CRISPR-dependent Base Editing Screens Identify Separation of Function Mutants of RADX with Altered RAD51 Regulatory Activity

RAD51 forms nucleoprotein filaments to promote homologous recombination, replication fork reversal, and fork protection. Numerous factors regulate the stability of these filaments and improper regulation leads to genomic instability and ultimately disease including cancer. RADX is a single stranded DNA binding protein that modulates RAD51 filament stability. Here, we utilize a CRISPR-dependent base editing screen to tile mutations across RADX to delineate motifs required for RADX function. We identified separation of function mutants of RADX that bind DNA and RAD51 but have a reduced ability to stimulate its ATP hydrolysis activity. Cells expressing these RADX mutants accumulate RAD51 on chromatin, exhibit replication defects, have reduced growth, accumulate DNA damage, and are hypersensitive to DNA damage and replication stress. These results indicate that RADX must promote RAD51 ATP turnover to regulate RAD51 and genome stability during DNA replication.     

A New Method for Estimation of Resource Selection Probability Function

ABSTRACT Weighted distributions can be used to fit various forms of resource selection probability functions (RSPF) under the use-versus-available study design (Lele and Keim 2006). Although valid, the numerical maximization procedure used by Lele and Keim (2006) is unstable because of the inherent roughness of the Monte Carlo likelihood function. We used a combination of the methods of partial likelihood and data cloning to obtain maximum likelihood estimators of the RSPF in a numerically stable fashion. We demonstrated the methodology using simulated data sets generated under the log—log RSPF model and a reanalysis of telemetry data presented in Lele and Keim (2006) using the logistic RSPF model. The new method for estimation of RSPF can be used to understand differential selection of resources by animals, an essential component of studies in conservation biology, wildlife management, and applied ecology.     

Comparison of different approaches for dose response analysis

Characterizing an appropriate dose‐response relationship and identifying the right dose in a clinical trial are two main goals of early drug‐development. MCP‐Mod is one of the pioneer approaches developed within the last 10 years that combines the modeling techniques with multiple comparison procedures to address the above goals in clinical drug development. The MCP‐Mod approach begins with a set of potential dose‐response models, tests for a significant dose‐response effect (proof of concept, PoC) using multiple linear contrasts tests and selects the “best” model among those with a significant contrast test. A disadvantage of the method is that the parameter values of the candidate models need to be fixed a priori for the contrasts tests. This may lead to a loss in power and unreliable model selection. For this reason, several variations of the MCP‐Mod approach and a hierarchical model selection approach have been suggested where the parameter values need not be fixed in the proof of concept testing step and can be estimated after the model selection step. This paper provides a numerical comparison of the different MCP‐Mod variants and the hierarchical model selection approach with regard to their ability of detecting the dose‐response trend, their potential to select the correct model and their accuracy in estimating the dose response shape and minimum effective dose. Additionally, as one of the approaches is based on two‐sided model comparisons only, we make it more consistent with the common goals of a PoC study, by extending it to one‐sided comparisons between the constant and alternative candidate models in the proof of concept step.     

Selective labeling of lipid droplets in aldehyde fixed cell monolayers by lipophilic fluorochromes

Abstract

We evaluated a number of lipophilic dyes and fluorochromes, including oxazone and thiazone derivatives of oxazine and thiazine dyes, scintillator agents, a carotenoid and a metal-porphyrin complex for visualization of lipid droplets within aldehyde fixed cultured HeLa and BGC-1 cells. Observation under ultraviolet, blue or green exciting light revealed selective fluorescence of lipid droplets, particularly after treatment with aqueous solutions of Nile blue and brilliant cresyl blue oxazones, toluidine blue thiazone, or propylene glycol solutions of canthaxanthin, ethyl-BAO, and ZnTPyP. Mounting in water was required to maintain the fluorescence of lipids; the use of glycerol, Mowiol or Vectashield was not adequate. The effect of dye structure on staining intensity was assessed with the aid of numerical structure parameters modeling lipophilicity (HI and log P), overall size (MW) and the size of the conjugated system (conjugated bond number; CBN). The best stains for lipid droplets were relatively lipophilic (HI > 4.0, log P > 5.0), of small size overall (MW < 370), with small conjugated systems (CBN < 24), and not significantly amphiphilic. The two hydrophobic-hydrophilic parameters (the classic log P and the hydrophobic index, HI; values calculated by molecular modeling software) were highly correlated; however, HI was a more suitable hydrophobicity index for the dyes studied here.     

Performance of pairwise shape dissimilarity morphometrics on nonmammalian taxa (Insecta: Neuroptera: Mantispidae)

Morphometric dissimilarity metrics aim to quantify the variation between compared specimens such that inferences about their relatedness and alpha taxonomy can be made. Recently, the technique has developed metrics that purport to quantify shape dissimilarity between specimens—employing the use of least squares regression analysis. These metrics have been well applied by studies in the hominin fossil record with an arguably unsubstantiated backing for the technique. Originally postulated was the log₁₀ se_m metric which subsequently led to the standard error test of the null hypothesis metric. Following this, the standard deviation of logged ratios (S_LR) metric arose as a pairwise dissimilarity metric that constrains the regression to a zero‐intercept, that is, a significant development in the robustness of the technique. This metric was tested on extant primates in order to evaluate its effectiveness alongside the two other metrics. It was shown to be the most reliable for comparisons between specimens of primates, but was unable to discriminate between heterospecific and conspecific comparisons. Arguably, an alternative model organism with which to compare the technique is lacking. This study considers shape dissimilarity metrics with respect to a group of nonmammalian organisms (mantidflies) and tests the metrics against three lines of evidence (morphology, CO1‐DNA, and geographic distribution) that can delimit the species‐level taxonomy for the group. It is shown that the metrics are unable to discriminate between pairwise comparisons of closely related species, resulting in biologically erroneous groupings, and contradicting the groupings derived from morphological, CO1‐DNA, and distributional comparisons. It is thus asserted that the technique is unsuitable for use in alpha taxonomy as an additional line of evidence in mantidflies. It is further supposed that morphometrics in general should be employed with caution in studies of evolutionary history as phylogeny is not the only information contained within morphometric data. J. Morphol. 276:1482–1494, 2015. © 2015 Wiley Periodicals, Inc.