Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform

Important insights into aging have been generated with the genetically tractable and short-lived budding yeast. However, it is still impossible today to continuously track cells by high-resolution microscopic imaging (e.g., fluorescent imaging) throughout their entire lifespan. Instead, the field still needs to rely on a 50-y-old laborious and time-consuming method to assess the lifespan of yeast cells and to isolate differentially aged cells for microscopic snapshots via manual dissection of daughter cells from the larger mother cell. Here, we are unique in achieving continuous and high-resolution microscopic imaging of the entire replicative lifespan of single yeast cells. Our microfluidic dissection platform features an optically prealigned single focal plane and an integrated array of soft elastomer-based micropads, used together to allow for trapping of mother cells, removal of daughter cells, monitoring gradual changes in aging, and unprecedented microscopic imaging of the whole aging process. Using the platform, we found remarkable age-associated changes in phenotypes (e.g., that cells can show strikingly differential cell and vacuole morphologies at the moment of their deaths), indicating substantial heterogeneity in cell aging and death. We envision the microfluidic dissection platform to become a major tool in aging research.

Background discrimination capabilities of a heat and ionization germanium cryogenic detector

The discrimination capabilities of a 70 g heat and ionization Ge bolometer are studied. This first prototype has been used by the EDELWEISS Dark Matter experiment, installed in the Laboratoire Souterrain de Modane, for direct detection of WIMPs. Gamma and neutron calibrations demonstrate that this type of detector is able to reject more than 99.6% of the background while retaining 95% of the signal, provided that the background events distribution is not biased towards the surface of the Ge crystal. However, the 1.17 kg.day of data taken in a relatively important radioactive environment show an extra population slightly overlapping the signal. This background is likely due to interactions of low energy photons or electrons near the surface of the crystal, and is somewhat reduced by applying a higher charge-collecting inverse bias voltage (-6 V instead of -2 V) to the Ge diode. Despite this contamination, more than 98% of the background can be rejected while retaining 50% of the signal. This yields a conservative upper limit of 0.7 event.day^{-1}.kg^{-1}.keV^{-1}_{recoil} at 90% confidence level in the 15-45 keV recoil energy interval; the present sensitivity appears to be limited by the fast ambient neutrons. Upgrades in progress on the installation are summarized.Comment: Submitted to Astroparticle Physics, 14 page

Mechanical response of plectonemic DNA: an analytical solution

We consider an elastic rod model for twisted DNA in the plectonemic regime. The molecule is treated as an impenetrable tube with an effective, adjustable radius. The model is solved analytically and we derive formulas for the contact pressure, twisting moment and geometrical parameters of the supercoiled region. We apply our model to magnetic tweezer experiments of a DNA molecule subjected to a tensile force and a torque, and extract mechanical and geometrical quantities from the linear part of the experimental response curve. These reconstructed values are derived in a self-contained manner, and are found to be consistent with those available in the literature.Comment: 14 pages, 4 figure

The EDELWEISS Experiment : Status and Outlook

The EDELWEISS Dark Matter search uses low-temperature Ge detectors with heat and ionisation read- out to identify nuclear recoils induced by elastic collisions with WIMPs from the galactic halo. Results from the operation of 70 g and 320 g Ge detectors in the low-background environment of the Modane Underground Laboratory (LSM) are presented.Comment: International Conference on Dark Matter in Astro and Particle Physics (Dark 2000), Heidelberg, Germany, 10-16 Jul 2000, v3 minor revision

Polarimetric Properties of Flux-Ropes and Sheared Arcades in Coronal Prominence Cavities

The coronal magnetic field is the primary driver of solar dynamic events. Linear and circular polarization signals of certain infrared coronal emission lines contain information about the magnetic field, and to access this information, either a forward or an inversion method must be used. We study three coronal magnetic configurations that are applicable to polar-crown filament cavities by doing forward calculations to produce synthetic polarization data. We analyze these forward data to determine the distinguishing characteristics of each model. We conclude that it is possible to distinguish between cylindrical flux ropes, spheromak flux ropes, and sheared arcades using coronal polarization measurements. If one of these models is found to be consistent with observational measurements, it will mean positive identification of the magnetic morphology that surrounds certain quiescent filaments, which will lead to a greater understanding of how they form and why they erupt.Comment: 22 pages, 8 figures, Solar Physics topical issue: Coronal Magnetis

First Results of the EDELWEISS WIMP Search using a 320 g Heat-and-Ionization Ge Detector

The EDELWEISS collaboration has performed a direct search for WIMP dark matter using a 320 g heat-and-ionization cryogenic Ge detector operated in a low-background environment in the Laboratoire Souterrain de Modane. No nuclear recoils are observed in the fiducial volume in the 30-200 keV energy range during an effective exposure of 4.53 kg.days. Limits for the cross-section for the spin-independent interaction of WIMPs and nucleons are set in the framework of the Minimal Supersymmetric Standard Model (MSSM). The central value of the signal reported by the experiment DAMA is excluded at 90% CL.Comment: 14 pages, Latex, 4 figures. Submitted to Phys. Lett.

Event categories in the EDELWEISS WIMP search experiment

Four categories of events have been identified in the EDELWEISS-I dark matter experiment using germanium cryogenic detectors measuring simultaneously charge and heat signals. These categories of events are interpreted as electron and nuclear interactions occurring in the volume of the detector, and electron and nuclear interactions occurring close to the surface of the detectors(10-20 mu-m of the surface). We discuss the hypothesis that low energy surface nuclear recoils,which seem to have been unnoticed by previous WIMP searches, may provide an interpretation of the anomalous events recorded by the UKDMC and Saclay NaI experiments. The present analysis points to the necessity of taking into account surface nuclear and electron recoil interactions for a reliable estimate of background rejection factors.Comment: 11 pages, submitted to Phys. Lett.

Single-molecule experiments in biological physics: methods and applications

I review single-molecule experiments (SME) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SME it is possible to: manipulate molecules one at a time and measure distributions describing molecular properties; characterize the kinetics of biomolecular reactions and; detect molecular intermediates. SME provide the additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SME it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level emphasizing the importance of SME to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SME from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOT), magnetic tweezers (MT), biomembrane force probe (BFP) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation), proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SME to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.Comment: Latex, 60 pages, 12 figures, Topical Review for J. Phys. C (Cond. Matt

Cell-Cycle Dependence of Transcription Dominates Noise in Gene Expression

The large variability in mRNA and protein levels found from both static and dynamic measurements in single cells has been largely attributed to random periods of transcription, often occurring in bursts. The cell cycle has a pronounced global role in affecting transcriptional and translational output, but how this influences transcriptional statistics from noisy promoters is unknown and generally ignored by current stochastic models. Here we show that variable transcription from the synthetic tetO promoter in S. cerevisiae is dominated by its dependence on the cell cycle. Real-time measurements of fluorescent protein at high expression levels indicate tetO promoters increase transcription rate ~2-fold in S/G2/M similar to constitutive genes. At low expression levels, where tetO promoters are thought to generate infrequent bursts of transcription, we observe random pulses of expression restricted to S/G2/M, which are correlated between homologous promoters present in the same cell. The analysis of static, single-cell mRNA measurements at different points along the cell cycle corroborates these findings. Our results demonstrate that highly variable mRNA distributions in yeast are not solely the result of randomly switching between periods of active and inactive gene expression, but instead largely driven by differences in transcriptional activity between G1 and S/G2/M.GM095733BBBE 103316MIT Startup Fun

Force spectroscopy in studying infection

Biophysical force spectroscopy tools - for example optical tweezers, magnetic tweezers, atomic force microscopy, - have been used to study elastic, mechanical, conformational and dynamic properties of single biological specimens from single proteins to whole cells to reveal information not accessible by ensemble average methods such as X-ray crystallography, mass spectroscopy, gel electrophoresis and so on. Here we review the application of these tools on a range of infection-related questions from antibody-inhibited protein processivity to virus-cell adhesion. In each case we focus on how the instrumental design tailored to the biological system in question translates into the functionality suitable for that particular study. The unique insights that force spectroscopy has gained to complement knowledge learned through population averaging techniques in interrogating biomolecular details prove to be instrumental in therapeutic innovations such as those in structure-based drug design