bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased severity of closed head injury or repetitive subconcussive head impacts enhances post-traumatic headache-like behaviors in a rat model
Authors: Bree Dara , Stratton Jennifer , Levy Dan
Published: March 2020

Introduction

Posttraumatic headache (PTH) is one of the most common, debilitating and difficult symptoms to manage after a traumatic head injury. The development of novel therapeutic approaches is nevertheless hampered by the paucity of preclinical models and poor understanding of the mechanisms underlying PTH. To address these shortcomings, we previously characterized the development of PTH-like pain behaviors in rats subjected to a single mild closed head injury using a 250 g weight drop. Here, we conducted a follow-up study to further develop this preclinical model by exploring the development of headache-like pain behaviors in male rats subjected to a single, but more severe head trauma (450 g) as well as following repetitive, subconcussive head impacts (150 g). In addition, we tested whether these behaviors involve peripheral CGRP signaling by testing the effect of systemic anti-CGRP monoclonal antibody (anti-CGRP mAb).

Methods

Adult male Sprague Dawley rats (total n=138) were subjected to diffuse closed head injury using a weight-drop device, or a sham procedure. Three injury paradigms were employed: a single hit, using 450 g or 150 g weight drop, and three successive 150 g weight drop events conducted 72 hours apart. Changes in open field activity and development of headache-related cephalic and extracephalic mechanical pain hypersensitivity were assessed up to 42 days post head trauma. Treatment included systemic administration of a mouse anti-calcitonin-gene-related peptide monoclonal antibody (30 mg/kg.).

Results

Rats subjected to 450 g closed head injury displayed an acute decrease in rearing and increased thigmotaxis, together with cephalic and extracephalic mechanical pain hypersensitivity that resolved by 6 weeks post-injury. Repetitive subconcussive head impacts using the 150 g weight drop, but not a single event, led to decreased vertical rearing as well as prolonged cephalic and extracephalic mechanical pain hypersensitivity. Early and prolonged anti-CGRP mAb treatment inhibited the development of the cephalic, but not extracephalic pain hypersensitivities in both the severe and repetitive subconcussive head impact models.

Conclusions

When compared to the data obtained from male rats in the previous study, a more severe head injury gives rise to a prolonged state of cephalic and extracephalic hyperalgesia. Such enhanced headache-like behaviors also occur following repetitive, subconcussive head impacts. Extended headache-like behaviors following severe and repetitive mild closed head injury are ameliorated by early and prolonged anti-CGRP mAb treatment, suggesting a mechanism linked to peripheral CGRP signaling.
Escherichia colimetabolism under short-term repetitive substrate dynamics: Adaptation and trade-offs
Authors: Vasilakou Eleni , van Loosdrecht Mark C. M. , Wahl S. Aljoscha
Published: March 2020

Background

Microbial metabolism is highly dependent on the environmental conditions. Especially, the substrate concentration, as well as oxygen availability, determine the metabolic rates. In large-scale bioreactors, microorganisms encounter dynamic conditions in substrate and oxygen availability (mixing limitations), which influence their metabolism and subsequently their physiology. Earlier, single substrate pulse experiments were not able to explain the observed physiological changes generated under large-scale industrial fermentation conditions.

Results

In this study we applied a repetitive feast-famine regime in an aerobicEscherichia coliculture in a time-scale of seconds. The regime was applied for several generations, allowing cells to adapt to the (repetitive) dynamic environment. The observed response was highly reproducible over the cycles, indicating that cells were indeed fully adapted to the regime. We observed an increase of the specific substrate and oxygen consumption (average) rates during the feast-famine regime, compared to a steady-state (chemostat) reference environment. The increased rates at same (average) growth rate led to a reduced biomass yield (30% lower). Interestingly, this drop was not followed by increased by-product formation, pointing to the existence of energy-spilling reactions and/or less effective ATP synthesis. During the feast-famine cycle, the cells rapidly increased their uptake rate. Within 10 seconds after the beginning of the feeding, the substrate uptake rate was higher (4.68 μmol/gCDW/s) than reported during batch growth (3.3 μmol/gCDW/s). The high uptake led to an accumulation of several intracellular metabolites, during the feast phase, accounting for up to 34 % of the carbon supplied. Although the metabolite concentrations changed rapidly, the cellular energy charge remained unaffected, suggesting well-controlled balance between ATP producing and ATP consuming reactions. The role of inorganic polyphosphate as an energy buffer is discussed.

Conclusions

The adaptation of the physiology and metabolism ofEscherichia coliunder substrate dynamics, representative for large-scale fermenters, revealed the existence of several cellular mechanisms coping with stress. Changes in the substrate uptake system, storage potential and energy-spilling processes resulted to be of great importance. These metabolic strategies consist a meaningful step to further tackle reduced microbial performance, observed under large-scale cultivations.
Evidence for face selectivity in early vision
Authors: Campana Florence , Martin Jacob G. , Bokeria Levan , Thorpe Simon , Jiang Xiong , Riesenhuber Maximilian
Published: March 2020

The commonly accepted “simple-to-complex” model of visual processing in the brain posits that visual tasks on complex objects such as faces are based on representations in high-level visual areas. Yet, recent experimental data showing the visual system’s ability to localize faces in natural images within 100ms (Crouzet et al., 2010) challenge the prevalent hierarchical description of the visual system, and instead suggest the hypothesis of face-selectivity in early visual areas. In the present study, we tested this hypothesis with human participants in two eye tracking experiments, an fMRI experiment and an EEG experiment. We found converging evidence for neural representations selective for upright faces in V1/V2, with latencies starting around 40 ms post-stimulus onset. Our findings suggest a revision of the standard “simple-to-complex” model of hierarchical visual processing.

Significance statement

Visual processing in the brain is classically described as a series of stages with increasingly complex object representations: early visual areas encode simple visual features (such as oriented bars), and high-level visual areas encode representations for complex objects (such as faces). In the present study, we provide behavioral, fMRI, and EEG evidence for representations of complex objects – namely faces – in early visual areas. Our results challenge the standard “simple-to-complex” model of visual processing, suggesting that it needs to be revised to include neural representations for faces at the lowest levels of the visual hierarchy. Such early object representations would permit the rapid and precise localization of complex objects, as has previously been reported for the object class of faces.
Simplified model assumptions artificially constrain the parameter range in which selection at the holobiont level can occur
Authors: Daybog Itay , Kolodny Oren
Published: March 2020

Van Vliet & Doebeli (1) (henceforth VVD) present a multi-level selection framework simulating host-microbiome evolutionary dynamics. The model explores the conditions under which the association between hosts and their “helper” microbiome – microbes that developed a trait that provides a benefit to the host at a cost to themselves – is strong enough to allow for selection at the holobiont level. The model is an important contribution to the holobiont debate, making the notion quantifiable and highlighting the involved parameters.

In the study, VVD conclude that the parameter space in which selection occurs at the level of the holobiont is extremely restricted (Fig 1). Here we show that this result stems from specific model assumptions. Although these assumptions are reasonable as a modeling starting point, slight biologically-reasonable modifications of the assumptions lead to qualitatively different outcomes.
Integrated Molecular Characterisation of the MAPK Pathways in Human Cancers
Authors: Sinkala Musalula , Nkhoma Panji , Mulder Nicola , Martin Darren Patrick
Published: March 2020

The mitogen-activated protein kinase (MAPK) pathways are a crucial regulator of the cellular processes that fuel the malignant transformation of normal cells. The genetic underpinnings of molecular aberrations which lead to cancer involve mutations in and, transcription variations of, various MAPK pathway genes. Here, we use datasets of 40,848 patient-derived tumours representing 101 distinct human cancers to identify cancer-associated mutations in MAPK signalling pathway genes. We identify the subset of these genes within which mutations tend to be associated with the worst disease outcomes. Furthermore, by integrating information extracted from various large-scale molecular datasets, we expose the relationship between the fitness of cancer cells after CRISPR mediated gene knockout of MAPK pathway genes, and their dose-responses to MAPK pathway inhibitors. Besides providing new insights into MAPK pathways, we unearth vulnerabilities in specific pathway genes that are reflected in the responses of cancer cells to MAPK drug perturbations: a revelation with great potential for guiding the development of innovative therapeutic strategies.
Locus coeruleus MRI contrast is associated with cortical thickness in older adults
Authors: Bachman Shelby L. , Dahl Martin J. , Werkle-Bergner Markus , Düzel Sandra , Forlim Caroline Garcia , Lindenberger Ulman , Kühn Simone , Mather Mara
Published: March 2020

There is growing evidence that neuronal integrity of the noradrenergic locus coeruleus (LC) is important for later-life cognition. Less understood is how LC integrity relates to brain correlates of cognition, such as brain structure. Here, we examined the relationship between cortical thickness and a measure reflecting LC integrity in older (n = 229) and younger adults (n = 67). Using a magnetic resonance imaging sequence which yields high signal intensity in the LC, we assessed the contrast between signal intensity of the LC and that of neighboring pontine reference tissue. The Freesurfer software suite was used to quantify cortical thickness. LC contrast was positively related to cortical thickness in older adults, and this association was prominent in parietal, frontal, and occipital regions. Brain regions where LC contrast was related to cortical thickness include portions of the frontoparietal network which have been implicated in noradrenergically modulated cognitive functions. These findings provide novel evidence for a link between LC structure and cortical brain structure in later adulthood.
Semantic and syntactic composition of minimal adjective-noun phrases in Dutch: an MEG study
Authors: Kochari Arnold , Lewis Ashley , Schoffelen Jan-Mathijs , Schriefers Herbert
Published: March 2020

The possibility to combine smaller units of meaning (e.g., words) to create new and more complex meanings (e.g., phrases and sentences) is a fundamental feature of human language. In the present project, we investigated how the brain supports the semantic and syntactic composition of two-word adjective-noun phrases in Dutch, using magnetoencephalography (MEG). The present investigation followed up on previous studies reporting a composition effect in the left anterior temporal lobe (LATL) when comparing neural activity at nouns combined with adjectives, as opposed to nouns in a non-compositional context. The first aim of the present study was to investigate whether this effect, as well as its modulation by noun specificity and adjective class, can also be observed in Dutch. A second aim was to investigate to what extent these effects may be driven by syntactic composition rather than primarily by semantic composition as was previously proposed. To this end, a novel condition was administered in which participants saw nouns combined with pseudowords lacking meaning but agreeing with the nouns in terms of grammatical gender, as real adjectives would. We failed to observe a composition effect or its modulation in both a confirmatory analysis (focused on the cortical region and time-window where it has previously been reported) and in exploratory analyses (where we tested multiple regions and an extended potential time-window of the effect). A syntactically driven composition effect was also not observed in our data. We do, however, successfully observe an independent, previously reported effect on single word processing in our data, confirming that our MEG data processing pipeline does meaningfully capture language processing activity by the brain. The failure to observe the composition effect in LATL is surprising given that it has been previously reported in multiple studies. Reviewing all previous studies investigating this effect, we propose that materials and a task involving imagery might be necessary for this effect to be observed. In addition, we identified substantial variability in the regions of interest analysed in previous studies, which warrants additional checks of robustness of the effect. Further research should identify limits and conditions under which this effect can be observed. The failure to observe specifically a syntactic composition effect in such minimal phrases is less surprising given that it has not been previously reported in MEG data.
Comparing basal dendrite branches in human and mouse hippocampal CA1 pyramidal neurons with Bayesian networks
Authors: Mihaljević Bojan , Larrañaga Pedro , Benavides-Piccione Ruth , DeFelipe Javier , Bielza Concha
Published: March 2020

Pyramidal neurons are the most common cell type in the cerebral cortex. Understanding how they differ between species is a key challenge in neuroscience. A recent study provided a unique set of human and mouse pyramidal neurons of the CA1 region of the hippocampus, and used it to compare the morphology of apical and basal dendritic branches of the two species. The study found inter-species differences in the magnitude of the morphometrics and similarities regarding their variation with respect to morphological determinants such as branch type and branch order. We use the same data set to perform additional comparisons of basal dendrites. In order to isolate the heterogeneity due to intrinsic differences between species from the heterogeneity due to differences in morphological determinants, we fit multivariate models over the morphometrics and the determinants. In particular, we use conditional linear Gaussian Bayesian networks, which provide a concise graphical representation of the independencies and correlations among the variables. We also extend the previous study by considering additional morphometrics and by formally testing test whether a morphometric increases or decreases with the distance from the soma. This study introduces a multivariate methodology for inter-species comparison of morphology.
SNAPflex: a paper-and-plastic device for instrument-free RNA and DNA extraction from whole blood
Authors: Kolluri Nikunja , Albarran Nikolas , Fan Andy , Olson Alex , Sagar Manish , Young Anna , Gomez-Marquez José , Klapperich Catherine M.
Published: March 2020

Nucleic acid amplification tests (NAATs), which amplify and detect pathogen nucleic acids, are vital methods to diagnose diseases, particularly in cases where patients exhibit low levels of infection. For many blood-borne pathogens such as HIV or Plasmodium, it is necessary to first extract pathogen RNA or DNA from patient blood prior to analysis with NAATs. Traditional nucleic acid extraction methods are expensive, resource-intensive and are often difficult to deploy to resource-limited areas where many blood-borne infections are widespread. Here, we describe a portable, paper-and-plastic device for instrument-free nucleic acid extraction from whole blood, which we call SNAPflex, that builds upon our previous work extracting RNA in a 2D platform from nasopharyngeal swabs. We demonstrated improved extraction of HIV RNA from simulated patient samples compared to traditional extraction methods and long-term stability of extracted RNA without the need for cold storage. We further demonstrated successful extraction and recovery of Plasmodium falciparum DNA from simulated patient samples with superior recovery compared to existing extraction methods. The SNAPflex device extracts and purifies DNA and RNA from whole blood which can be amplified with traditional NAATs, and was designed to easily manufacture and integrate into existing health systems.
Genome editing in mammals using CRISPR type I-D nuclease
Authors: Osakabe Keishi , Wada Naoki , Murakami Emi , Osakabe Yuriko
Published: March 2020

Adoption of the CRISPR-Cas system has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I—the most abundant CRISPR system in bacteria—has been less developed. Type I systems in which Cas3 nuclease degrades the target DNA are known; in contrast, for the sub-type CRISPR type I-D (TiD), which lacks a typical Cas3 nuclease in its cascade, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d—a nuclease in TiD—is multi-functional in PAM recognition, stabilization and target DNA degradation. TiD can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. TiD off-target effects, which were dependent on the mismatch position in the protospacer of TiD, were also identified. Our findings suggest TiD as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells.
CHD8Suppression Impacts on Histone H3 Lysine 36 Trimethylation and Alters RNA Alternative Splicing
Authors: Kerschbamer Emanuela , Tripathi Takshashila , Erdin Serkan , Salviato Elisa , Di Leva Francesca , Sebestyén Endre , Arnoldi Michele , Benelli Matteo , Gusella James F. , Piazza Silvano , Demichelis Francesca , Talkowski Michael E. , Ferrari Francesco , Biagioli Marta
Published: March 2020

Disruptive mutations in the chromodomain helicase DNA binding protein 8 (CHD8) have been recurrently associated with Autism Spectrum Disorders (ASD). In normal cellular physiology, CHD8 co-purifies with MLL1 and MOF transcriptional activation complex, with elongating RNAPII and directly binds to DNA promoters and enhancers regions, thus a regulatory role in transcriptional initiation and elongation could be postulated.

Here we investigated how chromatin landscape reacts toCHD8suppression by analyzing a panel of histone modifications in induced pluripotent stem cell-derived neural progenitors. We interrogated transcriptionally active and repressed regions, as well as active and poised enhancers.

CHD8suppression led to significant reduction (47.82%) in histone H3K36me3 peaks at gene bodies, particularly impacting on transcriptional elongation chromatin states. H3K36me3 reduction specifically affects highly expressed, CHD8-bound genes. Histone H3K36me3 reduction associated toCHD8-suppression does not functionally impact on global transcriptional levels, but correlated with altered alternative splicing patterns of ∼ 2000 protein coding genes implicated in “RNA splicing”, “mitotic cell cycle phase transition” and “mRNA processing”, especially affecting alternative first exon and exon skipping events.

In summary, our results point toward broad molecular consequences ofCHD8suppression, implicating altered histone deposition/maintenance and RNA processing regulation as important regulatory processes in ASD.
Rational Design of Aptamer Switches with Programmable pH Response
Authors: Thompson Ian A.P. , Zheng Liwei , Eisenstein Michael , Soh H. Tom
Published: March 2020

Aptamer switches that respond sensitively to local variations in pH could enable the development of molecular devices that greatly enhance diagnostic accuracy and therapeutic efficacy. Previous design strategies relied on inserting a known pH-sensitive DNA motif into the aptamer structure. Unfortunately, their performance was ultimately governed by the intrinsic pH-response of the inserted motif and could not be readily tuned to operate across arbitrary pH ranges. In this work, we present a general methodology for converting virtually any aptamer into a pH-responsive switch that can be modulated to undergo a strong change in affinity - in acidic, neutral, or alkaline conditions. This was achieved by inserting two orthogonal motifs that can be manipulated in parallel to tune sensitivity to different pH conditions without altering the core sequence of the aptamer itself. Using a single ATP aptamer, we achieve pH-dependent target binding and release under diverse pH conditions, producing as much as 1,000-fold pH-induced change in affinity. Importantly, we also demonstrate the novel capability to predictably engineer aptamer switches that only exhibit strong target affinity within a narrowly defined pH range. Our approach should offer a highly generalizable strategy for integrating pH-responsiveness into molecular devices.
Histone dynamics within the nucleosome play a critical role in SNF2h-mediated nucleosome sliding
Authors: Gamarra Nathan , Narlikar Geeta J.
Published: March 2020

Elucidating the mechanisms by which ATP-dependent chromatin remodeling enzymes disrupt nucleosome structure is essential to understanding how chromatin states are established and maintained. A key finding informing remodeler mechanism is the observation that the dynamics of protein residues buried within the histone core of the nucleosome are used by specific remodelers to mobilize the nucleosome1. Recently, a study obtaining cryo-electron microscopy (cryo-EM) structures of ISWI-family remodelernucleosome complexes failed to observe stable conformational rearrangements in the histone octamer2. The authors of this study also failed to replicate the earlier finding that site-specifically restraining histone dynamics inhibits nucleosome sliding by ISWI-family remodelers1,2. In contrast, a recent cryo-EM structure detected asymmetric histone dynamics in an ISWI-nucleosome complex3. Here, using two different protocols, we replicate the original finding in Sinha et al.1that dynamics within the histone core are important for nucleosome sliding by the human ISWI remodeler, SNF2h. These results firmly establish histone dynamics as an essential feature of ISWI-mediated nucleosome sliding and highlight the care required in designing and performing biochemical experiments investigating nucleosome dynamics using disulfide linkages.
Efferent Synaptic Transmission at the Vestibular Type II Hair Cell Synapse
Authors: Yu Zhou , McIntosh J. Michael , Sadeghi Soroush , Glowatzki Elisabeth
Published: March 2020

In the vestibular peripheral organs, type I and type II hair cells (HCs) transmit incoming signals via glutamatergic quantal transmission onto afferent nerve fibers. Additionally, type I HCs transmit via ‘non-quantal’ transmission to calyx afferent fibers, by accumulation of glutamate and potassium in the synaptic cleft. Vestibular efferent inputs originating in the brainstem contact type II HCs and vestibular afferents. Here, we aimed at characterizing the synaptic efferent inputs to type II HCs using electrical and optogenetic stimulation of efferent fibers combined within vitrowhole-cell patch clamp recording from type II HCs in the rodent vestibular crista. Properties of efferent synaptic currents in type II HCs were similar to those found in cochlear hair cells and mediated by activation of α9/α10 nicotinic acetylcholine receptors (AChRs) and SK potassium channels. While efferents showed a low probability of release at low frequencies of stimulation, repetitive stimulation resulted in facilitation and increased probability of release. Notably, the membrane potential of type II HCs measured during optogenetic stimulation of efferents showed a strong hyperpolarization even in response to single pulses and was further enhanced by repetitive stimulation. Such efferent-mediated inhibition of type II HCs can provide a mechanism to adjust the contribution of signals from type I and type II HCs to vestibular nerve fibers. As a result, the relative input of type I hair cells to vestibular afferents will be strengthened, emphasizing the phasic properties of the incoming signal that are transmitted via fast non-quantal transmission.

New and Noteworthy

Type II vestibular hair cells (HCs) receive inputs from efferent fibers originating in the brainstem. We usedin vitrooptogenetic and electrical stimulation of efferent fibers to study their synaptic inputs to type II HCs. Efferent inputs inhibited type II HCs, similar to cochlear efferent effects. We propose that efferent inputs adjust the contribution of signals from type I and type II HCs that report different components of the incoming signal to vestibular nerve fibers.
Structures of a P4-ATPase lipid flippase in lipid bilayers
Authors: He Yilin , Xu Jinkun , Wu Xiaofei , Li Long
Published: March 2020

Type 4 P-type ATPases (P4-ATPases) are a group of key enzymes maintaining lipid asymmetry of eukaryotic membranes. Phospholipids are actively and selectively flipped by P4-ATPases from the exoplasmic leaflet to the cytoplasmic leaflet. How lipid flipping is coupled with ATP-hydrolysis by P4-ATPases is poorly understood. Here, we report the electron cryo-microscopy structures of a P4-ATPase, Dnf1-Cdc50 fromChaetomium thermophilum, which had been reconstituted into lipid nanodiscs and captured in two transport intermediate states. The structures reveal that transmembrane segment 1 of Dnf1 becomes highly flexible during lipid transport. The local lipid bilayers are distorted to facilitate the entry of the phospholipid substrates from the exoplasmic leaflet to a cross-membrane groove. During transport, the lipid substrates are relayed through four binding sites in the groove which constantly shields the lipid polar heads away from the hydrophobic environment of the membranes.
Identification and functional analysis of long non-coding RNAs in autism spectrum disorders
Authors: Tong Zhan , Zhou Yuan , Wang Juan
Published: March 2020

Background

Genetic and environmental factors, alone or in combination, contribute to the pathogenesis of autism spectrum disorder (ASD). Although many protein-coding genes have now been identified as disease risk genes for ASD, a detailed illustration of long non-coding RNAs (lncRNAs) associated with ASD remains elusive. In this study, our aim was to identify ASD-related lncRNAs and explore their functions and associated biological pathways in autism.

Methods

ASD-related lncRNAs were identified based on genomic variant data of individuals with ASD from a twin study, and further validated using an independent copy number variant (CNV) dataset. The functions and associated biological pathways of ASD-related lncRNAs were explored by enrichment analysis of three different types of functional neighbor genes (i.e. genomic neighbors, competing endogenous RNA (ceRNA) neighbors and gene co-expression neighbors in the cortex). The differential functions of ASD-related lncRNAs in distinct brain regions were demonstrated by using gene co-expression network analysis based on tissue-specific gene expression profiles. Moreover, a functional network analysis were conducted for highly reliable functional neighbor genes of ASD-related lncRNAs. Finally, several potential drugs were predicted based on the enrichment of drug-induced pathway sets in ASD-altered biological pathway list.

Results

In total, 532 ASD-related lncRNAs were identified, and 86.7% of these ASD-related lncRNAs were further validated by a copy number variant (CNV) dataset. Most of functional neighbor genes of ASD-related lncRNAs were enriched in several functions and biological pathways, including nervous system development, inflammatory response and transcriptional regulation. As a set, ASD-related lncRNAs were mainly associated with nervous system development and dopaminergic synapse in the cortex, but associated with transcriptional regulation in the cerebellum. Moreover, all highly reliable functional neighbor genes were connected in a single functional network. Finally, several potential drugs were predicted and partly supported by the previous reports.

Conclusions

We concluded that ASD-related lncRNAs participate in the pathogenesis of ASD through various known biological pathways, which may be differential in distinct brain regions. And detailed investigation of ASD-related lncRNAs also provided clues for developing potential ASD diagnosis biomarker and therapy.
Structural and developmental principles of neuropil assembly inC. elegans
Authors: Moyle Mark W. , Barnes Kristopher M. , Kuchroo Manik , Gonopolskiy Alex , Duncan Leighton H. , Sengupta Titas , Shao Lin , Guo Min , Santella Anthony , Christensen Ryan , Kumar Abhishek , Wu Yicong , Moon Kevin R. , Wolf Guy , Krishnaswamy Smita , Bao Zhirong , Shroff Hari , Mohler William , Colón-Ramos Daniel A.
Published: March 2020

Neuropil is a fundamental form of tissue organization within brains1. In neuropils, densely packed neurons synaptically interconnect into precise circuit architecture2,3, yet the structural and developmental principles governing nanoscale precision in bundled neuropil assembly remain largely unknown4–6. Here we use diffusion condensation, a coarse-graining clustering algorithm7, to identify nested circuit structures within theC. eleganscerebral neuropil (called the nerve ring). We determine that the nerve ring neuropil is organized into four tightly bundled strata composed of related behavioral circuits. We demonstrate that the stratified architecture of the neuropil is a geometrical representation of the functional segregation of sensory information and motor outputs, with specific sensory organs and muscle quadrants mapping onto particular neuropil strata. We identify groups of neurons with unique morphologies that integrate information across strata and that create a sophisticated honeycomb-shaped scaffold that encases the strata within the nerve ring. We resolve the developmental sequence leading to stratified neuropil organization through the integration of lineaging and cell tracking algorithms with high resolution light-sheet microscopy, and reveal principles of cell position, migration and hierarchical outgrowth that guide neuropil organization. Our results uncover conserved design principles underlying nerve ring neuropil architecture and function, and a pioneer neuron-based, temporal progression of outgrowth that guides the hierarchical development of the layered neuropil. Our findings provide a blueprint for using structural and developmental approaches to systematically understand neuropil organization within brains.
NOSA, an analytical toolbox for multicellular optical electrophysiology
Authors: Oltmanns Sebastian , Abben Frauke Sophie , Ender Anatoli , Aimon Sophie , Kovacs Richard , Sigrist Stephan J , Storace Douglas A , Geiger Jörg R P , Raccuglia Davide
Published: March 2020

Understanding how neural networks generate activity patterns and communicate with each other requires monitoring the electrical activity from many neurons simultaneously. Perfectly suited tools for addressing this challenge are genetically encoded voltage indicators (GEVIs) because they can be targeted to specific cell types and optically report the electrical activity of individual, or populations of neurons. However, analyzing and interpreting the data from voltage imaging experiments is challenging because high recording speeds and properties of current GEVIs yield only low signal-to-noise ratios, making it necessary to apply specific analytical tools. Here, we present NOSA (Neuro-Optical Signal Analysis), a novel open source software designed for analyzing voltage imaging data and identifying temporal interactions between electrical activity patterns of different origin.

In this manuscript we explain the challenges that arise during voltage imaging experiments and provide hands-on analytical solutions. We demonstrate how NOSA’s baseline fitting, filtering algorithms and movement correction can compensate for shifts in baseline fluorescence and extract electrical patterns from low signal-to-noise recordings. Moreover, NOSA contains powerful features to identify oscillatory frequencies in electrical patterns and extract neuronal firing characteristics. NOSA is the first open-access software to provide an option for analyzing simultaneously recorded optical and electrical data derived from patch-clamp or other electrode-based recordings. To identify temporal relations between electrical activity patterns we implemented different options to perform cross correlation analysis, demonstrating their utility during voltage imaging inDrosophilaand mice. All features combined, NOSA will facilitate the first steps into using GEVIs and help to realize their full potential for revealing cell-type specific connectivity and functional interactions. If you would like to test NOSA, please send an email to the lead contact.
The antidepressant effects of asperosaponin VI are mediated by the suppression of microglial activation and reduction of TLR4/NF-ĸB induced IDO expression
Authors: Zhang Jinqiang , Yi Saini , Li Yahui , Xiao Chenghong , Liu Chan , Jiang Weike , Yang Changgui , Zhou Tao
Published: March 2020

Aim

Indoleamine 2, 3-dioxygenase (IDO) is responsible for the progression of the kynurenine pathway, which has been implicated in the pathophysiology of inflammation-induced depression. It has been reported that asperosaponin VI (ASA VI) could play a neuroprotective role through anti-inflammatory and antioxidant. In this study, we examined the antidepressant effect of ASA VI in LPS-treated mice and further explored its molecular mechanism by insight into the microglial kynurenine pathway.

Methods

To produce the model, lipopolysaccharide (LPS) (0.83 mg/kg) was administered intraperitoneally to mice. The mice received ASA VI (10 mg/kg, 20mg/kg, 40mg/kg and 80mg/kg, i.p.) thirty minutes prior to LPS injection. Depressive-like behaviors were evaluated based on the duration of immobility in the forced swim test. Microglial activation and inflammatory cytokines were detected by immunohistochemistry, real-time PCR and ELISA. The TLR4/NF-ĸB signaling pathway and the expression of IDO, GluA2, and CamKIIβ were measured by western blotting.

Results

ASA VI demonstrated significant antidepressant activity in the presence of LPS on immobility and latency times in the forced swim test. The LPS-induced activation of microglia and inflammatory response were inhabited by ASA VI in a dose-dependent manner. TLR4/NF-κB signaling pathway also was suppressed by ASA VI in the hippocampus and prefrontal cortex of LPS-treated mice. Furthermore, ASA VI inhibited the increase in IDO protein expression and normalized the aberrant glutamate transmission in the hippocampus and prefrontal cortex as a result of LPS administration.

Conclusion

Our results propose a promising antidepressant effect for ASA VI possibly through the downregulation of IDO expression and normalization of the aberrant glutamate transmission. This remedying effect of ASA VI could be attributed to suppress microglia-mediated neuroinflammatory response via inhibiting the TLR4/NF-κB signaling pathway.
Cropping systems impact changes in soil fungal, but not prokaryote, alpha-diversity and community composition stability over a growing season in a long-term field trial
Authors: Finn D.R. , Lee S. , Lanzén A. , Bertrand M. , Nicol G.W. , Hazard C.
Published: March 2020

Crop harvest followed by a fallow period can act as a disturbance on soil microbial communities. Cropping systems intended to improve alpha-diversity of communities may also confer increased compositional stability during succeeding growing seasons. Over a single growing season in a long-term (18 year) agricultural field experiment incorporating conventional (CON), conservation (CA), organic (ORG) and integrated (INT) cropping systems, temporal changes in prokaryote, fungal and arbuscular mycorrhizal fungi (AMF) communities were investigated overwinter, during crop growth and at harvest. While certain prokaryote phyla were influenced by cropping system (e.g. Acidobacteria), the community as a whole was primarily driven by temporal changes over the growing season as distinct overwinter and crop-associated communities, with the same trend observed regardless of cropping system. Species- rich prokaryote communities were most stable over the growing season. Cropping system exerted a greater effect on fungal communities, with alpha-diversity highest and temporal changes most stable under CA. CON was particularly detrimental for alpha-diversity in AMF communities, with AMF alpha-diversity and stability improved under all other cropping systems. Practices that promoted alpha-diversity tended to also increase the similarity and temporal stability of soil fungal (and AMF) communities during a growing season, while prokaryote communities were largely insensitive to management.
Structure of a Single Chain H2A/H2B Dimer
Authors: Warren Christopher , Bonanno Jeffrey B. , Almo Steven C. , Shechter David
Published: March 2020

Chromatin is the complex assembly of nucleic acids and proteins that makes up the physiological form of the eukaryotic genome. The nucleosome is the fundamental repeating unit of chromatin, composed of ~147bp of DNA wrapped around a histone octamer formed by two copies of each core histone: H2A, H2B, H3 and H4. Prior to nucleosome assembly, and during histone eviction, histones are typically assembled into soluble H2A/H2B dimers and H3/H4 dimers and tetramers. A multitude of factors interact with soluble histone dimers and tetramers, including chaperones, importins, histone modifying enzymes, and chromatin remodeling enzymes. It is still unclear how many of these proteins recognize soluble histones; therefore, there is a need for new structural tools to study non-nucleosomal histones. Here we created a single-chain, taillessXenopusH2A/H2B dimer by directly fusing the C-terminus of H2B to the N-terminus of H2A. We show that this construct (termed scH2BH2A) is readily expressed in bacteria and can be purified under non-denaturing conditions. A 1.31Å crystal structure of scH2BH2A shows that it adopts a conformation nearly identical to nucleosomal H2A/H2B. This new tool will facilitate future structural studies of a multitude of H2A/H2B-interacting proteins.
A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy
Authors: McGrath Kaitlyn , Dergai Mykola , Agarwal Shivani , Chung Daayun , van Rossum Damian B. , Shevade Aishwarya , Kuchin Sergei , Zaichick Sofia , Savas Jeffrey N , Fasshauer Dirk , Caraveo Gabriela
Published: March 2020

Membrane fusion, an essential process in all eukaryotes, is driven by SNARE proteins. Ykt6 is an essential SNARE that plays critical roles throughout the secretory, endocytic, and autophagy pathways. Ykt6 activity is thought to be regulated by a conformational change from a closed cytosolic form to an open membrane-bound form, yet the mechanism that regulates this transition is unknown. Through genetic, pharmacologic, and structural modeling approaches in mammalian cells, we found that phosphorylation regulates Ykt6 conversion from a closed to an open state. The phosphorylation site we identified is highly conserved in evolution and is regulated by the Ca2+-dependent phosphatase, calcineurin. We found that phosphorylation is a key determinant for intracellular localization of Ykt6 and its function in macroautophagy. Our studies reveal a novel mechanism by which Ykt6 conformation and activity is regulated by Ca2+signaling with implications in Parkinson’s Disease in which Ykt6 has been shown to play a role.
Whole slide imaging is a high-throughput method to assessCandidabiofilm formation
Authors: Raas Maximilian W. D. , Silva Thiago P. , Freitas Jhamine C. O. , Campos Lara M. , Fabri Rodrigo L. , Melo Rossana C.N.
Published: March 2020

New strategies that enable fast and accurate visualization ofCandidabiofilms are necessary to better study their structure and response to antifungals agents. Here, we applied whole slide imaging (WSI) to study biofilm formation ofCandidaspecies. Three relevant biofilm-formingCandidaspecies (C. albicansATCC 10231, C. glabrataATCC 2001, andC. tropicalisATCC 750) were cultivated on glass coverslips both in presence and absence of widely used antifungals. Accumulated biofilms were stained with fluorescent markers and scanned in both bright-field and fluorescence modes using a WSI digital scanner. WSI enabled clear assessment of both size and structural features ofCandidabiofilms. Quantitative analyses readily detected reductions in biofilm-covered surface area upon antifungal exposure. Furthermore, we show that the overall biofilm growth can be adequately assessed across both bright-field and fluorescence modes. At the single-cell level, WSI proved adequate, as morphometric parameters evaluated with WSI did not differ significantly from those obtained with scanning electron microscopy, considered as golden standard at single-cell resolution. Thus, WSI allows for reliable visualization ofCandidabiofilms enabling both large-scale growth assessment and morphometric characterization of single-cell features, making it an important addition to the available microscopic toolset to image and analyze fungal biofilm growth.
Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning & reduces neurodegeneration
Authors: Ryan Veronica H. , Perdikari Theodora Myrto , Naik Mandar T. , Saueressig Camillo F. , Lins Jeremy , Dignon Gregory L. , Mittal Jeetain , Hart Anne C. , Fawzi Nicolas L.
Published: March 2020

mRNA transport in neurons is a ubiquitous process but has been often overlooked as a contributor to disease. Mutations of transport granule protein hnRNPA2 cause hereditary proteinopathy of neurons, myocytes, and bone. Here, we examine transport granule component specificity, assembly/disassembly, and the link to neurodegeneration. hnRNPA2 transport granule components hnRNPF and ch-TOG interact weakly with hnRNPA2 yet they each partition specifically into hnRNPA2 liquid phases. hnRNPA2 tyrosine phosphorylation dissociates granule interactions by reducing hnRNPA2 phase separation and preventing partitioning of hnRNPF and ch-TOG; tyrosine phosphorylation also decreases aggregation of hnRNPA2 disease mutants. AC. elegansmodel of hnRNPA2 D290V-associated neurodegeneration exhibits TDP-43 ortholog-dependent glutamatergic neurodegeneration. Expression of the tyrosine kinase that phosphorylates hnRNPA2 reduces glutamatergic neurodegeneration. The evidence for specific partitioning of granule components as well as disruption of these interactions and reduction of neurodegeneration by tyrosine phosphorylation suggest transport granule biology has a role in the pathogenesis of neurodegeneration.
Expression of Toll like Receptor 4 in the ductal epithelial cells of the Breast tumor microenvironment is correlated with the invasiveness of the tumor
Authors: Moirangthem Anuradha , Mukherjee Mala , Bondhopadhyay Banashree , Bandyopadhyay Arghya , Mukherjee Narendranath , Konar Karabi , Das Tanya , Dutta Pran K , Basu Anupam
Published: March 2020

Toll like receptors are expressed by variety of cells, mainly immune cells and also found to have role in the tumor microenvironment. Among them, Toll like receptors-4 is found to modulate tumor progression. But definitive action of TLR4 in tumor progression is not well understood. In the present study, in breast tumor samples, expression of TLR4 was studied by immunohistochemistry method while MMP2 and MMP9 expression were studied by gelatin zymography. Kaplan Meier plotter was used to test survivability. Breast cancer cells - MCF7, MDA MB 231, T47D were studied in the presence of TLR4 lignd LPS, with the help of MTT assay, BrdU incorporation assay, scratch wound healing assay and invasion assay. Activation of TLR4 in MCF7 which is TP53 wild type has no significant effect in proliferative rate, adhesiveness and invasiveness. While in MDA-MB-231 and T47D which are TP53 mutant, there were a significant increase in adhesiveness and migratory ability, observed., TLR4 had been expressed in breast tumor of invasive ductal carcinoma (IDC) and was found to be significantly correlated with lymph node involvement. Kaplan Meier plotter analysis revealed that high TLR4 expression might serve as an immune-protectant in invading cancer cells of TP53 wild state. It has been revealed that activation of TLR4 in breast cancer cells leads to higher expression of EMT related genes along with matrix metalloproteinases helping in migration and invasion of cells. Kaplan Meier plotter analysis revealed that TP53 wild status of the patient along with high TLR4 expression has a good overall survival of the patients.
Multiplex single-molecule kinetics of nanopore-coupled polymerases
Authors: Palla Mirkό , Punthambaker Sukanya , Stranges P. Benjamin , Vigneault Frederic , Nivala Jeff , Wiegand Daniel J. , Ayer Aruna , Craig Timothy , Gremyachinskiy Dmitriy , Franklin Helen , Sun Shaw , Pollard James , Trans Andrew , Arnold Cleoma , Schwab Charles , Mcgaw Colin , Sarvabhowman Preethi , Dalal Dhruti , Thai Eileen , Amato Evan , Lederman Ilya , Taing Meng , Kelley Sara , Qwan Adam , Fuller Carl W. , Roever Stefan , Church George M.
Published: March 2020

DNA polymerases have revolutionized the biotechnology field due to their ability to precisely replicate stored genetic information. Screening variants of these enzymes for unique properties gives the opportunity to identify polymerases with novel features. We have previously developed a single-molecule DNA sequencing platform by coupling a DNA polymerase to a α-hemolysin pore on a nanopore array. Here, we use this approach to demonstrate a single-molecule method that enables rapid screening of polymerase variants in a multiplex manner. In this approach, barcoded DNA strands are complexed with polymerase variants and serve as templates for nanopore sequencing. Nanopore sequencing of the barcoded DNA reveals both the barcode identity and kinetic properties of the polymerase variant associated with the cognate barcode, allowing for multiplexed investigation of many polymerase variants in parallel on a single nanopore array. Further, we develop a robust classification algorithm that discriminates kinetic characteristics of the different polymerase mutants. As a proof of concept, we demonstrate the utility of our approach by screening a library of ~100 polymerases to identify variants for potential applications of biotechnological interest. We anticipate our screening method to be broadly useful for applications that require polymerases with unique or altered physical properties.