<i>Track-A-Worm</i>, An Open-Source System for Quantitative Assessment of <i>C. elegans</i> Locomotory and Bending Behavior

<div><p>A major challenge of neuroscience is to understand the circuit and gene bases of behavior. <i>C. elegans</i> is commonly used as a model system to investigate how various gene products function at specific tissue, cellular, and synaptic foci to produce complicated locomotory and bending behavior. The investigation generally requires quantitative behavioral analyses using an automated single-worm tracker, which constantly records and analyzes the position and body shape of a freely moving worm at a high magnification. Many single-worm trackers have been developed to meet lab-specific needs, but none has been widely implemented for various reasons, such as hardware difficult to assemble, and software lacking sufficient functionality, having closed source code, or using a programming language that is not broadly accessible. The lack of a versatile system convenient for wide implementation makes data comparisons difficult and compels other labs to develop new worm trackers. Here we describe <i>Track-A-Worm</i>, a system rich in functionality, open in source code, and easy to use. The system includes plug-and-play hardware (a stereomicroscope, a digital camera and a motorized stage), custom software written to run with Matlab in Windows 7, and a detailed user manual. Grayscale images are automatically converted to binary images followed by head identification and placement of 13 markers along a deduced spline. The software can extract and quantify a variety of parameters, including distance traveled, average speed, distance/time/speed of forward and backward locomotion, frequency and amplitude of dominant bends, overall bending activities measured as root mean square, and sum of all bends. It also plots worm travel path, bend trace, and bend frequency spectrum. All functionality is performed through graphical user interfaces and data is exported to clearly-annotated and documented Excel files. These features make <i>Track-A-Worm</i> a good candidate for implementation in other labs.</p></div

The <i>Analyze</i> and <i>Batch Analyze</i> modules.

<p>These two modules are used to quantify worm movement and bending behavior. <b>A</b>. The <i>Analyze</i> module is for quantifying bending and movement behavior of one worm at a time although the results may be appended and saved in a single file. <b>B</b>. In the <i>Batch Analyze</i> module, multiple recordings can be quantified and exported to one file. This is ideal for rapidly comparing and summarizing large groups of worms. All functionality of the <i>Analyze</i> module except for graph plotting is replicated in the <i>Batch Analyze</i> module.</p

Reconstruction of travel path, and determination of movement direction and body amplitude.

<p><b>A & B</b>. The traveling paths of a wild-type worm reconstructed based on the positions of the centroid (<b>A</b>) and the nose/1^st marker point (<b>B</b>). <b>C</b>. Directionality is determined by comparing the directions of a velocity vector and a head vector. The velocity vector is obtained by connecting the centroid positions of two consecutive frames (red circles “1” and “2”) whereas the head vector by connecting the current centroid and head positions. The green circle labeled “a” and the blue circle labeled “b” show that the head vector is projected to the positive and negative sides of the velocity vector, respectively. The worm is moving forward if the projection of the head vector onto the velocity vector is positive. <b>D</b>. The amplitude is determined by first finding the velocity vector, and then drawing a box in alignment with the velocity vector to enclose the widest points of the spline with the width of the box being the worm amplitude.</p

The <i>Launcher, Calibrate</i>, and <i>Record</i> modules.

<p><b>A</b>. The <i>Launcher</i> interface is for launching the various modules of <i>Track-A-Worm</i>. <b>B</b>. The <i>Calibrate</i> module determines the correct conversion factor from pixels to micrometers. <b>C</b>. The <i>Record</i> module is used to track and record a freely moving worm.</p

The <i>Playback</i> module.

<p>This module is for viewing recorded images (either as a movie or as separate frames), cutting out undesired frames, and evaluating the brightness threshold for binary conversion. It may display either original (<b>A</b>) or binary (<b>B</b>) images. It also has the option to display the 13 markers after spline fitting.</p

Image binarization and bending activity analyses.

<p><b>A</b>. <i>Track-A-Worm</i> converts a gray worm image (<i>left</i>) into a binary image (<i>middle</i>), identifies the head (indicated by “x”), and places 13 markers along a deduced spline at equal intervals (<i>right</i>). <b>B</b>. Diagram of the first bend, which is the complementary angle formed by the two straight lines between markers #1 (the nose) and #2, and between markers #2 and #3. <b>C</b>. Bend trace of the first bend of a wild-type worm. <b>D</b>. Bend frequency spectrum generated by Fourier transformation of the bend trace, which appears as a mirror image. The user should disregard the second half of the graph. The main peak of this graph indicates that the dominant bending frequency is ∼0.4 Hz. <b>E</b>. Quantification of the maximum bend from the bend trace. The bend trace shows large-amplitude bends (alternating between approximately +50 to −50 degrees) as well as many smaller oscillations. The maximum bend is the difference between the average of the most positive and negative values of the dominant bends in a bend trace.</p

Regional Practice Variation and Outcomes in the Standard Versus Accelerated Initiation of Renal Replacement Therapy in Acute Kidney Injury (STARRT-AKI) Trial: A Post Hoc Secondary Analysis.

ObjectivesAmong patients with severe acute kidney injury (AKI) admitted to the ICU in high-income countries, regional practice variations for fluid balance (FB) management, timing, and choice of renal replacement therapy (RRT) modality may be significant.DesignSecondary post hoc analysis of the STandard vs. Accelerated initiation of Renal Replacement Therapy in Acute Kidney Injury (STARRT-AKI) trial (ClinicalTrials.gov number NCT02568722).SettingOne hundred-fifty-three ICUs in 13 countries.PatientsAltogether 2693 critically ill patients with AKI, of whom 994 were North American, 1143 European, and 556 from Australia and New Zealand (ANZ).InterventionsNone.Measurements and main resultsTotal mean FB to a maximum of 14 days was +7199 mL in North America, +5641 mL in Europe, and +2211 mL in ANZ (p p p p p p p p = 0.007).ConclusionsAmong STARRT-AKI trial centers, significant regional practice variation exists regarding FB, timing of initiation of RRT, and initial use of continuous RRT. After adjustment, such practice variation was associated with lower ICU and hospital stay and 90-day mortality among ANZ patients compared with other regions

A Bayesian reanalysis of the Standard versus Accelerated Initiation of Renal-Replacement Therapy in Acute Kidney Injury (STARRT-AKI) trial

Background Timing of initiation of kidney-replacement therapy (KRT) in critically ill patients remains controversial. The Standard versus Accelerated Initiation of Renal-Replacement Therapy in Acute Kidney Injury (STARRT-AKI) trial compared two strategies of KRT initiation (accelerated versus standard) in critically ill patients with acute kidney injury and found neutral results for 90-day all-cause mortality. Probabilistic exploration of the trial endpoints may enable greater understanding of the trial findings. We aimed to perform a reanalysis using a Bayesian framework. Methods We performed a secondary analysis of all 2927 patients randomized in multi-national STARRT-AKI trial, performed at 168 centers in 15 countries. The primary endpoint, 90-day all-cause mortality, was evaluated using hierarchical Bayesian logistic regression. A spectrum of priors includes optimistic, neutral, and pessimistic priors, along with priors informed from earlier clinical trials. Secondary endpoints (KRT-free days and hospital-free days) were assessed using zero–one inflated beta regression. Results The posterior probability of benefit comparing an accelerated versus a standard KRT initiation strategy for the primary endpoint suggested no important difference, regardless of the prior used (absolute difference of 0.13% [95% credible interval [CrI] − 3.30%; 3.40%], − 0.39% [95% CrI − 3.46%; 3.00%], and 0.64% [95% CrI − 2.53%; 3.88%] for neutral, optimistic, and pessimistic priors, respectively). There was a very low probability that the effect size was equal or larger than a consensus-defined minimal clinically important difference. Patients allocated to the accelerated strategy had a lower number of KRT-free days (median absolute difference of − 3.55 days [95% CrI − 6.38; − 0.48]), with a probability that the accelerated strategy was associated with more KRT-free days of 0.008. Hospital-free days were similar between strategies, with the accelerated strategy having a median absolute difference of 0.48 more hospital-free days (95% CrI − 1.87; 2.72) compared with the standard strategy and the probability that the accelerated strategy had more hospital-free days was 0.66. Conclusions In a Bayesian reanalysis of the STARRT-AKI trial, we found very low probability that an accelerated strategy has clinically important benefits compared with the standard strategy. Patients receiving the accelerated strategy probably have fewer days alive and KRT-free. These findings do not support the adoption of an accelerated strategy of KRT initiation