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Abstract Background TDP-43 proteinopathies are characterized by loss of nuclear TDP-43 expression and formation of C-terminal TDP-43 fragmentation and accumulation in the cytoplasm. Recent studies have shown that TDP-43 can accumulate in RNA stress granules (SGs) in response to cell stresses and this could be associated with subsequent formation of TDP-43 ubiquinated protein aggregates. However, the initial mechanisms controlling endogenous TDP-43 accumulation in SGs during chronic disease are not understood. In this study we investigated the mechanism of TDP-43 processing and accumulation in SGs in SH-SY5Y neuronal-like cells exposed to chronic oxidative stress. Cell cultures were treated overnight with the mitochondrial inhibitor paraquat and examined for TDP-43 and SG processing. Results We found that mild stress induced by paraquat led to formation of TDP-43 and HuR-positive SGs, a proportion of which were ubiquitinated. The co-localization of TDP-43 with SGs could be fully prevented by inhibition of c-Jun N-terminal kinase (JNK). JNK inhibition did not prevent formation of HuR-positive SGs and did not prevent diffuse TDP-43 accumulation in the cytosol. In contrast, ERK or p38 inhibition prevented formation of both TDP-43 and HuR-positive SGs. JNK inhibition also inhibited TDP-43 SG localization in cells acutely treated with sodium arsenite and reduced the number of aggregates per cell in cultures transfected with C-terminal TDP-43 162-414 and 219-414 constructs. Conclusions Our studies are the first to demonstrate a critical role for kinase control of TDP-43 accumulation in SGs and may have important implications for development of treatments for FTD and ALS, targeting cell signal pathway control of TDP-43 aggregation.

Objective: Given the high disease and cost burden of ischemic stroke, evaluating the clinical efficacy and cost-effectiveness of new approaches to prevent and treat ischemic stroke is critical. Effective ischemic stroke management depends on timely administration of thrombolytics after stroke onset. This study evaluates the cost-effectiveness associated with the use of mobile stroke units (MSUs) to expedite tissue plasminogen activator (tPA) administration, as compared with standard management through emergency medical services (EMS)., Methods: This study is a prospective, multicenter, alternating-week, cluster-controlled trial of MSU versus EMS. One-year and life-time cost-effectiveness analyses, using the incremental cost-effectiveness ratio (ICER) method, were performed from the perspective of CMS's Medicare. Quality-adjusted life years (QALYs) estimated using patient-reported EQ-5D-5L data were used as the effectiveness measure. Health care utilizations were converted to costs using average national Medicare reimbursements. ICERs excluding patients with pre-existing disability, and limited to stroke-related costs were also calculated., Results: The first-year ICER for all tPA-eligible patients using total cost differences between MSU and EMS groups was $238,873/QALY; for patients without pre-existing disability was $61,199/QALY. The lifetime ICERs for all tPA-eligible patients and for those without pre-existing disability were $94,710 and $31,259/QALY, respectively. All ICERs were lower when restricted to stroke-related costs and were highly dependent on the number of patients treated per year in an MSU., Interpretation: MSUs' cost-effectiveness is borderline if we consider total first-year costs and outcomes in all tPA-eligible patients. MSUs are cost-effective to highly cost-effective when calculations are based on patients without pre-existing disability, patients' lifetime horizon, stroke-related costs, and more patients treated per year in an MSU. ANN NEUROL 2024., (© 2024 American Neurological Association.)

Purpose: The global cancer burden and mortality rates are increasing, with significant disparities in access to care in low- and middle-income countries (LMICs). This study aimed to identify radiology and radiation therapy needs in LMICs from the perspective of departmental and institutional leaders., Methods and Materials: A survey was developed and conducted by the American Association of Physicists in Medicine Global Needs Assessment Committee and the American Association of Physicists in Medicine International Council. The survey, organized into 5 sections (Introduction, Infrastructure Needs, Education Needs, Research Needs, and General Information), was open to respondents from March 1, to August 16, 2022., Results: A total of 175 responses were received from 6 global regions: Africa (31.4%), the Americas (17.7%), the Eastern Mediterranean (14.3%), Europe (9.1%), Southeast Asia (23.4%), and the Western Pacific (4.0%). The greatest reported need was for new or updated equipment, particularly positron emission tomography/computed tomography imaging technology. There was also a high demand for clinical and equipment training. Approximately 25% of institutions reported a lack of radiology-based cancer screening programs because of high health care costs and a shortage of specialized equipment. Many institutions that expressed interest in research face funding and grant challenges., Conclusions: The findings highlight critical areas where organizations can support LMICs in enhancing radiology and radiation therapy services to mitigate the growing cancer burden., (© 2024 The Author(s).)

Objective: This study was undertaken to examine averted stroke in optimized stroke systems., Methods: This secondary analysis of a multicenter trial from 2014 to 2020 compared patients treated by mobile stroke unit (MSU) versus standard management. The analytical cohort consisted of participants with suspected stroke treated with intravenous thrombolysis. The main outcome was a tissue-defined averted stroke, defined as a final diagnosis of stroke with resolution of presenting symptoms/signs by 24 hours attributed to thrombolysis and no acute infarction/hemorrhage on imaging. An additional outcome was stroke with early symptom resolution, defined as a final diagnosis of stroke with resolution of presenting symptoms/signs by 24 hours attributed to thrombolysis., Results: Among 1,009 patients with a median last known well to thrombolysis time of 87 minutes, 159 (16%) had tissue-defined averted stroke and 276 (27%) had stroke with early symptom resolution. Compared with standard management, MSU care was associated with more tissue-defined averted stroke (18% vs 11%, adjusted odds ratio [aOR] = 1.82, 95% confidence interval [CI] = 1.13-2.98) and stroke with early symptom resolution (31% vs 21%, aOR = 1.74, 95% CI = 1.12-2.61). The relationships between thrombolysis treatment time and averted/early recovered stroke appeared nonlinear. Most models indicated increased odds for stroke with early symptom resolution but not tissue-defined averted stroke with earlier treatment. Additionally, younger age, female gender, hyperlipidemia, lower National Institutes of Health Stroke Scale, lower blood pressure, and no large vessel occlusion were associated with both tissue-defined averted stroke and stroke with early symptom resolution., Interpretation: In optimized stroke systems, 1 in 4 patients treated with thrombolysis recovered within 24 hours and 1 in 6 had no demonstrable brain injury on imaging. ANN NEUROL 2024;95:347-361., (© 2023 American Neurological Association.)

Background: Few data exist on acute stroke treatment in patients with pre-existing disability (PD) since they are usually excluded from clinical trials. A recent trial of mobile stroke units (MSUs) demonstrated faster treatment and improved outcomes, and included PD patients., Aim: To determine outcomes with tissue plasminogen activator (tPA), and benefit of MSU versus management by emergency medical services (EMS), for PD patients., Methods: Primary outcomes were utility-weighted modified Rankin Scale (uw-mRS). Linear and logistic regression models compared outcomes in patients with versus without PD, and PD patients treated by MSU versus standard management by EMS. Time metrics, safety, quality of life, and health-care utilization were compared., Results: Of the 1047 tPA-eligible ischemic stroke patients, 254 were with PD (baseline mRS 2-5) and 793 were without PD (baseline mRS 0-1). Although PD patients had worse 90-day uw-mRS, higher mortality, more health-care utilization, and worse quality of life than non-disabled patients, 53% returned to at least their baseline mRS, those treated faster had better outcome, and there was no increased bleeding risk. Comparing PD patients treated by MSU versus EMS, 90-day uw-mRS was 0.42 versus 0.36 (p = 0.07) and 57% versus 46% returned to at least their baseline mRS. There was no interaction between disability status and MSU versus EMS group assignment (p = 0.67) for 90-day uw-mRS., Conclusion: PD did not prevent the benefit of faster treatment with tPA in the BEST-MSU study. Our data support inclusion of PD patients in the MSU management paradigm., Competing Interests: Declaration of conflicting interestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: J.C. Grotta has received grants from Patient-Centered Outcomes Research Institute (PCORI), NIH, Genentech, CSL Behring, and Chiesi; is a consultant for Frazer, advison the scientific advisory board for Haemonetics and Acticor, and on the DSMB for Prolong Pharma. J.L. Saver receives contracted hourly payments from Bayer, Biogen, Roche, Genentech, Medtronic, Novo Nordisk, and Occlutech for service on clinical trial steering committees and DSMBs. The remaining authors have no disclosures.

Background: Treatment of patients with acute ischemic stroke on mobile stroke units (MSUs) improves outcomes compared with management by standard emergency medical services ambulances and is associated with more patients treated with intravenous tPA (tissue-type plasminogen activator) in the first golden hour after last known normal. We explored the predictors and outcomes of first-hour treatment (FHT) compared with later treatment in an alternating-week cluster-controlled trial of MSUs., Methods: We analyzed all patients treated with intravenous tPA in the BEST-MSU Study (Benefits of Stroke Treatment Delivered by a Mobile Stroke Unit Compared to Standard Management by Emergency Medical Services). After stratifying by treatment timeframe, we identified factors associated with FHT. We performed adjusted analyses of the association between FHT and clinical outcome and modeled the shape of the relationship between last known normal-to-treatment time and excellent outcome., Results: Among 941 tPA-treated patients, 206 (21.8%) had lytic started within 60 minutes. Treatment on the MSU, older age, male sex, alert by 911, faster arrival on-scene and imaging, more severe stroke, atrial fibrillation, and absence of heart failure and pretreatment antihypertensive treatment were associated with FHT. Compared with later treatment, FHT was associated with higher adjusted odds ratio for 90-day modified Rankin Scale score of 0 to 1 (odds ratio, 1.87 [95% CI, 1.25-2.84]; P =0.003). Among FHT patients, 68% achieved a 90-day modified Rankin Scale of 0 or 1 or returned to their baseline status. FHT was not associated with higher risk of hemorrhage and was associated with reduced risk of treating neurovascular mimics., Conclusions: FHT almost doubles the odds of excellent clinical outcome without increased risk compared with later treatment, which supports the use of MSUs.

Background: Hematoma enlargement (HE) after intracerebral hemorrhage (ICH) is a therapeutic target for improving outcomes. Hemostatic therapies to prevent HE may be more effective the earlier they are attempted. An understanding of HE in first 1 to 2 hours specifically in the prehospital setting would help guide future treatment interventions in this time frame and setting., Methods: Patients with spontaneous ICH within 4 hours of symptom onset were prospectively evaluated between May 2014 and April 2020 as a prespecified substudy within a multicenter trial of prehospital mobile stroke unit versus standard management. Baseline computed tomography scans obtained <1, 1 to 2, and 2 to 4 hours postsymptom onset on the mobile stroke unit in the prehospital setting were compared with computed tomography scans repeated 1 hour later and at 24 hours in the hospital. HE was defined as >6 mL if baseline ICH volume was < 20 mL and 33% increase if baseline volume >20 mL. The association between time from symptom onset to baseline computed tomography (hours) and HE was investigated using Wilcoxon rank-sum test when time was treated as a continuous variable and using Fisher exact test when time was categorized. Kruskal-Wallis and Wilcoxon rank-sum tests evaluated differences in baseline volumes and HE. Univariable and multivariable logistic regression analyses were conducted to identify factors associated with HE and variable selection was performed using cross-validated L1-regularized (Lasso regression). This study adhered to STROBE guidelines (Strengthening the Reporting of Observational Studies in Epidemiology) for cohort studies., Results: One hundred thirty-nine patients were included. There was no difference between baseline ICH volumes obtained <1 hour (n=43) versus 1 to 2 hour (n=51) versus >2 hours (n=45) from symptom onset (median [interquartile range], 13 mL [6-24] versus 14 mL [6-30] versus 12 mL [4-19]; P =0.65). However, within the same 3 time epochs, initial hematoma growth (volume/time from onset) was greater with earlier baseline scanning (median [interquartile range], 17 mL/hour [9-35] versus 9 mL/hour [5-23]) versus 4 mL/hour [2-7]; P <0.001). Forty-nine patients had repeat scans 1 hour after baseline imaging (median, 2.3 hours [interquartile range. 1.9-3.1] after symptom onset). Eight patients (16%) had HE during that 1-hour interval; all of these occurred in patients with baseline imaging within 2 hours of onset (5/18=28% with baseline imaging within 1 hour, 3/18=17% within 1-2 hour, 0/13=0% >2 hours; P =0.02). HE did not occur between the scans repeated at 1 hour and 24 hours. No association between baseline variables and HE was detected in multivariable analyses., Conclusions: HE in the next hour occurs in 28% of ICH patients with baseline imaging within the first hour after symptom onset, and in 17% of those with baseline imaging between 1 and 2 hours. These patients would be a target for ultraearly hemostatic intervention.

Background and Purpose: Tissue plasminogen activator (tPA) requires a one-hour infusion after the bolus. The frequency of delay or interruption of the tPA infusion may be useful in weighing the advantages of Tenecteplase (TNKase, TNK) which does not require an infusion., Methods: Utilizing the Benefits of Stroke Treatment Delivered Using a Mobile Stroke Unit Compared to Standard Management by Emergency Medical Services study database, we calculated the frequency and magnitude of tPA infusion delay or interruption., Results: Of 497 patients treated with tPA on the Houston Mobile Stroke Unit (MSU), 41 (8.3%) had delay or interruption of the infusion for reasons that did not reflect a side effect of, or contraindication to, tPA. Nine received less than 90% of their calculated dose (median 62%, range 28-88%), and eleven had more than a 10% prolongation of their infusion (median 19 min, range 7-210 min). Six patients (1.2%) had infusion stopped for a valid concern for tPA side effect or contraindication., Conclusions: Interruption or discontinuation of the tPA infusion occurs in 8% of patients treated on a MSU providing an opportunity for more complete and faster treatment with TNK., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: The available literature on mobile stroke units (MSU) has focused on clinical outcomes, rather than operational performance. Our objective was to establish normalized metrics and to conduct a meta-analysis of the current literature on MSU performance., Methods: Our MSU in upstate New York serves 741,000 people. We present prospectively collected, retrospectively analyzed data from the inception of our MSU in October of 2018, through March of 2021. Rates of transportation/dispatch and MSU utilization were reported. We also performed a meta-analysis using MEDLINE, SCOPUS, and Cochrane Library databases, calculating rates of tPA/dispatch, tPA-per-24-operational-hours ("per day"), mechanical thrombectomy (MT)/dispatch and MT/day., Results: Our MSU was dispatched 1,719 times in 606 days (8.5 dispatches/24-operational-hours) and transported 324 patients (18.8%) to the hospital. Intravenous tPA was administered in 64 patients (3.7% of dispatches) and the rate of tPA/day was 0.317 (95% CI 0.150-0.567). MT was performed in 24 patients (1.4% of dispatches) for a MT/day rate of 0.119 (95% CI 0.074-0.163). The MSU was in use for 38,742 minutes out of 290,760 total available minutes (13.3% utilization rate). Our meta-analysis included 14 articles. Eight studies were included in the analysis of tPA/dispatch (342/5,862) for a rate of 7.2% (95% CI 4.8-9.5%, I 2 = 92%) and 11 were included in the analysis of tPA/day (1,858/4,961) for a rate of 0.358 (95% CI 0.215-0.502, I 2 = 99%). Seven studies were included for MT/dispatch (102/5,335) for a rate of 2.0% (95% CI 1.2-2.8%, I 2 = 67%) and MT/day (103/1,249) for a rate of 0.092 (95% CI 0.046-0.138, I 2 = 91%)., Conclusions: In this single institution retrospective study and meta-analysis, we outline the following operational metrics: tPA/dispatch, tPA/day, MT/dispatch, MT/day, and utilization rate. These metrics are useful for internal and external comparison for institutions with or considering developing mobile stroke programs., Competing Interests: 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., (Copyright © 2022 Ellens, Schartz, Rahmani, Akkipeddi, Kelly, Benesch, Parker, Burgett, Proper, Pilcher, Mattingly, Grotta, Bhalla and Bender.)

Objectives: Studies face challenges with missing 5-level EQ-5D (EQ-5D-5L) data, often because of the need for longitudinal EQ-5D-5L data collection. There is a dearth of validated methodologies for dealing with missing EQ-5D-5L data in the literature. This study, for the first time, examined the possibility of using retrospectively collected EQ-5D-5L data as proxies for the missing data., Methods: Participants who had prospectively completed a 3rd month postdischarge EQ-5D-5L instrument (in-the-moment collection) were randomly interviewed to respond to a 2nd "retrospective collection" of their 3rd month EQ-5D-5L at 6th, 9th, or 12th month after hospital discharge. A longitudinal single imputation was also used to assess the relative performance of retrospective collection compared with the longitudinal single imputation. Concordances between the in-the-moment, retrospective, and imputed measures were assessed using intraclass correlation coefficients and weighted kappa statistics., Results: Considerable agreement was observed on the basis of weighted kappa (range 0.72-0.95) between the mobility, self-care, and usual activities dimensions of EQ-5D-5L collected in-the-moment and retrospectively. Concordance based on intraclass correlation coefficients was good to excellent (range 0.79-0.81) for utility indices computed, and excellent (range 0.93-0.96) for quality-adjusted life-years computed using in-the-moment compared with retrospective EQ-5D-5L. The longitudinal single imputation did not perform as well as the retrospective collection method., Conclusions: This study demonstrates that retrospective collection of EQ-5D-5L has high concordance with "in-the-moment" EQ-5D-5L and could be a valid and attractive alternative for data imputation when longitudinally collected EQ-5D-5L data are missing. Future studies examining this method for other disease areas and populations are required to provide more generalizable evidence., (Copyright © 2021 ISPOR–The Professional Society for Health Economics and Outcomes Research. Published by Elsevier Inc. All rights reserved.)

Background: Mobile stroke units (MSUs) are ambulances with staff and a computed tomographic scanner that may enable faster treatment with tissue plasminogen activator (t-PA) than standard management by emergency medical services (EMS). Whether and how much MSUs alter outcomes has not been extensively studied., Methods: In an observational, prospective, multicenter, alternating-week trial, we assessed outcomes from MSU or EMS management within 4.5 hours after onset of acute stroke symptoms. The primary outcome was the score on the utility-weighted modified Rankin scale (range, 0 to 1, with higher scores indicating better outcomes according to a patient value system, derived from scores on the modified Rankin scale of 0 to 6, with higher scores indicating more disability). The main analysis involved dichotomized scores on the utility-weighted modified Rankin scale (≥0.91 or <0.91, approximating scores on the modified Rankin scale of ≤1 or >1) at 90 days in patients eligible for t-PA. Analyses were also performed in all enrolled patients., Results: We enrolled 1515 patients, of whom 1047 were eligible to receive t-PA; 617 received care by MSU and 430 by EMS. The median time from onset of stroke to administration of t-PA was 72 minutes in the MSU group and 108 minutes in the EMS group. Of patients eligible for t-PA, 97.1% in the MSU group received t-PA, as compared with 79.5% in the EMS group. The mean score on the utility-weighted modified Rankin scale at 90 days in patients eligible for t-PA was 0.72 in the MSU group and 0.66 in the EMS group (adjusted odds ratio for a score of ≥0.91, 2.43; 95% confidence interval [CI], 1.75 to 3.36; P<0.001). Among the patients eligible for t-PA, 55.0% in the MSU group and 44.4% in the EMS group had a score of 0 or 1 on the modified Rankin scale at 90 days. Among all enrolled patients, the mean score on the utility-weighted modified Rankin scale at discharge was 0.57 in the MSU group and 0.51 in the EMS group (adjusted odds ratio for a score of ≥0.91, 1.82; 95% CI, 1.39 to 2.37; P<0.001). Secondary clinical outcomes generally favored MSUs. Mortality at 90 days was 8.9% in the MSU group and 11.9% in the EMS group., Conclusions: In patients with acute stroke who were eligible for t-PA, utility-weighted disability outcomes at 90 days were better with MSUs than with EMS. (Funded by the Patient-Centered Outcomes Research Institute; BEST-MSU ClinicalTrials.gov number, NCT02190500.)., (Copyright © 2021 Massachusetts Medical Society.)

Abstract: BACKGROUND: Prehospital tissue plasminogen activator dosing in a mobile stroke unit (MSU) is estimated by the paramedic and nurse. We aimed to determine the accuracy of the estimated weight method compared with the actual weight of patients treated with tissue plasminogen activator on the MSU. METHODS: We prospectively collected the estimated weight used on the MSU for treatment and the first-documented hospital-measured weight (bed scale) within 24 hours of hospital arrival. Median absolute and percent difference in weights were calculated; less than 10% of difference in weights was considered acceptable. To compare the estimated and measured weights, we conducted a Wilcoxon signed rank test and Fisher exact test to explore the association between weight difference of greater than 10% and patient outcomes. RESULTS: Among 337 patients, median estimated and hospital-measured weights were 79.0 kg (interquartile range [IQR], 66.0-94.5) and 78.5 kg (IQR, 65.0-91.7), respectively. The median of the absolute value of the difference in estimated versus measured weight was 2.7 kg (IQR, 0.6-7.6; P < .0001). The median percent difference in weight was 3.6% (IQR, 0.8%-9.4%). The median difference between the tissue plasminogen activator dosage administered on the MSU and the recommended dose based on the actual weight was 1.3 mg (IQR, 0.06-4.8) in absolute value. In 56 patients (16.6% of the entire sample) with overestimation of weight by greater than 10%, there were no symptomatic intracerebral hemorrhages. There was no association between weight difference and discharge modified Rankin score (P = .59). CONCLUSION: Weight estimation on an MSU can lead to similar tissue plasminogen activator dosing for 83.4% of subjects compared with if dosing were determined based on actual weight. Weight overestimation or underestimation had no detected impact on tissue plasminogen activator outcomes., Competing Interests: S.A.P. is a consultant/advisory board member at Frazer Ltd. J.C.G. receives research grant from Genentech, CSL Behring, National Institutes of Health, and Patient Centered Outcomes Research Institute and is a consultant/advisory board member at Frazer Ltd. The other authors declare no conflicts of interest., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association of Neuroscience Nurses.)

Most clinical research stopped during COVID due to possible impact on data quality and personnel safety. We aimed to assess the impact of COVID on acute stroke clinical trial conduct at sites that continued to enroll patients during the pandemic. BEST-MSU is an ongoing study of Mobile Stroke Units (MSU) vs standard management of tPA-eligible acute stroke patients in the pre-hospital setting. MSU personnel include a vascular neurologist via telemedicine, and a nurse, CT technologist, paramedics and emergency medicine technicians on-board. During COVID, consent, 90-day modified Rankin Scale (mRS) and EQ5D were obtained by phone instead of in-person, but other aspects of management were similar to the pre-COVID period. We compared patient demographics, study metrics, and infection of study personnel during intra- vs pre-COVID eras. Five of 6 BEST-MSU sites continued to enroll during COVID. There were no differences in intra- (n = 57) vs pre- (n = 869) COVID enrolled tPA eligible patients' age, sex, race (38.6% vs 38.0% Black), ethnicity (15.8% vs 18.6% Hispanic), or NIHSS (median 11 vs 9). The percent of screened patients enrolled and adjudicated tPA eligible declined from 13.6% to 6.6% (p < .001); study enrollment correlated with local stay-at-home and reopening orders. There were no differences in alert to MSU arrival or arrival to tPA times, but MSU on-scene time was 5 min longer (p = .01). There were no differences in ED door to CT, tPA treatment or thrombectomy puncture times, hospital length of stay, discharge disposition, or remote vs in-person 90-day mRS or EQ5D. One MSU nurse tested positive but did not require hospitalization. Clinical research in the pre-hospital setting can be carried out accurately and safely during a pandemic. tPA eligibility rates declined, but otherwise there were no differences in patient demographics, deterioration of study processes, or serious infection of study staff. Trial registration: NCT02190500., Competing Interests: The authors have declared that no competing interests exist.

Background: The 90-day modified Rankin Scale is a widely used outcome after stroke but is sometimes hard to ascertain due to loss to follow-up. Missing outcomes can result in biased and/or inefficient estimates in clinical trials. The aim of this study is to assess the validity of acquiring the 90-day modified Rankin Scale at a later point of time when the patient has been lost at 90 days to impute the missing value., Methods: Participants who had prospectively completed a 90-day modified Rankin Scale questionnaire on their own in the Benefits of Stroke Treatment Using a Mobile Stroke Unit study were randomly interviewed to recall the 90-day modified Rankin Scale at 6, 9, or 12 months after hospital discharge over the phone. Concordance between the two scores was assessed using kappa and weighted kappa statistics. Logistic regression was used to identify factors associated with inconsistent reporting of the 90-day modified Rankin Scale., Results: Substantial agreement was observed between in-the-moment and retrospective 90-day modified Rankin Scale recalled at 6, 9, or 12 months (weighted kappa = 0.93, 95% confidence interval: 0.89-0.98; weighted kappa = 0.93, 95% confidence interval: 0.85-1.00 and weighted kappa = 0.89, 95% confidence interval: 0.82-0.95, respectively)., Conclusion: Retrospective recall of 90-day modified Rankin Scale at a later time point is a valid means to impute missing data in stroke clinical trials.

Introduction: Mobile Stroke Units (MSUs) deliver acute stroke treatment on-scene in coordination with Emergency Medical Services (EMS). One criticism of the MSU approach is the limited range of a single MSU. The Houston MSU is evaluating MSU implementation, and we developed a rendezvous approach as an innovative solution to expand the range and number of patients treated., Methods: In addition to direct 911 dispatch of our MSU to the scene within our 7-mile catchment area, we empowered more distant EMS units to activate the MSU. We also monitored EMS radio communications to identify possible patients. For these distant patients, the MSU met the EMS unit en route to the stroke center and treated the patient at that intermediate location. The distribution of the distance from MSU base station to site of stroke and time from 911 alert to tissue plasminogen activator (tPA) bolus were compared between patients treated on-scene and by rendezvous using Wilcoxon rank sum test., Results: Over 4 years, 338 acute ischemic stroke patients were treated with tPA on our MSU. Of these, 169 (50%) were treated on-scene after MSU dispatch at a median of 6.4 miles (IQR 6.4 miles) from MSU base station. 169 (50%) were treated by 'rendezvous' pathway with assessment and treatment of stroke a median of 12.4 miles from base (IQR 5.5 miles) (p< 0.0001). Time (min) from MSU alert to tPA bolus did not differ: 36.0 ± 10.0 for on-scene vs 37.0 ± 10.0 with rendezvous (p=0.65). 13% of patients alerted via direct 911 dispatch were treated vs 44% of rendezvous patients., Conclusion: Adding a rendezvous approach to an MSU dispatch pathway doubles the range of operations and the number of patients treated by an MSU in an urban area, without incurring delay., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr Grotta and Ms Parker receiving consulting fees from Frazer Ltd, a manufacturer or Mobile Stroke Units. Dr Grotta receives research support from the Patient Centered Outcomes Research Institute, Genentech, and CSL Behring, (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background and Purpose- Endovascular thrombectomy (ET) door-to-puncture time (DTPT) is a modifiable metric. One of the most important, yet time-consuming steps, is documentation of large vessel occlusion by computed tomography angiography (CTA). We hypothesized that obtaining CTA on board a Mobile Stroke Unit and direct alert of the ET team shortens DTPT by over 30 minutes. Methods- We compared DTPT between patients having CTA onboard the Mobile Stroke Unit then subsequent ET from September 2018 to November 2019 and patients in Mobile Stroke Unit from August 2014 to August 2018, when onboard CTA was not yet being used. We also correlated DTPT with change in National Institutes of Health Stroke Scale between baseline and 24 hours. Results- Median DTPT was 53.5 (95% CI, 35-67) minutes shorter with onboard CTA and direct ET team notification: 41 minutes (interquartile range, 30.0-63.5) versus 94.5 minutes (interquartile range, 69.8-117.3; P <0.001). Median on-scene time was 31.5 minutes (interquartile range, 28.8-35.5) versus 27.0 minutes (interquartile range, 23.0-31.0) ( P <0.001). Shorter DTPT correlated with greater improvement of National Institutes of Health Stroke Scale (correlation=-0.2, P =0.07). Conclusions- Prehospital Mobile Stroke Unit management including on-board CTA and ET team alert substantially shortens DTPT. Registration- URL: https://clinicaltrials.gov; Unique identifier: NCT02190500.

Background and Purpose- The impact of a mobile stroke unit (MSU) on access to intraarterial thrombectomy (IAT) is a prespecified BEST-MSU substudy (Benefits of Stroke Treatment Delivered Using a Mobile Stroke Unit Compared to Standard Management by Emergency Medical Services). On the MSU, IAT decision-making steps, such as computed tomography, neurological exam, and tPA (tissue-type plasminogen activator) treatment are completed before emergency department arrival. We hypothesized that such pre-ED assessment of potential IAT patients on an MSU improves the time from ED arrival to skin puncture time (door-to-puncture-time, DTPT). Methods- BEST-MSU is a prospective comparative effectiveness study of MSU versus standard management by emergency medical services (EMS). We compared ED DTPT among the following groups of MSU and EMS patients: all IAT patients, IAT patients post-tPA, and IAT patients post-tPA meeting thrombolytic adjudication criteria over the first 4 years of the study. Results- From August 2014 to July 2018, a total of 161 patients underwent IAT. Ninety-four patients presented to the ED via the MSU and 67 by EMS. One hundred forty patients received tPA before IAT, 85 in the MSU arm, and 55 in the EMS arm. One hundred twenty-six patients received tPA within thrombolytic adjudication criteria: 76 MSU and 50 EMS. DTPT in minutes was shorter for MSU patients (all IAT MSU versus EMS 89 versus 99, P=0.01; IAT post-tPA MSU versus EMS 93 versus 100, P=0.03; and IAT post-tPA within adjudicated criteria MSU versus EMS 93 versus 99.5, P=0.03). From 2014 to 2018, DTPT decreased at a faster rate for EMS compared with MSU-managed patients, improving by about an hour. Conclusions- Pre-ED IAT evaluation on an MSU results in faster DTPT compared with arrival by EMS. Since 2014, dramatic improvement in ED IAT metrics has attenuated this difference. However, DTPT in all groups indicates substantial room for improvement.

Peptoids are versatile peptidomimetic molecules with wide-ranging applications from drug discovery to materials science. An understanding of peptoid sequence features that contribute to both their three-dimensional structures and their interactions with lipids will expand functions of peptoids in varied fields. Furthermore, these topics capture the enthusiasm of undergraduate students who prepare and study diverse peptoids in laboratory coursework and/or in faculty led research. Here, we present the synthesis and study of 21 peptoids with varied functionality, including 19 tripeptoids and 2 longer oligomers. We observed differences in fluorescence spectral features for 10 of the tripeptoids that correlated with peptoid flexibility and relative positioning of chromophores. Interactions of representative peptoids with sonicated glycerophospholipid vesicles were also evaluated using fluorescence spectroscopy. We observed evidence of conformational changes effected by lipids for select peptoids. We also summarize our experiences engaging students in peptoid-based projects to advance both research and undergraduate educational objectives in parallel., (© 2019 The Authors. Biopolymers published by Wiley Periodicals, Inc.)

Cyclic polymers were produced by end-to-end coupling of telechelic linear polymers under dilute conditions in THF, using intramolecular atom transfer radical coupling or click chemistry. In addition to the expected shift to longer elution times on gel permeation chromatography (GPC) indicative of the formation of cyclic product, lower molecular weight species were consistently observed upon analysis of the unpurified cyclization reaction mixture. By systematically removing or altering single reaction components in the highly dilute cyclization reaction, it was found that THF itself was responsible for the low-molecular-weight material, forming oligomeric chains of poly(THF) regardless of the other reaction components. When the reactions were performed at higher concentrations and for shorter time intervals, conducive to intermolecular chain-end-joining reactions, the low-molecular-weight peaks were absent. Isolation of the material and analysis by ¹H NMR confirmed that poly(THF) was being formed in the highly dilute conditions required for cyclization by end-to-end coupling. When a series of mock cyclization reactions were performed with molar ratios of the reactants held constant, but concentrations changed, it was found that lower concentrations of reactants led to higher amounts of poly(THF) side product.

Background and Purpose: Mobile stroke units (MSUs) can speed treatment with intravenous tPA (tissue-type plasminogen activator). We previously showed substantial agreement between a telemedicine-based vascular neurologist (TM-VN) and an onboard vascular neurologist (OB-VN) for the evaluation of patients with stroke for tPA eligibility on an MSU. However, the time efficiency of the telemedicine-based evaluation remained uncertain. In this study, we examined the speed of decision and treatment from MSU arrival for the TM-VN compared with an OB-VN., Methods: In 50 consecutive situations, the TM-VN served as the primary decision maker. Times from MSU arrival to tPA decision and tPA bolus were compared with the same metrics for when the OB-VN served as the primary decision maker., Results: Time to tPA decision for the TM-VN was 21 minutes (interquartile range, 16.25-26) versus 18 minutes (interquartile range, 14-22) for the OB-VN ( P =0.01). Initiation of tPA bolus was 24 minutes (interquartile range, 19.75-30) for the TM-VN versus 24 minutes (interquartile range, 19-27.75) for the OB-VN ( P =0.5)., Conclusions: Assessment by a TM-VN is comparable with an OB-VN in making decisions about tPA administration on an MSU and does not lead to treatment delays., Clinical Trial Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02190500., (© 2018 American Heart Association, Inc.)

Rationale Mobile stroke units speed treatment for acute ischemic stroke, thereby possibly improving outcomes. Aim To compare mobile stroke unit and standard management clinical outcomes, healthcare utilization, and cost-effectiveness in tissue plasminogen activator-eligible acute ischemic stroke patients calling 911. Sample size 693. Eighty percent power with 0.05 type I error rate to detect a difference of 0.09 in mean utility-weighted modified Rankin scale between groups. Design Phase III, multicenter, prospective cluster-randomized (mobile stroke unit versus standard management weeks) comparative effectiveness study in tissue plasminogen activator-eligible patients. Outcomes Primary: Ninety-day mean utility-weighted modified Rankin scale. Coprimary: cost-effectiveness based on EQ5D quality of life and one year poststroke costs. Analysis Two-sample t-test and linear regression adjusting for covariates; incremental cost-effectiveness ratio and net benefit regression. Results As of March 2017, 288 tissue plasminogen activator-eligible patients have been enrolled (173 in the mobile stroke unit arm and 115 in the standard management arm). Two new centers start in early 2017 with target end of recruitment September 2019. Conclusion This is the first randomized study to test for disability, healthcare utilization, and cost-effectiveness of a mobile stroke unit. The progress of the study suggests that it is feasible. Management of tissue plasminogen activator eligible acute ischemic stroke patients by a mobile stroke unit could potentially result in less disability and healthcare utilization, and be cost effective. Mobile stroke units are very costly. This trial may determine if the fixed cost can be justified by a reduction in disability and healthcare utilization. Clinical Trial Registration NCT02190500.

Insufficient mixing in laminar flow reactors due to diffusion-dominated flow limits their use in applications where narrow residence time distribution (RTD) is required. The aim of this study was to design and characterize a laminar flow (Re 187.7-375.5) tubular reactor for low pH viral inactivation with enhanced radial mixing via the incorporation of curvature and flow inversions. Toward this aim, the reactor described here, Jig in a Box (JIB), was designed with a flow path consisting of alternating 270° turns. The design was optimized by considering the strength of secondary flows characterized by the Dean No., the corresponding secondary flow development length, and the reactor turn lengths. Comprehensive CFD analysis of the reactor centerline velocity profile, cross-sectional velocity, and secondary flow streamlines confirmed enhanced radial mixing due to secondary flows and changes in flow direction. For initial CFD and experimental studies the reactor was limited to a 16.43 m length. Pulse tracer studies for the reactor were computationally simulated and experimentally generated to determine the RTD, RTD variance, and minimum residence time for the tracer fluid elements leaving the reactor, as well as to validate the computational model. The reactor was scaled length wise to increase incubation time and it was observed that as the reactor length increases the RTD variance increases linearly and the dimensionless RTD profile becomes more symmetrical and tighter about the mean residence time., (© 2017 Wiley Periodicals, Inc.)

Background and Purpose: The BEST-MSU study (Benefits of Stroke Treatment Delivered Using a Mobile Stroke Unit) is a comparative effectiveness trial in patients randomized to mobile stroke unit or standard management. A substudy tested interrater agreement for tissue-type plasminogen activator eligibility between a telemedicine vascular neurologist and onboard vascular neurologist., Methods: On scene, both the telemedicine vascular neurologist and onboard vascular neurologist independently evaluated the patient, documenting their tissue-type plasminogen activator treatment decision, National Institutes of Health Stroke Scale score, and computed tomographic interpretation. Agreement was determined using Cohen κ statistic. Telemedicine-related technical failures that impeded remote assessment were recorded., Results: Simultaneous and independent telemedicine vascular neurologist and onboard vascular neurologist assessment was attempted in 174 patients. In 4 patients (2%), the telemedicine vascular neurologist could not make a decision because of technical problems. The telemedicine vascular neurologist agreed with the onboard vascular neurologist on 88% of evaluations (κ=0.73)., Conclusions: Remote telemedicine vascular neurologist assessment is reliable and accurate, supporting either telemedicine vascular neurologist or onboard vascular neurologist assessment on our mobile stroke unit., Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02190500., (© 2017 American Heart Association, Inc.)

When a blood clot blocks the blood supply to the brain or when a blood vessel bursts, resulting in brain cell death, the medical condition is referred to as a "stroke." Stroke is a main cause of death worldwide and is a common cause of disability. A common form of stroke, called ischemic stroke, is when blood flow to the brain is decreased. Clinical research has revealed that treatment within the very first hours of symptom onset is key for ischemic stroke with recanalization of occluded arteries by thrombolysis with alteplase. Computed tomography (CT) is one of the diagnostic tools used to determine if this treatment path is appropriate. To determine if health outcomes of possible stroke patients can be improved by decreasing the time from symptom presentation to treatment, the first mobile stroke ambulance unit in the United States was deployed by The University of Texas Health Science Center at Houston (UTHealth) in 2014, equipped with a computed tomography imaging system. The mobile stroke unit shortens the time to treatment for stroke patients by allowing pre-hospital treatment. Having completed its first year of operation, radiation-monitoring data describing the doses delivered to various entities have been characterized. The CT operator's cumulative deep dose equivalent for 1 y of operation was 1.14 mSv resulting from the care of 106 patients. Area monitors were deployed and measurements performed demonstrating that general public doses did not exceed 0.02 mSv h⁻¹ or 1.0 mSv year.

Thrombelastography (TEG) measures coagulation in venous blood. We hypothesized that TEG, by reflecting clot subtype and ex vivo fibrinolysis, might predict fibrinolytic response to tPA as reflected by rapid clinical improvement or hemorrhagic transformation of the infarct. 171 acute ischemic stroke patients treated with tPA were prospectively enrolled. Venous blood for TEG was drawn before and 10 min after tPA bolus. We measured rapid clinical improvement (RCI = 8 point improvement on NIHSS or total NIHSS of 0, 1 at 36 h), Hemorrhagic transformation (HT = any blood on imaging within 36 h), and hyperdense middle cerebral artery sign (HDMCA = biomarker for erythrocyte-rich clot). Multivariable regression models compared TEG parameters after adjusting for potential confounders. No differences in pre- or post-tPA TEG were found between patients with or without RCI. Also, there was no correlation between TEG and HDMCA. Clotting was slightly prolonged in patients with HT (p = 0.046). We failed to find a robust association between TEG and clinical response to tPA. It is likely that arterial clot lysis is determined by factors unrelated to coagulation status as measured by TEG in the venous circulation. It is unlikely that TEG will be useful to predict clinical response to tPA, but may help predict bleeding.

The production of norovirus virus-like particles (NoV VLPs) displaying NY-ESO-1 cancer testis antigen in Pichia pastoris BG11 Mut(+) has been enhanced through feed-strategy optimization using a near-infrared bioprocess monitor (RTBio(®) Bioprocess Monitor, ASL Analytical, Inc.), capable of monitoring and controlling the concentrations of glycerol and methanol in real-time. The production of NoV VLPs displaying NY-ESO-1 in P. pastoris has potential as a novel cancer vaccine platform. Optimization of the growth conditions resulted in an almost two-fold increase in the expression levels in the fermentation supernatant of P. pastoris as compared to the starting conditions. We investigated the effect of methanol concentration, batch phase time, and batch to induction transition on NoV VLP-NY-ESO-1 production. The optimized process included a glycerol transition phase during the first 2 h of induction and a methanol concentration set point of 4 g L(-1) during induction. Utilizing the bioprocess monitor to control the glycerol and methanol concentrations during induction resulted in a maximum NoV VP1-NY-ESO-1 yield of 0.85 g L(-1) . © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:518-526, 2016., (© 2016 American Institute of Chemical Engineers.)

Background and Purpose: Recently, the Mobile Stroke Unit (MSU) concept was introduced in Germany demonstrating prehospital treatment of more patients within the first hour of symptom onset. However, the details and complexities of establishing such a program in the United States are unknown. We describe the steps involved in setting up the first MSU in the United States., Methods: Implementation included establishing leadership, fund-raising, purchase and build-out, knitting a collaborative consortium of community stakeholders, writing protocols to ensure accountability, radiation safety, purchasing supplies, licensing, insurance, establishing a base station, developing a communication plan with city Emergency Medical Services, Emergency Medical Service training, staffing, and designing a research protocol., Results: The MSU was introduced after ≈1 year of preparation. Major obstacles to establishing the MSU were primarily obtaining funding, licensure, documenting radiation safety protocols, and establishing a smooth communication system with Emergency Medical Services. During an 8 week run-in phase, ≈2 patients were treated with recombinant tissue-type plasminogen activator per week, one-third within 60 minutes of symptom onset, with no complications. A randomized study to determine clinical outcomes, telemedicine reliability and accuracy, and cost effectiveness was formulated and has begun., Conclusion: The first MSU in the United States has been introduced in Houston, TX. The steps needed to accomplish this are described., (© 2015 American Heart Association, Inc.)

Background: Norovirus virus-like particles (NoV VLPs) have recently been explored as potential vaccine platforms due to their ability to produce an effective immune response. Expression of the main structural protein, VP1, leads to formation of self-assembled particles with similar characteristics to the original virus. These NoV VLPs have been expressed in Escherichia coli, yeast and insect cells. Expression in E. coli and insect cells share downstream processing issues due to the presence of inclusion bodies or the need for numerous purification steps. NoV VLPs have also been produced in the yeast P. pastoris; however the protein was only expressed intracellularly., Results: We have successfully expressed and secreted the VP1 protein in the novel P. pastoris strain, Bg11, using the methanol inducible pJ912 expression vector, containing the cDNA of NoV VP1. Expression of the VP1 protein in Bg11 was carried out in a 1.5 L bioreactor resulting in a total yield of NoV VLPs greater than 0.6 g/L. NoV VLPs obtained from the culture supernatant were purified via ion-exchange chromatography, resulting in particles with a purity over 90%. The average size of the particles after purification was 40 nm. Transmission electron microscopy was used to visualize the morphology of the particles and saliva-binding assay confirmed that the NoV VLPs bind to Histo-Blood Group Antigens (HBGA)., Conclusions: In this study we describe the expression and characterization of fully assembled Norovirus virus-like particles obtained from P. pastoris. The particles are similar in size, morphology and binding capacity, as previously described, for the original NoV. Our results detail the successful expression and secretion of VLPs in P. pastoris, improving their candidacy as a vaccine platform.

Biopolymer-based multi-functional nanoparticles have been developed through a one-step enzymatic polymerization reaction using engineered polyhydroxyalkanoate (PHA) synthase fused with green fluorescent protein (GFP) and a single chain variable fragment antibody (A33scFv) specific to colon cancer. PHA synthase possesses unique catalytic characteristics, namely covalent catalysis, by which the synthesized polyhydroxybutyrate chain remains covalently attached to the enzyme. The amphiphilic nature of the resulting protein-polymer hybrid gives rise to spontaneous self-assembly into a micellar structure with GFP and A33scFv displayed on the surface (AGPHB nanoparticle). A model compound, Nile red, was loaded into the hydrophobic core of the AGPHB nanoparticle during the polymerization and self-assembly process. The specificity of the fluorescent multi-functional AGPHB nanoparticle towards the colon cancer cell lines SW1222 (A33+) and HT29 (A33-) was confirmed and analysed quantitatively in vitro. This new biological approach provides a simple means of producing nanocarriers with a range of surface functionality and the sizes desired for imaging and targeted drug delivery.

Background and Purpose: Factor Xa inhibitors are prescribed for stroke prevention in atrial fibrillation. Managing such patients is challenging especially if they are eligible for thrombolysis because there is no rapidly available test to detect the effect of such medications. Thrombelastography analyzes the dynamics of coagulation and can be rapidly performed. We sought to determine whether thrombelastography can detect the anticoagulation effect of factor Xa inhibitors in patients with stroke., Methods: Blood from 10 patients with stroke was analyzed by thrombelastography at baseline and 2 to 18 hours after rivaroxaban administration., Results: Increased R, K, and δ were seen at 2, 4, and 6 hours, while G, maximum amplitude, α-angle, and LY30 were decreased. Baseline R was 5.8±0.5 when compared with 11.4±1.0 at 2 hours. R remained prolonged at 18 hours. Other thrombelastography parameters were normal by 18 hours., Conclusions: Thrombelastography can detect the anticoagulant effect of factor Xa inhibitors in patients with stroke and might be useful in the emergency management of those eligible for thrombolysis.

Background and Purpose: Intracerebral hemorrhage (ICH) has high morbidity, and hematoma enlargement (HE) causes worse outcome. Thrombelastography (TEG) measures the dynamics of clot formation and dissolution, and might be useful for assessing bleeding risk. We used TEG to detect changes in clotting in patients with and without HE after ICH., Methods: This prospective study included 64 patients with spontaneous ICH admitted from 2009 to 2013. TEG was performed within 6 hours of symptom onset and after 36 hours. Brain imaging was obtained at baseline and at 36±12 hours, and HE was defined as total volume increase>6 cc or >33%. TEG was also obtained from 57 controls., Results: Compared with controls, patients with ICH demonstrated faster and stronger clot formation; shorter R and delta (P<0.0001) at baseline; and higher MA and G (P<0.0001) at 36 hours; 11 patients had HE. After controlling for potential confounders, baseline K and delta were longer in HE+ compared with HE- patients, indicating that HE+ patients had slower clot formation (P<0.05). TEG was not different between HE+ and HE- patients at 36 hours., Conclusions: TEG may detect important coagulation changes in patients with ICH. Clotting may be faster and stronger in immediate response to ICH, and a less robust response may be associated with HE. These findings deserve further investigation.

Background and Purpose: Iodinated contrast agents used for computed tomography angiography (CTA) may alter fibrin fiber characteristics and decrease fibrinolysis by tissue plasminogen activator (tPA). Thromboelastography (TEG) measures the dynamics of coagulation and correlates with thrombolysis in acute ischemic stroke patients. We hypothesized that receiving CTA before tPA will not impair thrombolysis as measured by TEG., Methods: Acute ischemic stroke patients receiving 0.9 mg/kg tPA <4.5 hours of symptom onset were prospectively enrolled. For CTA, 350 mg/dL of iohexol or 320 mg/dL of iodixanol at a dose of 2.2 mL/kg was administered. TEG was measured before tPA and 10 minutes after tPA bolus. CTA timing was left to the discretion of the treating physician., Results: Of 136 acute ischemic stroke patients who received tPA, 47 had CTA before tPA bolus, and 42 had either CTA after tPA and post-tPA TEG draw or no CTA (noncontrast group). Median change in clot lysis (LY30) after tPA was 95.3% in the contrast group versus 95.0% in the noncontrast group (P=0.74). Thus, tPA-induced thrombolysis did not differ between contrast and noncontrast groups. Additionally, there was no effect of contrast on any pre-tPA TEG value., Conclusions: Our data do not support an effect of iodinated contrast agents on clot formation or tPA activity.

A single chain variable fragment (J591 ScFv) that recognizes the extracellular glyco-protein prostate specific membrane antigen (PSMA) was designed, constructed, and expressed in Pichia pastoris. Construction of the J591 ScFv was based on the reported complementarity-determining region (CDR) of the PSMA specific J591 monoclonal antibody (mAb). The nucleotide sequence encoding the J591-derived ScFv was codon-optimized for expression in P. pastoris and a 6× his-tag was added to facilitate affinity purification. A down-scale 2L methanol-induced P. pastoris fermentation yielded 330mg of total protein following a 96h induction. Following Immobolized Metal Affinity Chromatography, functionality of the J591 ScFv was confirmed via Western blot, immunoblot, binding studies, and flow cytometry analysis. The J591 ScFv showed binding affinity and specificity to cell extracts containing PSMA and PSMA-expressing prostate cancer cells. Our results demonstrate that functional J591 ScFv can be produced in P. pastoris for use in diagnostic and targeted therapeutic applications., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Phosphorylation is a universal mechanism for regulating cell behavior in eukaryotes. Although protein kinases target short linear sequence motifs on their substrates, the rules for kinase substrate recognition are not completely understood. We used a rapid peptide screening approach to determine consensus phosphorylation site motifs targeted by 61 of the 122 kinases in Saccharomyces cerevisiae. By correlating these motifs with kinase primary sequence, we uncovered previously unappreciated rules for determining specificity within the kinase family, including a residue determining P-3 arginine specificity among members of the CMGC [CDK (cyclin-dependent kinase), MAPK (mitogen-activated protein kinase), GSK (glycogen synthase kinase), and CDK-like] group of kinases. Furthermore, computational scanning of the yeast proteome enabled the prediction of thousands of new kinase-substrate relationships. We experimentally verified several candidate substrates of the Prk1 family of kinases in vitro and in vivo and identified a protein substrate of the kinase Vhs1. Together, these results elucidate how kinase catalytic domains recognize their phosphorylation targets and suggest general avenues for the identification of previously unknown kinase substrates across eukaryotes.

Ephrin receptor tyrosine kinase A3 (EphA3, EC 2.7.10.1) is a member of a unique branch of the kinome in which downstream signaling occurs in both ligand- and receptor-expressing cells. Consequently, the ephrins and ephrin receptor tyrosine kinases often mediate processes involving cell-cell contact, including cellular adhesion or repulsion, developmental remodeling and neuronal mapping. The receptor is also frequently overexpressed in invasive cancers, including breast, small-cell lung and gastrointestinal cancers. However, little is known about direct substrates of EphA3 kinase and no chemical probes are available. Using a library approach, we found a short peptide sequence that is a good substrate for EphA3 and is suitable for co-crystallization studies. Complex structures show multiple contacts between kinase and substrates; in particular, two residues undergo conformational changes and by mutation are found to be important for substrate binding and turnover. In addition, a difference in catalytic efficiency between EPH kinase family members is observed. These results provide insight into the mechanism of substrate binding to these developmentally integral enzymes.

The mammalian ortholog of the retroviral oncogene v-Eyk, and a receptor tyrosine kinase upstream of antiapoptotic and transforming signals, Mer (MerTK) is a mediator of the phagocytic process, being involved in retinal and immune cell clearance and platelet aggregation. Mer knockout mice are viable and are protected from epinephrine-induced pulmonary thromboembolism and ferric chloride-induced thrombosis. Mer overexpression, on the other hand, is associated with numerous carcinomas. Although Mer adaptor proteins and signaling pathways have been identified, it remains unclear how Mer initiates phagocytosis. When bound to its nucleotide cofactor, the high-resolution structure of Mer shows an autoinhibited alphaC-Glu-out conformation with insertion of an activation loop residue into the active site. Mer complexed with compound-52 (C52: 2-(2-hydroxyethylamino)-6-(3-chloroanilino)-9-isopropylpurine), a ligand identified from a focused library, retains its DFG-Asp-in and alphaC-Glu-out conformation, but acquires other conformational changes. The alphaC helix and DFGL region is closer to the hinge region and the ethanolamine moiety of C52 binds in the groove formed between Leu593 and Val601 of the P-loop, causing a compression of the active site pocket. These conformational states reveal the mechanisms of autoinhibition, the pathophysiological basis of disease-causing mutations, and a platform for the development of chemical probes.

Systematic and quantitative analysis of protein phosphorylation is revealing dynamic regulatory networks underlying cellular responses to environmental cues. However, matching these sites to the kinases that phosphorylate them and the phosphorylation-dependent binding domains that may subsequently bind to them remains a challenge. NetPhorest is an atlas of consensus sequence motifs that covers 179 kinases and 104 phosphorylation-dependent binding domains [Src homology 2 (SH2), phosphotyrosine binding (PTB), BRCA1 C-terminal (BRCT), WW, and 14-3-3]. The atlas reveals new aspects of signaling systems, including the observation that tyrosine kinases mutated in cancer have lower specificity than their non-oncogenic relatives. The resource is maintained by an automated pipeline, which uses phylogenetic trees to structure the currently available in vivo and in vitro data to derive probabilistic sequence models of linear motifs. The atlas is available as a community resource (http://netphorest.info).

Mitogen-activated protein kinases (MAPKs) mediate cellular responses to a wide variety of extracellular stimuli. MAPK signal transduction cascades are tightly regulated, and individual MAPKs display exquisite specificity in recognition of their target substrates. All MAPK family members share a common phosphorylation site motif, raising questions as to how substrate specificity is achieved. Here we describe a peptide library screen to identify sequence requirements of the DEF site (docking site for ERK FXF), a docking motif separate from the phosphorylation site. We show that MAPK isoforms recognize DEF sites with unique sequences and identify two key residues on the MAPK that largely dictate sequence specificity. Based on these observations and computational docking studies, we propose a revised model for MAPK interaction with substrates containing DEF sites. Variations in DEF site sequence requirements provide one possible mechanism for encoding complex target specificity among MAPK isoforms.

Protein kinase autophosphorylation of activation segment residues is a common regulatory mechanism in phosphorylation-dependent signalling cascades. However, the molecular mechanisms that guarantee specific and efficient phosphorylation of these sites have not been elucidated. Here, we report on three novel and diverse protein kinase structures that reveal an exchanged activation segment conformation. This dimeric arrangement results in an active kinase conformation in trans, with activation segment phosphorylation sites in close proximity to the active site of the interacting protomer. Analytical ultracentrifugation and chemical cross-linking confirmed the presence of dimers in solution. Consensus substrate sequences for each kinase showed that the identified activation segment autophosphorylation sites are non-consensus substrate sites. Based on the presented structural and functional data, a model for specific activation segment phosphorylation at non-consensus substrate sites is proposed that is likely to be common to other kinases from diverse subfamilies.

The activation segment of protein kinases is structurally highly conserved and central to regulation of kinase activation. Here we report an atypical activation segment architecture in human MPSK1 comprising a beta sheet and a large alpha-helical insertion. Sequence comparisons suggested that similar activation segments exist in all members of the MPSK1 family and in MAST kinases. The consequence of this nonclassical activation segment on substrate recognition was studied using peptide library screens that revealed a preferred substrate sequence of X-X-P/V/I-phi-H/Y-T*-N/G-X-X-X (phi is an aliphatic residue). In addition, we identified the GTPase DRG1 as an MPSK1 interaction partner and specific substrate. The interaction domain in DRG1 was mapped to the N terminus, leading to recruitment and phosphorylation at Thr100 within the GTPase domain. The presented data reveal an atypical kinase structural motif and suggest a role of MPSK1 regulating DRG1, a GTPase involved in regulation of cellular growth.