631 research outputs found
Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells.
Many key regulatory proteins in bacteria are present in too low numbers to be detected with conventional methods, which poses a particular challenge for single-cell analyses because such proteins can contribute greatly to phenotypic heterogeneity. Here we develop a microfluidics-based platform that enables single-molecule counting of low-abundance proteins by mechanically slowing-down their diffusion within the cytoplasm of live Escherichia coli (E. coli) cells. Our technique also allows for automated microscopy at high throughput with minimal perturbation to native physiology, as well as viable enrichment/retrieval. We illustrate the method by analysing the control of the master regulator of the E. coli stress response, RpoS, by its adapter protein, SprE (RssB). Quantification of SprE numbers shows that though SprE is necessary for RpoS degradation, it is expressed at levels as low as 3-4 molecules per average cell cycle, and fluctuations in SprE are approximately Poisson distributed during exponential phase with no sign of bursting
No imminent quantum supremacy by boson sampling
It is predicted that quantum computers will dramatically outperform their
conventional counterparts. However, large-scale universal quantum computers are
yet to be built. Boson sampling is a rudimentary quantum algorithm tailored to
the platform of photons in linear optics, which has sparked interest as a rapid
way to demonstrate this quantum supremacy. Photon statistics are governed by
intractable matrix functions known as permanents, which suggests that sampling
from the distribution obtained by injecting photons into a linear-optical
network could be solved more quickly by a photonic experiment than by a
classical computer. The contrast between the apparently awesome challenge faced
by any classical sampling algorithm and the apparently near-term experimental
resources required for a large boson sampling experiment has raised
expectations that quantum supremacy by boson sampling is on the horizon. Here
we present classical boson sampling algorithms and theoretical analyses of
prospects for scaling boson sampling experiments, showing that near-term
quantum supremacy via boson sampling is unlikely. While the largest boson
sampling experiments reported so far are with 5 photons, our classical
algorithm, based on Metropolised independence sampling (MIS), allowed the boson
sampling problem to be solved for 30 photons with standard computing hardware.
We argue that the impact of experimental photon losses means that demonstrating
quantum supremacy by boson sampling would require a step change in technology.Comment: 25 pages, 9 figures. Comments welcom
Cmr1/WDR76 defines a nuclear genotoxic stress body linking genome integrity and protein quality control
DNA replication stress is a source of genomic instability. Here we identify ​changed mutation rate 1 (​Cmr1) as a factor involved in the response to DNA replication stress in Saccharomyces cerevisiae and show that ​Cmr1—together with ​Mrc1/​Claspin, ​Pph3, the chaperonin containing ​TCP1 (CCT) and 25 other proteins—define a novel intranuclear quality control compartment (INQ) that sequesters misfolded, ubiquitylated and sumoylated proteins in response to genotoxic stress. The diversity of proteins that localize to INQ indicates that other biological processes such as cell cycle progression, chromatin and mitotic spindle organization may also be regulated through INQ. Similar to ​Cmr1, its human orthologue ​WDR76 responds to proteasome inhibition and DNA damage by relocalizing to nuclear foci and physically associating with CCT, suggesting an evolutionarily conserved biological function. We propose that ​Cmr1/​WDR76 plays a role in the recovery from genotoxic stress through regulation of the turnover of sumoylated and phosphorylated proteins
Classification of river morphology and hydrology to support management and restoration
The work leading to this paper has received funding from the European Union’s FP7 programme under Grant Agreement No. 282656 (REFORM
CRISPR transcriptional repression devices and layered circuits in mammalian cells
A key obstacle to creating sophisticated genetic circuits has been the lack of scalable device libraries. Here we present a modular transcriptional repression architecture based on clustered regularly interspaced palindromic repeats (CRISPR) system and examine approaches for regulated expression of guide RNAs in human cells. Subsequently we demonstrate that CRISPR regulatory devices can be layered to create functional cascaded circuits, which provide a valuable toolbox for engineering purposes.National Institutes of Health (U.S.) (Grant 5R01CA155320-04)National Institutes of Health (U.S.) (Grant P50 GM098792)Korea (South). Ministry of Science, Information and Communication Technolgy. Intelligent Synthetic Biology Center of Global Frontier Project (2013M3A6A8073557
Neutrophils in cancer: neutral no more
Neutrophils are indispensable antagonists of microbial infection and facilitators of wound healing. In the cancer setting, a newfound appreciation for neutrophils has come into view. The traditionally held belief that neutrophils are inert bystanders is being challenged by the recent literature. Emerging evidence indicates that tumours manipulate neutrophils, sometimes early in their differentiation process, to create diverse phenotypic and functional polarization states able to alter tumour behaviour. In this Review, we discuss the involvement of neutrophils in cancer initiation and progression, and their potential as clinical biomarkers and therapeutic targets
Evaluation of methods for detection of fluorescence labeled subcellular objects in microscope images
<p>Abstract</p> <p>Background</p> <p>Several algorithms have been proposed for detecting fluorescently labeled subcellular objects in microscope images. Many of these algorithms have been designed for specific tasks and validated with limited image data. But despite the potential of using extensive comparisons between algorithms to provide useful information to guide method selection and thus more accurate results, relatively few studies have been performed.</p> <p>Results</p> <p>To better understand algorithm performance under different conditions, we have carried out a comparative study including eleven spot detection or segmentation algorithms from various application fields. We used microscope images from well plate experiments with a human osteosarcoma cell line and frames from image stacks of yeast cells in different focal planes. These experimentally derived images permit a comparison of method performance in realistic situations where the number of objects varies within image set. We also used simulated microscope images in order to compare the methods and validate them against a ground truth reference result. Our study finds major differences in the performance of different algorithms, in terms of both object counts and segmentation accuracies.</p> <p>Conclusions</p> <p>These results suggest that the selection of detection algorithms for image based screens should be done carefully and take into account different conditions, such as the possibility of acquiring empty images or images with very few spots. Our inclusion of methods that have not been used before in this context broadens the set of available detection methods and compares them against the current state-of-the-art methods for subcellular particle detection.</p
Multisite Phosphorylation of the Guanine Nucleotide Exchange Factor Cdc24 during Yeast Cell Polarization
BACKGROUND:Cell polarization is essential for processes such as cell migration and asymmetric cell division. A common regulator of cell polarization in most eukaryotic cells is the conserved Rho GTPase, Cdc42. In budding yeast, Cdc42 is activated by a single guanine nucleotide exchange factor, Cdc24. The mechanistic details of Cdc24 activation at the onset of yeast cell polarization are unclear. Previous studies have suggested an important role for phosphorylation of Cdc24, which may regulate activity or function of the protein, representing a key step in the symmetry breaking process. METHODOLOGY/PRINCIPAL FINDINGS:Here, we directly ask whether multisite phosphorylation of Cdc24 plays a role in its regulation. We identify through mass spectrometry analysis over thirty putative in vivo phosphorylation sites. We first focus on sites matching consensus sequences for cyclin-dependent and p21-activated kinases, two kinase families that have been previously shown to phosphorylate Cdc24. Through site-directed mutagenesis, yeast genetics, and light and fluorescence microscopy, we show that nonphosphorylatable mutations of these consensus sites do not lead to any detectable consequences on growth rate, morphology, kinetics of polarization, or localization of the mutant protein. We do, however, observe a change in the mobility shift of mutant Cdc24 proteins on SDS-PAGE, suggesting that we have indeed perturbed its phosphorylation. Finally, we show that mutation of all identified phosphorylation sites does not cause observable defects in growth rate or morphology. CONCLUSIONS/SIGNIFICANCE:We conclude that lack of phosphorylation on Cdc24 has no overt functional consequences in budding yeast. Yeast cell polarization may be more tightly regulated by inactivation of Cdc42 by GTPase activating proteins or by alternative methods of Cdc24 regulation, such as conformational changes or oligomerization
On-chip pressure measurements and channel deformation after oil absorption
Microfluidic channels moulded from the soft polymer poly(dimethylsiloxane) (PDMS) are widely used as a platform for mimicking biological environments, and can be used for the simulation of fluid filled structures such as blood and lung vessels. The control of pressure and flow rate within these structures is vital to mimic physiological conditions. The flexibility of PDMS leads to pressure-induced deformation under flow, leading to variable flow profiles along a device. Here, we investigate the change in Young’s modulus of microfluidic channels due to infiltration of mineral oil, a PDMS permeable fluid, and how this affects the resulting pressure profile using a novel pressure measurement method. We found a 53% decrease in Young’s modulus of PDMS due to mineral oil absorption over the course of 3 h accounted for lower internal pressure and larger channel deformation compared to fresh PDMS at a given flow rate. Confocal fluorescence microscopy used to image channel profiles before and after the introduction of mineral oil showed a change in pressure-induced deformation after infiltration of the oil. Atomic force microscopy (AFM) nanoindentation was used to measure Young’s modulus of PDMS before (2.80±0.032.80±0.03 MPa) and after (1.32±0.041.32±0.04 MPa) mineral oil absorption. Raman spectroscopy showed the infiltration of mineral oil into PDMS from channel walls and revealed the diffusion coefficient of mineral oil in PDMS
Selective Molecular Sieving through Porous Graphene
Membranes act as selective barriers and play an important role in processes
such as cellular compartmentalization and industrial-scale chemical and gas
purification. The ideal membrane should be as thin as possible to maximize
flux, mechanically robust to prevent fracture, and have well-defined pore sizes
to increase selectivity. Graphene is an excellent starting point for developing
size selective membranes because of its atomic thickness, high mechanical
strength, relative inertness, and impermeability to all standard gases.
However, pores that can exclude larger molecules, but allow smaller molecules
to pass through have to be introduced into the material. Here we show
UV-induced oxidative etching can create pores in micrometre-sized graphene
membranes and the resulting membranes used as molecular sieves. A pressurized
blister test and mechanical resonance is used to measure the transport of a
variety of gases (H2, CO2, Ar, N2, CH4, and SF6) through the pores. The
experimentally measured leak rates, separation factors, and Raman spectrum
agree well with models based on effusion through a small number of
angstrom-sized pores.Comment: to appear in Nature Nanotechnolog
- …