Sampling methods for NMR-based metabolomics of Staphylococcus aureus

To select an appropriate sampling method for comparison of metabolite profiles between planktonic and biofilm Staphylococcus aureus using NMR techniques, we evaluated three methods: quenching-centrifugation (QC), filtration-quenching (FQ) and filtration-quenching-lyophilization (FQL). We found differences in metabolite loss, yield, reproducibility and metabolite profile. QC caused severe metabolite leakage and possible decomposition of nucleotides. FQ achieved high yields and reproducibility, although it had the disadvantages of long filtration and rinse times before quenching. FQL resulted in a loss of a few metabolites and a lower yield due to lyophilization. Although the biomarkers discovered by each method were nearly the same and seemed insensitive to technical variances, we conclude that FQ is the most appropriate sampling method because of its high yield and reproducibility.     

Organophosphorus compound detection on a cell chip with yeast coexpressing hydrolase and eGFP

Organophosphorus compounds (OPs) such as pesticides, fungicides, and herbicides are highly toxic but are nevertheless extensively used worldwide. To detect OPs, we constructed a yeast strain that co-displays organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (EGFP) on the cell surface using a Flo1p anchor system. OP degradation releases protons and causes a change in pH. This pH change results in structural deformation of EGFP, which triggers quenching of its fluorescence, thereby making this cell useful for visual detection of OPs. Fluorescence microscopy confirmed the high-intensity fluorescence displayed by EGFP on the cell surface. The yeast strain possessed sufficient OPH hydrolytic activities for degrading OPs, as measured by incubation with 1 mM paraoxon for 24 h at 30°C. In addition, with 20 mM paraoxon at 30°C, fluorescence quenching of EGFP on the single yeast cell was observed within 40 s in a microchamber chip. These observations suggest that engineered yeast cells are suitable for simultaneous degradation and visual detection of OPs.     

Flow-regulated glucose and lipid metabolism in adipose tissue,endothelial cell and hepatocyte cultures in a modular bioreactor

Static cell culture has serious limitations in its ability to represent cellular behaviour within a live organism. In vivo, cells are constantly exposed to the flow of bodily fluids and contact with other cell types. Bioreactors provide the opportunity to study cells in an environment that more closely resembles the in vivo setting because cell cultures can be exposed to dynamic flow in contact with or in proximity to other cell types. In this study we compared the metabolic profile of a dynamic cell culture system to that of a static cell culture in three different cellular phenotypes: adipocytes, endothelial cells and hepatocytes. Albumin, glucose, free fatty acids, glycerol, and lactate were measured over 48 h. We show that all three cell types have increased glucose uptake in the presence of flow; lactate release was also significantly affected. We provide robust evidence that the presence of flow significantly modifies cellular metabolism. While flow provides a more uniform nutrient distribution and increases metabolite turnover, our results indicate that different cell types have specific metabolic responses to flow, suggesting cell-specific flow-regulated activation of metabolite signalling pathways.     

Biotemplates in the green synthesis of silver nanoparticles

This article recapitulates the scientific advancement towards the greener synthesis of silver nanoparticles. Applications of noble metals have increased throughout human civilization, and the uses for nano-sized particles are even more remarkable. “Green” nanoparticle synthesis has been achieved using environmentally acceptable solvent systems and eco-friendly reducing and capping agents. Numerous microorganisms and plant extracts have been applied to synthesize inorganic nanostructures either intracellularly or extracellularly. The use of nanoparticles derived from noble metals has spread to many areas including jewelery, medical fields, electronics, water treatment and sport utilities, thus improving the longevity and comfort in human life. The application of nanoparticles as delivery vehicles for bactericidal agents represents a new paradigm in the design of antibacterial therapeutics. Orientation, size and physical properties of nanoparticles influences the performance and reproducibility of a potential device, thus making the synthesis and assembly of shape- and size-controlled nanocrystals an essential component for any practical application. This need has motivated researchers to explore different synthesis protocols.     

Responses to cadmium intoxication in the liver of the wall lizard Podarcis sicula

This study examined the cytological and molecular effects of cadmium, a toxic heavy metal, in the liver of the Italian wall lizard Podarcis sicula. Cadmium was administered in single dose, by diet, to induce a concentration comparable with that measured in animals living in contaminated sites. For comparison, cadmium was also administered in multiple doses by food (chronic) or in a single dose intraperitoneally (i.p.); the effects were followed at regular time intervals up to 30 days post treatments. Atomic absorption spectrometry analysis demonstrated cadmium ion uptake and accumulation in the parenchyma with an estimated half-life of approximately 8 days. Cytological analyses revealed that the metal induced oedema, activated metallothionein expression in Kupffer cells and extracellular matrix production in fat storing cells. It also caused swelling and alteration in lipid and sugar metabolism in hepatocytes. In conclusion, in the wall lizard cadmium is toxic to the liver even at very low concentrations, the response is not strictly dose and time dependent and almost no recovery occurs in short (30 days) time periods.     

The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase

Hsp70-Hsp40-NEF and possibly Hsp100 are the only known molecular chaperones that can use the energy of ATP to convert stably pre-aggregated polypeptides into natively refolded proteins. However, the kinetic parameters and ATP costs have remained elusive because refolding reactions have only been successful with a molar excess of chaperones over their polypeptide substrates. Here we describe a stable, misfolded luciferase species that can be efficiently renatured by substoichiometric amounts of bacterial Hsp70-Hsp40-NEF. The reactivation rates increased with substrate concentration and followed saturation kinetics, thus allowing the determination of apparent V(max)' and K(m)' values for a chaperone-mediated renaturation reaction for the first time. Under the in vitro conditions used, one Hsp70 molecule consumed five ATPs to effectively unfold a single misfolded protein into an intermediate that, upon chaperone dissociation, spontaneously refolded to the native state, a process with an ATP cost a thousand times lower than expected for protein degradation and resynthesis.     

A minimal model of metabolism-based chemotaxis

Since the pioneering work by Julius Adler in the 1960's, bacterial chemotaxis has been predominantly studied as metabolism-independent. All available simulation models of bacterial chemotaxis endorse this assumption. Recent studies have shown, however, that many metabolism-dependent chemotactic patterns occur in bacteria. We hereby present the simplest artificial protocell model capable of performing metabolism-based chemotaxis. The model serves as a proof of concept to show how even the simplest metabolism can sustain chemotactic patterns of varying sophistication. It also reproduces a set of phenomena that have recently attracted attention on bacterial chemotaxis and provides insights about alternative mechanisms that could instantiate them. We conclude that relaxing the metabolism-independent assumption provides important theoretical advances, forces us to rethink some established pre-conceptions and may help us better understand unexplored and poorly understood aspects of bacterial chemotaxis.     

The nuclear bodies inside out: PML conquers the cytoplasm

The promyelocytic leukemia (PML) protein is the core component of nuclear substructures that host more than 70 proteins, termed nuclear domains 10 or PML-nuclear bodies. PML was first identified as the gene participating in the translocation responsible for the pathogenesis of acute promyelocytic leukemia (APL). The notion that PML is a tumor suppressor gene was soon extrapolated from leukemia to solid tumors. The last decade has radically changed the view of how this tumor suppressor is regulated, how it can be therapeutically targeted, and how it functions. Notably, one of the most recent and striking features uncovered is how PML regulates cellular homeostasis outside its original niche in the nucleus. These new findings open an exciting new area of research in extra-nuclear PML functions.     

Absence of the CD1 molecule up-regulates antitumor activity induced by CpG oligodeoxynucleotides in mice

The role of NKT cells on antitumor activity of CpG oligodeoxynucleotides (ODNs) was evaluated by peritumoral injections of CpG-ODNs in s.c. melanoma-bearing mice of strains differing in the number of NKT cells (athymic nude mice, recombination-activating gene(-/-)/transgenic V(alpha)14/Vbeta8.2 mice that generate NKT cells; J(alpha)281(-/-) mice and CD1(-/-) mice, which both have a strongly reduced number of NKT cells; and C57BL/6 wild-type mice). Tumor growth was significantly inhibited in strains enriched or depleted of NKT cells. The two murine strains having a reduced number of NKT cells differed significantly in the CpG-dependent tumor growth inhibition: in J(alpha)281(-/-) mice this inhibition was superimposable to that observed in C57BL/6 mice, while in CD1(-/-) mice the inhibition was dramatic. The increased tumor inhibition in CD1(-/-) correlated with a significantly higher ratio of IFN-gamma-IL-4 production in response to CpG as compared with C57BL/6 and J(alpha)281(-/-) mice. Experiments in which preparations of APCs and lymphocytes of the three strains were mixed showed that in the presence of APCs not expressing CD1, the production of CpG-ODN-induced type 1 cytokines was higher. Phenotype analysis of IFN-gamma- and IL-4-producing cells revealed that the differences between CD1(-/-) and C57BL/6 in the production of these two cytokines were mainly due to CD3(+) T lymphocytes. These data point to a regulatory role for the CD1 molecule in antitumor activity induced by danger signals, independently of V(alpha)14 NKT cells. The identification of a CD1-dependent suppressive subpopulation(s) might have important implications for the study of tolerance in the context of cancer, autoimmunity, and transplantation.     

Gene expression profiling identifies eleven DNA repair genes down-regulated during mouse neural crest cell migration

Neural Crest Cells (NCCs) are transient multipotent migratory cells that derive from the embryonic neural crest which is itself derived from the margin of the neural tube. DNA repair genes are expressed in the early stages of mammalian development to reduce possible replication errors and genotoxic damage. Some birth defects and cancers are due to inappropriate or defective DNA repair machinery, indicating that the proper functioning of DNA repair genes in the early stages of fetal development is essential for maintaining DNA integrity. We performed a genome-wide expression analysis combining laser capture microdissection (LCM) and high-density oligo-microarray of murine NCCs at pre-migratory embryonic days 8.5 (E8.5), and at E13.5, as well as on neural crest-derived cells from the adrenal medulla at postnatal day 90. We found 11 genes involved in DNA repair activity (response to DNA damage stimulus, DNA damage checkpoint, base-excision repair, mismatch repair), over-expressed in the early stages of mouse embryo development. Expression of these 11 genes was very low or undetectable in the differentiated adrenal medulla of the adult mouse. Amongst the 11 genes, 6 had not been previously reported as being over-expressed during mouse embryonic development. High expression of DNA repair genes in enriched NCCs during early embryonic development may contribute to maintaining DNA integrity whilst failure of some of these genes may be associated with the onset of genetic disease and cancer. Our model of enriched murine NCCs and neural crest-derived cells can be used to elucidate the key roles of genes during normal embryonic development and in cancer pathogenesis.