Vitamin E Decreases Cytotoxicity and Mitigates Inflammatory and Oxidative Stress Responses in a Ferret Organotypic Brain Slice Model of Neonatal Hypoxia-Ischemia.

The gyrencephalic ferret brain is an excellent model in which to study hypoxia-ischemia (HI), a significant contributor to neurological injury in neonates. Vitamin E, an essential fat-soluble antioxidant, reduces oxidative stress and inflammation in both animal models and human infants. The aim of this study was to assess the effects of vitamin E after oxygen-glucose deprivation (OGD) in an organotypic ferret brain slice model of neonatal HI. We hypothesized that vitamin E would decrease cytotoxicity, inflammation, and oxidative stress in OGD-exposed brain slices. Term-equivalent ferrets were sacrificed at postnatal (P) day 21-23 and 300 µM whole-hemisphere brain slices were obtained. During a 24-h rest period, slices were cultured in either nontreated control conditions or with erastin, a promotor of oxidative stress. Slices were then exposed to 2 h of OGD followed by vitamin E (25-100 IU/kg), erastin (10 µM), or ferrostatin (1 µM), an inhibitor of ferroptosis. Relative cytotoxicity was determined using a lactate dehydrogenase assay, cell death was quantified via nuclear propidium iodide staining, oxidative stress was quantified via cellular glutathione (GSH) levels, and target genes responsive to oxidative stress and inflammation were evaluated by qRT-PCR. OGD increased cytotoxicity, which was significantly reduced by treatment with vitamin E. Vitamin E also preserved GSH after OGD and decreased amplification of certain markers of oxidative stress (CHAC1, SLC7A11) and inflammation (TNF-alpha, IL-8). Vitamin E remained protective after pretreatment with erastin and was more protective than ferrostatin, presumably due to its added anti-inflammatory properties. Results from the ferret whole-hemisphere OGD model support the premise that vitamin E neuroprotection is mediated by restoring GSH and acutely decreasing inflammation and oxidative stress after neonatal HI.

Mycorrhiza: genotype assignment using phylogenetic networks.

MOTIVATION: The genotype assignment problem consists of predicting, from the genotype of an individual, which of a known set of populations it originated from. The problem arises in a variety of contexts, including wildlife forensics, invasive species detection and biodiversity monitoring. Existing approaches perform well under ideal conditions but are sensitive to a variety of common violations of the assumptions they rely on. RESULTS: In this article, we introduce Mycorrhiza, a machine learning approach for the genotype assignment problem. Our algorithm makes use of phylogenetic networks to engineer features that encode the evolutionary relationships among samples. Those features are then used as input to a Random Forests classifier. The classification accuracy was assessed on multiple published empirical SNP, microsatellite or consensus sequence datasets with wide ranges of size, geographical distribution and population structure and on simulated datasets. It compared favorably against widely used assessment tests or mixture analysis methods such as STRUCTURE and Admixture, and against another machine-learning based approach using principal component analysis for dimensionality reduction. Mycorrhiza yields particularly significant gains on datasets with a large average fixation index (FST) or deviation from the Hardy-Weinberg equilibrium. Moreover, the phylogenetic network approach estimates mixture proportions with good accuracy. AVAILABILITY AND IMPLEMENTATION: Mycorrhiza is released as an easy to use open-source python package at github.com/jgeofil/mycorrhiza. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Spatiotemporal functional assembly of split protein pairs through a light-activated SpyLigation.

Proteins provide essential functional regulation of many bioprocesses across all scales of life; however, new techniques to specifically modulate protein activity within living systems and in engineered biomaterials are needed to better interrogate fundamental cell signalling and guide advanced decisions of biological fate. Here we establish a generalizable strategy to rapidly and irreversibly activate protein function with full spatiotemporal control. Through the development of a genetically encoded and light-activated SpyLigation (LASL), bioactive proteins can be stably reassembled from non-functional split fragment pairs following brief exposure (typically minutes) to cytocompatible light. Employing readily accessible photolithographic processing techniques to specify when, where and how much photoligation occurs, we demonstrate precise protein activation of UnaG, NanoLuc and Cre recombinase using LASL in solution, biomaterials and living mammalian cells, as well as optical control over protein subcellular localization. Looking forward, we expect that these photoclick-based optogenetic approaches will find tremendous utility in probing and directing complex cellular fates in both time and three-dimensional space.

Organotypic whole hemisphere brain slice models to study the effects of donor age and oxygen-glucose-deprivation on the extracellular properties of cortical and striatal tissue.

BACKGROUND: The brain extracellular environment is involved in many critical processes associated with neurodevelopment, neural function, and repair following injury. Organization of the extracellular matrix and properties of the extracellular space vary throughout development and across different brain regions, motivating the need for platforms that provide access to multiple brain regions at different stages of development. We demonstrate the utility of organotypic whole hemisphere brain slices as a platform to probe regional and developmental changes in the brain extracellular environment. We also leverage whole hemisphere brain slices to characterize the impact of cerebral ischemia on different regions of brain tissue. RESULTS: Whole hemisphere brain slices taken from postnatal (P) day 10 and P17 rats retained viable, metabolically active cells through 14 days in vitro (DIV). Oxygen-glucose-deprivation (OGD), used to model a cerebral ischemic event in vivo, resulted in reduced slice metabolic activity and elevated cell death, regardless of slice age. Slices from P10 and P17 brains showed an oligodendrocyte and microglia-driven proliferative response after OGD exposure, higher than the proliferative response seen in DIV-matched normal control slices. Multiple particle tracking in oxygen-glucose-deprived brain slices revealed that oxygen-glucose-deprivation impacts the extracellular environment of brain tissue differently depending on brain age and brain region. In most instances, the extracellular space was most difficult to navigate immediately following insult, then gradually provided less hindrance to extracellular nanoparticle diffusion as time progressed. However, changes in diffusion were not universal across all brain regions and ages. CONCLUSIONS: We demonstrate whole hemisphere brain slices from P10 and P17 rats can be cultured up to two weeks in vitro. These brain slices provide a viable platform for studying both normal physiological processes and injury associated mechanisms with control over brain age and region. Ex vivo OGD impacted cortical and striatal brain tissue differently, aligning with preexisting data generated in in vivo models. These data motivate the need to account for both brain region and age when investigating mechanisms of injury and designing potential therapies for cerebral ischemia.

Effect of Azide Preservative on Thermomechanical Aggregation of Purified Reference Protein Materials.

Protein aggregation can affect the quality of protein-based therapeutics. Attempting to unravel factors influencing protein aggregation involves systematic studies. These studies often include sodium azide or similar preservatives in the aggregation buffer. This work shows effects of azide on aggregation of two highly purified reference proteins, both a bovine serum albumin (BSA) as well as a monoclonal antibody (NISTmAb). The proteins were aggregated by thermomechanical stress, consisting of simultaneous heating of the solution with gentle agitation. Protein aggregates were characterized by asymmetric flow field flow fractionation (AF4) with light scattering measurements along with quantification by UV spectroscopy, revealing strong time-dependent generation of aggregated protein and an increase in aggregate molar mass. Gel electrophoresis was used to probe the reversibility of the aggregation and demonstrated complete reversibility for the NISTmAb, but not so for the BSA. Kinetic fitting to a commonly implemented nucleated polymerization model was also employed to provide mechanistic details into the kinetic process. The model suggests that the aggregation of the NISTmAb proceeds via nucleated growth and aggregate-aggregate condensation in a way that is dependent on the concentration (and presence) of the azide anion. This work overall implicates azide preservatives as having demonstrable effects on thermomechanical stress and aggregation of proteins undergoing systematic aggregation and stability studies.

Nanoparticle-microglial interaction in the ischemic brain is modulated by injury duration and treatment.

Cerebral ischemia is a major cause of death in both neonates and adults, and currently has no cure. Nanotechnology represents one promising area of therapeutic development for cerebral ischemia due to the ability of nanoparticles to overcome biological barriers in the brain. ex vivo injury models have emerged as a high-throughput alternative that can recapitulate disease processes and enable nanoscale probing of the brain microenvironment. In this study, we used oxygen-glucose deprivation (OGD) to model ischemic injury and studied nanoparticle interaction with microglia, resident immune cells in the brain that are of increasing interest for therapeutic delivery. By measuring cell death and glutathione production, we evaluated the effect of OGD exposure time and treatment with azithromycin (AZ) on slice health. We found a robust injury response with 0.5 hr of OGD exposure and effective treatment after immediate application of AZ. We observed an OGD-induced shift in microglial morphology toward increased heterogeneity and circularity, and a decrease in microglial number, which was reversed after treatment. OGD enhanced diffusion of polystyrene-poly(ethylene glycol) (PS-PEG) nanoparticles, improving transport and ability to reach target cells. While microglial uptake of dendrimers or quantum dots (QDs) was not enhanced after injury, internalization of PS-PEG was significantly increased. For PS-PEG, AZ treatment restored microglial uptake to normal control levels. Our results suggest that different nanoparticle platforms should be carefully screened before application and upon doing so; disease-mediated changes in the brain microenvironment can be leveraged by nanoscale drug delivery devices for enhanced cell interaction.