GRANAR, a new computational tool to better understand the functional importance of root anatomy, bioRxiv, 2019-05-23

AbstractRoot hydraulic conductivity is an important determinant of plant water uptake capacity. In particular, the root radial conductivity is often thought to be a limiting factor along the water pathways between the soil and the leaf. The root radial conductivity is itself defined by cell scale hydraulic properties and anatomical features. However, quantifying the influence of anatomical features on the radial conductivity remains challenging due to complex, and time-consuming, experimental procedures.We present a new computation tool, the Generator of Root ANAtomy in R (GRANAR) that can be used to rapidly generate digital versions of root anatomical networks. GRANAR uses a limited set of root anatomical parameters, easily acquired with existing image analysis tools. The generated anatomical network can then be used in combination with hydraulic models to estimate the corresponding hydraulic properties.We used GRANAR to re-analyse large maize (Zea mays) anatomical datasets from the literature. Our model was successful at creating virtual anatomies for each experimental observation. We also used GRANAR to generate anatomies not observed experimentally, over wider ranges of anatomical parameters. The generated anatomies were then used to estimate the corresponding radial conductivities with the hydraulic model MECHA. This enabled us to quantify the effect of individual anatomical features on the root radial conductivity. In particular, our simulations highlight the large importance of the width of the stele and the cortex.GRANAR is an open-source project available here <jatsext-link xmlnsxlink=httpwww.w3.org1999xlink ext-link-type=uri xlinkhref=httpgranar.github.io>httpgranar.github.io<jatsext-link>One-Sentence summaryGenerator of Root ANAtomy in R (GRANAR) is a new open-source computational tool that can be used to rapidly generate digital versions of root anatomical networks.

biorxiv plant-biology 0-100-users 2019

Allelic polymorphism shapes community function in evolving Pseudomonas aeruginosa populations, bioRxiv, 2019-05-22

AbstractPseudomonas aeruginosa is an opportunistic pathogen that chronically infects the lungs of individuals with cystic fibrosis (CF) by forming antibiotic resistant biofilms. Emergence of phenotypically diverse isolates within CF P. aeruginosa populations has previously been reported, however, the impact of heterogeneity on social behaviors and community function is poorly understood. Here we describe how this heterogeneity impacts on behavioral traits by evolving the strain PAO1 in biofilms grown in a synthetic sputum medium for 50 days. We measured social trait production and antibiotic resistance, and used a metagenomic approach to analyze and assess genomic changes over the duration of the evolution experiment. We found that (i) evolutionary trajectories were reproducible in independently evolving populations; (ii) over 60% of genomic diversity occurred within the first 10 days of selection. We then focused on quorum sensing (QS), a well-studied P. aeruginosa trait that is commonly mutated in strains isolated from CF lungs. We found that at the population level (i) evolution in sputum medium selected for decreased production of QS and QS-dependent traits; (ii) there was a significant correlation between lasR mutant frequency, the loss of protease and the 3O-C12-HSL signal, and an increase in resistance to clinically relevant β-lactam antibiotics, despite no previous antibiotic exposure. Overall, our findings provide insights into the impact of allelic polymorphism on community functions in diverse P. aeruginosa populations. Further, we demonstrate that P. aeruginosa population and evolutionary dynamics can impact on traits important for virulence and can lead to increased tolerance to β-lactam antibiotics.SignificancePseudomonas aeruginosa is a major cause of chronic infection of the lungs in individuals with cystic fibrosis (CF). Epidemic P. aeruginosa strains dominate and displace others in the lung and become phenotypically and genotypically diverse over time. How this heterogeneity impacts on clinically relevant traits such as virulence or antibiotic resistance has received little attention. Here we demonstrate that the accumulation of genetic variants within heterogenous P. aeruginosa populations, results in population level changes in important traits including quorum sensing and antimicrobial resistance. Our work suggests that in the future, we should consider metagenomic and metaphenotypic assessments of P. aeruginosa populations collected from CF patients, rather than focusing on single random colonies isolated from infection.

biorxiv microbiology 0-100-users 2019

Next-generation sequencing of double stranded RNA is greatly improved by treatment with the inexpensive denaturing reagent DMSO, bioRxiv, 2019-05-21

AbstractDouble stranded RNA (dsRNA) is the genetic material of important viruses and a key component of RNA interference-based immunity in eukaryotes. Previous studies have noted difficulties in determining the sequence of dsRNA molecules that have affected studies of immune function and estimates of viral diversity in nature. Dimethyl sulfoxide (DMSO) has been used to denature dsRNA prior to the reverse transcription stage to improve RT-PCR and Sanger sequencing. We systematically tested the utility of DMSO to improve sequencing yield of a dsRNA virus (Φ6) in a short-read next generation sequencing platform. DMSO treatment improved sequencing read recovery by over two orders of magnitude, even when RNA and cDNA concentrations were below the limit of detection. We also tested the effects of DMSO on a mock eukaryotic viral community and found that dsRNA virus reads increased with DMSO treatment. Furthermore, we provide evidence that DMSO treatment does not adversely affect recovery of reads from a single-stranded RNA viral genome (Influenza ACalifornia072009). We suggest that up to 50% DMSO treatment be used prior to cDNA synthesis when samples of interest are composed of or may contain dsRNA.Data SummarySequence data was deposited in the NCBI Short Read Archive (accession numbers PRJNA527100, PRJNA527101, PRJNA527098). Data and code for analysis is available on GitHub (<jatsext-link xmlnsxlink=httpwww.w3.org1999xlink ext-link-type=uri xlinkhref=httpsgithub.comawilcox83dsRNA-sequencing>httpsgithub.comawilcox83dsRNA-sequencing<jatsext-link>. doi10.5281zenodo.1453423). Protocol for dsRNA sequencing is posted on protocols.io (doi10.17504protocols.io.ugnetve).

biorxiv microbiology 0-100-users 2019

Npas1+-Nkx2.1+ Neurons Are an Integral Part of the Cortico-pallido-cortical Loop, bioRxiv, 2019-05-21

AbstractWithin the basal ganglia circuit, the external globus pallidus (GPe) is critically involved in motor control. Aside from Foxp2+ neurons and ChAT+ neurons that have been established as unique neuron types, there is little consensus on the classification of GPe neurons. Properties of the remaining neuron types are poorly-defined. In this study, we leverage new mouse lines, viral tools, and molecular markers to better define GPe neuron subtypes. We found that Sox6 represents a novel, defining marker for GPe neuron subtypes. Lhx6+ neurons that lack the expression of Sox6 were devoid of both parvalbumin and Npas1. This result confirms previous assertions of the existence of a unique Lhx6+ population. Neurons that arise from the Dbx1+ lineage were similarly abundant in the GPe and displayed a heterogeneous makeup. Importantly, tracing experiments revealed that Npas1+-Nkx2.1+ neurons represent the principal non-cholinergic, cortically-projecting neurons. In other words, they form the pallido-cortical arm of the cortico-pallido-cortical loop. Our data further described that pyramidal-tract neurons in the cortex collateralized within the GPe, forming a closed-loop system between the two brain structures. Overall, our findings reconcile some of the discrepancies that arose from differences in techniques or the reliance on pre-existing tools. While spatial distribution and electrophysiological properties of GPe neurons reaffirm the diversification of GPe subtypes, statistical analyses strongly support the notion that these neuron subtypes can be categorized under the two principal neuron classes—i.e., PV+ neurons and Npas1+ neurons.Significance statementThe poor understanding of the neuronal composition in the GPe undermines our ability to interrogate its precise behavioral and disease involvements. In this study, twelve different genetic crosses were used, hundreds of neurons were electrophysiologically-characterized, and over 100,000 neurons were histologically- andor anatomically-profiled. Our current study further establishes the segregation of GPe neuron classes and illustrates the complexity of GPe neurons in adult mice. Our results support the idea that Npas1+-Nkx2.1+ neurons are a distinct GPe neuron subclass. By providing a detailed analysis of the organization of the cortico-pallidal-cortical projection, our findings establish the cellular and circuit substrates that can be important for motor function and dysfunction.

biorxiv neuroscience 0-100-users 2019

 

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