Elevated pentose phosphate pathway flux supports appendage regeneration.

A fundamental step in regeneration is rapid growth to replace lost tissue. Cells must generate sufficient lipids, nucleotides, and proteins to fuel rapid cell division. To define metabolic pathways underlying regenerative growth, we undertake a multimodal investigation of metabolic reprogramming in Xenopus tropicalis appendage regeneration. Regenerating tissues have increased glucose uptake; however, inhibition of glycolysis does not decrease regeneration. Instead, glucose is funneled to the pentose phosphate pathway (PPP), which is essential for full tail regeneration. Liquid chromatography-mass spectrometry (LC-MS) metabolite profiling reveals increased nucleotide and nicotinamide intermediates required for cell division. Using single-cell RNA sequencing (scRNA-seq), we find that highly proliferative cells have increased transcription of PPP enzymes and not glycolytic enzymes. Further, PPP inhibition results in decreased cell division specifically in regenerating tissue. Our results inform a model wherein regenerating tissues direct glucose toward the PPP, yielding nucleotide precursors to drive regenerative cell proliferation.

Active learning narrows achievement gaps for underrepresented students in undergraduate science, technology, engineering, and math.

We tested the hypothesis that underrepresented students in active-learning classrooms experience narrower achievement gaps than underrepresented students in traditional lecturing classrooms, averaged across all science, technology, engineering, and mathematics (STEM) fields and courses. We conducted a comprehensive search for both published and unpublished studies that compared the performance of underrepresented students to their overrepresented classmates in active-learning and traditional-lecturing treatments. This search resulted in data on student examination scores from 15 studies (9,238 total students) and data on student failure rates from 26 studies (44,606 total students). Bayesian regression analyses showed that on average, active learning reduced achievement gaps in examination scores by 33% and narrowed gaps in passing rates by 45%. The reported proportion of time that students spend on in-class activities was important, as only classes that implemented high-intensity active learning narrowed achievement gaps. Sensitivity analyses showed that the conclusions are robust to sampling bias and other issues. To explain the extensive variation in efficacy observed among studies, we propose the heads-and-hearts hypothesis, which holds that meaningful reductions in achievement gaps only occur when course designs combine deliberate practice with inclusive teaching. Our results support calls to replace traditional lecturing with evidence-based, active-learning course designs across the STEM disciplines and suggest that innovations in instructional strategies can increase equity in higher education.

Morphogenesis of neurons and glia within an epithelium.

To sense the outside world, some neurons protrude across epithelia, the cellular barriers that line every surface of our bodies. To study the morphogenesis of such neurons, we examined the C. elegans amphid, in which dendrites protrude through a glial channel at the nose. During development, amphid dendrites extend by attaching to the nose via DYF-7, a type of protein typically found in epithelial apical ECM. Here, we show that amphid neurons and glia exhibit epithelial properties, including tight junctions and apical-basal polarity, and develop in a manner resembling other epithelia. We find that DYF-7 is a fibril-forming apical ECM component that promotes formation of the tube-shaped glial channel, reminiscent of roles for apical ECM in other narrow epithelial tubes. We also identify a requirement for FRM-2, a homolog of EPBL15/moe/Yurt that promotes epithelial integrity in other systems. Finally, we show that other environmentally exposed neurons share a requirement for DYF-7. Together, our results suggest that these neurons and glia can be viewed as part of an epithelium continuous with the skin, and are shaped by mechanisms shared with other epithelia.

Empirical assessment of the impact of sample number and read depth on RNA-Seq analysis workflow performance.

BACKGROUND: RNA-Sequencing analysis methods are rapidly evolving, and the tool choice for each step of one common workflow, differential expression analysis, which includes read alignment, expression modeling, and differentially expressed gene identification, has a dramatic impact on performance characteristics. Although a number of workflows are emerging as high performers that are robust to diverse input types, the relative performance characteristics of these workflows when either read depth or sample number is limited-a common occurrence in real-world practice-remain unexplored. RESULTS: Here, we evaluate the impact of varying read depth and sample number on the performance of differential gene expression identification workflows, as measured by precision, or the fraction of genes correctly identified as differentially expressed, and by recall, or the fraction of differentially expressed genes identified. We focus our analysis on 30 high-performing workflows, systematically varying the read depth and number of biological replicates of patient monocyte samples provided as input. We find that, in general for most workflows, read depth has little effect on workflow performance when held above two million reads per sample, with reduced workflow performance below this threshold. The greatest impact of decreased sample number is seen below seven samples per group, when more heterogeneity in workflow performance is observed. The choice of differential expression identification tool, in particular, has a large impact on the response to limited inputs. CONCLUSIONS: Among the tested workflows, the recall/precision balance remains relatively stable at a range of read depths and sample numbers, although some workflows are more sensitive to input restriction. At ranges typically recommended for biological studies, performance is more greatly impacted by the number of biological replicates than by read depth. Caution should be used when selecting analysis workflows and interpreting results from low sample number experiments, as all workflows exhibit poorer performance at lower sample numbers near typically reported values, with variable impact on recall versus precision. These analyses highlight the performance characteristics of common differential gene expression workflows at varying read depths and sample numbers, and provide empirical guidance in experimental and analytical design.

Microtubule Acetylation Is Required for Mechanosensation in Drosophila.

At the cellular level, α-tubulin acetylation alters the structure of microtubules to render them mechanically resistant to compressive forces. How this biochemical property of microtubule acetylation relates to mechanosensation remains unknown, although prior studies have shown that microtubule acetylation influences touch perception. Here, we identify the major Drosophila α-tubulin acetylase (dTAT) and show that it plays key roles in several forms of mechanosensation. dTAT is highly expressed in the larval peripheral nervous system (PNS), but it is largely dispensable for neuronal morphogenesis. Mutation of the acetylase gene or the K40 acetylation site in α-tubulin impairs mechanical sensitivity in sensory neurons and behavioral responses to gentle touch, harsh touch, gravity, and vibration stimuli, but not noxious thermal stimulus. Finally, we show that dTAT is required for mechanically induced activation of NOMPC, a microtubule-associated transient receptor potential channel, and functions to maintain integrity of the microtubule cytoskeleton in response to mechanical stimulation.

Prior activity of olfactory receptor neurons is required for proper sensory processing and behavior in Drosophila larvae.

Animal responses to their environment rely on activation of sensory neurons by external stimuli. In many sensory systems, however, neurons display basal activity prior to the external stimuli. This prior activity is thought to modulate neural functions, yet its impact on animal behavior remains elusive. Here, we reveal a potential role for prior activity in olfactory receptor neurons (ORNs) in shaping larval olfactory behavior. We show that prior activity in larval ORNs is mediated by the olfactory receptor complex (OR complex). Mutations of Orco, an odorant co-receptor required for OR complex function, cause reduced attractive behavior in response to optogenetic activation of ORNs. Calcium imaging reveals that Orco mutant ORNs fully respond to optogenetic stimulation but exhibit altered temporal patterns of neural responses. These findings together suggest a critical role for prior activity in information processing upon ORN activation in Drosophila larvae, which in turn contributes to olfactory behavior control.

TrpA1 activation in peripheral sensory neurons underlies the ionic basis of pain hypersensitivity in response to vinca alkaloids.

Chemotherapy induced peripheral neuropathy (CIPN), a side effect of many anti-cancer drugs including the vinca alkaloids, is characterized by a severe pain syndrome that compromises treatment in many patients. Currently there are no effective treatments for this pain syndrome except for the reduction of anti-cancer drug dose. Existing data supports the model that the pain associated with CIPN is the result of anti-cancer drugs augmenting the function of the peripheral sensory nociceptors but the cellular mechanisms underlying the effects of anti-cancer drugs on sensory neuron function are not well described. Studies from animal models have suggested a number of disease etiologies including mitotoxicity, axonal degeneration, immune signaling, and reduced sensory innervations but these outcomes are the result of prolonged treatment paradigms and do not necessarily represent the early formative events associated with CIPN. Here we show that acute exposure to vinca alkaloids results in an immediate pain syndrome in both flies and mice. Furthermore, we demonstrate that exposure of isolated sensory neurons to vinca alkaloids results in the generation of an inward sodium current capable of depolarizing these neurons to threshold resulting in neuronal firing. These neuronal effects of vinca alkaloids require the transient receptor potential ankyrin-1 (TrpA1) channel, and the hypersensitization to painful stimuli in response to the acute exposure to vinca alkaloids is reduced in TrpA1 mutant flies and mice. These findings demonstrate the direct excitation of sensory neurons by CIPN-causing chemotherapy drugs, and identify TrpA1 as an important target during the pathogenesis of CIPN.

Empirical assessment of analysis workflows for differential expression analysis of human samples using RNA-Seq.

BACKGROUND: RNA-Seq has supplanted microarrays as the preferred method of transcriptome-wide identification of differentially expressed genes. However, RNA-Seq analysis is still rapidly evolving, with a large number of tools available for each of the three major processing steps: read alignment, expression modeling, and identification of differentially expressed genes. Although some studies have benchmarked these tools against gold standard gene expression sets, few have evaluated their performance in concert with one another. Additionally, there is a general lack of testing of such tools on real-world, physiologically relevant datasets, which often possess qualities not reflected in tightly controlled reference RNA samples or synthetic datasets. RESULTS: Here, we evaluate 219 combinatorial implementations of the most commonly used analysis tools for their impact on differential gene expression analysis by RNA-Seq. A test dataset was generated using highly purified human classical and nonclassical monocyte subsets from a clinical cohort, allowing us to evaluate the performance of 495 unique workflows, when accounting for differences in expression units and gene- versus transcript-level estimation. We find that the choice of methodologies leads to wide variation in the number of genes called significant, as well as in performance as gauged by precision and recall, calculated by comparing our RNA-Seq results to those from four previously published microarray and BeadChip analyses of the same cell populations. The method of differential gene expression identification exhibited the strongest impact on performance, with smaller impacts from the choice of read aligner and expression modeler. Many workflows were found to exhibit similar overall performance, but with differences in their calibration, with some biased toward higher precision and others toward higher recall. CONCLUSIONS: There is significant heterogeneity in the performance of RNA-Seq workflows to identify differentially expressed genes. Among the higher performing workflows, different workflows exhibit a precision/recall tradeoff, and the ultimate choice of workflow should take into consideration how the results will be used in subsequent applications. Our analyses highlight the performance characteristics of these workflows, and the data generated in this study could also serve as a useful resource for future development of software for RNA-Seq analysis.

Trimming of sequence reads alters RNA-Seq gene expression estimates.

BACKGROUND: High-throughput RNA-Sequencing (RNA-Seq) has become the preferred technique for studying gene expression differences between biological samples and for discovering novel isoforms, though the techniques to analyze the resulting data are still immature. One pre-processing step that is widely but heterogeneously applied is trimming, in which low quality bases, identified by the probability that they are called incorrectly, are removed. However, the impact of trimming on subsequent alignment to a genome could influence downstream analyses including gene expression estimation; we hypothesized that this might occur in an inconsistent manner across different genes, resulting in differential bias. RESULTS: To assess the effects of trimming on gene expression, we generated RNA-Seq data sets from four samples of larval Drosophila melanogaster sensory neurons, and used three trimming algorithms--SolexaQA, Trimmomatic, and ConDeTri-to perform quality-based trimming across a wide range of stringencies. After aligning the reads to the D. melanogaster genome with TopHat2, we used Cuffdiff2 to compare the original, untrimmed gene expression estimates to those following trimming. With the most aggressive trimming parameters, over ten percent of genes had significant changes in their estimated expression levels. This trend was seen with two additional RNA-Seq data sets and with alternative differential expression analysis pipelines. We found that the majority of the expression changes could be mitigated by imposing a minimum length filter following trimming, suggesting that the differential gene expression was primarily being driven by spurious mapping of short reads. Slight differences with the untrimmed data set remained after length filtering, which were associated with genes with low exon numbers and high GC content. Finally, an analysis of paired RNA-seq/microarray data sets suggests that no or modest trimming results in the most biologically accurate gene expression estimates. CONCLUSIONS: We find that aggressive quality-based trimming has a large impact on the apparent makeup of RNA-Seq-based gene expression estimates, and that short reads can have a particularly strong impact. We conclude that implementation of trimming in RNA-Seq analysis workflows warrants caution, and if used, should be used in conjunction with a minimum read length filter to minimize the introduction of unpredictable changes in expression estimates.

Functions of the SLC36 transporter Pathetic in growth control.

Neurons exhibit extreme diversity in size, but whether large neurons have specialized mechanisms to support their growth is largely unknown. Recently, we identified the SLC36 transporter Pathetic (Path) as a factor required for extreme dendrite growth in neurons. Path is broadly expressed, but only neurons with large dendrite arbors or small neurons that are forced to grow large require path for their growth. To gain insight into the basis of growth control by path, we generated additional alleles of path and further examined the apparent specificity of growth defects in path mutants. Here, we confirm our prior finding that loss of path function imposes an upper limit on neuron growth, and additionally report that path likely limits overall neurite length rather than dendrite length alone. Using a GFP knock-in allele of path, we identify additional tissues where path likely functions in nutrient sensing and possibly growth control. Finally, we demonstrate that path regulates translational capacity in a cell type that does not normally require path for growth, suggesting that path may confer robustness on growth programs by buffering translational output. Altogether, these studies suggest that Path is a nutrient sensor with widespread function in Drosophila.

Bilateral consequences of chronic unilateral deafferentation in the auditory system of the cricket Gryllus bimaculatus.

The auditory system of the cricket has the unusual ability to respond to deafferentation by compensatory growth and synapse formation. Auditory interneurons such as ascending neuron 2 (AN-2) in the cricket Gryllus bimaculatus possess a dendritic arbor that normally grows up to, but not over, the midline of the prothoracic ganglion. After chronic deafferentation throughout larval development, however, the AN-2 dendritic arbor changes dramatically, and medial dendrites sprout across the midline where they form compensatory synapses with the auditory afferents from the contralateral ear. We quantified the extent of the effects of chronic, unilateral deafferentation by measuring several cellular parameters of 3 different neuronal components of the auditory system: the deafferented AN-2, the contralateral (or nondeafferented) AN-2 and the contralateral auditory afferents. Neuronal tracers and confocal microscopy were used to visualize neurons, and double-label experiments were performed to examine the cellular relationship between pairs of cells. Dendritic complexity was quantified using a modified Sholl analysis, and the length and volume of processes and presynaptic varicosities were assessed under control and deafferented conditions. Chronic deafferentation significantly influenced the morphology of all 3 neuronal components examined. The overall dendritic complexity of the deafferented AN-2 dendritic arbor was reduced, while both the contralateral AN-2 dendritic arbor and the remaining, intact, auditory afferents grew longer. We found no significant changes in the volume or density of varicosities after deafferentation. These complex cellular changes after deafferentation are interpreted in the light of the reported differential regulation of vesicle-associated membrane protein and semaphorin 2a.