{"text": "In the present study, a participation of RhoA-mediated Ca2+ sensitization in the augmented bronchial smooth muscle (BSM) contraction in a murine model of allergic asthma was examined.It has recently been suggested that RhoA plays an important role in the enhancement of the CaOvalbumin (OA)-sensitized BALB/c mice were repeatedly challenged with aerosolized OA and sacrificed 24 hours after the last antigen challenge. The contractility and RhoA protein expression of BSMs were measured by organ-bath technique and immunoblotting, respectively.+-depolarization. In \u03b1-toxin-permeabilized BSMs, ACh induced a Ca2+ sensitization of contraction, which is sensitive to Clostridium botulinum C3 exoenzyme, indicating that RhoA is implicated in this Ca2+ sensitization. Interestingly, the ACh-induced, RhoA-mediated Ca2+ sensitization was significantly augmented in permeabilized BSMs of OA-challenged mice. Moreover, protein expression of RhoA was significantly increased in the hyperresponsive BSMs.Repeated OA challenge to sensitized mice caused a BSM hyperresponsiveness to acetylcholine (ACh), but not to high K2+ sensitizing effect, probably via an up-regulation of RhoA protein, might be involved in the enhanced BSM contraction in antigen-induced airway hyperresponsiveness.These findings suggest that the augmentation of Ca Increased airway narrowing in response to nonspecific stimuli is a characteristic feature of human obstructive diseases, including bronchial asthma. This abnormality is an important symptom of the disease, although the pathophysiological variations leading to the hyperresponsiveness are unclear now. Several mechanisms have been suggested to explain the airway hyperresponsiveness (AHR), such as alterations in the neural control of airway smooth muscle , increas2+ concentration in myocytes. Recently, additional mechanisms have been suggested in agonist-induced smooth muscle contraction by studies in which the simultaneous measurements of force development and intracellular Ca2+ concentration, and chemically permeabilized preparations in various types of smooth muscles were used. It has been demonstrated that agonist stimulation increases myofilament Ca2+ sensitivity in permeabilized smooth muscles of the rat coronary artery  = 5.67 \u00b1 0.04, Emax = 26.7 \u00b1 1.2 mg; N = 6) and repeatedly OA-challenged (pEC50[Ca2+ (M)] = 5.78 \u00b1 0.15, Emax = 22.8 \u00b1 5.9 mg; N = 6) groups. In both groups, when the Ca2+ concentration was clamped at pCa 6.0, application of ACh (10-5\u201310-3 M) in the presence of GTP (10-4 M) caused a further contraction, i.e., ACh-induced Ca2+ sensitization, in an ACh concentration-dependent manner . However, the ACh -induced Ca2+ sensitizing effect was inhibited by treatment with C3 in both the sensitized control and OA-challenged groups . In the Pro-Q Diamond dye-stained gels, there were several positive bands, i.e., phosphorylated proteins [bottom panel). The ACh-induced phosphorylation of MLC in BSMs of OA-challenged mice was markedly augmented as compared with those of control animals. A Pro-Q Diamond dye-positive 140 kD band probably corresponding to MYPT1, i.e., phosphorylated MYPT1, was also found in the ACh-stimulated BSM samples and was increased in the OA-challenged group (data not shown).Figure proteins , in the in vivo AHR accompanied by increased IgE production and pulmonary eosinophilia has been demonstrated in the actively sensitized and repeatedly OA-challenged BALB/c strain of mice [+-depolarized (without receptors stimulation), intact muscle strips of the repeatedly OA-challenged mice . These findings suggest that the C3-sensitive, RhoA-mediated Ca2+ sensitization might be augmented in BSMs of the OA-challenged AHR mice. Indeed, the current study also demonstrated a marked increase in the expression and activation of RhoA protein in BSMs of the AHR mice  of \u03b1-toxin-permeabilized BSMs was observed between groups (see Result section), indicating that the contents of typical contractile elements such as calmodulin, myosin light chain  BSMs induced by high K+ depolarization was not changed after OA challenge also support our speculation. Thus, the baseline Ca2+ sensitivity of contractile elements themselves in BSM cells is unlikely to change in AHR.In the present study, no significant difference in the Ca2+ itself, the ACh-stimulated contraction of intact BSM strips from the OA-challenged mice was significantly augmented as compared to that from the sensitized control animals  and receptor-operated (nicardipine-insensitive) Ca2+ channels [2+ mobilization in BSM cells. However, the possibility might be denied by the current result that the ACh-induced contraction of \u03b1-toxin-permeabilized BSMs from the OA-challenged mice was significantly augmented as compared with that from the control animals even at a constant Ca2+ concentration  of up-regulation of RhoA in OA-challenged BSMs is not known here, inflammatory cytokines such as tumor necrosis factor-\u03b1 [One of the important findings in the present study is that the C3-sensitive, RhoA-mediated Cace Figs. . Thus, tfactor-\u03b1 , which ifactor-\u03b1 ,35. It i2+ level was within normal level even in the hyperresponsive BSMs [2+ sensitization, although the mechanism(s) for activation of RhoA by ACh is still unclear. If RhoA proteins are activated by receptors other than muscarinic receptor, it might account for the 'non-specific' AHR, which is a common feature of allergic asthmatics. Indeed, the BSMs of the OA-challenged mice also have hyperresponsiveness to endothelin-1 [An increase in responsiveness to muscarinic agonists of airway smooth muscle has been demonstrated in animal models of AHR ,22,36,37ive BSMs ,30. Takethelin-1 , which hthelin-1 .An upregulation of RhoA/Rho-kinase associated with the augmented smooth muscle contractility has also been reported in rat myomertium during pregnancy ,43, arte2+ sensitization in murine BSM contraction. An augmentation of the Ca2+ sensitizing effect, probably by the upregulation of RhoA protein, might be involved in the enhanced BSM contraction observed in the antigen-induced AHR in mice.In conclusion, the current study demonstrated an ACh-induced, RhoA-mediated CaYC conceived of the study, participated in its design and coordination, and drafted the manuscript. AU carried out the intact smooth muscle studies. KS, HT and HS carried out the skinned fiber studies and immunoblot analysis. SN carried out the analysis of active RhoA. MM participated in the direction of the study as well as writing and preparing the manuscript. All authors read and approved the final manuscript."}
{"text": "The brain-derived protein S100B has been shown to be a useful marker of brain injury of different etiologies. Cognitive dysfunction after cardiac surgery using cardiopulmonary bypass has been reported to occur in up to 70% of patients. In this study we tried to evaluate S100B as a marker for cognitive dysfunction after coronary bypass surgery with cardiopulmonary bypass in a model where the inflow of S100B from shed mediastinal blood was corrected for.56 patients scheduled for coronary artery bypass grafting underwent prospective neuropsychological testing. The test scores were standardized and an impairment index was constructed. S100B was sampled at the end of surgery, hourly for the first 6 hours, and then 8, 10, 15, 24 and 48 hours after surgery. None of the patients received autotransfusion.In simple linear analysis, no significant relation was found between S100B levels and neuropsychological outcome. In a backwards stepwise regression analysis the three variables, S100B levels at the end of cardiopulmonary bypass, S100B levels 1 hour later and the age of the patients were found to explain part of the neuropsychological deterioration .In this study we found that S100B levels 1 hour after surgery seem to be the most informative. Our attempt to control the increased levels of S100B caused by contamination from the surgical field did not yield different results. We conclude that the clinical value of S100B as a predictive measurement of postoperative cognitive dysfunction after cardiac surgery is limited. Despite the fact that incidence figures between 4\u201379% have been reported for cognitive dysfunction after cardiac surgery , diagnosSeveral studies in humans suffering from stroke of different ethiologies, have shown a rather strong correlation between serum levels of S100B and size of lesion(s) as well as outcome -17.Lately a number of studies have addressed the question whether S100B can be considered as a marker for cognitive dysfunction after cardiac surgery; however the conclusions presented are disparate. One major reason for this could be the fact that S100B is present in high concentrations in shed mediastinal blood that is retransfused to the patient by cardiotomy suction and autotransfusion, thus obscuring the measured levels of S100B early after surgery. To date, only one study have been published where this contamination was taken into account . We receThe study group comprised 56 patients who underwent coronary artery bypass graft (CABG) surgery at the Division of Cardiac Surgery, University Hospital MAS, Malmoe, Sweden and Department of Cardiothoracic Surgery, Lund University Hospital, Lund, Sweden. Only patients planned for elective CABG with cardiopulmonary bypass (CPB) as their sole procedure were included. The study protocol was approved by the local ethics committee, and patients gave a written informed consent before the study protocol was initiated. Patients with a history of stroke, transient ischemic attack (TIA), reversible neurological disorder (RIND), known carotid artery disease or other brain diseases were excluded. In order to avoid possible influence of renal disorder on the elimination of S100B, patients with known renal failure (Creatinin > 160 \u03bcmol/L) were excluded.The patients were examined for signs of neurological dysfunctions daily during the hospital stay by either experienced cardiac anesthesiologists or by experienced cardiac surgeons.\u00ae, Roche, Basel, Switzerland) or propofol  10 mg/kg. It was subsequently maintained with fentanyl 10 \u03bcg/kg , a continuous infusion of propofol 3\u20136 mg/kg/h or inhalation of isoflurane 0,5\u20131% . Nitrous oxide  was used before CPB but not during or after CPB.Anesthesia was induced with midazolam 3\u20135 mg iv  and administered in the ascending aorta and the anastomosed vein-grafts intermittently.CABG surgery was performed during aortic cross clamping with the distal anastomosis preceding the proximal anastomosis. A tangential occluder replaced the cross-clamp during the proximal anastomosis. Antegrade cold S:t Thomas crystalloid cardioplegia was used (Cardioplegi2 at normothermia. The perfusate was cooled to approximately 32\u00b0C. Heparin (400 U/kg bodyweight) was given prior to cannulation and reversed with equal doses of protamine sulphate at decannulation.Perfusion was performed with a roller pump . The perfusion catheters and circuit were made of polyvinylchloride in the line and silicon in the pumphead. The arterial cannulation was made in the ascending aorta and venous cannulation in the right atrium by a two-stage venous cannula. All circuits contained a heparin-coated 40 \u03bcm arterial filter  and a membrane oxygenator . The circuit was primed with approximately 1000 ml of Ringer's lactate (Pharmacia-Upjohn), 250 ml 15% Mannitol (Pharmacia-Upjohn) and 75 mmol Addex tromethamine (Pharmacia-Upjohn). Perfusion flow was non-pulsatile with a flow rate of 2.4 l/min/mAfter surgery, the patients patient were transferred to the ICU for recovery and enrolled in the sampling scheme for S100B analysis. None of the patients received autotransfusion.Serum for S100B analysis was sampled before surgery, at the end of CPB, and then 1, 2, 3, 4, 5, 6, 8, 10, 15, 24 and 48 hours after surgery. The S100B levels at these time points will be referred to as T0, T1, T2....T48. Blood samples, both arterial and venous samples, were cooled and centrifuged within 5 hours. All samples were measured by a monoclonal two-site immunoluminometric assay .Since early levels of S100B are contaminated by S100B from cardiotomy suction, an attempt was made to exclude this S100B from the levels measured one and two hours after surgery. Assuming that all of the measured S100B at the termination of CPB was a contamination, this non-cerebral S100B was eliminated with a half-life of 25 minutes, as illustrated in figure e is the estimated true levels of S100B at time t, Ct = the measured concentration of S100B at T1 or T2, C0 the concentration at the end of CPB, t the time after T0 and t1/2 the half-life of S100B.where CWe have earlier suggested that the elimination rate could be used as another measure of cerebral release , and theThe patients underwent neuropsychological testing by the same trained neuropsychologist 1\u20132 days before and 5\u20137 days after surgery. The tests used were: Mental Control, Figural Memory, Logical Memory (A/B), Visual Reproduction, Rey Auditory/Verbal Learning Test (RAVLT), Trail Making A, Trail Making B, Digit Symbol, Digit Span, Visual Memory Span, Visual Paired Associates II or Verbal Paired Associates I and RAVLT, Delayed Retention. The tests were chosen from the Wechsler Memory Scale-Revised (WMS-R), the Wechsler Adult Intelligence Scale (WAIS \u2013 R) and the Halsted-Reitan Neuropsychological Battery ,21.Differences for each sub-test were first calculated and then standardized to z-values. All sub-tests were then aggregated to create an impairment index . The impAll results were analyzed with the Statistica version 5.0 for PC. Regression analysis was performed with the least square method with a casewise deletion of missing data. For the multiple regression a backwards stepwise regression was performed to determine which variables to include in the final analysis. The initial variables included in this analysis were age, gender, perfusion time, years of education, and S100B levels at all sampling times. A p-value < 0,05 was considered significant. Unless otherwise stated numerical values are presented as mean \u00b1 1 standard deviation.Patient demographics are presented in table The appearance of S100B protein followed the same pattern in all patients with high levels at the end of CPB and a decrease or slight increase the first hour figure . ThereafWe found a correlation between patient age and S100B levels measured up to 24 hours after bypass Table . NeitherIn multiple regression analysis, measured S100B levels at the end of CPB (T0), one hour later (T1) and age were found to explain part of the neuropsychological impairment  declare that they have no competing interests.HJ: Principal investigator recruited, enrolled patients analyzed and wrote the paperPJ: Had an integral role in the planning and the analysis, also help with writingMB: Designed neuropsychological test battery. Analyzed neuropsychological dataCB: Performed all neuropsychological testing with patientsCA: Did the S100B-analyzesSB: Mentor an principal leader of the project, who also helped with the writing.The pre-publication history for this paper can be accessed here:"}
{"text": "Pulmonary oedema and impairment of oxygenation are reported as common consequences of haemorrhagic shock and resuscitation (HSR). Surprisingly, there is little information in the literature examining differences in crystalloid type during the early phase of HSR regarding the development of pulmonary oedema, the impact on oxygenation and the haemodynamic response. These experiments were designed to determine if differences exist because of crystalloid fluid type in the development of oedema, the impact on oxygenation and the haemodynamic response to fluid administration in early HSR.Twenty anaesthetised swine underwent a grade V liver injury and bled without resuscitation for 30 minutes. The animals were randomised to receive, in a blinded fashion, either normal saline  or lactated Ringer's solution . They were then resuscitated with study fluid to, and maintained at, the preinjury mean arterial pressure (MAP) for 90 minutes.P = 0.027). After 150 ml/kg of fluid, EVLWI increased from 9.5 \u00b1 0.3 ml/kg to 11.4 \u00b1 0.3 ml/kg NS and from 9.3 \u00b1 0.2 ml/kg to 10.8 \u00b1 0.3 ml/kg LR (P = 0.035). Despite this, oxygenation was not significantly impacted /fraction of inspired oxygen (FiO2) \u2264 100) until approximately 250 ml/kg of either fluid had been administered. Animals resuscitated with NS were more acidaemic (with lower lactates), pH 7.17 \u00b1 0.03 NS vs. 7.41 \u00b1 0.02 LR (P < 0.001).Extravascular lung water index (EVLWI) began to increase immediately with resuscitation with both fluid types, increasing earlier and to a greater degree with NS. A 1 ml/kg increase in EVLWI from baseline occurred after administartion of (mean \u00b1 standard error of the mean) 68.6 \u00b1 5.2 ml/kg of normal saline and 81.3 \u00b1 8.7 ml/kg of LR (This study suggests that early resuscitation of haemorrhagic shock with NS or LR has little impact on oxygenation when resuscitation volume is less than 250 ml/kg. LR has more favourable effects than NS on EVLWI, pH and blood pressure but not on oxygenation. Pulmonary oedema has been reported to be a common consequence of haemorrhagic shock and resuscitation (HSR) . The earThe EVLWI at any time represents a dynamic balance between factors that cause fluid to accumulate in the lungs and those that carry it out of the lungs. An increase in fluid leaving the vascular space and remaining in the pulmonary parenchyma following HSR can result from: endothelial injury with increased pulmonary capillary permeability; alveolar epithelial injury and decreased alveolar fluid clearance; dilutional decreases in serum oncotic pressure; increased pulmonary capillary pressure; coagulation abnormalities with increased extravasation of fluid; and increased inflammation ,3. But b2)/fraction of inspired oxygen (FiO2) and parameters of haemodynamics during early resuscitation from haemorrhagic shock with LR or NS and determine if there are differences on the development of oedema, the impact on oxygenation and the haemodynamic response to fluid administration because of fluid type.These experiments were designed to measure the EVLWI, the partial pressure of arterial oxygen , intubated with an oral endotracheal tube and placed on mechanical ventilation. Tidal volume was set at 12 \u00b1 2 ml/kg, and respiratory rate was adjusted to maintain end-tidal carbon dioxide and partial pressure of arterial carbon dioxide (PaCO2) of 40 \u00b1 4 mmHg. Anaesthesia was maintained using 1 to 3% isoflurane as needed. To assess adequacy of anaesthesia, we monitored jaw tone. Monitoring devices were placed after establishing anaesthesia, including an oesophageal thermometer and external jugular vein catheter. Animal temperature was maintained at 38.0 \u00b1 1.5\u00b0C using external warming devices and warmed fluids. Femoral artery cut down was performed to place a 4-F femoral catheter with an integrated thermistor tip  for continuous blood pressure monitoring, blood sampling and transpulmonary thermodilution determinations of cardiac output (CO), EVLWI, global end-diastolic volume (GEDV) and intrathoracic blood volume (ITBV). MAP and heart rate (HR) were continuously recorded.Twenty Yorkshire crossbred pigs weighing approximately 35 kg underwent a 16-hour fast preoperatively with water The transpulmonary thermodilution method using the single-indicator transpulmonary thermodilution technique  was originally developed in swine and has been previously validated by comparison with the postmortem gravimetric technique, and with the double dilution (thermo-dye) technique in swine, sheep and humans in a variety of disease states -18. The We examined the precision of this technique for measuring EVLWI in this species of swine using repeated measures of EVLWI at baseline in three animals in separate experiments. We found an average coefficient of variation (standard deviation/mean \u00d7 100) of less than 5%. As an example, repeated measurements (n = 20) were made in one animal at baseline yielding a mean EVLW (\u00b1 standard deviation) of 9.1 \u00b1 .45 ml/kg.For these experiments, a 15 ml bolus of iced randomised fluid (0 to 6\u00b0C) was injected via a central venous catheter into the right atrium. The thermodilution curve was recorded with a femoral artery thermistor and used to determine CO, the volume of blood in the heart at the end of filling or the GEDV, the ITBV and EVLWI, as previously described ,20. The The animals underwent a midline coeliotomy, suprapubic urinary catheter placement and splenectomy. Splenectomies are performed in swine haemorrhage models because of the spleen's distensibility and the resultant variable amounts of sequestered blood. The spleen was weighed and randomised fluid was infused to replace three times the spleen weight. Following a 15-minute stabilisation period, a standardised grade V liver injury was created with a specially designed clamp. The clamp was directed centrally over the liver and created a consistent pattern of injury involving one or more central hepatic veins. Injuries met criteria for grade V liver injuries as described by the American Association for the Surgery of Trauma Organ Injury Scaling system . This moAfter 30 minutes of uncontrolled haemorrhage, blood was evacuated from the abdomen and measured. Blinded resuscitation was begun with either LR or NS. Both fluids were purchased from Baxter  and were unmodified . A 30-minute delay before beginning resuscitation allowed the animals to reach their nadir blood pressure, replicating the civilian trauma scenario. Fluid was delivered at 165 mL/minute. This rate was chosen because it is one-half of the rate delivered by the level 1 infuser to humans, and the pigs were approximately one-half the weight of a normal adult human. The goal of resuscitation was to achieve and maintain the baseline MAP for 90 minutes post injury. Blood loss was determined by placing pre-weighed laparotomy sponges into the pelvis and inferior right and left pericolic gutters before creating the liver injury and collecting active haemorrhage by suction or with the sponges, while avoiding manipulation of the liver injury. The sponges were removed before abdominal closure. Following resuscitation and prior to euthanasia, the abdomen was opened and any additional blood loss was collected by suction. Blood loss (mL/kg) was reported as a mean for each resuscitation group. To ensure comparable injuries between the study groups, we removed the liver and identified the number of hepatic vessels injured.t tests using a statistical software package for personal computers . Significance was defined as p < 0.05. All data are presented as means \u00b1 standard error of the mean. Linear regression analysis was performed to determine the effect of fluid type, independent of volume, as shown by differences in the slopes of the regression lines. Boxplots comparing differences in means with confidence intervals of MAP, CO, systemic vascular resistance (SVR), GEDV, stroke volume (SV) and HR were performed to examine differences in those values with NS vs. LR resuscitation.Comparisons between groups were made with independent-sample One animal in the NS group did not survive the two-hour study. All the animals in the LR group survived. Composite MAP of the two groups throughout the study are shown in Figure Mean urine output and blood loss were greater in the NS group than in the LR group. Urine output was 44.1 ml/kg \u00b1 8.1 in the NS group vs. 19.4 ml/kg \u00b1 3.4 ml/kg in the LR group (p = 0.012), and total blood loss was 34.3 \u00b1 2.9 ml/kg vs. 23.7 \u00b1 2.1 ml/kg (p = 0.009). The NS group required almost twice as much fluid  over the 90-minute resuscitation period in an attempt to maintain goal MAP. There was no difference in blood loss prior to fluid resuscitation.2/FiO2 ratio as a function of resuscitation fluid volume over the entire study are plotted in Figure 2/FiO2 of 100  did not occur until approximately 250 ml/kg of either fluid had been administered.EVLWI and the PaO2/FiO2 plotted as a function of resuscitation fluid volume administered early in the study, limited to 250 ml/kg. This was performed to examine the effect of fluid type early in the resuscitation at similar volumes. Linear regression analysis revealed a larger increase in EVLWI in the NS group as compared with the LR group occurring early in the resuscitation. There was no significant difference in baseline EVLWI for the two groups. A 1 ml/kg increase in EVLWI from baseline occurred after 68.6 \u00b1 5.2 ml/kg of NS and 81.3 \u00b1 8.7 ml/kg of LR (p = 0.027). After 150 ml/kg of fluid EVLWI increased from 9.5 \u00b1 0.3 ml/kg to 11.4 \u00b1 0.3 ml/kg for NS and from 9.3 \u00b1 0.2 mg/g to 10.8 \u00b1 0.3 ml/kg for LR (p = 0.035). Significant changes in oxygenation, defined as a drop in baseline PaO2/FiO2 to 100 or greater, did not occur over this range for either fluid type.Figure 3 vs.1177.6 \u00b1 34.6 dyne \u00d7 sec/m3; p < 0.001). As shown in Figure Figure Figure Laboratory values at the end of the study are shown in Table 2/FiO2 of 100 or more , until approximately 250 ml of either fluid had been infused. This corresponds to 17.5 litres in a 70 kg human \u2013 well in excess of what would be normally administered during most human resuscitations prior to the arrival of blood products.In this swine model of traumatic haemorrhagic shock, resuscitation with NS as compared with LR resulted in greater EVLWI formation at similar amounts of resuscitation volume. In addition, the blood pressure response to fluid was considerably lower in the NS group  despite requiring more total fluid over the entire study period  in an attempt to maintain goal MAP. The lower blood pressure with NS administration was due to a greater systemic vasodilatation as compared with LR. Surprisingly neither fluid resulted in significant changes in oxygenation, as defined by a drop in baseline PaOWe observed differences in EVLWI early in the resuscitation when similar volumes of the fluids had been given, more NS than LR. This supports the idea that a fluid specific effect, independent of volume, was present. To evaluate this we performed two separate regression analyses: Figure 2/FiO2 vs. volume of resuscitation for two reasons. First, previous studies have shown a stepwise increase in the rate of EVLWI formation as a function of the interstitial matrix filling and then abruptly 'giving way' with a subsequent increased rate of fluid extravasation into the matrix and then alveoli. One could expect an initial improvement in PaO2/FiO2 with improved CO early in the resuscitation, followed by a curvilinear decline as the matrix 'lets go' with a subsequent increase rate in fluid extravasation. Secondly a curvilinear line dramatically improved the regression coefficient (R2); from 0.620 to 0.815 for NS, and from 0.15 to 0.60 for LR. We found significant differences in EVLWI formation at similar volumes of resuscitation, more NS than LR early in the resuscitation. The cause of the greater EVLWI accumulation in the NS group at similar resuscitation volumes as compared with the LR group is not known.A second order polynomial was used as the best fit line for PaOEVLWI is defined as the extravascular fluid in the lung at any moment including the intracellular fluid of inflammatory, endothelial and epithelial cells in the extravascular space, as well as alveolar and interstitial fluid and intrapulmonary lymph . The amoHSR results in increased expression of inflammatory cytokines, increased neutrophil sequestration in the lung, increased generation of reactive oxygen and nitrogen species, and activation of coagulation. All of these factors can cause endothelial and epithelial cell dysfunction and increased EVLWI ,26-28. HThe NS group developed a non-anion gap metabolic acidosis whereas the LR group remained pH neutral. Several studies have shown potential lung protective effects of hypercapnic acidosis in acute lung injury -32. HoweDilutional decreases in serum oncotic pressure are likely to have contributed to the rise in EVLWI seen with both fluid types in this study. It is likely that the differences in the total EVLWI measured at study end were in large part due to dilutional effects from the greater volume of NS required. Twice the volume of NS was required to maintain target MAP. This has clinical relevance in regards to selecting fluid type, in that a greater volume of NS will need to be administered to maintain blood pressure goals and will result in greater EVLWI formation and acidosis.Figure As shown in Figure Total blood loss was greater in the NS group than the LR group . It is known that HSR with NS attenuates the hypercoagulable response seen with haemorrhage as compared with LR . IncreasPrevious studies examining the effects of NS vs. LR on EVLWI, oxygenation and the haemodynamic response have been conducted in controlled haemorrhage models. Many re-infused shed blood with the resuscitation fluid. Our study used a more clinically relevant model by adding tissue injury to uncontrolled haemorrhage and initiating early resuscitation with crystalloids alone and resuscitating to a goal blood pressure. Although this complicated the analysis of the effect of fluid type independent of volume, we felt by doing so our results could be more reliably extrapolated to the human clinical scenario. Further work is needed to examine late effects of crystalloid type and volume on EVLWI, oxygenation and haemodynamics in a living model of HSR.Our study did have limitations. By study end the animals had received extremely large resuscitation volumes that exceeded those typically seen in human scenarios. We believe this reflects the severity of the model as well as the choice to resuscitate to the baseline MAP. This resulted in resuscitation beyond normal perfusion as evidenced by supranormal urine outputs especially in the NS group. Despite this fact, differences between the fluids manifested very early, prior to exceeding resuscitation volumes typically used clinically. We have also previously shown that differences in pH and coagulation between LR and NS occur very early after initiation of fluid resuscitation . This inA tidal volume of 12 ml/kg was used in this study. This tidal volume may have caused some lung injury. Although this may have played a role in the development of increased EVLWI, both animal groups were exposed to the same tidal volume making it an unlikely cause of a difference between groups.This study suggests that early resuscitation of haemorrhagic shock with NS or LR prior to the arrival of blood products is safe in terms of the immediate impact on oxygenation when resuscitation volume is limited to less than 250 ml/kg. Resuscitation with LR has more favourable effects on EVLWI formation, pH, coagulation and haemodynamics but not on oxygenation. Further work is needed to understand the cause of these fluid-specific differences.\u2022 When resuscitating haemorrhagic shock prior to the arrival of blood products significantly more NS is required to maintain goal blood pressure than LR.\u2022 LR has more favourable effects on EVLWI formation, pH, coagulation and haemodynamics than does NS.\u2022 Further investigation is warranted to attempt to explain these fluid-specific differences.2: fraction of inspired oxygen; GEDV: global end-diastolic volume; HR: heart rate; HSR: haemorrhagic shock and resuscitation; IL: interleukin; ITBV: intrathoracic blood volume; LR: lactated Ringer's solution; MAP: mean arterial pressure; NS: normal saline; PaCO2: partial pressure of arterial carbon dioxide; PaO2: partial pressure of arterial oxygen; PBV: pulmonary blood volume; PVPI: pulmonary vascular permeability index; SV: stroke volume; SVR: systemic vascular resistance; TNF: tumour necrosis factor.CO: cardiac output; EVLWI: extravascular lung water index; FiOCRP has acted as an advisor to Pulsion Medical Systems, makers of the PiCCO device that measures EVLW. There are no other potential conflicts from any of the authors.CRP participated in study design and coordination and helped draft the mauscript. KV helped with data collection and analysis. DSH participated in executing the experiments, and helped with data collection. RSS participated in the design of the study and participated in executing the experiments. JAD participated in the design and coordination of the study, in study execution, data collection and analysis. JMW participated in the design of the study and participated in executing the experiments. MAS conceived of the study, participated in study design and coordination and helped draft the mauscript. All authors read and approved the final manuscript."}
{"text": "Objective: Over the last 15\u2009years, researchers from around the world have developed instruments for assessing the risk of conversion to psychosis. The objective of this article is to review the literature on these instruments by focusing on genealogy links and on their performance in predicting conversion to psychosis.Method: A systematic review of articles published since 1980 relating to risk assessment instruments for conversion to psychosis by manual search and consultation of electronic databases MEDLINE, EMBASE, and PsycINFO.Results: Three hundred ninety one (391) publications were selected and analyzed. Among these, 22 instruments were identified. These instruments are briefly described and placed on a timeline according to their year of publication. A code of positions, patterns, and forms is used to schematize the characteristics of each instrument. A table is presented to show changes in rates of conversion to psychosis within cohorts of subjects considered at risk according to the instruments. A second code of shades and outlines is used to schematize the characteristics of each cohort of patients. The two graphics set the stage for a discussion about the major strategies that were adopted to improve the performance of risk assessment instruments.Conclusion: These graphics allow a better understanding of the origin, evolution, current status, strengths, shortcomings, and future prospects of research on risk assessment instruments.Clinical ImplicationsThe integration of theoretical approaches, the multicenter studies, and the pre-selection of patients with short questionnaires were the main strategies to improve the performance of instruments assessing the risk of conversion to psychosis.These instruments are better at predicting conversion to psychosis than conventional variables within a more limited time span and can therefore enable the evaluation of various risk factors and biomarkers that may be associated with psychosis.LimitationsThe studies selected for this review of literature were not classified according to their methodological quality.These studies are based on heterogeneous populations and this must be taken into account when comparing the rates of conversion to psychosis.This review of literature was based on published data only and they were no direct communication with the authors of these instruments. Beyond its economic implications, psychosis is a disease that all too often has a lasting and painful impact on the patient\u2019s life. In the hope of avoiding the most dramatic consequences, research groups around the world have been working on psychosis prevention since the early 1990s , the development of instruments to evaluate the risk of conversion to psychosis would make it possible to identify individuals who will develop psychosis before the disease sets in and therefore before their lives are disrupted. These people could then receive treatments to mitigate, delay, or even prevent the undesirable consequences. For more than 20\u2009years, such instruments have been developed and tested around the world, starting with the scales created by Chapman and Chapman , who souHowever, the conversion rate in the cohorts of individuals identified as being at risk of developing psychosis are relatively low, ranging from less than 10% to slightly over 50% within follow-up periods ranging from 6\u2009months to 10\u2009years. In many centers, it has also been observed that conversion rates decline with time . The publications retained had to concern one or more instruments for evaluating the risk of conversion to psychosis and to be written in English or French. Particular attention was paid to previously published literature reviews on these instruments to better understand their theoretical bases.To do this, a review of the literature on instruments designed to evaluate the risk of conversion to psychosis was done by our group from November 2009 to July 2011. The keywords The scales and other tools on which they were based were placed on a timeline Figure , and claThe search in PsycINFO returned 240 publications, in EMBASE, 249, and in MEDLINE, 63. After adding the publications found with a manual search and applying the inclusion and exclusion criteria, we retained a total of 391 publications. Of this number, 181 publications (46.3%) came from Europe, 123 (31.5%) from North America, 51 (13.0%) from Australia, 14 (3.6%) from Asia, and 2 (0.5%) from Israel. Certain publications featured contributions by authors from several research centers around the world, and 20 (5.1%) were classified as the outcome of a multicenter cooperative study. Several of these publications concerned the follow-up of cohorts of patients at risk of conversion to psychosis (155 or 39.6%). As well, 71 reviews of the literature (or 18.2% of the publications retained) enabled us to consolidate our knowledge of the topic.Through this search, we identified 22 instruments for evaluation of the risk of conversion to psychosis. These instruments were placed on the timeline based on their publication dates , which occurs at the very start of the development of psychosis , cognitive problems, disorders affecting cenesthesia (the sense of bodily existence), central neurovegetative disorders, and coping strategies  were first operationalized by the research team at the PACE Clinic in Melbourne, VIC, Australia. They refer to three groups of patients: patients who present attenuated psychotic symptoms ; patients who present what are known as Brief Limited Intermittent Psychotic Symptoms or BLIPS ; and patients who present risk factors combined with an at-risk state or trait and state risk factors  . According to this research strategy, patients are at risk of conversion to psychosis if they present attenuated positive symptoms (or CHR+), but also if they manifest attenuated negative symptoms (or CHR\u2212) , was developed by a Japanese team that added a section on the duration of symptoms to improve its specificity (symptoms present for a longer time are considered to be more representative of being at risk of conversion to psychosis)  more specifically indicate an increased risk of conversion to psychosis. In a sample of 501 subjects with at least one DSM-III-R criterion for prodrome, the D3R identified 20 subjects as being at risk of conversion to psychosis; 3 of them (15%) developed a psychosis after 6\u2009months of follow-up  in Switzerland led to the development of several instruments inspired by the DSM-III-R criteria, but also by the literature on prodromal psychosis . The PROD-screen is a 29-question screening questionnaire inspired by the SIPS, the IRAOS, and the BSABS . This is well represented in Figure But this reconciliation of the two approaches is not sufficient to mitigate the differences between the various research teams. Differences remain between the characteristics of the basic population, the recruitment of patients, the evaluation process, the follow-up, the treatments provided, etc. This is why there was the creation of several multicenter studies . In addition to enabling researchers to test evaluation instruments on a much larger population, these studies are pursuing the homogenization of the process of evaluating subjects at risk of conversion to psychosis. This homogenization makes it easier to compare the results of research on these subjects, which had formerly been difficult and risky.Figure We should point out that the way subjects get into programs for evaluating the risk of conversion probably has a great influence on the proportion of subjects who will develop psychosis out of the total cohort of patients considered to be at risk. Yung et al.  hypothesDespite their relatively low positive predictive value, it should be emphasized that these evaluation instruments are better at predicting conversion to psychosis than conventional variables  within a more limited time span Cannon, . They maThese results show how these instruments are evolving. They are constantly being revised as a function of the clinical and ethical challenges represented by research on the prodrome of psychosis. This is what we have attempted to schematize in Figure One of the next steps could be to push the analysis further and to consider changes in these patients\u2019 distress levels and functioning. As Ruhrmann et al.  emphasizThe 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": "The first is presented to a scientist who has made distinguished contributions over many years in research, teaching, service, or any combination of the three. This year, the ISCB Accomplishment by a Senior Scientist Award goes to Michael Ashburner in the department of genetics at the University of Cambridge. The second, known as the Overton Prize, honours a young scientist in the early to mid-stage of his or her career who has already achieved significant and lasting impact in the field of computational biology. In 2011, the Overton Prize is awarded to Olga Troyanskaya of Princeton University in New Jersey.Each year, the International Society for Computational Biology  in Madrid. The winners will receive their awards at the ISCB's annual meeting, where they will also deliver keynote talks. This meeting, ISMB/ECCB 2011 If computational biology seems challenging in the second decade of the 21st century, spare a thought for those who pioneered the discipline in the 1980s. Michael Ashburner  at the UDrosophila and, in particular, polytene chromosomes, which form when certain specialised cells undergo repeated rounds of DNA replication. Polytene chromosomes have a characteristic banded structure. In Drosophila there are some 5,000 bands and a subset of these undergo, during development, a reversible structural modification as the result of transcription; this is know as puffing and can be considered an analog of gene activity. In the late 1960s and early 1970s, Ashburner studied puffing patterns and inferred the existence of a cascade of genetic controls under the influence of the hormone ecdysone during larval development.Ashburner began his career with a degree in genetics from the University of Cambridge in 1964. He stayed on to do a PhD, studying Alcohol dehydrogenase gene and its environs. By the mid-1980s, he had the most detailed analysis in full genetic terms of any small chromosome region of any multi-cellular organism, and had the Adh gene sequences from several different species of Drosophila. \u201cThat drew me into bioinformatics because we needed a way of comparing sequences,\u201d he says. \u201cThere was almost no software available to help.\u201dIn the late 1970s, Ashburner turned his attention to the study of the Two people came to his aid. The first was Walter Bodmer, director of the Imperial Cancer Research Fund, who gave Ashburner the use of a DEC computer with access to the early network. \u201cWe could access this machine by dial-up and do some analysis,\u201d he says. The second was Doug Brutlag at Stanford University, who was developing MOLGEN, an early bioinformatics system, which he allowed Ashburner to access.That presented a significant obstacle, however. Getting a computer in the United Kingdom to speak to one in Stanford was not straightforward. Today, everybody uses the Internet, defined by the TCP/IP protocol. But in the early \u201880 s, the UK and United States used different systems. The US was pioneering TCP/IP while the UK had a standard called the Coloured Book protocols. \u201cThe only place that had an interface between the two protocols was University College, London, and they were very helpful,\u201d says Ashburner, \u201cgiving us 5 kb of disk space.\u201dThe process of connecting to Stanford was far from simple. \u201cThe way you did it was to dial up your local packet switching exchange at the Post Office and connect to the Rutherford Appleton Laboratory. You then typed in some code which connected you to UCL where you could use TCP/IP,\u201d he says. The signal was routed via Goonhilly satellite station in Cornwall to Carnegie Mellon University and from there to Stanford. \u201cI had a dumb terminal, that is a box with no memory, so everything had to be captured by a printer in parallel.\u201d Ashburner was far from deterred, however.At about that time, the European Molecular Biology Laboratory (EMBL) in Heidelberg and GenBank in the US released the first nucleotide sequence libraries in quick succession. Using his network access, Ashburner and his colleagues, collaboratively with MOLGEN, set up one of the first bulletin boards, called BioNet, to keep people informed of changes to the library and to software. \u201cThis became well used and things evolved from there,\u201d he says.As the field of bioinformatics grew, the need for an institution to house the data and conduct research increased. So in 1992, the EMBL decided to set up an institute of bioinformatics that would house this library and carry out research. This organisation became known as the European Bioinformatics Institute, based in Hinxton, UK, with Ashburner and John Sulston having led the UK bid to host it. \u201cI was persuaded to become the first program coordinator and took half-time leave from Cambridge to do that,\u201d he says. He eventually took over as joint-director, a post he held until 2001. \u201cAt first, the finances were sticky and the politics were horrendous. But it has since gone from strength to strength,\u201d he says.Drosophila genetics. This is a field with a rich and long history of collecting and sharing mutations. The first catalogue of mutations was published in 1925 and it was still being revised in paper form in the late 1980s. But the field was beginning to expand quickly and the books were out of date as soon as they were published. \u201cIt became clear to me that we couldn't carry on publishing in paper form every 10 or 20 years,\u201d he recalls.At the same time, Ashburner continued his interest in So in 1989 he proposed that the community set up an electronic database to take over the role of the printed one. In 1992, the NIH funded the project that became known as FlyBase, one of the first genetic and now genomic databases.FlyBase was a crucial factor in triggering Ashburner's interest in a structured, controlled vocabulary, a formal representation of knowledge about genes and gene products. He began to define terms for gene products by their biological processes, such as wing development, and then defined the data structure in which these terms were related to each other. \u201cIt occurred to me that if you were able to do this for several model species, you'd have a fantastic tool,\u201d he says.Drosophila, yeast, and mouse databases. They called it the Gene Ontology, and it is now a major bioinformatics project that covers over 1,800 species. Their original paper on the idea in Nature Genetics is one of the most highly cited in the field. \u201cHis achievement is not just to have built this system but also to have organised the consortium behind it. It is now one of the most used resources in all of biology,\u201d says Valencia.But this insight initially met with little interest. \u201cMy first presentation, at ISMB in Greece in 1997, went down like a lead balloon,\u201d he recalls. Eventually, he and three like-minded colleagues settled the matter in a bar at the Montreal ISMB in 1998. Together, they decided to set up a cross-species ontology to be used by the Drosophila genome in 1999. \u201cThe process turned me into a nervous wreck,\u201d he jokes. He published his account of this roller-coaster experience in a short but entertaining book called Won for All: How the Drosophila Genome was Sequenced .He went on to collaborate with Gerry Rubin and Craig Venter in sequencing the \u201cWe're lucky to have such an inspirational figure in the community,\u201d says Valencia. \u201cThis award has been well deserved for a number of years.\u201dIn the spring of 1997, Olga Troyanskaya  was workAnd so began the career of one of the most promising young researchers in bioinformatics, and a deserving winner of this year's Overton Prize. \u201cShe is one of these forces of nature, full of energy,\u201d says Alfonso Valencia, chair of the ISCB awards committee.Troyanskaya herself talks with infectious enthusiasm about her work. \u201cI've always been fascinated by the problems of biology,\u201d she says. \u201cI was just better at computer science and math than the wet lab research. And it seemed to me that there had to be a lot you could contribute with computer science that you couldn't do with experimental techniques alone.\u201dFrom the University of Richmond, Troyanskaya moved to Stanford University to complete a PhD in biomedical informatics, under the supervision of Russ Altman, a bioinformatician, and David Botstein, a geneticist. \u201cI wanted a setup that was close to real biological problems, and I got exactly that. I learned a great deal from both of them,\u201d she says.In 2003, she moved to Princeton University as an assistant professor in the Department of Computer Science and the Lewis-Sigler Institute for Integrative Genomics. \u201cI am fortunate that the computer science department appreciates the impact of computing in biology, and that I have many wonderful colleagues at both the department and in the Institute. I found several amazing collaborators, and this allowed me to begin a number of interesting projects.\u201dOne of the key problems she focuses on is making better use of the vast but unwieldy biological datasets in databases around the world. \u201cSo instead of focusing on one study, we can take the entirety of published data. That allows you to ask very specific questions in a data-driven way and to develop novel biological hypotheses,\u201d she says.An important goal is to predict the function of genes or proteins. There have been many experimental approaches to determine what genes do and how they are controlled inside the cell. But this work tends to produce datasets that are large and noisy. Troyanskaya's approach is to develop new ways for extracting useful information from these datasets using techniques from computer science such as machine learning and data mining.\u201cComputation by itself is often not enough to discover new biology but it can direct experimental work,\u201d she says. And she has set up a wet lab to help test and validate the hypotheses that the computer science helps generate. In 2009, for example, she used this approach to identify 109 new proteins involved in mitochondrial biogenesis in yeast.This combined approach is one of the things that sets Troyanskaya apart, says Valencia. \u201cShe is one of the first to have come from the computational side and then moved into the experimental area to combine both,\u201d he says.Understanding the function of individual genes is only a small part of a much bigger story. Many genes and proteins play multiple roles within a cell as parts of various networks of biological processes. Mapping out these networks and understanding how they work and interact with each other is yet another strand of her research. \u201cShe has made important contributions to systems biology,\u201d says Valencia.The process of evaluating and validating computational predictions is an area requiring a broad collaboration to develop standards and methods that can be used to achieve a consensus about the results. To this end, Troyanskaya is collaborating with the curators of model organism databases and members of the Gene Ontology Consortium.Another problem that many researchers face is handling the data avalanches currently being generated. So Troyanskaya, in collaboration with Princeton colleagues Kai Li and Moses Charikar, is looking at ways to better search and visualise these huge datasets, something that is challenging because of high noise levels and the enormous volume of the data. \u201cWe are developing better ways to do this,\u201d she says.PLoS Computational Biology and Bioinformatics. And she is involved in conferences: organizing, chairing tracks and program committees. \u201cThat is something that is very much appreciated,\u201d says Valencia. \u201cWe are lucky to have her.\u201dThe awards committee was also impressed by Troyanskaya's service for the community. She is involved in the Society's two official journals, And there is surely more to come. Troyanskaya points to numerous questions that are driving her research forward. She wants to know, for example, how we can predict which genes are involved in kidney disease, to understand their function and their clinical role on a molecular level. She works on these questions in close collaboration with experimental researchers, such as Matthias Kretzler and his group from the University of Michigan, Ann Arbor. And she is passionate about finding ways to ask questions in a data-driven way, not just in a knowledge-driven way that relies on what we already know about biology. \u201cThese are the questions that I'm really interested in,\u201d she says. \u201cAnd we really haven't yet harnessed the full potential of our data collections.\u201dhttp://www.iscb.org/ismbeccb2011. The conference will also feature parallel tracks for Proceedings of original research papers, Highlights of recently published papers, Special Sessions on emerging topics, Late Breaking Research of peer-reviewed abstract submissions, and Technology demonstrations and workshops presented by academic researchers and commercial vendors. The conference also displays a unique \u201cArt and Science\u201d exhibit of scientifically based artistic visual images and videos submitted, and offers a commercial and non-profit vendor exhibition.The full conference agenda and registration information for ISMB/ECCB 2011, where these ISCB award winners, along with four other distinguished Keynote lecturers, can be found on the conference Web site at http://www.iscb.org/iscb-awards.For a review of past ISCB award winners, please see"}
{"text": "We evaluated the potentiality of plasma microRNAs  that were considered as novel biomarkers for acute coronary syndrome (ACS), including acute myocardial infarction (AMI) and unstable angina (UA).We initially identified plasma miR-122, -140-3p, -144, -720, -1225-3p, -2861, and -3149 as candidate miRNAs associated with AMI (\u22652 fold and P < 0.05) by comparing expression differences of miRNAs among AMI, non-coronary heart disease (non-CHD) and stable angina (SA) groups, using miRNA microarrays (n = 8 independent arrays in each group). Those seven plasma miRNAs were further examined with qRT-PCR analyses in two replications including 111 and 428 patients separately, and the results demonstrated that plasma miR-122, -140-3p, -720, -2861, and -3149 were elevated in the ACS group vs. the non-ACS (non-CHD + SA) group (P < 0.01). The area under the receiver operating characteristic curve (AUC) of the five miRNAs for ACS classification was 0.838, 0.818, 0.865, 0.852, and 0.670, respectively , while the values reached 0.843 and 0.925 when simultaneously with miR-122 and -3149 or with miR-122, -2861, and -3149 together . In plasma of pigs after coronary ligation, miR-122 was increased from 180 min to 240 min and miR-3149 was augmented from 30 min to 240 min compared with the sham pigs .Plasma miR-122, -140-3p, -720, -2861, and -3149 were associated with and potentially novel biomarkers for ACS. Acute coronary syndrome (ACS), including acute myocardial infarction (AMI) and unstable angina (UA), is one of the most serious cardiovascular diseases and a major cause of mortality and morbidity worldwide. Timely revascularization therapy within 3 h after the onset of symptoms is recommended by guidelines to rescue the ischemic myocardium and, thereafter, could eventually reduce the mortality . TherefoRecently, microRNAs  have been demonstrated to be involved in the mechanism of ACS, including rupture of the atherosclerotic plaque , 5, platFor detailed methods, see Blood samples were obtained in the cardiac catheterization laboratory from 16 healthy individuals , 72 high-risk individuals (Highrisk), 81 stable angina (SA), 287 UA, and 115 AMI (including STEMI and NSTEMI) patients.The protocol of this study was approved by the Ethics Committee of Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, China. Written informed consent was obtained from each patient before enrolment.Thirty six Bama male minipigs (10 months old) were fed a high-cholesterol diet for 20 weeks. Then pigs were randomly assigned to Sham (n = 6) and AMI (n = 30) groups, and the AMI group was further divided into AMI with ventricular fibrillation  and AMI without VF (AMI-NVF) groups after the end of the operation. All pigs were anesthetized with a mixture of ketamine hydrochloride 700 mg and diazepam 30 mg intramuscularly and were continuously infused with the mixture (2 mg/kg per hour) intravenously. At the end of the experiment, the deeply anesthetized pig was killed by an injection of 15% KCl (1 ml/kg). Blood samples were collected before the operation and after 30, 60, 90, 120, 180, and 240 min of coronary ligation.All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, USA. The animal experimental protocols and procedures were approved by the Care of Experimental Animals Committee of Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, China.Peripheral blood samples (5 mL) were collected in the cardiac catheterization laboratory, and were separated into plasma and cellular fractions within 2 h of collection.Total RNA was isolated from plasma with TRIzol LS Reagent  according to the manufacturer\u2019s protocol.http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE66752). The candidate miRNAs that differentially expressed in AMI patients were further validated by miRNA stem loop quantitative real-time polymerase chain reaction (qRT-PCR) technology.miRNAs profile in human plasma of AMI, Health, Highrisk and SA groups was determined by the human miRNA array (Agilent miRNAs microarray Version 16.0). The microarray dataset is publicly available at GEO database .-\u0394\u0394ct method. All values of miRNAs are expressed as mean \u00b1 SE. Comparisons of parameters among \u2265 3 groups were analyzed by oneway ANOVA, followed by post hoc testing with Bonferroni correction. Independent-sample T test was used for 2 group comparisons. All P values were two-sided and differences were considered statistically significant at a value of P < 0.05. The receiver operating characteristic (ROC) curves were established for classification of ACS and non-ACS patients. All statistical calculations were performed by the SPSS 20.0.Patients\u2019 baseline characteristics of different groups were compared by One-way ANOVA and Fisher\u2019s exact test. For microarray analysis, the Mann-Whitney unpaired test was used for the three pairwise comparisons . For the data obtained by qRT-PCR, a widely used method to present relative expression of miRNA is the 2Expression profiles of 1205 human miRNAs were initially screened by microarray between the Health, Highrisk, SA, and AMI patients (n = 8 independent arrays in each group). No significant differences of clinical characteristics were found among the four groups .Quantification revealed that plasma miR-122, -140-3p, -144, -720, -1225-3p, -2861, and -3149 were up-regulated \u22652 fold in the AMI patients compared with the Health, Highrisk, and SA patients . These 7 miRNAs were selected as candidate miRNAs for further testing via qRT-PCR in human samples . BecauseThe seven candidate miRNAs were first tested with qRT-PCR in 111 human plasma samples, including 21 non-CHD, 30 SA, 30 UA, and 30 AMI patients, and no significant differences of clinical characteristics were found among the four groups . The resThe expression profiles of miR-122, -140-3p, -720, -2861, and -3149 were further examined with qRT-PCR in 428 additional subjects\u2019 plasma samples, including 51 non-CHD, 43 SA, 257 UA, and 77 AMI patients, and no significant differences of clinical characteristics were found among the four groups . The resA total of 539 human plasma samples from the two replications, including 394 ACS patients and 145 non-ACS patients, were used as the resource of training data set for the construction of the miRNA panel for the classification of ACS and non-ACS patients. The expression of miR-122, -140-3p, -720, -2861, and -3149 was increased respectively by 20.7, 14.7, 49.3, 27.6, and 12.5 fold in the ACS group compared with the non-ACS group  . The accAnimal experiments were conducted to assess whether the effects of ACS on miRNAs plasma levels in humans were observed in a swine model of AMI. Evans blue and triphenyltetrazolium chloride (TTC) staining indicated the success of AMI model , Panel AIschemic heart disease accounts for approximately 30% of all deaths, causing 1.4 million deaths in the developed world and 5.7 million deaths in developing regions . While aIn this study, we reported that the levels of circulating miR-122, -140-3p, -720, -2861, and -3149 were significantly up-regulated in human ACS, in comparison with the non-ACS patients. The ability of the five miRNAs to distinguish the ACS patients from the non-ACS patients was shown by the ROC curves with the AUC of 0.838, 0.818, 0.865, 0.852, and 0.670, respectively , and themiR-122, regarded as liver-specific , plays cmiR-140-3p has been reported to be increased in CHD . miR-720In addition, our animal study found that the plasma levels of miR-122 and -3149 were not only higher in the AMI pigs in comparison with the sham pigs, but also higher in the AMI-VF pigs versus the AMI-NVF pigs. The upregulated miR-21 has been reported to promote atrial fibrillation (AF) in MI-induced heart failure in rats . miR-29 It should be noted that the levels of cTnI were not significantly different within 240 min after coronary ligation between the sham and AMI pigs with the success of AMI model documented clearly by the triphenyltetrazolium chloride (TTC) staining. Previous studies have demonstrated that the cTnI levels in blood of AMI patients began to rise after 4\u20138 h of myocardial injury . But in The potential limitations should be addressed. First, the sample size of current study is relatively small. Second, the generalizability of our findings is limited by the demographic differences and treatment characteristics of ACS patients. In addition, it would be ideal to have information on the time course for corresponding miRNA quantification in AMI patients. Therefore, further studies with larger sample size and patients with various CHD subtypes are needed to validate the diagnostic value of plasma miR-122 and -3149 for ACS.In conclusion, plasma miR-122 and miR-3149 are associated with ACS and might be potentially novel biomarkers for accelerating the diagnosis of ACS patients in emergency department.S1 Appendix(DOCX)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file."}
{"text": "Topological states of matter support quantised nondissipative responses and exotic quantum particles that cannot be accessed in common materials. The exceptional properties and application potential of topological materials have triggered a large-scale search for new realisations. Breaking away from the popular trend focusing almost exclusively on crystalline symmetries, we introduce the Shiba glass as a platform for amorphous topological quantum matter. This system consists of an ensemble of randomly distributed magnetic atoms on a superconducting surface. We show that subgap Yu\u2013Shiba\u2013Rusinov states on the magnetic moments form a topological superconducting phase at critical density despite a complete absence of spatial order. Experimental signatures of the amorphous topological state can be obtained by scanning tunnelling microscopy measurements probing the topological edge mode. Our discovery demonstrates the physical feasibility of amorphous topological quantum matter, presenting a concrete route to fabricating new topological systems from nontopological materials with random dopants. Apart from the intensive efforts to explore topological properties in crystalline materials, the study of such properties in amorphous materials has been rare. Here, P\u00f6yh\u00f6nen et al. predict a topological superconducting phase in an ensemble of randomly distributed magnetic atoms on a superconducting surface. While topological properties can be studied independently of local order, spatial symmetries play a central role in virtually all material realisations. This is emphasised by the fact that the theoretical search for new topological materials extensively employs band structures and reciprocal space. While topological states are generically robust to disorder which breaks spatial symmetries, this is typically established by treating the disorder as an additional feature in a well-defined clean system. Even topological Anderson insulators4, where moderate disorder actually gives rise to nontrivial topological properties, rely crucially on a specific band structure of the clean system. The concept of disorder, almost by definition, implies the existence of an underlying ordered reference state.Topological states are characterised by integer-valued invariants7. The wires have the advantage of potentially allowing topological computation, but to carry out this function they must be almost defect free, which illustrates a generic complication in top-down fabrication strategies. On the other hand, fabrication of topological matter with randomly distributed constituents, if possible, would avoid that obstacle and offer new opportunities. A recent discovery of a mechanical gyroscopic metamaterial8, albeit a purely classical system, suggests that also amorphous topological quantum matter could be achievable. By studying the properties of long-range hopping toy models, Agarwala and Shenoy9 pointed out the mathematical possibility of topological states with randomly localised states.The role of spatial symmetries in topological materials raises the question of how much spatial order is necessary for topological states to persist. In addition to the fundamental interest, possible realisations have far-reaching practical implications. The search for topological states has already moved beyond the elements found in nature to artificial man-made structures such as Majorana wiresIn this work, we propose the Shiba glass, depicted in Fig.\u00a018 with supporting experimental evidence21. More recently, ferromagnetic 2D lattices have emerged as a promising platform for chiral superconductivity23 with a rich topological phase diagram25. Classical magnetic moments embedded in a gapped s-wave superconductor give rise to Yu\u2013Shiba\u2013Rusinov (YSR) subgap states26, localised subgap states which decay algebraically for distances smaller than the superconducting coherence length. In 2D superconductors, such as layered systems, thin films and surfaces, the decay of the wavefunctions from the deep-lying impurity has a functional form \u03be and kF are the superconducting coherence length and the Fermi wave vector of the underlying bulk. The Shiba glass results from a hybridisation of randomly distributed YSR states. To model the system, we consider deep-lying YSR states with energies \u03b50 located in the vicinity of the gap centre J is the magnetic coupling, S is the magnitude of the magnetic moment and \u03b50\u2009\u2248\u2009\u0394(1\u2009\u2212\u2009\u03b1). As outlined in the Methods section, the low-energy properties of the coupled impurity moments are modelled by a tight-binding Bogoliubov-de Gennes Hamiltonian24rn. The entries hmn, \u0394mn for arbitrary configuration of magnetic moments is lengthy and given in Supplementary Note\u00a0The studied amorphous topological superconductor is comprised of randomly distributed magnetic moments on a superconducting surface with a Rashba spin-orbit coupling. The moments can arise from magnetic atoms, molecules or nanoparticles. Regular 1D structures of this type have been predicted to host Majorana statesxmn and ymn are components of rm\u2009\u2212\u2009rn\u2009\u2261\u2009. The hopping elements are expressed in terms of the functionsJn and Hn are Bessel and Struve functions of order n. The Rashba spin-orbit coupling induces two helical Fermi surfaces with density of states \u03bb\u2009=\u2009\u03b1R/(\u0127vF) is the dimensionless Rashba coupling and kF,vF the Fermi wavenumber and velocity in the absence of spin-orbit coupling. The Rashba coupling also slightly modifies the superconducting coherence length ij vanishes with vanishing Rashba coupling \u03b1R\u2009=\u20090. The low-energy Hamiltonian  oscillates as a function of the position as cos(2kFr). While for distances \u03c0/4\u2009<\u2009kFr\u2009<\u20093\u03c0/4 the interaction is effectively ferromagnetic. Therefore, in the large part of the topological region BSZ would drive the system towards an out-of-plane polarisation.In our work, we have not addressed the question of the magnetic ordering, but rather show that a finite polarisation perpendicular to the plane gives rise to a topological phase. The nature of the ordering would likely depend sensitively on the specific physical realisation; however, there exist a number of mechanisms driving the system to a polarised state. For example, ignoring the modifications arising from superconductivity at large distancesd semiconductors with Rashba spin-orbit coupling are promising candidate systems. Another candidate system is the layered superconductor NbSe2 where 2d YSR states29 and their coupling have been observed30 recently. The most direct experimental probe is provided by STM measurement of the LDOS. As shown above, in the topological phase the Shiba glass system exhibits a significant concentration of the subgap LDOS at the sample boundaries, which can be directly observed by STM. This signal is clearly detectable at temperatures below the mobility gap scale which can be of the order of kBT\u2009=\u20090.1\u0394\u2009\u2212\u20090.3\u0394 as shown in Fig.\u00a0The studied Shiba glass system could be realised by decorating an effective 2D or a layered 3D superconductor with magnetic atoms or molecules. Considering the requirement In summary, we introduced the Shiba glass as a platform for amorphous topological superconductivity and elucidated the general properties of such systems. Our results illustrate the physical feasibility of amorphous topological quantum matterials and provide a concrete prescription to experimentally realise and observe them. Our discovery motivates expanding the search for topological materials beyond crystalline systems and paves the way for fabricating topological matter from nontopological materials with random dopants.k-space, but there are various methods of computing it in real space as well32. A comparison shows that these methods are generally of similar computational efficiency and yield the same values for the topological invariant.To find the topological phase diagram, we need to evaluate topological invariants in real space. The relevant topological index for 2D systems with broken time-reversal symmetry is the Chern number. This is generally obtained in 32 proceeds by defining the coupling matrices C\u03b1,\u03b1 + 1, with elementsR is the position operator, q\u03b1\u2009=\u2009\u03c0 for \u03b1\u2009=\u20090, \u2026, 3, and where \u03c8m are the eigenfunctions of the system with periodic boundary conditions. By use of these matrices, the Chern number is then obtained through the equation\u03bbm being the complex eigenvalues of the matrix C01C12C23C30.The real-space Chern number method of ref. Data sharing not applicable to this article as no datasets were generated or analysed during the current study.Supplementary InformationPeer Review File"}
{"text": "Diabetic nephropathy (DN) is the most serious microvascular complication of diabetes and the largest single cause of end-stage renal disease (ESRD) in many developed countries. DN is also associated with an increased cardiovascular mortality. It occurs as a result of interaction between both genetic and environmental factors. Hyperglycemia, hypertension, and genetic predisposition are the major risk factors. However, the exact mechanisms of DN are unclear. Despite the benefits derived from strict control of glucose and blood pressure, as well as inhibition of renin-angiotensin-aldosterone system, many patients continue to enter into ESRD. Thus, there is urgent need for improving mechanistic understanding of DN and then developing new and effective therapeutic approaches to delay the progression of DN. This review focuses on recent progress and future perspective about mechanistic insight and management of DN. Some preclinical relevant studies are highlighted and new perspectives of traditional Chinese medicine (TCM) for delaying DN progression are discussed in detail. These findings strengthen the therapeutic rationale for TCM in the treatment of DN and also provide new insights into the development of novel drugs for the prevention of DN. However, feasibility and safety of these therapeutic approaches and the clinical applicability of TCM in human DN need to be further investigated. Diabetic nephropathy (DN) is a major complication of diabetes and the largest single cause of end-stage renal disease (ESRD) in many developed countries. DN is also associated with an increased cardiovascular mortality. Immunologic derangement is the cornerstone of pathogenesis of CKD . Of courDN is one of the most frequent and severe complications of diabetes mellitus and a worldwide public health problem affecting millions of people. Currently, based on the criteria of the kidney disease, Improving Global Outcomes (KDIGO) Clinical Practice Guideline, DN was diagnosed by renal biopsy or medical history . DN is tThe prevalence of DN has been dramatically increased in recent decades; especially in China, the adjusted prevalence of diabetes mellitus and prediabetes among Chinese adults is 9.7% and 15.5%, respectively , 19. In DN occurs as a result of interaction between both genetic and environmental factors. Hyperglycemia, hypertension, and genetic predisposition are the major risk factors. The aggravating hyperglycemia is also a high risk factor for the development of microvascular complications . In othePodocyte damage occurs at a relatively early stage in CKD . PodocytThe management of diabetes mellitus hinges on \u201cfive carriages,\u201d which are health education, diet, exercise, weight control, and drug treatment, respectively, of which the former four therapies are described as nonpharmacological measures. And the pharmacological agents currently approved for treatment of diabetes mellitus include sulfonylureas, metformin, acarbose, and insulin . Nonphar\u03b2-cells function of pancreatic island [\u03b2-cells of pancreatic island [There is no doubt that glycemic control plays the most momentous role in management of DN; keeping blood glucose within normal limits is a foundation and prerequisite for the treatment of DN . Severalc island , 38. Henc island . Moreovec island . Sulfonyc island . In addic island \u201344. TakeProteinuria is considered as a hallmark and sensitive marker of DN, which is mainly caused by the severe podocyte injury . But whaAt the same time, many patients of DN still show merging symptoms, such as dyslipidemia and hypertension. Poorly controlled blood glucose levels in patients with DN are associated with dyslipidemia which constitutes an additional risk factor. Dyslipidemia consists of elevated levels of low density lipoprotein cholesterol and low levels of high-density lipoprotein cholesterol and increased levels of apolipoproteins B . In viewCutting-edge evidence shows that there are certain trends of the future therapies for DN, including comprehensive exploration of existing medicines, application of molecular biology discoveries, progress in stem cell therapy, and the applicability of traditional Chinese medicine (TCM) in the treatment of DN.As mentioned previously, The RAAS pathway is very important in the progression of DN. Several studies indicate that the combination of spironolactone with an ACEI or ARB could significantly mitigate proteinuria compared with the placebo , 54. Fur\u03b2-cell receptors, stimulate insulin release, and improve glycemic control and then play a protecting role in management of DN [\u03baB is a ubiquitous nuclear transcription factor that regulates expression of a large number of genes that are critical for the regulation of inflammation [Studies performed have discovered several key signaling pathways in succession, such as endoplasmic reticulum (ER) stress, which already have become a therapeutic target of much concern for DN . Besidesnt of DN . Moleculammation . The conammation .Stem cells that are identified nearly half a century ago exhibited great potential for the repair of damaged tissues and organs and provided new hope for a means to change the track of DN . Multipl\u03b13\u03b21 integrin upregulation and astragaloside IV could inhibit podocyte apoptosis by downregulation of PERK-ATF4-CHOP pathway [TCM has long histories in China and has emerged and influenced hundreds of thousands of people. Historically, conventional medicines, such as ACEI or ARB, are not able to totally prevent the development of DN . Thus, t pathway , 72. Fur pathway . Yiqi Ya pathway . These fIt is a long time that the relationship between hypertension and DN has been the subject of controversy, and after diabetes mellitus, hypertension is the second most commonly reported etiology of ESRD in the United States Renal Data System, and hypertension treatment targets in patients with DN remain important clinical concerns . Recent Despite the benefits derived from strict control of glucose and blood pressure, many patients continue to enter into ESRD. Thus, there is urgent need for improving mechanistic understanding of DN and then developing new and effective therapeutic approaches to delay the progression of DN. Many studies strengthen the therapeutic rationale for TCM in the treatment of DN. However, feasibility and safety of these therapeutic approaches and the clinical applicability of TCM in human DN need to be further investigated."}
{"text": "We propose a novel mechanism of enhancement of turbulence by energetic-particle-driven geodesic acoustic modes (EGAMs). The dynamics of drift-wave-type turbulence in the phase space is investigated by wave-kinetic equation. Spatially inhomogeneous turbulence in the presence of a transport barrier is considered. We discovered that trapping of turbulence clumps by the EGAMs is the key parameter that determines either suppress or enhance turbulence. In regions where turbulence is unstable, EGAM suppresses the turbulence. In contrast, in the stable region, EGAM traps clumps of turbulence and carries them across the transport barrier, so that the turbulence can be enhanced. The turbulence trapped by EGAMs can propagate independent of the gradients of density and temperature, which leads to non-Fickian transport. Hence, there appear a new global characteristic velocity, the phase velocity of GAMs, for turbulence dynamics, in addition to the local group velocity and that of the turbulence spreading. With these effect, EGAMs can deteriorate transport barriers and affect turbulence substantially. This manuscript provides a basis to consider whether a coherent wave breaks or strengthen transport barriers. The coexistence of global-coherent Alfven wave and micro-turbulence has been observed at the surface of the sun, which could link with the coronal heating problem3. Turbulence in planetary atmospheres generates zonal flows2, such as jet stream on the earth, stripes of Jupiter, super rotation on Venus, and tachocline on the sun4. In magnetically confined plasmas, geodesic acoustic modes (GAMs), which are oscillatory zonal flows, have attracted much attention as coherent waves, because GAMs are expected to suppress turbulence by their velocity shear1. Actually, the suppression of turbulence and transport have been reported in turbulence simulations5. Experimental study has shown that the transition to high confinement state is accompanied by GAMs6. However, recently, the enhancement of turbulence by GAMs has been observed in first principle simulations, with the subsequent destruction of a transport barrier9. In this way, GAMs can either mitigate or enhance the turbulence. This dual effect of the GAMs on turbulence requires theoretical investigation.Problems including interactions between micro-turbulence and coherent waves are ubiquitous in a variety of systems10 but also by energetic particles (EPs), which are called EGAM14. Turbulence driven GAMs suppress turbulence, which can be understood in terms of energy conservation . In contrast, the impacts of EGAMs on turbulence is not clear, in particular because EGAMs obtain their energy from EPs, not from turbulence. Actually, EGAMs have been reported to enhance turbulence in spite of the fact that EGAMs have velocity shears8. In order to clarify the impact of finite-frequency zonal flows on turbulence, we focus on EGAMs. The phase-space dynamics results in trapping of turbulence wave-packets by EGAMs15. We found that the trapped turbulence wave-packets leak across the transport barrier. As a result, turbulence is enhanced by EGAMs in the stable region, while turbulence suppression is obtained in the unstable region. We discovered that trapping of turbulence clumps by the EGAMs is the key parameter that determines either suppress or enhance turbulence. The propagation of the turbulence is ballistic, with the phase velocity of the EGAM. Thus, the turbulence propagation is in some sense independent from the background profiles such as the gradients of density and temperature. Thus, turbulence carried by the EGAMs shows non-Fickian transport properties. The propagation of trapped turbulence is different from processes such as turbulence spreading18, avalanches21 and others23. Hence, there appear a new global characteristic velocity, the phase velocity of GAMs, for turbulence dynamics, in addition to the local group velocity and that of the turbulence spreading.We investigate the phase-space dynamics of spatially inhomogeneous turbulence in the presence of GAMs. GAMs are driven not only by turbulencex-direction. We consider a situation where the turbulence unstable region faces the stable region, and a transport barrier is localized at the boundary, as shown in Fig.\u00a0qy\u2009\u2248\u2009011 and another has a steep poloidal structure24. In this study, we focus on the branch with qy\u2009\u2248\u20090, which is unaffected by the doppler-shift due to the mean sheared flow. This branch is driven by the resonance with the toroidally passing EPs, where we neglect the effect of the magnetically trapped EPs. In such a situation, the turbulence is governed by the wave-kinetic equation25,Nk and \u03c9k are the normalized action and frequency of the turbulence, respectively25. The linear growth rate and the nonlinear decorrelation rate of turbulence are denoted by \u03b3L, and \u0394\u03c9, respectively. Here time and space are normalized by \u03c1s, where Vd is the diamagnetic drift velocity, and \u03c1s is the ion gyro-radius measured by the sound velocity. It is noted that the typical frequency of the turbulence satisfies the relation, \u03c9EP is the transit frequency of EPs. Thus, we assume that the resonance of the EPs with the turbulence is weaker than that with EGAMs. We treat \u03b3L as an independent parameter on EPs. We consider drift-wave-type turbulence, so Nk is given as \u03c9k iskx, ky are the turbulence wavenumbers. The turbulence frequency \u03c9k includes the doppler shift due to We consider the dynamics of spatially inhomogeneous turbulence with a transport barrier in the presence of EGAMs. We focus on the impact of EGAM on turbulence. As the first step, in order to simplify the problem, we neglect the direct effect of EPs on turbulence, as shown in Fig.\u00a026. Here, we compare the assumptions and results between these previous studies26 and the present work. In these studies, an analytic solution for a nonlinear wave pattern for zonal flows was obtained by considering the interaction between turbulence driven zonal flows and turbulence. The following assumptions were used; spatially homogeneous turbulence with a marginal stability condition was assumed  are shown in Fig.\u00a0VG is the amplitude of the E\u2009\u00d7\u2009B velocity of the EGAM. The kx-spectrum of the turbulence in the unstable region x\u2009<\u200920 has positive-negative symmetry with respect to kx. In the stable region, the kx-spectrum becomes asymmetric, and the turbulence exists only for kx\u2009<\u20090, where the group velocity of the turbulence is positive. This outflow of the turbulence into the stable region can be understood by considering the motion of the quasi-particles (QPs). The equation of motion for the QPs is governed by Eq. , turbulence trapped by the EGAM forms islands in the phase space. As seen from the time evolution of the turbulence intensity in Fig.\u00a0\u03c9k becomes an adiabatic constant of motion. From Eq.  in space, with the typical scales, VG varies. The island in the case of n is an integer, and kx-spectra of ensemble averaged Nk at X-point, VG, the intensity of the turbulence increases inside the island We describe two mechanisms by which the turbulence propagates in the stable region; one occurs without the EGAM (transit loss), and another is due to the turbulence trapping by the EGAM (trapped loss). We investigate conditions for trapped loss to be dominant. Figure\u00a0For our parameters, this condition is When both conditions, Eqs  and 10)10), are x\u2009=\u20090 (the unstable region) on the EGAM amplitude. The intensity N is calculated from the ensemble average, and the error bar is estimated from the standard deviation. The intensity N, which is calculated from the whole region in kx-space, decreases with VG, whose amplitude dependence is fitted to x\u2009=\u200927, is shown in Fig.\u00a0VG, which clearly shows that the EGAM can enhance turbulence. The total N in the stable region scales as \u03b1 is a constant parameter, VG and the spectral width increases with We study the dependence of the turbulence intensity 27. In the case of the gyrokinetic simulations by Zarzoso and others8, the shear layer is much narrower than the EGAM wavelength, The present mechanism of turbulence enhancement is discussed below in the contexts of experiments, and first-principle simulations. In the LHD, large amplitude EGAMs are observed with amplitude 19. The turbulence intensity is determined by that of the trapped turbulence Itrap, while the theoretical expression of the turbulence intensity of the avalanche has not been not reported. The radial flux of the turbulence clump by the trapped turbulence by the EGAM is qx is small, and/or the amplitude is large. The propagation distance of the trapped turbulence is Finally, the propagation property of the trapped turbulence is compared with the other processes. The propagation characteristics are summarized in Table\u00a0In conclusion, two novel effects of the EGAM on turbulence are proposed by studying the phase-space dynamics of turbulence. Spatially inhomogeneous turbulence in the presence of transport barrier is considered. In the turbulence unstable region, the shear of the EGAM suppresses the turbulence. On the other hand, the turbulence trapped by EGAM leaks into the stable region across the transport barrier. Hence, turbulence is enhanced by the EGAM. The condition for the wave-trapping effect to be dominant is obtained, which is consistent with the simulations. The trapped turbulence by the EGAM can propagate independently from the gradients of density and temperature, which leads to the non-Fickian transport. Hence, there appear a new global characteristic velocity for turbulence dynamics, in addition to the local group velocity and that of the turbulence spreading. This manuscript provides a basis to consider whether a coherent wave breaks or strengthen transport barriers.ky-spectrum is assumed for simplicity26, and the time evolution of Nk is calculated in the phase space of A monochromatic VG is a given parameter, since the EGAM survives regardless of the presence of turbulence. We implicitly assume that the scale length of the EP-density is much longer than the wavelength of the EGAM. The calculation region is Nk with respect to x and kx are set to be zero at the boundaries. The simulations are performed with the set of the parameters;Nk numerically.Here,"}
{"text": "V2, an engineered voltage-gated Na+ channel harboring a selenocysteine in its inactivation motif, as a non-photonic, sensitive, gateable, and reversible sensor for membrane-delimited reactive species. roNaV2 allows for the assessment of chemical modification induced in fluorescence microscopy settings with high sensitivity and time resolution and it demonstrates the usefulness of ion channels as highly sensitive reporters of membrane processes.Photonic experiments are of key importance in life sciences but light-induced side effects are serious confounding factors. Here we introduce roNa Therefore, there is a clear demand for sensitive probes to precisely monitor cellular RS with spatio-temporal resolution to understand the multifaceted role of cellular RS in cell physiology. Recently, mutants and fusions of genetically encoded GFP (green fluorescent protein), for example, reduction and oxidation sensitive variants roGFP2These fluorescent reporters offer several useful features, such as the choice of localized sub-cellular targeting, real-time RS detection, and ratiometric observation that overcome artifacts arising due to photo-bleaching and inhomogeneous distribution of the sensors. However, there is the inherent problem of phototoxicity that can lead to lasting irreversible destruction of cellular structures and may even result in immediate alteration of molecular function. Despite the obvious detrimental consequences of visible light in cell physiological studies, the issue of phototoxicity has been only infrequently considered in life science research891112+ (NaV) channel with a cysteine residue in its inactivation domain  as a non-photonic RS sensor delimited to the cell membraneV channels activate (open) and inactivate (close) rapidly in response to membrane depolarization, thus yielding transient Na+ inward current. Rapid and essentially complete inactivation of the NaV channel, which is crucial for maintaining cellular excitability, is mediated by the inactivation motif \u201cIFM\u201d (Ile-Phe-Met), located in the intracellular linker between domains III and IV of the NaV proteinV1 exhibits an RS sensitivity comparable to roGFP2, including a relatively slow response to changes in the cellular redox milieuPreviously, we have reported a genetically engineered voltage-gated NaV1 response is limited by the kinetics by which the cysteine residue harbored in the inactivation motif of the channel is modified. We therefore thought to create a second-generation genetically engineered NaV channel containing selenocysteine (Sec or U) in its inactivation motif, also referred as roNaV2. As outlined below, it may serve as a non-photonic tool for monitoring RS delimited to the cell membrane \u2013 in particular those generated by light exposure \u2013 in a switchable and ratiometric manner.The speed of the roNaV1, a voltage-gated NaV channel with a cysteine residue in its inactivation domain, responds to local RS by diminished fast inactivation  at position 1305. Selenocysteine, the 21st natural amino acid, differs from cysteine by the single substitution of selenium for sulfur. Selenium is a better nucleophile than sulfur and, under physiological conditions, selenocysteine residues are ionized whereas cysteine residues are typically protonated16V1.4 channels with a TGA stop codon at the position equivalent to M1305, and followed by a selenocysteine insertion sequence (SECIS) element downstream of the channel gene  in HEK 293 cells, currents were measured in the whole-cell mode of the patch-clamp technique. The peak amplitude at \u221220\u2009mV for roNaV2 was much smaller  than for roNaV1  and wild-type rat NaV1.4 . Supplementation of the cell culture medium with sodium selenate , however, increased the functional expression of roNaV2 about 8-fold , while the mean current amplitudes of roNaV1  and NaV1.4  were unaffected  or high concentrations (25\u2009mM) of glucose as to affect endogenous RS production. The inactivation time course of roNaV2 was noticeably slower in the cells cultured in high glucose, while no difference was detected for roNaV1 (I) was about 2.5-fold larger in high glucose compared to low glucose. Furthermore, treatment of the cells with the mitochondrial uncoupler BAM15 (5\u2009\u03bcM), which does not depolarize the plasma membraneV1  was described with single-exponential functions. While the oxidant chloramine T  had a negligible effect on roNaV1 within 10\u2009min, roNaV2 responded rapidly and robustly ; roGFP2 was intermediate (218\u2009\u00b1\u20092\u2009s). Even 1\u2009\u03bcM H2O2 application could be readily detected, whereas the response of roGFP2 was about 100-fold slower  of a single amino-acid residue, either Cys (roNaV1) or Sec (roNaV2), located in the inactivation motif of the channel, i.e. a site right at the interface of membrane protein and cytosol. In particular roNaV2 may thus serve as a physiologically important and convenient tool for investigating real-time oxidative cellular events as exemplified for the response to mitochondria uncoupling  resulted in progressive loss of inactivation, characterized by a mean time constant of 0.377\u2009\u00b1\u20090.042\u2009s (n\u2009=\u200927), compared to 549\u2009\u00b1\u2009112\u2009s for roNaV1 and 5010\u2009\u00b1\u2009610\u2009s for the wild-type NaV1.4 channel  and GFP mutant S147C (i.e. lacking the partner for forming a disulfide bridge with C204 as present in roGFP2) was even slower . Even insertion of a selenocysteine at position 147 (eGFP:147U) showed a very weak and slow response , and in all three cases no reversibility was obtained. Only eGFP mutant 147U-204C showed a fast reaction on blue-light illumination , which was fully reversible  and hence comes close to the performance of roNaV2  by the membrane voltage allowing for complex experimental settings in which membrane-delimited occurrence of reactive species has to be monitored. Complementing other methods of RS determination in living cellsV2 adds a non-photonic variant with strict membrane delimitation and demonstrates the power of recombinant selenoproteins as sensitive sensors for redox processes.In summary, roNa+ channel construct used in this study was based on rat NaV1.4  downstream sequence and inserted after the original stop codon of the sequence of rNaV1.4, as shown in V1.4 construct.The wild-type Na2 incubator at 37\u2009\u00b0C. Cells were trypsinized, diluted with culture medium, and grown in 35-mm dishes. HEK 293 cells were transfected with the respective plasmids using the Roti\u00ae-Fect transfection reagent  following the instructions of the supplier. Grown cells were supplemented with sodium selenate  for 12\u2009hours from the day of transfection. After 12\u2009hours, transfected cells were maintained in fresh cell culture media without sodium selenate. Electrophysiological experiments were performed 1\u20135 days after plating. Cells not expressing a fluorescence protein were cotransfected with CD8 to identify transfected cells by means of anti-CD8-coated beads .HEK 293 cells  were maintained in Dulbecco\u2019s Modified Eagle\u2019s Medium (DMEM) mixed 1:1 with Ham\u2019s F12 medium and supplemented with 10% fetal calf serum in a 5% COWhole-cell voltage-clamp experiments were performed as described previouslyp/4 method with a leak holding voltage of \u2212120\u2009mV. Currents were low-pass filtered at 5\u2009kHz and sampled at a rate of 25\u2009kHz. Most experiments were performed at constant temperature of 19\u201321\u2009\u00b0C; 32\u2009\u00b0C were chosen for the assessment of H2O2 effects on HEK cells.A patch-clamp amplifier EPC10 was operated by PatchMaster software . Holding potential was \u2212120\u2009mV. Leak and capacitive currents were corrected with a 2, 1 MgCl2, 10 HEPES (pH 7.4 with NaOH).The patch pipettes contained (mM): 35 NaCl, 105 CsF, 10 EGTA, 10 HEPES (pH 7.4 with CsOH). The bath solution contained (mM): 150 NaCl, 2 KCl, 1.5 CaClm\u2009=\u20093 activation gates and a single-channel conductance according to the Goldman-Hodgkin-Katz equation.Channel activation and inactivation was assessed by depolarizing pulses to \u221260 through 60\u2009mV in steps of 10\u2009mV at an interval of 2\u2009s from a holding potential of \u2212120\u2009mV. The peak current-voltage relationships were fit according to a Hodgkin-Huxley formalism with m is the voltage of half-maximal gate activation and km the corresponding slope factor. \u0393 is the maximal conductance of all channels and Erev the reversal potential.VSteady-state inactivation was measured from a holding potential of \u2212120\u2009mV with conditioning pulses of 500\u2009ms at voltages ranging from \u2212120 to \u221245\u2009mV in steps of 5\u2009mV. Subsequently, peak current was determined at \u221220\u2009mV and normalized to a control peak current measured before conditioning. The repetition interval was 10\u2009s. The normalized peak current plotted versus the conditioning voltage was described with a two-component Boltzmann function:h, and the corresponding slope factors, kh, which indicate the voltage dependence of inactivation.with the half-maximal inactivation voltages, V5) or 10\u2009ms (I10) after the depolarization onset and the peak current (Ip) elicited by pulses to \u221220\u2009mV (RI). 5\u2009ms was chosen for cases were a direct comparison with wild-type channels and roNaV1 was intended because the inactivation of those channels is faster that that of roNaV2. The remaining non-inactivating remaining current fraction, RI(t), was plotted as a function of time (t) and fit with the following function:The time course of oxidation-induced loss of inactivation was monitored by measuring the ratio of current at 5\u2009ms . Groups of data were compared with a two-sided Student\u2019s t-test or ANOVA followed by a post hoc Bonferroni correction when appropriate.Data were analyzed with FitMaster (HEKA Elektronik) and IgorPro . If not stated otherwise, averaged data are presented as mean\u2009\u00b1\u2009s.e.m. (2O2) were diluted in the respective bath solution immediately before application. The oxidant was applied extracellularly with a fine-tipped application pipette. In some experiments the reducing agent TCEP (tris(2-carboxyethyl) phosphine hydrochloride) was added to the internal patch pipette solution at 1\u2009mM concentration with readjustment of the pH. BAM15 was from TimTec .Chemicals. Chloramine-T (ChT) and hydrogen peroxide . Excitation light of 400 and 470\u2009nm (width about 15\u2009nm) from a PolyChrome-V light source (TILL Photonics) was applied for 10\u2009ms each. Fluorescence ratio (F400/F470) was formed based on the mean fluorescence values of the trailing 5-ms intervals. Filter set: 492/SP, FT 495, HC 520/35 .RS were generated by epifluorescence excitation light. This was achieved with light from the monochromator set to 470\u2009nm, using a 63x/1.4 oil objective and the GFP filter set as mentioned above.How to cite this article: Ojha, N. K. et al. Non-photonic sensing of membrane-delimited reactive species with a Na+ channel protein containing selenocysteine. Sci. Rep.7, 46003; doi: 10.1038/srep46003 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."}
{"text": "For some time, estrogen has been suspected to play a negative role in anterior cruciate ligament (ACL) fibroblast biosynthesis; however, reports on this issue have been controversial. In a recent study, our group demonstrated a negative combined effect of estrogen and mechanical loading on the gene expression of major extracellular matrix component molecules in ACL fibroblasts."}
{"text": "Fiducial markers are used in correlated light and electron microscopy (CLEM) to enable accurate overlaying of fluorescence and electron microscopy images. Currently used fiducial markers, e.g. dye\u2010labelled nanoparticles and quantum dots, suffer from irreversible quenching of the luminescence after electron beam exposure. This limits their use in CLEM, since samples have to be studied with light microscopy before the sample can be studied with electron microscopy. Robust fiducial markers, i.e. luminescent labels that can  withstand electron bombardment, are interesting because of the recent development of integrated CLEM microscopes. In addition, nonintegrated CLEM setups may benefit from such fiducial markers. Such markers would allow switching back from EM to LM and are not available yet.2O3:Eu3+ particles. Robustness is studied by measuring the luminescence of (single) NPs after various cycles of electron beam exposure. The gold\u2010core rhodamine B\u2010labelled silica NPs and QDs are quenched after a single exposure to 60 ke\u2212\u00a0nm\u20132 with an energy of 120 keV, while Y2O3:Eu3+ NPs are robust and still show luminescence after five doses of 60 ke\u2212 nm\u20132. In addition, the luminescence intensity of Y2O3:Eu3+ NPs is investigated as function of electron dose for various electron fluxes. The luminescence intensity initially drops to a constant value well above the single particle detection limit. The intensity loss does not depend on the electron flux, but on the total electron dose. The results indicate that Y2O3:Eu3+ NPs are promising as robust fiducial marker in CLEM.Here, we investigate the robustness of various luminescent nanoparticles (NPs) that have good contrast in electron microscopy; 130 nm gold\u2010core rhodamine B\u2010labelled silica particles, 15 nm CdSe/CdS/ZnS core\u2013shell\u2013shell quantum dots (QDs) and 230 nm YLuminescent particles are used as fiducial markers in correlative light and electron microscopy (CLEM) to enable accurate overlaying of fluorescence and electron microscopy images. The currently used fiducial markers, e.g. dyes and quantum dots, loose their luminescence after exposure to the electron beam of the electron microscope. This limits their use in CLEM, since samples have to be studied with light microscopy before the sample can be studied with electron microscopy. Robust fiducial markers, i.e. luminescent labels that can withstand electron exposure, are interesting because of recent developments in integrated CLEM microscopes. Also nonintegrated CLEM setups may benefit from such fiducial markers. Such markers would allow for switching back to fluorescence imaging after the recording of electron microscopy imaging and are not available yet.\u2212 nm\u20132 with an energy of 120 keV, while lanthanide\u2010doped inorganic NPs are robust and still show luminescence after five doses of 60 ke\u2212 nm\u20132. In addition, the luminescence intensity of lanthanide\u2010doped inorganic NPs is investigated as function of electron dose for various electron fluxes. The luminescence intensity initially drops to a constant value well above the single particle detection limit. The intensity loss does not depend on the electron flux, but on the total electron dose. The results indicate that lanthanide\u2010doped NPs are promising as robust fiducial marker in CLEM.Here, we investigate the robustness of various luminescent nanoparticles (NPs) that have good contrast in electron microscopy; dye\u2010labelled silica particles, quantum dots and lanthanide\u2010doped inorganic particles. Robustness is studied by measuring the luminescence of (single) NPs after various cycles of electron beam exposure. The dye\u2010labelled silica NPs and QDs are quenched after a single exposure to 60 ke The use of integrated CLEM is relatively new and is mainly used to investigate biological samples  and integrated CLEM systems that allow CL detection . Up to now, the robustness of fiducial markers for combined light and electron microscopy has not been studied in detail.Robust fiducial markers, i.e. luminescent labels that can withstand bombardment with electrons, are interesting, especially in view of recent developments in integrated CLEM systems (Zonnevylle 3+) ions incorporated into an inorganic host are promising as robust fiducial marker. Inorganic NPs have a high stability and the luminescence of lanthanide ions is insensitive to local structural changes as the luminescence involves optical transitions within the 4fn shell. The 4f orbitals are shielded by filled outer 5s and 5p orbitals. As a result, the influence of the environment on the intraconfigurational 4fn transitions is small, which gives Ln3+ ions several advantages as luminescent marker. First, the chemical and temperature stability of Ln3+ luminescence is high  lifetime. As a result, the lanthanide luminescence can be discriminated from autofluorescence by performing time\u2010gated detection  by measuring the luminescence intensity of (single) NPs after exposure to various electron doses. The luminescence of 130 nm gold\u2010core rhodamine B\u2010labelled silica NPs and 15 nm CdSe/CdS/ZnS core\u2013shell\u2013shell QDs is quenched after exposure to a single electron dose of 60 ke\u2212 nm\u20132 with energy of 120 keV. In contrast, the Y2O3:Eu3+ NPs are robust and show luminescence after five electron doses of 60 ke\u2212 nm\u20132. To further investigate the stability after electron beam exposure, the luminescence intensity of Y2O3:Eu3+ NPs is investigated as function of electron dose at various electron fluxes. The luminescence intensity initially drops, but rapidly reaches a constant intensity of \u223c20% of the initial intensity. The luminescence intensity loss is not dependent on the electron flux, but on the total electron dose. The 200 nm diameter Y2O3:Eu3+ NPs retain their bright luminescence after electron doses as high as 66 Me\u2212\u00a0nm\u20132 at a level that allows single NP detection. The results indicate that Y2O3:Eu3+ NPs are suitable as robust fiducial marker, down to the single particle level.In this report, the robustness of lanthanide\u2010doped inorganic NPs, 230 nm Yet\u00a0al., et\u00a0al.  were synthesised according to the Turkevich method  with a diameter of 3 nm were prepared using a method described by Montanarella , et\u00a0al.  and Mont  was diluted with water. Next, the dispersion was sonicated for 10 min and 25 \u03bcL of the dispersion was placed on a Formvar coated copper TEM grid. After evaporation of the solvent, the grid was washed with water. The as\u2010synthesised QD dispersion was diluted 10\u00a0000 times with cyclohexane to a concentration of 0.01 nM and dropcasted on a Formvar coated copper TEM\u2010grid. The Yet\u00a0al. (\u20132 on sample) and an Omicron 100 mW 532 nm diode laser (25 W\u00a0cm\u20132 on sample) as excitation sources. The laser light was guided to the sample through a Chroma ZT532rdc single dichroic and a Nikon CF IC EPI Plan ELWD 0.55 NA, 50x air objective. Various bandpass filters were used to select the emission from the samples: a Chroma ET585/65m filter for the gold\u2010core rhodamine B\u2010labelled silica NPs; a ET645/75m filter for CdSe/CdS/ZnS core\u2013shell\u2013shell quantum dots; and a Semrock FF01\u2010610/5m filter for the Y2O3:Eu3+ NPs. The three\u2010part number in the filter code represents the wavelength in the middle of the band and the last number indicates roughly the band width. The emission was detected with a PCO sCMOS camera PCO 4.2 edge. For typical measurement shown in the figures, 20 frames with a frame time of 3 s were accumulated. The data was analysed using the ThunderStorm Fiji plugin , i.e. 133\u00a0\u00b1 6 nm gold\u2010core rhodamine B\u2010labelled silica NPs, 15\u00a0\u00b1 2 nm CdSe/CdS/ZnS core\u2013shell\u2013shell quantum dots (QDs) and 228\u00a0\u00b1\u00a023 nm Y\u03bbem = 580 nm) of the gold\u2010core rhodamine B\u2010labelled silica NPs shows a band centred at 555 nm corresponding to the singlet\u2013singlet (S0\u2013S1) transition, see Figure\u00a0\u03bbexc = 532 nm) shows a broad band originating from the reverse transition, see Figure\u00a0et\u00a0al., The excitation spectrum (glet S0\u2013S transitiglet S0\u2013S transiti\u03bbexc = 405 nm) is shown in Figure\u00a0et\u00a0al.  of Y2O3:Eu3+ NPs is shown in Figure\u00a06 levels are observed. The excitation lines are assigned to 7F0,1 \u2192 5H3, 5H6 (300\u2013330 nm), 7F0,1 \u2192 5L6, 5D3 (360\u2013420 nm), 7F0,1 \u2192 5D2 (460\u2013480 nm) and 7F0,1 \u2192 5D1 (520\u2013540 nm) transitions. The emission spectrum (\u03bbexc = 260 nm) shows several sharp emission peaks between 580 and 630 nm, see Figure\u00a06 transitions, are assigned to 5D0 \u2192 7F0 (580 nm), 5D0 \u2192 7F1 (585\u2013600 nm) and 5D0 \u2192 7F2 (605\u2013630 nm) transitions. The spectral positions of the luminescence lines of Y2O3:Eu3+ NPs agree well with values reported previously in literature  NPs, the NPs were excited at 532 nm and the emission was collected from 607.5 to 612.5 nm using a narrow band filter. A typical luminescence image of the NPs is shown in Figure\u00a05D0 \u2192 7F2 emission of Eu3+ ions incorporated in the Y2O3 NPs. Next, the sample was exposed to an electron dose of 60 ke\u2212 nm\u20132 and the luminescence image of the NPs was recorded again. Figure\u00a02O3:Eu3+ NPs survives direct electron beam exposure. To further investigate the robustness of the Y2O3:Eu3+ NPs, the NPs were exposed to four additional electron doses of 60 ke\u2212 nm\u20132. In between the electron exposures luminescence images were recorded. Figures 2O3:Eu3+ NPs is still observed after an electron dose of 300 ke\u2212 nm\u20132. The intensity of the NPs after the various electron doses was calculated using the ThunderSTORM plugin in ImageJ software. This software is typically used to determine the position of single luminescent molecules, but it also calculates the intensities and background levels. The results are shown by the blue dots in Figure\u00a02O3:Eu3+ NPs show good robustness compared to the gold\u2010core rhodamine B\u2010labelled silica NPs and QDs. This can be in part related to the size of the Y2O3:Eu3+ NPs, 228 \u00b1 23 nm, which is significant larger than the 133 \u00b1 6 nm gold\u2010core rhodamine B\u2010labelled silica NPs and the 15 \u00b1 2 nm CdSe/CdS/ZnS quantum dots. As a result, the electron beam has to travel a longer distance through the Y2O3:Eu3+ NP to fully penetrate the NP. Monte Carlo simulations (Casino v3.3) were performed to estimate the penetration depths of 120 keV electrons. Simulations obtained considering a 230 nm Y2O3 sphere with density 5.01 g\u00a0cm\u20133 and threshold displacement energy of 50 eV . In addition, the interaction volume and penetration depth of the electron beam is dependent on the material properties. For example, the interaction volume increases for materials with a lower density  than atoms inside the NP , was investigated by recording the luminescence of (single) NPs before and after various cycles of electron bombardments. The luminescence of the rhodamine B dye and QDs was completely quenched after a single exposure to 60 ke\u2212\u00a0nm\u20132 with energy of 120 keV, while the Y2O3:Eu3+ NPs are robust, i.e. luminescence is observed after exposure to 120 keV electrons, even after an electron dose of 66 Me\u2212\u00a0nm\u20132. The intensity of Y2O3:Eu3+ NPs initially decreases, but rapidly reaches a constant value. Approximately 20% of the original intensity is maintained. The intensity loss is independent on the electron flux and determined by the total electron dose. The cause of the drop in intensity is probably the creation of defects in the surface layers of the NP or the interaction with carbon deposits accumulating on the NPs during electron beam exposure.The robustness of various types of nanoparticles (NPs), namely gold\u2010core rhodamine B\u2010labelled silica, CdSe/CdS/ZnS core\u2013shell\u2013shell QDs and Y2O3:Eu3+ makes the NPs promising as fiducial marker in correlative light and electron microscopy. In addition, the sharp and characteristic line emission can be used to create a variety of luminescent labels by changing and combining the type of dopant ion. The various labels can be detected down to the single particle level by recording the emission spectra of these labels.The robust character of the Y"}
{"text": "Background: Non-binary and genderqueer (NBGQ) people are those who do not identify within the gender binary system , not falling exclusively in man/male or woman/female normative categories. A higher proportion of NBGQ people is usually found within young persons. This population is marginalized and, as such, is at risk of stigmatization and of developing negative health outcomes. As literature on the health of NBGQ people is sparse, this study aims at systematically review the limited studies on this field.Methods: The research questions which guided the systematic review were: (1) What are the differences in the health levels between NBGQ and binary transgender (BT) individuals? (2) What are the differences in the health levels between NBGQ and cisgender individuals? (3) Which medical and psychological interventions are most suitable for improving NBGQ health? According to PRISMA guidelines, a systematic search was conducted in PubMed, PsycInfo, Web of Science, and Google Scholar.Results: Eleven studies met the inclusion criteria for the current systematic review. Among them, 9 were focused on the health differences between NBGQ and BT individuals, 4 of the latter and 1 individually were focused on the health differences between NBGQ and cisgender individuals, and 1 was focused on the evaluation of health outcomes related to medical procedures. No studies assessed psychological interventions aimed at improving health in NBGQ individuals. All studies were cross-sectional, did not generally recruit a large sample of NBGQ individuals, and used non-probability sample design. Results related to the difference in health between NBGQ and BT were mixed; indeed, some found a better health status while others a worse one. Results related to the differences in health between NBGQ and cisgender highlighted higher health needs in NBGQ than in BT individuals. The only study analyzing the effects of medical interventions on health found that NBGQ female-assigned at birth individuals improved their quality of life after chest surgery.Conclusions: Although scholars are starting to pay attention to the NBGQ health, research needs to be expanded both in terms of methodology and research contents. Clinical, health-related social policies, and research recommendations in this field are reported. Transgender is an umbrella term referring to individuals who have a gender that differs from that normatively expected of their assigned sex [American Psychological Association (APA), non-binary and genderqueer (NBGQ) refers to individuals who have a gender identity that does not fall exclusively in man/male or woman/female normative categories. NBGQ individuals identify themselves with a neither exclusively masculine nor feminine gender, and their gender identity is situated beyond the gender binary, fluctuates between genders, or rejects the gender binary  of 14.320 transgender respondents identified themselves as NBGQ  What are the differences in the health levels between NBGQ and BT individuals? (2) What are the differences in the health levels between NBGQ and cisgender individuals? (3) Which medical and psychological interventions are most suitable for improving health in NBGQ population?A systematic review was performed to identify published papers on the health of NBGQ individuals using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines , qualitative studies, mixed-method studies, and longitudinal studies were considered eligible. Reviews, meta-analyses, letters to the editor, books or book chapters, commentaries, and abstracts were excluded.Two reviewers (CS and FM) extracted the relevant data and assessed titles and abstracts identified in the literature search. They also excluded duplicates from the dataset. Disagreements between the two reviewers were solved through the involvement of two additional reviewers (PV and RV). All studies which matched the inclusion criteria were reviewed by the authors (CS and FM) and any disagreement was settled through a discussion involving two other reviewers (PV and RV).n = 1); 2) no research designs were performed (n = 4); (3) the article did not specifically address health outcomes (n = 11); (4) the journal was not peer-reviewed (n = 1), and (5) the identity of participants was not clearly defined  (n = 2).The initial search identified 218 records, as shown in the PRISMA flow diagram . Only 2 The 11 papers included in the current systematic review are listed in N = 7), while 2 were conducted in UK, 1 in Spain, and 1 in Canada. Among the studies selected, 9 (81.81%) were focused on the health differences between NBGQ and BT individuals (first research domain), 4 of the latter and 1 individually (45.45%) were focused on the health differences between NBGQ and cisgender individuals (second research domain), while only 1 (9.09%) was focused on the evaluation of health outcomes related to medical procedures . No studies assessed psychological interventions aimed at improving health in NBGQ individuals.Overall, studies were published between 2016 and 2019 and all of them used a cross-sectional design with quantitative methods, except for one study which supported quantitative data with focus groups. Considering the number of NBGQ individuals within the general sample recruited, the NBGQ subsample size was not generally large, as it ranged from 28 to 380 participants. However, considering the cross-sectional nature of the studies, some of them seem to have acceptable subsample size  were NBGQ, all FAAB. Among them, 56% (N = 33) completed the post-operative questionnaire. NBGQ participants agreed that surgery improved their quality of life, comfort with exercise, sex life, and comfort in physical appearance with and without clothes. Thus, this study shows the benefits that also NBGQ individuals might experience from undergoing medical procedures. As reported before, no studies addressing specific psychological interventions  for the improvement of health in NBGQ individuals were found.Only one study addressing health outcomes related to medical interventions for NBGQ individuals was found. Among 458 patients undergoing gender-affirming chest surgery, Esmonde et al.  found thThe aim of this study was to systematically review the literature related to the health of NBGQ individuals, exploring in particular 3 domains: (1) difference in health between NBGQ and BT individuals; (2) difference in health between NBGQ and cisgender individuals; and (3) medical and psychological interventions addressed to the improvement of NBGQ health. A total of 11 studies met the inclusion criteria and were used for this systematic review.As regards the characteristics of the selected studies, papers were recently published (from 2016), prevalently used a cross-sectional design with quantitative methods, did not generally recruited large NBGQ subsample . This is particularly noteworthy as it seems that NBGQ individuals analyzed in the studies selected, except for the NBGQ subsample recruited by Jones et al.  whose meAs regards the differences in health between NBGQ and BT individuals , studies selected reported mixed findings, some finding a better health status  are quite clearer than the previous ones. Indeed, although Warren et al.  did not Finally, the only study analyzing the effects of medical interventions on health of NBGQ individuals  found that NBGQ FAAB individuals, contrary to the belief that they would not need to medically affirm their gender, improved their quality of life and health after chest surgery , From a health-related social policy perspective, our findings shed light on the urgency of implementing policies to address health needs of NBGQ people, overcoming discriminatory practices and promoting health equity. First, NBGQ identities should be recognized by healthcare service systems and providers as existing and healthy identities. To this end, healthcare providers could benefit from specialized training aimed at improving the knowledge on NBGQ identities, as well as the related specific health needs  NBGQ identity development and specific challenges, matching theoretical perspectives on gender diverse population  with theoretical frameworks of developmental psychology ; (b) causal relationships between stigmatizing processes , protective factors  and health outcomes across different life stages;To expand studies to geographical contexts different from USA, UK, Canada, and Spain, as well as to deepen the role of socio-economic status in health disparities in order to build culturally competent studies on NBGQ population;To make every effort to recruit probability samples to allow generalization of the results to the specific population from which the sample was recruited. Although recruiting probability sample in LGBT community is a challenging task ;To build randomized controlled trial experiments to assess the efficacy of specific psychological interventions  in improving health in NBGQ people.This systematic review shows that the scientific interest on NBGQ health is growing but, at the same time, needs to be expanded both in terms of methodology and research contents. Indeed, it was hard to obtain a clear picture of the NBGQ population in terms of health, as all selected studies used a cross-sectional design and reported data on non-probabilistic samples. Notwithstanding these limitations, studies provided valuable information on the health of NBGQ people, starting to run an innovative research field which unhooks transgender population from a gender binary system that is often reproduced in scientific research. Thus, this review may represent one of the references for future studies, which could hopefully follow the research recommendations to increase the knowledge on NBGQ health building an increasingly relevant research from both the cultural and methodological point of view, as well as informing affirmative clinical practice and social policies to reduce the health equity gap.CS, FM, and RV conceived and designed the study and drafted the manuscript. CS and FM managed the literature searches and analyses. NM, MB, and VB refined the literature search. PV and RV solved disagreements between two main reviewers (CS and FM). All authors revised the article critically and read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
{"text": "The \u201cF-words in Childhood Disability\u201d  are an adaptation and an attempt to operationalize the World Health Organization\u2019s (2001) International Classification of Functioning, Disability and Health (ICF) framework. Since the paper was published (November 2011), the \u201cF-words\u201d have attracted global attention . Internationally, people have adopted the \u201cF-words\u201d ideas, and many families and service providers have expressed a need for more information, tools, and resources on the \u201cF-words\u201d.This paper reports on the development and pilot evaluation of a Web-based knowledge translation (KT) resource, the \u201cF-words\u201d Knowledge Hub that was created to inform people about the \u201cF-words\u201d and to provide action-oriented tools to support the use of the \u201cF-words\u201d in practice.CanChild Centre for Childhood Disability Research collaborated to develop, implement, and evaluate the Knowledge Hub. A pilot study design was chosen to assess the usability and utility of the Web-based hub before implementing a larger evaluation study. Data were collected using a brief anonymous Web-based survey that included both closed-ended and open-ended questions, with the closed-ended responses being based on a five-point Likert-type scale. We used descriptive statistics and a summary of key themes to report findings.An integrated research team of families and researchers at From August to November 2017, the Knowledge Hub received >6,800 unique visitors. In 1 month (November 2017), 87 people completed the survey, of whom 63 completed the full survey and 24 completed 1 or 2 sections. The respondents included 42 clinicians and 30 family members or individuals with a disability. The majority of people visited the Knowledge Hub 1-5 times (n=63) and spent up to 45 minutes exploring (n=61) before providing feedback. Overall, 66 people provided information on the perceived usefulness of the Knowledge Hub, of which 92% (61/66) found the Knowledge Hub user-friendly and stated that they enjoyed exploring the hub, and a majority (n=52) reported that the Knowledge Hub would influence what they did when working with others. From the open-ended responses (n=48), the \u201cF-words\u201d videos (n=21) and the \u201cF-words\u201d tools (n=15) were rated as the best features on the Knowledge Hub.The \u201cF-words\u201d Knowledge Hub is an evidence-informed Web-based KT resource that was useful for respondents, most of whom were seen as \u201cearly adopters\u201d of the \u201cF-words\u201d concepts. Based on the findings, minor changes are to be made to improve the Knowledge Hub before completing a larger evaluation study on the impact at the family, clinician, and organizational levels with a wider group of users. Our hope is that the \u201cF-words\u201d Knowledge Hub will become a go-to resource for knowledge sharing and exchange for families and service providers. The \u201cF-elopment . The inielopment . Since iCanChild Centre for Childhood Disability Research focused on disseminating the \u201cF-words\u201d into practice. At that time, several parents  had learned about the \u201cF-words,\u201d liked the ideas, and were interested in how to share the \u201cF-words\u201d message with more families. Recognizing the potential impact of an integrated approach to this work , we partnered with family stakeholders to develop and evaluate knowledge translation (KT) strategies tailored to meet the families\u2019 needs and preferences.In 2014, based on considerable interest in the paper, we formed an integrated research team at The first project involved the development, dissemination, and evaluation of a three-minute awareness video . A videoCanChild website was identified by 65.7% of respondents (90/137) as the most popular strategy for sharing further information on the \u201cF-words\u201d concepts. A complete report of our findings and the lessons learned from this project are published [We evaluated the video by tracking its reach and asking viewers to complete an anonymous Web-based survey. In the first 2 months, there were 715 views and 137 survey responses. Overall, we learned that 97.8% (134/137) of people \u201cextremely liked\u201d or \u201cliked\u201d the \u201cF-words\u201d ideas, 87.5% (120/137) indicated they would share the video, and 92.7% (127/137) wanted to learn more. The The awareness video was only the first step toward moving the \u201cF-words\u201d into practice. By January 2015, we had given >30 international presentations and the \u201cF-words\u201d ideas had continued to spread over social media. We were gratified by the uptake of these ideas around the world and were excited to see the imaginative ways in which people were adapting and adopting the \u201cF-words\u201d to local contexts. We were also learning a great deal about the application of the \u201cF-words\u201d by connecting and working with families and other stakeholders such as service providers and health care administrators around the world. Therefore, as a research team, we were acting as \u201cknowledge brokers\u201d  by workiFrom our conversations with the families and service providers, it was evident that there was significant interest in having more information on the \u201cF-words\u201d as well as action-oriented resources and tools to assist with the application of the \u201cF-words\u201d into practice. Furthermore, as the \u201cF-words\u201d ideas continued to spread, we recognized the need (and opportunity) to compile and share all that was being done on the \u201cF-words\u201d ideas by building a centralized Web-based community for knowledge sharing and exchange. Therefore, in 2015, our research team decided to develop, implement, and evaluate the usability and utility of a Web-based KT resource: a website called \u201cThe \u2018F-words\u2019 in Childhood Disability Knowledge Hub.\u201dCanChild\u2019s website [CanChild website is world-renowned in the field of childhood disability and receives over 12,000 unique visitors each month from over 205 countries [The purpose of the \u201cF-words\u201d Knowledge Hub was to inform families and service providers about \u201cF-words\u201d/ICF concepts and to provide action-oriented tools to support the uptake and use of the \u201cF-words\u201d in practice. The Knowledge Hub is currently hosted on  website  and is mountries .In the last several years, there has been increasing interest in using the internet as a platform for KT and the use of Web-based KT resources as a strategy for disseminating health research evidence in the field of childhood disability -10. LevaWhile the current evidence base for Web-based KT strategies is limited, some studies have shown promising findings ,13; howeaction cycle: \u201cselect, tailor, and implement the intervention,\u201d \u201cmonitor knowledge use,\u201d and \u201cevaluate outcomes.\u201d This study was part of a larger research program that had already addressed the earlier stages of the action cycle [This paper reports on the development process, usability, and utility of the Knowledge Hub. The Knowledge-to-Action (KTA) framework was used as the guiding theoretical underpinning for this research . KT theoon cycle .We implemented a formal integrated knowledge translation (iKT) strategy to develop, implement, and evaluate the Knowledge Hub. iKT involves the collaboration of researchers and knowledge users  throughout all stages of the research or KT process  and has All team members were involved in each stage of the project: (1) participating in initial planning stages; (2) providing feedback on the content and design of the Knowledge Hub; (3) creating and sharing tools/resources; (4) assisting with evaluation; and (5) disseminating the hub across their social networks. During the initial planning stages, team meetings were held by teleconference. We initially planned to develop an \u201cF-words\u201d Tool Kit as a paper-based resource designed to share knowledge and provide tools/resources to support the use of the \u201cF-words\u201d in practice; however, after extensive conversations with stakeholders and a review of the literature, we turned toward Web-based KT strategies . AC led the development of the hub, but feedback was sought and received from all team members throughout the development process. Most team correspondence was done through email.CanChild students we developed draft tool templates that could be distributed to all team members for feedback. When all team members had approved the tools, they were then posted on the Knowledge Hub.An area in which all three family stakeholders were heavily involved was the creation of the \u201cF-words\u201d tools; the \u201cF-words\u201d agreements, photo collage, goal sheet, and profile. Many of the ideas for the tools came from family stakeholders\u2019 personal experiences of working with service providers and their perceptions of how the \u201cF-words\u201d could be used in practice. As an integrated research team, we discussed the purpose and goals for each tool, and then with the support of To help with the planning and development of the Knowledge Hub, we used Levac et al\u2019s  best praThe purpose of the Knowledge Hub is to have a single site where people can go to learn about and share ideas for utilizing the \u201cF-words\u201d concepts in practice. The Web-based hub  includesCanChild\u2019s social networks. The recruitment poster is provided in A pilot study design was used to assess the usability and utility of the Knowledge Hub, and to make any necessary changes, before implementing a larger evaluation study. Usability was measured with \u201cusefulness\u201d questions  and utility was measured using \u201cuse\u201d questions . Usability and utility testing is a critical component to the success of KT interventions ,23. VisiThe survey included both closed-ended and open-ended questions. The closed-ended responses had a five-point Likert-type scale that evaluated the visitors\u2019 prior familiarity with the \u201cF-words\u201d, the perceived usefulness, and reported or intended use of the Knowledge Hub. Adaptive questioning was used  to reduce the complexity of the survey. There were 37 questions in the survey. Google analytics evaluated the reach by tracking the number of visitors to the hub over a four-month period. Descriptive statistics were used to analyze the quantitative information, and descriptive content analysis was used to identify and synthesize the key themes from the open-ended questions. Ethics approval was obtained from Hamilton Integrated Research Ethics Board (Project# 2017-0977).CanChild newsletters featuring the Knowledge Hub) and spread of the Knowledge Hub by people who liked and were sharing it within their communication channels and social networks.Over the four-month evaluation period (August-November 2017), there were over 6,800 unique visitors to the Knowledge Hub, with the number of visitors increasing each month . This coThe survey went live on November 3, 2017, and data were collected for 1 month. A total of 87 respondents provided information, with 63 completing the full survey and 24 partially completing the survey , providing a completion rate of 72%. Most people visited the Knowledge Hub 1-5 times (n=63) and spent up to 45 minutes exploring the hub (n=61) prior to providing feedback. The following results were based on the survey data.Just under half the respondents that completed the survey lived in Canada . The only other country with >10 respondents was the United States . The remainder of respondents came from 13 countries. Respondents were asked to state the perspective from which they were viewing the Knowledge Hub . Of the 87 people who completed the survey, 42 were clinicians and 30 were family members (n=20) or individuals with a disability (n=10). There was a wide distribution of perspectives with many respondents (n=36) falling into >1 stakeholder category .The majority of people  had heard of the \u201cF-words\u201d prior to visiting the Knowledge Hub and either \u201cextremely liked the ideas\u201d  or \u201cliked the ideas\u201d . Of the 62 people who were familiar with the \u201cF-words\u201d, 43 (69%) felt confident identifying and explaining the \u201cF-words\u201d ideas, 37 (60%) had shared them with others, and 35 (56%) indicated that they had used or applied them in practice prior to exploring the hub. To understand how people were using or applying the \u201cF-words,\u201d we asked for open-ended feedback. The majority of people who provided written responses were clinicians, researchers, people with disabilities, or family members. Depending on the stakeholder group, the use of the \u201cF-words\u201d concepts varied. Examples of how the \u201cF-words\u201d concepts have been used by each stakeholder group are shown in To evaluate the usefulness of the Knowledge Hub, respondents were asked to rate their overall satisfaction. Of the 87 people who started the survey, 66 people completed this section. Therefore, the following data are based on these 66 responses. Overall, 86% (57/66) of respondents felt the purpose was clear, 92% (61/66) found the Knowledge Hub user-friendly, and 92% (61/66) and 94% (62/66) perceived the content to be meaningful and relevant for families and service providers, respectively. The average scores ranged from 4.23 to 4.39 out of 5 for each category .Respondents were also asked to indicate which sections of the Knowledge Hub they liked and what could be improved. A total of 65 people answered this question, all of whom indicated they liked at least one section of the Knowledge Hub, 57% (37/65) indicated that they liked all sections and 45% (29/65) indicated they had no further suggestions for improvements. The survey also collected open-ended feedback to gain a better understanding of what were perceived to be the best features of the Knowledge Hub (48 respondents) and which areas needed improvement (25 respondents). The best features and areas for improvement were categorized into two aspects: content and format or design of the Knowledge Hub. The key themes within these areas were then identified based on the number of responses. The final section of the survey explored the use or intended use of the Knowledge Hub and the \u201cF-words\u201d concepts. Among the people who started the survey, 72% (63/87) completed this final section. The following data are based on responses from these 63 people .Overall, 97% (61/63) people indicated that they either \u201cextremely liked\u201d  or \u201cliked\u201d  the \u201cF-words\u201d concepts, 92% (58/63) people reported that the hub increased their understanding of the \u201cF-words\u201d, and 78% (49/63) people reported that the hub influenced their thinking about the \u201cF-words\u201d. We were also interested in participants\u2019 confidence in identifying and explaining the F-words after exploring the Knowledge Hub. Overall, 90% (57/63) people indicated that they were either \u201cextremely confident\u201d  or \u201cconfident\u201d . When asked whether the Knowledge Hub would be useful to them, 83% (52/63) people reported that it would influence the way they did things when working with others.Lastly, respondents were asked to rate the Knowledge Hub as a KT tool for sharing information with families and service providers. Overall, 90% (57/63) people rated it 4 or 5  as a KT tool for families, 98% (60/63) people rated it as 4 or 5 as a KT tool for service providers, and 97% (58/63) people planned to share the Knowledge Hub.From the beginning, it was important to us that the Knowledge Hub be cocreated with stakeholders. While our integrated team of families and researchers led the development process, many stakeholders outside of our research team were involved. For example, we worked with clinicians and health care administrators, who we knew were applying the \u201cF-words\u201d to share examples of how they were using the \u201cF-words\u201d in their organizations. These examples then served as examples of application for other service providers.We believe early stakeholder involvement was crucial not only to the development of a meaningful and relevant resource but also to the dissemination of knowledge regarding the Knowledge Hub. Individuals interested in the Web-based hub were more likely to share it with their own communities, thus increasing its reach and potential impact as it was spread through broad communication channels and social networks . The impAnother key feature of the Knowledge Hub was its promotion of knowledge sharing and exchange . In compCanChild\u2019s resources  to design and maintain the Knowledge Hub. Creating and collating content for the Knowledge Hub also took a lot more time than initially expected. The development process involved iterative rounds of feedback from various stakeholders. We did not follow a structured system or timeline for collecting feedback, which led to a longer process. In the future, we would recommend the use of a structured process tailored to collecting feedback from a diverse group of stakeholders [One common barrier reported in the literature was the time and resources needed to develop and implement KT interventions ,25,30. Weholders .A key facilitator for this project was the use of theory and best practice guidelines to inform the KT intervention ,31. The The main aim of the Knowledge Hub is to inform families and service providers about the \u201cF-words\u201d/ICF concepts and to provide action-oriented tools to support the uptake and use of the \u201cF-words\u201d in practice. As such, the goal of this pilot evaluation was to evaluate the usability and utility of the Knowledge Hub. The findings from this study revealed that these self-assigned goals were attained. Overall, the respondents reported that the Knowledge Hub was informative and useful and the \u201cF-words\u201d tools were one of the best features of the Knowledge Hub.perceived usefulness and potential use. While mixed-model analyses between groups were not completed, the high ratings given by all participants implied that the Knowledge Hub was perceived to be a meaningful resource for both service providers and families. This finding was consistent with earlier research from CanChild that found that when educational materials were clearly written and user-friendly, they were useful and impactful for multiple target audiences  [In general, the hub received high ratings with regard to both its oviders) ,33. Furtoviders) ,35.early adopters\u2014 those who are already using the \u201cF-words\u201d\u2014to the late adopters\u2014 those to whom the \u201cF-words\u201d are new) [We recognize that prior to exploring the Knowledge Hub, over 70% of people who completed the survey had previously heard of the \u201cF-words.\u201d Of these respondents, the majority felt confident identifying and explaining the \u201cF-words\u201d ideas, and about half of them indicated that they had used or applied the \u201cF-words\u201d in practice. Despite many respondents already being familiar with the \u201cF-words\u201d concepts, the majority stated that the Knowledge Hub increased their understanding of the \u201cF-words\u201d ideas. This is an important finding as it implies that the Knowledge Hub can increase perceived knowledge even if individuals have prior familiarity with the concepts. This probably occurred because the resources provide tangible materials that move beyond simple concept familiarity. Unfortunately, due to the low response rate from people for whom the \u201cF-words\u201d concept was new, it is not possible to say whether the Knowledge Hub is useful across all adopter categories  .early adopters of the \u201cF-words\u201d concepts) and what was needed to broaden the applicability of the Knowledge Hub to a wider audience . These findings will both inform and complement future evaluations of the Knowledge Hub. Recognizing that experimental evaluations only identify whether an intervention is effective, process evaluations such as this are recommended to understand the reasons why interventions are (or are not) effective [Conducting a pilot evaluation of the usability and utility of the Knowledge Hub is an important step toward ensuring its overall impact and sustainability ,23. Thisffective ,37.Based on the respondents\u2019 positive feedback, we anticipate that the Knowledge Hub will be a useful resource for both families and service providers. A limitation to this work is that feedback was gained from only a small sample of the people who visited the hub during this period. It is important to remember that the majority of people who provided feedback were those who were already familiar with the \u201cF-words\u201d concepts and also liked the \u201cF-words\u201d ideas. Thus, their potential biases must be recognized.In order to reach a broader audience, more time is needed to actively disseminate the Knowledge Hub. While the preliminary findings after a one-month evaluation were reported here, in order to overcome selection bias , the evaluation will remain posted on the Knowledge Hub and further feedback will be monitored. The hope is that over time more people  will complete the survey.The next step is to evaluate the impact of the Knowledge Hub and \u201cF-words\u201d concepts at the family, clinician, and organizational levels. As active implementation strategies are useful in supporting the dissemination and uptake of educational materials, we plan to combine the Knowledge Hub intervention with tailored invitational outreach visits to local children\u2019s treatment centers (CTCs). Once again, this is a stakeholder-driven strategy as CTCs have contacted us and expressed a need for in-person educational training on the \u201cF-words\u201d concepts. Based on our positive experiences of working with families and service providers to develop the Knowledge Hub, this project will continue to be informed by an iKT strategy.early adopters  [early majority), further active implementation strategies are needed.Working with families and service providers, we designed a theory-informed and evidence-informed Web-based KT resource that was perceived to be relevant and meaningful to families raising children with disabilities and to service providers working in the field. To date, the Knowledge Hub has mainly reached rmation) ; therefoKT is not only the doing but also the studying of the KT process and outcomes. From the evaluation of the usability and utility of the Knowledge Hub, we now have a good understanding of what was done well and what can be improved. Based on the findings from this pilot study, we intend to make minor changes to the Knowledge Hub before conducting a larger evaluation study of the impact at the family, clinician, and organizational levels. Knowledge gained from this study is transferrable to other KT initiatives involving families and service providers. We hope that the findings and lessons learned from this integrated KT project will assist others in advancing iKT science and practice in other areas of childhood disability research."}
{"text": "P\u2010value of .05, the distribution of TIICs in 73 PCa tissues and 11 normal tissues was illustrated. Activated CD4+ T cells and M0 macrophages account for a high proportion in PCa tissues, while neutrophils and monocytes were found at a high density in normal tissues. Further results showed that the density of plasma cells, Treg cells and resting mast cells were associated with advanced PCa. Additionally, M2 macrophages affected the RFS of PCa patients, and AR was also involved. In the current study, we first evaluated the immune infiltration among PCa and revealed that M2 macrophages could predict the prognosis of PCa patients. Meanwhile, based on the LASSO regression analysis, we established a novel nomogram to assess the recurrence risk of PCa based on immune cell proportions and clinical features.Prostate cancer (PCa), a severe health burden for males, accounts for the second frequent cancer and fifth tumor specific death cancer around the world. Several studies on tumor\u2010infiltrating immune cells (TIICs) have shown inconsistent and controversial results to PCa. We downloaded a gene expression matrix and clinical information from TCGA, and CIBERSORT was used to identify the proportion of TIICs. Wilcoxon's Sign Rank Test evaluated different gene expression levels in PCa and normal tissues. Kaplan\u2010Meier curves were used to evaluate the associations of TIICs and recurrence\u2010free survival (RFS). Finally, based on the preset  This effect was accompanied with increased expression of PD\u2010L1 from tumor cells. Philippe et alTumor\u2010infiltrating immune cells (TIICs) are essential components of the tumor microenvironment and can alter the immune status of the tumor. Several studies have demonstrated therapeutic strategies against tumors by targeting TIICs.Due to the limitations of methods and techniques, prior studies focused on finite areas of the immune response, whereas the full view of the immune response is based on the infiltration of different TIICs. Alternatively, the immunohistochemistry (IHC)\u2010based labels of TIICs are not precise, and several TIICs may express the same makers on the membrane, leading to the inaccurate measurements of TIIC density in PCa tumor tissues. In the present study, the proportion of 22 TIICs in PCa tissues was illustrated by a newly developed deconvolution algorithm, CIBERSORT, which can predict the density of TIICs through the expression of 547 key genes, and revealed the potential effect of TIICs in PCa prognosis.22.1In the current study, TCGA database of PCa tissues and control tissues were enrolled. We downloaded the overall gene expression profiles and clinical information of patients from TCGA up to June 31, 2019. Finally, the gene expression profiles of 499 PCa samples and 52 controls were prepared for subsequent analysis. We performed the stable multi\u2010array average algorithm to preprocess the recorded raw data. The mRNA sequencing data of TCGA database were first converted to a microarray\u2010similar result using the voom method . Then, we annotated the gene ID, and the mRNA sequencing results were normalized with the \u201climma\u201d package of R, to average the data of repeated genes and delete results that were not available. In Figure 2.2+ T cells, na\u00efve CD4+ T cells, resting memory CD4+ T cells, activated memory CD4+ T cells, follicular helper T cells, regulatory T cells (Treg cells), gamma delta T cells, resting NK cells, activated NK cells, monocytes, M0 macrophages, M1 macrophages, M2 macrophages, resting dendritic cells, activated dendritic cells, resting mast cells, activated mast cells, eosinophils, and neutrophils, with the preset signature matrix at 1000 permutations. After using the CIBERSORT program, the distribution of 22 subtypes of TIICs was presented, along with the results of correlation coefficient, P\u2010value and root mean squared error (RMSE), which can evaluate the accuracy of the results in each sample. The P\u2010value\u00a0\u2264\u00a0.05 reflects a statistical connotation of the results of deconvolution across all cell subsets for each sample and is useful for excluding results with less accuracy. Finally, 73 PCa samples and 11 control samples were selected for later analysis because they met the required P\u2010value.The analytical tool called CIBERSORT was used in the study, which can quantify the percentage of different types of TIICs accurately, under the complex \u201cgene signature matrix\u201d based on 547 genes.2.3http://www.r-project.org). ROC and AUC were completed to assess the discriminatory ability of the nomogram, while decision curve analysis was also performed to evaluate the usability of the nomogram. STRING, an online tool that could reveal the functional protein association networks, was employed to identify androgen receptor associated proteins.P\u00a0<\u00a0.05 was considered statistically significant.For each type of TIICs, we assessed the different levels in patients and controls with Wilcoxon's Sign Rank Test and displayed the violin plot. Using the Pearson correlation coefficient, correlations between two immune cell subsets were evaluated. The different distribution of TIICs in several subgroups of clinical features were evaluated with Wilcoxon's Sign Rank Test in parameters divided into two groups and with the Kolmogorov\u2010Smirnov test in parameters separated into more than two groups. Kaplan\u2010Meier (K\u2010M) curves were constructed to analyze and evaluate the associations of immune cell infiltrate and corresponding recurrence\u2010free survival (RFS) using the log\u2010rank test. The median of the proportion of each cell type was computed to divide the patients into high\u2010 and low\u2010density groups. The nomogram was constructed using the rms package in R (33.1P\u00a0<\u00a0.05). The flowchart of study design and the process of samples enrolled is shown in Figure + memory T cells were highly present in normal tissues (P\u00a0=\u00a0.029), as well as monocytes (P\u00a0<\u00a0.001), resting dendritic cells (P\u00a0=\u00a0.013), resting mast cells (P\u00a0=\u00a0.001) and neutrophils (P\u00a0<\u00a0.001)  and M0 macrophages (P\u00a0=\u00a0.004) was higher in PCa tissues compared to normal tissues , and neutrophils were linked with monocytes (R\u00a0=\u00a00.44). In contrast, the existence of resting CD4+ memory T cells was negatively associated with follicular helper T cells (R\u00a0=\u00a0\u22120.44), activated NK cells, activated CD4+ memory T cells (R\u00a0=\u00a0\u22120.38), resting CD4+ memory T cells and M0 macrophages (R\u00a0=\u00a0\u22120.38). Figure To determine the underlying relationships of different immune types, we evaluated the correlation between TIICs among PCa tissues. Figure s R\u00a0=\u00a0\u22120.8, restins R\u00a0=\u00a0\u22120.8, restin3.2P\u00a0=\u00a0.076), M2 macrophage in Gleason 9\u00a0+\u00a010 group (P\u00a0=\u00a0.008) , T3\u00a0+\u00a0T4 stage (P\u00a0=\u00a0.36). Treg cells were high in tumor tissues among severe stages, including the 9\u00a0+\u00a010 Gleason score group (P\u00a0=\u00a0.009), T3\u00a0+\u00a0T4 stage (P\u00a0=\u00a0.044), while resting mast cells showed a lower density among the 9\u00a0+\u00a010 Gleason score group (P\u00a0=\u00a0.004), T3\u00a0+\u00a0T4 stage (P\u00a0=\u00a0.027)  Figure A. Additi) Figure .P\u00a0=\u00a0.025) , while a lower proportion indicated a more prolonged RFS  Figure C. There 3.3To establish a convenient method to predict the recurrence risk of PCa, we designed a nomogram based on clinical information and density of TIICs. First, the subtypes of TIICs were selected by LASSO regression analysis, including M2 macrophages, follicular helper T cells and resting dendritic cells Figure . In addi3.4P\u00a0<\u00a0.001), after adjusting for tumor purity, and similar results were illustrated for NCOA2  and NCOA4 . In contrast, the expression of FOXA1 , KDM1A  and KLK3  were negatively associated with the distribution of macrophages in PCa tissues.AR plays pivotal roles in the biochemical recurrence and drug resistance of PCa. As a high proportion of M2 macrophage infiltration is positively associated with the shorter RFS, we evaluated the relationship of AR and M2 macrophages infiltration, as well as AR\u2010related genes. The five main AR\u2010related proteins were identified with STRING, an online website that illustrates the correlation between proteins. All of the information was based on curated databases, experimentally determined or text mining. Finally, we recorded KLK3, KDM1A, NCOA2, NCOA4, and FOXA1 Figure . FurtherP\u00a0<\u00a0.001), VSIG4 , MS4A4A , MRC1 , CCL24 , and CD209   Figure . These r4+ cells, and na\u00efve CD4+ T cells, while Treg cells and NK cells increased. Regarding macrophages, Shimura et alImmune infiltration plays a critical role in tumor progression. Immune evasion is now regarded as a cancer hotspot. One of the critical mechanisms is the programmed cell death protein (PD\u20101) receptor on cytotoxic T lymphocytes (CTLs) and its ligand (PD\u2010L1) on cancer cells or host TIICs. Immunotherapies targeting PD1/PD\u2010L1, which can reverse immune tolerance and yielded remarkable clinical responses, have been widely studied in tumors.+ T cells did not appear in any patient or control. We found that activated CD4+ T cells and M0 macrophages are found at high levels in tumor tissues, while neutrophil and monocyte levels are lower, and these results are consistent with previous studies.In previous studies, most researchers detected the density of TIIC proportion based on fewer specific markers. However, several papers demonstrated the overlap of molecular markers for TIICs.+ T cells are positively associated with activated CD4+ memory T cells, while resting CD4+ memory T cells are negatively associated with follicular helper T cells. Connie et al+ memory T cells, are required to enhance the primary activated process of CD8+ T cells in immune responses, while Edith et al+ T cells are required for secondary expansion and memory of CD8+ T cells. Follicular helper T cells are distinguished from CD4+ T cells, which function in the formation of germinal centres and promote the proliferation and differentiation of B cells. Hale et al+ T cell populations that commit to follicular helper T cell lineages, and CD4+ memory T cells can recall their previous lineage of effectors provided by follicular T cells.In the second part of the study, we illustrated the TIICs in PCa tissues and their internal correlation. The features of patients are divided into two parts with unsupervised hierarchical clustering, meaning that the different proportions of TIICs in PCa are associated with the clinical results. We also determined that CD8+ Treg cells was associated with advanced PCa tumor stage. Erlandsson et alSubsequently, the analysis of TIICs and clinical features was performed, and we found that the proportion of plasma cells and mast cells was downregulated in severe PCa, compared to mild PCa, while the density of Treg cells was upregulated. Lohr et alAfter assessing the association between TIICs and RFS of PCa patients, M2 macrophages were singled out. Patients whose M2 macrophage density is higher had a shorter RFS time. Although the K\u2010M curve of M0 and M1 macrophages showed a similar trend, no statistical significance was found. Feng et alIn summary, using the deconvolution algorithm of CIBERSORT, we first obtained the distribution of 22 TIICs in PCa patients based on the expression of 547 core genes. The proportion of immune infiltration was significantly different between PCa and control tissues. Plasma cells, Treg cells, and mast cells are involved in the progression of PCa, while M2 macrophages could predict the prognosis of PCa patients. Meanwhile, based on the LASSO regression analysis, we established a novel nomogram to assess the recurrence risk of PCa based on immune cell proportions and clinical features, of which need to be verified by a large size cohort in the feature.All authors declared no competing interests.JLM, MZ, and CZL conceived and designed the study. MZ, YL, and SF performed the data extraction. JLM, MZ, and SYG analyzed the data. YL, SF, and ZYH contributed materials and analysis tools. JLM, JZ, JZ, and CZL wrote the manuscript. All authors have read and approved the final version of the manuscript and agreed with the order of presentation of the authors.\u00a0Click here for additional data file."}
{"text": "Single-anastomosis duodenoileal bypass with sleeve gastrectomy (SADI-S) is a simplified biliopancreatic diversion. The objective of this study was to develop a reproducible animal model for SADI-S. We used three techniques for duodenal exclusion and duodenoileal anastomosis: (a) surgical clip and side-to-side anastomosis, (b) ligation and side-to-side anastomosis and (c) sectioning the duodenum, closing the duodenal stump and end-to-side anastomosis. We recorded the surgical technique and complications for each method. Twenty-five of 31 rats survived to the end of the study period. One death occurred from accidental anaesthesia overdose and the others from anastomosis leak. Four duodenal exclusions had repermeabilised at necropsy. Our murine model of SADI-S can be consistently reproduced. Sectioning the duodenum is preferable to avoid repermeabilisation of the duodenum. Single-anastomosis duodenoileal bypass with sleeve gastrectomy (SADI-S) was proposed by S\u00e0nchez-Pernaute A. et al.  as a modThirty-one adult male Winstar rats (12\u201316\u00a0weeks old) were included in this study. All animals were housed in individual cages under constant ambient temperature (23\u00a0\u00b0C) and humidity, and were maintained in a 12-h light/dark cycle room.The night before surgery, the rats were kept on raised wire platforms to avoid coprophagy and fasted for approximately 12\u00a0h; only water was allowed ad libitum. An isoflurane anaesthetic chamber was used for anaesthesia induction, which was changed to nose cone flow for maintenance of anaesthesia during the procedure. Enrofloxacin 2.5%, 25\u00a0mg/kg, was administered intramuscularly for antibacterial prophylaxis, and 20\u00a0mL of normal saline solution was given subcutaneously for hydration (10\u00a0mL before surgery and 10\u00a0mL postoperatively).linea alba, a midline incision was made in the abdominal wall using an electrical scalpel. The abdominal cavity was then exposed using a surgical retraction system .Following induction, the abdomen was shaved with electric hair clipper from sternum to groin, and the rat was placed on a heating pad in dorsal recumbency. The skin was disinfected using an aqueous iodophor solution, and after identifying the xiphoid process, a 5-cm midline abdominal incision was made using a #11 surgical scalpel. After identifying the We performed a complete gastrectomy of the greater curvature using surgical scissors. The gastrosplenic ligament was sectioned after ligation with 4.0 absorbable sutures . We continue with dissection of the duodenum Fig.\u00a0. Next, wLigation of the duodenum 5\u00a0mm from the pylorus using 3.0 nonabsorbable suture  followed by side-to-side duodenoileal anastomosis using a continuous pattern with absorbable 6.0 suture .Ligation of the duodenum 5\u00a0mm from the pylorus using a nonabsorbable surgical clip  followed by side-to-side duodenoileal anastomosis using a continuous pattern with absorbable 6.0 suture .Sectioning the duodenum 5\u00a0mm from the pylorus and closure of the duodenal stump in a continuous pattern with nonabsorbable 6.0 suture  followed by end-to-side duodenoileal anastomosis in a continuous pattern with absorbable 6.0 suture . It is interesting to note that duodenal sectioning can be an alternative method of gastric tube calibration.Three methods were used for duodenal exclusion and duodenoileal anastomosis:Following the anastomosis Fig.\u00a0, we retuImmediately postoperatively, each rat recovered under a heat lamp with continuous monitoring until regaining consciousness. After recovery, the animals were transferred to a cage in isolation, kept on a raised wire platform, and fasted for the first 24\u00a0h. Liquid diet  was provided for the next 3\u00a0days. Antibiotics (enrofloxacin) were administered for 72\u00a0h. Full solid diet was administered on day 7 with a transition time of 3\u00a0days during which we provided liquid and solid diet simultaneously. At the end of the study period, the rats were sacrificed by decapitation after exploratory laparotomy under isoflurane anaesthesia. The brain was removed for immunohistochemistry studies measuring neuronal activity (quantification of cfos which is a marker of activated neurons).Operative time and anaesthetic death were recorded, and the study period, as our goal was to validate the technique in rats, was 2\u00a0weeks in duration. Body weight was recorded before surgery and every postoperative day, with each death investigated to identify complications. After 2\u00a0weeks, an exploratory laparotomy was performed to identify fistula, intestinal occlusion, or repermeabilisation of the duodenal exclusion. Data analyses are expressed as the mean \u00b1 standard deviation.p\u2009<\u00a00,001) , and the mean postoperative weight was 261\u2009\u00b1\u200942\u00a0g (range 195\u2013391\u00a0g) , and 4 cases of repermeabilisation of the duodenal exclusion .p\u2009=\u20090.34). To decrease duodenal stump complications, we performed duodenal exclusion with either suture ligation or surgical clip ligation. However, four cases of repermeabilisation of the duodenal exclusion were seen, and these occurred with duodenal exclusion without sectioning the duodenum. In contrast, we encountered no duodenal stump complications when we sectioned the duodenum.To our knowledge, ours is the first study evaluating the feasibility of an animal model for SADI-S. We chose this strain of Wistar rats because it is the strain most used in preclinical studies focused on obesity and diabetes. This strain is also highly sensitive to weight gain on hyperlipidic diet and quickly becomes insulin resistance. We experienced low overall mortality  and seven anastomotic leaks: four in the side-to-side anastomosis group and three in the end-to-side anastomosis group (To create a larger gastric tube, we used an 8-FR calibration catheter Fig. , contrarIn the original technique described by Sanchez-Pernaute , a 200-cIn conclusion, this study represents the first report of a preclinical model for SADI-S. This technique will allow researchers to evaluate postoperative metabolic homeostasis and compare their findings with other standard surgical bariatric techniques."}
{"text": "Substantial improvement of ULM is further demonstrated on a chicken embryo tumor xenograft model and a chicken brain, showing both morphological and functional microvasculature details at super-resolution within a short acquisition time . The proposed technique allows more robust MB localization and tracking at relatively high MB concentrations, alleviating the need for dilute MB injections, and thereby shortening the acquisition time of ULM imaging and showing great potential for clinical translation.Super-resolution ultrasound localization microscopy (ULM), based on localization and tracking of individual microbubbles (MBs), offers unprecedented microvascular imaging resolution at clinically relevant penetration depths. However, ULM is currently limited by the requirement of dilute MB concentrations to ensure spatially sparse MB events for accurate localization and tracking. The corresponding long imaging acquisition times  to accumulate sufficient isolated MB events for full reconstruction of microvasculature preclude the clinical translation of the technique. To break this fundamental tradeoff between acquisition time and MB concentration, in this paper we propose to separate spatially overlapping MB events into sub-populations, each with sparser MB concentration, based on spatiotemporal differences in the flow dynamics (flow speeds and directions). MB localization and tracking are performed for each sub-population separately, permitting more robust ULM imaging of high-concentration MB injections. The superiority of the proposed MB separation technique over conventional ULM processing is demonstrated in flow channel phantom data, and in the chorioallantoic membrane of chicken embryos with optical imaging as an  Contrast MBs can be considered as acoustic point scatterers, since they are substantially smaller than the typical ultrasound wavelength, but also provide strong acoustic backscatter, permitting the application of signal processing methods that can overcome the inherent compromise between ultrasound frequency and attenuation. ULM has been shown by numerous research groups18 to break the diffraction limit of conventional ultrasound, resulting in an approximately tenfold improvement in imaging resolution while maintaining imaging penetration depth. Furthermore, as an ultrasound-based imaging modality, ULM is safe, non-invasive, low-cost, and lacks ionizing radiation, thereby promising great clinical significance by providing capillary-level imaging resolution at acoustic-level imaging penetrations. As such, ULM has rapidly gained traction and is applicable to a wide spectrum of preclinical and clinical applications, including the diagnosis and characterization of cancer8, cardiovascular disease19, and the monitoring of neurological activity20.Super-resolution ultrasound localization microscopy (ULM), the acoustic analogue to optical sub-diffraction-limit techniques such as PALM5. The successful reconstruction of microvasculature via ULM requires an adequate number of MBs in circulation to fully traverse all vasculature of interest. However, MBs must also be sufficiently spatially isolated to allow for accurate and unequivocal localization of signal centroids\u2014thus, poorly isolated MB centroids are typically excluded from analysis. This represents a challenging trade-off for imaging biologically relevant vascular beds: The imaging of small vessels, which typically have slower flow speeds, benefits from higher MB concentrations that increase the probability of MB traversal; however, this is at the expense of more MB signal overlap in larger vessels. Lowering the concentration of MBs by dilution will result in sparser signal events but will also significantly increase the data acquisition time needed to reconstruct a vascular bed. A long acquisition time is challenging to implement in vivo as both tissue- and operator-induced motion will degrade the final super-resolution image. The dilution method also requires a constant infusion of MBs or multiple bolus injections, both of which are challenging in a clinical setting. Phase-change droplets, which can change phase from liquid to gas when activated by ultrasound, have been utilized as a contrast agent for ULM and have recently demonstrated the potential for real-time super-resolution imaging in vitro18. Distinct from clinically used MBs, the droplet-based super-resolution method can sparsely activate droplets for localization, allowing for high droplet concentrations, and does not necessarily require frame-to-frame droplet flow for contrast-agent detection. However, further in vivo evaluation and validation may be required for this technique.However, ULM is currently challenged by technical and clinical difficulties including long data acquisition times, low temporal resolution, limited clinical dosages of contrast MBs, and inaccurate localization and tracking of MBs due to ultrasound noise and phase aberrationTo break this tradeoff between acquisition time and MB concentration, we propose leveraging the spatiotemporal characteristics of MBs in circulation to improve the performance of MB localization and tracking. Spatiotemporal filtering (e.g.: SVD filtering) is already extensively used in ultrasound blood flow imaging and ULM pre-processing, as a clutter filter to separate MB signals from tissue background. We propose an additional step of signal post-processing to separate MBs into sub-populations based on differences in spatiotemporal flow dynamics, such as movement speed, flow direction, and signal decorrelation. Although MB signals often spatially overlap over the course of an injection bolus, they may be still separable through the judicious use of spatiotemporal filtering. This paper focuses on the use of 3D conical filters in the Fourier domain for detecting and extracting signal differences to separate MB datasets into distinct subsets, each of which can be processed independently. We apply this technique to MB signal acquisitions taken from the chorioallantoic membrane (CAM) of chicken embryos and use optical imaging as validation. We posit that this type of filtering increases the possibility of observing isolated MBs for high-concentration injections and therefore alleviates the need for MB dilution; as a result, it also shortens the corresponding long acquisition time, which is critical for the clinical translation of ULM.The proposed MB separation method divides an original, densely populated MB dataset into subsets of data with a sparser effective MB concentration for more robust localization and tracking. This is demonstrated in the workflow diagram presented in Fig.\u00a0Separate ultrasound datasets were acquired from a flow channel phantom running at a constant flow rate with a relatively low concentration of MBs . A total of four different flow rates  and two different flow directions were combined frame-by-frame to generate a synthetic ultrasound data set. This combined data set was used to test the performance of the MB separation technique, as the data set represents a complicated flow scenario where MBs are flowing at different speeds and directions simultaneously. To serve as a reference standard, each individual low-concentration MB dataset was processed using conventional ULM super-resolution processing  and more consistent flow direction. ULM was performed on each data subset individually, producing separate super-resolution images Sup Fig.\u00a0. A finalIt should be noted that generating synthetic data by adding up different individual datasets to simulate high MB concentration does not represent a true physiological blood flow situation. For a more realistic situation, the proposed method was also applied to one individual flow channel data, where different flow velocities exist naturally along the radial direction. The results are shown in Fig.\u00a0ex ovo CAM provides a thin vascular bed with complex branching patterns and a minimal amount of tissue motion  chicken embryo through intact skull bone; a stack of 5000 frames of data (corresponding to data length of 16.6\u2009s) was used to generate super-resolved images. As with tumor imaging, conventional ULM produced vascular localization maps . Likewise, MB separation applied to CAM imaging revealed better delineation of a broader spectrum of vascular bed features than conventional ULM alone , as validated with optical imaging. Furthermore, MB separation could produce velocity maps that retained complex flow patterning, such as the counter-directional blood flow of arterial capillaries . Finally, the technique was applied to high-MB concentration contrast-enhanced ultrasound (CEUS) datasets acquired from multifaceted three-dimensional networks\u2014namely the chicken CAM tumor xenograft model and chick brain. In both cases, MB separation produced well-defined microvascular features  while also retaining the highly perfused vessel lumen of feeding vasculature. Thus, the proposed technique offers a simple post-processing solution that alleviates the tradeoff between acquisition time and MB concentration for ULM imaging. This permits the widespread utilization of the method without the need to adjust experimental parameters and even allows for retrospective analysis of previously gathered datasets.We have demonstrated that the proposed MB separation technique, which leverages the spatiotemporal characteristics of MBs in circulation to allow for independent analysis of sub-populations of MBs, improves the performance of MB localization and tracking for ULM imaging at high MB concentrations. This technique was tested ies Fig.\u00a0. Finallyk-\u03c9 domain). With these assumptions in mind, we designed a set of conical k-\u03c9 domain filters to isolate MB subpopulations that are flowing at different speeds and trajectories. Applying this set of 3D cone-shaped filters to MB k-\u03c9 domain data separates the MB signal space into multiple subsets (15 subsets for in vivo data in this study) containing spatially sparser MB subpopulations with less flow complexity. We posit that this MB separation technique allows for useful ULM imaging at relatively high MB concentrations and is therefore preferable to the clinically relevant solutions of MB where MB signals may be spatially overlapping. Given that Hingot et al.21 report that the long acquisition times for ULM imaging are dictated by rare MB flow events in the microvasculature, it follows that higher MB injection concentrations would allow for reduced acquisition times. We observed that MB separation improved the performance of established MB localization and tracking algorithms7 and thereby resulted in better super-resolution images of vasculature.An underlying assumption of the proposed MB separation technique is that MBs will follow physiologically relevant blood flow trajectories and will therefore be constantly changing their relative positions and directions to one another. However, groups of MBs that are flowing in large vessels will exhibit high flow speeds and complicated flow dynamics, resulting in a low degree of correlation. In contrast, MBs in capillary beds will flow more slowly and stably and will maintain a high degree of correlation in their motion. These differences in movement speed, flow direction, and signal decorrelation produce tangible shifts in the spatiotemporal flow dynamics of MBs, which then correspond to distinct energy concentrations within different regions of the 3D Fourier domain , but it should be noted that this number can be arbitrary, and the only requirement is to avoid significant MB signal smearing or distortion by using relatively large passbands. MB signals of each subset can be reviewed separately to empirically determine an appropriate passband for each filter. In general, a tradeoff exists between the number of separated datasets and computation cost. Although increasing the number of subsets can potentially provide a larger number of localized MBs, it also dramatically increases the computational cost. The optimal number of subsets and passband range would need to consider MB concentration(s), complexity of the flow dynamics for specific organ, flow speed range, and ultrasound center frequency, which would be varying from case to case.Ultimately, the goal of the proposed MB separation algorithm is to produce multiple spatially sparse MB subsets from overlapping signals, thereby alleviating the need for dilution of indicator, rejection of overlapping MB signals, and long acquisition times. However, we acknowledge that our proposed method does not produce perfect MB separation and, indeed, such a goal may be impossible using only post-processing techniques. One salient concern with the proposed method is the risk of MB signal distortion resulting from splitting the MBs into two or more filters. To minimize the risk of MB signal distortion, we use relatively large passbands with smooth transitions and large overlaps between adjacent filters, which would avoid hard splitting of single MB signals. The remaining undesired signals can be further suppressed via intensity thresholding and correlation thresholding in the proceeding ULM processing steps (as detailed in Methods). As a result of filter overlap, MBs may be included within the passband of two or more filters and could therefore be localized and paired multiple times. This \u201cMB doubling\u201d effect would lead to an overestimate of vascular density value  but should not influence the imaging the vasculature structure or morphology. Instead, \u201cMB doubling\u201d might be beneficial for maintaining the intensity of the ULM density image at the big vessel lumen, where the MBs signals would have been discarded without MB separation  will be the subject of a future study.This study has some limitations that should be addressed. The synthetic flow channel phantom data were combined under the assumption of purely linear interference, whereas an actual high concentration MB injection may have more complex and nonlinear interference characteristics. The purpose of using such a synthetic data was to artificially generate an extremely complicated MB flow dataset that contains different MB velocities and directions, in order to validate the capability of the proposed MB separation method in the differentiation of different MB sources. However, the synthetic combination of flow channel data produces conditions that are physiologically impossible, such as simultaneous counter-directional flow in a single vessel and MB boundaries that are truly spatially overlapping. We thus applied the proposed method to an individual flow channel data, which represented a realistic flow situation for a large vessel, and the results Fig.\u00a0 were in k-\u03c9 domain, increases the possibility of observing spatially sparse MB events and therefore alleviates the need for MB dilution. This method could substantially reduce the acquisition times required for the reconstruction of microvasculature, which is critical for the clinical translation of ULM.In this paper we demonstrate a substantial improvement in the performance of ULM at relatively high MB concentrations by using a post-processing technique to separate MB subsets based on their spatiotemporal characteristics. The application of the proposed MB separation algorithm, which consists of a series of 3D cone-shaped filters in the r. A MB moving in a straight line with a constant velocity v (and speed r0 at t\u2009=\u20090 can be described as:Assume B(k) is the Fourier transform of k-\u03c9 domain), where k-\u03c9 domain . Since MB signal is modulated by the center frequency (f0) of the transmitted ultrasound, its energy is centered at c is the sound speed, deviating from the baseband in k domain. This facilitates the reduction of overlap between MBs with different speeds. Therefore, by applying a bandpass cone-shape filter with frequency response:The spatiotemporal Fourier transform of the moving MB can then be described as:mentclass2pt{minimk-\u03c9 domain. Instead, we are extracting a group of MBs whose majority of spatiotemporal movement energies are concentrated inside the cone-shaped filter defined above, which may include energy from other MBs moving at other velocities. The hypothesis is that this undesired energy is suppressed inside the cone-shaped filter and, by subsequent post-processing like intensity thresholding and de-noising, this energy should be further reduced.It should be noted that we are not separating all MBs moving with all possible velocities, which is very difficult due to the theoretical overlap of MBs in k-\u03c9 domain also indicates the direction of moving MBs, with those moving towards or away from the transducer allocated in distinct quadrants. Therefore, MBs extracted from each cone-shaped filter  of the MB ultrasound data. It is hypothesized that MBs with different velocities correspond to different frequency components in the temporal direction . The amount of Doppler frequency shift is proportional to MB moving velocity along the ultrasound beam direction, where a positive frequency indicates the MB moving towards the transducer, and a negative frequency indicates the MB moving away from the transducer. Therefore, the MB signal within a certain speed range can be extracted by applying a bandpass filter with frequency response as follows:A custom-made flow channel phantom , with a 2-mm inner diameter straight flow channel which has an oblique angle of 30 degrees with respect to the surface of the phantom was used in this study. The outlet of the flow channel was connected to a syringe pump  that provided constant flow through the channel. Lumason MB suspension  was diluted with saline to approximately 1/2000 times the original concentration to provide a solution with adequately isolated MB signals. Ultrasound in-phase quadrature (IQ) data were acquired separately from the longitudinal view of the flow channel at four different flow rates  using a Verasonics Vantage ultrasound system  and a GE 9\u2009L linear array probe . A 10-angle coherent compounding plane-wave imaging (step angle = 1\u00b0) was implemented for data acquisition with a center frequency of 6.25\u2009MHz (single cycle pulse) for each transmit angle. The transmit voltage was lowered to 10\u2009V (one-side voltage) to minimize MB destruction. The frame rate was 500\u2009Hz, and for each flow rate setting, a stack of 6000 ultrasound frames (12\u2009s) was acquired. For each flow rate, the flow direction was reversed by switching the outlet connected to the syringe pump, and the data acquisition was repeated. Thus, a total of 8 independent datasets were acquired (4 flow rates and two directions) at a relatively low MB concentration. All 8 IQ datasets were then linearly added frame-by-frame to generate a synthetic ultrasound dataset, which represents a complicated flow scenario at higher MB concentration, to validate the proposed MB separation method for ULM. The 8 independent datasets, each at low MB concentration, can also be processed separately to produce 8 ULM images, and the combination can be used as a reference standard.No IACUC approval was required for the chicken embryo experiments presented in this paper, as under NIH PHS policy avian embryos are not considered to be live vertebrate animals.oC, 5% CO2, humidified incubator.Renca cells (ATCC CRL-2947) were obtained from the American Type Culture Collection Inc.  and cultured in accordance with ATCC guidelines. Growth medium was RPMI 1640 media  supplemented with 10% FBS , non-essential amino acids (0.1\u2009mM), sodium pyruvate (1\u2009mM), and L-glutamine (2\u2009mM). Cells were subcultured at a subcultivation ratio of 1:5 when >80% confluence. All cells were kept in a 37\u200929, with slight modification to facilitate co-registered acoustic-optical imaging. Fertilized chicken eggs were obtained from Hoover\u2019s Hatchery  and placed into a turning humidified incubator . For some preparations, acoustic windows were opened in the plastic weigh boat with a razor blade and sealed with saran wrap. On the fourth day of embryonic development (EDD-04) egg shells were opened with a rotatory dremel tool and contents were carefully transferred into the prepared weigh boats. Embryos were then transferred into a second humidified incubator (Caron model 7003-33) until ultrasound imaging on EDD-18.We followed the CAM preparation and tumor engraftment procedures outlined in5 cells per 10\u2009\u00b5L. This mixture was kept on ice until implantation into the CAM. On the engraftment day, EDD-09, a small disk of autoclaved Whatman No.1 filter paper was used to abrade the surface of the CAM, and 10\u2009\u00b5L of the cell mixture was placed in the wound. Embryos were then returned to the incubator.Renca cells of high confluence (>80%) were trypsinized  and pelleted (centrifuged for 5\u2009minutes at 300\u2009g) on the day of engraftment (EDD-09). Matrigel (BD Bioscience) was combined with the cell pellet to obtain an inoculation dose of 4 \u00d7 109 MBs/mL.All CEUS imaging was performed using an intravenous injection of the commercially available Bracco Lumason microbubble solution. For this study, MBs were reconstituted with 1.0\u2009mL sterile saline yielding a solution of approximately 1.8 \u00d7 10For CAM injection, an 18\u2009G \u00d7 1.5-inch beveled needle tip was attached to 8\u2009cm of Tygon R-3603 laboratory tubing. The open end of tubing was fitted with a glass capillary needle. With the aid of a dissecting microscope, the glass capillary needle tip was manually cannulated into a high-order vein on the CAM surface for contrast injection. A total volume of 70\u2009\u03bcL of the MB solution was injected into each embryo.For CAM membrane and tumor imaging, a 15-angle compounding plane-wave imaging was performed (step angle = 1\u00b0) using a Verasonics Vantage ultrasound system equipped with an L35-16vX high-frequency linear array transducer  operating at a center frequency of 25\u2009MHz. A two-cycle pulse was used for each transmit angle and the transmit voltage was set to 5\u2009V (one-side voltage). The post-compounded frame rate was 500\u2009Hz. For the CAM membrane, a total of 21 IQ datasets were collected from one bolus injection, with each dataset containing 720 ultrasound frames (corresponding to 1.44\u2009seconds data length per dataset for the given frame rate). For each CAM tumor, a total of 5 acquisitions were gathered at 720 frames of IQ data each, for a total length of 3600 frames (7.2\u2009seconds).Chick embryo brain imaging was performed using a Vevo 3100 high-frequency ultrasound system  equipped with an MX250S linear array transducer  transmitting at 21\u2009MHz (single-cycle pulse). Line-by-line focused imaging was used to scan the chick brain through the intact skull bone with a 16\u2009mm by 6\u2009mm field-of-view (FOV) and one fixed transmit focus, leading to a frame rate of 301 frames per second  at 4x magnification, and images were digitized with an integrated Nikon DS-Ri2 digital camera (Nikon). Optical images were acquired from the top of the weigh boat housing the embryo, and an L35-16vX ultrasound transducer was placed to image the CAM surface through a lateral acoustic window in the side of the weigh boat  using a 2-D spline interpolation. Following interpolation, an intensity threshold was applied to the envelope of MB signals to remove the noisy background, which would also help suppress undesired energy leaking from other subsets of MBs. A point-spread function (PSF) specific to the imaging system and settings was derived from a multivariate Gaussian function. A 2-D normalized cross-correlation between each frame of the interpolated MB signal and the derived PSF was performed for MB localization. A threshold was applied to the cross-correlation map to remove pixels with correlation coefficient values lower than 0.6, which further suppresses unwanted signals from other MB subsets while preserving high confidence MB signals for the given subset. Then, MB centroids were localized using a regional maximum search of the cross-correlation peaks for each frame. A bipartite graph-based pairing and tracking algorithm was then applied to the identified MB centroids, with a minimum persistence of 7 frames22. A Kalman-filter-based algorithm was then applied to each MB trace to further reject unreliable MB tracks based on the MB movement acceleration and direction constraints, and improve in-painting of the MB trace and MB speed measurement22.A spatiotemporal SVD-based clutter filter was first applied to the ultrasound IQ dataset to extract moving MB signals by rejecting background tissue signals and stationary MB signalsSupplementary information.Supplementary Figure S1Supplementary Figure S2Supplementary Figure S3"}
{"text": "Lactobacillus delbrueckii strain A01, Lactobacillus delbrueckii subsp. bulgaricus strain D01, Lactobacillus delbrueckii subsp. bulgaricus strain E01, Lactococcus lactis strain G01, Lactobacillus delbrueckii strain C01, and Lactobacillus delbrueckii subsp. bulgaricus strain F01 were identified using 16S rDNA sequencing, morphological and biochemical traits. All strains have been registered in the National Center for Biotechnology Information (NCBI) with accession numbers MN611241.1, MN611300.1, MN611301.1, MN611303.1, MN611241.1, and MN611299.1, respectively. Having found \u03b5-Poly-L-Lysine (\u03b5-PL) in all strains isolated, Lactobacillus delbrueckii strain A01 was identified as an active producer of \u03b5-Poly-L-Lysine (\u03b5-PL). The one-factor-at-a-time method and central composite design were applied to optimize \u03b5-Poly-L-Lysine (\u03b5-PL). A predicted 200\u2009ppm of \u03b5-PL was obtained in the medium containing the lowest level of glucose, 25\u2009g/l, and yeast extract, 6\u2009g/l.New strains of lactic acid bacteria (LAB) were isolated from different traditional dairy products. Six new strains named  Lactobacillus acidophilus reduced the cholesterol and triglycerides of rabbit blood , w, wA) andppm \u03b5-PL , consideLactobacillus delbrueckii strain A01, in optimal condition, could be regarded as a promising source of \u03b5-PL. The highest content of \u03b5-PL was attained in the medium containing the lowest content of protein and glucose. To conclude, a preserving activity of LAB in fermented dairy products could be somehow related to the \u03b5-PL produced by the wild LAB in traditional dairy products.The first attempt has been made to isolate and characterize LAB from the traditional fermented milk of the rural area near Shahrood in Iran. Six strains of LAB isolated from dairy products were identified and published with GenBank accession numbers. The result indicated that isolated strains named"}
{"text": "ADORA2A gene, coding for adenosine receptor type 2A (A2AR), has been involved in aberrant immune activation. Here we aimed to assess the prevalence of this SNP in 279 MCS patients and 238 healthy subjects, and its influence on ADORA2A, IFNG and IL4 transcript amounts in peripheral blood mononuclear cells of randomly selected patients (n = 70) and controls (n = 66) having different ADORA2A genotypes. The ADORA2A rs2298383 TT mutated genotype, significantly more frequent in MCS patients than in controls, was associated with a three-fold increased risk for MCS , while the CT genotype, highly prevalent among controls, resulted to be protective . Notably, ADORA2A mRNA levels were significantly lower, while IFNG, but not IL4, mRNA levels were significantly higher in TT MCS patients compared with controls. A significant negative correlation was found between ADORA2A and both IFNG and IL4, while a significant positive correlation was found between IFNG and IL4. These findings suggest that A2AR defective signaling may play a relevant role in PBMC shift towards a pro-inflammatory phenotype in MCS patients.Systemic inflammation and immune activation are striking features of multiple chemical sensitivity (MCS). The rs2298383 SNP of  Multiple chemical sensitivity (MCS), also called environmental sensitivity illness (ESI) or toxicant-induced loss of tolerance (TILT), involves an aberrant susceptibility response to a broad range of chemical substances present in daily life . In the Despite the absence of validated diagnostic biomarkers, the epidemiological evidence has led the individual countries to at least partially recognize MCS as a pathological state. In Europe, in particular, Germany and Austria classified MCS under the ICD-10 code T78.4  [d-aspartate (NMDA)-nitric oxide/peroxynitrite triggered by various chemical agents, that ultimately lead to oxidative stress and activation of pro-inflammatory transcription factors [To date the proposed etiopathogenetic mechanism involves an aberrant activation of the vicious cycle N-methyl- factors . Indeed, factors ,12,13,14 factors ,19,20,21ADORA2A gene, encoding for A2AR, is a functional variant that may affect the rate of gene transcription due to its location within a regulatory sequence of the gene [The release of pro-inflammatory cytokines, as well as the activation and proliferation of T cells, can be inhibited by adenosine receptor activation. Adenosine receptor 2A (A2AR), one of four adenosine receptor sub-types, is present in almost all immune cells, including lymphocytes, monocytes, macrophages and dendritic cells, and its activation increases the production of anti-inflammatory cytokines . The sinthe gene ,24. Notathe gene .ADORA2A gene, and the consequent effects on the pro-inflammatory phenotype shift of peripheral blood mononuclear cells (PBMC) isolated from both MCS patients and healthy controls.We here aimed to assess the prevalence of ADORA2A rs2298383 polymorphism in MCS patients as well as age- and gender-matched healthy subjects, in order to establish a possible association of this SNP with MCS syndrome. Moreover, we evaluated the influence of the rs2298383 SNP on the transcription levels of ADORA2A rs2298383 SNP, and results are shown in p = 0.928), and with those reported for Italian population in the 1000 Genomes project (http://asia.ensembl.org/Homo_sapiens/Variation/Population?db=core), while they were found to deviate from the expected value by HWE (p = 0.000000) in the group of MCS patients. The T mutated allele was more frequent among MCS patients than among healthy subjects, and was predominant in homozygous state, while in controls it was more frequently found in heterozygous state (Both patients and controls were genotyped for us state . The mutus state . The wilp < 0.0001). Instead, a negative association with MCS was observed for the heterozygous CT genotype, that was significantly more frequent among healthy subjects than in MCS group. Individuals that were carriers of this genotype had a three fold-decreased risk for MCS .The mutated TT genotype resulted to be associated with MCS disorder, since the Odds Ratio calculation showed that the risk of developing MCS syndrome increases by about three folds in individuals who have this genotype , and in comparison with MCS patients having other genotypes . Significant differences were also found when MCS having either CC or CT genotype were compared with healthy subjects carriers of the same genotypes . No statistically significant differences were found among controls having different ADORA2A genotypes , while no statistically significant differences were found in comparison with MCS patients having other genotypes. No statistically significant differences in IFNG mRNA levels were found when comparing the different genotypes within the healthy cohort. Interestingly, statistically significant differences were observed between either CC or CT MCS subjects and their healthy counterpart .The IL4 mRNA levels were observed between MCS cases and controls or between different genotype subgroups within the two cohorts , while IL-4 concentrations were 1.5 fold-increased in MCS cases compared with controls . However, only IFN-\u03b3 protein levels resulted to be significantly different between the two groups, likely due to the small number of subjects examined.The assessment of secreted cytokine amounts showed that the mean IFN-\u03b3 serum concentrations were three-fold increased in MCS patients compared with healthy subjects  and IL4  mRNA expression levels, while a significant positive correlation was found between IFNG and IL4 mRNA expression levels .Notably, a significant negative correlation was found between In the last decade, literature data provided evidence for the association of MCS with increased oxidative/nitrosative stress and inflammation ,12,13,14ADORA2A rs2298383 SNP, that has been shown to affect A2AR signaling [ADORA2A rs2298383 TT mutated genotype, being largely prevalent in MCS patients, is associated with MCS and represents an unfavorable genetic background for this disorder. Instead, the heterozygous CT genotype, more frequent among healthy subjects, is negatively associated with MCS and displays a protective effect due to a three-fold decreased disease risk. The ADORA2A rs2298383 genotype distributions observed in our control group agree with those previously reported for Caucasian population [http://asia.ensembl.org/Homo_sapiens/Variation/Population?db=core), an optimal reference population which contains allelic frequencies for a sample of 107 subjects from Tuscany, Italy.In this study, we first investigated the prevalence of ignaling ,30, in Cpulation  and in 1ADORA2A transcript levels in MCS cases. These findings are apparently in contrast with previously made assumptions. Indeed, on the basis of in silico analysis it has been hypothesized that the wild-type CC genotype is associated with a drastic decrease in ADORA2A transcription levels and amount of A2AR molecules, with functional consequences comparable to the receptor blocking by antagonists [ADORA2A haplotype including the rs2298383 CC genotype, showed higher amounts of ADORA2A mRNA and A2AR protein compared with those having different haplotypes [Also interestingly, the TT genotype was associated with a dramatic reduction in agonists ,32. Howeplotypes . The preplotypes , and sugInterestingly, given that A2AR is abundantly present in olfactory bulb , it is pIFNG and IL4 were higher in subjects carriers of the ADORA2A rs2298383 TT mutated genotype than in subjects having either CC or CT genotype. Moreover, the highest serum concentrations of these cytokines were found in TT MCS patients that also had the lowest amount of ADORA mRNA transcripts. These observations are supported by the significant negative correlation found between ADORA2A mRNA and both IFNG and IL4. Our data partially replicated the findings of Dantoft et al. [We here also first provided a confirmation of this hypothesis by showing that mRNA levels of t et al. , that deADORA2A expression in PBMC of MCS patients.IFN-\u03b3 plays a key role in macrophage activation, inflammation, and host defense against intracellular pathogens. The inhibition of expression and function of anti-inflammatory molecules represents a key mechanism of IFN-\u03b3-mediated priming of enhanced innate immune responses . The higIL-4 is a cytokine associated with the activation of Th2 cells, and has long been considered as one of the tolerogenic cytokines in many autoimmune animal models and clinical settings; however, its overproduction has mainly been associated with the development of allergic reactions and atopy . NotablyADORA2A homozygous mutated genotypes were shown to have lower habitual caffeine consumption compared with wild-type ones, likely due to a mechanism of negative feedback [ADORA2A heterozygous genotype in the general Caucasian population supports the hypothesis that this genetic trait was selected as a result of adaptive evolution to the daily intake of caffeine, the excess of which may display well known toxic side effects for the body. Thus, it is possible to speculate that the ADORA2A rs2298383 TT mutated genotype has been subjected to positive selection because it is protective against the long lasting effects of caffeine resulting from its slowed metabolism. A side effect of this natural selection is the increased susceptibility to the onset of inflammatory processes and dysregulation of immune system, as observed in MCS patients that have a defective metabolism of xenobiotics.It is well known that adenosine effects are reduced by caffeine and its metabolites, acting as competitive inhibitors at adenosine receptors throughout the body . Individfeedback ,31,42,43ADORA2A mRNA expression in a dose- and time-dependent manner [These observations suggest that recommendations of family practitioners to MCS patients should include drastic reduction or even elimination of caffeine, that has been shown to reduce t manner . Moreovet manner ,28, coulOur preliminary findings suggest the important role of A2AR signaling in the chronic inflammatory process associated with MCS. Further replication studies will be useful to confirm the present observations.This study conforms with the Declaration of Helsinki (amend. 2013). All subjects recruited for this study provided written informed consent to the collection of anamnestic data and peripheral blood sampling in EDTA tubes aimed to carry out research investigations.This study was carried out using a biobank of whole blood samples collected from 279 Italian MCS patients , and 238 Italian healthy volunteers . The enrollment of MCS patients was mostly carried out at Department of Medical Pathophysiology, University of Rome \u201cLa Sapienza\u201d, Polyclinic Umberto I, and at IDI IRCCS .All subjects, presenting with symptoms compatible with MCS and QEESI score > 20 ,18,19,43Healthy subjects were recruited either among staff members of Polyclinic Umberto I, University \u201cLa Sapienza\u201d and IDI IRCCS , or among staff members and volunteer blood donors at Polyclinic Hospital University of Messina , according to the following established inclusion criteria: a) absence of any clinically diagnosed disease; b) absence of allergic or immunologic disturbances; c) absence of supplementation with drugs or nutraceutical compounds in the last six weeks, at the time of blood sampling; d) absence of oxidative stress, i.e., whole blood total production of ROS/RNS below 500 cps/\u03bcL, as previously reported on the basis of a luminol-dependent chemiluminescent response to phorbol 12-myristate 13-acetate ; e) non-Genomic DNA (gDNA) was isolated from peripheral white blood cells using the Gentra Systems PureGene-DNA purification kit , according to manufacturer\u2019s instructions. The DNA was quantified by spectrophotometric measurement at 260 nm using a Biophotometer . DNA quality was considered acceptable for samples having a 260/280 ratio \u2265 1.6. DNA integrity and the presence of contaminant RNA were checked by electrophoresis on 0.8% agarose gel, and subsequent UV detection of DNA bands using a gel photodocumentation system .The screening for the presence of ADORA2A rs2298383 (C > T) SNP was performed by Real-time PCR-based allelic discrimination using a pre-designed TaqMan-based Genotyping Assay , as previously described . PCR rean = 70; 18M, 52F) and healthy subjects , having different ADORA2A rs2298383 genotypes . PBMC were isolated from 300 \u00b5L of whole blood samples, after red blood cells lysis by RBC buffer available from Gentra Systems PureGene-DNA purification kit . The isolated RNA was quantified by spectrophotometric measurement at 260 nm using a Biophotometer , then, 2 \u03bcg RNA were reverse-transcribed to cDNA using the High-Capacity cDNA Archive kit  according to manufacturer\u2019s instructions.Total RNA was extracted and purified from PBMC of randomly selected MCS patients (BACT) as endogenous control. PCR reactions were set up in a 96-well plate on a 7900HT Fast Real-Time PCR System , and were carried out in a final volume of 20 \u03bcL, containing Master mix (2\u00d7) Power up SYBR Green PCR , 0.2 \u00b5M for each primer, and 40 ng of cDNA. The used primer sequences were 5\u2032-GGCTGCCCCTACACATCATC-3\u2032 (fwd) and 5\u2032-GCCAGGTACATGAGCCAGAGA-3\u2032(rev) for ADORA2A, 5\u2032-GCAGCCAACCTAAGCAAGAT-3\u2032 (fwd) and 5\u2032-TCACCTGACACATTCAAGTTC TG-3\u2032 (rev) for IFNG, 5\u2032-CCTCTGTTCTTCCTGCTA-3\u2032 (fwd) and 5\u2032-AGATGTCTGTTACGGTC-3\u2032 (rev) for IL4, and 5\u2032-TTGTTACAGGAAGTCCCTTGCC-3\u2032 (fwd) and 5\u2032-ATGCTATCACCTCCCCTG TGTG-3\u2032 (rev) for BACT. A final dissociation curve analysis was carried out to evaluate the amplification specificity.mRNA transcript levels were quantified by SYBR green-based Real-time PCR, using \u03b2-actin , due to unavailability of serum samples. In order to minimize gender variations only female patients were included in these analyses. The measurements of cytokine serum levels were performed using a commercially available assay  as described by De Luca and co-workers .ADORA2A rs2298383 genotype distributions to HWE, based on a Web program (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl).Continuous data are expressed as means \u00b1 standard deviation (S.D.), and the categorical variables as number and percentage. The Fisher\u2019s exact test was applied to test the difference between cases and controls in terms of categorical variables, and the compliance of ADORA2A mRNA levels as the primary outcome variable. Taking into account a power of 80% and an alpha of 0.05, and a number of groups equal to three (as per three different genotypes), it was determined that a minimum number of 69 MCS patients would be needed. Thus, we chose to analyze gene expression levels in 70 MCS subjects.A sample size analysis was performed to calculate the minimum sample size required for gene expression studies. The sample size was calculatedconsidering ADORA2A, IFNG and IL4 between MCS group and control group were analysed by Mann-Whitney test, while differences between subgroups having different ADORA2A genotypes within either the MCS cohort or healthy cohort were analyzed by Kruskall-Wallis test. A Spearman correlation analysis was performed to test the relationships between the examined continuous variables.The distribution of values obtained by gene expression studies was tested by Kolmogorov-Smirnov test, and was not found normal. As a consequence, non-parametric tests were used for subsequent statistical analyses. Differences in the mean mRNA total levels of p-value \u2264 0.05 was considered statistically significant for all the analyses.Statistical analyses were performed using the software GraphPad Prism 5 . A"}
{"text": "Overall, the results identified purchase/transport time and purchase order as the emerging and unchanged risk factors, respectively. Consumers\u2019 preferences into channels for purchase  and transport (using cars or delivery) implied the convenience as the noticeable trend. Whereas, unexpected increases in purchase/transport time highlighted the underestimated risks in long-term exposure of foods under inadequate temperature. Food should not be exposed to danger zones > 1\u20132 h, but consumers might be unaware of the risk especially for preferred channels . In the case of unchanged risky behavior, more than half of consumers in both surveys did not follow proper purchasing orders. Our findings highlight the necessity for novel countermeasures and the improvement of current consumer guidelines against emerging and unchanged risky behaviors, respectively.Although consumers\u2019 food purchase/transport have been reported as causes of food safety risks, there is a lack of empirical data that are feasible to identify persistent and emerging risky behaviors of consumers. This longitudinal trend study consists of individual consumer surveys in 2010 ( Consumers\u2019 food and meal preparation behaviors from shopping to consumption have been associated with various human health issues, including foodborne diseases ,2,3. FooBecause consumers\u2019 improper behaviors from the first step of the food preparation affect the risk level at following stages  , food puThe present study aims to conduct consumer surveys on risky behaviors during food purchase/transport and their changes over time. Comprehensive analysis on time-temperature control is needed in order to estimate the actual level of risks derived from consumers\u2019 food purchase/transport behaviors, as follows: (1) consumers\u2019 preferences for food purchase and transport methods, including where and how temperature abuse can occur; 2) food purchase and transport time, including whether the exposure of food to ambient environments exceeds the recommended limit; and, (3) risk perceptions of food purchasing, namely, whether consumers aware of the proper purchasing order and which factors affect the purchased foods. However, there has been no survey research on food purchase and/or transport to analyze not only consumers\u2019 preferences, but also the time that is required for each step and consumers\u2019 risk perceptions regarding time-temperature control and microbiological risk factors ,19,20. M food purn = 605) and 2010 (n = 609) with the same questionnaires regarding the following determinant factors for risky behaviors during food purchase/transport: consumer preferences for purchase/transport methods, time that is required for purchasing/transporting food products, and risk perceptions. A comparative analysis of the surveys was conducted to identify the major changes in each factor over time. Results from longitudinal analysis are expected to reveal the changes in the consumers\u2019 behaviors during a decade and the insights whether those changes are positive or negative with the view to the proper consumer guidelines. The identification of emerging behaviors that can raise the risk level will imply the direction for establishing feasible countermeasures taken into consideration to drive the alteration of the behaviors of contemporary food consumers.In this study, quantitative surveys of primary food handlers were conducted in 2019  who were the main people involved in food preparation at home (hereafter defined as primary home food handlers) were recruited as the interviewees. Previous studies regarding home food safety have reported that hygienic perceptions and behaviors vary according to sociodemographic characteristics  ; these cA questionnaire was used as the research instrument for the survey and a same questionnaire was used as the research instrument for the longitudinal trend surveys that were conducted in 2010 and 2019. A consulting committee organized by a government institution  with experts in consumer surveys , consumer organization, and food safety and hygiene laboratory  developed the questionnaire. All members of the consulting committee participated in composing a draft questionnaire and then revising the final version to verify its applicability. In addition, Gallup Korea reviewed the survey instrument for clarity and validity.The questionnaires were developed for the identification of consumers\u2019 behaviors with perceptions linked to the risks of food purchase/transport. Those behaviors and the questionnaire contents were mainly organized based on the internationally recognized food safety guidelines provided by major institutions, including Food and Drug Administration (FDA), Food Safety and Inspection Service in U.S. Department of Agriculture (USDA FSIS) ,15,17. Gn = 15; randomly selected) and expert researchers . The pretesters were asked to evaluate the questionnaire with the perspectives to the terminologies that needed to be revised to improve clarity, unclear, and/or difficult expressions, and contents that might induce the respondents to feel displeasure or resistance during the survey [To revise the draft questionnaire for clarity and validity, a pilot test was conducted by the pretesters who were consumers , food purchase/transport time (Q3\u2013Q4), and risk perceptions (Q5\u2013Q7). The specific questions were, as follows: Q1. Where do you buy each food ? Q2. How do you transport purchased food from each place to your home? Q3. How long does it take you to buy food at each place? Q4. How long does it take you to transport the food? Q5. What do you buy first between food and nonfood items? Q6. What do you buy first between refrigerated/frozen foods or foods that can be stored at room temperature? Q7. Which factors do you consider when purchasing food?n = 609) and 2019 . The, same questionnaires were and method were used for sociodemographic group from each survey to examine how behaviors with risk perceptions from socio-demographic groups of primary food handlers have changed over time [The present study is constructed with two individual surveys conducted in 2010  was utilized to analyze the obtained data.Consumers\u2019 preferences regarding transport methods also showed distinct changes toward more convenient methods in survey 2 (2019) when compared with survey 1 (2010): (1) an increase in the use of cars or delivery rather than walking to traditional markets or supermarkets/small markets near home, and (2) an increase in the use of delivery from large discount stores and department stores . GrowingConsumers should buy nonfood stuffs and foods that can be stored at room temperature earlier, followed by refrigerated and frozen foods. However, as shown in Respondents from both surveys indicated the \u2018shelf life\u2019 as the information of highest interest, as shown in Problems in time-temperature control during consumers\u2019 food purchase and transport have also been linked to the potential risks in food quality and safety, as the occurrence of the deterioration of food products at the post-harvest or post-processing levels are mostly attributed to inadequate infrastructure for storage and/or transport . This trConsumers\u2019 preferences into purchase and transport channels implied the convenience as the noticeable trend ,47. The Increases in total food purchase and transport time suggest the importance of time-temperature control. Although food safety guidelines suggest following the \u201c2-h rule and 1-h rule\u201d  ,15,17,52Perceptions of microbiological risks that are derived from purchasing orders are representative unchanged risky behavior over time. These results indicate that many consumers overlook the importance of time-temperature control of food at the purchasing step. Although previous studies regarding consumers\u2019 risky behaviors of food preparation have reported improved risk perceptions and knowledge  ,55, imprThis study newly identified risk factors of food purchase/transport, highlighting the impact of consumers\u2019 behavior studies which have been mainly focused on hygienic practices during the food preparation steps after the food purchase/transport ,60,61,62Our longitudinal trend study implied the necessity of the improvement on conventional consumer guidelines to cover contemporary trends in food purchase/transport with the perspectives to the time-temperature control during food purchase/transport: (1) chilling of perishable foods during transportation, especially in car trunks; (2) handling of delivered groceries from receipt to storage; (3) purchasing of food in the proper order; and, (4) compliance with the \u201c2-h rule and 1-h rule\u201d by considering the temperature of the environment to which foods are exposed and the total time required for food purchase/transport. Since unchanged and emerging risky behaviors observed in this study also highlighted the limitation of current risk management represented by the guideline, the development of novel consumer education materials and programs  should be considered. To sum up, the implications from this research are expected to suggest food purchase/transport as underestimated topic in the research area of the link between food preparation behaviors and consumers\u2019 health. Because this study focused on the identification of risky behaviors rather than the establishments of the causes and/or factors for those behaviors, further researches should be followed in order to identify the major determinants of identified risks , especially for the multiple factors that can increase risk levels."}
{"text": "We propose here a method to analyze whether financial and macroeconomic shocks influence the entropy of financial networks. We derive a measure of entropy using the correlation matrix of the stock market components of the DOW Jones Industrial Average (DJIA) index. Using VAR models in different specifications, we show that shocks in production or the DJIA index lead to an increase in the entropy of the financial markets. The use of networks to analyze economic and financial phenomena is developing fast. There have been numerous recent significant contributions that have further developed our understanding about the potential of using networks or network-based perspectives in studying financial phenomena see ,6,7,8,9),7,8,96,7Among the approaches used, the entropy-based methods have provided valuable results. We can include here the contribution of  who apprAnother strand of research using entropy has focused on its capacity to reveal the state of the financial markets. For example, Ref.  has showA related research looked at the long run relationship between stock and commodities (see ). The maIn a recent paper, Ref.  has showIn this paper, we aim at building upon recent contributions on the use of entropy in modelling financial markets. Specifically, we aim at looking at whether and how macroeconomic and financial shocks contributed to the variation in entropy of financial markets. To achieve this, we built on previous studies that also used time-varying measures of entropy derived from correlation matrices (see  or 15]))15]), anThere are several contributions that we make in this paper. First, we propose one of the first studies that approaches the changes of the structure of networks following macroeconomic and financial shocks. Second, we characterize the changes in the networks through the use of entropy derived on the basis of matrix correlations of stocks composing the network.This section details the methods and tools used in the paper. The techniques are quite standard; however, the presentation might be of interest to those not familiar with it.Our approach to construct correlation-based matrices is standard. We look at the correlation between the stock components of the DOW Jones Industrial Average index  properties of the correlation matrices, as seen from the next sections.The singular value decomposition , when applied to a matrix allows us to obtain the following decomposition:A, with m rows and n columns. The matrix U has m rows and k columns, while V has n rows and k columns. The matrix S can then be written by:k is determined through the expression S has two important properties, namely positive elements as well as a decreasing order for the values of Here, we applied the decomposition to a given matrix It must be underscored (we thank a reviewer for pointing this out) that the initial matrix is squared and we can just define the E, throughout the text. The construction of the E measure of entropy is based on previous contributions by ..dot-com In this section, we take a first step towards answering the main research question of the paper: how do macroeconomic and financial shocks affect the entropy of the financial markets? We test here for Granger causality between interest rate (as well as shadow interest rate), the inflation, industrial production index as well as DJIA, on one hand, and the entropy (computed either using a 2-year or a 3-year window), on the other hand.The role of this step is to establish, in a bivariate setting, whether the selected macroeconomic and financial variables have predictive power with respect to the entropy of financial markets. While this test is limited through the simple use of a bivariate setting and the lack of a structural model, it is still an important step before using a VAR model as we do in the next section.The results are shown below in For the case of the industrial production, the evidence is the strongest, with evidence of Granger causality at almost all the lags. In contrast, there is no evidence for Granger causality from inflation to entropy. Finally, for the case of the DJIA index, the evidence that supports the Granger causality is limited to just one lag.The evidence presented here, although not enough per se, justifies considering a more extensive econometric framework in the next section.In this section, we aim at answering the main research question in this paper: if and how financial and macroeconomic shocks affect the entropy of financial markets. To answer it, we build a VAR model consisting of monthly frequency series. We use the following set of variables: c is a vector of constants Formally, the VAR model is written as follows:The variables, except the interest rate (as it is a custom in the literature), are log-differenced, since they have a unit root see . We testWe consider a baseline estimation, with the federal funds rate as the measure for the interest rate, and, additionally, the shadow policy rate, for an alternative estimation. Furthermore, for robustness, we also consider an alternative measure of entropy based on a three-year moving window.To identify the structural shocks, including the monetary policy shocks, we use a Choleski ordering of the variables, with the following ordering: industrial production, consumer price index, interest rate, entropy, DOW 30. This is a rather typical ordering, with the variables moving the fastest, i.e., the stock market variables (including the entropy) ordered as last. The setting implies that the industrial production and the consumer price index do not contemporaneously react to interest rate changes, while the interest change does not respond to contemporaneous changes in the entropy measure or in the stock market.This approach allows us to uncover whether the macroeconomic and financial variables considered here have any statistically significant impact on the main variable of interest, i.e., the entropy of the financial markets. The approach relies on a certain setting for identifying the structural shocks, see above, and thus it is better fit to answer the main question of the paper as compared to the simple Granger test for causality.x-axis indicates the horizon of the response in months. log_entropy2y is the log-difference for entropy using a two-year window, while log_entropy3y is computed using a 3-year window, log_ip is the log-difference for industrial production, intrate is the interest rate used in the model and log_dow is the log-difference of the DJIA index.The results for the estimated baseline VAR models using the federal funds rate as the interest rate are shown below. We present the impact of the different structural shocks on entropy. The We also performed a robustness exercise, using a similar setting and the same variables, except the interest rate, for which the shadow rate is used. The alternative estimates are shown in This section provides a tentative answer to the main question of the paper: if and how do the macroeconomic and financial variables influence the entropy measure of financial markets. We found that there is a statistically significant role for industrial production and the DJIA index. While we would have expected a statistically significant impact of the stock market dynamics, the finding related to the industrial production index raises interesting issues that are worth studying further.This finding could imply that the connections between the nodes of the financial market also reflect a strong connection with the real economy. On one hand, we know that stocks tend to organize themselves in financial networks in clusters following the specific of their activity . On theThe main purpose of this study was to extend the previous literature regarding the modelling of the entropy within a macro-finance framework. We started by considering the derivation of entropy based on a moving window consisting of the DOW 30 index components for which we computed the correlations. Using a singular value decomposition, and a sliding window of 2 or 3 years of observations, we were able to derive a time-varying measure of entropy.The principal contribution of the paper was to use the derived measure of entropy within a VAR framework including the industrial production, the inflation, the interest rate, the entropy as well as the DOW 30 returns. In this modelling framework, we were able to determine the impact of financial and macroeconomic shocks on entropy.In our VAR framework, entropy responds positively to shocks in the monetary policy, although the impact is hardly statistically significant. However, there are positive responses that are statistically significant following industrial production shocks (after 2\u20133 months), as well as a positive response after a shock in the stock market (DJIA).Furthermore, the results are robust to the use of different interest rate measures , as well as to the different ways to construct the entropy (by varying the size of the moving window).Compared to the results when using the simpler bivariate Granger causality tests, we see that the industrial production leads again to significant responses in entropy, while the inflation rate does not significantly impact it. There is a positive impact by the interest rate, although barely statistically significant. However, the DJIA index has a significant impact, although the results for the Granger causality tests were quite weak. The two frameworks are, however, not fully comparable since the VAR framework is multivariate and uses a certain procedure for identifying the structural shocks, while the Granger causality test is a simpler bivariate framework.This is one of the first contributions not only to employ the entropy of a financial network in a typical macro-financial model, but also to obtain evidence regarding the movement of entropy following macroeconomic and financial shocks. The results are significant, since they show that the networks are not static at all, but they do react to changing in the macroeconomic and financial conditions.The contributions here can be further developed and tested in many ways, and they show that there is a significant potential for entropy measures of networks to be used within standard macroeconomic and financial models."}
{"text": "Evidence suggests that microRNAs (miRNAs) regulate the expression of genes involved in bone metabolism. This study aimed to investigate the role of miR-505 in the osteogenic differentiation of MC3T3-E1 cells.We performed miRNA sequencing to identify differentially expressed miRNAs between MC3T3-E1 cells treated with osteogenic induction medium (OIM) and control cells. Bioinformatics analysis was performed by using the TargetScan and miRDB databases. The expression of miR-505 in MC3T3-E1 cells was detected during osteogenic differentiation. After transfection with miR-505 mimic or miR-505 inhibitor, MC3T3-E1 cells were induced to differentiate into osteoblasts, and the expression of osteogenic differentiation markers (Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN), and osterix (OSX)) was detected.miR-505 was the most downregulated miRNA among the differentially expressed miRNAs. The RUNX2 gene was identified as a potential target of miR-505 using the target prediction program. miR-505 expression was downregulated during osteogenic differentiation of MC3T3-E1 cells. The expression of osteogenic marker genes was inhibited in MC3T3-E1 cells after transfection with miR-505. However, the expression of osteogenic marker genes was upregulated after transfection with miR-505 inhibitor.This study is the first to report miR-505 could bind to the RUNX2 gene and thus regulate partly the dysfunction of osteoblasts differentiation, which is expected to be targets for the treatment of osteoporosis. Osteoporosis is a chronic systemic bone disease manifesting as lower bone mass, which eventually contributes to increased risk of fractures , 2. SevemiRNAs are a class of multifunctional noncoding small RNAs that regulate cell functions and signaling pathways by inhibiting the expression of target mRNAs , 7. miRNTo clarify involvement of miRNAs in osteogenic differentiation of MC3T3-E1 cells, we performed microRNA array and found that miR-505 was the significantly differentially expressed miRNA. In experiment study, we found that miR-505 was downregulated in the process of osteogenic differentiation of MC3T3-E1 cells. Moreover, we also determined that miR-505 could directly bind to RUNX2 and thus inhibit osteogenic differentiation.The following reagents were used in this study: \u03b1-MEM medium ; fetal bovine serum (FBS) and Opti-MEM medium ; 0.25% trypsin, dexamethasone, ascorbic acid, and \u03b2-glycerol phosphate ; Lipofectamine RNAiMAX reagent, TRIzol reagent, and protein marker ; PrimeScript RT reagent Kit with gDNA Eraser kit and SYBR Green PCR Master Mix ; miRNA quantitation and U6 calibration qRT-PCR kit, miR-505 mimics, inhibitor, and negative control (NC) ; Alizarin red staining reagent ; RUNX2, OSX, OPN, ALP, GAPDH, miR-505, and U6 primers ; BCA protein assay kit , RIPA lysis buffer, and SDS-PAGE Gel configuration Kit ; PVDF membrane and chemiluminescence ECL solution ; anti-OPN, anti-OCN, and anti-GAPDH antibodies ; anti-RUNX2 antibody ; anti-ALP antibody ; and other reagents generated by our group.2 and 37\u2009\u00b0C, and the cells were subcultured once a day. A single cell suspension was prepared from MC3T3-E1 cells growing under steady-state conditions and inoculated in a 6-well culture plate. The density of MC3T3-E1 cells was more than 80% after plating in a 6-well culture plate, and the osteogenic induction medium , 50\u2009\u03bcg/mL ascorbic acid , and 10\u2009mmol/L \u03b2-glycerophosphate sodium ) was replaced every 2\u2009days.Mouse progenitor osteoblasts MC3T3-E1 were purchased from the Cell Bank of the Chinese Academy of Sciences. The cell culture medium was 10% FBS in MEM containing 0.35\u2009g/L glutamine. The cells were cultured in a constant-temperature cell culture incubator at 5% CO5 cells per well) were initially seeded in a six-well plate, at 70% confluence; the cells were treated with growth medium (control) or OIM for 10\u2009days, respectively.The RiboArray miDETECT mouse array  was used to detect the microRNA expression levels in 3 independent replicates for MC3T3-E1 without osteoblast induction and 3 independent replicates for MC3T3-E1 which underwent for 10\u2009days in OIM. Briefly, MC3T3-E1 cells  according to the manufacturer\u2019s instructions. NanoDrop ND-1000 (Thermo) was used to measure the concentration of purified RNA and detect the quality of RNA. RNA labeling, microarray chip hybridization, and scanning were performed according to the method and instructions provided by the Affymetrix Multispecies miRNA-2 Array.http://www.targetscan.org/vert_71/) and miRDB (http://mirdb.org) databases. Then, a Venn diagram of the results from these two databases was generated using Venny 2.1 (http://bioinfogp.cnb.csic.es/tools/venny/). Overlapping genes were then submitted to DAVID 6.8 (https://david.ncifcrf.gov/tools.jsp) to identify Gene Ontology (GO) categories , molecular function (MF), and cellular component (CC)) and perform KEGG pathway analysis.First, we predicted miR-505 targets using the TargetScan  of each specimen and the internal reference gene U6 were obtained. The relative content of miR-505 was calculated relative to housekeeping U6 with the 2equation . The relequation . The expMC3T3-E1 cells were induced to undergo osteogenesis for 21\u2009days, after which the culture medium was discarded, and the cells were washed with PBS 2 times, fixed with 4% paraformaldehyde for 30\u2009min, washed with PBS 2 times, stained with alizarin red 10\u2009min, washed completely with PBS, and observed by inverted microscopy.5/mL. MC3T3-E1 cells were randomly divided into 4 groups: miR-505 mimics, miR-505 mimics-NC, miR-505 inhibitor, and miR-505 inhibitor-NC. Lipofectamine RNAiMAX liposomes were used for transfection. Then, miRNA duplexes at a concentration of 30\u2009nmol/L were mixed with 150\u2009\u03bcL Opti-MEM medium. Concurrently, Lipofectamine RNAiMAX reagent was mixed with 150\u2009\u03bcL Opti-MEM medium at 7.5\u2009\u03bcL per well. After incubation for 5\u2009min, the miRNA and Lipofectamine RNAiMAX solutions were mixed and incubated at room temperature for 20\u2009min. The medium in the 6-well plate was discarded, and 700\u2009\u03bcL Opti-MEN culture medium was added to each well. Then, the incubated miRNA/Lipofectamine RNAiMAX mixture was added to each well and gently mixed. After the cells were incubated in a constant-temperature cell culture incubator at 37\u2009\u00b0C for 6\u2009h, \u03b1-MEM with 10% FBS was added, and the cells continued to be cultured. Then, the osteogenic induction medium was added for 3\u2009days.MC3T3-E1 cells were digested by trypsin and resuspended in \u03b1-MEM containing 10% FBS. The cells were inoculated in 6-well culture plates at a density of 2.5 \u00d7 10To determine the success of miR-505 transfection, the expression of miR-505 was detected by real-time quantitative RT-PCR. After transfection with 15, 30, or 60\u2009nmol/L concentration of miR-505 mimics, miR-505 mimics-NC, miR-505 inhibitor, or miR-505 inhibitor-NC for 24\u2009h, total RNA was extracted to detect the mRNA expression and miR-505 expression levels. Optimal concentration was identified as used for further experiments. All of the transfections were performed by using Lipofectamine 3000  based on the provided directions [The cells were transfected with miR-505 mimics, miR-505 mimics-NC, miR-505 inhibitor, or miR-505 inhibitor-NC for 24\u2009h and cultured for 3 additional days after replacement of the medium with osteogenic induction medium. The mRNA expression of RUNX2, OSX, ALP, and OPN in cells was detected by collecting and extracting total RNA.MC3T3-E1 cells were transformed with miR-505 mimics, miR-505 mimics-NC, miR-505 inhibitor, or miR-505 inhibitor-NC and induced to undergo osteogenesis for 3\u2009days. Total protein was collected and lysed with RIPA buffer. The protein concentration was determined by BCA protein quantitative test. The total protein was transferred to a PVDF membrane by SDS-PAGE. After blocking with skimmed milk powder, 1:1000-diluted anti-RUNX2, anti-OPN, anti-OCN, anti-ALP, and anti-GAPDH antibodies were added overnight at 4\u2009\u00b0C. After rinsing, the secondary antibody was incubated at 1:2000, at room temperature for 2\u2009h.5 cells per well. 3\u2032 UTR of the RUNX2 gene containing putative miR-505 targeting site was amplified by chemical synthesis and was inserted into the psiCHECK2 vector . When the confluence was up to 70%, MC3T3-E1 were transfected with related mixtures including 50\u2009ng RUNX2 wild-type or RUNX2 mutant-type 3\u2032 UTR reporter plasmids, miR-505 mimics or miR-505 mimics NC in a final concentration of 20\u2009nM, and Lipofectamine 2000 for 48\u2009h. Luciferase activity was detected using the dual-luciferase reporter gene kit .MC3T3-E1 cells were firstly inoculated into 12-well plates at a density of 1 \u00d7 10t test. The results were considered to be statistically significant when P < 0.05. All statistical analyses were performed using SPSS 19.0 .The experimental data are expressed as the mean \u00b1 standard deviation. Differences between two groups were analyzed by two-sided Student\u2019s P value \u2264 0.05). Among the differentially expressed miRNAs, miR-550 was the most downregulated miRNA. A heatmap of differentially expressed miRNAs between Con and OIM groups is shown in Fig. We used volcano plot to show the inter-relationships between differentially expressed mRNAs in MC3T3-E1 cells in the control (Con) and OIM groups . Compared with that in miR-505 inhibitor-NC-treated cells, the expression of miR-505 in miR-505 inhibitor-treated cells was significantly downregulated in a concentration-dependent manner , and the expression of miR-505 was also downregulated in a concentration-dependent manner .The expression of miR-505 transfected at concentrations of 15, 30, or 60\u2009nmol/L in MC3T3-E1 cells was detected by RT-PCR. The results showed that the expression of miR-505 in MC3T3-E1 cells of the miR-505 mimic group was significantly upregulated in a concentration-dependent manner compared with that in the corresponding NC group. The higher the concentration of miR-505 mimics, the higher the expression of miR-505, and the difference was statistically significant (Fig. To verify the effect of miR-505 on the osteogenic differentiation of MC3T3-E1 cells, miR-505 mimics, miR-505 mimics-NC, miR-505 inhibitor, and miR-505 inhibitor-NC were transfected into cells at a final concentration of 30\u2009nmol/L, and then, the cells were treated with osteogenic induction medium for 3\u2009days. The expression of osteogenic-related genes was detected by RT-PCR and Western blot. The results showed that compared with that in the miR-505 mimics-NC group, the mRNA Fig. a\u2013c and pThe predictive complementary sequences are shown in Fig. To further determine whether miR-505 can regulate osteogenic differentiation by targeting the RUNX2 gene, we detected the expression of the Runx2 gene by RT-PCR after transfection with miR-505 mimics or miR-505 inhibitor. The results showed that compared with that in the miR-505 mimic-NC group, the mRNA expression level of the RUNX2 gene in cells overexpressing miR-505 was significantly downregulated, while the expression level of the RUNX2 gene in the miR-505 inhibitor group was significantly upregulated Fig. c. In addIn this study, we found that miR-505 was downregulated during osteogenic differentiation of MC3T3-E1 cells. Moreover, miR-505 negatively regulated osteogenic differentiation of MC3T3-E1 cells by targeting RUNX2.Osteoporosis is a relatively prevalent disease that mainly leads to loss of bone mass and microstructural deterioration of bone tissues and eventually increases the susceptibility of patients to bone fractures , 17. It In this study, we found that miR-505 was downregulated during osteogenic differentiation of MC3T3-E1 cells. Moreover, we found that miR-505 directly binds to the RUNX2 3\u2032 UTR, thus inhibiting the osteogenic differentiation of MC3T3-E1 cells. Kapora et al.  demonstrA large number of studies have confirmed that miRNAs are involved in the regulation of osteoblast differentiation. miR-29a modulates the expression of RUNX2 in osteoblasts by regulating the Wnt signaling pathway, ERK1/2 pathway, and Akt phosphorylation . Sun et Software prediction analysis showed that miR-505 could target the RUNX2 gene sequence. After overexpression of miR-505 mimics in MC3T3-E1 cells, the expression of RUNX2 was downregulated, suggesting that RUNX2 could be a target gene of miR-505. The RUNX2 gene is a landmark gene of osteogenic differentiation and bone formation . RUNX2 cIn summary, the results obtained in the present study indicated that miR-505 was downregulated during osteogenic differentiation in vitro and that its overexpression could inhibit osteogenic differentiation and suppress the growth of osteoblast cells by targeting RUNX2. Therefore, miR-505 may be a promising therapeutic target for the promotion of new bone regeneration."}
{"text": "In addition, we validate DFTB simulated far-IR spectra for several phosphine- and thiolate-ligated gold clusters against experimental and DFT spectra. The transferability of the parameter set is evaluated using DFT and DFTB potential energy surfaces resulting from the chemisorption of a PH3 molecule on the gold (111) surface. To demonstrate the potential of the DFTB method for quantum chemical simulations of metalloid gold clusters that are challenging for traditional DFT calculations, we report the predicted molecular geometry, electronic structure, ligand binding energy, and IR spectrum of Au108S24(PPh3)16.We report a parameterization of the second-order density-functional tight-binding (DFTB2) method for the quantum chemical simulation of phosphine-ligated nanoscale gold clusters, metalloids, and gold surfaces. Our parameterization extends the previously released DFTB2 \u201cauorg\u201d parameter set by connecting it to the electronic parameter of phosphorus in the \u201cmio\u201d parameter set. Although this connection could technically simply be accomplished by creating only the required additional Au\u2013P repulsive potential, we found that the Au 6p and P 3d virtual atomic orbital energy levels exert a strong influence on the overall performance of the combined parameter set. Our optimized parameters are validated against density functional theory (DFT) geometries, ligand binding and cluster isomerization energies, ligand dissociation potential energy curves, and molecular orbital energies for relevant phosphine-ligated Au We report a parameterization of the density-functional tight-binding (DFTB) method for the accurate prediction of molecular, electronic and vibrational structure of phosphine-ligated nanoscale gold clusters, metalloids, and gold surfaces. Density functional theory (DFT) methods are most often employed in theoretical investigations, as they are capable to accurately describe electronic, geometrical, and vibrational structure of gold clusters and nanoparticles.16\u201329 In particular, the DFT-based simulation of IR, Raman, and UV-vis spectra has been achieved for a range of gold clusters, thus providing useful theoretical fingerprints to distinguish between bonding arrangements and orientations between gold atoms and ligands, and ligand\u2013ligand interactions within clusters.23,30\u201334Atomically precise ligated gold clusters of nanometer dimension receive continued attention due to their unique catalytic properties35 and routine theoretical investigations are limited to moderate system sizes. One way of reducing the computational cost is to entirely neglect the ligands and only perform DFT calculations on the gold cores. An alternative way is to employ simplified ligand models where e.g. triphenylphosphine (PPh3) is replaced by phosphine (PH3) or trimethylphosphine (PMe3).17,18,22,25,26,28,29,36 This, as with the complete ligand removal approach, has the additional benefit that conformational searching is simplified, as the torsions of the three phenyl groups per ligand give rise to a large number of local minima with similar energies. Nevertheless, it is well known that the electronic effects of the larger ligands are different from those of the smaller ones, for instance inductive effects,36\u201339 and a computational truncation of the ligands will influence the chemistry and therefore description of the catalytic properties in calculations. Integrated schemes such as ONIOM40 may be used to capture such electronic effects in calculations on the untruncated \u201creal\u201d systems; however, the choice for high and low levels of theory and the definition of the interface between them is not straightforward. A rigorous ONIOM study requires benchmarks of the selected methods against a high-level calculation for the \u201creal\u201d system,41 which is often computationally unfeasible. In practice therefore, integrated methods are often difficult to employ in the context of ligated nanoscale metal clusters. Besides the computational effort related to the proper modeling of the ligands, the size of the metal cluster itself can become problematic for conventional DFT studies, severely impacting the size range of gold clusters to be investigated for property control and fine-tuning. Exacerbating this problem is the recent emergence of larger, so called metalloid, clusters such as Au108S24(PPh3)16.25 To make matters even worse, interactions of deposited gold clusters with substrate surfaces such as SiO2 and TiO2 may play an important role in the catalytic reaction,10\u201314,42 which further increases the computational expense of DFT studies. A recent review on connections between theory and experiment for gold nanoclusters has thus posed the question as to how theoretical calculations can be expanded to treat larger sizes and length scales.15Unfortunately, DFT calculations can become prohibitively expensive with system size,ab initio or first principles methods.43\u201348 Among them, density-functional tight-binding (DFTB),45,49\u201353 an approximation to DFT formulated in the framework of non-orthogonal tight binding, has emerged as one of the most accurate, and potentially versatile choices. DFTB is capable of simulating systems containing many thousands of atoms with an accuracy comparable to traditional DFT methods.46,54,55 As the DFTB method takes advantage of the two-center approximation, tabulated Hamiltonian and overlap integrals within the Slater\u2013Koster scheme,51,56 it is two to three orders of magnitudes faster than DFT. In order to apply DFTB into the theoretical study of ligated gold clusters, it is necessary to provide accurate parameters for all binary chemical element interactions, most notably Au\u2013S and Au\u2013P. The Au\u2013S interaction in combination with the \u201cmio\u201d parameter set51,52,57 was included in the \u201cauorg\u201d parameter set for gold\u2013thiolates clusters,58,59 but the parameters for the Au\u2013P interaction have not been developed.Semi-empirical electronic structure methods offer the capability to simulate large systems with explicit inclusion of electronic structure by introducing empirical parameters and methodological approximations to rigorous i.e. ligand binding energies, relative isomer energies, and ligand dissociation energy profiles, (3) electronic structure, (4) vibrational normal modes of Au\u2013P containing clusters, and (5) the adsorption of a PH3 molecule on the gold (111) surface. Finally, we present DFTB-based predictions for structural, energetic, and vibrational properties of the recent experimentally reported metalloid gold cluster Au108S24(PPh3)16.25In this work, we report a parameterization of the Au\u2013P interactions for the second-order DFTB (DFTB2) method for the sake of compatibility with the previously developed parameters. The accuracy of these new DFTB parameters is probed by comparing the corresponding properties against DFT-calculated values and available experimental results in terms of: (1) root mean square deviations of optimized geometries, (2) energetic properties, 22.160 and will not be repeated here. In this work, we only focus on generating parameters for the DFTB2 method, which is also referred to as self-consistent-charge (SCC)-DFTB.51 The DFTB2 total energy can be viewed as a 2nd order Taylor expansion of the Kohn\u2013Sham energy with respect to a reference initial electron density \u03c10 and electron density fluctuations \u0394\u03c1 is the initial Hamiltonian constructed from the superposition of neutral atomic densities in a two-center approximation,49 and the |\u03a8i\u3009 are occupied valence molecular orbitals (MOs), expanded as a linear combination of optimized pseudo-atomic orbitals |\u03d5\u03bc\u3009. The optimization of these pseudo-atomic valence orbitals and orbital densities for a given chemical element constitutes the determination of the electronic parameters. \u0394qA is a point charge61 on atom A, and \u03b3AB\u0394qA\u0394qB represents the Coulomb interaction energy between the two point charges;51 when A = B, \u03b3AA is the chemical hardness or second derivative of the total energy with respect to the charge on atom A. \u03b3AB is defined asS is an exponentially decaying short-range function that depends on the distance between the two atoms rAB = |rA \u2212 rB| and their chemical hardness, given in form of the so-called Hubbard parameter U. The latter is calculated prior to molecular DFTB calculations for each chemical element using the DFT method, typically employing the Perdew\u2013Burke\u2013Ernzerhof (PBE) functional.62A comprehensive review of DFTB methods can be found elsewhere and the overlap integrals \u3008\u03d5\u03bc|\u03d5\u03bd\u3009 are pre-tabulated for each chemical element pair. As in all tight binding approaches, the DFTB2 total energy consists of an electronic energy, which is the sum of the first two terms in ErepAB. The latter are formulated as a two-center term that depends only on the chemical element type of atoms A and B and their interatomic distance rAB.60 In principle, these pairwise repulsive potentials can be pre-calculated analytically from DFT for diatomic molecules. However, it was found that the performance of such DFT-based repulsive potentials is usually not sufficiently accurate for general purposes.59 Therefore, in practice, one computes DFT- or wave function theory (WFT)-based reference relative energies for model systems that contain various bond lengths of the chemical element pair in question, and fits the total repulsive energy such as to minimize the difference between reference and resulting DFTB relative energies. For this purpose, the repulsive potentials are approximated by a combination of exponential and spline functionsr0nAB,\u201d is a spline knot at the nth interval, and the \u201canAB,\u201d are the polynomial spline coefficients. These variables are considered the free empirical parameters and optimized either by hand or automatically63\u201365 to minimize the difference between reference and DFTB relative energy for a series of training systems. In addition to relative energies, repulsive potentials can also be optimized to fit molecular geometries by minimizing the difference between the reference DFT or WFT energy gradient and the DFTB energy gradient for a set of equilibrium and non-equilibrium geometries in the training set. The possible constraints on the repulsive potentials include a cutoff radius, a limit on the number of allowable extrema, and a continuity requirement up to a given derivative order.In the framework of the two-center approximation, the Hamiltonian integrals 2.2rwf used in the definition of atomic confinement potentials for generating pseudo-atomic orbitals |\u03d5\u03bc\u3009 as well as the confinement radii rdens for the atomic density; additional electronic parameters are the atomic orbital energies and the atomic Hubbard parameters U for each chemical elements.51 While the AO energies and the Hubbard parameters U are normally taken from DFT calculations of the free atom, the other electronic parameters and pairwise repulsive potentials are subject to optimization with the goal to reproduce certain desired properties; for instance, electronic band structure, atomization energies, reaction energies, and geometries (energy gradients or atomic forces). In order to parameterize the Au and P interactions for DFTB2, we adopted the Au electronic parameters from the \u201cauorg\u201d set published by Fihey et al. (referred to as auorg\u03b1),58 and a modified version of \u201cauorg\u201d by Oliveira et al. (referred to as auorg\u03c7).59 The difference between these two parameter sets lies in the Au 6p-orbital energy; in the auorg\u03b1 set it was taken as the true PBE orbital energy, while in the auorg\u03c7 set it was empirically shifted upward by \u2248+0.0279 hartree. The main purpose for this orbital energy shift was to obtain improved values for cohesive energies of pure gold nanoclusters with respect to PBE.59 Following the work of \u201cauorg\u201d, only 5d and 6 s valence electrons are considered, in total 11 valence electrons per Au atom. The parameters of the other elements in the auorg\u03b1 and auorg\u03c7 sets were taken from the \u201cmio\u201d parameter set.51,52 Consequently, we adopted the electronic parameters for P taken from the \u201cmio\u201d parameter set as well. It is important to mention that the 3d orbital energy of the P atom had been shifted by +0.5 hartree from its PBE-calculated value of 0.02044 hartree, according to Gaus et al. to reduce the overbinding in phosphate compounds, as the shift had also been adopted in the \u201c3ob\u201d parameter set for the DFTB3 method.52,66 However, in the case of Au\u2013P interactions, such a drastic shift of the P 3d virtual orbital energy introduces significant underbinding. We therefore decided to investigate the effect of the P 3d orbital energy level in detail. We systematically increased the value of the P 3d orbital energy by increments of 0.1 hartree from its PBE computed and the \u201cmio\u201d shifted value, and used our genetic algorithm (GA) optimization tool65 to automatically generate repulsive potentials for the Au\u2013P interaction with these orbital energy shifts. In this way we tested the performance of the resulting Au\u2013P parameterization with special consideration of geometries and binding energies for the adsorption of a PH3 on Au (111). An energy value of 0.12044 hartree was determined as the optimal compromise for the P 3d orbital. We refer to Table S1 in the ESIAs indicated above, there are two groups of DFTB2 parameters that need to be determined: (1) the electronic parameters, and (2) the repulsive potentials for pairs of chemical elements. The electronic parameters are comprised of the radii et al.59 we denote the original PBE Au 6p energy value by \u2018\u03b1\u2019 and its modified value by \u2018\u03c7\u2019. The shifted \u201cmio\u201d P 3d energy will leave these notations unchanged, while the use of our new optimized P 3d orbital energy value of 0.12044 hartree will be denoted with a prime, \u2018\u2032\u2019. Hence, auorg\u03b1 coupled with the original \u201cmio\u201d P is unchanged, while the auorg\u03b1 and auorg\u03c7 coupled with the new P 3d-orbital energy are referred to as auorg\u03b1\u2032 and auorg\u03c7\u2032, respectively. Because auorg\u03c7 exhibits the largest underestimation of the Au\u2013P electronic binding energy , we did not generate the Au\u2013P repulsive potential for this orbital energy combination. We decided to introduce a nomenclature for denoting the different choices of Au 6p and P 3d orbital energies in our parameters. Following Oliveira 65 After several preliminary tests, we employed a cutoff radius of 4 \u00c5 and 5 spline knots  surface. Surprisingly, auorg\u03b1\u2032 and auorg\u03c7\u2032 optimized Au\u2013P repulsive potentials are almost identical, see Fig. S1 in the ESI.\u03b1 can also be optimized in a similar way to maximize its performance, however, for the sake of simplicity and transferability we decided to use the same Au\u2013P repulsive potential for auorg\u03b1\u2032 and auorg\u03b1.The Au\u2013P repulsive potentials were optimized on the basis of shell-resolved self-consistent charge electronic energies to fit ligand binding energies and forces for the training set listed in 2.358 the generalized gradient approximation (GGA) PBE density functional was selected to generate reference geometries and cluster binding energies. In this work, we opted for the TPSS density functional67 because it was noted by Kepp68 and Goel et al.69 that this meta-GGA functional reproduces experimental or high-level theory bond energies and ligand\u2013gold distances better than the standard GGA PBE functional. For the training set of small clusters in 70 No dispersion correction was employed in the calculations generating the training set in order to avoid complications originating from a possible convolution of DFTB repulsive energy terms and the long-distance dispersion term.In the original parameterization of the auorg set,3, PMe3, PPh3 and small- to moderate-sized gold clusters. For the larger complexes, which are listed in 3), 1,1-bis(diphenylphosphino) propane (dppm), 1,3-bis(diphenylphosphino) propane (dppp), methyldiphenylphosphine (PMePh2), tris(2-(diphenylphosphino) ethyl) phosphine (PP3) and 1,8-bis(diphenylphosphino) octane (dppo). Additionally, there are thiol-containing ligands in some clusters comprised of reduced S2\u2212 and meta-methylbenzenethiol (m-MBT). For these test sets, the reference calculations were performed at the TPSS/def2-SVP70 level of theory. Here we included the empirical D3 dispersion contribution71,72 in both DFT and DFTB2 calculations.73 The D3 dispersion correction was used to improve description of ligand\u2013ligand interactions and ligand effects which are deemed to be important factors on the structural, electronic, and vibrational properties of ligated gold clusters.23 In the DFT calculations we employed an effective core potential (ECP)74 for the Au atoms. In order to accelerate the DFT calculations, we employed the resolution-of-the-identity (RI) approximation with the corresponding auxiliary basis sets.75 All non-periodic DFT calculations were carried out using the implementation of the ORCA code.76 DFTB2 single point energy and geometry optimization calculations were performed with the DFTB+ code.77 All DFTB calculations were carried out with the shell-resolved SCC option \u201cOrbitallyResolvedSCC = Yes\u201d. The auorg parameter was designed such that this option mostly affects the charge distribution on the gold atoms themselves and their interactions with the other elements.In order to benchmark the accuracy of the new parameters, ligand binding energies and optimized geometries were compared to their TPSS counterparts for various complexes of PH6(dppp)4]2+, [Au8(PPh3)8]2+ and [Au9(PPh3)8]3+ clusters. The DFTB2 IR vibrational spectroscopy calculations were computed using the GAMESS-US code.87,88 The simulated IR spectra were obtained by convoluting the calculated stick spectra using a Lorentzian line shape function with 3 cm\u22121 full width at half maximum.To test the accuracy of the DFTB parameters for the prediction of vibrational spectroscopic data, we compared DFTB- and PBE-calculated far-IR spectra to the experimental spectra for +, Au22(PH3)12 and [Au20(PMe3)16]4+ complexes exhibit RMSD values larger than 0.7 \u00c5. While the RMSD of auorg\u03b1\u2032 and auorg\u03c7\u2032 for these three clusters, and auorg\u03b1 for Au22(PH3)12 [Au20(PMe3)16]4+ remain in an acceptable range around \u22480.7 \u00c5, the auorg\u03b1 RMSD value of 1.18 \u00c5 appears problematic for [Au7(PH3)7]+.7 core shape that can only be stabilized when larger ligands such as PMe3 or PPh3 are used. It is worth mentioning that these model systems are experimentally not stable in general, because the gold core prefers a planar structure rather than a 3D geometry, unless ligands are present.92,93 The model geometries here were constructed by replacing the experimentally used ligands 7]+ cluster, lower RMSDs were observed for auorg\u03b1\u2032 and auorg\u03c7\u2032 because these two parameters increase the contribution of the P 3d-orbital in stabilizing the complex due to its lower orbital energy.After closer inspection, we conclude that this large value results from a strongly distorted Au\u03b1\u2032 and auorg\u03c7\u2032 parameters, with energy deviations as high as \u221218 kcal mol\u22121 for the smallest PH3-ligated clusters  in complexes with their experimentally used larger ligands including PPh3, PMePh2, PP3, dppp, dppm, dppo, and m-MBT, see \u03b1 and auorg\u03b1\u2032 parameters is presented and discussed in the main text. The performance of the auorg\u03c7\u2032 parameter is presented in the ESI.\u2020The accuracy of the new DFTB2 parameters for geometries and ligand binding energies was further assessed for a series of gold clusters 4]2+ (BOTSOS), [Au6(PPh3)6]2+ (CATPAO10), and [Au8S2(dppm)4]2+ (LEVKIJ). In the previous section, [Au7(PH3)7]+, Au22(PH3)12 and [Au20(PMe3)16]4+ model clusters were found to be the most problematic cases. Here, with the experimentally used ligands, DFTB-optimized structures of [Au7(PPh3)7]+, [Au20(PP3)4]4+, and Au22(dppo)6 have lower RMSD values than their previously described truncated model clusters with PH3 and PMe3. auorg\u03b1\u2032 slightly outperforms auorg\u03b1 in most of the cases (by only up to 0.1 \u00c5 for the [Au22(dppo)6] neutral complex). It is important to keep in mind that the RMSDs over atom position convolutes deviations in angles and torsions with the bond lengths. If one only considers bond length comparison, Au\u2013Au and Au\u2013P deviations are within 0.06 \u00c5 from experimental values.The RMSDs between experimental and computed cluster geometries shown in \u03c7\u2032 geometries, 6(dppp)4]2+ (BOTSOS), [Au7(PPh3)7]+ (BIXZAK), [Au8(PPh3)8]2+ (OPAUPF), and [Au9(PPh3)8]3+ (MIVPOX-D2h). The analogous comparison for auorg\u03b1 and auorg\u03c7\u2032 is shown in Fig. S3 in the ESI.94 The figure highlights the good performance of the theoretical methods in the description of the cluster core geometries relative to experiment. We note that ligand orientations can be strongly impacted by crystal field effects that are not present in our gas phase theoretical calculations, and thus we will not discuss differences in ligand geometries.In order to graphically illustrate the differences between experimental and computed DFT and auorg\u03b1 and auorg\u03b1\u2032, and Fig. S4 in the ESI\u03c7\u2032.The evaluation of predicted averaged and normalized ligand binding energies follows the schemes used above for the smaller-sized and moderate-sized clusters. The ligand binding energies are listed in Table S4 in the ESI,6(dppp)4]2+ up to [Au13(dppm)6]5+, the auorg\u03b1\u2032 binding energy deviations of \u22645 kcal mol\u22121 are in very good agreement with the DFT reference. auorg\u03b1 trends towards strong underbinding in all cases. In the case of clusters with more core Au atoms (n \u2265 20), auorg\u03b1 and auorg\u03b1\u2032 have both noticeable underbinding as high as 20 and 15 kcal mol\u22121, respectively. The results are consistent with the overall trend that was already seen in the small-sized and moderate-sized ligand complexes above. To further investigate the effect of geometry on the ligand binding energies, TPSS single point energy calculation using the DFTB optimized geometries were carried out, and the corresponding deviations in ligand binding energies are shown in \u22121. It follows that, if highly accurate ligand binding energies are required, DFTB geometry optimizations followed by TPSS single point energy calculations can provide a reasonable \u201cshortcut\u201d over straightforward DFT calculations. In addition to the binding energies, DFTB isomerization energies are compared to the DFT values as well for [Au9(PPh3)8]3+ and [Au11(PMePh2)10]3+ clusters, see Fig. S5 in the ESIAll predictions by DFTB tend towards underbinding, with only three minor exceptions. From the smallest 2+ cluster as a representative for the experimentally relevant complexes with larger ligands. Starting from the DFT optimized geometries of the cluster, the Au\u2013P bond of the ligand in question was simply stretched up to a distance of 20 \u00c5. The relative energy \u0394E of this practically dissociated geometry was defined in all methods as 0 kcal mol\u22121. In reality, complex structural relaxation of the cluster and ligand would occur obscuring methodological differences, which is the reason for presenting rigid scans. Ligand removal of nanoscale gold clusters is a key step in making the clusters catalytically more active by increasing the gold core interaction with the substrate or reactants. Previous studies have reported the partial removal of ligands of atomically-precise gold clusters Aucf.\u22121  surface.Overall, the DFTB curves mimic the TPSS curves closely in both energy of the binding region and the shape of the energy curves. The rigid scan does not include a barrier and converges within about 12 \u00c5 to the dissociation limit. As for the DFTB curves, we observe underbinding in the region from 2.5 to 6 \u00c5 in all of the plots cf.. DFTB is3.4\u03b1\u2032 parameter, but show the corresponding performance of the other parameters in the ESI.\u2020\u03b1\u2032 calculated energy levels of the frontier molecular orbitals for selected gold clusters. The plots showing frontier orbitals computed with auorg\u03b1\u2032 and auorg\u03c7\u2032 are shown in Fig. S5 in the ESI.\u03b1\u2032 tends to underestimate those for the singly charged [Au7(PPh3)7]+ (BIXZAK), the doubly charged [Au8(PPh3)8]2+ (OPAUPF), and the triply charged [Au11(PMePh2)10]3+ (ZUCMEP) clusters. In these particular three clusters, the DFTB HOMO energy shifts are \u22120.28, \u22120.32, and \u22120.40 eV for Au7, Au8 and Au11 respectively, while their LUMO shifts are \u22120.63, \u22120.89, \u22120.92 eV, obviously quantitatively larger. This imbalance results in their smaller HOMO\u2013LUMO gap values. Nonetheless, the HLG values of DFTB/auorg\u03b1\u2032 are quantitatively close to the TPSS calculated values with deviations no greater than 0.6 eV.To evaluate the ability of DFTB to accurately describe electronic structures of phosphine-stabilized gold clusters, we investigated the frontier orbital energies  and HOMO\u2013LUMO gaps (HLGs) of a number of medium-sized gold clusters using DFT and DFTB methods. In this discussion, we concentrate on the auorg6(dppp)4]2+, [Au7(PPh3)7]+ (BIXZAK), [Au8(PPh3)8]2+ (OPAUPF) and [Au9(PPh3)8]3+ (MIVPOX-D2h) clusters for the TPSS and DFTB2/auorg\u03b1\u2032 methods. Despite some inevitable quantitative differences in the orbital topology, the amplitudes of the HOMO and LUMO orbitals obtained with the DFTB2/auorg\u03b1\u2032 method are very close to the ones obtained by TPSS/def2-SVP. Noticeable qualitative differences are the LUMOs of Au7 and Au8 where DFTB both overestimated the contribution from the gold core and less contributions from the phosphine ligands, possibly providing a rationale for the previously discussed large LUMO energy shifts of Au7 and Au8. The HOMO and LUMO plots of DFTB2/auorg\u03b1 and DFTB2/auorg\u03c7\u2032 are shown in Fig. S8 and S9 in the ESI.\u03b1\u2032 orbitals; with DFTB2/auorg\u03b1 also overestimating contribution from the gold core while DFTB2/auorg\u03c7\u2032 underestimates the gold and overestimates the contribution from phosphine ligands in Au7. We note that these differences within the different DFTB parameter sets originate mostly from the different choices of the virtual orbital energies 4]2+ (BOTSOS), [Au8(PPh3)8]2+ (OPAUPF), and [Au9(PPh3)8]3+ (MIVPOX-D2h) clusters have been studied previously both experimentally and computationally using DFT19 and therefore represent good choices for the performance of our DFTB parameters for predicting vibrational spectra. The calculated DFTB IR spectra were evaluated by comparing them with experimental data and DFT-calculated IR, see \u22121 and Au\u2013P modes above 400 cm\u22121. Both PBE/def2-SVP and DFTB2/auorg\u03b1\u2032-calculated IR spectra match the previously reported experimental and M06/LANL2DZ predicted spectra, having the same mode description for each vibration. Also, the DFTB and PBE normalized spectral shapes are in good agreement, especially concerning the main peaks involving Au\u2013P interactions. A noticeable difference between the experimental and calculated spectra of Au8 and Au9 is the presence of an experimentally distinct peak at 398 cm\u22121. While all DFT and DFTB methods are able to predict peaks that are comparable with the position of this feature, the calculated intensities in DFTB are considerably weaker. Nevertheless, both methods have shown that this peak can be attributed to phenyl group twisting vibrations. Another difference is the presence of a peak at 317 cm\u22121 in the Au8 and Au9 DFTB calculated spectra that is missing in the DFT spectra. The DFTB peak matches a seemingly broad experimental peak in the same region. In the previous study,19 this peak was not assigned to any vibrational mode, as DFT was not able to predict this feature. DFTB revealed that these peaks near 317 cm\u22121 of Au8 are related to vibrations with weaker intensity assigned to a phenyl rocking vibration that is attached to protruding Au1\u2013P8. In the case of Au9, this peak has several contributing vibrations that are also assigned to a phenyl rocking of the complex's several phosphine ligands. The summary of contributing transitions for all clusters are given in Tables S7\u2013S9 in the ESI.58,59The IR spectra of \u2212 clusters, and six Au18 clusters protected with various types of ligands to the corresponding DFT and experimental spectra in the case of Au18(S-c-C6H11)14.23,31,32 The comparison is presented in the ESI, Fig. S13 and S14.108S24(PPh3)16. We focus our attention on the far-IR region because it is of particular interest in understanding the core vibrations of Au, S, and P atoms. The calculated IR spectrum has several large peaks in the 500\u2013600 cm\u22121 region which are attributed to PPh3 distortions. The multiple peaks in the 200\u2013350 cm\u22121 region are caused by various normal modes of both Au\u2013P and Au\u2013S vibrations (see Tables S10\u2013S12 in the ESI4S4 planar rings: (1) Au\u2013S stretching at 241.1 cm\u22121, (2) S\u2013Au\u2013S\u2013Au\u2013S symmetric stretching at 277.2 cm\u22121 and (3) S\u2013Au\u2013S symmetric stretch at 517.05 cm\u22121. The inset of \u22121 which are mostly attributed to Au44 core distortion.The calculation of IR spectra of large-scale systems within the normal mode approximation, requiring the calculation of the Hessian matrix, is beyond the capability of most contemporary DFT codes on current computer systems within a reasonable amount of time. In this case, DFTB can be particularly useful as it has been shown in the benchmark sections that DFTB can reproduce the experimental IR spectra very well. In this case study, we carried out DFTB2/auorg4S4 planar ring motif of Au108S24(PPh3)16 has not yet been studied using IR spectroscopy, there are no experimental validations so far about its normal modes. The calculated Au\u2013S stretches found in this planar ring motif are compared with that of the Au4(SCH3)4, [Au25(SCH3)4]\u2212 and [Au37(PPh3)10(SR)10Cl2]+ clusters.31\u201333 The S\u2013Au\u2013S\u2013Au\u2013S asymmetric and symmetric stretching values are found to be 274 cm\u22121 and 277 cm\u22121 (see Peak 2 of the inset in \u22121) of Au4(SCH3)4 cluster. Other IR studies of thiolate-containing Au nanoclusters such as [Au25(SCH3)18]\u2212 and [Au37(PPh3)10(SR)10Cl2]+ have reported their Au\u2013S symmetric stretches at 293 cm\u22121 and 239 cm\u22121, respectively, which are relatively close to our DFTB-calculated Au\u2013S stretch values; 241 cm\u22121 as shown as Peak 1 in the inset of \u22121 as presented in Tables S11 and S12,108S24(PPh3)16 cluster. Peak 3 in the inset of \u22121, which is another characteristic S\u2013Au\u2013S symmetric stretch. One can depict the similarity of the Au\u2013S stretches in the 200\u2013300 cm\u22121 regions with that of other sulfur-capped Au clusters.20,23,31\u201333,96,97 Based on the good agreement between our DFTB calculated IR spectra with DFT calculated as well as the experimental spectra for gold clusters in the benchmark, the predicted IR spectrum of Au108S24(PPh3)16 is reliable and provides a useful fingerprint to identify this cluster in future studies.Since the novel Au458,59 which were themselves based on the mio parameter set.51,52,57 The present effort thus expands the applicability of the DFTB2 method to organometallic gold complexes with ligands containing the full set of chemical elements contained in the mio parameter set: H, C, N, O, P, and S. In the construction of the repulsive potentials we considered three different combinations of the gold 6p and phosphorus 3d virtual atomic orbital energies. The performance of our parameters was evaluated using density functional theory (DFT) geometries, ligand binding and cluster isomerization energies, ligand dissociation potential energy curves, molecular orbital energies, and simulated far-IR spectra. We further compare predicted geometries with X-ray crystallographic structures and experimental far-IR spectra, and evaluate parameter transferability for phosphine chemisorption on a gold surface.Parameters for the density-functional tight-binding (DFTB) method were generated to describe gold\u2013phosphorus interactions for simulations of phosphine-stabilized nanoscale gold clusters. We build on preceding works reporting second-order DFTB (DFTB2) parameters for hybrid gold\u2013thiolates compounds,i.e. auorg\u03b1 < auorg\u03c7\u2032 \u2248 auorg\u03b1\u2032. The total ligand binding energy increases both with cluster and ligand size. This means that for a given gold cluster, the variation in the ligand binding energy in DFTB needs to be almost completely described by the electronic energy, as the Au\u2013P bond distances only marginally change with different ligands. However, the current DFTB electronic parameters are not flexible enough to accurately describe the variations in the electronic ligand binding energies. Similarly, the same trend holds for increasing gold cluster size. According to the benchmark results, we found that DFTB2/auorg\u03b1 is the best option to study small ligands and small Au clusters. DFTB2/auorg\u03b1\u2032 is the best option to study large ligands, large Au clusters, or Au surfaces. For the moderate-sized ligands or gold clusters, both DFTB2/auorg\u03b1 and DFTB2/auorg\u03b1\u2032 are good options to be used. The performance of auorg\u03c7\u2032 for Au\u2013P interactions is similar to that of auorg\u03b1\u2032 because the effects of shifting the Au 6p orbital energy from \u22120.02786 to \u22120.00001 hartree is less significant than shifting the P 3d orbital energy from 0.12044 to 0.52044 hartree. However, the effect of shifting the Au 6p orbital energy has not been investigated for Au\u2013 interactions, thus the use of auorg\u03b1\u2032 is more preferable over auorg\u03c7\u2032. Besides consideration of cluster and ligand sizes, the geometrical environment of the Au\u2013P bond also influences the performance of our parameters. Surface-like Au atoms present a challenge while more pyramidalized Au atoms are better described. Possible reasons are the minimum basis set or the missing multipolar charge contribution. Such situations are rare, however, and our auorg\u03b1\u2032 parameter performs overall well in theoretical studies of relevant nanoscale gold clusters that experimentally feature large capping ligands. In particular, our DFTB parameterization enables the simulation of ligand dissociation, reliably predicting how post-treatment can be done in experiment without inducing concomitant side-effects such as agglomeration. The auorg\u03b1\u2032 parameter set will be publicly distributed via the http://www.dftb.org website. To switch from DFTB2/auorg\u03b1\u2032 to DFTB2/auorg\u03b1, one can simply modify the P 3d-orbital energy according to In general it is found that the absolute ligand binding energies increase with decreased virtual orbital energies, 108S24(PPh3)16 nanocluster25 and investigate its molecular and electronic structure. The optimized Cartesian coordinates of Au108S24(PPh3)16 are provided in the ESI.108S24(PPh3)16 nanocluster will serve as useful reference for future studies.We employed the new DFTB parameters to determine the geometric structure of an AuDFTB approaches with appropriately developed parameters for the description of molecular and electronic structure and energetics as well as vibrational spectra are promising to provide insight as to how to tune catalyst reactivity for both product selectivity and reaction specificity. Further development of DFTB parameters for the interaction of gold clusters with various substrate surfaces will advance the development of transformative catalytic systems. This work is vital to herald the promised potential of unprecedented reaction tunability using cluster-based catalysts.http://energy.gov/downloads/doe-public-access-plan).This manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (There are no conflicts to declare.SC-011-D0SC04514D-s001SC-011-D0SC04514D-s002SC-011-D0SC04514D-s003SC-011-D0SC04514D-s004SC-011-D0SC04514D-s005"}
{"text": "Urinary biomarkers for organ dysfunction could predict the outcomes of severe trauma patients. However, the use of neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker of trauma is not well studied.To evaluate the association between the short-term prognosis of trauma patients and NGAL levels.We conducted a single center study and compared predictive performances between NGAL levels and the trauma severity.A total of 104 patients were included in the study. Patients were divided into two groups based on ISS score of 16. There was no significant difference in patient characteristics based on trauma severity. However, the lactate level was significantly higher in the more severe group. There was a significant association between urinary NGAL levels and trauma severity indicators, such as intensive care unit stay (ICU) (p = 0.005) and emergency care unit (ECU) stay (p = 0.049). In addition, receiver operating curve analysis showed that as a predictor, NGAL could be used for detecting severity with moderate precision, especially for short-term outcomes (specificity 70.6 for ICU and 69.0 for ECU stay).In this study, we revealed that the level of NGAL could predict the degree of invasiveness in trauma patients with moderate precision and estimate the duration of treatment during the acute phase. It is necessary to examine the validity of the findings of this study using a prospective, cohort, and multi-center collaborative study design. To this date, trauma treatment is still difficult and challenging, and early severity prediction is important for improving prognosis in trauma patients . Fast evUrinary biomarkers for organ dysfunction could predict outcomes of severe trauma patients, such as those involved in battle fields . Among tThis is a single center study at the Yokosuka Kyosai Hospital, Japan. The study was carried out for a year, between October 1, 2017 and September 30, 2018. The facility has a critical care center with a 740-bed hospital in Yokosuka City which has a population of 395,903 . This stWe obtained patient characteristics and clinical information from the visiting hospital, such as age in years, sex, lactate level, creatinine at admission and at day 5, ISS score, previous history of chronic kidney disease, CKD, and heart disease. Further we chose large blood transfusion, surgery, and transcatheter arterial embolization (TAE) as trauma-specific treatment. The primary outcomes were intensive care unit (ICU) stay period, the emergency care unit (ECU) stay period, and length of hospital stay (LOS) because no mortality was observed during the study period. Additionally, urinary NGAL was measured at the time of visit of the patient as a routine blood test procedure and reexamined the day after admission. We measured NGAL with the CLIA (chemiluminescent immunoassay) technique, using the ARCHITECT uNGAL assay from the Abbott Japan, Tokyo, Japan. We did not utilize creatinine correction to urinary NGAL, because original values and corrected ones are highly correlated and a few previous studies have used the former values . Inclusip < 0.05 was considered statistically significant. For statistical analysis software, IBM-SPSS Statistics 23  and MedCalc for Windows, version 15.0  were used.Univariate analysis was performed on the items according to the severity of trauma . The Mann-Whitney U test was used for continuous variables and Fisher\u2019s exact test for categorical variables. Furthermore, in order to examine the relationship between the outcome and NGAL levels, multiple regression analysis was performed using all measurement items. Collinearity was checked with the variation inflation factor (VIF). The VIF value larger than four was considered to violate a premise of collinearity and to cast multi-collinearity which determined if a variable should be excluded from the model . A two-sThere were 246 trauma patients who visited the institution and were hospitalized during the study period. After the exclusion criteria were applied, a total of 104 patients were included for further analysis. There was no significant difference in patient characteristics based on trauma severity, except that lactate level was significantly high in the more severe group with ISS \u226516 . Urinary NGAL could differentiate AKI and no AKI within the high resource utilization cohort, indicating that it could be a confounder if using NGAL to indicate injury severity \u201324. ThusIn this study, we revealed that the level of NGAL could moderately predict the degree of invasiveness in trauma patients and estimate the duration of treatment during the acute phase. It is necessary to examine the validity of the findings of this study by a prospective, cohort, and multi-center collaborative study design.S1 Data(XLSX)Click here for additional data file."}
{"text": "The higher alcohols 2-phenylethanol, tryptophol, and tyrosol are a group of yeast-derived compounds that have been shown to affect the aroma and flavour of fermented beverages. Five variants of the industrial wine strain AWRI796, previously isolated due to their elevated production of the \u2018rose-like aroma\u2019 compound 2-phenylethanol, were characterised during pilot-scale fermentation of a Chardonnay juice. We show that these variants not only increase the concentration of 2-phenylethanol but also modulate the formation of the higher alcohols tryptophol, tyrosol, and methionol, as well as other volatile sulfur compounds derived from methionine, highlighting the connections between yeast nitrogen and sulfur metabolism during fermentation. We also investigate the development of these compounds during wine storage, focusing on the sulfonation of tryptophol. Finally, the sensory properties of wines produced using these strains were quantified at two time points, unravelling differences produced by biologically modulating higher alcohols and the dynamic changes in wine flavour over aging. Saccharomyces cerevisiae performs a wide range of industrial fermentations that functionally depend upon its ability to convert sugars to ethanol and carbon dioxide efficiently. While performing this primary function, S. cerevisiae also produces a range of secondary metabolites, such as esters, volatile fatty acids, higher alcohols, and volatile sulfur compounds (VSCs), which contribute substantially to the flavour and aroma of wine [ of wine , beer [2 of wine . Of thes of wine .Higher alcohols, also known as fusel alcohols, are compounds with more than two carbon atoms. These alcohols are derived from yeast amino acid metabolism via the Ehrlich pathway . Amino a2), which is widely used in winemaking as an additive due to its antimicrobial and antioxidant effects, to yield the tryptophol-2-sulfonate (TOL-SO3H) adduct [3H from TOL seems to be increased by bottle aging, particularly for white wines [3H on the taste and mouthfeel properties of fermented beverages remains unclear, it has recently been linked with bitterness [Except for 2-phenylethanol (2-PE), which is derived from phenylalanine and associated with a \u2018rose-like\u2019 odour quality , the ind) adduct . This re) adduct , and thete wines . Althougtterness .Higher alcohols are substrates for acetate ester production, a reaction catalysed by yeast alcohol acetyltransferases. Many acetate esters are associated with \u2019fruity\u2019 and \u2018floral\u2019 aromas in wine . For exaS. cerevisiae strains that improve the organoleptic properties of sake, wine, and bread [Due to the ability of 2-PE and 2-PEA to impart floral notes, these compounds present an opportunity to shape wine style. The ability to influence the production of 2-PE and 2-PEA in white wines would be beneficial because, although naturally occurring, they are usually present at relatively low concentrations and are unlikely to impart a definitive character ,21. The nd bread ,24,25.2S). Similarly, a range of compounds derived from methionine by either enzymatic or non-enzymatic reactions are also associated with negative attributes in wine, such as methanethiol (MeSH) (\u2018sewage and rubber\u2019 aromas), methional (\u2018cooked potato\u2019 aroma) and methyl thioacetate (MeSAc) (\u2018sulfurous and cheesy\u2019 aromas) [2S by yeast has been extensively studied, and commercial low H2S producing strains have been generated [Conversely, and with the exception of a small group of polyfunctional thiols associated with \u2018fruity\u2019 and \u2018tropical\u2019 characters , most VS aromas) ,8,27. Whenerated , little 3H and TOL during wine ageing are also reported. Formal sensory analysis was conducted on the Chardonnay wines produced by each strain at two time points, and the links between the resulting changes to chemical composition and sensory properties are presented and explored.This paper explores the influence of compounds derived from the metabolism of both aromatic and sulfur-containing amino acids in the sensory profile of Chardonnay wine over time. For this purpose, we characterised five variants of a commercially available wine yeast strain (AWRI796), which were previously isolated because of their high 2-PE production phenotype. Here we show that these variants influence not only the production of 2-PE and 2-PEA but also the concentration of the higher alcohols TOL, TyrOH, and methionol, as well as other odoriferous VSCs derived from the amino acid methionine. The progression of these compounds and the equilibrium shift between TOL-SOPreviously, we isolated a range of variants from the commercial wine yeast AWRI796 that were resistant to toxic fluorinated analogues of phenylalanine. These variants were shown to overproduce 2-PE and 2-PEA to different extents in laboratory-scale fermentations . A singlp < 0.0001, The effects of variations in 2-PE, TyrOH and TOL concentration on wine sensory properties were determined using five mutants of AWRI796 together with parent strain (control) to produce Chardonnay wine. The variant strains harbour distinct mutations in two of the enzymes involved in aromatic amino acid metabolism: Tyr1p and Aro4p , while an exponential model was a better fit when all strains were included (R2 = 0.97)  . In cont = 0.97) . While t = 0.97) . Notably3H adduct. At the end of alcoholic fermentation, only wines made with the highest TOL producer (AWRI2940) contained TOL-SO3H (0.26 mg L\u22121), representing a 0.3% molar conversion of TOL into TOL-SO3H. The post-fermentation concentration of total SO2 in the wines averaged 21 mg L\u22121, with no detectable free SO2 . Before bottling, free SO2 concentration was adjusted to between 35 and 40 mg L\u22121 (\u22121 TOL) the yield of TOL-SO3H was at least 80%, while a 26% yield was observed in wines made using the high TOL producer (AWRI2940)  .3H species was reached, with longer storage times (12 and 15 months) having little or no effect on the conversion of the higher alcohol into the sulfonated adduct   the yield was only 36%  . For theonly 36% .3H formation was a significant contributor to SO2 loss during bottle storage. On the day of bottling, the concentration of free SO2 averaged 39 mg L\u22121 across all wines  than those made with the parent strain (AWRI796), whereas the low 2-PE producer and the high TyrOH-producing strains  accumulated similar concentrations of methionol to the parent . The con2S was observed at any time point  while showing a slightly elevated concentration of dimethylsulfide (DMS). No statistical evidence supporting differences between the strains in the concentration of Hme point .To further confirm the effects that mutations in either Tyr1p or Aro4p might have on methionine catabolism, two of the variants were used to ferment a synthetic grape medium (SGM) with grape-like concentrations of methionine . The twop < 0.05) supports differences between strains in three attributes rated by the panel: \u2018yellow colour intensity\u2019, \u2018floral aroma\u2019 and \u2018grassy flavour\u2019 (p < 0.15) alluded to possible trends among the strains for the attributes \u2018grassy aroma\u2019, \u2018cooked vegetable/potato aroma\u2019, \u2018sweetness\u2019, \u2018bitterness\u2019, \u2018stone fruit flavour\u2019 and \u2018flint flavour\u2019. In particular, wines made with AWRI796 and AWRI4124 were rated highly in the attributes \u2018cooked vegetable/potato aroma\u2019 and \u2018sweetness\u2019, while there was a trend for \u2018bitterness\u2019 to be rated lowest for the low TOL-/TOL-SO3H-producing strains AWRI2936 and AWRI796 compared to the other strains.Sensory descriptive analysis of the wines was performed twice, after 3 and 15 months in-bottle. The analyses were compared to assess the effect of bottle storage on wine sensory attributes. The mean scores for a subset of sensory attributes are summarised in flavour\u2019 . Wines fp < 0.001) that strains produced wines with different intensities of \u2018yellow colour\u2019, \u2018cooked vegetable/potato aroma\u2019, \u2018sourness\u2019 and \u2018sweetness\u2019. There was evidence (p < 0.05) for differences in \u2018pungency\u2019, \u2018rose aroma\u2019 and \u2018stone fruit flavour\u2019. Notably, at this time point, judges chose to rate a specific \u2018rose aroma\u2019 quality rather than the more general \u2018floral\u2019 aroma attribute used to describe the wines at three months. Wine from strain AWRI2940 was notably higher in \u2018rose aroma\u2019, as well as in \u2018yellow colour\u2019 and \u2018sourness\u2019, while lower in \u2018cooked vegetable/potato\u2019 and \u2018sweetness\u2019. Strains AWRI2936, AWRI2965, and AWRI4124 were rated with intermediate values for \u2018rose aroma\u2019  was used to investigate the relationships between wine composition and sensory attributes for both time points at which the wines were evaluated . Chemica3H, methionol), and the compounds 2-PEA, ethyl butanoate, 2- and 3-methylbutyl acetate, 2-methylpropanol, 3-methylbutanol were significant contributors to the sensory differences and were heavily loaded on Factor 1.Both the 3-month and 15-month PLS-R models indicated three optimum factors, explaining 50% and 54% of the variance of the sensory data, respectively. 2 calibration 0.69 and R2 validation 0.51), \u2018floral aroma\u2019 , \u2018cooked vegetable/potato aroma\u2019 , \u2018sweetness\u2019 , \u2018viscosity\u2019 , and \u2018stone fruit flavour\u2019  were relatively well modelled but not so well predicted, indicated by the low R2 validation. TOL and TOL-SO3H were significantly associated with \u2018yellow colour\u2019. No compounds were significant for \u2018floral aroma\u2019 but two monoterpenes, cis-rose oxide and \u03b1-terpineol, and several esters had moderately sized positive regression coefficients  for this attribute, while 2-PE and 2-PEA were only weakly positively associated. \u2018Cooked vegetable/potato aroma\u2019 was associated with several sulfur compounds, notably H2S and MeSH. \u2018Bitterness\u2019 was most strongly related to volatile acidity and 2-phenylacetaldehyde, with TyrOH and TOL-SO3H weakly associated. TOL-SO3H and volatile acidity, in addition to 2-PE and 2-PEA, were significantly negatively associated with \u2018sweetness\u2019.Of the sensory attributes at the 3-month time-point, \u2018yellow colour\u2019 , \u2018stone fruit aroma\u2019 , \u2018rose aroma\u2019 , \u2018cooked vegetable/potato aroma\u2019 , \u2018sweetness\u2019 , \u2018stone fruit flavour\u2019 , \u2018rose flavour\u2019 , and \u2018sourness\u2019  were relatively well modelled, while \u2018banana confection aroma\u2019, \u2018citrus flavour\u2019, \u2018pungent aroma\u2019, and \u2018bitterness\u2019 were not modelled well. Similar to the model at three months, TOL and TOL-SO3H were again significantly associated with \u2018yellow colour\u2019, together with several other compounds. Of the many compounds associated with \u2018rose aroma and flavour\u2019, 2-PE and 2-PEA had the largest positive regression coefficients, while the volatiles 2-phenylacetaldehyde, methionol, hexyl acetate, 2- and 3-methylbutyl acetate, ethyl butanoate, 2-methylpropyl acetate, ethyl 2-methyl propanoate, H2S, and non-volatiles TOL and TOL-SO3H were also associated with these attributes. There was strong evidence for an association between \u2018cooked vegetable/potato aroma\u2019 and the compounds methional (regression coefficient 0.09) and MeSH (regression coefficient 0.07); although the association with H2S was weak, it had a relatively high regression coefficient of 0.04. There was strong evidence for a negative association between the compounds TOL and TOL-SO3H and \u2018sweetness\u2019, and a positive association with \u2018sourness\u2019. Although \u2018bitterness\u2019 was not well modelled, TOL and TOL-SO3H were positively associated with this attribute, with relatively high regression coefficients.In the 15-month PLS model, similar links between compounds and sensory attributes emerged, but this model was generally stronger and predicted most attributes well. From the three-factor model, \u2018yellow colour\u2019  is consistent with that reported in a range of commercial white wines of different ages [2 and TOL was between 13 and 132 in wines with low to moderate TOL concentrations favouring the formation of TOL-SO3H. The high conversion yields are therefore not unexpected. In the wines made with the highest TOL producer, AWRI2940, this molar ratio was as low as 1.8, explaining the lower conversion yield. The TOL concentration found in Chardonnay wines fermented with the low and moderate TOL-producing strains was in the range of those reported in the literature for commercial white wines, typically below 5 mg L\u22121 [2 due to this reaction would be less than 2 mg L\u22121. A survey of a large number of commercial bottled Australian white wines [\u22121 of free SO2 in the most recent vintages assessed, a concentration similar to that found in our Chardonnay wines at bottling (39 mg L\u22121). These data suggest that the conversion of TOL into TOL-SO3H makes only a limited contribution to the losses of SO2 observed during wine development, which are primarily driven by reaction with dissolved O2 in wines after bottling and by sulfonation of other species [3H concentration and SO2 loss in wine observed here.Our results demonstrate that the time required to reach equilibrium between TOL and its sulfonated adduct, typically between 3 and 6 months, is independent of the initial amount of TOL. Longer storage times have little or no effect on the yield of TOL-SOent ages . The mol5 mg L\u22121 ,34,35. Ate wines  found an species ,36. This3H. This high accumulation allowed us to assess the possible effects of TOL and TOL-SO3H on white wine sensory properties. After 15 months in-bottle, wines made with AWRI2940 were rated higher in \u2018sourness\u2019 and lower in \u2018sweetness\u2019, and tended to be more bitter for some assessors even though these wines had slightly elevated fructose and glycerol concentrations  relative to the control. The PLS-R results suggest that both TOL and TOL-SO3H may impart a degree of \u2018bitterness\u2019 to these wines while decreasing \u2018sweetness\u2019 and increasing \u2018sourness\u2019 ratings, probably through well-established taste\u2013taste interactions [Wines made with AWRI2940 accumulated exceptionally high concentrations of both TOL and TOL-SOractions , especia3H in a wine matrix have been published, but Van Gemert [\u22121) eliciting bitter taste was reported in water [\u22121) [\u22121) have been reported to impart bitterness in wine [\u22121 of TyrOH to white wine and observed no influence on sensory properties, and only when unrealistically high concentrations were added (500 mg L\u22121) did TyrOH seem to depress the overall \u2018flavour quality\u2019 of wine. The fact that our wines made with AWRI2965, with a concentration of TyrOH exceeding 120 mg L\u22121, were not rated differently to the control wine in palate attributes such as \u2018astringency\u2019, \u2018bitterness\u2019 or \u2018sourness\u2019, suggest a limited effect of this higher alcohol on white wine in-mouth sensory properties.To our knowledge, no sensory recognition or detection thresholds for TOL-SOn Gemert  lists a n Gemert ,14,30, cn Gemert . Consequn Gemert . Differein water , lower ater [\u22121) . Even th in wine , to the  in wine  added 50Over time in-bottle, the sensory differences among the wines produced by each yeast became larger. Although the magnitude of the strain effect on \u2018floral/rose aroma\u2019 was similar across the two time points , an aromAnother aspect related to the effect of aging is the formation of aldehydes from oxidation of the analogous higher alcohols. These aldehydes can have a much lower sensory threshold than their corresponding higher alcohols; for example, the sensory threshold for methional is about 1000-times lower than that of methionol ,42. TherARO8 in a wine strain, encoding for the aromatic transaminase Aro8p, decreased methionol formation after fermentation of a synthetic grape must [ARO8 mutants indicates that by blocking the aromatic Ehrlich pathway, the catabolism of methionine might also be impaired.The production of methionol and other negative VSCs has been shown to be affected by the branch of the Ehrlich pathway responsible for the production of aromatic higher alcohols . Recentlape must . The decARO9 and ARO10 genes are highly induced by aromatic amino acids [ARO9-promoter-BFP reporter gene  it can be described as contributing \u2018blackcurrant\u2019 and \u2018red fruit\u2019 aromas, and it is considered to enhance the bouquet of some wine styles [The idea that there is competition for methionine between the Erhlich and demethiolation pathways is supported by the VSC profile observed in the pilot-scale wines made with the three homozygous Tyr1p mutants, particularly with the most active strain AWRI2940. Again, AWRI2965, which harbours a mutation (Aro4p) earlier in the amino acid biosynthetic pathway, behaved somewhat differently than the Tyr1p variants. Higher concentrations of methionol were not observed in the wines produced using AWRI2965 but decreased concentrations of other undesirable VSCs such as MeSH, MeSAc or methional were observed. Interestingly, AWRI2965 also produced slightly higher concentrations of DMS in these conditions. Even though DMS is also associated with reductive off-odours and \u2018vegetal\u2019 aromas, at low concentrations  culture collection . These v\u22121 of methionine. SGM was filtered through 0.22 \u00b5m Stericup filters (Millipore). Yeast starter cultures were prepared by growing cells aerobically in YPD medium for 24 h to stationary phase at 22 \u00b0C. Then, 1 \u00d7 106 cells mL\u22121 were inoculated into 50% diluted SGM medium and grown for another 48 h at 22 \u00b0C. The acclimatised cells were inoculated into 100 mL of SGM at a density of 1 \u00d7 106 cells mL\u22121. Fermentations were conducted at 17 \u00b0C in 100 mL glass bottles (Schott Duran), fitted with stir bars and stirred at 200 rpm using a magnetic stirrer. The lids of the bottles were fitted with selective H2S detector tubes  to measure the release of H2S during fermentation. Fermentation progress was followed by CO2 weight loss, measured every 24 h. After fermentation, the wines were cold settled at 4 \u00b0C for 5 days and sampled for different volatile and non-volatile compound analyses.Laboratory-scale fermentations were performed in triplicate in a synthetic grape medium (SGM) , with a \u22121, and pH 3.29. A concentration of 25 mg L\u22121 of SO2 was added to the grape must at the crusher. Yeast strains were grown for 48 h in filter-sterilised neutral grape concentrate , which had been previously diluted to ~6 \u00b0Baume and pH adjusted to 3.5. Cells were inoculated at a density of approximately 2 \u00d7 106 cells mL\u22121 in 19 L of the Chardonnay juice, and fermentation was conducted at 15 \u00b0C in 20 L stainless steel kegs in triplicate. When \u00b0Baum\u00e9 was below 3, wines were moved to 20 \u00b0C. Irrespective of the starter culture used, wines got stuck around 1 \u00b0Baum\u00e9 (day 16\u201318 of fermentation). Ferments were then rescued at day 26 by the addition of the commercial yeast Lalvin EC 1118 . Once alcoholic fermentation had finished (day 31), wines were sulfured with 80 mg L\u22121 of SO2, and cold-stabilised for approximately 2 months at 0 \u00b0C. Before bottling, SO2 concentration was adjusted to between 35\u201340 mg L\u22121 of free SO2. Screw-cap sealed bottled wines (375 mL) were stored in the dark at a constant temperature of 15 \u00b0C.The pilot-scale winemaking trial with Chardonnay juice was performed by the Wine Innovation Cluster (WIC) winemaking services, according to a standardised white winemaking protocol. Hand-harvested Chardonnay grapes from the McLaren Vale region  were used. The basic chemical parameters of the Chardonnay juice were: 12.7 \u00b0Baum\u00e9, yeast assimilable nitrogen 217 mg LTargeted analyses of fermentation-derived compounds  were performed by Metabolomics Australia (Adelaide) by GC-MS using a stable isotope dilution assay  at the ecis-rose oxide, \u03b1-terpineol, nerol, geraniol) and C13-norisoprenoids (\u03b2-damascenone and \u03b2-ionone) was performed at 3 months post-bottling by GC-MS on an Agilent 6890 gas chromatograph equipped with a Gerstel MPS2 autosampler and coupled to an Agilent 5973N mass selective detector. Sample preparation was as follows: 10 mL of wine was transferred into a 20 mL crimp-cap, headspace-SPME vial (Grace Davison) with 3 g of NaCl followed by 50 \u00b5L of a combined d4-\u03b2-damascenone, d3-\u03b1-ionone and d3-\u03b2-ionone internal standard solution. Instrument control was performed with Agilent G1701EA Revision E.02.02 ChemStation software. The gas chromatograph was fitted with an Agilent DB-5ms 30 m \u00d7 0.25 mm \u00d7 0.5 um. Helium (Ultra High Purity) was used as the carrier gas with linear velocity 46 cm/s, flow rate 1.6 mL/min in constant flow mode. The oven temperature was started at 40 \u00b0C, held at this temperature for 2 min, then increased to 190 \u00b0C at 8 \u00b0C/min and held at this temperature for 5.25 min. The vial and its contents were heated to 60 \u00b0C for 10 min in the heater/agitator with the agitator on for 5 s and off for 2 s at 500 r.p.m. A Supelco grey 2 cm SPME fibre was exposed to the sample during this heating time through the septum. The fibre was then injected into a split/splitless inlet in splitless mode. The analytes were desorbed into a Supelco 0.75 mm ID sleeveless SPME liner for 10 min, which was held at 200 \u00b0C. The purge flow to the split vent was 50 mL/min at 2.1 min with the septum purge flow turned off. The mass spectrometer quadrupole temperature was set at 150 \u00b0C, the source was set at 230 \u00b0C and the transfer line was held at 250 \u00b0C. EMV Mode was set to Gain Factor = 1.00 and spectra were recorded in SIM mode.Analysis of monoterpenoids , as described previously [2S and MeSH, respectively. Ethylmethyl sulfide and propyl thioacetate were used as internal standards. Analytes were identified by comparison of their retention times with those of the corresponding pure reference compounds.The VSCs Heviously . ReferenO-hydroxylamine hydrochloride (Sigma-Aldrich). Reference standards for these compounds of the highest purity were purchased from Sigma-Aldrich. Isotopically labelled analogues for furfural, methionol, methional, benzaldehyde, and 2-phenylacetaldehyde were used as internal standards for accurate quantification of these compounds. For the quantitation of 2-methylpropanal and 3-methylbutanal, d5-benzaldehyde was used as an internal standard. Similarly, d4-furfural was used for the determination of 5-methylfurfural. With the exception of d4-furfural , the synthesis of the other isotopically labelled standards was carried out in-house as described in [Analysis of the sulfur-containing compounds methionol and methional, as well as 2-methylpropanal, 3-methylbutanal, furfural, 5-methylfurfural, benzaldehyde, and 2-phenylacetadehyde was performed by GC-MS/MS, as described in . Aldehydribed in .VSCs and aldehydes were analysed 3 and 15 months post-bottling.The concentrations of sugars, ethanol, glycerol, and organic acids  were measured by HPLC using a Bio-Rad HPX-87H column, as described previously . Referen3H were analysed on an Agilent 1200SL HPLC using a Phenomenex Kinetex PFP column  at different time points . The injection volume was 5 \u00b5L. The column was eluted at 45 \u00b0C with a gradient of 0.1% formic acid in Milli-Q water (A) and 0.1% formic acid in acetonitrile (B) at a flow rate of 0.4 mL min\u22121. The gradient was as follows: an initial isocratic hold (0% B) for 8 min, then gradient to 5% B over 32 min, gradient to 25% B over 9 min, then gradient to 80% B over 3 min, held isocratically at 80% B for 3 min, and dropped to 0% B and held for another 15 min. Absorbance at 280 nm was monitored with an Agilent 1260 Series G7117C DAD, while fluorescence was monitored at excitation and emission wavelengths of 280 and 350 nm, respectively, with an Agilent 1260 Series G7121B FLD. Quantification of TOL and TyrOH was performed using the absorbance detector, while TOL-SO3H was quantified using the fluorescence detector. Reference standards for TyrOH and TOL were obtained from Sigma-Aldrich. TOL-SO3H was synthesised as previously described by Arapitsas, Guella and Mattivi [2O) with stirring and reacted at room temperature for 48 h. The reaction product was dried under a vacuum (30 \u00b0C) and dissolved in H2O. The product was purified using preparative HPLC with a Dionex UltiMate 3000 system, a C18 Synergi Hydro RP column , and solvent system of 100% H2O (A) and 100% acetonitrile (B). Gradient: 0\u201310 min 0% B, 10\u201325 min 50% B, 25\u201335 min 100% B, 8 mL/min flow rate. The structure of TOL-SO3H was confirmed using HRMS and NMR  with samples in D2O at 300 K. Results were processed using Topspin software. M-H mass (m/z): 240.0325; Chemical shifts for 1H-NMR  and 13C-NMR (D2O) concur with those previously reported [TyrOH, TOL, and TOL-SO Mattivi  with modreported .Quantitative descriptive analysis (QDA) sensory studies  were conp values were determined by a two-tailed Student\u2019s t test. For the sensory data, ANOVA was carried out using Minitab 19. The effects of the yeast strain treatment, judge, judge by strain, ferment replicate nested into strain, judge by ferment replicate nested into strain, presentation replicate nested into strain, and ferment replicate were assessed, treating judge as a random effect. Following ANOVA, a protected HSD value was calculated using the mean square term of the judge \u00d7 strain interaction at a 95% confidence level for attributes with a significant (p < 0.05) treatment effect. To explore the relationship between wine chemical composition and sensory attributes, PLS-R was conducted for each wine replicate, as described in [p < 0.10) signalled a treatment effect or a high F-ratio was found indicating potential treatment effects which may have been overshadowed by judge, fermentation, or presentation replicate variation.Minitab 19  was used for statistical analysis of the compositional data which were analysed by one-way analysis of variance (ANOVA). Multiple comparisons of the analyte concentration with respect to treatment were undertaken using Tukey\u2019s honestly significant difference (HSD) test , and ribed in  with somThis study confirmed that the higher alcohol overproduction phenotype of five variants derived from the commercial wine strain AWRI796 is maintained in pilot-scale white winemaking conditions. This overproduction was associated with meaningful changes in wine sensory profiles, especially after some period of bottle storage. The effect of these strains on wine chemical composition was not just limited to the overproduction of 2-PE but also to an increase in the concentration of the higher alcohols TyrOH and/or TOL and to the formation of VSCs. Associations between these compounds and \u2018sweet\u2019, \u2018sour\u2019 and \u2018bitter\u2019 tastes, and \u2018cooked vegetable/potato aroma\u2019, were identified. These results highlight the intricate connections between the metabolism of aromatic amino acids and the sulfur-containing amino acid methionine during fermentation, ultimately influencing wine flavour.The various yeast strains isolated in this study provide novel tools for winemakers to adjust and preserve wine style. In particular, AWRI2965 has excellent potential as a white wine winemaking yeast, imparting accentuated and lasting rose/floral aromas to wines.3H, needs to be elucidated along with the physico-chemical conditions such as pH, temperature, storage time, and SO2 concentration, which might influence the equilibrium between these compounds in the finished wine.More research will be needed to understand the compositional drivers of bitterness. In particular, the role of higher alcohols derived from the metabolism of aromatic amino acids TyrOH, TOL, and its sulfonated derivative TOL-SO"}
{"text": "Class III peroxidases constitute a plant-specific multigene family, where 73 genes have been identified in Arabidopsis thaliana. These genes are members of the reactive oxygen species (ROS) regulatory network in the whole plant, but more importantly, at the root level. In response to abiotic stresses such as cold, heat, and salinity, their expression is significantly modified. To learn more about their transcriptional regulation, an integrative phenotypic, genomic, and transcriptomic study was executed on the roots of A. thaliana Pyrenean populations. Initially, the root phenotyping highlighted 3 Pyrenean populations to be tolerant to cold (Eaux), heat (Herr), and salt (Grip) stresses. Then, the RNA-seq analyses on these three populations, in addition to Col-0, displayed variations in CIII Prxs expression under stressful treatments and between different genotypes. Consequently, several CIII Prxs were particularly upregulated in the tolerant populations, suggesting novel and specific roles of these genes in plant tolerance against abiotic stresses. The CI duce ROS .Therefore, they are involved in a myriad of physiological processes throughout the plant\u2019s life cycle because of their genetic and catalytic diversities . They coPrx62 was found to be implicated in salt tolerance during germination [CIII Prxs enhanced cold  [Prx08) [Moreover, several CIII Prxs were characterized for their significant roles in providing tolerance to plants against abiotic stresses. For instance, mination . Additio [Prx08) . Indeed,A. thaliana [The presence of 73 members in thaliana  suggeststhaliana . The attthaliana , and thethaliana .CIII Prxs. Notably, these transcriptional regulations are not linear circuits but are rather integrated networks involving multiple actors, where thousands of genes are differentially regulated under abiotic stresses [With the increasing manifestations of climate change, plants are facing more frequent and severe abiotic stresses that drastically affect their growth and development ,12,13. Pstresses .A. thaliana has been adopted as a model species [A. thaliana Pyrenean ecotypes to analyze the transcriptional regulation of CIII Prxs in response to multiple abiotic stresses and in different populations. By definition, natural intraspecific variation is the inherent genetic diversity among individual organisms making up a population within a species. It originates from the accumulation and natural selection of spontaneous mutations that improve the fitness of their hosts to their direct environment [ species  because  species . This stironment . It was ironment ,23,24,25A. thaliana populations, distributed along an altitudinal gradient in the French Pyrenees, not covered yet in the 1001 Genomes Project [CIII Prxs regulation under multiple abiotic stresses. The objective of this study is to provide a detailed view of the roles of CIII Prxs in response to abiotic stresses in A. thaliana Pyrenean populations.Therefore, this study implemented the natural intraspecific variation of 30  Project , in addiThe root development of the populations was studied under control conditions \u201cctrl\u201d (22 \u00b0C) and under three stresses: cold stress \u201cC\u201d (16 \u00b0C), heat stress \u201cH\u201d (28 \u00b0C), and salt stress \u201cS\u201d (22 \u00b0C + 50 mM NaCl). Accordingly, three stress-tolerant populations were identified based on their root phenotypes under each treatment. Then, the seed germination of these selected populations was tested to mark the initiation of their root development. Afterward, transcriptional analyses were performed on Col-0 and the selected stress-tolerant populations to highlight the differential regulation of CIII Prxs in response to abiotic constraints.A. thaliana populations, and on the other hand, the differential impacts of temperature and salt stress on this trait among the studied populations.The primary root growth of the multiple populations under different growth conditions was described by the root growth rates \u201cRGRs\u201d (mm/day) and the primary root lengths \u201cPRLs\u201d (mm). The aim was to highlight, on the one hand, the natural plasticity of the root development in the different p-values less than 0.0001  of the populations under the four designed growth conditions were summarized using principal component analysis (PCA). The principal component 1 (PC1) explained 54% of the phenotypic variables, while PC2 explained 20%. The populations were differentially distributed along the PCA plot, and they mostly clustered apart from Col-0 and Sha, which highlighted their plasticity in response to abiotic stresses d. PC1 isThe development of the populations\u2019 primary roots was tracked over 8 to 14 days, and it was contrasted in a genotype-dependent as well as treatment-dependent manner . For insAt 16 \u00b0C, Eaux, Belc, and Roch developed the longest roots  at the fastest rates , whereas Mari, Biel, and Bedo grew the shortest ones  most slowly . Interestingly, certain populations, such as Hosp and Mere, had moderate PRLs (10.14 and 10.34 mm) but with fast growth rates (0.88 and 0.86 mm/day). This phenomenon was due to the slow rates of seed germination of these populations, which delayed the root development initiation .To further highlight the impacts of the applied stress on the populations, the PRL of each population under each treatment was compared to that at 22 \u00b0C (ctrl). Under cold stress, the PRLs of all the populations decreased, yet the magnitudes of these reductions were differential according to the genotype. For instance, the PRLs of Eaux, Hern, and Lave were the least reduced at 16 \u00b0C compared to Arag, Argu, and Bedo . AccordiAt 28 \u00b0C, the most elongated roots and fastest growth rates were observed in Urdo, Jaco, and Herr ; however, Biel, Mari, and Hern had the shortest roots  with slowest RGRs . These observations revealed that the temperature differentially affected the root growth of the studied populations and that its effects were influenced by the different genotypes (populations) .Under heat stress, several populations developed longer roots compared with the ctrl, such as Herr, Urdo, and Jaco, while others developed shorter ones, such as Col-0, Roch, and Hosp . Herr waMoreover, Hosp, Prad, and Guch developed the longest roots under salt stress , with higher growth rates for Hosp and Guch (1.46 and 1.40 mm/day) but a lower one for Prad (1.01 mm/day). Yet, the roots of Bier, Mere, and Mari were reduced because of salinity  . AltogetFurthermore, the salt treatment globally decreased the PRLs and RGRs; however, these reductions were variable, depending on the population. To exemplify, the PRLs were moderately diminished in populations such as Savi, Prad, and Grip compared with other severely reduced populations such as Gedr, Bier, and Mere . CorrespTo sum up, the root phenotyping showed that the populations had varying root growth under control conditions which could be related to their genetic architecture. Additionally, their roots were generally reduced because of the applied treatments, except under heat for some populations. Most importantly, they unevenly responded to the applied stresses, where the root development of sensitive populations was more reduced compared with other tolerant ones. Accordingly, the root phenotyping allowed identifying some populations that had fairly advanced root growth under cold (Eaux), heat (Herr), and salt (Grip) stresses compared with Col-0. Then, the seed germination of the selected populations was tested to benchmark the beginning of their root development, aiming to know if they developed longer roots because they formerly germinated earlier or because they were truthfully tolerant to the stresses.Germination tests were performed on the cold-, salt-, and heat-tolerant populations  in addition to Col-0 under different combinations of treatments. These experiments highlighted the existing natural plasticity of this trait among the selected candidates and featured how cold, heat, and salt altered it. In addition, it was essential to assess the germination rates of the populations to normalize the effects of any significant variations that may advance or delay the initiation of root growth. For this aim, the testa rupture (TR) and the endosperm rupture (ER) were tracked over a time course . The gerIn Col-0, 100% of the seeds successfully germinated under the four conditions; however, the speed of their germination increased with the temperature but decreased with the salt a,b. To iThese results suggested that the germination of Col-0 was fastest at 28 \u00b0C, but it decelerated at 16 \u00b0C and under salt stress. Additionally, the low temperature slowed the germination of Col-0 more than the salinity. Moreover, the cold stress reduced the TR more than the ER; the heat stress enhanced the TR and ER almost equally, whereas the salt stress decelerated the ER more than the TR.The seeds of Eaux, the cold-tolerant population, fully germinated under the ctrl and cold conditions. At 22 \u00b0C, the TR and ER t50s recorded 43.46 and 55.92 h. They both increased under the cold treatment by 45% and 56%. Furthermore, the germination of Eaux was constantly slower than that of Col-0 under the two tested conditions. To illustrate, the TR t50s of Eaux were prolonged by 20.47 and 39.9 h compared with those of Col-0 at 22 \u00b0C and 16 \u00b0C, and its ER t50s were extended by 26.07 and 57.61 h under ctrl and cold conditions compared with Col-0 c,d.Herr, the heat-tolerant population, entirely germinated at 22 \u00b0C and 28 \u00b0C. The TR and ER t50s recorded 51.67 and 65.03 h at the control conditions. The heat treatment (H) did not significantly decrease the TR t50 (6%), but it diminished the ER t50 by 16%. In addition, the germination of Herr was always lagging that of Col-0. For instance, the TR t50s in Herr were greater than those in Col-0 by 28.68 and 25.8 h at 22 \u00b0C and 28 \u00b0C, respectively, and its ER t50s were longer by 35.18 and 24.65 h under ctrl and heat conditions compared with Col-0 c,d.Grip, the salt-tolerant population, completely germinated under the ctrl and salt conditions, although salinity delayed its germination. To illustrate, its TR and ER t50s were 41.13 and 35.29 h at 22 \u00b0C. Upon salt application, they increased by 20% and 65%, respectively. In reference to Col-0, Grip had slower germination under both conditions. To illustrate, the TR t50s were prolonged by 18.14 h and 26.16 h, and the ER t50s by 5.44 h and 28.24 h, respectively, under the control and salt stress c,d.To sum up, the tests showed that each of the selected populations  had slower seed germination compared with Col-0 under both the control and the corresponding stress condition. However, the slowness in their germination was extended because of the different stresses, causing a subsequent delay in their root development initiation. However, despite these delays, the roots of Eaux, Grip, and Herr were less reduced compared with Col-0 under cold, salt, and heat treatments, respectively. Hence, their choice as convenient candidates for cold, salt, and heat tolerance was validated.CIII Prxs were extracted  under control conditions (22 \u00b0C) and under the corresponding stress for each, i.e., cold for Eaux, heat for Herr, and salt for Grip. The overall results were summarized in gene-level expression tracks (GEs) holding information about the expression values (TPMs) for each gene in the multiple populations grown under different conditions . The expxtracted .CIII Prxs under control conditions (22 \u00b0C) varied within and among the studied populations. For instance, a subset of five CIII Prxs was highly expressed in all populations , with an average of 95  were significantly upregulated, while 15 genes were downregulated because of the cold treatment compared with the ctrl. However, nine members of this multigene family were upregulated in Eaux under cold, where only Prx71 was mutual with Col-0. In addition, 10 genes were downregulated in Eaux under low temperature, where Prx38, Prx52, Prx53, Prx54, and Prx65 were exclusively suppressed in this population. Comparing genotypes, 17 upregulated CIII Prxs, and 14 downregulated ones were present in Eaux versus Col-0  was specifically assayed using guaiacol/H2O2  in Col-0These results showed that CIII Prxs had basal enzymatic activity in all studied populations under control conditions, proving that these proteins performed vital processes during cellular homeostasis. Their activity was importantly enhanced by heat in Col-0, which displayed their role in responding to temperature changes. Additionally, the low-temperature treatment in Eaux induced their activity, suggesting that they could contribute to cold tolerance. However, it did not increase in Herr, the heat-tolerant population, when exposed to elevated temperatures.CIII Prxs expression and their peroxidase activity was noticed. To illustrate, a subset of 7 CIII Prxs was upregulated in Eaux, whereas only two of these genes were upregulated in Col-0 under the cold treatment. In addition, Eaux had more upregulated CIII Prxs at 16 \u00b0C when compared with Col-0 based on their genotypic difference. Coherently, this transcriptional pattern was reflected in the peroxidase activity, which was enhanced in Eaux under cold stress. Similarly, under hot conditions, 11 CIII Prxs were upregulated in Col-0, but only the expression of Prx65 was enhanced in Herr. Moreover, the genotype-based DE analyses detected 12 upregulated genes in Col-0 versus Herr at 28 \u00b0C. Consistently, the peroxidase activity was triggered by elevated temperatures in Col-0 but not in Herr.A correlation between the A. thaliana, where numerous genomic maps of climate adaptations were established for populations at macro and microgeographic scales [On an evolutionary scale, plants adapted to their local environments by selecting genes that enhanced their fitness  and expac scales .A. thaliana, so they could serve as new tools to understand the genetic variation and the plant adaptations to abiotic stresses between the Iberian Peninsula and the rest of Europe. The particularity of this sampling was derived from their close geographical distribution but in highly contrasted environments due to the mountainous nature of their collection sites. To illustrate, these populations spanned an altitudinal gradient, and hence they were naturally exposed to variable climatic conditions such as annual minimum, maximum, and mean temperatures (\u00b0C), in addition to annual precipitation (mm) and total annual UV radiations (kWh/m2). Such variations would certainly trigger the evolutionary divergence among these populations.The Pyrenean populations in this study filled a gap in the geographic distribution of In the Pyrenees Mountains, the local environmental conditions are highly contrasted, where temperature, precipitation, and UV radiation patterns are correlated with the altitude. In a previous study, the genetic structure of the populations was established, by which they were segregated into three clusters, and specific genetic lineages were detected among them. This genetic diversity was translated into contrasted phenotypes of these populations when exposed to suboptimal temperatures .In this study, the populations\u2019 roots phenotypes were also contrasted between different abiotic stresses. The root lengths and growth rates were used as indices to estimate the root development in the populations. In fact, roots play fundamental roles in the plant by foraging underground resources such as minerals and water. They are also known for their enormous plasticity by which they can respond to extrinsic cues via modulating their growth . TherefoTo sum up, differential root lengths and growth rates were observed between different treatments and various populations. The multivariate analyses of the root phenotypic data allowed the classification of the populations into cold-, heat-, and salt-tolerant or sensitive. For instance, Arag and Bedo were cold-sensitive populations since they developed tiny roots at 16 \u00b0C, but Eaux and Lave were cold-tolerant populations having relatively longer and faster-growing roots under cold conditions. These cold-sensitive and tolerant populations were formerly characterized to accumulate low and high anthocyanin content, i.e., stress indicator, in their rosettes under cold stress .Moreover, the seed germination of Col-0 and selected stress-tolerant populations  was studied under control and stressful conditions, which determined their germination timing and hence the beginning of their root development. This experiment enabled distinguishing whether the stress-tolerant populations developed longer roots because of early germination and growth initiation or because of their potential to develop properly under stress.In fact, seed germination is a crucial event that marks the beginning of the plant\u2019s life cycle and ensures its survival . It is rThe seeds of Col-0, Eaux, Grip, and Herr fully germinated under control and under stressful conditions, but their germination speed varied between genotypes and treatments. For instance, Col-0 had the fastest germination rate compared with the selected Pyrenean populations under all tested conditions. However, the selected populations developed longer roots under stressful conditions compared with Col-0 despite the delay in their growth initiation caused by the delay in their seed germination. This observation validated that these populations were stress tolerant regarding their root development since they developed longer roots faster compared with Col-0. Indeed, these observations highlighted the phenotypic plasticity among the Pyrenean populations, which could be related to their genetic diversity.RNA-seq was adopted as a high-throughput technique to study the whole transcriptome of an organism at once . TranscrCIII Prxs in three stress-tolerant Pyrenean populations, in addition to Col-0, under control and stressful conditions. In fact, CIII Prxs is a larger gene family including a plurality of members compared with ascorbate peroxidase  and glutathione peroxidase  [CIII Prxs were previously characterized as extensively involved in many physiological processes and play key roles during the plant\u2019s response to environmental constraints [This study focused on the expression of the amilies) . In addistraints , during straints .CI Prxs, only a few genes were found significantly differentially expressed in the Pyrenean populations. To exemplify, the treatment-based DE analyses showed that GPx02 and GPxO6 were upregulated in Eaux under cold stress compared with the optimal condition, and APx01 was enhanced in Grip under salinity. Yet, none were triggered in Herr when exposed to heat. Additionally, the genotype-based analyses highlighted other CI Prxs with higher expression in the Pyrenean populations compared with Col-0, such as APx01 and GPx07 in Eaux at low temperature, APx01 and APx04 in Herr at high temperature, and APx01 in Grip at saline conditions. Despite such significant changes in the expression of APxs and GPxs between genotypes and in response to abiotic stresses, the variations detected in CIII Prxs were more multitudinous.As for CIII prxs transcriptomic data under control conditions vividly demonstrated the natural variation in their expression between the Pyrenean populations, which could be attributed to their underlying genetic diversity. Moreover, the treatment-based differential expression (DE) analyses showed that the expression of many CIII Prxs was altered in response to the applied stress in each population. Additionally, the genotype-based DE analyses displayed that their regulation was variable between the stress-sensitive Col-0 and the stress-tolerant Pyrenean populations under each condition. Consequently, their contrasted expression between treatments and genotypes allowed the characterization and specification of novel roles of these genes in providing tolerance to plants.Initially, CIII Prxs in Col-0 and Eaux, with Prx71 mutually upregulated in both populations. These results suggested novel roles in cold tolerance of the Eaux-exclusive eight upregulated genes . Furthermore, another subset of 17 upregulated CIII Prxs in Eaux compared with Col-0 was identified by the genotype-based DE analyses, suggesting putative roles of 9 additional genes to be involved in cold tolerance   analyses highlighted subsets of two and nine upregulated d Prx70) a.CIII Prxs in Col-0 and Herr, with Prx65 mutually upregulated in both populations, signifying that these genes were involved in heat tolerance . Additionally, the genotype-based DE analyses, which highlighted a subset of six CIII Prxs to be upregulated in the heat-tolerant population \u201cHerr\u201d, suggested additional putative roles of these genes in response to elevated temperatures   a.CIII Prxs in Col-0 and Grip, with Prx49 mutually upregulated in both populations, proposing new functions of the Grip-exclusive upregulated genes in the response against salty conditions (Prx15 and Prx62). Furthermore, the genotype-dependent DE analyses, which exposed a subset of 11 upregulated CIII Prxs in the salt-tolerant population \u201cGrip\u201d compared with Col-0, suggested additional putative roles of these genes in response to salinity   a.CIII Prxs. For instance, some CIII Prxs were significantly upregulated in the cold-tolerant population Eaux compared with Col-0 under low temperature  or in Eaux at 16 \u00b0C compared with 22 \u00b0C (Prx55 and Prx57), suggesting that they were particularly involved in cold tolerance. These genes were previously characterized for other roles such as Prx16, which was involved in seed germination [Compiling these results allowed a functional specification of temperature- and salt-related mination , Prx27 imination , Prx47 imination , Prx66 imination . Additiomination  and Prx5mination  , or in either Col-0 or Herr at 28 \u00b0C compared with 22 \u00b0C  were associated to the response against heat stress. Formerly, Prx61 and Prx67 were not associated with any functions in any context. However, Prx17 was a direct target of the AGAMOUS-LIKE15 transcription factor and contributed to lignification [However, genes that were upregulated in the heat-tolerant population Herr compared with Col-0 under high temperature , in Grip at salty conditions compared with ctrl conditions (Prx15 and Prx62), or in Col-0 (Prx49 and Prx52) had specific roles in responding against salinity. The corresponding proteins were formerly known for playing distinct roles in plants, except for Prx14. For instance, Prx40 was found essential to proper anther and pollen development [Nevertheless, other elopment . As for elopment , while Pelopment . Moreoveelopment  and in lelopment , respectPrx01, Prx37, and Prx60 had higher expression under both cold and hot temperatures, and they hence played roles in responding to temperature variations. Formerly, Prx01 was identified as an actor in pathogenesis, cold tolerance, and extension regulation during root hair growth [Botrytis cinerea [In addition, some genes were upregulated under different treatments in the Pyrenean populations suggesting that they were involved in numerous stress-induced physiological responses. To exemplify, r growth , Prx37 i cinerea , and Prx cinerea  and the  cinerea   gene regulating root hair development [Furthermore, elopment . Prx10 welopment , while Pelopment   was triggered by all applied stresses, i.e., cold, heat, and salinity. These genes were previously identified in other contexts, such as Prx08, which was involved in regulating epidermal differentiation in Arabidopsis roots [Finally, a subset of three is roots . Moreoveis roots , while Pis roots   and Shahdara . Plant individuals of these populations were previously gathered from different locations in the French Pyrenees, the mountainous physical barrier separating the Iberian Peninsula from France. Their names, climatic data, and taxonomic relevance to A. thaliana were previously reported [Thirty new-found reported . To mini2\u00b7s\u22121).The primary roots lengths of the 32 studied populations were measured over a timeline under four growth conditions. Plants were grown in vitro in vertical positions inside 12 \u00d7 12 square Petri dishes at four contrasted conditions on half Murashige and Skoog basal medium (\u00bd MS) with sucrose and agar  and with the addition of 50 mM NaCl  when salt stress was applied. The seeds were sterilized with a bleach solution , and 0.1% Triton), followed by three successive washes with autoclaved water. For each biological replicate, 16 to 18 seeds per population were sown along two lanes with even spacing; each lane contained 8 seeds from 2 populations to maximize homogeneity and randomness. Afterward, a cold stratification treatment was performed at 4 \u00b0C for 24 h in the dark to synchronize and promote germination before transfer to long day (16 h light/8 h dark) growth chambers (light intensity = 150 \u00b5mol/mThe plants were grown at three different temperatures designating a control \u201cCtrl\u201d (22 \u00b0C), cold stress \u201cC\u201d (16 \u00b0C), and heat stress \u201cH\u201d (28 \u00b0C). They were grown with 50 mM NaCl at 22 \u00b0C . The plants were scanned on a flat-bed scanner  at 1200 dpi over a timeline adapted for each temperature. The scanning time points were 0, 3, 6, 8, 10, and 14 days at 16 \u00b0C; 0, 2, 3, 4, 6, 8, and 10 days at 22 \u00b0C; and 0, 2, 3, 4, 6, and 8 days at 28 \u00b0C.The primary roots were measured using the NeuronJ plug-in in ImageJ. The experiments were carried out in 3 to 12 replicates. Most data analyses were performed using the R software (Version 1.2.1335). First, the root growth rates RGRs (mm/day) were computed as the slopes of the linear regression lines of the primary root lengths (PRLs) measurements (mm) as a function of time (days). Second, the arithmetic means and the standard errors of means . Then, the differences among the PRLs recorded on the last day of growth were measured in percentage to evaluate their variation in each population and under each treatment in reference to the control (22 \u00b0C). Additionally, the variations in the PRLs and the RGRs due to the different treatments in the populations were statistically tested using the analysis of variance, two-way ANOVA, from the \u201cmultcomp\u201d R package .Germination tests were performed on seeds of three selected cold-, heat-, and salt-tolerant populations , in addition to Col-0, to estimate the effects of the corresponding treatments and different genotypes on seed germination. The germinating seeds were quantified up to 144 h. The three stages of germination were no rupture of the seed envelope (NR), rupture of the external testa envelope (TR), and rupture of the internal endosperm envelope (ER) marked by the embryo\u2019s radicle protrusion. The statistical analyses were performed using the \u201cgerminationmetrics\u201d R package .2\u00b7s\u22121). Col-0 germination was tested under all conditions, whereas each of the three selected Pyrenean populations was tested under the control (22 \u00b0C) and the stress condition to which it was tolerant. The quantification was performed using a binocular loupe . The experiments were conducted in triplicates.The germination tests were performed in vitro inside 12 \u00d7 12 square Petri dishes on \u00bd MS +/\u2212 50 mM NaCl. At least 100 sterilized seeds of each population were put on the media surface, stratified for 48 h at 4 \u00b0C, and then placed in a growth chamber  and under cold stress \u201cC\u201d (16 \u00b0C), the heat-tolerant population Herr grown at the ctrl and under heat stress \u201cH\u201d (28 \u00b0C), and finally the salt-tolerant population Grip grown at the ctrl and under salt stress \u201cS\u201d. The roots were harvested into microcentrifuge tubes containing metallic beads after 14, 10, or 8 days for plants grown at 16 \u00b0C, 22 \u00b0C, and 28 \u00b0C, respectively, and immediately frozen in liquid nitrogen.The plants were ground via RETSCH Mixer Mill MM 400 for 2 min at 30 Hertz. The RNA was extracted using the Qiagen RNeasy Plant Mini kit according to the manufacturer\u2019s instructions and dissolved into 50 \u03bcL elution buffer. The yield of RNA was determined by taking spectrophotometric readings by a Denovix DS-11 Nanodrop spectrophotometer. RNA sequencing and the preparation of RNA libraries were performed at Genewiz in Leipzig, Germany.A. thaliana cDNA library (TAIR10_cdna_20101214_updated) in batches, one reference sequence per transcript. The mapping process was parameterized by a match score = 1, a mismatch score = 2, a linear gap cost with insertion and deletion costs = 3, length fraction = 0.95, a similarity fraction = 0.98 to maximize stringency, and a maximum number of hits per read = 10.The obtained data were treated using Qiagen CLC Genomics Workbench software . First, the sequenced reads were trimmed prior to mapping using quality-scores-based trimming , trimming of ambiguous nucleotides (maximum number of ambiguities = 2), sequencing adaptors trimming , and length trimming by which short reads less than 50 nucleotides were discarded. Then, the trimmed reads were mapped against the TPM or \u201cTranscripts per million\u201d, a normalization method for RNA-seq, was selected as the expression value  less than or equal to 0.05 was used to detect the significant differentially expressed genes (DEGs).The analyses of the genes differential expression (DE) were performed on the whole transcriptome using statistical differential expression tests for the set of expression tracks with the associated metadata by multifactorial statistics based on a negative binomial linear model (GLM) while assuming that the read counts follow a negative binomial distribution . The effhttps://github.com/FelixKrueger/TrimGalore) with -q 30 --length 20 options. The cleaned reads were mapped onto the TAIR10 A. thaliana reference genome Col-0 using bowtie2 (v 2.3.5.1) [p-value 0.01 \u2013strand-filter 1 \u2013output-vcf 1. All polymorphic sites were then identified among the populations. Finally, an SNP calling based on all accessions was performed on all polymorphic sites to differentiate null values (NA) from the reference allele. The single nucleotide polymorphisms (SNPs) identified were then located on the Col-0 genome and have been classified as (i) upstream, (ii) downstream, (iii) in the coding, or (iv) in the noncoding regions of each gene. In the frame of this study focused on CIII Prxs, only SNPs variation in those genes has been presented.Genomic DNA was extracted from a pool of individuals for each population. The rosette leaves of 3- to 4-week-old plants were harvested and frozen in collection microtubes  containing metallic beads. They were then ground for 2 min at 30 Hertz using the RETSCH Mixer Mill MM 400. The obtained powder in each well was incubated with 400 \u03bcL of a DNA extraction buffer 0.5 M NaCl, 100 mM Tris, 50 mM EDTA, 1.25% sodium dodecyl sulfate (SDS) , 1% PVP, 1% sodium metabisulfite , 10 \u03bcg/mL RNase A , and 0.5 mg/mL proteinase K ) at 65 \u00b0C for 30 min. After cooling and centrifugation, 130 \u03bcL of 4 M potassium acetate  and 0.35 M acetic glacial acid  mixture were added and the plate was incubated at \u221220 \u00b0C for 30 min. The resulting supernatants were transferred into a 96-well column plate , where 600 \u03bcL of the fixation buffer  and 100% ethanol ) were added to each sample. After centrifugation, the new supernatants were transferred into a 96-well AcroPrepTM Advance filter plate  fixed above a new 96-well column plate. The plates\u2019 block was centrifuged, and the effluents were discarded. The filters were washed twice with 650 \u03bcL of a washing buffer . Finally, DNA was eluted into an elution plate  with 50 \u03bcL of Tris-EDTA buffer added to each well. The extracted genomic DNA from 1 to 5 individuals corresponding to each population were pooled together into equimolar DNA pools that were sent for Illuminas sequencing by NovaSeq 6000 sequencer at the GeT-PlaGe GenoToul platform. The raw fastq reads were cleaned by removing the adapters and the low-quality sequences using cutadapt (v2.1)  and Trim2.3.5.1)  with -no2.3.5.1)  markdup 2O2. The total proteins from selected populations grown under contrasting conditions were extracted with 10 mM Tris Buffer , containing 20 mM EDTA , and 5% PVP . The protein concentrations were measured following Bradford\u2019s protein quantification assay in 96-well plates . After the addition of the diluted Bradford reagent (1X) , the optical densities of the samples were recorded at 590 nm wavelength. Formerly, the spectrophotometer . To estimate the peroxidase activity, an 8:1 ratio of 200 mM phosphate buffer pH = 6 containing 0.125% guaiacol  was added to 11 mM H2O2  and incubated immediately with the protein extracts. OD470 was successively registered 1 and 2 min after the reaction started. The peroxidase-specific activity was calculated . Two-way ANOVA tests for multiple independent samples were carried out to determine the effects of the different treatments and the populations on the peroxidase activity [The peroxidase activity was assayed using guaiacol/Hactivity .A. thaliana Pyrenean populations that developed contrasted root phenotypes under thermal and saline stresses. This contrast allowed the identification of three stress-tolerant populations from the Pyrenean collection\u2014Eaux against cold, Herr against heat, and Grip against salinity.This study benefitted from the phenotypic plasticity displayed by CIII Prxs in these stress-tolerant populations was elaborately analyzed via RNA-seq. As expected, a myriad of genes had different expressions among populations under control conditions, reflecting their genetic diversity. Additionally, the expression of these genes significantly changed because of stressful treatments. Interestingly, these changes were contrasted between the Pyrenean populations and Col-0.Afterward, the transcriptional regulation of CIII Prxs regulations. To illustrate, several CIII Prxs were exclusively upregulated in the Pyrenean populations under stressful conditions, and hence novel roles in stress tolerance were associated with these genes. Furthermore, the compilation of these comparisons upgraded the available knowledge about the functional specificity of different CIII Prxs, where particular genes were identified to be involved in response to specific stress.The treatment- and genotype-based differential expression (DE) analyses revealed additional aspects of the Indeed, the novel and specific putative roles in stress tolerance that were associated with cIII genes opened future perspectives to study tolerance mechanisms. Since such mechanisms rely on complex and interconnected regulatory pathways, further studies should integrate all biological aspects and scales from the molecule to the organism to achieve a better understanding of plant tolerance."}
{"text": "This study was conducted to achieve the following three aims. Firstly, to understand the solubility mechanism of granular sodium metasilicate pentahydrate (Na2SiO3.5H2O) when used in a one-part mixing method. Secondly, to investigate the properties of AAMs when a sodium metasilicate aqueous solution is used as an alkaline material and as a source of silica. Lastly, to study the retardation effect of sucrose on AAMs. This research used aluminum silicate precursors, such as low-calcium fly ash, slag, and micros silica, alkali activators, such as NaOH pellets and Na2SiO3.5H2O, and standardized sand. The alkaline activators were first dissolved in water using a water bath shaker to achieve the alkaline solution. Sucrose, which is about 2% of the weight of the solid precursors, was added to modify the reaction process between the precursors and the alkaline materials. Four types of samples were prepared: M1, M2, M3, and M4, with the fly ash, slag, and silica fume ratios of 80:20:0, 70:30:0, 75:20:5, and 100:0:0, respectively. The research conducted solubility test of the alkaline materials, flowability, 7-, 28-, 56-day compressive and flexural tests, drying shrinkage test of mortar samples, and the setting tests of pastes with and without sucrose. The results show that the dissolution time of the NaOH was much shorter, whereas Na2SiO3.5H2O needed a solvent with a temperature of around 40 \u00b0C to be fully dissolved. This problem of solubility decreases the quality of AAMs formed using the one-part mixing method. Among the mortar samples, the M4 had the highest flow rate, while M3 had the lowest flow rate. M2 had the highest compressive and flexural strength of 43.4 MPa and 6.1 MPa, respectively. The setting time test shows that sucrose retards the reaction process in AAM.This research investigates the properties of alkali-activated materials (AAMs) using sodium metasilicate, with the ratio of SiO Alkali-activated material, Sodium metasilicate, Solubility, Sucrose, Setting time test. Annual global production of ordinary Portland cement (OPC) reaches 4 billion tons . It is nmissions . The pro2 to the atmosphere [When aluminosilicate materials, such as fly ash and slag, react with an alkali source, it produces a material that has binding properties , 6. Suchmosphere , 8. Moremosphere . Besidesmosphere , 11. Furmosphere , 13, 14.2SiO3, and K2SiO3, is used as an alkali source in AAMs, as geopolymerization and activation of AAM occur in an alkaline environment. Water glass (Na2SiO3) provides such an environment [2SiO3, such as potassium silicate. However, using potassium silicate has some disadvantages, such as increased specific surface area values, a lower degree of crystallinity, and lower resistance to attack by HCL [Aqueous alkaline solution, such as NaOH, KOH, Naironment , and theironment . Moreoveironment , and sodironment . Some stk by HCL . Therefok by HCL .AAMs are made conventionally by the reaction of aluminosilicate precursors, such as fly ash and slag, and aqueous alkaline solution, such as hydroxides, silicate, and carbonate. This method for making the materials is called the two-part mixing method , 20, 21.2SiO3.5H2O), a type of sodium metasilicate, were used as alkaline activator (AA). The result showed that the strength of AAMs made using the one-part mixing method was far less than the one made by the two-part mixing method. Moreover, some other researchers have also reported that the AAMs made using the one-part mixing method had lower mechanical strength than the one made using the two-part mixing method [2SiO3.5H2O in different solvent temperatures. The result will help the further development of the method.The author of this study investigated the properties of AAM made by both one-part and two-part mixing methods and compared both results in previous unpublished research. Sodium hydroxide (NaOH) pellets and sodium metasilicate pentahydrate  of around 2, which makes the AAM viscose and causes application problems [2SiO3 is decreased when reducing the SiO2:Na2O ratio.The granular Nag method , 28, 29.g method , 31 and problems , 33, 34.problems , the visFly ash-based AAMs, especially those with a small amount of Ca+ in their composition, have a much higher setting time due to their slow reactivity. Therefore, they are cured at higher temperatures , 37. IncThe authors observed the problem of quick setting in a one-part mixed AAM mortar, which had a significant amount of slag in their composition in the previous unpublished experiment. Using sucrose delayed the solidification of the mortar, thereby making ease in the casting of the mortar. Furthermore, sucrose increased the strength of the mortar. This may be due to the modification of the reaction process caused by the addition of sucrose and better dispersion of the precursors and alkaline material throughout the material because of the improved workability. This research also studies the retardation behavior of sucrose on AAMs through setting time test since the retarding behavior of sucrose on OPC materials is reported by some researchers. Some researchers have concluded that sucrose retards the setting time and improves the microstructure of OPC concrete , 41. Bes22.12/gr, and microsilica were used as aluminum silicate precursors. Standardized sand compatible with the ISO 679:2009 was used as fine aggregate, and the sand to binder ratio was 2.97. The chemical composition of the materials is shown in 2SiO3.5H2O that consists of 30% Na2O, 29% SiO2, and 41% H2O. Sucrose, which is about 2% of the weight of the solid precursors, was added to modify the reaction process between aluminosilicate precursors and alkaline materials.Fly ash type II, ground granulated blast-furnace slag (GGBFS) with a Blaine size of 6000 cm2SiO3: NaOH) was 1.5 for all types of mortar and paste samples. The liquid to binder ratio was set to 0.33.Overall, four types of mortar samples were made in this experiment. Tables\u00a02.22.2.1To improve the reaction process of making the AAM, fly ash, slag, and silica were premixed in a mixer called Omni mixer.An Omni mixer with a 5 L capacity and rotation speed of 2.5 Hz (2.5 spin or round per second) was used to mix the materials. The mixing duration was first set to 2 h. However, when the materials were not appropriately mixed and some white slag particles were visible, the mixing duration was increased to 3 h. After the 3-hour mixing, the materials were well mixed than before. The premixed materials were covered by plastic bags and put inside a bucket with a lid. The bucket was then covered with a lid.2.2.22SiO3.5H2O cannot easily dissolve in water at room temperature and need a solvent with a temperature of around 40 \u00b0C or above. This was found by the solubility test of the alkaline materials conducted in this study. The details and results of the experiment are explained in the result section.NaOH pellets can quickly dissolve in water at room temperature, but granular Na2SiO3.5H2O was placed inside the flasks in 3 equal groups of shaking. During the dissolution of NaOH pellets in water, the solution's temperature increased. It helped the first group of granular Na2SiO3.H2O to be well dissolved in the NaOH solution. When the temperature decreased, the granular Na2SiO3.5H2O could not be dissolved well. Therefore, in the second and third sets, the tub water temperature was increased to 40\u00b0C\u201345 \u00b0C, respectively.A water bath shaker was used to make an AA solution. It was equipped with a heater to control the temperature of the water inside, wires to hold the flasks, and a shaking mechanism. The making of the alkaline solution was as follows. First, the shaker's tub was half-filled with water. Then, flasks with a specified amount of distilled water were placed inside it and tightened by its wires. The temperature of the water inside the tub was set to 30 \u00b0C. The shaking rate was 120 rounds/min. A specific NaOH powder was put inside each flask in two sets, then shook for 3 min for complete dissolution. Granular Na2SiO3.5H2O dissolved entirely in NaOH solution, it was put in a larger flask and stored at room temperature. After some time, it was noticed that the solution was susceptible at normal temperature, and solution crystallization could quickly occur inside the solution. Therefore, the AA solution was kept in an incubator at 45 \u00b0C immediately after its making. After that, the crystallization did not occur, and the solution was transparent, smooth, not viscose, and looked like water.After Na2.3The experiment prepared prisms of 160 \u00d7 40 \u00d7 40 mm for both compressive and flexural strength and shrinkage tests. The materials were mixed in a paddle mixer.During the mixing procedure, as the solution's temperature decreased due to the solute crystallization, the flow and workability of the mortars decreased. Therefore, it was too difficult to cast them into molds. This mixing condition was called the ambient mixing condition.Then, it was decided to keep the mixing materials in the incubator at 45 \u00b0C for at least 24 h, and then mix the materials at a constant temperature of 24\u00b0C\u201326 \u00b0C. This mixing condition was named the hot mixing condition. The mixing procedure was done inside a tent at above mentioned temperature. The study observed a significant improvement in the flow and rheology of the mortar.The flowability was measured according to JIS R5201. A flow table was used to test the flowability. The mortar was put in the cone in two equal layers. Each layer was tamped 15 times by a tamped rod. Then, excess mortar material was cut off and the surface of the cone was flattened. Then, the cone was slowly lifted upward. The plate was then repeatedly risen and dropped 15 times. Next, the mortar was spread. The maximum direction and the opposed direction, which was perpendicular to the maximum direction were measured. This procedure was done twice and the average value in millimeter (mm) was taken as the flow value of the mortar. After that, the mixtures were poured inside prisms and vibrated for 2 min. To avoid crystallization of the solutes inside the mix and facilitate the reaction process between aluminum silicate precursors and alkaline solution, the prisms were put at 45 \u00b0C and 94% relative humidity inside an incubator for two days. After that, they were cured in the control room at 18\u00b0C\u201320 \u00b0C and 30%\u201350% relative humidity.In this research, the solubility test of the solid alkaline materials, flow test, 7-, 28-, 56-day compressive and flexural strength test, and drying shrinkage of mortar samples were studied. Moreover, to investigate the retarding effect of sucrose on AAM, the mixed proportion of the raw precursors of M2, which has the highest amount of slag, and M4 were chosen to make pastes for studying the initial and final setting time. The pastes were studied in the presence and absence of 2% sucrose.33.1The reaction between aluminosilicate precursors and alkaline materials is vital for producing geopolymers and AAMs. Therefore, in a one-part mixing method, solid alkaline materials, such as NaOH and sodium metasilicate need to be well dissolved to produce the desired AAM.2SiO3.5H2O, and water was put inside a flask. The Na2SiO3.5H2O: NaOH ratio was set to 1.5. A water bath shaker was used to mix the materials. The shaking rate was set to 120 rounds/min.A solubility test of the materials was conducted to better understand the alkaline materials\u2019 solubility mechanism in the one-part mixing method. One batch mixing amount of NaOH pellets, granular Na2SiO3.5H2O were put inside the flask and shook for 5 min. The duration of shaking was chosen according to the estimated mixing timing of making mortar or concrete in a mixer. After 5 min of mixing, the temperature of the solution increased to about 34 \u00b0C. As the dissolution of NaOH in water is an exothermic reaction, it rose the temperature of the solution to about 8 degrees Celsius. Most particles could not be dissolved in water, and those particles were the sodium metasilicate since NaOH quickly dissolved in water. For confirmation, a similar experiment was performed, in which it took less than 5 min for the NaOH to be fully dissolved in water.The temperature of the water before mixing with the alkaline materials was 26 \u00b0C. NaOH pellets and granular NaTherefore, the solution was mixed for two more minutes, but there was still no improvement observed in the dissolution rate, and the temperature of the solution decreased to 31 \u00b0C. Therefore, to increase the temperature of the solution, the temperature of the shaker's tub water was set to 35 \u00b0C and then to 40 \u00b0C, and each was mixed for 5 min. Thus, the temperature of the solution increased to 32 \u00b0C and 36.5 \u00b0C, respectively, and a significant improvement in the dissolution of the sodium metasilicate was observed. However, still some particles remained undissolved. When the temperature of the solution increased to 41 \u00b0C, total dissolution of the particles occurred.2SiO3.5H2O by a solvent with a lower temperature is difficult, and it could not be fully dissolved when adopting the one-part or the drying mixing method to make geopolymers and AAMs. For making 32% sodium metasilicate solution, a temperature of around 40 \u00b0C is required for the solvent to dissolve the solid material thoroughly. Concerning increment of some degrees of temperature by NaOH, a solvent with a temperature slightly lower than 40 \u00b0C can also be enough for the solubility of the sodium metasilicate. The temperature of the aluminum silicate precursors and aggregates may also influence the solubility of the materials. Therefore, the hot mixing condition will have a significant effect on the one-part mixing method. Moreover, besides maintaining the proposed temperature, increasing the mixing duration and reducing the Na2SiO3.5H2O: NaOH ratio may also play a significant part in the dissolution of the material in the one-part mixing method.This study shows that the dissolution of the granular Na3.2The flowability test was conducted to observe the influence of the aluminosilicate precursors and the change in the mixing conditions on the flow of the samples. Moreover, the flow and rheology of the alkaline solution formed by the sodium metasilicate are discussed in this section.2SiO3.5H2O was smooth and fluent and there was no sign of viscosity. But as mentioned earlier, the alkali solution was very sensitive to the temperature variation as the crystallization of the AA solution occurred at a temperature lower than 30 \u00b0C. Therefore, the workability and flowability of the mortars were decreased at the aforementioned temperature. The same phenomena of crystallization of the water glass or sodium silicate can also happen while adopting the one-part mixing method using granular Na2SiO3.5H2O. The crystallization in the solution may reduce the reaction process between the alkali and the aluminosilicate precursors. Hence, the properties of the AAMs formed can be reduced.The alkaline solution formed by NaOH pellets and granular NaIn this study, it was decided to change the temperature of the materials and the mixing condition. All materials were kept at 45 \u00b0C for 24 h, and mixing was done at a controlled temperature of 24\u00b0C\u201326 \u00b0C. When the mixing condition was changed to the hot mixing condition, significant improvement in the workability of fresh mortar was observed and crystallization did not occur.Before applying the hot mixing condition, the M1 sample was mixed in an ambient mixing condition, and the flow of the sample was so low that it was not easily put inside the molds. The flow value of the M1 was 102.5 mm. There was about a 15% increment in the flow value of the mixture when the hot mixing condition was applied. A higher flow value was achieved due to a reduction in the mix's cohesiveness at a higher temperature.Among the samples, the M4 sample had the highest flowability, and its flow value could not be measured due to its overflow from the flow table. It was due to the low reactivity of low-calcium fly ash or fly ash type II  which caPrevious studies , 34, 43 3.3Compressive and flexural strength are an important characterization of AAM. As the sodium silicate improves the reaction process between the precursors and alkali , so the Slag had a significant influence on both the compressive and flexural strength of the samples due to the presence of a high amount of Ca + that increases the reactivity of the material . The M2 The M4 sample, made of 100% fly ash, had the lowest compressive strength of 17.8, 20.6, and 21.2 MPa at the ages of 7, 28, and 56 days, respectively. The lowest compressive strength for all ages was due to the extremely low reaction between the aluminosilicate precursor and alkaline solution.Following the compressive strength result, the M2 sample had the highest 7-, 28-, and 56-day flexural strength, which is approximately 6 MPa for all ages, followed by the M1 and M3 samples, which both had the flexural strength of approximately 5 MPa for all ages. The M4 sample had a flexural strength of 3.7, 4.2, and 4.7 MPa at the ages of 7, 28, and 56 days, respectively.All samples were cured at 45 \u00b0C and 94% relative humidity inside an incubator for two days. Afterward, they were cured at 20 \u00b0C and 30%\u201350% relative humidity. Therefore, they gain most of the strength within a short time. The buildup in strength was due to the increase in the reaction or hydration rate due to heat and steam inside an incubator , 45, 46.Compressive strength of more than 40 MPa and flexural strength of 6 MPa were achieved using 32% concentrated sodium metasilicate solution, which is much lower than the concentration of sodium silicate solution used in making traditional geopolymer and AAMs , 48, 49.3.4Drying shrinkage of the samples was studied according to . The tesThe M1, M2, and M3 samples had higher amounts of shrinkage compared to M4 due to the presence of the slag and silica fume in their composition, which caused the quick reaction and early loss of water in the mixture. The shrinkage of the samples was increasing until 50 days. After that, it was decreased and then flattened. In contrast, the shrinkage of the 100% fly ash sample was nearly 0 microstrain at the age of around 90 days. This is attributed to the slow reactivity of the precursor . The dryThe shrinkage of all samples was below 0.07%, but it was within the range of allowable shrinkage defined by the ACI 209R, which is 0.05%\u20130.078% . This ma3.5The setting time test was conducted to understand the retarding influence of sucrose on AAMs. The initial and final setting times of P2 and P4 were studied according to JIS R 5201. The mixed proportion of aluminosilicate precursors of M2 and M4 was used to make pastes, which are named P2 and P4, respectively. The liquid to the binder ratio was kept to 0.19 and 0.17 for the P2 and P4 pastes, respectively. Figures\u00a0The P4 pastes, P4S0% and P4S2%, followed the same trend: sucrose increased the initial setting time of P4 from 310 to 515 min and the final setting time from 433 min to 990 min.The initial and final setting time test results of the pastes revealed that the retarding behavior of sucrose can also be observed on AAMs. Many researchers have confirmed its retarding effect on OPC materials in the past , 41. The41)2SiO3.5H2O increases with an increase in temperature. Total dissolution of the material for making 32% sodium metasilicate solution occurs at a temperature of around 40 \u00b0C. If the aforementioned type and concentration of water glass or sodium silicate are changed, then a different temperature of a solvent will be required to totally dissolve the material. Therefore, it is essential to check the solubility of sodium silicate before using it in a one-part mixing method.The dissolution of granular Na2)2 and concentration of sodium metasilicate solution. However, the alkaline solution is more sensitive to the lower temperature. Crystallization of the solution can easily happen at a temperature below 30 \u00b0C. The same phenomena of crystallization of water glass can also occur while adopting a dry mixing method or one-part mixing method. This crystallization can reduce the reaction process between alkali and aluminum silicate precursors that result in reduction in the quality of the alkali-activated product. To avoid crystallization and to keep the solution smooth and fluent, storing it at a higher temperature is recommended.The alkaline solution formed by the sodium metasilicate is more fluent and smoother. This is due to the lower amount of SiO3)A compressive strength of around 40 MPa can be achieved by combined fly ash and slag-based AAM activated by a low concentrated sodium metasilicate and NaOH solution.4)2:Na2O = 2) sodium silicate solution.Using sodium metasilicate solution in the two-part mixing method can solve the viscosity problem created by a higher modulus (SiO5)Sucrose retards the reaction process between aluminosilicate precursors and AA solutions in AAM, hence, the setting time is delayed.One-part mixing method was studied by many researchers to make ease in the production of AAMs and geopolymers and to generalize the materials. None of the researchers checked the solubility of the alkaline materials used in the aforementioned mixing method. Therefore, this research studied the dissolution mechanism and solubility of the alkaline materials used in a one-part or dry mixing method. Moreover, the aqueous solution from sodium metasilicate and NaOH was made to overcome workability and rheological problems. Furthermore, the retardation behavior of sucrose on AAM was studied by setting time test. This study concludes as follow:Mohammad Idris Rasuli: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.Yuyun Tajunnisa: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data.Akifumi Yamamura: Performed the experiments.Mitsuhiro Shigeishi: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data.10.13039/501100012009Hitachi Global Foundation Ref.\u00a0RS-15, E-1 March 8, 2019, 10.13039/501100004091Kumamoto University, and 10.13039/501100013355Institut Teknologi Sepuluh Nopember.This work was supported by the Data related to our research is not publicly available at this time, but will be deposited in a publicly accessible repository after the dissertation, including the contents of this thesis, is reviewed.The authors declare no conflict of interest.No additional information is available for this paper."}
{"text": "The human body requires energy to function. Adenosine triphosphate (ATP) is the cellular currency for energy-requiring processes including mechanical work . ATP used by the cells is ultimately derived from the catabolism of energy substrate molecules\u2014carbohydrates, fat, and protein. In prolonged moderate to high-intensity exercise, there is a delicate interplay between carbohydrate and fat metabolism, and this bioenergetic process is tightly regulated by numerous physiological, nutritional, and environmental factors such as exercise intensity and duration, body mass and feeding state. Carbohydrate metabolism is of critical importance during prolonged endurance-type exercise, reflecting the physiological need to regulate glucose homeostasis, assuring optimal glycogen storage, proper muscle fuelling, and delaying the onset of fatigue. Fat metabolism represents a sustainable source of energy to meet energy demands and preserve the \u2018limited\u2019 carbohydrate stores. Coordinated neural, hormonal and circulatory events occur during prolonged endurance-type exercise, facilitating the delivery of fatty acids from adipose tissue to the working muscle for oxidation. However, with increasing exercise intensity, fat oxidation declines and is unable to supply ATP at the rate of the exercise demand. Protein is considered a subsidiary source of energy supporting carbohydrates and fat metabolism, contributing to approximately 10% of total ATP turnover during prolonged endurance-type exercise. In this review we present an overview of substrate metabolism during prolonged endurance-type exercise and the regulatory mechanisms involved in ATP turnover to meet the energetic demands of exercise. Humans are capable of performing extraordinary feats; with both natural endowment and sport-specific training, athletes can perform various activities with astonishing power, speed, skill and tolerance . Even foResearch into exercise metabolism and the substrates necessary to support different activities has been extensive and spans just over a century ,23,24,25In exercise physiology, exercise can be broadly characterized as either low load, high repetition (endurance activities) or high load, low repetition (weight or resistance training) ,41, and \u22121 [\u22121 [2max lasting >45 min, while fat stores are abundant in the body  and may theoretically support many hours of exercise at the same intensity [Endogenous carbohydrates are mostly stored as glycogen in the skeletal muscle and liver ,48. Skel\u22121 . Manipul\u22121 [\u22121 . Glycogentensity ,44,53.2+) release and an adrenaline-mediated increase in cyclic AMP (cAMP), thereby activating phosphorylase kinase (PK) and the resultant activation of glycogen phosphorylase [Muscle glycogen degradation depends on the activation of glycogen phosphorylase and debranching enzymes, which ultimately split glucose residues from the glycogen chain . Activathorylase ,57.Muscular contraction also activates AMP-activated protein kinase (AMPK) by an increase in the cellular AMP/ATP ratio when there is a drain on ATP . AMPK is2+ signal into a cellular process [2+ concentration in the cells, via activation of phosphorylase kinase, triggers glycogen degradation [Calmodulin, the \u03b4-subunit of phosphorylase kinase, associates with the protein troponin that stimulates skeletal muscle contraction . It is a process . An incrradation ,66.An acute exercise bout\u2019s intensity and duration predominantly influences muscle glycogen degradation rates. As a consequence, muscle glycogen degradation exponentially increases with exercise intensity and, over time, progressively declines due to the finite nature of this carbohydrate store within skeletal muscles . AnotherThe mode of exercise and whether or not carbohydrates were ingested during exercise seem to be interrelated determinants of equal importance on utilising muscle glycogen during physical work . For exaSex differences may play a role in the discrepancies in the utilisation of endogenous carbohydrate stores ,83. It h2max and estimated physical activity level can only account for 34% in the variability in peak fat oxidation and thus the inter-individual variation in fat oxidation remains largely unexplained [Training status affects the selection of substrate use during exercise as a result of an enhancement in lipid oxidation and a concurrent improved insulin-stimulated glucose uptake capability when compared with untrained individuals ,89,90. Explained . Howeverxplained . Other fxplained ,95,96,972max) exercise intensities, whereby splanchnic glucose production was sufficient to deplete 75% of the liver glycogen stores [Although muscle glycogen plays a central role in energy metabolism during moderate to high intensity exercise, the importance of other extra-muscular carbohydrate sources  is profound when performing prolonged exercise . The remn stores . These tn stores . The livn stores , which in stores  and physn stores . The impn stores  and a sun stores .In healthy individuals, the liver ensures the maintenance of glycaemia within a tight range under widely divergent physiological conditions. This is achieved through the dynamic equilibrium of hepatic glucose production mechanisms (namely glycogenolysis and gluconeogenesis) by which the relative contribution is determined by the intensity and duration of exercise, in addition to the absorptive state of subjects ,104. UndSimilarly, during exercise, the contribution of both glycogenolysis and gluconeogenesis cannot be overstated in the absence of nutrient (carbohydrate) ingestion. In fact, an appreciable amount (15\u201330%) of the energy required for moderate exercise is obtained from blood glucose , while s2max, [Glucose output is almost entirely derived from liver glycogenolysis at the initial stages of prolonged moderate to high-intensity exercise secondary to glucagon and noradrenaline-mediated activation of hepatic glycogen phosphorylase ,102. Ind2max, . The latGlucoregulation is accomplished by a combination of regulatory controls, namely through feedback  and feed2max) endurance exercise [2max, while 10% of lactate was indirectly oxidised via gluconeogenesis [Lactate is a crucial intermediate in energy metabolism and it is known to be produced in many tissues including the skeletal muscles and liver . Lactateexercise . Most laexercise ,122,123.exercise ,124. Thiexercise ,126. Recogenesis . The metogenesis ,120. Durogenesis ,128. In ogenesis . An accuogenesis .Fat oxidation is the second dominant substrate for endurance exercise ,130. AltThe use of fat as an energy substrate involves hydrolysis of triacylglycerols (TG) to FA and glycerol , and the delivery of the released FA for oxidation in skeletal muscle mitochondria. TG can be derived from three sources: adipose tissue TG, intra-myocellular TG (IMTG) and circulating plasma TG . During 2max) the contribution of fat oxidation is blunted by \u224834% compared with fat utilisation rates during lower intensities (\u224855% VO2max) [2max) becomes down-regulated compared with moderate intensities (\u224865% VO2max) [TG may practically serve as an unlimited energy source to support the energetic requirements during endurance exercise, given their quantitative superiority to be stored over carbohydrates, and their higher energy density than carbohydrates . On the  VO2max) . In a si VO2max) . The sup VO2max) .A number of factors have been suggested for the down-regulation of fat oxidation at higher exercise intensities. Failure in adipose tissue to supply the exercising muscle with sufficient FA may be related to an inhibition of fat mobilisation or an inadequate perfusion of the adipose tissue . SecretiWhen focussing on the limitations of FA oxidation from the transition from moderate to high-intensity exercise, the failure of the muscle to oxidise FA in the mitochondria may also be a candidate. The oxidation of long chain FA  have to be converted to their acylcarnitine form to enter the mitochondria for \u03b2-oxidation by a reaction catalysed by carnitine palmitoyltransferase 1 (CPT-1) . Indeed,2max [Malonyl-CoA is an intermediate in the de novo synthesis of FA and is an allosteric inhibitor of CPT-1, thereby inhibiting fat oxidation . It is f2max , with gr2max , althoug2max . At high2max ,142.Protein metabolism serves as an auxiliary fuel source during prolonged endurance exercise by contributing to \u22645% of ATP provision . Althoug2max) leucine oxidation and BCOAD activation were lower in both males and females [2max [During exercise, the branched-chain amino acids (BCAA) are preferentially oxidised to other forms of amino acids through a transamination process to become transferred into keto-acids by the rate-limiting enzyme branched-chain oxo-acid dehydrogenase (BCOAD) ,172. The females . However females ,171. Thees [2max  and thates [2max .The role of protein metabolism during exercise has also been linked to the provision of precursors for tricarboxylic acid (TCA) reaction; a major common pathway for the oxidation of carbohydrates, fats and amino acids ,181. TheAlanine is a dispensable amino acid that can be synthesized endogenously by the liver, acting as an subsidiary source of energy during extreme circumstances such as starvation and prolonged endurance exercise . This prGlycerol is a hydrophilic carbon skeleton of sugar alcohol that can be easily transported as a free molecule in the bloodstream in response to triglyceride hydrolysis and FA mobilization. Lipolytic activity within adipose tissue and intramuscular fat is highly active during moderate-intensity endurance exercise by the action of catecholamines, leading to elevated circulating glycerol levels ,188. GlyKetone bodies are lipid-derived low molecular weight hydrophilic molecules, and are normally found in circulation in relatively small quantities ranging between 0.1 to 0.5 mmol/L . They arAfter over a century of research, the regulation of metabolism in endurance-type activities remains an area of interest for exercise physiologists, biologists and nutritionists ,198,199.Endurance exercise improves overall health and enhances sport performance through a shared mechanism of increased metabolic activity in response to a high energetic and/or oxygen demand of the contracting skeletal muscles; as a result, different bodily systems respond in an integrative fashion to meet this homeostatic challenge. If said exercise stimulus is repeated over time, chronic structural and functional changes occur ,200. Ind"}
{"text": "Picha. pastoris strain X33. This FCP promotes cell migration and adhesion and maintains rBMSC stemness by binding integrin \u03b23. Its effects were blocked by both integrin \u03b23 siRNA and the integrin \u03b23 inhibitor Cilengitide. A template-independent ab initio prediction modeling approach is the best approach to construct a stable FCP protein model, which predicts the binding sites between FCP and integrin \u03b23. FCP may be used in the in vitro culture and clinical regeneration of stem cells that highly express integrin \u03b23, such as hematopoietic stem cells. The study provides information on the molecular structure of FCP and its bioactivity, which can be used to design new compounds.Constructing bionic extracellular matrix (ECM) is an attractive proposition for tissue engineering and clinical regeneration therapy involving the stemness of stem cells. Here, a novel recombinant protein fibronectin-collagen peptide (FCP) was designed to modulate the function of ECM expressed by Design a novel recombinant fibronectin-collagen peptide biomimetic ECM.\u2022 FCP promotes cell adhesion, migration, and proliferation.\u2022 Predicted and verified FCP structure and affinity with integrin \u03b23.\u2022 FCP binds integrin \u03b23 to maintain rBMSC stemness.\u2022 The online version contains supplementary material available at 10.1007/s00253-022-11965-4. Stem cells play an important role in tissue engineering and clinical regeneration therapy due to their ability to self-replicate and potential to differentiate into multiple cell lines, called stemness Keating . The cliExtracellular matrix (ECM), a noncellular three-dimensional macromolecular network composed of collagens, fibronectin, elastin, and several other glycoproteins  and P. pastoris strain X33  were used for cloning and heterologous expression. PCR purification kits, gel extraction kits, and micro preparation kits were purchased from Tiangen . CC  was artificially synthesized by GL Biochem . FN10 was recombinantly expressed in our Lab  and ECV304-eGFP  cells were purchased from the Chinese Academy of Sciences . They were cultured in Roswell Park Memorial Institute 1640 medium  containing 10% fetal bovine serum, penicillin (100 I.U./mL), and streptomycin (100\u00a0\u03bcg/mL) . Rat bone marrow mesenchymal stem cells (rBMSCs) were extracted in our laboratory approved by the Animal Care Committee of Jinan University (JNU20200826-11) and were performed in accordance with animal ethics guidelines of Agricultural Animals for Research and Teaching at Jinan University. rBMSCs were cultured in Dulbecco\u2019s modified Eagle\u2019s medium  supplemented with 10% fetal bovine serum, penicillin (100 I.U./mL), and streptomycin (100\u00a0\u03bcg/mL).pPICZ\u03b1\u0391 vector to give a recombinant plasmid named pPICZ\u03b1\u0391-FCP; then, the pPICZ\u03b1\u0391-FCP was transformed into P. pastoris strain X33. Zeocin-resistant clones were selected on YPD plates containing 0.1\u00a0mg/mL of Zeocin to obtain different copies of the integrated DNA of FCP. All strains were cultivated overnight at 30\u00a0\u00b0C in 5\u00a0mL of YPD medium, and 1% of the culture was transferred to 25\u00a0mL of YPG in a 250\u00a0mL baffled flask. The cells were grown at 30\u00a0\u00b0C in a shaking incubator until the OD600 reached 1.5. The cell pellets were then induced in YPM. At the same time, methanol  was added to the culture, and 1-mL samples were collected at 0, 24, 48, and 72\u00a0h after induction. The expressions of proteins were monitored by SDS-PAGE. FCP proteins were purified using the Ni Sepharose 6 Fast Flow column combined with gel filtration Sephadex G-25. Polymerase chain reaction , agarose gel electrophoresis, and western blot , etc., were used to verify the authenticity of FCP. Expanded culture of highly expressing P. pastoris strain X33 in 2L YPD media was performed. Additionally, the circular dichroism (CD) spectrum of FCP was measured with a Chirascan plus Circular Dichroism Spectrophotometer .The cDNA encoding FCP was inserted into the An in vitro scratch wound healing model was used to assess the cell migration induced by FCP. Briefly, ECV304 cells were plated in 12\u2010well plates and cultured until approximately 95% confluence before the scratch study was undertaken. A scratch was created with a 1\u00a0ml sterile pipette tip. After scratching, cells were washed using PBS to remove cell debris caused by the scratch. Another 1\u00a0mL treatment solution  was added to cells. Three images of the scratch area were photographed using an inverted microscope  at 0, 18, and 36\u00a0h. The ImageJ software was used to determine percentage closure (%).In another separate experiment, ECV304 cells were treated with or without FCP, integrin \u03b23 siRNA, and NC group (negative control: cells transfected with scrambled siRNA), respectively. All operations are performed as above.5 cells/mL) were seeded on tissue culture plates (96-well) and allowed to attach for 4\u00a0h at 37\u00a0\u00b0C. After incubation, non-attached cells were removed by three rinses with PBS. The remaining cells were fixed with 2% paraformaldehyde for 20\u00a0min and stained with 1% crystal violet  for 20\u00a0min.Crystal violet staining was performed to assess the adhesion of FCP. Briefly, culture plates were coated with 250\u00a0nmol/L of FCP or Cilengitide overnight at 4\u00a0\u00b0C. Next, the plates were washed three times with PBS. Subsequently, ECV304 (1.0\u2009\u00d7\u2009105 cells/mL) were seeded on plates (24-well) and calculated approximately 95% confluence. Then, after being treated with different test substances for 24\u00a0h, we discarded culture supernates and washed ECV304 cells using PBS 3 times, following fixed them in 4% paraformaldehyde. In the next moment, washed with PBS and then permeabilized with ice-cold 0.5% Triton X-100. The cells were blocked with PBS containing 2% bovine serum albumin, then incubated with primary antibody of vinculin  overnight at 4\u00a0\u00b0C; after that, Alexa Fluor 488 goat anti-rabbit IgG  were co-incubated at room temperature for 30\u00a0min. Next, TRITC Phalloidin  was incubated at room temperature for 10\u00a0min. Finally, the sections were stained with 4\u2032,6-diamidino-2- phenylindole (DAPI)  and imaged with a confocal laser scanning fluorescence microscope .After adhesion, cell spread areas were characterized by vinculin and TRITC Phalloidin and detected by immunofluorescence staining assays via a confocal microscope. Briefly, ECV304 . ECV304 cells were treated with FCP, integrin \u03b23 siRNA, or scramble siRNA for 24\u00a0h separately in the second separated assays. In the last separate experiment, ECV304 cells were treated with FCP, Cilengitide, and FCP combined with Cilengitide (250\u00a0nmol/L) for 24\u00a0h. All test corresponding control groups were given.4) were added to each well and incubated at 37\u00a0\u00b0C and 5% CO2 for 12\u00a0h. Cells were respectively treated with or without FCP, FN10, and CC (250\u00a0nmol/L) for 4\u00a0h. Then, tube formation was observed and captured with an inverted microscope .The tube formation assay was performed with ECV304-eGFP. Briefly, 96-well plates were coated with 50 \u03bcL/well matrigel, then polymerized at 37\u00a0\u00b0C for 30\u00a0min. Then, ECV304-eGFP (3\u2009\u00d7\u200910https://swissmodel.expasy.org/)  (Yang and Zhang https://robetta.bakerlab.org/) .The structural modeling of FCP was performed using bioinformatic webservers such as SWISS-MODEL (http://bioinfo3d.cs.tau.ac.il/PatchDock/php.php)  to screen the docking results from PatchDock. Finally, 3D and 2D plot analyses of the protein interaction surface through PyMoL v.2.5.1 and Ligplot+v.2.2 respectively.Utilizing the FireDOCK server   three different target sequences of the siRNA as presented in Table 5 cells per well) in the logarithmic growth phase were seeded in 6-well plates and cultured overnight, then transfected with integrin \u03b23 siRNA or scramble siRNA, respectively according to the manufacturer\u2019s protocol of Ribio. Transfected cells were incubated for another 24\u00a0h and treated with FCP; then, scratch wound assay and immunofluorescence staining assay were used to examining the effects of FCP.Shortly, the ECV304 cells (2\u2009\u00d7\u2009103 cells per well) were inoculated into a 6-well plate precoated with FCP, Cilengitide, and FCP combined with Cilengitide (250\u00a0nmol/L), respectively; the group that did not precoat with anything as the blank control. The cells were cultured continuing for 14\u00a0days. After that, the cells were washed three times with PBS and incubated with 1\u00a0mL of crystal violet staining solution  for 30\u00a0min. After being washed three times, the cells were observed under an optical microscope  and photographed. At the same time, cells on another independent 6-well plates were collected to detect stemness-related genes: NANOG; REX1; PPAR-\u03b32; ALP; SOX9, and integrin \u03b23.rBMSCs . To obtain cDNA, 1\u00a0\u03bcg of RNA was reverse transcribed by using the reverse transcription kit . An equal volume of cDNA was then used for RT-qPCR using the SYBR-Green Quantitative PCR kit  and the CFX96 Touch Real-Time PCR Detection System . The rat-specific primers were as follows: NANOG; REX1; PPAR-\u03b32; ALP; SOX9; and integrin \u03b23. All primers were purchased from BGI . Relative expression levels of genes were examined by 2in Table .SD) of at least three independent experiments. Statistical analyses were performed using the GraphPad Prism 6 software . Differences between more than two groups were analyzed using one-way ANOVA followed by Tukey s HSD comparison test. Statistical significance was set at P\u2009<\u20090.05.All data are expressed as mean\u2009\u00b1\u2009standard deviation . A similar trend was observed in the number of attached cells . After 36\u00a0h of FCP treatment, the wound was almost completely closed in the wound healing assay, while others were still open  of wound closure was observed in the FCP groups (99.32%\u2009\u00b1\u20090.69%) than in the control groups (50.68%\u2009\u00b1\u20092.31%), , Fig.\u00a0. Similar%), Fig.\u00a0.Fig. 2CeDifferent prediction models of FCP were validated using Ramachandran Plot, Whatcheck, and Verify3D by the SAVES 6.0 online server. Validation statistics showed that RoseTTAFold best predicted the structure of FCP compared with the other methods, evidenced by the presence of the maximum residues (83.6%) in the most favored regions of the Ramachandran plot, 100% of the residues with an average 3D-ID score\u2009\u2265\u20090.2, and the largest proportion of green regions (68.1%) in Whatcheck Fig.\u00a0. The besBased on these results, the RoseTTAFold model was used for molecular docking. After protein\u2013protein docking via PatchDock and filtering the docking results by FireDock, the stable docking complex of FCP-integrin \u03b23, with the highest binding energy of\u2009\u2212\u200915.68\u00a0kcal/mol, was obtained. The 3D plot of the docking complex showed that the C-terminal FN10 region of FCP formed a relatively stable complex with integrin \u03b23 Fig.\u00a0. More sp\u20138\u00a0M . Gene expression levels of both SOX and ALP, gene markers of chondrogenic and osteogenic differentiation abilities of rBMSCs, were higher than others, especially compared to gene expression levels in the control group (P\u2009<\u20090.05). However, gene expression of PPAR-\u03b32, an adipogenic gene, was similar in FCP-treated and control groups (P\u2009>\u20090.05). When Cilengitide was used to inhibit integrin \u03b23, the expression of all genes was significantly downregulated. After treatment with both FCP and Cilengitide simultaneously, the related genes were upregulated compared with those in the Cilengitide groups . The SPR technique is a powerful tool in the study of target molecule interaction and has the advantages of low cost and direct and quantifiable results. However, because SPR is sensitive to interfering factors within the sample, it represents an indirect interaction between proteins.The high affinity of FCP for integrin \u03b23 was verified by SPR assay. Integrin \u03b23 protein, captured on an NTA chip, bound FCP with an affinity constant of 5.37\u2009\u00d7\u200910P\u2009>\u20090.05). This finding is difficult to explain, but we speculate that it is related to the function of integrin \u03b23. Further study of these results was outside the scope of the current study due to laboratory constraints. For a sample, we are unable to test karyotypic stability after transfer generation.Collectively, these data suggest that integrin \u03b23, an important receptor on the surface of ECV304 cells, may be a key target for FCP to exert its biological effects. By knocking down integrin \u03b23 expression in ECV304 cells and competitively blocking integrin \u03b23 binding sites with Cilengitide, FCP-induced angiogenesis, migration, and adhesion of ECV304 cells were reduced. rBMSCs were chosen as the model system in the present studies due to ethical constraints and other difficulties in obtaining hHSCs. Considering that rBMSCs are one of the most widely used stem cells in the field of tissue engineering  and CC (a kind of human-like collagen) proteins, which have an ideal domain to provide mechanical support for cells. The FCP bionic ECM may provide a microenvironment to support stem cell localization, proliferation, differentiation, and stemness maintenance. In the future, FCP may be used in the in vitro culture of stem cells, such as hematopoietic stem cells and other stem cells with high expression of integrin \u03b23, or even in the field of tissue regeneration engineering.Supplementary file1 (PDF 564 KB)Below is the link to the electronic supplementary material."}
{"text": "We quantified both viral stocks using droplet digital PCR (ddPCR), which revealed that the Omicron stock had equivalent copies of the N gene to Delta at a one log lower infectious virus. The Abbott BinaxNow and Orasure InteliSwab had the highest analytical sensitivity for Omicron while the Orasure InteliSwab and the Quidel QuickVue had the highest analytical sensitivity for Delta. When 14 SARS-CoV-2 real-time PCR positive nasal/nasopharyngeal swab samples , were tested by the four assays, only the QuickVue detected all samples. Antigen test positivity correlated with recovery of infectious virus on cell culture in 9 out of 13 tested specimens from symptomatic, asymptomatic, unvaccinated, and vaccinated individuals. Although our study confirms the reduced analytical sensitivity of antigen testing compared to molecular methods, the Omicron variant was detectable by the four evaluated rapid antigen tests.Ensuring SARS-CoV-2 diagnostics that can reliably detect emerging variants has been an ongoing challenge. Due to the rapid spread of the Omicron variant, point-of-care (POC) antigen tests have become more widely used. This study aimed at (i) comparing the analytical sensitivity (LOD) of 4 POC antigen assays, BD Veritor, Abbott BinaxNow, Orasure InteliSwab and Quidel QuickVue, for the Omicron versus the Delta variant and (ii) verifying the reproducible detection of Omicron by the 4 antigen assays. The LOD for all four assays were evaluated using Omicron and Delta virus stocks quantified for infectivity and genome copies. The four assays detected all replicates of Omicron and Delta dilutions at 10IMPORTANCE In the manuscript, we report an evaluation of the capability of 4 point of care (POC) antigen assays, the BD Veritor, Abbott BinaxNow, Orasure InteliSwab and Quidel QuickVue to detect the Omicron variant of SARS-CoV-2, and we compared their analytical sensitivity for Omicron versus Delta. In this analysis we found that all four assays detected Omicron and Delta at 104 and 105 TCID50/mL, respectively. We further quantified the viral stocks used by droplet digital (ddPCR) and found that the Omicron stock had equivalent copies of the N gene to Delta at a one log lower infectious virus titer and that an increased RNA to infectious virus ratio may be contributing to discrepancies in limit of detection in Omicron compared to Delta. We evaluated 14 SARS-CoV-2 real-time PCR positive nasal/nasopharyngeal swab samples (12 Omicron and 2 Delta), with an average cycle threshold value of 19.1, and only the QuickVue showed 100% agreement. Since the beginning of the SARS-CoV-2 pandemic, the importance of accurate diagnostic assays has been highlighted . As new 2\u20136\u20135 TCID50/mL. For the Omicron variant, all assays were able to detect all replicates at 1\u2009\u00d7\u2009104 TCID50/mL , BinaxNow and InteliSwab detected 92% (11/12), and QuickVue detected 100% (12/12) of Omicron positives . The 12 Our data indicate that the lower clinical sensitivity of some POC tests is not associated with the recovery of infectious virus, or antibody levels in the upper respiratory samples and are likely related to the analytical sensitivity of the assays for these particular variants. Our LOD results were similar to other studies , 16 illuFor our clinical sample evaluation, our results are similar to other studies , 4 partiThis study has limitations, notably the small clinical samples size evaluated (N\u2009=\u200914), and the relatively high concentration of virus within the clinical samples evaluated (mean N Ct =19.12). However, for our study, lower Ct value clinical samples were intentionally chosen to assess the performance of each assay for the detection of Omicron without the added variable of assay sensitivity associated with lower viral load. Additional limitations include the use of simulated anterior nasal swab samples for testing spiked and clinical specimens; even though this is a nonvalidated approach for sample collection and testing, most of our tested samples were positive and all the runs were valid  endorsing the validity of this approach. Lastly, LOD comparison studies were performed using relatively limited number of replicates for each tested concentration.Our data confirm that antigen tests have lower sensitivity than nucleic-acid amplification tests. The four antigen tests that we evaluated were similar in their ability to detect Omicron variant. Antigen assays had a lower LOD with the Omicron variant which correlated with the higher number of nucleocapsid genomic copies per infectious virus concentration compared to Delta. Evaluations of new variants should include quantification of infectious virus and genomic viral RNA copies for comparing sensitivities of diagnostic tests.We performed a limit of detection (LOD) study for each assay with Delta and Omicron SARS-CoV-2 viral stocks that were quantified for infectious virus concentration by a tissue culture infectious dose (TCID50) assay and viral genome copy number ddPCR . Delta aNasal/nasopharyngeal (N\u2009=\u200914) samples with Ct values\u2009<\u200920 were selected from SARS-CoV-2 positive samples identified as Omicron at the Johns Hopkins Virology Laboratory as a part of whole-genome sequencing for surveillance in December 2021. The clinical diagnosis was performed by the NeuMoDx SARS-CoV-2 assay  and the 23\u2013Viral stock dilutions and clinical specimens were tested by each POC antigen test as per manufacturer instructions with a modification where the provided swab in the assay was immersed in each spiked or clinical specimen.Serology was performed with the EUROIMMUN Anti-SARS-CoV-2 ELISA (IgG) following the package insert  on undilThe Delta  and Omicron  variants used in this study were isolated from clinical specimen as previously described ."}
{"text": "In the abnormal circumstances caused by the COVID-19 pandemic, patient portals have supported patient empowerment and engagement by providing patients with access to their health care documents and medical information. However, the potential benefits of patient portals cannot be utilized unless the patients accept and use the services. Disparities in the use of patient portals may exacerbate the already existing inequalities in health care access and health outcomes, possibly increasing the digital inequality in societies.The aim of this study is to examine the factors associated with nonuse of and dissatisfaction with the Finnish nationwide patient portal My Kanta Pages among the users of health care services during the COVID-19 outbreak. Several factors related to sociodemographic characteristics, health, and the use of health care services; experiences of guidance concerning electronic services; and digital skills and attitudes were evaluated.A national population survey was sent using stratified sampling to 13,200 Finnish residents who had reached the age of 20 years. Data were collected from September 2020 to February 2021 during the COVID-19 pandemic. Respondents who had used health care services and the internet for transactions or for searching for information in the past 12 months were included in the analyses. Bivariate logistic regression analyses were used to examine the adjusted associations of respondent characteristics with the nonuse of My Kanta Pages and dissatisfaction with the service. The inverse probability weighting (IPW) method was applied in all statistical analyses to correct for bias.In total, 3919 (64.9%) of 6034 respondents were included in the study. Most respondents  used My Kanta Pages, and 2841 (85.3%) of them were satisfied. Nonusers  were a minority among all respondents, and only 489 (14.7%) of the 3330 users were dissatisfied with the service. Especially patients without a long-term illness , those who were not referred to electronic health care services by a professional , and those in need of guidance using online social and health care services  were more likely nonusers of the patient portal. Perceptions of poor health  and security concerns  were associated with dissatisfaction with the service.Patients without long-term illnesses, those not referred to electronic health care services, and those in need of guidance on the use of online social and health care services seemed to be more likely nonusers of the Finnish nationwide patient portal. Moreover, poor health and security concerns appeared to be associated with dissatisfaction with the service. Interventions to promote referral to electronic health care services by professionals are needed. Attention should be targeted to information security of the service and promotion of the public\u2019s confidence in the protection of their confidential data. The worldwide COVID-19 pandemic limited the provision of nonurgent health care services . During Patient portals offer transparent information about the patients\u2019 health and well-being  and enhaThe potential benefits of patient portals cannot be utilized unless the patients accept and adopt the service ,19. AccoPrevious research has examined differences in patient portal use in different contexts and patient populations. Several studies have reported an association between portal use and sociodemographic background ,23,25,26Factors associated with the patients\u2019 satisfaction with patient portals have been less studied. Mainly descriptive research on the portal users\u2019 experiences exists, and only little research has been conducted with quantitative methods about factors associated with satisfaction . Kong etThe aim of this study is to examine factors associated with the nonuse of and dissatisfaction with the Finnish nationwide patient portal My Kanta Pages (My Kanta) during the COVID-19 pandemic. Only respondents who had used the internet in the past 12 months were included to examine nonuse beyond the first-level digital divide  caused bFinland is a sparsely populated country with 5.5 million residents. The health care system is decentralized, and until the end of 2022, municipalities n=311) are responsible for organizing health care services, which are funded by taxes, state transfers, and user fees 1 are res. One of My Kanta was launched step-by-step starting from 2010  to promoThe number of My Kanta users has grown steadily since its launch , and theThis study was conducted in Finland as part of the FinSote 2020 National Survey of Health, Wellbeing, and Service Use . The queAltogether, 6034 Finnish residents  responded to the questionnaire (response rate 46.5%). In total, 3919 (65.0%) respondents were included in the study sample as they had used health care services and the internet in the past 12 months. The sample was weighted using inverse probability weighting (IPW) correction . The weiParticipation in the study was completely voluntary. Ethical approval was obtained from the Ethics Committee of the Finnish Institute for Health and Welfare (THL/637/6.02.01/2017).nonuse of My Kanta was evaluated with the question \u201cHave you used My Kanta in the past 12 months?\u201d Respondents were asked to respond (1) no or (2) yes. For the analyses, the measure was binary-coded , and the users of My Kanta were set as the reference group.The dissatisfaction with My Kanta was evaluated with a question concerning satisfaction: \u201cIf you have used the service, assess the quality of the service using a school grade (4-10).\u201d In the Finnish education system, grades 8-10 represent grades from good to excellent and grades 4-7 from fail to satisfactory [The sfactory . For thesfactory  between Independent variables included characteristics concerning (1) sociodemographic background, (2) health and the use of health care services, (3) experiences of guidance concerning electronic services, and (4) digital skills and attitudes. All the used variables are presented in degree of urbanization was determined according to the municipal classification and divided into 3 categories according to the proportion of people living in urban settlements and the population of the largest urban settlement: urban, semiurban, and rural municipalities [educational level was first divided into 10-year age groups by gender. Each group was divided into 3 categories based on their years of education, with approximately one-third of the respondents in each category: low, median, and high. Hence, the education-level variable had hardly any interaction with age and gender.The respondents\u2019 sociodemographic characteristics included their age, gender, education, and degree of urbanization. Age was used as a categorical variable in the descriptive statistics and as a continuous variable in all analyses. The palities . BecauseSelf-rated health was evaluated with a widely used, single-item measure of self-perceived health status. A subjective assessment of own health has been reported as a more sensitive measure in health monitoring than external measures of health, since it includes biological, psychological, and social dimensions. [good and (2) fairly good were combined to represent good health, and the remaining options represented average or poor health. Long-term illness was binary-coded as (1) yes and (2) no. The use of health care services was binary-coded according to the number of annual outpatient appointments with a physician; 8 or more annual appointments were considered a high use of health care services, and less than 8 were counted as low or average use [Variables concerning the respondents\u2019 health and the use of health care services included self-rated health, long-term illness, and the use of health care services. ensions. . A scalerage use .referral to electronic services was evaluated with the question \u201cIf you have used social or health care services in the traditional way  in the past 12 months, were you referred to electronic services ?\u201d For the analyses, option (1) yes, I was referred represented the respondents who were referred to electronic health care services. Option (2) was for those who were not referred to electronic health care services.Variables concerning the experiences of guidance concerning electronic services included referrals to electronic services and the need for guidance on how to use online social or health care services. The need for guidance on using online social and health care services was evaluated with the statement \u201cI need help with using online social and health care services.\u201d In the analyses, the options (1) completely agree and (2) somewhat agree were combined as (1) yes and the remaining options as (2) no or no opinion.The Digital skills were evaluated with 6 validated statements [good skills (mean\u22642.5) and (2) poor skills (mean>2.6). The same coding has previously been used in national research [Variables related to digital skills and attitudes included digital skills, perceived benefits of electronic social and health care services, and security concerns. atements . Based oatements . A 5-poiresearch .perceived benefits of electronic social and health care services were measured with 8 statements. A 5-point Likert scale was used to answer the statements (1=completely agree to 5=strongly disagree). Cronbach \u03b1 for the statements was .91. In the analyses, missing values were coded as neither agree nor disagree. A mean variable from 1 to 5 was calculated for each respondent, and the measure was binary-coded as (1) beneficial (mean\u22642.5) and (2) unbeneficial (mean>2.6). The same coding has previously been used in national research [The research .Security concerns were evaluated with the statement \u201cI am concerned about information security when it comes to my personal details\u201d. In the analyses, options (1) completely agree and (2) somewhat agree were combined as (1) yes and the remaining options as (2) no.In all statistical analyses, the IPW method  was applP value of <.10. This cut-off for the P value was used for including the variables in the multivariable model, because the purpose was to identify potential independent variables rather than to test a hypothesis [P value of <.05 was considered statistically significant. Statistical methods suitable for weighted data were used, and SPSS Statistics version 27 was applied for the analyses.Bivariate logistic regression analyses were used to examine the adjusted associations of respondent characteristics with the nonuse of My Kanta and dissatisfaction with the service . First, univariate analyses, adjusted for age, gender, and education, were conducted at a time to examine the association of the dependent variable with each independent variable. Second, a multivariable model was formed, including only those independent variables with a pothesis . In the The weighted majority  of the respondents had used My Kanta in the past 12 months. Most of the My Kanta users  were satisfied with the service. A minority of respondents  had not used My Kanta in the past 12 months.The IPW weighted characteristics of the respondents representative of the Finnish population are presented in Based on the results of age-, gender-, and education-adjusted univariate logistic regression analysis , the folThe results of the fully adjusted logistic regression analysis regarding the nonuse of My Kanta are presented in Based on the results of the age-, gender-, and education-adjusted univariate analyses , the folIn the fully adjusted multivariable model, respondents who were younger, were male, and had a high level of education were more likely to be dissatisfied with My Kanta compared to their counterparts. Respondents with average or poor self-rated health were over 2 times more likely to be dissatisfied with My Kanta compared to respondents with a good perception of their own health. In addition, respondents who perceived electronic services as unbeneficial, who needed guidance, and who had security concerns were more likely to be dissatisfied with My Kanta compared to their counterparts.Most respondents of this nationally representative survey study had used the nationwide Finnish patient portal My Kanta in the previous 12 months and were satisfied with the service. However, more than every 10th user of health care services and the internet were nonusers of the national patient portal, and approximately the same number of users were dissatisfied with the service. Males and those in a need of guidance were more likely to be nonusers of the patient portal and dissatisfied with the service compared to women and those not needing guidance. Not having a long-term illness and low or average use of health care services were associated with the increased likelihood of nonuse of the My Kanta portal. In addition, respondents who were not referred to electronic services and who had poor digital skills were more likely to be nonusers of My Kanta compared to their counterparts. A younger age, higher education, and poor self-rated health were associated with an increased likelihood of dissatisfaction with the service. In addition, respondents who did not perceive electronic health care services to be beneficial and who had security concerns were more likely to be dissatisfied with the service compared to their counterparts.Finland is 1 of the forerunners of digitalization and ranked highest in information exchange and patient-centered information processing in an international comparative study . By presA nationally presentative sample of Finnish residents was included in the analysis. The applied IPW method has previously been reported to improve the accuracy and generalizability of results . HoweverSince only respondents who had used the internet for transactions or for searching for information were included in the study, the results are only applicable when considering the nonuse of patient portals beyond the first-level digital divide caused by the lack of necessary devices and access to the internet. Some respondents who did not use My Kanta used additional patient portals provided by private or public providers of health care services. It is also noteworthy that some respondents might not be referred to electronic health care services, because their transactions in health care do not require further action or electronic services cannot provide support in their situation. The research concerning the users and nonusers of nationwide patient portals is sparse, and comparison is difficult as the properties provided in the portals vary, in addition to the differing patient populations and adjustments in the analyses.The use of nationwide patient portals varies by country and portal ,10. ThisThe results of this study suggested that younger and more educated respondents were more likely to be dissatisfied with My Kanta compared to older and less educated respondents. The findings of this study were supported by the fact that younger generations have grown up with technology and were thus more comfortable using electronic services , which mThis study found that respondents without any long-term illness and with low or average use of health care services are more likely to be nonusers of My Kanta, which is consistent with previous research ,25,27,64Over half of the respondents were not referred to electronic health care services by their care providers, and the nonreferred respondents were less likely to use the nationwide patient portal My Kanta compared to referred respondents. S\u00e4\u00e4skilahti et al  and KongAlthough patients have prior experience in the use of electronic services and information technology tools, the ability to review and manage medical records on patient portals should not be assumed . ResultsThe previous literature has suggested different ways in which the use of patient portals could be promoted, including ease of entry ,30,72, eIn addition to easy accessibility of the service, assistance on the use should be available at a low threshold. Because patients have previously been reported to seldom seek help from family members, friends, service support, or health care providers , electroDigital skills are necessary for wider patient adoption and use of patient portals ,37,81,82Attitudes about the usefulness, appropriateness, and potential downsides of electronic services may encourage or impede the use . In thisAccording to this study, respondents who had security concerns were more likely to be dissatisfied with My Kanta compared to respondents who felt more secure. Similar to the results of Woods et al , securitAccording to the results of this population-based cross-sectional survey study in the era of COVID-19, patients without long-term illnesses, those not referred to electronic health care services, and those in need of guidance on the use of online social and health care services seem to be more likely nonusers of the Finnish nationwide patient portal My Kanta. Moreover, poor health and security concerns seem to be associated with dissatisfaction with the service. Interventions to promote referral to electronic health care services by professionals are needed. Attention must be paid to information security of the service as well as the alleviation of the patients\u2019 privacy concerns."}
{"text": "BMPR2 (bone morphogenetic protein (BMP) receptor type II) cause pulmonary arterial hypertension. BMPRII is a receptor for over 15 BMP ligands, but why BMPR2 mutations cause lung-specific pathology is unknown. To elucidate the molecular basis of BMP:BMPRII interactions, we report crystal structures of binary and ternary BMPRII receptor complexes with BMP10, which contain an ensemble of seven different BMP10:BMPRII 1:1 complexes. BMPRII binds BMP10 at the knuckle epitope, with the A-loop and \u03b24 strand making BMPRII-specific interactions. The BMPRII binding surface on BMP10 is dynamic, and the affinity is weaker in the ternary complex than in the binary complex. Hydrophobic core and A-loop interactions are important in BMPRII-mediated signalling. Our data reveal how BMPRII is a low affinity receptor, implying that forming a signalling complex requires high concentrations of BMPRII, hence mutations will impact on tissues with highest BMPR2 expression such as the lung vasculature.Heterozygous mutations in  Mutations in BMPR2 is the major genetic cause for pulmonary arterial hypertension (PAH). Here by solving crystal structures of BMPRII in binary and ternary receptor complexes with BMP10, the authors report the molecular recognition between BMPRII and BMP10, and its implication in PAH. Upon signalling complex formation, the constitutively active type II receptor phosphorylates and activates the type I receptor; subsequently the type I receptor phosphorylates Smad1/5 or Smad2/3 to regulate transcriptional responses.Transforming growth factor \u03b2 (TGF-\u03b2) family cytokines control many fundamental biological processes, from the establishment of body patterning during embryonic development, to the maintenance of adult homeostasis in vasculature, wound repair, and immune response. The TGF-\u03b2 ligands are dimers and initiate signal transduction by forming a signalling complex with two copies of a type I receptor and two copies of a type II receptor; all are single-pass transmembrane proteins containing a small 3. Although BMPRII binds BMP10 with the highest affinity among different ligands1, its affinity is still at least 10-fold weaker than other high-affinity cognate receptor:ligand interactions in the TGF-\u03b2 family1, and around 10-fold weaker than ActRIIB binding to BMP102. How BMPRII regulates BMP signalling through low-affinity interactions is not known.There are more than 30 genes encoding TGF-\u03b2 ligands which can be broadly divided into three subfamilies, the TGF-\u03b2s, the activins, and the bone morphogenetic proteins (BMPs). Their signalling is mediated by only 7 type I receptors (Activin receptor-like kinase 1-7 (ALK1-7)) and 5 type II receptors (TGF-\u03b2 receptor type II (TGF\u03b2RII), Activin receptor type 2\u2009A and 2B (ActRIIA and ActRIIB), BMP receptor type II (BMPRII) and Anti-Mullerian hormone (AMH) receptor type II (AMHRII)). Although some ligand:receptor interactions are of high affinity and specificity, such as TGF\u03b2RII for TGF-\u03b21 and TGF-\u03b23, ALK1 for BMP9 and BMP10, many are promiscuous. For example, ActRIIA/B bind to and mediate the signalling from both activin ligands and some BMP ligands, whereas BMPRII is the low-affinity type II receptor for over 15 BMP ligandsBMPR2 are the major genetic cause for pulmonary arterial hypertension (PAH)5, the consequences of which include remodelling of the pulmonary arteries, elevated right ventricular pressure, right ventricle hypertrophy and eventually heart failure. There is currently no cure for PAH and it represents a significant unmet medical need. Of note, no mutations in genes encoding ActRIIA or ActRIIB have been reported in PAH patients.BMPRII is unique among TGF-\u03b2 family receptors because it possesses a long carboxy-terminal tail of more than 500 amino acids after the kinase domain. Although both ActRIIA/B and BMPRII can mediate signalling from different BMPs, the phenotypes of knockout mice, tissue distribution, and the effects of mutations in human diseases are very different among these three type II receptors. Loss of function mutations in BMPR2 mutations have been identified in PAH cohorts to date6. Most mutations result in haploinsufficiency, but around 25% are missense mutations in the ECD and ICD. Some missense mutations, including many cysteine substitutions, cause protein misfolding and retention in the endoplasmic reticulum8. Other mutations in the ECD do not affect cell surface localisation and their impact on ligand binding and signalling activity remains unclear.Around 668 BMPR2 mutations cause PAH despite the receptor being rather ubiquitously expressed in the body is intriguing, suggesting a pivotal role for BMPRII in the lung vasculature. Of note, BMPRII is particularly highly expressed in lung vascular endothelial cells, where it mediates the signalling from circulating BMP9 and BMP10. The unique features of these two ligands include binding with very high affinity and specificity to the endothelial-specific type I receptor ALK1, and the co-receptor endoglin (ENG). The importance of ALK1 and ENG in the vasculature is underpinned by human genetics showing that autosomal dominant loss-of-function mutations in these two genes cause Hereditary Haemorrhagic Telangiectasia (HHT)10, a vascular abnormality characterised by telangiectases (broken capillaries) in the nasal mucosa, gastrointestinal tract and skin, and larger arteriovenous malformations in brain, lungs and liver, which can be life-threatening. Mutations in ALK1, ENG, GDF2 (encoding BMP9) and BMP10 have also been reported in PAH patients11. Thus, human genetics strongly supports a role for BMPRII and BMPRII/ALK1-mediated BMP9 and 10 signalling in lung endothelial cells in the pathogenesis of PAH. In accordance with this, we have recently shown that endogenous BMP9 plays a critical role in the maintenance of endothelial integrity, particularly in the pulmonary vasculature12.Why BMPR2 mutations in PAH, crystal structures of BMPRII signalling complexes are yet to be solved, perhaps due to the difficulty of obtaining crystals from the low affinity receptor:ligand complexes. Here we report crystal structures of BMP10:BMPRII complex in two crystal forms, and the ALK1:BMP10:BMPRII signalling complex. We show that although BMPRII utilises the same hydrophobic core as ActRIIA/B and binds to BMP10 at the knuckle epitope, the longer A-loop and finger 3 loop (F3-loop) in BMPRII make unique interactions. Importantly, we show that BMPRII makes fewer interactions with BMP10 and has lower binding affinity for BMP10 in the ternary complex than in the binary complex, suggesting a mechanism for the transient nature of the BMPRII-mediated signalling. Surprisingly, we found that BMP10 fingertip 3/4 preferentially adopts either an extended or a bent conformation in different protein-protein interaction contexts. Such conformational plasticity is also present in BMP9; which may be another unique feature of BMP9 and 10 contributing to their signalling specificity.Despite two decades of extensive research on BMPRII since the discovery of We solved crystal structures of the BMPRII binary complex with BMP10 in two different crystal forms, at 1.48\u2009\u00c5 and 2.4\u2009\u00c5, respectively , which provided a BMP10 conformation in yet another protein interaction context. Two crystal forms  because the expression level is very low, and transfection did not lead to an increase in BMP signalling  by Uniprot32. Once it is made in a recombinant expression system, it can bind to BMP10 with similar affinity to WT BMPRII  to undergo the bent-to-extended conformational change in fingertip 3/4. The insertion in the A-loop in BMPRII comes at a slight cost of complex stability because the loop truncation mutant \u0394GDP binds to BMP10 with both a slower on-rate and a slower off-rate.1. Sequence alignment revealed a conserved IAP motif across all BMPRII-binding ligands , mouse 2H-11 endothelial cells (cat. No. CRL-2163) and human embryonic kidney (HEK) EBNA cells (cat. No. CRL-10852) were all purchased from American Type Culture Collection (ATCC). All cell lines were cultured in Dulbecco\u2019s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 100\u2009U/mL penicillin-streptomycin and were frequently tested for the absence of mycoplasma contamination.31. Briefly, cDNA of full-length open reading frame of human BMP10 (NM_014482) was cloned into pCEP4 and transfected into HEK EBNA in DMEM supplied with 5% FBS. Human full-length FURIN cDNA in pCEP4 was co-transfected to facilitate processing. Serum-free chemically defined (CD) CHO  medium was applied from day 2 and conditioned media were harvested every 3\u20134 days for 5 harvests. To purify Pro:BMP10, conditioned medium was loaded onto 5\u2009ml Hitrap Q columns, selected fractions containing Pro:BMP10 were concentrated and loaded onto a HiLoad Superdex 200\u2009pg 16/600 gel filtration column. Peak fractions were further purified using a MonoP 5/50 GL column pre-equilibrated with 20\u2009mM Tris, 50\u2009mM NaCl, pH 7.4 and eluted with a NaCl gradient. Target fractions were concentrated and loaded onto an Superdex 200 column pre-equilibrated in 20\u2009mM Tris, 150\u2009mM NaCl, pH 7.4. To facilitate crystallisation, a mutant form of Pro:BMP10 was produced. Using the UCLA MBI-SERp server34, residues K267-E269 and E296-E297 were suggested as residues with high entropy at the protein surface, and mutating which may enhance the protein\u2019s crystallisability via the generation of crystal contacts. Therefore these residues were all mutated to alanine using site-directed mutagenesis. The mutant Pro:BMP10 protein was expressed and purified using the same method as described for the WT Pro:BMP10.Pro:BMP10 was expressed and purified following the published method13. In brief, human ACVRL1  ECD cDNA (amino acids 22-118) was cloned into a pET39b vector. A TEV (Tobacco Etch Virus nuclear inclusion A endopeptidase) cleavage site was introduced at the N-terminus of ALK1 ECD. The plasmid was transformed into Rosetta DE3 bacteria and cultured in 2x YT medium at 37 \u00b0C to mid-log phase followed by IPTG  induction at 22\u2009\u00b0C overnight. The fusion protein DsbA-(His)6-ALK1 ECD was extracted from the periplasmic compartment according to the pET System Manual (Novagen) followed by purification using a 5\u2009ml HisTrap Excel column . The fusion protein was incubated with a His-tagged TEV protease and dialysed in Tris-buffered saline (TBS) overnight. The cleaved DsbA-(His)6 and TEV were removed from ALK1 ECD using a 5\u2009ml HisTrap Excel column. ALK1 ECD was further purified by a Superdex 75\u2009pg 16/600 column .ALK1 ECD was expressed and purified following the published methodBMPR2 cDNA (NM_001204) encoding amino acids 27-150 was cloned into a pET39b plasmid to create a fusion WT protein DsbA-(His)6-BMPRII ECD. All the mutations and deletions were introduced using a Q5\u00ae Site-Directed Mutagenesis Kit (New England Biolabs) following the manufacturer\u2019s protocol and were confirmed by sequencing. The WT and mutant proteins were expressed and purified following the method described above for ALK1 ECD.Human KD was determined by the ratio of binding rate constants kd/ka.Surface plasmon resonance (SPR) experiments were undertaken using the Biacore T200 system . Recombinant human BMP10 growth factor (GF)-domain  was immobilised onto a Series S research grade CM5 sensor chip  by amine-coupling at a density of 1300 resonance units. For kinetic measurements, a series of concentrations of ALK1-Fc , BMPRII-Fc , monomeric BMPRII WT and mutant ECDs were injected in duplicate over the flow cells at a flow rate of 40 ul/min in a buffer containing 10\u2009mM HEPES, pH 7.4, 150\u2009mM NaCl, 3.4\u2009mM EDTA, 0.5\u2009mg/ml BSA  and 0.005% (v/v) surfactant P20 at 25\u2009\u00b0C. For ALK1-Fc and BMPRII-Fc binding experiments, the surface was regenerated with 4\u2009M Guanidine Hydrochloride while for BMPRII WT and mutant ECD experiments, no regeneration was needed. The kinetic rate constants were obtained by fitting the corrected data to a 1:1 interaction model using Biacore T200 Evaluation Software . The equilibrium binding constant P212121 to 1.48 \u00c5 using DIALS35 and AIMLESS36 in the CCP4 suite37. The structure was solved by molecular replacement using Phaser38, with BMP10 from PDB entry 6SF3 and BMPRII (PDB entry 2HLQ) as the search models. Model building was performed using Coot39 and refinement using REFMAC540. The final model was validated using MolProbity41. A second crystal form was obtained in 14% PEG 3350, 0.14\u2009M KCl, pH 7.0 in a month and data were collected at I04, DLS at a wavelength of 0.97950 \u00c5. The data were processed in C2 space group to 2.40 \u00c5. The structure was determined by molecular replacement in Phaser using the 1.48 \u00c5 BMP10:BMPRII structure as a search model. As above, model building was carried out using Coot, refinements using REFMAC5 and phenix.refine42 and validation using MolProbity. All the data collection, data reduction, structure determination and refinement statistics are summarised in Supplementary Table\u00a0Pro:BMP10 was denatured in 7\u2009M urea solution overnight and the denatured protein was loaded onto a 5\u2009ml HiTrap S column pre-equilibrated in a binding buffer of 20\u2009mM Tris, 25\u2009mM NaCl, 6\u2009M urea, pH 7.4 followed by elution using a NaCl gradient. Fractions containing the denatured BMP10 GF-domain were concentrated to 1\u2009ml followed by a rapid dilution in 19\u2009ml cold refolding buffer  dimethylammonio)-1-propanesulfonate), 2.5% glycine, 5\u2009mM glutamic acid, pH 4.0). Excess of BMPRII ECD was then added in the refolding buffer and left on a roller at 4\u2009\u00b0C for 6 days. The refolded mixture was concentrated and further purified using a Superdex 200 16/600 column pre-equilibrated in 20\u2009mM Tris, 150\u2009mM NaCl, pH 7.4. BMP10:BMPRII ECD was concentrated to 4.8\u2009mg/ml for crystallisation trials using the hanging-drop method with 1\u2009\u03bcl protein and 1\u2009\u03bcl reservoir solution. Crystals were obtained over 4 days at 21 \u00b0C in 14% polyethylene glycol (PEG) 3350, 0.19\u2009M ammonium citrate dibasic, 0.02\u2009M sodium citrate tribasic dihydrate, pH 5.8. Crystals were cryo-protected in 30% glycerol in crystallisation reservoir solution and vitrified in liquid nitrogen. Data collection was undertaken at 100\u2009K at Diamond Light Source  on Beamline I04 at a wavelength of 0.91589 \u00c5, and processed in space group P21 to 3.60 \u00c5 using DIALS and AIMLESS in CCP4 suite. The structure was solved by molecular replacement using Phaser. The ALK1:BMP10 complex from 6SF3 was used as the first search model which gave 4 copies of ALK1:BMP10 1:1 complex in the asymmetric unit forming two copies of ALK1:BMP10 2:2 complex. The BMPRII ECD from the 1.48 \u00c5 BMP10:BMPRII structure was used as the second search model and only 1 copy of BMPRII was found by Phaser using the default settings. The second copy of BMPRII was found by searching more deeply in the rotation list, using all peaks above 50% of the top peak in the translation search in Phaser. The last 2 copies were found by using the Brute rotation function generating all orientations within 15 degrees of their expected values (found by superposing the BMP10:BMPRII complex structure on BMP10) and using all these orientations for the following translation search. Since the data were highly anisotropic, anisotropic correction of data used for refinement was performed using the STARANISO Server . Model building, refinement and validation were performed as described for the BMP10:BMPRII complexes. All the data collection, data reduction, structure determination and refinement statistics are summarised in Supplementary Table\u00a0To make the ALK1:BMP10:BMPRII complex, ALK1 was mixed with preformed BMP10:BMPRII complex in a 1.2:1 ratio and the mixture was concentrated to 3.9\u2009mg/ml. Crystallisation was performed by hanging drop method using 1\u2009\u03bcl protein and 1\u2009\u03bcl reservoir solution. A single crystal cluster was obtained after 15 days at 21 \u00b0C in 20% PEG 3350, 0.2\u2009M calcium acetate hydrate, pH 7.5. Crystals were cryo-protected in 30% glycerol in crystallisation reservoir solution and vitrified in liquid nitrogen. Data collection was undertaken at 100\u2009K at Diamond Light Source on Beamline I04-1 at a wavelength of 0.91188 \u00c5 and processed in space group C2221 to 2.90 \u00c5 using DIALS and AIMLESS in CCP4 suite. The structure was solved by molecular replacement using Phaser, with BMP10 from PDB entry 6SF3 and BMP9 prodomain from PDB entry 4YCI as the search models. Model building, refinement and validation were performed as described above.Pro:BMP10, with mutations to promote crystal contacts, was concentrated to 5\u2009mg/ml and subjected to a crystal screen at 21 \u00b0C with 1.5\u2009\u03bcl protein and 1\u2009\u03bcl reservoir solution. Crystals  were obtained in 19% PEG 3350, 0.15\u2009M ammonium tartrate dibasic, 0.02\u2009M sodium cacodylate trihydrate, pH 6.6 after 15 days. Crystals were cryoprotected in 30% glycerol in crystallisation reservoir solution and vitrified in liquid nitrogen. Data collection was undertaken at 100\u2009K at Diamond Light Source on Beamline I04-1 at a wavelength of 0.91589 \u00c5 and processed in space group C2221 to 3.5 \u00c5. The structure was solved by molecular replacement using Phaser, with the crystal form 1 structure as the search model. Model building, refinement and validation were performed as described above for the other structures. All the data collection, data reduction, structure determination and refinement statistics are summarised in Supplementary Table\u00a0WT Pro:BMP10 was concentrated to 8.4\u2009mg/ml and crystallised in 20% PEG3350, 0.2\u2009M ammonium tartrate dibasic, pH 6.6 by mixing 1\u2009\u03bcl protein and 1\u2009\u03bcl reservoir solution at 21 \u00b0C for about 100 days . Data collection was at 100\u2009K at I04-1, DLS at a wavelength of 0.91188 \u00c5, and processed in space group 43 was used for sequence alignment. Buried interface area were calculated using QtPISA 2.1.0 in CCP4-7.1.Structural analyses were performed using Coot and Pymol , and figures generated using Pymol. Clustal OmegaPro:BMP10 was pre-mixed with BMPRII WT or mutant ECDs (at 1:2 and 1:5 molar ratio) in 20\u2009mM Tris, 150\u2009mM NaCl, pH 7.4 in a final volume of 8 ul for 30\u2009minutes at room temperature before fractionation on a 12% native PAGE. After staining with Coomassie Blue, band intensities were quantified by ImageJ (version 1.51\u2009s). To identify the native gel bands, bands were cut out from the native gel and inserted directly into the wells of a 12% gel for SDS-PAGE using a standard protocol.2, pH 9.8 and absorbance was measured at 405\u2009nm. The experiment was repeated three times, with technical duplicates each time. All values are presented as the ratio of OD405nm of samples to Pro:BMP10 only sample.A high binding 96-well plate was coated with 0.25\u2009\u03bcg/well of anti-human BMP10 GF-domain antibody  in phosphate-buffered saline (PBS) and incubated in a humidified chamber at 4 \u00b0C overnight. The wells were washed with PBS containing 0.05% Tween 20 (PBST) followed by blocking with 1% BSA in PBS (BSA/PBS) at room temperature for 2\u2009hours. In parallel, 25\u2009ng Pro:BMP10 samples were premixed with BMPRII ECD WT  or mutants (at 1:125 molar ratio) in 1% BSA/PBS at room temperature for 30\u2009minutes. The plate was washed with PBST before samples were added. After incubation for 2\u2009hours, the plate was washed, and antihuman BMP10 propeptide detection antibody  in 1% BSA/PBS was added. After washing, ExtrAvidin\u00ae-Alkaline phosphatase  diluted 1:400 in 1% BSA/PBS was added. The assay was then developed with 0.67\u2009mg/ml 4-Nitrophenyl phosphate disodium salt hexahydrate  in 1\u2009M Diethanolamine, 0.5\u2009mM MgCl44, \u03b2-gal expression plasmid (transfection control) and mutant or WT pcDEF-BMPR2 plasmids were transfected per well in triplicate. Forty-eight hours post-transfection, the cells were serum-starved overnight before luciferase activity was measured.HepG2 and 2H-11 cells were seeded in 24-well plates and transfected, respectively, with PEI  and Lipofectamine 3000. BRE-luc transcriptional reporter45.Cell lysate was fractionated on 10 or 12% SDS-PAGE and transferred to PVDF membrane. For anti-FLAG blots, after blocking in BSA/PBS, the membrane was incubated with anti-FLAG antibody , followed by wash and horseradish peroxidase (HRP)-conjugated secondary antibody . For phosphor-Smad1 blot, an in-house made phosphor-Smad1 antibody (1:1000 dilution) was used which has been validated previouslyBMPR2 plasmid for 24\u2009hours using Lipofectamine\u2122 LTX with PLUS\u2122 Reagent . Cells were washed twice with ice-cold PBS (containing Ca2+/Mg2+) and incubated at 4\u2009\u00b0C for 30\u2009min with 0.33\u2009mg/ml Thermo EZ-Link Sulfo-NHS-SS-Biotin in PBS . Cells were quenched thrice with 50\u2009mM glycine in PBS before lysed on ice for 45\u2009min in 320\u2009\u03bcl lysis buffer . Cell lysate was sonicated briefly and centrifuged at 21,000\u2009g for 10\u2009min at 4\u2009\u00b0C. Lysate supernatant was loaded (100 \u03bcl) in duplicate into 96-well Pierce\u2122 NeutrAvidin\u2122 Coated High Capacity Plates  and incubated for 2\u2009hours at 4\u2009\u00b0C. The plate was blocked with 3% BSA in PBS and incubated with anti-FLAG antibody  overnight at 4\u2009\u00b0C and then 1\u2009hour at room temperature with anti-mouse HRP secondary antibody . Peroxidase activity was detected by incubating with 100 \u03bcl/well SIGMAFAST\u2122 OPD (o-Phenylenediamine dihydrochloride) for 30\u2009minutes at room temperature and absorbance was read at 450\u2009nm. Total FLAG-tagged BMPRII in the cell lysate was assessed by immunoblotting.HEK EBNA cells were seeded in a 24-well plate in duplicate 2 days prior experiment and transfected with 500\u2009ng pcDEF-BMPR2 plasmid. Total RNA was extracted using RNeasy Mini Kit buffers  and Silica Membrane Mini Spin Columns (EconoSpin) following the manufacturer\u2019s instructions. Equal amounts of RNA (~1\u2009\u03bcg) were then reverse transcribed into cDNA using a High Capacity Reverse Transcriptase kit (Applied Biosystems). 2\u2009\u03bcl cDNA, 1.8\u2009\u03bcl associated premixed primer sets , 5\u2009\u03bcl 2X SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich), 0.2\u2009\u03bcl ROX reference dye (Invitrogen) and 1\u2009\u03bcl DEPC-treated water were prepared into one well of a MicroAmp\u00ae Optical 384-Well Reaction Plate (Applied Biosystems) which was then put on a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). Amplification reactions were initiated with a 2-minute pre-incubation at 95\u2009\u00b0C, followed by 50 amplification cycles of 30-second denaturation at 95\u2009\u00b0C, 30\u2009seconds annealing at 55\u2009\u00b0C and 30-seconds extension at 72\u2009\u00b0C. The following primers were used for the qPCR reactions: human ID1: 5\u2032-CTGCTCTACGACATGAACGGC-3\u2032, 5\u2032-TGACGTGCTGGAGAATCTCCA-3\u2032; human \u03b22 microglobulin (B2M): 5\u2032-CTCGCGCTACTCTCTCTTTCT-3\u2032, 5\u2032-CATTCTCTGCTGGATGACGTG-3\u2032. The relative expression levels of ID1 were calculated using the \u0394\u0394Ct method by normalizing to B2M. Differences in gene expression are presented as the fold change relative to control.HEK EBNA were seeded at 200,000 cell/well into a 6-well plate followed by 24-hour transfection with 1\u03bcg of pcDEF-P\u2009<\u20090.05 were considered significant.Data analyses were performed using GraphPad Prism 6.0 (GraphPad Software). Results are shown as means \u00b1 SEMs. Statistical significance was analysed using One-way ANOVA with Dunnett\u2019s post-test comparing with appropriate controls as indicated in figure legends. Values of Further information on research design is available in the\u00a0Supplementary InformationDescription of Additional Supplementary FilesSupplementary movie 1Reporting Summary"}
{"text": "Octodonta nipae, is a pest of palm trees in Sothern China. To address its increasing damage, environmentally friendly control methods are required. This study aimed to test efficacy of Heterorhabditis bacteriophora and Steinernema carpocapsae on O. nipae and investigated the influence of secondary metabolites, nematodes, and their isolated cuticles on the activation of O. nipae\u2019s prophenoloxidase system using qPCR analysis. Our data revealed that O. nipae were less susceptible to H. bacteriophora than S. carpocapsae and penetrations of infective juveniles were higher with S. carpocapsae treatment than H. bacteriophora. Moreover, expression levels of the serine protease P56, prophenoloxidase activation factor 1, PPO and serine protease inhibitor 28 upon S. carpocapsae and H. bacteriophora infections were generally downregulated at all times. However, upon heating, the cuticles lost their inhibitory effects and resulted in upregulation of the PPO gene. Similarly, the addition of arachidonic acid reversed the process and resulted in the upregulation of the PPO gene compared to the control. Further work is needed to identify toxic substances secreted by these EPNs to evade O. nipae\u2019s immune system.Entomopathogenic nematodes are biocontrol agents of invasive insect pests in soil and cryptic habitats. Nipa palm hispid,  Octodonta nipae (Maulik) is not an exception [Tetrastichus brontispae Ferri\u00e8re (Hymenoptera: Eulophidae) [Steinernema carpocapsae [Metarrhizium anisopliae [O. nipae are needed.The movement of destructive pest species from one country or region to another is on the rise. Nipa palm hispid xception ,6,7,8,9.xception ,14,15,16xception ,18,19. Oophidae) ,26,27,28pocapsae ,18,19 anisopliae ,30,31,32Diabrotica virgifera LeConte [Phyllotreta spp. [Leptinotarsa decemlineata (Say) found on leaf surfaces [O. nipae larvae was conducted in our laboratory using S. carpocapsae and found to be virulent at different concentrations and time points, as published in Sanda et al. [O. nipae larvae have yet to be conducted.Heterorhabditidae and Steinernematidae are families of entomopathogenic nematodes (EPNs) mainly used for biological control of different economically invasive pests at field and laboratory levels ,35,36,37 LeConte , flea beeta spp. , and Colsurfaces . Similara et al. . HoweverO. nipae, three full-length cDNAs of PPAFs (OnPPAFs) were cloned, namely OnPPAF3, OnPPAF1, and OnPPAF2. They were highly expressed in hemolymph except OnPPAF2, which shows low transcript abundance. However, knockdown of OnPPAF1 and OnPPAF3 showed a reduction in hemolymph phenoloxidase activity and inhibition of hemolymph melanization [Insects employ both humeral and cellular immune reactions in response to attacks by pathogens such as bacteria, fungi, and nematodes. These innate immune mechanisms include nodulation, phagocytosis, encapsulations, production of antimicrobial peptides, and activation of PPO system, which leads to melanization and subsequent pathogen death by septicemia ,47. Insenization ,52,53,54S. carpocapsae use their body surface cuticle to escape host hemocyte encapsulation and at the same time inhibit its proPO system activity [S. carpocapsae and are involved in host immune modulation [Moreover, different insect hosts responded differently to EPNs infections depending on the species and strain. Each nematode species uses different strategies to evade and suppress melanization and escape cellular encapsulation. Our previous study showed that EPNs of the family Steinernematids, activity . These aactivity . These Edulation ,56,57.H. bacteriophora and S. carpocapsae interact with O. nipae humoral immune system remains unknown. The present study, therefore, was conducted to determine the survival rates of O. nipae larvae at different concentrations of H. bacteriophora. Similarly, we evaluated the penetration abilities of both H. bacteriophora and S. carpocapsae against the third instar larvae of O. nipae. Further, we tested the differences in expression levels of four selected genes involved in proPO activation in O. nipae larvae. Finally, the effects of some selected secondary metabolites and isolated cuticles from nematodes on O. nipae proPO gene expression were investigated.Eicosanoids are other insect cellular and humoral immune mediators against several microbial infections ,59. TheyH. bacteriophora H06 (Poinar) and S. carpocapsae All (Weiser) were obtained from the Guangdong Institute of Applied Biological Resources, China [Galleria mellonella [O. nipae adults were collected from Hainan Island, China, and reared with small pieces of fortunes windmill palm, Trachycarpus fortunei (Hook) in the laboratory for many generations, after which healthy third instar larvae were selected and used for this study. The insects were maintained at 25 \u00b1 1 \u00b0C, relative humidity of 80 \u00b1 5%, and a photoperiod of 12 light: 12 dark hours, as previously described by Sanda et al. [s, China . Nematodllonella . Infectillonella . O. nipaa et al. .2 inhibitor, dexamethasone -9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3), the eicosanoid precursor, arachidonic acid , serine proteases inhibitor, phenylmethanesulfonyl (PMSF) fluoride, and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich  Trading Co., Ltd. .The phospholipase AO. nipae larvae were explored. In addition, the ability of the IJs to penetrate the O. nipae larvae at 0, 25, 50 and 100 IJs/larva concentrations was also experimented, and complete randomized design (CRD) was used as experimental design. Bioassay was conducted to determine the survival of O. nipae larvae at different concentrations of H. bacteriophora as fully described in our previous experiment using S. carpocapsae [H. bacteriophora and S. carpocapsae. After 72 h of inoculation, ten dead larvae (cadavers) were picked from the well plates, rinsed with distilled water, and dissected under dissecting stereo-microscope -Nikon company, Japan. The number of penetrated IJs were counted and recorded. Distilled water was used as control and each treatment contained 30 larvae, replicated three times to confirm the results.Here, the effects of EPN concentrations on mortality of pocapsae . In the O. nipae larvae infected with the two nematode species. These include Serine Protease P56 (SPP56), prophenoloxidase activation factor 1 (PPAF1), PPO, and serine protease inhibitor 28 (SPI28) genes  genes . Third ia et al. . RibosomO. nipae PPO gene, H. bacteriophora and S. carpocapsae body cuticles were isolated and purified as described in our previous study [O. nipae larvae were injected with 112 nL of purified isolated cuticles from H. bacteriophora and S. carpocapsae and the same amount of phosphate buffered saline (PBS) as control. Another set of the cuticles were subjected to heat treatment at 100 \u00b0C for 20 min. The heat-treated cuticles were injected in the same way as mentioned above. Total RNA isolation and qRT-PCR analysis were performed in triplicate for each biological replicate according to Sanda et al. [PPO gene expression levels due to the untreated cuticle, heat-treated cuticle, and control treatments.To determine whether nematode body cuticle have an influence on immune expression of us study . The O. a et al.  to checkO. nipae larvae, as described in our previous study [\u22121, and at 20 \u00b0C. The experiments were run in triplicate to confirm the results.We determined the inhibition effects of Dexamethasone\u2019s presence on the phenoloxidase activity in an in vitro assay with hemolymph of us study . Secondlus study . For conO. nipae larvae with eicosanoid biosynthesis inhibitor, dexamethasone (DEX) and precursor, arachidonic acid (AA) individually and in combination with H. bacteriophora and S. carpocapsae to ascertain their roles on the expression of PPO gene. The inhibitor, DEX, was dissolved in 100% dimethyl sulfoxide (DMSO) at an initial concentration of 1 M and subsequently diluted to 5 mM concentration. Three treatment groups were prepared for injection into the O. nipae larvae at 112 nL using a Nanoliter 2010 injection system . The first group was injected with DEX only to test its inhibitory effects on O. nipae proPO activation system, and PBS was used as control. The second group of the larvae was initially infected with nematodes and then injected with DEX after 8 h post-infection. The last group was infected with nematodes + AA to recover the effects of nematode inhibition. Samples were taken at 24 h after treatment for RNA isolation. Total RNA isolation and qRT-PCR analysis were performed in triplicate for each biological replicate according to Sanda et al. [PPO gene expression levels of each group.We injected the a et al.  to invesp \u02c2 0.05), means were compared or separated using least significance differences (LSD). The level of mRNA expression of some selected genes in proPO activation system of O. nipae was transformed by Logarithmic function and analyzed using the Student\u2019s t-test. Differences between mean values were analyzed and considered significant when p < 0.05 or considered extremely significant when p < 0.0001 concerned the control values.All statistical analyses were performed using IBM SPSS Statistics version 22  . Data were corrected for control mortality using Abbott formula  and percS. carpocapsae at different concentrations by plotting the survival curve at different time points [O. nipae larvae at different concentrations were reported and at 24 and 48 h post-treatments [H. bacteriophora on the survival of O. nipae larvae was evaluated. The results showed that the survival ability of O. nipae larvae was significant at different time points and H. bacteriophora IJs concentrations . The O. nipae larvae survived for 140 h longer at lower concentrations of 25 and 50 IJs/larva than at 100 IJs/larvae, as shown in We previously evaluated the pathogenicity of e points . Signifieatments . In thisS. carpocapsae and H. bacteriophora penetrated the hemocoel of the larvae of O. nipae at the concentration of 25, 50, and 100 IJs/larva  than that of H. bacteriophora  in O. nipae larvae, as shown in S. carpocapsae and H. bacteriophora.Similarly, the results for the penetration assay revealed that the IJs of Js/larva . HoweverO. nipae prophenoloxidase enzymes  between H. bacteriophora- and S. carpocapsae-treated larvae at three distinct time intervals. Our qRT-PCR results reveals that SPP56 was significantly upregulated  after H. bacteriophora challenge and insignificantly upregulated  upon S. carpocapsae treatment at 8 h post-infection  of the gene  and H. bacteriophora  treatments  but significantly downregulated at 24 h  after injection of the isolated cuticles from the two nematodes  and S. carpocapsae  heat-treated cuticles injections. At 16 h after injection of S. carpocapsae cuticle, insignificance  upregulation of PPO gene was recorded . However, the addition of AA rescued the phenoloxidase-inhibitory activities caused by DEX treatments .In this study, phenoloxidase-inhibitory activities of eicosanoid biosynthesis inhibitors and DEX treatments compared to controls were observed. There were significant decreases in phenoloxidase enzyme activities in hemolymph of eatments . There wPPO gene in treated larvae samples. Our results revealed that the expression level of PPO gene was downregulated significantly upon treatment of DEX  compared with control  and S. carpocapsae plus DEX  treatments at 24 h. Interestingly, injection of AA (precursor of eicosanoid biosynthesis) to nematode-treated larvae reverses the inhibition effects of both DEX and nematode plus DEX-treated larvae on the PPO gene expression. The mRNA level of PPO gene in H. bacteriophora plus AA  and S. carpocapsae plus AA  treatment were highly significant at 24 h after treatments.Secondly, injections of DEX to nematodes-treated larvae further suppressed the expression level of O. nipae larvae were more susceptible to H. bacteriophora. Compared to our previous results, the virulence of S. carpocapsae [H. bacteriophora can be attributed to their differences in host-searching strategies, as reported by Grewal et al. [G. mellonella hemocytes can recognize H. bacteriophora IJs, but not S. carpocapsae and S. glaseri nematodes [S. carpocapsae efficiently resists being encapsulated and melanized within the O. nipae\u2019s hemolymph and most of the nematodes were observed moving freely in the hemolymph even at 24 h post-incubation, whereas H. bacteriophora was identified, encapsulated and subsequently melanized by O. nipae\u2019s hemolymph [H. bacteriophora was less virulent to carob moth Ectomyelois ceratoniae Zeller (LC50 = 426.9 IJs larva\u22121) when compared to both S. carpocapsae and S. feltiae (LC50 = 2 IJs/larva) at concentrations similar to this study. Additionally, Sharifi et al. [S. carpocapsae caused higher mortality of Osphranteria coerulescens than H. bacteriophora, as evident by their LC50 of 2.7 and 9.0 IJs larva\u22121, respectively. This is consistent with quite a number of reports for different insect pest control [Entomopathogenic nematodes modulate their hosts differently, depending on the strategies used to escape the host defense mechanisms. For example, Zang and Maizels  describepocapsae  over H. l et al.  and Koppl et al. . Additioematodes . This isemolymph . Moreoveemolymph  reportedi et al.  have rep control ,78,79,80S. carpocapsae and H. bacteriophora with regards to the mean penetration potential within the host larvae. This is also similar to a report by Susurluk et al. [S. carpocapsae into turf pest, Dorcadion pseudopreissi, and lower in H. bacteriophora treatments. Similarly, higher penetration rates were also reported for Steinernematids in previous works by Phan et al. [In any successful biological control program using EPNs as control agents, the nematodes should have the ability to not only penetrate their hosts but also reproduce inside them. The routes of entry of IJs into the host body cavity differ among nematode species and insect hosts. These require some mechanical processes by the nematodes to bypass the insects\u2019 natural barriers, such as hairs . Accordik et al. , which en et al. , Salari n et al. , Salem en et al. , Koppenhn et al. , and Carn et al. .self\u2019 as in S. carpocapsae. They also suppress the hosts\u2019 immune responses after being recognised as \u2018non-self\u2019 [O. nipae infected with S. carpocapsae and H. bacteriophora. From our results, we found that nearly all the genes involved were downregulated in both nematodes and at all time points, except in a few cases. Initially, when the serine protease SPP56 was majorly downregulated, the whole enzymes involved in the activation of proPO cascades are shut down. This is consistent with our previous study where O. nipae\u2019s phenoloxidase activity was found to be slightly suppressed due to infection of both H. bacteriophora and S. carpocapsae at early stages of infection [During host infection, nematodes contend the humoral and cellular host immune responses through passive and active strategies, in which nematodes avoid triggering an immune response by being recognised as \u2018on-self\u2019 ,87. In aon-self\u2019 ,89,90. Aon-self\u2019 . Howevernfection .S. carpocapsae isolated cuticles escaped being melanized by O. nipae\u2019s hemolymph in vitro and were found to be completely melanized, however, when isolated cuticles were heat-killed before treatment. This is in line with some previous reports that show success in parasitization of Steinernematids is aided by their body surface cuticles [S. feltiae was shown to prevent the antibacterial immune response in G. mellonella by their body surface [S. carpocapsae or H. bacteriophora cuticles caused suppressions of humoral and cellular immune processes, such as phenoloxidase activity, haemocytes vitality, phagocytosis and antimicrobial activity. In this study, we confirm that the purified, isolated cuticles suppress the expression level of PPO gene in both nematodes\u2019 treatments. However, upon heating, the cuticles lost their inhibitory effects on O. nipae\u2019s immune responses and resulted in the upregulation of the PPO gene at both 16 and 24 h after treatment.Additionally, the cuticles ,91,92,93 surface . Akhurst surface  suggest  surface . Yi et a surface  showed tPPO gene in O. nipae, similar to that caused by treatment with H. bacteriophora and S. carpocapsae. In contrast, the addition of AA reversed the process and resulted in the upregulation of the PPO gene compared to the control treatment. In a study by Yi et al. [G. mellonella. However, injection of larvae with AA reverses the inhibitory effects of these inhibitors. Similarly, benzaldehyde and its derivative treatments resulted in the inhibition of PO activity in Pieris rapae larvae [G. mellonella larvae. Similarly, treatments with BZA (benzylideneacetone) in another study resulted in the immunosuppression of PO activities in Plutella xylostella and S. exigua [Furthermore, eicosanoids mediate basic humoral and cellular immune response mechanisms in insects . These ii et al. , injectie larvae . This ise larvae  in which. exigua ,98,99. WH. bacteriophora and S. carpocapsae for biological control of O. nipae larvae at different concentrations in the laboratory. We further dissected the expression levels of four selected proPO activation enzymes infected with H. bacteriophora and S. carpocapsae and found out that all of the genes were downregulated from both nematodes\u2019 treatments. These were further confirmed by the counter experiments of heat-treated cuticles and AA treatment, which reversed the inhibitory effects caused by the nematodes and the eicosanoid inhibitor. Therefore, we speculated that these nematodes devised some means to evade and suppress the immune system of O. nipae, either by secretion of toxins or by manipulation of the host system. Future work will focus on the identification and functional analysis of the secretions used by S. carpocapsae and H. bacteriophora against O. nipae immune system.Overall, this study provides the first data on the use of"}
{"text": "Worldwide, up to 8.8 million excess deaths/year have been attributed to air pollution, mainly due to the exposure to fine particulate matter (PM). Traffic-related noise is an additional contributor to global mortality and morbidity. Both health risk factors substantially contribute to cardiovascular, metabolic and neuropsychiatric sequelae. Studies on the combined exposure are rare and urgently needed because of frequent co-occurrence of both risk factors in urban and industrial settings. To study the synergistic effects of PM and noise, we used an exposure system equipped with aerosol generator and loud-speakers, where C57BL/6 mice were acutely exposed for 3d to either ambient PM (NIST particles) and/or noise (aircraft landing and take-off events). The combination of both stressors caused endothelial dysfunction, increased blood pressure, oxidative stress and inflammation. An additive impairment of endothelial function was observed in isolated aortic rings and even more pronounced in cerebral and retinal arterioles. The increase in oxidative stress and inflammation markers together with RNA sequencing data indicate that noise particularly affects the brain and PM the lungs. The combination of both stressors has additive adverse effects on the cardiovascular system that are based on PM-induced systemic inflammation and noise-triggered stress hormone signaling. We demonstrate an additive upregulation of ACE-2 in the lung, suggesting that there may be an increased vulnerability to COVID-19 infection. The data warrant further mechanistic studies to characterize the propagation of primary target tissue damage  to remote organs such as aorta and heart by combined noise and PM exposure. \u2022Traffic noise and particulate matter (PM) always co-localize in urban environments.\u2022New prototype exposure system for combined exposure of mice to traffic noise and PM.\u2022First mechanistic insights in multi-organ damage by combined exposure to noise and PM.\u2022Characterization of brain damage by noise, lung damage by PM and vascular/systemic damage by combination.\u2022Evaluation of multi-organ gene expression by RNA sequencing and cytokine profiles by array technique. Diseases caused by pollution caused an estimated 9 million premature deaths/year worldwide . The WHO3 and \u2022NO2) markedly contribute to cardiovascular and cerebrovascular disease and excess mortality ) and reactive gases . Forto PM2.5 . In 2020to PM2.5 ,14.In addition to the well-established health risks of air pollution , environAccording to the WHO, at least 1.6 million healthy life years are lost annually from traffic noise in Western Europe . StudiesDespite the urgent need for multi-exposure models, most studies on combined effects of noise and air pollution thus far rely on mathematical rather than experimental models, warranting studies under controlled laboratory conditions. Only a few studies have addressed the mutual effects of traffic noise and air pollution due to their interconnected nature . Here, w1)What is the ideal suspension of PM (NIST) to study the effects of air pollution? Ideally, it should contain a high fraction of PM with a diameter of 2.5\u00a0\u03bcm and lead to oxidative stress, inflammation and endothelial dysfunction, representing the main adverse effects of PM .2)What are the health effects of individual exposures to noise and air pollution?3)Are there additional health effects from the co-exposure to noise and air pollution related to the cardiovascular and cerebral system and the lungs?4)What is the impact of co-exposure to both environmental stressors on the expression of genes in various affected organs?Specifically, we addressed the following questions:22.1The system for the combined PM and noise exposure is illustrated in suppl. 2.23, as explained in the supplement, which is justified by the short exposure protocol and real world particulate matter concentrations reaching up to 200\u00a0\u03bcg/m3 in highly polluted cities [All animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the U.S. National Institutes of Health and approval was granted by the Ethics Committee of the University Medical Center Mainz and the Landesuntersuchungsamt Rheinland-Pfalz . For the main study, a total of 172 male C57BL/6J mice were exposed to either PM only (n\u00a0=\u00a047), noise only (n\u00a0=\u00a040), combination of PM and noise (n\u00a0=\u00a042) or to fresh air and no noise (n\u00a0=\u00a043) , a number of highly toxic (nitro) polycyclic aromatic hydrocarbons (PAHs) such as pyrenes and anthracenes as well as several polychlorinated biphenyls (PCBs) and chlorinated pesticides (see SRM1648a datasheet in the supplement). The content of biogenic (e.g. endotoxins) and anthropogenic contaminations  on the particles, partially taken-up in urban space or generated by photochemical reactions during particle atmospheric aging, seems to represent a major determinant of their biological toxicity . The met2.3All other methodological details, e.g., for blood pressure measurements by tail cuff, vasoreactivity studies by isometric tension technique, immunohistochemistry, Western blotting, RT-PCR, envisaging of reactive oxygen species (ROS) formation by fluorescent dihydroethidium microtopography, cytokine array, RNA sequencing and bioinformatical analysis, are provided in the Extended Methods in the online supplement and also in our previous work related to environmental risk factors .33.1We have developed an exposure protocol to study the effects of noise on the cardiovascular system of mice ,42. We f3.2Nox2 and Hmox1 mRNA was mostly driven by PM exposure alone and was highest in the combined exposure group  were only observed in the PM\u00a0+\u00a0noise exposure group suppl. . Gene re4Our study yields a novel co-exposure model of noise and ambient PM pollution. NIST2 particles were chosen since the exposure to this PM preparation causes significant endothelial dysfunction, oxidative stress and inflammation comparable to that observed with single exposure to aircraft noise ,42. Our 4.1Nox2 knockout [It is believed that the cognitive perception of noise triggers cortical activation and the release of stress hormones leading to high blood pressure, increased vascular and cerebral inflammation and oxidative stress that in the long turn can enhance cardiovascular risk factors such as diabetes, high cholesterol levels, activation of blood coagulation factors and hypertension that subsequently manifests as cardiovascular disease  ,51. Receknockout  and by hknockout , pointinOxidative stress is also a hall-mark and trigger of the adverse vascular health effects of air pollution ,57. Ultr\u2022NO bioavailability, endothelial cell activation (adhesion molecule expression) and inflammation [Nox2 knockout mice [p47phox knockout mice [Receptors, such as the transient receptor potential cation channel subfamily A member 1 (TRPA1) receptors, in airway sensory neurons can also sense environmental toxicants and aerogenic oxidants, resulting in neurogenic inflammation . Activatammation ,57. Impoout mice  and p47pout mice . These pout mice ,62.In summary, noise and air pollution share a number of pathophysiological aspects, e.g. the dominant role for reactive oxygen species and inflammation in causing endothelial dysfunction and high blood pressure. We exposed our animals in an experimental approach separately as well as simultaneously to these environmental stressors because noise and air pollution share a number of urban/industrial sources.4.2Nox2 and Hmox1 mRNA was mostly driven by PM exposure alone and was highest in the combined exposure group. MCP-1 was also additively upregulated by co-exposure, indicating additive effects of the combination of both stressors on inflammatory processes. Importantly, the protein and mRNA expressions of pulmonary ACE-2, which represents the docking and entry mechanism for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [Exposure to PM is known to increase pulmonary oxidative stress  while thnfection , showed nfection ,66. A renfection . In addinfection . Polymornfection ,68,69. Tnfection .We have previously reported that exposure to noise alone increased systolic and diastolic blood pressure via stimulating the release of neurohormones and by activating the HPA-axis ,42. Sing\u2022NO donor sodium nitroprusside was not modified. Vascular oxidative stress as determined by fluorescence of oxidized dihydroethidium products was increased by the single exposures and showed a trend of additive augmentation in the co-exposure group along with higher nitrotyrosine levels. In addition, the oxidative stress response protein, heme oxygenase-1, was upregulated in an additive manner and the expression levels of the antioxidant enzymes Cu,Zn- and Mn-superoxide dismutase were lower in an additive manner. Thus taken together, a higher degree of oxidative stress may be at least in part responsible for the severe degree of endothelial dysfunction in the co-exposure group.In contrast, endothelial dysfunction of conductance (aorta) and resistance vessels  showed a substantial additive negative effect on vascular function, while vasodilation to the endothelium independent Transportation noise exerts its detrimental health effects to a large extent via the brain, where it initiates stress responses by activating direct and indirect pathways accompanied by increases in circulating catecholamine and cortisol concentrations . AccordiPreviously, we demonstrated significant changes of genes in part responsible for the regulation of vascular function, circadian rhythm, vascular remodeling, and cell death using comparative Illumina sequencing of transcriptomes of aortic tissues from aircraft noise-treated animals . The traThe lung represents one organ for which we identified possible synergistic negative interactions. We detected a robust induction of pathways related to a DNA-damage response upon PM exposure as exemplified by the activation of p53 signaling, mismatch repair, cell cycle changes and apoptosis. In addition, phagocytosis and lysosomal activation are likely to be induced to clear small particles lodged in the lung tissue. PM clearly induces oxidative stress and inflammation , but also appears to induce metabolic responses. We detected altered signaling in the AMPK pathway, one of the major regulators of cellular energy metabolism. Accordingly, we observed that metabolic pathways downstream of AMPK signaling were also regulated by PM. It included both PPAR signaling, which is a crucial regulator of lipid metabolism, as well as the induction of proteins which are involved in oxidative phosphorylation, or the TCA cycle. In addition, we have identified that glutathione synthesis is induced as a response that is likely due to heightened levels of oxidative stress. Intriguingly, the induction of these PM-induced stress pathways is further exaggerated by noise stress, despite the fact that noise exposure alone causes a nearly completely independent transcriptional response. Transcriptomic analysis in the brain, on the other hand, indicated that the combination of noise and PM exposure elicited a transcriptional response that was comparable to the noise-stress response only, with many of the PM-only dependent transcriptional responses being absent in the combination treatment.trans-synaptic signaling and \u201cbehavior\u201d, all of which are intimately linked to neuropathological pathways. In the aorta, noise-only exposure triggered the expression of a hypoxic signature. We detected that numerous genes reported to be induced by the hypoxia inducible factor (HIF)-driven transcription were upregulated. This included the induction of genes in the glycolytic pathway, and the downregulation of genes involved in the oxygen-dependent production of ATP, the TCA cycle, lipolysis and the mitochondrial electron transport chain. Suggesting that noise stress may affect the metabolic state of the cardiovascular system, in a similar way to hypoxia. Remarkably, the combination of PM\u00a0+\u00a0noise reduced this response, for which we currently have no explanation.Overall, stressors induced multiple signaling pathways that are of relevance for cardiovascular and neurological diseases. In the brain, the most prominent GO cluster induced by noise or co-exposure included ion transport, 5Previous animal studies have shown additive/synergistic adverse cardiovascular health effects via exaggerated oxidative stress by noise and pre-existing arterial hypertension  as well 10.13039/501100008454Boehringer Ingelheim Foundation for the collaborative research group \u201cNovel and neglected cardiovascular risk factors: molecular mechanisms and therapeutics\u201d and through continuous research support from Foundation Heart of Mainz (mostly dedicated to the purchase of the exposure system). M.K., I.K., M.T.B.J., K.V.-M. and S.K. hold or held TransMed PhD stipends funded by the 10.13039/501100008454Boehringer Ingelheim Foundation. L.S. and H.U. hold TransMed PhD stipends funded by the Else-Kr\u00f6ner-Fresenius Foundation. S.S. holds an excellence stipend of the Else-Kr\u00f6ner-Fresenius Foundation (2021_EKES.04). T.J. holds a MD stipend funded by the Heart Foundation Mainz. T.M. is PI and A.D. and P.S. and S.D. are (Young) Scientists of the 10.13039/100010447DZHK (German Center for Cardiovascular Research), Partner Site Rhine-Main, Mainz, Germany. Also financial support by 10.13039/501100000289Cancer Research UK (CRUK Edinburgh Centre C157/A255140) and 10.13039/100010269Wellcome Trust  is gratefully acknowledged. The collaboration of the authors was supported by European 10.13039/501100000921COST Action CA20121: Bench to bedside transition for pharmacological regulation of NRF2 in noncommunicable diseases (BenBedPhar). Webpage: https://benbedphar.org/about-benbedphar/.A.D. and T.M. were supported by vascular biology research grants from the The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper."}
{"text": "Nitric oxide is a ubiquitous signaling radical that influences critical body functions. Its importance in the cardiovascular system and the innate immune response to bacterial and viral infections has been extensively investigated. The overproduction of NO is an early component of viral infections, including those affecting the respiratory tract. The production of high levels of NO is due to the overexpression of NO biosynthesis by inducible NO synthase (iNOS), which is involved in viral clearance. The development of NO-based antiviral therapies, particularly gaseous NO inhalation and NO-donors, has proven to be an excellent antiviral therapeutic strategy. The aim of this review is to systematically examine the multiple research studies that have been carried out to elucidate the role of NO in viral infections and to comprehensively describe the NO-based antiviral strategies that have been developed thus far. Particular attention has been paid to the potential mechanisms of NO and its clinical use in the prevention and therapy of COVID-19. Unfortunately, the availability of effective antiviral drugs is limited. Although a great deal of effort has been devoted to the development of new therapeutic approaches since the first approval of an antiviral drug in 1963, only 90 drugs were formally approved up to 2016 [The enormous cost of viral infections has been made abundantly evident over the last two years, in which we have observed the worldwide spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which affects every aspect of human life and all economic sectors . After t to 2016 . Further to 2016 ,6, and i to 2016 . Its use to 2016 . The aimNO is a radical species that is involved in a variety of physiological and pathological processes. It is ubiquitous in mammalian tissues, where it is produced from the conversion of L-arginine into L-citrulline in a reaction catalyzed by three isoforms of the enzyme NO-synthase (NOS). The three isoforms differ in terms of the regulation, amplitude and duration of NO production, and their tissue localization . NeuronaNO has direct effects in that it can block the functions of target molecules by reacting with the metal centers . It also has indirect effects that are mediated by its reaction with oxygen and the superoxide anion to give reactive nitrogen species (RNS), which then oxidize, nitrate and nitrosate target molecules .The balance between the physiological and pathological effects of NO depends on its concentration, meaning that any proposal to use NO for therapeutic purposes should take its concentration and localization into careful consideration.S-nitroso-N-acetylpenicillamine (SNAP) was demonstrated to have in vitro activity against SARS-CoV-1 [While endogenous NO is one of the major mediators in the body\u2019s defense against microorganisms in the airway, its antiviral role has been less extensively studied. The non-specific action that NO exerts against viruses was first described in the early 1990s, and, since these pioneering studies, further evidence has demonstrated that NO exerts an action against all types of viruses, including the coronavirus, with broad activity ,15. ReseRS-CoV-1 . SubsequRS-CoV-1 ,18. StudMany studies have shown that a number of diseases caused by viral infection are characterized by an increase in NO levels. Indeed, following infection, the large amounts of NO that are necessary to fight viruses are produced thanks to the upregulation of iNOS via the activation of several pathways that are mediated by: (i) toll-like receptors (TLRs); (ii) signal transducer and activator of transcription 1 (STAT-1); and, (iii) protein kinase-R (PKR) , as summiNOS activation results in the increased production of NO, which is effective against both DNA and RNA viruses, including SARS-CoV-2 . NO actiS-nitrosylation of the cysteine residues that are directly involved in catalysis or present near the active site [S-nitrosylation of the cysteine in the enzyme active site [(1) The inactivation of viral proteins via nitrosylation reduces or inhibits enzyme activity. Target proteins include proteases, reverse transcriptases, ribonucleotide reductases, transcription factors and tyrosine-containing enzymes . The mecive site . For SARive site . In factive site . Host prive site . Anotherive site .(2) Deamination of viral DNA following nitrosative stress. NO induces DNA damage both to viral and host genetic material, although host DNA can be repaired by the DNA repair machinery .(3) NO increases the clearance of pathogens, including DNA and RNA viruses, by enhancing ciliary beat frequency ,25. NO aThe high levels of NO produced following iNOS induction also play a role in the inflammatory response. Acute inflammation is a defense mechanism against pathogens and has the aim of destroying or inactivating the invading microorganisms. If microbes are killed and cellular debris removed, inflammation is resolved and the normal tissue is restored. However, when excessive and non-regulated inflammation persists, it leads to tissue damage. During sustained inflammation, NO is produced in high quantities and regulates the activity of a variety of inflammatory mediators, including NF-kB . Other eThe endothelial cells that line vessels control several vascular functions, including blood flow, blood fluidity and vascular permeability. For an in-depth description, see the review by Pober and Sessa .The vascular endothelium acts in response to changes in the microenvironment  and induces the release of specific mediators that act in the control of the tone of smooth muscle cells . These m2+ concentration and mediators such as acetylcholine and bradykinin [Under physiological conditions, the maintenance of blood fluidity, via the inhibition of coagulation is another role of the vascular endothelium . NO alsoadykinin . NO workadykinin .Impaired endothelial function, with reduced NO availability, is found in diseases such as diabetes mellitus, obesity and hypertension . The redThe maintenance of endothelium functionality is essential as it allows for controlled exchanges between blood and the surrounding tissue and corrects vascular tone, while also avoiding the formation of a pro-thrombotic environment. Alterations in the vascular endothelium are therefore associated with pathological conditions, such as those related to viral infections. HIV infection is associated with oxidative stress, which reduces NO availability, leading to endothelium dysfunctions . Similar2+-dependent K+ channels [NO is produced in the human airway by the three isoforms of NOS . A reducchannels  and the channels .S-nitrosylation of surfactant proteins can induce a conformational change, leading to inflammatory response [Another factor in the respiratory system is the lung surfactant, a complex of proteins and phospholipids with the function of reducing surface tension at the air/liquid interface: evidence suggests a role for NO in surfactant secretion and the preservation of its function . At low response .iNOS, which is constitutively expressed in the airway epithelium, also contributes to the production of NO, which, among other functions, helps the regulation of ciliary beat, a defense mechanism of airway epithelium . The effThe use of NO in the treatment of pulmonary diseases is derived from this evidence .Although the role of NO in inhibiting viral replication in host defense has been demonstrated, NO-based antiviral strategies have not yet been extensively investigated. To date, no systematic approach has been adopted for the study of NO-based antiviral therapeutics, apart from those active in the cardiovascular system ,47. DespNO-based antiviral strategies can be mainly classified into two groups: gaseous NO (gNO) inhalation-based therapies and NO-donors. As well as chronic inflammatory diseases, viral infections of the upper respiratory tract are related to the presence of higher levels of NO in the air exhaled by human subjects. This is due to an increase in NO production as part of the host response to infection , with thTM; the recommended dosage was 20 ppm and the length of treatment was up to two weeks.The greatest efficacy found in gNO therapy to date has been demonstrated in the treatment of oxygen desaturation in infants with hypoxic respiratory failure. The treatment has been approved as a drug by the FDA under the trade name INOMAXIn the antiviral field, gNO inhalation-based therapies have resulted in quite discordant data, with one of the reasons for this being the gNO dosage sensitivity threshold. This limitation has been highlighted in gNO treatments following acute respiratory distress syndrome (ARDS), which is a particular pathological condition with a number of different etiologies such as SARS-related coronavirus infection (SARS-CoV), pulmonary ischemia-reperfusion (I/R) injury and bacterial/viral pneumonia. The role of NO in animal and human subjects affected by ARDS has been described in a work by Chen et al. .In a Beijing rescue study of fourteen patients with ARDS, which manifested as a result of SARS-CoV infection, gNO-based treatments performed for at least three days at a dosage of 30 ppm led to a marked improvement in arterial oxygenation in patients and a concomitant decrease in pneumonia infiltrates . On the Although the dosage of gNO in antiviral treatments must be significantly higher than in other applications, e.g., antimicrobial treatment ,66,70,71Other discrepancies in gNO inhalation-based antiviral therapies have been identified in dosage and the dependence of the therapeutic efficacy on the mode of gNO replenishment. In the antimicrobial field, Hall et al. observed a huge gap in the dosage between gNO and a soluble NO-releasing chitosan oligosaccharide . In otheN-diazeniumdiolates and S-nitrosothiols (RSNOs). Their general chemical structures are reported in There are three classes of NO-donors with antiviral effects; nitrites, N-diazeniumdiolates class and that has antiviral effects in Human Papillomavirus (HPV) raft cultures [NVN1000  d, which cultures , also dicultures ,53,76.SNAP e, a deriS-nitrosoglutathione (GSNO), have been incorporated into polymeric platforms [Several NO-donors, including SNAP and latforms ,81,82. Tlatforms ,84. The latforms ,86,87,88Endogenous NO-regulating drugs may be considered another potential class of antiviral drugs. These derivatives are described separately from gNO inhalation and NO-donors because of several discordances in the data collected in the literature. Reducing endogenous NO production is undoubtedly a therapeutic antiviral strategy and can be achieved by modulating iNOS activity. Pyrazole derivatives are examples of selective iNOS inhibitors that are active against the vaccinia virus ,90,91; cAlready-known drugs, such as ribavirin g have beNot always regulating iNOS activity in order to decrease endogenous NO production has led to the arrest of viral replication. In specific cases, instead of regressing, viral infection advanced if iNOS activity was downregulated. An example of this can be found in a study carried out by Santangelo et al. ; the virIn addition to these discrepancies, the use of drugs that can modulate iNOS activity has caused viral immunopathogenesis and, thus, the occurrence of numerous side effects . For thiCoronaviruses (CoVs) are a family of single-stranded positive-sense RNA viruses that contain a genome of approximately 30 kilobases and are named after their crown-like spike protrusions on the virus surface . After cBecause of the overlap of genetic structures and pathological features between SARS-CoV-1 and 2, knowledge of SARS-CoV-1 has provided suggestions for understanding SARS-CoV-2, including the role, potential mechanism and therapeutic application of NO. The promising results from the rescue trial during the SARS-CoV-1-caused epidemic in the years 2002\u20132004 , promptePathway 1\u2014Although the main targets of SARS-CoV-2 are bronchial hair epithelial cells and type II lung cells, viral particles have also been found in endothelial cells. This is because SARS-CoV-2, SARS-CoV-1 and MERS-CoV lead to endothelial cell apoptosis with a consequent decrease in endothelial NO production [Pathway 2\u2014SARS-CoV-2 invades host cells via the binding of its surface glycoprotein-S to angiotensin-converting enzyme 2 (ACE2) [Pathway 3\u2014NO/ROS imbalance in the body triggers the considerable release of proinflammatory cytokines and chemokines, which lead to the overstimulation of the inflammatory system, causing significant damage to tissues and organs. In the meantime, ROS production, especially in the mitochondria, generates mitochondrial dysfunctions because of increased mitochondrial membrane permeability [Pathway 4\u2014In order to restore the physiological ROS/NO balance, NO reacts with oxygen and its derivatives to form toxic reactive nitrogen species (RNS), such as peroxynitrite. The high concentration of ROS and RNS contributes to vascular oxidative stress, causing a change in vascular tones and leading to decreased NO bioavailability [There are four etiological pathways for COVID-19 that have been identified and described, and the level of endogenous NO and its bioavailability are quite low in each. oduction . Pathway2 (ACE2) . The dow2 (ACE2) ,106. Pateability . Pathwaylability . TherefoOn the basis of the outcomes obtained from the rescue clinical study following SARS-CoV-1 infection , gNO-bashttps://clinicaltrials.gov/ct2/show/NCT04305457), gNO is administered at 140\u2013180 ppm concentration for 20\u201330 min, twice a day and for 14 consecutive days. The primary goal is to reduce the incidence of patients requiring intubation and mechanical ventilation, as a marker of deterioration from mild to severe COVID-19. The secondary goal is both the reduction of the mortality rate and the clinical recovery time. Clinical recovery means normalization of fever, respiratory rate, alleviation of cough and resolution of hypoxia. All these improvements must be maintained for 72 h. Other outcome measures are the increase in the percentage of patients with the negative conversion of RT-PCR from the oropharyngeal or oropharyngeal swab. The estimated study completion date is 1 April 2022.In the first clinical trial  is to investigate whether gNO treatment can improve oxygenation in mechanically ventilated patients. The treatment involves inhalation of 80 ppm nitric oxide for 48 h, followed by 40 ppm, followed by weaning before stopping. If a patient dies during the first 48 h of treatment, the last available blood gas analysis will be used. Secondary outcomes are the calculation of the time to reach normoxemia, the percentage of SARS-nCoV-2 free patients during the first 28 days after enrollment, and the survival rate both 28 days and 90 days after treatment. The estimated study completion date is 21 December 2022.The primary outcome of the second clinical trial  focuses on hospital-associated transmission among healthcare workers. 470 healthcare workers scheduled to work with COVID-19 patients were randomly selected and divided into two groups: the first received nitric oxide gas  while the second did not . The primary endpoint of this study is the incidence of subjects with COVID-19 disease 14 days after enrollment. Secondary endpoints are the percentage of caregivers presenting with a positive real-time RT-PCR test for SARS-CoV2 14 days after enrollment, the percentage of caregivers requiring quarantine, and the total number of days of quarantine in the two groups.The third clinical trial , which ended 30 June 2021, aimed to investigate whether gNO therapy could reduce both mechanical ventilator intervention and oxygen therapy use [The fourth clinical trial . Similarly, COViNOX is another NO-releasing drug that is in the third phase of clinical trials. In this trial, subjects were treated by means of an INOpulse device using an INOpulse nasal cannula .To overcome the use of gNO therapy, Kalytera Therapeutics Inc. announced, on June 29, 2020, that it has entered into a binding Letter of Intent to license R-107 from the Salzman Group for the treatment of CoVs and COVID-19 infection. R-107 is a non-gaseous, liquid prodrug of NO. Following a single administration by injection, R-107 slowly releases NO into the lung tissues over 48 h (17 February 2022, SNAP e has beeDespite decades of studies, fighting viral infections is still a great medical challenge, as the SARS-CoV-2 spread has recently demonstrated. Since the discovery of NO, many studies have described its role on the respiratory system, even if some aspects still need clarification. NO improves oxygenation, regulates lung vasculature and surfactant function, improves ciliary motility and is involved in host defense.As the role of NO in the defense against viral infections is becoming clearer and a positive role for this radical has been proposed in the treatment of respiratory infections, progress has been made in the development of NO-based therapies. gNO and NO-donors have been studied and some treatments have already been commercialized or are in ongoing clinical trials. Indeed, it should be considered that, depending on the concentration, NO can induce nitrosative stress and damage also host tissues.In the case of gNO therapy, more studies are needed to define the amount of the radical that is able to reach the local tissues in order to maximize clinical outcomes while reducing the potential side effects. In the case of NO-donors, the use of nanosystems is a particularly promising field as it allows drug delivery and targeting to be improved. For this reason, further studies are important as they can contribute to the development of innovative therapeutic approaches."}
{"text": "Anxiety disorders rank among the most prevalent mental disorders worldwide. Anxiety symptoms are typically evaluated using self-assessment surveys or interview-based assessment methods conducted by clinicians, which can be subjective, time-consuming, and challenging to repeat. Therefore, there is an increasing demand for using technologies capable of providing objective and early detection of anxiety. Wearable artificial intelligence (AI), the combination of AI technology and wearable devices, has been widely used to detect and predict anxiety disorders automatically, objectively, and more efficiently.This systematic review and meta-analysis aims to assess the performance of wearable AI in detecting and predicting anxiety.Relevant studies were retrieved by searching 8 electronic databases and backward and forward reference list checking. In total, 2 reviewers independently carried out study selection, data extraction, and risk-of-bias assessment. The included studies were assessed for risk of bias using a modified version of the Quality Assessment of Diagnostic Accuracy Studies\u2013Revised. Evidence was synthesized using a narrative  and statistical  approach as appropriate.Of the 918 records identified, 21 (2.3%) were included in this review. A meta-analysis of results from 81% (17/21) of the studies revealed a pooled mean accuracy of 0.82 (95% CI 0.71-0.89). Meta-analyses of results from 48% (10/21) of the studies showed a pooled mean sensitivity of 0.79 (95% CI 0.57-0.91) and a pooled mean specificity of 0.92 (95% CI 0.68-0.98). Subgroup analyses demonstrated that the performance of wearable AI was not moderated by algorithms, aims of AI, wearable devices used, status of wearable devices, data types, data sources, reference standards, and validation methods.Although wearable AI has the potential to detect anxiety, it is not yet advanced enough for clinical use. Until further evidence shows an ideal performance of wearable AI, it should be used along with other clinical assessments. Wearable device companies need to develop devices that can promptly detect anxiety and identify specific time points during the day when anxiety levels are high. Further research is needed to differentiate types of anxiety, compare the performance of different wearable devices, and investigate the impact of the combination of wearable device data and neuroimaging data on the performance of wearable AI.PROSPERO CRD42023387560; https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=387560 Anxiety is defined as an unpleasant emotional state whose cause is either not easily defined or considered to be uncontrollable or unavoidable, resulting in tension and physiological manifestations . AnxietyThe diagnosis of ADs is a very complicated and challenging task. Currently, ADs are diagnosed and screened primarily through clinical observations of patients\u2019 mental states, clinical histories, and self-report questionnaires  for anxiety . HoweverAdvances in digital technologies and wireless sensors have led to the proliferation of wearable health care devices, which can be particularly useful for the diagnosis and prediction of anxiety. Wearable devices offer a convenient way for people with anxiety to monitor, examine, track, and share their health features, such as physical activities, heart rates, sleep patterns, blood oxygen, and respiratory rate. Wearable devices are made in different forms to meet their use requirements and can be classified into 4 types: on-body devices (fixed directly on the body or skin), near-body devices (fixed close to the body with no direct contact with the body or skin), in-body devices (implantable electronics), and electronic textiles (textiles with integrated electronics).Wearable devices have undergone a significant transformation over the last few years, reflecting the rapid advancement of technology in the field. Early iterations of smartwatches and activity trackers were primarily focused on basic monitoring and display functions. Many of these devices lacked connectivity options, limiting their ability to interact with other technologies. However, the introduction of Bluetooth components marked a turning point in the evolution of wearables, allowing for synchronization with smartphones and other wireless devices. This integration not only enhanced the user experience but also paved the way for more advanced functionalities.wearable AI technology. Wearable AI is the fusion of data obtained from wearables and sophisticated machine learning algorithms [More recent versions of wearable devices have embraced cutting-edge innovations by incorporating artificial intelligence (AI) and machine learning components, thus introducing what we call gorithms . Machinegorithms -16. If eIn the past few years, numerous studies have examined the performance of wearable AI devices for the detection of anxiety. In an effort to summarize these studies, several reviews have been conducted, but they had the following limitations. First, most extant reviews have largely focused on general wearable devices rather than wearable AI devices ,17-21. SThe authors conducted and reported this systematic review in accordance with the PRISMA-DTA  . The PRITo find relevant studies, the first author searched the following 8 electronic databases on October 3, 2022: MEDLINE (via Ovid), Embase (via Ovid), PsycINFO (via Ovid), CINAHL (via EBSCO), ACM Digital Library, Scopus, IEEE Xplore, and Google Scholar. An automated search was set up with biweekly alerts for 3 months . Owing to the large number of results retrieved from Google Scholar, only the first 100 hits  were checked for this review. To identify additional studies, we screened the reference lists of the included studies  and reviewed studies that cited the included studies .artificial intelligence, machine learning, and deep learning), those related to wearable devices , and those related to anxiety . The search queries used in this review are presented in The search terms used in this review were compiled after consulting with 3 experts in digital mental health and after reviewing relevant reviews. The search query was composed of 3 groups of terms: those related to AI . We excluded studies that used AI to predict the outcome of an anxiety intervention or treatment. The data acquisition had to be via noninvasive on-body wearables, such as smartwatches, smart glasses, smart wristbands, smart clothes, and smart rings. We excluded studies that used the following devices to collect the data: nonwearable devices, handheld devices , near-body wearable devices , in-body wearable devices , wearable devices wired to nonwearable devices, and wearable devices requiring expert supervision . This review included studies that collected data using other methods  along with wearable devices. We included peer-reviewed journal articles, conference papers, and dissertations with full text regardless of study settings, reference standards, and the country in which the study was conducted. Considering our focus on modern technology and the fact that the domain of wearable AI devices is under constant development, only articles from 2015 onward were included. Studies published in a language other than English or structured as review articles, editorials, conference abstracts, preprints, posters, protocols, and research highlights were excluded. Articles demonstrating a theoretical foundation for wearable AI devices for anxiety were disregarded.Relevant studies were identified through the following 3 steps. First, all the retrieved studies were imported into EndNote X9  to identify and eliminate duplicate items. Second, 2 reviewers independently screened the titles and abstracts of all the retrieved studies. Finally, the remaining articles were subsequently sourced in full text and inspected by the 2 reviewers independently. Any disagreements in the second and third steps were resolved through discussion. The Cohen \u03ba was used to calculate interrater agreement, which was 0.90 for title and abstract screening and 0.94 for full-text reading.Using Excel (Microsoft Corp), 2 reviewers independently extracted metadata, wearable devices, AI algorithms, and results of the studies. The data extraction form used in this review was pilot-tested with 5 studies . Any disTo carefully assess the quality of the included studies, we adapted a well-known tool   for our meta package in R  [Narrative and statistical approaches were used to synthesize the data extracted from the included studies. In our narrative synthesis, we used text and tables to summarize and describe the characteristics of the included studies . With regard to the statistical approach, DerSimonian-Laird random-effects models  using thmputing) .metafor package in R (version 4.2.2) [In this review, some studies reported multiple effect sizes. Such studies will have a larger effect on the results of the meta-analysis than studies reporting only one effect size. Therefore, we used a multilevel meta-analysis ,31 to acn 4.2.2) .P value of <.05.When applicable, subgroup multilevel meta-analyses were conducted to assess for a possible association between outcome measures and different moderators . TheQ statistic (P<.05 indicated heterogeneity), between-study variance was assessed using \u03c42, and the magnitude of between-study variation because of true difference in effect sizes rather than chance was assessed using I2 [I2 ranged from 0% to 40%, moderate when it ranged from 30% to 60%, substantial when it ranged from 50% to 90%, or considerable when it ranged from 75% to 100% [Between-study heterogeneity was assessed using the Cochran using I2 ,33. The  to 100% .The results of the systematic search are presented in The key characteristics of the studies included in the review are presented in Among the included studies, 8 different wearable devices were used. Approximately a quarter of all studies  did not indicate what type of wearable device they used. The most common wearable devices used were the Fitbit series , the Empatica series , and Muse  did not provide adequate information to identify whether an appropriate consecutive or random sample of eligible patients was used. Most of the included studies  avoided inappropriate exclusions. The number of patients in the subgroups was appropriately balanced across half  of the studies. A sufficient sample size was reported in 43% (9/21) of the studies, whereas there was no clear indication of whether a sufficient sample size was used in the remaining studies . Consequently, the risk of bias resulting from the \u201cselection of participants\u201d was rated as low in only half  of the studies . A low lAlmost all studies  described the AI models in detail. Most of the included studies  provided a clear description of the features (predictors) used in the models, and the features in nearly all studies  were assessed in the same way for all participants. In all the included studies , features were collected without knowledge of outcome data. Thus, the risk of bias owing to the \u201cindex test\u201d was rated as low in most of the included studies  was assessed using appropriate tools in 81% (17/21) of the included studies. The outcome was defined in a similar way for all participants in almost all studies  and was determined without knowledge of predictor information in all studies . An adequate interval was used between the index test and the reference standard in most studies . Accordingly, the risk of bias because of the \u201creference standard\u201d was low in 90% (19/21) of the studies . All theAll participants enrolled in the study were included in the data analysis in 62% (13/21) of the studies. In 90% (19/21) of the studies, the data preprocessing was carried out appropriately, and in 86% (18/21) of the studies, the breakdown of the training, validation, and test sets was adequate. In 71% (15/21) of the studies, suitable measures were used to evaluate the performance of the models. According to these judgments, 76% (16/21) of the studies had a low risk of bias in the \u201canalysis\u201d domain . MultimeMeta-analyses were carried out for the highest accuracy, sensitivity, and specificity. Furthermore, when applicable, subgroup meta-analyses were performed to assess the performance of wearable AI based on different AI algorithms, aims of AI, wearable devices used, status of wearable devices, data types, data sources, reference standards, and validation methods. The following sections present the aforementioned results.P<.001; I2=99.9%). P>.05) was found in the highest accuracy between subgroups in all groups.Wearable AI accuracy, which is the ability of the AI to correctly classify patients with and without anxiety, was examined in 81% (17/21) of the studies. From these investigations, we extracted 40 accuracy estimates as multiple algorithms were often assessed in a single study. The highest accuracies observed spanned 0.50 to 1.00. As displayed in P<.001; I2=99.9%). P>.05) in the highest sensitivity was revealed between subgroups in all groups.In 48% (10/21) of the studies, the sensitivity of wearable AI, referring to the AI\u2019s capacity to accurately identify patients with anxiety, was examined. From these studies, we extracted 24 sensitivity estimates as many studies assessed sensitivity for more than one algorithm. The highest sensitivity in these studies ranged from 0.21 to 1.00. A meta-analysis of the 24 estimates, involving 97,794 participants from the 48% (10/21) of the studies, revealed a pooled mean sensitivity of 0.79 (95% CI 0.57-0.91), as displayed in P<.001; I2=100%). P>.05) was found in the highest specificity between subgroups in all groups.The specificity of wearable AI, which refers to the AI\u2019s capacity to accurately identify patients without anxiety, was examined in 48% (10/21) of the studies. From these studies, we extracted 24 specificity estimates as many studies assessed specificity for more than one algorithm. The highest specificity observed spanned 0.52 to 1.00. As displayed in This review aimed to assess the performance of wearable AI in detecting and predicting anxiety. The results of our meta-analyses showed that wearable AI has a good but not optimal performance in detecting and predicting anxiety. To be more precise, the review revealed that wearable AI was able to correctly classify patients with and without anxiety in 81% of cases. Furthermore, we found that wearable AI has a slightly better performance in detecting individuals who do not have anxiety (92%) compared with those who do (79%). This may be attributed to the fact that the number of controls  was larger than the number of cases  in 78% (14/18) of the studies that reported the number of cases and controls. Therefore, the algorithms were trained on imbalanced data with more representation of control samples. This review also demonstrated that the performance of wearable AI was not moderated by algorithms, aims of AI, wearable devices used, status of wearable devices, data types, data sources, reference standards, and validation methods. This finding should be interpreted carefully given that the number of studies in most subgroup analyses was small (\u22655).As mentioned earlier, no previous reviews have examined the performance of wearable AI in detecting or predicting anxiety. However, a recent systematic review investigated the performance of wearable AI in detecting or predicting depression . AlthougAlthough this review showed that wearable AI is a promising tool for diagnosing anxiety, wearable AI is not ready to be implemented in clinical practice for the following reasons: (1) its performance in detecting patients with anxiety is not optimal at present, (2) the sample size was small (\u2264100) in two-thirds of the studies , and (3) only 29% (6/21) of the studies were judged to have a low risk of bias in all domains. Consequently, it is advisable to use wearable AI in conjunction with other clinical assessments and diagnostic criteria  to detect and predict anxiety.None of the commercial wearable devices in this review had AI embedded into them to detect anxiety. Instead, AI was embedded in a host device  where the data collected by wearable devices were stored. Therefore, there is a need to develop wearable devices that can promptly identify and predict anxiety, similar to those that detect stress , and are also capable of identifying specific time points during the day when anxiety levels are high, which could help users and health care providers identify causes of anxiety. We expect that this scenario could materialize in the near future, particularly with the advancements in wearable technology and the development of new chips that augment computing power.The studies included in this review did not use neuroimaging data in addition to wearable device data to detect or predict anxiety. Neuroimaging can play an essential role in the diagnosis of anxiety by visualizing the brain and identifying structural or functional changes that may be associated with ADs -59. ThroMost studies  included in this review focused on the performance of wearable AI in identifying current anxiety status rather than forecasting the likelihood or severity of anxiety in the future. Predicting the occurrence of anxiety in the future is as important as or more important than detecting the current anxiety state as it can help develop and deliver more effective, timely, and personalized interventions. Thus, we encourage researchers to conduct additional investigations on the performance of wearable AI in predicting the occurrence of anxiety in the future.None of the studies included in this review assessed the performance of wearable AI in distinguishing anxiety from other mental health conditions  or distinguishing types of anxiety . Typically, clinical practitioners rely on intricate and error-prone diagnostic methods to differentiate between various patient groups rather than merely distinguishing them from healthy individuals. As a result, additional research is necessary to examine the performance of wearable AI in distinguishing different types of anxiety and distinguishing individuals with anxiety from those with other mental disorders that exhibit comparable signs and symptoms of anxiety.As previously stated, the sample size of two-thirds of the studies  was limited to \u2264100 participants. This may have hindered the detection of potential variations in the efficacy of wearable AI technology in subgroup analyses. In addition, it may have restricted the use of certain algorithms that require a considerable amount of data to be trained and tested. We encourage researchers to undertake additional studies with larger sample sizes and extended durations to ensure adequate statistical power and enable the use of more sophisticated and efficient algorithms that require greater quantities of data.Although the included studies used some common wearables , they did not assess the performance of other common wearables such as Google Pixel Watch, Galaxy Watch, and Oura Ring. Furthermore, none of the included studies compared the performance of different wearable devices. Therefore, it is recommended that researchers evaluate the performance of other wearable devices and compare their efficacies.The discrepancy between the wearable AI accuracy in detecting individuals with and without anxiety highlights the need for refining the AI algorithms to improve their performance. This could involve gathering more diverse and representative data, refining feature selection, or implementing advanced techniques to enhance the detection of anxiety among users.There are many challenges associated with the integration of AI into wearable devices for mental disorders in general and ADs in particular. First, obtaining high-quality data is difficult with wearable technology owing to differences in spatial, temporal, and data resolution. This becomes more challenging when multiple devices have to be combined to collect multiple types of data to generate a comprehensive picture of the body. Therefore, the quality of wearable data should be emphasized to improve the performance of the algorithms. To achieve this, there is a need for more practical standards for wearable device development that are necessary to ensure the consistent measurement of different signals generated from wearable devices. Second, the presence of missing data, outliers, signal noise, and artifacts can also lead to large variations and inaccurate algorithms . For exaThe transition of wearable AI into existing clinical practice for anxiety detection and management is an intricate process that requires careful consideration. A robust framework must be devised that outlines how wearable AI technologies can complement traditional methods such as interviews, self-report surveys, and existing diagnostic criteria. Such an integration framework would involve validating AI algorithms using established clinical guidelines, ensuring data privacy and security compliance, training health care providers in the interpretation of AI-generated insights, and creating a clear protocol for incorporating these insights into patient care. The integration of wearable AI into existing practices could offer a more refined, real-time understanding of anxiety levels, allow for tailored interventions, and foster collaboration between health care providers and technology developers. Efforts toward these integrations could form a promising direction for future research and innovation, contributing to a more effective and patient-centric approach to anxiety management.t test and ANOVA based on their power spectral density. The highest accuracies of 94.9% and 92.74% using an RF classifier were achieved from the 2 and 4 levels in the power spectral density of the EEG recording, respectively. In a study carried out by Arsalan and Majid [F1-score of 92.27% and 92.13%, respectively.Recently, various studies have proposed statistical and AI approaches for wearable devices to study the effectiveness of various parameters and biosignals  in differentiating patients with ADs from healthy individuals ,63-66. And Majid , EEG datnd Majid -70. In and Majid , a consund Majid , a wearaThis review cannot comment on (1) the performance of wearable AI in diagnosing other mental disorders ; (2) the performance of wearable AI in managing anxiety or predicting outcomes of anxiety treatment; and (3) the performance of nonwearable devices, handheld devices, near-body wearable devices, in-body wearable devices, wearable devices connected to nonwearable devices using wires, and wearable devices that require an expert to be applied on users. This is because such disorders, outcomes, and wearable devices were beyond the scope of this review, thus limiting the generalizability of our findings to these contexts. In addition, the results of our meta-analyses are likely to be overestimated or underestimated for 2 reasons. First, it is probable that we overlooked some studies as our search was limited to research published in the English language from 2015 onward and we did not use terms related to types of anxiety . Second, several studies in this review were not included in the meta-analyses as they did not provide findings suitable for meta-analysis.Although wearable AI shows promise in detecting and predicting anxiety, it is not yet advanced enough to be used in clinical practice. As such, wearable AI should be used along with other clinical assessments and diagnostic criteria to provide a more comprehensive understanding of a patient\u2019s condition until further evidence shows an ideal performance of wearable AI. Wearable device companies should develop devices that can promptly detect anxiety and identify specific time points during the day when anxiety levels are high. There is a need to investigate the effect of using a combination of wearable device data and neuroimaging data on the performance of wearable AI in detecting and predicting anxiety. In addition, further studies are needed to differentiate among types of anxiety and differentiate patients with anxiety from those with other mental disorders. We urge researchers to compare the performance of different wearable devices in detecting anxiety."}
{"text": "Trust is essential for establishing stable and fulfilling romantic relationships between partners. Development of trust, however, can be assumed to depend on many factors related to an individual's earlier experiences and relationship-related beliefs. This study aimed to investigate how adult attachment style , experiences about parents' divorce and breakdown of one's own romantic relationship, and relationship beliefs are related to the level of dyadic trust in romantic relationships.t-tests, Pearson correlations, regression analyses and mediation analyses.The present study included 131 Turkish undergraduate university students (55.7% women) from different faculties. The research instrument had questions about parents' and respondents' own relationship status, Dyadic Trust Scale (DTS), Experiences in Close Relationships Inventory-Revised (ECR-R), and Inventory of Close Relationship Beliefs (ICRB), in addition to background questions. The data were analyzed using descriptive statistics, Respondents whose parents had divorced or who had experienced a relationship breakdown had lower dyadic trust scores than those without these experiences. The trust scores correlated negatively with anxious and avoidant attachment styles and positively with relationship belief scales, although the correlations to \u201cexternal factors\u201d were not statistically significant. In regression analysis, anxious and avoidant attachment styles explained 42% and relationship beliefs 25% of the variance in trust. The only significant predictor among beliefs was \u201cindividuality.\u201d Mediation analysis showed that the effects of anxious attachment style on trust were fully mediated by the relationship belief in \u201cindividuality.\u201d The avoidant attachment style had a direct relationship to trust.The results show that anxious attachment style influences trust via relationship beliefs, while avoidant attachment style has a strong direct effect on trust as well as weaker effects via beliefs. The results are discussed in the context of Turkish culture and horizontal collectivism. Trust is a key factor in successful romantic relationships. Trust evolves throughout the various stages of dating, flirting, engagement, and marriage , experiences about parents' divorce and breakdown of one's own romantic relationship, and relationship beliefs influence the level of dyadic trust in romantic relationships. Since heterosexual romantic relationships are very much based on sex and gender roles, we expected that there might be differences between men and women in relationships between attachment, relationship beliefs and dyadic trust. We hypothesized the following relationships:Participants who had experienced parental divorce or had separated themselves from a close relationship would score lower in trust than those whose parents are married or who have not experienced a breakdown of a romantic relationship.An anxious and avoidant attachment style would have a negative relationship with interpersonal trust.Positive relationship beliefs would have a positive relationship with interpersonal trust.The effects of attachment style would be at least partly mediated by relationship beliefs, i.e., attachment style would influence the beliefs, which, in turn, would be related to interpersonal trust.*power . The estimated sample size was n=111. The sample consisted of 131 undergraduate students of various majors , of whom 55.7% were women. The participants were student volunteers who completed a 20-min survey during their class hour. The participants did not receive any benefit from participating in the study. Participants were informed of their rights to voluntary participation and the option to stop answering at any time.The sample size estimation was conducted with GEthical approval was obtained from the Near East University Ethical Committee and the University of Kyrenia Ethical Committee (protocol number: YDU/SB/2020/615).n = 105, 80.2%) were married to each other, 21 (16.0%) were divorced or living separately, and two (1.5%) were widows or single parents not having been married. Since the number of widows or single parents was low, they were excluded from the analysis related to parental relationship status. Most participants reported being in a romantic relationship , 46 (35.1%) reported being single because of a breakdown of a relationship, and 21 (16.0%) reported never having been in a romantic relationship.The demographic information form included questions about gender , age (full years), relationship status of the parents  and relationship status of the respondent . Most parents , developed by Fraley and Shaver , measureThe Inventory of Close Relationship Beliefs, developed by Fletcher and Kininmonth , measuret-tests, reliability statistics, correlations and regression analyses. JASP was used for mediation analysis.IBM SPSS 28.0 was used for calculating descriptive statistics, t-tests were used. The only hypothesis (H1) was that a gender difference occurs in the variable concerned.Since gender is an important factor in adult heterosexual relationships, gender differences were calculated for the two attachment styles, the four relationship belief scales, and the dyadic trust scores. The tests of gender differences were considered exploratory and therefore direction of gender differences was not specified; consequently, two-tailed t-tests for gender difference are presented in t-test values for gender differences. Men scored higher than women in anxious attachment style, while women scored higher on the individuality scale of the ICRB. Women seem to value individuality  in relationships more than men. No gender difference was found in the other variables.The descriptive statistics separately for men and women and t-test was employed to investigate the mean difference in trust between respondents with parents who had divorced or separated and those with parents who remained together. Respondents with married parents scored higher  on the Dyadic Trust Scale than those with divorced parents , t(22.93) = 2.46, p = 0.011, Cohen's d = 0.82. This suggests that experiencing parental divorce might be associated with reduced trust in relationships. However, it's essential to note that the sample size for respondents with divorced parents was small (n = 21), which limits the generalizability of the results. These results confirmed Hypothesis 1, that respondents having experienced parental divorce experienced less trust in relationships.An independent samples  = 9.82, p < 0.001, \u03b72 = 0.13. Bonferroni corrected pairwise comparisons indicated that respondents in an ongoing relationship had higher trust scores  than both those who had ended a relationship , p < 0.001, and those who had never been in a romantic relationship , p = 0.019. There was no statistically significant difference in trust scores between respondents who had ended their relationship and those who had never been in one. These results suggest that positive experiences in a romantic relationship may bolster interpersonal trust. Moreover, it can be inferred that respondents who had never been in a romantic relationship based their trust responses on their beliefs about romantic relationships in general. These results confirmed Hypothesis 1, which posited that respondents who had experienced a relationship breakdown would have less trust in relationships. No hypothesis was formed about not having been in a romantic relationship and trust.In addition to parental divorce, respondents also provided information about their current relationship status, choosing from the options: no relationship, relationship ended, or in an ongoing romantic relationship. A one-way ANOVA revealed a statistically significant main effect of relationship status, FCorrelations among study variables are displayed in p < 0.001) direct effect of avoidant attachment style on trust, whereas the direct effect of anxious attachment style on trust was not statistically significant (p = 0.054). The indirect effect of anxiety on trust via individuality was statistically significant . Similarly, the indirect effect of avoidance on trust via individualism was statistically significant . The total effects of both anxiety  and avoidance  on trust were statistically significant. The model explained 46% of the variance in trust scores.Hypothesis 4 proposed that the effects of attachment style on dyadic trust would be at least partly mediated by relationship beliefs. Consequently, The mediation model results show that the effects of anxious attachment on trust were fully mediated by individuality, whereas the direct effect of avoidant attachment style on trust was stronger than the mediation effect of individuality. We can, therefore, conclude that Hypothesis 4 was confirmed.When evaluating the results of the mediation analysis, it should be borne in mind that these analyses are based on a theoretical model, and no causal relationships can be confirmed based on cross-sectional data.This study aimed to investigate how attachment style , experiences about parents' divorce and breakdown of one's own romantic relationship, and relationship beliefs are related to the level of dyadic trust in romantic relationships. The findings were consistent with previous research showing that experiencing one's own relationship breakdown , demonstrated more conventional perspectives compared to their counterparts from less conservative locales and those with less vertical collectivism tendencies (Bugday et al., The mediation model indicated that the path from anxious attachment style to trust was fully mediated by individuality, while the direct relationship from avoidant attachment style to trust was stronger than the mediated relationship. An anxious attachment style reduces the belief in individuality and, hence, leads to lower trust. It seems that people with anxious attachment styles perceive a romantic partner's need for independence as a threat to the relationship. Avoidance is directly related to lower trust because, for an avoidant person, trust is simply not important in the relationship. In this manner, the mediation model illustrates two distinct pathways through which attachment style is related to trust.The study has some limitations. Firstly, it was based on volunteer participation. This might lead to self-selection bias, whereby participants scoring high in avoidant attachment style might also avoid participating. However, since the study was conducted during class hours and not online, the potential for self-selection bias should be less than in internet-based studies. Moreover, the issue of self-selection is inherent in all attachment studies based on self-reports, as participation in psychological studies must always be voluntary. In addition to possible self-selection, it should be noted that attachment in adult romantic relationships might be lower that the attachment theory suggests (Fraley et al., This study offers a preliminary examination of the mechanisms by which anxious and avoidant attachment styles may influence dyadic trust through relationship beliefs. It is noteworthy that beliefs about independence seem to play a pivotal role in dyadic trust, with these beliefs holding different degrees of importance for men and women. This observation, which might be particularly relevant for Turkish and other semi-collectivistic cultures, has profound implications for couple therapists and counselors. Divergent views between spouses regarding a woman's role and equality within relationships can lead to reduced trust, heightened emotional and behavioral jealousy, and, tragically, to relationship breakdowns and instances of domestic violence. As such, our initial findings should catalyze further extensive research and alert family therapists and couple counselors to this issue. If independence proves to be a cornerstone in building dyadic trust, it is imperative to communicate this insight to the broader public. Fostering mutual understanding between partners about a woman's role in marriage might enhance trust and overall relationship quality, especially in societies where collectivistic perspectives on marriage and relationships prevail.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving humans were approved by Near East University Ethical Committee and the University of Kyrenia Ethical Committee. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.TL: Conceptualization, Formal analysis, Methodology, Writing\u2014review & editing. CDY: Conceptualization, Data curation, Investigation, Methodology, Writing\u2014original draft. MJMS: Writing\u2014review & editing."}
{"text": "Epigenetics studies the impact of environmental and behavioral factors on stable phenotypic changes; however, the state of the science examining epigenomic mechanisms of regulation related to secondary health conditions (SHCs) and neuroepigenetics in chronic spinal cord injury (SCI) remain markedly underdeveloped.This scoping review seeks to understand the state of the science in epigenetics and secondary complications following SCI.A literature search was conducted, yielding 277 articles. The inclusion criteria were articles (1) investigating SCI and (2) examining epigenetic regulation as part of the study methodology. A total of 23 articles were selected for final inclusion.Of the 23 articles 52% focused on histone modification, while 26% focused on DNA methylation. One study had a human sample, while the majority sampled rats and mice. Primarily, studies examined regeneration, with only one study looking at clinically relevant SHC, such as neuropathic pain.The findings of this scoping review offer exciting insights into epigenetic and neuroepigenetic application in SCI research. Several key genes, proteins, and pathways emerged across studies, suggesting the critical role of epigenetic regulation in biological processes. This review reinforced the dearth of studies that leverage epigenetic methods to identify prognostic biomarkers in SHCs. Preclinical models of SCI were genotypically and phenotypically similar, which is not reflective of the heterogeneity found in the clinical population of persons with SCI. There is a need to develop better preclinical models and more studies that examine the role of genomics and epigenomics in understanding the diverse health outcomes associated with traumatic SCI. Clinical Relevance: Identification of the mechanisms and relevant prognostic biomarkers that regulate the epigenome can support the development of targeted clinical decision making tools, and interventions. This article highlights the need to advance translational and precision applications in SCI research, bringing personalized care into the clinical environment. The SCI population is highly heterogenous in both American Injury Scale (AIS) level and the extent of permanent neurologic deficit. No two patients will have an identical pathophysiologic progression or clinical course post injury. However, functional recovery continues to be hampered by a paucity of clinical treatment options. Regenerative approaches are ongoing with small progress; treatment strategies remain exploratory, and few have translated to patients (T2), practice(T3), or community (T4) on the translational research spectrum. Post-injury management of secondary health conditions (SHCs) and complications is the key to longevity with good health and acceptable quality of life.Spinal cord injury (SCI) is a devastating condition that often yields permanent disability and challenges in medical care and symptom management. This once \u201cailment to not be treated\u201d has over the years seen increased life expectancy resulting from advances in adaptive technology, pharmacologic intervention, and more robust understanding of SCI pathophysiology. The literature lacks consensus on how chronicity is defined likely due to the interindividual differences in functional recovery between human and preclinical pathophysiology. Thus, for the current review, we define chronicity as starting between 6\u2009months to 1\u2009year following injury.There are 3 generally accepted phases of recovery following traumatic SCI: (1) acute (hours to days), (2) subacute (weeks to months), and (3) chronic (>1\u2009year) states. However, it is also accepted that more than 6\u2009months post-injury is also considered chronic and may be more commonly reflected in preclinical studies. This is likely to be due to the interest in time-dependent inflammatory and immune responses post trauma. There is also increasing recognition that the long-term impact of traumatic SCI may be characterized as a sub-clinical inflammatory disease. A review by Noller and colleagues discussed the contribution of persistent chronic inflammation in SCI resulting in common SHCs. Chronic inflammation is significant for individuals living with SCI because of the increased risk of cardiometabolic disorders that are the leading cause of morbidity and mortality in this population. Several researchers have shown SCI-specific inflammatory and structural markers found in the blood/cerebrospinal fluid (CSF) linked to injury and neurologic loss may be predictive of functional recovery in SCI.10It has been found that most of the research on SCI has focused on acute and chronic phases.12 Disruption of gene expression patterns governed by hyper, or hypo methylation may result in autoimmune diseases, cancers, and various other illnesses, in contrast to genetic changes, which are difficult to reverse. However, environmental  influences on DNA methylation may be reversed if reduced or eliminated , and pharmaceutical interventions have also been used to reverse effects.16 The field of epigenetic research seeks to discover how lived environment, social condition, psychosocial factors, and nutrition affect an individual\u2019s expression of genetic information. The 3 central mechanisms regulating epigenetics are (a) alteration of histone modification, (b) DNA methylation (DNAm), and (c) RNA regulation.Epigenetics is broadly defined as processes that may regulate gene expression and do not involve changes to the underlying DNA sequence: a change in phenotype without a change in genotype. However, the central regulating mechanisms remain the same. As more is being understood about environmental influences on disease development, multimorbidity, and health outcomes, epigenetic methodology opens the door to leverage biological and clinical data to inform SHC phenotypes in persons with SCI. Hence, to better understand the evolving science of epigenetics and its application in SCI research, we conducted a scoping review.Neuroepigenetics is distinguished from classic epigenetics due to its specific application to neurons, which means modifications are not propagated to progeny cells as seen in non-neuronal cells. A scoping approach was chosen to guide this review to map and characterize studies investigating epigenetic regulation and their links to SHCs after acute and chronic SCI. We hypothesize that given the paucity of literature on this topic, the influence of epigenetics remains an understudied area for this population. The specific purpose of this scoping review was to examine the state of the science focusing on epigenetics that attempts to explain SHC and other conditions associated with chronic SCI.Scoping reviews differ from systematic reviews in that they do not aim to produce a critically appraised and synthesized result/answer to a particular question, but rather focus on (1) identifying certain characteristics/concepts in papers or studies and (2) mapping, reporting, or discussing these characteristics/concepts.A librarian-assisted literature search was conducted across 4 databases  using the search terms \u201cspinal cord injury,\u201d \u201cspinal cord injuries,\u201d \u201cepigenetics,\u201d \u201cepigenome,\u201d \u201chistone modification,\u201d and \u201cchromatin modification.\u201d An Endnote library was shared with 277 articles from the years 2010 to 2020.LYG, T-FW), and 135 studies were retained for full text review. Three reviewers completed the full text review and final article abstraction . One author served to resolve conflicts if they arose (author LYG). After the removal of articles that did not meet inclusion criteria, a total of 23 articles was selected for final inclusion  was used to facilitate abstract and full text review. For inclusion, articles (1) investigated SCI and (2) examined a mechanism of epigenetic regulation as part of the study methodology. Articles were further excluded if they were reviews, commentaries, book chapters, or unavailable in English. Articles were uploaded to the Covidence software, and 30 duplicates were removed, resulting in 247 articles remaining for title and abstract review. Two reviewers completed the abstract review  and 9% using RNA regulation as epigenetic mechanisms. The studies occurred with mice (43%) and rats (44%) as the most commonly studied species. Only one human study was identified in this review. Most of the studies focused primarily on central nervous system (CNS) regeneration and plasticity, with only one article considered clinically relevant for SHC, such as neuropathic pain. We have divided and will discuss the included articles by epigenetic mechanism. Methyltransferase enzymes , are responsible for the transfer of the methyl group to DNA and are responsible for the mediation and maintenance of DNAm patterns  was used to analyze changes in folate receptor (Folr) expression, global methylation, and methyltransferase levels in wild-type and Folr knock-out adult male Sprague-Dawley rats and male mice. They found that injury-induced augmentation of Folr receptor expression is important to the regeneration effects of folic acid on the spinal cord. Changes in the Folr expression were related to the suppression of donor methyl groups to enable synthesis of S-adenosylmethionine (SAM), the primary donor in neurons and all cells of the CNS. DNA methyltransferases (Dnmts) were directly involved in the process via the methylation of hemi-methylated sites by Dnmt 1 and de novo methylation of previously unmethylated sites by Dnmt3a and Dnmt3b, resulting in detectable effects on suppressed Dnmt3a and Dnmt3b protein levels, returning to baseline following folic acid treatment. Global methylation decreased by 40% in the presence of injury (SCI) with the use of folic acid supplementation.In an earlier study by Iskandar et al used a contusion SCI model in adult male Sprague-Dawley rates to analyze the effect of folic acid on matrix metalloproteinase 2 (MMP2) expression and neuropathic pain. MMP2 is a protein coding gene found to be associated with diverse function, such as the induction of mitochondrial-nuclear stress signaling with activation of the pro-inflammatory NF-\u03baB, NFAT, and IRF transcriptional pathways. They found that locomotor function improved among the folic-acid-treated group during mid and late phases of the injury, that is, comparable to subacute and chronic phases of SCI in humans, compared with the control (water) group. Moreover, those rats in the folic acid treatment group also demonstrated reduction in neuropathic pain compared to the control (water) group. In summary, folic acid and the folate pathway are mediated by epigenetic mechanisms. The use of folic acid supplementation appears to promote endogenous axonal regeneration and spinal cord healing.Miranpuri et al used Wistar rats to examine the role of abnormal DNAm in axonal regeneration and cell proliferation through histological observation, motor function assessment, and whole-genome bisulfate sequencing (WGBS) to detect gene methylation with Gene Ontology (GO) and KEGG pathway analyses. Histologically, they found several cavities, tissue necrosis, and glial scaring, with poor motor function in behavioral analysis. Bioinformatic analyses revealed 96 differentially methylated genes (DMG\u2019s). Biological processes of the hypermethylated genes (50) were related to cell shape regulation and neurogenesis, while the biological processes enriched by the hypomethylated genes were related to brain development, protein phosphorylation, and ethanol response.Shi et al reported results of a bioinformatic analysis of data to identify common hypermethylated and demethylated genes from DNAm sequencing of samples taken before and after sciatic nerve injury in rats. Although the focus of this paper examined common DMG\u2019s among SCI and non-CNS injury (sciatic nerve injury), one area of interest is identifying protein-protein interaction networks specific to SCI that may have shared pathways significant to SCI-related SHC. The findings of this study indicated 12 DMGs that are involved in biological regulation processes, such as cellular signaling and molecular functions of protein binding. Of particular interest was the Hippo signaling pathway given its activation by many demethylated genes important to neuroregeneration.Another study by Shi et al examined the perturbations of hydropymethylcytosine (5hmC), an oxidation product of DNAm, on gene expression related to cell death after SCI. In other neurological conditions such as TBI, stroke, and sporadic amyotrophic lateral sclerosis (ALS), 5hmC has been found to play an important role in ischemia and perfusion to the brain and spinal cord. A contusion SCI model was used in rats at the T9-T11, followed by quantitative real-time polymerase chain reaction analysis (qRT-PCR) and hydroxymethylated DNA immunoprecipitation (hMeDIP) assays. Results revealed that global change in DNA 5hmC levels significantly increased at 6, 12, and 24\u2009hours after SCI. Additionally, gene expression changes were found to increase in the presence of ten-eleven translocation family (Tet2) enzyme and worsened SCI in the presence of SC-1, a Tet2 expression suppressor, suggesting the importance of 5hmC in the expression of genes related to cell death and survival in SCI and other neurologic conditions.Sun et al, Doherty et al focused on brain derived neurotrophic factor (BDNF), a known target of epigenetic modifications in the brain resulting from environmental influence. Male Sprague-Dawley pup rats underwent low-thoracic spinal cord transection and behavioral testing. This study was among the first to examine the epigenome of the developing spinal cord and the environmental influence on motor outcomes. The findings of this study support that SCI is linked to large increases in methylation throughout the spine.Lastly, DNAm was used to examine the developing spinal cord and its response to environmental input during locomotor development related to hindlimb activity.29 Six studies focused on histone acetylation, 6 examined histone deacetylation, and 3 examined histone demethylations. Histone acetylation is regulated by HAT and HDAC enzymes that add or remove acetyl groups to the N-terminal histone tails and together maintain acetylation homeostasis and play a role in plasticity transcription and regeneration-associated genes. Evidence demonstrates that downregulation of HDACs has positive effects on learning, memory, and synaptic plasticity. Seira and colleagues assessed the potential effect of trichostatin A (TSA), a known promotor of synaptic plasticity, neurogenesis, neuroprotection, and neurite branching in Pten knockout mice. They found that TSA had inhibitory effectors on HDAC, allowing for more acetylation and supporting axonal regeneration in young mice with Pten deletion.The bulk of the studies examined histone modification, the covalent, post-translational modification of histones. This epigenetic regulatory process impacts gene expression by altering chromatin structure  or recruiting histone modifiers, such as histone acetyltransferase (HAT), histone methyl transferase (HMT), histone deacetylase (HDAC), histone demethylase (HDMT), kinases, and E3-ubiquitin .28,29 Si measured glial fibrillary acidic protein (GFAP), S100 calcium-binding protein B (S100B), and global histone h4 levels in Wistar rats. They found that global histone H4 acetylation levels in the perilesional tissue are time-related after SCI, with the 72-hour (acute) timepoint revealing an increase in global histone H4 acetylation levels.De Menezes et al examined the influence of environmental enrichment versus standard housing (control) on Cbp-dependent histone acetylation-mediated axon regeneration. Enriched environment (EE) offers opportunities for increased motor, social, and sensory activity and has been found to enhance neural connections and improve functional recovery on behavioral testing.35 They found that by exposing mice to EE, cAMP-response element binding protein had a mediation effect on histone acetylation; thus, changes in chromatin that increase regenerative capacity of neurons result in increased sensory and motor functions.Hutson et al They examined the impact of a single body weight support gait training session on oxidative stress parameters, BDNF modulation, and global histone H3 and H4 acetylation levels in SCI participants. No changes were observed in BDNF levels, nor histone acetylation levels; however, there was an observed increase in levels of oxidative stress markers  in both treadmill and walker groups.The only human study in this scoping review was conducted by Goldhardt et al examined the role of bromodomain and extraterminal (BET) domain-containing proteins (epigenetic readers that bind acetylated lysines) on neuroinflammation after contusive SCI. The first finding was that BETs were expressed in all major CNS cell populations and their mRNA levels were not modulated in response to SCI. They also found that BET inhibition (via treatment with BET inhibitor JQ1) attenuated pro-inflammatory cytokine expression in activated primary CNS cells, astrocytes, oligodendrocytes, and microglia. S\u00e1nchez-Ventura et al also found that BET inhibition was effective in reducing pro-inflammatory cytokine production, promoting neuroprotection, and reducing acute neuropathic pain in a mouse contusion model.Rudman et al tested valproic acid (VPA), a known HDAC inhibitor, in rats after SCI. HDAC inhibition results in transcriptional reactivation by inducing histone acetylation through HDAC enzyme suppression. Their findings support earlier studies which also report neuroprotective effects of VPA and increased BDNF and GDNF mRNA levels in neurodegenerative conditions. Kim et al investigated changes in the expression of the main regulators of neuronal survival and death. Following contusion injury to young male mice, they found the upregulation of BDNF, GDNF, and HDAC1 expressions, with HDAC1 being an important regulator of neuron apoptosis. Another study of changes in the main regulators of neuronal development, survival, and death in a contusion SCI rat found similar results. Neurotrophic factors BDNF, NGG, GDNF, and HDAC1 were examined, and BDNF was found to be significantly elevated in the SCI group compared to the control, while the other factors were not found highly expressed. Although increased levels of HDACs were found to be expressed in the brain, their role in brain reorganization after injury remains poorly understood.Abdanipour et al examined class IIa HDACs, which differ from class I and class IIb HDACs by their sequence and structural organization, in that they rarely interact with histone tails or the removal of acetylated lysine groups. To better understand the epigenetic mechanism of macrophages , a male mice contusion model was tested with TMP269, a highly selective class IIa HDAC inhibitor. They found that the TMP269-treated group had higher levels of pro-inflammatory cytokines , suggesting the critical role of class IIa HDACs as powerful inhibitors of pro-inflammatory cytokines in macrophages. Sanchez et al specifically examined HDAC3. They hypothesized that HDAC3 inhibition suppresses the phenotype M1 (pro-inflammatory) and promotes the anti-inflammatory phenotype M2 to improve functional SCI recovery. By introducing scriptaid and RGFP966, known HDAC3 inhibitors to female mice with T-cut spinal cord hemisection injury, they found that histone 3 and 4 acetylation were increased, and macrophage polarization was skewed toward M2 phenotype (anti-inflammatory). Scriptaid treatment also decreased M1 macrophage foaminess, while RGFP966 significantly reduced the formation of foamy macrophages in both. They also found that foamy macrophages became proinflammatory and hindered regeneration. Taken together, HDAC inhibitors are strong targets for macrophage polarization and creating an anti-inflammatory environment for SCI recovery.Qi and Wang also studied HDAC3 to determine its role as a novel mechanism to discriminate between axonal regeneration and regenerative failure in dorsal root ganglia (DRG) sensory neurons. Conducting both in vivo and bioinformatic analysis, they found that reduced HDAC3 activity allowed for increased histone acetylation and regenerative gene expression in DRG. However, HDAC3 phosphorylation is calcium dependent and spinal injury does not induce the calcium-dependent protein phosphate 4 (PP4) activity needed for this process. The finding of both analyses supports the role of HDAC3 in restricting the regeneration program needed to enhance DRG neurite growth and expression of regeneration-associated genes including JUN, MYC, STAT3, ATF3, and FOS with key signaling pathways of MAPK, insulin, and JAK-STAT.Hervera et al46 and Ni et al investigated the epigenetic regulation of angiogenesis and vascular regeneration in SCI. The study of post-SCI vascular regeneration by Ni et al  examined mRNA levels of UTX, EZH2, Jmjd3, CBX8, CBX4, CBX6, and CBX7 at the site of the spinal lesion post-SCI contusion in C57BL/6J wild-type and UTX knockout male mice. They found that UTX was elevated at the day 3 timepoint and remained elevated at days 7 and 14 and was found to occur in CD31+ endothelial cells, implicating the potential role of UTX in vascular regeneration post SCI. They went on to demonstrate that UTX regulated expression of miR-24 (increased expression during myogenesis) by binding to the promoter and directly decreasing the level of methylation in several CpG positions in the miR-24 promoter, thereby promoting the expression of miR-24. In summary, UTX deletion can epigenetically promote vascular regeneration and functional recovery post SCI via a regulatory network with miR-24.Lee et al46 histone H3K27 demethylation Jmjd3 was characterized for its role in acute inflammatory response regulation. Lee et al used adult Sprague Dawley male rats subjected to T10 contusion. They found that Jmjd3 gene expression was increased within the first 8 hours after SCI and was more highly expressed in blood vessels at or near the injured spinal cord lesion site. This suggested that it is acutely upregulated following injury with specific functions in acute immune response and/or the blood-spinal cord barrier (BSCB). Moreover, given its role as a transcription activator through demethylation of trimethylated histone H3K27, it is also associated with inflammation-specific markers. The Lee et al study found that IL-6 showed a similar upregulation pattern as Jmjd3, which indicated that in the absence of Jmjd3, the upregulation of IL-6 is inhibited. Lastly, transcription activators known to bind to IL-6 gene promoters, such as NF-kB, were examined for interaction with Jmjd3 using both Western blot and chromatin immunoprecipitation (ChIP) assays. They found that Jmjd3 was directly involved in IL-6 gene activation by demethylation of H3K27me3 at the promoter in cooperation with NF-kB and C/EBP.In both Lee et al studies, contribute findings to the critical role of histone H3K27 demethylase Jmjd3 in the regulation of matrix metalloprotease (MMP) gene expression and BSCB integrity. MMP activation is known to contribute to permanent neurological disability after SCI because of blood cell infiltration, inflammation, and apoptosis.MMP-2, MMP-3, and MMP-9 are specifically known to be involved in SCI-induced BSCB disruption, but the mechanisms following SCI are not well understood. Again, using adult Sprague-Dawley male rats, SCI was induced using a contusion model at T9-T10. They found that Jmjd3 activated MMP-2, MMP-3, and MMP-9 expression in brain endothelial cells upon OGD/reperfusion injury. Using Jmjd3 siRNA to deplete the Jmjd3 transcript, they determined that depletion of Jmjd3 expression inhibited the upregulation of MMP-2, MMP-3, and MMP-9 expression and activities. Like their previous work that found NF-kB to be associated with Jmjd3 activation of IL-6 gene expression, they found in this study that NF-kB was also required for Jmjd3-mediated MMP-2, MMP-3, and MMP-9 gene activations. These 2 studies reinforce the significance of NF-kB involvement as a mediator of gene expression both in DNA methylation and histone demethylation. The combined results of all 3 studies provide potential therapeutic clues for targeting vascular regeneration in the treatment of SCI.Lee et al Using small interfering RNAs (siRNAs) to inhibit the interphase phase of the cell cycle, Wang et al studied epigenetic silencing of cyclinD1 in bone-marrow-derived mesenchymal stem cells (BMSCs) for protective effects in rats after SCI. Cyclins are proteins that function to progress the cell cycle by binding a group of enzymes called cyclin-dependent kinases (CDKs) which phosphorylate target proteins. CyclinD1 specifically is responsible for progressing the cell cycle from G1 to S phase. Inhibition of CyclinD1 expression is associated with downregulation of pro-inflammatory cytokines, which may be a potential therapeutic target for pro-inflammatory conditions such as SCI.miRNA is a class of non-coding small RNA that functions to downregulate gene expression through mRNA cleavage, translational inactivation, and deadenylation .16 UsiGFAP-positive and NGF-positive cells were significantly increased in the si-cyclinD1 + BMSCs and BMSC groups compared with the control group. GFAP and NGF regulation were associated with enhanced astrocyte proliferation. These findings, along with higher BBB scores in groups with si-CyclinD1-engineered BMSCs, support that by silencing CyclinD1, BMSC may promote functional recovery.In female Sprague Dawley rats, cyclinD1 was transplanted with BMSCs into the lesion area of the SCI using siRNA plasmids. CyclinD1 was found nominally expressed by Western blotting and qRT-PCR, confirming that siRNA transfection was successful. Observations of histological changes confirmed decreased tissue edema, inflammatory cell infiltration at the site of the SCI lesion, and smaller syrinx in groups with si-cyclinD1 + BMSCs and BMSC groups compared with the control and sham groups. Additionally,  sought to use bioinformatic analysis to construct and integrate ceRNA network of critical genes, miRNAs, long non-coding RNAs (lncRNA), transcription factors, and signaling pathways. Gene expression data sets were used from the gene expression omnibus (GEO) database to define the \u201cSCI group\u201d as mice with SCI acquired by contusion. DEG analysis was performed using GO and KEGG enrichment. They found 4 genes , 4 transcription factors , 2 miRNAs (miR-16-5p and miR-325-3p), and 3 lncRNAs  that are considered significant contributors to the pathogenesis of subacute SCI.In another study investigating the subacute phase of SCI, Wang et al  Current evidence suggests that in the presence of lost neuronal acetylation homeostasis, short- and long-term chromatin architecture changes, resulting in the development of neurodegenerative disorders, such as Parkinson\u2019s disease (PD), multiple sclerosis (MS), stroke (CVA), and Alzheimer\u2019s disease (AD), which has significance post-SCI neurodegenerative changes. Notably, the histone acetylation-deacetylation balance modulates different cell types, such as inflammatory and immunological cells, which influence the severity of blood-brain barrier dysfunction, axonal demyelination, oxidative stress, and injury impact.54 Applying neuroepigenetics to SCI research could lead to new approaches in rehabilitation where re-learning motor activities could improve functional outcomes.The results of this scoping review suggest that there is an increasing enthusiasm for epigenetics and neuroepigenetics in SCI research, especially given the increased complexity when considering the different cell types that exist in the brain and spine. Further, the findings from this review offer exciting insights into neural circuits and synaptic plasticity involved in learning new information. The emerging sub-discipline of neuroepigenetics describes the \u201cdiscoveries of a wide variety of roles for epigenetic molecular mechanisms in the central nervous system (CNS) regarding acquired behaviors, CNS disorders, neural plasticity, neurotoxicity, and drug addiction.\u201d presented the Rehabilomics research framework, offering a translational path for rehabilitation research that combines omics technology with personal biology, physiological responses to environmental exposures, and biopsychosocial outcomes based on the World Health Organization International Classification of Functioning, Disability, and Health domains. However, this adapted model primarily focuses its application on traumatic brain injury (TBI) research. While TBI and SCI share similarities in primary and secondary injury cascades following mechanical injury, the study of SCI and the chronic SHCs that follow remain understudied. Individuals living with SCI are living longer and have increased and/or unpredictable levels of risk for developing a wide range of SCI-driven SHCs. The identification and use of prognostic biomarkers are major steps in developing decision trees, predictive tools, personalized interventions, and treatments.This scoping review also reinforces the dearth of studies that examine the potential of clinically applied precision health within the rehabilitation setting, particularly for individuals living with SCI and managing SCI-associated SHCs. Many of the studies included in this review examined changes in the acute or subacute post-SCI phase. None of the studies looked at these gene expression changes in chronic SCI, thus leaving a larger gap in the significance of chronicity on these mechanisms. Understanding gene expression and its regulation translates to more accurate diagnosis and treatment for clinically significant changes in phenotype. Wagner These clinical features are reinforced by the findings of studies included in this scoping review, particularly as key genes, proteins, and pathways that were present in multiple studies, including brain derived neurotrophic factor (BDNF), glial cell derived neurotrophic factor (GDNF), neuronal growth factor (NGF), histone deacetylase 1 (HDAC1), NF-kappaB signaling pathway, and the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathways. These key genes and pathways offer an opportunity to develop our understanding of mechanisms underlying SCI-triggered SHC that result from gene expression activation and/or silencing.The subacute phase after SCI is categorized by neuronal apoptosis, axonal demyelination, Wallerian degeneration, axonal remodeling, and glial scar formation, while the chronic phase is characterized by cystic cavity, axonal dieback, and maturation of glial scar.BDNF is well known for its glucose and energy metabolism regulation, as well as neuroplasticity and synaptogenesis. The JAK-STAT pathway is an essential signaling pathway for several cellular response functions including inflammatory responses, hypoxia/reperfusion, cellular stress, and more. Saligan found that the BDNF Val66MET single nucleotide polymorphism (SNP) genotype was protective against cancer-related fatigue. This could be a significant target in the fatigue experienced by individuals living with SCI and chronic multimorbidity. BDNF is also implicated in stress-related phenotypes, which given its role in brain-based gene regulation, has implications for shared mechanisms of other stress-related conditions, such as depression and anxiety.GDNF is a protein coding gene specifically associated with SCI and is also involved in the RAF/MAP kinase cascade super pathway that, like JAK-STAT, regulates cellular processes related to differentiation, survival, senescence, and cell movement.60 has shown the genetic influence of candidate DNA variants and single nucleotide polymorphism (SNPs) on intramuscular adipose tissue (IMAT) accumulation and recurrent pressure injury (PrI) risk. They have previously shown that varying levels of circulatory biomarkers may be indicative of recurrent PrI risk.60 Furthermore, genetic biomarkers, specifically those related to fatty metabolism, are indicative of persons with SCI at highest risk for recurrent PrI. Histone H3K27me3 demethylase Jmjd3 has been found to be involved in wound repair and the inflammation process,63 highlighting the value of incorporating both genetic and epigenetic regulation of gene expression, as it may hold significance for the study of pressure injury and other SHCs after SCI. Thus, defining the epigenetic mechanisms linked to remodeling neural tissue would help researchers design therapeutic strategies to promote wound healing and tissue functionality after SCI among other potential targets.65Preliminary work by Bogie and colleagues, and recognizes the \u201ccomplex relationships within and between symptoms both in a single chronic condition and among chronic conditions .\u201d Symptoms serve as critical clues to changes in health status  and are the primary reason urgent or emergent care is sought, which may (or may not) lead to hospitalization and/or diagnosis of a newly developed SHC. This is of particular importance for SCI research, where individuals are likely to suffer higher rates of multimorbid SHCs and the management of accompanying symptoms.There is a key discussion absent in SCI research around epigenetics, chronic multimorbidity, and SHCs. Symptom science is \u201cfocused on quantifying subjective symptom experiences and measuring the biologic, physiologic, and omics underpinnings of symptoms and sequela common to health conditions and their treatments\u201d While we present the effects of epigenetics leading to the development of multimorbid SHC, that is, the gene-environment, it is very probable that multimorbid SHC directly lead to remodeling in the epigenome. Studies looking at the influence of changes due SHC on the epigenome need to address the dynamic variability inherent in epigenetic studies. Prospective studies to measure and compare pre-multimorbidity states are also challenging. Multimorbidity as previously described carries a significant burden for individuals living with SCI and is influenced by age and injury characteristics . These influences also carry significant complexity in the presence of the injury. Although the relationship between multimorbid SHC and epigenetic change is even less understood given the dearth of research in this field, we wanted to acknowledge it as an important consideration as more work is being done to develop this field.Diseases can often be due to multiple factors including routine environmental exposures.72 Development of a Rehabilomics theoretical model specific to SCI symptomology would provide guidance that would support the translation of bench findings to clinical management of chronic multimorbidity resulting from SCI.Theoretical models have been developed to improve our understanding of the mechanisms that contribute to symptom behavior . However, symptom behavior and associated mechanisms has primarily been evaluated in other conditions, such as cancer and cardiovascular disease; only one study addresses symptom behavior in SCI research.This scoping review has revealed some key epigenetic regulators of gene expression and adjacent pathways that appear to overlap many critical biological, molecular, and cellular processes following SCI. However, it also elucidates the need for more translational/applied clinical research as a next step from the bench to begin impacting the patient. Given the paucity of research that studies the concepts of epigenetics, SHCs, and/or their associated symptoms in SCI research , more work in this area is critical to continue advancing our understanding of SCI-specific biobehavioral outcomes."}
{"text": "The formation of atherosclerotic plaques is one of the main sources of cardiovascular disease. In addition to known risk factors such as dyslipidemia, diabetes, obesity, and hypertension, endothelial dysfunction has been shown to play a key role in the formation and progression of atherosclerosis. Peroxisome proliferator-activated receptor-gamma (PPAR\u03b3), a transcription factor belonging to the steroid superfamily, is expressed in the aorta and plays a critical role in protecting endothelial function. It thereby serves as a target for treating both diabetes and atherosclerosis. Although many studies have examined endothelial cell disorders in atherosclerosis, the role of PPAR\u03b3 in endothelial dysfunction is still not well understood. In this review, we summarize the possible mechanisms of action behind PPAR\u03b3 regulatory compounds and post-translational modifications (PTMs) of PPAR\u03b3 in the control of endothelial function. We also explore the potential use of endothelial PPAR\u03b3-targeted agents in the prevention and treatment of atherosclerosis. Endothelial dysfunction plays important roles in atherosclerosis initiation and progression. Atherosclerosis poses a major threat to human health and is one of the leading causes of morbidity and mortality in type 2 diabetes (T2DM) patients ,2. Much Peroxisome proliferator-activated receptor gamma (PPAR\u03b3), a nuclear receptor of the steroid superfamily, is a potent target for counteracting risk factors of atherosclerosis. Thiazolidinediones (TZDs), synthetic ligands of PPAR\u03b3, have been shown to reduce the incidence of atherosclerosis . PPAR\u03b3 hPPAR\u03b3, increasing attention has been directed towards these PTMs [PPAR\u03b3 can be regulated by altering the activity of PPAR\u03b3 or modulating its downstream pathways. With regard to the former, the bioactivity of PPAR\u03b3 can be regulated by ligands that bind to peroxisome proliferator response elements (PPREs), the corepressor complex, and the co-activator complex through different domains ,23. PPARese PTMs ,25,26,27The cardiovascular system is responsible for distributing nutrients and oxygen throughout the body , and oneIn terms of physiologic function, the endothelial barrier is responsible for the exchange of substances entering or exiting the blood ,34. The ECs also play critical roles in maintaining the appropriate hemostasis by interacting with coagulation and fibrinolytic system . ActivatSimilarly, certain substances in the blood are involved in regulating the vascular function of endothelium. For example, high levels of Thyroid Stimulating Hormone (TSH) in blood can trigger excessive opening of the mitochondrial permeability transition pore as well as increased mitochondrial oxidative damage in ECs . BlockinECs secrete connective tissue components, collagen molecules, proteoglycans, and elastin to regulate cell-to-cell or matrix binding, which plays an important role in the stability of early plaques. However, with progressive age, rupture of collagen fibers and degradation of the extracellular matrix occurs, resulting in plaque rupture and subsequent cardiovascular events. Reducing monocyte adhesion to the aortic endothelium and modulating levels of vascular cell adhesion molecules have demonstrated beneficial effects ,50.The function of ECs can be evaluated by testing the impairment of vascular dilation with acetylcholine ,52, and Atherosclerosis is widely recognized as a chronic inflammatory disease ,56. PlaqThe term \u201cendothelial dysfunction\u201d can be used to refer to the impairment of vascular function caused by abnormal production or activity of endothelium-derived NO . Here, hPPAR\u03b3 has several PTM patterns, including phosphorylation, SUMOylation, and acetylation . These PPPAR\u03b3 is involved in several molecular pathways that promote endothelial function , as outlPhosphorylation and inactivation of PPAR\u03b3 is directly catalyzed by extracellular regulated protein kinase 1/2 (ERK1/2), a pro-inflammatory kinase . FurtherNO production and eNOS activity also play integral roles in the effect of PPAR\u03b3 on endothelial function ,69,70,71PPAR\u03b3 participates in the recruitment of leukocytes; in particular, recruitment of monocytes to activated vascular ECs is a critical step in the inflammatory cascade . PPAR\u03b3 a2O2-induced injury [NF-\u03baB is involved in PPAR\u03b3-mediated endothelial protection ,81,82,83d injury . Susceptd injury .Lectin-like receptor for oxidized LDL (LOX-1), located on the membrane of ECs, can recognize oxLDL, TNF\u03b1, and other stimuli, and plays an important role in the progression of atherosclerosis ,86. In bOther mechanisms of endothelial protection by PPAR\u03b3 have been identified, including its role in apoptosis. Upregulation of PPAR\u03b3 was found to induce the upregulation of p53 and promote apoptosis of HUVECs . Activat\u2212/\u2212) mice, endothelial PPAR\u03b3 demonstrated anti-inflammatory and hypopermeability properties, thereby contributing to a protective effect against atherogenesis [Furthermore, gene knockout and overexpression techniques have facilitated research efforts into the endothelial-protective role of PPAR\u03b3. In a comparison between endothelial-specific and macrophage-specific PPAR\u03b3 disruption in LDL receptor-knockout  signal pathway, thus producing anti-inflammatory effects . BerberiNotoginsenoside Fc, ginsenoside-Rb1, and 7,8-didehydrocimigenol  are members of the saponin and triterpene class. Notoginsenoside Fc was found to attenuate high glucose (HG)-induced EC injury through the inhibition of pro-inflammatory cytokines and apoptosis as well as increased PPAR\u03b3 proliferation in rat aortic ECs . ActivatGenistein and formononetin are examples of isoflavones. Genistein inhibits monocyte adhesion to the endothelium via PPAR\u03b3 and the physical forces associated with blood flow, rather than by regulating the expression of adhesion molecules . Activat2-methoxyestradiol and urolithin A(UA) are biological metabolites. 2-methoxyestradiol is an endogenous estradiol metabolite with antiatherogenic properties. This molecule has been found to promote the release of NO primarily through the PPAR\u03b3/PI3K/Akt/eNOS cascade, thereby encouraging endothelial-dependent vasodilation . UA, an Dual PPAR\u03b1/\u03b3 agonists include TAK-559 and conjugated linoleic acid ,109. TAKThis category consists of compounds that do not fall into any of the aforementioned groups.15-deoxy-Delta -prostaglandin J2 (15d-PGJ2) is a prostaglandin that is a strong, naturally existing PPAR\u03b3 agonist . 15d-PGJTelmisartan, an angiotensin II-receptor blocker (ARB), was recently reported to have PPAR\u03b3 partial agonist properties ,70. TelmKR-62980, a new PPAR\u03b3 agonist, impaired angiogenesis in HUVECs and promoted apoptosis through PPAR\u03b3-mediated downregulation of VEGF receptor 2 (VEGFR-2) and upregulation of chromosome 10 (PTEN) .Tert-Butylhydroquinone is an activator of Nrf2 that may increase the expression of eNOS by up-regulating the transcription and translation of PPAR\u03b3, subsequently increasing endothelial NO production .Magnolol is a dual agonist of RXR\u03b1 and PPAR\u03b3. In cultured HUVECs, incubation with magnolol enhanced the release of NO and promoted insulin-induced vasodilation through the PPAR\u03b3-dependent Akt/eNOS/NO cascade .Cyclic phosphatidic acid (cPA) potentially inhibits neointima formation by attenuating VEGF-mediated growth and migration through upregulation of the cPA-PPAR\u03b3 axis in human coronary artery ECs from patients with diabetes .1,8-Cineole may protect the endothelium from inflammatory infiltration by upregulating PPAR\u03b3 expression and inhibiting NF-\u03baB p65 phosphorylation .Carbon monoxide derived from CO-releasing molecule-2 (CORM-2) exhibited anti-atherogenic effects through attenuation of HG-induced endothelial ICAM-1 via the AMPK/PPAR\u03b3 pathway .Simvastatin, a member of the statin class, demonstrated benefits in maintaining thrombotic homeostasis of saphenous vein ECs challenged by advanced glycation endproducts. Decreased PPAR\u03b3 expression was significantly improved by simvastatin in saphenous vein ECs from non-diabetic patients in vitro. There was also a slight, but not statistically significant, increase in PPAR\u03b3 expression in ECs obtained from diabetic patients treated with simvastatin .PTMs of PPAR\u03b3 play critical roles in finely regulating its activity. PTMs of PPAR\u03b3, such as acetylation, phosphorylation, and SUMOylation, are known to regulate glucose and lipid metabolism ,27. Here\u2212/\u2212 mice [PPAR\u03b3 acetylation regulates PPAR\u03b3 activity, and the dynamics of acetylation are disturbed in obesity and aging . PPAR\u03b3 i\u2212/\u2212 mice ,124. Thi\u2212/\u2212 mice . PPAR\u03b3 d\u2212/\u2212 mice . Adminis\u2212/\u2212 mice . As suchSUMOylation is another PTM that can selectively regulate PPAR\u03b3 activity. PPAR\u03b3-K107R-mutant mice (K107R prevents SUMOylation) were found to have increased insulin sensitivity without the associated increase in adiposity that typically accompanies TZD treatment . DeSUMOyGiven the importance of PPAR\u03b3 in EC dysfunction and the role of EC disorders in atherosclerosis progression, it is essential that we understand the mechanisms behind PPAR\u03b3 signaling in endothelial function. This can serve as a springboard for the development of novel PPAR\u03b3-targeted drug therapies in the future. Furthermore, understanding how PPAR\u03b3-modulating compounds and PTMs alter EC function may aid in identifying new therapeutic targets, such as deacetylation or deSUMOylation processes.A PTM we did not cover in this review is PPAR\u03b3 phosphorylation, as few studies have been conducted on this topic and its significance remains unclear. Based on existing studies, PPAR\u03b3 phosphorylation at various sites has demonstrated a range of glucose and lipid metabolic effects. For example, phosphorylation at S273 by cyclin-dependent kinase 5 (CDK5) has been shown to increase proinflammatory factors including TNF-\u03b1, IL-1\u03b2, and foam cell formation, which worsen atherosclerosis . Both PPTo conclude, we can reasonably anticipate: (1) The use of next-generation sequencing technologies, such as RNA sequencing, and other omics techniques to further evaluate the molecular basis of PPAR\u03b3 regulation of EC dysfunction in the context of atherosclerosis. This will allow us to establish a more comprehensive regulatory network incorporating upstream and downstream targets of PPAR\u03b3. (2) There is growing evidence that selective activation of PPAR\u03b3 may reveal novel therapeutic targets for future generations of drugs in the treatment of both T2DM and atherosclerosis, and PTMs of PPAR\u03b3 have shown great potential in this regard. (3) Given the many different PTMs and regulatory sites of PPAR\u03b3, along with their apparent involvement in EC dysfunction, increasing attention will continue to be directed towards PPAR\u03b3 regulation in other metabolic and cardiovascular diseases."}
{"text": "Analysis of legumain mutant zebrafish shows a requirement for early ECM remodeling and efficient ligament regeneration. Our study establishes a new model of adult scar-free ligament regeneration and highlights roles of immune-mesenchyme cross-talk in ECM remodeling that initiates regeneration.After traumatic injury, healing of mammalian ligaments is typically associated with fibrotic scarring as opposed to scar-free regeneration. In contrast, here we show that the ligament supporting the jaw joint of adult zebrafish is capable of rapid and complete scar-free healing. Following surgical transection of the jaw joint ligament, we observe breakdown of ligament tissue adjacent to the cut sites, expansion of mesenchymal tissue within the wound site, and then remodeling of extracellular matrix (ECM) to a normal ligament morphology. Lineage tracing of mature ligamentocytes following transection shows that they dedifferentiate, undergo cell cycle re-entry, and contribute to the regenerated ligament. Single-cell RNA sequencing of the regenerating ligament reveals dynamic expression of ECM genes in neural-crest-derived mesenchymal cells, as well as diverse immune cells expressing the endopeptidase-encoding gene  Healed ligaments fail to recapitulate the highly organized structure of the uninjured tissue. Foundational research using non-regenerative models has highlighted that the failure to regenerate ligaments without scarring is common among mammals5. Early studies investigating repair of the medial collateral ligament (MCL) following rupture showed that the healed ligament undergoes more deformation under the same load, coincident with disorganization of the collagen fibers at the wound site6. The first 10 days of ligament healing in mammals are characterized by an invasion of fibroblasts, vasculature, and pro-inflammatory immune cells along the ligament7. While ligament repair continues for 14 weeks, an incomplete remodeling of both the cellular and extracellular matrix (ECM) composition occurs, ending with a fibrotic scar bridging the injury. Similarly, in human and murine tendon and ligament injuries, the stubs are bridged by \u03b1-Smooth Muscle Actin-positive scarring myofibroblasts11. Notably, these myofibroblasts do not express tendon and ligament marker Scleraxis (Scx), indicating that they are not true ligamentocytes8. Existing studies on adult connective tissue injury highlight the importance of the reestablishment of cell fate and ligament ECM through regeneration.Ligament injuries typically heal with biomechanically inferior scar tissue that compromises joint stability and increases the risk of developing painful and debilitating osteoarthritis8. In this neonatal repair context, Scx-positive tenocytes retain their developmental potency to proliferate, migrate to the wound site, and heal without forming a fibrotic scar8. Further studies on developmental regenerative potential have been performed using juvenile zebrafish, which described that BMP-dependent activation of perichondrial progenitors is necessary for tendon regeneration after tendon cell ablation injury12. However, while these models interrogate scar-free tendon regeneration in the context of development, there are currently no models to study scar-free ligament regeneration in mature adult ligaments. To address this need, we utilized the highly regenerative zebrafish, as they are a well-established model for heart, spinal cord, and tail fin regeneration15 and moreover possess regenerative synovial joints supported by ligaments18.Recent advances in tendon injury modeling showed that neonatal mice are capable of scar-free regeneration after Achilles tendon transection18. Here we characterize the cellular and molecular events underlying the three overlapping phases of ligament healing: inflammation, proliferation, and remodeling. We also used single-cell RNA sequencing (scRNAseq) to characterize the dynamics of cell populations during regeneration and identified legumain (lgmn) as a gene highly enriched in macrophages that invade the injury site. Legumain is a member of the C13 family of cysteine proteases that regulate ECM factors such as cathepsins, fibronectin1 (Fn1), and matrix metalloprotease-2 (MMP-2)20. Through injury analyses in a zebrafish mutant for lgmn, we uncovered an essential role in regulating ECM remodeling and scar-free ligament regeneration. Our findings highlight the important role of immune-mediated ECM remodeling for successful scar-free regeneration.In the course of establishing a post-traumatic osteoarthritis model, we had observed rapid regeneration of the craniofacial interopercular-mandibular (IOM) ligament, which supports the zebrafish jaw joint, following complete transection in one-year-old fishThe IOM ligament connects the interopercular bone to the retroarticular bone of the mandible to control movement of the zebrafish jaw joint. Following complete transection of the IOM ligament in adult zebrafish, a time-course of histological analysis revealed dynamic morphological changes during ligament regeneration Fig. . The uniscxa:mCherry transgenic line. In these fish, mCherry expression is driven by the regulatory region surrounding the scleraxis a (scxa) gene, which encodes a transcription factor highly expressed in ligament and tendon cells22. In uninjured adults, live imaging revealed scxa:mCherry fluorescence in each of the bilateral IOM ligaments  sequencing data of zebrafish cranial neural crest-derived cells24 identified a 635\u2009bp region within intron 13 of the thrombospondin 4a (thbs4a) gene that was selectively accessible in the tendon and ligament cluster  to DsRed fluorescent protein -inducible CreERT2 (thbs4a_p1:CreERT2) in combination with the ubiquitously-expressed actb2:loxP-BFP-STOP-loxP-DsRed reporter. Treatment of adult double transgenic fish with 3 doses of 4-OHT resulted in DsRed fluorescence throughout the IOM ligaments  and collagen 12 alpha 1a (col12a1a), which label injury mesenchyme in zebrafish spinal cord and heart regeneration28. While fn1a is minimally expressed in the uninjured ligament, we see abundant fn1a transcripts in DsRed+ thbs4a-lineage cells in both the injured ligament stubs, as well as in the bridging regenerative mesenchyme . These data support typically quiescent ligamentocytes re-entering the cell cycle upon injury and contributing to the regenerated ligament.In addition, we observed extensive proliferation, based on immunofluorescence staining for Proliferating Cell Nuclear Antigen (PCNA), within both the regenerative mesenchyme and ligament stubs adjacent to the transection site. Compared to only minimal PCNA+ cells prior to injury and at 1 dplt, 39% of cells within the ligament stub domain and 47.5% cells within the regenerative mesenchyme domain were PCNA+ at 3 dplt  on micro-dissected IOM ligaments, including adjacent joint and mesenchymal tissues, before and during early stages of regeneration. We used FACS in ent Fig. . After pkrt5+), superficial cells , and mucosal cells   and macrophages , and Immune 2 cluster consisted primarily of neutrophils . We also detected an endothelial cluster . Two superclusters, enriched for DsRed+ cells  or the chondrocyte genes col2a1a and mia (NC-Derived 2) , and endothelial cells Fig. . Epithellls Fig. , represe 2) Fig. .ifitm5+), chondrocytes (col2a1a+), superficial chondrocytes and synoviocytes (prg4b+), ligamentocytes , two types of dermal fibroblasts (pah+), periosteal cells (mmel1+), three types of stromal cells , and glial cells (mbpa+)24  and fibronectin 1a (fn1a) , a marker of pro-scarring fibroblasts in mammals. These data are consistent with regenerative mesenchyme cells producing new ligamentocytes as opposed to scar tissue during regeneration.Re-clustering of the two neural crest-derived superclusters revealed clusters of osteoblasts , macrophages (mpeg1.1+/mfap4+), dendritic cells (mpeg1.1+/mfap4-), two types of T helper cells (il21r.2+ or il4+), proliferative cells (mki67+), and four types of lymphocytes (lck+), including two T cell-containing clusters (cd8a+), NK cells (s1pr5a+)29, and activated NK cells (ifng1+/gzmk+)  Fig. . The Neumpeg1.1+/mfap4+ macrophage cluster, we noted a subpopulation of macrophages with enriched composition of 1 dplt cells and selective expression of fn1a   are anatomically similar to wild types with no overt differences in the gross morphology of their uninjured ligaments  Fig. . Homozygnts Fig. . To charnts Fig. . In wildlgmn mutants, we first measured apoptosis and proliferation using TUNEL and PCNA immunofluorescence assays, respectively. After transection in wild types, we observed 7.4% TUNEL+ ligamentocyte nuclei at 1 dplt and 2.5% TUNEL+ at 3 dplt  and a trend toward lower proliferation in ligament tissue adjacent to the transection site (p\u2009=\u20090.4660)  Fig. .lgmn mutants, we first quantified neutrophils at 1 and 3 dplt using immunofluorescence for the neutrophil marker, Mpx   were immunostained for eGFP and PCNA. At 1 dplt, we observed that ligamentocytes within the ligament stub re-enter the cell cycle without coincident macrophages 51, Tg(actab2:loxP-BFP-STOP-loxP-dsRed)sd2752, Tg(Mmu.Sox10-Mmu.Fos:Cre)zf38453, Tg(mpx:mCherry)uwm754, and Tg(mpeg1:EGFP)gl2255. Embryos were raised in a methylene blue salt solution at 28.5\u2009\u00b0C. Juvenile and adult fish were housed in groups of 15-30 individuals. All surgical injuries were performed in size-matched 3-6 months post fertilization (mpf) adult zebrafish using standard body length (SL) measurements.The Institutional Animal Care and Use Committees of Columbia University and the University of Southern California approved all zebrafish experiments. Published Zebrafish lines used in this study include lgmnsa22350 mutant allele containing a G\u2009>\u2009T point mutation resulting in a premature stop codon was used to generate homozygous lgmn mutants (referred to as lgmnMUT). Zebrafish were genotyped by PCR with primers flanking intron 1 (lgmn forward primer: AGATCTTATGATCCCAGATCCAATGACT) and end of exon 2 (lgmn reverse primer: GGTGAGAAAATGAAACCCGAAACTAGTCT). The 500\u2009bp PCR product was digested with NspI  that recognizes a RCATG/Y cut site only present in mutants. Mutant alleles were then identified by presence of the cleaved 358\u2009bp and 142\u2009bp bands.Tg(thbs4a_p1:CreERT2)el913 and Tg(thbs4a_p1:eGFP)el912 alleles were generated using Gateway cloning and Tol2 transgenesis. p5E plasmid including the 635\u2009bp thbs4a_p1 enhancer and minimal E1B promoter sequence was generated using synthesized gBlock DNA (IDT) in a BP cloning reaction (see Supplementary Methods thbs4a_p1 gBlock sequence). The thbs4a_p1:eGFP transgene was generated by combining p5E-thbs4a_p1:E1B, pME-eGFP, p3E-polyA, and pDestTol2AB2. The thbs4a_p1:CreERT2 transgene was generated by combining p5E-thbs4a_p1:E1B, pME-CreERT2, p3E-polyA, and pDestTol2CG2. Transgenes were injected into one-cell stage zebrafish embryos at 30\u2009ng/ul plasmid DNA with 50\u2009ng/ul Tol2 mRNA. Injected F0 animals were raised to adulthood and outcrossed to T\u00fcbingen or Tg(actab2:loxP-BFP-STOP-loxP-dsRed)sd27 animals. F1 founders were screened for the presence of eye CFP (thbs4a_p1:eGFP) or heart GFP and successful 4-OHT conversion in progeny (thbs4a_p1:CreERT2), respectively.The gthbs4a_p:CreERT2e18. Briefly, adult zebrafish were anesthetized with Tricaine MS-222 and restrained in a damp sponge. Using 3-mm Vannas spring scissors , the IOM ligament was severed with a single cut and a pull test performed on the IOP bone to confirm transection. Fish were then revived in clean system water, housed on-system, and received daily post-operative health checks for three days. At experimental endpoints, zebrafish were euthanized using a lethal dose of Tricaine MS-222 with rapid chilling on ice.Interopercular-mandibular (IOM) ligament transection surgery was adapted from Smeeton et al.Adult zebrafish samples were fixed in 4% PFA at 4\u2009\u00b0C overnight. Following fixation, samples were washed twice for 30\u2009minutes in 1xPBS and cut to facilitate embedding and sectioning of the head. Samples were decalcified in 0.5\u2009M EDTA solution for 10 days rocking at room temperature. For embedding, the tissue was dehydrated in 20\u2009min increments with a series of ethanol dilutions . The water was then replaced with Hemo-De Xylene Substitute  in a series  of 15\u2009min washes. Then the samples were incubated in a 1:1 Hemo-De:Paraffin solution at 65 \u00b0C for an hour before overnight incubation in 100% paraffin at 65 \u00b0C. Samples were then embedded in freshly melted paraffin. Paraffin sectioning was performed using Thermo HM355S automatic microtome to collect 5 \u03bcm sections.For AFOG staining, 5\u03bcm paraffin sections were deparaffinized with Hemo-De and re-hydrated through an ethanol series  diluted with water. Sections were then fixed in 4% PFA for 5\u2009min at RT, then washed 3x for 5\u2009min with 1xPBS and then washed 1x for 5\u2009min in water. Following washes, samples were incubated in Bouin\u2019s solution  that was preheated to 60 \u00b0C for 2-hours and cooled to room temperature for another hour. Slides were then washed 5-6x for 5\u2009min with water and then rinsed in 1% Phosphomolybdic acid  for 10\u2009min and rinsed in water for 5\u2009min. Slides were then incubated in AFOG staining solution  for 7\u2009min at RT and then rinsed 1x for 2\u2009min with water. Samples were dehydrated with a series of ethanol dilutions  and 1x in Hemo-De for 5\u2009min before mounted with Cytoseal .For Toluidine Blue/Fast Green staining, zebrafish tissue was fixed and paraffin-embedded according to the aforementioned paraffin embedding protocol. Paraffin blocks were sectioned using HM 355\u2009S automated microtome and MX35 microtome blades at 5\u03bcm. Sections were floated in DEPC-treated water  in a waterbath and collected onto Leica adhesive slides, then allowed to air dry at room temperature at least overnight. Slides were then processed for toluidine blue staining with the following procedure: two 5-minute washes in xylene substitute Hemo-De, two 1-minute washes in 100% ethanol; serial washes  in EtOH/H2O for 1\u2009minute per wash; and three 1-minute washes in tap water. Slides were then placed in 0.04% toluidine blue solution  for 10\u2009minutes, followed by three 1-minute washes in tap water. Slides were then placed in 0.1% fast green solution  for 3\u2009minutes, followed by three 1-minute washes in tap water. Stained slides were then given two 1-minute washes in 100% isopropanol and two 1-minute washes in Hemo-De. Lastly, coverslips were mounted on the slides using CytoSeal and allowed to air dry for 30\u2009minutes to overnight.56) for use with zebrafish. Whole adult zebrafish were fixed overnight in the dark at 4\u2009\u00b0C in 4% PFA. Tissue was then washed twice in PBS for at least 30\u2009minutes per wash and heads were separated from the trunk of the body. At a ratio of 5 zebrafish heads per 50\u2009mL of solution, tissue was incubated in the dark at 37\u2009\u00b0C in CUBIC-1 solution with gentle rocking for at least 3 days. Tissue was then transferred to new CUBIC-1 to incubate until tissue was transparent and iridophores were cleared (2-4 days). Tissue was then washed twice in PBS for at least 30\u2009minutes per wash, then immersed in 20% sucrose in PBS at room temperature until tissue was no longer floating in solution and once again opaque, 2\u2009hours to overnight. Lastly, the tissue was immersed in CUBIC-2 solution and incubated in the dark at room temperature with gentle rocking for at least 1 day before imaging. Fluorescent microscopy images were captured using a Leica SP8 confocal microscope and processed in Fiji. CUBIC-1 was prepared as a mixture of 25\u2009wt% urea, 25\u2009wt% N,N,N\u2019,N\u2019-tetrakis(2-hydroxypropyl) ethylenediamine  and 15\u2009wt% Triton X-100. To minimize water evaporation, solution was prepared with heat not exceeding 100\u2009\u00b0C and inside bottles with caps just screwed on. Deionized water was heated prior to the addition of urea. Once urea was dissolved and the solution was no longer cold, N,N,N\u2019,N\u2019-Tetrakis(2-hydroxypropyl)ethylenediamine was mixed until homogenous. Solution was removed from heat and allowed to cool to room temperature, then Triton-X-100 was added with gentle stirring to minimize the production of bubbles. CUBIC-1 solution was stored in the dark at room temperature and used within 1 week.CUBIC tissue clearing protocol was adapted from , and 0.1% (v/v) Triton X-100. To minimize water evaporation, solution was prepared with heat not exceeding 100\u2009\u00b0C and inside bottles with caps just screwed on. Deionized water was heated prior to the addition of urea. Once urea was dissolved and the solution was no longer cold, 2,2\u2032,2\u2032\u2032-Nitrilotriethanol was mixed until homogenous. Sucrose was mixed with continued heat until the solution was clear with a pale yellow tint. Solution was removed from heat and allowed to cool to room temperature, then Triton-X-100 was added with gentle stirring to minimize the production of bubbles. CUBIC-2 solution was stored in the dark at room temperature and used within 1 week.We collected 5\u03bcm formalin-fixed paraffin-embedded (FFPE) tissue sections. For immunofluorescence, sections were deparaffinized with Hemo-De and rehydrated through an ethanol series, then subjected to antigen retrieval in sodium citrate buffer, blocked with Agilent Dako protein block (cat. X090930-2), and incubated with primary antibody   ,  overnight at 4\u2009\u00b0C, washed, and incubated with secondary antibody . TUNEL staining was performed as per manufacturer\u2019s instructions using the Apoptag Fluorescein In Situ Apoptosis Detection Kit . Slides were mounted and counterstained with Fluoromount-G\u2009+\u2009DAPI . Single-molecule fluorescence in situ hybridization was performed on 5\u03bcm FFPE sections according to manufacturer guidelines using RNAScope Multiplex Fluorescent Reagent Kit v2 Assay (ACD Bio). Slides were treated with 1x Target Retrieval Reagent for 4\u2009min in a steamer. RNAScope probes used were Dr-acta2 (508581-C4), Dr-col12a1a (556481-C2), DsRed-C2 (481361-C2), Dr-fn1a (1097911-C1), Dr-scxa (564451), Dr-lgmn (1003381-C2), Dr-mpeg1.1 (536171-C3), and Dr-thbs4a (812151) from ACD Bio. Opal 520, 570, and 690 fluorophores were used to visualize expression . Sections were counterstained and mounted with Fluoromount-G\u2009+\u2009DAPI.thbs4a_p1:CreERT2 zebrafish lines were converted with three 5\u2009\u00b5M (Z)-4-Hydroxytamoxifen, \u226598% Z isomer  overnight treatments in the dark. Following drug treatment, adults were screened for conversion using Leica M165FC stereo microscope and then washed 2 \u00d71\u2009hour in system water before being re-housed on-system.Adult n\u2009=\u200922 joints per 1 dplt and 3 dplt time points), sham surgery (n\u2009=\u200922 joints per 1 d and 3 d time point) was performed on size-matched Sox10Cre;actb2:loxP-BFP-loxP-DsRed fish at 3-4 mpf. The jaw joint region of each fish and uninjured control (25 joints) was microdissected and incubated in protease solution  with mechanical dissociation by nutation and agitation with a P1000 pipette every 5\u2009min for 1\u2009h at 28 \u00b0C. Cells were FACS sorted and immediately processed to create single-cell RNA Sequencing libraries using the Chromium Single Cell 3\u2032 Library & Gel Bead Kit v2 (10X Genomics), according to the manufacturer\u2019s recommendations. To determine cell count, libraries were sequenced at the CHLA Center for Personalized Medicine Genomics Core using a MiSeq Nano v2  with 26\u2009bp sequencing for Read1, 8\u2009bp sequencing for Index1, and 120\u2009bp sequencing for Read2. Libraries were pooled for even coverage per cell and were sequenced at the CHLA Center for Personalized Medicine Molecular Genomics Core on the NextSeq 550  with 26\u2009bp sequencing for Read1, 8\u2009bp sequencing for Index1, and 120\u2009bp sequencing for Read2. All samples were sequenced to an average read depth of greater than 130,000 reads per cell. CellRanger v3.0.0 (10X Genomics) was used with default parameters for alignment to GRCz11 to generate gene-by-cell count matrices.Ligament transection (p\u2009<\u20090.05). Principal components 1-68 were used for K-nearest-neighbor graph construction (KNN). Clustering was performed at low resolution (r\u2009=\u20090.02) to identify broad superclusters for downstream analysis . Principal components 1-38 were used for KNN and graphed using UMAP-non-linear dimensional reduction before clustering at r\u2009=\u20090.95. Cluster 13, which showed uncharacteristically high unique feature counts and markers for epithelial cells , were excluded as a likely doublet cluster. Markers for each cluster were identified with function FindAllMarkers using a Wilcoxon Rank Sum test for features expressed in at least 10% of the cluster and a minimum of 0.2 log-fold-change higher expression than the other cells in the neural crest-derived superclusters.Analysis of scRNAseq data from neural crest-derived cells were performed by subsetting and merging the superclusters containing cells from the DsRed-enriched samples Fig. . Using tAnalysis of scRNAseq data from immune cells were performed by subsetting and merging the superclusters Immune 1 and Immune 2 Fig. . Using tmpeg1.1:eGFP+ macrophages and PCNA+ cells were generated from z-stacks obtained from 63x confocal imaging using the LASX-3D software v3.5.7.23225.Images of histological sections were captured using a Keyence BZ-X810 microscope and software, then processed in Fiji. Live imaging was performed using a Leica M165FC stereo microscope with Leica K5 sCMOS camera and LASX software v3.7.4.23463. Fluorescence imaging was performed using a Leica SP8 confocal microscope with LASX software v3.5.7.23225. The 3-D projections of lgmn mutant PCNA staining and quantification were performed concurrently and wild type quantification is shown both in Fig. fn1a expression levels from smFISH images. Ligament domains and mesenchyme domains were drawn based on regional nuclear morphology in the tissue section. To calculate the corrected total fluorescence, an ROI was drawn to measure the integrated density for each channel using the same ROI for each channel. To account for tissue autofluorescence and background signal, 3 background regions of each image were measured, and the average mean gray value was calculated. To calculate the corrected total fluorescence (CTF), we used the following formula CTF = Integrated Density-(area of ROI*background mean). ImageJ was used on smFISH images to quantify the proportion of mpeg1.1+ cells that were also positive for fn1a and/or lgmn that surrounded the ligament stub by a blinded observer. The ROI for quantification was drawn to include 2-3 cell layers most proximal to the ligament stub excluding skin cells. ImageJ was used to quantify the number of Mpx+ cells by a blinded observer. The region of interest for quantification of Mpx+ cells was drawn to include the entire injury area.Proliferation was quantified as a percentage PCNA+ cells of total DAPI+ cells in a region of interest (ROI) in the uninjured ligament domain, ligament stub domain, or regenerative mesenchyme domain and scored by an observer blinded to treatment and/or genotype. Wild type and lgmn mutant proliferation, TUNEL, and fn1a transcript expression were each performed by 2-way ANOVA with \u0160\u00edd\u00e1k\u2019s multiple comparisons test. Comparison of wild type to lgmn mutant Mpx+/ROI was performed by 2-way ANOVA with Tukey\u2019s multiple comparisons test. Comparison of wild type and lgmn mutant macrophage expression of fn1a and/or lgmn, and mpeg1.1:eGFP+ cell abundance were performed with Student\u2019s T-test.All statistical analysis was carried out using GraphPad Prism 9. Proliferation in wild type animals was performed by ordinary 1-way ANOVA with Tukey\u2019s multiple comparison test. Comparison of wild type to Further information on research design is available in the Supplementary InformationSupplementary Table 1Supplementary Table 2Supplementary Table 3Reporting SummarySupplementary Video 1Supplementary Video 2Supplementary Video 3Supplementary Video 4"}
{"text": "This study aimed to investigate the interaction of the healthy eating index (HEI) and the dietary approach to stop hypertension (DASH) diet scores with FTO polymorphisms in relation to change in obesity traits. A total of 4480 subjects aged\u2009\u2265\u200918 years were selected from participants of the Tehran lipid and glucose study and followed-up 3 years. Selected polymorphisms  were genotyped and genetic risk score (GRS) was computed. HEI and DASH scores were computed based on dietary data. Changes in body mass index (BMI), waist circumference (WC), waist to hip ratio (WHR) and visceral adiposity index (VAI) were measured. Higher adherence to both DASH and HEI scores were increased with higher ages. Individuals with high GRS had a lower change in BMI when they had higher adherence to HEI, compared to subjects with lower HEI score (P trend\u2009=\u20090.01). Change in WC in participants in the fourth quartile of HEI score in minor allele carriers of FTO variants was lower compared to the first quartile; conversely, higher adherence to the DASH score by this genotypic group was related to increase in WC. No significant interaction was seen between FTO polymorphisms and both diet scores regarding changes in any of obesity traits. In conclusion, in individuals with high GRS higher adherence to HEI score was associated with lower change in BMI and WC, while higher adherence to DASH diet was associated with higher change in WC, compared to individuals with lower adherence to both scores.The online version contains supplementary material available at 10.1186/s13104-023-06463-3. Obesity is a condition in which excess fat accumulates in the body and could adversely affect one\u2019s health . AccordiMainstream medicine views obesity as a result of chronic energy imbalance. In recent decades, changes in dietary and physical activity patterns have contributed to the increased rate of obesity , affirmiAdvances in genetic technology have helped us to identify several common genetic variants associated with obesity. The most commonly implicated gene is fat mass and obesity associated gene (FTO) . There aOf lifestyle factors affecting body composition, diet is of utmost importance. Dietary approaches to stop hypertension (DASH) which is advised for preventing and treating high blood pressure, has been attributed to lower risk of cardiovascular diseases (CVD), cancer, insulin resistance, and blood lipid markers . HealthySubjects of this cohort study were chosen from participants of the Tehran Lipid and Glucose Study (TLGS), a population-based ongoing study performed to determine risk factors for non-communicable diseases in a group of residents of District 13 of Tehran, the capital of Iran. The first survey was done from 1999 to 2001 on 15,005 individuals aged\u2009\u2265\u20093 years, using the multistage stratified cluster random sampling technique, and follow-up surveys were performed every 3 years; 2002\u20132005 (survey 2), 2005\u20132008 (survey 3), 2008\u20132011(survey 4), and 2012\u20132015 (survey 5) to find out recently non-communicable diseases .Of 12,823 individuals attending the fourth phase of the TLGS (2008\u20132011) 8843 adults were \u2265\u200918 years. Subjects were removed due to anthropometric and nutritional missing data (n\u2009=\u20091961), so the data of 6882 subjects entered in this study as the baseline population and were followed-up to the survey 5 (2011\u20132014). Exclusion criteria were in these ways; subjects who had not DNA samples or lacking DNA purification in the range of 1.7\u2009<\u2009A260/A280\u2009<\u20092 or incomplete follow up data (n\u2009=\u20091600), pregnant (n\u2009=\u200980) or lactating women (n\u2009=\u200985), those with under- or over-reporting of energy intake  (n\u2009=\u2009637). Overall, data provided by 4480 subjects were entered in this study.A written informed consent form was signed by all participants before entering the study. The ethics committee of the Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences accepted the study protocol. All methods were carried out according to their relevant regulations and guidelines.A valid and reliable 168-item semi-quantitative food frequency questionnaire (FFQ) was applied for dietary assessment . SkilledThe DASH diet primarily developed to prevent hypertension. The DASH index is a posteriori-dietary pattern which calculates diet quality score. The index score (ranging 8\u201340) was based on eight food groups or nutrients including fruit, vegetable, nuts and legumes, low-fat dairy products, whole grains, sodium, sweetened beverages, red and processed meats intakes. Individual dietary food groups were calculated per 1000\u00a0kcal intake for each item, and then were categorized into quintiles. Subjects were given a score of 1 to 5 for each item. Subjects in the highest quintile intake of sodium, red and processed meat, and sweetened beverage were given a score of 0, and those in the lowest quintile of these food items were given a score of five. For fruit, vegetable, whole grain, low fat dairy, nuts and legumes, those in the highest quintile was given a score of 5 .The HEI is based on key recommendations of the 2015\u20132020 dietary guidelines for Americans which is composed of 13 dietary integrals; nine of them are adequacy components, including total fruits, whole fruits, total vegetables, greens and beans, whole grains, dairy, total protein foods, seafood and plant proteins, and fatty acids. Four of them are moderation components (those that should be limited) including refined grain, sodium, added sugars, and saturated fats. The HEI score is depend on density, so the amount per 1000\u00a0kcal of food groups and ratio of fatty acids was calculated. Recommendations are in the range of 1200\u20132400\u00a0kcal. Six components of adequacy components, including total fruit, whole fruit, total vegetables, greens and beans, total protein foods, seafood and plant proteins, each acquired a score of 0 and 5 for the lowest and highest intake, respectively. The other three adequacy components  were scored from 0 to 10 for the lowest and highest intake, respectively. The four moderation components including refined grains, sodium, added sugars, and saturated fats (SFA), gave a score of 0 and 10, respectively for the highest and lowest intakes. The score of intermediate intakes (between the minimum and maximum) were estimated. The sum of 13 integral scores was computed for a total HEI score ranging from 0 to 100. Individuals with a higher total HEI score had greater adherence to dietary guideline recommendations [Trained technician measured participants\u2019 body weight with minimal clothing using a calibrated digital scale  and rounded to the nearest 0.1\u00a0kg. They measured heights (cm) by a stadiometer  and rounded to the nearest 0.5\u00a0cm, while the subjects standing without shoes in a normal position. Waist circumference (WC) was measured over light clothing, without exerting any pressure on the umbilicus using an un-stretched tape meter. Hip circumference was assessed at the level of maximal protrusion of the gluteal muscles. By dividing WC (cm) to hip circumference (cm), the waist to hip ratio (WHR) was calculated.VAI was estimated by the following formulas for men and women separately .\\documenAnthropometric changes were considered as the outcomes or dependent variables in this study. BMI change was calculated by subtracting the BMIs obtained at the following survey from its corresponding at the baseline. The increase or decrease in BMI was determined if the BMI change was >\u20090 or \u2264\u20090 respectively. WHR, WC, and VAI change was computed using the same principle.Trained interviewer questioned physical activity with the Persian-translated modifiable activity questionnaire (MAQ). MAQ has high reliability and moderate validity. Data on the time and frequency of light, moderate, hard and very hard intensity typical activities over the last year were collected. The level of physical activity levels were estimated based on metabolic equivalent (MET)-hours/week (MET/h/week) .\u2212\u20097. Six FTO single nucleotide polymorphisms (SNPs) were selected which were related to both dietary patterns and obesity phenotypes, including rs1421085, rs1121980, rs17817449, rs8050136, rs9939973, and rs3751812. There were a strong correlation between rs8050136, rs1421085, and rs1121980 (r2\u2009>\u20090.8) with the other three SNPs in our analyses, so we applied these SNPs in our study. Also, there were a moderate relationship (r2\u2009<\u20090.7) between these three SNPs [The region of the FTO gene was evaluated based on the Phenotype-Genotype Integrator and the authenticated catalog of published genome-wide related studies. FTO SNPs were chosen based on the available data, minor allele frequency\u2009>\u20090.2, and P values\u2009<\u200910ree SNPs .Genomic DNA was derived from non-coagulated whole blood samples, using a standard proteinase K, salting-out method. The evaluation of DNA quality was done by a Thermo Scientific NanoDrop 1000 Spectrophotometer. DNA purification outside the range of 1.7\u2009<\u2009A260/A280\u2009<\u20092 were excluded due to low quality. Extracted DNAs were aliquoted into 1.5-ml tubes and kept in ultra-freezers at -80\u00a0C, for preservation. HumanOmniExpress-24-v1-0 bead chips, containing 649,932 SNP loci was used to genotype the portions of DNA samples according to the manufacturer\u2019s qualifications , an average mean distance of 4\u00a0kb at the deCODE genetics company  was regarded. PLINK program (V 1.07) and R statistic (V 3.2) were used to evaluate the quality, with a total genotyping rate of 0.9774. Finally, genotyping data of FTO SNPs  were analyzed [The weighted method was applied for GRS calculation based on 3 selected SNPs. Based on the existence of risk alleles , the coefficients of 0, 1, and 2 were allocated to each SNP. The weight of each SNP was obtained from the logistic regression analysis performed on the study population. GRS score is calculated by multiplying the coefficient of its risk alleles with the weight of each SNP.GRS = (OR1 \u00d7 SNP1\u2009+\u2009OR2 \u00d7 SNP2\u2009+\u2009OR3 \u00d7 SNP3) \u00d7 (n/sum of the ORs).OR is the odds ratio of each individual SNP on BMI. The logistic regression analysis was used to determine coefficients for the standardized weighted GRS in our population. The coefficients for risk alleles of rs1121980, rs1421085 and rs8050136 were respectively 0.21, 0.23, 0.24. Three SNPs (dominant model) were considered as independent variables and obesity (BMI\u2009\u2265\u200930 and BMI\u2009<\u200930) as dependent variable .Data were analyzed using SPSS version 21, and the statistical significance was considered as P\u2009<\u20090.05. To evaluate the qualitative and quantitative variables across quartiles of DASH and HEI scores, Chi-square and ANOVA tests were used respectively. The Pearson\u2019s Chi-square test was applied to test The Hardy-Weinberg equilibrium.The interaction of diet quality scores with FTO gene variants (dominant model) or GRS concerning WHR, WC, VAI, and BMI alteration was estimated using a general linear model (ANCOVA). Participants were categorized into 8 groups based on three SNP polymorphisms (dominant model) and quartiles of dietary scores (HEI and DASH) to estimate mean\u2009\u00b1\u2009SEM changes of obesity phenotypes. Participants were categorized into two groups based on the median of GRS (>\u20096.81 and \u2264\u20096.81). General linear models were performed to estimate the interactions of GRS with quartiles of DASH and HEI score concerning changes of WHR, WC, VAI, and BMI.The potential confounders, including smoking status , age, gender, physical activity , education levels (>\u200914 and \u2264\u200914 years), and energy intake were taken into account in all models.Baseline characteristics of participants across quartiles of HEI and DASH scores are presented in Table\u00a0There was no statistically significant deviation from the Hardy\u2013Weinberg equilibrium for the three polymorphisms. The median of GRS among participants was 6.81.Interactions of HEI and DASH scores by FTO SNPs  and GRS concerning changes in BMI and WHR were described in Table\u00a0The interaction between HEI and DASH scores by FTO SNPs  and GRS concerning changes in VAI and WC were shown in Table\u00a0In the present study, no significant interactions between DASH and HEI scores and genetic predisposition in relation to changes in obesity phenotypes including BMI, WC, WHR, or VAI were found. To the best of our knowledge, few studies have investigated the interactions between HEI and DASH diets and multiple FTO genetic variants in relation to adiposity features in a population. Moreover, participants with higher GRS and minor allele carriers of FTO SNPs 1,121,980 had a lower increase in BMI and WC in the highest quartile of HEI score, compared to the lowest quartile. Adherence to the DASH diet was also associated with higher increase in WHR and WC in participants with high GRS and minor allele carriers of FTO SNPs.Our findings are important for public health because the SNPs of FTO gene are common in our population and a high percentage of people carry these risk alleles. Moreover, Previous studies more emphasized on the interaction of certain food groups or nutrients with genetic predisposition in relation to obesity. The human diet contains numerous chemical compounds which make it difficult to investigate their separate effects on diseases; therefore, determining posteriori or priori dietary patterns and their association with obesity has been recommended. Identifying the best posteriori dietary pattern which modifies the association of FTO polymorphisms with obesity can help people adhere preventive recommendations especially in individuals with greater genetic susceptibility to obesity.FTO is identified as a gene of interest for obesity and has been subject to many investigations, most of which confirming that there is a notable association between FTO polymorphism and obesity traits , 27. StuPrevious studies showed that SIRT1 potentially prevented excessive accumulation of fat in adipose tissue. The interesting point is that recent studies have shown that sirtuins exert their role in energy metabolism in response to nutrient signals. Giving resveratrol to rats fed a high-fat diet protected them from high-fat-induced obesity, and this protective effect was due to increased activation of sirtuins by resveratrol , 37. TheInvestigating a whole dietary pattern could provide a better insight into the progression of a certain condition, compared to evaluating the effects of a single nutrient or food . It is wIn the current study, lower adherence to HEI in minor allele carriers of FTO variants led to a higher increase in BMI and WC. Moreover, individuals in the highest quartile of the DASH diet had a lower increase in WHR and WC compared to the individuals in the lowest quartile. However, no significant interaction was seen between FTO polymorphisms and DASH and HEI diets regarding changes in obesity indices. Recent studies regarding the gene-diet interaction reported controversial findings; in line with our results, a recent meta-analysis did not detect any interactions between protein intake and genetic predisposition to obesity on BMI, WC, or WHR . LivingsOn the other hand, there are a number of studies in which significant diet-gene interplay had been discovered. A recent study reported a notable interaction between GRS and intakes of energy, protein, total fat, SFA, poly-unsaturated fat (PUFA), and carbohydrate on BMI, body fat mass, and WC . A signiResults of the present study on DASH score and WC/ WHR in individuals with minor allele of FTO variants are opposite of the reports of previous studies, which suggested that high adherence to DASH diet is associated with reduction in the risk of abdominal obesity . This coDifferent findings might also be explained by the different definition of the DASH diet among studies. For example, in our study, nuts and legumes were in the same group and they are scored together. Also DASH dietary pattern depends on energy intake reduction to exert protective effects on abdominal obesity management and if this reduction in caloric intake does not occur, abdominal obesity may increase, so DASH score was not a good choice for weight management in minor allele  carriers of rs1121980, rs1421085 and rs8050136.The strengths of our study include its prospective design and utilizing a pre-defined dietary pattern analysis to better investigate the effect of overall dietary composition. Detailed information on physical activity, BMI, smoking status allowed extensive adjustment for obesity risk factors.There are some limitations that should be noted; our study population was highly homogenous as the subjects reside roughly in the same part of the town. These findings should be determined in other race and ethnicities. Moreover, there might be other potential confounders like economical influence and other social factors that could not be measured or adjusted. Our study included only three SNPs of the FTO gene whereas there are certainly more polymorphisms of FTO and other genes like MC4R, OLFM4, TCF7L2, ADCY3, GNPDA2, MAP2K5, and NRXN3 to be investigated. Besides, despite FFQ benefits, is not a robust tool to measure an individual\u2019s exact dietary assessment. The major limitation of our study is calculating the DASH score, as the quintile levels of each food group was based on individuals\u2019 dietary intake, so the range of each food group intake in each quintile of our study may be inconsistent with other studies. Finally, in order to have more conclusive findings, further studies with longer follow-up period are warranted.Our study revealed that there was no notable interaction between adherence to DASH diet or HEI and genetic predisposition on the obesity indices. However, adherence to HEI and DASH diets modified the association between FTO genetic variations and obesity phenotypes. In minor allele  carriers of FTO polymorphisms, low change in BMI and WC were seen with high adherence to the HEI. Conversely, high adherence to the DASH diet by this genotypic group was related to increasing WC.Below is the link to the electronic supplementary material.Supplementary Table 1. STROBE-nut: An extension of the STROBE statement for nutritional epidemiology"}