Enrichable consortia of microbial symbionts degrade macroalgal polysaccharides in Kyphosus fish.

Coastal herbivorous fishes consume macroalgae, which is then degraded by microbes along their digestive tract. However, there is scarce genomic information about the microbiota that perform this degradation. This study explores the potential of Kyphosus gastrointestinal microbial symbionts to collaboratively degrade and ferment polysaccharides from red, green, and brown macroalgae through in silico study of carbohydrate-active enzyme and sulfatase sequences. Recovery of metagenome-assembled genomes (MAGs) from previously described Kyphosus gut metagenomes and newly sequenced bioreactor enrichments reveals differences in enzymatic capabilities between the major microbial taxa in Kyphosus guts. The most versatile of the recovered MAGs were from the Bacteroidota phylum, whose MAGs house enzyme collections able to decompose a variety of algal polysaccharides. Unique enzymes and predicted degradative capacities of genomes from the Bacillota (genus Vallitalea) and Verrucomicrobiota (order Kiritimatiellales) highlight the importance of metabolic contributions from multiple phyla to broaden polysaccharide degradation capabilities. Few genomes contain the required enzymes to fully degrade any complex sulfated algal polysaccharide alone. The distribution of suitable enzymes between MAGs originating from different taxa, along with the widespread detection of signal peptides in candidate enzymes, is consistent with cooperative extracellular degradation of these carbohydrates. This study leverages genomic evidence to reveal an untapped diversity at the enzyme and strain level among Kyphosus symbionts and their contributions to macroalgae decomposition. Bioreactor enrichments provide a genomic foundation for degradative and fermentative processes central to translating the knowledge gained from this system to the aquaculture and bioenergy sectors.IMPORTANCESeaweed has long been considered a promising source of sustainable biomass for bioenergy and aquaculture feed, but scalable industrial methods for decomposing terrestrial compounds can struggle to break down seaweed polysaccharides efficiently due to their unique sulfated structures. Fish of the genus Kyphosus feed on seaweed by leveraging gastrointestinal bacteria to degrade algal polysaccharides into simple sugars. This study reconstructs metagenome-assembled genomes for these gastrointestinal bacteria to enhance our understanding of herbivorous fish digestion and fermentation of algal sugars. Investigations at the gene level identify Kyphosus guts as an untapped source of seaweed-degrading enzymes ripe for further characterization. These discoveries set the stage for future work incorporating marine enzymes and microbial communities in the industrial degradation of algal polysaccharides.

Enrichable consortia of microbial symbionts degrade macroalgal polysaccharides in Kyphosus fish.

Coastal herbivorous fishes consume macroalgae, which is then degraded by microbes along their digestive tract. However, there is scarce foundational genomic work on the microbiota that perform this degradation. This study explores the potential of Kyphosus gastrointestinal microbial symbionts to collaboratively degrade and ferment polysaccharides from red, green, and brown macroalgae through in silico study of carbohydrate-active enzyme and sulfatase sequences. Recovery of metagenome-assembled genomes (MAGs) reveals differences in enzymatic capabilities between the major microbial taxa in Kyphosus guts. The most versatile of the recovered MAGs were from the Bacteroidota phylum, whose MAGs house enzymes able to decompose a variety of algal polysaccharides. Unique enzymes and predicted degradative capacities of genomes from the Bacillota (genus Vallitalea) and Verrucomicrobiota (order Kiritimatiellales) suggest the potential for microbial transfer between marine sediment and Kyphosus digestive tracts. Few genomes contain the required enzymes to fully degrade any complex sulfated algal polysaccharide alone. The distribution of suitable enzymes between MAGs originating from different taxa, along with the widespread detection of signal peptides in candidate enzymes, is consistent with cooperative extracellular degradation of these carbohydrates. This study leverages genomic evidence to reveal an untapped diversity at the enzyme and strain level among Kyphosus symbionts and their contributions to macroalgae decomposition. Bioreactor enrichments provide a genomic foundation for degradative and fermentative processes central to translating the knowledge gained from this system to the aquaculture and bioenergy sectors.

Tropical Red Macroalgae Cultivation with a Focus on Compositional Analysis.

To create carbon efficient sources of bioenergy feedstocks and feedstuff for aquaculture and terrestrial livestock, it is critical to develop and commercialize the most efficient seaweed cultivation approach with a sustainable nutrient input supply. Here, we present data for a novel, onshore tropical macroalgae cultivation system, based on influent deep seawater as the nutrient and carbon sources. Two red algal species were selected, Agardhiella subulata and Halymenia hawaiiana, as the basis for growth optimization. Highest productivity in small-scale cultivation was demonstrated with A. subulata in the 10% deep seawater (64.7 µg N L-1) treatment, growing at up to 26% specific growth rate day-1 with highest yields observed at 247.5 g m-2 day-1 fresh weight. The highest yields for H. hawaiiana were measured with the addition of 10% deep seawater up to 8.8% specific growth rate day-1 and yields at 63.3 g fresh weight m-2 day-1 equivalent. Biomass should be culled weekly or biweekly to avoid density limitations, which likely contributed to a decrease in SGR over time. With a measured 30-40% carbon content of the ash-free dry weight (20-30% of the dry weight) biomass, this translates to an almost 1:1 CO2 capture to biomass ratio. The compositional fingerprint of the high carbohydrate content of both Agardhiella and Halymenia makes for an attractive feedstock for downstream biorefinery applications. By focusing on scaling and optimizing seaweed farming technologies for large-scale onshore farms, the opportunities for yield potential, adaptability to cultivation conditions, and meeting global sustainability goals through novel, carbon-negative biomass sources such as seaweed can be realized.

Herbivorous Fish Microbiome Adaptations to Sulfated Dietary Polysaccharides.

Marine herbivorous fish that feed primarily on macroalgae, such as those from the genus Kyphosus, are essential for maintaining coral health and abundance on tropical reefs. Here, deep metagenomic sequencing and assembly of gut compartment-specific samples from three sympatric, macroalgivorous Hawaiian kyphosid species have been used to connect host gut microbial taxa with predicted protein functional capacities likely to contribute to efficient macroalgal digestion. Bacterial community compositions, algal dietary sources, and predicted enzyme functionalities were analyzed in parallel for 16 metagenomes spanning the mid- and hindgut digestive regions of wild-caught fishes. Gene colocalization patterns of expanded carbohydrate (CAZy) and sulfatase (SulfAtlas) digestive enzyme families on assembled contigs were used to identify likely polysaccharide utilization locus associations and to visualize potential cooperative networks of extracellularly exported proteins targeting complex sulfated polysaccharides. These insights into the gut microbiota of herbivorous marine fish and their functional capabilities improve our understanding of the enzymes and microorganisms involved in digesting complex macroalgal sulfated polysaccharides. IMPORTANCE This work connects specific uncultured bacterial taxa with distinct polysaccharide digestion capabilities lacking in their marine vertebrate hosts, providing fresh insights into poorly understood processes for deconstructing complex sulfated polysaccharides and potential evolutionary mechanisms for microbial acquisition of expanded macroalgal utilization gene functions. Several thousand new marine-specific candidate enzyme sequences for polysaccharide utilization have been identified. These data provide foundational resources for future investigations into suppression of coral reef macroalgal overgrowth, fish host physiology, the use of macroalgal feedstocks in terrestrial and aquaculture animal feeds, and the bioconversion of macroalgae biomass into value-added commercial fuel and chemical products.

Investigation into the Effect of Interlock Volume on SPR Strength.

During the design of automotive structures assembled using Self-Piercing Rivets (SPRs), a rivet and die combination is selected for each joint stack. To conduct extensive physical tensile testing on every joint combination to determine the range of strength achieved by each rivet-die combination, a great deal of lab technician time and substrate material are required. It is much simpler and less material-consuming to select the rivet and die solution by examining the cross sections of joints. However, the current methods of measuring cross sections by measuring the amount of mechanical interlock in a linear X-Y direction, achieved with the flared rivet tail, do not give an accurate prediction of joint strength, because they do not measure the full amount of material that must be defeated to pull the rivet tail out of the bottom sheet. The X-Y linear interlock measurement approach also makes it difficult to rapidly rank joint solutions, as it creates two values for each cross section rather than a single value. This study investigates an innovative new measurement method developed by the authors called Volumelock. The approach measures the volume of material that must be defeated to pull out the rivet. Creating a single measurement value for each rivet-die combination makes it much easier to compare different rivet and die solutions; to identify solutions that work well across a number of different stacks; to aid the grouping of stacks on one setter for low-volume line; and to select the strongest solutions for a high-volume line where only one or two different stacks are made by each setter. The joint stack results in this paper indicate that there is a good predictive relationship between the new Volumelock method and peel strength, measured by physical cross-tension testing. In this study, the Volumelock approach predicted the peel strength within a 5% error margin.

Fine scale transitions of the microbiota and metabolome along the gastrointestinal tract of herbivorous fishes.

BACKGROUND: Gut microorganisms aid in the digestion of food by providing exogenous metabolic pathways to break down organic compounds. An integration of longitudinal microbial and chemical data is necessary to illuminate how gut microorganisms supplement the energetic and nutritional requirements of animals. Although mammalian gut systems are well-studied in this capacity, the role of microbes in the breakdown and utilization of recalcitrant marine macroalgae in herbivorous fish is relatively understudied and an emerging priority for bioproduct extraction. Here we use a comprehensive survey of the marine herbivorous fish gut microbial ecosystem via parallel 16S rRNA gene amplicon profiling (microbiota) and untargeted tandem mass spectrometry (metabolomes) to demonstrate consistent transitions among 8 gut subsections across five fish of the genus of Kyphosus. RESULTS: Integration of microbial phylogenetic and chemical diversity data reveals that microbial communities and metabolomes covaried and differentiated continuously from stomach to hindgut, with the midgut containing multiple distinct and previously uncharacterized microenvironments and a distinct hindgut community dominated by obligate anaerobes. This differentiation was driven primarily by anaerobic gut endosymbionts of the classes Bacteroidia and Clostridia changing in concert with bile acids, small peptides, and phospholipids: bile acid deconjugation associated with early midgut microbiota, small peptide production associated with midgut microbiota, and phospholipid production associated with hindgut microbiota. CONCLUSIONS: The combination of microbial and untargeted metabolomic data at high spatial resolution provides a new view of the diverse fish gut microenvironment and serves as a foundation to understand functional partitioning of microbial activities that contribute to the digestion of complex macroalgae in herbivorous marine fish.

Differential effects of the cell cycle inhibitor, olomoucine, on functional recovery and on responses of peri-infarct microglia and astrocytes following photothrombotic stroke in rats.

BACKGROUND: Following stroke, changes in neuronal connectivity in tissue surrounding the infarct play an important role in both spontaneous recovery of neurological function and in treatment-induced improvements in function. Microglia and astrocytes influence this process through direct interactions with the neurons and as major determinants of the local tissue environment. Subpopulations of peri-infarct glia proliferate early after stroke providing a possible target to modify recovery. Treatment with cell cycle inhibitors can reduce infarct volume and improve functional recovery. However, it is not known whether these inhibitors can influence neurological function or alter the responses of peri-infarct glia without reducing infarction. The present study aimed to address these issues by testing the effects of the cell cycle inhibitor, olomoucine, on recovery and peri-infarct changes following photothrombotic stroke. METHODS: Stroke was induced by photothrombosis in the forelimb sensorimotor cortex in Sprague-Dawley rats. Olomoucine was administered at 1 h and 24 h after stroke induction. Forelimb function was monitored up to 29 days. The effects of olomoucine on glial cell responses in peri-infarct tissue were evaluated using immunohistochemistry and Western blotting. RESULTS: Olomoucine treatment did not significantly affect maximal infarct volume. Recovery of the affected forelimb on a placing test was impaired in olomoucine-treated rats, whereas recovery in a skilled reaching test was substantially improved. Olomoucine treatment produced small changes in aspects of Iba1 immunolabelling and in the number of CD68-positive cells in cerebral cortex but did not selectively modify responses in peri-infarct tissue. The content of the astrocytic protein, vimentin, was reduced by 30% in the region of the lesion in olomoucine-treated rats. CONCLUSIONS: Olomoucine treatment modified functional recovery in the absence of significant changes in infarct volume. The effects on recovery were markedly test dependent, adding to evidence that skilled tasks requiring specific training and general measures of motor function can be differentially modified by some interventions. The altered recovery was not associated with specific changes in key responses of peri-infarct microglia, even though these cells were considered a likely target for early olomoucine treatment. Changes detected in peri-infarct reactive astrogliosis could contribute to the altered patterns of functional recovery.

Delayed Treatment with Human Dental Pulp Stem Cells Accelerates Functional Recovery and Modifies Responses of Peri-Infarct Astrocytes Following Photothrombotic Stroke in Rats.

Dental pulp contains multipotent mesenchymal stem cells that improve outcomes when administered early after temporary middle cerebral artery occlusion in rats. To further assess the therapeutic potential of these cells, we tested whether functional recovery following stroke induced by photothrombosis could be modified by a delayed treatment that was initiated after the infarct attained maximal volume. Photothrombosis induces permanent focal ischemia resulting in tissue changes that better reflect key aspects of the many human strokes in which early restoration of blood flow does not occur. Human dental pulp stem cells (approximately 400 × 103 viable cells) or vehicle were injected into the infarct and adjacent brain tissue of Sprague-Dawley rats at 3 days after the induction of unilateral photothrombotic stroke in the sensorimotor cortex. Forepaw function was tested up to 28 days after stroke. Cellular changes in peri-infarct tissue at 28 days were assessed using immunohistochemistry. Rats treated with the stem cells showed faster recovery compared with vehicle-treated animals in a test of forelimb placing in response to vibrissae stimulation and in first attempt success in a skilled forelimb reaching test. Total success in the skilled reaching test and forepaw use during exploration in a Perspex cylinder were not significantly different between the 2 groups. At 28 days after stroke, rats treated with the stem cells showed decreased immunolabeling for glial fibrillary acidic protein in tissue up to 1 mm from the infarct, suggesting decreased reactive astrogliosis. Synaptophysin, a marker of synapses, and collagen IV, a marker of capillaries, were not significantly altered at this time by the stem-cell treatment. These results indicate that dental pulp stem cells can accelerate recovery without modifying initial infarct formation. Decreases in reactive astrogliosis in peri-infarct tissue could have contributed to the change by promoting adaptive responses in neighboring neurons.

Early treatment with minocycline following stroke in rats improves functional recovery and differentially modifies responses of peri-infarct microglia and astrocytes.

BACKGROUND: Altered neuronal connectivity in peri-infarct tissue is an important contributor to both the spontaneous recovery of neurological function that commonly develops after stroke and improvements in recovery that have been induced by experimental treatments in animal models. Microglia and astrocytes are primary determinants of the environment in peri-infarct tissue and hence strongly influence the potential for neuronal plasticity. However, the specific roles of these cells and the timing of critical changes in their function are not well understood. Minocycline can protect against ischemic damage and promote recovery. These effects are usually attributed, at least partially, to the ability of this drug to suppress microglial activation. This study tested the ability of minocycline treatment early after stroke to modify reactive responses in microglia and astrocytes and improve recovery. METHODS: Stroke was induced by photothrombosis in the forelimb sensorimotor cortex of Sprague-Dawley rats. Minocycline was administered for 2 days after stroke induction and the effects on forelimb function assessed up to 28 days. The responses of peri-infarct Iba1-positive cells and astrocytes were evaluated using immunohistochemistry and Western blots. RESULTS: Initial characterization showed that the numbers of Iba1-positive microglia and macrophages decreased in peri-infarct tissue at 24 h then increased markedly over the next few days. Morphological changes characteristic of activation were readily apparent by 3 h and increased by 24 h. Minocycline treatment improved the rate of recovery of motor function as measured by a forelimb placing test but did not alter infarct volume. At 3 days, there were only minor effects on core features of peri-infarct microglial reactivity including the morphological changes and increased density of Iba1-positive cells. The treatment caused a decrease of 57% in the small subpopulation of cells that expressed CD68, a marker of phagocytosis. At 7 days, the expression of glial fibrillary acidic protein and vimentin was markedly increased by minocycline treatment, indicating enhanced reactive astrogliosis. CONCLUSIONS: Early post-stroke treatment with minocycline improved recovery but had little effect on key features of microglial activation. Both the decrease in CD68-positive cells and the increased activation of astrogliosis could influence neuronal plasticity and contribute to the improved recovery.

Nutritional Attributes, Substitutability, Scalability, and Environmental Intensity of an Illustrative Subset of Current and Future Protein Sources for Aquaculture Feeds: Joint Consideration of Potential Synergies and Trade-offs.

Aquaculture is anticipated to play an increasingly important role in global food security because it may represent one of the best opportunities to increase the availability of healthy animal protein in the context of resource and environmental constraints. However, the growth and sustainability of the aquaculture industry faces important bottlenecks with respect to feed resources, which may be derived from diverse sources. Here, using a small but representative subset of potential aquafeed inputs (which we selected to highlight a range of relevant attributes), we review a core suite of considerations that need to be accommodated in concert in order to overcome key bottlenecks to the continued development and expansion of the aquaculture industry. Specifically, we evaluate the nutritional attributes, substitutability, scalability, and resource and environmental intensity of each input. On this basis, we illustrate a range of potential synergies and trade-offs within and across attributes that are characteristic of ingredient types. We posit that the recognition and management of such synergies and trade-offs is imperative to satisfying the multi-objective decision-making associated with sustainable increases in future aquaculture production.

Investigation of Mature BDNF and proBDNF Signaling in a Rat Photothrombotic Ischemic Model.

Treatment with mature brain-derived neurotrophic factor (mBDNF) promotes functional recovery after ischemia in animal trials but the possible role of its precursor protein proBDNF and its receptors or the factors responsible for the conversion of proBDNF to mBDNF in ischemic stroke are not known. The main aim of this study was to characterize the time-dependent expression of genes and/or proteins related to BDNF processing and signaling after ischemia as well as the sensorimotor behavioral dysfunction in a photothrombotic ischemic model in rats. Characterization of different genes and proteins related to BDNF processing and signaling was performed using qPCR, immunoblotting and enzyme-linked immunosorbent assays. We showed in this study that some sensory and motor functional deficiencies appeared in the ischemic group at day 1 and persisted until day 14. Most changes in gene expression of BDNF and its processing enzymes occurred within the first 24 h in the ipsilateral cortex, but not in the contralateral cortex. At the protein level, proBDNF expression was increased at 6 h, mBDNF expression was increased between 15 h and 1 day while p75 receptor protein expression was increased between 6 h and 3 days in the ipsilateral cortex, but not in the contralateral cortex. Therefore, cerebral ischemia in rats led to the up-regulation of genes and/or proteins of BDNF, proBDNF and their processing enzymes and receptors in a time-dependent manner. We propose that the balance between BDNF and proBDNF and their associated proteins may play an important role in the pathogenesis and recovery from ischemia.

Spectral separability and mapping potential of cassava leaf damage symptoms caused by whiteflies (Bemisia tabaci).

BACKGROUND: This study examines whether leaf spectra can be used to measure damage to cassava plants from whitefly (Bemisia tabaci), and the potential to translate measurements from leaf to landscape scale in eastern Africa. Symptoms of the cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) viruses, and sooty mould (SM) blackening of lower leaves from whiteflies feeding on the upper leaves, were measured at the leaf scale with a high-resolution spectroradiometer and a single photon avalanche diode (SPAD) meter, which retrieves relative chlorophyll concentration. Spectral measurements were compared to the five-level visual scores used to assess the severity of each of the three damaging agents in the field, and also to leaf chemistry data. RESULTS: Leaves exhibiting severe CBSD and CMD were spectrally indistinguishable from leaves without any symptoms. Severe SM was spectrally distinctive but is likely to be difficult to map because of its occurrence in the lower crown. SPAD measurements were highly correlated with most foliar chemistry measurements and field scores of disease severity. Regression models between simulated Sentinel 2 bands, field scores and SPAD measurements were strongest using wavelengths with high importance weightings in random forest models. CONCLUSION: SPAD measurements are highly correlated to many foliar chemistry parameters, and should be considered for use in mapping disease severity over larger areas. Remaining challenges for mapping relate to the subtle expression of symptoms, the spatial distribution of disease severity within fields, and the small size and complex structure of the cassava fields themselves. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Reactive astrogliosis in stroke: Contributions of astrocytes to recovery of neurological function.

Alterations in neuronal connectivity, particularly in the "peri-infarct" tissue adjacent to the region of ischemic damage, are important contributors to the spontaneous recovery of function that commonly follows stroke. Peri-infarct astrocytes undergo reactive astrogliosis and play key roles in modulating the adaptive responses in neurons. This reactive astrogliosis shares many features with that induced by other forms of damage to the central nervous system but also differs in details that potentially influence neurological recovery. A subpopulation of astrocytes within a few hundred micrometers of the infarct proliferate and are centrally involved in the development of the glial scar that separates the damaged tissue in the infarct from surrounding normal brain. The intertwined processes of astrocytes adjacent to the infarct provide the core structural component of the mature scar. Interventions that cause early disruption of glial scar formation typically impede restoration of neurological function. Marked reactive astrogliosis also develops in cells more distant from the infarct but these cells largely remain in the spatial territories they occupied prior to stroke. These cells play important roles in controlling the extracellular environment and release proteins and other molecules that are able to promote neuronal plasticity and improve functional recovery. Treatments manipulating aspects of reactive astrogliosis can enhance neuronal plasticity following stroke. Optimising these treatments for use in human stroke would benefit from a more complete characterization of the specific responses of peri-infarct astrocytes to stroke as well as a better understanding of the influence of other factors including age, sex, comorbidities and reperfusion of the ischemic tissue.

Towards the development of a sustainable soya bean-based feedstock for aquaculture.

Soya bean (Glycine max (L.) Merr.) is sought after for both its oil and protein components. Genetic approaches to add value to either component are ongoing efforts in soya bean breeding and molecular biology programmes. The former is the primary vegetable oil consumed in the world. Hence, its primary usage is in direct human consumption. As a means to increase its utility in feed applications, thereby expanding the market of soya bean coproducts, we investigated the simultaneous displacement of marine ingredients in aquafeeds with soya bean-based protein and a high Omega-3 fatty acid soya bean oil, enriched with alpha-linolenic and stearidonic acids, in both steelhead trout (Oncorhynchus mykiss) and Kampachi (Seriola rivoliana). Communicated herein are aquafeed formulations with major reduction in marine ingredients that translates to more total Omega-3 fatty acids in harvested flesh. Building off of these findings, subsequent efforts were directed towards a genetic strategy that would translate to a prototype design of an optimal identity-preserved soya bean-based feedstock for aquaculture, whereby a multigene stack approach for the targeted synthesis of two value-added output traits, eicosapentaenoic acid and the ketocarotenoid, astaxanthin, were introduced into the crop. To this end, the systematic introduction of seven transgenic cassettes into soya bean, and the molecular and phenotypic evaluation of the derived novel events are described.

Automated in-situ laser scanner for monitoring forest Leaf Area Index.

An automated laser rangefinding instrument was developed to characterize overstorey and understorey vegetation dynamics over time. Design criteria were based on information needs within the statewide forest monitoring program in Victoria, Australia. The ground-based monitoring instrument captures the key vegetation structural information needed to overcome ambiguity in the estimation of forest Leaf Area Index (LAI) from satellite sensors. The scanning lidar instrument was developed primarily from low cost, commercially accessible components. While the 635 nm wavelength lidar is not ideally suited to vegetation studies, there was an acceptable trade-off between cost and performance. Tests demonstrated reliable range estimates to live foliage up to a distance of 60 m during night-time operation. Given the instrument's scan angle of 57.5 degrees zenith, the instrument is an effective tool for monitoring LAI in forest canopies up to a height of 30 m. An 18 month field trial of three co-located instruments showed consistent seasonal trends and mean LAI of between 1.32 to 1.56 and a temporal LAI variation of 8 to 17% relative to the mean.

The mitochondrial T1095C mutation increases gentamicin-mediated apoptosis.

We have previously reported a heteroplasmic mtDNA mutation (T1095C) in the 12SrRNA gene of an Italian family with features of maternally-inherited parkinsonism, antibiotic-mediated deafness and peripheral neuropathy. In the present study, we demonstrate that a transmitochondrial cybrid line derived from the proband of this family shows selective depletion of mitochondrial glutathione and decreases in the activity of complex II/III. Moreover, when exposed to an aminoglycoside antibiotic these cells responded with a ten-fold increase in the number of apoptotic cells compared to controls. These results support a pathogenic role for the T1095C mutation and indicate that the mutation increases the risk for aminoglycoside-induced toxicity.

Isolation of brain mitochondria from neonatal mice.

Mitochondria are key contributors to many forms of cell death including those resulting from neonatal hypoxic-ischemic brain injury. Mice have become increasingly popular in studies of brain injury, but there are few reports evaluating mitochondrial isolation procedures for the neonatal mouse brain. Using evaluation of respiratory activity, marker enzymes, western blotting and electron microscopy, we have compared a previously published procedure for isolating mitochondria from neonatal mouse brain (method A) with procedures adapted from those for adult rats (method B) and neonatal rats (method C). All three procedures use Percoll density gradient centrifugation as a key step in the isolation but differ in many aspects of the fractionation procedure and the solutions used during fractionation. Methods A and B both produced highly enriched fractions of well-coupled mitochondria with high rates of respiratory activity. The fraction from method C exhibited less preservation of respiratory properties and was more contaminated with other subcellular components. Method A offers the advantage of being more rapid and producing larger mitochondrial yields making it useful for routine applications. However, method B produced mitochondria that were less contaminated with synaptosomes and associated cytosolic components that suits studies that have a requirement for higher mitochondrial purification.

Mitochondria, oxidative metabolism and cell death in stroke.

Stroke most commonly results from occlusion of a major artery in the brain and typically leads to the death of all cells within the affected tissue. Mitochondria are centrally involved in the development of this tissue injury due to modifications of their major role in supplying ATP and to changes in their properties that can contribute to the development of apoptotic and necrotic cell death. In animal models of stroke, the limited availability of glucose and oxygen directly impairs oxidative metabolism in severely ischemic regions of the affected tissue and leads to rapid changes in ATP and other energy-related metabolites. In the less-severely ischemic "penumbral" tissue, more moderate alterations develop in these metabolites, associated with near normal glucose use but impaired oxidative metabolism. This tissue remains potentially salvageable for at least the first few hours following stroke onset. Early restoration of blood flow can result in substantial recovery of energy-related metabolites throughout the affected tissue. However, glucose oxidation is markedly decreased due both to lower energy requirements in the post-ischemic tissue and limitations on the mitochondrial oxidation of pyruvate. A secondary deterioration of mitochondrial function subsequently develops that may contribute to progression to cell loss. Mitochondrial release of multiple apoptogenic proteins has been identified in ischemic and post-ischemic brain, mostly in neurons. Pharmacological interventions and genetic modifications in rodent models strongly implicate caspase-dependent and caspase-independent apoptosis and the mitochondrial permeability transition as important contributors to tissue damage, particularly when induced by short periods of temporary focal ischemia.

Alterations in membrane potential in mitochondria isolated from brain subregions during focal cerebral ischemia and early reperfusion: evaluation using flow cytometry.

Mitochondria isolated from brain tissue following middle cerebral artery occlusion or during early reperfusion were tested for their ability to generate a membrane potential under standard conditions in vitro. Membrane potential was evaluated based on rhodamine 123 fluorescence in the mitochondria as detected using flow cytometry. Compared with equivalent samples from the contralateral hemisphere, the geometric mean fluorescence was significantly lower in mitochondria prepared from the striatum and perifocal tissue in the cortex at 3 h ischemia. During reperfusion, this property was decreased in mitochondria from tissue in the striatum and cortex that had been part of severely ischemic core tissue during the arterial occlusion. These findings provide additional evidence that mitochondria develop changes during ischemia and reperfusion that are likely to limit their ability to respond to changing energy requirements and contribute to cell dysfunction and cell death. It also demonstrates the ability to gain a sensitive measure of these mitochondrial changes using flow cytometry.

Mitochondrial glutathione uptake: characterization in isolated brain mitochondria and astrocytes in culture.

Glutathione in the mitochondria is an important determinant of cellular responses to oxidative stress. Mitochondrial glutathione is maintained by uptake from the cytosol, a process that has been little studied in brain cells. In the present study, measurements using isolated rat brain mitochondria showed a rapid uptake of [3H]-glutathione that was strongly influenced by the mitochondrial glutathione content. [3H]-glutathione incorporated into the mitochondria was not rapidly released. Uptake was inhibited by substrates and inhibitors for several known mitochondrial anion transporters. Citrate, isocitrate and benzene-1,2,3-tricarboxylate were particularly effective inhibitors, suggesting a possible role for a tricarboxylate carrier in the glutathione transport. The properties of uptake differed greatly from those reported previously for mitochondria from kidney and liver. In astrocytes in primary culture, diethylmaleate or hydrogen peroxide treatment resulted in depletion of cytosolic and mitochondrial glutathione. The pattern of restoration of glutathione content in the presence of glutathione precursors following treatment with diethylmaleate was consistent with uptake into mitochondria being controlled primarily by the glutathione gradient between the cytosol and mitochondria. However, following hydrogen peroxide treatment, recovery of glutathione in the mitochondria initially preceded comparable proportional restoration in the cytosol, suggesting the possibility of additional controls on glutathione uptake in some conditions.