Comparison of the mouse spleen cell assay and a radioimmunoassay for the measurement of serum erythropoietin

The mouse spleen cell assay (MSCA) has been compared with a radioimmunoassay for the measurement of serum erythropoietin (Ep). In 20 normal subjects the serum values ranged from 15 to 73 mU/ml for the MSCA compared with 5-30 mU/ml for the RIA. For normal sera there was no correlation between the results of the two assays. In 37 patients with anaemias of differing aetiologies and at various stages of treatment values ranged from 10 to 3645 mU/ml for the MSCA and 13-10,000 mU/ml for the RIA. Although patient values from the two assays were highly correlated (r = 0.98, P less than 0.001), the MSCA results were generally lower. These discrepancies can be largely accounted for by two factors. Firstly the MSCA is sensitive to non-specific matrix effects. Secondly, heat inactivation of serum, a prerequisite for the MSCA, but not for the RIA, destroys a variable and unpredictable proportion of the Ep in the test sera leading to an underestimation of Ep in the MSCA. We conclude that the RIA is more reliable than the MSCA which, in its present form, cannot be recommended for the accurate measurement of serum erythropoietin.

Produção de "single cell oil" por Yarrowia lipolytica : seleção de meios de cultura

Com o crescimento económico e populacional, as necessidades energéticas globais aumentam diariamente. Juntamente com a percepção de que os combustíveis fósseis são finitos e com uma progressiva consciência ambiental, os biocombustíveis são vistos como a alternativa a seguir. A produção de biodiesel a partir da transesterificação de óleos vegetais aumenta a deflorestação e o custo de bens alimentares. Como alternativa, surgem os óleos de origem microbiana – single cell oil (SCO) - acumulados principalmente como triacilgliceróis, a mesma forma dos óleos vegetais, podendo ser utilizados para biodiesel ou como lípidos de alto valor acrescentado. Contudo, a utilização de microrganismos para síntese de lípidos está limitada pelos custos associados ao processo de produção. Este bioprocesso torna-se economicamente mais favorável quando resíduos obtidos a baixo custo são utilizados como fonte de carbono. Assim, o objectivo deste trabalho foi estudar a possibilidade de utilização de subprodutos de diversas indústrias (glicerol bruto e banha de porco) e glicerol puro na produção de SCO, utilizando a levedura Yarrowia lipolytica. Foram realizados ensaios em batch em matraz de 500 mL, matraz de 1000 mL e biorreator de 2 L, de modo a estudar qual o melhor substrato e concentração para produção de SCO. Verificou-se que a banha é o substrato mais eficaz para a acumulação de gordura pela estirpe Y. lipolytica W29, seguida do glicerol bruto, enquanto o glicerol puro mostrou ser a fonte de carbono menos adequada. Encontrou-se um intervalo de concentração de glicerol bruto para produção de SCO, enquanto que a concentração de banha no meio não afeta a acumulação de gordura pelas células. Os ácidos gordos preferencialmente acumulados pelas células em meio com banha e glicerol bruto foram o ácido oleico e o linoleico, respetivamente. Nos meios com glicerol puro e bruto observou-se a produção de ácido cítrico, o que não se verificou em meio com banha.

Single-cell gene-expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines.

CD8 T cells play a key role in mediating protective immunity against selected pathogens after vaccination. Understanding the mechanism of this protection is dependent upon definition of the heterogeneity and complexity of cellular immune responses generated by different vaccines. Here, we identify previously unrecognized subsets of CD8 T cells based upon analysis of gene-expression patterns within single cells and show that they are differentially induced by different vaccines. Three prime-boost vector combinations encoding HIV Env stimulated antigen-specific CD8 T-cell populations of similar magnitude, phenotype, and functionality. Remarkably, however, analysis of single-cell gene-expression profiles enabled discrimination of a majority of central memory (CM) and effector memory (EM) CD8 T cells elicited by the three vaccines. Subsets of T cells could be defined based on their expression of Eomes, Cxcr3, and Ccr7, or Klrk1, Klrg1, and Ccr5 in CM and EM cells, respectively. Of CM cells elicited by DNA prime-recombinant adenoviral (rAd) boost vectors, 67% were Eomes(-) Ccr7(+) Cxcr3(-), in contrast to only 7% and 2% stimulated by rAd5-rAd5 or rAd-LCMV, respectively. Of EM cells elicited by DNA-rAd, 74% were Klrk1(-) Klrg1(-)Ccr5(-) compared with only 26% and 20% for rAd5-rAd5 or rAd5-LCMV. Definition by single-cell gene profiling of specific CM and EM CD8 T-cell subsets that are differentially induced by different gene-based vaccines will facilitate the design and evaluation of vaccines, as well as enable our understanding of mechanisms of protective immunity.

In situ evaluation of single-cell lysis by cytosol extraction observation through fluorescence decay and dielectrophoretic trapping time

We present a new method for lysis of single cells in continuous flow, where cells are sequentially trapped, lysed and released in an automatic process. Using optimized frequencies, dielectrophoretic trapping allows exposing cells in a reproducible way to high electrical fields for long durations, thereby giving good control on the lysis parameters. In situ evaluation of cytosol extraction on single cells has been studied for Chinese hamster ovary (CHO) cells through out-diffusion of fluorescent molecules for different voltage amplitudes. A diffusion model is proposed to correlate this out-diffusion to the total area of the created pores, which is dependent on the potential drop across the cell membrane and enables evaluation of the total pore area in the membrane. The dielectrophoretic trapping is no longer effective after lysis because of the reduced conductivity inside the cells, leading to cell release. The trapping time is linked to the time required for cytosol extraction and can thus provide additional validation of the effective cytosol extraction for non-fluorescent cells. Furthermore, the application of one single voltage for both trapping and lysis provides a fully automatic process including cell trapping, lysis, and release, allowing operating the device in continuous flow without human intervention.

Direct binding of peptides to MHC class I molecules on living cells. Analysis at the single cell level.

To directly assess the binding of exogenous peptides to cell surface-associated MHC class I molecules at the single cell level, we examined the possibility of combining the use of biotinylated peptide derivatives with an immunofluorescence detection system based on flow cytometry. Various biotinylated derivatives of the adenovirus 5 early region 1A peptide 234-243, an antigenic peptide recognized by CTL in the context of H-2Db, were first screened in functional assays for their ability to bind efficiently to Db molecules on living cells. Suitable peptide derivatives were then tested for their ability to generate positive fluorescence signals upon addition of phycoerythrin-labeled streptavidin to peptide derivative-bearing cells. Strong fluorescent staining of Db-expressing cells was achieved after incubation with a peptide derivative containing a biotin group at the C-terminus. Competition experiments using the unmodified parental peptide as well as unrelated peptides known to bind to Kd, Kb, or Db, respectively, established that binding of the biotinylated peptide to living cells was Db-specific. By using Con A blasts derived from different H-2 congenic mouse strains, it could be shown that the biotinylated peptide bound only to Db among > 20 class I alleles tested. Moreover, binding of the biotinylated peptide to cells expressing the Dbm13 and Dbm14 mutant molecules was drastically reduced compared to Db. Binding of the biotinylated peptide to freshly isolated Db+ cells was readily detectable, allowing direct assessment of the relative amount of peptide bound to distinct lymphocyte subpopulations by three-color flow cytometry. While minor differences between peripheral T and B cells could be documented, thymocytes were found to differ widely in their peptide binding activity. In all cases, these differences correlated positively with the differential expression of Db at the cell surface. Finally, kinetic studies at different temperatures strongly suggested that the biotinylated peptide first associated with Db molecules available constitutively at the cell surface and then with newly arrived Db molecules.

Dynamic single cell measurements of kinase activity by synthetic kinase activity relocation sensors.

BACKGROUND: Mitogen activated protein kinases (MAPK) play an essential role in integrating extra-cellular signals and intra-cellular cues to allow cells to grow, adapt to stresses, or undergo apoptosis. Budding yeast serves as a powerful system to understand the fundamental regulatory mechanisms that allow these pathways to combine multiple signals and deliver an appropriate response. To fully comprehend the variability and dynamics of these signaling cascades, dynamic and quantitative single cell measurements are required. Microscopy is an ideal technique to obtain these data; however, novel assays have to be developed to measure the activity of these cascades. RESULTS: We have generated fluorescent biosensors that allow the real-time measurement of kinase activity at the single cell level. Here, synthetic MAPK substrates were engineered to undergo nuclear-to-cytoplasmic relocation upon phosphorylation of a nuclear localization sequence. Combination of fluorescence microscopy and automated image analysis allows the quantification of the dynamics of kinase activity in hundreds of single cells. A large heterogeneity in the dynamics of MAPK activity between individual cells was measured. The variability in the mating pathway can be accounted for by differences in cell cycle stage, while, in the cell wall integrity pathway, the response to cell wall stress is independent of cell cycle stage. CONCLUSIONS: These synthetic kinase activity relocation sensors allow the quantification of kinase activity in live single cells. The modularity of the architecture of these reporters will allow their application in many other signaling cascades. These measurements will allow to uncover new dynamic behaviour that previously could not be observed in population level measurements.

Marine yeasts as source of single cell protein and immunostimulant for application in penaeid prawn culture systems

The study revealed the potential of marine yeasts as a source of single cell protein and immunostimulant for prawns. Prawns fed with the selected marine yeasts were showing more growth compared to the control feed and commercial feed. Yeasts being rich with proteins, vitamins and carbohydrates serve as a growth promoter for prawns as being evidenced in this study. The better performance of marine yeasts, D. hansenii S8 and S100 and C. tropicalis S186 compared to S. cerevisiae S36 as a feed supplement is worth investigating. Besides being a rich nutritional source, yeasts act as immunostimulants by virtue of its high carbohydrate (Beta, 1-3 glucan) and RNA content. Beta, 1-3 glucan, a cell wall component of yeasts /fungi is the most commonly used immunostimulant in aquaculture. The present study shows that even the whole cell yeast could serve as a good immunostimulant when supplied through diet. Extraction of Beta-1,3 glucan results in the removal of nutrients like proteins, vitamins etc. from the cell biomass.Utilization of the yeast biomass as such in the diet would help perform a dual role as nutritional component and immunostimulant for aquaculture applications.

Marine Actinomycetes as Source of Antimicrobial Compounds and as Probiotics and Single Cell Protein for Application in Penaeid Prawn Culture Systems

This thesis Entitled Marine actinomycetes as source of antimicrobial compounds and as probiotics and single cell protein for application in penaeid peawn culture systems. Ocean harbours more than 80% of all life on earth and remains our greatest untapped natural resource. The study revealed the potential of marine actinomycetes as a source of antimicrobial compounds. The selected streptomycetes were found to be capable of inhibiting most of the pathogenic vibrios, whichis a major problem both in hatcheries and grow out systems. The bioactive principle can be incorporated with commercial feeds and applied as medicated diet for the control of vibrios in culture systems.The hydrolytic potential inhibitory property against pathogens and non—pathogenicity to penaeid prawns make the selected Streptomycesspp.an effective probioic in aquaculture. Since there is considerably less inhibition to the natural in pond ecosystem the microbial diversityis being maintained and thereby the water quality. Actinomycetes was found to be a good source of single cell protein as an ingredient inaquaculture feed formulations. Large amount of mycelial waste (actinomycete biomassO is produced from antibiotic industries and this nutrient rich waste can be effectively used as a protein source in aquaculture feeds.This study reveals the importance of marine actinomycetes as a source of antimicrobial compounds and as a probiotic and single cell protein for aquaculture applications.

Selection of Marine Yeasts for the Generation of Single Cell Protein from Prawn-shell Waste

Marine yeasts (33 strains) were isolated from the coastal and offshore waters off Cochin. The isolates were identified and then characterized for the utilization of starch, gelatin, lipid, cellulose, urea, pectin, lignin, chitin and prawn-shell waste. Most of the isolates were Candida species. Based on the biochemical characterization, four potential strains were selected and their optimum pH and NaCI concentration for growth were determined. These strains were then inoculated into prawn-shell waste and SCP (single cell protein) generation was noted in terms of the increase in protein content of the final product.

Overview of mathematical approaches used to model bacterial chemotaxis I: the single cell

Mathematical modeling of bacterial chemotaxis systems has been influential and insightful in helping to understand experimental observations. We provide here a comprehensive overview of the range of mathematical approaches used for modeling, within a single bacterium, chemotactic processes caused by changes to external gradients in its environment. Specific areas of the bacterial system which have been studied and modeled are discussed in detail, including the modeling of adaptation in response to attractant gradients, the intracellular phosphorylation cascade, membrane receptor clustering, and spatial modeling of intracellular protein signal transduction. The importance of producing robust models that address adaptation, gain, and sensitivity are also discussed. This review highlights that while mathematical modeling has aided in understanding bacterial chemotaxis on the individual cell scale and guiding experimental design, no single model succeeds in robustly describing all of the basic elements of the cell. We conclude by discussing the importance of this and the future of modeling in this area.

Modeling the variability of single-cell lag times for Listeria innocua populations after sublethal and lethal heat treatments

Optical density measurements were used to estimate the effect of heat treatments on the single-cell lag times of Listeria innocua fitted to a shifted gamma distribution. The single-cell lag time was subdivided into repair time ( the shift of the distribution assumed to be uniform for all cells) and adjustment time (varying randomly from cell to cell). After heat treatments in which all of the cells recovered (sublethal), the repair time and the mean and the variance of the single-cell adjustment time increased with the severity of the treatment. When the heat treatments resulted in a loss of viability (lethal), the repair time of the survivors increased with the decimal reduction of the cell numbers independently of the temperature, while the mean and variance of the single-cell adjustment times remained the same irrespective of the heat treatment. Based on these observations and modeling of the effect of time and temperature of the heat treatment, we propose that the severity of a heat treatment can be characterized by the repair time of the cells whether the heat treatment is lethal or not, an extension of the F value concept for sublethal heat treatments. In addition, the repair time could be interpreted as the extent or degree of injury with a multiple-hit lethality model. Another implication of these results is that the distribution of the time for cells to reach unacceptable numbers in food is not affected by the time-temperature combination resulting in a given decimal reduction.

Single-cell proteomic analysis of glucosinolate-rich S-cells in Arabidopsis thaliana

Single-cell analysis is essential for understanding the processes of cell differentiation and metabolic specialisation in rare cell types. The amount of single proteins in single cells can be as low as one copy per cell and is for most proteins in the attomole range or below; usually considered as insufficient for proteomic analysis. The development of modern mass spectrometers possessing increased sensitivity and mass accuracy in combination with nano-LC-MS/MS now enables the analysis of single-cell contents. In Arabidopsis thaliana, we have successfully identified nine unique proteins in a single-cell sample and 56 proteins from a pool of 15 single-cell samples from glucosinolate-rich S-cells by nanoLC-MS/MS proteomic analysis, thus establishing the proof-of-concept for true single-cell proteomic analysis. Dehydrin (ERD14_ARATH), two myrosinases (BGL37_ARATH and BGL38_ARATH), annexin (ANXD1_ARATH), vegetative storage proteins (VSP1_ARATH and VSP2_ARATH) and four proteins belonging to the S-adenosyl-l-methionine cycle (METE_ARATH, SAHH1_ARATH, METK4_ARATH and METK1/3_ARATH) with associated adenosine kinase (ADK1_ARATH), were amongst the proteins identified in these single-S-cell samples. Comparison of the functional groups of proteins identified in S-cells with epidermal/cortical cells and whole tissue provided a unique insight into the metabolism of S-cells. We conclude that S-cells are metabolically active and contain the machinery for de novo biosynthesis of methionine, a precursor for the most abundant glucosinolate glucoraphanine in these cells. Moreover, since abundant TGG2 and TGG1 peptides were consistently found in single-S-cell samples, previously shown to have high amounts of glucosinolates, we suggest that both myrosinases and glucosinolates can be localised in the same cells, but in separate subcellular compartments. The complex membrane structure of S-cells was reflected by the presence of a number of proteins involved in membrane maintenance and cellular organisation.

Co-ordinate single-cell expression of LEE4- and LEE5-encoded proteins of Escherichia coli O157:H7

Escherichia coli O157:H7 is a zoonotic pathogen that can express a type III secretion system (TTSS) considered important for colonization and persistence in ruminants. E. coli O157:H7 strains have been shown to vary markedly in levels of protein secreted using the TTSS and this study has confirmed that a high secretion phenotype is more prevalent among isolates associated with human disease than isolates shed by healthy cattle. The variation in secretion levels is a consequence of heterogeneous expression, being dependent on the proportion of bacteria in a population that are actively engaged in protein secretion. This was demonstrated by indirect immunofluorescence and eGFP fusions that examined the expression of locus of enterocyte effacement (LEE)-encoded factors in individual bacteria. In liquid media, the expression of EspA, tir::egfp, intimin, but not map::egfp were co-ordinated in a subpopulation of bacteria. In contrast to E. coli O157:H7, expression of tir::egfp in EPEC E2348/69 was equivalent in all bacteria although the same fusion exhibited variable expression when transformed into an E. coli O157:H7 background. An E. coli O157:H7 strain deleted for the LEE demonstrated weak but variable expression of tir::egfp indicating that the elements controlling the heterogeneous expression lie outside the LEE. The research also demonstrated the rapid induction of tir::egfp and map::egfp on contact with bovine epithelial cells. This control in E. coli O157:H7 may be required to limit exposure of key surface antigens, EspA, Tir and intimin during colonization of cattle but allow their rapid production on contact with bovine gastrointestinal epithelium at the terminal rectum.