Nox2 redox signaling maintains essential cell populations in the brain.

Reactive oxygen species (ROS) are conventionally classified as toxic consequences of aerobic life, and the brain is particularly susceptible to ROS-induced oxidative stress and damage owing to its high energy and oxygen demands. NADPH oxidases (Nox) are a widespread source of brain ROS implicated in seizures, stroke and neurodegeneration. A physiological role for ROS generation in normal brain function has not been established, despite the fact that mice and humans lacking functional Nox proteins have cognitive deficits. Using molecular imaging with Peroxyfluor-6 (PF6), a new selective fluorescent indicator for hydrogen peroxide (H(2)O(2)), we show that adult hippocampal stem/progenitor cells (AHPs) generate H(2)O(2) through Nox2 to regulate intracellular growth signaling pathways, which in turn maintains their normal proliferation in vitro and in vivo. Our results challenge the traditional view that brain ROS are solely deleterious by demonstrating that controlled ROS chemistry is needed for maintaining specific cell populations

HSC70/HSP40 overexpression in viral vector-producing Sf9 cells impacts AAV productivity and product quality

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Proteomic characterisation of endoplasmic reticulum-derived protein bodies in tobacco leaves

<p>Abstract</p> <p>Background</p> <p>The N-terminal proline-rich domain (Zera) of the maize storage protein γ-zein, is able to induce the formation of endoplasmic reticulum (ER)-derived protein bodies (PBs) when fused to proteins of interest. This encapsulation enables a recombinant fused protein to escape from degradation and facilitates its recovery from plant biomass by gradient purification. The aim of the present work was to evaluate if induced PBs encapsulate additional proteins jointly with the recombinant protein. The exhaustive analysis of protein composition of PBs is expected to facilitate a better understanding of PB formation and the optimization of recombinant protein purification approaches from these organelles.</p> <p>Results</p> <p>We analysed the proteome of PBs induced in <it>Nicotiana benthamiana </it>leaves by transient transformation with Zera fused to a fluorescent marker protein (DsRed). Intact PBs with their surrounding ER-membrane were isolated on iodixanol based density gradients and their integrity verified by confocal and electron microscopy. SDS-PAGE analysis of isolated PBs showed that Zera-DsRed accounted for around 85% of PB proteins in term of abundance. Differential extraction of PBs was performed for in-depth analysis of their proteome and structure. Besides Zera-DsRed, 195 additional proteins were identified including a broad range of proteins resident or trafficking through the ER and recruited within the Zera-DsRed polymer.</p> <p>Conclusions</p> <p>This study indicates that Zera-protein fusion is still the major protein component of the new formed organelle in tobacco leaves. The analysis also reveals the presence of an unexpected diversity of proteins in PBs derived from both the insoluble Zera-DsRed polymer formation, including ER-resident and secretory proteins, and a secretory stress response induced most likely by the recombinant protein overloading. Knowledge of PBs protein composition is likely to be useful to optimize downstream purification of recombinant proteins in molecular farming applications.</p

Deploying a Top-100 Supercomputer for Large Parallel Workloads: the Niagara Supercomputer

Niagara is currently the fastest supercomputer accessible to academics in Canada. It was deployed at the beginning of 2018 and has been serving the research community ever since. This homogeneous 60,000-core cluster, owned by the University of Toronto and operated by SciNet, was intended to enable large parallel jobs and has a measured performance of 3.02 petaflops, debuting at #53 in the June 2018 TOP500 list. It was designed to optimize throughput of a range of scientific codes running at scale, energy efficiency, and network and storage performance and capacity. It replaced two systems that SciNet operated for over 8 years, the Tightly Coupled System (TCS) and the General Purpose Cluster (GPC). In this paper we describe the transition process from these two systems, the procurement and deployment processes, as well as the unique features that make Niagara a one-of-a-kind machine in Canada.Comment: PEARC'19: "Practice and Experience in Advanced Research Computing", July 28-August 1, 2019, Chicago, IL, US

Complement membrane attack complex is an immunometabolic regulator of NLRP3 activation and IL-18 secretion in human macrophages

The complement system is an ancient and critical part of innate immunity. Recent studies have highlighted novel roles of complement beyond lysis of invading pathogens with implications in regulating the innate immune response, as well as contributing to metabolic reprogramming of T-cells, synoviocytes as well as cells in the CNS. These findings hint that complement can be an immunometabolic regulator, but whether this is also the case for the terminal step of the complement pathway, the membrane attack complex (MAC) is not clear. In this study we focused on determining whether MAC is an immunometabolic regulator of the innate immune response in human monocyte-derived macrophages. Here, we uncover previously uncharacterized metabolic changes and mitochondrial dysfunction occurring downstream of MAC deposition. These alterations in glycolytic flux and mitochondrial morphology and function mediate NLRP3 inflammasome activation, pro-inflammatory cytokine release and gasdermin D formation. Together, these data elucidate a novel signalling cascade, with metabolic alterations at its center, in MAC-stimulated human macrophages that drives an inflammatory consequence in an immunologically relevant cell type

Serum microrna biomarkers for detection of non-small cell lung cancer

Non small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality world-wide and the majority of cases are diagnosed at late stages of disease. There is currently no cost-effective screening test for NSCLC, and the development of such a test is a public health imperative. Recent studies have suggested that chest computed tomography screening of patients at high risk of lung cancer can increase survival from disease, however, the cost effectiveness of such screening has not been established. In this Phase I/II biomarker study we examined the feasibility of using serum miRNA as biomarkers of NSCLC using RT-qPCR to examine the expression of 180 miRNAs in sera from 30 treatment naive NSCLC patients and 20 healthy controls. Receiver operating characteristic curves (ROC) and area under the curve were used to identify differentially expressed miRNA pairs that could distinguish NSCLC from healthy controls. Selected miRNA candidates were further validated in sera from an additional 55 NSCLC patients and 75 healthy controls. Examination of miRNA expression levels in serum from a multi-institutional cohort of 50 subjects (30 NSCLC patients and 20 healthy controls) identified differentially expressed miRNAs. A combination of two differentially expressed miRNAs miR-15b and miR-27b, was able to discriminate NSCLC from healthy controls with sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 100% in the training set. Upon further testing on additional 130 subjects (55 NSCLC and 75 healthy controls), this miRNA pair predicted NSCLC with a specificity of 84% (95% CI 0.73-0.91), sensitivity of 100% (95% CI; 0.93-1.0), NPV of 100%, and PPV of 82%. These data provide evidence that serum miRNAs have the potential to be sensitive, cost-effective biomarkers for the early detection of NSCLC. Further testing in a Phase III biomarker study in is necessary for validation of these results. © 2012 Hennessey et al

Across‐vendor standardization of semi‐LASER for single‐voxel MRS at 3T

The semi‐adiabatic localization by adiabatic selective refocusing (sLASER) sequence provides single‐shot full intensity signal with clean localization and minimal chemical shift displacement error and was recommended by the international MRS Consensus Group as the preferred localization sequence at high‐ and ultra‐high fields. Across‐vendor standardization of the sLASER sequence at 3 tesla has been challenging due to the B1 requirements of the adiabatic inversion pulses and maximum B1 limitations on some platforms. The aims of this study were to design a short‐echo sLASER sequence that can be executed within a B1 limit of 15 μT by taking advantage of gradient‐modulated RF pulses, to implement it on three major platforms and to evaluate the between‐vendor reproducibility of its perfomance with phantoms and in vivo. In addition, voxel‐based first and second order B0 shimming and voxel‐based B1 adjustments of RF pulses were implemented on all platforms. Amongst the gradient‐modulated pulses considered (GOIA, FOCI and BASSI), GOIA‐WURST was identified as the optimal refocusing pulse that provides good voxel selection within a maximum B1 of 15 μT based on localization efficiency, contamination error and ripple artifacts of the inversion profile. An sLASER sequence (30 ms echo time) that incorporates VAPOR water suppression and 3D outer volume suppression was implemented with identical parameters (RF pulse type and duration, spoiler gradients and inter‐pulse delays) on GE, Philips and Siemens and generated identical spectra on the GE ‘Braino’ phantom between vendors. High‐quality spectra were consistently obtained in multiple regions (cerebellar white matter, hippocampus, pons, posterior cingulate cortex and putamen) in the human brain across vendors (5 subjects scanned per vendor per region; mean signal‐to‐noise ratio [less than] 33; mean water linewidth between 6.5 Hz to 11.4 Hz). The harmonized sLASER protocol is expected to produce high reproducibility of MRS across sites thereby allowing large multi‐site studies with clinical cohorts

Frequency drift in MR spectroscopy at 3T

Purpose: Heating of gradient coils and passive shim components is a common cause of instability in the B-0 field, especially when gradient intensive sequences are used. The aim of the study was to set a benchmark for typical drift encountered during MR spectroscopy (MRS) to assess the need for real-time field-frequency locking on MRI scanners by comparing field drift data from a large number of sites.Method: A standardized protocol was developed for 80 participating sites using 99 3T MR scanners from 3 major vendors. Phantom water signals were acquired before and after an EPI sequence. The protocol consisted of: minimal preparatory imaging; a short pre-fMRI PRESS; a ten-minute fMRI acquisition; and a long post-fMRI PRESS acquisition. Both pre- and post-fMRI PRESS were non-water suppressed. Real-time frequency stabilization/adjustment was switched off when appropriate. Sixty scanners repeated the protocol for a second dataset. In addition, a three-hour post-fMRI MRS acquisition was performed at one site to observe change of gradient temperature and drift rate. Spectral analysis was performed using MATLAB. Frequency drift in pre-fMRI PRESS data were compared with the first 5:20 minutes and the full 30:00 minutes of data after fMRI. Median (interquartile range) drifts were measured and showed in violin plot. Paired t-tests were performed to compare frequency drift pre- and post-fMRI. A simulated in vivo spectrum was generated using FID-A to visualize the effect of the observed frequency drifts. The simulated spectrum was convolved with the frequency trace for the most extreme cases. Impacts of frequency drifts on NAA and GABA were also simulated as a function of linear drift. Data from the repeated protocol were compared with the corresponding first dataset using Pearson's and intraclass correlation coefficients (ICC).Results: Of the data collected from 99 scanners, 4 were excluded due to various reasons. Thus, data from 95 scanners were ultimately analyzed. For the first 5:20 min (64 transients), median (interquartile range) drift was 0.44 (1.29) Hz before fMRI and 0.83 (1.29) Hz after. This increased to 3.15 (4.02) Hz for the full 30 min (360 transients) run. Average drift rates were 0.29 Hz/min before fMRI and 0.43 Hz/min after. Paired t-tests indicated that drift increased after fMRI, as expected (p &lt; 0.05). Simulated spectra convolved with the frequency drift showed that the intensity of the NAA singlet was reduced by up to 26%, 44 % and 18% for GE, Philips and Siemens scanners after fMRI, respectively. ICCs indicated good agreement between datasets acquired on separate days. The single site long acquisition showed drift rate was reduced to 0.03 Hz/min approximately three hours after fMRI.Discussion: This study analyzed frequency drift data from 95 3T MRI scanners. Median levels of drift were relatively low (5-min average under 1 Hz), but the most extreme cases suffered from higher levels of drift. The extent of drift varied across scanners which both linear and nonlinear drifts were observed.</p