Patients with physiologic disorders, such as hypoxemia or hypotension, are at high risk of peri-intubation cardiac arrest. Standardization improves emergency tracheal intubation safety, but no published reports describe initiatives to reduce the risk of cardiac arrest. This initiative aims to improve the care of children at risk of peri-intubation cardiac arrest in a pediatric emergency department (PED). We specifically aimed to increase the number of patients between those with peri-intubation cardiac arrest by 50%, from a baseline of 11–16, over 12-months.Our multidisciplinary team outlined a theory of improvement and designed interventions aimed at key drivers. The primary intervention was creating a PICU-ED Team (PET) and a checklist to guide the assessment and mitigation of risk for peri-intubation arrest and rapid consultation of the pediatric intensivists. The PET was iteratively refined, and we collected data by a video review of tracheal intubations.Fifty-one patients with risk factors for peri-intubation arrest underwent tracheal -intubation in the PED from January 2016 to March 2020: 14 with PET activation since PET go-live in April 2019. None of the 14 PET patients had a peri-intubation cardiac arrest. Ninety-three percent (13/14) of PET patients were intubated in the PED, and 78% (10/13) of these patients had the first intubation attempt completed by PED physicians .We successfully developed the PET to mitigate the risk of peri-intubation cardiac arrest without significantly reducing key procedural opportunities for the PED. Initial data are promising, but further refinement is needed. Anatomically difficult airways are known to be associated with adverse events.1 Patients can also have physiologically difficult airways, with physiologic derangements sufficiently severe to increase the risk of cardiovascular collapse during airway management.2 Emerging evidence suggests the physiologically difficult airway is also associated with increased risk of adverse events including cardiac arrest.3Peri-intubation cardiac arrest is the most feared complication of emergency tracheal intubation, but the incidence in children is incompletely described. Peri-intubation cardiac arrest accompanies approximately 1.7% of intubations in the pediatric intensive care unit (PICU) setting,5 Identifying these risk factors is necessary but insufficient to mitigate the risk of peri-intubation cardiac arrest. Since physiologically high-risk pediatric intubations are low-frequency events,6 a system-level improvement process is needed to decrease the risk of mortality. Some investigators have shown that quality improvement initiatives can reduce peri-intubation adverse events, specifically hypoxemia.9 The use of QI methodology in the PED to mitigate the risk of peri-intubation cardiac arrest has not been reported.Cardiac disease, hypoxemia, and hypotension have previously been identified as risk factors for peri-intubation cardiac arrest.10 While this rate is similar to published data for the PICU, due to issues identified in the case review and the high mortality associated with peri-intubation arrest, we felt it to be unacceptable. We embarked on improvement initiatives to eliminate this phenomenon.In 2018, an unfortunate case of peri-intubation cardiac arrest in our institution’s PED prompted further investigation. Before this case, previous studies at our institution identified an intubation-associated cardiac arrest rate of 1.8% (2/114) over 12 months.The long-term objective of this improvement initiative is to minimize the risk of peri-intubation cardiac arrest in our PED. We theorized that the infrequent nature of these patients was a critical barrier to pre-intubation recognition and mitigation. We also theorized that a standardized approach would facilitate the early identification of high-risk patients and mitigate the risk of arrest. We intend to describe the PICU-ED Team (PET) development, a novel response system designed to improve the care of patients at high-risk for peri-intubation cardiac arrest. Our primary aim is to decrease the frequency of peri-intubation cardiac arrests, as measured by a 50% increase in the number of high-risk patients between patients with peri-intubation arrest, over 12 months. Our secondary aim is to assess the impact of the intervention on post-intubation cardiac arrest at any time in PED care and monitor the discipline of the proceduralist and location of tracheal intubation (PED versus PICU) as balancing measures.We conducted this project in the resuscitation area [shock trauma suite (STS)] of a high-volume academic PED with an annual volume of 62,000 visits per year. The STS has approximately 4,500 patient encounters per year or 7% of the total patient volume. The parent institution is a level I trauma center, with approximately 85% of the regional pediatric admissions from a population base of more than 2,000,000 people.The institution trains approximately 4 pediatric emergency medicine (PEM) and 4 critical care (CC) fellows per year. Our institutional review board determined the project to be non-human subjects research and, therefore, did not require institutional review board review and approval.Approximately 100 patients per year are intubated in the PED. All tracheal intubations occur in the STS. Each of the 4 STS bays is equipped with ceiling-mounted digital video cameras and microphones that record continuously. Video recordings of patient encounters are available for review using a proprietary software program . Patients and families provide consent for video recording in the general PED consent to treat form.9 Multiple prior projects have demonstrated sustained improvement in the safety of tracheal intubation in our PED.11Since 2013, the PED has had a well-established process of both quality assurance through structured video review and a clinical pathway, including the use of rapid sequence intubation (RSI) checklist. Video-based studies have found the checklist, and other vital processes are performed for more than 90% of patients undergoing RSI.In August 2018, PEM and CC faculty physicians initiated a project to reduce the risk of peri-intubation cardiac arrest among high-risk patients in our PED. The improvement team consisted of 6 faculty (4 PEM and 2 CC), 2 fellows (1 PEM and 1 CC), and 3 nurses (2 ED and 1 CC).The improvement team first developed a theory of improvement based on extensive knowledge of our airway management system and additional video review of actual cases, including the index patient. We theorized the risk of peri-intubation cardiac arrest could be minimized by interventions that addressed the following key drivers: 1) improved situational awareness of higher-risk patients by the PED team; (2) thoughtful optimization of patient hemodynamic status before tracheal intubation; and (3) facilitation of efficient PICU arrival to the PED and effective communication between PED and PICU clinicians. The improvement team constructed a key driver diagram to make explicit the theory for improvement  hypotension for age, (2) clinical concern for cardiac dysfunction, (3) persistent hypoxemia (pulse oximetry < 90%) despite supplemental oxygenation or positive pressure, (4) severe metabolic acidosis (pH < 7.1), (5) post-return of spontaneous circulation, and (6) status asthmaticus.The improvement team determined situational awareness of risk factors was a key driver of peri-intubation cardiac arrest. To address this driver, the improvement team developed high-risk criteria that would trigger PET activation. The improvement team drafted an initial list of criteria based on existing literature;9 A prompt was added to the RSI checklist to assess for high-risk status.The improvement team designed a process checklist Fig.  that incTo promote effective communication with PICU, the CC members of our improvement team identified the critical aspects of a patient’s care they need to understand quickly upon arrival to the PED. Based on this input, we added the checklist prompts for communication to the PICU providers regarding the most recent blood pressure and venous blood gas results. To mitigate risk factors for cardiac arrest, we added checklist prompts that optimize hemodynamics and suggest cardiac arrest precaution measures . The improvement team believed high-risk patients have complex and varied pathology leading to their physiologic derangements. Patients may need different therapies to optimize hemodynamics before intubation, depending on the underlying pathology. Therefore, the checklist does not contain specific hemodynamic interventions. Instead, the checklist includes prompts to promote a thoughtful patient-specific discussion before intubation.The PET elements were iteratively tested and refined through a series of ten in situ simulations conducted over 6 months. In concert with our institution’s Center for Simulation and Research, simulation scenarios were designed to test the identification of high-risk patients and PET processes. Improvement team members facilitated each simulation, and recorded observations were discussed by the larger team to refine the process.We focused educational efforts on critical groups including, PEM and CC fellows, PED respiratory therapists, and PED and CC nurses through regular divisional meetings and emails. Debriefings following in situ simulations increased awareness of and reinforced the goals of the PET.PET-eligible patients were children presenting with a critical illness needing emergency tracheal intubation. We excluded critically injured (trauma) patients since the institutional trauma system includes PICU, trauma surgery, and anesthesiology team members. Additionally, we excluded children with known cardiac disease, as they are typically co-managed with cardiac intensivists. Finally, we excluded crash or no-medication intubations, as these patients are typically already in cardiac arrest.10 The lead improvement team nurse (M.F.) screened the database for eligible patients and PET activations. We reviewed all eligible RSI cases to determine if any PET criteria were present. If a likely missed eligible case was identified, an improvement team physician (P.D.) reviewed the corresponding patient video. Two members of the improvement team (P.D. and G.L.G.) performed structured video-based data collection for each PET activation. The improvement team then met monthly to review activation data and potentially missed cases, discussed issues with each activation, and made necessary revisions to the PET criteria/system. After each activation, the team emailed a feedback form to the care team members, requesting additional feedback on the PET process.We identified eligible patients and PET activations through our existing STS quality assurance program, including a database of all STS activations. The STS database is based on daily reports generated from our institution’s electronic health record, capturing patients undergoing tracheal intubation in the PED with high reliability.To establish a baseline for the frequency of peri-intubation arrest among patients meeting high-risk criteria, we queried the STS database for all patients meeting above inclusion and exclusion criteria for the 39 months from January 1, 2016 to March 31, 2019. We conducted an additional structured review of the electronic health record to collect data not available in the STS quality assurance database. We identified a subset of patients considered high-risk intubations if they met one or more previously defined high-risk PET criteria.14 The secondary outcome measure is a post-intubation cardiac arrest in the PED. Post-intubation cardiac arrest is defined as a cardiac arrest that occurred while the patient was still in the PED, regardless of time elapsed after intubation. This term includes all peri-intubation arrests (within 10 minutes of intubation) plus patients with cardiac arrest outside of that timeframe but while still in the PED.Our global aim is to improve the care provided to critically ill children needing emergency airway management by reducing the risk of peri-intubation cardiac arrest. The primary outcome measure, though rare, is a peri-intubation cardiac arrest in a patient meeting one or more high-risk criteria. Peri-intubation cardiac arrest is defined as cardiac arrest  within 10 minutes of tracheal intubation. This timeframe is consistent with definitions in the literature, which vary from 5 to 20 minutes.As process measures, we tracked both instances when a patient met PET criteria in which the team was not activated and those in which a team activation was initiated without any PET criteria being met. Balancing measures were (1) the discipline of the proceduralist and (2) the location of tracheal intubation (PED versus PICU). These balancing measures were chosen due to the concern that the PET’s implementation may have the undesired consequence of limiting opportunities for intubation by PED physicians.15 We used standard rules for interpreting a Shewhart chart to determine special cause variation indicating the association between PET development and significant changes in the process or outcomes.15We tabulated all activation data and generated standard descriptive statistics. All PET activations, any missed eligible activations, and key clinical features and outcomes of these cases were described. Annotated control charts were developed and updated regularly to monitor outcome measures. We used G charts to track these measures as high-risk intubations are in themselves rare; even more rare is the occurrence of either peri- or post-intubation cardiac arrest. G charts track the number of successful events between rare-event failures. When using G charts, data are skewed, and there is no lower control limit; thus, a theoretical median is used for the centerline, which is calculated by the equation 0.693 × mean.Fifty-one patients with risk factors for peri-intubation arrest underwent tracheal intubation in the PED from January 2016 to March 2020: 36 in the historical cohort and 15 since PET go-live in April 2019. Ninety-three percent (14/15) of high-risk patients intubated after go-live had PET activation  of PET-activation patients were successfully intubated in the PED. Seventy-eight percent (10/13) of patients intubated in the PED had a first intubation attempt by a PED physician . Otorhinolaryngology or anesthesiology physicians intubated the remaining 3 patients. The PICU intubation resulted from a transfer to the PICU for tracheal intubation to have extracorporeal membrane oxygenation therapy available if needed; PICU fellow successfully intubated this patient.Sixty-five percent (9/14) of PET-activation patients had a least 1 cardiac arrest precaution measure  in place before intubation.Peri-intubation cardiac arrest is a rare but potentially fatal complication of tracheal intubation. We recognized the need for a systematic approach to hemodynamic optimization for children with critical physiologic derangements. We established a multidisciplinary improvement team and a new clinical process to decrease the risk of peri-intubation cardiac arrest. Implementation of this process has resulted in 14 successful intubations of children at high-risk for peri-intubation cardiac arrest and has not displaced PED physicians as the primary proceduralists. We feel the critical reasons for this initiative’s success are the well-established institutional RSI process that provided a foundation to build upon, and the PET checklist which may have improved situational awareness and effective communication for high-risk patient-specific mitigation strategies. This novel process may be easily extrapolated to other settings with an established RSI process but does not include physiologic risk factors for peri-intubation cardiac arrest. Similarly, for setting starting an RSI process, incorporating physiologic risk factors into the RSI checklist may be beneficial to the most high-risk patients.8 demonstrated a 59% reduction in airway related cardiac arrest in pediatric patients undergoing airway management with anesthesia over 2.5 years. While this study demonstrates the potential for QI interventions to minimize patient risk, the clinical setting in which it takes place  is very different from emergency airway management in the PED. A PED based study conducted by Long et al,16 demonstrated an increase in first-pass success without hypoxemia or hypotension following the implementation of a QI bundle for emergent airway management. Our study differs in that we are specifically looking at a physiologically high-risk patient population and expanding on an already well-established RSI process.Similar quality improvement studies have demonstrated a reduction in intubation related adverse events or cardiac arrest in the pediatric population. Spaeth et alThis work occurred at a single large pediatric center with specialized resources that may limit generalizability. Our institutional culture, including a well-established RSI checklist and in situ simulation program, supported successful implementation but may pose challenges in other settings. Finally, the low frequency of both physiologically high-risk pediatric intubations and peri-intubation cardiac arrest limits our ability to evaluate changes in mortality and draw definitive conclusions regarding the effectiveness of our interventions. It also limits our ability to demonstrate sustainability over time. However, due to the high mortality associated with peri-intubation cardiac arrest, there is value in disseminating this ongoing work that promises to reduce mortality.We successfully developed a novel approach to mitigate the risk for peri-intubation cardiac arrest in a PED without significantly reducing key procedural opportunities for the PED team. The PET may have resulted in zero cardiac arrests during the study period, but further refinement and long-term data monitoring are needed.The authors have no financial interest to declare in relation to the content of this article.


Advances in machine learning and artificial intelligence have led to many promising solutions for challenging issues in agriculture. One of the remaining challenges is to develop practical applications, such as an automatic sorting system for after-ripening crops such as tomatoes, according to ripeness stages in the post-harvesting process. This paper proposes a novel method for detecting tomato ripeness by utilizing multiple streams of convolutional neural network (ConvNet) and their stochastic decision fusion (SDF) methodology. We have named the overall pipeline as SDF-ConvNets. The SDF-ConvNets can correctly detect the tomato ripeness by following consecutive phases: (1) an initial tomato ripeness detection for multi-view images based on the deep learning model, and (2) stochastic decision fusion of those initial results to obtain the final classification result. To train and validate the proposed method, we built a large-scale image dataset collected from a total of 2712 tomato samples according to five continuous ripeness stages. Five-fold cross-validation was used for a reliable evaluation of the performance of the proposed method. The experimental results indicate that the average accuracy for detecting the five ripeness stages of tomato samples reached 96%. In addition, we found that the proposed decision fusion phase contributed to the improvement of the accuracy of the tomato ripeness detection. The quality of tomatoes depends on appearance  and nutritional value . These properties are commonly related to ripeness . As tomaIn recent years, artificial intelligence technologies have become popular in the food and agriculture research field. In several recent studies related to food and agricultural production, machine learning and deep learning applications have shown great success. In the food domain, deep learning has shown promising performance in a variety of tasks, such as food recognition , caloriePrevious related works on fruit defect or grade detection in computer vision can be categorized into two types: approaches based on hand-crafted features and those using deeply learned features. Most existing studies belong to the former class ,15,16,17This paper aims to develop an accurate tomato ripeness detection method based on deep learning, called stochastic decision fusion of convolutional neural networks (SDF-ConvNets). The proposed method is expected to be applied in the form of a sorting system that classifies fruits according to those ripeness degrees in the post-harvest stage. Since the ripeness detection process is conducted by observing images from various viewpoints, excellent synergy can be expected if the sorting module can be designed based on a conveyor structure capable of rotating fruit objects and transporting them. To train and evaluate the model, we constructed a large-scale image dataset collected by our customized image acquisition system. Experiments for evaluating our system have shown superior performance to other methods. In summary, the key contributions of our work are: (a) developing a deep-learning-based robust and accurate ripe tomato detector, (b) increasing the accuracy of ripening stage classification using the stochastic decision fusion method, and (c) collecting a large-scale image dataset that captures the five ripening stages of tomatoes from different viewpoints.The tomato images used in this study were captured using a JAI 3CCD camera and a light source chamber, as shown in We designed the customized image acquisition system in conjunction with an annotation labeling software that can manually generate ground truth labels for training the proposed deep learning model at the same time as image acquisition. The ground truth labels consisted of the spatial information of tomatoes in the image space as well as the ripening stages. We selected tomatoes of the “Dafnis” variety for building a large-scale image dataset. These were classified into five stages according to the USDA color chart. The dataset was collected from a minimum of 500 samples for each ripeness stage to contain as many atypical features as possible, as shown in We concentrated on an approach for classifying the ripening stage of tomatoes, considering practical scenarios such as an automated sorting application in the post-harvesting process.We built a sequential process consisting of an initial tomato ripeness detection phase and a stochastic decision fusion phase . A standThe initial tomato ripeness detection task consisted of two main parts: localizing the spatial region of the target tomato sample and classifying those ripening stages from tomato images viewed from the stem-/flower-end. Even when the observed environment is constrained, classifying ripening stages is a difficult fine-grained problem, in which the variation between consecutive stages is low and the variation between tomatoes belonging to the same group is relatively high. Recently, deep-learning-based approaches have been used as a solution to this type of problem. The typical ConvNet architecture consists of two main parts: a set of convolutional layers that perform feature extraction, and classification layers. The frontal layers of the network mainly focus on obtaining deeper domain features of the input, and the extracted features are transferred to the classification layers to discriminate between classes, such as the ripening stages by using fully connected  or globaS × S grid cells, and B proposal regions are located on each cell. The detector generates an output tensor consisting of five elements, such as the spatial information and class score for each region. If there is a We applied a one-stage object detector based on YOLOv3 to resolve the tomato ripeness detection problem. The YOLOv3 starts with an assumption that the entire input image can be divided into Equation (3) defines the loss Finally, the sub-loss functions are defined in Equation (4).The weights applied to losses (1) and (2) were set as ectively . By optiWe tried to arrange the backbone network architecture by piling multiple residual modules with a simple shortcut connection . The ConWe trained the model with the hyperparameter configuration of the YOLOv3 . The optLearning rate: 0.01 ;Max. training iteration: 50,000;Size of mini-batch/subdivision: 32/8;Weight decay: 0.0005;Learning momentum: 0.9;Total number of convolution layers: 79;Scales of final feature map: 8, 16, 32.To accurately classify the final ripening stages of target tomato samples, we tried to apply two types of weighted-fusion-based approaches. The first was to assign equal weight to both ConvNet stream results. We assigned the equivalent scalar value as the weight for each stream, as shown in Equation (5):Second, we hypothesized that weighting the superior one among the streams would increase the accuracy of the final decision. In this paper, a multi-label confusion matrix For the element with The final ripening stage of the input tomato sample was computed as shown in Equation (7):® Core™ i7-4790K 4.00 GHz CPU, 32 GB of RAM, and an NVIDIA GeForce GTX Titan Xp GPU processor. We utilized the deep learning framework Darknet [c. Then, the per-class F1-score can be computed by Equation (9).The proposed SDF-ConvNets was verified with our tomato image dataset through experiments in this section. The experiments were conducted on a computer equipped with an Intel Darknet . Three mIn this paper, we used two decision fusion approaches based on stochastic metrics for the final decision on the tomato ripeness stage of the target sample. As a result of comparing the ripeness detection performance according to the proposed decision fusion method with the results in We also used the precision–recall (PR) curve to compare the SDF-ConvNets to other recent models, such as SVM  and YOLOThese experimental results demonstrate that the proposed SDF-ConvNets outperformed other methods. In this paper, we proposed an accurate tomato ripeness detection methodology called SDF-ConvNets. The overall ripeness detection pipeline consisted of two major steps: the initial tomato ripeness detection phase based on ConvNet streams, and the stochastic decision fusion phase to obtain a more precise ripeness classification result. Even if the initial ripeness classification fails for the stem-end or flower-end tomato image, the proposed decision fusion phase can compensate for the misclassified stage into the correct stage. To train, test, and verify the proposed method, a large-scale image dataset was collected and labeled. The scale of the tomato image dataset is larger than any dataset used in recent related works. The dataset consisted of 2166 tomato samples for training ConvNets and 546 tomato samples used to evaluate the SDF-ConvNets. The experimental results were obtained by averaging 5-fold cross-validation and evaluated in terms of the three statistical metrics  of the tomato ripeness detection task. The SDF-ConvNets successfully achieved accurate and fine-grained tomato ripeness detection compared with other deep-learning-based approaches. The F1-score of the tomato ripeness detection using the SDF-ConvNets was 96.5%. The proposed method was compared with the recent achievement of related ripeness detection tasks and its superiority was demonstrated.In future work, a follow-up study will be conducted to develop an integrated framework that can determine the appropriate harvest time and monitor the crop growth status by recognizing and estimating the ripening stage in real-time through the observation of tomatoes before harvest.


Reality monitoring is defined as the ability to distinguish internally self-generated information from externally-derived information. Functional imaging studies have consistently found that the medial prefrontal cortex (mPFC) is a key brain region subserving reality monitoring. This study aimed to determine a causal role for mPFC in reality monitoring using navigated repetitive transcranial magnetic stimulation (nrTMS). In a subject-blinded sham-controlled crossover design, healthy individuals received either active or sham nrTMS targeting mPFC. Active modulation of mPFC using nrTMS at a frequency of 10 Hz, significantly improved identification of both self-generated and externally-derived information during reality monitoring, when compared to sham or baseline. Targeted excitatory modulation of mPFC also improved positive mood, reduced negative mood, and increased overall alertness/arousal. These results establish optimal nrTMS dosing parameters that maximized tolerability/comfort and induced significant neuromodulatory effects in the mPFC target. Importantly, this is a proof-of-concept study that establishes the mPFC as a novel brain target that can be stimulated with nrTMS to causally impact both higher-order reality monitoring and mood. Reality monitoring is defined as the ability to distinguish internally self-generated information from externally-derived information  studies, deep brain stimulation and neuroimaging studies, all strongly suggest that mPFC is also a critical node for mediating mood state  to establish the causal role of a novel neural target in the mPFC on impacting higher-order reality monitoring and mood in healthy controls (HC).We and others have also demonstrated that the mPFC plays a critical function in mediating interactions between mood and higher-order cognition during reality-monitoring  nrTMS targeting mPFC to increase its activity  at the University of California San Francisco (UCSF) and all research was performed following IRB regulations at UCSF. Participants were evaluated by a clinical psychologist and completed questionnaires to meet the following inclusion criteria. Inclusion criteria for healthy participants were: no psychiatric/neurological disorders, including no personality disorders, no current or history of substance dependence or substance abuse, meets MRI criteria, good general physical health , age between 20 and 40 years, right-handed, and English as the first language. All participants  gave written informed consent and then completed the reality monitoring task and mood and alertness/arousal ratings at baseline . After bstove provided theThe  ___ ) or was externally-derived as it was provided by the experimenter . For each sentence, participants were told to pay attention to the underlined words and to vocalize the final word of each sentence. Participants then completed the reality-monitoring retrieval task where they were randomly presented with the noun pairs from the sentences , and had to identify with a button-press whether the second word was previously self-generated or externally-derived  at baseline, and also immediately before and after each nrTMS active and sham session. The visual analog scale is a 10 cm line with numbers marked from 0 to 9, but with labels only on 0  and 9 (“Vey high”), to minimize subject scale rating bias. The experimenter does not influence subjects’ self-reported ratings since subjects rated their mood and arousal levels on their own without any prompting from the experimenter. Mood and alertness/arousal ratings for each condition are shown in d-prime analyses for the reality monitoring task were conducted on overall accuracy in identification of word items by calculating the hit rate and the false alarm rate for self-generated and externally-presented items, then converting each measure to z-scores, and subtracting the false alarm rate from the hit rate to differentiate sensitivity during accurate performance from response bias. Self-generated and externally-derived accuracy was computed as a percentage of the total number of self-generated and externally-presented trials in each condition  for each subject, and then averaged across all subjects. Repeated-measures ANOVAs with follow-up pairwise comparisons were implemented to examine differences in reality monitoring performance and mood ratings between baseline, active nrTMS, and sham nrTMS conditions. Outliers were defined as values above/below two standard deviations from the mean. We did not find any outliers in the behavioral data. For repeated measures designs, effect sizes are computed using Cohen’s d as below:Signal detection theoretic r is the correlation between the two conditions. Mean accuracy in each condition is illustrated for overall d-prime accuracy, and for correctly identified self-generated and externally-derived information, averaged across all participants  20 Hz nrTMS for 2 s trains at 110% of their resting motor threshold (RMT) with an ITI of 28 s; (ii) 10 Hz nrTMS for 4 s trains at 110% of their RMT with an ITI of 26 s; (iii) 10 Hz nrTMS for 2 s trains at 120% of their RMT with an ITI of 28 s; and (iv) 10 Hz nrTMS for 2 s trains at 110% of their RMT with an ITI of 28 s . The firvia state-of-the-art Nexstim Navigated Brain Stimulation . This system integrates the TMS figure-8 coil with a software-based frameless stereotactic navigational system that allows for highly accurate cortical targeting that is individualized for each subject. Nexstim uses in-plane coil geometry to simulate the induced electric field and to make reliable predictions of electromagnetic field strength at the target location of neuronal activation  that were based on our prior functional localization of mPFC peak activity mediating reality monitoring  MRI to Head Registration. The nrTMS session began with aligning the MRI images to the subject’s head via the MRI to the Head Registration process. Using the bridge of the nose and the crus of the helix of the ear, the 3D-locations of the landmarks (visible on both the subject’s MRI and the head) were measured using a digitizing pen with an optical tracking system. The optical tracking system used two cameras to triangulate the location in 3D space of infrared reflectors attached to the coil and subject’s head.(II) Resting Hand Motor Threshold Determination. Once the co-registration was completed, subjects completed single-pulse TMS mapping of the hand motor region. The RMT was obtained by delivering single TMS pulses to the left motor cortex hand area. RMT was defined as the minimum stimulation intensity at which motor evoked potentials (thresholded at >50 microvolts) were observed 50% of the time from surface muscle recordings from the first dorsal interosseous muscle of the right hand in half of the trials.(III) nrTMS Session. The target region for the nrTMS stimulation was the mPFC, located using MNI coordinates  based on functional localization of mPFC peak activity mediating reality monitoring in HC, which promisingly also showed peak activity increase  in schizophrenia (SZ) patients after behavioral cognitive training interventions . Follow-up comparisons indicated that active nrTMS targeting of mPFC at 10 Hz significantly improved reality monitoring performance, indexed by increased d-prime scores for overall accuracy when compared to baseline (p = 0.007) and sham conditions  and sham conditions (Cohen’s d = 1.1). Medium to large effect sizes showing improvements in retrieval accuracy after active nrTMS targeting mPFC compared to baseline and sham were observed for both self-generated  and externally-derived information  and externally-derived information (p = 0.002). When compared to the sham condition, subjects also accurately identified significantly more self-generated (p = 0.04) and externally-derived information (p = 0.03). There were no significant differences in reality monitoring performance between the sham nrTMS and baseline conditions or an order effect in nrTMS condition assignment on reality monitoring performance , indicating that any changes resulting from the active TMS were not due to practice effects and did not result from subjects being un-blinded/aware of the study design. Indeed, before un-blinding at study completion, no participant was aware of the study design.A repeated-measures ANOVA showed a significant effect on overall reality monitoring accuracy  of 0.7 between reality monitoring scores at baseline and after sham nrTMS, showed high test-retest validity and reliability of the reality-monitoring assessments. A sample size of p = 0.03) and marginal improvements in a positive mood (p = 0.08) when compared to baseline . They also showed significant improvements in overall alertness/arousal levels after active nrTMS when compared to baseline (p = 0.05) and sham conditions (p = 0.046). Large effect sizes were found after active nrTMS targeting mPFC in positive and negative mood and arousal levels, compared to baseline  and compared to sham conditions . There were no significant differences in mood/arousal levels between sham and baseline conditions or an order effect in nrTMS condition assignment on mood/arousal levels .After active nrTMS of mPFC, subjects had significant reductions in their negative mood  on a scale of 0–9 . No seizures or other serious adverse events occurred in any participant. Only one subject experienced referred pain at the back of her head  after the first single nrTMS train for a 2 s duration. We ramped down the intensity after every single train in steps ; however, this subject still experienced pain even at 80% of RMT. This subject reported no adverse events or side effects beyond the duration of every single train of nrTMS but was eliminated from the study and further analyses.In the current subject-blinded sham-controlled crossover study, we found that the active high-frequency 10 Hz nrTMS targeting this functionally localized focal peak of mPFC  activityx, y, z co-ordinates = −16, 48, 6), which also showed peak increase after cognitive training interventions in psychosis  before and after nrTMS to investigate the neural plasticity of how cortical networks are reconfigured after nrTMS-induced mPFC modulation. Improvements in reality monitoring that are related to enhanced mPFC activity will confirm the necessary/causal role of mPFC in the effects of stimulation in these future studies.N = 10 requires an effect size of Cohen’s d = 0.75 to attain a significant treatment effect for repeated-measures designs, thus providing the go-decision criteria for the main findings of this study. Findings must be interpreted with caution for results that show effect sizes that are below a Cohen’s d of 0.75. Future studies will involve larger efficacy trials, in which we will test whether the same effects are observed with a larger sample size, including multiple repeated sessions per participant using the same protocol and assessments of multiple covariates.Despite having repeated-measures of 29 data points altogether at pre-and post-treatment, the main limitation is the small sample size. As is stated in our RFA MH-18-704 grant that supports this proof-of-concept study, studies during the R61 phase are required to focus on a preliminary evaluation of a treatment effect on manipulating a target, and are required to be pilot studies that should not be powered as strong tests of clinical efficacy, but should rather test the preliminary impact of target engagement on a clinical or cognitive outcome . To attain a power of 0.7 our sample size of In sum, this is a tightly-controlled study, with each subject completing assessments at three time points, that provides the first direct evidence to establish the mPFC as a novel neural target that can be modulated with nrTMS to causally impact both mood and higher-order reality monitoring (Subramaniam et al., All datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by UCSF IRB approval: 10-02932. The patients/participants provided their written informed consent to participate in this study.KS recruited all the subjects and designed the reality monitoring experiment, acquired, analyzed and interpreted all the data, and wrote and edited the manuscript. HK helped with subject recruitment and acquisition of the nrTMS data. LH provided advice on the acquisition of reality monitoring data. PT conducted the nrTMS sessions on all subjects, and edited the manuscript. SN edited the manuscript and provided advice on the overall acquisition, analyses, and interpretation of all the data.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.


The National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) established a harmonized method for large-scale clinical proteomic studies. SWATH-MS, an instance of data-independent acquisition (DIA) proteomic methods, is an alternate proteomic approach. In this study, we used SWATH-MS to analyze remnant peptides from the original retrospective TCGA samples generated for the CPTAC ovarian cancer proteogenomic study. The SWATH-MS results recapitulated the confident identification of differentially expressed proteins in enriched pathways associated with the robust Mesenchymal high-grade serous ovarian cancer subtype and the homologous recombination deficient tumors. Hence, SWATH/DIA-MS presents a promising complementary or orthogonal alternative to the CPTAC proteomic workflow, with the advantages of simpler and faster workflows and lower sample consumption, albeit with shallower proteome coverage. In summary, both analytical methods are suitable to characterize clinical samples, providing proteomic workflow alternatives for cancer researchers depending on the context-specific goals of the studies.  •SWATH-MS and iTRAQ-DDA are used to classify 103 high-grade serous ovarian cancer•SWATH-MS re-capitulates differentially expressed proteins in ovarian cancer subtypes•SWATH-MS is a robust proteomic approach for large-scale clinical proteomic studies Biological Sciences; Proteomics; Cancer Systems Biology Advances in sample preparation workflows, mass spectrometry instrumentation, and data processing software have positioned proteomics to provide comprehensive insights into complex biological processes at a level close to the underlying biochemical mechanisms. Indeed, it is currently possible to routinely quantify >10,000 proteins in human cell proteomes  and humam/z values were recorded in a survey scan are selected for fragmentation using a pre-determined set of rules  Clinical Proteomic Tumor Analysis Consortium (CPTAC) studies. CPTAC was formed to accelerate the understanding of the molecular basis of cancer through the application of large-scale proteogenomic analyses. Several hundred tumor tissue specimens from breast, ovarian, and colorectal cancer tissues previously analyzed by NCI's The Cancer Genome Atlas (TCGA) have also been characterized using proteomics, informed by genomics, resulting in the identification and quantification of proteins and phosphoproteins in cancer-associated cell signaling pathways and networks . These sof rules . DDA-basof rules .A relatively newer method termed data-independent acquisition (DIA) mass spectrometry has been gaining traction in large-scale proteomic studies . DIA masSaccharomyces cerevisiae as a model system when incorporating offline peptide fractionation into the LC-MS workflow  have been conducted . Basak eworkflow . In theiIn the current study, we utilized SWATH-MS to analyze peptides from 103 HGSOC tumors that were previously analyzed by iTRAQ DDA as part of the NCI CPTAC study . The iTRTaken together, compared with the DDA analysis of HGSOC using iTRAQ labeling and 2D fractionation, SWATH-MS is a robust proteomic method that can be used to re-capitulate common differentially expressed proteins in enriched pathways associated with the HGSOC Mesenchymal subtype . The SWALudwig et al. provide an in-depth overview of the advantages and limitations of SWATH-MS in comparison with DDA . There aProteomic measurements of clinically annotated HGSOC previously characterized by TCGA  were conProtein was extracted from each tumor specimen followed by enzymatic digestion with trypsin. For the iTRAQ DDA workflow, the resulting peptides were labeled with 4-plex iTRAQ reagents followed by combination into analysis sets comprising the peptides from three tumors, each labeled with a distinct iTRAQ tag, and an iTRAQ-labeled reference pool comprising the peptides from most of the tumors. Each analysis set was subjected to offline fractionation into 24 concatenated fractions, and the fractions from each analytical set were sequentially analyzed using a DDA method on an LTQ-Orbitrap Velos mass spectrometer. An unfractionated aliquot of each analytical set was also analyzed by DDA-MS without fractionation. In comparison, the SWATH-MS workflow did not require stable isotope labeling. However, for the purpose of generating a spectral library to facilitate the targeted protein identification, each sample from 103 tumors was pooled, fractionated to 48 fractions and subjected to DDA analysis, which is an optional step for this workflow. We note that, although we chose in this study to create a sample-specific reference library, several publicly available reference libraries exist . The impThe CV of SWATH-MS data was assessed using two QC approaches . For the2 intensity ratios compared with the reference iTRAQ channel of each iTRAQ set for the iTRAQ data, and relative protein abundances in the SWATH-MS dataset were determined based on the log2 intensity of each protein as represented by the mean intensity of the constituent peptide ratios (peptide intensity divided by the mean peptide intensity)  A. The mefication   and the differences in protein quantification wherein multiple peptides from the same protein were used to quantify each protein in the dataset. Additionally, for the majority of the quantified proteins, different constituent peptides and different numbers of unique peptides were identified in the iTRAQ DDA workflow compared with SWATH-MS. A direct comparison of the CV of each method was not possible because replicate analyses of the samples were not conducted; therefore, a CV-like score was calculated, which represents the biological variability of the sibling peptides from the quantified proteins. Because aliquots of identical samples were used for this analysis, the displayed variation reflects technical differences in the measurements and data handling of both methods. The median variability of the SWATH-MS data was higher than the iTRAQ DDA data . As shown in the violin plots, the variability of the iTRAQ DDA data has a wider distribution than that of the SWATH-MS data  with the ECM organization Reactome pathway were assigned to the Mesenchymal subtype. A significantly positive correlation  was also observed among the proteins assigned to the gene expression and translation ontology.The correlation of the WGCNA-derived protein modules with the proteomic subtypes derived from the iTRAQ DDA and SWATH-MS datasets is shown in Based on the observation highlighted by the dashed box in Because the protein and mRNA components of the same ovarian tumors were analyzed, we were able to compare the robustness of the Mesenchymal subtype across different analytical methods. We used the Adjusted Rand Index (ARI) to quantitatively assess the agreement among the classification of the tumors based on the proteomic analyses (iTRAQ DDA and SWATH-MS) B. ARI vaWe further examined the unexpected lack of similarity when comparing the subset of the iTRAQ data to the full dataset by adopting a systematic bootstrapping approach. Bootstrapping was used to randomly select a fraction of either the 103 samples analyzed by iTRAQ DDA and SWATH-MS A and 4B The sensitivity of molecular subtype clustering to the number of samples included in the analysis has been shown previously ; howeverNevertheless, in our SWATH-MS analysis, we were able to extract one robust HGSOC subtype, which was characterized by the increased relative abundance of proteins with extracellular matrix functions A. The ro2-fold changes of these proteins in the iTRAQ DDA and SWATH-MS datasets were determined as well as the associated p value  inhibitor treatment . Homolog2-fold changes obtained from the group comparison approach were mapped on this network, and these signals were propagated over the network to identify signals accumulating within a subnetwork. The top 5% (221 proteins) of the resulting positive and negative scores were used for further investigation and functional enrichment.A group comparison of HRD versus non-HRD patients using mapDIA  revealedin vitro studies using yeast two-hybrid screening, affinity purification followed by western blotting, and co-immunoprecipitation purification followed by LC-MS/MS (in vitro studies indicating the interaction of these proteins with PARP and BRCA1 support the well-known role of these proteins in the control of homologous recombination during DNA repair.Further verifying that proteins with roles in histone acetylation differentiate HRD from non-HRD tumors , DNA repLC-MS/MS . The ideIn summary, the analysis of the SWATH data was able to provide not only confirmation of some previously identified signatures from the iTRAQ DDA data in a larger untargeted network approach, but also important mechanistic insights into HRD-related pathways as discovered by iTRAQ labeling and LC-MS/MS using DDA .HGSOC is among the cancer types that have been proteogenomically characterized by CPTAC. The large-scale proteome analytical workflow employed by CPTAC uses isobaric tagging, offline peptide fractionation, and LC-MS/MS. This workflow is considered a reference method for the comparison of tissue protein relative abundance across large sample cohorts. However, recently, DIA proteomic methods exemplified by SWATH-MS have been developed, which are simpler, cheaper, and consume less sample than the reference method but provide shallower proteome coverage.Although HGSOC is the most common histological subtype of ovarian cancer, there is a considerable amount of tumor heterogeneity , thus unDDA mass spectrometry workflows have been employed to comprehensively characterize HGSOC  with varIn this study, we compared the results obtained by the reference large-scale DDA proteomic method versus SWATH-MS using aliquots of peptide samples generated for the CPTAC HGSOC study . The resIn addition to its association with worse survival and low rates of resection, the Mesenchymal subtype of HGSOC is associated with a significant contribution from the cancer-associated stroma . Cancer-The notion of discrete HGSOC subtypes that are mutually exclusive is not universally accepted. Verhaak et al. suggested that an individual tumor could be represented by multiple signatures based on different levels of pathway activation . This coBRCA1/2 mutations (Our SWATH-MS analysis confirmed the proteomic signature of HRD established by iTRAQ DDA analysis wherein a sub-network of BRCA1- or BRCA2-related proteins displayed co-expression patterns differentiating HRD from non-HRD patients . Many ofutations . Thus, tOne of the strengths of the iTRAQ DDA workflow is the ability to achieve deep proteome coverage that exceeds that of the SWATH workflow by almost 3-fold. A total of 8,597 proteins were quantified by iTRAQ DDA compared with 2,914 proteins by SWATH-MS. These numbers represent the aggregate numbers of quantified proteins. However, after employing filtering strategies to restrict the data to only the proteins that were quantified across all 103 tumors for each workflow, these numbers decreased to 4,363 and 1,659, respectively. The group of 1,599 proteins that were quantified by both proteomic workflows was used to compare the performance of iTRAQ DDA and SWATH-MS.In addition to their analytical performance, there are also considerable differences in the resource characteristics of iTRAQ DDA and SWATH-MS, including sample requirement, sample throughput, and cost. Hence, it is clear that iTRAQ DDA for comprehensive proteome profiling is a substantially more resource-intense workflow compared with SWATH-MS.Based on the concordance between the iTRAQ DDA and SWATH-MS results that we have shown in this study, SWATH-MS, which is considerably less resource intense than iTRAQ DDA, can be reliably deployed in the proteomic analysis of clinical specimens. The clinical utility of future large-scale translational proteomic studies, regardless of the employed analytical methodology, can be strengthened by the use of large sample cohorts that have undergone comprehensive pathology review.Although our study was focused on the performance of SWATH-MS, other DIA methods are currently available. Our SWATH-MS analysis was conducted using a 5600+ TripleTOF mass spectrometer; however, a newer generation of this mass spectrometer platform exists with an increased linear dynamic range and enhanced detection system, which could result in improved instrument performance with respect to the number of quantifiable proteins per analytical run. A recent DIA study conducted by Bruderer et al. using a quadrupole ultra-high field Orbitrap mass spectrometer resulted in the identification of more than 6,000 proteins in human cell lines and more than 7,000 proteins in mouse tissues . Of noteIn contrast to the original CPTAC HGSOC study, we did not assess protein phosphorylation in the current study. However, it should be noted that SWATH/DIA-MS has been shown to be compatible with the analysis of protein phosphorylation patterns and specific software tools supporting such analyses have been developed .As expected, the development of new analytical instruments and methods often enables an expanded breadth and/or depth of analytical measurement. After the performance of these instruments and methods has been optimized and validated, the short-comings of previously existing methods become evident. It is as yet unknown whether SWATH-MS and other DIA proteomic methods will have an increased prevalence in clinical proteomic analyses. However, our current study provides compelling orthogonal evidence that SWATH-MS elucidates some of the common biological signatures of the Mesenchymal subtype of HGSOC.Novel biological insights beyond those that were initially gleaned from the iTRAQ DDA data  and confAll methods can be found in the accompanying


Melanoma is a skin cancer with great metastatic potential, which is responsible for the major deaths in skin cancer. Although the prognosis of melanoma patients has been improved with the comprehensive treatment, for patients with metastasis, the complexity and heterogeneity of diffuse diseases make prognosis prediction and systematic treatment difficult and ineffective. Therefore, we established a novel personalized immune-related gene pairs index (IRGPI) to predict the prognosis of patients with metastatic melanoma, which was conducive to provide new insights into clinical decision-making and prognostic monitoring for metastatic melanoma. Through complex analysis and filtering, we identified 24 immune-related gene pairs to build the model and obtained the optimal cut-off value from receiver operating characteristic curves, which divided the patients into high and low immune-risk groups. Meantime, the Kaplan–Meier analysis, Cox regression analysis and subgroup analysis showed that IRGPI had excellent prognostic value. Furthermore, IRGPI was shown that was closely associated with immune system in the subsequent tumor microenvironment analysis and gene set enrichment analysis. In addition, we broken through the data processing limitations of traditional researches in different platforms through the application of gene pairs, which would provide great credibility for our model. We believe that our research would provide a new perspective for clinical decision-making and prognostic monitoring in metastatic melanoma. It accounts for only two percent of all skin cancers but causes the most deaths3. Through the combination of treatments, the 5-years relative survival rate for persons with melanoma is good, at 92%4. However, there are still a small number of patients with poor prognosis due to the propensity of melanoma to spread5. The prognosis prediction and systemic treatment are difficult and not always productive in diffusion disease due to its complexity and heterogeneity7. Accordingly, it is urgent to find out novel personalized therapeutic strategies and biomarkers to improve and monitor the unfavourable prognosis of metastatic melanoma.Melanoma is one of the most immunogenic tumors due to its high genomic mutational load10. Tumor cells express certain antigenic components, and immune cells can penetrate into tumor tissues through chemotaxis for immune clearance11. However, when tumor microenvironment is disordered, tumor cells would evade immune elimination and suppress immune response, thus leading to tumor progression13. In melanoma, existed studies have also shown that the initiation and progression of melanoma are closely related to tumor immunity12. What's more, immunotherapy in melanoma has been widely studied on this basis16.Growing studies reveals that the immune system plays a key role in the development and progression of cancers and the relationship between body immune and tumor is complicated17. Interleukin-21 (IL-21) was mainly produced by T helper cell 17 (Th17) and played a key role in the development of Th17. It had strong anti-tumor activity18. Petrella et al. proved the activity of IL-21 in metastatic melanoma through a multicenter phase II study, and it had a certain curative effect on metastatic melanoma19. High expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) was closely related to antigen-specific T cell dysfunction in metastatic melanoma. Relevant preclinical studies showed that the introduction of inhibitory antibodies to CTLA-4 could eliminate downstream inhibitory signals, thus avoiding the dysfunction of antigen-specific T cell, which could produce a cytotoxic anti-tumor response21. Meanwhile, a clinical trial in the antitumor activity of programmed cell death ligand 1/programmed cell death 1(PD-L1/PD-1) signaling blocking was confirmed useful in multiple types of cancers, including advanced melanoma22. These studies proved that tumor immunity was closely related to the progression and treatment of metastatic melanoma, so a new personalized comprehensive prognosis index based on immunity would be very promising.ENK et al. applied interleukin-2 (IL-2) inhalation therapy to patients with pulmonary metastatic melanoma by transferring cytokines to the tumor site, which reduced the toxicity associated with systemic IL-2 administration and achieved good efficacyIn our study, we attempt to establish a novel prognosis index to predict the prognosis of patients with metastatic melanoma, which is based on the screening of gene pairs and could greatly reduce the biological heterogeneity and technical bias of different cross-sequencing platforms. We hope to provide a novel insight for prognosis prediction and clinical decision-making for metastatic melanoma patients.https://cancergenome.nih.gov/). The samples with no metastatic foci, no follow-up information, or follow-up time less than 30 days were excluded. Then, 354 metastatic melanoma samples were reserved for training dataset. Additionally, the expression profiles of GSE65904 were downloaded for testing dataset through GEOquery R package. The same sample filtering standard was used for GSE65904 dataset and 186 metastatic melanoma samples were retained. Finally, 1811 unique immune-related genes (IRGs) were acquired from ImmPort database (https://immport.niaid.nih.gov) to construct immune-related prognostic signature.The flowchart of IRGPI establishment and validation is presented in Fig. http://asia.ensembl.org/index.html). The probe IDs of GSE65904 expression profiles were converted into gene symbols through the illuminaHumanv4.db R package. Ensembl IDs or probe IDs were retained on basis of the mean overall expression of each gene. Next, only IRGs measured by all platforms with relatively high variation  were selected for further analysis.The Ensembl IDs of RNA-FPKM data were transformed into gene symbols based on the Ensembl database . The IRGPs of training dataset with a small variation and imbalanced distribution (MAD = 0) were excluded from the analysis. Then, we conducted 1000 times Lasso Cox proportion hazards regression analysis into training dataset, in which the model with the most occurrences was identified as the most stable gene pairs model and was used for the development of IRGPI.The immune-related gene pairs (IRGPs) were constructed by pairwise comparison of the gene expression level in a specific sample or profile. If the expression level of the first IRG was higher than that of the second IRG, the score of this IRGP was 1; otherwise, the score was 0. Next, the establishment of immune-related gene pairs index (IRGPI) was based on previous descriptionThe prognosis risk of metastatic melanoma patients was distinguished based on IRGPI and the time-dependent receiver operating characteristic (ROC) curve analysis within five years was performed to determine the optimal cut off in the training dataset. KM curve analysis and subgroup analysis were further used to evaluate the ability of IRGPI to distinguish survival risk of metastatic melanoma. Univariate and multivariate Cox analyses were conducted to compare the survival impact of IRGPI with other clinical characteristics. Further, the prognosis ability of IRGPI was validated in the independent GSE65904 testing cohort by KM curve analysis.24. The immune score of each sample was calculated by ESTIMATE R package9. We used the t test to identify the difference of tumor immune microenvironment between the high and low-IRGPI risk groups.The underlying tumor microenvironment mechanisms of different survival prediction impact of IRGPI on metastatic melanoma were explored by the single-sample gene-set enrichment analysis (ssGSEA). It quantified 29 tumor microenvironment immune cell infiltrating score in each sample by using 29 immune gene sets25 was conducted on the gene with false discovery rate (FDR) less than 0.05 to identify biological processes that were differently activated between the high and low-IRGPI risk groups. The number of random sample permutations were set at 1000 and the min size of gene set was set at 160. The biological processes with FDR < 0.01 in both cohorts indicated a statistically significant difference.The ordered gene lists of two cohorts were identified through corresponding R packages  and Gene Set Enrichment Analysis (GSEA)http://www.r-project.org/) and its corresponding R packages. The Area Under Curve (AUC) of ROC curve was performed using the survialROC R package. KM curve analysis was completed using log-rank test from survminer R package. GSEA analysis was completed using clusterProfiler R package. The c5.bp.v7.1.entrez.gmt file from the Molecular Signatures Database  was obtained for the GSEA to identify biological processes. All P value of less than 0.05 indicated a statistically significant difference in all analysis.All statistical analyses were conducted using the R software (version 3.6.3)  and disease-free survival  in training cohort . The subgroup analysis also showed that the prediction impact of IRGPI on OS of metastatic melanoma was promising in training cohort .The IRGPI was also performed into the independent GSE65904 testing cohort to validate its accuracy and prediction ability. The patients of testing cohort were divided into high and low-IRGPI risk group based on the same cut off. The KM curve analysis indicated that patients with the high-IRGPI risk also had poor OS compared with the low-IRGPI risk group .The relative scores of 29 immune cells for each patient were estimated by ssGSEA algorithm. The comparative summary of ssGSEA output result was performed in these two risk groups and a wide variety of differential immune infiltration cells existed. Moreover, both the training cohort and the testing cohort showed the low-IRGPI risk group had a better total immune score than the high-IRGPI risk group by ESTIMATE algorithm  played an important role in the metastasis of melanoma41. Leukocyte mediated immunity could inhibit the metastasis of melanoma, such as cytotoxic T cell (CTL), which could reduce the metastatic foci39. This was consistent with the previous understanding. In addition, the immune response regulating signaling pathway may play a role in tumor progression and metastasis through the regulation of PD-L1 expression42. These results suggested that immune-related signaling pathways were an important aspect in affecting tumor progression. Our study elucidated the relevance of IRGPI in multiple immune biological processes, providing theoretical support for the application of IRGPI and the immune-related studies of subsequent metastatic melanoma.We conducted GSEA to explore the biological processes of IRGPI acting on metastatic melanoma and demonstrated that large number of immune-related processes were differentially enriched between high and low IRGPI risk groups. Existing studies have shown that positive regulation of the immune response could reduce the risk of death from lymphatic metastasis of melanoma, and it was closely related to the increase and activation of tumor infiltrating lymphocytes (TIL)The study breaks the limitations of traditional studies and reduces the biological heterogeneity and technical bias of different cross-sequencing platforms through the application of the gene pairs, which provided great convenience and reliability for the establishment of the prognostic index. Although our research showed advantages in data processing, there were still some limitations to consider. First, due to the lack of in vitro or in vivo experiments, the reliability of our molecular mechanism analysis results was limited. Second, this study was a retrospective study and a well-designed clinical trial was needed to further verify our results, though the mechanism of action of IRGPI in metastatic melanoma has been effectively elucidated through multiple methods. Therefore, the findings would provide new insights into the clinical decision-making and prognostic monitoring of metastatic melanoma and provide theoretical support for further researches.


COVID-19 pandemic is a rapidly spreading virus that is changing the World and the way doctors are practicing medicine. The huge number of patients searching for medical care and needing intensive care beds led the health care system to a burnout status especially in places where the care system was already overloaded. In this setting, and also due to the absence of a specific treatment for the disease, health authorities had to opt for recommending or imposing social distancing to relieve the health system and reduce deaths. All other medical specialties nondirectly related to the treatment of COVID-19 had to interrupt or strongly reduce their activities in order to give room to seriously ill patients, since no one knows so far the real extent of the virus damage on human body and the consequences of doing non deferrable procedures in this pandemic era.Despite not been a urological disease, the urologist needs to be updated on how to deal with these patients and how to take care of himself and of the medical team he works with.The aim of this article is to review briefly some practical aspects of COVID-19 and its implications in the urological practice in our country. COVID-19 is a rapidly spreading virus whose first manifestations as a viral pneumonia occurred in Wuhan, China, in December 2019 . Human CCOVID-19 is highly contagious through SARS-CoV-2 virus-containing respiratory droplets of infected personnel resulting from coughing or sneezing. This is considered the main source of infection as they are propelled for approximately one to two meters and then they can be deposited on the oral, nasal or ocular mucous membranes of a nearby person. The virus can also be transmitted by human to human contact or still, contact with infected objects . The incth, 2020 , Center for Diseases Control (CDC) and Brazilian Council of Medicine (CFM). The protocols of the Brazilian Ministry of Health were also accessed. The terms searched were COVID-19; SARS-CoV-2; surgery; urology, operation, laparoscopy, pandemic, renal transplantation.Effects of SARS Cov-2 on the genitourinary tract SARS-CoV-2 has a spike protein that binds to the epithelial cells through the angiotensin-converting enzyme 2 (ACE2) receptors. Therefore, the human cells that express ACE 2, like the heart, esophagus, urinary bladder and kidney can be potential target cells for the SARS-COV-2 infection . AdditioThe exact mechanism of kidney involvement is unclear: postulated mechanisms include sepsis leading to cytokine storm syndrome or a direct cellular injury due to the virus . HistopaThe blood-testis barrier does not protect against SARS-CoV-2 infection. As other viruses like mumps, HIV, hepatitis B and C, Epstein- Barr and papilloma can cause viral orchitis, also SARS-COVID 2 can lead to orchitis and even lead to male infertility . HistopaDespite no clinical cases of orchitis related to COVID-19 have been described so far, a fertility evaluation of young men interested in having children after recovery from COVID-19 is advisable .It is highly recommended that patients suspect or positive for COVID-19 do not have any contact with the other patients. This way many hospitals have created special areas for treatment of such patients reducing the other activities only to non-deferrable procedures.Routine deferrable appointments should be postponed for at least six months and electronic consultations should be performed when possible. Medical prescriptions should be delivered outside the hospital or preferably electronically once the Brazilian Council of Medicine and the Health Ministry authorized validated electronic prescriptions during the pandemic period .Outpatients diagnostic and elective procedures should also be postponed especially those requiring general anesthesia or sedation. Ficarra et al. proposed deferring diagnostic urodynamic studies, cystoscopy for lower grade bladder tumors, replacement of ureteral stents and nephrostomy tubes, prostatic biopsies and intravesical therapy for low or intermediate grade bladder tumors for up to six months . On the Recent studies reported the occurrence of SARS-CoV 2 in urine, blood, anal swabs and stool and failed to demonstrate viral particles in the semen of patients with positive oro-pharyngeal swabs , 22 corrConsent discussion with patients must cover the risk of COVID-19 exposure and the potential consequences;It is highly recommendable to have a dedicated operating room (OR) with negative pressure and a separated access from the other ORs. Also, a separate anesthesia machine and experienced team of anesthesiologists and operating room nurses are important.Testing asymptomatic patients for COVID-19 before surgery with the aim of protecting them and the surgical team is controversial, once the nasopharyngeal and oropharyngeal testing PCR-RT has a 60 to 70% sensitivity . The CenOnly those considered essential staff should be participating in the surgical procedure and unless there is an emergency, there should be no exchange of room staff;All members of the OR staff should use Protective Personal Equipment (PPE). Everyone in the operating-room (OR) should wear caps, personal protective glasses, N95 or PF2 mask, protective gowns for contacts, procedure gloves and shoe covers . The recSurgical equipment used during procedures with COVID-19 positive or persons under investigation/suspected COVID-19 patients should be cleaned separately from other surgical equipment .Generally, all patients with non-life threating pathologies should have their surgeries and other procedures re-evaluated and delayed. Lei et al. reported the results of 34 asymptomatic patients for COVID-19 operated in Wuhan for various types of surgeries ranging from cesarean and appendectomies to orthopedic and cancer surgeries and who developed COVID-19 in the postoperative period. All patients in this group developed pneumonia, 15 (44%) were recovered at ICU and seven (20.5%) died from COVID-19 related complications . These aUrologic emergencies must be treated without delay but avoiding invasive procedures is recommended. The main emergencies and treatments during COVID-19 pandemic are summarized in Cancer patients are considered immunocompromised patients due to the nature of their disease and to the treatment they are submitted . Due to more advanced age, the impossibility of receiving adequate medical care and to the fact that cancer patients have 3.5 folds risk of developing COVID-19 related serious events, all elective surgeries, chemotherapy and radiotherapy procedures in stable patients should be deferred –38. The Cancer surgeries can be classified in four categories according to their urgencies , 38, 39.Non-deferrable surgeries: procedures whose delay can jeopardize cancer-related outcomes:Renal cancer: radical or partial nephrectomy for clinical T2-4 tumors;Urothelial cancer: radical nephroureterectomy for high grade upper tract urothelial carcinoma;Bladder cancer: transurethral resection for high-risk non-muscle invasive bladder cancer, any high-grade bladder cancer, or tumors more than 2cm at the time of diagnosis. Radical cystectomy and urinary diversion for muscle-invasive bladder cancer or refractory carcinoma in situ;Prostate cancer: radical prostatectomy with pelvic lymph node dissection for high risk or locally advanced prostatic carcinoma; Cancer of the testis: radical orchiectomy; Adrenal cancer: adrenalectomy for tumor >6cm;Penile cancer: partial penectomy for clinical >T1G3 penile cancer.Partial non-deferrable surgeries: are those that when postponed probably will not cause harm to patients:Renal cancer: partial or radical nephrectomy for clinical T1b tumors;Bladder cancer: Endoscopic resection for small low-grade bladder tumors;Prostate cancer: radical prostatectomy for intermediate or high-grade prostate cancer.Deferrable surgeries: those that when postponed will cause minimal harm to patients:Renal cancer: partial nephrectomy for small clinical T1 b renal tumors;Bladder cancer: transurethral resection for small low-grade bladder tumors;Prostate cancer: radical prostatectomy for low/intermediate prostate cancer.Replaceable surgeriesSmall low-grade renal tumors can be replaced by cryotherapy or radiofrequency ablation.These data are summarized in Renal colic and urinary stones management accounts for approximately 30% of the daily practice of the general urologist. Renal colic is one of the most frequent pathologies in the emergency room of every hospital. During pandemic, renal colic should be managed conservatively as much as possible . AdequatPatients should be kept home whenever possible avoiding admission to the ward and monitored for refractory pain and development of infection and sepsis. In the case of infection associated to an obstructive ureteral stone, decompression with a ureteral stent or a percutaneous nephrostomy or even with ureteroscopy and intracorporeal lithotripsy or stone removal with baskets in favorable cases should be performed promptly once these patients can progress to sepsis in up to 25% of the cases , 43. ExtPercutaneous nephrolithotomy (PCNL) is generally a bigger procedure requiring general anesthesia and intensive care especially in older patients with co-morbidities like obesity, diabetes, hypertension or other cardiac diseases or in cases of big staghorn stones .In order to spare ICU beds and respirators these procedures should be postponed except in very selected cases. In the pandemic setting Extracorporeal Shockwave Lithotripsy (SWL) appears as an interesting option once it can be performed ambulatory under local anesthesia or sedation. Both the American Urological Association/Endourological Society (AUA/ES) and European Association of Urology (EAU) guidelines recommend SWL as an effective and safe therapy for ureteral stones <10mm, once the median stone-free rates for SWL is 66.5% against 85% for ureteroscopy but the complication rate is lower for SWL , 42. ForThe peak of the pandemic is decreasing in several countries which does not mean that the World will get rid of the virus soon and that protective measures will be relaxed in the next months. Thus, changing some practice patterns and not simply postponing procedures are of utmost importance once stone patients suffer with pain and repeated infections. Therefore, SWL can be a very interesting alternative in this moment.Proietti et al. proposed a classification for endourologic procedures regarding their urgency . We propGrade 1 - Surgical emergencies: patients that should be operated in 24-48 hours: obstruction associated to infection, obstructive anuria in solitary anatomic or functional kidney or bilateral obstructive ureteral stones, obstructive ureteral stones in immunocompromised patients or in those with complications of previous endourologic procedures presenting with urinary fistula, abscess or obstruction.Grade 2 - Urgencies: patients that should be operated in no more than two weeks, including obstructive ureteral stones with mild or no pain and obstructive renal pelvic stone with nephrostomy.Grade 3 - Semi-elective surgeries: patients that should be operated in no more than 8 weeks: patients previously stented due to ureteral or renal stones or in cases of renal stones with afebrile UTI or chronic renal function loss.Grade 4 - Elective surgeries: asymptomatic non-obstructive caliceal stones in non-solitary kidneys.One of the characteristics of SARS-CoV-2 is its high infectivity and capacity to remain viable and infective for up to four hours in aerosols and days in stainless steel and plastic, similarly to SARS-CoV-1 . These fIn order to avoid or minimize the risk of contamination during laparoscopic and robotic procedures, some rules must be followed. The trocars should be adequate to the incision to avoid carbon dioxide (CO2) leakage; an exclusive 5mm trocar should be used for insufflation and deflation and a smoke evacuation filter used, the pneumoperitoneum should be reduced to the lowest possible as well as the electrocautery power, the pneumoperitoneum should be evacuated slowly and only by the exclusive trocar , 49, 50.No evidence of transmission of SARS-CoV-2 by organ donation has been demonstrated but it has been shown the presence of the virus in the blood of 15% of patents with COVID-19; therefore, all solid organs are at risk of transmission , 51, 52.Like other elective surgeries, renal transplants, particularly those with live donors should be deferred especially in places with a high incidence of COVID-19 . Renal tRegarding donors, it is recommendable and mandatory that all potential live and deceased donors be screened with PCR-RT nasal and oropharynx swab test and also with the rapid test. Additionally, a clinical and laboratorial screening should be performed regarding the previous occurrence of symptoms  also including a chest CT . Living Regarding the safety of the transplant surgical team, it is mandatory the use of PPE as discussed above for other surgeries. Organ procurement teams should avoid travelling to COVID-19 high incidence zones and work in parallel with other procurement teams in order avoid contact among them .There are only few reports on the outcomes of transplanted patients that developed COVID-19; in one case, the patient recovered with initial cessation and subsequent reduction of immunosuppressive agents  and anotSARS-CoV-2 induces a renal tubular lesion compatible with acute tubular necrosis that can lead to acute renal insufficiency and a testicular lesion similar to other viral orchitis but with no clinical cases reported. Presential outpatient visits must be replaced by electronic consultations and the majority of invasive outpatient diagnostic and therapeutic procedures shall be postponed. Urgent surgeries in general urology, oncology and endourology are well defined and exceptions should be discussed case by case. Elective surgeries must be postponed. Patients undergoing undeferrable surgeries shall be screened for SARS-CoV-2 infection before their procedures and positive cases should have their surgeries postponed unless an organ or life-threatening emergency occurs. Medical team must protect themselves by wearing appropriate PPE and by adopting surgical techniques to reduce spreading of the virus. Kidney transplantation can be done in particular situations with very special concerns to the live donor, receptor and medical team health.


The fabrication of smart biocomposites from sustainable resources that could replace today’s petroleum-derived polymer materials is a growing field of research. Here, we report preparation of novel biocomposites using nanocellulose networks extracted from food residue (onion skin) and a vegetable oil-based bioresin. The resin was synthesized via the Diels-Alder reaction between furfuryl methacrylate and tung oil at various ratios of the components. The onion-skin-extracted cellulose nanofiber and cellulose nanocrystal networks were then impregnated with the resins yielding biocomposites that exhibited improved mechanical strength and higher storage modulus values. The properties of the resins, as well as biocomposites, were affected by the resin compositions. A 190–240-fold increase in mechanical strength was observed in the cellulose nanofiber (CNF) and cellulose nanocrystal (CNC)-reinforced biocomposites with low furfuryl methacrylate content. The biocomposites exhibited interesting shape-memory behavior with 80–96% shape recovery being observed after 7 creep cycles. Commercial plastic products are derived from petrochemical resources and therefore, replacing them with renewable biobased materials sourced by sustainable means fits with our current strive towards circular economy. In this context, biocomposites composed of bioresins, thermosetting resins typically derived from vegetable oils , and natcis-9, trans-11, trans-13-octadecatrienoic acid) units, allows it to undergo various polymerization reactions, including thermal, cationic, free radical and the Diels-Alder (DA) reaction with different olefinic co-monomers . T. T−1 (−1nd T1F9) c confirm1H and 13C NMR analysis of the resins. A tentative structure of the acrylated TO resin is shown in 1H NMR of the synthesized resin  facilitates the diffusion of the resins through the porous network structures of CNF and CNC. It is hypothesized that resin impregnation procedure followed by in-situ curing process of these composites might help to control the moisture absorbance tendency by the nanocellulose networks, which is a major drawback of these kind of natural fiber reinforced biocomposites. In this context, FTIR spectra of the biocomposites were recorded to verify if the surface hydroxyl groups of CNF and CNC networks participate in the chemical reaction during in-situ curing process. The FTIR spectra of CNF and CNC-reinforced biocomposites exhibited peaks similar to those of the cured resins, which indicates weak or no chemical interaction between the nanocellulose and the resin in the biocomposites . Only thTg) in the materials: one at a lower temperature (~50 °C) due to the long hydrocarbon chain (a soft segment), and the other at a higher temperature (~175 °C) originating from the crosslinked network in the resins (a rigid segment) [Tgs. The absence of any exothermic transition in the cured resins and biocomposites indicates complete curing of the samples. The presence of sharp endothermic transition between 90–100 °C in CNF and CNC  ,21. The  and CNC b indicat and CNC . The preThe thermal stability of the resins and composites was assessed by TGA, and the results are given in Contact angle measurements of the materials showed a significant increase in the degree of hydrophobicity of the biocomposite films compared with CNF and CNC, as can be seen in T = Tg + 398 K) [DMA was performed to gain insight into the thermo-mechanical behavior of the cured resins and the composite materials. It can be seen from + 398 K) ,54. Low + 398 K) . Incorpo+ 398 K) . Resin cTg ~40–60 °C and ~90–100 °C). This implies the presence of two different polymer phases in the resins and biocomposites consisting of soft and rigid segments [Tan δ signals provided the information regarding viscoelastic damping of the materials a–c. Signsegments ,52,56, wDMA experiments were performed in controlled force mode to investigate the flexibility of cured resins and biocomposite films. As can be seen from The prepared biocomposites exhibited interesting shape recovery properties. The shape-memory behavior of a CNF-reinforced biocomposite film is shown in a video clip attached as Video SI. At room temperature, a hand-folded biocomposite film exhibited very slow recovery, whereas the deformed film regained its original shape much more quickly upon heating. Short-term creep measurements were performed to characterize this shape recovery behavior at different temperatures. UV-Vis transmittance spectra obtained from the samples revealed that T1F3 biocomposites had higher transparency  than other composites with higher FMA contents a. OveralIncreasing the FMA content in the biocomposites increased the concentration of chromophores (furans) in the material, leading to more coloured and opaque films . BiocompIn comparison to metals and ceramics, low strength and stiffness of the shape-memory polymers often affect their shape-recovery property. Thus, addition of fillers or reinforcements to these polymers is required to increase the stiffness, as well as recovery force to meet certain application needs . EarlierFuran derivative- (FMA) and vegetable oil- (TO)-based bioresins were synthesized via the Diels-Alder reaction with various TO:FMA ratios. Food-waste (onion skin)-derived nanocellulose networks (CNF and CNC) were incorporated into the bioresins to fabricate biocomposites, which exhibited significantly improved storage modulus, strength  and increased hydrophobicity. The incorporation of CNC networks led to greater improvements in the properties of the biocomposites due to its high degree of crystallinity and less dense structure in comparison to CNF. Increasing the FMA content in the resins led to phase-separated, brittle biocomposite films with poor transparency, suggesting that the properties of these composite materials could be tuned by altering the ratios of the raw materials. Notably, the biocomposite films exhibited interesting shape-memory behavior resulting from the presence of soft and rigid segments due to the acrylated TO resin. A significant degree of shape recovery (~80–96%) was observed in the biocomposites, which is comparable to shape recovery properties of polyurethane. This study demonstrates a convenient method for fabricating lightweight, flexible, and hydrophobic shape-memory composite materials with tailored properties in a sustainable manner using renewable components, household food wastes, and a clean synthesis protocol. These types of materials are in high demand in packaging, smart textile, biomedical, and electronic industries, where thermally activated shape-memory properties are required.


In the intensive care unit (ICU), patients with thrombocytopenia are at high risk for bleeding and should be assessed for their thrombogenic potential. However, the analytical conditions of conventional hemostatic tests are unsuitable for the evaluation of low-platelet samples. Here we aimed to establish suitable analytical conditions with the Total Thrombus-formation Analysis System (T-TAS) for quantitative assessment of thrombogenic potential in patients with thrombocytopenia and to investigate how T-TAS values relate to bleeding symptoms and the effects of platelet transfusion.Modified chips with a different chamber depth were developed for the analysis of low-platelet samples in the T-TAS. We included 10 adult patients admitted to the ICU of Kagoshima University Hospital who required platelet transfusion. Patients were divided into major and minor bleeding groups according to their bleeding scale before platelet transfusion. The thrombogenic potential of these patients before and after platelet transfusion was assessed with hemostatic function tests, including rotational thromboelastometry, multiplate aggregometry, and the T-TAS.P <  0.01). The recovery of thrombogenic potential after platelet transfusion was confirmed with the T-TAS and correlated with the function, rather than the count, of transfused platelets. Among all evaluated parameters in hemostatic function tests, only the T-TAS showed significant differences in occlusion time and area under the curve both between the minor and major bleeding groups and between pre- and post-platelet transfusion.Analysis of low-platelet samples revealed that, compared with the conventional chip (80-μm-deep chamber), the modified chip (50-μm-deep chamber) achieved higher sensitivity in detecting elevation of flow pressure caused by growth of an occlusive thrombus in the T-TAS analytical chamber. All patients in the minor bleeding group retained thrombogenic potential that occluded the modified chip (occlusion time 16.3 ± 3.3 min), whereas most patients in the major bleeding group were unable to occlude the modified chip during the 30-min measurement (We developed a modified microchip-based flow chamber system that reflects the hemostatic function of patients with thrombocytopenia. Patients in the intensive care unit (ICU) often experience thrombocytopenia. Causes of thrombocytopenia include loss of platelets resulting from hemorrhage, dilution resulting from fluid resuscitation, consumption resulting from platelet adhesion to the vascular wall or extracorporeal devices, and insufficient production resulting from hematopoietic disease or adverse drug effects –3. To maPractice criteria for platelet transfusion are controversial. Platelets may be transfused to patients with thrombocytopenia during treatment of hematopoietic disease, before insertion of a central venous catheter, before diagnostic lumbar puncture, and before surgery. Practice criteria are not standardized and depend to a large extent on the circumstances mentioned above –8. RandoThromboelastometry and multiplate impedance aggregometry are widely used for analyzing hemostatic function. Thromboelastometry, which monitors changes in the viscoelasticity of whole blood during formation of the fibrin clot, is mainly used to evaluate hemostatic function during cardiovascular surgery –18. MultWe developed the Total Thrombus-formation Analysis System (T-TAS), which comprehensively evaluates hemostatic function under conditions similar to those of thrombus formation in vivo . The T-TThis single-center observational study was approved by the Ethics Committee of Kagoshima University Hospital . The study was conducted in compliance with the Declaration of Helsinki, and written informed consent to participate was obtained from patients or their close relatives. We included 10 adult patients admitted to the ICU of Kagoshima University Hospital between November 2017 and October 2019 who required a platelet transfusion. We excluded patients administered drugs that affect the hemostatic system, such as antiplatelet agents or anticoagulants. The use of anticoagulants for the maintenance of arterial lines or extracorporeal devices was permitted. The need for platelet transfusion was determined in accordance with our standard clinical practice, independent of the present study.Blood was drawn from the radial arterial line before and after platelet transfusion. The samples were anticoagulated with EDTA , 3.2% sodium citrate , or hirudin  and were used for blood cell counts, thromboelastometry, T-TAS, and multiplate aggregometry. After platelet transfusion, platelet concentrates remaining in the transfusion tube were collected and analyzed for their thrombogenic potential.Before platelet transfusion, bleeding symptoms were assessed with the modified WHO bleeding scale . Grades Platelet counts were measured with an XN-9000 automated blood cell analyzer . General coagulation tests, including measurement of prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen, were performed with the Automated Coagulation System-CP3000  or a STACIA .Blood anticoagulated with sodium citrate was used for ROTEM (Instrumentation Laboratory). Tissue factor-induced blood coagulation (EXTEM) and ellagic acid-induced blood coagulation (INTEM) were analyzed with ROTEM according to the protocol recommended by the manufacturer, and clotting time and maximum clot firmness were evaluated.Blood anticoagulated with hirudin was used for multiple electrode aggregometry with a Multiplate Analyzer (Roche). Platelet aggregation induced by collagen, adenosine diphosphate (ADP), thrombin receptor activating peptide-6 (TRAP-6), or ristocetin was analyzed according to the protocol recommended by the manufacturer, and the area under the curve (AUC) was evaluated.− 1 in the 50-, 60-, and 80-μm-deep chambers, respectively. The intra-assay coefficient of variation of the AUC was 1.24% when using conventional flow chambers and whole blood from healthy volunteers [Blood anticoagulated with sodium citrate was used for T-TAS analysis . Calcium chloride and corn trypsin inhibitor were added at the start of the measurement. In some experiments, platelet concentrates (240 μL) mixed with pooled normal plasma (240 μL)  and a reagent mix (20 μL) containing calcium chloride, corn trypsin inhibitor, aprotinin, and heparan sulfate were used for T-TAS analysis to evaluate the hemostatic function of platelet concentrates. Thrombus formation in the flow chamber, which was coated with tissue factor and collagen, was analyzed according to the protocol recommended by the manufacturer, and occlusion time and the AUC were evaluated. For the analysis of low-platelet samples, newly developed flow chambers  were used instead of conventional flow chambers . The flow rate was set at 10 μL/min, which corresponds to initial wall shear rates of approximately 1500, 1100, and 600 slunteers  .Immediately after the T-TAS analysis, immunofluorescence analysis was performed to determine the composition of thrombi formed in the flow chamber . Platelet test. The significance of differences before versus after platelet transfusion was evaluated with the paired t test. Relationships between hemostatic function test values were evaluated with Pearson’s correlation coefficients and P values. All statistical analyses were performed with IBM SPSS version 23 , and a P value < 0.05 was considered to indicate a significant difference.The significance of differences between the major and minor bleeding groups was evaluated with the Student When we used conventional T-TAS chips with a chamber depth of 80 μm to analyze low-platelet samples, the flow pressure did not increase during the 30-min measurement Fig. a. This fP <  0.01). When the T-TAS HD chip was used, all four patients in the minor bleeding group retained thrombogenic potential to occlude the flow chamber. In contrast, patients in the major bleeding group showed delayed or defective thrombus formation in the T-TAS HD chips  varied from one case to another, we examined factors closely related to this rate. The ΔAUC of the T-TAS in patients significantly correlated with the AUC of the T-TAS of the platelet concentrate, but did not correlate with the number of platelets in the platelet concentrate  We developed modified T-TAS HD chips suitable for quantitatively evaluating hemostatic function in patients with thrombocytopenia. (2) T-TAS HD chips discriminated between hemostatic function of the major and minor bleeding groups. (3) T-TAS HD chips detected the recovery of hemostatic function following platelet transfusion. (4) The recovery of hemostatic function following platelet transfusion may depend on the function of transfused platelets, rather than the count. The advantages of T-TAS HD chips do not necessarily suggest that the T-TAS is superior to conventional hemostatic function tests, such as those obtained with the Multiplate Analyzer and ROTEM, because suitable analytical conditions for low-platelet samples are indispensable, not only for the T-TAS but also for the Multiplate Analyzer and ROTEM.It is important to determine whether evaluation of hemostatic function with the T-TAS supports clinical decision-making. For example, the ability of the T-TAS to indicate the need for platelet transfusion should be clarified in the future. Answering this critically important clinical question requires prospective investigation of how the frequency of bleeding events and platelet transfusion differ when criteria based on the T-TAS versus platelet counts are applied. Such interventional studies may not be approved at this time because insufficient data are available to ensure that the T-TAS may be safely used to make clinical decisions. We therefore consider that the present study is a first step in this direction.Efficient hemostasis requires sufficient numbers of functional platelets as well as coagulation factors , 27. If The recovery of thrombogenic potential after platelet transfusion was confirmed in most patients, with the exception of patient #9. Table The 10 patients tested in the present study had different underlying diseases. It is important to consider whether the cut-off values must be changed according to the underlying disease when making clinical decisions. Although it is possible that thrombogenic potential can be uniformly determined with the T-TAS regardless of underlying disease, the T-TAS is unable to evaluate the vulnerability of blood vessels, which is one factor that contributes to bleeding –31. ThusWe developed a modified microchip (HD chip)-based flow chamber system suitable for evaluating the thrombogenicity of patients with thrombocytopenia. For analysis of blood samples with normal platelet counts, we still recommend using conventional AR chips.