A novel flow cytometry-based assay for the quantification of Staphylococcus aureus adhesion to and invasion of eukaryotic cells

Flow cytometry is a powerful tool for analyzing the adhesion to and invasion of Staphylococcus aureus (S. aureus) to eukaryotic cells. Established techniques have used bacteria that have been genetically modified to express fluorescent proteins or directly labeled with fluorochromes prior to infection. Such approaches are appropriate in most cases; however, the use of genetically or chemically altered bacteria could introduce a bias when measuring fine differences in adhesion and invasiveness. Here, we describe a combined flow cytometry-based invasion and adhesion assay that does not require the processing of bacteria prior to internalization. This method was performed on osteoblastic MG-63 cells infected with S. aureus reference strain 8325-4 and its invasion-deficient isogenic mutant, which carries deletions in the genes encoding fibronectin-binding proteins A and B. The data from this assay were compared to those obtained using the standard gentamicin protection assay. The results obtained by the two methods were consistent. Moreover, quantification of internalized bacteria was more reproducible using the flow cytometry-based assay than the gentamicin protection assay, which allowed for the simultaneous quantification of host cell adhesion and invasion.     

Effects of binding of S-peptide and 2′-cytidine monophosphate on hydrogen exchange from the S-protein component of ribonuclease S: The amide protons of serine 123 and valine 124

The hydrogen exchange rates of amide protons from many chain segments in S-protein, previously shown to be dramatically affected on association with S-peptide, are essentially unchanged, over a period of 150 hours at least, on binding the inhibitor 2′CMP to RNAase S. An exception was the C-terminal tetrapeptide (121–124), which is close to, but not in actual contact with, substrates or inhibitors bound at the active site of the enzyme. Due to the effect of the intrinsic exchange rates within the fragment during analysis only two protons are observed, those of serine 123 and valine 124. The intrinsic rates of these two sites are also sufficiently different that their rates of exchange in the intact protein can be separately monitored. Both rates decrease on binding S-peptide and decrease by an additional factor of ten on binding 2′CMP. The amide of Vall24 is hydrogen bonded as part of a β-sheet region in the native structure. The amide of Ser123 is not hydrogen-bonded, is directed towards the solvent, but is not accessible to it in the time average structure of the intact enzyme. The main-chain motion required to permit exchange is such that both amides are affected in the same way in spite of their different environments.     

A cytoskeletal demolition worker: myosin II acts as an actin depolymerization agent

Myosin II motors play several important roles in a variety of cellular processes, some of which involve active assembly/disassembly of cytoskeletal substructures. Myosin II motors have been shown to function in actin bundle turnover in neuronal growth cones and in the recycling of actin filaments during cytokinesis. Close examination had shown an intimate relationship between myosin II motor adenosine triphosphatase activity and actin turnover rate. However, the direct implication of myosin II in actin turnover is still not understood. Herein, we show, using high-resolution cryo-transmission electron microscopy, that myosin II motors control the turnover of actin bundles in a concentration-dependent manner in vitro. We demonstrate that disassembly of actin bundles occurs through two main stages: the first stage involves unbundling into individual filaments, and the second involves their subsequent depolymerization. These evidence suggest that, in addition to their “classical” contractile abilities, myosin II motors may be directly implicated in active actin depolymerization. We believe that myosin II motors may function similarly in vivo (e.g., in the disassembly of the contractile ring by fine tuning the local concentration/activity of myosin II motors).     

Minireview: Cardiolipins and Mitochondrial Proton-Selective Leakage

The proton-selective leak (State 4 respiratory rate) but not , in mitochondria from thyroid-sensitive tissues, responds to in vivo stimuli in unique correlation with changes in cardiolipins, saturated and mono-unsaturated (extended) fatty acyl contents, cardiolipins/phospholipids ratios, and/or membrane outer-sidedness. Liver mitochondrial State 4 respiration, basal in fasted rats, contributes little to resting metabolic rate in fed rats, where State 3 depresses . In a proposed model, an essential inner-membrane outer-surface proton antenna collects protons and donates them, via a water-shuttle, to transmembrane porters: transient water-molecule-chains between extended phospholipid acyls; protonophores, and uncoupling proteins. Only cardiolipin microdomains can donate, from an anomalously-dissociating phosphate group in each headgroup; unadapted cardiolipins have few conducting water chains. Thyroid states regulate each cardiolipin property, and are permissive, via the proton antenna, for proton leaks, including those through adapted and possibly constitutive BAT and ectopic uncoupling proteins. Slow leakage in liposomes may reflect insufficient cardiolipin proton antennas.     

Syntrophin isoforms play specific functional roles in the α1D-adrenergic receptor/DAPC signalosome

α_1D-Adrenergic receptors, key regulators of cardiovascular system function, are organized as a multi-protein complex in the plasma membrane. Using a Type-I PDZ-binding motif in their distal C-terminal domain, α_1D-ARs associate with syntrophins and dystrophin-associated protein complex (DAPC) members utrophin, dystrobrevin and α-catulin. Three of the five syntrophin isoforms (α, β₁ and β₂) interact with α_1D-ARs and our previous studies suggest multiple isoforms are required for proper α_1D-AR function in vivo. This study determined the contribution of each specific syntrophin isoform to α_1D-AR function. Radioligand binding experiments reveal α-syntrophin enhances α_1D-AR binding site density, while phosphoinositol and ERK1/2 signaling assays indicate β₂-syntrophin augments full and partial agonist efficacy for coupling to downstream signaling mechanisms. The results of this study provide clear evidence that the cytosolic components within the α_1D-AR/DAPC signalosome significantly alter the pharmacological properties of α₁-AR ligands in vitro.     

Detection of synthetic glucocorticoid residues in cattle tissue and hair samples after a single dose administration using LC-MS/MS

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the detection of several synthetic glucocorticoids in kidney, muscle and hair samples of cattle after a single intramuscular injection is described. After a dichloromethane wash of the hair samples, analytes were released from the hair matrix by enzymatic digestion. Muscle samples were also digested enzymatically using proteinase, while kidney samples were deconjugated by Helix pomatia juice. These preliminary steps were followed by a methanol extraction and a solid phase extraction (SPE) clean up step for all matrices. Chromatographic separation was achieved on a Hypersil Hypercarb column and MS/MS data were obtained in the multiple reaction monitoring mode using negative electrospray ionization. The developed protocols were evaluated by assessing residue concentrations in muscle, kidney and hair samples of thirteen calves, treated with a particular intramuscular injection of glucocorticoid. The lowest residue levels were found in muscle samples (approximately 5% of the residue levels in kidney), while high residue levels were obtained in hair samples. Hair is an interesting matrix since the sampling is non-invasive and the drugs may stay incorporated for a longer period of time.     

Origin matters: Using a local reference genome improves measures in population genomics

Genome sequencing enables answering fundamental questions about the genetic basis of adaptation, population structure and epigenetic mechanisms. Yet, we usually need a suitable reference genome for mapping population-level resequencing data. In some model systems, multiple reference genomes are available, giving the challenging task of determining which reference genome best suits the data. Here, we compared the use of two different reference genomes for the three-spined stickleback (Gasterosteus aculeatus), one novel genome derived from a European gynogenetic individual and the published reference genome of a North American individual. Specifically, we investigated the impact of using a local reference versus one generated from a distinct lineage on several common population genomics analyses. Through mapping genome resequencing data of 60 sticklebacks from across Europe and North America, we demonstrate that genetic distance among samples and the reference genomes impacts downstream analyses. Using a local reference genome increased mapping efficiency and genotyping accuracy, effectively retaining more and better data. Despite comparable distributions of the metrics generated across the genome using SNP data (i.e. π, Tajima's D and F_ST), window-based statistics using different references resulted in different outlier genes and enriched gene functions. A marker-based analysis of DNA methylation distributions had a comparably high overlap in outlier genes and functions, yet with distinct differences depending on the reference genome. Overall, our results highlight how using a local reference genome decreases reference bias to increase confidence in downstream analyses of the data. Such results have significant implications in all reference-genome-based population genomic analyses.     

Age-specific reproductive success in a long-lived bird: do older parents resist stress better?

In many vertebrates, reproductive performance increases with advancing age but mechanisms involved in such a pattern remain poorly studied. One potential mechanism may be the hormonal stress response, which shifts energy investment away from reproduction and redirects it towards survival. In birds, this stress response is achieved through a release of corticosterone and is also accompanied by a decrease in circulating prolactin, a hormone involved widely in regulating parental cares. It has been predicted that, when the value of the current reproduction is high relative to the value of future reproduction and survival, as it is expected to be in older adults, the stress response should be attenuated to ensure that reproduction is not inhibited. We tested this hypothesis by measuring the corticosterone and prolactin responses of known-age (8-36 years old) incubating snow petrels (Pagadroma nivea) to a standardized capture/handling stress protocol. We also investigated whether an attenuation of the stress responses will correlate with a lower occurrence of egg neglect, a frequently observed behaviour in snow petrels. The probability of successfully fledging a chick increased from 6 years to 12 years before stabilizing after 12 years of age. Corticosterone response to stress was unaffected by age. Prolactin response to stress, however, was influenced clearly by age: in both sexes older breeders had higher stress-induced prolactin levels than younger ones. This was due to an increasing attenuation of the prolactin response to stress with advancing age in females, and in males this was due to a probably higher intrinsic capacity of older males to secrete prolactin. Moreover, higher stress-induced prolactin levels were correlated with a lower probability of neglecting the egg. In young breeders, the combination of a robust corticosterone increase with a lower ability to maintain prolactin secretion during acute stress is probably one of the functional causes of their lower incubation commitment. We suggest that the ability to maintain a threshold level of prolactin during a stressful situation may be an important physiological mechanism involved in the improvement of reproductive performance with advancing age in long-lived birds.