Detection of quorum sensing signals in the haloalkaliphilic archaeon Natronococcus occultus

Bacteria communicate at high cell density through quorum sensing, however, there are no reports about this mechanism in archaea. The archaeon Natronococcus occultus produces an extracellular protease at the end of growth. Early production of protease activity was observed when a low density culture was incubated with late exponential conditioned medium suggesting the presence of factor(s) inducing this activity. Conditioned medium and ethyl acetate extracts corresponding to the transition from exponential to stationary phase showed a positive signal in Agrobacterium biosensor. We report the detection of potential autoinducer molecules of the acylated homoserine lactone type in the archaeon N. occultus. These molecules may be responsible for the production/activation of extracellular protease.     

A method for efficient isotopic labeling of recombinant proteins

A rapid and efficient approach for preparing isotopically labeled recombinant proteins is presented. The method is demonstrated for ¹³C labeling of the C-terminal domain of angiopoietin-2, ¹⁵N labeling of ubiquitin and for ²H/¹³C/¹⁵N labeling of the Escherichia coli outer-membrane lipoprotein Lpp-56. The production method generates cell mass using unlabeled rich media followed by exchange into a small volume of labeled media at high cell density. Following a short period for growth recovery and unlabeled metabolite clearance, the cells are induced. The expression yields obtained provide a fourfold to eightfold reduction in isotope costs using simple shake flask growths.     

Glycine: An important potential component of spinal shock

Amino acid neurotransmitters (AANTs) play a major role in maintenance of muscle tone. Abnormal AANT concentrations are associated with hyper- or hypotonic states. Flaccidity from spinal shock commonly occurs after spinal cord injury (SCI) and may be associated with changes in AANT concentrations. Ischemic SCIs created in the lumbar region of rabbits by intraaortic balloon occlusion produced spastic or flaccid injuries. Microdialysis sampling of AANTs from the injured segmental structures was done 3 days after SCI. Evoked potentials were used to monitor spinal cord stability. No significant changes in AANT levels occurred in the spastic or flaccid group after 4 hour sampling. However, flaccid animals had baseline glycine levels 2–3 times higher (p<0.001) than spastic animals or controls. High concentrations of the inhibitory AANT glycine is associated with flaccidity following SCI, or spinal shock, but not spasticity. Glycinergic compounds directed toward suppression of excess muscle tone deserve further study.     

Detection of microRNA Expression in Human Peripheral Blood Microvesicles

Background

MicroRNAs (miRNA) are small non-coding RNAs that regulate translation of mRNA and protein. Loss or enhanced expression of miRNAs is associated with several diseases, including cancer. However, the identification of circulating miRNA in healthy donors is not well characterized. Microvesicles, also known as exosomes or microparticles, circulate in the peripheral blood and can stimulate cellular signaling. In this study, we hypothesized that under normal healthy conditions, microvesicles contain miRNAs, contributing to biological homeostasis.

Methodology/Principal Findings

Microvesicles were isolated from the plasma of normal healthy individuals. RNA was isolated from both the microvesicles and matched mononuclear cells and profiled for 420 known mature miRNAs by real-time PCR. Hierarchical clustering of the data sets indicated significant differences in miRNA expression between peripheral blood mononuclear cells (PBMC) and plasma microvesicles. We observed 71 miRNAs co-expressed between microvesicles and PBMC. Notably, we found 33 and 4 significantly differentially expressed miRNAs in the plasma microvesicles and mononuclear cells, respectively. Prediction of the gene targets and associated biological pathways regulated by the detected miRNAs was performed. The majority of the miRNAs expressed in the microvesicles from the blood were predicted to regulate cellular differentiation of blood cells and metabolic pathways. Interestingly, a select few miRNAs were also predicted to be important modulators of immune function.

Conclusions

This study is the first to identify and define miRNA expression in circulating plasma microvesicles of normal subjects. The data generated from this study provides a basis for future studies to determine the predictive role of peripheral blood miRNA signatures in human disease and will enable the definition of the biological processes regulated by these miRNA.     

Conformational Sampling and Binding Site Assessment of Suppression of Tumorigenicity 2 Ectodomain

Suppression of Tumorigenicity 2 (ST2), a member of the interleukin-1 receptor (IL-1R) family, activates type 2 immune responses to pathogens and tissue damage via binding to IL-33. Dysregulated responses contribute to asthma, graft-versus-host and autoinflammatory diseases and disorders. To study ST2 structure for inhibitor development, we performed the principal component (PC) analysis on the crystal structures of IL1-1R1, IL1-1R2, ST2 and the refined ST2 ectodomain (ST2^ECD) models, constructed from previously reported small-angle X-ray scattering data. The analysis facilitates mapping of the ST2^ECD conformations to PC subspace for characterizing structural changes. Extensive coverage of ST2^ECD conformations was then obtained using the accelerated molecular dynamics simulations started with the IL-33 bound ST2^ECD structure as instructed by their projected locations on the PC subspace. Cluster analysis of all conformations further determined representative conformations of ST2^ECD ensemble in solution. Alignment of the representative conformations with the ST2/IL-33 structure showed that the D3 domain of ST2^ECD (containing D1-D3 domains) in most conformations exhibits no clashes with IL-33 in the crystal structure. Our experimental binding data informed that the D1-D2 domain of ST2^ECD contributes predominantly to the interaction between ST2^ECD and IL-33 underscoring the importance of the D1-D2 domain in binding. Computational binding site assessment revealed one third of the total detected binding sites in the representative conformations may be suitable for binding to potent small molecules. Locations of these sites include the D1-D2 domain ST2^ECD and modulation sites conformed to ST2^ECD conformations. Our study provides structural models and analyses of ST2^ECD that could be useful for inhibitor discovery.     

Pancreatic beta-cell lipoprotein lipase independently regulates islet glucose metabolism and normal insulin secretion

Lipid and glucose metabolism are adversely affected by diabetes, a disease characterized by pancreatic beta-cell dysfunction. To clarify the role of lipids in insulin secretion, we generated mice with beta-cell-specific overexpression (betaLPL-TG) or inactivation (betaLPL-KO) of lipoprotein lipase (LPL), a physiologic provider of fatty acids. LPL enzyme activity and triglyceride content were increased in betaLPL-TG islets; decreased LPL enzyme activity in betaLPL-KO islets did not affect islet triglyceride content. Surprisingly, both betaLPL-TG and betaLPL-KO mice were strikingly hyperglycemic during glucose tolerance testing. Impaired glucose tolerance in betaLPL-KO mice was present at one month of age, whereas betaLPL-TG mice did not develop defective glucose homeostasis until approximately five months of age. Glucose-simulated insulin secretion was impaired in islets isolated from both mouse models. Glucose oxidation, critical for ATP production and triggering of insulin secretion mediated by the ATP-sensitive potassium (KATP) channel, was decreased in betaLPL-TG islets but increased in betaLPL-KO islets. Islet ATP content was not decreased in either model. Insulin secretion was defective in both betaLPL-TG and betaLPL-KO islets under conditions causing calcium-dependent insulin secretion independent of the KATP channel. These results show that beta-cell-derived LPL has two physiologically relevant effects in islets, the inverse regulation of glucose metabolism and the independent mediation of insulin secretion through effects distal to membrane depolarization.     

NK cell activation by dendritic cell vaccine: a mechanism of action for clinical activity

Recent reports revealed that dendritic cell (DC)–natural killer (NK) cell interaction plays an important role in tumor immunity, but few DC vaccine studies have attempted to evaluate the non-specific, yet potentially clinically relevant, NK response to immunization. In this study, we first analyzed in vitro activation of NK cells by DCs similar to those used in clinical trials. Subsequently, NK cell responses were analyzed in a phase I clinical trial of a vaccine consisting of autologous DCs loaded with a fowlpox vector encoding CEA. The data were compared with the clinical outcome of the patients. DC enhances NK activity in vitro, partly by sustaining NK cell survival and by enhancing the expression of NK-activating receptors, including NKp46 and NKG2D. Among nine patients in our clinical trial, NK cytolytic activity increased in four (range 2.5–5 times greater lytic activity) including three who had increased NK cell frequency, was stable in two and decreased in three. NKp46 and NKG2D expression showed a good correlation with the patients’ NK activity. When patients were grouped by clinical activity (stable disease/no evidence of disease (stable/NE, n=5) vs progressive disease (N=4) at 3 months), the majority in the stable/NE group had increases in NK activity (P=0.016). Anti-CEA T cell response was enhanced in all the nine patients analyzed, but was not significantly different between the two groups (P=0.14). Thus, NK responses following DC vaccination may correlate more closely with clinical outcome than do T cell responses. Monitoring of NK response during vaccine studies should be routinely performed.