Monitoring Dynamic Protein Expression in Single Living E. Coli. Bacterial Cells by Laser Tweezers Raman Spectroscopy

Laser tweezers Raman spectroscopy (LTRS) is a novel, nondestructive, and label-free method that can be used to quantitatively measure changes in cellular activity in single living cells. Here, we demonstrate its use to monitor changes in a population of E. coli cells that occur during overexpression of a protein, the extracellular domain of myelin oligodendrocyte glycoprotein (MOG(1-120)) Raman spectra were acquired of individual E. coli cells suspended in solution and trapped by a single tightly focused laser beam. Overexpression of MOG(1-120) in transformed E. coli Rosetta-Gami (DE3)pLysS cells was induced by addition of isopropyl thiogalactoside (IPTG). Changes in the peak intensities of the Raman spectra from a population of cells were monitored and analyzed over a total duration of three hours. Data was also collected for concentrated purified MOG(1-120) protein in solution, and the spectra compared with that obtained for the MOG(1-120) expressing cells. Raman spectra of individual, living E. coli cells exhibit signatures due to DNA and protein molecular vibrations. Characteristic Raman markers associated with protein vibrations, such as 1257 cm{sup -1}, 1340 cm{sup -1}, 1453 cm{sup -1} and 1660 cm{sup -1}, are shown to increase as a function of time following the addition of IPTG. Comparison of these spectra and the spectra of purified MOG protein indicates that the changes are predominantly due to the induction of MOG protein expression. Protein expression was found to occur mostly within the second hour, with a 470% increase relative to the protein expressed in the first hour. A 230% relative increase between the second and third hour indicates that protein expression begins to level off within the third hour. It is demonstrated that LTRS has sufficient sensitivity for real-time, nondestructive, and quantitative monitoring of biological processes, such as protein expression, in single living cells. Such capabilities, which are not currently available in flow cytometry, open up new possibilities for analyzing cellular processes occurring in single microbial and eukaryotic cells

Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii

Lauersen KJ, Baier T, Wichmann J, et al. Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii. Metabolic Engineering. 2016;38:331-343

Subcellular heterogeneity of ryanodine receptor properties in ventricular myocytes with low T-tubule density

Rationale: In ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI). Objective: To characterize RyR functional properties in relation to TT proximity, at baseline and after MI. Methods: Myocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca2+ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca2+ release, F>F50 within 20 ms) or their absence (delayed areas). Spontaneous Ca2+ release events during diastole, Ca2+ sparks, reflecting RyR activity and properties, were subsequently assigned to either category. Results: In CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na+/Ca2+ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca2+ influx via Ca2+ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca2+ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca2+ removal by NCX at the membrane was significantly lower in MI. Conclusion: TT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca2+ loss and raise SR Ca2+ content, but may promote Ca2+ waves

Dirty Spin Ice: The Effect of Dilution on Spin Freezing in Dy2Ti2O7

We have studied spin freezing in the diluted spin ice compound Dy2-xYxTi2O7 where the non-magnetic Y ions replace the magnetic Dy ions on the frustrated pyrochlore lattice. Magnetic a.c. and d.c. susceptibility data are presented with an analysis of relaxation times for dilutions of x = 0, 0.1, 0.2, and 0.4. Site dilution apparently decreases the relative number of spins participating in the ice-like freezing near 16 K while leaving the freezing temperature unchanged. Correspondingly the distribution of relaxation times associated with the freezing is broadened only slightly with increasing dilution, suggesting that the freezing process observed near T = 16 K involves local correlations among the spins.Comment: 15 pages, 6 figures, submitted to Physical Review

Improving the physician-patient cardiovascular risk dialogue to improve statin adherence

<p>Abstract</p> <p>Background</p> <p>The purpose of this study was to evaluate the effectiveness of a patient education program developed to facilitate statin adherence.</p> <p>Methods</p> <p>A controlled trial was designed to test the effectiveness of a multifaceted patient education program to facilitate statin adherence. The program included a brief, in-office physician counseling kit followed by patient mailings. The primary end point was adherence to filling statin prescriptions during a 120-day period. Patients new to statins enrolled and completed a survey. Data from a national pharmacy claims database were used to track adherence.</p> <p>Results</p> <p>Patients new to statin therapy exposed to a patient counseling and education program achieved a 12.4 higher average number of statin prescription fill days and were 10% more likely to fill prescriptions for at least 120 days (<it>p </it>= .01).</p> <p>Conclusion</p> <p>Brief in-office counseling on cardiovascular risk followed by patient education mailings can be effective in increasing adherence. Physicians found a one-minute counseling tool and pocket guidelines useful in counseling patients.</p

X-ray Conversion Efficiency of high-Z hohlraum wall materials for indirect drive ignition

We measure the conversion efficiency of 351 nm laser light to soft x-rays (0.1-5 keV) for Au, U and high Z mixtures 'cocktails' used for hohlraum wall materials in indirect drive ICF. We use spherical targets in a direct drive geometry, flattop laser pulses and laser smoothing with phase plates to achieve constant and uniform laser intensities of 10{sup 14} and 10{sup 15} W/cm{sup 2} over the target surface that are relevant for the future ignition experiments on NIF. The absolute time and spectrally-resolved radiation flux is measured with a multichannel soft x-ray power diagnostic. The conversion efficiency is then calculated by dividing the measured x-ray power by the incident laser power from which the measured laser backscattering losses is subtracted. After {approx}0.5 ns, the time resolved x-ray conversion efficiency reaches a slowly increasing plateau of 95% at 10{sup 14} W/cm{sup 2} laser intensity and of 80% at 10{sup 15} W/cm{sup 2}. The M-band flux (2-5 keV) is negligible at 10{sup 14} W/cm{sup 2} reaching {approx}1% of the total x-ray flux for all target materials. In contrast, the M-band flux is significant and depends on the target material at 10{sup 15} W/cm{sup 2} laser intensity, reaching values between 10% of the total flux for U and 27% for Au. Our LASNEX simulations show good agreement in conversion efficiency and radiated spectra with data when using XSN atomic physics model and a flux limiter of 0.15, but they underestimate the generated M-band flux

Use of mitogenic cascade blockers for treatment of C-Raf induced lung adenoma in vivo: CI-1040 strongly reduces growth and improves lung structure

BACKGROUND: Signaling networks promoting cell growth and proliferation are frequently deregulated in cancer. Tumors often are highly dependent on such signaling pathways and may become hypersensitive to downregulation of key components within these signaling cascades. The classical mitogenic cascade transmits stimuli from growth factor receptors via Ras, Raf, MEK and ERK to the cell nucleus and provides attractive molecular targets for cancer treatment. For example, Ras and Raf kinase inhibitors are already in a number of ongoing phase II and phase III clinical trials. In this study the effect of the Raf kinase inhibitor BAY 43-9006 and of the MEK inhibitor CI-1040 (PD184352) on a Raf dependent lung tumor mouse model was analyzed in detail. METHODS: We have generated a lung cancer mouse model by targeting constitutively active C-Raf kinase to the lung. These mice develop adenomas within 4 months of life. At this time-point they received daily intraperitoneal injections of either 100 mg/kg BAY 43-9006 or CI-1040 for additional 21 days. Thereafter, lungs were isolated and the following parameters were analyzed using histology and immunohistochemistry: overall lung structure, frequency of adenoma foci, proliferation rate, ERK activity, caspase-3 activation, and lung differentiation. RESULTS: Both inhibitors were equally effective in vitro using a sensitive Raf/MEK/ERK ELISA. In vivo, the systemic administration of the MEK inhibitor CI-1040 reduced adenoma formation to a third and significantly restored lung structure. The proliferation rate of lung cells of mice treated with CL-1040 was decreased without any obvious effects on differentiation of pneumocytes. In contrast, the Raf inhibitor BAY 43-9006 did not influence adenoma formation in vivo. CONCLUSION: The MEK inhibitor CI-1040 may be used for the treatment of Ras and/or Raf-dependent human malignancies