Preparation of Functional, Fluorescently Labeled mRNA Capped with Anthraniloyl-m(7)GpppG.

Fluorescent mRNA molecules offer a wide range of applications for studying capping/decapping reactions, translation, and other biophysical studies. Furthermore, fluorescent tags prove invaluable for tracking RNA molecules in cells. Here, we describe an efficient synthesis of a fluorescent cap analog, anthranioyl-GTP, its purification, and in vitro cap labeling of transcribed mRNA catalyzed by the recombinant vaccinia capping enzyme to produce anthranioyl-m(7)GpppG-capped RNA.

Specification of matrix cleanup goals in fractured porous media.

Semianalytical transient solutions have been developed to evaluate what level of fractured porous media (e.g., bedrock or clay) matrix cleanup must be achieved in order to achieve compliance of fracture pore water concentrations within a specified time at specified locations of interest. The developed mathematical solutions account for forward and backward diffusion in a fractured porous medium where the initial condition comprises a spatially uniform, nonzero matrix concentration throughout the domain. Illustrative simulations incorporating the properties of mudstone fractured bedrock demonstrate that the time required to reach a desired fracture pore water concentration is a function of the distance between the point of compliance and the upgradient face of the domain where clean groundwater is inflowing. Shorter distances correspond to reduced times required to reach compliance, implying that shorter treatment zones will respond more favorably to remediation than longer treatment zones in which back-diffusion dominates the fracture pore water response. For a specified matrix cleanup goal, compliance of fracture pore water concentrations will be reached sooner for decreased fracture spacing, increased fracture aperture, higher matrix fraction organic carbon, lower matrix porosity, shorter aqueous phase decay half-life, and a higher hydraulic gradient. The parameters dominating the response of the system can be measured using standard field and laboratory techniques.

The prevalence of methicillin resistant organisms among pacemaker and defibrillator implant recipients.

INTRODUCTION: Pacemaker and defibrillator infections are an uncommon, but catastrophic complication of device implantation. The present study examined the prevalence of device-related infections, the patterns of antibiotic resistance, and the presence of methicillin resistant staphylococcus aureus (MRSA) nares colonization in device implant recipients. METHODS: Two protocols were employed using a retrospective and a prospective analysis. A retrospective chart review of 218 patients with suspected device infection from 1/2000 to 1/2011 was performed. Demographics, infection rates, and patterns of antibiotic resistance were compared. The prospective analysis enrolled one hundred eighty two patients undergoing device implantations or generator replacements. The nares were swabbed and analyzed for the presence of staphylococcus aureus, and tested for methicillin sensitivity. RESULTS: Over a period of ten years, 12,771 device implants/generator changes/system revisions were performed, with an infection rate of 1.2%. Methicillin resistance (MR) was identified in 98/218 (44.9%) of patients. Those with MR infection had more diabetes and cardiomyopathy. There was no significant increase in methicillin resistance over time (p=0.30). Our prospective analysis included 110 men. A total of 32 patients (17.6%) had positive cultures for SA: 6.6% with MRSA. Patients positive for MRSA nares colonization had a statistically significant greater length of hospital stay 8.5 days (mean) versus 4.4 days (P=0.049). CONCLUSIONS: Methicillin resistant organisms appear to be emerging and persistent pathogens in device implants. The screening of MRSA colonization may identify new populations at risk. Further studies and analysis are needed to determine the cost effectiveness of a screening protocol.

ATF3 protects against atherosclerosis by suppressing 25-hydroxycholesterol-induced lipid body formation.

Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipid-loaded macrophages in the arterial wall. We demonstrate that macrophage lipid body formation can be induced by modified lipoproteins or by inflammatory Toll-like receptor agonists. We used an unbiased approach to study the overlap in these pathways to identify regulators that control foam cell formation and atherogenesis. An analysis method integrating epigenomic and transcriptomic datasets with a transcription factor (TF) binding site prediction algorithm suggested that the TF ATF3 may regulate macrophage foam cell formation. Indeed, we found that deletion of this TF results in increased lipid body accumulation, and that ATF3 directly regulates transcription of the gene encoding cholesterol 25-hydroxylase. We further showed that production of 25-hydroxycholesterol (25-HC) promotes macrophage foam cell formation. Finally, deletion of ATF3 in Apoe(-/-) mice led to in vivo increases in foam cell formation, aortic 25-HC levels, and disease progression. These results define a previously unknown role for ATF3 in controlling macrophage lipid metabolism and demonstrate that ATF3 is a key intersection point for lipid metabolic and inflammatory pathways in these cells.

A microfluidic device for multiplexed protein detection in nano-liter volumes.

We describe a microfluidic immunoassay device that permits sensitive and quantitative multiplexed protein measurements on nano-liter-scale samples. The device exploits the combined power of integrated microfluidics and optically encoded microspheres to create an array of approximately 100-microm(2) sensors functionalized with capture antibodies directed against distinct targets. This strategy overcomes the need for performing biochemical coupling of affinity reagents to the device substrate, permits multiple proteins to be detected in a nano-liter-scale sample, is scalable to large numbers of samples, and has the required sensitivity to measure the abundance of proteins derived from single mammalian cells. The sensitivity of the device is sufficient to detect 1000 copies of tumor necrosis factor (TNF) in a volume of 4.7nl.

Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron.

Although iron is required to sustain life, its free concentration and metabolism have to be tightly regulated. This is achieved through a variety of iron-binding proteins including transferrin and ferritin. During infection, bacteria acquire much of their iron from the host by synthesizing siderophores that scavenge iron and transport it into the pathogen. We recently demonstrated that enterochelin, a bacterial catecholate siderophore, binds to the host protein lipocalin 2 (ref. 5). Here, we show that this event is pivotal in the innate immune response to bacterial infection. Upon encountering invading bacteria the Toll-like receptors on immune cells stimulate the transcription, translation and secretion of lipocalin 2; secreted lipocalin 2 then limits bacterial growth by sequestrating the iron-laden siderophore. Our finding represents a new component of the innate immune system and the acute phase response to infection.