Toward an Optogenetic Autonomous Nerve Control (OANC) System

Optogenetics is a developing field in neuroscience, where nerves are stimulated through optical instead of electrical signals, allowing for a more selective action of nerve populations [Aravanis et al, 2007]. In the context of electrical stimulation, the autonomous nerve control (ANC) system has been used successfully, first to map the response of different nerve fibers to create a Nerve Activation Profile, and then to use this profile in recruiting specific nerve fibers in a controlled way [MP Ward et al, unpublished manuscript in preparation]. The objective of the present project is to apply the ANC system to optical stimulation thus creating what we could call an optically-driven ANC system (OANC). With this in mind, we have designed an optical cuff that delivers an optical signal through LEDs whose light intensity is controlled by the ANC system. Using this cuff electrode, we have performed preliminary experiments (at The Jackson Laboratory, Bar Harbor, Maine) on genetically modified mice which include a light sensitive protein on the cell membranes that allows them to convert the optical stimulation in to electrical signals on the sensory nerve fibers in the sciatic nerve. These preliminary experiments showed very encouraging sensory nerve response, suggesting that further work is warranted in order to develop a closed loop OANC that measures the degree of nerve stimulation and controls the input signals accordingly

Focused ultrasound-mediated brain genome editing.

Gene editing in the brain has been challenging because of the restricted transport imposed by the blood-brain barrier (BBB). Current approaches mainly rely on local injection to bypass the BBB. However, such administration is highly invasive and not amenable to treating certain delicate regions of the brain. We demonstrate a safe and effective gene editing technique by using focused ultrasound (FUS) to transiently open the BBB for the transport of intravenously delivered CRISPR/Cas9 machinery to the brain

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK.

BACKGROUND: A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. METHODS: This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. FINDINGS: Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0-75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4-97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8-80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3-4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. INTERPRETATION: ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials. FUNDING: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, Bill & Melinda Gates Foundation, Lemann Foundation, Rede D'Or, Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK

Background A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. Methods This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. Findings Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0–75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4–97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8–80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3–4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. Interpretation ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials

Light sheet theta microscopy for rapid high-resolution imaging of large biological samples

Abstract Background Advances in tissue clearing and molecular labeling methods are enabling unprecedented optical access to large intact biological systems. These developments fuel the need for high-speed microscopy approaches to image large samples quantitatively and at high resolution. While light sheet microscopy (LSM), with its high planar imaging speed and low photo-bleaching, can be effective, scaling up to larger imaging volumes has been hindered by the use of orthogonal light sheet illumination. Results To address this fundamental limitation, we have developed light sheet theta microscopy (LSTM), which uniformly illuminates samples from the same side as the detection objective, thereby eliminating limits on lateral dimensions without sacrificing the imaging resolution, depth, and speed. We present a detailed characterization of LSTM, and demonstrate its complementary advantages over LSM for rapid high-resolution quantitative imaging of large intact samples with high uniform quality. Conclusions The reported LSTM approach is a significant step for the rapid high-resolution quantitative mapping of the structure and function of very large biological systems, such as a clarified thick coronal slab of human brain and uniformly expanded tissues, and also for rapid volumetric calcium imaging of highly motile animals, such as Hydra, undergoing non-isomorphic body shape changes

Video 5: Visualization of an image stack of vasculature stained rat brain tissue.

This video visualizes an image stack acquired from a large rat brain slice (stained for vasculature with tomato lectin) using LSTM in 2-AS (2-axes scan) mode. The bounding box is 1mm x 1mm x 5mm. Uniform high-resolution imaging of the entire tissue under LSTM is presented.<div><div><br></div><div>This video relates to the development of the imaging technique LSTM: Light Sheet Theta Microscopy for rapid high-resolution imaging of large biological samples, an approach designed to overcome the limitations of 'standard' Light Sheet microscopy LSM in terms of lateral dimensions and imaging quality, while preserving the benefits of LSM in terms of imaging resolution, depth and speed.</div><div><br></div><div>LSTM uses two symmetrically-arranged oblique light-sheets, generated using independent illumination objectives, for rapid high-resolution imaging of large samples.</div><div><br></div><div>View the full collection of related videos at: </div><p><a href="https://doi.org/10.6084/m9.figshare.c.4072160">https://doi.org/10.6084/m9.figshare.c.4072160</a></p></div

Video 8: Neuronal activity traces of representative neurons.

A visualization of the neuronal traces shown in Figure 7b of the related publication; presenting rapid volumetric calcium imaging of highly motile Hydra.<div><br><div>An advantage of the LSTM imaging technique (see below for details) is the capacity for rapid volumetric live imaging of samples undergoing substantial non-isomorphic rearrangements in their body shape and cellular density, resulting in continuously changing local optical properties.<br><div><br></div><div>This video relates to the development of the imaging technique LSTM: Light Sheet Theta Microscopy for rapid high-resolution imaging of large biological samples, an approach designed to overcome the limitations of 'standard' Light Sheet microscopy LSM in terms of lateral dimensions and imaging quality, while preserving the benefits of LSM in terms of imaging resolution, depth and speed.</div><div><br></div><div>LSTM uses two symmetrically-arranged oblique light-sheets, generated using independent illumination objectives, for rapid high-resolution imaging of large samples.</div><div><br></div><div>View the full collection of related videos at: </div><p><a href="https://doi.org/10.6084/m9.figshare.c.4072160">https://doi.org/10.6084/m9.figshare.c.4072160</a></p></div></div

Video 1: 3D model of LSTM implementation.

<div>The 3D modelling and rendering was performed using Autodesk Inventor 2017, and the animation was performed using Autodesk Fusion 360 2017 and MATLAB. The components labelled are LS (laser source), collimator, ND (neural density) filter mount, iris, ETL (electrically tunable lens), slit, CL (cylindrical lens), Galvo scanner, SL (scan lens), iris and TL (tube lens).</div><div><br></div><div>The LSTM illumination and detection arms are implemented as rigid assemblies (using the caging system from Thorlabs), connected to a vertically mounted breadboard via x-y manual translation stages for finer adjustments.</div><div><div><br></div><div>This video relates to the development of the imaging technique LSTM: Light Sheet Theta Microscopy for rapid high-resolution imaging of large biological samples, an approach designed to overcome the limitations of 'standard' Light Sheet microscopy LSM in terms of lateral dimensions and imaging quality, while preserving the benefits of LSM in terms of imaging resolution, depth and speed.<br></div><div><br></div><div>LSTM uses two symmetrically-arranged oblique light-sheets, generated using independent illumination objectives, for rapid high-resolution imaging of large samples.</div><div><br></div><div>View the full collection of related videos at: </div><p><a href="https://doi.org/10.6084/m9.figshare.c.4072160">https://doi.org/10.6084/m9.figshare.c.4072160</a></p></div

Video 2: Comparison of image volumes acquired with LSTM in 1-AS and 2-AS modes.

<div><p>The 3D rendering visualizes the image stacks acquired from the same sample (human brain section stained with DAPI) with LSTM in 1-Axis Scan (1-AS) and simultaneous 2-Axes Scan (2-AS) mode.</p><p><br></p> <p>This video relates to the development of the imaging technique LSTM: Light Sheet Theta Microscopy for rapid high-resolution imaging of large biological samples, an approach designed to overcome the limitations of 'standard' Light Sheet microscopy LSM in terms of lateral dimensions and imaging quality, while preserving the benefits of LSM in terms of imaging resolution, depth and speed.</p></div><div><br></div><div>LSTM uses two symmetrically-arranged oblique light-sheets, generated using independent illumination objectives, for rapid high-resolution imaging of large samples.</div><div><br></div><div>View the full collection of related videos at: </div><p><a href="https://doi.org/10.6084/m9.figshare.c.4072160">https://doi.org/10.6084/m9.figshare.c.4072160</a></p

Video 7: Rapid volumetric calcium imaging of highly motile Hydra.

GCaMP6s expressing Hydra was imaged using LSTM with 10x/0.6NA objective. Maximum intensity projections are shown for the two halves of the volume. First occurrences of longitudinal and radial contractions are annotated. The scale Bar is 100 microns.<div><br></div><div>An advantage of the LSTM imaging technique (see below for details) is the capacity for rapid volumetric live imaging of samples undergoing substantial non-isomorphic rearrangements in their body shape and cellular density, resulting in continuously changing local optical properties.<div><div><br></div><div>This video relates to the development of the imaging technique LSTM: Light Sheet Theta Microscopy for rapid high-resolution imaging of large biological samples, an approach designed to overcome the limitations of 'standard' Light Sheet microscopy LSM in terms of lateral dimensions and imaging quality, while preserving the benefits of LSM in terms of imaging resolution, depth and speed.</div><div><br></div><div>LSTM uses two symmetrically-arranged oblique light-sheets, generated using independent illumination objectives, for rapid high-resolution imaging of large samples.</div><div><br></div><div>View the full collection of related videos at: </div><p><a href="https://doi.org/10.6084/m9.figshare.c.4072160">https://doi.org/10.6084/m9.figshare.c.4072160</a></p></div></div