Effect of reduced irradiance on 13C uptake, gene expression and protein activity of the seagrass Zostera muelleri.

Photosynthesis in the seagrass Zostera muelleri remains poorly understood. We investigated the effect of reduced irradiance on the incorporation of 13C, gene expression of photosynthetic, photorespiratory and intermediates recycling genes as well as the enzymatic content and activity of Rubisco and PEPC within Z. muelleri. Following 48 h of reduced irradiance, we found that i) there was a ∼7 fold reduction in 13C incorporation in above ground tissue, ii) a significant down regulation of photosynthetic, photorespiratory and intermediates recycling genes and iii) no significant difference in enzyme activity and content. We propose that Z. muelleri is able to alter its physiology in order to reduce the amount of C lost through photorespiration to compensate for the reduced carbon assimilation as a result of reduced irradiance. In addition, the first estimated rate constant (Kcat) and maximum rates of carboxylation (Vcmax) of Rubisco is reported for the first time for Z. muelleri.

Zinc and Cadmium Mapping in the Apical Shoot and Hypocotyl Tissues of Radish by High-Resolution Secondary Ion Mass Spectrometry (NanoSIMS) after Short-Term Exposure to Metal Contamination.

Zinc (as an essential phytonutrient) and cadmium (as a toxic but readily bioavailable nonessential metal for plants) share similar routes for crossing plant biomembranes, although with a substantially different potential for translocation into above-ground tissues. The in situ distribution of these metals in plant cells and tissues (particularly intensively-dividing and fast-growing areas) is poorly understood. In this study, 17-day-old radish (Raphanus sativus L.) plants grown in nutrient solution were subjected to short-term (24 h) equimolar contamination (2.2 µM of each 70Zn and Cd) to investigate their accumulation and distribution in the shoot apex (leaf primordia) and edible fleshy hypocotyl tissues. After 24-h exposure, radish hypocotyl had similar concentration (in µg/g dry weight) of 70Zn (12.1 ± 1.1) and total Cd (12.9 ± 0.8), with relatively limited translocation of both metals to shoots (concentrations lower by 2.5-fold for 70Zn and 4.8-fold for Cd) as determined by inductively-coupled plasma mass spectrometry (ICP-MS). The in situ Zn/Cd distribution maps created by high-resolution secondary ion mass spectrometry (NanoSIMS, Cameca, Gennevilliers, France) imaging corresponded well with the ICP-MS data, confirming a similar pattern and uniform distribution of 70Zn and Cd across the examined areas. Both applied techniques can be powerful tools for quantification (ICP-MS) and localisation and visualisation (NanoSIMS) of some ultra-trace isotopes in the intensively-dividing cells and fast-growing tissues of non-metalophytes even after short-term metal exposure. The results emphasise the importance of the quality of (agro)ecosystem resources (growing media, metal-contaminated soils/waters) in the public health risk, given that, even under low contamination and short-term exposure, some of the most toxic metallic ions (e.g., Cd) can relatively rapidly enter the human food chain.

Zinc and cadmium mapping by NanoSIMS within the root apex after short-term exposure to metal contamination.

Zinc as a micronutrient and cadmium as a nonessential toxic element share similar pathways for entering plant tissues and thus may be antagonistic. In nutrient solution culture, 17-day-old radish (Raphanus sativus L) plants were exposed to short-term (24 h) equimolar metal contamination (2.2 µM of each 70Zn and total Cd) to investigate the in situ Zn/Cd distribution in the apical root tissues using high-resolution secondary ion mass spectrometry (NanoSIMS) imaging. Inductively-coupled plasma mass spectrometry analysis of bulk root tissue confirmed large root uptake of both metal elements. After 24-h exposure the total root concentration (in µg/g DW) of 70Zn was 180 ±â€¯24 (mean±SE) and of total Cd 352 ±â€¯11. NanoSIMS mapping was performed on the cross sections of the radish root apex as a crucial component in root growth and uptake of water and nutrients from soil. Elemental maps of 70Zn and 114Cd isotopes revealed greater enrichment of both metals in the outer epidermal root layer than in cortical tissues and especially stele, confirming the epidermal root cells as preferential sites of metal uptake, and indicating relatively slow and less-intensive metal transport into other parts (edible hypocotyl, shoot) of metal-sensitive radish. NanoSIMS has been confirmed as a powerful tool for spatial detection and visualisation of some ultra-trace metal isotopes (e.g. 70Zn) in the fast-growing root tips. However, precise (sub)cellular mapping of diffusible metallic ions (Cd, Zn) remains a technically-challenging task in plant specimens given an unavoidable compromise between optimising methodology for structural preservation vs. authentic in vivo ion localisation.

Intracellular speciation of gold nanorods alters the conformational dynamics of genomic DNA.

Gold nanorods are one of the most widely explored inorganic materials in nanomedicine for diagnostics, therapeutics and sensing1. It has been shown that gold nanorods are not cytotoxic and localize within cytoplasmic vesicles following endocytosis, with no nuclear localization2,3, but other studies have reported alterations in gene expression profiles in cells following exposure to gold nanorods, via unknown mechanisms4. In this work we describe a pathway that can contribute to this phenomenon. By mapping the intracellular chemical speciation process of gold nanorods, we show that the commonly used Au-thiol conjugation, which is important for maintaining the noble (inert) properties of gold nanostructures, is altered following endocytosis, resulting in the formation of Au(I)-thiolates that localize in the nucleus5. Furthermore, we show that nuclear localization of the gold species perturbs the dynamic microenvironment within the nucleus and triggers alteration of gene expression in human cells. We demonstrate this using quantitative visualization of ubiquitous DNA G-quadruplex structures, which are sensitive to ionic imbalances, as an indicator of the formation of structural alterations in genomic DNA.

Oligodendroglia Are Particularly Vulnerable to Oxidative Damage after Neurotrauma In Vivo.

Loss of function following injury to the CNS is worsened by secondary degeneration of neurons and glia surrounding the injury and is initiated by oxidative damage. However, it is not yet known which cellular populations and structures are most vulnerable to oxidative damage in vivo Using Nanoscale secondary ion mass spectrometry (NanoSIMS), oxidative damage was semiquantified within cellular subpopulations and structures of optic nerve vulnerable to secondary degeneration, following a partial transection of the optic nerve in adult female PVG rats. Simultaneous assessment of cellular subpopulations and structures revealed oligodendroglia as the most vulnerable to DNA oxidation following injury. 5-Ethynyl-2'-deoxyuridine (EdU) was used to label cells that proliferated in the first 3 d after injury. Injury led to increases in DNA, protein, and lipid damage in oligodendrocyte progenitor cells and mature oligodendrocytes at 3 d, regardless of proliferative state, associated with a decline in the numbers of oligodendrocyte progenitor cells at 7 d. O4+ preoligodendrocytes also exhibited increased lipid peroxidation. Interestingly, EdU+ mature oligodendrocytes derived after injury demonstrated increased early susceptibility to DNA damage and lipid peroxidation. However, EdU- mature oligodendrocytes with high 8-hydroxyguanosine immunoreactivity were more likely to be caspase3+ By day 28, newly derived mature oligodendrocytes had significantly reduced myelin regulatory factor gene mRNA, indicating that the myelination potential of these cells may be reduced. The proportion of caspase3+ oligodendrocytes remained higher in EdU- cells. Innovative use of NanoSIMS together with traditional immunohistochemistry and in situ hybridization have enabled the first demonstration of subpopulation specific oligodendroglial vulnerability to oxidative damage, due to secondary degeneration in vivoSIGNIFICANCE STATEMENT Injury to the CNS is characterized by oxidative damage in areas adjacent to the injury. However, the cellular subpopulations and structures most vulnerable to this damage remain to be elucidated. Here we use powerful NanoSIMS techniques to show increased oxidative damage in oligodendroglia and axons and to demonstrate that cells early in the oligodendroglial lineage are the most vulnerable to DNA oxidation. Further immunohistochemical and in situ hybridization investigation reveals that mature oligodendrocytes derived after injury are more vulnerable to oxidative damage than their counterparts existing at the time of injury and have reduced myelin regulatory factor gene mRNA, yet preexisting oligodendrocytes are more likely to die.

Corrigendum: Using Aiptasia as a Model to Study Metabolic Interactions in Cnidarian-Symbiodinium Symbioses.

[This corrects the article on p. 214 in vol. 9, PMID: 29615919.].

Using Aiptasia as a Model to Study Metabolic Interactions in Cnidarian-Symbiodinium Symbioses.

The symbiosis between cnidarian hosts and microalgae of the genus Symbiodinium provides the foundation of coral reefs in oligotrophic waters. Understanding the nutrient-exchange between these partners is key to identifying the fundamental mechanisms behind this symbiosis, yet has proven difficult given the endosymbiotic nature of this relationship. In this study, we investigated the respective contribution of host and symbiont to carbon and nitrogen assimilation in the coral model anemone Aiptaisa. For this, we combined traditional measurements with nanoscale secondary ion mass spectrometry (NanoSIMS) and stable isotope labeling to investigate patterns of nutrient uptake and translocation both at the organismal scale and at the cellular scale. Our results show that the rate of carbon and nitrogen assimilation in Aiptasia depends on the identity of the host and the symbiont. NanoSIMS analysis confirmed that both host and symbiont incorporated carbon and nitrogen into their cells, implying a rapid uptake and cycling of nutrients in this symbiotic relationship. Gross carbon fixation was highest in Aiptasia associated with their native Symbiodinium communities. However, differences in fixation rates were only reflected in the δ13C enrichment of the cnidarian host, whereas the algal symbiont showed stable enrichment levels regardless of host identity. Thereby, our results point toward a "selfish" character of the cnidarian-Symbiodinium association in which both partners directly compete for available resources. Consequently, this symbiosis may be inherently instable and highly susceptible to environmental change. While questions remain regarding the underlying cellular controls of nutrient exchange and the nature of metabolites involved, the approach outlined in this study constitutes a powerful toolset to address these questions.

Three dimensional electron microscopy reveals changing axonal and myelin morphology along normal and partially injured optic nerves.

Following injury to the central nervous system, axons and myelin distinct from the initial injury site undergo changes associated with compromised function. Quantifying such changes is important to understanding the pathophysiology of neurotrauma; however, most studies to date used 2 dimensional (D) electron microscopy to analyse single sections, thereby failing to capture changes along individual axons. We used serial block face scanning electron microscopy (SBF SEM) to undertake 3D reconstruction of axons and myelin, analysing optic nerves from normal uninjured female rats and following partial optic nerve transection. Measures of axon and myelin dimensions were generated by examining 2D images at 5 µm intervals along the 100 µm segments. In both normal and injured animals, changes in axonal diameter, myelin thickness, fiber diameter, G-ratio and percentage myelin decompaction were apparent along the lengths of axons to varying degrees. The range of values for axon diameter along individual reconstructed axons in 3D was similar to the range from 2D datasets, encompassing reported variation in axonal diameter attributed to retinal ganglion cell diversity. 3D electron microscopy analyses have provided the means to demonstrate substantial variability in ultrastructure along the length of individual axons and to improve understanding of the pathophysiology of neurotrauma.

Dissecting the Re-Os molybdenite geochronometer.

Rhenium and osmium isotopes have been used for decades to date the formation of molybdenite (MoS2), a common mineral in ore deposits and the world's main source of molybdenum and rhenium. Understanding the distribution of parent 187Re and radiogenic daughter 187Os isotopes in molybdenite is critical in interpreting isotopic measurements because it can compromise the accurate determination and interpretation of mineralization ages. In order to resolve the controls on the distribution of these elements, chemical and isotope mapping of MoS2 grains from representative porphyry copper-molybdenum deposits were performed using electron microprobe and nano-scale secondary ion mass spectrometry. Our results show a heterogeneous distribution of 185,187Re and 192Os isotopes in MoS2, and that both 187Re and 187Os isotopes are not decoupled as previously thought. We conclude that Re and Os are structurally bound or present as nanoparticles in or next to molybdenite grains, recording a complex formation history and hindering the use of microbeam techniques for Re-Os molybdenite dating. Our study opens new avenues to explore the effects of isotope nuggeting in geochronometers.

Managing the excessive proliferation of glycogen accumulating organisms in industrial activated sludge by nitrogen supplementation: A FISH-NanoSIMS approach.

Defluviicoccus vanus-related glycogen accumulating organisms (GAO) regularly proliferate in industrial wastewater treatment plants handling high carbon but nitrogen deficient wastes. When GAO dominate, they are associated with poor performance, characterised by slow settling biomass and turbid effluents. Although their ecophysiology has been studied thoroughly in domestic waste treatment plants, little attention has been paid to them in aerobic industrial systems. In this study, the effect of nitrogen addition on GAO carbon metabolism was investigated during an 8h cycle. Activated sludge dominated by GAO from a winery wastewater sequencing batch reactor was incubated under different carbon to nitrogen (COD:N) ratios (100:1, 60:1 and 20:1) using 13C - acetate and 15N - urea. GAO cell assimilation was quantified using FISH-NanoSIMS. The activated sludge community was assessed by 16S rRNA gene profiling, DNA and storage polymer production. Carbon and nitrogen quantification at the cellular level by NanoSIMS revealed that low (COD:N of 100:1) or null nitrogen concentrations enhanced GAO carbon uptake. COD:N ratios of 60:1 and 20:1 reduced GAO carbon uptake and promoted whole microbial community DNA production. Nitrogen dosing at COD:N ratios of 60:1 or higher was demonstrated as feasible strategy for controlling the excessive GAO growth in high COD waste treatment plants.

Subcellular tracking reveals the location of dimethylsulfoniopropionate in microalgae and visualises its uptake by marine bacteria.

Phytoplankton-bacteria interactions drive the surface ocean sulfur cycle and local climatic processes through the production and exchange of a key compound: dimethylsulfoniopropionate (DMSP). Despite their large-scale implications, these interactions remain unquantified at the cellular-scale. Here we use secondary-ion mass spectrometry to provide the first visualization of DMSP at sub-cellular levels, tracking the fate of a stable sulfur isotope (34S) from its incorporation by microalgae as inorganic sulfate to its biosynthesis and exudation as DMSP, and finally its uptake and degradation by bacteria. Our results identify for the first time the storage locations of DMSP in microalgae, with high enrichments present in vacuoles, cytoplasm and chloroplasts. In addition, we quantify DMSP incorporation at the single-cell level, with DMSP-degrading bacteria containing seven times more 34S than the control strain. This study provides an unprecedented methodology to label, retain, and image small diffusible molecules, which can be transposable to other symbiotic systems.

High-resolution sub-cellular imaging by correlative NanoSIMS and electron microscopy of amiodarone internalisation by lung macrophages as evidence for drug-induced phospholipidosis.

Correlative NanoSIMS and EM imaging of amiodarone-treated macrophages shows the internalisation of the drug at a sub-cellular level and reveals its accumulation within the lysosomes, providing direct evidence for amiodarone-induced phospholipidosis. Chemical fixation using tannic acid effectively seals cellular membranes aiding intracellular retention of diffusible drugs.

Characterization of polymeric nanoparticles for treatment of partial injury to the central nervous system.

Before using nanoparticles for therapeutic applications, it is necessary to comprehensively investigate nanoparticle effects, both in vitro and in vivo. In the associated research article [1] we generate multimodal polymeric nanoparticles functionalized with an antibody, that are designed to deliver an anti-oxidant to astrocytes. Here we provide additional data demonstrating the effects of the nanoparticle preparations on an indicator of oxidative stress in an immortalized Müller cell line in vitro. We provide data demonstrating the use of nanoscale secondary ion mass spectroscopy (NanoSIMS) to identify specific ions in bulk dried NP. NanoSIMS is also used to visualize 40Ca microdomains in the z dimension of optic nerve that has been subjected to a partial optic nerve transection. The associated article [1] describes the use of NanoSIMS to quantify 40Ca microdomains in optic nerve from animals treated with various nanoparticle preparations and provides further interpretation and discussion of the findings.

Light microenvironment and single-cell gradients of carbon fixation in tissues of symbiont-bearing corals.

Recent coral optics studies have revealed the presence of steep light gradients and optical microniches in tissues of symbiont-bearing corals. Yet, it is unknown whether such resource stratification allows for physiological differences of Symbiodinium within coral tissues. Using a combination of stable isotope labelling and nanoscale secondary ion mass spectrometry, we investigated in hospite carbon fixation of individual Symbiodinium as a function of the local O2 and light microenvironment within the coral host determined with microsensors. We found that net carbon fixation rates of individual Symbiodinium cells differed on average about sixfold between upper and lower tissue layers of single coral polyps, whereas the light and O2 microenvironments differed ~15- and 2.5-fold, respectively, indicating differences in light utilisation efficiency along the light microgradient within the coral tissue. Our study suggests that the structure of coral tissues might be conceptually similar to photosynthetic biofilms, where steep physico-chemical gradients define form and function of the local microbial community.

Imaging the uptake of nitrogen-fixing bacteria into larvae of the coral Acropora millepora.

Diazotrophic bacteria are instrumental in generating biologically usable forms of nitrogen by converting abundant dinitrogen gas (N2) into available forms, such as ammonium. Although nitrogen is crucial for coral growth, direct observation of associations between diazotrophs and corals has previously been elusive. We applied fluorescence in situ hybridization (FISH) and nanoscale secondary ion mass spectrometry to observe the uptake of (15)N-enriched diazotrophic Vibrio sp. isolated from Acropora millepora into conspecific coral larvae. Incorporation of Vibrio sp. cells was observed in coral larvae after 4-h incubation with enriched bacteria. Uptake was restricted to the aboral epidermis of larvae, where Vibrio cells clustered in elongated aggregations. Other bacterial associates were also observed in epidermal areas in FISH analyses. Although the fate and role of these bacteria requires additional investigation, this study describes a powerful approach to further explore cell associations and nutritional pathways in the early life stages of the coral holobiont.

Exploring the transfer of recent plant photosynthates to soil microbes: mycorrhizal pathway vs direct root exudation.

Plants rapidly release photoassimilated carbon (C) to the soil via direct root exudation and associated mycorrhizal fungi, with both pathways promoting plant nutrient availability. This study aimed to explore these pathways from the root's vascular bundle to soil microbial communities. Using nanoscale secondary ion mass spectrometry (NanoSIMS) imaging and (13) C-phospho- and neutral lipid fatty acids, we traced in-situ flows of recently photoassimilated C of (13) CO2 -exposed wheat (Triticum aestivum) through arbuscular mycorrhiza (AM) into root- and hyphae-associated soil microbial communities. Intraradical hyphae of AM fungi were significantly (13) C-enriched compared to other root-cortex areas after 8 h of labelling. Immature fine root areas close to the root tip, where AM features were absent, showed signs of passive C loss and co-location of photoassimilates with nitrogen taken up from the soil solution. A significant and exclusively fresh proportion of (13) C-photosynthates was delivered through the AM pathway and was utilised by different microbial groups compared to C directly released by roots. Our results indicate that a major release of recent photosynthates into soil leave plant roots via AM intraradical hyphae already upstream of passive root exudations. AM fungi may act as a rapid hub for translocating fresh plant C to soil microbes.

Viruses as new agents of organomineralization in the geological record.

Viruses are the most abundant biological entities throughout marine and terrestrial ecosystems, but little is known about virus-mineral interactions or the potential for virus preservation in the geological record. Here we use contextual metagenomic data and microscopic analyses to show that viruses occur in high diversity within a modern lacustrine microbial mat, and vastly outnumber prokaryotes and other components of the microbial mat. Experimental data reveal that mineral precipitation takes place directly on free viruses and, as a result of viral infections, on cell debris resulting from cell lysis. Viruses are initially permineralized by amorphous magnesium silicates, which then alter to magnesium carbonate nanospheres of ~80-200 nm in diameter during diagenesis. Our findings open up the possibility to investigate the evolution and geological history of viruses and their role in organomineralization, as well as providing an alternative explanation for enigmatic carbonate nanospheres previously observed in the geological record.

Changes in subtypes of Ca microdomains following partial injury to the central nervous system.

Rapid changes in Ca(2+) concentration and location in response to injury play key roles in a range of biological systems. However, quantitative analysis of changes in size and distribution of Ca(2+) microdomains in specific cell types in whole tissue samples has been limited by analytical resolution and reliance on indirect Ca(2+) indicator systems. Here, we combine the unique advantages of nanoscale secondary ion mass spectrometry (NanoSIMS) with immunohistochemistry to directly quantify changes in number, size and intensity of Ca microdomains specific to axonal or glial regions vulnerable to spreading damage following neurotrauma. Furthermore, using NanoSIMS allows separate quantification of Ca microdomains according to their co-localization with areas enriched in P. We rapidly excise and cryopreserve optic nerve segments from adult rat at time points ranging from 5 minutes to 3 months after injury, allowing assessment of Ca microdomains dynamics with minimal disruption due to tissue processing. We demonstrate significantly more non-P co-localized Ca microdomains in glial than axonal regions in normal optic nerve. The density of Ca microdomains not co-localized with areas enriched in P rapidly, selectively and significantly decreases after injury; densities of Ca microdomains co-localized with P enriched areas are unchanged. An efflux of Ca(2+) from microdomains not co-localized with P may contribute to the structural and functional deficits observed in nerve vulnerable to spreading damage following neurotrauma. NanoSIMS analyses of Ca microdomains allow quantitative and novel insights into Ca dynamics, applicable to a range of normal, as well as diseased or injured mammalian systems.

Elemental and isotopic imaging of biological samples using NanoSIMS.

With its low detection limits and the ability to analyze most of the elements in the periodic table, secondary ion mass spectrometry (SIMS) represents one of the most versatile in situ analytical techniques available, and recent developments have resulted in significant advantages for the use of imaging mass spectrometry in biological and biomedical research. Increases in spatial resolution and sensitivity allow detailed interrogation of samples at relevant scales and chemical concentrations. Advances in dynamic SIMS, specifically with the advent of NanoSIMS, now allow the tracking of stable isotopes within biological systems at subcellular length scales, while static SIMS combines subcellular imaging with molecular identification. In this chapter, we present an introduction to the SIMS technique, with particular reference to NanoSIMS, and discuss its application in biological and biomedical research.

Competition between plant and bacterial cells at the microscale regulates the dynamics of nitrogen acquisition in wheat (Triticum aestivum).

The ability of plants to compete effectively for nitrogen (N) resources is critical to plant survival. However, controversy surrounds the importance of organic and inorganic sources of N in plant nutrition because of our poor ability to visualize and understand processes happening at the root-microbial-soil interface. Using high-resolution nano-scale secondary ion mass spectrometry stable isotope imaging (NanoSIMS-SII), we quantified the fate of ¹5N over both space and time within the rhizosphere. We pulse-labelled the soil surrounding wheat (Triticum aestivum) roots with either ¹5NH4⁺ or ¹5N-glutamate and traced the movement of ¹5N over 24 h. Imaging revealed that glutamate was rapidly depleted from the rhizosphere and that most ¹5N was captured by rhizobacteria, leading to very high ¹5N microbial enrichment. After microbial capture, approximately half of the ¹5N-glutamate was rapidly mineralized, leading to the excretion of NH4⁺, which became available for plant capture. Roots proved to be poor competitors for ¹5N-glutamate and took up N mainly as ¹5NH4⁺. Spatial mapping of ¹5N revealed differential patterns of ¹5N uptake within bacteria and the rapid uptake and redistribution of ¹5N within roots. In conclusion, we demonstrate the rapid cycling and transformation of N at the soil-root interface and that wheat capture of organic N is low in comparison to inorganic N under the conditions tested.