Development of bioluminescent sensors for interrogating cyclic di-nucleotide signaling

Bioluminescence is the spectacular natural phenomenon in which living organisms produce and emit light. Natural bioluminescent protein systems have been discovered and characterized in over 30 species, ranging from fireflies to fungi and bacteria. Significant effort has been put towards engineering and improving these natural systems so that they may be used as tools for interrogating biological processes. As opposed to fluorescence, because bioluminescence requires no excitation light to produce a signal, it has proven extremely useful for imaging in highly autofluorescent samples, such as within deep tissues of whole organisms. To date, however, most bioluminescent imaging systems have been developed solely for the study of eukaryotic systems, and few have focused on the study of bacteria. Bacteria naturally colonize highly diverse and complex environments, from gastrointestinal tracts to soil and plant surfaces, that have proven difficult to study with currently available fluorescent tools. To allow for the study of bacterial signaling within these complex environments, new bioluminescent sensors developed specifically for bacterial signaling are needed.Here, we describe the development and application of bioluminescent sensors for the bacterial cyclic di-nucleotide (CDN) signaling molecule cyclic di-GMP. Cyclic di-GMP is nearly ubiquitous in bacteria and plays a key role in the controlling motility and biofilm formation. As a first-generation bioluminescent sensor system, we developed intensity-based bioluminescent sensors for cyclic di-GMP. These sensors proved useful as in vitro tools for studying cyclic di-GMP, however were not amenable to live cell imaging. To move beyond purely in vitro systems, we developed next-generation ratiometric bioluminescent sensors for cyclic di-GMP. This next-generation system led to significantly improved sensor properties and allowed for the imaging of small numbers of live bacterial cells in an animal tissue-like model system. Finally, to expand these sensor systems to CDNs other than cyclic di-GMP, we applied a novel directed evolution approach to find sensors that respond to cyclic GMP-AMP. The first round of directed evolution was not successful, but work is ongoing on this front. Collectively, the work presented here lays the groundwork for using bioluminescent sensor systems to interrogate signaling in bacteria in their natural environments, which was previously not possible

Improving neural network representations using human similarity judgments

Deep neural networks have reached human-level performance on many computer vision tasks. However, the objectives used to train these networks enforce only that similar images are embedded at similar locations in the representation space, and do not directly constrain the global structure of the resulting space. Here, we explore the impact of supervising this global structure by linearly aligning it with human similarity judgments. We find that a naive approach leads to large changes in local representational structure that harm downstream performance. Thus, we propose a novel method that aligns the global structure of representations while preserving their local structure. This global-local transform considerably improves accuracy across a variety of few-shot learning and anomaly detection tasks. Our results indicate that human visual representations are globally organized in a way that facilitates learning from few examples, and incorporating this global structure into neural network representations improves performance on downstream tasks.Comment: Published as a conference paper at NeurIPS 202

Patients’ Perspectives Regarding Digital Health Technology to Support Self-management and Improve Integrated Stroke Care:Qualitative Interview Study

Background: Digital technologies such as mobile apps and robotics have the potential to involve stroke patients better in the care process and to promote self-management. However, barriers exist that constrain the adoption and acceptance of technology in clinical practice. Examples of barriers are privacy concerns, challenges regarding usability, and the perception that there is no need for health-related technology. To address these barriers, co-design can be used to enable patients to reflect on their experiences of a service and to tailor digital technologies to the needs and preferences of end users regarding content and usability. Objective: This study aims to explore the perspectives of stroke patients toward how digital health technology could support self-management regarding health and well-being, as well as integrated stroke care. Methods: A qualitative study was conducted to understand patient perspectives. Data were collected in co-design sessions during the ValueCare study. Patients from a Dutch hospital who experienced an ischemic stroke (n=36) within the past 18 months were invited to participate. Data collection took place between December 2020 and April 2021 via one-to-one telephone interviews. A short self-report questionnaire was used to collect data on sociodemographics, disease-specific information, and technology use. All interviews were audio-taped and transcribed verbatim. The interview data were analyzed using a thematic approach. Results: Patients held mixed attitudes toward digital health technologies. Some patients viewed digital technology as a convenient product or service, while others expressed no desire or need to use technology for self-management or care. Digital features suggested by stroke patients included (1) information about the causes of stroke, medication, prognosis, and follow-up care; (2) an online library with information regarding stroke-related health and care issues; (3) a personal health record by which patients can retrieve and manage their own health information; and (4) online rehabilitation support to empower patients to exercise at home. Regarding the user interface of future digital health technology, patients emphasized the need for easy-to-use and simple designs. Conclusions: Stroke patients mentioned credible health information, an online library with stroke-related health and care information, a personal health record, and online rehabilitation support as the main features to include in future digital health technologies. We recommend that developers and designers of digital health for stroke care listen to the “voice of the stroke patients” regarding both functionality and the characteristics of the interface.</p

Management of Mild Traumatic Brain Injury at the Emergency Department and Hospital Admission in Europe: A Survey of 71 Neurotrauma Centers Participating in the CENTER-TBI Study.

Previous studies have indicated that there is no consensus about management of mild traumatic brain injury (mTBI) at the emergency department (ED) and during hospital admission. We aim to study variability between management policies for TBI patients at the ED and at the hospital ward across Europe. Centers participating in the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study received questionnaires about different phases of TBI care. These questionnaires included 71 questions about TBI management at the ED and at the hospital ward. We found differences in how centers defined mTBI. For example, 40 centers (59%) defined mTBI as a Glasgow Coma Scale (GCS) score between 13 and 15 and 26 (38%) defined it as a GCS score between 14 and 15. At the ED various guidelines for the use of head computed tomography (CT) in mTBI patients were used; 32 centers (49%) used national guidelines, 10 centers (15%) local guidelines, and 14 centers (21%) used no guidelines at all. Also, differences in indication for admission between centers were found. After ED discharge, 7 centers (10%) scheduled a routine follow-up appointment, whereas 38 (54%) did so only after ward admission. In conclusion, large between-center variation exists in policies for diagnostics, admission, and discharge decisions in patients with mTBI at the ED and in the hospital. Guidelines are not always operational in centers, and reported policies systematically diverge from what is recommended in those guidelines. The results of this study may be useful in the understanding of mTBI care in Europe and show the need for further studies on the effectiveness of different policies on outcome

Predicting outcome in acute stroke with large vessel occlusion:application and validation of MR PREDICTS in the ESCAPE-NA1 population

Background: Predicting outcome after endovascular treatment for acute ischemic stroke is challenging. We aim to investigate differences between predicted and observed outcomes in patients with acute ischemic stroke treated with endovascular treatment and to evaluate the performance of a validated outcome prediction score. Patients and methods: MR PREDICTS is an outcome prediction tool based on a logistic regression model designed to predict the treatment benefit of endovascular treatment based on the MR CLEAN and HERMES populations. ESCAPE-NA1 is a randomized trial of nerinetide vs. placebo in patients with acute stroke and large vessel occlusion. We applied MR PREDICTS to patients in the control arm of ESCAPE-NA1. Model performance was assessed by calculating its discriminative ability and calibration. Results: Overall, 556/1105 patients (50.3%) in the ESCAPE-NA1-trial were randomized to the control arm, 435/556 (78.2%) were treated within 6 h of symptom onset. Good outcome (modified Rankin scale 0–2) at 3 months was achieved in 275/435 patients (63.2%), the predicted probability of good outcome was 52.5%. Baseline characteristics were similar in the study and model derivation cohort except for age (ESCAPE-NA1: mean: 70 y vs. HERMES: 66 y), hypertension (72% vs. 57%), and collaterals (good collaterals, 15% vs. 44%). Compared to HERMES we observed higher rates of successful reperfusion (TICI 2b-3, ESCAPE-NA1: 87% vs. HERMES: 71%) and faster times from symptom onset to reperfusion (median: 201 min vs. 286 min). Model performance was good, indicated by a c-statistic of 0.76 (95%confidence interval: 0.71–0.81). Conclusion: Outcome-prediction using models created from HERMES data, based on information available in the emergency department underestimated the actual outcome in patients with acute ischemic stroke and large vessel occlusion receiving endovascular treatment despite overall good model performance, which might be explained by differences in quality of and time to reperfusion. These findings underline the importance of timely and successful reperfusion for functional outcomes in acute stroke patients.</p

Rapid alteplase administration improves functional outcomes in patients with stroke due to large vessel occlusions

Background and Purpose: We report the relation of onset-to-treatment time and door-to-needle time with functional outcomes and mortality among patients with ischemic stroke with imaging-proven large vessel occlusion treated with intravenous alteplase. Methods: Individual patient-level data from the HERMES (Highly Effective Reperfusion Evaluated in Multiple Endovascular Stroke Trials) collaboration were pooled from 7 trials that randomized patients to mechanical thrombectomy added to best medical therapy versus best medical therapy alone. Analysis was restricted to patients who received alteplase directly at the endovascular hospital. The primary outcome was disability defined on the modified Rankin Scale at 3 months. Results: Among 601 patients, mean age was 66.0 years (SD, 13.9), 50% were women, and median National Institutes of Health Stroke Scale score was 17. Onset-to-treatment time was median 125 minutes (interquartile range, 90–170). Door-to-treatment time was median 38 minutes (interquartile range, 26–55). Each 60-minute onset-to-treatment time delay was associated with greater disability at 90 days; the odds of functional independence (modified Rankin Scale, 0–2) at 90 days was 0.82 (95% CI, 0.66–1.03). With each 60-minute delay in door-to-needle time; the odds of functional independence was 0.55 (95% CI, 0.37–0.81) at 90 days. The absolute decline in the rate of excellent outcome (modified Rankin Scale, 0–1 at 90 days) was 20.3 per 1000 patients treated per 15-minute delay in door-to-needle time. The adjusted absolute risk difference for a door-to-needle time &lt;30 minutes versus 30 to 60 minutes was 19.3% for independent outcome (number-needed-to-treat ≈5 to gain 1 additional good outcome). Symptomatic intracranial hemorrhage occurred in 3.4% of patients, without a significant time dependency: odds ratio, 0.74 (95% CI, 0.43–1.28). Conclusions: Faster intravenous thrombolysis delivery is associated with less disability at 3 months among patients with large vessel occlusion

Efficacy of endovascular thrombectomy in patients with M2 segment middle cerebral artery occlusions: meta-analysis of data from the HERMES Collaboration

Background: The Society of Neurointerventional Surgery revised its operational definition of emergent large vessel occlusion (ELVO) recently to include proximal M2 segment middle cerebral artery (MCA) occlusions. We sought to assess the benefit of endovascular thrombectomy (EVT) over best medical care for M2 segment MCA occlusion. Methods: Patient level data from trials in the HERMES Collaboration were included. The HERMES core laboratory identified patients with M2 segment MCA occlusions and further classified them as proximal versus distal, anterior versus posterior division, and dominant versus co-dominant versus non-dominant. Primary outcome was modified Rankin Scale (mRS) score 0–2 at 90 days. Secondary outcomes were modified Thrombolysis in Cerebral Infarction (mTICI) rates at end of procedure, 90-day mRS shift, 90-day mRS 0–1, 24 hours National Institute of Health Stroke Scale (NIHSS) score 0–2, symptomatic intracerebral hemorrhage (ICH), and death. Results: 130 patients with M2 MCA (proximal location n=116 vs distal n=14, anterior division n=72 vs posterior n=58, dominant n=73 vs co-dominant n=50 vs non-dominant n=7) were included. Successful reperfusion (mTICI 2b or 3) among those undergoing EVT was seen in 59.2% of patients. Treatment effect favored EVT (adjusted OR 2.39, 95% CI 1.08 to 5.28, p=0.03) for 90-day mRS 0–2 (58.2% EVT vs 39.7% control). Direction of benefit favored EVT for other outcomes. Treatment effect favoring EVT was maximal in patients with proximal M2 segment MCA occlusions (n=116, adjusted OR 2.68, 95% CI 1.13 to 6.37) and in dominant M2 segment MCA occlusions (n=73, adjusted OR 4.08, 95% CI 1.08 to 15.48). No sICH (0%) was observed in patients treated with EVT compared with five (7.9%) in the control arm. Conclusion: Patients with proximal M2 segment MCA occlusions eligible for EVT trial protocols benefited from EVT

Confirmatory study of time-dependent computed tomographic perfusion thresholds for use in acute ischemic stroke

Background and Purpose: Computed tomographic perfusion (CTP) thresholds associated with follow-up brain infarction may differ by time from symptom onset to imaging and reperfusion. We confirm CTP thresholds over time to imaging and reperfusion in patients with acute ischemic stroke from the HERMES collaboration (Highly Effective Reperfusion Evaluated in Multiple Endovascular Stroke Trials) data. Methods: Patients with occlusion on CT angiography were acutely imaged with CTP. Noncontrast CT and magnetic resonance-diffusion weighted imaging at 24 to 48 hours defined follow-up infarction. Reperfusion was assessed on conventional angiogram. Tmax, cerebral blood flow (CBF), and cerebral blood volume maps were derived from delay-insensitive CTP postprocessing. These parameters were analyzed using receiver operator characteristics to derive optimal thresholds based on time from stroke onset-to-CTP or to reperfusion. ANOVA and linear regression were used to test whether the derived CTP thresholds were different by time. Results: One hundred thirty-seven patients were included. Tmax thresholds of &gt;15.7 s and &gt;15.8 s and absolute CBF thresholds of &lt;8.9 and &lt;7.5 mL·min−1·100 g−1 for gray matter and white matter respectively were associated with infarct if reperfusion was achieved &lt;90 minutes from CTP with stroke onset-to-CTP &lt;180 minutes. The discriminative ability of cerebral blood volume was modest. There were no statistically significant relationships between stroke onset-to-CTP time and Tmax, CBF, and cerebral blood volume thresholds (all P&gt;0.05). A statistically significant relationship was observed between CTP-to-reperfusion time and the optimal thresholds for Tmax (P&lt;0.001) and CBF (P&lt;0.001). Similar but more modest relationship was noted for onset-to-reperfusion time and optimal thresholds for CBF (P≤0.01). Conclusions: CTP thresholds based on stroke onset and imaging time and taking into account time needed for reperfusion may improve infarct prediction in patients with acute ischemic stroke