Substrate analyses in single cells: I. Determination of ATP

Subfemtomole quantities of ATP were determined using luminescence analysis with firefly extracts. The methodological advances include development of microtechniques for sample handling, and recording of the time profile of the light emission by photon counting in connection with multichannel techniques. The amount of ATP in isolated single cells of the microorganisms Paramecium, Peridinium, and of macrophages from the abdominal cavity of mice were assayed. These cells represented a range from 0.07 to 2.5 ng dry mass as assessed with automatic interferometric scanning.     

Metabolite and target transcript analyses during Crocussativus stigma development

Saffron, the desiccated stigmas of Crocussativus, is highly appreciated for its peculiar colour, flavour and aroma. Several studies have been conducted with the spice, but little is known about the evolution of volatile and non-volatile compounds generated during the development of the stigma. In this study, we have followed these compounds, with special attention to those of isoprenoid origin (carotenoids and monoterpenes), which are responsible for the organoleptic properties of saffron. The main compounds that accumulated throughout stigma development in C.sativus were crocetin, its glucoside derivatives and picrocrocin, all of which increased as stigmas reached a fully developed stage. The volatile composition of C.sativus stigmas changed notably as stigmas developed with each developmental stage being characterized by a different volatile combination. In red stigmas, β-cyclocitral, the 7,8 cleavage product of β-carotene, was highly produced, suggesting the implication of both β-carotene and zeaxanthin in crocetin formation. As stigmas matured, hydroxy-β-ionone and β-ionone were produced while safranal, the most typical aroma compound of the processed spice, was only detected at low levels. However, a safranal-related compound 2,2,2-trimethyl-2-cyclohexene-1,4-dione (4-oxoisophorone) increased rapidly at the anthesis stage and also in senescent stigmas. Monoterpenes were mainly emitted at the time of anthesis and the emission patterns followed the expression patterns of two putative terpene synthases CsTS1 and CsTS2. Fatty acid derivates, which predominated at the earlier developmental stages, were observed at low levels in later stages.     

Metabolite profiling of CHO cells with different growth characteristics

Mammalian cell cultures are the predominant system for the production of recombinant proteins requiring post-translational modifications. As protein yields are a function of growth performance (among others), and performance varies greatly between culture medium (e.g., different growth rates and peak cell densities), an understanding of the biological mechanisms underpinning this variability would facilitate rational medium and process optimization, increasing product yields, and reducing costs. We employed a metabolomics approach to analyze differences in metabolite concentrations of CHO cells cultivated in three different media exhibiting different growth rates and maximum viable cell densities. Analysis of intra- and extracellular metabolite concentrations over the course of the cultures using a combination of HPLC and GC-MS, readily detected medium specific and time dependent changes. Using multivariate data analysis, we identified a range of metabolites correlating with growth rate, illustrating how metabolomics can be used to relate gross phenotypic changes to the fine details of cellular metabolism.     

Metabolite and mineral analyses of cotton near-isogenic lines introgressed with QTLs for productivity and drought-related traits

Quantitative trait loci (QTLs) for yield and drought-related traits were exchanged via marker-assisted selection between elite cultivars of two cotton species, Gossypium barbadense (GB) cv. F-177 and Gossypium hirsutum (GH) cv. Siv'on. Three of the resultant near-isogenic lines (NILs), each introgressed with a different QTL region, expressed an advantage in osmotic adjustment (OA) and other drought-related traits relative to their recipient parents. These NILs and the parental genotypes were field-grown under well-watered and water-limited conditions, and characterized for their metabolic and mineral compositions. Comparisons were then made between (1) GB and GH genotypes, (2) the contrasting water regimes and (3) each NIL and its recipient parent. Hierarchical clustering analysis clearly distinguished between GB and GH genotypes based on either metabolite or mineral composition. Comparisons between well-watered and water-limited conditions in each of the genotypes showed differing trends in the various solutes. The greater concentrations of potassium, magnesium and calcium under water stress, when compared with well-watered conditions, may have enhanced OA or osmoprotection. All NILs exhibited significantly modified solute composition relative to their recipient parents. In particular, increased levels of alanine, aspartic acid, citric acid, malic acid, glycerol, myoinositol, threonic acid, potassium, magnesium and calcium were found under drought conditions in one or more of the NILs relative to their recipient parents. The increased values of these solutes could contribute to the superior capacity of these NILs to cope with drought.     

A simple and reliable method for the localization in cell culture of single identifiable cells for ultrastructural analyses

A simple method for relocating single cells in monolayer cultures for subsequent morphological or ultrastructural analysis is reported. This consists of producing, on the culture dish surface, a nontoxic carbon grid that is preserved during processing for either transmission (TEM) or scanning (SEM) electron microscopy. For TEM studies these grids are readily transferred along with the cells into the embedding plastic, and thus individual grid squares containing a cell(s) of interest can be quickly located, remounted, and sectioned. These grids may be useful for ultrastructural analyses of single cells previously studied electrophysiologically or after microinjection of macromolecules.     

Metabolite profiling of CHO cells: Molecular reflections of bioprocessing effectiveness

Whilst development of medium and feeds has provided major advances in recombinant protein production in CHO cells, the fundamental understanding is limited. We have applied metabolite profiling with established robust (GC‐MS) analytics to define the molecular loci by which two yield‐enhancing feeds improve recombinant antibody yields from a model GS‐CHO cell line. With data across core metabolic pathways, that report on metabolism within several cellular compartments, these data identify key metabolites and events associated with increased cell survival and specific productivity of cells. Of particular importance, increased process efficiency was linked to the functional activity of the mitochondria, with the amount and time course of use/production of intermediates of the citric acid cycle, for uses such as lipid biosynthesis, precursor generation and energy production, providing direct indicators of cellular status with respect to productivity. The data provide clear association between specific cellular metabolic indicators and cell process efficiency, extending from prior indications of the relevance of lactate metabolic balance to other redox sinks (glycerol, sorbitol and threitol). The information, and its interpretation, identifies targets for engineering cell culture efficiency, either from genetic or environmental perspectives, and greater understanding of the significance of specific medium components towards overall CHO cell bioprocessing.     

A single molecule array for digital targeted molecular analyses

We present a new random array format together with a decoding scheme for targeted multiplex digital molecular analyses. DNA samples are analyzed using multiplex sets of padlock or selector probes that create circular DNA molecules upon target recognition. The circularized DNA molecules are amplified through rolling-circle amplification (RCA) to generate amplified single molecules (ASMs). A random array is generated by immobilizing all ASMs on a microscopy glass slide. The ASMs are identified and counted through serial hybridizations of small sets of tag probes, according to a combinatorial decoding scheme. We show that random array format permits at least 10 iterations of hybridization, imaging and dehybridization, a process required for the combinatorial decoding scheme. We further investigated the quantitative dynamic range and precision of the random array format. Finally, as a demonstration, the decoding scheme was applied for multiplex quantitative analysis of genomic loci in samples having verified copy-number variations. Of 31 analyzed loci, all but one were correctly identified and responded according to the known copy-number variations. The decoding strategy is generic in that the target can be any biomolecule which has been encoded into a DNA circle via a molecular probing reaction.     

Metabolite extraction from suspension-cultured mammalian cells for global metabolite profiling

Metabolite profiling of industrially important suspension-cultured mammalian cells is being increasingly used for rational improvement of bioprocesses. This requires the generation of global metabolite profiles that cover a broad range of metabolites and that are representative of the cells at the time of sampling. The protocol described here is a validated method for recovery of physiologically relevant amounts of key metabolites from suspension-cultured mammalian cells. The method is a two-step process consisting of initial quenching of the cells (to stop cellular metabolism and allow isolation of the cells) followed by extraction of the metabolites. The cells are quenched in 60% methanol supplemented with 0.85% (wt/vol) ammonium bicarbonate at -40 °C. Metabolites are then extracted from the quenched cells using two 100% methanol extractions followed by a single water extraction. Metabolite samples generated using this protocol are amenable to analysis by mass spectrometry-based techniques (e.g., gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry), NMR spectroscopy and enzymatic assays.     

Pluripotent-related gene expression analyses in single porcine recloned embryo

The developmental ability among embryos produced by three different techniques were examined: there were no significant differences in the developmental rate in porcine embryos produced by in vitro fertilization (IVF) and first generation of somatic cell nucleus transfer (SCNT), but the developmental rate dropped sharply at the 2- to four-cell stage in recloned (second generation of SCNT) embryos. In most recloned embryos, Oct4 and Klf4 were under-expressed at all stages, whereas Sox2 and Nanog were over-expressed at the two-cell stage. In contrast, Nanog was absent in IVF and SCNT embryos at the two-cell stage. The recloned embryos were treated with valproic acid to enhance developmental capacity and this led to an increase in the rate of blastocyst formation and total cell number compared with the findings for untreated recloned embryos (29.8 vs. 12.4 %, 39 vs. 25, respectively, p < 0.05).     

Metabolic responses of Thellungiella halophila/salsuginea to biotic and abiotic stresses: Metabolite profiles and quantitative analyses

The metabolite profiles of the model crucifer Thellungiella salsuginea (salt cress) ecotype Shandong subjected to various biotic and abiotic stresses were analyzed using HPLC-DAD-ESI-MS. Two different cruciferous microbial pathogens, Albugo candida, a biotrophic oomycete, and Leptosphaeria maculans, a necrotrophic fungus, elicited formation of the phytoalexins wasalexins A and B without causing visual damage on inoculated leaves. Analyses of non-polar and polar metabolites led to elucidation of the chemical structures of five metabolites: 4′-O-(E)-sinapoyl-7-methoxyisovitexin-2″-O-β-d-glucopyranoside, 4′-O-(E)-sinapoylisovitexin-2″-O-β-d-glucopyranoside, 4-O-β-d-glucopyranosyl-7-hydroxymatairesinol, 5′-O-β-d-glucopyranosyldihydroneoascorbigen and 3-O-β-d-glucopyranosylthiane. 3-O-β-d-glucopyranosylthiane, an unique metabolite for which we suggest the name glucosalsuginin, is proposed to derive from the glucosinolate glucoberteroin. In addition, the identification of a broad range of polar metabolites identical to those of other crucifers was carried out. Quantification of several metabolites over a period of eight days showed that concentrations of the polar phytoanticipin 4-methoxyglucobrassicin increased substantially in leaves irradiated with UV light (λ_max 254 nm) relative to control leaves, but not in leaves subjected to other stresses.     

Microarray analyses of transdifferentiated mesenchymal stem cells

The molecular events associated with the age-related gain of fatty tissue in human bone marrow are still largely unknown. Besides enhanced adipogenic differentiation of mesenchymal stem cells (MSCs), transdifferentiation of osteoblast progenitors may contribute to bone-related diseases like osteopenia. Transdifferentiation of MSC-derived osteoblast progenitors into adipocytes and vice versa has previously been proven feasible in our cell culture system. Here, we focus on mRNA species that are regulated during transdifferentiation and represent possible control factors for the initiation of transdifferentiation. Microarray analyses comparing transdifferentiated cells with normally differentiated cells exhibited large numbers of reproducibly regulated genes for both, adipogenic and osteogenic transdifferentiation. To evaluate the relevance of individual genes, we designed a scoring scheme to rank genes according to reproducibility, regulation level, and reciprocity between the different transdifferentiation directions. Thereby, members of several signaling pathways like FGF, IGF, and Wnt signaling showed explicitly differential expression patterns. Additional bioinformatic analysis of microarray analyses allowed us to identify potential key factors associated with transdifferentiation of adipocytes and osteoblasts, respectively. Fibroblast growth factor 1 (FGF1) was scored as one of several lead candidate gene products to modulate the transdifferentiation process and is shown here to exert inhibitory effects on adipogenic commitment and differentiation.     

Complementation analyses of differentiation-defective embryonal carcinoma cells

We have generated cell hybrids by fusing embryonal carcinoma (EC) cells which fail to differentiate in response to retinoic acid (RA) and/or hexamethylenebisacetamide (HMBA). The first two classes of hybrids were between an RA- line (also unresponsive to HMBA) that lacks cellular RA binding protein (cRABP) activity and HMBA- lines which possess cRABP and differentiate in the presence of RA. All of the hybrid clones possessed cRABP and differentiated normally upon exposure to either RA or HMBA. When the aforementioned RA- mutant was fused with a second mutant which was refractory to RA and HMBA but possessed cRABP activity, the resultant hybrid clones were responsive to both RA and HMBA and had cRABP activity. These results suggest that all of these mutants were recessive and complementary. Tumors from these hybrid lines differentiated extensively, in some instances much more so than the mutant parental lines and even the wild-type lines from which the mutants were derived. Based upon these observations, we propose that various EC lines might differentiate poorly in tumor form for different reasons. Hybrids between two differentiation-defective, cRABP- lines appeared to be at least partially complemented for responsiveness to RA and HMBA. These hybrids contained low but detectable levels of cRABP. This is not a consequence of tetraploidy since fusions between cells from the same mutant line retained their differentiation-defective phenotype and possessed little or no cRABP activity. Unlike tumors from the other hybrids described above, tumors from these hybrid lines expressed a very restricted pattern of differentiated cell types. This might be because the mutant lines in the latter hybrids originally derived from the same wild-type EC line.     

Enzyme activity analyses along ragged-red and normal single muscle fibres

Summary Mitochondrial myopathies are morphologically characterized by ragged-red fibres (RRF). Serial cross-section revealed that the ragged-red appearance was only focal. This is in agreement with a partial cytochromec oxidase (COX) deficiency in chronic progressive external ophthalmoplegia (CPEO). Since most of these patients show deletions of the mitochondrial genome single fibre analyses were performed determining COX and succinate dehydrogenase (SDH) in serial muscle sections from two patients with CPEO. High SDH activity was demonstrated in RRF; in contrast COX activity was lower in RRF in a patient, possibly representing a focal assembly of mitochondria with deletions in their genomes. The variation of enzyme activities along the muscle fibre was especially high in RRF. This study presents the first quantitative evidence that enzyme activities vary considerably along fibres in muscle from patients with a mitochondrial myopathy.     

Creep indentation of single cells

An apparatus for creep indentation of individual adherent cells was designed, developed, and experimentally validated. The creep cytoindentation apparatus (CCA) can perform stress-controlled experiments and measure the corresponding deformation of single anchorage-dependent cells. The apparatus can resolve forces on the order of 1 nN and cellular deformations on the order of 0.1 micron. Experiments were conducted on bovine articular chondrocytes using loads on the order of 10 nN. The experimentally observed viscoelastic behavior of these cells was modeled using the punch problem and standard linear solid. The punch problem yielded a Young's modulus of 1.11 +/- 0.48 kPa. The standard linear solid model yielded an instantaneous elastic modulus of 8.00 +/- 4.41 kPa, a relaxed modulus of 1.09 +/- 0.54 kPa, an apparent viscosity of 1.50 +/- 0.92 kPa-s, and a time constant of 1.32 +/- 0.65 s. To our knowledge, this is the first time that stress-controlled indentation testing has been applied at the single cell level. This methodology represents a new tool in understanding the mechanical nature of anchorage-dependent cells and mechanotransductional pathways.