Mitochondria regulate critical components of cellular function via ATP production, reactive oxygen species production, Ca2+ handling and apoptotic signaling. Two classical methods exist to study mitochondrial function of skeletal muscles: isolated mitochondria and permeabilized myofibers. Whereas mitochondrial isolation removes a portion of the mitochondria from their cellular environment, myofiber permeabilization preserves mitochondrial morphology and functional interactions with other intracellular components. Despite this, isolated mitochondria remain the most commonly used method to infer in vivo mitochondrial function. In this study, we directly compared measures of several key aspects of mitochondrial function in both isolated mitochondria and permeabilized myofibers of rat gastrocnemius muscle. Here we show that mitochondrial isolation i) induced fragmented organelle morphology; ii) dramatically sensitized the permeability transition pore sensitivity to a Ca2+ challenge; iii) differentially altered mitochondrial respiration depending upon the respiratory conditions; and iv) dramatically increased H2O2 production. These alterations are qualitatively similar to the changes in mitochondrial structure and function observed in vivo after cellular stress-induced mitochondrial fragmentation, but are generally of much greater magnitude. Furthermore, mitochondrial isolation markedly altered electron transport chain protein stoichiometry. Collectively, our results demonstrate that isolated mitochondria possess functional characteristics that differ fundamentally from those of intact mitochondria in permeabilized myofibers. Our work and that of others underscores the importance of studying mitochondrial function in tissue preparations where mitochondrial structure is preserved and all mitochondria are represented.

SummaryMitochondria regulate cellular bioenergetics and apoptosis and have been implicated in aging. However, it remains unclear whether age-related loss of muscle mass, known as sarcopenia, is associated with abnormal mitochondrial function. Two technically different approaches have mainly been used to measure mitochondrial function: isolated mitochondria and permeabilized myofiber bundles, but the reliability of these measures in the context of sarcopenia has not been systematically assessed before. A key difference between these approaches is that contrary to isolated mitochondria, permeabilized bundles contain the totality of fiber mitochondria where normal mitochondrial morphology and intracellular interactions are preserved. Using the gastrocnemius muscle from young adult and senescent rats, we show marked effects of aging on three primary indices of mitochondrial function (respiration, H 2 O 2 emission, sensitivity of permeability transition pore to Ca 2+ ) when measured in isolated mitochondria, but to a much lesser degree when measured in permeabilized bundles. Our results clearly demonstrate that mitochondrial isolation procedures typically employed to study aged muscles expose functional impairments not seen in situ. We conclude that aging is associated with more modest changes in mitochondrial function in sarcopenic muscle than suggested previously from isolated organelle studies.

SummaryTo determine whether mitochondrial dysfunction is causally related to muscle atrophy with aging, we examined respiratory capacity, H 2 O 2 emission, and function of the mitochondrial permeability transition pore (mPTP) in permeabilized myofibers prepared from four rat muscles that span a range of fiber type and degree of age-related atrophy. Muscle atrophy with aging was greatest in fast-twitch gastrocnemius (Gas) muscle ()38%), intermediate in both the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (Sol) muscles ()21%), and non-existent in adductor longus (AL) muscle (+47%). In contrast, indices of mitochondrial dysfunction did not correspond to this differential degree of atrophy. Specifically, despite higher protein expression for oxidative phosphorylation (oxphos) system in fast Gas and EDL, state III respiratory capacity per myofiber wet weight was unchanged with aging, whereas the slow Sol showed proportional decreases in oxphos protein, citrate synthase activity, and state III respiration. Free radical leak (H 2 O 2 emission per O 2 flux) under state III respiration was higher with aging in the fast Gas, whereas state II free radical leak was higher in the slow AL. Only the fast muscles had impaired mPTP function with aging, with lower mitochondrial calcium retention capacity in EDL and shorter time to mPTP opening in Gas and EDL. Collectively, our results underscore that the age-related changes in muscle mitochondrial function depend largely upon fiber type and are unrelated to the severity of muscle atrophy, suggesting that intrinsic changes in mitochondrial function are unlikely to be causally involved in aging muscle atrophy.

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Atomistic computer simulation methods have been employed to model the structure of the surfaces of (101 6 4) calcite dolomite and magnesite Our calculations show that, under). anhydrous vacuum conditions, calcite undergoes the greatest degree of surface relaxation with rotation and distortion of the carbonate group accompanied by movement of the calcium ion. The magnesite surface is the least distorted of the three carbonates, with dolomite being intermediate to the two end members. When water molecules are placed on the surface to produce complete monolayer coverage, the surfaces of all three carbonate minerals are stabilized and the amount of relaxation in the surface layers substantially reduced. Of the three phases, dolomite shows the strongest and highest number of interfacial hydrogen bonds between water and the carbonate mineral surface. These calculations suggest that the equilibrium H 2 O ] CO 3 2~H will favour the production of hydrogen carbonate ions most strongly for dolomite, less HCO 3 ~] OHs trongly for calcite, and least likely for magnesite. Computational methodsAtomistic simulation methods are based on the Born model of solids where interatomic potential functions are deÐned to model the long-and short-range forces acting between atoms or ions in the solid. Oxygen ion polarizability is described by the shell model13 while directionality of the bonding is

The thermally induced transformation of kaolinite to metakoalin is simulated using molecular dynamics through a step-wise dehydroxylation approach. The simulation shows that the removal of structural water through dehydroxylation produces a distortion or buckling effect in the 1:1 Al-Si layers, which is due to the migration of the aluminium into vacant sites provided by the inter-layer spacing. The structural change is characterized by a loss of crystallinity and a concomitant change in aluminium coordination from octahedral to tetrahedral, with this study confirming the presence of 5-fold aluminium within the metakaolin structure. The degree and probability of Al migration is proportional to the amount of local disorder within the structure, which is governed by the degree of local hydroxyl group loss. This results in the formation of aluminium clusters within the layers. This study proposes that instead of a uniform structure, metakaolin exhibits regions of differing aluminium concentrations, which can have major effects in the reaction chemistry at those sites.

FeS 2 pyrite can incorporate large amounts of arsenic (up to ca. 10 wt%) and hence has a strong impact on the mobility of this toxic metalloid. Focussing on the lowest arsenic concentrations for which the incorporation occurs in solid solution, the substitution mechanisms involved have been investigated by assuming simple incorporation reactions in both oxidising and reducing conditions. The solution energies were calculated by Density Functional Theory (DFT) calculations and we predict that the formation of AsS dianion groups is the most energetically favourable mechanism. The results also suggest that the presence of arsenic will accelerate the dissolution and thus the generation of acid drainage, when the crystal dissolves in oxidising conditions.

Jarosite [KFe 3 (SO 4 ) 2 (OH) 6 ] is a mineral that is common in acidic, sulphate-rich environments, such as acid sulphate soils derived from pyrite-bearing sediments, weathering zones of sulphide ore deposits and acid mine or acid rock drainage (ARD/AMD) sites. The structure of jarosite is based on linear tetrahedral-octahedral-tetrahedral (T-O-T) sheets, made up from slightly distorted FeO 6 octahedra and SO 4 tetrahedra. Batch dissolution experiments carried out on synthetic jarosite at pH 2, to mimic environments affected by ARD/AMD, and at pH 8, to simulate ARD/AMD environments recently remediated with slaked lime (Ca(OH) 2 ), suggest first order dissolution kinetics. Both dissolution reactions are incongruent as revealed by non-ideal dissolution of the parent solids and, in the case of the pH 8 dissolution, because of a secondary goethite precipitate on the surface of the dissolving jarosite grains. The pH 2 dissolution yields only aqueous K, Fe and SO 4 . Aqueous, residual solid and computational modelling of the jarosite structure and surfaces using the GULP and MARVIN codes, respectively, show for the first time that there is selective dissolution of the A-and T-sites, which contain K and SO 4 , respectively, relative to Fe, which is located deep within the T-O-T jarosite structure. These results have implications for the chemistry of ARD/AMD waters, and for understanding reaction pathways of ARD/AMD mineral dissolution.