CRISPR-Based Methods for Caenorhabditis elegans Genome Engineering

The advent of genome editing techniques based on the clustered regularly interspersed short palindromic repeats (CRISPR)–Cas9 system has revolutionized research in the biological sciences. CRISPR is quickly becoming an indispensible experimental tool for researchers using genetic model organisms, including the nematode Caenorhabditis elegans. Here, we provide an overview of CRISPR-based strategies for genome editing in C. elegans. We focus on practical considerations for successful genome editing, including a discussion of which strategies are best suited to producing different kinds of targeted genome modifications

Nox2 redox signaling maintains essential cell populations in the brain.

Reactive oxygen species (ROS) are conventionally classified as toxic consequences of aerobic life, and the brain is particularly susceptible to ROS-induced oxidative stress and damage owing to its high energy and oxygen demands. NADPH oxidases (Nox) are a widespread source of brain ROS implicated in seizures, stroke and neurodegeneration. A physiological role for ROS generation in normal brain function has not been established, despite the fact that mice and humans lacking functional Nox proteins have cognitive deficits. Using molecular imaging with Peroxyfluor-6 (PF6), a new selective fluorescent indicator for hydrogen peroxide (H(2)O(2)), we show that adult hippocampal stem/progenitor cells (AHPs) generate H(2)O(2) through Nox2 to regulate intracellular growth signaling pathways, which in turn maintains their normal proliferation in vitro and in vivo. Our results challenge the traditional view that brain ROS are solely deleterious by demonstrating that controlled ROS chemistry is needed for maintaining specific cell populations

Measuring Protein Binding to F-actin by Co-sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique – the actin co-sedimentation, or pelleting, assay – to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin

MRCK-1 Drives Apical Constriction in C. elegans by Linking Developmental Patterning to Force Generation

Apical constriction is a change in cell shape that drives key morphogenetic events including gastrulation and neural tube formation. Apical force-producing actomyosin networks drive apical constriction by contracting while connected to cell-cell junctions. The mechanisms by which developmental patterning regulates these actomyosin networks and associated junctions with spatial precision are not fully understood. Here, we identify a myosin light chain kinase MRCK-1 as a key regulator of C. elegans gastrulation that integrates spatial and developmental patterning information. We show that MRCK-1 is required for activation of contractile actomyosin dynamics and elevated cortical tension in the apical cell cortex of endodermal precursor cells. MRCK-1 is apically localized by active Cdc42 at the external, cell-cell contact-free surfaces of apically constricting cells, downstream of cell fate determination mechanisms. We establish that the junctional components α-catenin, β-catenin, and cadherin become highly enriched at the apical junctions of apically-constricting cells, and that MRCK-1 and myosin activity are required in vivo for this enrichment. Taken together, our results define mechanisms that position a myosin activator to a specific cell surface where it both locally increases cortical tension and locally enriches junctional components to facilitate apical constriction. These results reveal crucial links that can tie spatial information to local force generation to drive morphogenesis

A computationally engineered RAS rheostat reveals RAS-ERK signaling dynamics.

Synthetic protein switches controlled with user-defined inputs are powerful tools for studying and controlling dynamic cellular processes. To date, these approaches have relied primarily on intermolecular regulation. Here we report a computationally guided framework for engineering intramolecular regulation of protein function. We utilize this framework to develop chemically inducible activator of RAS (CIAR), a single-component RAS rheostat that directly activates endogenous RAS in response to a small molecule. Using CIAR, we show that direct RAS activation elicits markedly different RAS-ERK signaling dynamics from growth factor stimulation, and that these dynamics differ among cell types. We also found that the clinically approved RAF inhibitor vemurafenib potently primes cells to respond to direct wild-type RAS activation. These results demonstrate the utility of CIAR for quantitatively interrogating RAS signaling. Finally, we demonstrate the general utility of our approach in design of intramolecularly regulated protein tools by applying it to the Rho family of guanine nucleotide exchange factors

New Hubble Space Telescope Discoveries of Type Ia Supernovae at z > 1: Narrowing Constraints on the Early Behavior of Dark Energy

We have discovered 21 new Type Ia supernovae (SNe Ia) with the Hubble Space Telescope (HST) and have used them to trace the history of cosmic expansion over the last 10 billion years. These objects, which include 13 spectroscopically confirmed SNe Ia at z > 1, were discovered during 14 epochs of reimaging of the GOODS fields North and South over two years with the Advanced Camera for Surveys on HST. Together with a recalibration of our previous HST-discovered SNe Ia, the full sample of 23 SNe Ia at z > 1 provides the highest-redshift sample known. Combined with previous SN Ia datasets, we measured H(z) at discrete, uncorrelated epochs, reducing the uncertainty of H(z>1) from 50% to under 20%, strengthening the evidence for a cosmic jerk--the transition from deceleration in the past to acceleration in the present. The unique leverage of the HST high-redshift SNe Ia provides the first meaningful constraint on the dark energy equation-of-state parameter at z >1. The result remains consistent with a cosmological constant (w(z)=-1), and rules out rapidly evolving dark energy (dw/dz >>1). The defining property of dark energy, its negative pressure, appears to be present at z>1, in the epoch preceding acceleration, with ~98% confidence in our primary fit. Moreover, the z>1 sample-averaged spectral energy distribution is consistent with that of the typical SN Ia over the last 10 Gyr, indicating that any spectral evolution of the properties of SNe Ia with redshift is still below our detection threshold.Comment: typos, references corrected, minor additions to exposition 82 pages, 17 figures, 6 tables. Data also available at: http://braeburn.pha.jhu.edu/~ariess/R06. Accepted, Astrophysical Journal vol. 656 for March 10, 200

Fat free mass explains the relationship between stunting and energy expenditure in urban Mexican Maya children

Background: Childhood stunting has been associated with an increased risk of obesity in adulthood, but the causes are unclear. This study hypothesizes that stunting significantly reduces both resting and activity energy expenditure. Aim: To assess and describe energy expenditure of low socio-economic Maya children and to determine whether stunting is independently related to energy expenditure after controlling for lean mass. Subjects and methods: Thirty-three urban Maya children, 17 boys, aged 7–9 years, living in Merida, Mexico, were measured for height, weight and bioelectrical impedance analysis (BIA). Body composition was estimated from BIA. Energy expenditure was measured for one week using the Actiheart (combined heart rate and accelerometer). Results: Stunting (height-for-age below the 5th percentile of NHANES III based references) affected 35% of these physically active children. Using multiple linear regression analysis, greater lean body mass predicted higher resting and activity energy expenditure. Stature was not a significant predictor of resting energy expenditure. A lower height-for-age z-score, but not stunting as a categorical variable, significantly predicted lower activity energy expenditure. Conclusion: The hypothesis that stunting reduces total energy expenditure (resting + active) in children is not supported. Rather, children with shorter stature and less lean body mass have lower total energy expenditure. Complex interactions between body size, body composition, and metabolic activity appear to elevate the risk for later life obesity in these Maya children

In Vivo Time- Resolved Microtomography Reveals the Mechanics of the Blowfly Flight Motor

Dipteran flies are amongst the smallest and most agile of flying animals. Their wings are driven indirectly by large power muscles, which cause cyclical deformations of the thorax that are amplified through the intricate wing hinge. Asymmetric flight manoeuvres are controlled by 13 pairs of steering muscles acting directly on the wing articulations. Collectively the steering muscles account for <3% of total flight muscle mass, raising the question of how they can modulate the vastly greater output of the power muscles during manoeuvres. Here we present the results of a synchrotron-based study performing micrometre-resolution, time-resolved microtomography on the 145 Hz wingbeat of blowflies. These data represent the first four-dimensional visualizations of an organism's internal movements on sub-millisecond and micrometre scales. This technique allows us to visualize and measure the three-dimensional movements of five of the largest steering muscles, and to place these in the context of the deforming thoracic mechanism that the muscles actuate. Our visualizations show that the steering muscles operate through a diverse range of nonlinear mechanisms, revealing several unexpected features that could not have been identified using any other technique. The tendons of some steering muscles buckle on every wingbeat to accommodate high amplitude movements of the wing hinge. Other steering muscles absorb kinetic energy from an oscillating control linkage, which rotates at low wingbeat amplitude but translates at high wingbeat amplitude. Kinetic energy is distributed differently in these two modes of oscillation, which may play a role in asymmetric power management during flight control. Structural flexibility is known to be important to the aerodynamic efficiency of insect wings, and to the function of their indirect power muscles. We show that it is integral also to the operation of the steering muscles, and so to the functional flexibility of the insect flight motor

Morphologies and Spectral Energy Distributions of Extremely Red Galaxies in the GOODS-South Field

Using U'- through Ks-band imaging data in the GOODS-South field, we construct a large, complete sample of 275 ``extremely red objects'' (EROs; K_s<22.0, R-K_s>3.35; AB), all with deep HST/ACS imaging in B_435, V_606, i_775, and z_850, and well-calibrated photometric redshifts. Quantitative concentration and asymmetry measurements fail to separate EROs into distinct morphological classes. We therefore visually classify the morphologies of all EROs into four broad types: ``Early'' (elliptical-like), ``Late'' (disk galaxies), ``Irregular'' and ``Other'' (chain galaxies and low surface brightness galaxies), and calculate their relative fractions and comoving space densities. For a broad range of limiting magnitudes and color thresholds, the relative number of early-type EROs is approximately constant at 33-44%, and the comoving space densities of Early- and Late-type EROs are comparable. Mean rest-frame spectral energy distributions (SEDs) at wavelengths between 0.1 and 1.2 um are constructed for all EROs. The SEDs are extremely similar in their range of shapes, independent of morphological type. The implication is that any differences between the broad-band SEDs of Early-type EROs and the other types are relatively subtle, and there is no robust way of photometrically distinguishing between different morphological types with usual optical/near-infrared photometry.Comment: Submitted to the ApJL. A version with full-resolution figures, all GOODS data and all GOODS collaboration papers may be found at http://www.stsci.edu/science/goods