Latent mitochondrial DNA deletion mutations drive muscle fiber loss at old age

With age, somatically derived mitochondrial DNA (mtDNA) deletion mutations arise in many tissues and species. In skeletal muscle, deletion mutations clonally accumulate along the length of individual fibers. At high intrafiber abundances, these mutations disrupt individual cell respiration and are linked to the activation of apoptosis, intrafiber atrophy, breakage, and necrosis, contributing to fiber loss. This sequence of molecular and cellular events suggests a putative mechanism for the permanent loss of muscle fibers with age. To test whether mtDNA deletion mutation accumulation is a significant contributor to the fiber loss observed in aging muscle, we pharmacologically induced deletion mutation accumulation. We observed a 1200% increase in mtDNA deletion mutation‐containing electron transport chain‐deficient muscle fibers, an 18% decrease in muscle fiber number and 22% worsening of muscle mass loss. These data affirm the hypothesized role for mtDNA deletion mutation in the etiology of muscle fiber loss at old age.     

Transformation of galena to pyromorphite produces bioavailable sulfur for neutrophilic chemoautotrophy

The aqueous concentration of lead [Pb(II)] in geochemical environments is controlled by the solubility of Pb‐bearing minerals and their weathering products. In contaminated soils, a common method for in situ stabilization of Pb(II) is the addition of phosphate to convert more redox sensitive sulfide minerals into sparingly soluble pyromorphite [Pb₅(PO₄)₃X]. In this study, we conducted experimental studies to investigate the fate of reduced sulfur during the conversion of galena [PbS] to chloropyromorphite [Pb₅(PO₄)₃Cl]. Powder X‐ray diffraction analysis indicated that the reaction of phosphate with galena under oxic conditions resulted in the oxidation of sulfide and formation of elemental sulfur [S₈]. Under oxic abiotic conditions, the S₈ was retained in the solid phase, and negligible concentrations of sulfur as sulfide and thiosulfate were detected in the aqueous phase and only a small amount of sulfate. When PbS reacted in the presence of the chemoautotrophic organism Bosea sp. WAO, the S₈ in the secondary mineral was oxidized to sulfate. Strain WAO produced significantly more sulfate from the secondary S₈ than from the primary galena. Microscopic analysis of mineral–microbe aggregates on mineral‐embedded slide cultures showed that the organism was colocalized and increased in biomass over time on the secondary mineral surface supporting a microbial role. The results of this study indicate that stimulation of sulfur‐oxidizing activity may be a direct consequence of phosphate amendments to Pb(II)‐contaminated soils.     

Spatio‐temporal gradients in food supply help explain the short‐term colonisation dynamics of the critically endangered central rock‐rat (Zyzomys pedunculatus)

Currently, the impact of introduced predators on small mammal population decline is a focal research direction in the Australian desert literature. In all likelihood though, single‐factor explanation of population dynamics is inadequate, leaving gaps in our knowledge of the multitude of potential influences on small mammal abundance and occupancy patterns in time and space. Here, we investigated floristic gradients across four potential refuge sites of the central rock‐rat, Zyzomys pedunculatus, a granivore rodent (50–120 g) that is endemic to central Australia and is categorised as critically endangered. The study took place in Tjoritja/West MacDonnell National Park in the MacDonnell Ranges bioregion. Floristic sampling was allocated across the four sites, the locations of which were predetermined by an established monitoring and management programme for the central rock‐rat. Our aim was to examine the relationship between environmental gradients and floristic composition across the four sites, and thereby test the extent to which the patterns of food type and food availability can inform central rock‐rat spatio‐temporal dynamics. We found high site‐scale floristic patterning that related foremost to elevation and then to antecedent rainfall and time‐since‐fire and fire‐severity effects. To interpret these results, we applied the principles of refuge theory and we described a gradient from core refuge habitat to intermittent and then marginal habitat within the current central rock‐rat stronghold area. Overall, our results implied a strong floristic basis to central rock‐rat site occurrence, and they thus compel us to take explicit account of spatial (elevation) and temporal (rainfall–productivity and fire‐disturbance) influences on the food axis of potential refuge sites of this critically endangered species.     

Pro-MMP-2 activation by the PPARgamma agonist, ciglitazone, induces cell invasion through the generation of ROS and the activation of ERK

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor modulating a variety of biological functions including cancer cell proliferation and differentiation. However, the role of PPARgamma and its ligands in tumor invasion is unclear. To evaluate a possible role for PPARgamma ligands in tumor invasion, we examined whether PPARgamma agonists including pioglitazone, troglitazone, rosiglitazone, and ciglitazone could affect the activity of matrix metalloproteinases (MMPs) in the HT1080 cell line, a well-studied and well-characterized cell line for MMP research. The gelatin zymography assay showed that ciglitazone activated pro-MMP-2 significantly. In addition, ciglitazone increased the expression of MMP-2, which was accompanied by an increase of membrane type 1-MMP (MT1-MMP) expression. The PPARgamma antagonist, GW9662 attenuated the ciglitazone-induced PPARgamma activation but it did not affect the pro-MMP2 activation by ciglitazone, suggesting that the action of ciglitazone on the pro-MMP-2 activation bypassed the PPARgamma pathway. Antioxidants and various inhibitors of signal transduction were used to investigate the mechanism of ciglitazone-induced pro-MMP-2 activation. We found that the sustained production of reactive oxygen species (ROS) was required for pro-MMP-2 activation by ciglitazone. We also found that PB98059, an inhibitor of MEK-ERK, significantly blocked ciglitazone-induced pro-MMP-2 activation and that extracellular signal-regulated kinase (ERK) was hyperphosphorylated by ciglitazone. Moreover, cell invasion was significantly increased by ciglitazone in the HT1080 cell lines, whereas cell motility was not affected. This study suggests that ciglitazone-induced pro-MMP-2 activation increases PPARgamma-independent tumor cell invasion through ROS production and ERK activation in some types of cancer cells.     

Measuring triamcinolone acetonide in aqueous humor by gas chromatography-electron-capture negative-ion mass spectrometry

Intravitreal triamcinolone acetonide (IVTA) injection has been used in the treatment of various posterior segment diseases. One of the side effects of IVTA is raised intraocular pressure, which may be secondary to triamcinolone acetonide (TAA)'s effects on the trabecular meshwork that affects aqueous outflow. In order to study the biological effects of TAA on the trabecular meshwork, we firstly need to reliably and accurately detect the concentration of TAA in tissue cells or fluids. In this study we have described a technique of using gas chromatography-electron-capture-negative-ion mass spectrometry (GC-NCI-MS) to develop a simple, sensitive, selective and validated method to detect TAA in aqueous humor (AH) of rabbits following IVTA and subconjunctival TAA injections. We derivatized TAA from extracted aqueous sample by acetic anhydride and BSTFA, respectively, and analyzed by GC-NCI-MS. The detection limit was 0.3ng/ml, linearity over 0.995 from 0 to 300ng/ml. The reproducibility ranged from 10.4 to 3.9 for concentrations from 3 to 300ng/ml, and recovery was over 95% for the concentrations 10, 60, and 200ng/ml. No interference was found from 159 aqueous samples. There was no TAA residue carried to the next injection from previously high concentration injection, 10,000ng/ml. We have provided an alternative, rapid, and robust method other than LC-MS-MS for TAA detection in AH.     

In situ proteolysis for protein crystallization and structure determination

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.