundus phenotype, however, further evidence would require cis-trans tests. Nonetheless, such studies are hampered by very low transformation efficiency in narrow-leafed lupin [In addition to Lup007628 and Lup007632, nine other nsferase . Moreove roseus) . Lup0176 pathway . Lup0326Ku locus . Genes showing the highest gene expression association and eQTL peak co-localization (≤2 cM) with Ku included Lup011808  and Lup015372 . Lup015372 encodes hypothetical uncharacterized protein, whereas Lup011808 a homolog of A. thaliana CALCIUM/CALMODULIN-REGULATED RECEPTOR-LIKE KINASE 1, CRLK1. CRLK1 confers cold responsiveness in plants via C-repeat binding factors (CBF) pathway [CBF in Arabidopsis delays flowering by promoting the expression of FLOWERING LOCUS C (FLC), indicating a link between cold signaling and flowering time regulation [INDUCER OF CBF EXPRESSION 1 (ICE1) which integrates cold signals into FLC-mediated flowering pathway [SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) gene was also identified [CBF pathway affects flowering time but does not affect vernalization response in Arabidopsis [NtCBK1) from N. tabacum functions as a negative regulator of flowering; high levels of NtCBK1 in the shoot apical meristem extended the vegetative phase of growth [CRLK1, in this study, was revealed to be positively associated with late flowering phenotype. These observations bring attention to the hypothetical involvement of calcium and calmodulin link with FLC pathway in flowering time regulation in L. angustifolius. Legume genomes do not contain FLC homologs but other genes from this pathway, including activators and repressors of FLC, are present [The P27255 parent is late flowering and requires vernalization for flowering induction, whereas the 83A:476 parent is early flowering and vernalization independent. In this study, seeds were subjected to vernalization procedure to ensure transition from vegetative to generative phase in all analyzed RILs. Such an approach could result in diminishing of some differences in expression profiles of vernalization-responsive genes between early and late flowering RILs. However, despite this partial pre-sowing vernalization, relatively a large number of genes revealed to have their eQTL peaks closely localized to Ku locus b. Contra pathway . Anotherentified . A recenbidopsis . Moreove present .Ku eQTLs include Lup011781 (OIW03144.1), Lup011739 (OIV96743.1), Lup011836 (OIW03199.1), and Lup002110 (OIW20134.1) sequences. Lup011781 has been identified as a homolog of MHM17-10 (AT5G56980) gene. It is pathogen-associated molecular pattern-induced gene with unknown function, putatively participating in jasmonic acid pathway [GALACTURONOSYLTRANSFERASE-LIKE 10, which is involved in cell wall organization and its expression is regulated by FLAVIN-BINDING KELCH REPEAT, F-BOX 1 (FKF1), blue light receptor, and well-known photoperiodic flowering time regulator [GTF2H2), performing basic functions in transcription and nucleotide excision repair of damaged DNA. Lup002110 has been annotated as a representative of the UDP-glycosyltransferases protein family. One of the A. thaliana UDP-glycosyltransferases, UGT87A2, was revealed to be involved in the regulation of flowering in vernalization and gibberellin pathways via the flowering repressor FLC [Other egulator . Lup01180 AT5G569 gene. ItL. angustifolius gene underlying vernalization responsiveness, a homolog of A. thaliana FLOWERING LOCUS T (FT), LanFTc1  [LanFTc1 revealed negative association with late flowering phenotype. Similar LOD values were also revealed for Lup018024 (OIV92673.1), Lup020573 (OIW03269.1), and Lup018485 (OIW19675.1) genes. Lup018024 encodes a homolog of bZIP transcription factor, FD, which mediates signals from the FT gene at the shoot apex and promotes plant flowering in general [FD expression revealed a positive association with late flowering phenotype. In Arabidopsis, the FT-FD complex induces the transcription of several floral-promoting genes, such as SOC1 and FRUITFULL (FUL), which accelerate flowering, as well as APETALA1 (AP1) and LEAFY (LFY), which control floral meristem identity [AGAMOUS-LIKE 8 (AGL8)/ FUL and AP1 MADS-box transcription factors and was found to be negatively associated with late flowering phenotype. Lup020573 was annotated as MYB60 transcription factor. MYB transcription factors perform various regulatory functions in plants in responses to biotic and abiotic stresses, development, differentiation, metabolism, defense, etc. [As plants in this study were subjected to a moderate vernalization procedure, differences in gene expression resulting from variation in vernalization responsiveness should be reduced as compared with those expected for nonvernalized plants. This reduction might be highlighted by a relatively low LOD value (as compared with other eQTLs from this region) of the major LOD 7.0) ,81. As e general . Howeveridentity . Indeed,Ku eQTLs shed light on vernalization pathway in L. angustifolius, providing transcriptomic evidence for the contribution of several genes acting upstream of FLC in CBF and UDP-glycosyltransferases pathways. The study also revealed transcriptomic contribution of conserved mechanism of FT-FD regulon on transition from vegetative to generative development phase in L. angustifolius.To summarize, analysis of lentus (le) allele changes the orientation of the sclerified endocarp in the pod, substantially reducing torsional forces after drying [lentus were identified to have major eQTL peaks localized in the proximity of this locus: Lup018336 , Lup018348 , and Lup018228  , MODIFYING WALL LIGNIN-1, and MODIFYING WALL LIGNIN-2 (At1g31720/MWL-1 and At4g19370/MWL-2) [DETOXIFICATION14, a member of the multidrug and toxic compound extrusion (MATE efflux) family [le allele affects a pod pigmentation, resulting in a purplish hue of young pods and a bright yellowish-brown color on the internal surface of mature pods. Lup018348 may be responsible for this pigmentation, whereas Lup018336 for pod shattering in L. angustifolius.The recessive r drying . In thisOD 13.0) c. All tha margin . Another0/MWL-2) ,87,88. L) family . MATE tr) family . Some MA) family ,92. Anthtardus (ta) allele affects the sclerenchyma strips of the dorsal and ventral pod seams, greatly increasing the fusion of two pod halves and moderately hampering their separation when drying [tardus, namely Lup002465 (OIW17837.1) and Lup002448 (OIW17820.1) (RCN1) is required for hypodermal suberization of roots [ABCG1 confers suberin formation in potato tuber periderm [tardus trait.The recessive n drying . Two gen17820.1) d. Lup002of roots . Similarperiderm . Some ABperiderm ,97. Moreperiderm . ABCG trmollis provides water permeable testa at maturity [A. thaliana DWARF5 gene)  e. Becausfluidity . Mutatiofluidity . Recentlthaliana . Mollis leucospermus allele confers white flower and bright seed pigmentation in L. angustifolius. A similar trait in pea was conferred by a basic-helix-loop-helix (bHLH) transcription factor [leucospermus locus. Two genes with expression positively associated with recessive allele revealed eQTL peaks close to leucospermus, namely Lup008087 (OIW21684.1) and (Lup017573) OIV97389.1  annotated as F-BOX/WD repeat-containing protein. Interestingly, a single mutation in an F-BOX domain-containing protein, OsFBX310, confers brown hull phenotype in rice resulting from a high content of total flavonoids and anthocyanins [Recessive n factor  but eQTLV97389.1 f. Lup008ocyanins , howeverL. angustifolius genome and transcriptome analysis. As a method for gene expression analysis, the MACE was first used in chronic kidney disease survey [Calliphora vicina pupae [Lolium perenne), highlighting a candidate LpPg1 resistance gene and yielding numerous SNPs which were further transformed into PCR-based molecular markers [Pisum sativum) providing single nucleotide variants subsequently converted into CAPS markers [Sym33 [Phaseolus vulgaris seeds and Solanum lycopersicum pollen [Triticum aestivum [This study is the first report on exploitation of the MACE for e survey  and in dna pupae . The MAC markers . The MAC markers . Furthers [Sym33 . The MACm pollen ,40. The aestivum .L. angustifolius genotyping approaches were based on diversity arrays technology (DArT) profiling. DArT studies have highlighted low genetic diversity in narrow-leafed lupin breeding material as compared with primitive and wild germplasm [Several previous ermplasm . This doermplasm ,110. Theermplasm ,112.L. angustifolius can serve as a reference model for such studies across the whole genus. Moreover, information about candidate genes identified in L. angustifolius can be translated to other legume species as these processes are generally conserved.In this study, the MACE was revealed to be an advantageous technique for marker development and gene expression profiling. The eQTL mapping highlighted numerous genes involved in the vernalization response and alkaloid biosynthesis, providing a valuable contribution for further advancement of knowledge on the complexity of molecular networks controlling these two biological processes. Taking into consideration the recent improvements in deciphering the molecular basis underlying early flowering and low-alkaloid phenotypes, as well as addressing results reported here, L. angustifolius domestication several agronomic traits were identified and transferred into improved germplasm by classical selection approaches. Current breeding materials and cultivars usually carry desired alleles of all major domestication traits in homozygous state . However, domestication process was highly focused on these traits and resulted in approximately threefold reduction in genome-wide diversity across domesticated accessions as compared with their wild relatives [LanFTc1, CRLK1, FD, UGT85A2, GAUT10, and MYB60. Moreover, a common issue related to dry and warm weather patterns, which are expected to occur more frequently due to changing climate, is pod dehiscence. Identified candidate genes for lentus (a homolog carrying DUF1218 domain) and tardus (an ABCG5 transporter) await further genotypic and phenotypic exploration in wide genetic background because mapping population represents only a small fraction of diversity existing in L. angustifolius germplasm.During the process of elatives . Furtherelatives . Novel on = 89, F8) of L. angustifolius [ku), pod shattering , hard seeded (Mollis), blue flower and dark seed color Leucospermus), high alkaloid (Iucundus) and anthracnose susceptible (lanr1). 83A:476 has an opposite allele combination . Both parental lines are homozygous in relation to these alleles.The reference 83A:476 × P27255 recombinant inbred line (RIL) population  were vernalized for 16 days at 4 °C in darkness on Petri dishes with moist filter paper. Filter paper  was changed every four days to maintain phytosanitary conditions. Following vernalization, plants were transferred to pots  and grown in controlled conditions  at the Wielkopolska Center of Advanced Technologies in Poznań, Poland. Tissue was sampled from young leaves two times a day, 4 h after beginning of photoperiod and 1 h before the end of photoperiod on the 28th, 36th, and 44th day from sowing. Five biological replicates were collected.L. angustifolius genome assembly [http://www.lupinexpress.org). Normalization procedure was as follows [Frozen plant tissue  was homogenized using TissueLyser II  and two stainless steel beads (ø 5 mm) placed in a 2 mL tube . RNA isolation was performed using SV Total RNA Isolation System  according to the protocol. The concentration of RNA was measured using NanoDrop 2000  and A260/A280 ratio. RNA quality was visualized by 1% agarose gel electrophoresis (1X TAE) of denaturated samples. RNA concentration was equalized to 400 ng/µL in nuclease-free water. Samples from particular line  were bulked together in equal aliquots. 10 µL of mixture (4 µg of RNA) was subjected to the MACE protocol. The MACE profiling and SNP calling was outsourced . The MACE reads were aligned to the assembly  (http:// follows : The ave2) values for Mendelian segregation were estimated using the expected 1:1 ratio. The calculation of probability was based on χ2 and 2 degrees of freedom. Based on the segregation distortion observed in recently published L. angustifolius linkage map versions [2p-value threshold of 1 × 10−7 was applied. The 83A:476-like scores were assigned as “a”, the P27255-like scores as “b”, and the heterozygotes as “h”. If several cosegregating MACE markers in particular gene were identified, the marker with the lowest percentage of missing data was chosen to infer consensus segregation representing each cluster. To provide a mapping file, heterozygote scores were removed. Accepted missing data threshold was 11%. Chi-square  and sequenced using ABI PRISM 3130 Genetic Analyzer XL . Allele-specific PCR (AS-PCR) polymorphisms were visualized by 1% agarose gel electrophoresis, whereas nucleotide substitution polymorphisms were revealed by the cleaved amplified polymorphic sequence (CAPS) and dCAPS approaches [Segregation data for domestication traits and tightly linked SSR-derived markers were included in the study ,25,52,53proaches ,116. Resproaches ,118. Digproaches .Ku, tardus, lentus, mollis, leucospermus, iucundus, and Lanr1 segregation data (binary trait) by t-Student test in two classes of polymorphism. Obtained p-values were false discovery rate (FDR) corrected [p-value ≤ 0.01 were subjected to composite interval mapping performed in Windows QTL Cartographer V2.5  using 5 background control markers, window size 10 cM, walk speed 0.5 cM, and backward regression method. LOD threshold for QTL calling was established by permutation test (N = 1000) using the same parameters. Linkage groups and LOD graphs were drawn in MapChart [p-value ≤ 0.01 were analyzed for gene ontology (GO) term enrichment by hypergeometric test with FDR correction in Bingo [L. angustifolius genes obtained from Ensembl Plants Genes database . Whole-genome annotation was used as reference set. Results were provided as −log10 .Normalized gene expression values (continuous traits) obtained for RILs and mapping population parental lines were associated with orrected . Genes wMapChart . Moreovein Bingo  using GOThe massive analysis of cDNA ends was revealed to be applicable for molecular marker development and linkage map construction, as well as for gene expression evaluation and expression quantitative trait loci mapping.Ku locus shed light on vernalization response via FLOWERING LOCUS T and FD regulon, providing transcriptomic evidence for contribution of several genes acting in C-repeat binding factor (CBF) cold responsiveness and in UDP-glycosyltransferases pathways. This information can be relevant to decipher vernalization pathway in legumes, because legume genomes do not contain a major vernalization-responsive gene FLOWERING LOCUS C (FLC) but other genes from this pathway, including activators and repressors of FLC, are present.The analysis of vernalization independence iucundus locus highlighted a high number of cis- and trans-regulated alkaloid biosynthesis genes with gene expression orchestrated by a regulatory agent localized at iucundus locus, supporting the concept that the ETHYLENE RESPONSIVE TRANSCRIPTION FACTOR RAP2-7 gene may control low-alkaloid phenotype in narrow-leafed lupin.The study of low-alkaloid lentus locus selected a DUF1218 domain homolog as a candidate gene controlling the orientation of the sclerified endocarp and a DETOXIFICATION14 homolog for purplish hue of young pods.Research on reduced pod shattering ABCG transporter gene was identified as a hypothetical contributor to sclerenchyma fortification underlying reduced pod shattering tardus locus.An"}
{"text": "SD = 1.9) receiving FFT to investigate whether outcomes in FFT are predicted by CU traits and to what extent reliable changes in CU traits can be observed. The results showed that although CU traits are related to increased problem severity at baseline, they predicted neither treatment dropout nor post-treatment externalizing behavior and family functioning. CU traits were related to diminished improvement ratings, in particular with respect to parental supervision. Reductions in CU traits were observed across the time of treatment, and these were most profound among adolescents with elevated levels of CU traits at baseline. Further research should investigate whether certain evidence-based treatment components are more suited for adolescents with CU, and if the addition of specific intervention elements for reducing CU-traits could further improve outcomes for this high-risk population.Despite the availability of evidence-based treatment models for adolescent behavior problems, little is known about the effectiveness of these programs for adolescents with callous-unemotional (CU) traits. Defined by lack of empathy, lack of guilt, flattened affect and lack of caring, CU traits have been linked to long-term anti-social behavior and unfavorable treatment outcomes and might be negatively related to outcomes in evidence-based programs such as Functional Family Therapy (FFT). This study used a single-group pre-post evaluation design with a sample of 407 adolescents (49.1% female, mean age = 14.4 years,  Problem behaviors in adolescence can range in severity from heated verbal arguments and breaking curfew to aggression, violence, criminal behavior and drug use . These bEngagement to ensure family members participation in sessions; (2) Motivation to decrease the level of blame and negativity in the family and increase hope and motivation for change; (3) Relational Assessment to assess how risk and protective factors and family relational dynamics relate to the behavior problems; (4) Behavior Change to improve family skills such as communication, conflict management and problem solving to reduce problem behavior; and (5) Generalization to maintain and extend skill use inside and outside the family, prevent relapse and refer the family to additional support and services, if needed. FFT has a primary focus on family level risk factors as the reduction of these have been linked to diminished behavior problems  and age (older scoring lower than younger) .Secondly, we sought to evaluate if CU traits predict completion status and treatment outcomes in the context of FFT treatment. We assessed for this both related to adolescent behavior problems and family level problems. In line with previous research by Lastly, we wanted to determine the level of change in CU traits over time in the context of FFT-treatment. Previous research has shown mixed and limited evidence for the therapeutic malleability of CU traits in adolescents, so we hypothesized that for the majority of the participants in our study, CU traits would not reliably change and that any observed effect-size in relation to changes in CU traits would be small. We also examined the relationship between change in CU traits and change in adolescent behavior problems and family level problems.SD = 1.9). See This study builds upon data from a single-group pre-post design evaluation of FFT in eleven municipalities and two private treatment agencies in Denmark . Inclusir(405) = 0.689, p < 0.001, and the factor structure of the ICU12 might mostly be due to method variance effects  is a 6-item questionnaire completed at the end of FFT-treatment. Adolescents, parent(s) and therapist rate the family’s change on a general level (one item) and on five specific areas (one item per area): family communication skills, adolescent behavior, parenting skills, parental ability to supervise and monitor adolescent and family conflict levels. Each item is rated on a 6-point scale ranging from 0 (Things are worse) to 5 (Very much better), with a no change anchor at scale point 1 (Things are no different). Reliability across rater groups was good with αs ranging from 0.90 to 0.95.The FFT quality assurance database provided data on the number of days between each of the first three sessions , the number of sessions (as a measure of treatment intensity) and treatment duration. In addition, we collected the therapist assigned closing category, defined as either completion , drop out (family discontinued treatment prior to treatment completion), or non-completion .The adolescents and their parents completed the study questionnaires prior to or during their first FFT sessions for the baseline assessment, and during their last FFT session or shortly thereafter for the post-treatment assessment. The time between baseline assessment and post-assessment ranged from 17 to 473 days, with a median of 169 days and interquartile range of 88 days. Therapists entered data for each case in the FFT quality assurance database in relation to session dates and treatment completion status.There is no formal agency for ethical approval of survey and register-based studies in Denmark. The present study was, however, presented to the regional research scientific ethical committee in Southern Denmark in February of 2017 and deemed not required to obtain formal ethical approval as the study only used questionnaire-based data, according to governing law in Denmark. Information about the survey was provided to potential participants in an invitation letter, and the participants’ voluntary completion and return of the survey questionnaires constituted implied consent. The University of Southern Denmark (SDU) is the controller for processing of personal data in connection with the project, which has been included in SDU’s internal record of processing activities under file number [18/28527], cf. GDPR Article 30.All statistical analyses were conducted in SPSS Version 26. The relationships of CU traits to pre-treatment variables were assessed by partial bivariate correlations controlling for pre-treatment within-rater scores on the SDQ externalizing problems scale. This allowed for an assessment of the unique contribution of CU traits, as co-occurring levels of conduct problems and hyperactivity/impulsivity constitute the central aspects of externalizing problems .The association between CU traits and treatment completion was assessed by binary logistic regression of the two non-completion categories combined against the completed category. The conditional predictive value of CU traits on treatment service parameters, e.g., treatment length, were assessed by bivariate partial correlation controlling for baseline parent-rated externalizing problems. The prediction of CU traits on the continuous outcome measures was assessed by multiple regression analyses. As we were interested in the additional predictive value of CU traits, we controlled for baseline externalizing behavior as they might predict treatment gains to a large extent . In addiGiven that CU traits are meant to identify a more severe subgroup amongst adolescents with CD , a lack dRM) was calculated as the average-based change statistics over the time of treatment  and handled by pairwise deletion in the correlation and regression analyses. There were 3 outliers in relation to treatment pacing with more than 60 days between sessions, and these values were treated as missing in our analyses.The baseline zero-order correlations between CU traits and the measures of adolescent and family functioning and covariates are presented in p = 0.067 in the full sample. In the clinically elevated subsample, CU traits did statistically significant predict treatment non-completion, OR: 1.053, 95% CI , p = 0.037. However, the sensitivity of the model in its ability to correctly predict treatment non-completion, was relatively poor, with only 12.2% correct identification of non-completers in the clinically elevated subsample.Among all cases, 268 (65.8%) completed, 47 (11.5%) dropped out, 75 (18.4%) were non-completers and 17 (4.2%) were missing therapist-reported completion data. The results of the binary logistic regression model including both baseline externalizing and baseline CU traits as predictors, showed that CU traits were not significantly related to treatment non-completion, OR: 1.036, 95% CI , SD = 8.2, the total number of sessions averaged 11.8, SD = 5.6, and treatment duration averaged 160.2 days, SD = 72.3. The partial correlations of CU traits to treatment parameters controlling for baseline parent-ratings on the SDQ-externalizing scale are presented in r(206) = −0.166, p = 0.016.Treatment parameters showed that initial treatment pacing between the first two sessions averaged 9.46 days, SD = 0.92, for adolescent-report, 3.50, SD = 0.89, for parent-report and 3.06, SD = 1.08 for therapist-report. The post-treatment improvement ratings averaged 3.39, To further explore the significant predictions of CU traits on post-treatment improvement ratings, dRM) for the group average reduction was 0.36, 95%CI . The threshold value of elevated CU traits generated as 0.5 SD above the mean of the ICU12 was 16 scale points, which coincided well with the mathematically derived cut-off value of 15 scale points. In the subgroup of 60 adolescents with elevated pre-treatment ICU12 scores and an available post-treatment score, 26 (43.3%) were reliably declining, 32 (53.3%) had no reliable change and 2 (3.3%) were reliably increasing, with a corresponding group average based dRM of 0.91, 95%CI . The computation of a reliable change index for CU traits from baseline to post-treatment showed that for the 216 adolescents with available post-treatment CU ratings, 37 (17.1%) were reliably declining, 166 (76.9%) had no reliable change and 13 (6.0%) were reliably increasing. The corresponding repeated measures effect size  of the study participants had no reliable change, as hypothesized, while 17.1% had a reliable decline and 6.0% had a reliable increase. The theoretical values of standardized reliable change scores beyond −1.96 and +1.96 within a normal distribution is 2.5% in each direction. This means that in the whole sample we observed more cases both reliably declining and reliably increasing than what would be expected by measurement error and chance. These proportions were markedly different in the subsample with elevated levels of CU traits at baseline, where 43.3% had a reliable decline, 53.3% had no reliable change and 3.3% had a reliable increase. This represents a much larger proportion of cases reliably declining and a similar proportion of cases reliably increasing, compared to what would be expected by chance. This therefore provides some support for the therapeutic malleability of CU traits in adolescence.The increased proportion of reliable reductions in CU traits observed for the high CU group could entail that baseline CU traits act as a moderator for the malleability of CU traits in the context of FFT. For those with higher levels of CU traits at the start of treatment, we are more likely to observe a reliable decline during treatment. This is clinically relevant, as one might argue that only those with higher scores are in need of reducing their CU traits. This potential moderating effect of baseline CU might explain the somewhat mixed results from previous research . In addiChanges in CU traits were positively correlated with changes in externalizing problems and family functioning as reported by parents. This indicates that, from a parental perspective, changes in CU traits coincide closely with changes in externalizing behavior and to some extent with changes in family functioning. By youth-report, however, there was only a weak positive correlation between changes in CU trait and the change in family functioning. This could be due to the lower level of cross-rater correlations between CU traits and externalizing behavior as demonstrated in the baseline correlations and other studies . The facThis study has its strengths in investigating the association between CU traits and treatment outcomes in a large clinical sample of adolescents with behavior problems that received a well-established evidence-based treatment. There are, however, some limitations of the study pertinent to the interpretation of its findings. In the first place, this study used a single group pre-post-treatment design, which limits causal inferences. Secondly, a good proportion of the sample did not complete the study questionnaires at post-treatment, which lowers the study’s statistical power and generalizability. Thirdly, this study assessed the affective dimension of psychopathy, but not the other dimensions of psychopathy which could have provided improved predictions . FourthlThe results of this study indicate that CU traits do not seem to impede treatment completion or improvements in externalizing behavior and family functioning observed in FFT treatment. However, a relationship between CU traits and lower improvements in parental supervision and a lower number of generalization sessions was observed. This suggests that for adolescents with CU traits these treatment areas might need additional interventions and tailoring, than what is already embedded within the FFT program as implemented at these Danish sites. With respect to the malleability of CU, this study demonstrated that a good portion of adolescents with high levels of CU traits at baseline gained reliable reductions in CU traits. Changes in CU traits were furthermore related to changes in externalizing behavior and family functioning.These findings raise several questions and ideas for future research. First of all, further research is needed to determine whether CU traits predict diminished improvements in parental supervision in other contexts, and identify interventions that might be more suited to increase parental supervision for adolescents with CU traits. Second, the potential long-term predictive effect of CU traits on treatment improvements should be studied, to see if CU traits can indicate reduced longevity of treatment results or increased long-term risk of relapse. Third, more research is needed to understand the therapeutic processes and interventions that can result in attenuation of CU traits. Although plausible explanations could be given for FFT , as has been given for a mental models approach (positive emotions and prosocial strategies; css@vive.dk. Requests to access the quality assurance dataset can be directed to DT, d.m.thogersen@nubu.no.The datasets generated for this study will not be made publicly available because: the data contains sensitive data related to personal mental health information that is considered pseudonymized data by the legal advisory board at the University of Southern Denmark. Requests to access the main dataset can be directed to CS, Ethical review and approval was not required for this study in accordance with the local legislation and institutional requirements. Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements. Participants received information about the survey in an invitation letter and voluntary completion and return of the survey questionnaires constituted an implied consent.CS, DT, ME, and GB contributed to the conception and design of the study. CS and DT organized the database. DT performed the statistical analysis with guidance from ME and GB. DT wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.DT is occasionally hired to provide training, supervision, and consultation in the Functional Family Therapy model in Denmark, which could be construed as a potential conflict of interest. The remaining 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": "Abnormally excessive growth results from perturbation of a complex interplay of genetic, epigenetic, and hormonal factors that orchestrate human growth. Overgrowth syndromes generally present with inherent health concerns and, in some instances, an increased risk of tumor predisposition that necessitate prompt diagnosis and appropriate referral. In this review, we introduce some of the more common overgrowth syndromes, along with their molecular mechanisms, diagnostics, and medical complications for improved recognition and management of patients affected with these disorders. Characteristic facial appearance includes hypertelorism (widely spaced eyes), large ears, and a “stuck on” protruding chin. Newborns and infants often have truncal hypotonia (~50%) and extremity hypertonia with limited elbow extension. Hypotonia is associated with poor feeding and may also be associated with a characteristic weak, low pitched cry affecting about 50% of patients. Developmental delays are common, both in gross motor  and fine motor skills (poor coordination). Ventriculomegaly is frequently seen on brain imaging. Advanced bone age is uniformly reported, exceeding the chronological age by a factor of 1.5–2 . It is inherited in an autosomal dominant manner. Interestingly, pathogenic variants in EZH2 have been found in individuals with overgrowth without other clinical features of Weaver syndrome, indicating a wide spectrum of the disorder  (617561)  and microretrognathia seen in WS. Intellectual disability has been reported in one out of three published cases  , 75 and (617561) , respected cases . In our 615879). It is caused by pathogenic variants in DNA methyltransferase 3A. Despite sharing a similar mechanism with Weaver syndrome, that is, a heterozygote mutation (pathogenic variant in one copy) in DNA methyltransferase, this syndrome bears more similarities to Sotos syndrome. Both include features of macrocephaly noticed at birth, joint hyperlaxity, scoliosis, hypotonia, and seizures. However, the facial dysmorphism is different and includes round facies, straight and thick (bushy) eyebrows, and prominent maxillary incisors. These features evolve over time and may not be present in early childhood. Moreover, unlike Sotos and Weaver syndromes, patients' length at birth is usually normal and increases to >2 SD only later in life. About 16% of individuals are reported to have congenital heart defects; therefore, an echocardiography should be considered upon diagnosis  syndrome, similar to Sotos and Weaver syndromes, is a DNMT3A-related overgrowth syndrome, also known as Tatton–Brown–Rahman syndrome OMIM 6158. It is cCompared with the Sotos and Weaver syndromes, the “average patient” is more intellectually impaired. A cohort of 55 patients from 41 families  found thDNMT3A can be found in patients with acute myeloid leukemia (AML), myelodysplastic syndrome, and myeloproliferative syndromes. Interestingly, 26 out of the 40 variants described (63%) in DNMT3A, (DNMT3A) were also documented in affected patients with the overgrowth syndrome. Two out of the reported 77 patients with this OGID syndrome developed AML at the ages of 12 and 15  is a syndrome characterized by macrosomia, macrocephaly, round facies, hypotonia, and visceromegaly. Visceromegaly most commonly involves the kidneys  or liver  but can also include the heart, spleen, and pancreatic islet cells . Nephromegaly, seen in 80–100% of affected individuals, is frequently accompanied by nephroblastomatosis (diffuse persistence of metanephric blastema), a characteristic finding in Perlman syndrome, that predisposes to Wilms tumor seen in about a third of the patients. Other common findings are cryptorchidism and inguinal hernias. Newborns commonly present with abdominal distention as a result of nephromegaly, hepatomegaly, ascites, and/or abdominal wall muscular hypoplasia. The abdominal distension induces, in turn, hypoplastic lungs. About 87% of the affected infants develop respiratory distress and/or renal failure and die within the first hours or days of life. In surviving patients, growth parameters typically decline rapidly to reach the lower end of normal.Perlman syndrome (OMIM DIS3L2 exoribonuclease (RNAase); DIS3L2 has been shown to lead to IGF2 overexpression, as seen in the Beckwith–Wiedemann syndrome and is strongly associated with tissue overgrowth and Wilms tumor development  is a complex syndrome with variable presentation consisting of progressive segmental overgrowth of the feet and/or hands, cutaneous connective tissue nevi, and cranial hyperostosis. Affected individuals have minimal manifestation at birth and are born with normal growth parameters. Starting around 6–18 months of age, they can develop excessive asymmetric growth that can reach twice its normal size by age 6 years. The growth is of the bones and the soft tissue, and although the hands and feet are most commonly involved, any bone can be affected. Leg length discrepancy of up to 20 cm and scoliosis of more than 90° have been reported. Final height is normal as skeletal growth plateaus at adolescence. In childhood, patients can develop cutaneous connective tissue nevi most commonly involving the feet, hands, abdomen, or nose. These nevi are highly collagenized connective tissues that are firm with their surface resembling the gyri and sulci of the brain, thus named cerebriform. These nevi are pathognomonic for Proteus syndrome however they should be differentiated from the neurofibromas seen in neurofibromatosis syndrome; neurofibromas are smooth, soft, movable, and rarely involve the plantar aspect of the foot. Another cutaneous finding seen in Proteus syndrome is epidermal nevi. These lesions can present as early as the first few months of life and tend to follow the lines of Blaschko. Adipose tissue overgrowth is common in this syndrome; focal growth tends to occur from early infancy to early adulthood. They are not encapsulated lipomas but their histology is almost always benign. Along with growth, there are areas of adipose atrophy giving a combined presentation of adipose dysregulation. Skeletal abnormalities can occur anywhere in the body and add further to the striking asymmetry. Restrictive pulmonary disease is therefore common. Other distinctive features of Proteus syndrome include cranial hyperostosis, condylar (mandibular) hyperplasia, and rarely craniosynostosis  complicates these vascular anomalies and can lead to early death.The majority of individuals with Proteus syndrome have normal intelligence; however, a subgroup (30%) of patients with dysmorphic facial features has seizures and intellectual disability. Dysmorphism includes dolichocephaly , long face, downslanting of the palpebral fissures , and open mouth at rest. Other organs that are commonly affected include the eye , lungs (13% with cystic changes), and kidneys  . It has AKT1 gene, which is part of the PI3KA/AKT1/mTor proliferation pathway  occurrence, and progressive course. Lesions can be either the pathognomonic cutaneous (cerebriform) connective tissue nevi, or other findings such as asymmetric growth, dysregulated adipose tissue growth, lung cysts, and/or the specific tumors . These criteria were constructed to avoid over-diagnosis of this syndrome. A review of 205 published cases of Proteus syndrome found that only 95 of them fulfilled these criteria, and 80 cases (39%) “clearly did not” . It is rPTEN (Phosphatase and tensin homolog)—characterized by unregulated cellular proliferation leading to the formation of hamartomas. PTEN is a phosphatase that removes a phosphate from the second messenger phosphatidylinositol triphosphate and, by doing so, inhibits the Akt (Protein kinase B) pathway, a cardinal pathway of cell proliferation and angiogenesis. Not surprisingly, somatic inactivating mutations in PTEN are found in breast, prostate, lung, endometrial carcinomas and glioblastoma. In cancer, PTEN mutations are acquired in adulthood in a single cell which proliferates to create a tumor (“somatic” mutations). Alternatively, PTEN mutations that are either inherited from affected parent or formed in the parental gamete prior to conception affect all cell populations (“germline” mutations). In such cases, cells with an additional PTEN mutation (a “second hit”) results in hamartomata and cancer predisposition. The two-hit theory, also referred to as Knudson hypothesis, also explain the nature behind the observed segmental overgrowth: affected individuals are susceptible to Akt overactivation with only one active allele of PTEN; a second spontaneous deactivating mutation in PTEN in a post-zygotic developing tissue may occur, leading to discretely affected areas. The segmental overgrowth is manifested differently in each phenotype of PHTS: newborns with Bannayan–Riley–Ruvalcaba have striking macrocephaly (≥4.5 SD), out of proportion to their birth weight and length; those with Proteus-like presentation exhibit mosaic pattern of rapidly progressive overgrowth of different tissue types; Cowden syndrome, typically manifests in the second decade of life, and is associated with hamartomata and macrocephaly. Cowden syndrome bears an increased lifetime risk of benign and malignant tumors in the breast , papillary or follicular thyroid carcinoma (35%), renal cell carcinoma (35%), endometrial (28%), colorectal (9%), melanoma (5%), and rarely Lhermitte–Duclos disease (cerebellar dysplastic gangliocytoma).Phosphatase and tensin homolog (PTEN) Hamartoma tumor syndrome (PHTS) is a disorder encompassing three seemingly distinct clinical syndromes—Cowden syndrome, Bannayan–Riley–Ruvalcaba syndrome, and Proteus-like syndrome—all of which are characterized by mutations in the tumor suppressor gene Bannayan–Riley–Ruvalcaba is further characterized by hypotonia, intellectual disability (50–70%), proximal myopathy (60%), scoliosis (50%), hamartomatous polys in colon (45%), which may cause intussusception or rectal bleeding, seizures (25%), and joint hypermobility. A distinctive clinical finding in most affected individuals is pigmented macules on the penile shaft and if seen on physical examination should raise immediate concern for PHTS. The risk for tumors and malignancy is currently perceived to be similar to Cowden syndrome, especially breast and thyroid cancers, and merits similar surveillance. Proteus-like syndrome is clinically similar to Proteus syndrome and exhibit vascular malformations, lipomas, connective tissue nevi, epidermal nevi, and cranial hyperostosis. Cowden syndrome, named after the first reported patient, is rarely expressed in children and has distinctive trichilemmomas (benign neoplasm derived from the outer root sheath epithelium of the hair follicle), papillomatous papules (benign neoplasm of epithelium), and acral and plantar keratosis seen in 99% of patients by the third decade of life.PTEN (10q23), can give rise to either one of the above phenotypes—making these three conditions allelic disorders. Furthermore, it has been shown that the very same PTEN variant in an affected family can be expressed as either Cowden or Bannayan–Riley–Ruvalcaba syndrome in different family members. Another allelic disorder is macrocephaly with autism. In fact, 10–20% of autistic children with macrocephaly harbor germline mutation in PTEN. Mutations in PTEN includes gene sequencing, analysis of deletions and duplications and the promoter region. PHTS is an autosomal dominant disorder, meaning that only one pathogenic variant in PTEN gene is enough to express the disorder. Affected individuals have a 50% chance of transmitting the pathogenic variant to each one of their children. We recommend referring to genetics post-pubertal individuals for discussion about family planning. In vitro fertilization (IVF) techniques with preimplantation genetic testing for monogenic disorder (PGT-M) for fetuses can be offered to significantly reduce the transmission risk.Genetic testing for Management of PHTS include symptomatic management for the mucocutaneous manifestations of CS  and close follow up on development with neuropsychological evaluation (including IQ test) if suspicion for intellectual disability arises. Genetic testing for other family members is also recommended , 89–91. BRCA1 or BRACA2 carriers: clinical breast examination starting at 25 years of age or 5–10 years before the earliest known breast cancer in the family (whichever comes first). Starting at age 30–35 or 5–10 years prior to first known breast cancer in the family, annual mammography, tomosynthesis (“3D mammography”), or MRI with contrast should be performed until the age of 75. A discussion about risk reduction with double mastectomy should be conducted (sparing oophorectomy). Endometrial cancer screening is symptom-based  only. For both men and women, colonoscopy should be performed at least every 5 years starting at 35 years of age or 5–10 years prior to first known familial case (whichever comes first). Neck ultrasound for thyroid cancer should be obtained at the age of 7, then if negative every 2 years. Renal US is recommended at 40, then every 1–2 years subsequently  of PTEN, while testing cells from affected tissue will show variants in the two alleles. On the contrary, proteins that participate in the growth-promoting pathway that PTEN inhibits, may harbor only one variant which allows their escape from inactivation, resulting in overgrowth. PTEN is the opposed enzyme of phosphatidylinositol 3-kinase: the former removes and the latter adds phosphate to the aliphatic second messenger phosphatidylinositol di/tri-phosphate. The catalytic subunit of phosphatidylinositol 3-kinase is p110α, encoded by the gene PIK3CA. Variants causing overactivation of p110α shifts the balance between p110α and PTEN toward creation of phosphatidylinositol -trisphosphate, which activates AKT  and the second is megalencephaly-capillary malformation (MCAP) syndrome. CLOVES is associated with complex lipomatous overgrowth of the thoracic and abdominal wall, macrodactyly (enlargement of a digit), and plantar or palmar overgrowth which results in wrinkling of the overlying palmar or plantar skin. The lipomatous masses can be found on the skin or extending into the paraspinal and intraspinal spaces which may cause compression of the cord or nerve roots. The lesions are commonly covered by capillary, venous, lymphatic, or arteriovenous malformations. Skeletal malformations can be severely deforming; it includes scoliosis and asymmetric bony overgrowth. Structural anomalies are found in the CNS  and kidneys. A very similar presentation of asymmetric overgrowth of bony, adipose, fibrous, and vascular overgrowth due to PIK3CA over-activation is called Fibroadipose hyperplasia. Milder presentation may include only asymmetric overgrowth of limbs with overlying vascular malformations and can be expressed merely as isolated macrodactyly  seen perinatally by elevated occipitofrontal circumference. It is accompanied by secondary overgrowth of specific brain structures, and ventriculomegaly with polymicrogyria  are common. Patients with MCAP are hypotonic and prone to seizures (30%). Similarly to CLOVES, vascular malformations are common  and asymmetric overgrowth of limbs may be seen. Not surprisingly, mutations causing CLOVES and MCAP syndromes are seen in the COSMIC database, as shed yet . Managemshed yet . Experimshed yet , 96. Sirshed yet . In two shed yet , 98. Sevshed yet . A dual in vitro .PIK3CA is expressed in gonads; therefore, transmission to next generation is expected to be unlikely. Similarly, because this is a post-zygotic mutation, the risk for recurrence in a family is not elevated.Genetic testing should be obtained from the affected tissue and not blood. Prognosis depends on the degree of the severity of the phenotype ranging from guarded to dismal . Rarely Somatic overgrowth with overlying vascular malformation is shared by many other syndromes making this clinical finding an important sign for an underlying disorder. Combined capillary, venous and lymphatic malformation  along with limb enlargement is seen in Klippel–Trenaunay syndrome (KTS); arteriovenous (fast-flowing) fistulae without lymphatic malformation along an enlarged limb are seen in Parkes–Weber syndrome . In KTS, the affected limb is the lower extremity in 95% of cases and upper extremity in close to 5%. It is uncommon to have hypertrophy of the trunk. Capillary malformations appear bluish-purplish in color and may extend to the trunk but rarely to the face. They are commonly accompanied by lymphatic malformation causing lymphatic leak and lymphedema of the involved extremity. In 80% of patients, significant varicosities extending from the dorsum of foot to the popliteal or even the gluteal venous system are noticeable beginning in infancy or early childhood. It is often complicated by thrombophlebitis (up to 50%) and pulmonary embolism (10%). Surgical correction may be complicated by incompetency of the deep vein system seen in some patients. The affected limb may exhibit bone and/or soft tissues hypertrophy and thus the limb can be asymmetric in both length and girth . Toes maybe significantly more affected than the rest of the limb. The sole may have wrinkled appearance to it but lacks the firm consistency of cerebriform connective tissue nevi seen in Proteus syndrome , 102. ClOvergrowth syndromes can present with excessive post-natal growth, tall stature that is out of proportion to the individual's genetic potential in a child, isolated increased head circumference, or somatic asymmetry. Clues for overgrowth syndrome can come from the following: (i) physical examination such as omphalocele, abnormal ear creasing, and macroglossia for BWSp, dolichocephaly and frontal bossing in Sotos syndrome or lipomas, cerebriform cutaneous nevi or vascular anomalies in segmented overgrowth syndrome; (ii) imaging findings such as accelerated bone age and visceromegaly in the prenatally presenting overgrowth syndromes, or hemi-megalencephaly and paraspinous lipomas seen in PIK3CA-related overgrowth; and (iii) neurobehavioral assessment showing learning difficulties, developmental delays, or autism as seen commonly in Sotos, Weaver, and PTEN-hamartoma syndromes. Overgrowth syndromes can predispose affected individuals to hypoglycemia, embryonal tumors, seizures, developmental delay, intellectual disability, and musculoskeletal complications; therefore, timely diagnosis is important. Careful history taking, physical examination, anthropometric measurements, and developmental follow-ups, all of which are tools used on routine office visits, can provide significant evidence for suspected overgrowth syndrome. Maintaining a high index of suspicion for these disorders can assist in timely referrals to genetics, which can assist in the evaluation and testing of these patients. We encourage the primary clinician to be alert of the “large” end of the weight, length and FOC measures, and become familiar with BWSp, which is the most common overgrowth syndrome requiring vigilant tumor surveillance see .Based on expert opinion, stature larger than 2 standard deviations from the mean should be considered tall and raise suspicion for an overgrowth syndrome.Based on expert opinion, workup for tall stature that exceeds the individual's mid-parental height should include assessment of growth velocity and should consider full blood counts, complete biochemical analysis, IGF-I, IGFBP-3, free T4, and TSH, as well as a karyotype and bone age.Based on expert opinion, newborns with either macroglossia, exophthalmos, lateralized overgrowth, persistent hyperinsulinism, multifocal or bilateral Wilms tumor, or adrenal cortex cytomegaly should be tested molecularly for Beckwith–Wiedemann syndrome.Based on published guidelines, individuals with BWSp or SGB syndrome should undergo routine cancer surveillance every 3 months for hepatoblastoma (until the age of 4) and Wilms tumor (until the age of 7).Based on expert opinion, individuals with FOC > 4 SD and autism should be tested molecularly for possible PHTS.Based on published guidelines individuals with PTEN-hamartoma Tumor syndrome should undergo breast cancer surveillance similar to BRCA 1/2, colonoscopy surveillance every 5 years, biennial thyroid US, renal US every 1–2 years.Based on expert opinion, individuals with intellectual disability and tall stature should be evaluated for suspected overgrowth syndrome.JM conceptualized, prepared, and wrote the manuscript and made the tables and figures. SL reviewed, edited, and revised the manuscript and provided the patients' figures.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": "Through experimental and theoretical results, it is discovered that the outstanding performance of V8O20·nH2O is ascribed to the Mn2+/Mn3+‐induced high electrical conductivity and Na+‐induced fast migration of Zn2+. Other cathode materials derived from V8O20·nH2O by substituting Mn with Fe, Co, Ni, Ca, and K are explored to confirm the unique advantages of transition metal ions. With an increase in Mn content in NaV8O20, V8O20 ·nH2O can deliver a reversible capacity of 150 mA h g−1 and a capacity retention of 99% after 1000 cycles, which may open new opportunities for the development of high‐performance aqueous ZIBs.Rechargeable aqueous zinc‐ion batteries (ZIBs) show promise for use in energy storage. However, the development of ZIBs has been plagued by the limited cathode candidates, which usually show low capacity or poor cycling performance. Here, a reversible Zn//V 8O20 is synthesized via a one‐step hydrothermal reaction. The Mott–Schottky plots, Tafel curves and calculation results are given to explain the improved cycling performance of Mn‐doped NaV8O20. Other cathode materials derived from V8O20·nH2O by substituting Mn with Fe, Co, Ni, Ca, and K are explored to confirm the unique advantages of transition metal ions in NaV8O20.Mn‐doped NaV However, the toxicity and flammability of organic electrolytes cause environmental problems and safety issues. Reliable strategies such as the utilization of solid‐state electrolytes or aqueous electrolytes seem to overcome these dilemma. Aqueous rechargeable batteries are competitive alternatives because aqueous electrolytes are characterized by lower cost, higher safety, greater environmental friendliness, and higher ionic conductivity (up to 1 S cm−1) than organic electrolytes or solid‐state electrolytes. Aqueous Zn‐ion batteries (ZIBs) using metallic Zn as the anode have attracted surging interest due to their high specific capacity of 819 mA h g−1 at a low redox potential (−0.762 V versus the standard hydrogen electrode). Unfortunately, exploiting suitable cathode materials with high specific capacity and stable cycling performance is still an urgent task, and it limits the practical and large‐scale applications of ZIBs.Given the looming energy crisis and environmental pollution, renewable green energy resources such as wind and solar have attracted widespread attention in recent years.3, 4 such as Mn‐based oxides, Prussian blue analogs, transition metal sulfides, and vanadium‐based compounds for aqueous ZIBs. MnO2 displays a high operating voltage around 1.3 V but suffers from the irreversible phase transition and Mn cations dissolution during the cycling. Prussian blue analogs deliver a low capacity of ≈100 mA h g−1 and are hindered by their sluggish kinetics. Layered vanadium‐based oxides have high capacity exceeding 300 mA h g−1 but suffer from structural degradation. Incorporating metal  cations and/or molecular water (H2O) into V2O5 has been proven to be an effective method for solving the poor long‐term cycling performance of vanadium‐based oxides. Structural H2O in bilayer V2O5 reduces the electrostatic interactions within the V2O5 framework, thus effectively promoting Zn2+ diffusion. Metal ion‐preintercalated V2O5 usually expands the interlayer spacing more than V2O5. The expanded interlayer spacing could provide fast channel for Zn2+ diffusion and improve the reaction kinetics, and the chemically connected layers become more robust to alleviate the structural degradation, resulting for a long‐term cycling stability as well as excellent rate capability. Apart from V2O5 derivatives, other layered vanadium‐based oxides, such as NaV3O8 composed of V3O8 layers and inserted sodium ions, delivers a capacity of 380 mA h g−1 at a current density of 0.05 A g−1. However, NaV3O8 gradually dissolved into the ZnSO4 electrolyte and caused a rapid degradation in capacity during cycling. The addition of high concentration Na2SO4 into the electrolyte can suppress the dissolution equilibrium of Na+ from NaV3O8 because of common‐ion effect. Apart from adding electrolyte additives, exploring other strategies for improving the structural stability of these cathodes during cycling is of essential importance in ZIBs. Until now, many metal ions pre‐intercalated strategies have focused on V2O5‐based cathodes. However, the different role of transition metal cations and alkali metal cations in improving the electrochemical performance of vanadium‐based materials is unclear. New compounds intended as cathode materials for ZIBs could be explored by intercalating transition metal ions such as Mn into NaV8O20, forming V8O20·nH2O.Considerable research efforts have been devoted to exploring cathode materials,33 suc8O20 nanobelts as cathode materials for ZIBs, and we show strong experimental and theoretical evidences that the outstanding performance of V8O20·nH2O is ascribed to the Mn2+/Mn3+‐induced high electrical conductivity and Na+‐induced fast migration of Zn2+. The Mn ions stabilize the NaV8O20·nH2O (NVO) structure during the charge/discharge process. As the positive electrode for aqueous ZIBs, NVO delivers a capacity of 140 mA h g−1 and exhibits a capacity retention of 69% after 1000 cycles at 4 A g−1, while V8O20·nH2O delivers a reversible capacity of 146 mA h g−1 and a capacity retention of 88% after 1000 cycles. With an increase in Mn content in NVO, V8O20·nH2O can deliver a reversible capacity of 150 mA h g−1 and a capacity retention of 99% after 1000 cycles. Other cathode materials that were derived from V8O20·nH2O by substituting Na with K, Li, and Zn are explored and deliver long‐term cycling stability. Furthermore, derivative cathode materials from V8O20·nH2O by substituting Mn with Fe, Co, Ni, Ca, and K are also explored to confirm the unique advantages of transition metal ions.Here we report a hydrated, monovalent and divalent/trivalent cations co‐pre‐inserted V28O20·nH2O with different amounts of Mn content was obtained by setting the molar ratio of MnSO4 and V2O5 to 1:13, 13:13, and 20:13 during the hydrothermal process. The as‐prepared Mn‐doped NaV8O20·nH2O was denoted as Mn1‐NVO, Mn2‐NVO, and Mn3‐NVO, respectively. Figure 8O20·nH2O with C2/m space group (JCPDS No. 45‐1363). With the intercalation of a small amount of Mn ion in the NVO, the resulting XRD peaks of Mn1‐NVO were in accord with the structure of NVO, suggesting that the intercalated Mn ions would not produce a new crystal structure. It can be speculated that Mn1‐NVO has two possible structures, which are V8O20·nH2O and Na8O20·nH2O. The average analytical formula can be written as V8O20·nH2O or Na0.97O20·nH2O for Mn1‐NVO, as the molar ratio of Na:Mn:V was calculated to be 0.97:0.02:8 based on the electron microprobe analysis (EPMA). The molar ratios of Na:Mn:V for Mn‐doped NVO are shown in Table S1, Supporting Information.The NVO and Mn‐doped NVO were synthesized via a one‐step hydrothermal method. Va = 11.699(6) Å, b = 3.647(8) Å, c = 11.119(2) Å, α = γ = 90°, and β = 104.05°. The refinement data in detail are shown in Tables S2 and S3, Supporting Information. After the Mn ion was doped, Na(1) moved away from their average positions and Mn(1) occupied the Na(1) positions, indicating that Mn1‐NVO had the crystal structure of V8O20·nH2O instead of Na8O20·nH2O , showing a mixture of 83% Mn3+ and 17% Mn2+ for Mn2‐NVO and Mn3‐NVO . Noted that the average valence of V in the synthesized NVO was 4.876. While for the Mn‐doped NVO, the average valence of V in V8O20·nH2O was 4.871, revealing an increased V4+:V5+ ratio in this structure. On the contrary, the average valence of V in Na0.97O20·nH2O was 4.883, suggesting an decreased V4+:V5+ ratio. From the XPS results , the V4+:V5+ ratio of NVO was 0.44:1, and the V4+:V5+ ratios of Mn1‐NVO, Mn2‐NVO, Mn3‐NVO were 0.47:1, 0.5:1, and 0.53:1, respectively. The increased V4+:V5+ ratio confirmed that the Mn ion doped NVO had the formula V8O20·nH2O. With a thermogravimetric‐differential thermal analysis study, the structural water content in Mn1‐NVO was obtained , and the stoichiometric formula for the prepared Mn1‐NVO was then confirmed as V8O20·0.32H2O.To confirm the position of the Mn ions in NVO, Rietveld refinement of Mn1‐NVO was conducted. The chemical composition of the compound was analyzed by inductively coupled plasma optical emission spectroscopy (ICP‐OES) before progress the Rietveld refinement. The molar ratio of Na:Mn:V was 1.1:0.03:8. Based on the Rietveld refinement results in Figure O Figure .34]34a = 2+ insertion/extraction in aqueous ZIBs.The scanning electron microscope (SEM) image in Figure ous ZIBs.2m aqueous Zn(CF3SO3)2 electrolyte, the electrochemical performance of the Mn1‐NVO nanobelts as the positive electrode were investigated. The Mn1‐NVO delivers a high capacity of 363 mA h g−1 at a current density of 0.1 A g−1  curves of Mn1‐NVO captured at a scan rate of 0.1 mV s−1 are shown in Figure 2+ insertion/extraction could be inferred, which is quite different from the reported V2O5 based cathodes that have two pairs of redox peaks. Some peaks disappear at a faster scan rate of 0.5 mV s−1, leading to one obvious cathode peak at 0.5 V and two anodic peaks at around 0.65 and 1.05 V. Figure b in the range of 0.5–1 represents the slope of the log(i) versus log(v) curve. Ionic diffusion controls the reaction process when the value of b is 0.5, and pseudocapacitance dominates the electrochemical process completely when the value of b is 1. The calculated b values of peaks 1, 2, and 3 are 0.69, 0.54, and 0.76, respectively . In addition, electrochemical impedance spectrum analysis of Mn1‐NVO and NVO batteries after 20 cycles was carried out . It shows lower charge‐transfer resistance of the Mn1‐NVO than the NVO (Rct 4.8 ohms for Mn1‐NVO versus 14 ohms for NVO) after 20 cycles, which suggests a higher surface electron mobility and improved electrochemical activity for Mn1‐NVO.Using a Zn negative electrode and Ti foil (20 µm) as the positive current collector in 3 1 Figure 2a. With 1 Figure . The speox peaks.23 Somy Figure , meaning2+ diffusion at the Mn1‐NVO and NVO electrodes were studied via the Galvanostatic Intermittent Titration Technique (GITT) measurement, and the corresponding Zn2+ diffusion coefficients for the Mn1‐NVO and NVO electrodes in the third cycle are illustrated in Figure 2+ diffusion coefficients in Mn1‐NVO ranged from 1.9 × 10−9 to 2.5 × 10−10 cm2 s−1, while the DZn2+ for NVO ranged from 1.2 × 10−10 to 7.0 × 10−10 cm2 s−1 upon discharge. Interestingly, the Zn2+‐diffusion coefficient in Mn2‐NVO and Mn3‐NVO  were only slightly reduced with an increase in Mn content . The high Zn2+ diffusion coefficient may have been responsible for the improved battery performance of Mn1‐NVO. Figure S9, Supporting Information shows Ragone plot of Zn//Mn1‐NVO and Zn//NVO battery. Mn1‐NVO and NVO electrodes achieve an energy density of 249 and 239 Wh kg−1 at 100 mA g−1, respectively. Even though the Zn//Mn1‐NVO battery cycled at 1 A g−1, the energy density still reaches up to 157 Wh kg−1 at an outstanding power density of 628 W kg−1, which is higher than that of Zn//NVO battery (144 Wh kg−1at the power density of 598 W kg−1).To determine the roles of introducing Mn, the kinetics of the ZnFigure −1, the diffraction peaks for Mn1‐NVO located at 8.3°, 25.6°, 33.5°, and 43.2° shift to lower angle at 8.2°, 25.3°, 33.8°, and 42.4°, respectively. Particularly, the calculated interlayer spacing of the (001) plane shifts from 10.62 Å to 10.70 Å during the initial Zn2+ insertion process, which is ascribed to the insertion of a certain amount of Zn2+ into the interlayer of Mn1‐NVO, expanding the interlayer spacing. Notably, the interlayer spacing of the (001) plane shifts to 10.46 Å and 10.31 Å upon the following discharge to 0.6 and 0.3 V, indicating that lattice contraction occurs. At the subsequent charging from 0.3 to 0.5 V, the diffraction peaks located at 8.6°, 25.8°, 34.6°, and 43.8° shift to a lower angle at 8.5°, 25.9°, 34.5°, and 43.6°, respectively, suggesting an increase in interlayer space corresponding to the deintercalation of Zn2+. When first charged to 1.25 V, the interlayer spacing of the (001) plane increased from 10.31 Å to 10.54 Å. The fully charged electrode at 1.25 V after 400 cycles showed the same XRD pattern as the electrode cycled at the tenth cycle , suggesting that a reversible phase transition occurred during long‐term cycling.To investigate the structural evolution and reaction mechanisms of the Mn1‐NVO, ex situ XRD was performed during the first discharge/charge process. As shown in −1 is ascribed to V‐O‐Zn vibrations in Mn1‐NVO upon discharge, and the Raman shifts at 253, 310 and 371 cm−1 are assigned to the Zn‐OH vibrations, implying the presence of Zn(OH)2 or Znx(OTf)y(OH)2x−y·nH2O upon fully discharge. The OH− in the compound is generated from the decomposition of water, thus accompany with the formation of H+, and H+ may intercalate in the Mn1‐NVO electrode to maintain a neutral charge state. Therefore, the reaction mechanism can be inferred that the H+ is co‐intercalated with Zn2+ into Mn1‐NVO upon discharge from the presence of the above precipitates. However, the low crystallinity and small quantity of the precipitates may be the reason why XRD cannot detect the phase. As shown in the enlarged XRD patterns in Figure S12, Supporting Information, there are no extra signals besides peaks of Mn1‐NVO, suggesting that the main structure is maintained during the charging/discharging process. The lowering of the relative intensity and the broadening of the XRD peaks during the discharge was mainly due to the formation of Zn(OH)2 and Znx(OTf)y(OH)2x−y·nH2O precipitates and the insertion of Zn2+ into the Mn1‐NVO. In addition, the peak associated with V—O—Zn bond and the characteristic Raman peaks of precipitates disappear at the fully charged state, and the spectra is similar with the pristine electrode, suggesting there is no Zn complex formed upon charge. Upon fully charged state, the recovery of the XRD peak was due to the extraction of Zn2+ and H+ from the Mn1‐NVO cathode, which is in accord with the previous studies. Noted that the energy storage mechanism of Mn1‐NVO involves mainly Zn2+ (de)intercalation with minor H+ (de)intercalation, which is in accord with the previous report. For Zn anodes, the Raman shifts at the fully charged/discharged state are similar with the pristine Zn anodes, and the peak associated with O‐Zn bond is stable during the cycling, suggesting there is no Zn complex formed upon charge.To confirm whether there is new phases formation upon discharge/charge, Raman spectra were provided to further show structural information on the Mn1‐NVO electrode at the fully discharged/charged states. As shown in Figure S11, Supporting Information, the strong Raman shift at around 846 cm2+ in the Mn1‐NVO electrode was further demonstrated by ex situ high‐resolution XPS analysis. Characteristic peaks at 517.5 and 516.2 eV in Figure S3b, Supporting Information showed the V5+ and V4+ for Mn1‐NVO. As shown in Figure 5+ become weak, while the peaks for V4+ and V3+ increase during the intercalation of Zn2+, indicating the electrochemical reduction of V cations in the V8O20 framework during the first discharge. Upon charging in Figure 5+ increases as V cations are oxidized to +5, and the molar ratio of V5+:V4+ recovers to that of the pristine V spectrum . Meanwhile, the Zn 2p peaks emerge at the discharge state of 0.3 V 2 or Znx(OTf)y(OH)2x−y·nH2O) formed on the cathode upon the fully discharge state. The Zn 2p signals located at 1022 eV at fully discharged state was caused by the intercalated Zn and the zinc hydroxide triflate precipitation. Upon charging, the intensity of the Zn peaks remarkably decreases  and absorbed Zn features remain . As a result, the NVO crystal structure consists of a small amount of lattice‐trapped Zn after the first cycle, which might be the reason for the rapidly declining capacity in the second cycle. Mn ions were detected by XPS in the fully discharged and charged states both in the first cycle and the 400th cycle , meaning Mn ions keep stable in the crystal structure during the cycling. The Mn ions existing in the NVO crystal structure can act as pillar and stabilize the structure during the long‐term cycling. Furthermore, the Mn ions can change the electronic structure by strengthening the chemical bonds, thus causing more stable cycling performance than that in NVO. To confirm the electronic structure change of Mn1‐NVO, conductivity of the samples was carried out. The Mn1‐NVO exhibits higher electrical conductivity than NVO . To show the role of Mn ion and Na ion in Mn1‐NVO more clearly, the theoretical calculations of density of states and Zn2+ diffusion pathways for the NaV8O20, V8O20, MnV8O20 (MnVO) are provided in Figure 2+ (de)intercalation, thus contributes to the excellent performance of Mn‐doped NVO. Possible migration pathways for Zn2+ in Mn1‐NVO is shown in Figure 2+ mainly diffuses along b axis. Energy barriers along Zn2+‐migration pathways for NVO and Mn1‐NVO is lower than that of MnVO  images in Figure  Upon charging, the lattice spacing increased to 0.21 nm with Zn2+ extraction, which was in accord with the ex situ XRD results.To gain better insight into the electrochemical process during charging/discharging, the morphologies after the first discharge and charge were observed by TEM. The nanobelts was maintained during the cycling, and the element distributions of V, O, Na, Mn, and Zn was collected by EDS mapping with TEM Figure 4a,b. Theanalysis.45 The4, CoSO4, NiSO4, CaSO4 and K2SO4, were used as raw materials to replace MnSO4, producing V8O20, V8O20, V8O20, V8O20, and V8O20 , respectively. The XRD results in Figure −1 was investigated. An initial decrease in capacity was observed for these cathodes, which might be attributable to the slight surface dissolution of materials and the irreversible reactions during the first few cycles. Taking Fe1‐NVO as an example . To further understand the enhanced cycle stability of NVO via transition metal insertion, Mott–Schottky plots and Tafel curves are shown in Figure  Furthermore, there is a decreasing trend in the absolute value of the slope for transition metal‐doped NVO compared with pristine NVO, Ca1‐NVO, and K1‐NVO V8O20·nH2O by substituting Na with Li, K, and Zn  were also explored to confirm the catalytic effect of Mn ion. As shown in Figure S19a,b, Supporting Information, the crystal structures of these derivates were similar to that of Mn1‐NVO, and these cathodes exhibited more outstanding cycling performances compared with that of NVO.To explore the reasons why the Mn ions stabilized the NVO structure, other metal sulfates, such as FeSOe Figure , a dischn Figure 40 FurFigure 0.33V2O5 appeared in the Mn3‐NVO. Interestingly, Mn2‐NVO as well as the mixture of V8O20 and Na0.33V2O5 as cathodes exhibited more outstanding cycling performances than Mn1‐NVO  lattice spacing, that is, the lattice spacing is 10.46 Å in Mn2‐NVO and 10.62 Å in Mn3‐NVO, respectively. Thus, the combined effects of proper lattice spacing, as well as the stable crystal structure are key factors that determine electrochemical performance. In addition, the capacitive contribution of Mn2‐NVO was calculated to be 37.2%, 45.5%, 50.6%, 56.9%, and 62.6% at the scan rate of 0.1, 0.2, 0.3, 0.5, and 0.8 mV s−1, respectively, which is higher than that of Mn1‐NVO or NVO . The capacitive contribution increased with the increase in Mn content in NVO, and this was also responsible for the excellent cycling performance of the Mn2‐NVO and Mn3‐NVO batteries.To further improve the cycling performance of the Zn//Mn1‐NVO battery, the content of Mn in NVO was carefully investigated. With an increase in Mn content in NVO , we found that the (003) plane disappear Figure 6a. AnothO Figure . ObviousO Figure . From th3O8 in the ZnSO4 electrolyte and the formation of Zn dendrites caused rapid capacity fading, we carefully studied the dissolution of NVO and Mn‐doped NVO electrodes. To confirm the dissolution of NVO and Mn‐doped NVO, the NVO and Mn1‐NVO electrodes were dipped in the 1 m ZnSO4 electrolyte for 12 h. ICP‐OES results confirmed the dissolution of NVO and Mn1‐NVO . Furthermore, the dissolution of Mn1‐NVO could be suppressed via the addition of 2 m Na2SO4 to the 1 m ZnSO4, and therefore the capacity retention of Zn//Mn1‐NVO is superior to that of the case without Na2SO4 , which was in accord with the previous report. Noted that a high level of Na2SO4 additive in the electrolyte decreases the energy density of the batteries when they are used in practical applications. As shown in Figure S21, Supporting Information, increasing the concentration of Na2SO4 can suppress the dissolution of Mn1‐NVO and show high capacity retention, but the capacity of Mn1‐NVO was decreased. The dissolution of Mn1‐NVO in 1, 2, and 3 m ZnSO4 electrolytes was also carried out. As shown in Figure S22a, Supporting Information, a high concentration of electrolytes can delay the dissolution process. More importantly, it was noted that the dissolution of Mn‐NVO in ZnSO4 electrolytes was suppressed with the increase in Mn content in NVO, which is likely attributed to the increased structural stability. The increasing Mn in NVO causes the electrolyte colors to become faint, as shown in Figure S22b, Supporting Information. Interestingly, the dissolution of Mn1‐LVO, Mn1‐KVO, and Mn1‐ZVO in 1 m aqueous ZnSO4 was slower than that of Mn1‐NVO , suggesting that Na ions play a significant role in the dissolution process. Furthermore, different kinds of doping transition metals also affect the dissolution process of NVO electrodes , as Fe1‐NVO and Ni1‐NVO have a slower dissolution rate than Mn1‐NVO or Co1‐NVO. The dissolution of Mn1‐NVO in 3 m aqueous Zn(CF3SO3)2 was also carried out to show the advantage of Zn(CF3SO3)2 electrolyte for Mn1‐NVO . As shown in Figure S23, Supporting Information, a large number of vertical and harsh Zn dendrites formed on the surface of Zn negative electrode in the ZnSO4 electrolyte after 500 cycles at 4 A g−1, while no obvious Zn dendrites in 3M Zn(CF3SO3)2 is observed. Therefore, compared with ZnSO4 electrolyte, 3M Zn(CF3SO3)2 can not only suppress dissolution of Mn1‐NVO, but also suppress the formation of vertical and harsh Zn dendrites during the cycling.Since the dissolution of NaVy fading,27 we 38O20 framework. The Mn1‐NVO used as a cathode delivered a high capacity retention of 88% after 1000 cycles at a high current density of 4 A g−1, which was much better than NVO, Ca1‐NVO, or K1‐NVO. With an increase in Mn content in NVO, V8O20 ·nH2O can deliver a reversible capacity of 150 mA h g−1 and a capacity retention of 99% after 1000 cycles. Importantly, the dissolution of Mn‐doped NVO was suppressed with an increase in the Mn ion content, providing another way to stabilize the cathodes in the electrolyte. When combined with improved ion diffusion kinetics, charge carrier concentration, and catalysis of transition metal ions, the electrochemical performance of NVO‐based cathodes significantly improved. This strategy may pave the way for the potential application of doping in cathode materials and expand the choice of cathodes for energy storage devices.In summary, we reported a series of transition metal‐doped NVO, in which transition metal ions were intercalated in the interlayers of V44.14.1.14.1.1.12O5, 1.99 g of Na2SO4, and 0.676 g of MnSO4·H2O were dispersed in 50 mL of DI H2O, then CH3OOH was used to adjust pH to 3. After that, the solution was transferred into a 100 mL Teflon‐lined stainless‐steel autoclave. The sealed autoclave was heated to 180 °C for 72 h. Finally, the Mn1‐NVO sample was collected by centrifugation and washed with water and ethanol for three times. The sample was dried at 60 °C in an electric oven for 6 h, then further dried at 60 °C in a vacuum oven for 12 h. NVO was synthesized with the same procedure and processing conditions without MnSO4 source.The Mn1‐NVO nanobelts were prepared by one step hydrothermal method. Briefly, 4.73 g of V4.1.1.2α radiation to determine the phase of samples. The morphologies were characterized by SEM (S‐4800) and TEM (JEM‐2100F). XPS (ESCALAB 250) with Al Kα X‐ray radiation was carried out to measure the oxidation states of the elements. The structural water was detected by the thermogravimetric analysis (LABSYS EVO) under nitrogen atmosphere with a rate of 10 °C min−1 from room temperature to 800 °C and holding at 800 °C for 30 min and then cooling naturally to room temperature. ICP (iCAP Q) performed to analyze the elementals ratio.XRD was performed using a SmartLab‐3KW with Cu K4.1.1.3−2. Zinc foil as the anode and glass fiber membrane was used as the separator. LAND battery testing system (CT2001A) was used to evaluate the electrochemical performance of the assembled cells with a voltage range from 0.3 to 1.25 V versus Zn/Zn2+. CV, Tafel plot, and Mott–Schottky plots were collected by using a CHI 600E electrochemical workstation.The electrodes were assembled into coin cells (CR2032) to investigate the electrochemical performance. Typically, the active material, acetylene black and polyvinylidene fluoride with a mass ratio of 7:1.5:1.5 were fully mixed in N‐methyl‐2‐pyrrolidone solvent for the electrode preparation. Then the slurry was pasted on a Ti foil and dried in a vacuum oven at 60 °C for 12 h. The active material mass loading was around 2 mg cm6The authors declare no conflict of interest.Supporting InformationClick here for additional data file."}
{"text": "Crocus sativus L. (Iridaceae), or saffron, has been used as food additives and spices. In the traditional medicine of Iran, C. sativus has been used for the treatment of liver disorders, coughs, and as an anti-inflammatory agent for eyes.C. sativus stigma and its petal flavonoid (kaempferol) on respiratory disorders with several mechanisms such as anti-inflammatory, and smooth muscle relaxant effects. The current study reviewed the possible therapeutic effects of Crocus sativus’, ‘saffron’, ‘kaempferol’, ‘airway inflammation’, and ‘smooth muscle relaxant’ were searched. This review article searched databases including PubMed, Google Scholar, and ScienceDirect, up to November 2019. The keywords including; ‘C. sativus reduced nitric oxide (NO), inducible nitric oxide synthase (iNOS) levels and inflammatory cytokines in the lung tissue. Saffron and kaempferol reduced white blood cells (WBCs) and the percentage of neutrophils and eosinophils in bronchoalveolar lavage fluid. Moreover, saffron reduced tracheal responsiveness to methacholine and ovalbumin on tracheal smooth muscles. In addition, kaempferol reduced the total leukocyte and eosinophil counts similar to the effect of dexamethasone and also showed relaxant effects on smooth muscle.Crocus sativus and its petal flavonoid, kaempferol, showed relatively potent therapeutic effects on respiratory disorders by relaxation of tracheal smooth muscles via stimulatory or blocking effects on β-adrenoceptor and muscarinic receptors, respectively. Saffron and kaempferol also decreased production of NO, inflammatory cytokines and chemokines in respiratory systems. Asthma is a complex inflammatory disorder characterised by airway inflammation and hyperresponsiveness, hypersecretion of mucus by goblet cells and eosinophilic inflammation -sensitised animal model , or saffron, is commonly cultivated in Iran, Afghanistan, Turkey and Spain  with several mechanisms, such as anti-inflammatory and smooth muscle relaxant effects on inflammatory respiratory diseases.The current study reviewed the potential therapeutic effects of Crocus sativus stigma and its petal flavonoid, kaempferol on respiratory disorders were selected in PubMed, Google Scholar, and ScienceDirect, up to November 2019. The keywords including; ‘Crocus sativus’, ‘saffron’, ‘Kaempferol’, ‘airway inflammation’, and ‘smooth muscle relaxant’ were searched individually or combined.All literature that reported the effects of C. sativus  on cell viability and cytokine release of stimulated peripheral blood mononuclear cells (PBMC) by phytohemagglutinin (PHA) and non-stimulated cells significantly inhibited cell viability of lymphocytes and secretion of interferon-γ (IFN-γ) in stimulated cells and also inhibited interleukin (IL)-10 secretion in stimulated and non-stimulated cells  with crocin  for 24 and 48 h reduced cell viability at dose dependently. Crocin also significantly decreased IL-8 secretion and protein levels of TNF receptor 1 (TNFR1) by HepG2 cells at 6 and 12 h after treatment  and its constituents safranal resulted in a decrease of nitric oxide (NO), inducible nitric oxide synthase (iNOS) levels, peroxynitrite ion generation, and prevented cytochrome c release  and safranal in the OVA-induced murine model of asthma (in vitro) study, reduced iNOS levels and inflammatory cytokines such as; L-5 and IL-13 levels in the lung tissue. Airway hyper‐responsiveness and airway cellular infiltration to the lungs as well as, bronchial epithelial cell apoptosis was decreased in treated mice with C. sativus and safranal  was higher effective than dexamethasone (50 µg/mL)  on OVA-sensitised induced asthma in guinea pigs significantly reduced serum levels of endotheline-1 (ET-1) and total protein (TP) compared to untreated OVA-sensitised guinea pigs  prevented the increase in total white blood cells (WBC), eosinophil and lymphocyte numbers in OVA-sensitised animals. The extract reduced WBC count similar to dexamethasone. In addition, the low concentration of extract (0.1 mg/mL) reduced eosinophil count more than dexamethasone treatment  also significantly decreased WBC count, eosinophil percentage, neutrophil percentage, red blood cell (RBC) and platelet count in the blood of sensitised rats. Lymphocyte percentage was increased in animals receiving 100 mg/kg of saffron hydroethanol extract  remarkably reduced tracheal responsiveness, as main characteristic of obstructive respiratory diseases especially asthma, to both methacholine and OVA and also decreased serum levels of inflammatory mediators compared to untreated sensitised animals. In addition, C. sativus extract (80 mg/kg) was more effective than those of dexamethasone (10 mg/kg). These findings indicated that the extract of the plant could attenuate serum levels of inflammatory mediators as well as increase tracheal responsiveness to methacholine and OVA  reduced neuropathic pain in the chronic constriction injury (CCI) model through attenuation levels of pro-inflammatory factors including: tumour necrosis factor α (TNF-α), IL-1 β and IL-6 , on the lumbar spinal cord  intraperitoneal administrated in mice showed antinociceptive activity against acetic acid-induced writhing. Also, the plant extracts showed mild to moderate effects against acute inflammation caused by xylene in mice ear edoema. In addition, both aqueous and ethanol C. sativus extracts, showed anti-inflammatory effects by formalin-induced edoema in rat paw in induced chronic inflammation  in the treatment of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice significantly reduced the clinical symptoms in C57BL/6 mice with EAE. Also, treated mice displayed a delayed disease onset compared with control mice. Antioxidant capacity production was significantly elevated in saffron treated mice. Furthermore, mice treated with saffron reduced typical spinal cord leukocyte infiltration compared with control mice. These results suggested that saffron is effective in the prevention of symptomatic EAE by inhibition of oxidative stress and leukocyte infiltration to the central naevus system and may be potentially useful for the treatment of multiple sclerosis (MS)  twice daily compared with patients receiving a placebo in schizophrenia in which WBC count was in the normal range. Moreover, the other haematologic components and markers did not change significantly during 3 months of the study  were significantly increased in patients receiving the C. sativus,trans-crocetin  enhanced amyloid-β 42 (Aβ42) degradation in AD monocytes through the up-regulation of the lysosomal protease cathepsin B  of both healthy donors and Alzheimer’s disease (AD) subject with a constituent of These results indicated that saffron and its components may be useful in treating CNS disorders by reducing oxidative stress and reducing inflammation. Anti-inflammatory effects of saffron is summarised in Kaempferol is a polyphenolic compound isolated from the fresh flower petals of saffron  also suppressed mRNA expression of MMP-2 to restrain the migration of oral cancer cells via inhibiting the c-Jun pathway and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation in a dose-dependent manner  inhibited secretion of β-hexosaminidase and histamine, and reduced the production and mRNA expression of inflammatory cytokines (IL-4 and TNF-α) in immunoglobulin E (IgE)-sensitised RBL-2H3 cells. Kaempferol also inhibited (IgE)-mediated phosphorylation of phospholipase Cγ, protein kinase C (PKC), and the mitogen-activated protein kinases, extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase  decreased the levels of TNF-ɑ and IL-1β in serum of high cholesterol-fed rabbits. In addition, kaempferol respectively down-regulated mRNA and protein expression of inflammatory molecules such as E-selectin (E-sel), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and MCP-1 in the aorta of rabbits  on inflammation in human airway epithelial cells (BEAS-2B) showed kaempferol inhibited the expression of Toll-like receptor 4 (TLR4) a promotor of inflammatory mechanisms which significantly increased by LPS. Kaempferol also decreased the cellular expression of IL-8 through stimulating  inhibited mast cell degranulation and prostaglandin release, leading to the development of aberrant airways in basophilic leukaemia (RBL-2H3) mast cells obtained from dinitrophenylated bovine serum albumin (DNP-BSA)-sensitised rat. Kaempferol suppressed β-hexosaminidase release and COX-2-mediated production of prostaglandin D2 (PGD2) and prostaglandin F2α (PGF2α) in sensitised mast cells. Kaempferol prevented the antigen-induced mast cell activation of cytosolic phospholipase A2 (cPLA2) responsive to protein kinase Cμ (PKCμ) and extracellular signal-regulated kinase (ERK). These results demonstrated that kaempferol inhibited ERK-cPLA2-COX-2 signalling in mast cells  significantly decreased LPS-induced cellular levels of transforming growth factor beta 1 (TGF-β1) in a dose-dependent manner in BEAS-2B cells. Furthermore, LPS stimulation significantly induced TGF-β RI and TGF-β RII, which was reversed by kaempferol. Epithelial E-cadherin expression was substantially increased by kaempferol, which was dampened by TGF-β. Kaempferol (1–20 μM) suppressed the epithelial production of collagen IV, which was enhanced by LPS in BEAS-2B cells. Thus, kaempferol may improve epithelial barrier function by cell-cell adhesion  pathway in human monocytic cell line THP-1 were reviewed  on lipopolysaccharide (LPS)-induced lung injury in BALB/c mice strongly reduced overproduction of pro-inflammatory cytokines in BALF, including TNF-α, IL-1β and IL-6. Kaempferol also significantly inhibited LPS-induced alveolar wall thickness, leukocytes infiltration and alveolar haemorrhage in lung tissue with evidence of reduced myeloperoxidase (MPO) activity and increased superoxide dismutase (SOD) activity. Furthermore, kaempferol significantly blocked the activation of mitogen-activated protein kinases (MAPKs) and NF-κB signalling pathways induced by LPS. These results suggested that kaempferol exhibits a protective effect on LPS-induced acute lung injury by suppressing MAPKs and NF-κB signalling pathways, involved the inhibition of oxidative injury and inflammatory process  and eotaxin-1 protein in the lung tissue were enhanced in OVA-exposed mice, but the supplementation of kaempferol  dose-dependently eliminated the production levels of CCR3 and eotaxin-1. OVA challenge also increased macrophage inflammatory protein 2 (MIP-2) and C-X-C chemokine receptor type 2 (CXCR2) production in mouse lung tissue which kaempferol supplemented markedly reduced MIP-2 and CXCR2 production  either as a preventive (administered 1 h before OVA-sensitisation) or curative (OVA-sensitisation at day 18–21) treatment in sensitised mouse models  and MAPKs signal pathways in aged rat kidney  markedly inhibited the passive cutaneous antigen-induced anaphylaxis (PCA) response in IgE-sensitised mice  suppressed bovine serum albumin inhalation-induced epithelial cell excrescence and hypertrophy in smooth muscle by attenuating the induced COX-2 and the formation of PGD2 and PGF2α, as well as reduced the expression of anti-α-smooth muscle actin in mouse airways. These results demonstrated that kaempferol inhibited airway wall thickening through disturbing Syk-PLCγ signalling and suggested it may be a potent anti-allergic compound that targeting of allergic asthma  to OVA-challenged mice induced pulmonary TGF-β1 induction reduced TGF-β RI and levels of the epithelial marker E-cadherin in lung tissues of OVA-challenged mice. In addition, kaempferol (10 or 20 mg/kg) significantly reduced the lung tissue level of fibrogenic collagen IV which increased in mice sensitised with 5% OVA  reduces omega-6 and ovalbumin-induced allergic reactions at lung and trachea in BALB/c mice. It also inhibited the increased histamine level and expression level of COX-2 which increased in the lung and trachea after OVA sensitisation. The results of this study suggested kaempferol might have a positive effect in mitigating allergic inflammatory response of the respiratory tract  1 h before caecal ligation and puncture surgery in mice significantly decreased water content in the lungs. Pre-treatment with kaempferol also reduced cytokines, such as IL-6, IL-1β, and TNF-α in the plasma and in the lung tissue compared to the untreated mice. Kaempferol increased SOD and catalase and non-enzymatic antioxidant glutathione (GSH) activities in septic mice. Additionally, kaempferol reduced the lung tissue nitrite level and iNOS level and down regulated mRNA expression of intercellular adhesion molecule 1 (ICAM-1) and iNOS in septic mice  activity and increased superoxide dismutase (SOD) activity. Kaempferol also reduced the total leukocyte and eosinophil counts similar to dexamethasone used for the treatment of allergic inflammatory diseases. The anti-inflammatory effects of kaempferol are summarised in C. sativus  showed a potent relaxant effect on guinea‐pig precontracted tracheal chains (in vitro) by methacholine (10 μM) when compared with saline treatment. Furthermore, there was a positive correlation between increasing concentrations of the extract and the relaxant effects  reduced tracheal responsiveness to methacholine in ovalbumin (OVA) sensitised guinea pigs  by reducing the number of cough was observed, which could be due to its relaxant effect on airway smooth muscle (Hosseinzadeh and Ghenaati C. sativus (0.1 and 0.2%), and 10 nM propranalol (β-blockers) precontracted isolated guinea pig tracheal smooth muscle showed clear leftward shifts in isoprenaline (β adrenoreceptor agonist) curves obtained in the presence of saffron extract compared to that of saline, while propranolol caused rightward shift in isoprenaline response curve. This result indicated a potent stimulatory effect for C. sativus extract on β2-adrenoreceptors  on muscarinic receptors of guinea pig tracheal chains showed the functional antagonistic effect of the extracts on muscarinic receptors on smooth muscle of tracheal chain  on 10 nM chlorpheniramine, precontracted isolated guinea pig tracheal smooth muscle showed inhibitory effect of the plant extract on histamine H1 receptors , in normotensive and hypertensive rat model, reduced the mean arterial blood pressure and heart rate in a dose‐dependent manner. Additionally, administration of 10 mg/kg of saffron reduced the mean systolic blood pressure (MABP) by 60 ± 8.7 mmHg. These results suggested that C. sativus extract and its two active components have hypotensive properties in an animal model of hypertensive  showed a concentration-dependent inhibitory effect on heart rate and contractility, which was comparable to the effect of diltiazem. The effect of plant extract on heart contractility could be due to its muscle relaxant effect  in desoxycorticosterone acetate (DOCA) salt induced hypertensive in rats resulting in reduced MSBP in a dose-dependent manner, but this hypotensive effect was not observed in normotensive rats. Data showed that antihypertensive effects of saffron did not last long, so it could be postulated that long-term blood pressure regulation systems are not affected by saffron , on isolated rat thoracic aorta rings (tissue) were contracted by 10−6 M phenylephrine (PE) or KCl 80 mM showed vasodilatory effect on intact and denuded endothelium aortic rings. The extract induced relaxation in a dose-dependent manner in endothelium-intact aortic rings precontracted with PE. The relaxation effects of plant extract in precontracted tissue with KCl was less than that of precontracted tissue with PE. The relaxant activity of C. sativus was abolished by incubation of tissue with L-NAME but not in the presence of indomethacin. These results suggested C. sativus induced relaxation in isolated rat aortic tissue due to its effect on endothelium via nitric oxide synthase and the effect on vascular smooth muscle cells via L-type voltage-dependent calcium channels  , kaempferol (3–60 µM) and quercetin (1–100 µM) in rat uterus incubated in medium in the presence of different inhibitors proposed that cyclic adenosine monophosphate (cAMP) contributed to the relaxant effect of quercetin and kaempferol on induced tonic contraction by KCL. Furthermore, polyamins contributed to the relaxant effects of kaempferol on KCL-induced tonic contraction but not on CaCl2-induced contraction in the depolarised uterus. The other results of this study also indicated that flavonoids have a calcium antagonist action  through cAMP produces transcriptional events and polyamines have a relaxant effect on KCL induced tonic contraction in isolated rat uterus  on KCl (60 mM)-induced contraction in isolated rat uterus showed relaxant effect in a dose-dependent manner. This relaxing effect was antagonised by cAMP-dependent protein kinase inhibitors (Rp-cAMPS and TPCK) and adenylyl cyclase inhibitor  . Thus, results suggested that kaempferol glycosides are not responsible for the extract induced relaxation  on isolated pulmonary artery of rats was investigated. Experiments were done using the isolated organ bath system by recording tension with the help of data acquisition system, Power lab. Results of this study demonstrated that kaempferol caused concentration-dependent relaxation of endothelium intact pulmonary artery. In endothelium-denuded arterial rings, relaxation produced by kaempferol was not different from the intact artery. L-NAME (NOS inhibitor), indomethacin had not any effect on kaempferol induced relaxation. Furthermore, the administration of other drugs together with kaempferol was done. The results suggested that kaempferol relaxes rat pulmonary artery in endothelium-independent manner through involvement of BKca channel, sGC, PKA pathways and inhibition of Ca2+-influx through l-type calcium channels  was constructed. Kaempferol at high concentration has significant relaxation but this effect was not shown at low concentration. At low concentration (10 µM), kaempferol enhanced relaxation produced by isoproternole, bradykinin, sodium nitroprusside and calcium ionophore A23187 in endothelium intact arteries. In endothelium disrupt rings, kaempferol (10 µM) enhanced relaxation caused by isoproternole, sodium nitroprusside and nifidipine. According to these results, kaempferol at low concentration (10 µM) has no significant vascular effect, but it can enhance endothelium-dependent and endothelium-independent relaxations  on inflammatory respiratory diseases is shown in in vivo and in vitro studies. Saffron and kaempferol showed smooth muscle relaxant effects on tracheal smooth muscle (in vitro studies) by stimulatory and inhibitory effects on β-adrenoceptor and muscarinic receptors, respectively. The results of this review article indicate C. sativus (saffron) and its constituent (kaempferol) have potent therapeutic effects that may be useful for attenuating inflammation in some respiratory diseases such as asthma and COPD.There are two types of common respiratory disorders, including asthma and COPD, which is characterized by airway inflammation, generates excess mucus and smooth muscle dysfunction, making it hard to breathe. Saffron and kaempferol reduced the production of NO, and inflammatory mediators including; IL-4, IL-1β, TNF-α, and MMP-9 in the serum and broncho-alveolar lavage fluid. This plant and its active constituent also increased anti-inflammatory mediators including IFN-γ and IL-10 in"}
{"text": "Type 2 diabetes mellitus (T2DM) is an age-related metabolic disease that is of increasing concern. Gut microbiota might have a critical role in the pathogenesis of T2DM. Additionally, Hippo signaling has been associated strongly with the progression of T2DM and the aging process. We adopted db/db male mice as a T2DM model, and the gut microbiota of db/db and m/m mice were transplanted successfully into pseudo germ-free mice. Furthermore, Hippo signaling, including mammalian sterile 20-like protein kinases 1 (MST1), large tumor suppressors 1 (LATS1), Yes-associated protein (YAP), and phosphorylation of YAP (p-YAP) in peripheral tissues were significantly altered and highly correlated with blood glucose in db/db mice. Interestingly, the host after gut microbiota transplantation from db/db mice showed decreased MST1 and LATS1 levels, and p-YAP/YAP ratio in the heart, liver, and kidney compared to those from m/m mice. Negative correlations between fasting blood glucose and Hippo signaling levels in selected peripheral tissues also were identified. These findings suggest that alterations in Hippo signaling in selected peripheral tissues may contribute to the development of T2DM, and that therapeutic interventions improving Hippo signaling by gut microbiota transplantation might be beneficial for the treatment of T2DM and other age-related metabolic diseases. Worldwide, type 2 diabetes mellitus (T2DM) in individuals older than 65 years is gradually becoming a prevalent public concern , 2 becauWe previously reported that abnormal composition of gut microbiota greatly contributed to cognitive decline in streptozotocin-induced diabetic mice and Alzheimer’s disease’s (AD) mouse model-senescence-accelerated mouse prone 8 mice , 10. AltThere also is a causal linkage between T2DM characterized by insulin resistance and obesity in pathology and gut microbiota dysbiosis –19. AccoHippo signaling is recognized as a key regulator of organ size and tissue homeostasis , 24. It Although recent work has uncovered novel mechanisms of mitochondrial dysfunction in peripheral tissues implicated in T2DM progression , the rolWe adopted db/db mice as a T2DM mouse model and m/m mice as control subjects. Metabolic parameters, including blood glucose, body weight, and food and water intake, between the db/db and m/m mice were evaluated after 1 week of acclimation. Fasting blood glucose, body weight as well as food and fluid intake were significantly increased in db/db mice than those of m/m mice –1D.Expressions of Hippo signaling, consisting of MST1, LATS1, p-YAP, and YAP were determined in selected peripheral tissues between db/db and m/m mice –2E. CompConsidering the pivotal role of fasting blood glucose in the progression of T2DM, correlations between fasting blood glucose and Hippo signaling levels were analyzed –3E. IntrAfter the establishment of pseudo germ-free mice, fecal transplantation were conducted from db/db and m/m mice . Then, mAfter fecal transplantation, MST1 and LATS1 levels, and p-YAP/YAP ratio in selected peripheral tissues were determined by Western blot in pseudo germ-free mice –5E. IntrA significant correlation was noted between fasting blood glucose and MST1, LATS1 levels, and p-YAP/YAP ratio in the heart, liver, and muscle tissues, as analyzed in selected peripheral tissues after fecal transplantation from db/db and m/m mice into pseudo germ-free mice , 6B, 6D.It is generally acknowledged that aging plays a predisposing role in the dysregulation of glucose metabolism . Age-relIn fact of growing incidence of T2DM in elderly populations, more attentions have focused on the intricate pathogenesis of T2DM that mainly attributed to genetic susceptibility and environmental risk factors , 43. RecPeripheral tissues, such as skeletal muscle and liver, are considered as vital tissues in regulation of glucose metabolism . In thisin vitro trials of multiple cell lines [It is acknowledged that Hippo signaling is mediated by G protein coupled receptor pathway, and epinephrine or glucagon can stimulate Gs-coupled receptors, thus activating Lats1/2 kinase and inhibiting YAP function ll lines . TherefoOur study has several limitations. First, we should adopt more mice to minimize the difference among the groups. Second, all age groups especially the aged ones are needed. Third, tissues, including adipose tissues and various brain tissues, also should be evaluated by various techniques, including immunohistochemistry and quantitative polymerase chain reaction. Moreover, antagonists of Hippo signaling, or interference plasmid with lentivirus vectors that knockdown Hippo signaling were not conducted in this study. It is therefore further investigations are urgently needed.To conclude, our results revealed that the aberrant expressions of Hippo signaling in selected peripheral tissues may trigger the onset of T2DM. Thus, therapeutic interventions improving Hippo signaling by gut microbiota transplantation might be beneficial to the treatment of T2DM, which provides a new insight into the study of age-related metabolic disorders in the near future.n = 6), m/m mice  of the C57 BL/KS strain, and C57BL/6J male mice (n = 40)  were housed 3-5 per cage under controlled lighting conditions (12 h light: 12 h darkness cycle), with free access to rodent feed and water. Housing conditions were in a specific pathogen–free (SPF) facility at a consistent temperature (22° C ± 2° C) combined with a relative humidity of 60% ± 5%. All experimental protocols and animal handling procedures were conducted strictly according to the recommendations in the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health . This study was approved by the Experimental Animal Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology .8-week-old male db/db mice  was measured from a tail vein blood sample via a OneTouch Ultra blood glucose meter .ad libitum for 2 weeks before fecal transplantation [C57BL/6J male mice (n = 40) weighing 20-25 g were treated with large doses of broad-spectrum antibiotics  by antation , 10, 20.antation , 52.During the establishment of pseudo germ-free mouse model, fecal samples can be prepared and stored at −80° C , 53. EacTo prepare total protein extracts, selected peripheral tissues  were quickly collected after sacrifice and lysed in RIPA buffer  mixed with protease and phosphatase inhibitors. The concentration of proteins in the supernatants was quantified through a BCA protein assay kit . Protein samples were denatured in loading buffer and resolved by 10% SDS-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene difluoride membranes . Membranes were blocked in 5% bovine serum albumin for 1 hour before incubation with primary antibody overnight at 4° C. Membranes were washed with TBST and incubated with secondary antibodies at room temperature for 1.5 hour. The signals of proteins were then visualized by an ChemiDocXRS chemiluminescence imaging system . The following antibodies were used: rabbit anti-MST1 , rabbit anti-LATS1 , rabbit anti-p-YAP  and rabbit anti-YAP , and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody .t-test. Correlation analyses were performed using Pearson’s product–moment coefficient. The P < 0.05 was considered statistically significant.Data are shown as mean ± standard error of the mean. Statistical analyses were conducted using GraphPad Prism 7 . Data were analyzed by 1- or 2-way analysis of variance or unpaired"}
{"text": "Discrepancies exist between healthcare provider and patient perceptions surrounding breast cancer treatment. Significant treatment changes in the last 10 years have made re-evaluation of these perceptions necessary.Physicians and nurses involved in breast cancer treatment, and patients who had received breast cancer chemotherapy (past 5 years), were questioned using an Internet survey. Participants ranked physical concerns (treatment side effects), psychological concerns, priorities for treatment selection, and side effects to be avoided during treatment. Patients were asked about desired treatment information/information sources. Rankings were calculated using the mean value of scores. Spearman’s rank correlation was used to determine the concordance of rankings among groups.Survey respondents included 207 patients, 185 physicians, and 150 nurses. Patients and nurses similarly ranked distressing physical concerns; physician rankings differed. Quality of life (QoL) and treatment response ranked high with physicians and patients when considering future treatment; nurses prioritized QoL. All three groups generally agreed on ranking of psychological concerns experienced during chemotherapy, explanation of treatment options, and how treatment decisions were made, although more patients thought treatment decisions should be made independently. Healthcare providers reported providing explanations of treatment side effects and information on physical/psychological support options while patients felt both were lacking. Concordance was calculated as 0.47 (patient–physician), 0.83 (patient–nurse), and 0.76 (physician–nurse). Patients desired additional information, preferring healthcare providers as the source.Specific areas for improvement in breast cancer patient care were identified; programs should be implemented to address unmet needs and improve treatment in these areas.The online version of this article (10.1007/s00520-019-05029-z) contains supplementary material, which is available to authorized users. The progress made in breast cancer treatment over the last decade, including new diagnostic methods and approval of targeted therapeutic agents, has improved patient survival rates . These tRegarding disease awareness in patients, a survey conducted in 2008 reported differences between major concerns of patients and those of physicians and nurses , 4. OtheIn Japan, the major target of the medical care system had been shifted to “reduction of burden among all cancer patients and their families and improvement of quality of life (QoL),” and “building a society in which cancer patients can live peacefully” . HoweverIn the current therapeutic environment, it is clinically important to clarify the priority of the unmet medical information needs of breast cancer patients and to detect discrepancies in perceptions between physicians or nurses. This will maximize the effectiveness of communication between patients and healthcare professionals, making the best use of limited resources. We conducted a survey of physical and psychological perceptions toward treatment between breast cancer patients and healthcare professionals using an Internet questionnaire that was given to patients, physicians, and nurses in Japan.For the survey, patients aged 20–69 years with a diagnosis of breast cancer who underwent chemotherapy within the past 5 years were recruited from a Medilead patient panel . This general consumer panel consisted of 2 million people, 300,000 of whom have verified medical conditions and diseases, including cancer. From this panel, we contacted Japanese patients with breast cancer who met our study criteria. Physicians and nurses were recruited from the M3 panel  which includes 250,000 physicians and 80,000 nurses in Japan. Doctors and nurses with a background suitable for this survey  received study questionnaires. Specifically, physicians (oncologists or surgeons) who belonged to a department of breast oncology/surgery, department of general surgery, or department of oncology, who had experience treating more than 10 breast cancer patients per month with chemotherapy, and nurses who had managed at least one breast cancer patient in the past month were recruited. Participant recruitment occurred between 14 May 2018 and 1 June 2018. Within this period, participants were recruited for each group until the target sample size for that group  was reached. The number of participants was defined by the feasibility of each research panel.An Internet survey aimed at thorough identification of circumstances surrounding common breast cancer treatments in Japan was implemented by Medilead, Inc. The survey questionnaire was developed with reference to previously published questionnaires , 10, 11 Background information and the following survey items were asked of all survey participants: (1) physical symptoms during chemotherapy, (2) psychological anxiety, (3) priorities at treatment selection, and (4) side effects considered most desirable to avoid when selecting treatment. Patients were surveyed regarding: (1) the most necessary information on breast cancer treatment (treatment-related), (2) the most necessary information on breast cancer treatment (other than treatment-related), and (3) desirable information sources and their usefulness.Hormone receptor and HER2 status were assessed based on the American Society of Clinical Oncology–College of American Pathologists guideline definitions  in cliniAnswers to questions, other than single-choice questions, were rated on a 5-point scale, and rank was calculated using the mean value of scores. The concordance of rankings among the three groups was examined using Spearman’s rank correlation, and side effects and concerns regarding chemotherapy were cross tabulated with screening items. Statistical analyses were performed by Medilead, Inc.Study participants comprised individuals who responded to screening questions. Informed consent was obtained from all individual participants included in the study as part of the survey. Although no personal information was collected, disease information was carefully handled. Medilead, Inc. stored the results of the survey on a server housed inside Medilead, Inc.n = 45); fluorouracil, epirubicin, and cyclophosphamide (FEC) (n = 33); paclitaxel (n = 29); nab-paclitaxel (n = 17); epirubicin and cyclophosphamide (EC) (n = 17); docetaxel and cyclophosphamide (TC) (n = 16); doxorubicin and cyclophosphamide (AC) (n = 10), all injections; and capecitabine (n = 12), an oral formulation.A total of 207 patients were included in the analyses. Their regional distribution reflected the general Japanese population. Mean age was 50.8 years (range 26–69 years), and approximately 80% of patients did not experience recurrence or metastasis Table . Fifty-sA total of 185 physicians including those practicing in breast oncology/surgery (81 [43.8%]), general surgery (81 [43.8%]), and oncology (23 [12.4%]) responded to the survey. Affiliated institutions included university hospitals (24.3%), designated cancer care hospitals (29.2%), and general hospitals (42.2%) , general outpatient units (48 [32.0%]), outpatient chemotherapy units (45 [30.0%]), and breast cancer outpatient units (13 [8.7%]). Some nurses had multiple affiliations  experienced by patients during chemotherapy, the most distressing was hair loss, followed by skin and nail problems, weariness/fatigue, taste disorder, numbness in hands and legs , and edema Fig. . The sevWhen asked about side effects most desirable to avoid when selecting treatment, nausea/vomiting and hair loss were ranked high by both patients and nurses. However, physicians most highly ranked pain, followed by nausea/vomiting, then fever; hair loss ranked low (data not shown). Skin and nail problems were a major concern for nurses but not for patients and physicians (data not shown).Regarding psychological concerns experienced during chemotherapy, patients were highly concerned about “changes in appearance and mood,” “fear for metastases and recurrence,” “uncertainty about the future,” and “vague anxiety and depressed mood.” This was similar to the results of physicians and nurses Fig. . ConcernThe results of rank correlation for concern analyzed by Spearman’s rank correlation coefficient Fig.  demonstrFor the questions regarding the priority of treatment attributes important in determining future treatments for the patient, approximately 47% of patients chose items related to QoL (QoL or mild side effects) and approximately 40% of patients chose items related to treatment response  (Online Resource provided by most physicians and nurses.Information related to chemotherapy that patients perceived as having been received included “treatment-associated side effects” (97%), “drugs to reduce treatment-associated side effects” (86%), “all available treatments” (78%), and “risk-benefit balance of treatment” (71%). These informational items were all expected to be received by patients and perceived as provided by physicians Fig. . HoweverMost physicians and nurses thought that treatment decisions should be made either through discussion between patients and physicians or by patients with consideration of the opinion of their treating physician. However, a higher percentage of patients (25%) thought that treatment should be determined by physicians (data not shown). This trend was higher in elderly patients (data not shown).Regarding the most desirable person for consultation regarding chemotherapy, most patients thought that the physician was the only person who should be consulted  between patients and physicians.Regarding physical concerns, “hair loss” and “skin and nail problems” were highly ranked by patients as both the most distressing chemotherapy side effects and most desirable to avoid when selecting treatment, but these were ranked as less important by physicians. This result was reflected in the higher ranking of “changes in appearance (hair loss and complexion) and mood” in patients and nurses regarding psychological anxiety. With an improved survival rate for breast cancer , more paDistress caused by changes in appearance is reported to be particularly intense in patients with breast cancer compared with patients with other types of cancer . For phyContrary to the concerns of patients and nurses, physicians perceived the most important side effects for concern as fever, numbness in hands and legs , and pain. Physicians consider these of high concern because fever may be a sign of serious complication and peripheral neuropathy may cause a dose reduction or discontinuation of chemotherapy. Nausea/vomiting was also recognized as important to manage because it can cause a reduction in both QoL and the continuation of chemotherapy. As a result of the high priority placed on the management of nausea/vomiting by physicians and nurses, these side effects are relatively controlled in clinical practice. The resulting decreased occurrence may have led to reduced concerns in patients.Regarding psychological concerns experienced during chemotherapy, patients were highly concerned about “changes in appearance and mood.” We speculate this may be related to hair loss; this point differed from the previous survey .Regarding treatment efficacy, concerns of “fear for metastases and recurrence,” “anxiety about dying,” and “anxiety about failure to achieve expected treatment responses” were ranked similarly high by patients, physicians, and nurses. In other words, these concerns might reflect longer OS as an attribute for future treatment and were consistent with the results of the previous survey . In the Regarding the difference between the information which patients perceived they had received and the information that was necessary for them to receive (information that should be provided), the core therapeutic information (risk and benefit of chemotherapy) was considered as necessary information, as information having been received by patients, and as information considered by physicians and nurses to have been provided to patients. However, patients felt there was a shortage of information regarding cost (treatment or other), pain relief, professional and social rehabilitation, and information provided during their recovery period despite the fact that patients felt this information should be provided to them. The types of information perceived to be lacking by the patients suggest that patients realized the need for this information after receiving chemotherapy.Over half of physicians and nurses believed they had fully explained physical and psychological support available to relieve symptoms, but patients reported that the information was inadequate. “Provision of palliative care from the time of diagnosis” is a mainstay of cancer control so it may be necessary to provide this information through multiple channels, encouraging patients to use palliative care and to liaise with palliative care specialists wherever possible. This study also showed that there is a need for better provision of information regarding the use of the social security system for help with treatment costs, particularly because the agents used to treat breast cancer are known to be expensive .The discrepancy between physician and nurse beliefs around their explanations of the support available and patient perceptions about these explanations could arise because the explanations really are insufficient. Alternatively, the mental state of the patient at the time of explanation could impact how well an explanation is understood. Regarding information other than that on treatment, patients strongly wished to obtain “information on how to communicate with healthcare professionals,” further suggesting that patients may have felt there was a communication gap. In terms of health information, relationships between health literacy and physical/mental activity have been reported , 20. ThiRegarding consultation for the choice of chemotherapy, patients thought that physicians were both the most appropriate and most important person to consult. Both physicians and nurses expressed a desire for patients to consult nurses and pharmacists more actively. This suggests that while healthcare professionals understand that nurses and pharmacists can provide information regarding the management of side effects, patients do not know that consulting with nurses and pharmacists is a viable option.Our study had several limitations resulting from the use of a questionnaire survey. These limitations included that the responses were chosen from a list, therefore limiting responses; the questionnaire required the time and effort of respondents; there was a possible bias in that respondents may not have disclosed inconvenient information; and there was uncertainty about the credibility of answers received. The survey respondents may have been limited to those who are highly conscious of breast cancer treatment.The results of this survey revealed some differences between patients and physicians in the perception of chemotherapy. While healthcare providers believed they were appropriately sharing information with patients, patients felt that they had not received adequate information regarding pain relief, treatment side effects, and options for physical and psychological support. Both patients and physicians were highly aware of the importance of shared decision-making about treatment selection and of the provision of necessary basic information for decision-making, suggesting that the overall awareness of these points in Japan is good.Specific areas to focus on for improving overall communication between healthcare providers and patients include the establishment of educational programs to inform healthcare professionals that more patients may need appropriate informational and psychological support. Healthcare providers should strive to provide a wide range of information and take time to fully understand and address the sentiments of patients regarding their treatment and care.Online Resource 1Questionnaire completed by participants. (PDF 94 kb)Online Resource 2Priority of treatment attributes for the future treatment of breast cancer. Answers chosen by < 3% of respondents are not listed. QoL: quality of life. (PDF 314 kb)Online Resource 3Preferred source of consultation regarding breast cancer chemotherapy. Patients with breast cancer who had received chemotherapy within the past 5 years, physicians (oncologists or surgeons) who had treated patients with breast cancer and nurses who had at least one breast cancer patient in their care were surveyed and asked who they considered to be the preferred source of consultation regarding breast cancer chemotherapy. Results are shown for each survey group and are represented as a percentage of the total responses. (PDF 320 kb)"}
{"text": "In partnership with communities of the Williams Treaties First Nations in southern Ontario (Canada), we describe an approach to work with communities, and highlight perspectives of food security and sustainability, including priorities and opportunities to revitalize local food systems as a pathway to food security and food sovereignty. The objectives of our project were: (1) to build a shared understanding of food security and sustainability; and (2) to document community priorities, challenges and opportunities to enhance local food access. Utilizing an Indigenous methodology, the conversational method, within the framework of community-based participatory research, formative work undertaken helped to conceptualize food security and sustainability from a community perspective and solidify interests within the four participating communities to inform community-led action planning. Knowledge generated from our project will inform development of initiatives, programs or projects that promote sustainable food systems. The community-based actions identified support a path towards holistic wellbeing and, ultimately, Indigenous peoples’ right to food security and food sovereignty. So if they’re hunting muskrat there’s no muskrat left here really. There might be a few beavers.”Community advisor 8: Concerns were shared related to the high cost of food within the community, including high costs associated with travelling to grocery stores outside of the community. In addition, the costs to engage in traditional food activities related to travel to hunting areas, and access to tools or equipment to hunt was a concern. Amplifying these challenges is the dependency on social assistance due to low employment opportunities and rates in the community.“Food insecurity is huge for us. What is the root problem? It’s income basically.”Community advisor 2: “I think also because we live in the cottage country that really determines the price especially meat and produce. I think sometimes we end up as during summer months like we are paying more for stuff that you know if you lived in the city you wouldn’t be paying that price. I think costs is the biggest factor.”Community advisor 6: The loss of traditional knowledge, language and skills to engage in traditional food acquisition practices was emphasized as a barrier to food security. In particular, reflections were made on how the loss of traditional knowledge is tied to colonization and the Indian Act which have contributed to a lack of sovereignty and control over access to land to hunt, fish and gather (The Constitution Act (1876), Canada has the responsibility for ‘Indians and Lands Reserved for Indians’ under the Indian Act . The Ind“I think we need to start with the younger generations because I think with the older generation we’re slowly losing that link to the past especially the ones that remember what it’s like way back when times were simpler, when people helped themselves, help each other, they didn’t have to rely with a lot of programs.”Community advisor 6: Emphasized, was the implication of limited traditional knowledge, in particular the lack of opportunities for traditional knowledge of food and culture to be passed down to younger generations on how to engage in community food gathering and harvesting practices, including knowing the location of hunting grounds and how to use tools or equipment to hunt.“I think there’s a gap that needs to be bridged somehow because we are losing our traditions and cultures within our own community. Because of that we are having to bring other people in that are teaching the same skills, but they are not our community’s skills.”Community advisor 9: Priorities and actions were identified within each community to enhance food security . These rAccess to grocery stores outside of the community was emphasized. Within each community, the importance of having access to means of transportation  was commonly expressed, as this was recognized as essential to accessing food, and especially healthy food of good quality.Having a community-based travel system in place with appropriate storage was identified as an opportunity to support access to grocery stores outside of the community. For example, having a community-based bus that could accommodate large grocery portions and preserve freshness of vegetables in the summer was discussed.“…my one Aunt she won’t even buy bananas in the winter because they are brown by the time they get on the boat across the bay, but I think too, a lot of people like having to transport and so many people are on the boat. So if the ice starts building up then we use our passenger ferry only, so if you have 70 people riding a ferry then all their food and stuff like you kinda have to watch yourself.”Community advisor 5: “But again it’s just our access has always been an issue because you know you have to bring stuff over. We do have farmers market in the summer, sometimes they are well received and sometimes they are not. It depends who’s bringing stuff over; sometimes it’s fresh, sometimes it’s not.”Community advisor 6: Also highlighted as key to supporting access to food, was ensuring sufficient income and employment opportunities within the community. Creating jobs within the community to support economic growth and security was emphasized.“And we know about [how] income and accessing affordable fresh produce in general, changes their body. Chronic disease goes down, you feel good. You’re not losing limbs because you’re eating sugary bread all the time.”Community advisor 2: The importance of making fresh produce available and affordable for communities to access at wholesale costs was emphasized. Having food markets in the community with a variety of vendors of locally produced food and other essential products was identified as an action that could support greater access to fresh food. Having stable and reliable access to enough healthy and affordable food year-round without worry was broadly understood by communities as having secure access to food.Support for health promoting activities to increase food literacy and knowledge of healthy foods and recipes was also identified as a priority that can support community in healthy eating practices. Developing incentives to have community members engaged at local markets and community-based food initiatives was also discussed.“I think with our community, I’ve heard from [a] few people that the issue too is education and equipment right. So, I mean, just finding out from people like, Ellen, you know getting that knowledge in order [to know] ‘where do I start?’. Some areas in our community are so sandy that you probably couldn’t get a garden, ‘so what do you have to do, okay fine you need to build up, so you have to build a crib right.’ ‘Who do I talk to?’ ‘Who has the skills to build this crib right?’Community advisor 7: “I think it’s trying to find ways to provide fresh foods and vegetables. Cause like I said, they have this program called, the good food box, but that’s only catered towards certain people right; it’s not available to everybody.”Community advisor 3: An existing initiative within one of the partnering communities, Nourish, provides affordable access to local produce and market foods, was identified as an opportunity to support local food production and enhance community food security.“Nourish is a collaboration from Peterborough and public health from the YMCA which we are housed out of. We are a small group. We try to bring people into the system to help with system change…It’s all about food. We have people entering into our programs so we do cooking, we do growing, we have community meals, and we do advocacy work. Basically, the bottom line is to empower people to make decisions for themselves in order to make system changes.”Community advisor 2: Accelerating the uptake of existing projects within the community to support scale-up efforts, including support for local business to enhance traditional food supply  ,46 is anRestoring and maintaining access to traditional food sources and distribution systems, such as wild rice production within the community, was emphasized. Communities shared the importance of promoting traditional food acquisition activities such as fishing, hunting and cultivating . This was discussed as an opportunity to re-establish connections to traditions, culture and the environment . This included promoting greater engagement with youth in traditional food teachings and encouraging younger generations to be advocates for local food projects and traditional food activities. In addition, utilizing social media platforms to engage youth and generate interest in learning more about traditional food and ways to contribute to food security in their community through gardening, gathering of traditional medicines, fishing, hunting, and harvesting and production , was emphasized. An approach identified to support this was having hunters and trappers from the community share their knowledge and skills to other members of the community , including teachings of food as medicine. In addition, having a communal resource for members of the community to access tools and equipment for hunting was also identified as an opportunity to enable greater engagement in traditional food acquisition practices.“I think if we are able somehow to find a way to get the kids excited about farming, gardening, and fishing that we might see a difference, you know? Them bringing that home to their own parents and then getting them interested as well, but I think we need to offer some, I don’t know some ways to get them involved.”Community advisor 8: Creating a sustainable food system, that can support healthy eating in the community and opportunities to achieve food security through approaches that enable local food production , was discussed. Communities highlighted the importance of having policies that enable better management, protection and preservation of the land from pesticides/herbicides, and industry related activities. This was identified as an important process to ensure food safety and minimize food system exposure to environmental contaminants.Regulations in place, with community input to support ongoing environmental assessment of water and soil quality, was identified as a priority to support local food production and a way to build legislative trust within the community. As invasive species can pose a potential threat to traditional food systems, programs and services in place that can remove invasive species were identified as an opportunity to support long-term environmental health in the community. Holding workshops that can enhance community members’ skills and knowledge to engage in local food production and activities that promote energy sustainability was also emphasized.“…well I think there is movement already to tell you the truth. From what I see from the people who have access to programs and their wants and their needs. I think even the younger are more [willing] now to try things. We’ve done canning workshops here. The only thing that I think of is if people were to grow their own food and they have all this extra produce. They get overwhelmed by okay, what do I do with all this kale? So, there’s education in that and how to do that or maybe have a barter system set up or whatever trading and things.”Community advisor 7: The formative work undertaken in this participatory project aimed to facilitate the building of shared understandings of food security and food sustainability, including opportunities to strengthen access to nutritious and sustainable food systems. The Indigenous approaches utilized for knowledge generation and sharing helped to ensure appropriate engagement that centred the priorities of First Nations communities at the core. In addition, the findings will support community-led action planning and will be used to inform next steps for implementation. Knowledge generated from our project is intended to help develop initiatives, programs or projects that promote sustainable food systems. Such community-based action supports a path towards holistic wellbeing and, ultimately, Indigenous peoples’ right to food security and food sovereignty.Food security was broadly understood from a holistic wellness perspective by all partnering communities. It was viewed as both an outcome and determinant of wellbeing. Participants highlighted the importance of access to food, knowledge of how to grow your own food, and the skills and resources to prepare food. Also emphasized was the importance of food procurement from the land, whether through hunting, fishing or gardening, should honour traditional practices and be carried out in ways that protect the environment.Conceptualizations of food security underscored the importance of considering unique dimensions as related to access, availability and use of market and traditional food systems in efforts aimed at enhancing food security in Indigenous communities . For IndFood sovereignty is closely aligned with how partnering communities described food security, food sustainability and opportunities to improve access to food ,49,50,51present day strategies that enable and support the ability of Indigenous communities to sustain traditional hunting, fishing, gathering, farming and distribution practices, the way we have done for thousands of years prior to contact with the first European settlers” .While partnering communities recognize the need to mobilize interests towards creating more sustainable and accessible food systems, moving from priorities to actions requires a level of readiness and capacity to plan, develop and implement initiatives. In addition, it requires working within the specific context of each community’s traditional and market food systems. Building on the momentum to better understand their local context as it relates to challenges and opportunities to strengthen access and availability of food, communities can begin thinking about how to create an environment for transformation and meaningful change. Existing implementation and change frameworks have highlighted the importance of understanding local context to build shared knowledge, assumptions and practices to define the change pursuits of interest as an initial step before effective implementation can take place ,52,53.The community-identified priorities from this formative work therefore present opportunities for change, whereby communities mobilize their interest for sustainable food systems, food sovereignty and food security by identifying and building on existing strengths within the community to enhance readiness and capacity for action. This may include identification of key system actors and champions, along with existing projects, initiatives and programs that can be scaled-up within the community. In addition, successes and lessons identified from existing or previous projects can also be leveraged. For example, an existing initiative, Nourish, was identified by community members as an opportunity for scale-up; extending its reach to nearby First Nations communities and incorporating traditional food could support their access to nutritious, culturally-relevant food. Strengthening relational capacities with key system actors has been identified as a critical process to support successful change pursuits ,53. WithOngoing engagement with key community members from this preliminary work will be essential to identifying key system actors and champions that can lead and sustain implementation efforts and ensure success is achieved in the community. Furthermore, such engagement, driven by the community, will be fundamental to ensuring that actions, including partner-support of implementation strategies, are taken in a culturally safe way that best serves the interests of communities.Community-based participatory work, though well respected and recognized as an appropriate approach to working with Indigenous communities, is not without challenges or shortcomings. Short-term funding cycles may not support additional efforts to plan and implement community-identified projects of interest. While time and resource constraints limited the immediate uptake of the current project, partners continue to identify opportunities to utilize and leverage the findings from this project to inform other areas of existing or future funded projects.We use Indigenous methodologies and western approaches to partner with communities and engage with community advisors to solicit input on priorities and opportunities to enhance food security, strengthen sustainability of food systems, and in doing so support the promotion of food sovereignty. The shared understanding of community perspectives, priorities, challenges and opportunities to strengthen food security will benefit participating Williams Treaties First Nations communities directly. Moreover, the approach taken in this formative initiative will help to inform other work to integrate robust Indigenous practices and approaches to community engagement critical to building shared knowledge and understanding as an initial step and requirement of implementation of programs intended to improve Indigenous health. Hence, the contributions made to understanding ways to support the wellbeing of partnering Williams Treaties First Nations by taking a community driven and participatory approach to identifying priorities and opportunities to revive local food systems, will promote greater food security, sustainability and food sovereignty that may resonate with other Indigenous communities.The current research can serve as an important foundation for planning Indigenous community-based projects and initiatives to strengthen food security, create more sustainable food systems and work towards food sovereignty."}
{"text": "Children with persistent obesity, newly developed obesity, or converting from obese to non-obese had significantly higher HBP risk. For children with GOB or AOB, reduction of <3.6% in BMI or <4.5% in WHtR could decrease the HBP risk.This study aimed to examine the impact of short-term adiposity change on risk of high blood pressure (HBP), and to assess the low limit range of body mass index (BMI) and waist-to-height ratio (WHtR) reduction proposed to decrease the HBP risk in children. Children were longitudinally surveyed at baseline and after a short-term follow-up. General obesity (GOB) is categorized by age and gender-specific BMI cut-off points, abdominal obesity (AOB) by WHtR. Logistic regression model was used to estimate relations between adiposity change and HBP risk with adjustment of covariates. A total of 28,288 children (median of baseline age:10 years) were involved with follow-up of 6.88±1.20 months. After the follow-up, 9.4% of the children had persistent general obesity (GOB), 2.8% converted from GOB to non-GOB, 0.9% had newly developed GOB. When compared with children remained non-GOB, children with continuous GOB status, newly developed GOB, converting from GOB to non-GOB had 5.03-fold (95%CI: 4.32~5.86), 3.35-fold (95%CI: 1.99~5.65), 2.72-fold (2.03~3.63) HBP risk, respectively. Similar findings were observed for abdominal obesity (AOB). Reduction of 0.21–0.88 kg/m The prevalence and magnitude of pediatric high blood pressure (HBP) increased dramatically in recent years, which poses a heavy burden for cardiovascular diseases (CVDs) prevention . As the The sharp increase of overweight and obesity prevalence contributes greatly to the epidemic of HBP among children . The assIn addition, a national secular trend study of BP and obesity among Chinese children showed that the magnitude of impact of obesity on HBP increased sharply in recent years . TherefoA previous weight management study among overweight children show that the reduction of just 5% of body mass index (BMI) is associated with improvements in lipid profiles and insulin sensitivity in six months , and a mhttps://www.clinicaltrials.gov/, Registration number: NCT02343588) which involved more than 70,000 children aged 6–17 years based on a random sampling method from 7 provinces  in China at autumn of 2013. A detailed description of the program was published before Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?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: YesReviewer #2: YesReviewer #3: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********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—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?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: YesReviewer #2: YesReviewer #3: Yes**********5. Review Comments to the AuthorPlease 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: The statistics used in this paper, both univariate and multivariate, are fairly routine and the sample size is certainly large enough. The paper, given the size of the sample, is more descriptive than inferential and the conclusions follow from the analyses performed.Reviewer #2: An important issue, the one the Authors decided to confront with, and given the big sample they could put together, I think their results are worth being widespread.Here just a few remarks:- It's a pity you used \"standard “BMI Reference for Screening for overweight and obesity among school-age children and adolescents” published by National Health Commission of the People‘s Republic of China\" instead of more internationally accepted ones: thinking of future research, it makes difficult to compare your results with other obtained in different countries. I believe that this point deserves to be added to the limitations of the study, and possibly counter-explained.- At lines 224-225 the sentence \"A reduction of at least 0.21~0.88 kg/m2 of BMI (only 0.86%~3.59% of the baseline BMI) in children with GOB...\" is not clear to me; specifically, what do you mean with \"(only 0.86%~3.59% of the baseline BMI)\"?- At lines 274-276, the meaning of the sentence \"those converted from obese to non-obese doubles the HBP risk compared with the persistent non-obese children\" is clear enough, but I believe it should be re-phrased.- There is some other minor revision of English to be made; in the attached file I highlighted the line numbers where some correction is needed, IMHO. There are possibly more than those ones, but they are the ones I easily spotted.Reviewer #3: 1. Did the Authors use some exclusion criteria to enrol the subjects?2. All the investigated variables were normally distributed? Which kind of test the Authors used to verify data normality?3. It would be better use BMI-SDS instead of BMI considering the age of subjects.4. Why the Authors decided to categorize the adiposity parameter (BMI or WHtR) change during the follow-up as quintiles?5. In logistic regression, in addition to OR they have to show and mention also the goodness of fit test of the model. To adjust a regression for multiple variables without to verify if these variable are possible confounders is not correct. Mantel–Haenszel method could be used to verify if a variable is a possible confounder.6. Could the Authors clarify the sentence a” reduction of at least 0.21-~0.88 kg of BMI (only 0.86%~3.59% of the baseline BMI) in children with GOB (baseline BMI=24.49 kg) at lines 224. It is not very clear: only 0.86%~3.59% of the baseline BMI.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, AttachmentPONE-D-21-04822 - Impact of short-term change of adiposity on risk of high blood pressure in children, follow-up study in China.pdfSubmitted filename: Click here for additional data file. 17 Aug 2021Response letter to the Editors’ suggestions1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfResponse: Thanks for the suggestion. We have revised our paper according to PLOS ONE’s style requirements.2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction noResponse: Thanks for the suggestion. We have checked all the references in the manuscript, no cited papers have been retracted.3. Please ensure you have included the registration number for the clinical trial referenced in the manuscript. Moreover, please clarify 1) the inclusion criteria used for recruitment, as we note that the trial registry indicates that children aged 7-18, not 6-17, were included; 2) where the study took place; 3) the length of the follow-up .https://www.clinicaltrials.gov/, Registration number: NCT02343588) in the Method Section (Line 89).Response: Thanks for the suggestion. We have added the registration number for this national multi-centered cluster randomized controlled trial (1) the inclusion criteria used for recruitmentFor the inclusion criteria used for our recruitment, the inclusion and exclusion criteria were listed as follows:Inclusion Criteria:Grade of 1 to 5 in primary schools and grade of 6 to 8 and 10 to 11 in secondary schools.Exclusion Criteria:Suffering or any history of disabilities, cardiovascular diseases, asthma, metabolic diseases, etc.Actually, before the study began, we planned to recruit grade 1 to 5 in primary schools, grade of 6 to 8 and 10 to 11 in secondary schools. For students in grade 1 to 5 in primary schools, the age of them were supposed to be 7-11 years old. But actually, in the real-life study, some children aged 6 years old also started primary school and the intervention is school based. Therefore, there is slight difference in the age range in the registration trial (7-18 years old) and the present study (6-18 years old), also the age range of our paper is same with another paper about the effectiveness of this lifestyle intervention study (1).https://doi.org/10.3389/fped.2021.615283.[1] Dong Y, Zou Z, Wang H, Dong B, Hu P, Ma Y et al. National School-Based Health Lifestyles Intervention in Chinese Children and Adolescents on Obesity and Hypertension. Front Pediatr. 2021; 9(615283. 2) where the study took placeThe study was conducted in 7 provinces  in China. We have added the description of study location in the Method Section (Line 98).3) the length of the follow-up The length of follow up was 6.88 months , which is shorter than the length of follow up described in the study registry (9 months). This is because our study was conducted in 7 provinces , the length of baseline physical examination and survey was almost a month, and the follow-up physical examination and survey also take a month to finish, so the range of the follow-up is set at 9 months, but for every participant at different schools the length of follow-up might be slightly different, and the mean value of follow-up is about 7 months.We have added more description of the length of follow-up and age of the study population in the Method section as follows (Line 104-119).For the enrollment of subjects, the inclusion criteria were as follows: grade of 1 to 5 in primary schools and grade of 6 to 8 and 10 to 11 in secondary schools. And the exclusion criteria were as follows: Suffering or any history of disabilities, cardiovascular diseases, asthma, etc. For students in grade 1 to 5 in primary schools, the age of them were supposed to be 7-11 years old. But actually, in the real life, some children aged 6 years old also started primary school. Therefore, the age range in the registration trial (7-18 years old) is slightly different that of the present study (6-18 years old), also the age range of our paper is same with another paper about the effectiveness of this lifestyle intervention study. The length of follow up was 6.88 months , which is shorter than the length of follow up described in the study registry (9 months). This is because our study was conducted in 7 provinces , the length of baseline physical examination and survey was almost a month, and the follow-up physical examination and survey also take a month to complete, so the range of the follow-up is set at 9 months, but for every participant at different schools the length of follow-up might be slightly different, and the mean value of follow-up is 6.88 months.4. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.Response: Thanks for the suggestion. We have thoroughly copyedited our manuscript for language usage, spelling, and grammar.5. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.Response: Thanks for the suggestion. We have revised the “Funding information” and “Financial Disclosure” accordingly.6. Thank you for stating the following in the Acknowledgments Section of your manuscript: We would like to thank all the children for their participation in our study, also we would like to thank all the head teachers of the schools for their help and cooperation in the research. This research was funded by the National Natural Science Foundation of China , the Hunan Provincial Natural Science Foundation of China , Scientific Research Project of Hunan Health Committee , and a Project Supported by Scientific Research Fund of Hunan Provincial Education Department . The funders had no role in the design, analysis or writing of this article.We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: This research was funded by the National Natural Science Foundation of China , the Hunan Provincial Natural Science Foundation of China , Scientific Research Project of Hunan Health Committee , and a Project Supported by Scientific Research Fund of Hunan Provincial Education Department . The funders had no role in the design, analysis or writing of this article.Please include your amended statements within your cover letter; we will change the online submission form on your behalf.Response: Thanks for the suggestion. We have deleted the funding information in the Acknowledgments Section. Please help revise the funding information in the online submission as follows:FundingThis research was funded by the National Natural Science Foundation of China (81903336), the Hunan Provincial Natural Science Foundation of China (2019JJ50376 & 2020JJ5386), Scientific Research Project of Hunan Provincial Health Commission (202112031516), Key Project of Hunan Provincial Science and Technology Innovation (2020SK1015-3) and a Project Supported by Scientific Research Fund of Hunan Provincial Education Department (18A0028). The funders had no role in the study design, data collection, data analysis, writing of this article or interpreting the results.http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. 7. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see In your revised cover letter, please address the following prompts:a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail  and who has imposed them . Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see We will update your Data Availability statement on your behalf to reflect the information you provide.Response: Thanks for the suggestion. The datasets generated and/or analyzed during the present studyare not publicly available, since ethics approval and participants’ consent does not allow public sharingbindong@bjmu.edu.cn) upon reasonable request.of data, only available from the institute , which involving six large nationally representative cross sectional growth studies  [1], which was also used in previous studies [2-4]. And also similar results based on the World Health Organization (WHO) standard were observed. Although lower prevalence of obesity was observed when two international definitions for child obesity was used (S2 Table), the associations between different obesity patterns with BP level or HBP risk were observed were similar with the results when Chinese BMI Reference was used . We have added these results as part of the sensitivity analysis, and the description of the method and results were added the text  as follows. In the Method section (Line 195-198)(4) We used an international definition of child obesity developed by the International Obesity Task Force (IOTF) and the World Health Organization (WHO) standard of obesity for 5-19 years old children to test the associations[18-24].In the result section (Line 212-217 and line 291-293)The prevalence of obesity based on the international definition of child obesity developed by IOTF was 6.1% at the baseline  and 5.1% at the follow-up survey . The prevalence of obesity based the WHO standard was 10.1%  at baseline and 8.8% at the follow-up survey .We also used two international definitions of child obesity (ITOF standard and WHO standard) to test the associations between BP level or HBP risk and obesity change pattern, similar results were observed . 18. Cole TJ, Bellizzi MC, Flegal KM, Dietz WH. Establishing a standard definition for child overweight and obesity worldwide: international survey. BMJ. 2000; 320: 1240-1243. 19. Deren K, Wyszynska J, Nyankovskyy S, Nyankovska O, Yatsula M, Luszczki E, Sobolewski M, Mazur A: Assessment of body mass index in a pediatric population aged 7-17 from Ukraine according to various international criteria-A cross-sectional study. PLoS One 2020, 15(12):e0244300.20. Oosterhoff M, Over EAB, van Giessen A, Hoogenveen RT, Bosma H, van Schayck OCP, Joore MA: Lifetime cost-effectiveness and equity impacts of the Healthy Primary School of the Future initiative. BMC Public Health 2020, 20(1):1887.21. Martos-Moreno GA, Martinez-Villanueva J, Gonzalez-Leal R, Barrios V, Sirvent S, Hawkins F, Chowen JA, Argente J: Ethnicity Strongly Influences Body Fat Distribution Determining Serum Adipokine Profile and Metabolic Derangement in Childhood Obesity. Front Pediatr 2020, 8:551103.https://www.who.int/tools/growth-reference-data-for-5to19-years/indicators/bmi-for-age]22. World Health Organization (WHO). Growth reference data for 5-19 years: WHO . Then for the obese children, we categorized the then into 5 quintiles with P20, P40, P60, and P80 of BMI SDS change from baseline to the follow-up, similar results were observed. When compared with the reference group (quintile 5), quintile 1 group had significantly low risk of HBP . For quintile 2 group, OR was also <1 , but it didn’t reach statistical significance. We have added this as a part of our sensitivity analysis as follows. In the method section (Line 187-190)(5) We also used BMI standard deviation score (BMI SDS) instead of BMI to compare the risk of HBP in different groups with multivariate logistic models. Age- and sex-specific BMI SDS was calculated using the World Health Organization (WHO) child growth charts[25-27].In the result section (Line 282-284)In addition, BMI SDS was used instead of BMI to test the association of different quintiles of BMI SDS change and risk of HBP, similar results were observed (S8 Table).25. McLaughlin EJ, Hiscock RJ, Robinson AJ, Hui L, Tong S, Dane KM, Middleton AL, Walker SP, MacDonald TM: Appropriate-for-gestational-age infants who exhibit reduced antenatal growth velocity display postnatal catch-up growth. PLoS One 2020, 15(9):e0238700.26. Zhang L, Huang L, Zhao Z, Ding R, Liu H, Qu W, Jia X: Associations Between Delivery Mode and Early Childhood Body Mass Index Z-Score Trajectories: A Retrospective Analysis of 2,685 Children From Mothers Aged 18 to 35 Years at Delivery. Front Pediatr 2020, 8:598016.27. Poh BK, Lee ST, Yeo GS, Tang KC, Noor Afifah AR, Siti Hanisa A, Parikh P, Wong JE, Ng ALO, Group SS: Low socioeconomic status and severe obesity are linked to poor cognitive performance in Malaysian children. BMC Public Health 2019, 19(Suppl 4):541.4. Why the Authors decided to categorize the adiposity parameter (BMI or WHtR) change during the follow-up as quintiles?Response: We thank the reviewer for this comment. We categorized (BMI or WHtR change) as quintiles [1] because we want to find the potential BMI or WhtR reduction range of obese children to get an improved BP profile. We further categorized the reduction range of obese children into ten equal groups with similar results observed.[1] Carthy P, Lyons S, Nolan A. Characterising urban green space density and footpath-accessibility in models of BMI[J]. BMC Public Health, 2020, 20(1).5. In logistic regression, in addition to OR they have to show and mention also the goodness of fit test of the model. To adjust a regression for multiple variables without to verify if these variables are possible confounders is not correct. Mantel–Haenszel method could be used to verify if a variable is a possible confounder.Response: Thanks for this comment. For logistic regression models exploring the association between risk of high blood pressure and different general or abdominal obesity status change, from model 1 to model 4, the goodness of fit test improved gradually, the Nagelkerke R2 of model 1 and model 4 for general obesity change pattern were 5.9% and 13.4%, and 4.8% and 13.2% for model 1 & 4 for abdominal obesity change patterns. For the categorical covariates in the logistic regression models, we used Mantel–Haenszel method to verify whether sex, age group(<=13 years old and >13 years old), and area  are potential cofounders, the results showed that they were possible confounders which were also found in previous studies[1-3]. These variables were adjusted when the risk factors for high blood pressure were analyzed.[1] Yang YD, Song J Y, Wang S, et al. Combined effects of the rs9810888 polymorphism in calcium voltage-gated channel subunit alpha1 D (CACNA1D) and lifestyle behaviors on blood pressure level among Chinese children[J]. PLoS ONE, 2019, 14(5):e0216950-.[2] Yang Y, Dong B , Wang S , et al. Prevalence of high blood pressure subtypes and its associations with BMI in Chinese children: a national cross-sectional survey[J]. BMC Public Health, 2017, 17(1):598.[3] Yang Y, Dong B , Zou Z , et al. Association between Vegetable Consumption and Blood Pressure, Stratified by BMI, among Chinese Adolescents Aged 13–17 Years: A National Cross-Sectional Study[J]. Nutrients, 2018, 10(4):451.6. Could the Authors clarify the sentence a” reduction of at least 0.21-~0.88 kg of BMI (only 0.86%~3.59% of the baseline BMI) in children with GOB (baseline BMI=24.49 kg/m2) at lines 224. It is not very clear: only 0.86%~3.59% of the baseline BMI.Response: We apologize for this and have clarified this sentence. This sentence means that a reduction of 0.21-0.88 kg/m2, and the average baseline BMI of children with general obesity was 24.49kg/m2, so 0.86%-3.59%  of the baseline BMI will beneficial to the BP profile. We have clarified it in the text as follows (Line 277-279).A reduction of at least 0.21~0.88 kg/m2 of BMI  in children with GOB (baseline BMI=24.49 kg/m2) could be significantly beneficial to the BP profile compared with the reference group (Table 4).AttachmentCover letter+Response letter to reviewers 20210816.docxSubmitted filename: Click here for additional data file. 25 Aug 2021Impact of short-term change of adiposity on risk of high blood pressure in children: Results from a follow-up study in China.PONE-D-21-04822R1Dear Dr. Yang,We’re 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’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll 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’ll 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,Raffaella Buzzetti, M.D.Academic EditorPLOS ONE 1 Sep 2021PONE-D-21-04822R1 Impact of short-term change of adiposity on risk of high blood pressure in children: Results from a follow-up study in China. Dear Dr. Yang: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 ofDr. Raffaella Buzzetti Academic EditorPLOS ONE"}
{"text": "Background: As a plant-derived polycyclic phenolic carboxylic acid isolated from Salvia miltiorrhiza, lithospermic acid (LA) has been identified as the pharmacological management for neuroprotection and hepatoprotection. However, the role and mechanism of lithospermic acid in the pathological process of myocardial ischemia-reperfusion injury are not fully revealed.Methods: C57BL/6 mice were subjected to myocardial ischemia and reperfusion (MI/R) surgery and pretreated by LA  for six consecutive days before operation. The in vitro model of hypoxia reoxygenation (HR) was induced by hypoxia for 24 h and reoxygenation for 6 h in H9C2 cells, which were subsequently administrated with lithospermic acid (100 μM). Nrf2 siRNA and dorsomorphin (DM), an inhibitor of AMPKα, were used to explore the function of AMPKα/Nrf2 in LA-mediated effects.Results: LA pretreatment attenuates infarct area and decreases levels of TnT and CK-MB in plasm following MI/R surgery in mice. Echocardiography and hemodynamics indicate that LA suppresses MI/R-induced cardiac dysfunction. Moreover, LA ameliorates oxidative stress and cardiomyocytes apoptosis following MI/R operation or HR in vivo and in vitro. In terms of mechanism, LA selectively activates eNOS, simultaneously increases nuclear translocation and phosphorylation of Nrf2 and promotes Nrf2/HO-1 pathway in vivo and in vitro, while cardioprotection of LA is abolished by pharmacological inhibitor of AMPK or Nrf2 siRNA in H9C2 cells.Conclusion: LA protects against MI/R-induced cardiac injury by promoting eNOS and Nrf2/HO-1 signaling via phosphorylation of AMPKα. Myocardial ischemia-reperfusion (MI/R) injury has a significant impact on the prognosis of patients with revascularization . MoreoveSalvia miltiorrhiza has been widely utilized to prevent cardiovascular diseases (CVDs) in China and Asia -induced hepatic oxidative damage  is implicated in antioxidant defense mechanisms in the myocardium, which is usually activated by increased reactive oxygen species (ROS) production . Nrf2 upIn the present study, we demonstrate that LA improves cardiac function and attenuates myocardial injury during MI/R. Moreover, LA suppresses oxidative stress and apoptosis by promoting activation of endothelial nitric oxide synthase (eNOS) and Nrf2/HO-1 pathway via phosphorylation of AMPKα in MI/R injury.TableAll animal experimental procedures followed the National Institutes of Health (NIH) guidelines and were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University. Lithospermic acid  was obtained from Shanghai Winberb Medical Technology Co., Ltd .n = 10), Sham-LA (n = 10), MI/R-NS (n = 15) and MI/R-LA (n = 15). Mice were pretreated with LA  for six consecutive days before MI/R surgery or sham, and poured into the normal saline control group processing.C57BL/6 male mice , obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences , were randomly separated into 4 groups: Sham-NS   was utilized to determine the area at risk and infarcted area following MI/R operation. More details are provided in the supplement material. The isolated heart tissue was immobilized with 4% paraformaldehyde and dehydrated with gradient alcohol. Then, 5 μm-thick sections were paraffin-embedded. Immunohistochemistry (IHC) of 4-hydroxynonenal and Nrf2 was performed to detect oxidative stress and the location of Nrf2 in the heart. Dihydroethidium (DHE) fluorescence was also performed to assess oxidative. Moreover, a terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay was used to determine apoptotic cells in the heart after MI/R surgery. The density of IHC and DHE staining was assessed using Image-Pro Plus version 6.0. More details are provided in the supplement material.in vivo and in vitro using the Nuclear and Cytoplasmic Protein Extraction kit  and protein concentration was determined by bicinchoninic acid assay . Subsequently, 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to separate protein samples (40 μg), and then proteins were transferred to polyvinylidene difluoride (PVDF) membranes. After incubation with blocking buffer and targeted antibodies overnight, the PVDF membranes were subsequently incubated with a corresponding secondary antibody. Afterward, targeted protein bands were examined using a chemiluminescence method, and the density of target bands was evaluated by AlphaEaseFC software processing system  and Image Lab software. Primary antibodies used in this study are provided in The heart sample was collected after reperfusion for 24 h. Protein was extracted from heart homogenates by radioimmunoprecipitation assay (RIPA) lysis buffer. Nuclear protein was obtained Total RNA was extracted from mouse hearts and H9C2 cells using Trizol reagent  and cDNA was produced using the Transcriptor First Strand cDNA synthesis kit . Meanwhile, SYBR Green (04707516001) was utilized to amplify transcripts, and GAPDH was the endogenous reference. All primers used are listed in 2 at 37°C, whereas for HR, cells were subjected to hypoxia for 24 h and reoxygenation for 6 h with 5% CO2, 94% N2 and 1% O2 at 37°C in tri-gas incubators. H9C2 cells in the LA group were subjected to LA (100 μM) treatment for 24 h after Nor or HR.H9C2 cells were obtained from the Cell Bank of the Chinese Academy of Sciences  and cultured in Dulbecco’s modified Eagle’s medium . Afterward, H9C2 cells in good growth condition were divided into 4 groups: Normoxia (Nor)-PBS group, Nor-LA group, hypoxia-reoxygenation (HR)-PBS group and HR-LA group. For Nor, cells were incubated under 5% COFor small interfering RNA (siRNA)-mediated knockdown experiments, Nrf2-siRNA (sc-156128) or Scr-siRNA was transfected with Lipofectamine 6,000  at 40 nM concentration in culture medium following the manufacturer’s protocol. Western blot analysis was performed to assess the efficiency of knockdown. For the AMPK inhibitor, dorsomorphin  was incubated with H9C2 cells for 18 h after Nor or HR. Immunoblotting was performed to assess the efficiency of blockage.After washing with phosphate-buffered saline (PBS), H9C2 cells were fixed by 4% paraformaldehyde for over 15 min. Afterward, permeabilization was performed with 0.1% Triton X-100 (Amresco) in PBS. Then cells were incubated with 10% goat serum and subsequently stained with anti-Nrf2 overnight at 4°C. Next, cells scrubbed with PBS were then subjected to the secondary antibody goat anti-rabbit Alexa Fluor™ 488  for 1 h. SlowFade® Gold anti-fade reagent with DAPI  was utilized for mounting as previously described .p-value <0.05 was considered statistically significant.Data are expressed as mean ± standard error of the mean (SEM) and evaluated using SPSS version 22.0 . Comparison of multiple groups was performed using one-way analysis of variance (ANOVA) and two group comparisons were analyzed by unpaired Student’s t-test. A MI/R injury is characterized by a significant increase in oxidative stress, inflammation and apoptosis in the heart tissues . LA has in vivo.To determine the effects of LA on cardiac function, echocardiography and hemodynamic assessment were performed in mice. It was found that LA attenuated MI/R-induced left ventricle systolic dysfunction ; Table 1To evaluate whether LA regulates oxidative stress and apoptosis in the MI/R heart, immunohistochemistry staining of 4-hydroxynonenal (4-HNE) in the heart section was performed. The results showed a higher expression of 4-HNE after MI/R operation, which was significantly suppressed by LA pretreatment . SimilarTUNEL staining was employed to determine the role of LA MI/R-induced apoptosis in heart tissues. TUNEL-positive cells were remarkably increased after MI/R surgery; however, LA treatment attenuated MI/R-induced apoptosis in the heart . Subsequin vitro experiments were performed with the HR model  production in heart tissues , which wowing HR , which wowing HR . Overallin vitro were evaluated. Congruous with in vivo experiments, Western blotting showed that LA promoted the activation of the Nrf2/HO-1 pathway in H9C2 cells under HR  kinase group, AMPK is widely distributed in various organs . AMPK isve genes . FurtherIn summary, this study demonstrates that LA improves cardiac function and attenuates myocardial injury in mice. It also suppresses oxidative stress and apoptosis following hypoxia-reoxygenation. Mechanistically, LA promotes the activation of the eNOS and Nrf2/HO-1 pathway by enhancing phosphorylation of AMPKα, providing a novel therapeutic strategy for reperfusion in AMI patients."}
{"text": "In the PRD group, a shorter LOP compared with the GA group was observed . It was noted that the PRD group had a lower total remifentanil consumption than the SPI group . In ASA I-III patients undergoing ESS, intraoperative monitoring based on state entropy and SPI values can optimize the CSF and reduce TEIBL, whereas monitoring based on state entropy and PRD measurements can optimize the cost effectiveness of anesthetic drugs and the use of the operation room.Inadequate intraoperative analgesia causes the deterioration of the condition of the surgical field (CSF) as a result of hemodynamic instability. Analgesia monitors are used to guide remifentanil) infusion to optimize intraoperative analgesia. The main aim of the current randomized controlled trial was to investigate the potential advantages of intraoperative analgesia monitoring using surgical Pleth index (SPI)- or pupillometry (PRD)-guided remifentanil administration for managing the volume of total intraoperative blood loss (TEIBL), CSF, and length of operation (LOP) in comparison with the standard practice in patients undergoing endoscopic sinus surgery (ESS). The 89 patients in our study were grouped as follows: 30 patients were assigned to the general analgesia (GA) group, 31 patients were assigned to the SPI group, and 28 patients were assigned to the PRD group. The speed of remifentanil infusion was accelerated by 50% when SPI, PRD, or BSS were increased by >15 points, >5%, or >2, respectively, in adjacent groups until their normalization. The SPI group showed significantly lower TEIBL in comparison to the GA group (165.2 ± 100.2 vs. 283.3 ± 193.5 mL;  Endoscopic sinus surgery (ESS) is currently the surgical treatment of choice for sinonasal disease, and its outcome is dependent on the intraoperative condition of the surgical field (CSF) . Marked A wide variety of maneuvers have been proposed to decrease the potential risk of harming the surrounding structures by reducing the amount of total intraoperative blood loss (TEIBL) during ESS performed under general anesthesia. Numerous perioperative regimens, including either premedication or the intraoperative intravenous infusion of beta-blockers ,4,5 or aSurgical maneuvers in the operation field provoke painful afferent stimulation (nociceptive effect), which can induce the release of stress hormones in the absence of sufficient intraoperative analgesia; trigger heart rate and arterial blood pressure increments, resulting in potentially excessive TEIBL; and lead to the administration of recall of awareness tests (ROA attenuates this activity (anti-nociceptive effect)). However, excessively high doses of intravenous intraoperative remifentanil may lead to hemodynamic depression with hazardous bradycardia and hypotension and an increased requirement for intravenous fluids with unwelcome untoward events ,14,15,16Currently, different techniques for monitoring the intraoperative efficacy of analgesia surgical pleth index (SPI), antinociception index (ANI), and pupillary dilatation reflex (PRD) are gaining increasing popularity , as in tThe main aim of the current randomized controlled trial was to investigate the potential advantages of intraoperative analgesia monitoring using SPI- or PRD-guided remifentanil administration for managing the volume of TEIBL, CSF, and LOP in comparison with the standard practice based on the observance of CSF.One hundred patients  score, I–III) with a history of chronic rhinosinusitis who were scheduled to undergo elective ESS without septoplasty with or without polyposis at the Department of Otolaryngology in Regional Hospital no. 5 in Sosnowiec, Poland, met the inclusion criteria for the present comparative analysis.The exclusion criteria were as follows: pregnancy, history of allergy to hypnotics, pre-existing cardiovascular diseases , risk of intraoperative hypotension (low left ventricle ejection fraction < 30%) requiring extensive fluid challenge and the administration of vasoactive drugs that may possibly influence hemodynamic monitoring, low platelet count , any pathology in coagulation tests, medication with drugs interfering with clotting.Randomization was performed by the principal investigator (M.S.) by opening sealed envelopes after each participant provided written informed consent to participate in the study and undergo general anesthesia for ESS. The study protocol was in compliance with the 1964 Helsinki Declaration and was approved by the Local Bioethics Committee at the Medical University of Silesia in Katowice (Poland) on 24 May 2016 . The study was registered in the Clinical Trials Registry .2 was maintained at 35–37 mmHg. The lungs were ventilated using a low flow  and lung-protective strategies with low tidal volumes of 6 mL/kg of ideal body weight.Patients fasted for 12 h before ESS. All the patients received 3.75–7.5 mg of midazolam  in premedication on the day of surgery. Immediately before the surgery, the patients received 10 mL/kg per body weight of Optylite Solution intravenously (i.v.). The patients were preoxygenated with 100% oxygen and intravenously administered fentanyl  in a single dose of 2 µg/kg body weight and propofol  in a single dose of 2.5 mg/kg body weight. When the ciliary reflex disappeared, patients in all groups were paralyzed with a standard intravenous dose of rocuronium  at 0.6 mg/kg body weight, and were intubated after approximately 1 min 20 s with a proper-sized cuffed endotracheal tube 7.0–8.5 in the supine position. During the co-induction of general anesthesia, CO2), fraction of inspired oxygen in the gas mixture (FiO2), and exhaled carbon dioxide concentration (etCO2). The depth of anesthesia was monitored using response and state entropy  and muscle relaxation was monitored using the E-NMT module in all patients, despite their group allocation. Surgical pleth index (SPI) measurements using a Carescape Monitor  in patients allocated to the SPI group only and PRD measurements using NeuroLight Algiscan  in patients allocated to the PRD group only were performed accordingly.Throughout the induction and the surgery itself, standard monitoring procedures for vital parameters were used, including non-invasive arterial pressure (NIBP) measurement every 5 min and measurements of systolic arterial pressure (SAP), mean arterial pressure (MAP), diastolic arterial pressure (DAP), heart rate (HR), standard ECG II, arterial blood saturation  technique. Propofol and remifentanil were administered in a constant intravenous  infusion to separate venous access. An extra dose of rocuronium was always administered when train of poor (TOF). The depth of anesthesia was maintained using propofol at a target SE value of around 40, since the depth of general anesthesia influences patients’ reactions to painful stimuli.After the end of the ESS, in order to avoid unacceptable postoperative pain perception a standard dose of non-steroidal anti-inflammatory drug was administered intravenously according to the guidelines and individual patients’ conditions . At the TEIBL measurements for each patient were performed by the same researcher (I.S.). The blood suction canisters, surgical pads, and irrigation totals were examined to calculate the final blood loss, similar to the technique adopted by DeMaria S et al.  All BBS Upon their admission to the operating theatre, a sensor of entropy EEG  was affixed onto the patients’ foreheads according to the manufacturer’s suggestions, a pulse oximeter was attached to their finger contralateral to that used for venous access, an NIBP cuff was attached to their left arm, and the first values were recorded.The patients were positioned at 15° in the reverse Trendelenburg position to reduce venous congestion. After the induction of TIVA, initial infusion of remifentanil 0.25 µg/kg body weight/minute was started. Randomization was performed simultaneously.On the basis of group allocation, remifentanil infusion was adjusted alongside the maintenance of TIVA as described below.The SPI value was measured online and recorded with a sampling frequency of 1 min. Each time the SPI value was >15 in comparison with the mean SPI value during stage 2, the remifentanil infusion speed was increased by 50% in the sequence 0.25/0.375/0.5/0.675/etc. µg/kg body weight/min. When the MAP was <65 mmHg, an extra fluid challenge of 5 mL/kg body weight was administered.Alternatively, to avoid unnecessary hemodynamic complications in cases with HR < 45/min, MAP < 65 mmHg with no reaction to fluid challenge, BBS < 2, and SPI < baseline value, the remifentanil infusion rate was decreased by 50% alongside the administration of a single 10 mg dose of ephedrine .The PRD was measured and recorded with a sampling frequency of 15 min. Each time the PRD value was >5%, the remifentanil infusion speed was increased by 50% in the sequence 0.25/0.375/0.5/0.675/etc. µg/kg body weight/min. When MAP was <65 mmHg, an extra fluid challenge of 5 mL/kg body weight was administered.Additionally, each time the BBS value was >2, extra PRD measurements were conducted.Alternatively, to avoid unnecessary hemodynamic complications in cases with HR < 45/min, MAP < 65 mmHg with no reaction to fluid challenge, BBS < 2, and PRD < 5%, the remifentanil infusion rate was decreased by 50% with the administration of a single 10 mg dose of ephedrine.BBS values were measured and recorded with a sampling frequency of 5 min. Each time the BBS value was >2, the remifentanil infusion speed was increased by 50% in the sequence 0.25/0.375/0.5/0.675/etc. µg/kg body weight/min.Alternatively, despite the group allocation, to avoid unwelcome bradycardia and hypotension, in cases with HR < 45/min or MAP < 65 mmHg with no reaction to fluid challenge (iv bolus of 5 mL/kg of body weight of Optylite solution), and BBS < 2 the infusion rate of remifentanil was reduced to 50% of the initial infusion speed alongside the administration of a single 10 mg dose of ephedrine.The sequence of events related to the adjustment of remifentanil infusion according to the group allocation  values is shown on the graph .After the induction of TIVA, pledgets impregnated with topical vasoconstrictor were administered to the nasal cavity for the duration of stage 2. All ESS procedures were performed by the same specialist in otolaryngology with over 10 years of experience in this field (P.W), who performed either sphenoethmoidectomy with medial antrostomy (the most severe procedure), total ethmoidectomy with medial antrostomy, anterior ethmoidectomy with medial antrostomy, or isolated antrostomy (the least severe procedure) using powered and manual instrumentation. When the patients showed bleeding that did not respond to topical vasoconstriction, bipolar cautery was administered and standard nasal dressings were placed within the middle meatus, as required.CSF estimations were performed every 5 min by the operator on the basis of the Boezaart bleeding score (BBS) ; 1, slight bleeding, no suctioning required; 2, slight bleeding, occasional suctioning required; 3, slight bleeding, frequent suctioning required and bleeding threatens the surgical field a few seconds after suction is removed; 4, moderate bleeding, frequent suctioning required and bleeding threatens the surgical field directly after suction is removed; 5, severe bleeding, constant suctioning required and bleeding appears faster than can be removed by suction, with the surgical field being severely threatened and surgery usually not possible) .The time of surgery was counted from the moment of first insertion into the nasal cavity until the last withdrawal of the endoscope from the nasal cavity.n = 135 per 1.5 years), a confidence level of 95%, and a margin of error of 5%. Statistical calculations were performed using MS Excel, STATISTICA 13, and Stat Soft Poland. The measured data were characterized using the mean and standard deviation (X ± SD), as well as median with interquartile range (M (IQR)). The normality of distribution was assessed using the Shapiro–Wilk W test. The significance of the differences between means was tested using a one-way ANOVA, and for skewed distributions the compatibility in groups was examined using the Kruskal–Wallis test. To explore differences between multiple groups, the appropriate post hoc tests were used. For nominal data, we used percentages. The relationships between nominal variables were verified by the χ2 test of independence. Statistical significance was set at p < 0.05.The sample size was estimated at 100 considering the total number of surgeries performed  women and 56 (62.9%) men, who were grouped as follows: GA, 30 patients (33.7%); SPI, 31 patients (34.8%); and PRD, 28 patients (31.5%). The detailed patient characteristics are shown in All operated patients were divided into four categories depending on the severity of the surgery performed: sphenoethmoidectomy with medial antrostomy (the most severe procedure), total ethmoidectomy with medial antrostomy, anterior ethmoidectomy with medial antrostomy, or isolated antrostomy (the least severe procedure). This classification provided the best representation of the difficulty and duration of the procedure, thus allowing the best assessment of the major factors influencing the bleeding. Another criterion of similar importance for the final result was the distinction between single- and double-sided procedures. This system of division allowed for the statistical estimation of homogeneity among the three groups tested  with respect to the surgical procedure performed .There were no significant differences in terms of the number of surgical procedures carried out. This allowed us to eliminate the possibility of the unwanted influence of the type of surgical procedure performed on intraoperative bleeding. No statistically significant intergroup differences were observed in the percentages of surgical procedures performed .The GA group showed a significantly higher intraoperative blood loss, length of surgery, propofol consumption, and maximum speed of remifentanil infusion, while the SPI group showed a higher total remifentanil consumption and higher minimum and mean speeds of remifentanil infusion . A compaObtaining a “dry” surgical field—assessed, for example, using the BBS—has become a matter of great concern for patients undergoing ESS, as excessive intraoperative bleeding in the surgical field can lead to a number of unwelcome untoward life-threatening complications, such as the disruption of neighboring structures  as a result of bleeding impairing visibility ,17,18. FFor PRD, the intraoperative guidance of opioid titration was based on objective measurements of pupil sizes and pupillary reflexes using portable infrared pupillometers reflecting the reactions of the human pupils to drugs and nociceptive stimulation. During the session, the patient’s eye was flooded with infrared light and the reflected images were measured on an infrared sensor, with the variations in pupil size, alongside the pupillary light reflex and pupillary reflex dilatation, being calculated by the instrument and displayed on a screen immediately after each time-stamped measurement. The monitoring of the quality of intraoperative analgesia using pupillometry has proven to be useful in the management of pain because it allows for the assessment of the effect of opioids and the titration of anesthetics ,29, and Karkanevatos et al.  analyzedIn turn, Sabourdin et al.  performeIn our study, despite the group allocation, we employed state entropy (SE) to achieve similar conditions for all subjects in terms of central nervous system suppression, which was achieved using propofol infusion. The depth of anesthesia markedly influences the intraoperative pain perception. SE within a range of 40–60 is known to reflect the proper surgical depth of anesthesia, and the target SE in our study was set at around 40  to provide comparable conditions for all subjects in the study. Although the mean SE in the PRD group was significantly higher than that in the SPI and GA groups , the three different anesthetic regimens could be reliably compared, since such differences showed little clinical relevance and because the majority of studies in the recent literature pay no attention to the depth of anesthesia in individual subjects undergoing ESS.We hypothesized that intraoperative surgical manipulations lead to afferent nociceptive stimulation, resulting in increased blood pressure and heart rate and eventually excessive bleeding impairing the CSF, the intensity of which would vary depending on the stage of ESS , with varied hemodynamic changes occurring according to the intensity of the surgical maneuvers performed.p < 0.05) and no significant difference from the PRD group. In our study, the online observation of SPI value changes alongside the online adjustment of remifentanil infusion speed in the case of a delta SPI increase >15 points over the baseline level supposedly resulted in the effective suppression of afferent nociceptive stimulation before the increase in heart rate and systolic blood pressure resulted in excessive blood loss. In contrast to SPI monitoring, PRD assessments could not have been performed online due to the manufacturer’s indications. Therefore, nociceptive afferent stimulation in the PRD group led to some hemodynamic decompensation, resulting in increased blood loss. However, this was subsequently suppressed by accelerating the FMFNT infusion after PRD measurement (sensitivity > 5%), and no statistically significant difference was noted between the PRD and GA groups.In the current study, the final analysis showed a statistically significant difference between the SPI and GA groups in terms of TEIBL (p = 0.111), the authors underlined the limited application of their anesthetic regimen to patients with ASA I-II status due to the subsequent risk of myocardial depression, whereas the current analysis also included 14 ASA III patients.According to the literature, the values of TEIBL and CSF vary according to the anesthetic regimen used. Lee et al.  comparedVery impressive TEIBL values were also achieved by Cardesín A et al. , with paThe overall cumulative dose of remifentanil and propofol given failed to reach statistical significance in the SPI guidance and optimized the intraoperative use of TIVA without sparring effect and influence on LOP. In the PRD group, the LOP was significantly shorter (63.1 ± 26.7 min) than that in the control group (82.6 ± 33.1 min). The introduction of PRD-guided remifentanil administration in our study resulted in a shorter LOP than that in the study conducted by Lee J et al. , who comp < 0.05), and they were also far from the definition of controlled hypotension, whereas the CSF and TEIBL values were surprisingly even more acceptable. Blood pressure reductions, which constitute an independent risk factor for the occurrence of myocardial ischemia in high-risk patients, were not as low in the SPI group as in the PRD and control groups . Thus, the anesthetic regimen used in the current study consisting of SPI-guided remifentanil infusion provided patient safety with a satisfactory TEIBL.Interestingly, the novel approach to the intraoperative monitoring of analgesia during ESS adopted in the current study appeared to be safer for patients in the SPI group compared with the control group in view of the fact that profound hypotension was reported to lead to severe postoperative complications, such as the impairment of cerebrovascular autoregulation and heart function, multi-organ failure, personality changes, and death ,35,36,37In the study of Nair S et al. , the autOur study has several limitations. Although the anesthetic regimen of SPI-guided remifentanil infusion is usually based on the assumption that any SPI value > 50 or intraoperative delta SPI > 10 constitutes an indication of the acceleration of remifentanil infusion , we adopOur results are similar to observations made by other researchers. This procedure may impose either a substantial cost or constitute a risk of potential cardiovascular complications while excluding numerous ASA III patients, whose population is growing. We recommend the introduction of adequacy of anesthesia (AoA) monitoring based on SE and SPI to optimize the CSF and reduce the total estimated intraoperative blood loss in a wider group of patients, who may otherwise receive either premedication with clonidine or an expensive intravenous infusion of esmolol. The introduction of intraoperative monitoring based on state entropy and PRD measurements will help us to optimize resources related to the cost-effectiveness of drugs and the use of an operation room."}
{"text": "VEGF-A knockdown (KDVEGF) in diabetic mice and in endothelial nitric oxide synthase knockout mice (−/−eNOS). Diabetes was induced with streptozotocin using the Animal Models of Diabetic Complications Consortium (AMDCC) protocol. Induction of podocyte KDVEGF led to diffuse glomerulosclerosis, foot process effacement, and GBM thickening in both diabetic mice with intact eNOS and in non-diabetic −/−:VEGFKDeNOS mice. KDVEGF diabetic mice developed mild proteinuria and maintained normal glomerular filtration rate (GFR), associated with extremely high NO and thiol urinary excretion. In −/−:VEGFKDeNOS (+dox) mice severe diffuse glomerulosclerosis was associated with microaneurisms, arteriolar hyalinosis, massive proteinuria, and renal failure. Collectively, data indicate that combined podocyte VEGF-A and eNOS deficiency result in diffuse glomerulosclerosis in mice; compensatory NO and thiol generation prevents severe proteinuria and GFR loss in KDVEGF diabetic mice with intact eNOS, whereas KDVEGF induction in −/−:VEGFKDeNOS mice causes massive proteinuria and renal failure mimicking DKD in the absence of diabetes. Mechanistically, we identify KDVEGF-induced abnormal S-nitrosylation of specific proteins, including β3-integrin, laminin, and S-nitrosoglutathione reductase (GSNOR), as targetable molecular mechanisms involved in the development of advanced diffuse glomerulosclerosis and renal failure.Vascular endothelial growth factor-a (VEGF-A) and nitric oxide (NO) are essential for glomerular filtration barrier homeostasis, and are dysregulated in diabetic kidney disease (DKD). While NO availability is consistently low in diabetes, both high and low VEGF-A have been reported in patients with DKD. Here we examined the effect of inducible podocyte  Diabetic kidney disease (DKD) is a major complication of both type 1 and type 2 diabetes that leads to renal failure, and the single most frequent cause of end-stage renal disease (ESRD) worldwide . An incoVEGF-A gain-of-function in diabetic mice leads to the development of Kimmelstiel-Wilson-like nodular glomerulosclerosis and massive proteinuria  and nitric oxide (NO) are essential for glomerular filtration barrier homeostasis, and both are disregulated in diabetic nephropathy . Unlike teinuria . Similare models . Moreovediabetes . These fdiabetes . Endotheult mice . Short tneurisms , and VEGse model .VEGF-A knockdown (KDVEGF) in diabetic mice and in −/−:VEGFKDeNOS mice. We determined that in the setting of NO deficiency, caused either by diabetic milieu or eNOS knockout, KDVEGF results in diffuse glomerulosclerosis and proteinuria, mimicking human diabetic diffuse glomerulosclerosis of increasing severity. This phenotype is linked to the generation of NO and thiol mediated by changes in S-nitrosoglutathione reductase (GSNOR) and β3-integrin S-nitrosylation that impairs their activity.Here we examined the effect of podocyte KDVEGF in eNOS KO mice (−/−:VEGFKDeNOS) by crossbreeding podocin-rtTA:tet-O-siVEGF mice (siVEGF) , and we backcrossed them >8 generations to a stable FVB background. In this study −/−:VEGFKDeNOS mice were fed standard or doxycycline-containing chow (Harlan-Teklad) for 1 month.We generated doxycycline-inducible podocyte (siVEGF)  with eNOeNOS−/−  (eNOS KOsiVEGF mice (KDVEGF) by intraperitoneal streptozotocin (STZ) using the low dose AMDCC  protocol, as previously described (KDVEGF (DM-KDVEGF) and non-diabetic (non-DM-KDVEGF) mice were fed standard (−dox) or doxycycline containing chow (+dox) for 12 weeks to induce VEGF-A knockdown. At the end of the study 24 h urine was collected in metabolic cages; blood and kidney samples were obtained under anesthesia, as we previously described , and BP was measured under anesthesia and analyzed using PowerLab/8SP system  as previously described . Plasma Kidneys were processed for light microscopy and TEM or frozen in isopentane, mounted in OCT (Sakura). Histology was assessed by hematoxylin/eosin and periodic acid–Schiff’s reagent (PAS) stains. TEM was performed using standard techniques, as previously described . A renalImmunohistochemistry (IHC) was performed in frozen kidney sections using primary antibodies against laminin, nephrin, podocin, and S-nitroso-cysteine and appropriate Cy2 and Cy3 fluorescent-tagged secondary antibodies (Jackson ImmunoResearch Laboratories), visualized by confocal microscopy , as previously described . QuantitImmunoblotting was performed using the following primary antibodies: podocin , nephrin , laminin , β3-integrin , VEGF receptor 2 ; actin (A2066. Sigma) or tubulin (Sigma) were used as a loading control. Signals were visualized by chemiluminescence, and quantified using ImageJ software .In situ Proximity Link Assays (PLA) were performed to identify specific S-nitrosylated proteins in kidney frozen sections using laminin rabbit polyclonal antibody  or β3-integrin antibody  and S-nitrosocysteine mouse monoclonal antibody (AG Scientific) and Duolink II fluorescence protocol  , as previously described . Ascorba Sweden) , as prev Sweden) .p < 0.05) was determined using Prism 8 software by unpaired t test with Welch’s correction and one-way Brown-Forsythe ANOVA to compare two or multiple experimental groups, respectively. Mann-Whitney test was used to analyze non-parametric variables.Data are expressed as mean ± SEM unless otherwise stated. Statistical significance (KDVEGF − dox mice (KDVEGF induction with doxycycline for 12 weeks prevented the development of glomerular hypertrophy in DM-KDVEGF + dox mice (KDVEGF (+dox) mice (KDVEGF (+dox) revealed diffuse glomerulosclerosis  mice  was simiox) mice , as quanclerosis  with lim−/−:VEGFKDeNOS (− dox) mice (KDVEGF (+dox) for 4 weeks in −/−:VEGFKDeNOS mice caused severe diffuse glomerulosclerosis (−/−:VEGFKDeNOS (+dox) mice. Significant tubulo-interstitial damage consisting of tubular atrophy and basement membrane thickening, tubular proteinaceous casts, and interstitial lymphocytic infiltrates were also observed in −/−:VEGFKDeNOS (+dox) (−/−:VEGFKDeNOS (−dox) kidneys (KDVEGF (+dox) kidneys (−/−:VEGFKDeNOS (−dox) kidneys, the severity and extension of the changes observed in −/−:VEGFKDeNOS (+dox) kidneys were obvious as demonstrated by highly significant score differences in all parameters.Glomerular histology was mostly normal in uninduced ox) mice , althougclerosis . Extensiclerosis , whereas (+dox)  but were kidneys  or in di kidneys . A semi-KDVEGF (+dox) mice (−/−:VEGFKDeNOS (−dox) kidneys showed normal glomeruliar filtration barrier ultrastructure (−/−:VEGFKDeNOS mice with podocyte KDVEGF (+dox) revealed extensive foot process effacement, GBM thickening, severe mesangial sclerosis, and endotheliosis (KDVEGF (+dox) kidneys  mice . eNOS−/−tructure . In contheliosis , a more  kidneys .KDVEGF in −/−:VEGFKDeNOS (+dox) and diabetic mice resulted in nephrin downregulation, assessed by immunoblotting and immunohistochemistry (−/−:VEGFKDeNOS and diabetic mice subjected to KDVEGF (+dox) (KDVEGF resulted in β3-integrin upregulation in −/−:VEGFKDeNOS (+dox) kidneys, whereas β3-integrin protein expression was not altered in diabetic mice with intact eNOS  . Podocytact eNOS . Collectact eNOS .KDVEGF in −/−:VEGFKDeNOS (+dox) mice caused massive albuminuria >30-fold higher than that measured in uninduced genetically identical mice −/−:VEGFKDeNOS (−dox), (−/−eNOS mice (data not shown). In contrast, mice with intact eNOS developed mild proteinuria when podocyte KDVEGF (+dox) was induced  mice , as reported in KDVEGF mice with intact eNOS  mice (KDVEGF) or intact eNOS (KDVEGF)  mice , red barVEGF-A and urinary excretion are similarly elevated in −/−:VEGFKDeNOS mice, irrespective of podocyte KDVEGF  than in non-diabetic mice (white bar), but KDVEGF did not significantly decrease plasma NO in any experimental group  in diabetic mice, irrespective of podocyte KDVEGF  and in DM-KDVEGF (+dox) kidneys, as detected by biotin-shift assay (BST) (−/−:VEGFKDeNOS − dox) or intact eNOS (KDVEGF − dox), but increased ∼2.5 fold when KDVEGF was induced in −/−:VEGFKDeNOS (+ dox) mice (KDVEGF (DM-KDVEGF −dox or + dox), than in non-diabetic mice (−/−:VEGFKDeNOS − dox or KDVEGF − dox) (−/−:VEGFKDeNOS (+ dox) mice and ∼5-fold in diabetic DM-KDVEGF mice than in non-diabetic mice (S-nitrosoglutathione (GSNO) is the major source of cellular NO not generated by NOS . GSNO rediabetes . In contay (BST) , This SNox) mice . In cont − dox) . Urine Gtic mice . The remKDVEGF (+dox) alters S-nitrosylation of glomerular proteins in −/−:VEGFKDeNOS mice. Using immunohistochemistry we determined that S-nitrosylated proteins localized to glomeruli are significantly increased in −/−:VEGFKDeNOS kidneys with KDVEGF (+dox) as compared to uninduced −/−:VEGFKDeNOS (− dox) kidneys . Glomerular laminin S-nitrosylation (SNO-laminin) was increased significantly in −/−:VEGFKDeNOS mice with KDVEGF (+dox) as compared to uninduced −/−:VEGFKDeNOS (− dox) mice (−/−:VEGFKDeNOS (+ dox) mice .Specific S-nitrosylated proteins were detected ox) mice . Consistin situ PLA we detected β3-integrin S-nitrosylation in glomeruli (−/−:VEGFKDeNOS (+dox) kidneys as compared to uninduced −/−:VEGFKDeNOS (−dox) kidneys (−/−:VEGFKDeNOS (+dox) mice, a phenotype that mimics human advanced DKD with low VEGF.Next, we examined β3-integrin, a transmembrane protein critically involved in maintaining glomerular filtration barrier integrity , whose alomeruli . Quantit kidneys . CollecteNOS knockout, podocyte VEGF-A knockdown results in diffuse glomerulosclerosis and proteinuria of increasing severity, leading to renal failure in −/−:VEGFKDeNOS mice. We show that podocyte KDVEGF and −/−eNOS induce severe diffuse glomerulosclerosis in the absence of diabetic milieu. Podocyte KDVEGF in diabetic mice prevents diabetes-induced glomerulomegaly but causes diabetic diffuse glomerulosclerosis. Mechanistically, we show that compensatory local NO and thiols generation prevent severe proteinuria and GFR loss in diabetic mice with intact eNOS, and we identify abnormal S-nitrosylation of specific proteins, including GSNOR, laminin, and β3-integrin, as novel molecular pathways potentially involved in advanced diffuse glomerulosclerosis.This study demonstrates that in the setting of bioavailable NO deficiency, caused by diabetic milieu or by KDVEGF abrogates VEGF-A-mediated glomerular hypertrophy, leading to diffuse glomerulosclerosis with modest albuminuria and normal creatinine clearance. In contrast, −/−:VEGFKDeNOS mice have a decreased ability to increase NO when podocyte KDVEGF is induced, despite similarly elevated circulating VEGF-A, thereby becoming more susceptible than diabetic mice to deleterious effects of local KDVEGF, resulting in mesangiolysis, extensive podocyte foot process effacement, GBM thickening, and a notably severe diffuse glomerulosclerosis phenotype reminiscent of advanced diabetic diffuse glomerulosclerosis  and leading to renal failure, consistent with the more severe morphologic phenotype.High circulating VEGF-A in diabetic mice stimulates NOS leading to NO production, protecting the integrity of the glomerular endothelium and attenuating functional abnormalities of the glomerular filtration barrier . VEGF-A clerosis . MoreoveVEGF knockdown results in decreased glomerular size in non-diabetic mice . This phenotype is partially similar to that described in diabetic VEGF-A knockout mice  mice is associated with increased S-nitrosylation of glomerular proteins. As opposed to loss of laminin S-nitrosylation in the setting of excess VEGF-A (VEGF-A knockdown increased laminin S-nitrosylation in −/−:VEGFKDeNOS (+dox) mice associates with severe diffuse glomerulosclerosis, suggesting that laminin nitrosylation might prevent the development of glomerular nodules, probably by regulating the secretion or polymerization of 521-laminin heterotrimers  and diabetic KDVEGF (+ dox) mice. Consistent with GSNOR de-nitrosylation, we detected several fold increase in urine NO, GSH-, and Cys-thiols excretion in −/−:VEGFKDeNOS (+dox) and DM-KDVEGF (+dox) diabetic mice. The precise cellular origin of urine NO and thiols  remains to be determined. We posit that GSNOR de-nitrosylation underlies the compensatory mechanism providing an alternative NO source in diabetic and −/−:VEGFKDeNOS (+dox) mice (KDVEGF (+dox) mice, but does not prevent the development of diffuse glomerulosclerosis. The SNO-GSNOR mediated alternate source of NO supports renal function in −/−:VEGFKDeNOS (− dox) mice, but it fails to do so when podocyte KDVEGF is induced (+ dox), leading to massive proteinuria and renal failure, as well as severe diffuse glomerulosclerosis, suggesting incomplete compensation or an additional KDVEGF related pathway, including iNOS activation, which we have not evaluated. GSNOR function is influenced by subcellular localization and modulated by VEGF and NOS signaling  mice . This coignaling .KDVEGF increases β3-integrin S-nitrosylation in −/−eNOS glomeruli might be linked to diffuse glomerulosclerosis. Laminin-521, the mature GBM laminin, binds αvβ3-integrin through interaction between α5-laminin and β3-integrin, transducing FGF and VEGF signals (KDVEGF decreases αvβ3-integrin activity in non-diabetic kidneys and cultured podocytes (KDVEGF increases glomerular β3-integrin S-nitrosylation in −/−:VEGFKDeNOS (+dox) mice, likely decreasing β3-integrin signaling. Decreased β3-integrin inside-out activation disrupts nephrin-VEGFR2-β3 integrin signaling in podocytes (−/−:VEGFKDeNOS (+ dox) mice.  mice. . Whetherx) mice.  and incrx) mice.  β3-integx) mice.  probablyx) mice. . Thus, w failure .Collectively, these findings suggest that S-nitrosylation contributes to the tight regulation of glomerular homeostasis by modulating several important signaling pathways in DKD models. Our findings support a model whereby laminin S-nitrosylation is instrumental to prevent glomerular nodule development, while GSNOR denitrosylation and increased β3-integrin S-nitrosylation lead to diffuse glomerulosclerosis in the setting of low podocyte VEGF-A.−/−:VEGFKDeNOS mice, perform a broad molecular phenotyping, confirm in cultured glomerular cell types the S-nitrosylation abnormalities identified in −/−:VEGFKDeNOS + dox kidneys and assess SNO-protein dysregulation in diabetic mice. Such additional studies will provide insight into how S-nitrosylation modulates several signaling pathways that are critical for glomerular homeostasis in DKD.Further studies are needed to address several limitations of this study: evaluate diabetic KDVEGF in −/−:VEGFKDeNOS mice causes renal failure, massive proteinuria, and severe diffuse glomerulosclerosis in the absence of diabetes. KDVEGF in diabetic mice with intact eNOS prevents diabetes-induced glomerulomegaly, causes diabetic diffuse glomerulosclerosis, and compensatory NO generation attenuates proteinuria and prevents GFR loss. Together, these models are reminiscent of human DKD phenotypes associated with low VEGF-A expression (KDVEGF in −/−:VEGFKDeNOS mice induces increased glomerular β3-integrin S-nitrosylation, likely disrupting nephrin-VEGFR2-β3-integrin signaling (In summary, pression . Mechaniignaling . Our obs"}
{"text": "Shape from focus is an accurate, but relatively time-consuming, 3D profilometry technique . This is the case because a large amount of data that needs to be captured and processed to obtain 3D measurements. In this paper, we propose a two-step shape-from-focus measurement approach that can improve the speed with 40%. By using a faster profilometry technique to create a coarse measurement of an unknown target, this coarse measurement can be used to limit the data capture to only the required frames. This method can significantly improve the measurement and processing speed. The method was tested on a 40 mm by 40 mm custom target and resulted in an overall 46% reduction of measurement time. The accuracy of the proposed method was compared against the conventional shape from focus method by comparing both methods with a more accurate reference. With the industrial integration of additive manufacturing into the standard production process comes the need for proper metrology on additive manufactured parts. Many techniques exist to recover the shape from an unknown object. Most techniques are developed for inspection and metrology of finished parts. To improve the quality of additive manufactured products it is of interest to develop these shape recovery techniques for use in at-line or even on-line metrology. In terms of precision and repeatability compared to other techniques like stereo vision or laser triangulation, shape from focus (SFF), otherwise known as depth from focus (DFF), is a good candidate for micrometer precision measurements ,2,3. AddThe following sections will first present the traditional method of shape from focus, i.e., the capturing of images and the processing of these images to generate a depth map. Next, the proposed method, combining coarse topography methods with SFF to speedup the SFF measurement process, will be discussed. Then last, the measurement results will be presented and discussed in the results and conclusion.For use as an on-line metrology technique, it is crucial that the measurement process is sufficiently fast, depending on the speed of the manufacturing process, in order not to slow down the manufacturing. As explained in ETL’s have an advantage over translation stages in terms of speed. The measurements in this paper were made with the Optotune EL-10-30C . With atThe relatively small FOV is thus required for μm accuracy measurements. For the purpose of this paper, we used a target with a size of 40 mm by 40 mm . In ordeTo create the depth-maps from the original images, we have used the Modified Gray Level Variance (GLVM) implementation in Matlab by Pertuz  and testSince the pixels sharpness value follows a Gaussian profile , it is pAs described above, the conventional method for shape from focus has a lot of benefits for use in additive manufacturing but is slower than other topography methods because of the large amount of data capturing and processing needed for it. By comparison, for stereovision, a minimum of two images is required to produce a disparity map albeit with more complex processing. For laser triangulation, all processing complexity can be accounted for by calibration which results in a direct conversion from the reflected laser line to the actual height of an object. By limiting the amount of data to capture and process, SFF’s measurement speed can be improved invariant of the hardware used. This can be done by limiting the data capture to the depth range of the object’s section that is in the field of view of the system. So, instead of capturing images across the whole 2.5 mm range, data capture is limited to a smaller range, e.g., 1 mm. For unknown objects, it is impossible to predetermine this smaller range with the SFF system alone. Another topography method capable of creating depth-maps much faster but also coarser can be used to get a rough estimate of the object’s profile. The lower accuracy of the coarse depth map can then be translated to a margin around the estimated depth range to reduce the possibility of incorrect measurements.A possible topography method to use for the purpose of creating rough depth-maps is Laser triangulation (LT) also known as Sheet of Light (SoL) or laser ranging sensors , but anyL and x and x and mined by (1)x=Lx′If we take The complete measurement system is thus composed of two individual measurement systems. One LT system for coarse measurements and one SFF system for precision measurements. The translation stages are common to both systems . As SFF The measurement principle is very simple . InitialOnce the section is scanned using SFF with the limited range, the reduced data-set is processed with the exact same focus measure algorithm. Next, the translation stages move the target, so another section of it is visible for the SFF system and the process restarts from selecting the same section on the coarse depth map generated by the LT system.This approach combines the benefits of the precision method with the speed of any other method. Additionally, developing the coarse method like LT to have a similar resolution as the SFF system would require an equally small FOV and therefore an increase in measurement time. The addition of extra components to a measurement setup also requires extra investment. However, the additional cost of the extra components is estimated to be small compared to the total measurement cost of measuring for prolonged periods of time. The cost of the laser triangulation system is in the order of magnitude of 10,000 euro. Whereas the measurement cost of running this equipment is estimated to be a minimum of 100 euro per hour. After already 200 h of measurement time, the extra investment cost would already be earned back. Adding a second measurement system for improvement of the original system, can in certain situations be a limitation. e.g., in mobile applications, adding the second measurement system introduces a lot of extra complexity. This method was specifically developed to be used for at-line or on-line metrology purposes, where the added complexity is not an issue.The gain in measurement time with this proposed two-step approach is dependent on two important parameters. Firstly due to the lower accuracy of the coarse measurement, a margin must be applied to the boundaries set to limit the capturing range. For example, if the coarse measurement has a depth resolution of 50 μm the margin should ideally be at least 50 μm above and 50 μm below. If we apply this to the example given above the actual measurement range is 1.1 mm instead of 1 mm. So with the margin applied the total amount of images to be captured is 66 for 1 mm. A larger margin leads to less gain in measurement time.The second important parameter is the ratio between the measurement range of the SFF system and the number of subimages of the full target that need to be measured across the full range. If a relatively flat object is measured, the gain in measurement time will be large because most of the subframes of the depth map will require a limited set of images to be created. If a very rough object is measured that requires every subframe to be measured across the entire measurement range, then there is no gain in measurement time.The reduction in measurement time would not be beneficial if it would also mean a reduction in measurement accuracy. To validate this, we performed reference measurements with a Keyence VK-X1000 using its shape from focus option. The reference measurements have an accuracy of approximately 500 nm and took about 3 h to complete due to the much higher resolution of the Keyence VK-X1000. The measurements of our system were then compared with these reference measurements using the software CloudCompare . For theFrom We have introduced a two-step approach to improve the measurement speed of shape from focus. We have shown that, by applying coarse depth information to shape from focus, the measurement time for stitched measurements can be reduced by limiting the measurement ranges of the individual sub-frames. This approach has led to an overall improvement in the measurement time of 43% on a test target. It has been shown that the proposed method does not impact the accuracy of the measurement. The method is independent of the hardware used. The total time reduction is, however, dependent on multiple parameters. First of all, the added margin to the limits set by the coarse measurement. The applied margin is limited by the accuracy of the fast 3D profilometry technique. Using a more accurate system for the coarse measurements allows us to use narrower margins and therefore a bigger reduction in measurement time. Secondly, the measurement time reduction is dependent on the ratio between the field of view of the shape from focus system and the height ranges of the target within this field of view. If the target has height variations over the full shape from focus range in every section to be imaged, the total reduction in measurement time can be zero. However, the field of view and measurement range of the shape from focus system could also be adapted or optimized for specific parts to provide the maximum reduction. Lastly, due to the lower resolution of the laser triangulation scan some objects of the target might go undetected. So those features will not be included while determining the measurement range for the shape from focus scan which might result in errors.A possible improvement for this method would be to use the measurement from the LT system as an initial value on a point basis when improving the accuracy by Gaussian fitting on the measurement points. The LT measurement could also be used to determine the validity of the depth estimation by shape from focus. To further improve upon SFF as a technique it should be possible to speed up processing significantly with the use of GPU. Using higher speed cameras and a different way of controlling the ETL can possibly also lead to additional gains in measurement time."}
{"text": "C. briggsae mutations orthologous to alleles of importantC. elegans genes. Both Cel-glp-4(bn2) andCbr-glp-4(v473) aretemperature-sensitive, causing sterility at 25°C. By contrast,Cel-fog-1(q253)is stronglyts, but its orthologCbr-fog-1(v442)causes a loss-of-function at all temperatures. Finally, theC. elegansglp-1allelesbn18ande2141are tssterile. However, theirC. briggsae orthologs,Cbr-glp-1(v429)andCbr-glp-1(v438)respectively, are wild-type at all temperatures. Thus, atsmutation in one species provides clues about how to designts alleles in another, but all theoretical outcomes are possible.To learn if orthologous mutations are temperature-sensitive in related species, we studied four C. eleganshas severaltsalleles of critical genes that are helpful for genetic and developmental studies. To determine whether the temperature-sensitive behavior of mutations inC. elegansand C. briggsaeis evolutionarily conserved, we constructed orthologs of fourC. elegans germlinetsmutations inC. briggsae using CRISPR-Cas9  or TALEN  mediated gene editing.Temperature-sensitive mutations have a wild-type phenotype at the normal (or permissive) temperature and a mutant phenotype at the restrictive temperature, and are versatile tools for studying gene function. The temperature-sensitive proteins may exhibit altered stability, failure to fold or aggregate, resistance to proteolysis, or be cleared more quickly because of partial unfolding, since any of these factors might reduce function at unfavorable temperatures .glp-4gene encodes a Valyl Aminoacyl tRNA Synthetase, essential for populating the germline with sufficient numbers of cells for gametogenesis . At the restrictive temperature of 25°C,C. elegans glp-4(bn2ts) mutants are sterile, because they produce very few germ cells, all of which arrest at meiotic prophase (Beanan and Strome 1992). By contrast, they are self-fertile hermaphrodites at the permissive temperature of 15°C. There is 87% sequence identity between Cel-GLP-4 and Cbr-GLP-4, and even higher amino acid sequence conservation (96%) within 50 amino acids of thebn2allele. This allele alters the Gly 296 residue to aspartic acid, and the orthologous CRISPR mutation inC. briggsaechanges Gly 293 to aspartic acid. This ortholog,C. briggsaeglp-4(v473) I, is also temperature-sensitive ; much like itsC. elegansortholog, it results in sterility and germ cells that fail to differentiate into gametes only when grown at 25°C . Thus,Cbr-glp-4(v473ts)provides a valuable new germline-proliferation-defectivetsallele forC. briggsae.TheC. elegans . Infog-1mutants, germ cells that would normally develop into sperm instead become oocytes. Furthermore,fog-1is required for spermatogenesis in bothXOmales andXXhermaphrodites. TheC. elegansfog-1alleleq253tsis a replacement of Thr 366 by Ile . It is strongly temperature sensitive, causingXXanimals to become self-sterile at 25°C but not at the permissive temperature of 15°. Although Cel-FOG-1 and Cbr-FOG-1 share only 54% sequence identity, they have higher conservation near the site ofq253. TheCbr-fog-1(v442) Iallele has an orthologous change (Thr 391 to Ile) that we made using TALEN gene editing. In contrast toC. elegansq253ts, theCbr-fog-1(v442) allele causesa loss of function at all temperatures . TheXXhermaphrodites made only oocytes  and theXOmales produced oocytes at both permissive and non-permissive temperatures. As inC. elegans,this mutation is semidominant in males , which implies conservation of howfog-1is regulated in this sex. Since the homozygotes are self-sterile, we balancedCbr-fog-1(v442)withunc-40(v270).FOG-1 is a Cytoplasmic Polyadenylation Element Binding (CPEB) protein that controls the sperm fate inC. elegansglp-1allelesbn18ande2141arestrongly temperature-sensitive, blocking germline proliferation at restrictive temperatures and causing sterility .C. elegans glp-1(e2141ts) is a missense allele that changes arginine 974 to cysteine, and is orthologous toCbr-glp-1(v438), which we made using CRISPR/Cas9. Although theC. briggsaeallele changes arginine 955 to cysteine, it is notts, since the mutants appear wild type at both permissive and restrictive temperatures .GLP-1 is a notch receptor protein that regulates the mitotic proliferation of germ cells . TheCel-glp-1(bn18ts) withC. briggsae. Thebn18ts allelereplaces alanine 1034 with threonine, and is orthologous toCbr-glp-1(v429), an alanine 1020 to threonine substitution that we made using CRISPR-Cas9. Unlikebn18ts, Cbr-glp-1(v429)is not sterile at either the restrictive or permissive temperatures, but instead develops like the wild type.We also comparedC. elegans glp-1(ts)alleles are sterile at the non-permissive temperature, neither of theC. briggsaeorthologues showed sterility or defective germline development. However, a loss of function allele located nearby in the transcript does exhibit a Glp phenotype.Cbr-glp-1(v439) is a frameshift mutation caused by the deletion of nucleotide 2867 T in the coding sequence, near the site ofv438. These mutant worms are sterile and display the Glp-1 defective phenotype at all temperatures . Thus, theglp-1 loss-of-function phenotypeis conserved betweenC. briggsae and C. elegans but not the temperature-sensitivity of key alleles.AlthoughC. eleganstsalleles we studied had similar behavior inC. briggsae. However, the other alleles were onlytsin one species. Thus, mutations orthologous to knowntsalleles can be temperature-sensitive in other species, and provide a promising guide for generating such alleles, but their behavior often differs. Although high levels of structural conservation might suggest that mutations will behave similarly in both species, we found it hard to predict which alleles will bets without experimental tests. We suspect thatC. elegansandC. briggsae sometimes differ because some genetic backgrounds are more sensitive to perturbation than others.Taken together, our findings show that one of the fourReagents"}
{"text": "Pulmonary sarcomatoid carcinoma (PSC) is a rare and aggressive disease without standardized treatment strategies. The efficacy of second-line or beyond immune checkpoint inhibitors (ICIs) has been proven in recent studies, whereas the evidence for first-line immunotherapy for PSC is still limited to case reports and remains poorly understood.This was a multicenter, retrospective analysis of 21 patients with a histological diagnosis of PSC who received ICI as first-line therapy from January 2019 to March 2022. The expression of PD-L1 was evaluated by immunohistochemistry (IHC) using the monoclonal antibody 22C3. Low and high PD-L1 expressions were defined using the tumor proportion score (TPS), with cutoffs of 1 and 50%, respectively.P = 0.86 and P = 0.34, respectively) and different immunotherapies . No serious adverse events (grade ≥ 3) were noted.All eight patients had PD-L1 positivity who underwent PD-L1 expression assessment, and six patients  had high PD-L1 expression. Among the 21 PSC patients, seven received tislelizumab, six received camrelizumab, four received sintilimab, three received pembrolizumab, and one received durvalumab. Among them, 18 PSCs received combination therapy, whereas another three PSCs received immunotherapy alone. Out of the 21 PSC patients, 12 (57.1%) achieved a partial response (PR), and five patients had stable disease (SD) as the best response, whereas four PSCs experienced dramatic progressive disease (PD). The median progression-free survival (PFS) was 9.2  months, and the median OS was 22.8  months. Among the three treatment groups , the median PFS was 8.0, 9.4, and 9.6 months, and the median OS was 19.0, 22.8, and 30.6 months, respectively. There was no difference in PFS and OS between the three treatment regimen groups (First-line immunotherapy has promising therapeutic potential in the treatment of PSC. More studies are warranted to confirm these findings. Pulmonary sarcomatoid carcinoma (PSC), a rare and aggressive disease, accounts for less than 1% of non-small cell lung cancer (NSCLC) . No stanStudies have shown higher frequencies of genetic mutations and PD-L1–positive expression in PSC than in conventional NSCLC, and PD-L1 positivity might lead to survival benefits from immunotherapy or even a favorable response in those harboring actionable mutations , 8, 9. IThe low number of patients who have undergone immunotherapy as a first-line treatment strategy makes it challenging to evaluate the specific safety and efficacy of first-line immunotherapy for PSC. The purpose of this paper is to report the largest study of first-line immunotherapy for PSC treatment to date.A multicenter, retrospective study was conducted at two tertiary medical institutions in the Nanchang region of China, namely The First and Second Affiliated Hospital of Nanchang University in China. The patients diagnosed with advanced PSC (III/IV) who received first-line immunotherapy from January 2019 to March 2022 were enrolled. Patients with pulmonary interstitial disease, systemic immunosuppression, autoimmune disease, or second primary malignancy were excluded from this study. Written informed consent was not required, as this was a retrospective review study. The last follow-up time was 30 April 2022. Each patient’s relevant clinical data were collected from hospital electronic medical records, including sex, age, smoking status, Eastern Cooperative Oncology Group (ECOG) score, histology, clinical stage, and distant metastasis. The best tumor response was evaluated according to RECIST version 1.1. Progression-free survival (PFS) was the time from the date of immunotherapy to the date of disease progression or death. Overall survival (OS) was the time from the date of immunotherapy to death from any cause or the last follow-up.The expression of PD-L1 protein was evaluated by immunohistochemistry (IHC) performed on 4-µm formalin-fixed paraffin-embedded (FFPE) tissue sections using a Dako PD-L1 22C3 pharmDx kit . PD-L1 protein expression was determined by using the tumor proportion score (TPS), and the cutoffs for low and high expressions were 1 and 50%, respectively .p < 0.05 was considered statistically significant.Kaplan–Meier curves were drawn to analyze the survival of PSC patients. The corresponding 95% confidence interval (95% CI) was calculated. Statistical tests were performed in IBM SPSS version 25 . In the end, 21 PSC patients who were treated with first-line immunotherapy were enrolled. The clinical characteristics are summarized in PD-L1 expression was assessed by IHC in the eight samples available. Six patients  had high expression (TPS > 49%) and the others had low expression (TPS 1–49%) , camrelizumab , sintilimab , and pembrolizumab . Of the seven patients who received tislelizumab combined with anlotinib treatment, two achieved partial response (PR) as the best response, one of them with tumor shrinkage of approximately 72%, and four achieved stable disease (SD), whereas rapid progression occurred in another patient for PFS of 1.7 months and OS of 5.4 months , sintilimab , or durvalumab . Both patients treated with the pembrolizumab combination achieved PR , and another did not have PD-L1 testing since there was no gene mutation. Two were treated with camrelizumab monotherapy. The OS was 12.6 months in one patient (PD-L1 95%) and more than 25.4 months in the other. Sintilimab monotherapy was adopted as a first-line treatment for one CS patient with PD-L1 expression of 20%, who had a PFS of more than 5 months and whose OS was not reached.The median follow-up time for this cohort was 8.5 (range: 0.3–30.6) months, and only seven of the 21 PSC patients (33.3%) were deceased at the last follow-up time. In the whole sample, the median PFS was 9.2  months , the median PFS was 8.0, 9.4, and 9.6 months, and the median OS was 19.0, 22.8, and 30.6 months, respectively. Nevertheless, there was no difference in PFS and OS between the three treatment regimens groups  was 57.2%, and a disease control rate (DCR) of 81.0% was achieved Table 2.Five patients (23.8%) experienced adverse events Table 2,Continuing efforts to find novel therapeutic strategies for PSC are imperative. Although the efficacy of immunotherapy after disease progression has been demonstrated , first-lImmunotherapy has substantially contributed to the treatment of conventional NSCLC and has improved patient outcomes –19, provAlong with accumulating research on immune therapies in PSC, the efficacy of first-line treatment has been gradually unveiled. Pembrolizumab monotherapy treatment in three PSC patients showed a response, with a PFS of more than 11.0 months . SimilarStudies have shown that harboring the BRAF V600E mutation was associated with greater clinical benefit from ICIs and significantly prolonged PFS in NSCLC . SimilarDisease progression is inevitable, and little is known about what contributes to dramatic progress without any response to immunotherapy; however, we cannot ignore the four patients who showed progress in our study with advanced IVB stages at immunotherapy initiation.KRAS and TP53 mutations have been demonstrated to be associated with high PD-L1 expression in NSCLC –26. FurtA phase Ib trial (NCT03628521) regarding combined sintilimab and anlotinib as first-line therapy in patients with advanced NSCLC saw decent success, with a median PFS of 15.0 months . AnotherThe limitations of this study are its small sample size, and we did not find an exact predictive factor or mechanism of dramatic progression. Additionally, this study had different treatment regimens and several immunotherapies with limited numbers of people on each treatment, and although the results showed no differences between groups, a larger sample of studies is needed to support this. Although remarkable outcomes have been obtained, they may suffer from selection bias (a low rate of incidence). Several clinical trials including first-line toripalimab combined therapy (NCT04725448) in the treatment of patients with advanced PSC are in progress. We hope that those studies can reveal more detail about the value of immunotherapy and help guide treatment decisions. One challenge is to be able to predict which patients are most likely to derive benefit from immune therapies and the mechanism of progression at initial treatment. Considering the rarity and complexity of PSC, clinical studies involving multiple centers, even globally, are needed to improve prognosis.Our study sheds light on the promising therapeutic potential of first-line immunotherapy in the treatment of PSC. Given these findings, a prospective study is warranted to explore the efficacy of immunotherapy with or without chemotherapy, and it is necessary to determine the reason for the dramatic progression at initial immunotherapy treatment.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.This study was reviewed and approved by the institutional ethics committee of the Second Affiliated Hospital of Nanchang University and the Medical Research Ethics Committee of the First Affiliated Hospital of Nanchang University.AL and YL co-designed the research. ZZ, XQ, and FL made a significant contribution to the data integration and analysis. ZZ provided patients’ information and data analysis. XQ drew up the manuscript. FL and YW processed the figures and tables. YYu, CF, XZ, and SY collected the outcome. RC, BY, TWand YYi followed up on the case. All authors contributed to the article and approved the submitted version.This study was supported by grants from the National Natural Science Foundation of China , the Surface project of the Natural Science Foundation of Jiangxi Province , Technology Supporting Program of Jiangxi Province (No.2015BBG70236), The Key Project of Education Department of Jiangxi Province (No.GJJ170012), and The Graduate Student Innovation Special Fund Project of Jiangxi Province (No. YC2022-s207).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": "Ramadan fasting was the only time-restricted eating regimen trialled in this population with no strong evidence of a significant effect on insulin levels.  As the systematic review retrieved only one study investigating time-restricted eating to reduce insulin in patients with PCOS, there is no evidence to suggest that this intervention is effective. From the narrative review, based on studies in other patient groups, time-restricted eating could improve insulin resistance in those with PCOS; however, well-designed studies are required before this intervention can be recommended. Polycystic ovarian syndrome (PCOS) affects up to 10% of premenopausal women and is characterised by multiple ovarian cysts, hyperandrogenism causing hirsutism, hyperinsulinemia, insulin resistance, obesity, infertility, and mood disturbances . Both phThe majority of women with PCOS have insulin resistance, independent of weight –20. ThisHyperinsulinemia is linked with persistent hyperandrogenism, as well as its clinical manifestations, by directly driving excessive androgen production . Due to Dietary interventions in PCOS are varied with many diets being unsustainable and most largely ineffective. Dietary regimens including energy  restriction , 28, MedTime-restricted eating involves a period of fasting, thereby allowing a decrease in insulin levels, an improvement in insulin sensitivity, and an improvement in glucose regulation. The short-term putative benefits of time-restricted eating include increased cell metabolic and oxidative stress resistance . A perioFasting-induced fuel switching has the potential to overcome the issues seen in patients with PCOS, as seen in other populations with underlying insulin resistance, including metabolic syndrome, obesity, and type 2 diabetes –44. TimeThis systematic review followed PRISMA reporting guidelines . This rePopulation: Females above the age of eighteen years, diagnosed with polycystic ovarian syndrome using Rotterdam criteria.Intervention: Any time-restricted feeding regimen, alone or in combination . To include 16 : 8 method, 18 : 6 method, 5 : 2 diet, alternate day fasting, intermittent fasting, Ramadan or other religious fasting methods deemed suitable.Comparator: Comparison to usual ad libitum diet or no dietary or fasting intervention, standard treatment-as-usual, and pharmacological therapy.Outcomes: Metabolic parameters which represent effect on insulin levels, C peptide concentrations, glucagon, insulin-like growth factor 1 (IGF-1), glycated haemoglobin (HbA1c), homeostatic model assessment for insulin resistance (HOMA-IR), fasting blood glucose, sex hormone binding globulin (SHBG), or oral glucose tolerance test (OGTT).The PICO model was employed to expand the return from the review and applicability of data collected.A preliminary scoping review was conducted to identify the nature and extent of the research available. A systematic search strategy was constructed with oversight of a medical librarian DM. Five search strategies were created for Medline, CINAHL , EMBASE, WOS (Web of Science), and Cochrane Library, the details of which are found in Timeline: Published before 10th of May 2021 when final searches were run.Patients: Female patients with a diagnosis of polycystic ovarian syndrome by Rotterdam criteria.Inclusion: All study designs considered which were published in peer-reviewed medical journals. Studies involving time-restricted eating, Ramadan fasting, and diets with any fasting regimen were considered, in addition to studies where time-restricted eating/intermittent fasting combined with concurrent medication use , metformin, spironolactone, etc.). Studies must have baseline and postintervention measures of outcomes. Pilot studies were assessed for inclusion if clear outcome effects were detailed. Only studies involving humans were included.Exclusion: Conference papers, unpublished reports, letters to the editor, papers not in English were excluded. Studies considering administration of supplements, studies without a control group, studies without a baseline compared to postintervention measures, and nonhuman, preclinical, and animal studies were also excluded.Results of included studies were extracted by two independent reviewers (R.F. and R.G.). Study characteristics were collected including details of participants, study methodology, intervention details, and effect on insulin and other metabolic parameters. Analysis to assess study quality and risk of bias was conducted in accordance with Cochrane Handbook for Systematic Reviews of Interventions.The studies included in the review were inherently heterogeneous due to varying interventions and outcomes. As a result, a descriptive presentation of the studies was used without pooling of outcomes for analysis.The one study included was a nonrandomised controlled study with a Ramadan fasting regimen. This study recruited women with a PCOS diagnosis. The details of the study, population characteristics, and outcomes were extracted and reviewed.Due to the lack of studies identified for inclusion and heterogeneity of study designs, it was not possible to conduct a meta-analysis.p=0.543), insulin (p=0.818), FSH, LH, testosterone, or adrenaline after undergoing Ramadan fasting. Significant effects of Ramadan fasting on reducing cortisol (p=0.049) and noradrenaline levels (p=0.047) were shown. Overall there was no benefit of Ramadan fasting shown in this study on insulin levels or glucose haemostasis [One small study was identified which looked at 40 women with PCOS, 20 of whom completed Ramadan fasting for a mean of 26 days and were compared to a nonfasting control group. Details of the length of fasting times were not included but rather details of eating patterns before sunrise, after sunset, and between sunset and sunrise. There were no significant differences in levels of beta-endorphin . The overall risk of bias was deemed to be low.A systematic review of the literature found that the effects of time-restricted eating on insulin levels in patients with PCOS has not been investigated to date, and therefore there is no evidence to suggest that this intervention would be effective in reducing insulin in patients with PCOS. The systematic review retrieved just one study  which inp=0.017), area under the curve for insulin (AUCIns) (p=0.007), ratio of AUCIns/AUCGlu (p=0.001), HOMA-IR (p=0.0025), SHBG (p < 0.001), body weight (p < 0.001), BMI (p < 0.001), body fat mass (p < 0.001), percentage of free androgen index (p=0.001), CRP (p=0.040), ALT (p=0.027), and IGF-1 (p=0.006). There were no significant differences in fasting glucose, area under the curve for glucose (AUCGlu), lipid profiles, and gonadal parameters including luteinizing hormone (LH), follicular stimulating hormone (FSH), and LH/FSH ratio or total testosterone. Overall, this study suggested that 16 : 8 time-restricted regimen improved parameters of glucose haemostasis and weight [The systematic review had excluded two studies investigating the effects of time-restricted eating as they were noncontrolled. The first of these  investigd weight  in womenp=0.439), BMI (p=0.646), fasting blood glucose (p=0.183), insulin (p=0.474), HOMA-IR (p=0.364), HOMA-B (p=0.736), QUICKI (p=0.308), or lipid profiles. There were significant decreases in C-reactive protein levels (p=0.072), nitric oxide (NO) levels (p=0.003), and total glutathione levels (p=0.011). This study corroborated the systematic review finding that Ramadan fasting exerted no benefit on parameters of glucose haemostasis but had modest benefits on markers of inflammation and cardiovascular health [The second study  investigulin p=0.4, HOMA-IThere is some evidence from small studies in other patient groups that Ramadan fasting may have positive benefits on insulin levels and glucose haemostasis. Ramadan improved HbA1c levels in 29 patients with type 2 diabetes  and was β cell responsiveness, independent of weight loss [In studies on time-restricted eating reporting weight loss , it is dght loss . HoweverLi et al. observed significant results in a short, time-restricted eating regimen suggesting that improvements in hyperinsulinemia and insulin resistance can be seen without energy restriction . This isThe optimum length of a time-restricted eating intervention to have a significant effect on insulin levels is undecided. Initially switching from standard dietary pattern to intermittent fasting, hunger, irritability, and reduced concentration ability while adapting to the new dietary regimen is expected . These iLi et al.  allowed Time-restricted eating reportedly causes a shift in fuel source from glucose to fatty acids during the fasting period . TypicalTypical ad libitum eating (with short fasting periods) might perturb normal glucose metabolism. Eating habits have largely changed with modern lifestyle. A longitudinal study of dietary habit changes over a 40-year timespan in USA showed higher daily energy intake, later breakfast and lunch times, and shorter time between dinner and a postdinner snack, with a significant increase in snacking among women in particular . There aIncreased recruitment of insulin receptors, internalised by the effects of persistent hyperinsulinemia as demonstrated in Upregulation of insulin receptor expression which is downregulated by hyperinsulinemia.Lowering insulin requirements with fasting. Fasting overcomes the issues seen in patients with PCOS including saturation of defected insulin receptors, beta cell dysfunction, and reduced hepatic insulin clearance –72.Reduced leptin levels and increased adiponectin with an overall improved balance improving insulin resistance , 73, 74.On a molecular level, the biochemical effects of time-restricted eating in PCOS are thought to include the following:α, and NRF2), AMP kinase, and deacetylases such as sirtuins (SIRTs) [On a cellular level, time-restricted eating reportedly contributes to the fluctuating ratios of NAD+ to NADH, ATP to AMP, and acetyl CoA to CoA. These result in downstream activation of proteins including transcription factors  , 40, as  (SIRTs) , 40. SIRFasting reportedly results in downregulation of the insulin-IGF-1 signalling pathway and reduced circulating amino acids causing reduced mTOR activity with subsequent inhibition of protein synthesis. All of the above processes allow activation of cellular repair and maintenance processes, stress resistance and mitochondrial biogenesis, cellular proteostasis, and autophagy with overall improved cell survival. The AMP to ATP ratio is increased by fasting, activating AMPK, which triggers repair and halts anabolic processes .A prospective randomised intervention study with crossover design would be best to determine the effect of a time-restricted eating pattern on insulin levels, androgens, fertility, and satiety. We suggest that a controlled feasibility study should be conducted to investigate the feasibility, safety, compliance, and acceptance of time-restricted eating in patients with PCOS, as well as its effects on insulin, androgens, and satiety, and have registered a study to investigate these outcomes (NCT05126199).The systematic review found no studies investigating the effect of time-restricted eating on insulin levels in patients with PCOS, with the exception of one study on Ramadan fasting which showed no effect. Although a narrative review discussed an uncontrolled study which showed improvement in glucose homeostasis, weight, and androgens with 16 : 8 time-restricted eating in PCOS, overall we conclude that there is insufficient evidence that time-restricted eating works to reduce insulin levels in PCOS, and, pending further studies, the intervention should not be recommended in this group."}
{"text": "Dolutegravir is being scaled up globally as part of antiretroviral therapy (ART), but for people with HIV and tuberculosis co‐infection, its use is complicated by a drug–drug interaction with rifampicin requiring an additional daily dose of dolutegravir. This represents a disadvantage over efavirenz, which does not have a major drug–drug interaction with rifampicin. We sought to describe HIV clinic practices for prescribing concomitant dolutegravir and rifampicin, and characterize virologic outcomes among patients with tuberculosis co‐infection receiving dolutegravir or efavirenz.Within the four sub‐Saharan Africa regions of the International epidemiology Databases to Evaluate AIDS consortium, we conducted a site survey (2021) and a cohort study (2015–2021). The cohort study used routine clinical data and included patients newly initiating or already receiving dolutegravir or efavirenz at the time of tuberculosis diagnosis. Patients were followed from tuberculosis diagnosis until viral suppression (<1000 copies/ml), a competing event  or administrative censoring at 12 months.n = 465), the cumulative incidence of viral suppression was 58.9% , switching ART regimen was 4.1% (95% CI: 2.6–6.2%) and loss to program/death was 23.4% (95% CI: 19.7–27.4%). Patients receiving dolutegravir had improved viral suppression compared with patients receiving efavirenz who had a tuberculosis diagnosis before site dolutegravir availability  and after site dolutegravir availability .In the survey, 86 of 90 (96%) HIV clinics in 18 countries reported prescribing dolutegravir to patients who were receiving rifampicin as part of tuberculosis treatment, with 77 (90%) reporting that they use twice‐daily dosing of dolutegravir, of which 74 (96%) reported having 50 mg tablets available to accommodate twice‐daily dosing. The cohort study included 3563 patients in 11 countries, with 67% newly or recently initiating ART. Among patients receiving dolutegravir (At a programmatic level, dolutegravir was being widely prescribed in sub‐Saharan Africa for people with HIV and tuberculosis co‐infection with a dose adjustment for the drug–drug interaction with rifampicin. Despite this more complex regimen, our cohort study revealed that dolutegravir did not negatively impact viral suppression. GlobFor patients with tuberculosis co‐infection, the use of dolutegravir is complicated by a drug–drug interaction with rifampicin , which iWe sought to (1) describe HIV clinic practices for prescribing concomitant dolutegravir and rifampicin and (2) characterize virologic outcomes among patients with tuberculosis co‐infection receiving dolutegravir and make comparisons with patients receiving efavirenz.2The data for this study came from the International epidemiology Databases to Evaluate AIDS (IeDEA) consortium, which collects routine clinical data from HIV care and treatment sites in 44 countries across seven geographical regions. This study, which focused on the four sub‐Saharan Africa regional cohorts  for drug‐susceptible tuberculosis . AdditioTo reduce potential misclassification of ART regimen at tuberculosis diagnosis, we excluded patients with conflicting ART regimen start dates (e.g. same start date for efavirenz and dolutegravir), likely resulting from data entry errors, as well as patients with a documented change in ART regimen (e.g. from dolutegravir to efavirenz) within 1 month after their tuberculosis diagnosis. ART regimens were defined as first‐ and second‐line regimens recommended by the World Health Organization (WHO) , consist2.4Our exposure was receiving either a dolutegravir‐ or efavirenz‐containing regimen at the time of tuberculosis diagnosis or initiating ART with such a regimen within 2 months after tuberculosis diagnosis. Patients receiving efavirenz comprised the control groups for comparison with dolutegravir and were stratified by timing of tuberculosis diagnosis, either before the introduction of dolutegravir at their site  or after the introduction of dolutegravir at their site  Figure . The incOur outcome was viral suppression, which was defined as the earliest documentation of a viral load (VL) measurement <1000 copies/ml during follow‐up. VL was based on documentation of a quantitative HIV RNA test using local site protocols and procedural standards. A threshold of 1000 copies/ml was chosen to reflect variation across sites in specimens used and assay lower limits of detection. Based on site testing capacity , we assuCompeting events were switching to a new ART regimen and loss to program or death, as these events could either modify the risk or prevent the occurrence of the viral suppression outcome. Switching to a new ART regimen was based on medication records, indicating the discontinuation of efavirenz or dolutegravir, that is switch from efavirenz to dolutegravir, a PI or another NNRTI, or switch from dolutegravir to an NNRTI or a PI. Reasons for regimen switches were not documented but were likely related to issues specific to the patient (e.g. tolerability) or program . Loss to program included loss to follow‐up (i.e. no record for ≥6 months immediately before administrative censoring at 12 months after tuberculosis diagnosis), known facility transfer or other documented reason for leaving care, with the last recorded date of contact used to determine the timing of this outcome. Determination of death was based on a site's usual practices for ascertainment.3 or CD4+ percentage <14, or WHO Clinical Stage 4 event), initial NRTI backbone, time of ART initiation, CD4 count closest to date of tuberculosis diagnosis (within 12 months before or 2 months after), most recent VL in the previous 12 months among those on ART for >6 months at tuberculosis diagnosis and site of tuberculosis disease . Documentation of an HRZE regimen included both patients with a proxy diagnosis and patients who initiated HRZE within 1 month of a documented tuberculosis diagnosis. As the collection and completeness of data on tuberculosis medications varied by site, we limited this variable to sites with ≥1 patient documented to receive HRZE.Baseline variables were age group, sex, underweight , documentation of a prior AIDS diagnosis  and Mann–Whitney U tests (numeric variables) to compare DTG with C‐EFV and H‐EFV.Patients were followed from their date of tuberculosis diagnosis until documentation of viral suppression, a competing event  or administrative censoring at 12 months after tuberculosis diagnosis. If the viral suppression outcome was documented on the same day as a competing event, the patient was considered to have the former outcome.p<0.05) in bivariate analyses, except for CD4 count, which was expected to be influenced by the decreasing use of CD4 testing over time ), eight (2%) were at sites reporting only once‐daily dosing and 25 (5%) were at sites that did not participate in the survey.Patients were categorized into the following exposure groups: DTG (3) versus DTG group (median 107 cells/mm3); however, patients in the DTG group less often had a CD4 test (34% vs. 41%). Although ART‐experienced patients in the DTG group were more likely than EFV groups to have a baseline VL test (81% vs. 59%), there were no significant differences in viral suppression. Documentation of HRZE was significantly lower in the DTG group (61%) compared with both EFV groups (73–75%).When comparing patient characteristics of DTG with both EFV groups, there were significant differences in age group, sex and site of tuberculosis disease, and when comparing DTG with the H‐EFV group, there were also significant differences in timing of ART initiation and CD4 count Table . CompareAt the end of 12‐month follow‐up, the cumulative incidence of viral suppression was 58.9% for DTG, 49.6% for C‐EFV and 44.9% for H‐EFV; the cumulative incidence of switching ART regimen was 4.1% for DTG, 10.0% for C‐EFV and 5.7% for H‐EFV; and the cumulative incidence of loss to program or death was 23.4% for DTG, 28.6% for C‐EFV and 28.8% for H‐EFV  . TherefoIn the cohort study, a large proportion (91%) of patients receiving dolutegravir who had VL ascertained in the 12 months after tuberculosis diagnosis had viral suppression, which was lower (59%) for the entire sample that fully considered loss to program or death, which could preclude VL testing, and patients who did not have VL testing for other reasons. For both samples, dolutegravir was significantly associated with improved viral suppression compared with efavirenz. When stratifying by subgroup, there were some differences in statistical significance for comparisons, but overall, results were similar in magnitude. In the INSPIRING trial, 48‐week viral suppression (<50 copies/ml) was similar in magnitude for both the dolutegravir (75%) and efavirenz (82%) arms among participants newly initiating ART; however, this trial was not powered to statistically compare viral suppression between treatment arms . Real‐woThe finding that viral suppression with dolutegravir was at least as good as (and possibly better) compared with efavirenz is supportive of HIV treatment programs already routinely prescribing dolutegravir to patients with tuberculosis co‐infection. In some settings, efavirenz remains preferred for patients initiating ART at the time of tuberculosis diagnosis to avoid the drug–drug interaction with rifampicin  and our The results from our cohort study should be interpreted in the context of some limitations. We attempted to adjust for potential confounders in regression analyses, which we considered to be patient characteristics that were significantly different between exposure groups. Nevertheless, because of the observational study design, unobserved confounders may be present. The comparison of DTG with H‐EFV allowed for potentially less selection bias than the comparison with C‐EFV, as dolutegravir was not yet available when these patients had a tuberculosis diagnosis. However, follow‐up in the H‐EFV group began as early as 2015 and thus, the lower viral suppression in this group may have been related to changes in VL testing availability and practices over time, including the use of targeted rather than routine VL monitoring . DespiteAs we leveraged routine clinical data, misclassification of some variables and missing data related to erroneous or incomplete documentation is anticipated. For the inclusion criteria, it may be possible that a tuberculosis diagnosis was incorrectly documented. It may also be possible that some patients had drug resistant tuberculosis and were not receiving rifampicin; however, such treatment is often provided off‐site in our setting . To examWe did not have information on dosing frequency of antiretroviral drugs, so we could not compare outcomes by dolutegravir dosing. Few patients in the DTG group were at sites that reported not using twice‐daily dosing, precluding a robust subgroup analysis. The necessity of dolutegravir 50 mg twice daily for all patients receiving concomitant rifampicin remains unresolved , 32, 33;5We documented the widespread programmatic implementation of dolutegravir for patients with tuberculosis co‐infection at HIV clinics in sub‐Saharan Africa and use of the recommended twice‐daily dosing to counteract the drug–drug interaction with rifampicin. Despite this more complex regimen, dolutegravir did not negatively impact viral suppression.The authors declare that they have no competing interests.MLR and DN conceived the study. MLR, EB, MY, LF, KKW‐K, NSS and DN contributed to the study design. EB, DM‐N, RDW, CS‐W, CC, WRM, GM, CK, TT, JAM, PL, AD, CT, IR, OCE and LD contributed to data collection. MLR analysed the data and wrote the manuscript. All authors participated in the interpretation of the results, revision of the manuscript, and have read and approved the final manuscript.Eunice Kennedy Shriver National Institute of Child Health and Human Development, the National Cancer Institute, the National Institute of Mental Health, the National Institute on Drug Abuse, the National Heart, Lung, and Blood Institute, the National Institute on Alcohol Abuse and Alcoholism, the National Institute of Diabetes and Digestive and Kidney Diseases, the Fogarty International Center and the National Library of Medicine: Central Africa, U01AI096299; East Africa, U01AI069911; Southern Africa, U01AI069924; West Africa, U01AI069919. Informatics resources are supported by the Harmonist project, R24AI24872.The International Epidemiology Databases to Evaluate AIDS (IeDEA) is supported by the U.S. National Institutes of Health's National Institute of Allergy and Infectious Diseases, the This work is solely the responsibility of the authors and does not necessarily represent the official views of the U.S. National Institutes of Health.Appendix S1. Members of the contributing IeDEA regions.Click here for additional data file.Appendix S2. Site survey of dolutegravir use among patients with tuberculosis co‐infection.Click here for additional data file.Supplemental Table 1. Characteristics of sites included in survey of dolutegravir use in tuberculosis co‐infection.Supplemental Table 2. Cumulative incidence of viral suppression redefining viral suppression from <1000 copies/mL to <400 copies/mL .Supplemental Table 3. Hazards regression models for the associations between dolutegravir and efavirenz groups with viral suppression redefined from <1000 copies/mL to <400 copies/mL .Supplemental Table 4. Cumulative incidence of viral suppression stratified by number of viral load tests in the 12 months after tuberculosis diagnosis .Supplemental Table 5. Hazards regression models for the associations between dolutegravir and efavirenz groups stratified by number of viral load tests in the 12 months after tuberculosis diagnosis .Supplemental Table 6. Cumulative incidence of viral suppression stratified by baseline recent viral load among patients initiating antiretroviral therapy >6 months before tuberculosis diagnosis .Supplemental Table 7. Hazards regression models for the associations between dolutegravir and efavirenz groups stratified by baseline recent viral load among patients initiating antiretroviral therapy >6 months before tuberculosis diagnosis .Click here for additional data file."}
{"text": "Diabetes mellitus is a growing worldwide health challenge especially in sub-Saharan Africa. While the use and effectiveness of diabetes self-management interventions is well documented in high-income countries, little information exists in sub-Saharan Africa. Therefore, this study attempted to synthesize information in the literature on the use and efficacy of peer support and social networking in diabetes self-management in Kenya and Uganda.The purpose of this scoping review is to summarize research on the extent of use and efficacy of peer support and social networking interventions in diabetes self-management in Kenya and Uganda.We searched PubMed, ScienceDirect and Cochrane Library databases for articles reporting peer support and social networking interventions in Kenya and Uganda published in English between 2000 and September 2021. Key words encapsulated three major themes: peer support, social networking and self-management. Hand searches were also conducted to select eligible papers. Data was extracted using a form prepared and piloted in line with PRISMA-ScR guidelines.Thirteen peer reviewed articles were selected for analysis. Eleven studies reported peer support interventions while two focused on social networks in diabetes self-management. Peer support and social networking interventions incorporated microfinance and group medical visits, diabetes self-management education, telephone support and Medication Adherence Clubs. Most interventions were delivered by multidisciplinary teams comprising nurses and other professionals, peer educators, peer leaders and community health workers. Most interventions were effective and led to improvements in HbA1c and blood pressure, eating behaviors and physical activity and social support.The limited studies available show that peer support and social networking interventions have mixed results on health and other outcomes. Importantly, most studies reported significant improvements in clinical outcomes. Further research is needed on the nature and mechanisms through which peer support and social network characteristics affect health outcomes. Diabetes is a growing worldwide health challenge especially in sub-Saharan Africa. By the end of 2019, an estimated 19.4 million adults aged 20–79 years were living with diabetes in the International Diabetes Federation (IDF) Africa region, representing a regional prevalence of 3.9% . Peer anThere are wide variations of content and structure, roles and interactions, and settings and activities of peer support, ranging from structured and unstructured models . HoweverDSME interventions are associated with improvements in self-care behaviors , diabetes knowledge, coping and stress relief –36; signWhereas the use and efficacy of peer support interventions has been established in high income countries (HIC), limited data restricts drawing similar conclusions in SSA. It remains unclear what kind of peer support and social networking interventions are used in Kenya and Uganda. Therefore, a scoping study was conducted to systematically map literature in this area and identify gaps in research and practice. The following research question was formulated: What is known from existing literature about the use of peer support and social networking interventions in diabetes self-management in Kenya and Uganda?Ethical approval was provided by the Kenya Medical Research Institute (KEMRI) Protocol No. 4295. Research approval was provided by the National Commission for Science, Technology and Innovation (NACOSTI) Permit number: NACOSTI/P/21/13911.We employed a scoping design to carry out an overview of research evidence on the use of peer support and social networking interventions for diabetes management in Kenya and Uganda. These interventions are relatively new in sub-Saharan Africa, and to the best of our knowledge, this study is the first attempt to systematically examine the breadth of research on the use of peer support and social networking interventions in the East African region. With little research evidence available and negligible randomized controlled trials done, it is difficult to undertake a systematic review, hence making this study exploratory , 43. ConWe searched PubMed, Sciencedirect and Cochrane Library using key terms and Boolean operators related to 1) diabetes/diabetes mellitus; 2) peer support/social support/peer group*/social group*/peer support group*; 3) peer network*/social network*/support network*/social support network*. The search strategies were drafted by a Librarian from Makerere University and refined by the research team. The final search strategy for PubMed can be found in Two researchers independently screened titles and abstracts for relevance based on the inclusion and exclusion criteria established a priori and refined in a pilot using the first 20 studies identified. Records were then consolidated in an Excel sheet where duplicates were removed. Two researchers verified the screening for accuracy. Any disagreements on study selection and data extraction were resolved by discussion. Citations were excluded if they did not appear to be relevant to the topic of peer support and social networking in diabetes management. Following the initial title/abstract screening and removal of duplicates, two investigators screened the full texts and articles that met the inclusion criteria qualified for data extraction while those that failed to meet the criteria were tagged indicating the reason for exclusion.The following inclusion criteria were used in study selection: 1) published peer reviewed papers describing peer support and/or social networking interventions in diabetes self-management; 2) examined peer support and/or social networking intervention outcomes on diabetes self-management; 3) available in English; 4) interventions described were carried out in Kenya or Uganda only. Articles were also included if the peer/social networking intervention targeted more than a single non communicable disease (NCD) in one setting. Qualitative, quantitative and mixed methods articles were included to capture characteristics of peer support and social networking interventions, outcomes, efficacy and challenges. Articles were excluded if they were commentaries, conference presentations, reviews, protocols, studies describing an intervention carried out in multiple countries including Kenya or Uganda, studies whose full-text article was unavailable, and studies that reported PHC-based interventions without any peer dimension. The most recent reviews were used for reference list search.One researcher used a detailed charting form to extract data from each eligible article. One researcher verified the data charted and any disagreements were resolved by consensus of the four researchers. The form captured the following key information: article characteristics , study characteristics  and intervention characteristics .To present an overview of all material reviewed a narrative account of existing literature was preferred in line with the recommendations of Arksey & O’Malley. First, a numerical analysis of the nature and distribution of interventions, populations and outcomes was summarized in a table  under thThe search identified 624 articles. 26 articles were added from hand searches. 234 duplicate articles were removed, yielding 416 articles for title and abstract screening . After sn = 11) and two in Uganda. Their designs were as follows: randomized controlled trials or studies with a pre-post design (n = 4), retrospective comparisons and cohort (n = 4), mixed methods (n = 1), cross-sectional (n = 1) and qualitative (n = 3). Eleven studies [A total of 13 studies were reviewed. Majority of the studies were conducted in Kenya , community settings (n = 5); and distributed in urban (n = 2), rural (n = 7) and peri-urban and rural (n = 4) settings.The interventions varied in length from 3 months to 12 months. Patients receiving “routine care” or “usual care” were designated as control groups. Care services were provided as follows: diabetes clinics in PHC facilities  , 56, micTwo studies assessed diabetes knowledge of patients as an indicator of learning outcomes. One study  reportedDiabetes-related behavioral outcomes reported were medication adherence and compliance with protocols, eating behaviors, physical activity and acceptability and loss to follow up (LTFU).Medication adherence and compliance with protocols were reported in a single study  which reTwo studies reported on physical activity. One  reportedEating behaviours were reported in three studies. Two , 55 repoAcceptability was reported in 10 studies; two , 48 repoRetention rate/loss to follow up was reported in eight studies –53. OnlyClinical outcome indicators assessed included: glycated hemoglobin (HbA1c), body mass index (BMI)/weight, systolic/diastolic blood pressure (SBP/DBP), blood sugar/glucose and hip circumference (HC) and waist circumference (WC). HbA1c was measured in four studies , 52, 55 BMI was reported in three studies; two , 58 founBlood pressure was assessed in five studies; four , 53, 55 Blood glucose or sugar was an outcome measure of three studies. Two studies , 53 repoOther clinical outcomes including lipid profiles, cholesterol and high-density lipoprotein and overall metabolic syndrome (MetS), were the outcome measures of one study  which reSocial support was reported by four studies. One  reportedThe efficacy of reviewed interventions was assessed using established benchmarks  where peSome studies reviewed demonstrate evidence of more practical aspects of diabetes self-management. For instance, patient groups supported each other in acquiring medicines, food and transport , and parFace-to-face meetings were complemented by telephone support , 53, 55 Peer supporters also provided specific services that encouraged daily self-management. In one study  peer supAn intervention using a reciprocal model  ensured Belonging to groups provided opportunities for social support and resolution of emotional issues. In one intervention  peer supReviewed interventions connected peer supporters with healthcare professionals as well as patients to clinical care beginning from screening. First, being based in diabetes clinics in primary healthcare facilities, or in local diabetes support groups increased linkage to clinical care and increased retention rates. For instance, one study  reportedCertain features of reviewed interventions show evidence of feasibility and sustainability. In one intervention , group mInterventions focusing on microfinance emphasize community-based recruitment approaches and leverage on CHWs who are part of the healthcare system on a long-term basis using them as group liaisons , 54. FurIn primary healthcare that is grappling with huge patient numbers requiring follow up, majority of whom are stable, MACs provide an opportunity to offload single provider clinic visits by coping with appointments that would ordinarily be included in the regular clinic. MACs therefore reduce the burden of regular follow up among stable patients hence allowing clinicians to deal with other pressing cases, save time, provide support and increase patient satisfaction and flexibility of care delivery , 56.The efficacy of assessed interventions is hampered by recruitment processes. In two studies assessing MACs , 56, theInterventions assessed reported that the lack of meeting places resulted in group members holding meetings in clinics which also serve other healthcare needs. On their part, MACs are constrained by clinic settings and opening hours in line with legal limitations of drug dispensing by non-medical professionals . FurtherReviewed studies reported that the efficacy of interventions is hampered by lack of homogeneity in methods of delivery, duration, frequency and content of interventions and training of peer supporters. For instance, the duration of interventions varied from 3 months to 12 months and training of peer supporters varied from one day to 4 weeks, with or without a trainers’ manual. Supervision of peer educators to assess the extent of fidelity to objectives was not consistent across interventions yet contributed to success/failure. In one study , two diaOne major impediment to NCD management is poverty as it limits the ability of social networks. As an emerging innovation, the microfinance model reviewed , 54 seekThe twin challenges of low literacy and poor eyesight hamper the dissemination of DSME. Some interventions reviewed managed the challenge by reading educational materials translated into local languages loudly in group meetings .We used a scoping review to identify 13 studies published between 2000 and 2021 addressing the use of peer support and social networking in the management of diabetes in Kenya and Uganda. Our findings show the existence of different forms and contexts of group-based peer support models. This supports existing literature on the increased use of cost-effective group-based models in sub-Saharan Africa in provision of DSME , 59–61.In line with literature from SSA, peer supporters were trained for short durations, often ranging from one day to four weeks , 28, 29.Though very few studies focused on social networks, available evidence points to the important role of SNCs including family and friends and other linkages to social capital in daily management of diabetes which supports existing literature , 64–66. The review also revealed challenges that hamper the efficacy of interventions including recruitment processes, duration and frequency of intervention, training of peer supporters, poverty, limited literacy and unavailability of meeting places among others. These factors were found to be responsible for differences in retention rates and effectiveness of interventions. Interventions that employed interactive peer-to-peer communication , 49, 50 In MACs, the flexibility in scheduling allowed judicious use of time for people living in urban areas who must juggle between earning economic livelihoods and seeking health care. This is especially important since literature  suggestsFinally, some interventions demonstrated evidence of sustainability by involving diverse stakeholders in conceptualization and execution; and by working within existing healthcare systems and networks, including community health workers, diabetes support groups, administrators and chronic disease networks. By leveraging on existing networks, such interventions cut costs, reduce stigma for NCDs and gain from synergy.First, our search was limited to articles published in English and indexed in three databases–PubMed, ScienceDirect and Cochrane Library. We may have missed important articles written in other languages and in other databases or other informal sources not reviewed, or not publicly available. Second, included studies employed diverse designs which were not subjected to methodological quality assessment. Third, the review is based on 13 studies majorly from Kenya which may limit the strength of conclusions derived. Fourth, the inability to determine which kinds of interventions were found to be effective draws from the broad aim of this scoping review to rapidly map evidence supporting peer support and social networking interventions.The limited number of studies provide insufficient evidence to make concrete conclusions on the efficacy of interventions. However, findings shed light on the formats and strategies used, and provide some evidence for the acceptability, feasibility and scalability of peer support and social networking in diabetes care. Although further research should identify the specific activities, processes and interventions that were highly successful, these findings provide a basis for debate among stakeholders in NCD care. As emerging evidence suggests, peer support and social networking may be promising approaches to NCD self-management.S1 Checklist(DOCX)Click here for additional data file.S1 Appendix(PDF)Click here for additional data file."}
{"text": "African Americans chronically managing their diabetes benefit from receiving support from peers with shared experiences. Peer support is known to improve the well-being of individuals receiving support, however, there is limited literature on the experiences of those providing the support. The Peers Supporting Health Literacy, Self-efficacy, Self-Advocacy, and Adherence (Peers LEAD) program pairs Peer Ambassadors who are adherent to their diabetes medication, with Peer Buddies who need support with their medication adherence. Peer Ambassadors engage with Peer Buddies as they receive diabetes information, develop the skills and motivation to address identified psychosocial/sociocultural issues to enhance their diabetes medication adherence. This study qualitatively explores the experiences of African Americans who provided peer support in the Peers LEAD medication adherence intervention.Two focus groups were conducted with twelve Peer Ambassadors to explore their experiences of providing peer support in the Peers LEAD medication adherence intervention. Qualitative content analysis was conducted using an inductive open coding approach.Emergent themes provided insight into Peer Ambassador’ rationale for providing peer support and the benefits and challenges they experienced in their roles. Themes regarding their rationale included: their desire to receive support for their diabetes self-management as well as to contribute to their communities in reducing the stigma associated with diabetes. The perceived benefits they gained centered on creating interpersonal connections, experiencing personal growth as they adapted to their roles, and experiencing opportunities to contribute to an intervention regardless of professional training. Peer Ambassadors reflected on the challenges which included difficulties on coming to terms with their role as Peer Ambassadors, seeing African Americans experience complications associated with diabetes, and navigating supporting Peer Buddies who are also burdened with the challenges their family members are experiencing with managing their diabetes.This study provides unique insight to what motivates individuals to provide peer support and what they gain from these experiences despite the challenges. Understanding the experiences of peers participating in such interventions may help inform the structure and content of programs that use peer support to focus on the benefits of and the motivation for participating in the program. An estimated 30.2 million people in the United States have diabetes, and the total annual cost associated with diagnosed diabetes is approximately $245 billion . ChronicPeer support interventions are cost-effective means of improving and sustaining the self-management of chronic diseases such as diabetes –10. PeerIncreasing attention is being given to racial and ethnic minority groups—including African-Americans--and development of diabetes self-management peer support programs that are culturally-adapted . Peers SPeer support is shown to be beneficial for those who need to improve on their diabetes self-management, as well as for peer leaders who provide the support , 21. DesThe objective of this paper is to qualitatively explore the experiences of African Americans who provided peer support in the Peers LEAD medication adherence intervention.Details about the Peers LEAD intervention can be found in a protocol paper that describes the theoretical and conceptual frameworks that informed the study design, the participant recruitment/training procedure, and the intervention outcomes . As suchPurposeful sampling was used to recruit Peer Ambassadors. The research team asked community stakeholders and individuals who were Peer Ambassadors in previous studies to help identify Peer Ambassador candidates , 18. TheProspective Peer Ambassadors were screened via a phone conversation or face-to-face to assess their eligibility. During the screening, the research team assessed a candidate’s interest in serving on an advisory board, offering peer support, and their personal diabetes experiences.Peer Ambassadors attested to completion of human subjects ethics training by signing an investigator responsibility form approved by the University’s Institutional Review Board and consented to participate in a focus group by reviewing a consent information sheet approved by the University’s Institutional Review Board.The training of Peer Ambassadors was facilitated by the research team and consultants from the Wisconsin Network for Research Support (WINRS), a patient and community engagement group with experience in facilitating stakeholder engagement meetings and training since 2010 .Peer Ambassadors attended an orientation session 3 weeks before the start of the eight-week intervention. The orientation lasted two-and-half hours. Peer Ambassadors were informed about the project goals and the roles of all project stakeholders. During the orientation, PAs also participated in a demonstration of a Peer Ambassador actively listening and providing support to their peer buddies. Then, 1 week before the eight-week intervention, Peer Ambassadors participated in a two-hour training session where they prepared for phone calls with Buddies, were provided with detailed phone call suggestions, and discussed a telephone call guide that the Peer Ambassadors adjusted to their conversation styles.Fifteen peer ambassadors were paired with 21 Peer Buddies to provide support throughout the intervention. Peer Ambassadors were paired with one or two Buddies. Peer ambassadors attended 8 h of education with their peer buddies, including a separate Peer Ambassador/Buddy orientation and three group education sessions. Peer Ambassadors worked with their Buddies for 11 weeks, during which they attended the education sessions and completed five phone calls with their Peer Buddies  years. Of the Peer Ambassadors that were trained and participated in the Peers LEAD program, 12 consented to participate in the focus group.When asked about their roles as Peer Ambassadors, three main topics emerged regarding the Peer Ambassador experiences: the benefits of being a Peer Ambassador, the challenges of being a Peer Ambassador, and the rationale for being a Peer Ambassador.Qualitative analysis of the focus groups revealed several personal benefits that the PAs attributed to serving in this role. Most PAs reported that serving as a PA offered an opportunity to make meaningful connections by opening up, building trust, and listening to their Buddies. The themes and subthemes are described below with verbatim quotes from the PAs. Table PAs felt a strong connection to their buddies in having a shared experience. The PAs perceived Peers LEAD as an opportunity for them to connect with their buddies and listen to the buddies share their own experiences of living with diabetes.“I would have to say the contact, speaking with them [Peer Buddies], letting them share their journey. That’s what was really beneficial to me” – PA2.Peer Ambassadors benefited from the growth they experienced in the Peers LEAD program. They identified how the program helped them contribute to participants’ experiences, adapt to their roles as Peer Ambassadors, and self-reflect on their own journey of living with and managing their type 2 diabetes.Some Peer Ambassadors who were also nurses shared that regardless of their professional training, being a Peer Ambassador was an opportunity for them to learn from others in the program.“Beneficial, I would have to say the contact, speaking with them, letting them share their journey. That’s what was really beneficial to me, and I agreed with [PA 1] I didn’t have to be a nurse.” – PA7.Peer Ambassadors shared about their experience with their buddy which in some instances started with conflicts. However, by participating in the program, Peer Ambassadors reflected upon how those conflicts led them to open up more and share about their own frustrating challenges of living with diabetes. By adapting to their role and providing support they benefited in building meaningful relationships.“But as time went on, I had a peer buddy who actually was, we just kept bumping heads. It just was not working. But as time went on, the peer buddy I had this time, was like a whole 360. You know, personally, myself, I opened more, and I’ve shared a lot, and I received a lot.” – PA12.Peer Ambassadors shared how the program allowed them to reflect on their own journey of living with diabetes.“I’m just going to say that being a Peer Ambassador helped me to think about how many years I’ve actually been a diabetic. And beginning when things were not. .. that first year, and I worked, you know, to be where I am right now. So it’s been an experience. It’s just been an experience. It’s something different, and I’ve enjoyed it.” – PA7.Peer Ambassadors described several challenges of providing peer support as: 1) Seeing Peer Buddies experience complications associated with diabetes, 2) Buddies want to share the burden of their family member’s experiences with diabetes, 3) Coming to terms with the responsibilities of being a Peer Ambassador and, 4) Building a relationship and sense of trust with their buddies.“You know, it was hard to see people suffer. It’s difficult to see, I mean, to see how some people have such, both of my Buddies have suffered. They were ill, very ill. Like one is post-stroke. Another one, she has lung disease along with her diabetes, and they’re young. And it was just hard for me to see such guilt in people of that age.” – PA6.Some Peer Ambassadors described that it was difficult to hear their PBs describe the medical effects associated with their poorly controlled diabetes.“And then my Peer Buddy, he had amputated legs, and he has cancer. “– PA8.Peer Ambassadors shared in detail the diabetes-related complications their buddies were experiencing. The Peer Ambassadors were concerned about their buddy’s blood glucose level and poor management of diabetes.“You know, to me, it’s unfathomable as far as how one could allow themselves, I’ve heard people say that they had an A1C of 15. I just can’t, for me, that’s hard for me to imagine. You know, I’m not saying that it’s not, I know it’s possible. You know, just to hear that someone has allowed themselves to get to that level, for whatever reason.” – PA8“He has a sister that was struggling with a lot of things, and a lot of times he would want to talk about his sister, and the things that she was going through, because she likewise was a diabetic and had gangrene. You just wouldn’t think that people would allow themselves to get to a certain level, but it happens. That was. .. hard, just hearing some of the stories.” – PA8.Peer Ambassadors expressed the uneasiness they felt after their buddy described the complications associated with a family member’s uncontrolled diabetes.“It was, in the very beginning when I started doing this [Being a Peer Ambassador], it was really difficult for me to start asking questions or saying stuff, because I would feel that I’m saying something stupid or something didn’t make sense. – PA7.Participants mentioned that adjusting to the role of a Peer Ambassador was more difficult than they had anticipated.“I feel, for me, the hardest part was the first couple of conversations with my peer buddy, to get him to buy in to trusting me. And then after he did that, it just took off from there. But getting him to put trust in what we were talking about that I would keep the confidentiality, and we went from there.” – PA3.Another PA found that forging a bond with their buddy was challenging at first, but it provided them with an opportunity to cultivate a meaningful relationship.The Peer Ambassadors described their rationale for participating in the Peers LEAD program. These reasons ranged from their desire to receive support for their diabetes self-management as well as to contribute to their communities and to reduce the stigma associated with diabetes. Table “The reason why I wanted to become an ambassador is, because, I wanted to educate myself on diabetes, and then, at the same time, be able to reach out and help someone in the community.” – PA8.Most of the participants agreed that being a Peer Ambassador allowed them to educate themselves about their diabetes in order to help their Peer Buddies, their family members, and/or their communities struggling with diabetes management. Several Peer Ambassadors highlighted that they wanted to gain personal knowledge about diabetes prior to assisting others with the disease.The reason I wanted to be an ambassador was because when I was approached about it. .. diabetes, something that was not exactly talked about in our community. So, therefore, I wanted to learn more, and I wanted to give out more to other people, be able to give it out to other peoples who have diabetes. Then they don’t have to be ashamed of it, and that, you know, we can talk freely about it. I wanted to be so that we can just talk so freely about it, you know, instead of hiding it.” – PA10.Some Peer Ambassadors described the lack of dialogue about diabetes in the African American community and their desire to make a difference.Other Peer Ambassadors indicated that they wanted to share their experiences and offer support to others with diabetes in a safe and welcoming environment. Similarly, to learn more about their diabetes, Peer Ambassadors saw their role as an opportunity for them to personally open up about their own experiences and receive the support they also needed.“I am a diabetic. I’m a nurse as well. And the reason why I agreed to do it is because I think that a lot of people, they have a lot of questions and a lot of concern. And I thought that maybe I could answer some of them on a personal level, you know, share my experiences, and let them know that they’re not alone. We all struggled in the beginning to get hold. I didn’t always have perfect A1Cs either, you know, so that’s why I decided to do it.” – PA7.Peer Ambassador who were nurses, identified how their role as Peer Ambassador differed from their profession and gave them the opportunity to receive support.The study results showed that peer supporters in diabetes self-management programs aim to provide encouragement for other people with diabetes and to receive support for themselves. They view providing peer support as a responsibility, and they also benefit from their peer support role. These benefits include creating interpersonal connections, experiencing opportunities for growth through self-awareness and self-reflection, and finding reward in the commitment that comes with supporting others. Notwithstanding the benefits, African Americans described some challenges associated with peer support, especially when supporting peers who were seriously ill or who struggled with medication management despite the education provided during the program. One initial challenge for some of peers was creating a trusting relationship. Others struggled with accepting the terms of their roles as trained supporters. Nonetheless, the reasons African Americans partake in peer support consist of educating themselves to help others, decreasing diabetes-related stigma in African American communities, and receiving support themselves.Our study adds to the existing literature on peer support in several ways. First, our study adds a new dimension to the benefits of providing peer support. Prior studies of peer support only emphasize the clinical benefits of the programs to the peers involved , 30, 31.Second, existing literature recognizes peer support as a mutually beneficial relationship where individuals give and receive support from each other . Our PeeThird, trust has been associated with reasons why African Americans living with type 2 diabetes do not seek out support from their peers. A study revealed that African Americans were distrustful about disclosing their type 2 diabetes status to their peers and seeking out support . One of Familial relationships are ingrained in cultural beliefs that impact medication adherence among African Americans . For AfrOur Peer Ambassadors discussed how the knowledge they gained about their diabetes is to help themselves as well as their families and their communities. Individuals’ rationale for participating in a peer support program to help their communities can be further explored especially in under-resourced communities. Studies evaluating the benefits of diabetes self-management programs to individuals and their families show that families may benefit directly from attending health education programs , 43. HowPeer Ambassadors had difficulties seeing their peers suffer from diabetes-related complications. Peer support programs can provide emotional support to peers in their roles of supporting individuals struggling with type 2 diabetes. Other studies can further explore in-depth the challenges peers face when providing support and design interventions to equip them with the necessary tools to cope and be efficient in their roles. These roles do not require professional expertise, and although two peers in our study identified as nurses, their professional training was not necessary to provide peer support.While African Americans experience challenges in their roles as peer ambassadors, they participate in peer support programs to benefit themselves and their communities. A study of peer support in low-income African Americans found that sharing illness experiences in the group setting gave participants practical ideas on how to manage their illness . Peer AmThere are several limitations to our study. Focus groups have the advantage of eliciting the views, feelings, and perceptions of a group , 46. HowThe study findings were based on perceptions of middle-aged African American adults with diabetes in one Midwestern state. Experiences of ambassadors providing peer support might differ among age groups and regional locations. Future studies should consider exploring these experiences among other age groups of African Americans in different geographic locations. It is important to note that the sample size was 12 participants, which is consistent with the acceptable sample size in qualitative research for focus groups. However, two Peer Ambassadors that participated in the program did not participate in the focus group due to time constraints. Despite these limitations, the findings of this study can inform future research, as well as peer support training and programs.As the number of people with diabetes grows exponentially, particularly in low-resource communities, the evidence continues to grow for peer support as a viable and compelling approach to lifelong diabetes self-management. African Americans providing peer support perceive their roles as beneficial to them and their peers. The challenges they experience in their roles are linked to the emotional difficulties of seeing peers experience diabetes-related complications. Peer support programs in African American communities should consider the benefits and rationale for participants’ involvement in their programs and address the challenges they face when providing support."}
{"text": "Current observational and interventional studies in nutritional psychiatry suggest that healthy dietary patterns rich in fresh whole foods could protect against depressive symptoms, and that unhealthy dietary patterns high in ultra-processed and refined foods could contribute to depressive symptoms. However, no studies have explored detailed subjective accounts behind the food and mood relationship. This study aimed to uncover unknown factors in the human experience with food and mood. Using a phenomenological framework, this focus group study applied thematic template analysis to accounts of over 50 Australians aged between 18 and 72. Three themes were identified from the transcript of the focus groups: (i) reactive and proactive relationships with food, (ii) acknowledgement of individual diversity relating to eating and mental health, and (iii) improving mood by removing food restriction and eating intuitively. The data highlights the complexity of the relationship between food and mood that extends beyond biological mechanisms which could be used to extend current epidemiological and intervention studies in the field of dietary patterns and depression. Nutritional psychiatry is an emerging field in mental health . The impDue to these inconsistent findings, it is important to seek more nuanced information regarding the interplay of diet and depression, which may be achieved by exploring individuals’ experiences with food and mood. Experiences of depression and depressive symptoms have been extensively reported within qualitative contexts ,13,14, ySome published qualitative papers focus on food intake and mood but explore consumer perspectives of functional foods—those specifically designed to prevent chronic disease . Others It has been well documented that individuals have a socially constructed idea of how food, and diet influence mental health ,18,19. IPhenomenology is the philosophical enquiry of the structure of human experience and consciousness; it details how individuals’ experience a specific phenomenon from their perspective, context and interpretation . A phenoThe extent to which individuals assign meaning to food and the impact on mood needs further research. A phenomenological perspective may assist such examination in a broader context, considering strong social representations about certain foods exist and have done so for many years . ConsideThis qualitative focus group study explores individuals’ experiences with food and mood using the consolidated criteria for reporting qualitative studies, the (COREQ) 32-item checklist  can be fNine focus groups, each of approximately 60 min duration, were conducted in regional Australian locations. The regional locations have a wide range of ethnic and cultural communities. Focus groups were conducted in seven face-to-face and two online formats (via Zoom). All students and staff at a multi-campus university in Australia received an invitation email to participate. Additionally, participation invitations were shared on social media platforms Facebook, Twitter, Instagram, and LinkedIn. Individuals interested in participating were asked to provide their telephone number and a preferred time to call to discuss the study’s details. A follow-up email was then sent which contained an information sheet that outlined the study and a consent form. The study protocol was approved by Southern Cross University Research Ethics Committee #19-083.Eighty-three emails were received from interested individuals; 50 were able to attend the arranged timeslots for one of the nine focus groups. No compensation apart from refreshments was offered for participation. The focus groups were facilitated by the lead researcher (ML), a female PhD candidate and psychology tutor at Southern Cross University. The first focus group was also attended by the primary PhD supervisor (Assoc Prof SS) as part of the qualitative training within the PhD program, while the following eight were attended solely by ML and the participants. All focus groups were audio-recorded and transcribed verbatim. Field notes were taken directly after each focus group discussion and a reflexive journal was kept, including thoughts, issues, concerns, feelings attitudes, beliefs, values, expectations, ideas, experiences, and potential biases to account for why certain elements were identified during data collection ,27.Before each focus group, the facilitator established rapport, explained the personal interests and potential biases that she brought to the research, and described that the purpose of the focus groups was to explore individuals’ experiences with food and mood. The facilitator then prompted group discussion, using questions such as ‘Can you describe how something you ate resulted in changing the way you felt?’, and ‘Can you describe how your feelings and emotions have influenced your eating?’. Participants were not given the list of questions prior, in order to the focus group discussion. A full list of semi-structured questions and prompts used in the focus groups can be found in the Of the 50 participants who attended the discussions, 36 were female (72%), and 14 were male (28%). Further demographic information was not collected to ensure minimal intrusiveness of the research and potentially increased information sharing during the discussions ). Some (https://otter.ai/ (accessed on 12 September 2019)) to upload audio recordings into text. Transcriptions were then read line by line against the audio, and corrections made to the text by the lead researcher. All participants were de-identified at this stage in the transcription. An email was sent offering participants the opportunity to read and return feedback on the transcripts. One participant reviewed the transcripts and returned no comments. The remainder of participants were happy not to review the transcripts. Each transcript was then read in its entirety by the lead researcher. During the initial reading, patterns in the text and initial participant quotes of interest were noted. The lead researcher continued to keep account of any thoughts, issues, concerns, feelings attitudes, beliefs, values, expectations, ideas, experiences, and potential biases that could influence data analysis in the reflexive journal.A six-stage thematic template analysis was used to explore the data . During During stage two, an iterative and inductive coding process was conducted. It began with open coding, in which all transcriptions were reread line by line. Codes—a short word or phrase assigned to label a portion of the data )—were orAn inductive thematic template analysis revealed three themes, (i) reactive and proactive relationships with food, (ii) acknowledgement of individual diversity relating to eating and mental health, and (iii) improving mood by removing food restriction and eating intuitively. These themes were found across all focus groups and included descriptions of participants’ relationships with food, the meaning they attached to food choices, the social context in which food choices were made and the overall relationship between mood and mental health.This theme explores participants’ knowledge of how different ways of eating influenced their mood. The theme has two subthemes: (i) values assigned to food, and (ii) agency and direction of choice.In this subtheme, participants explored the values that they assigned to food and how this influenced their mood. Discussions were not limited to mood as a result of food consumption or intention to shift mood. On several occasions, participants suggested that their mood was influenced by values assigned to the food before it is eaten. Josephine describes how the values she assigns to the foods she eats directly influence her mood:It’s about what you’ve assigned to the salad. If you’ve assigned to the salad, this is healthy, and I’m doing this because I haven’t done this much, then it’s a good thing. However, if I’ve assigned to salad, I’m doing this because I’m restricting, then a salad is the worst thing like for me it will ruin my mood.(Josephine)Josephine suggests that if she believes that she is eating a salad because it is good for her health and what she wants to eat, this enhances her mood. However, if she eats a salad because she is restricting food to lose weight and eating it under duress, this worsens her mood. Josephine describes that fluctuations in mood are less about the food components and more about the value ascribed to the things eaten, possibly inferring that the biological mechanisms of food are secondary to the psychological value placed on what she eats.Negative values assigned to food were reported across the focus groups. One participant suggests a fear of food learned from childhood. In this extract, Mary describes the fear of food she developed as a young ballerina:Well, I think possibly, I am terrified of food from the early ballet years. I’m never just going to eat carelessly unless I’m madly drunk. One time I did cry eating some chocolate mousse at a big meeting in _____, a long, long, long time ago. Other than that, it’s a decision driven occupation. Sometimes it’s good. Sometimes it’s great. Sometimes it’s not quite as good as you thought it might be. The creme brulee that you had to have. I guess. Terrified. I think we’re coming from a position of terror. However, then perhaps in an extremely different way., I’m just not going to put it in my mouth If I’m going to gain weight.(Mary)Strict food restrictions imposed by parents and trainers lead to an unhealthy relationship with food; Mary uses the word ‘terrified’ more than once in her extract to emphasise this. Mary explains that she continues to restrict food in the same way that she was forced to when she was a child, and believes this is entrenched in her adult behaviour. Her description of these behaviours as an ‘occupation’ further highlights the seriousness of the value she assigns to food and its influence on her mood. Another factor is that she strongly controls her food intake unless she is under the influence of alcohol. She suggests that drinking alcohol may help her loosen her food inhibitions. Mary explains that the terror surrounding her relationship with food is deeply entrenched in her fear of gaining weight, which seems to be a side-effect of childhood eating habits. Elizabeth shares a similar view to Mary regarding a negative relationship with food:I went on an alkalising diet last year, and it was very strict. I lost way too much weight really quickly. It was too strict. However, you find, food is company, and when you cut it out, you have no comfort anymore. So, all these emotions come out…. I think that’s why things like chocolate and cakes and all the yummy things are quite comforting. I know through years of sort of struggling with weight, that eating is to push the emotions down. If you remove all of that, the emotions are coming out.(Elizabeth)Elizabeth considers a recent diet she tried, and due to the strictness of the food restriction, she had adverse symptoms such as excessive weight loss. Elizabeth suggests the diet impacted her mood because she finds comfort and solace in the foods she enjoys. When she could no longer have these foods, she was deprived of the comfort and company that food would normally provide for her. Elizabeth then acknowledges that this deprivation would increase her negative emotions. Like Mary, Elizabeth also describes that the guilt she experiences with food and her food relationship relates to a societal thin ideal and that she must always be trying to lose weight, which lowers her mood.Participants described how low mood could make them turn to unhealthy food options, making their mood worse. The interconnection between mood, mental health and food intake was commonly discussed across the focus groups. For example, Amy describes how she feels when eating refined and processed grains, and how this influences her mood:If I eat bread in any form, whether it be organic or mass-produced grain that has no nutrition, then I start to get feelings of lethargy, and I start to lose the impulse to have anything energetic happen. I will perpetuate more and more reasons to not get up and do anything in a hurry. It will make me feel like I’ve been less accomplished. Therefore, contributing to more feelings about low worth because I haven’t achieved anything. If I compound the wheat intake with dairy (and I love ice cream), then I know that that also helps get me into a downward spiral of low energy, lethargy, lack of enthusiasm towards the things I need to do, which makes me feel guilt. We totally are affected by what we put into our bodies.(Amy)A vicious cycle is reported between eating what is considered unhealthy food, lack of motivation, energy, lowered mood and feelings of insignificance and lowered self-esteem. Amy describes being stuck in a negative cycle and how difficult it is to stop once it begins. However, Amy also uses the word ‘accomplished’ when describing food intake, indicating that she may link food to a sense of achievement. Although she only describes the negative cycle of unhealthy food intake, it suggests that she may also be aware of what foods to eat that help maintain a positive mood and lead to feeling more accomplished.Much of the discussion about reactive relationships with food focused on reward, comfort, or commiseration. Many participants commented that no matter what their mood, reasons always existed to use food to either elevate mood or comfort negative mood. Jesse describes food as a constant reward or commiseration:It’s like an excuse in my brain. You’ve done really well. You should go and treat yourself to something nice. Or you’re upset. I should eat something. Whatever mood, I have an excuse to eat, and I use food as my emotional crutch.(Jesse)When Jesse does well, she rewards herself with food. Conversely, when she is upset, she turns to food for comfort and solace. This implies that there is always time to use food to celebrate or commiserate. She acknowledges needing to give herself a reason or an excuse to use food as a reward or comfort. The relationship between food and mood is not always a conscious choice to elevate mood. Food may also be used to wallow, grieve, commiserate, or when bored, despite the health implications of eating that food.Sugary and convenient snack foods were commonly associated with lowered mood. However, while participants acknowledged this, they disclosed eating them frequently. This cognitive dissonance is acknowledged by Alexa, who describes how her way of dealing with low mood and swings in emotion is by eating more chocolate than she considers wise:There would be times where I would hit like an emotional low, and my way of coping with that was going and buying a block of chocolate and just eating it. Like the whole thing. However, then I feel guilt and shame for eating more than I should, and this makes me feel worse than I was feeling before.(Alexa)Alexa suggests that she overindulges when feeling emotional by eating a whole chocolate block rather than a square or row. The consequence of this is that she is not choosing healthy foods to improve her mood but making an unhealthy choice that negatively influences her mood. Despite her knowing that her pattern of chocolate consumption lowers her mood, she seems unable to control her intake, which may be a negative coping mechanism for dealing with her emotions.Discussions of the bidirectional association between food and mood were common among the participants. Most suggested that their food choices influenced their mood, but their mood also influenced food choices. On several occasions, participants suggested that feeling lazy or tired made them turn to unhealthier, convenient options which they knew would lower their mood.Participants in each focus group described that the values assigned to the foods they consumed contributed to their mood and overall mental health. They articulated how these values signified rewards, comforts or commiserations, alongside reasons to treat themselves with foods of lower nutritional value. The discussions also included aspects of the role of food guilt. Assigning the value that a portion of food was detrimental to health led to guilt, blame and shame for eating these foods, which in turn was described as lowering mood.In theme two, participants suggested that every person is different physiologically and psychologically, which influences food and mood experiences. Participants discussed how there should not be one uniform set of guidelines for food and mood recommendations. Matilda suggests that each person’s food and mood experiences differ:It’s very individualistic. Everybody’s body processes are different. Some people react to one food in one way, and other people react to that same food in a different way. So, what is considered to be healthy, and what’s considered to be unhealthy may not be the same for every single person.(Matilda)Matilda wrestles with the idea that health may mean different things to different people because of the various ways that food impacts people physically and mentally.In the following extract, Su describes how her individual reactions to food contribute to guilt due to not eating the way deemed healthy or acceptable by society:I think it’s also hard not to beat yourself up if you’re doing something that is not working. Like, what is the best? Is it the food pyramid? Is it Paleo? Is it primal? Is it no grains? Not beating yourself up that you’re eating something different than everyone else. Recognising that everyone has their own types of food that works best for their body. I think everyone needs to come to that conclusion. It’s not a one shop fits all for everyone. To me, it’s just confusion, even though I know sort of what works best for me. Sometimes I eat well. Sometimes I don’t. You just do the best you can and not trying to realise you’ve got to understand it all.(Su)Su acknowledges that individual differences in food consumption and mood are common in society. However, Su explains she does not feel guilty about relinquishing a style of eating that does not work for her. The implication of this is that the acceptance of societal diversity enables contentment with her individual choices. She illustrates a catalogue of different media messages regarding changing dietary advice, fad diets, and government guidelines, but finds it important to recognise that food choice and what works for each person are different. She suggests it is difficult to determine what foods to choose on an individual level because uniform guidelines for everyone may not suit one person.The perception that uniform guidelines are not suitable for whole populations of people of different sizes, shapes, genders, physical activity levels and food tolerances was commonly addressed. Emma agrees with Su that the current population-based dietary guidelines in Australia may not be useful advice for everyone but are the default from most health professionals:I know when I’ve been like to see a dietitian in the past, and you’re going there because you’re wanting to get some healthy assistance. It is very much the dietary guidelines and diet pyramid. In the end, you just stop going because this is not what I want. Even with doctors in a way you feel like you aren’t being heard. I don’t want the grains, I don’t want the dairy, I’m looking at an alternative, you know, healthier. It’s frustrating, sometimes you go to the doctor about feeling sad. They don’t know about nutrition, they offer you a pill, you try and do something a little bit different. Go see a dietitian, it’s still that food pyramid and it seems that everyone’s still very behind the eight ball with all of this.(Emma)Emma describes frustrations with health professionals who always provide advice in line with dietary guidelines. Emma describes not feeling heard by health professionals in that she does not want the same advice that has not worked for her in the past, and ultimately, she chooses not to engage with health services because she feels their nutrition advice is outdated and not supported by evidence or a lack of individualised nutrition advice. Emma implies that medication may not be the only option for the treatment of mental ill-health symptoms, and that diet and nutrition could be a lifestyle factor that may be used as an alternative to current treatments for low mood.Throughout the focus groups, participants described how individuals have different eating patterns and consume different foods. They also suggested that each person’s biological system is different and that foods may react differently for each person. Descriptions of food intolerances and individual dietary needs were discussed, which presented specific physiological and psychological reactions.Participants reflected on the influence of food restriction and dieting compared to eating intuitively and the influence that such consumption patterns had on mood. In this theme, participants discussed intuitive eating and its association with mood—Intuitive eating is defined as an eating pattern that removes food restriction, weight focused thinking and promotes listening to internal hunger and satiety cues, accepting the body’s natural size and shape, eating mindfully, removing food guilt and ending food preoccupation . ParticiThe detrimental impacts of dieting and food restriction were commonly acknowledged across the focus groups. In the following extract, Shae describes her struggle in the past with food restriction:I had orthorexia for a while, without realising it. Because I wanted to be healthy, and then, you know, like, no that food’s good, that food’s bad…….However, I was really anxious and panicky around food. I was protective around food, if I’m cooking, don’t come and pick at it when I’m eating, because that’s less food for me, this may be my only chance to eat. It did change my mood and my mindset, and I still am struggling with myself to try and eat intuitively.(Shae)Shae describes her experience with orthorexia nervosa. Orthorexia nervosa is defined as a disordered obsession with eating only foods that are considered to be healthy. People who experience symptoms of orthorexia avoid any foods they deem as unhealthy, which can create disordered eating patterns over the long term . Shae deAs the mother of a newborn, Katie describes the urgency to lose the weight she gained during pregnancy and how food restriction lowered her mood:I went through postnatal depression. I had a huge period that I didn’t realise was disordered eating, like very disordered. Going on the diet bandwagon, lose the baby weight while having postnatal depression. I went on that food restriction, and I felt good losing weight. Then, it’s not sustainable. So, then the binge happens; and then I’m already low mood with the depression, and dealing with the baby, and then adding to it, because food was the coping mechanism or the control factor, I suppose. And then that body image stuff came into play.(Katie)The consequences of restrictive food behaviours during the onset of postnatal depression contributed to Katie’s negative eating patterns and exacerbated her symptoms. She explains that despite this, she continued dieting throughout this time which she believed helped her lose weight, but the weight loss was only for a short time and her deprivation mindset ultimately led to bingeing on the foods she loves. This cycle of dieting and bingeing resulted in an even lower mood, exacerbating her already compromised psychological well-being and the added pressures of parental caretaking responsibilities.The cycle of dieting and restricting foods is also described by Jane, who suggests that she finds it very hard to stop such a pattern:Because without having a good relationship with food and seeing food as like the enemy, or bad or feel guilty about it. You feel bad about yourself because you’re gaining weight. It can really affect my mental health and then the whole dieting cycle begins, which I think is really a big part of like the whole food and mood thing as well. However, when I eat the foods that I like, that my body tells me are good for me, and that I want without restriction—intuitively—when I’m not on a diet that makes me feel good.(Jane)Jane describes the impacts of the cycle of dieting and food restriction as a contributing factor to mood and mental health levels. She explains that viewing particular foods as restricted make her feel more negatively about herself. She implicates food guilt with weight gain and body image problems leading to lowered mood and negative mental health. She also describes that when she is not restricting foods and eating intuitively her mood and mental health improves.Intuitive eating was a term that was discussed across the focus groups. Participants suggested that dieting and restricting foods was detrimental to positive mood and increased negative mental health. They described eating the foods they enjoyed without guilt and listening to their bodies about which foods made them feel good. This intuitive way of eating was described as elevating mood for many of the focus group participants. They suggested that removing weight-focused thinking and focusing instead on the health foods brought to their body was uplifting for their mood, and that focusing on food guilt, losing weight, and dieting worsened their mood.A recurring theme across the focus groups was reactive and proactive relationships with food. Within this theme, many participants suggested a bidirectional link between food choices and mood. It was acknowledged that food choices influenced mood, but mood also influenced food choices. These findings suggest that reverse causality could be a factor in the food and mood relationship. For example, ultra-processed, refined and sugary foods are correlated with lowered mood and increased mood disorder symptoms  (but it values assigned to food, participants described that although the nutritional components of the food they ate were important for their mood, their relationships with, and the values they assigned to food were of greater importance than the nutrients consumed in the diet. These findings suggest that “there is more to food than just nutrition” [In the subtheme of trition” . There atrition” ,44, and trition” . Researctrition” . Mood hatrition” .acknowledgement of individual diversity relating to eating and mental health suggests that participants did not support a uniform approach to food and mood guidelines and health promotion. These findings support epigenetic studies that emphasise flexible and integrative approaches to nutrition, with recent recommendations for the future direction of nutrition advice to be personalised to diet type, genes and phenotype [Participants also perceived that individual differences determined the efficacy of recommended dietary patterns. This finding in the theme of henotype . Recent henotype . It is phenotype ’s asserthenotype .Participants also reported frustrations of not knowing which foods were good or bad for physical and mental health due to being surrounded by many different media messages, advertising, and population-based guidelines, such as the national dietary guidelines. This aligns with previous research exploring individuals’ knowledge of dietary guidelines, sources of nutrition information, and factors influencing food choices , which fImproving mood by removing food restriction and eating intuitively, there was a consensus among the groups that intuitive eating and food restriction were important elements in the food and mood relationship. Intuitive eating is a non-diet approach to eating known to positively influence body image, self-esteem and psychological well-being and reduce disordered eating behaviours [In the final theme of haviours . Intuitihaviours . The fochaviours ,55,56. Ohaviours ,58,59. TThis is the first study that has explored individuals’ experiences with food and mood in depth, specifically focusing on mental health. This is also the only paper that has applied a phenomenological lens to individual food and mood experiences and socially constructed representational contexts. There were also limitations of the study that should be addressed. Most of the participants were observed to be health or diet conscious. The participants’ dietary needs were acknowledged before the focus groups took place when asked if there were any dietary restrictions for the provided refreshments. Most participants discussed health-related dietary restrictions such as vegetarianism, veganism, paleolithic or ketogenic diets, or that they adhered to gluten or dairy-free dietary patterns. Future focus group research could ensure the voices of participants who were not as diet-conscious are included. Other limitations common to focus group studies may also have influenced the findings .Overall, the data from the focus groups point to the diversity of experience related by participants in their consideration of the bidirectional relationship between food intake and mood. More specifically, the data emphasise the need for more detailed phenomenological and social representational research in this area. The prevalence of depression is widespread, and the importance of good mental health maintenance is now greatly emphasised. If dietary patterns are to be promoted to influence and improve mental health, research must extend beyond a positivist paradigm. Despite growing statistical evidence supporting the role of nutrition in mental health, high levels of heterogeneity raise more questions relating to subjective perceptions of the links between nutrition and mental health. The demand for more experiential inquiry to fully explore the nuanced relationships between food and mood must continue."}
{"text": "Participants suggested that nutrients, food components or food patterns may not be the only way that food impacts mood. Rather, they described the social context of who, with, and where food is eaten, and that time, finances, and access to healthy fresh foods and bittersweet memories of foods shared with loved ones all impacted their mood. Findings suggest that quantitative studies examining the links between diet and mood should look beyond nutritional factors and give increased attention to the cultural, social, economic, and identity aspects of diet.Epidemiological and intervention studies in nutritional psychiatry suggest that the risk of mood disorders is associated with what we eat. However, few studies use a person-centred approach to explore the food and mood relationship. In this qualitative study of 50 Australian participants, we explored individuals’ experiences with food and mood as revealed during focus group discussions. Using a thematic template analysis, we identified three themes in the food and mood relationship: (i)  Over 350 million people annually experience symptoms of mood disorders such as depression . AlthougAlthough several qualitative studies have explored food and mood relationships, these focused on consumer preferences, rather than impacts on individuals’ mental health ,19. For A phenomenological viewpoint may provide broader insight on social contexts within which the food and mood relationships exist . PhenomeThe concepts of healthy/unhealthy eating are shaped by individuals’ unique perceptions and meanings assigned to food choices . LikewisNine focus groups, seven face-to-face and two online via Zoom, were conducted to explore individuals’ experiences with food choices and mood/mental health using the Consolidated criteria for Reporting Qualitative research (COREQ) . See theParticipants (N = 50) from Australian regional locations attended one of nine 60 min discussions. Excluding gender and age range—which were revealed during the group discussions—participant demographic information was not collected to facilitate comfort and promote open discussion. Participants consisted of 36 females (72%) and 14 males (28%). During the discussion, ages were revealed to be between 18 and 72 years of age, and all participants identified as either holding a current academic qualification or working towards a university degree. Participants were provided refreshments but no further compensation for participation. At this stage, participants self-disclosed dietary requirements for the refreshments. Most participants identified as being vegan, vegetarian, gluten-free, pescatarian, ketogenic or dairy-free. A total of 83 participants were recruited by email invitations circulated throughout a multicampus Australian university, or by social media advertisements. Participants responded by nominating their telephone number and preferred time to call. All respondents were called and informed of the study’s context. Thirty-three participants were unable to make the available times. A follow-up email was sent to the remaining 50 participants, including an information sheet and a consent form to participate.a priori semi-structured questions were asked, including “Can you describe how your feelings and emotions have influenced your eating” and “How do you think food and mood are related?” , a female PhD candidate and psychology tutor. The first focus group was supervised by the primary PhD supervisor as part of qualitative PhD training (Assoc. Prof. SS). The lead researcher jotted field notes directly after the commencement of each focus group and kept a reflexive journal noting thoughts, ideas, feelings, values, concerns, attitudes, beliefs, and any biases throughout the data collection and analysis stages .https://otter.ai/, accessed on 10 January 2022), cross-checking for accuracy, deidentifying participant information, and making corrections as necessary. Initial notes were made on first reading. Of the 50 participants invited to review the transcripts, one returned no adjustments, and the remainder declined the invitation.Data were explored utilising a six-stage thematic template analysis, as demonstrated in During the second stage, the lead researcher utilised NVIVO software, version 12  to condusocial context: familial and cultural influences in food and mood, (ii) social economics: time, finance, and food security, and (iii) food nostalgia: unlocking memories that impact mood. These themes identified the importance of social context, life experiences, beliefs, expectations, and values in the food and mood relationship.Three themes were identified during thematic template analysis: (i) In this theme, participants acknowledged their childhood, upbringing, family structures, and food culture as essential parts of the social context surrounding food and the connection to mood. Most of the recollections of family and food highlighted the importance of social context for mood. Mary suggests that her mood may not be impacted by the components in her food but that her mood is more highly influenced by the social context or environment within which she eats:Maybe it’s not just the food? There’s a whole environment and the context in which you’re in. I think it really makes a difference if you’re in some kind of a family system, or you’re not. I think part of mood, if I’m not being too analytical, is, maybe with feeling ready, feeling prepared, feeling organised. To me, that’s where the comfort level is. Way beyond my own personal tastes for food. Eating with my family makes me happy, despite what foods are being served. Preparing and sharing meals together is a way we express our love and gratitude towards each other.(Mary)Much of the extract describes the social context of family systems, the preparation and consumption of food, and how this is linked to feelings of comfort and security. A possible consequence of this is that Mary may eat foods that she does not like or does not think are healthy but are preferred /shared by her family. She has a sense of family duty and the role that food plays in this, knowing her family is organised and prepared with food makes her feel positive. She suggests that creating a loving family environment when eating influences her mood positively, and for her, food and mood relationships centre around social environments and context rather than the specific foods or nutrients eaten.Similarly, Vicki describes that eating a healthy meal in solitude may not provide the psychological benefits of eating in an environment that supports social connection. She describes eating lunch at her work desk and the resultant isolation impacting her mood negatively, compared with the feelings of wellbeing she receives eating lunch outside or with her co-workers:As far as mood is concerned, I can eat the healthiest diet in the world, in complete isolation. Say if I pack myself this beautiful organic lunch, and it’s got probiotics from the kefir and sauerkraut and all my organic greens, and I go to work in a cubicle, and I don’t talk to anyone. I just sit down at my desk all day and eat lunch at my computer. I just think I’m going to get sick. I don’t think. I’m going to be well. When I eat away from my desk surrounded by others, even if the food itself is not as healthy, I tend to feel more uplifted once I return to my desk.(Vicki)Vicki suggests that eating healthily while on her own may not benefit her psychologically as when eating in a less healthy manner with others. A potential implication of this is that her work lunchtime routine, eating without stopping or moving away from her workstation, could adversely influence her mood negatively compared with eating in the lunchroom (or away from the workplace) with co-workers. The value Vicki ascribes to eating in isolation suggests that her mood may benefit from eating in a social context with friends or co-workers and may not necessarily come from components of the food she eats.Another way that family and cultural influences were addressed during the focus group discussions was how current moods manifest by historical eating attitudes linked to childhood, upbringing, and parental eating exemplars. Amy reflects on her mother’s role modelling and the relationship between emotion and food:Every time I come into a situation that’s highly emotional. I just stuff my face. My Mum doesn’t deal with emotions. If you’re upset, she’ll bring around a cheesecake (laughs) and two spoons. I think a lot of times they don’t know how to comfort and that’s where my Mum brings a cake…... She won’t even give you a hug, so uncomfortable. But she will eat a cake with you. That’s her hug. We know where it’s coming from, a place of love. But it was the way she was raised as well.(Amy)This extract describes the use of food as a mechanism for emotional expression in a family, where conventional emotional displays were less favoured. Amy suggests a physical and emotional disconnection from her mother, which her mother tries to compensate by using food in times of trouble or sadness. She reflects that this method of using food to show love may have been learned from her grandparents. Amy acknowledges that these learned behaviours passed down from generation to generation are reflected in her adult relationship with food and its link to comfort eating and using food for emotional regulation in her adult life.Participants reflected on childhood eating and how foods eaten with friends and family increased or decreased mood. The discussions also included descriptions of how eating in a social context with family and friends impacted mood and wellbeing either positively or negatively. Participants discussed the importance of preparing and eating foods in the social environment on mood rather than the nutrients or components in the food providing mental wellbeing. Participants also referred to how families used food to show love towards each other, particularly if this emotion was difficult to express within the family.In this theme, participants reflected on how time constraints and other food security issues—such as cost—influenced their food choices and mood. They expressed that organisation, foresight in preparation, and planning helped them make healthier food decisions, which elevated their mood. Others described lack of time, tight finances, feelings of procrastination, lethargy, and decision fatigue as responsible for making less healthier choices, and this lowered their mood. Katherine acknowledges her frustrations with a lack of time to create healthy meals for her family:I’d make homemade pizzas, we did the base, and we could all put what we liked on them. But that would take me like an hour and a half of making the dough and rolling it out. It’s about time restraints as well, families who work and then pick up kids and then get home, and it’s not just about cost, it’s about the cost of time.(Katherine)In this extract, Katherine reflects the positive social context of cooking together as a family, but that this was not achievable every night. Time constraints from work and school make it difficult for her family to get together and cook in this way. An implication of this lies in that eating healthily and in a social way that elevates her family’s mood may not always be about the financial cost of the ingredients, but that lack of time and other priorities are also a significant contributor.Time constraints were a commonly discussed barrier to healthy eating, and having time to prepare food was seen as a luxury to improving mood and health. This acknowledgement of time as a luxury is reflected in Sarah’s description of how food preparation is an important part of her day that helps her limit unhealthy, convenient food options:I wouldn’t say I’m a food prepper…… sometimes I try, but I find that too boring and too overwhelming. I tend to make lunch at the same time as dinner. If I’m making dinner for my boyfriend. I’m going to make our lunches……… If I’m making this now, I’m just gonna do it double. I always make sure there’s leftovers, put it in the lunch boxes, ready to go. I found that if I didn’t, it would be too hard in the morning. I wouldn’t even be able to stomach the idea of lunch at that time. I would just end up wasting all this money buying stuff out and not feeling that good about it.(Sarah)Feelings of preparedness were a mediating factor in the food and mood relationship. Sarah reveals high levels of preparation that compensate for her mood. She feels that preparing food in advance helps her control her eating habits. Her mood is elevated by feeling prepared and being in control. Sarah reflects that a lack of preparation would leave her feeling low and lazy, and she fears a lack of control over the types of foods that would end up in her lunch if she was not well prepared. A potential positive consequence of this is that Sarah does not have to waste money buying lunch or being left to deal with food preparation in the morning. Sarah uses the words “overwhelming” and “boring” to describe traditional meal preparation, but she describes how her way of preparing lunches from dinners makes her feel ready and organised for the next day, and this feeling of preparedness elevates her mood.Participants across the focus group concurred that substantial time, effort, and preparation translated into eating well, but that this investment in time and health delivers rewards in heightened mood levels and greater wellbeing. Mary compares the differences between planning and being prepared and choosing fast, convenient options and their role in her mood:It takes extra time if you’re going to eat well and prepare well and plan well. I think we all know; if you’re tired or fatigued, it’s too hard to think, you can’t be bothered. You just go and grab whatever, which keeps you in that cycle, and when you’re feeling down, and then you’re tired, you’re like, “I’ll just grab something else.” Now you’re more tired and down. I’ve made all these decisions during the day. I think that’s the hardest part. The whole day like, tick, tick, tick, tick, tick, tick and then you just get to the night and you’re like—well I just had to eat the whole packet of chips.(Mary)When Mary describes “eating well” in this context, it appears that she means eating healthily. She suggests that, for her, preparation and planning are important aspects of healthy eating. As with Sarah, Mary finds it difficult to be well-planned and prepared when feeling exhausted at the end of the day. She acknowledges the role of decision fatigue at the end of a hard day—after making many healthy decisions throughout the day her ability to make healthy choices in the evening is diminished. Potential implications of her fatigue could result in it being easier to eat an unhealthier, more convenient option, promoting a cycle of unhealthier choices. It is possible that the cognitive dissonance of wanting to eat healthily but instead choosing an unhealthy option lowers her mood and exacerbates her weariness.In the following extract, Sophie highlights her frustrations with balancing work, study, personal, and family responsibilities and explains that time is an important factor in her and her family’s food choices:When do you find time to cook? To go shopping, get your whole foods and then go home and cook them and participate in some social activity like soccer? It’s hard, working full time, studying full time, being a parent, and managing time. It’s not so much an emotional thing. It’s more like, I just am spread thin. I’ve got the kids, and I’m like okay, you want frozen pizza for dinner? Hey, let’s just have frozen pizza for dinner. It’s the time thing for me. I would love if I had the energy to always walk into a perfectly clean kitchen. I would love to cook a healthy meal and have healthy snacks around for the kids, me, and everybody, but it’s the time really.(Sophie)Time constraints negatively impact Sophie’s energy and emotions. She illustrates maintaining a difficult balance between competing responsibilities that detract from her ability to think about, cook, and prepare healthy meals. Sophie discusses how succumbing to unhealthier food choices is sometimes the easiest option due to her time constraints and visualises what a perfect world would look like for her to provide her family with healthy meals, if only she had the time.Descriptions of social economic stressors for busy, working families included both financial and time pressures. Participants recounted that they did not always provide the healthy, nutritious meals for families as intended to because of these constraints. A common narrative was the influence of decision fatigue. Participants reflected on the many decisions made throughout the day, making it difficult to make healthy choices in the evening, where it is easier to turn to convenient, less healthy choices.In this theme, lines of discussion within the focus groups turned towards the role that food and nostalgic memories had in the experience of happy and bittersweet mood. The following extracts explore how certain foods connected participants with fond memories of loved ones who had passed on, creating feelings of nostalgia, and this reconnected them to happy times through the consumption of common foods eaten in their past:I think there are certain things that are comfort, whether it’s a particular food group, or it’s a memory of a food. If you had something that was wonderful when you were a kid, and you associate food with a wonderful time and you just want to go back there. My Mum always loved jam and cream. You’d have a slice of bread, no butter, jam, and then whipped cream on top, and that was her go-to. I lost her 18 months ago, so I’ve been having this periodically, because it’s what Mum wanted. So that’s the other element of food and mood—because it is memory that connects you to those you love.(Sarah)Sarah comments on how she can transport herself back to these feelings of her late mother through the food they would eat together. She uses the emotive word “wonderful” when reminiscing about the foods she shared with her mother when she was a child. Sarah recalls her mother’s love for comfort foods and how these foods now instil comfort in her. Food provides her with a connection to memories that inspire a positive mood and help her remember happy times. Similarly, Layla reminisces about food memories from her childhood that bring back nostalgic memories and impact her mood:Growing up, my mother, who has now passed, cooked desserts. I have strong feelings associated with these foods, and they take me back to my childhood and make me feel comforted. I just need cream. If I have any sort of cake even if it’s cheesecake creamy, I’ll have to have a big glob of cream. This was my mother as well. She was the same.(Layla)Layla reflects on how she now chooses the same foods she witnessed her late mother eating as a child in an attempt to reconnect with those memories and feelings of comfort and solace. In another discussion, the role of food and nostalgia in mood was addressed by the male participant Jonas. He describes how he developed a love for cooking and preparing food, which ties him to his family who live overseas:I love cooking. So, I get really excited when I start cooking …. Because my father lives in Greece and I cook there with my Mum and my Dad. It brings me in touch with those moments. So that’s really motivating. I get really happy when I’m cooking. I love having a glass of wine and chuck on the music and there’s a great atmosphere. It’s a big event for me every time I cook, because it makes me feel like I am there with them.(Jonas)The process of cooking food and being absorbed in the experience of cooking connects Jonas with his family, who all live abroad. The physical act reminds him of cooking with his family and connects him with happy memories. He finds that his family does not need to be directly in the room where he is cooking and eating. Rather, he benefits from the social context through actions, smells, texture, sound, and sights of the food; this connects him with his family when they are not physically present and contributes to elevated mood.Participants recollected and shared bittersweet memories of their childhood, families, and loved ones both passed and still alive which were inspired by certain types of foods they remember their loved ones eating. Food provided an instant connection and portal to past feelings and emotions which elevated mood. Participants shared that they would consciously seek out specific foods to help with a reconnection to memories of the past and happy times with their loved ones.social context: familial and cultural influences in food and mood, participants expressed that the social context in which they consumed food had a greater influence on their mood than the nutrients in or components of the food itself. They also considered how family circumstances and cultural upbringing influenced the relationship between food and mood. In the second theme, social economics: time, finance and food security, participants described how financial and time restraints influenced food choices that subsequently impacted mood. In the third theme, food nostalgia: unlocking memories that impact mood, participants suggested that they could reconnect with bittersweet memories from the past by choosing to eat specific foods from their childhood or that loved ones preferred.A thematic template analysis of participants’ accounts of food and mood experiences highlighted three themes. In the first theme, The focus group findings highlight the influence of social context and social economics on food choices and mood. Participants suggested that the social context of how food is eaten influenced mood positively and negatively, and past and present familial and cultural factors influenced the relationship between food and mood. It has been proposed that there is a social influence on individuals’ eating behaviours . People’Individuals experiencing mental ill-health are more likely to encounter social disconnect, stigma, and loneliness ,45, whicfamilial and cultural influences in food and mood was supported by research on the psychosocial benefits of cooking interventions, which found that cooking had positive effects on quality of life, social function, self-esteem, and influence [Participants who identified that they noticed a positive mood increase when they prepared meals at home suggested this was due to the deep connection with their family, culture, and the origins of the food they sourced and consumed. The finding within the focus groups of preparing and cooking elevating participants’ mood in the theme nfluence .social economics: time, finance, and food security, participants suggested that food insecurity, time, and financial constraints influenced their food choices, indirectly impacting their mood and mental wellbeing. Food insecurity is defined as the limited availability of nutritional foods and is linked to the stress and uncertainty of not knowing where the next healthy meal comes from [In the theme mes from . Food inmes from . Furthermes from . Those wmes from ,51. Ultimes from , which imes from . People mes from . This limes from ,16,55,56food nostalgia: unlocking memories that impact mood, participants described a recollection of memories prompted by the consumption of foods from their past or that loved ones’ preferred. Nostalgia is described as a bittersweet recollection of memories and sentimental longing for the past [In the novel and surprising theme the past . Food nothe past . Howeverthe past . Additiothe past  describethe past . The resIn the current study, participants described social context, food security, and nostalgia as important constructs that impacted their food and mood relationship. These three constructs are tightly coupled: there is a direct link between social connection and nostalgia, and food enhances this connection . There iThis study was the first of its kind to explore food and mood in a group of Australian participants. However, it is noted that, due to recruitment through a multicampus Australian university, all participants were well-educated and self-identified as being health conscious during the discussions and when nominating dietary requirements for refreshments. Selection bias may have influenced the discussion, as the voices of those who are less health-conscious were not explored . AdditioThis study aimed to explore individuals’ experiences with the food and mood relationship by conducting focus groups with Australian participants. Using thematic template analysis, three themes were identified that suggested that social context, food security, and food nostalgia were elements that impacted the food and mood relationship outside the food components or nutrients that are consumed. This research expands on the current quantitative nutritional psychiatry research and suggests that quantitative studies examining the links between diet and mood should look beyond the nutritional factors and give increased attention to the cultural, social, economic, and identity aspects of diet. This type of inquiry could broaden the current field of nutritional psychiatry and expand the context and understanding surrounding the diet and depression relationship."}
{"text": "Zingiber officinale Roscoe (ginger) has long been used as an herbal medicine to treat various diseases, and its main sub-components, [6]-gingerol and [6]-shogaol, were also reported to have anti-inflammatory, anti-oxidant, and anti-tumor effects. However, their effects on various types of pain and their underlying mechanisms of action have not been clearly analyzed and understood yet. Thus, in this review, by analyzing 16 studies that used Z. officinale, [6]-gingerol, and [6]-shogaol on mechanical, spontaneous and thermal pain, their effects and mechanisms of action have been analyzed. Pain was induced by either nerve injury or chemical injections in rodents. Nine studies analyzed the analgesic effect of Z. officinale, and four and three studies focused on [6]-gingerol and [6]-shogaol, respectively. Seven papers have demonstrated the underlying mechanism of action of their analgesic effects. Studies have focused on the spinal cord and one on the dorsal root ganglion (DRG) neurons. Involvement and change in the function of serotonergic receptors , transient receptor potential vanilloid 1 (TRPV1), N-methyl-D-aspartate (NMDA) receptors, phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2), histone deacetylase 1 (HDAC1), voltage-gated sodium channel 1.8 (Nav1.8), substance P (SP), and sciatic nerve’s morphology have been observed. Zingiber officinale Roscoe is a perennial herb from a member of the Zingiberaceae family -gingerol and [6]-shogaol are the main compounds -gingerol, and [6]-shogaol has been summarized and analyzed along with the underlying mechanisms of action. This review study includes a total of 16 studies.Thus, in this review, the effect of Z. officinale -shogaol. In addition, in their study, intrathecal pre-treatment of nonselective nitric oxide (NO) synthase inhibitor, inhibitor of guanylate cyclase, and ATP-sensitive K+ channels channel blocker also inhibited the [6]-gingerol-induced anti-allodynic effect.Gauthier et al.  has repoZ. officinale. First, the acute oral administration of Z. officinale significantly increased the threshold to mechanical stimuli, which was reduced after surgery. In their second experiment, the repeated oral administration of Z.officinale for 7 days starting from 3 days after surgery significantly decreased the pain induced by mechanical stimuli. Among the three doses used in the study , the anti-allodynia effect of 200 mg/kg was greater, which was similar to 30 mg/kg of pregabalin. The increase in histone deacetylase 1 (HDAC1) in BV2 cell and spinal cord after nerve injury were not shown in single and repeated Z. officinale treated rodents. Moreover, acute Z. officinale application decreased both phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) activation in BV2 cell and spinal cord, respectively; however, repeated Z. officinale treatments decreased pERK2 activation in the spinal cord. Montserrat-de la Paz et al. -shogaol increased spinal GABA and glutamate decarboxylase 65 (GAD65) protein concentration in the spinal dorsal horn of L4–5 segments. Altogether, these results suggest that Z. officinale and its sub-components use spinal serotonergic pathways to induce an analgesic effect.Contrasting to the above-mentioned studies, Lee et al.  demonstrm et al.  also repm et al. . In thisZ. officinale and [6]-shogaol significantly attenuated mechanical pain induced by 110 mg/kg of STZ injection. Moreover, both Z. officinale and [6]-shogaol demonstrated less damage in the sciatic nerve’s morphology compared to the STZ group. In their second study [Z. officinale and [6]-shogaol significantly decreased mechanical pain induced by STZ injection. They reported that both Z. officinale and [6]-shogaol could significantly reduce upregulated spinal TRPV1 and N-methyl D-aspartate receptor subtype 2B (NMDAR2B) mRNA expression after STZ treatment.In two studies conducted by Fajrin et al. ,44, STZ-v1.8 currents and decreased substance P (SP) release induced by KCL and veratridine in CHO cells.Hitomi et al.  assessedZ.officinale and its main physiological active indicators, [6]-gingerol and [6]-shogaol, could significantly attenuate mechanical pain that has been induced by various animal models of pain. In summary, the above-mentioned studies demonstrate that Spontaneous pain includes sensations of stabbing, shooting, burning and paroxysmal pain associated with dysesthesia or paresthesia . ParesthIn this section, various types of chemicals, such as acetic acid ,32,39, fZ. officinale could prevent acetic acid-induced spontaneous pain in mice. Spontaneous pain was induced by intraperitoneal injection of 3% acetic acid (i.p.), and increase in the number of abdominal constrictions (writhing) and stretching with a jerk of the hind limb were shown after the injection. Z. officinale was intraperitoneally injected 1 h before acetic acid administration, and it significantly prevented acetic acid-induced writhing. The effect of Z. officinale was similar to the effect of 150 mg/kg of aspirin, which was used as a positive control. In the work of Ojewole -gingerol could be used to attenuate the spontaneous pain induced with acetic acid and formalin injection as the writhing and licking the affected limb decreased as much as the conventionally used drugs, such as aspirin -shogaol were evaluated through a tail-flick test in the STZ-induced heat pain in mice. Oral and intraperitoneal administration of Z. officinale and [6]-shogaol, decreased thermal hyperalgesia, respectively. In their subsequent study [Z. officinale and [6]-shogaol. The results show that both Z. officinale and [6]-shogaol treated group mice showed significantly longer latency time toward thermal stimulus compared to the diabetic control group.Chemotherapy treatment is also known to induce thermal pain both in humans and rodents ,76. In tm et al.  also repm et al. , the effnt study , STZ wasZ. officinale in electrical and radiant heat-induced thermal pain using a hot plate test and a tail flick test, respectively. In the study of Ojewole, Z. officinale was intraperitoneally administrated 20 min before the hot-plate test, and jumping-out of the beaker was considered a response to heat-induced pain. Z. officinale treatment significantly delayed the reaction time induced by electrical heat. Sepahvand et al. -gingerol and [6]-shogaol can effectively attenuate thermal pain  induced by nerve injury and chemotherapy treatment.Altogether, the results demonstrated in the included studies clearly show that Z. officinale, [6]-gingerol and [6]-shogaol on different types of pain have been summarized. A total of 16 studies that focused on Z.officinale -gingerol and [6]-shogaol have been analyzed on mechanical, spontaneous, and thermal allodynia or hyperalgesia , Mata-Bermudez et al. have also focused on spinal 5-HT1A, B, D, and 5A receptors and demonstrated that the analgesic effect of intrathecal injection of [6]-gingerol is mediated by these receptors. In addition, in the study of Kim et al. -shogaol act on spinal 5-HT1A and 5-HT3 receptors and [6]-gingerol on spinal 5-HT1A, B, D, and 5A receptors. Seven families of serotonin recipients are divided into 15 subtypes -shorneurons . Altogetsubtypes , and aresubtypes . Among t (GPCRs) . 5-HT1, tivation ,95,96. AZ. officinale and [6]-shogaol modulate the expression of spinal TRPV1 to induce analgesia. They reported that both Z. officinale and [6]-shogaol decrease the expression of TRPV1 in the spinal cord. Compared to the relatively well understood role of the TRPV1 present on the peripheral nervous system, the role of spinal TRPV1 has not been clearly understood yet -Shogaol”, “[6]-Gingerol”, “Allodynia” and “Hyperalgesia” “Pain”. After the initial search, duplicates, bibliographies, study protocols, clinical trials, and non-English studies were excluded. Sixteen animal studies were included in this study.A search was conducted on all studies on the effect of  Scholar . Extensi"}
{"text": "Acinetobacter baumannii (AB) is a bacterium that causes infections, particularly in immunocompromised patients. Treatment is challenging due to biofilm formation by AB strains, which hinders antibiotic effectiveness and promotes drug resistance. The aim of our study was to analyze the biofilm-producing capacity of AB isolates from various forms of infections in relation to biofilm-related genes and their drug resistance. We tested one hundred isolates for biofilm formation using the crystal violet microplate method. Drug resistance analyses were performed based on EUCAST and CLSI guidelines, and biofilm genes were detected using PCR. All tested strains were found to form biofilms, with 50% being ICU strains and 72% classified as strong biofilm producers. Among these, 87% were extensively drug-resistant (XDR) and 2% were extra-extensively drug-resistant (E-XDR). The most common gene set was bap, bfmS, csuE, and ompA, found in 57% of all isolates. Our research shows that, regardless of the form of infection, biofilm-forming strains can be expected among AB isolates. The emergence of E-XDR and XDR strains among non-ICU infections highlights the necessity for the rational use of antibiotics to stop or limit the further acquisition of drug resistance by A. baumannii. Acinetobacter baumannii (AB) is an opportunistic pathogen, dominant in Central and Southern Europe in healthcare-associated infections (HAIs). It is considered by the WHO as a critical-priority pathogen, for which there is an urgent need to search for new therapeutic solutions, primarily due to acquired resistance mechanisms [chanisms ,2. It cachanisms ,4.According to the European Antimicrobial Resistance Surveillance Network (EARS Net), the prevalence of Acinetobacter isolates resistant to at least one of the antimicrobial groups varies greatly according to country, ranging from below 1% to 98.6%. In 2021, 3 countries  reported a prevalence of less than 1%, while in 25 European countries, it was above 50%—the highest level of resistant isolates is recorded in Eastern and Southern Europe, including Poland [In Polish hospitals, especially in ICUs, the dominance of extensively drug-resistant (XDR) AB, defined as those strains that are susceptible to no more than two antimicrobial classes, has been noted for many years, and it is one of the major therapeutic problems associated with Gram-negative bacilli. The prevalence of AB isolates resistant to carbapenems in 2021 reached 82.7%, while 67% were resistant to carbapenems, fluoroquinolones, and aminoglycosides together ,6,7,8.Multidrug resistance in AB is associated with numerous mechanisms—enzymatic degradation, modification of antibiotics, reduction in membrane permeability, and increased efflux. Carbapenem resistance of AB is conferred by carbapenem-hydrolyzing class D oxacillinases (OXA): OXA-23-like, OXA-24/40-like, OXA-58-like, and intrinsic OXA-51-like. Permeability to beta-lactams, efflux pumps, and ISAba1 elements located upstream of the blaOXA-51-like gene also contributes to carbapenem resistance. Often, the production of carbapenemases coexists with overexpression of efflux pumps .The connection between biofilms and antibiotic resistance is of a considerable interest to biomedical researchers. The ability to form a biofilm, which is possessed by a large percentage of AB strains (significantly higher than in the case of other Acinetobacter species ), is conIn the hospital environment, biofilm formation promotes the long-term persistence of AB on abiotic surfaces . Under uBiofilms, as organized multicellular communities of bacteria, are surrounded by self-produced exopolysaccharide matrices. The ability to form both biofilms and genes involved in this process has been studied extensively in recent years. The formation and development of the biofilm involve many virulence factors, such as the outer membrane protein A (OmpA), biofilm-associated protein (Bap), chaperon-usher pilus (Csu), extracellular exopolysaccharide (EPS), and two-component regulatory system (BfmS/BfmR) ,17,23. CThe aim of our research was to investigate the biofilm-forming ability of AB strains recovered from bloodstream infections, pneumonia, skin and soft tissue infections, and colonizing patients in relation to the presence of biofilm-related genes and antimicrobial resistance.The studied AB strains were isolated from clinical materials collected from patients hospitalized in 2019–2021 in 7 hospitals of the Silesian Province in Southern Poland  and 2 hospitals of the Opolskie Province  and from materials collected as part of screening tests at the University Hospital in Krakow. The collection was stored in the laboratory of the Department of Microbiology at −70 °C.One hundred AB isolates were randomly selected applying the principle of one isolate from one patient, taking into account three clinical forms of infection, such as bloodstream infection (BSI), pneumonia (PNEU), skin and soft tissue infections (SSTIs), and colonization. Patients were hospitalized in ICU and non-ICU units . In this way, 25 isolates from BSI, 25 from PNEU, 25 from SSTIs, and 25 from patients without symptoms of infection (colonization) were collected.The strains were collected in accordance with the consent of the bioethics committee of the Jagiellonian University KBET 1072.6120.274.2021 and KBET 1072.6120.2.2021.® 19606™, was used in the study as a positive control in the experiment on the production of biofilm by bacterial isolates [Isolates were identified using automated systems , depending on the hospital laboratory. In addition, the intensively producing biofilm reference strain, AB ATCCisolates .Antibiotic susceptibility was determined based on the results of an automated system, MIDITECH Analyser v.12. The results were interpreted using the clinical breakpoints defined in the latest EUCAST guidelines—v. 13.0  For cefoThe pattern of antimicrobial resistance was defined as the set of antibiotics to which at least two strains are resistant. For all strains, the multi-antibiotic-resistant (MAR) index was calculated. The MAR index is the number of antibiotics that an isolate is resistant to divided by the total number of antibiotics utilized in the study .MAR InThe Genomic Mini AX Bacteria Kit  was used to extract genomic DNA from AB isolates following the manufacturer’s protocol. The concentration and purity of the isolated DNA was assessed using a Nano Drop Lite spectrophotometer . DNA extracted from pure cultures was stored at −20 °C for further study.blaOXA-23,blaOXA-40, blaOXA-58, and blaNDM [blaOXA-23 (718 bp), blaOXA-40 (413 bp), blaNDM (517 bp), and blaOXA-58 (303 bp) genes were identified .Quantification of biofilm formation was performed as previously described by Stepanovic  with som8 CFU/mL) in saline was prepared from colonies on Tryptic Soy Agar (TSA) plates . Twenty microliters of the prepared bacterial suspension was mixed with 1980 μL of Tryptic Soy Broth (TSB)  and applied to 24-well flat-bottomed plates  yielding a titer of approximately 1.5 × 106 CFU/mL.Briefly, a bacterial suspension of approx. 0.5 MacFarland  was used as a negative control. ATCC 19606 strain was used as a positive control. After incubation, the medium with unbound cells was gently removed, then carefully rinsed three times with PBS and fixed with methanol for 30 s. The plate was dried overnight at 37 °C upside down, and then the biofilm was stained with crystal violet (1000 µL) for ~15 min at room temperature. After this time, the dye was poured off and rinsed with distilled water until the water in the wells was colorless. Again, it was dried overnight at 37 °C upside down. After the plate was completely dried, the optical density absorbance was measured at 570 nm on the surface of the biofilm formed at 225 points of each well. For each strain, two replicates were analyzed to detect the biofilm formation ability.Using the i-control software (ver. 2.0.10.0), the mean optical density (OD) and standard deviation (SD) values were calculated for each test isolate and all replicates. The cut-off point (ODc) was calculated using the following formula: ODc = mean OD of the negative control + (3 × standard deviation (SD) of the negative control). The averaged OD value of the tested isolates was reduced by the ODc value. The ODc value was calculated for each 12-well plate separately.The strains were categorized according to Stepanović  into fouGenomic DNA was extracted as described above.bap, csuE, ompA, bfmS, and espA were identified  was used to find significance between associations for the different combination of the biofilm genes and the ODs and significance between the MDR or XDR and the type of the produced biofilm. The results were considered significant at p ≤ 0.05.In statistical analyses, the determination of significant differences between the groups of isolates with low and moderate biofilm production and the group with intensive biofilm production and the clinical types of infection or colonization  was demonstrated in the cross-analysis in the Fisher–Freeman–Halton exact test with two-sided exact significance A total of 100 AB isolates were tested following the principle of 1 strain per patient, including 25 from PNEU, 25 from SSTI, 25 from BSI, and 25 from colonization. Fifty-three percent of the strains came from ICU patients, including all colonization strains from ICU, 56% from PNEU infections, and 44% from BSI infections. In contrast, only 12% of SSTIs were from ICU. The morbidity and distribution of AB in ICU and non-ICU isolates are shown in p = 0.023) and meropenem (p = 0.023) between strains from the colonization and SSTI groups and for gentamicin between the colonization and SSTI; In the BSI and PNEU groups , no significant differences were observed in the case of other antimicrobials and the other groups of strains (The highest rate of resistance was for cefoperazone/sulbactam (95%) and the lowest was for colistin (8%). Strains resistant to both carbapenems (imipenem and meropenem) were 69%, and 45% were resistant to the carbapenems, fluoroquinolones, and aminoglycosides tested. A significant difference was found for resistance to imipenem  . Among tblaOXA-40 (42%); considering the clinical form of infection, it was detected in 72% of strains from SSTI and 56% of strains from colonization. In turn, in BSI strains, the blaOXA-23 gene was found more often (44%). Three strains isolated from patient colonization had the blaNDM gene (BlaNDM gene was carried together with the blaOXA-23 gene. Two strains (from PNEU and colonization) had a combination of OXA-51/ISAba-I upstream. None of the isolates carried the blaOXA-58 gene. Further, 17 of the 31 strains that were carbapenem-sensitive in the phenotypic study, nevertheless, had one of the oxacillinase genes.The most frequently detected gene among all AB strains was NDM gene . The BlaQuantification of biofilm production showed that all tested isolates (n = 100) produce a biofilm: 3% were classified as weak producers of biofilm, 25% as moderate, and 72% as strong . Among tA group of strains characterized by weak and moderate biofilm production was combined for statistical analyses, due to the small number of strains in the group of weak biofilm producers (n = 3 and n = 25).p < 0.001) were found between the type of infection and the ability to form a biofilm among the isolates from the colonization group in relation to BSI and SSTI. No differences were found between isolates from different clinical forms of infection (Most of the strains strongly producing a biofilm were in the group of colonization and PNEU. Significant differences (two-sided exact significance nfection .p = 0.012) was found between the number of isolates resistant to gentamicin in the group of strong biofilm producers  and in the group of weak and moderate biofilm producers .The correlation between biofilm formation and resistance to specific antibiotics was also analyzed. All colistin-resistant strains (n = 8) were strong biofilm producers. A statistically significant difference  and groups of different biofilm producers  in different groups of origin of the strains  is shown in a radar chart . There wAB strains, regardless of the origin of the strains, were characterized by a similar distribution of XDR resistance. Among SSTI isolates, it was 68% (n = 17), BSI 76% (n = 19) and PNEU 76% (n = 19), and colonization was 80% (n = 20).All weak biofilm-producing strains (n = 3) were isolated from SSTI, while moderate biofilm-producing strains were present in all other groups, and most of them  were isolated from BSI.ompA 99%, while the least frequently detected was the epsA gene 26%.Among the tested isolates, the most frequently detected gene was bap/bfmS/csuE/ompA, observed in 57% of isolates. Among them, 66.67% (n = 38) belonged to the group of strong biofilm producers.The most common characteristic genotype was bap/bfmS/csuE/ompA/epsA, was less frequent (19%), but 78.9% (n = 15) of isolates with this set were strong producers of a biofilm. The third most common set of genes, bfmS/csuE/ompA, present in 12% of isolates, concerned 74% (n = 9) of strong biofilm producers.The set of all five tested genes, bap/bfmS/csuE/ompA, more than half of the isolates were isolates from the colonization group . The same genotype was the least frequent in the group of isolates from BSI . Moreover, the same set of genes was found among all E-XDR strains  and in 89.47% (n = 34) of XDR strains . Also, most belonged to the XDR group (75%) and were resistant to imipenem and meropenem (69%), noting that only half of the isolates were from ICU. It is alarming to note eight colistin-resistant strains that were isolated during screening tests from asymptomatic patients.In our study, the vast majority of AB strains (72%) strongly produced a biofilm and were characterized by the presence of four genes associated with biofilm formation  . Other ablaOXA-40 (42%) and blaOXA-23 (26%), which confirms our previous studies as well as other reports from Poland [BlaNDM gene was also detected in three strains from colonized patients. Slightly over 26% of carbamenem-resistant strains did not carry any of the oxacillinase genes tested; however, carbapenem-hydrolyzing class D β-lactamases (CHLD) have so many types of oxacillinase that it is possible that they have other genes not detected in our study. Resistance in AB also results from non-enzymatic mechanisms of resistance, e.g., activity of efflux pumps. One family of efflux pumps is the RND family. This efflux pump is also involved in biofilm formation and maturation. Yoon et al. [The predominant carbapenemases genes in the studied AB strains were m Poland ,39,40. Tn et al.  showed tlaOXA-40 % and blan et al.  indicateThe vast majority of studied strains tested in this publication were biofilm-producing strains, including strong producers, which accounted for 72%. In studies, where, like in ours, the association between biofilm formation and antimicrobial resistance was checked, regardless of the mechanism, a positive correlation was found much more often; strains strongly producing biofilms were characterized by higher resistance, primarily to antibiotics from the group of B-lactams and aminoglycosides, or the XDR phenotype in general ,45,46,47In our earlier research , the vasIn our current study, most of the strains were classified as strong biofilm producers. We introduced—in relation to previous experiments—a modification of the study and assessment of biofilm intensity; namely, we read the absorbance of crystal violet on the surface of the created biofilm instead of in the solution, and, additionally, the measurement was made at 225 points. This approach allows for the classification of strains into a given group of biofilm producers with high accuracy. We showed a significant relationship in strains isolated from patients without clinical symptoms of infection and strains isolated from patients with pneumonia and between AB strong biofilm producers. Most of these patients were hospitalized in ICUs. The vast majority of strains strongly producing biofilms were XDR type, including all of them being resistant to colistin. The only antibiotic for which those isolates were more likely to produce less biofilm was gentamicin.AB survival on often-touched surfaces may have an impact on the spread of AB strains in hospital environments. It is extremely important to understand the impact of biofilm formation and antibiotic resistance on AB survival in the hospital environment. Greene et al.  indicatebap, bfmS, csuE and ompA), from which 87.2% are XDR. Other studies report a high frequency of csuE, bap, and ompA genes [epsA, which codes for extracellular exopolysaccharide, which is consistent with the reports of Thummeepak et al. and inconsistent with Zeighami et al. [ompA and bap gene products may contribute to the drug-resistant AB phenotype, especially OmpA, an outer membrane porin.The presence of genes that are mainly associated with biofilm production was confirmed in both strong and moderate biofilm producers. The strong biofilm-producing AB represents 70% of the most common set of genes  and highly biofilm-forming strains. These strains pose a serious threat to patients and a challenge for physicians in treatment ,53. In tNew strategies are also being sought to combat biofilm-forming and multidrug-resistant strains, such as new antibiotics, e.g., synthetic lipopeptides , naturalRegardless of the search for new solutions in the fight against multidrug-resistant AB, infection prevention and control are important in reducing AB infections. These include hand hygiene, environmental cleaning, provision and appropriate use of personal protective equipment, appropriate training of healthcare staff, and promotion of antimicrobial stewardship programs ,6,64. InCurrent research on biofilm-forming AB, including ours, focuses mainly on the assessment of biofilm formation and its impact on bacterial resistance and survival in the environment; however, research on agents that destroy biofilms and interact with antibiotic therapy is also needed. Therefore, in our future studies, we plan to research, among others, the influence of bacteriophages on biofilms.In this study, we did not detect the presence of efflux pumps from the RND family in the tested strains. Also, the effectiveness of cefiderocol on the tested strains was not tested. Strain genotyping was not performed. We have no information about the type of resistance to colistin  and about the association of BSIs when using catheters."}
{"text": "Automated surveillance methods that re-use electronic health record data are considered an attractive alternative to traditional manual surveillance. However, surveillance algorithms need to be thoroughly validated before being implemented in a clinical setting. With semi-automated surveillance patients are classified as low or high probability of having developed infection, and only high probability patients subsequently undergo manual record review. The aim of this study was to externally validate two existing semi-automated surveillance algorithms for deep SSI after colorectal surgery, developed on Spanish and Dutch data, in a Swedish setting.The algorithms were validated in 225 randomly selected surgeries from Karolinska University Hospital from the period January 1, 2015 until August 31, 2020. Both algorithms were based on (re)admission and discharge data, mortality, reoperations, radiology orders, and antibiotic prescriptions, while one additionally used microbiology cultures. SSI was based on ECDC definitions. Sensitivity, specificity, positive predictive value, negative predictive value, and workload reduction were assessed compared to manual surveillance.Both algorithms performed well, yet the algorithm not relying on microbiological culture data had highest sensitivity , which was comparable to previously published results. The latter algorithm aligned best with clinical practice and would lead to 57% records less to review.The results highlight the importance of thorough validation before implementation in other clinical settings than in which algorithms were originally developed: the algorithm excluding microbiology cultures had highest sensitivity in this new setting and has the potential to support large-scale semi-automated surveillance of SSI after colorectal surgery. Healthcare-associated infections (HAIs) pose a major burden on the healthcare system, and result in increased morbidity, mortality, prolonged hospital stay, and additional costs –3. HAIs Continuous surveillance with feedback to healthcare personnel and stakeholders is essential to allocate the required resources and assess the effect of interventions to prevent HAIs. Traditional HAI surveillance is often based on time-consuming and resource-intensive manual review of patient records, which is also prone to subjective interpretation and surveillance bias –7. AutomIn this study, the aim was to externally validate two existing semi-automated surveillance algorithms for deep SSI after colorectal surgery, developed based on Spanish and Dutch data , 9, in aThis retrospective study used EHR data from the Karolinska University Hospital (KUH) stored in the 2SPARE (2020 started Stockholm/Sweden Proactive Adverse Events REsearch) database. KUH is a tertiary care academic center with 1,100 beds divided between two hospitals (Huddinge and Solna), which serves the population of Region Stockholm (2.3 million inhabitants). The study was approved by the Regional Ethical Review Board in Stockholm (no. 2018/1030-31).With semi-automated surveillance patients are divided in low- and high-probability cases where low-probability cases are automatically regarded as no SSI while high-probability cases undergo manual record review to determine SSI status . Two exiOriginal classification algorithm of van Rooden et al., i.e., probability of having a deep SSI based on (re)admission and discharge data, mortality, reoperations, radiology orders, antibiotic prescriptions, and microbiology cultures ;Adapted classification algorithm according to Verberk et al., i.e., probability of having a deep SSI based on the original classification algorithm without the microbiology component .The algorithms’ performance was assessed in a validation cohort of 225 colorectal surgeries selected via simple random sampling from the 2,675 performed surgeries in the period January 1, 2015 until August 31, 2020  and Python (version 3.7), and in accordance with current regulations concerning privacy and ethics. For algorithm performance, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were assessed. The confidence interval (CI) for these estimates were calculated using the asymptotic variance with Wilson score method.Within the validation cohort the median age was 66 year (IQR 55–75), 48.9% (n = 110) were female and the median body mass index was 25.6 (IQR 22.3–29.4). Most surgeries were primary , open procedures  and related to malignancy . The surgeries had a median duration of 316 min (IQR 206–427) and in 41.8% (n = 94) of surgeries a stoma was created. In 36.5% (n = 82) of surgeries the patient had an ASA class ≥ 3 and 24.0% (n = 54) of surgeries had a contaminated or dirty-infected wound classification. Within 30-days after surgery 18.2% (n = 41) of patients developed a deep SSI and 2.2% (n = 5) of the patients died.Both semi-automated algorithms were applied to the validation cohort. Ordered radiology  and receiving antibiotic therapy for ≥ 3 consecutive days  were the most common components. In 38.7% (n = 87) of surgeries ≥ 14 days length-of-stay, readmission and/or mortality was present and in 31.1% (n = 70) a microbiological culture was taken. A reoperation  was the least common component.Of the 41 patients with a deep SSI, 34 were classified as high probability by the original algorithm  while 40 were classified as high probability by the adapted algorithm  admission and discharge dates, mortality, reoperations, radiology orders and antibiotic prescriptions, performed best and outperformed the original classification algorithm which also included microbiology culture data. Only one mild case of deep SSI was missed, which would be hard to detect using only structured data as it was assessed merely based on a free-text medical note.The original algorithm using microbiology culture data was less useful for this specific clinical setting as microbiological culture practices are not standard in suspected deep SSIs after colorectal surgery. However, this might be different in other settings and highlights the importance of thoroughly validation before implementation and pre-emptively investigating if algorithms correspond with clinical practices . It shouAlthough the original semi-automated classification algorithm was developed based on Spanish and Dutch data, and validated and adapted in the Netherlands, also within Sweden the sensitivity was high and comparable with previous results , 9. ThesStrengths of our study were the independent external validation with the extensive availability of EHR data through which the performance of epidemiological surveillance using real-world, real-time data could be mimicked. Limitations were the usage of data from only one center in Sweden, focus of algorithms on only deep SSI and that absence of active post-discharge surveillance of SSI could result in missed SSI.In conclusion, the results from this study in Sweden, in conjunction with previous studies in the Netherlands and Spain, indicate that a classification algorithm based on (re)admission and discharge dates, mortality, reoperations, radiology orders and antibiotic prescriptions, could be widely implemented for semi-automated surveillance of SSI after colorectal surgery."}
{"text": "This article has been retracted by Hindawi, as publisher, following an investigation undertaken by the publisher . This inPlease note that this notice is intended solely to alert readers that the peer-review process of this article has been compromised.Wiley and Hindawi regret that the usual quality checks did not identify these issues before publication and have since put additional measures in place to safeguard research integrity.We wish to credit our Research Integrity and Research Publishing teams and anonymous and named external researchers and research integrity experts for contributing to this investigation.The corresponding author, as the representative of all authors, has been given the opportunity to register their agreement or disagreement to this retraction. We have kept a record of any response received."}