In situ 3D visualization of biomineralization matrix proteins.

Calcium biominerals occur in all major animal phyla, and through biomolecular control, exhibit such diverse structures as exoskeletons, shells, bones, teeth and earstones (otoliths). Determining the three-dimensional expression of key biomineral proteins, however, has proven challenging as typical protein identification methods either lose spatial resolution during dissolution of the mineral phase or are costly and limited to two-dimensional expression of high abundance proteins. Here we present a modification of the CLARITY and ACT-PRESTO protocols to visualize and confirm, for the first time, the timing of expression and function of two key regulators of biomineralization.

Choice of mobile phase: Implications for size exclusion chromatography-inductively coupled plasma-mass spectrometry analyses of copper, zinc and iron metalloproteins.

The correct identification of the metalloproteins present in human tissues and fluids is essential to our understanding of the cellular mechanisms underpinning a host of health disorders. Separation and analysis of biological samples are typically done via size exclusion chromatography hyphenated with inductively coupled plasma-mass spectrometry (SEC-ICP-MS). Although this technique can be extremely effective in identification of potential metalloproteins, the choice of mobile phase may have a marked effect on results, results by adversely affecting metal-protein bonds of the metalloproteins of interest. To assess the choice of mobile phase on SEC-ICP-MS resolution and the resulting metalloproteome pattern, we analysed several different sample types (brain homogenate; Cu/Zn-superoxide dismutase (SOD1); a molecular weight standard mix containing ferritin (Ft), ceruloplasmin (Cp), cytochrome c (CytC), vitamin B12 (B12) and thyroglobulin (Tg) using six different mobile phase conditions (200 mM, pH 7.5 solutions of ammonium salts nitrate, acetate, and sulfate; HEPES, MOPS and Tris-HCl). Our findings suggest that ammonium nitrate, ammonium acetate and Tris-HCl are optimal choices for the mobile phase, with the specific choice being dependent on both the number of samples and method of detection that is hyphenated with separation. Furthermore, we found that MOPS, HEPES and ammonium sulfate mobile phases all caused significant changes to peak resolution, retention time and overall profile shape. MOPS and HEPES, in particular, produced additional Fe peaks that were not detected with any of the other mobile phases that were investigated. As well as this, MOPS and HEPES both caused significant concentration dependent matrix suppression of the internal standard.

Detection of small molecule concentration gradients in ocular tissues and humours.

The eye is an elegant organ consisting of a number of tissues and fluids with specialised functions that together allow it to effectively transmit and transduce light input to the brain for visual perception. One key determinant of this integrated function is the spatial relationship of ocular tissues. Biomolecular distributions within the main ocular tissues cornea, lens, and retina have been studied extensively in isolation, yet the potential for metabolic communication between ocular tissues via the ocular humours has been difficult to visualise. To address this limitation, the current study presents a method to map spatial distributions of metabolites and small molecules in whole eyes, including ocular humours. Using a tape-transfer system and freeze-drying, the spatial distribution of ocular small molecules was investigated in mouse, rat, fish (black bream), and rabbit eyes using negative ion mode MALDI imaging mass spectrometry. Full-scan imaging was used for discovery experiments, while MS/MS imaging for identification and localisation was also demonstrated. In all eyes, metabolites such as glutathione and phospholipids were localised in the main ocular tissues. In addition, in rodent eyes, major metabolites were distributed relatively uniformly in ocular humours. In contrast, both uniform and spatially defined ocular metabolite distributions were observed in the black bream eye. Tissue and ocular humour distributions were reproducible, as demonstrated by the three-dimensional analysis of a mouse eye, and able to be captured with high spatial resolution analysis. The presented method could be used to further investigate the role of inter-tissue metabolism in ocular health, and to support the development of therapeutics to treat major ocular diseases.

The inner ear proteome of fish.

The mechanisms that underpin the formation, growth and composition of otoliths, the biomineralized stones in the inner ear of fish, are largely unknown, as only a few fish inner ear proteins have been reported. Using a partial transcriptome for the inner ear of black bream (Acanthopagrus butcheri), in conjunction with proteomic data, we discovered hundreds of previously unknown proteins in the otolith. This allowed us to develop hypotheses to explain the mechanisms of inorganic material supply and daily formation of growth bands. We further identified a likely protein mediator of crystal nucleation and an explanation for the apparent metabolic inertness of the otolith. Due to the formation of both daily and annual increments, otoliths are routinely employed as natural chronometers, being used for age and growth estimation, fisheries stock assessments, and the reconstruction of habitat use, movement, diet and the impacts of climate change. Our findings provide an unprecedented view of otolith molecular machinery, aiding in the interpretation of these essential archived data.

Trace element-protein interactions in endolymph from the inner ear of fish: implications for environmental reconstructions using fish otolith chemistry.

Otoliths, the biomineralised hearing "ear stones" from the inner ear of fish, grow throughout the lifespan of an individual, with deposition of alternating calciferous and proteinaceous bands occurring daily. Trace element : calcium ratios within daily increments measured by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) are often used in fisheries science to reconstruct environmental histories. There is, however, considerable uncertainty as to which elements are interacting with either the proteinaceous or calciferous zones of the otolith, and thus their utility as indicators of environmental change. To answer this, we used size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) of endolymph, the otolith growth medium, to determine the binding interactions for a range of elements. In addition, we used solution ICP-MS to quantify element concentrations in paired otolith and endolymph samples and determined relative enrichment factors for each. We found 12 elements that are present only in the proteinaceous fraction, 6 that are present only in the salt fraction, and 4 that are present in both. These findings have important implications for the reconstruction of environmental histories based on changes in otolith elemental composition: (1) elements occurring only in the salt fraction are most likely to reflect changes in the physico-chemical environment experienced during life; (2) elements occurring only in the proteinaceous fraction are more likely to reflect physiological rather than environmental events; and (3) elements occurring in both the salt and proteinaceous fractions are likely to be informative about both endogenous and exogenous processes, potentially reducing their utility in environmental reconstructions.

Smell no evil: Copper disrupts the alarm chemical response in a diadromous fish, Galaxias maculatus.

Fish, at all life stages, utilize olfactory information in the decision-making processes essential to survival. Olfaction is a sensitive sensory process, and toxicants within urban aquatic environments can have destructive or depreciating effects. In the present study, the authors exposed Galaxias maculatus, a native fish commonly found in urban waterways throughout southeastern Australia, to 1 of 5 ecologically relevant copper (II) chloride concentrations (<1 µg/L, 1 µg/L, 6 µg/L, 8 µg/L, 18 µg/L) for 16 h. After exposure, the authors tested the response of individual fish to 1 of 3 stimuli: a conspecific skin extract containing a stress-inducing alarm chemical odor, a conspecific odor, and distilled water as a control. Stress responses were quantified through behavioral assays. The authors found evidence for distinct changes in behavioral response with increasing copper concentration and a marked difference in response between control fish and fish exposed to the alarm chemical odor. Copper, even at relatively low concentrations, can have a significant effect on the stress response behavior shown by G. maculatus. Environ Toxicol Chem 2016;35:2209-2214. © 2016 SETAC.