ABSTRACT
Stramenopile algae contribute significantly to global primary productivity, and one class, Eustigmatophyceae, is increasingly studied for applications in high-value lipid production. Yet much about their basic biology remains unknown, including the nature of an enigmatic, pigmented globule found in vegetative cells. Here, we present an in-depth examination of this "red body," focusing on Nannochloropsis oceanica. During the cell cycle, the red body forms adjacent to the plastid, but unexpectedly it is secreted and released with the autosporangial wall following cell division. Shed red bodies contain antioxidant ketocarotenoids, and overexpression of a beta-carotene ketolase results in enlarged red bodies. Infrared spectroscopy indicates long-chain, aliphatic lipids in shed red bodies and cell walls, and UHPLC-HRMS detects a C32 alkyl diol, a potential precursor of algaenan, a recalcitrant cell wall polymer. We propose that the red body transports algaenan precursors from plastid to apoplast to be incorporated into daughter cell walls.
Subject(s)
Cell Wall , Plastids , Stramenopiles , Cell Wall/metabolism , Stramenopiles/metabolism , Plastids/metabolismABSTRACT
Non-invasive methods of detecting radiation exposure show promise to improve upon current approaches to biological dosimetry in ease, speed, and accuracy. Here we developed a pipeline that employs Fourier transform infrared (FTIR) spectroscopy in the mid-infrared spectrum to identify a signature of low dose ionizing radiation exposure in mouse ear pinnae over time. Mice exposed to 0.1 to 2 Gy total body irradiation were repeatedly measured by FTIR at the stratum corneum of the ear pinnae. We found significant discriminative power for all doses and time-points out to 90 days after exposure. Classification accuracy was maximized when testing 14 days after exposure (specificity > 0.9 with a sensitivity threshold of 0.9) and dropped by roughly 30% sensitivity at 90 days. Infrared frequencies point towards biological changes in DNA conformation, lipid oxidation and accumulation and shifts in protein secondary structure. Since only hundreds of samples were used to learn the highly discriminative signature, developing human-relevant diagnostic capabilities is likely feasible and this non-invasive procedure points toward rapid, non-invasive, and reagent-free biodosimetry applications at population scales.
Subject(s)
Radiation Exposure , Radiometry , Humans , Mice , Animals , Spectroscopy, Fourier Transform Infrared , Fourier Analysis , Radiometry/methods , Proteins , Radiation, Ionizing , Radiation Exposure/analysis , Radiation DosageABSTRACT
Cesium (Cs+) enters environments largely because of global release into the environment from weapons testing and accidents such as Fukushima Daiichi and Chernobyl nuclear waste. Even at low concentrations, Cs+ is highly toxic to ecological receptors because of its physicochemical similarity to macronutrient potassium (K+). We investigated the uptake and accumulation of Cs+ by Arthrobacter globiformis strain 151B in reference to three similar alkali metal cations rubidium (Rb+), sodium (Na+), and potassium (K+). The impact of hexavalent chromium (Cr+6) as a co-contaminant was also evaluated. A. globiformis 151B accumulated Cs+ and Cr6+ in a time-dependent fashion. In contrast, the uptake and accumulation of Rb+ did not exhibit any trends. An exposure to Cs+, Rb+, and Cr+6 triggered a drastic increase in K+ and Na+ uptake by the bacterial cells. That was followed by the efflux of K+ and Na+, suggesting a Cs+ substitution. Two-dimensional gel-electrophoresis of bacterial cell proteomes with the following mass-spectrometry of differentially expressed bands revealed that incubation of bacterial cells with Cs+ induced changes in the expression of proteins involved in the maintenance of cellular homeostasis and reactive oxygen species removal. The ability of A. globiformis 151B to mediate the uptake and accumulation of cesium and hexavalent chromium suggests that it possesses wide-range bioremediation potential.(AU)
Subject(s)
Humans , Biodegradation, Environmental , Arthrobacter , Ions , Cesium , Toxicity , MicrobiologyABSTRACT
Cesium (Cs+) enters environments largely because of global release into the environment from weapons testing and accidents such as Fukushima Daiichi and Chernobyl nuclear waste. Even at low concentrations, Cs+ is highly toxic to ecological receptors because of its physicochemical similarity to macronutrient potassium (K+). We investigated the uptake and accumulation of Cs+ by Arthrobacter globiformis strain 151B in reference to three similar alkali metal cations rubidium (Rb+), sodium (Na+), and potassium (K+). The impact of hexavalent chromium (Cr+6) as a co-contaminant was also evaluated. A. globiformis 151B accumulated Cs+ and Cr6+ in a time-dependent fashion. In contrast, the uptake and accumulation of Rb+ did not exhibit any trends. An exposure to Cs+, Rb+, and Cr+6 triggered a drastic increase in K+ and Na+ uptake by the bacterial cells. That was followed by the efflux of K+ and Na+, suggesting a Cs+ "substitution." Two-dimensional gel-electrophoresis of bacterial cell proteomes with the following mass-spectrometry of differentially expressed bands revealed that incubation of bacterial cells with Cs+ induced changes in the expression of proteins involved in the maintenance of cellular homeostasis and reactive oxygen species removal. The ability of A. globiformis 151B to mediate the uptake and accumulation of cesium and hexavalent chromium suggests that it possesses wide-range bioremediation potential.
Subject(s)
Metals, Alkali , Radioactive Waste , Alkalies , Arthrobacter , Biodegradation, Environmental , Cations/analysis , Cesium/analysis , Cesium/metabolism , Chromium , Potassium/analysis , Potassium/metabolism , Proteome , Radioactive Waste/analysis , Reactive Oxygen Species , Rubidium/analysis , Rubidium/metabolism , Sodium/metabolismABSTRACT
Coralline algae are worldwide carbonate builders, considered to be foundational species and biodiversity hotspots. Coralline habitats face increasing pressure from human activities and effects related to Global Change, yet their ecological properties and adaptive responses remain poorly understood. The relationships of the algal microbiota with the mineral bioconstructions, as well as plasticity and resilience of coralline holobionts in a changing environment, are of particular interest. In the Gulf of California, Neogoniolithon trichotomum (Rhodophyta) is the main carbonate builder in tidal pools. We performed a multi-disciplinary assessment of the N. trichotomum microstructure using XRD, SEM microscopy and SR-FTIR spectromicroscopy. In the algal perithallus, magnesium-calcite and aragonite were spatially segregated and embedded in a polysaccharide matrix (rich in sulfated polysaccharides). Mg-calcites (18-19 mol% Mg) were the main mineral components of the thallus overall, followed by iron carbonates related to dolomite (ankerite) and siderite. Minerals of late evaporitic sequences (sylvite and bischofite) were also present, suggesting potential halophilic microenvironments within the algal thalli. The diverse set of abundant halophilic, halotolerant and oligotrophic taxa, whose abundance increase in the summer, further suggests this condition. We created an integrated model, based on environmental parameters and the microbiota distribution, that identified temperature and nutrient availability (particularly nitrate and silicate) as the main parameters related to specific taxa patterns. Among these, Hahella, Granulossicoccus, Ferrimonas, Spongiibacteraceae and cyanobacterial Xenococcaceae and Nostocaceae change significantly between seasons. These bacterial components might play relevant roles in algal plasticity and adaptive responses to a changing environment. This study contributes to the understanding of the interplay of the prokaryotic microbiota with the mineral microenvironments of coralline algae. Because of their carbonates with potential resistance to dissolution in a higher pCO2 world and their seasonally dynamic bacteria, coralline algae are relevant targets to study coastal resilience and carbonated systems responses to changing environments.
Subject(s)
Microbiota , Rhodophyta , Biodiversity , Humans , Minerals , TemperatureABSTRACT
Rainbow light trapping in plasmonic devices allows for field enhancement of multiple wavelengths within a single device. However, many of these devices lack precise control over spatial and spectral enhancement profiles and cannot provide extremely high localised field strengths. Here we present a versatile, analytical design paradigm for rainbow trapping in nanogroove arrays by utilising both the groove-width and groove-length as tuning parameters. We couple this design technique with fabrication through multilayer thin-film deposition and focused ion beam milling, which enables the realisation of unprecedented feature sizes down to 5 nm and corresponding extreme normalised local field enhancements up to 103. We demonstrate rainbow trapping within the devices through hyperspectral microscopy and show agreement between the experimental results and simulation. The combination of expeditious design and precise fabrication underpins the implementation of these nanogroove arrays for manifold applications in sensing and nanoscale optics.
ABSTRACT
Non-invasive and label-free spectral microscopy (spectromicroscopy) techniques can provide quantitative biochemical information complementary to genomic sequencing, transcriptomic profiling, and proteomic analyses. However, spectromicroscopy techniques generate high-dimensional data; acquisition of a single spectral image can range from tens of minutes to hours, depending on the desired spatial resolution and the image size. This substantially limits the timescales of observable transient biological processes. To address this challenge and move spectromicroscopy towards efficient real-time spatiochemical imaging, we developed a grid-less autonomous adaptive sampling method. Our method substantially decreases image acquisition time while increasing sampling density in regions of steeper physico-chemical gradients. When implemented with scanning Fourier Transform infrared spectromicroscopy experiments, this grid-less adaptive sampling approach outperformed standard uniform grid sampling in a two-component chemical model system and in a complex biological sample, Caenorhabditis elegans. We quantitatively and qualitatively assess the efficiency of data acquisition using performance metrics and multivariate infrared spectral analysis, respectively.
Subject(s)
Hyperspectral Imaging/methods , Image Processing, Computer-Assisted/methods , Animals , Caenorhabditis elegans/metabolism , Databases, Factual , Gene Expression Regulation , Models, Biological , Time FactorsABSTRACT
Sediment-hosted CO2-rich aquifers deep below the Colorado Plateau (USA) contain a remarkable diversity of uncultivated microorganisms, including Candidate Phyla Radiation (CPR) bacteria that are putative symbionts unable to synthesize membrane lipids. The origin of organic carbon in these ecosystems is unknown and the source of CPR membrane lipids remains elusive. We collected cells from deep groundwater brought to the surface by eruptions of Crystal Geyser, sequenced the community, and analyzed the whole community lipidome over time. Characteristic stable carbon isotopic compositions of microbial lipids suggest that bacterial and archaeal CO2 fixation ongoing in the deep subsurface provides organic carbon for the complex communities that reside there. Coupled lipidomic-metagenomic analysis indicates that CPR bacteria lack complete lipid biosynthesis pathways but still possess regular lipid membranes. These lipids may therefore originate from other community members, which also adapt to high in situ pressure by increasing fatty acid unsaturation. An unusually high abundance of lysolipids attributed to CPR bacteria may represent an adaptation to membrane curvature stress induced by their small cell sizes. Our findings provide new insights into the carbon cycle in the deep subsurface and suggest the redistribution of lipids into putative symbionts within this community.
Subject(s)
Carbon Dioxide/metabolism , Groundwater/microbiology , Archaea/genetics , Autotrophic Processes , Bacteria/genetics , Carbon/metabolism , Carbon Cycle , Colorado , Ecosystem , Lipids/analysis , Metagenome , PhylogenyABSTRACT
Plant material falling into the ultra-basic (pH 11.5-11.9) springs within The Cedars, an actively serpentinizing site in Sonoma County, California, is subject to conditions that mimic the industrial pretreatment of lignocellulosic biomass for biofuel production. We sought to obtain hemicellulolytic/cellulolytic bacteria from The Cedars springs that are capable of withstanding the extreme alkaline conditions wherein calcium hydroxide-rich water removes lignin, making cell wall polysaccharides more accessible to microorganisms and their enzymes. We enriched for such bacteria by adding plant debris from the springs into a synthetic alkaline medium with ground tissue of the biofuel crop switchgrass (Panicum virgatum L.) as the sole source of carbon. From the enrichment culture we isolated the facultative anaerobic bacterium Cellulomonas sp. strain FA1 (NBRC 114238), which tolerates high pH and catabolizes the major plant cell wall-associated polysaccharides cellulose, pectin, and hemicellulose. Strain FA1 in monoculture colonized the plant material and degraded switchgrass at a faster rate than the community from which it was derived. Cells of strain FA1 could be acclimated through subculturing to grow at a maximal concentration of 13.4% ethanol. A strain FA1-encoded ß-1, 4-endoxylanase expressed in E. coli was active at a broad pH range, displaying near maximal activity at pH 6-9. Discovery of this bacterium illustrates the value of extreme alkaline springs in the search for microorganisms with potential for consolidated bioprocessing of plant biomass to biofuels and other valuable bio-inspired products.
Subject(s)
Biofuels/microbiology , Cellulomonas/isolation & purification , Cellulomonas/metabolism , Endo-1,4-beta Xylanases/metabolism , Lignin/metabolism , Base Composition/genetics , Biomass , Cellulose/metabolism , Endo-1,4-beta Xylanases/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Ethanol/metabolism , Panicum/chemistry , Panicum/genetics , Panicum/metabolism , Pectins/metabolism , Phylogeny , Plants/metabolism , Polysaccharides/metabolism , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNAABSTRACT
The idea that original soft tissue structures and the native structural proteins comprising them can persist across geological time is controversial, in part because rigorous and testable mechanisms that can occur under natural conditions, resulting in such preservation, have not been well defined. Here, we evaluate two non-enzymatic structural protein crosslinking mechanisms, Fenton chemistry and glycation, for their possible contribution to the preservation of blood vessel structures recovered from the cortical bone of a Tyrannosaurus rex (USNM 555000 [formerly, MOR 555]). We demonstrate the endogeneity of the fossil vessel tissues, as well as the presence of type I collagen in the outermost vessel layers, using imaging, diffraction, spectroscopy, and immunohistochemistry. Then, we use data derived from synchrotron FTIR studies of the T. rex vessels to analyse their crosslink character, with comparison against two non-enzymatic Fenton chemistry- and glycation-treated extant chicken samples. We also provide supporting X-ray microprobe analyses of the chemical state of these fossil tissues to support our conclusion that non-enzymatic crosslinking pathways likely contributed to stabilizing, and thus preserving, these T. rex vessels. Finally, we propose that these stabilizing crosslinks could play a crucial role in the preservation of other microvascular tissues in skeletal elements from the Mesozoic.
Subject(s)
Collagen Type I/chemistry , Dinosaurs/metabolism , Fossils , Proteins/chemistry , Animals , Bone and Bones/chemistry , Bone and Bones/metabolism , Collagen Type I/metabolism , Humans , Preservation, Biological , Proteins/metabolismABSTRACT
The contribution of planktonic cyanobacteria to burial of organic carbon in deep-sea sediments before the emergence of eukaryotic predators ~1.5 Ga has been considered negligible owing to the slow sinking speed of their small cells. However, global, highly positive excursion in carbon isotope values of inorganic carbonates ~2.22-2.06 Ga implies massive organic matter burial that had to be linked to oceanic cyanobacteria. Here to elucidate that link, we experiment with unicellular planktonic cyanobacteria acclimated to high partial CO2 pressure (pCO2) representative of the early Paleoproterozoic. We find that high pCO2 boosts generation of acidic extracellular polysaccharides (EPS) that adsorb Ca and Mg cations, support mineralization, and aggregate cells to form ballasted particles. The down flux of such self-assembled cyanobacterial aggregates would decouple the oxygenic photosynthesis from oxidative respiration at the ocean scale, drive export of organic matter from surface to deep ocean and sustain oxygenation of the planetary surface.
Subject(s)
Carbon Dioxide/analysis , Carbon/chemistry , Cyanobacteria/metabolism , Geologic Sediments/chemistry , Oxygen/metabolism , Plankton/metabolism , Seawater/chemistry , Carbon Isotopes/analysis , Carbonates/chemistry , Geologic Sediments/analysis , Oceans and Seas , Photosynthesis , Polysaccharides, Bacterial/chemistryABSTRACT
Microbialites are modern analogs of ancient microbial consortia that date as far back as the Archaean Eon. Microbialites have contributed to the geochemical history of our planet through their diverse metabolic capacities that mediate mineral precipitation. These mineral-forming microbial assemblages accumulate major ions, trace elements and biomass from their ambient aquatic environments; their role in the resulting chemical structure of these lithifications needs clarification. We studied the biogeochemistry and microbial structure of microbialites collected from diverse locations in Mexico and in a previously undescribed microbialite in Cuba. We examined their structure, chemistry and mineralogy at different scales using an array of nested methods including 16S rRNA gene high-throughput sequencing, elemental analysis, X-Ray fluorescence (XRF), X-Ray diffraction (XRD), Scanning Electron Microscopy-Energy Dispersive Spectroscopy (SEM-EDS), Fourier Transformed Infrared (FTIR) spectroscopy and Synchrotron Radiation-based Fourier Transformed Infrared (SR-FTIR) spectromicroscopy. The resulting data revealed high biological and chemical diversity among microbialites and specific microbe to chemical correlations. Regardless of the sampling site, Proteobacteria had the most significant correlations with biogeochemical parameters such as organic carbon (Corg), nitrogen and Corg:Ca ratio. Biogeochemically relevant bacterial groups (dominant phototrophs and heterotrophs) showed significant correlations with major ion composition, mineral type and transition element content, such as cadmium, cobalt, chromium, copper and nickel. Microbial-chemical relationships were discussed in reference to microbialite formation, microbial metabolic capacities and the role of transition elements as enzyme cofactors. This paper provides an analytical baseline to drive our understanding of the links between microbial diversity with the chemistry of their lithified precipitations.
ABSTRACT
The human skin microbiome acts as an important barrier protecting our body from pathogens and other environmental influences. Recent investigations have provided evidence that Archaea are a constant but highly variable component of the human skin microbiome, yet factors that determine their abundance changes are unknown. Here, we tested the hypothesis that the abundance of archaea on human skin is influenced by human age and skin physiology by quantitative PCR of 51 different skin samples taken from human subjects of various age. Our results reveal that archaea are more abundant in human subjects either older than 60 years or younger than 12 years as compared to middle-aged human subjects. These results, together with results obtained from spectroscopy analysis, allowed us gain first insights into a potential link of lower sebum levels and lipid content and thus reduced skin moisture with an increase in archaeal signatures. Amplicon sequencing of selected samples revealed the prevalence of specific eury- and mainly thaumarchaeal taxa, represented by a core archaeome of the human skin.
Subject(s)
Archaea/classification , Archaea/genetics , Microbiota , Skin Physiological Phenomena , Skin/microbiology , Age Factors , Biodiversity , Female , High-Throughput Nucleotide Sequencing , Humans , Male , Middle Aged , Phylogeny , RNA, Ribosomal, 16S , Spectroscopy, Fourier Transform InfraredABSTRACT
Climate model projections for tropical regions show clear perturbation of precipitation patterns leading to increased frequency and severity of drought in some regions. Previous work has shown declining soil moisture to be a strong driver of changes in microbial trait distribution, however, the feedback of any shift in functional potential on ecosystem properties related to carbon cycling are poorly understood. Here we show that drought-induced changes in microbial functional diversity and activity shape, and are in turn shaped by, the composition of dissolved and soil-associated carbon. We also demonstrate that a shift in microbial functional traits that favor the production of hygroscopic compounds alter the eï¬ux of carbon dioxide following soil rewetting. Under drought the composition of the dissolved organic carbon pool changed in a manner consistent with a microbial metabolic response. We hypothesize that this microbial ecophysiological response to changing soil moisture elevates the intracellular carbon demand stimulating extracellular enzyme production, that prompts the observed decline in more complex carbon compounds (e.g., cellulose and lignin). Furthermore, a metabolic response to drought appeared to condition (biologically and physically) the soil, notably through the production of polysaccharides, particularly in experimental plots that had been pre-exposed to a short-term drought. This hysteretic response, in addition to an observed drought-related decline in phosphorus concentration, may have been responsible for a comparatively modest CO2 eï¬ux following wet-up in drought plots relative to control plots.
ABSTRACT
A long-standing desire in biological and biomedical sciences is to be able to probe cellular chemistry as biological processes are happening inside living cells. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectral microscopy is a label-free and nondestructive analytical technique that can provide spatiotemporal distributions and relative abundances of biomolecules of a specimen by their characteristic vibrational modes. Despite great progress in recent years, SR-FTIR imaging of living biological systems remains challenging because of the demanding requirements on environmental control and strong infrared absorption of water. To meet this challenge, microfluidic devices have emerged as a method to control the water thickness while providing a hospitable environment to measure cellular processes and responses over many hours or days. This paper will provide an overview of microfluidic device development for SR-FTIR imaging of living biological systems, provide contrast between the various techniques including closed and open-channel designs, and discuss future directions of development within this area. Even as the fundamental science and technological demonstrations develop, other ongoing issues must be addressed; for example, choosing applications whose experimental requirements closely match device capabilities, and developing strategies to efficiently complete the cycle of development. These will require imagination, ingenuity and collaboration.
Subject(s)
Cells/chemistry , Microfluidics/instrumentation , Spectroscopy, Fourier Transform Infrared/instrumentation , Animals , Microfluidics/methods , Spectroscopy, Fourier Transform Infrared/methods , Synchrotrons , Water/chemistryABSTRACT
The CELLULOSE SYNTHASE-LIKE F6 (CslF6) gene was previously shown to mediate the biosynthesis of mixed-linkage glucan (MLG), a cell wall polysaccharide that is hypothesized to be tightly associated with cellulose and also have a role in cell expansion in the primary cell wall of young seedlings in grass species. We have recently shown that loss-of-function cslf6 rice mutants do not accumulate MLG in most vegetative tissues. Despite the absence of a structurally important polymer, MLG, these mutants are unexpectedly viable and only show a moderate growth compromise compared to wild type. Therefore these mutants are ideal biological systems to test the current grass cell wall model. In order to gain a better understanding of the role of MLG in the primary wall, we performed in-depth compositional and structural analyses of the cell walls of 3 day-old rice seedlings using various biochemical and novel microspectroscopic approaches. We found that cellulose content as well as matrix polysaccharide composition was not significantly altered in the MLG deficient mutant. However, we observed a significant change in cellulose microfibril bundle organization in mesophyll cell walls of the cslf6 mutant. Using synchrotron source Fourier Transform Mid-Infrared (FTM-IR) Spectromicroscopy for high-resolution imaging, we determined that the bonds associated with cellulose and arabinoxylan, another major component of the primary cell walls of grasses, were in a lower energy configuration compared to wild type, suggesting a slightly weaker primary wall in MLG deficient mesophyll cells. Taken together, these results suggest that MLG may influence cellulose deposition in mesophyll cell walls without significantly affecting anisotropic growth thus challenging MLG importance in cell wall expansion.
ABSTRACT
In the genus Azolla rapid abscission of roots from floating fronds occurs within minutes in response to a variety of stresses, including exposure to nitrite. We found that hydrogen peroxide, though itself not an inducer of root abscission, modulates nitrite-induced root abscission by Azolla pinnata in a dose-dependent manner, with 2 mM H2O2 significantly diminishing the responsiveness to 2 mM NaNO2, and 10 mM H2O2 slightly enhancing it. Hypoxia, which has been found in other plants to result in autogenic production of H2O2, dramatically stimulated root abscission of A. pinnata in response to nitrite, especially for plants previously cultivated in medium containing 5 mM KNO3 compared to plants cultivated under N2-fixing conditions without combined nitrogen. Plants, including Azolla, produce the small signaling molecule nitric oxide (NO) from nitrite using nitrate reductase. We found Azolla plants to display dose-dependent root abscission in response to the NO donor spermine NONOate. Treatment of plants with the thiol-modifying agents S-methyl methanethiosulfonate or glutathione inhibited the nitrite-induced root abscission response. Synchrotron radiation-based Fourier transform infrared spectromicroscopy revealed higher levels of carbonylation in the abscission zone of dropped roots, indicative of reaction products of polysaccharides with potent free radical oxidants. We hypothesize that metabolic products of nitrite and NO react with H2O2 in the apoplast leading to free-radical-mediated cleavage of structural polysaccharides and consequent rapid root abscission.
ABSTRACT
We present the genome of the cellulose-degrading Cellulomonas sp. strain FA1 isolated from an actively serpentinizing highly alkaline spring. Knowledge of this genome will enable studies into the molecular basis of plant material degradation in alkaline environments and inform the development of lignocellulose bioprocessing procedures for biofuel production.
ABSTRACT
Spatially resolved infrared spectroscopy is a label-free and nondestructive analytical technique that can provide spatiotemporal information on functional groups in biomolecules of a sample by their characteristic vibrational modes. One difficulty in performing long-term FT-IR measurements on live cells is the competition between the strong IR absorption from water and the need to supply nutrients and remove waste. In this proof of principle study, we developed an open-channel membrane device that allows long-term continuous IR measurement of live, adherent mammalian cells. Composed of a gold-coated porous membrane between a feeding channel and a viewing chamber, it allows cells to be maintained on the upper membrane surface in a thin layer of fluid while media is replenished from the feeding channel below. Using this device, we monitored the spatiotemporal chemical changes in living colonies of PC12 cells under nerve growth factor (NGF) stimulation for up to 7 days using both conventional globar and high-resolution synchrotron radiation-based IR sources. We identified the primary chemical change cells undergo is an increase in glycogen that may be associated with secretion of glycoprotein to protect the cells from evaporative stress at the air-liquid interface. Analyzing the spectral maps with multivariate methods of hierarchical cluster analysis (HCA) and principal component analysis (PCA), we found that the cells at the boundary of the colony and in a localized region in the center of the colony tend to produce more glycogen and glycoprotein than cells located elsewhere in the colony and that the degree of spatial heterogeneity decreases with time. This method provides a promising approach for long-term live-cell spectromicroscopy on mammalian cell systems.
Subject(s)
Microfluidic Analytical Techniques , Animals , Cell Adhesion , Cluster Analysis , PC12 Cells , Principal Component Analysis , Rats , Spectroscopy, Fourier Transform InfraredABSTRACT
As a means to improve carbon uptake in the cyanobacterium Synechocystis sp. strain PCC6803, we engineered strains to contain additional inducible copies of the endogenous bicarbonate transporter BicA, an essential component of the CO2-concentrating mechanism in cyanobacteria. When cultured under atmospheric CO2 pressure, the strain expressing extra BicA transporters (BicA(+) strain) grew almost twice as fast and accumulated almost twice as much biomass as the control strain. When enriched with 0.5% or 5% CO2, the BicA(+) strain grew slower than the control but still showed a superior biomass production. Introducing a point mutation in the large C-terminal cytosolic domain of the inserted BicA, at a site implicated in allosteric regulation of transport activity, resulted in a strain (BicA(+)(T485G) strain) that exhibited pronounced cell aggregation and failed to grow at 5% CO2. However, the bicarbonate uptake capacity of the induced BicA(+)(T485G) was twice higher than for the wild-type strain. Metabolic analyses, including phenotyping by synchrotron-radiation Fourier transform Infrared spectromicroscopy, scanning electron microscopy, and lectin staining, suggest that the excess assimilated carbon in BicA(+) and BicA(+)(T485G) cells was directed into production of saccharide-rich exopolymeric substances. We propose that the increased capacity for CO2 uptake in the BicA(+) strain can be capitalized on by re-directing carbon flux from exopolymeric substances to other end products such as fuels or high-value chemicals.