RNA–protein binding kinetics in an automated microfluidic reactor

Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA–protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic ‘Riboreactor’ has been designed and constructed to facilitate the study of kinetics of RNA–protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA–protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome

Preparation of Isotopically Labeled Ribonucleotides for Multidimensional NMR Spectroscopy of RNA

A general method for large scale preparation of uniformly isotopically labeled ribonucleotides and RNAs is described. Bacteria are grown on isotopic growth medium, and their nucleic acids are harvested and degraded to mononucleotides. These are enzymatically converted into ribonucleoside triphosphates, which are used in transcription reactions in vitro to prepare RNAs for NMR studies. For 15N-labeling, E.coli is grown on15N-ammonium sulfate, whereas for 13C-labeling, Methylophilus methylotrophus is grown on 13C-methanol, which is more economical than 13C-glucose. To demonstrate the feasibility and utility of this method, uniformly 13C-labeled ribonucleotides were used to synthesize a 31 nucleotide HIV TAR RNA that was analyzed by 3D-NMR. This method should find widespread use in the structural analysis of RNA by NMR

Oilheat research agenda: A ten year blueprint for residential oilheat research and development in the Twenty-First Century

This report summarizes a joint research agenda planned for the US Department of Energy and the National Oilheat Research Alliance (NORA) under a cooperative effort between the Federal government and the private sector industries involved in oilheat marketing. The objective of the oilheat research program is to develop the technical basis for improved equipment designs and operating strategies based on an enhanced understanding of oil-burning fundamentals, heat transfer, and associated environmental factors. The program will continue to provide the oil-fueled heating equipment industry with the basis for developing a new, modern generation of equipment and provide the oil marketers, equipment installers, and consumers with improved knowledge of how best to install, maintain, and operate such equipment for maximum performance and minimum fuel use and environmental impact

Operando and High-throughput multicscale-tomography

We report about multiscale tomography with high throughput at the Diamond beamline I13L. The beamline has the purpose of multi-scale and operando imaging and consists of two independent branchlines operating in real and reciprocal space. The imaging branch -called Diamond-Manchester branchline- hosts micro-tomography, grating interferometry and a full-field microscope. For rapid recording a broad spectrum of the undulator radiation is used either with band-passing the light with a combination of a filter and a deflecting mirror or using a multilayer monochromator. For all the methods similar recording times can be achieved, with typical scanning times of some minutes and covering the resolution range from microns to the 100nm range. Most recently a robot arm has been installed to increase the throughput to 300 samples per day. The system is now implemented for user operation in remote operation mode for the micro-tomography setup and can be expanded to the two other experiments. The instrumental capabilities are applied on various topics such as the study of biodiversity of insects or the structural variations of electrode materials in batteries. Fast recording with dedicated sample environments (not using the sample changing robot) enables operando studies in many areas, the charging/discharging cycles on batteries, the degradation of teeth enamel under various conditions or loading brine sandstone mixtures with CO2, to name some examples. For imaging with highest spatial resolution we managed to improve significantly the recording speed of ptycho-tomography, which is now in the order of hours and will be reduced further. We demonstrated in the past 2-D recording with 10kHz and expand the instrumental capability with specific hardware dependent triggering and scanning schemes. We expand the research program for multi-scale imaging across both branchlines (imaging and coherence branchlines) with first studies such as batteries, brain research, concrete

Porosity and Structure of Hierarchically Porous Ni/Al2O3 Catalysts for CO2 Methanation

CO2 methanation is often performed on Ni/Al2O3 catalysts, which can suffer from mass transport limitations and, therefore, decreased efficiency. Here we show the application of a hierarchically porous Ni/Al2O3 catalyst for methanation of CO2. The material has a well-defined and connected meso- and macropore structure with a total porosity of 78%. The pore structure was thoroughly studied with conventional methods, i.e., N2 sorption, Hg porosimetry, and He pycnometry, and advanced imaging techniques, i.e., electron tomography and ptychographic X-ray computed tomography. Tomography can quantify the pore system in a manner that is not possible using conventional porosimetry. Macrokinetic simulations were performed based on the measures obtained by porosity analysis. These show the potential benefit of enhanced mass-transfer properties of the hierarchical pore system compared to a pure mesoporous catalyst at industrially relevant conditions. Besides the investigation of the pore system, the catalyst was studied by Rietveld refinement, diffuse reflectance ultraviolet-visible (DRUV/vis) spectroscopy, and H2-temperature programmed reduction (TPR), showing a high reduction temperature required for activation due to structural incorporation of Ni into the transition alumina. The reduced hierarchically porous Ni/Al2O3 catalyst is highly active in CO2 methanation, showing comparable conversion and selectivity for CH4 to an industrial reference catalyst

Factors associated with severity of hepatic fibrosis in people with chronic hepatitis C infection

The document attached has been archived with permission from the editor of the Medical Journal of Australia. An external link to the publisher’s copy is included.OBJECTIVE: To determine factors associated with hepatic fibrosis development in people with chronic hepatitis C virus (HCV) infection. METHODS: As a requirement for access to interferon therapy through the S100 scheme in Australia, individual pretreatment demographic and clinical information was collected on 2986 patients from 61 hospital-based liver clinics from 1 October 1994 through 31 December 1996. Patients with both a hepatic fibrosis score and an estimated duration of HCV infection (910) were divided into 540 with no or minimal hepatic fibrosis (stage 0–1) and 370 with moderate to severe hepatic fibrosis (stage 2–3). Seven factors were examined: age at HCV infection, sex, ethnicity, source of infection, duration of infection, alcohol intake, and mean ALT level. A further analysis was performed for all 1135 patients with a hepatic fibrosis score disregarding age at and duration of HCV infection. RESULTS: In multivariate analysis, four factors were significantly associated with moderate to severe hepatic fibrosis: age at infection (OR, 2.33 for age 31–40 years, 5.27 for age > 40 years, and 0.20 for age 30 years, compared with 3 times, compared with 1.5–2 times the upper limit of normal). In the analysis disregarding age at HCV infection and duration of HCV infection, older age was strongly associated with moderate to severe hepatic fibrosis (OR, 2.32 for age 36–40 years, 2.46 for age 41–50 years, 7.87 for age 51–60 years, and 7.15 for age > 60 years, compared with 16–30 years). There was no association in either analysis with sex or source of HCV infection. CONCLUSION: These factors may assist in targeting patients for both liver biopsy-based investigation and therapeutic intervention.Mark Danta, Gregory J Dore, Lisa Hennessy, Yueming Li, Chris R Vickers, Hugh Harley, Meng Ngu, William Reed, Paul V Desmond, William Sievert, Geoff C Farrell, John M Kaldor and Robert G Bate

Optical autocollimator for vibration measurements at Diamond I13 beamline

I13 is a 250 m long hard X ray beamline for imaging and coherence experiments at the Diamond Light Source. The beamline comprises two independent experimental branches one for imaging in direct space using X ray microscopy and one for imaging in reciprocal space using coherent imaging techniques. The mechanical stability is very important for implementation of increased capabilities at latest generation of long beamlines. Therefore, the beam stability monitoring is essential part of the day to day operation of the beamlines as well as for analysis of mechanical instability sources for the Diamond II upgrade. In this paper we present the setup developed to measure mechanical stability of beamline based on optical autocollimato