Background: The limitation to the use of ElectroMyoGraphy (sEMG) in rehabilitation services is in contrast with its potential diagnostic capacity for rational planning and monitoring of the rehabilitation treatments, especially the overground Robot-Assisted Gait Training (o-RAGT).Objective: To assess the barriers to the implementation of a sEMG-based assessment protocol in a clinical context for evaluating the effects of o-RAGT in subacute stroke patients.Methods: An observational study was conducted in a rehabilitation hospital. The primary outcome was the success rate of the implementation of the sEMG-based assessment. The number of dropouts and the motivations have been registered. A detailed report on difficulties in implementing the sEMG protocol has been edited for each patient. The educational level and the working status of the staff have been registered. Each member of staff completed a brief survey indicating their level of knowledge of sEMG, using a five-point Likert scale.Results: The sEMG protocol was carried out by a multidisciplinary team composed of Physical Therapists (PTs) and Biomedical Engineers (BEs). Indeed, the educational level and the expertise of the members of staff influenced the fulfillment of the implementation of the study. The PTs involved in the study did not receive any formal education on sEMG during their course of study. The low success rate (22.7%) of the protocol was caused by several factors which could be grouped in: patient-related barriers; cultural barriers; technical barriers; and administrative barriers.Conclusions: Since a series of barriers limited the use of sEMG in the clinical rehabilitative environment, concrete actions are needed for disseminating sEMG in rehabilitation services. The sEMG assessment should be included in health systems regulations and specific education should be part of the rehabilitation professionals' curriculum.Clinical Trial Registration:www.ClinicalTrials.gov, identifier: NCT03395717. In neurorehabilitation services, new devices have been widely used for both assessment and rehabilitation. The technology-based assessment includes instrumentation for medical imaging, for measuring electrophysiological signals  and biomechanics . The technology for rehabilitation includes a wide range of devices such as: electrical stimulators, mechanical vibrators, robotics, virtual reality-based systems, etc. While the diffusion of technology for rehabilitation has been simplified by the great clinical interest in maximizing the outcomes of the rehabilitative treatment, the technology for assessing electrophysiological signals (EEG and sEMG) has been usually confined to research studies. As a consequence, the clinical acceptance of measuring tools, like sEMG, is low and such devices are not included in the usual clinical practice . On the The limitation to the use of sEMG in rehabilitation services is in contrast with its potential diagnostic capacity for rational planning and monitoring of the rehabilitation treatments –27. A nuThe aim of this work is to assess the barriers faced during the implementation of a sEMG protocol in a clinical study on o-RAGT in subacute stroke patients. The analysis of difficulties encountered during the protocol will allow us to discuss strategies and options which could help to reduce them.An observational study was conducted in a rehabilitation hospital (IRCCS San Raffaele Pisana of Rome) in order to investigate the barriers in implementing a sEMG-based assessment protocol in subacute stroke patients who underwent o-RAGT.The primary outcome was the success rate of the implementation of the sEMG-based assessment, expressed as the percentage of successfully assessed patients relative to the total number of recruited ones. The number of dropouts and the motivations were registered. A detailed report on difficulties in implementing the sEMG protocol was edited for each patient.The educational level and the working status of the staff involved in each phase of the study  were registered.Each member of staff completed a brief survey indicating their level of knowledge of sEMG, using a five-point Likert scale: very poor (no knowledge about sEMG); poor (basic knowledge about muscle electrophysiology); fair (good knowledge of muscle electrophysiology and basic knowledge of detection/interpretation techniques); good ; excellent .ClinicalTrials.gov by the unique identifier number: NCT03395717, and all subjects gave informed written consent in accordance with the Declaration of Helsinki.A pilot clinical trial was carried out on a group of subacute stroke subjects who underwent o-RAGT with a WPE. This study was a subgroup analysis from a large multicenter clinical trial assessing the effects of o-RAGT , 16. The1). Two gait tasks for each walking trial were collected. The total duration of the experimental session was about 2 h. An eight-channel wireless sEMG device  was used to acquire  the activity of the agonist/antagonist muscles of the distal and proximal compartment of lower limbs. The skin was abraded and cleaned with alcohol, then electrodes were placed on Tibialis Anterior (TA), Gastrocnemius Medialis (GM), Rectus Femoris (RF), and Biceps Femoris caput longus (BF) muscles of each leg according to the SENIAM guidelines (The subjects were screened at the beginning (T1) and at the end (T2: three weeks after T1) of the o-RAGT with both clinical measures and sEMG-based assessment. The sEMG-based assessment was carried out for assessing electromyographic activity during the following walking trials: at T1 and T2 during ecological overground gait at a self-selected speed along a 10-m walkway with assistance ; and during the first session of o-RAGT  and full-wave rectified. For standardization, the sEMG data were normalized to 100% of a gait cycle based on the temporal events (heel strikes and toe-offs) extracted from the kinematic data. The sEMG signals of each gait cycle were processed as follows: (1) the sEMG envelope was obtained using a moving average filter  and normalized at the maximum sEMG amplitude level; (2) the activation threshold identifying onset and offset status of muscle activity was detected as the 20% of minimum-maximum amplitude level distance , when keDescriptive statistics were computed in order to appropriately explain the clinical and demographic characteristics of the sample. Data were represented for each recruited stroke subject. The sEMG outcomes were averaged from the two gait tasks for each walking trial and were used for subsequent analyses. The one-way ANalysis Of VAriance (ANOVA) was applied between T1 and T2 in order to test the treatment effect of o-RAGT on sEMG outcomes.The study was carried out by a multidisciplinary team composed of two Medical Doctors (MDs), four Physical Therapists (PTs), and two Biomedical Engineers (BEs). The MDs were Physical Medicine & Rehabilitation specialists. The sEMG acquisition and analysis were conducted by PTs and BEs. Indeed, the educational level and the expertise of the members of staff influenced the accomplishment of the implementation of the study. The PTs involved in the study did not receive any formal education on sEMG during their course of study. Two of them (PT3 and PT4) were part of the research staff and studied the sEMG recording procedures  as a self-taught. However, no PTs had enough knowledge to recognize artifacts and interferences or process the signals. The BEs, on the other hand, were responsible for the entire sEMG procedure from signal acquisition to processing. The limitation of sufficient knowledge in sEMG among PTs was one of the reasons for the limited diffusion of electromyography in the rehabilitation hospital, and specifically in the clinical study on o-RAGT.1 of a sample composed of 8 patients  were not available. Specifically, the experimental setup for data acquisition during o-RAGT1 was partially influenced by the presence of the WPE: the electrode application procedure did not always comply with the SENIAM guidelines . The effects of the 15 sessions of o-RAGT improved the BS in TA  and moderately in RF . The proximal muscles registered a decrease in CC between T1 and o-RAGT1  and between T1 and T2 , while the distal muscles did not. In the future, an advanced analysis of sEMG (by means of a time-varying multi-muscle coactivation function) could help to investigate the o-RAGT effects, in terms of simultaneous coactivation of a group of muscles during gait  the BS coefficient; (ii) the CC coefficient; (iii) and the RMS value . The resing gait –39. The This paper focuses primarily on the difficulties encountered in the investigation of the o-RAGT technique by mean of sEMG, and secondarily on the results obtained in the o-RAGT study. This experience evidences a number of barriers limiting the implementation of the study on the effects of o-RAGT in terms of muscle activation. The low success rate (22.7%) of the protocol has been caused by several factors which can be grouped in: patient-related barriers; cultural barriers; technical barriers; and administrative barriers.Patient-related barriers are mainly caused by low patient compliance. Except for two drop-outs caused by medical issues Cultural barriers depend on the insufficient knowledge of sEMG among PTs. The role of PTs in the study was mainly as assistants of BEs during the sEMG acquisition phase. Specifically, PT3 and PT4 placed the electrodes and helped the patients to conduct the requested motor tasks. This situation is representative of the condition of PTs with respect to sEMG in Italy. In fact, the Italian academic curriculum in Physical Therapy does not include any courses on electromyography, and thus the professional figure of PT is usually focused on conventional therapy. Indeed, considering the dramatic introduction of new technologies in rehabilitation services, the role of PT should be drastically changed, and this change should start from the university curricula . While t1. The sEMG signal was affected by the change of electrode location (1 was altered and the sEMG outcomes were unreliable. This barrier could be overcome in future studies by using high-density sEMG (Technical barriers have been encountered during o-RAGTlocation , 42, dueity sEMG , 44 or mAdministrative barriers related to management and time-related issues had a negative impact on the dissemination of sEMG assessment in our clinical environment: a limited amount of time was available for the sEMG acquisition, because of the intensive schedule of rehabilitative treatments. These time-related barriers have been highlighted also by Feldner et al.  and by SIn conclusion, this paper offers an insight into the barriers limiting the use of sEMG during o-RAGT in subacute stroke patients. Certainly, since a series of barriers limited the application of sEMG in the clinical rehabilitative environment, concrete actions are needed for disseminating sEMG in rehabilitation services. The sEMG assessment should be included in health systems regulations and specific education should be part of the rehabilitation professionals' curriculum.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by IRCCS San Raffaele Pisana, Via della Pisana, 235, 00163 Rome, Italy. The patients/participants provided their written informed consent to participate in this study.MG, FI, and MF have made substantial contributions to conception and design. SP, DGal, and DGab participated in the enrollment phase and carried out the o-RAGT treatment. SP, MG, and PR carried out the clinical and sEMG assessments. MO and PR designed the algorithm for sEMG data analysis. LP, PR, MO, and AG participated in the study design and coordination, and statistical analysis. SP and FI participated in the manuscript revisions. MF and FI gave the final approval of the version. All authors contributed to the article and approved the submitted version.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.


Nasal bridle is a feeding tube retaining device that is now increasingly used worldwide. While common complications tend to be minor, it is important to remain vigilant for newer adverse events. We hereby delineate the case of an elderly female who required nasoenteric feeding tube following simultaneous liver-kidney transplantation. Nasal bridle placement was warranted owing to her significant frailty and poor mentation. Due to her extreme agitation during the procedure, bridle insertion could not be completed. Upon removal of the probe, unprompted detachment of the magnetic tip was noted. Radiological workup revealed the dislodged magnet in the sphenoid sinus. Subsequently, she underwent an uneventful endoscopic sinus surgery, resulting in successful retrieval of the magnet. This paper highlights the spontaneous magnet avulsion from a bridling system and serves the purpose of community awareness regarding this unusual procedural complication. Additionally, we aim to evaluate the efficacy of the nasal bridle, further accentuating its advantages and possible complications. In 1980, McGuirt and Strout first described securing nasoenteric tubes by employing a nasal bridle . AMT BriNasal bridle effectively reduces the tube dislodgement rates compared to traditional modalities used to this end . ConsequA 66-year-old female underwent an uneventful simultaneous liver-kidney transplantation 10 days prior to the current presentation. Her medical history included steatohepatitis-related cirrhosis and chronic renal insufficiency following diabetic nephropathy. Owing to her significant baseline frailty, the patient showed a prolonged postoperative recovery. She continued to have a poor appetite and fluctuating mentation after the surgery. A nasoenteric tube was thus placed through the left nostril to ameliorate the caloric deficit. After the procedure, magnetic nasal bridle  insertion was attempted. However, she became extremely agitated during the bridle placement and intermagnetic linkage could not be achieved. Upon removal of the probe, it was noted that the magnet was missing from its distal end. The otorhinolaryngology service was consulted. An urgent nasal endoscopy was performed, but the magnet could not be visualized. A plain radiograph of the paranasal sinuses showed a radiopaque object lodged at the sphenoid os  of the head and neck confirmed the magnet with the associated metallic artifact, originating from the sphenoid sinus Figure .The artifact appeared as small, well-circumscribed, hyperdense opacity in the superomedial aspect of the left sphenoid sinus Figure .A multidisciplinary team planned a CT-guided endoscopic sinus surgery. The surgical intervention resulted in the successful retrieval of the magnet. Thereafter, she completely recovered without the need for a percutaneous gastrostomy tube (PEG) placement.Enteral nutrition plays a vital role in the nourishment of patients who are unable to maintain their volitional intake. This technique ensures the appropriate supplementation of required nutrients, which in turn hastens the recovery in such patients . Due to The data regarding the use of nasal bridles show that it effectively decreases the feeding tube dislodgement rate ,6. ThereWhile the efficacy of nasal bridle in yielding ameliorated nasoenteric feeding outcomes is indubitable, perceptions about associated complications can limit its uptake. Due to the fact that a probe is initially required, the bridling procedure can elicit minor complications such as epistaxis and nasal ulceration ,14. InitRecently, several newer adverse events of bridling have been documented. Saunders and Osborne reported a rare case of flexible guidewire retention, which was initially used to insert a nasal bridle . The guiThe minor complications associated with bridling can be circumvented with proper insertional technique. In order to reduce the cutaneous complications, the external placement of the bridle should be evaluated and finalized in such a way that it does not exert undue traction on the nose of adjacent structures. A standard-check procedure should be adhered to upon the initial insertion of the bridle, ensuring that the guidewire is safely retracted after the administration of the bridle so that no component is left in situ. In terms of dealing with these rarer complications, such as magnet detachment, the bridle system should be assembled in accordance with the manufacturer’s guidelines. A multitude of manufacturers produce these bridles, making it important to adhere to the instructions detailed in the respective manual. Furthermore, the clinical staff should ensure that the magnet is securely fitted into the bridle prior to insertion. Although it is generally believed that the magnet is well embedded into the bridle, it can sometimes come loose. Therefore, pre-procedural checks can help avert this unusual complication. We also recommend bridle placement prior to nasoenteric tube insertion as this will leave adequate space in the nasal cavity for catheter passage. This sequence has also been authorized by the bridle manufacturer’s insertion guidelines. It is expected that patients may still develop such problems in clinical practice. Therefore, we emphasize that magnetic resonance imaging should not be performed as a part of the radiological workup in patients with suspected bridle magnetic avulsion.Additional measures might also help in evading the procedural complications of bridle placement. Physicians and ancillary staff must ensure that they have thoroughly perused the training manuals and that at the time of insertion, they closely follow a standard checklist. Certain anatomical observations might also contribute towards the uneventful bridle insertion. Puricelli et al. posited that insertion of the bridle at the level of the nasal floor is likely to proceed unhindered, and it is pivotal to ensure that the bridle is parallel to the nasal floor upon insertion . FurtherNasal bridles help to provide sustained enteral nutrition in patients incapable of maintaining volitional feeding. The benefits afforded by this device significantly outweigh the associated minor complications. Therefore, this technique is now increasingly being used for optimal enteral feeding across the United States. This study presents an important clinical update regarding unusual adverse events associated with bridle insertion. Physicians should be aware of procedural complications like spontaneous magnet detachment due to the risk of aspiration or its lodgment into unusual locations, potentially culminating in a devastating outcome or unnecessary surgical intervention.


Coronary slow flow (CSF) refers to the phenomenon of delayed distal flow in the absence of lesions detected on coronary angiography. Although the detection rate of CSF has been increasing in clinical practice, early diagnosis is difficult and the factors contributing to this condition remain unclear. Given the increasing demonstration of the roles of microRNAs (miRNAs) in disease and as diagnostic biomarkers, the aim of this study was to analyze the expression of serum miRNA-22 in patients with CSF detected using coronary angiography and its diagnostic efficacy.  A retrospective analysis including 44 patients with CSF and 42 patients with normal coronary flow (control group) was conducted. Additionally, all included patients either did not have visually estimated coronary artery stenosis or had <50% stenosis. Plasma samples were collected from patients in these two groups, and the levels of miRNA-22 were detected. The receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic efficiency of serum miRNA-22 in the context of CSF. P < 0.0001). The area under the ROC curve for miRNA-22 in diagnosing CSF was 0.8293 , with a sensitivity of 75.0% and specificity of 88.1%.  The expression of serum miRNA-22 was significantly higher in the CSF patients than in the control subjects ( The expression of serum miRNA-22 in CSF is upregulated compared to that in subjects with normal coronary flow and shows relatively high clinical diagnostic efficiency, suggesting a new potential biomarker for the early diagnosis of CSF. In 1972, Tambe et al. first reported cases of patients with symptoms of chest pain accompanied by a significantly reduced blood flow rate in the absence of lesions detected on coronary angiographs . CSF hasMicroRNAs (miRNAs) are endogenous noncoding small RNAs (22–23 nucleotides in length) that have recently been identified as having a wide range of functions with respect to regulating gene expression for influencing cell proliferation, differentiation, and migration –9. The mWe retrospectively analyzed the data of 1256 patients who underwent elective coronary angiography from March 2018 to March 2019 at the Department of Internal Medicine of the Xiangya Hospital. The inclusion criteria of this study were patients who either did not have visually estimated coronary artery stenosis or had <50% stenosis, which was defined as insignificant coronary artery stenosis, being consistent with many other studies [All patients underwent coronary angiography with the standard Judkins method. Coronary flow rates were assessed by TFC, a classical method described by Gibson et al. . StandarBefore coronary angiography, 10 mL of venous blood was collected from all study subjects. The Hitachi 7600 fully automated biochemistry analyzer was used to measure the levels of fasting plasma glucose, total cholesterol, triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol.−ΔΔCt method was used to calculate the relative miRNA-22 expression in exosomes, in which ΔCt = CtmiRNA-21–CtU6 and ΔΔCt = ΔCtCSF group–ΔCtcontrol group, where Ct number is the number of amplification cycles for the fluorescence reaction intensity.Five milliliters of whole venous blood was collected in ethylenediaminetetraacetic acid tubes and left to stand for 30 min at room temperature. The tubes were centrifuged at 4°C and 1200 ×g for 15 min and the serum in the upper layer was aspirated into 1.5 mL EP tubes. TRIzol  was used for sample lysis and total RNA extraction before reverse transcription. Complementary DNA (cDNA) was synthesized under the following reaction conditions: 16°C for 15 min, 42°C for 60 min, 85°C for 5 min, and 4°C for 5 min. qPCR was performed using the SYBR Premix Ex Taq kit (Takara) on an ABI 7500 real-time PCR system . Proprietary qPCR primers were used, which were designed and validated by a commercial company . qPCR was performed in triplicate for each sample. The 2t-test was employed for intergroup comparisons and one-way analysis of variance was employed for multiple group comparisons. Qualitative data are expressed as the number of subjects and percentage, and the chi-square test was employed for intergroup comparisons of these variables. The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic performance of miRNA-22 in CSF. A difference with P < 0.05 was considered to be statistically significant.SPSS 19.0 statistical software  was used for data analysis. Quantitative data are presented as mean ± standard deviation. Student's P > 0.05).P < 0.001).As shown in As shown in Univariate linear regression analysis showed that miRNA-22 expression was negatively correlated with the CSF TIMI grade .P < 0.001) with 75.0% sensitivity and 88.1% specificity (The area under the ROC curve was 0.8293 (95% confidence interval: 0.7313–0.9272, cificity , suggestTo our knowledge, this is the first study to identify miRNA-22 as a potential early diagnostic biomarker of CSF. The expression of miRNA-22 was found to be upregulated in patients with CSF, and ROC analysis and univariate linear regression analysis of the TIMI frame count further indicated that miRNA-22 had significant diagnostic power for CSF.CSF refers to a phenomenon whereby normal or near-normal major branches of the coronary arteries are observed on coronary angiography but there is delayed blood flow in distal blood vessel upon imaging –4. TheremiRNAs are highly conserved with the primary function of negatively regulating gene expression at the posttranscription level. miRNAs are broadly expressed in tissues and demonstrate high tissue specificity; approximately 60% of the human genes are regulated by miRNAs. miRNAs are also present in many types of body fluids such as plasma and serum, and extracellular circulating miRNAs are found enclosed within various vesicles, such as microvesicles, exosomes, and apoptosomes . CirculaMALAT1 protects the endothelium against oxidized low-density lipoprotein-induced dysfunction via upregulating the expression of the miRNA-22-3p target genes CXCR2 and AKT [AKT3 [Although the pathophysiological mechanism of CSF remains unclear, several potential mechanisms have been suggested. Inflammation , vasocon and AKT . Zheng eKT [AKT3 . HoweverHuang et al. recently showed that the serum level of miRNA-155 was upregulated in patients with CSF compared with that of the control group, indicating that miRNA-155 could be another potential biomarker for CSF . AlthougAlthough we detected a strong association between serum miRNA-22 levels and CSF, key limitations of the study need to be mentioned. First, only 86 patients were enrolled in this study, which is an insufficient number to make a substantial conclusion. Therefore, a large-scale study is required to substantiate our results and to further validate the specificity and sensitivity of the candidate biomarkers. Second, as a retrospective study, bias may arise from many factors. For example, though a common and useful method of selection and detection was applied in our study, selection bias or detection bias may lead to a spurious association between the predictor variable and outcome in the study sample, but not in the population, which is unavoidable. Finally, an experimental model should be established to further verify the targets of miRNA-22 and its role in the progression of CSF.In summary, the relative expression of serum miRNA-22 is increased in patients with CSF, suggesting that changes in miRNA-22 expression may be involved in the occurrence of CSF. Serum miRNA-22 may serve as a biomarker for the early diagnosis of CSF. Further studies with a larger sample size are required to validate the association between serum miRNA levels and CSF to provide a reference for the clinical diagnosis and treatment of CSF.


Pythium insidiosum is a widespread pathogen that causes pythiosis in mammals. Recent increase in cases reported in North America indicates a need to better understand the distribution and persistence of the pathogen in the environment. In this study, we reconstructed the distribution of P. insidiosum in the Chincoteague National Wildlife Refuge, located on Assateague Island, Virginia, and based on 136 environmental water samples collected between June and September of 2019. The Refuge hosts two grazing areas for horses, also known as the Chincoteague Ponies. In the past 3 years, 12 horses have succumbed to infection by P. insidiosum. Using an ecological niche model framework, we estimated and mapped suitable areas for P. insidiosum throughout the Refuge. We found P. insidiosum throughout much of the study area. Our results showed significant monthly variation in the predicted suitability, where the most influential environmental predictors were land-surface water and temperature. We found that June, July, and August were the months with the highest suitability for P. insidiosum across the Refuge, while December through March were less favorable months. Likewise, significant differences in suitability were observed between the two grazing areas. The suitability map provided here could also be used to make management decisions, such as monitoring horses for lesions during high risk months. Pythium insidiosum is the only etiologic agent of pythiosis that affects mainly mammals in tropical and subtropical countries   –10. Zoossidiosum , 4. Prevsidiosum , 12. Zoosidiosum , 14. Zooike 37°C .P. insidiosum resemble the typical plant pathogens of the genus Pythium sensu lato, which tend to be ubiquitous with broad host ranges  data collection, considering the definition of sampling sites for water sample collection, isolation, and identification of sidiosum , (ii) exsidiosum , (iii) Esidiosum , followesidiosum .The water samples were collected from moist soil management units , localized ephemeral ponds and watering holes, and tidal salt marsh located within both grazing paddocks throughout the Virginia portion of Assateague Island . SamplinPythium proceeded as in July.At each sampling site , 500 ml P. insidiosum exhibits a distinct colony morphology compared to other common Pythium species. PCR identification was also used for all isolated Pythium colonies. A small amount of mycelia was collected from 2 day-old Pythium cultures and genomic DNA extracted from the mycelia using the REDExtract-N-Amp kit (Sigma-Aldrich): mycelia were placed in 50 μl extraction solution in a 0.2 ml sterile PCR tube, vortexed well, and then incubated at 95°C for 10 min. After incubation, 50 μl dilution solution was added and vortexed again. Extracted genomic DNA was stored at 4°C. To confirm successful DNA extraction, the ITS region was first amplified using ITS1 and ITS4 primers  of each paddock: 18,797 cells in the north, and 3,086 in the south paddock. To assess the difference between suitability distributions between the paddocks we used the Two-sample Welch T-statistic  consortium  in July, and we did not detect P. insidiosum from any water with salinity >7 ppt. In June and July, P. insidiosum was detected at water temperatures ranging from 25 to 38°C. In September, P. insidiosum was detected in water of 21 and 22°C.In total, 30 spatially unique sites were determined positive for P. insidiosum samples  was quadratic + threshold + hinge with a regularization multiplier of 1.9 . The ovee island . When coe island . While te island . The pree island .Ecological niche models showed substantial monthly variation in the predicted suitable areas across the island. We observed that the monthly fluctuation in the proportion of suitability affected both paddocks similarly, while the southern paddock produced a higher proportion of suitable areas, except during November .P. insidiosum distribution were land-surface water, assessed through NDWI (median = 39.4%), minimum temperature (median = 26.8%), followed by mean annual temperature (median = 19.3%)  . However= 19.3%) .To visualize the variation in the probability of presence (suitability) as each environmental variable is varied, we looked at the MaxEnt response curves of the annual model . Our resmean=−37, p < 0.01). Our results also showed that the lowest suitability values were observed during the cold months, between December and March, while the highest levels during June, July, and August , followed by woody wetlands (30.7%), and barren land (21.1%). These types of land cover are widely distributed across the island, with 38.1, 24.6, and 26.5% of coverage in the study area, respectively, being mostly distributed along the western side .P. insidiosum than the northern paddock, and the summer months more suitable than the winter months. These observations could help guide exposure management and pathogen surveillance.Our results showed that the majority of the refuge exhibits moderate to high suitability, although we observed a cold-spot between the two grazing areas. Proportionally, the southern paddock was more suitable for P. insidiosum in the Chincoteague National Wildlife Refuge, Assateague Island Unit located in the state of Virginia, United States. The distribution of positive water samples indicated a widespread prevalence of P. insidiosum swimming zoospores in the Refuge. Our ecological niche model predicted suitable areas for P. insidiosum zoosporulation across the Refuge, beyond the areas where the disease has been detected within the horse population. Novel areas predicted at risk include ephemeral wetlands associated with maritime forest and adjacent emergent freshwater/brackish marsh. The most dominant plant species associated with these habitats is Spartina patens, however, emergent vegetation within freshwater impoundments include, Eleocharis sp., Bidens sp., Polygonum sp., Scirpus sp., Bacopa monnieri, and Phragmites australis. We observed a higher potential exposure of the horse population to suitable conditions for the presence of P. insidiosum during June, July, and August, especially in the southern paddock.This study reconstructed the potential distribution of P. insidiosum   , 30, ourr months . Our resP. insidiosum zoospores and high-resolution satellite imagery data. ENMs have been widely applied in epidemiology to predict species' potential distributions aiming to anticipate the future emergence of pathogens  approach using the occurrences of athogens –66. Thisathogens –69. NeveFinally, this study highlights the imperative need to improve our ability to adequately define risk areas for pythiosis, given its severe and often fatal impact on infected animals despite its overall low incidence. Thus, the information generated here is a valuable first step toward informing pythiosis prevention and control plans in the Chincoteague National Wildlife Refuge and a first step toward defining high risk environments elsewhere.P. insidiosum, by identifying its geographic and environmental distribution in the Chincoteague National Wildlife Refuge Island. Using an ecological niche model approach, we produced an annual high-resolution map to visualize the most suitable areas for the presence of infectious P. insidiosum zoospores. The suitability map could be used to identify areas of higher risk for surveillance and of lower risk for grazing. We also found that June, July, and August were the months that showed the highest suitability for P. insidiosum in both grazing areas, especially in the southern paddock, with potential elevated suitability in isolated wet areas throughout the summer months. The information generated in this study could be useful to design dynamic management plans aimed at reducing the risk of exposure to P. insidiosum.In this study, we examined the ecological conditions favoring gmachad@ncsu.edu.The datasets presented in this article are not readily available. Requests to access the datasets should be directed to EG and GM conceived the study, designed the study, and secured the funding. EG, GM, and KH conducted field sampling. XW, KH, and EG processed the samples and did the laboratory isolations. MJ conducted data processing, cleaning, and data analysis. MJ, XW, EG, KH, and GM wrote and edited the manuscript. All authors discussed the results and critically reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Glioblastoma is the most aggressive form of primary brain cancer, with a median survival of 12–15 months. The P2X receptor 7 (P2X7R) is upregulated in glioblastoma and is associated with increased tumor cell proliferation. The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is also upregulated in glioblastoma and has been shown to have both pro- and anti-tumor functions. This study investigates the potential mechanism linking P2X7R and GM-CSF in the U251 glioblastoma cell line and the therapeutic potential of P2X7R antagonism in this setting. P2X7R protein and mRNA was demonstrated to be expressed in the U251 cell line as assessed by immunocytochemistry and qPCR. Its channel function was intact as demonstrated by live cell confocal imaging using a calcium indicator Fluo-4 AM. Inhibition of P2X7R using antagonist AZ10606120, decreased both GM-CSF mRNA (P < 0.05) and protein (P < 0.01) measured by qPCR and ELISA respectively. Neutralization of GM-CSF with an anti-GM-CSF antibody did not alter U251 cell proliferation, however, P2X7R antagonism with AZ10606120 significantly reduced U251 glioblastoma cell numbers (P < 0.01). This study describes a novel link between P2X7R activity and GM-CSF expression in a human glioblastoma cell line and highlights the potential therapeutic benefit of P2X7R inhibition with AZ10606120 in glioblastoma. They are derived from glial cells, with an overall incidence between 3 and 6 per 100,000 persons5. They are classified into grades I–IV based on the criteria of histology and molecular features, as outlined by the World Health Organization6. Grade IV glioma, also known as glioblastoma, is the most common and most aggressive form, representing ~ 55% of all gliomas with an incidence rate between 0.59 and 3.69 per 100,000 persons7.Gliomas are the most common primary malignancies of the central nervous system (CNS), representing 81% of malignant brain tumors in adults1. The current standard therapy for glioblastoma includes surgical resection of the tumor, followed by radiotherapy and adjuvant chemotherapy using temozolomide (TMZ)8. Despite these treatments, glioblastoma is associated with high mortality: the average survival time is 14.6 months, and 5 year survival is < 5%9. Hence, targeted therapies that can specifically inhibit the growth of the glioblastoma cells and improve prognostic outcomes are urgently needed.Glioblastoma commonly affects younger individuals, with the median age of presentation between 30 and 40 years11, with recruitment of large numbers of microglia and macrophages to the tumor site. These responses increase the release of bioactive factors that promote tissue invasion and tumor cell proliferation13, which can result in infiltration of glioblastoma cells into surrounding healthy tissue11. Therefore neuroinflammatory mediators are of key interest in understanding the link between neuroinflammation and glioblastoma propagation14.Neuroinflammation plays a key role in the pathogenesis of glioblastoma. It involves the activation and recruitment of immune cells such as microglia, monocytes and lymphocytes that can release a variety of bioactive factors that influence the tumor microenvironment. In glioblastoma, neuroinflammation is integral to the ongoing proliferation of the tumor15. It is expressed on a number of immune cells, such as monocytes, macrophages and microglia. P2X7R is upregulated in a number of human cancers, including both glioma cells and microglia in glioblastoma16 and its inhibition decreases tumor growth19. It has been shown that P2X7R activation interacts with multiple inflammatory intracellular signaling cascades including the NLRP3 inflammasome, the PI3K/Akt pathway, NFκB activation and subsequent release of Interleukin 1 beta (IL-1β)25. The P2X4 receptor has also been shown to potentially interact with the P2X7 receptor to activate these inflammatory pathways26. Recent studies have highlighted the role of P2X7R in glioblastoma tumor cell proliferation, the recruitment of microglia and the promotion of an inflammatory microenvironment29. Inhibition of this receptor has been demonstrated to decrease tumor cell number in human glioma cultures30. These results support the notion that P2X7R plays a trophic role in glioblastoma and highlights the potential of P2X7R as a therapeutic target to reduce the progression of glioblastoma.The P2X purinoceptor 7 (P2X7R) is a key component in the neuroinflammatory cascade. P2X7R is a purinergic signaling receptor which binds adenosine triphosphate (ATP) to form a transmembrane cation channel31. It is produced in response to pro-inflammatory cytokines, and functions as a cytokine promoting proliferation, migration and development of several myeloid populations including neutrophils, eosinophils and macrophages through well characterized inflammatory pathways such as Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling and NFκB activation34. In the cancer setting, GM-CSF has been described to have dual roles, showing both pro-tumor and anti-tumor effects35. It is currently a key cytokine being investigated in the emerging field of cancer immunotherapy, due to its broad range of immunostimulatory effects acting on several different immune cells36. Recent cancer vaccines utilize this by inducing GM-CSF secretion from inactivated tumor cells to stimulate dendritic cells and macrophages to increase antigen presentation and the immune system’s ability to recognize and destroy tumor cells38. However, GM-CSF has also been shown to promote tumor progression, possibly via activation of Signal transducer and activator of transcription 3 (STAT3) or upregulation of vascular endothelial growth factor (VEGF)35, and decreasing GM-CSF levels has been shown to suppress glioblastoma cell growth indicating a potential stimulatory effect on glioblastoma progression39.GM-CSF, also known as colony-stimulating factor 2 (CSF2), is a glycoprotein secreted, for example by several innate and adaptive immune cells, including macrophages41. The human U251 glioblastoma cell line is one of the most thoroughly investigated glioblastoma cell lines and allows for specific pathways and mediators to be investigated in vitro42, with previous studies having already investigated aspects of P2X7R biology in this model43. Use of the U251 cell line allows for rapid and repeatable investigations and can provide valuable insight into tumor characteristics in culture42.To investigate the underlying causes and pathological processes in glioblastoma, several experimental models have been utilized, the most common being cell lines. Whilst there are limitations in the use of cell lines such as genetic changes occurring upon culture and inherent differences to the in vivo conditions, cell lines are still useful as initial experimental models, where results can be subsequently replicated in animal models or human samplesFrom the above, both P2X7R and GM-CSF have been implicated in tumor pathogenesis; however, a connection between them has not been investigated. It is also unclear if GM-CSF could have direct functional effects on tumor cells in the absence of immune cells. Therefore, the objective of this study was to investigate the potential link between P2X7R and GM-CSF and determine if antagonism of P2X7R and GM-CSF neutralization could serve as possible means of reducing tumor cell proliferation.Immunocytochemistry was used to show expression of P2X7R and GM-CSFα in U251 cells   and protein expression (P < 0.001) compared to controls Fig. A,B. mRNATreatment with AZ10606120 and anti-GM-CSF mAb were compared to treatment with Temozolomide, the conventional chemotherapy agent, to determine the effects on cell proliferation. Treatment with anti-GM-CSF mAb had no significant effect on tumor proliferation compared to untreated cells. Conversely, treatment with AZ10606120 (P2X7R antagonist) significantly reduced cell proliferation compared to both untreated and anti-GM-CSF mAb treatment (P > 0.01). Treatment with Temozolomide also significantly reduced cell proliferation compared to untreated and anti-GM-CSF treatment mAb, though to a lesser extent than the reduction seen with AZ10606120 treatment Fig. .Figure 4The results of this study identified expression of both P2X7R and GM-CSFRα in the U251 glioblastoma cell line and established that the P2X7R ion channel is functional in this setting. It highlights a previously undescribed mechanistic link between P2X7R and GM-CSF through changes in mRNA expression and protein levels. Lastly, it demonstrates the potential therapeutic benefit of the P2X7R antagonist AZ10606120 at reducing tumor cell proliferation . The channel responses observed in the U251 cell line were P2X7R specific as they were significantly limited by the specific antagonist of the receptor, AZ1060612051. To our knowledge, this is also the first study to demonstrate expression of GM-CSFRα and functional P2X7R in the U251 glioblastoma cell line.The P2X7R ion channel was identified as functional, measured by monitoring intracellular Ca52. Reduced levels of GM-CSF have been associated with suppression of tumor growth in glioblastoma in an immune-independent manner39. Interestingly, GM-CSF administration has also shown anti-tumor effects, being able to regulate immune responses through recruitment and activation of dendritic cells to increase tumor antigen presentation leading to immune-mediated removal of cancer cells35. This immunostimulatory function of GM-CSF is also currently being investigated for use in cancer vaccines, with GM-CSF-transduced inactive glioma cells having been shown to increase survival in glioma mouse models, by stimulating immune mediated clearance of tumor antigens53. Thus the role of GM-CSF in the glioblastoma tumor setting is dependent on the context of the surrounding microenvironment, and its role in cancer pathology, or specifically in glioblastoma progression, is not fully understood.GM-CSF gene and protein expression were significantly decreased upon treatment with AZ10606120. Whilst the exact relationship between P2X7R and GM-CSF is not described, these results confirm that P2X7R activity can influence expression of GM-CSF Fig. . Convers54. The GM-CSFR has also been reported to induce NFκB production; however the lack of GM-CSFR signalling could indicate why no changes were seen56 , resulting in degradation of the inhibitor of κB to release NFκB; therefore their unchanged mRNA expression via P2X7R inhibition is in line with this mechanism59, was used to lower GM-CSF concentrations in the U251 cell culture, mimicking one downstream consequence of P2X7R inhibition. Reducing GM-CSF in this way caused no significant changes whilst treatment of cells with AZ10606120 significantly reduced cell proliferation. Therefore, whilst AZ10606120 reduces tumor cell proliferation and GM-CSF concentration, removing GM-CSF itself is not sufficient to cause this effect. Thus, it is likely that other mediators are responsible for this effect of P2X7R antagonism on cell proliferation. These results are consistent with previous research demonstrating that P2X7R is associated with a number of downstream pro-inflammatory pathways25 and hence effects on GM-CSF could be one of many such mechanisms. The effect of P2X7R antagonism in other cancer settings have been demonstrated in vivo where AZ10606120 treatment inhibited tumor growth mouse models of pancreatic cancer19 and mesothelioma17. Temozolomide, the conventional therapy used in glioblastoma, also resulted in decreased cell proliferation, but with less efficacy when compared to treatment with AZ10606120. Considering the toxic side effects of Temozolomide treatment, these results highlight the potential for AZ10606120 as a possible safer, more efficacious treatment for glioblastoma.A neutralizing anti-GM-CSF mAb, previously demonstrated to neutralize GM-CSF induced cell proliferationThis study describes, for the first time, a link between P2X7R and GM-CSF and the effect of P2X7R antagonism on cell proliferation in human glioblastoma cell line U251. In U251 glioblastoma cell line, P2X7R is expressed and functional, and inhibition of the receptor with AZ10606120 caused a decrease in GM-CSF mRNA and protein expression. Importantly, and compared to conventional chemotherapy Temozolomide, treatment of the U251 glioblastoma cell line with P2X7R antagonist AZ10606120 resulted in a significant decrease in glioma cell proliferation. This study highlights an important link between P2X7R and GM-CSF in glioblastoma and revealed a potentially exciting new therapy in AZ10606120.2/95% O2 and routinely passaged at 80% confluency.The U-251 MG human glioblastoma cell line  was maintained in Dulbecco’s modified Eagle’s Medium , supplemented with 10% heat-inactivated Fetal Bovine Serum , penicillin–streptomycin , 1% non-essential amino acids (Sigma) and sodium pyruvate . Cells were cultured in a humidified incubator at 37 °C, 5% COCells were grown to 70% confluency and then treated for 72 h unless specified otherwise with the following reagents: human GM-CSF , AZ10606120 , anti-human GM-CSF monoclonal antibody  and Temozolomide .60) Alexa Fluor 488 conjugate , anti-GM-CSFRα Alexa 594 conjugate , mouse anti-human GM-CSFRα non conjugate , anti-P2X7R FITC (Fluorescein isothiocyanate) conjugate , goat anti-P2X7R non-conjugated  and rabbit anti-GFAP non conjugated . The following secondary antibodies were used: Alexa Fluor 488 , Texas Red 633 anti-goat  and Texas Red 594 anti-rabbit . Samples were washed and counterstained with 5 μM of 4′,6-diamidino-2-phenylindole (DAPI) nuclear stain (Invitrogen), then mounted and imaged using a Nikon A1r confocal microscope. All experiments were conducted with negative controls .Cultured cells were fixed with 50% acetone/methanol at − 20 °C for 15 min, washed and immersed in 2% Bovine Serum Albumin  for 1 h at 37 °C. Samples were incubated with the following primary antibodies for 48 h at 4 °C: anti-GFAP -1-piperazineethanesulfonic acid (HEPES) buffer and incubated with 5 μM of Fluo-4 AM  for 1 h at room temperature. The cells were then imaged using a Ziess LSM 510 M META fluorescence confocal microscope. To elicit P2X7R channel activity the cells were stimulated with P2X7R agonist BzATP  and the change in fluorescence intensity was measured using the TimeSeries component of the software where images were captured every 15 s. To make sure that the changes in fluorescence were P2X7R specific some coverslips were preincubated with P2X7R antagonist AZ10606120 (15 µM) for 15 min prior to the above. Channel function was quantified as change in fluorescence before and after addition of BzATP (ΔF) and expressed as the relative change in fluorescence intensity (ΔF/Fmin) which correlates with changes in Ca2+ concentration as a function of ion movement through the P2X7R ion channel.Cells were grown on 18 mmT method with relative expression compared to housekeeping genes61.Cultured cells were treated with human GM-CSF (15 ng/mL), AZ10606120 (15 μM) or left as untreated controls. At termination cells were gently scraped, centrifuged and frozen at − 80 °C. Total RNA was isolated using a QIAGEN RNeasy Plus Mini Kit (QIAGEN) and a QIAcube robotic workstation (QIAGEN) following the manufacturer’s protocol. 2 μg of total RNA per sample were converted to cDNA using the QuantiTect Reverse Transcription Kit (QIAGEN) containing Quantiscript Reverse Transcriptase, Quantiscript RT Buffer and RT primer Mix following the manufacturer’s protocol. 50 ng cDNA per sample was analyzed using catalogued human Taqman gene expression assays (Life Technologies) for P2X7R (assay ID Hs00175721_m1), GM-CSF , NF-kappa B Nuclear Factor subunit 1 , NF-kappa B Nuclear Factor subunit 2 , GM-CSF receptor subunit α  GM-CSF receptor subunit β  and 5 housekeeping genes, namely Glyceraldehyde 3-phosphate dehydrogenase , β-actin (assay ID Hs01060665_g1), Succinate dehydrogenase complex, subunit A , Peptidylprolyl isomerase A  and 60S ribosomal protein L13a . qPCR was carried out using a QuantStudio7 Flex Real-Time PCR system (Thermo-Fisher). Relative assessment of mRNA was performed using the ΔΔCCultured cells were treated with AZ10606120 (15 µM) or left as untreated controls for 72 h, and supernatant collected. ELISA was performed using the Human GM-CSF Quantikine ELISA Kit (R&D Systems) with 200 μL per sample (in duplicate) loaded into the ELISA plate following manufacturer’s protocol. Optical density was measured using a Multiskan Spectrum microplate reader (Thermo-Fisher Scientific) set to 450 nm (with correction at 540 nm) with SkanIt Software 2.4.2. GM-CSF protein levels were determined from a standard curve constructed from a dilution series of standard absorbance values.Samples treated with AZ10606120 (15 μM), anti-human GM-CSF (20 ng/mL), TMZ (50 μM) or control (untreated) were fixed on 18 mm coverslips with 50% acetone/methanol for 15 min at − 20 °C, washed with Phosphate Buffered Saline , incubated with 5 μM of DAPI nuclear stain  and then washed and mounted. Images were taken using an Olympus IX-81 fluorescence microscope with a 40× air objective lens. Twenty-five random fields were imaged per sample, and cells counted using Metamorph Imaging Software .All statistical tests were completed using GraphPad Prism 7 software. D'Agostino & Pearson normality test was used to determine normality. Non-parametric data was analyzed by Mann Whitney U test and Kruskal–Wallis test with post hoc Dunn’s multiple comparisons depending on the number of comparisons. Unpaired t-tests and one-way Analysis of Variance (ANOVA) with post hoc Tukey’s HSD tests were utilized for parametric data. Data was expressed as mean ± S.E.M with statistical significance set at P < 0.05.


Agitated patients in the prehospital setting pose challenges for both patient care and emergency medical services (EMS) provider safety. Midazolam is frequently used to control agitation in the emergency department setting; however, limited data exist in the prehospital setting. We describe our experience treating patients with midazolam for behavioral emergencies in a large urban EMS system. We hypothesized that using midazolam for acute agitation leads to improved clinical conditions without causing significant clinical deterioration.We performed a retrospective review of EMS patient care reports following implementation of a behavioral emergencies protocol in a large urban EMS system from February 2014–June 2016. For acute agitation, paramedics administered midazolam 1 milligram (mg) intravenous (IV), 5 mg intramuscular (IM), or 5 mg intranasal (IN). Results were analyzed using descriptive statistics, Levene’s test for assessing variance among study groups, and t-test to evaluate effectiveness based on route.In total, midazolam was administered 294 times to 257 patients. Median age was 30 (interquartile range 24–42) years, and 66.5% were male. Doses administered were 1 mg (7.1%) and 5 mg (92.9%). Routes were IM (52.0%), IN (40.8%), and IV (7.1%). A second dose was administered to 37 patients. In the majority of administrations, midazolam improved the patient’s condition (73.5%) with infrequent adverse events (3.4%). There was no significant difference between the effectiveness of IM and IN midazolam .A midazolam protocol for prehospital agitation was associated with reduced agitation and a low rate of adverse events. Agitated patients pose challenges for both patient care and emergency medical services (EMS) provider safety, but there is no consensus regarding the optimal medication to manage prehospital agitation.4Previous studies in the emergency department (ED) setting demonstrate that benzodiazepines and antipsychotics can be effective agents todecrease patient’s level of agitation.7In this study, we report the rate of clinical improvement, need for repeat dosing, and rate of adverse events for a prehospital behavioral emergencies protocol using midazolam. Secondarily, we compare the effectiveness and safety of IN midazolam to IM midazolam for treating behavioral emergencies.We performed a retrospective chart review of patients who were administered midazolam by EMS for behavioral emergencies from February 2014 through June 2016. We conducted this study in the Chicago EMS System, which serves an estimated 2.7 million residents and covers 237 square miles. The Chicago EMS System is a regional collaborative of hospital-based, EMS physicians and nurses who provide medical oversight for EMS provider agencies within the system, including the Chicago Fire Department (CFD), which provides emergency response to all 9-1-1 calls. CFD is an urban, fire-based EMS agency with over 280,000 annual transports, of which approximately 3% are for behavioral or psychiatric emergencies and related complaints.In 2014, the Chicago EMS system implemented a new protocol for management of patients with behavioral emergencies using midazolam. Paramedics had previous training on the use of IN medications and on the use of IV midazolam for other indications. They underwent additional training for the use of midazolam for behavioral emergencies including IN delivery. Per protocol, paramedics attempted verbal de-escalation techniques and physical restraint, but if a patient remained combative and physically dangerous to themselves and others, paramedics could administer midazolam. Midazolam dosing was 1 milligram (mg) IV or 5 mg either IM or IN (repeating once as needed), guided by prior studies.What do we already know about this issue?While prehospital agitation poses a significant challenge to emergency medical services providers, medications can help improve safety of care and transport.What was the research question?Is a prehospital protocol using midazolam for agitation safe and effective?What was the major finding of the study?Use of this protocol was associated with decreased agitation and had a low rate of complications.How does this improve population health?Most of the agitated patients in our study were from racial minorities. Identifying the optimal treatment for agitation is important for this at-risk population.Using SafetyPAD software , we extracted all EMS patient care reports from February 2014 through June 2016 of patients for whom 9-1-1 was called and in which midazolam was administered. We included all cases in which midazolam was administered to adult patients for behavioral emergency via the IM, IN, or IV route. We excluded all cases in which midazolam was given for indications other than behavioral emergency, cases in which midazolam was administered other than via IM, IN, or IV routes, and cases with dosages outside the range prescribed in the protocol. Per EMS patient care protocol, we excluded patients less than 18 years of age or greater than 60 years of age. Additionally, we excluded cases if key data elements were missing, such as dose, route, or patient response.Patient demographic information including age, gender, and race was collected and included in analysis. Additionally, an unblinded abstractor (RH) reviewed all charts for complications, predefined as systolic blood pressure < 100, oxygen saturation < 95%, use of airway intervention, and mention of provider injury. RH was trained in chart abstraction by another author (CTR). Meetings were held weekly during chart abstraction to answer questions and review results.We evaluated the dose, route, need for repeat dosing, and clinical effect of midazolam administrations, and we performed descriptive statistics for administrations of midazolam for behavioral emergencies. Levene’s test was used to assess variance between aggregate groups of “any improvement” (significant and slight improvement) and “no improvement.” Using t-tests, we compared effectiveness between the routes and rate of adverse events for IN and IM administrations. We used Stata 15  to create descriptive statistics, calculate confidence intervals (CI), and perform the t-tests.Northwestern University’s institutional review board approved the study.During the study period, 478 patients received midazolam. We excluded 221 cases for indications other than behavioral emergency, deviations from dosing protocol, or missing data . After eIncluding all administrations of midazolam (n = 294), paramedics noted improvement in 73.5% of cases, 34.5%  of which had substantial improvement in level of agitation and 39.3%  had slight improvement. No improvement was noted in 25.5%  of cases, and 0.6%  had clinical deterioration. Of all administrations, 52.0% were IM, 40.8% were IN, and 7.1% were IV. The doses administered were 1 mg (7.1%) and 5 mg (92.9%).In the subset of first-dose midazolam administrations (n = 257), paramedics reported substantial improvement in 32.7% , slight improvement in 39.3% , no change in 27.2% , and deterioration in 0.8%  . The rouIn those patients requiring a repeat dosage (n = 37), paramedics reported improvement in 83.7% of cases, with substantial improvement noted in 43.2%  of patients, and slight improvement documented for 40.5%  of patients. No change was noted in 16.2%  of patients . The rouIM and IN routes of midazolam administration were compared for effectiveness after applying Levene’s test to compare “any improvement”  to “no improvement” . The datasets were found to be homogenous (p = 0.18). Using a t-test, we found no significant difference between “any improvement” after IN midazolam (71.0%) and IM . There was also no significant difference found between the documented adverse events of IM midazolam (3.9%) and IN . Additionally, we found no significant difference between the rates of reported EMS provider injury for IM (3.9%) and IN  doses. The majority of these injuries were kicks or bites by the patient, with no needlestick injuries reported.Paramedics reported six adverse events thought to be due to midazolam administration, and an additional three adverse events were identified upon chart review. Adverse events included hypotension (systolic blood pressure < 100 millimeters of mercury [mmHg]) (n = 3); hypoxia with airway intervention required (n = 1); hypoxia without airway intervention (n = 1); unresponsiveness (n = 2); traumatic cardiac arrest (n =1); and worsening agitation (n = 2). For the nine cases, all happened after a single 5 mg dose administered via IM (n = 6) or IN (n = 3) routes. The patient who received an airway intervention had an oropharyngeal airway placed and bag-valve-mask ventilation performed. In the two cases of hypotension with systolic pressures less than 100 mmHg, none of the cases had a systolic blood pressure less than 90 mmHg systolic. Of patients experiencing unresponsiveness, two were given midazolam for agitation after naloxone was administered for suspected opioid overdose. The patient who experienced traumatic cardiac arrest sustained blunt trauma injuries after a four-story fall. This patient received 5 mg of midazolam IM to facilitate safe and timely transport in the setting of severe trauma and experienced cardiac arrest during transport.To our knowledge, this study represents the largest cohort of prehospital patients administered midazolam as a single agent for behavioral emergency. Paramedics reported clinical improvement in a majority of patients following midazolam administration. Only 14.4% of patients required a second dose, after which, the majority were assessed by paramedics to have a clinical improvement. Over 294 administrations, adverse events were noted in 3.1%, all with IM or IN dosing. Based on this improvement rate and low complication rate, a prehospital behavioral emergencies protocols using midazolam to control agitation may be considered for use in EMS systems.One notable advantage of midazolam over alternative agents is the possibility for IN administration. In this study, IN administration represented 40.8% of the midazolam administrations, suggesting a preference of IN route by EMS providers. Additionally, we found IN midazolam to be no less effective than IM midazolam. These results suggest that IN administration may represent a preferable route of delivering midazolam, particularly as the IN route eliminates the risk of needlestick provider injury.Adverse events after midazolam use for behavioral emergency were rare in this study, with only 3.1% of patients experiencing an adverse event, all after initial administration of midazolam. Hypoxia and apnea were also rare, with only one patient requiring any airway intervention and no patients requiring intubation in the field. For the patient administered midazolam in order to facilitate transport in the setting of blunt trauma, they likely experienced cardiac arrest due to injuries rather than midazolam administration. Further studies are needed to investigate the safety of midazolam administration in the setting of known or suspected opioid use as prior studies have demonstrated that concurrent benzodiazepine and opioid administration increases the risk of respiratory depression.19Prehospital providers are frequently exposed to agitated patients, and improved strategies are needed to safely care for these patients.21An alternate agent for prehospital agitation that has attracted attention over the last decade is ketamine. Despite being used since the 1960s, ketamine has only recently been evaluated for use in treating agitation in the prehospital and ED settings and has been shown to be effective in controlling agitated patients in several studies.10Using paramedic impression as an outcome limits the results of this study. While paramedic impression is certainly important for prehospital treatments and has been used in prior studies, using a standardized aggression scoring systems may more accurately measure effectiveness of midazolam in treating agitation and improve external validity.We did not limit midazolam administration to excited delirium, so these results may not accurately represent the effects of midazolam on excited delirium. Excited delirium represents a small portion of agitated patients though, and protocols are necessary for control of agitation in a variety of clinical scenarios. Additionally, despite 3% of transports being for behavioral complaints, less than 0.1% of patients received midazolam during the study period. This likely suggests that non-pharmacological approaches may be adequate to address the majority of behavioral emergencies.Adverse events were limited to paramedic documentation and chart review, which may not capture all adverse events, particularly paramedic injuries. We excluded more than a third of cases for dosing deviations, the large portion of which were due to online medical control. Excluding these deviations might bias the results by missing adverse events, but on analysis of all midazolam administrations, the adverse rate was similar at 2.9% for all administrations compared to 3.1% for per protocol. Lastly, this study was performed in a single EMS system, and further studies could verify effectiveness in other settings.In a large urban EMS system, we found that a prehospital behavioral emergencies protocol using midazolam was associated with improved agitation and a limited number of adverse events. Additionally, we demonstrate the effective use of IN midazolam for use in a prehospital behavioral emergencies protocol. Further studies will be needed to validate these findings in other EMS systems and to compare midazolam to other pharmacological options to help determine the ideal agent for the agitated prehospital patient.


Surface electromyography (sEMG) is the main non-invasive tool used to record the electrical activity of muscles during dynamic tasks. In clinical gait analysis, a number of techniques have been developed to obtain and interpret the muscle activation patterns of patients showing altered locomotion. However, the body of knowledge described in these studies is very seldom translated into routine clinical practice. The aim of this work is to analyze critically the key factors limiting the extensive use of these powerful techniques among clinicians. A thorough understanding of these limiting factors will provide an important opportunity to overcome limitations through specific actions, and advance toward an evidence-based approach to rehabilitation based on objective findings and measurements. Walking is one of the most essential activities of daily living (ADL) . The stuInstrumented gait analysis provides comprehensive data on normal and pathological gait, which are useful in clinical practice producing objective information about time-distance variables , joint motions (kinematics), and joint moments and powers (kinetics) . In the Indeed, the knowledge about the dynamic contractile activity of the muscles during pathological gait may provide unique information to help clinicians in the following activities:to support diagnosis , 23to design complex surgical interventions to design personalized rehabilitation protocols and objectively prove their effectiveness , including new rehabilitation trends exploiting exoskeletons, e.g., in acute stroke patients , neuroreto support clinical decision  – 35for the production of quantitative reports to optimize patient's follow-up or to conduct longitudinal studies to evaluate muscle fatigue  –39to support forensic medicine with objective outcomes  , 41.Despite the wide variety of possible clinical applications described above and their unquestionable relevance, clinicians underutilize instrumented gait analysis (GA) , especiaIn the following, we will indicate with the acronym sEMG-GA gait analysis when it includes the recording of sEMG signals for sensing muscle activity during locomotion. SEMG-GA requires the acquisition of sEMG signals from the main lower limb muscles and, in some cases, from the trunk . The armIn a standard sEMG-GA session –54, sEMGA sEMG-GA test requires, overall, from 15 to 30 min (including sensor positioning). It is well-tolerated by children, adults, and the elderly, and by patients affected by a wide variety of pathologies altering locomotion patterns , 56–63. Yet, although there is a relevant number of studies supporting the use of sEMG in clinical gait analysis , 60, 67,In this section, we discuss the following key factors limiting the widespread use of the sEMG signals in clinical gait analysis:lack of normative (reference) data of sEMG patterns [section Lack of Normative (Reference) Data Regarding sEMG Patterns]low intra-operator repeatability and inter-operator reproducibility in the collection of high-quality sEMG signals inappropriate use of treadmill  ]difficulties in the sEMG-GA interpretability due to the large intra-subject variability of myoelectric patterns (section Difficulties in the sEMG-GA Interpretability Due to the Large Intra-subject Variability of Myoelectric Patterns)lack of reliable/compact/unique clinical scores obtainable from sEMG-GA and discuss their characteristics and criticalities. The pinpointing of these limiting factors are the result of a 15-year experience of cooperative work and tight collaboration with clinicians of different specialties , in different gait analysis laboratories , with the aim of solving the research and clinical questions they had through sEMG-GA systems.For each of these limiting factors, we also present, when available, possible solutions to overcome the described criticalities. When solutions are not currently available, we suggest future developments that might help bridging the gap between academic knowledge and clinical practice.When a physician has available a sEMG-GA report, the first question that comes to his/her mind is: “How do I interpret this exam?” To provide a satisfying answer to this fundamental question, normative (reference) data on healthy populations are necessary. These normative data should be available, for each age class , differentiated by gender and body mass index (BMI). However, there is a lack of open databases of “physiological” sEMG activations patterns. One study analyzed 100 typically developing children aged 6–11 . AnotherThe authors suggest that the sEMG-GA systems to be used in clinics should be designed to automatically support clinicians with reliable reference data. Just as reference ranges  appear on a blood test report, reference ranges should appear on a sEMG-GA report. Consequently, it is strongly advisable to integrate sEMG reference datasets into newly designed systems for clinical gait analysis. It would be ideal to produce these embedded reference datasets following the recommendation guidelines established through worldwide accepted standards, specifically developed for clinical gait analysis .In a gait analysis laboratory, different professional figures may perform the acquisitions, such as biomedical engineers, gait analysis experts, physiatrists, physical therapists, and students. They have different expertise and some of them may lack experience in sEMG probe positioning, in recognizing the presence of detrimental artifacts in the signals, or in being aware of signal saturation or very low signal-to-noise ratio (SNR). This can lead to low inter-operator reproducibility. However, in clinical gait analysis, it is fundamental to guarantee that the different operators alternating in the various shifts do not affect the outcome measures derived from sEMG-GA. It follows that user-independent systems are required. Furthermore, it is also fundamental that the same operator is able to provide repeatable outcome measures, at different time-points, for a specific patient .A key factor to promote intra-operator repeatability and inter-operator reproducibility is the automatic assessment of the quality of the sEMG signals acquired, performed during the acquisition itself. However, there is a lack of systems designed to provide this essential feature . We sugg- GREEN: ok, good signal quality;- YELLOW: sufficient, signal quality should be improved if possible;- RED: completely inadequate, please stop the acquisition and check the electrodes.Frequently, sEMG signals are collected while the patient walks on the treadmill , 69, 70.However, the use of treadmill to study gait in pathological subjects may be rather inappropriate. Indeed, patients affected by neurological or musculoskeletal pathologies are not always able to walk on a treadmill, or it could be unsafe testing them on a treadmill, since additional balance skills are required to walk on a treadmill with respect to walk overground, naturally, at self-selected speed. Furthermore, when on the treadmill, the use of harnesses, or the fact that the patient, to maintain balance, leans on the treadmill horizontal bars or grasps vertical bars alters patient's perception, proprioception and muscle activation patterns. In addition, also if dynamic balance can be properly maintained without any external help, the muscle activations patterns during natural and treadmill gait are not the same . It was Already 15 years ago, our research team designed a multichannel recording system for clinical gait analysis that integrated this design concept. The system was technologically transferred to an Italian company to reach the market  and it is being sold mainly in Italy and Spain. Thanks to this device, the possibility to perform sEMG-GA in the clinical setting, during overground walking, was fully demonstrated by several works , 75, 76.In recent years, the market revolution around wearable sensors based on Inertial Measurements Units (IMUs) has taken hold and is trying to substitute traditional motion capture systems with new devices, allowing for out-of-the-lab and low-cost motion analysis –79. We eIt is well-known that human locomotion is characterized by a high intra-subject variability . Each gaextrinsic and intrinsic sources of sEMG variability.In the following, we will analyze various issues related to the sEMG gait variability and how it can make it difficult to interpret sEMG-GA, if not properly handled. In particular, we will distinguish between Among the problems to tackle for analyzing a natural walk lasting several minutes, there is the fact that the acquisition should be performed, at least in theory, along a straight walking track between 200 and 500 m of length. However, this is unfeasible in many practical situations, for both technical and logistic issues, and it would require outdoor pathways. A reasonable solution is to have available, indoor, a large room or a long corridor (of length 10–15 m), which is not difficult to obtain in a hospital setting. Therefore, the patient can walk continuously, without interruptions, back and forth along the corridor. When arrived at the end of the walking track, the patient simply turns, reverses his/her direction, and keeps on walking, for many rounds. At each round, the patient travels for 10–12 gait cycles along the straight path, at an approximately steady velocity. Walking uninterruptedly for several minutes allows the patient to walk naturally, as in everyday life. Indeed, after a few rounds, the patient feels at ease and walks at his/her natural pace. Then, the signal acquisition can start.indirect measurements, by using stereophotogrammetric systems or wearable IMU sensors; (2) direct measurements, by using sensorized mats, foot-switches or foot-pressure insoles.To process gait signals during overground walking, the first step is to segment gait cycles occurred during straight steady-state locomotion, separating them from the cycles relative to the direction changes, including decelerations before, and accelerations after the U-turns. As mentioned above, sterephotogrammetric systems have been historically considered the gold standard in gait analysis, both with and without an associated sEMG investigation. However, they never truly succeeded to help medical practitioners in clinical gait analysis, and most of the research-work done remained confined to academic studies. Indeed, stereophotogrammetric systems are expensive, they require a dedicated gait analysis laboratory and technical personnel, their sample volume is intrinsically limited to a few cube-meters, and they are complex to use, necessitating highly trained experts  to manage the system calibration and acquisition procedures. On the other hand, IMU systems are experiencing a “market boom” in many different applications, since they are lightweight, low-cost, and wearable, allowing for out-of-the-lab applications. Researchers, as well as medical-device producers, are actually trying to improve the performances of IMU systems on the reconstruction of joint angle measurements and 3D biomechanical models, and to mitigate drift errors observed during gait analysis , 83, 84.T-square test)  , 87. It More specifically, placing 3 foot-switches under the heel, the first, and the fifth metatarsal heads  it is possible to obtain a 4-level basography, as shown by the green lines in H = 6.6 ± 2% GCF = 26.4 ± 4% GCP = 22.6 ± 4% GCS = 44.4 ± 4% GCHowever, other gait cycle typologies are also observed, especially (but not exclusively) during U-turns. A markedly different sEMG activity is expected in these cases. Furthermore, the specific duration of gait phases, within a specific cycle typology, depends on the individual subject and gait speed, and it slightly changes from cycle to cycle. For each subject, and for each gait cycle type , the mean, or the median value (more robust against outliers), of each gait phase duration can be calculated. In correspondence of the U-turns/outlier epochs, a relevant change in the sequence or in the duration of the gait phases appears in the basography and can be automatically detected. In pathological gait, the number of gait phases, their sequence, and duration can change with respect to normal gait, as well as the overall intra-subject variability. As an example, hemiplegic children after cerebral palsy, with a foot drop on the affected lower limb, typically strike the floor with the forefoot instead of the heel. They mainly show PFPS and/or PS sequence of gait phases instead of HFPS . NeverthWe would like to stress that, if the subject contacts the floor differently in different gait cycles , it is evident that different sEMG patterns are produced. These differences are more pronounced in the distal part of the lower limb, e.g., for the ankle flexo-extensor muscles (TA and LGS). If this source of extrinsic sEMG variability is not properly handled, the results of the analysis cannot be accurate. Hence, a fundamental step before analyzing sEMG patterns is to group together only those patterns belonging the same typology of gait cycle.While a gait analysis expert can select the subject's most representative gait cycles, choosing them one-by-one “manually,” this is unfeasible in clinical applications, requiring a reliable and repeatable analysis of hundreds of gait cycles, in a user-independent manner. Therefore, in summary, it is advisable that systems designed for clinical sEMG-GA incorporate algorithms to:- remove U-turns and outlier epochs- segment and classify gait cycles and their frequency of occurrence- focus sEMG analysis on representative gait cycles of the same type.Even if sEMG signals are processed separately for each class of representative gait cycles, there are other sources of intra-subject variability that must be accounted for. More specifically, literature reports that, even in normal HFPS gait cycles of healthy subjects, a specific subject's muscle does not show a single “preferred” pattern of activation. Instead, from 3 to 5 distinct sEMG patterns are usually observed, each characterized by a different number of activation intervals occurring within the gait cycle. These are called “activation modalities” . Figure In recent years, a clustering algorithm was proposed and validated, both on healthy and pathological subjects, to manage intrinsic sources of within-subject sEMG variability in overground locomotion. This algorithm, named CIMAP (Clustering for Identification of Muscle Activation Patterns) allows grouping together sEMG patterns sharing similar timing patterns , 81, 89.The use of this kind of algorithms would enormously simplify the interpretation of sEMG signals during locomotion. Indeed, a single (but representative) PA-string might be compared before and after a rehabilitation program, or a therapeutic intervention, helping clinicians in their work. Notice that this process is scalable, and can be repeated to obtain unique PA from a cohort population (instead of a single patient) to ease the interpretation of randomized clinical trials using sEMG measurements .However, the described advanced sEMG processing tools, aimed at managing the intrinsic sources of sEMG variability, are currently not available to clinicians. Presently, there are no medical system integrating these important features. Future systems designed for clinical sEMG-GA should incorporate, cascaded to the algorithms mentioned in the previous section, CIMAP-like algorithms.After many years of tight collaborative work with clinicians, and fervent requests of help in the interpretation of sEMG signals during gait, we understood that an essential point is providing simple/compact/unique clinical scores for easily comparing the patient outcome at different time points (and with reference data).The great majority of research papers typically presents results from dozens of different parameters, typically estimated from signals of each specific muscle under study. This makes the application to patient management difficult. A few attempts can be found in literature to summarize the information obtained from sEMG-GA test in a unique and representative value of the patient's locomotion performance. One successful recent example is the introduction of a sEMG-based “asymmetry index” . This inHowever, none of the available systems for sEMG-GA integrates this index or similar ones.One of the fathers of modern sEMG analysis, Prof. C.J. De Luca, already 30 years ago warned that the sEMG signal, if not properly analyzed, could become “a seductive muse.” In the last decade, there have been intense efforts to find reliable methods to process and correctly interpret muscle activation patterns in locomotion. Nevertheless, there is still an evident gap between literature findings and clinical practice. In this contribution, we critically analyzed the main key factors limiting the widespread use of sEMG signals in clinical gait analysis.In synthesis:There is a lack of open databases related to reference populations  containing normative activation patterns as well as raw sEMG signals, collected during gait. Furthermore, there are no accepted standards on how to report muscle activation patterns.There is a lack of systems for clinical gait analysis that integrate quality information about the collected sEMG signals, in real-time, to improve intra-operator repeatability and inter-operator reproducibility.There is a lack of (wearable and wireless) systems for sEMG detection that integrate algorithms for the study of gait in natural conditions.There is a lack of systems that integrate algorithms aimed at managing the high intra-subject variability of sEMG patterns in human gait (both of extrinsic and intrinsic nature).There is a lack of systems that integrate simple scores or indexes, calculated from sEMG-GA data, to help clinical interpretation.Therefore, the authors believe that it is fundamental to rethink this research field, organizing debates, consensus meetings, interdisciplinary projects and other initiatives to provide a critical view of the topic and, last but not least, redesign user-friendly systems for sEMG-GA, usable in clinics. In addition, it would be important to offer training on sEMG-GA techniques to clinicians and health practitioners, including open education and open data resources. If the proposed “positive actions” will be successful, good clinical practices will benefit from new evidence-based approach to rehabilitation.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.VA and MK designed the article structure and topics covered. VA drafted the article, with the contribution of MG for the production of some of the figures. MG, SR, GB, and MK critically revised the article. All authors contributed to the article and approved the submitted version.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.


This secondary analysis of a randomized clinical trial evaluates the testing of high-sensitivity cardiac troponin in clinical practice. The index diagnosis was independently adjudicated by 2 physicians on review of all clinical information, according to the Universal Definition of Myocardial Infarction.6 Patients with type 1 ST-segment elevation myocardial infarction (STEMI) were stratified according to cardiac troponin concentration at presentation using a validated risk-stratification threshold (5 ng/L),1 the sex-specific 99th percentile, and the European Society of Cardiology (ESC) 0 of 1 hour pathway rule-in threshold (52 ng/L).4 Posterior STEMI was defined as those with STEMI and an acute occlusion of the circumflex, obtuse marginal, or posterior left ventricular artery on angiography. Time from symptom onset was recorded prospectively by attending clinicians. The trial was approved by the National Health Service Scotland A Research Ethics Committee, the Public Benefit and Privacy Panel for Health and Social Care, and by each local National Health Service Health Board. Because randomization was at the hospital level, individual patient consent was not sought.5 Comparisons between groups were performed using the χ2 test for categorical variables and an unpaired t test or the Kruskal-Wallis test for continuous variables. Statistical analysis was performed using R, version 3.6.1 (R Foundation).Between June 2013 and March 2016, consecutive patients with suspected acute coronary syndrome were recruited across 10 hospitals in the High-Sensitivity Troponin in the Evaluation of Patients With Acute Coronary Syndrome (High-STEACS) cluster randomized clinical trial.P < .001) and were more likely to have concentrations at less than the 99th percentile , compared with those presenting later. Posterior STEMI was more common in patients presenting with troponin at less than the 99th percentile .The trial enrolled 48 282 consecutive patients, of whom 925 had an adjudicated diagnosis of STEMI . At presentation, the median troponin concentration was 196 ng/L , with 2.2% (n = 20 of 925) and 14.4% of patients (n = 133 of 925) having concentrations less than 5 ng/L and the 99th percentile, respectively . Just 73Despite significant advances in the sensitivity of cardiac troponin testing, more than 1 in 4 patients with STEMI have troponin concentrations at less than the ESC-recommended rule-in threshold at presentation. Patients presenting within 2 hours were more likely to have a troponin concentration at less than the 99th percentile; however, even in those who presented later, 1 in 6 had troponin concentrations at less than the diagnostic threshold. During myocardial infarction, abrupt coronary occlusion may prevent the release of troponin into the circulation until reperfusion has occurred. Our observations are an important reminder of the limited role of troponin testing in the early assessment of patients with ST-segment elevation. Where clinical suspicion is high, troponin concentrations within the reference range should not delay the initiation of therapeutic agents or urgent coronary angiography. This is particularly relevant in patients with electrocardiographic changes suspicious of posterior myocardial infarction, but our findings are relevant to a wider group of patients with conduction abnormalities, such as bundle branch block or ventricular pacing, where interpretation of the electrocardiogram is challenging.