Sparse interleaved sampling for high resolution focal construct geometry X-ray tomography.

We demonstrate interleaved sampling by multiplexing conical subshells within the tomosynthesis and raster scanning a phantom through a 150 kV shell X-ray beam. Each view comprises pixels sampled on a regular 1 mm grid, which is then upscaled by padding with null pixels before tomosynthesis. We show that upscaled views comprising 1% sample pixels and 99% null pixels increase the contrast transfer function (CTF) computed from constructed optical sections from approximately 0.6 line pairs/mm to 3 line pairs/mm. The driver of our method is to complement work concerning the application of conical shell beams to the measurement of diffracted photons for materials identification. Our approach is relevant to time-critical, and dose-sensitive analytical scanning applications in security screening, process control and medical imaging.

Highly efficient CRISPR-Cas9-mediated editing identifies novel mechanosensitive microRNA-140 targets in primary human articular chondrocytes.

OBJECTIVE: MicroRNA 140 (miR-140) is a chondrocyte-specific endogenous gene regulator implicated in osteoarthritis (OA). As mechanical injury is a primary aetiological factor in OA, we investigated miR-140-dependent mechanosensitive gene regulation using a novel CRISPR-Cas9 methodology in primary human chondrocytes. METHOD: Primary (passage 1/2) human OA chondrocytes were isolated from arthroplasty samples (six donors) and transfected with ribonuclear protein complexes or plasmids using single guide RNAs (sgRNAs) targeting miR-140, in combination with Cas9 endonuclease. Combinations of sgRNAs and single/double transfections were tested. Gene editing was measured by T7 endonuclease 1 (T7E1) assay. miRNA levels were confirmed by qPCR in chondrocytes and in wild type murine femoral head cartilage after acute injury. Predicted close match off-targets were examined. Mechanosensitive miR-140 target validation was assessed in 42 injury-associated genes using TaqMan Microfluidic cards in targeted and donor-matched control chondrocytes. Identified targets were examined in RNAseq data from costal chondrocytes from miR-140-/- mice. RESULTS: High efficiency gene editing of miR-140 (90-98%) was obtained when two sgRNAs were combined with double RNP-mediated CRISPR-Cas9 transfection. miR-140 levels fell rapidly after femoral cartilage injury. Of the top eight miR-140 gene targets identified (P < 0.01), we validated three previously identified ones (septin 2, bone morphogenetic protein 2 and fibroblast growth factor 2). Novel targets included Agrin, a newly recognised pro-regenerative cartilage agent, and proteins associated with retinoic acid signalling and the primary cilium. CONCLUSION: We describe a highly efficient CRISPR-Cas9-mediated strategy for gene editing in primary human chondrocytes and identify several novel mechanosensitive miR-140 targets of disease relevance.

Combined X-ray diffraction and absorption tomography using a conical shell beam.

We combine diffraction and absorption tomography by raster scanning samples through a hollow cone of pseudo monochromatic X-rays with a mean energy of 58.4 keV. A single image intensifier takes 90x90 (x,y) snapshots during the scan. We demonstrate a proof-of-principle of our technique using a heterogeneous three-dimensional (x,y,z) phantom (90x90x170 mm3) comprised of different material phases, i.e., copper and sodium chlorate. Each snapshot enables the simultaneous measurement of absorption contrast and diffracted flux. The axial resolution was ~1 mm along the (x,y) orthogonal scan directions and ~7 mm along the z-axis. The tomosynthesis of diffracted flux measurements enable the calculation of d-spacing values with ~0.1 Å full width at half maximum (FWHM) at ~2 Å. Thus the identified materials may be color-coded in the absorption optical sections. Characterization of specific material phases is of particular interest in security screening for the identification of narcotics and a wide range of homemade explosives concealed within complex "everyday objects." Other potential application areas include process control and biological imaging.

Confocal energy-dispersive X-ray diffraction tomography employing a conical shell beam.

We introduce a new high-energy X-ray diffraction tomography technique for volumetric materials characterization. In this method, a conical shell beam is raster scanned through the samples. A central aperture optically couples the diffracted flux from the samples onto a pixelated energy-resolving detector. Snapshot measurements taken during the scan enable the construction of depth-resolved dark-field section images. The calculation of d-spacing values enables the mapping of material phase in a volumetric image. We demonstrate our technique using five ~15 mm thick, axially separated samples placed within a polymer tray of the type used routinely in airport security stations. Our method has broad analytical utility due to scalability in both scan size and X-ray energy. Additional application areas include medical diagnostics, materials science, and process control.

X-ray absorption tomography employing a conical shell beam.

We demonstrate depth-resolved absorption imaging by scanning an object through a conical shell of X-rays. We measure ring shaped projections and apply tomosynthesis to extract optical sections at different axial focal plane positions. Three-dimensional objects have been imaged to validate our theoretical treatment. The novel principle of our method is scalable with respect to both scan size and X-ray energy. A driver for this work is to complement previously reported methods concerning the measurement of diffracted X-rays for structural analysis. The prospect of employing conical shell beams to combine both absorption and diffraction modalities would provide enhanced analytical utility and has many potential applications in security screening, process control and diagnostic imaging.

Differences in the properties of mammalian ribonucleotide reductase toward its substrates.

These studies, using three different reagents, show that the substrate properties of ribonucleotide reductase are specific but can be variable depending upon the nature of the interaction of the reagent with the holoenzyme or the individual subunit. Etheno-CDP, which acts as a competitive inhibitor with respect to CDP, interacts with the active site of the holoenzyme. This interaction was the result of rather tight structural requirements as epsilon-ADP did not result in a similar level of inhibition of either CDP or ADP reductase activities. The YL 1/2 antibody which binds very tightly to the NHI subunit has a much greater effect on CDP reductase activity than ADP reductase activity. The nonapeptide that corresponds to the C-terminus amino acid sequence of the NHI subunit and which binds to the EB subunit and aborts the formation of the NHI-EB active complex has a greater effect on ADP reductase activity than on CDP reductase activity. The use of reagents such as these can be helpful in dissecting the subtle but important differences in the substrate properties of mammalian ribonucleotide reductase.

Substituted 2-acylpyridine-alpha-(N)-hetarylhydrazones as inhibitors of ribonucleotide reductase activity and L1210 cell growth.

A series of substituted 2-acylpyridine-alpha-(N)-hetarylhydrazones was prepared and studied for their effects on mammalian ribonucleotide reductase activity using a highly purified enzyme preparation from Ehrlich tumor cells and on mouse leukemia L1210 cell growth in culture. Pyridine-2-aldehyde-2-pyridylhydrazone (PH 22), ethyl-2-pyridylketone-I-phthalazinylhydrazone (PH 22-25) and pyridine-2-aldehyde-2'-quinolylhydrazone (PQ 22) inhibited purified ribonucleotide reductase activity and inhibited L1210 cell growth in culture. PH 22-25 inhibited [3H]thymidine incorporation into DNA and inhibited ribonucleotide reductase activity in situ (as measured bvy [14C]cytidine metabolism and as a result inhibited DNA synthesis. There was no effect on RNA synthesis. These data indicate that these substituted hydrazones are potent inhibitors of tumor cell growth through the inhibition of ribonucleotide reductase.

5-hexyl-2'-deoxyuridine inhibition of nucleoside transport in L1210 cells.

Previous studies have shown that 5-hexyl-2'-deoxyuridine (HdUrd) blocked the cytotoxic effects of 5-fluorodeoxyuridine and deoxyadenosine in L1210 cells. HdUrd had no effect in preventing the inhibitory effects of 5-fluorouracil. These data suggested that HdUrd was an inhibitor of nucleoside transport in L1210 cells (Cory, J. G.; Halley, M. C.; Janey, A.; Lapis, K. Cancer Res. 50:4552-4556; 1990). Studies have now been carried out which show that HdUrd inhibits nucleoside transport as measured by [3H]uridine or [3H]formycin B transport into L1210 cells in culture. The IC50 for HdUrd inhibition of total [3H]uridine uptake was approximately 20 microM in wild-type L1210 cells. Since wild-type L1210 cells have three distinct nucleoside transporters, the effect of HdUrd on each transporter was examined using the non-metabolized nucleoside analog, formycin B. The nitrobenzylmercaptopurine riboside (NBMPR)-sensitive transporter, es, was most sensitive to HdUrd with an IC50 of 1.0 +/- 0.1 microM; the NBMPR-insensitive transporter, ei, was much less sensitive to HdUrd with an IC50 of 32 +/- 2 microM; the sodium ion-dependent transporter, cif, was the least sensitive transporter to HdUrd with an IC50 of 130 +/- 5 microM. These data support the concept that HdUrd, a relatively non-cytotoxic agent, could be useful in increasing the potency of antitumor inhibitors directed at the de novo pathways for nucleotide synthesis through the blockage of the salvage pathways for nucleosides.

Immunolocalization of glucose transporter GLUT4 within human skeletal muscle.

To investigate the cellular and subcellular distribution of glucose transporters in skeletal muscle, the glucose transporter isoform GLUT4 was localized in human muscle by electron microscopy via immunogold labeling with monoclonal (1F8) or COOH-terminal peptide polyclonal (ECU4) antibody and in isolated rat membranes by Western blot. There was no labeling of GLUT4 in endothelial cells of the capillaries. There also was no labeling of GLUT4 on the surface plasma membrane (sarcolemma) under either basal or insulin-stimulated conditions. Specific labeling for GLUT4 was clearly observed in two compartments: within the triad (on terminal cisternae and transverse tubules) and on an intracellular compartment, possibly sarcoplasmic tubules. Isolated triad membranes from rat muscle also contained substantial quantities of GLUT4 transporter, but there was no detectable GLUT4 protein in isolated sarcolemmal membranes. These data suggest a possible mechanism that involves glucose transport across the muscle cell at the transverse tubule membrane, not the sarcolemma.

Insulin resistance induced by high-fat feeding is only partially reversed by exercise training.

Diets high in saturated fat and simple carbohydrate result in an insulin-resistant state, while training increases insulin sensitivity. Insulin resistance was induced by feeding a high-fat, high-sucrose (HFS) diet to 4-week-old female Sprague-Dawley rats. A control diet (low-fat, complex-carbohydrate) was fed to another group for comparison. During the 4-week dietary treatment, half of each group was trained by treadmill running (2 h day-1, 6 days week-1m 30 m min-1, 0% grade). At the end of this 4-week experimental period, hindquarter perfusions were performed at either basal (0) or maximal (100 nM) insulin concentrations to determine glucose uptake, glycogen synthesis, total glycogen content and the activity of several enzymes. Insulin (100 nM) significantly increased glucose uptake and glycogen synthesis in all four groups (CON-UN, CON-TR, HFS-UN, HFS-TR, where CON, UN and TR refer to control, untrained and trained respectively). HFS feeding significantly decreased (P less than 0.002) glucose uptake (mumol g-1 h-1) with maximal insulin stimulation, while training significantly increased uptake (P less than 0.01) at both insulin concentrations. Glycogen synthesis was also increased by training (P less than 0.05) at both insulin concentrations, but accounted for only 25-28% of the glucose uptake. Although training improved the insulin resistance caused by the HFS diet, glucose uptake in the HFS-TR group was still significantly lower than the CON-TR group. Changes in glycogen synthesis are not great enough to account for the decrease or increase in glucose uptake found in the HFS-fed or trained animals.

Effect of a high-fat-sucrose diet on in vivo insulin receptor kinase activation.

Insulin-stimulated glucose uptake into muscle is depressed by high-fat-sucrose (HFS) feeding of rats. To investigate the mechanism of this insulin resistance, the in vivo activation of the insulin receptor kinase in liver and muscle of control and HFS-fed rats was determined. Rats were injected with glucose and insulin and killed 0, 5, 15, and 30 min after injection. Insulin binding was not changed in partially purified receptors from muscle of HFS rats. In control rats insulin receptor kinase activity was maximally stimulated threefold in liver at 5 min and fourfold in muscle at 15 min after insulin-glucose injection. The insulin-stimulated tyrosine kinase activity of receptors isolated from the liver of rats fed the HFS diet was decreased by 30% in comparison with the controls. In contrast, receptors isolated from muscle did not show any difference in basal or insulin-stimulated kinase activity between HFS-fed and control rats. Decreased in vivo activation of the insulin receptor kinase may be at least partially responsible for insulin resistance in liver. Because insulin binding and insulin stimulation of receptor kinase were normal in muscle of HFS-fed animals, it is concluded that the insulin resistance of glucose uptake into muscle is caused by a defect distal to the insulin receptor.

Fish oil supplementation does not lower plasma cholesterol in men with hypercholesterolemia. Results of a randomized, placebo-controlled crossover study.

STUDY OBJECTIVE: To determine the effects of fish oil supplementation on plasma cholesterol in middle-aged men with isolated hypercholesterolemia. DESIGN: Randomized double-blind placebo-controlled (safflower oil) two-period crossover trial with 12-week treatment periods. SETTING: Outpatient general medicine clinic at a university-affiliated Veterans Affairs hospital. PATIENTS: Thirty-eight men with plasma cholesterol between 5.68 and 7.76 mmol/L (220 to 300 mg/dL), triglyceride levels less than 3.39 mmol/L (300 mg/dL), and free of coexisting diseases. INTERVENTIONS: Fish oil and placebo (safflower oil) supplementation. After basal measurements and a 4-week lead-in period, twenty 1-g capsules of either fish oil or placebo oil were provided for 12 weeks (period 1). After a 4-week washout phase participants then received the other oil for an additional 12 weeks (period 2). MEASUREMENTS AND MAIN RESULTS: Blood was drawn at the beginning and end of each study period and analyzed for levels of total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, apolipoprotein A1, and apolipoprotein B. Low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald equation. Total and LDL cholesterol increased from the before treatment values by 4.8% and 9.1%, respectively, after ingestion of fish oil. Compared with placebo, LDL cholesterol was significantly higher (4.5 compared with 4.1 mmol/L, P = 0.01) and triglycerides lower (1.3 compared with 1.8 mmol/L, P = 0.01) after fish oil. Total and HDL cholesterol and apolipoprotein A1 and B levels did not differ. CONCLUSIONS: Fish oil supplements do not lower plasma cholesterol levels in middle-aged men with hypercholesterolemia without elevated triglycerides. They should not be recommended as a method to lower plasma cholesterol in these patients.