bluuebunny/crossref_metadata_2025 huggingface dataset:
SopB- and SifA-dependent shaping of theSalmonella-containing vacuole proteome in the social amoebaDictyostelium discoideum
Authors: Valenzuela Camila , Gil Magdalena , Urrutia Ítalo M. , Sabag Andrea , Enninga Jost , Santiviago Carlos A.
Published: December 2019

The ability ofSalmonellato survive and replicate within mammalian host cells involves the generation of a membranous compartment known as theSalmonella-containing vacuole (SCV).Salmonellaemploys a number of effector proteins that are injected into host cells for SCV formation using its type-three secretion systems encoded in SPI-1 and SPI-2 (T3SSSPI-1and T3SSSPI-2, respectively). Recently, we reported thatS. Typhimurium requires T3SSSPI-1and T3SSSPI-2to survive in the model amoebaDictyostelium discoideum. Despite these findings, the involved effector proteins have not been identified yet. Therefore, we evaluated the role of two majorS. Typhimurium effectors SopB and SifA duringD. discoideumintracellular niche formation. First, we established thatS. Typhimurium resides in a vacuolar compartment withinD. discoideum. Next, we isolated SCVs from amoebae infected with wild type or the ΔsopBand ΔsifAmutant strains ofS. Typhimurium, and we characterized the composition of this compartment by quantitative proteomics. This comparative analysis suggests thatS. Typhimurium requires SopB and SifA to modify the SCV proteome in order to generate a suitable intracellular niche inD. discoideum. Accordingly, we observed that SopB and SifA are needed for intracellular survival ofS. Typhimurium in this organism. Thus, our results provide insight into the mechanisms employed bySalmonellato survive intracellularly in phagocytic amoebae.

Importance

The molecular mechanisms involved inSalmonellasurvival to predation by phagocytic amoebae, such asD. discoideum, remains poorly understood. Although we established thatS. Typhimurium requires two specialized type-three secretion systems to survive inD. discoideum, no effector protein has been implicated in this process so far. Here, we confirmed the presence of a membrane-bound compartment containingS. Typhimurium inD. discoideum, and purified theD. discoideumSCV to characterize the associated proteome. In doing so, we established a key role for effector proteins SopB and SifA in remodeling the protein content of the SCV that ultimately allow the intracellular survival ofS. Typhimurium inD. discoideum. We also discuss similarities and differences with the proteomes of the human SCV. These findings contribute to unravel the mechanisms used bySalmonellato survive in the environment exploiting phagocytic amoebae as a reservoir.
Probing behavior ofDiaphorina citri(Hemiptera: Liviidae) on Valencia orange influenced by sex, color, and size
Authors: Ebert Timothy A , Rogers Michael E
Published: December 2019

CandidatusLiberibacter asiaticus is vectored by the psyllidDiaphorina citriKuwayama (Hemiptera: Liviidae) and putatively causes Huanglongbing disease in citrus. Huanglongbing has reduced yields by 68% relative to pre-disease yields in Florida. Disease management is partly through vector control. Understanding vector biology is essential in this endeavor. Our goal was to document differences in probing behavior linked to sex. Based on both a literature review and our results we conclude that there is either no effect of sex or that identifying such an effect requires a sample size at least four times larger than standard methodologies. Including both color and sex in statistical models did not improve model performance. Both sex and color are correlated with body size, and body size has not been considered in previous studies on sex inD. citri. An effect of body size was found wherein larger psyllids took longer to reach ingestion behaviors and larger individuals spent more time ingesting phloem, but these relationships explained little of the variability in these data. We suggest that the effects of sex can be ignored when running EPG experiments on healthy psyllids.
Double dissociation of fMRI activity in the caudate nucleus supportsde novomotor skill learning
Authors: Choi Yera , Shin Emily Yunha , Kim Sungshin
Published: December 2019

Motor skill learning involves a complex process of generating novel movement patterns typically guided by evaluative feedback such as reward. Many studies have suggested that two separate circuits in the basal ganglia, rostral and caudal, are implicated in early goal-directed and later automatic stages of motor skill learning, respectively. However, there remains much to be elucidated about the respective involvement of the basal ganglia circuits in learning motor skills from scratch, which requires learning arbitrary action-outcome associations. To investigate this issue, we conducted a novel human fMRI experiment in which the participants learned to control a computer cursor on a screen by manipulating their right fingers. The experiment consisted of two fMRI sessions separated by five behavioral training sessions over multiple days. We discovered a double dissociation of fMRI activities in the rostral and caudal caudate nucleus, which were associated with skill performance in the early and late stages of learning. Moreover, we found that cognitive and sensorimotor cortico-caudate interactions distinctively predicted individual learning performance. In line with recent non-human primate studies, our results support the existence of parallel cortico-caudate networks involved in goal-directed and automatic stages ofde novomotor skill learning.
Spontaneous and Stimulus-Driven Rhythmic Behaviors in ADHD Adults and Controls
Authors: Amrani Anat Kliger , Golumbic Elana Zion
Published: December 2019

Many aspects of human behavior are inherently rhythmic, requiring production of rhythmic motor actions as well as synchronizing to rhythms in the environment. It is well-established that individuals with ADHD exhibit deficits in temporal estimation and timing functions, which may impact their ability to accurately produce and interact with rhythmic stimuli. In the current study we seek to understand the specific aspects of rhythmic behavior that are implicated in ADHD. We specifically ask whether they are attributed to imprecision in the internal generation of rhythms or to reduced acuity in rhythm perception. We also test key predictions of the Preferred Period Hypothesis, which suggests that both perceptual and motor rhythmic behaviors are biased towards a specific personal ‘default’ tempo. To this end, we tested several aspects of rhythmic behavior and the correspondence between them, including spontaneous motor tempo (SMT), preferred auditory perceptual tempo (PPT) and synchronization-continuations tapping in a broad range of rhythms, from sub-second to supra-second intervals. Moreover, we evaluate the intra-subject consistency of rhythmic preferences, as a means for testing the reality and reliability of personal ‘default-rhythms’. We used a modified operational definition for assessing SMT and PPT, instructing participants to tap or calibrate the rhythms most comfortable for them to count along with, to avoid subjective interpretations of the task.

Our results shed new light on the specific aspect of rhythmic deficits implicated in ADHD adults. We find that individuals with ADHD are primarily challenged in producing and maintaining isochronous self-generated motor rhythms, during both spontaneous and memory-paced tapping. However, they nonetheless exhibit good flexibility for synchronizing to a broad range of external rhythms, suggesting that auditory-motor entrainment for simple rhythms is preserved in ADHD, and that the presence of an external pacer allows overcoming their inherent difficulty in self-generating isochronous motor rhythms. In addition, both groups showed optimal memory-paced tapping for rhythms near their ‘counting-based’ SMT and PPT, which were slightly faster in the ADHD group. This is in line with the predictions of the Preferred Period Hypothesis, indicating that at least for this well-defined rhythmic behavior (i.e., counting), individuals tend to prefer similar time-scales in both motor production and perceptual evaluation.
Transcription levels of a long noncoding RNA orchestrate opposing regulatory and cell fate outcomes in yeast
Authors: Moretto Fabien , Wood N. Ezgi , Chia Minghao , Li Cai , Luscombe Nicholas M. , van Werven Folkert J.
Published: December 2019

Many long noncoding RNAs (lncRNAs) actin cisthrough transcription-coupled chromatin alterations that drive changes in local gene expression. How somecis-acting lncRNAs promote and others repress gene expression remains poorly understood. Here we report that inS. cerevisiaetranscription levels of the lncRNAIRT2, located upstream in the promoter of the inducer of meiosis gene, regulate opposing chromatin and transcription states. LowIRT2transcription displays enhancer RNA-like features. At these levels,IRT2promotes histone exchange delivering acetylated histone H3 lysine 56 to chromatin thereby facilitating recruitment of a transcription factor and consequently activating transcription. Conversely, increasingIRT2transcription enhances chromatin assembly and transcriptional repression. The opposing functions ofIRT2direct a regulatory circuit, which ensures that cells expressing opposite, but not one of either, mating-type loci enter meiosis. Our data demonstrate that the transcription levels of an lncRNA are key to controlling gene expression and cell fate outcomes.
CNV-BAC: Copy Number Variation Detection in Bacterial Circular Genome
Authors: Wu Linjie , Wang Han , Xia Yuchao , Xi Ruibin
Published: December 2019

Motivation

Whole genome sequencing (WGS) is widely used for copy number variation (CNV) detection. However, for most bacteria, their circular genome structure and high replication rate make reads more enriched near the replication origin. CNV detection based on read depth could be seriously influenced by such replication bias.

Results

We show that the replication bias is widespread using ~200 bacterial WGS data. We develop CNV-BAC that can properly normalize the replication bias as well as other known biases in bacterial WGS data and can accurately detect CNVs. Simulation and real data analysis show that CNV-BAC achieves the best performance in CNV detection compared with available algorithms.

Availability and implementation

CNV-BAC is available athttps://github.com/LinjieWu/CNV-BAC.

Contact

ruibinxi@math.pku.edu.cn
Haloperidol-induced impairment on working memory capacity affecting long term memory performance: the binding hypothesis
Authors: Guarnieri Regina V. , Alves Marcus Vinicius , Nogueira Ana Maria Lemos , de Souza Silva Tudesco Ivanda , Galduróz José Carlos F. , Covolan Luciene , Bueno Orlando F. A.
Published: December 2019

The dopaminergic system is implicated in several cognitive processes including memory, attention and executive functions. This study was a double-blind, placebo-randomized trial designed to investigate the effect of dopamine D2receptor blockade on episodic and working memory and particularly the relationship between executive functions, working memory capacity and long-term memory (LTM). Subjects ingested a single oral dose (4 mg) of haloperidol, a dopamine D2receptor antagonist or placebo. Multiple linear regression using generalized linear models and a generalized estimating equation were used for statistical analyses. The results demonstrated that haloperidol impaired episodic memory (free recall of words and prose recall), working memory capacity-WMC (operation span task-OSPAN) and highly demanding executive functions (random number generation - RNG). In addition, it demonstrated that despite the large impairment in the RNG task performance in the haloperidol group, it did not affect episodic memory. The OSPAN task is predictive of episodic memory impairment, suggesting that memory impairments produced by haloperidol could be due in part to the impairment of WMC. As WMC partly relies on the appropriate functioning of the medial temporal lobe, probably the haloperidol-induced impairment on episodic memory through the decrease in the performance of WMC may depend on the activation of this area of the brain. The present study is relevant because it provides data on dopaminergic modulation of memory systems; suggesting that the major cause of deficits in episodic memory may be due to hippocampal function and WMC impairments, the latter more specifically with regard to controlled search and binding.
Visual feature tuning of superior colliculus neural reafferent responses after fixational microsaccades
Authors: Khademi Fatemeh , Chen Chih-Yang , Hafed Ziad M.
Published: December 2019

The primate superior colliculus (SC) is causally involved in microsaccade generation. Moreover, visually-responsive SC neurons across this structure’s topographic map, even at peripheral eccentricities much larger than the tiny microsaccade amplitudes, exhibit significant modulations of evoked response sensitivity when stimuli appear peri-microsaccadically. However, during natural viewing, visual stimuli are normally stably present in the environment and are only shifted on the retina by eye movements. Here we investigated this scenario for the case of microsaccades, asking whether and how SC neurons respond to microsaccade-induced image jitter. We recorded neural activity from two male rhesus macaque monkeys. Within the response field (RF) of a neuron, there was a stable stimulus consisting of a grating of one of three possible spatial frequencies. The grating was stable on the display, but microsaccades periodically jittered the retinotopic RF location over it. We observed clear short-latency visual reafferent responses after microsaccades. These responses were weaker, but earlier (relative to new fixation onset after microsaccade end), than responses to sudden stimulus onsets without microsaccades. The reafferent responses clearly depended on microsaccade amplitude, as well as microsaccade direction relative to grating orientation. Our results indicate that one way for microsaccades to influence vision is through modulating how the spatio-temporal landscape of SC visual neural activity represents stable stimuli in the environment. Such representation strongly depends on the specific pattern of temporal luminance modulations expected from the relative relationship between eye movement vector (size and direction), on the one hand, and spatial visual pattern layout, on the other.

Significance statement

Despite being diminutive, microsaccades still jitter retinal images. We investigated how such jitter affects superior colliculus (SC) activity. We found that SC neurons exhibit short-latency visual reafferent bursts after microsaccades. These bursts reflect not only the spatial luminance profiles of visual patterns, but also how such profiles are shifted by eye movement size and direction. These results indicate that the SC continuously represents visual patterns, even as they are jittered by the smallest possible saccades.
Anterograde Trans-Synaptic AAV Strategies for Probing Neural Circuitry
Authors: Zingg Brian , Peng Bo , Huang Junxiang J. , Tao Huizhong W. , Zhang Li I.
Published: December 2019

Elucidating the organization and function of neural circuits is greatly facilitated by viral tools that spread transsynaptically. Adeno-associated virus (AAV) has been shown to exhibit anterograde transneuronal spread. However, the synaptic specificity of the spread and its broad application in various neural circuits remain to be explored. Here, using anatomical, functional, and molecular approaches, we provide strong evidence for the specifically preferential spread of AAV1 to post-synaptically connected neurons. Besides glutamatergic synapses made onto excitatory and inhibitory neurons, AAV1 also transsynaptically spreads through GABAergic synapses and effectively tags spinal cord neurons receiving long-distance projections from various brain regions, but exhibits little or no spread through neuromodulatory projections (e.g. serotonergic, cholinergic, and noradrenergic). Combined with newly designed intersectional and sparse labeling strategies, AAV1 can be utilized to categorize neurons according to their input sources, morphological and molecular identities. These properties make AAV a unique anterograde transsynaptic tool for establishing a comprehensive cell-atlas of the brain.
A Machine Learning Approach to the Recognition of Brazilian Atlantic Forest Parrot Species
Authors: Padovese Bruno Tavares , Padovese Linilson Rodrigues
Published: December 2019

Avian survey is a time-consuming and challenging task, often being conducted in remote and sometimes inhospitable locations. In this context, the development of automated acoustic landscape monitoring systems for bird survey is essential. We conducted a comparative study between two machine learning methods for the detection and identification of 2 endangered Brazilian bird species from the Psittacidae species, theAmazona brasiliensisand theAmazona vinacea. Specifically, we focus on the identification of these 2 species in an acoustic landscape where similar vocalizations from other Psittacidae species are present. A 3-step approach is presented, composed of signal segmentation and filtering, feature extraction, and classification. In the feature extraction step, the Mel-Frequency Cepstrum Coefficients features were extract and fed to the Random Forest Algorithm and the Multilayer Perceptron for training and classifying acoustic samples. The experiments showed promising results, particularly for the Random Forest algorithm, achieving accuracy of up to 99%. Using a combination of signal segmentation and filtering before the feature extraction steps greatly increased experimental results. Additionally, the results show that the proposed approach is robust and flexible to be adopted in passive acoustic monitoring systems.
Object novelty and value coding segregate across functionally connected brain networks
Authors: Ghazizadeh Ali , Fakharian MohammadAmin , Amini Arash , Griggs Whitney , Leopold David A. , Hikosaka Okihide
Published: December 2019

Novel and valuable objects are motivationally attractive for animals including primates. However, little is known about how novelty and value processing is organized across the brain. We used fMRI in macaques to map brain activity to fractal patterns varying in either novelty or value dimensions in the context of functionally connected brain networks determined at rest. Results show unique combinations of novelty and value coding across the brain networks. Networks in the ventral temporal cortex and in the parietal cortex showed preferential coding of novelty and value dimensions, respectively, while a wider network composed of temporal and prefrontal areas (TP network), along with functionally connected portions of the striatum, amygdala, and claustrum, responded to both dimensions with similar activation dynamics. Our results support emergence of a common currency signal in the TP network that may underlie the common attitudes toward novel and valuable objects.
Identifying triplex binding rulesin vitroleads to creation of a new synthetic regulatory toolin vivo
Authors: Kaufmann Beate , Willinger Or , Eden Noa , Kermas Lisa , Anavy Leon , Solomon Oz , Atar Orna , Yakhini Zohar , Goldberg Sarah , Amit Roee
Published: December 2019

Nature provides a rich toolbox of dynamic nucleic acid structures that are widespread in cells and affect multiple biological processes1. Recently, non-canonical structures gained renewed scientific and biotechnological interest2,3. One particularly intriguing form of such structures are triplexes4in which a single-stranded nucleic acid molecule interacts via Hoogsteen bonds with a DNA/RNA double helix5. Despite extensive researchin vitro6–9, the underlying rules for triplex formation remain debated and evidence for triplexesin vivois circumstantial10–12. Here, we demonstrate the development of a deep-sequencing platform termed Triplex-Seq to systematically refine the DNA triplex code and identify high affinity triplex forming oligo (TFO) variants. We identified a preference for short G-rich motifs using an oligo-library with a mix of all four bases. These high-information content motifs formed specific high-affinity triplexes in a pH-independent manner and stability was increased with G-rich double-stranded molecules. We then conjugated one high-affinity and one low-affinity variant to a VP48 peptide and studied these synthetic biomolecules in mammalian cells. Using these peptide-oligo constructs (POCs), we demonstrated possible triplex-induced down-regulation activity in 544 differentially expressed genes. Our results show that deep-sequencing platforms can substantially expand our understanding of triplex binding rules, which in turn has led to the development of a functional non-genetically encoded regulatory tool forin vivoapplications.
Elaboration of the corticosteroid synthesis pathway in primates through a multi-step enzyme
Authors: Olson-Manning Carrie F.
Published: December 2019

Metabolic networks are complex cellular systems dependent on the interactions among, and regulation of, the enzymes in the network. However, the mechanisms that lead to the expansion of networks are not well understood. While gene duplication is a major driver of the expansion and functional evolution of metabolic networks, the effect and fate of retained duplicates in a network is poorly understood. Here, I study the evolution of an enzyme family that performs multiple subsequent enzymatic reactions in the corticosteroid pathway in primates to illuminate the mechanisms that shape network components following duplication. The products of the pathway (aldosterone, corticosterone, and cortisol) are steroid hormones that regulate metabolism and stress in tetrapods. These steroids are synthesized by a multi-step enzyme Cytochrome P450 11B (CYP11B) that performs subsequent steps on different carbon atoms of the steroid derivatives. Through ancestral state reconstruction andin vitrocharacterization, I find the ancestor of the CYP11B1 and CYP11B2 paralogs (in primates) had moderate ability to synthesize cortisol and aldosterone. Following duplication in the primate lineage the CYP11B1 homolog specialized on the production of cortisol while its paralog, CYP11B2, maintained its ability to perform multiple subsequent steps as in the ancestral pathway. Unlike CYP11B1, CYP11B2 could not specialize on the production of aldosterone because it is constrained to perform earlier steps in the corticosteroid synthesis pathway to achieve the final product aldosterone. These results suggest that pathway context, along with tissue-specific regulation, both play a role in shaping potential outcomes of metabolic network elaboration.
Multi-Parametric Profiling of Endothelial Cell Networks Reveals Functional Role of Glutamate Receptors in Angiogenesis
Authors: Sailem Heba Z. , Haj Zen Ayman Al
Published: December 2019

Angiogenesis plays a key role in several diseases including cancer, ischemic vascular disease, and Alzheimer’s disease. High throughput screening of endothelial tube formation provides a robust approach for identifying drugs that impact microvascular network formation and morphology. However, the analysis of resulting imaging datasets has been limited to a few phenotypic features such as the total tube length or the number of branching points. Here we developed a high content analysis framework for detailed quantification of various aspects of network morphology including network complexity, symmetry and topology. By applying our approach to a high content screen of 1,280 drugs, we found that many drugs that result in a similar phenotype share the same mechanism of action or common downstream signalling pathways. Our multiparametric analysis revealed a group of drugs, that target glutamate receptors, results in enhanced branching and network connectivity. Using an integrative meta-analysis approach, we validated the link between these receptors and angiogenesis. We further found that the expression of these genes correlates with the prognosis of Alzheimer’s patients. In conclusion, our work shows that detailed image analysis of complex endothelial phenotypes can reveal new insights into biological mechanisms modulating the morphogenesis of endothelial networks and identify potential therapeutics for angiogenesis-related diseases.
A Novel Chloroplast Super-Complex Consisting of the ATP Synthase and Photosystem I Reaction Center
Authors: Bhaduri Satarupa , Singh Sandeep K , Cohn Whitaker , Saif Hasan S. , Whitelegge Julian P. , Cramer William A.
Published: December 2019

Several ‘super-complexes’ of individual hetero-oligomeric membrane protein complexes, whose function is to facilitate intra-membrane electron and proton transfer and harvesting of light energy, have been previously characterized in the mitochondrial cristae and chloroplast thylakoid membranes. The latter membrane is reported here to also be the location of an intra-membrane super-complex which is dominated by the ATP-synthase and photosystem I (PSI) reaction-center complexes, defined by mass spectrometry, clear-native PAGE and Western Blot analyses. This is the first documented presence of ATP synthase in a super-complex with the PSI reaction-center located in the non-appressed stromal domain of the thylakoid membrane.
Exploring the Conformational Transition Between the Fully Folded and Locally Unfolded Substates of theEscherichia colithiol peroxidase
Authors: Vazquez Diego S. , Zeida Ari , Agudelo William A. , Montes Mónica , Ferrer-Sueta Gerardo , Santos Javier
Published: December 2019

Thiol peroxidase fromEscherichia coli(EcTPx) is a peroxiredoxin that catalyzes the reduction of different hydroperoxides. During the catalytic cycle ofEcTPx, the peroxidatic cysteine (CP) is oxidized to a sulfenic acid by peroxide, then the resolving cysteine (CR) condenses with the sulfenic acid of CPto form a disulfide bond, which is finally reduced by thioredoxin. PurifiedEcTPx as dithiol and disulfide behaves as a monomer in close to physiological conditions. Although secondary structure rearrangements are present when comparing different redox states of the enzyme, no significant differences in unfolding free energies are observed under reducing and oxidizing conditions. A conformational change denominatedfully folded (FF) to locally unfolded (LU) transition, involving a partial unfolding of αH2 and αH3 helices, must occur to enable the formation of the disulfide bond since the catalytic cysteines are 12 Å apart in the FF conformation ofEcTPx. To explore this crucial process, the mechanism of the FF→LU and the LU→FF transitions were studied using long time scale conventional molecular dynamic simulations and an enhanced conformational sampling technique for different oxidation and protonation states of CPand/or CR. Our results suggest that the FF→LU transition has a higher associated energy barrier than the refolding LU→FF process in agreement with the relatively slow experimental turnover number ofEcTPx. Furthermore,in silicodesigned single-point mutants of the αH3 enhanced locally unfolding events, suggesting that the native FF interactions in the active site are not evolutionary optimized to fully speed-up the conformational transition of wild-typeEcTPx.
Opponent regulation of action performance and timing by striatonigral and striatopallidal pathways
Authors: Bakhurin Konstantin I. , Li Xiaoran , Friedman Alexander D. , Lusk Nicholas A. , Watson Glenn D.R. , Kim Namsoo , Yin Henry H.
Published: December 2019

The basal ganglia have been implicated in action selection and timing, but the relative contributions of the striatonigral (direct) and striatopallidal (indirect) pathways to these functions remain unclear. We investigated the effects of optogenetic stimulation of D1+ (direct) and A2A+ (indirect) neurons in the ventrolateral striatum in head-fixed mice on a fixed time reinforcement schedule. Direct pathway stimulation initiates licking, whereas indirect pathway stimulation suppresses licking and results in rebound licking after stimulation. Moreover, direct and indirect pathways also play distinct roles in timing. Direct pathway stimulation produced a resetting of the internal timing process, whereas indirect pathway stimulation transiently paused timing, and proportionally delayed the next bout of licking. Our results provide evidence for the continuous and opposing contributions of the direct and indirect pathways in the production and timing of reward-guided behavior.
For running or burying—The toe fringe ofPhrynocephalus mystaceusis important
Authors: Zheng Peng , Liang Tao , An Jing , Shi Lei
Published: December 2019

Toe fringe is the most typical morphological feature of lizards adapted to sandy environments, and toe fringe is suitable for testing the adaptive convergence suggested by form-environment correlations.Phrynocephalus mystaceusmainly lives in dune habitats, has a developed bilateral toe fringe, and exhibits fast sand-diving behavior for predator avoidance. We tested the effects of resecting the medial and bilateral toe fringes on the locomotor performance and sand-diving performance ofP. mystaceus.The results showed that the individuals that had their medial toe fringe removed exhibited significantly faster sprint speeds than the unresected and all resected individuals (P<0.05). The results of stepwise regression analysis show that the relative area of the toe fringe is an important morphological feature that affects locomotor performance. The sand-burial performance scores of the unresected individuals were significantly greater than those of the resected individuals (P<0.05). The results of stepwise regression analysis show that the relative area of toe fringe and the axilla-groin length are the main morphological features that affect the sand-diving performance ofP. mystaceus. After the bilateral toe fringe was removed, a significant negative correlation between locomotor and sand-diving performance was observed (P<0.05). Taken together, these results provide experimental evidence that toe fringe is positively associated with the locomotor and sand-diving performance ofP. mystaceus.
Antigen-driven PD-1+TOX+EOMES+and PD-1+TOX+BHLHE40+synovial T lymphocytes regulate chronic inflammationin situ
Authors: Maschmeyer Patrick , Anne Heinz Gitta , Skopnik Christopher Mark , Lutter Lisanne , Mazzoni Alessio , Heinrich Frederik , von Stuckrad Sae Lim , Wirth Lorenz Elias , Tran Cam Loan , Riedel René , Lehmann Katrin , Sakwa Imme , Cimaz Rolando , Giudici Francesco , Mall Marcus Alexander , Enghard Philipp , Vastert Bas , Chang Hyun-Dong , Durek Pawel , Annunziato Francesco , van Wijk Femke , Radbruch Andreas , Kallinich Tilmann , Mashreghi Mir-Farzin
Published: December 2019

Introduction/Abstract

T lymphocytes accumulate in inflamed tissues of patients with chronic inflammatory diseases (CIDs) and express pro-inflammatory cytokines upon re-stimulationin vitro1–29. Further, a significant genetic linkage to MHC genes suggests that T lymphocytes play an important role in the pathogenesis of CIDs including juvenile idiopathic arthritis (JIA)30–33. However, the functions of T lymphocytes in established disease remain elusive. Here we dissect the heterogeneity of synovial T lymphocytes in JIA patients by single cell RNA-sequencing. We identify subpopulations of T lymphocytes expressing genes reflecting recent activation by antigenin situ. A PD-1+TOX+EOMES+population of CD4+T lymphocytes expressed immune regulatory genes and chemoattractant genes for myeloid cells. A PD-1+TOX+BHLHE40+population of CD4+, and a mirror population of CD8+T lymphocytes expressed genes driving inflammation, and genes supporting B lymphocyte activation. This analysis points out that multiple types of T lymphocytes have to be targeted for therapeutic regeneration of tolerance in arthritis.
Nanopore sequencing of native adeno-associated virus (AAV) single-stranded DNA using a transposase-based rapid protocol
Authors: Radukic Marco T. , Brandt David , Haak Markus , Müller Kristian M. , Kalinowski Jörn
Published: December 2019

Next-generation sequencing of single-stranded DNA (ssDNA) enables transgene characterization of gene-therapy vectors such as adeno-associated virus (AAV), but current library generation uses complicated and potentially biased second-strand synthesis. We report that libraries for nanopore sequencing of ssDNA can be conveniently created without second-strand synthesis using a transposase-based protocol. We show for bacteriophage M13 ssDNA that the MuA transposase has unexpected residual activity on ssDNA, explained in part by transposase action on transient double-stranded hairpins. In case of AAV, library creation is additionally aided by genome hybridization. We demonstrate the power of direct sequencing combined with nanopore long reads by characterizing AAV vector transgenes. Sequencing yielded reads up to full genome length, including GC-rich inverted terminal repeats. Unlike short-read techniques, single reads covered genome-genome and genome-contaminant fusions and other recombination events, while additionally providing information on epigenetic methylation. Single-nucleotide variants across the transgene cassette were revealed and secondary genome packaging signals were readily identified. Moreover, comparison of sequence abundance with qPCR results demonstrated the technique’s future potential for quantification of DNA impurities in AAV vector stocks. The findings promote direct nanopore sequencing as a fast and versatile platform for ssDNA characterization, such as AAV ssDNA in research and clinical settings.
DNA methylation across the genome in aged human skeletal muscle tissue and stem cells: The role of HOX genes and physical activity
Authors: Turner DC , Gorski PP , Maasar MF , Seaborne RA , Baumert P , Brown AD , Kitchen MO , Erskine RM , Dos-Remedios I , Voisin S , Eynon N , Sultanov RI , Borisov OV , Larin AK , Semenova EA , Popov DV , Generozov EV , Stewart CE , Drust B , Owens DJ , Ahmetov II , Sharples AP
Published: December 2019

Skeletal muscle tissue demonstrates global hypermethylation with aging. However, methylome changes across the time-course of differentiation in aged human muscle derived stem cells, and larger coverage arrays in aged muscle tissue have not been undertaken. Using 850K DNA methylation arrays we compared the methylomes of young (27 ± 4.4 years) and aged (83 ± 4 years) human skeletal muscle and that of young/aged muscle stem cells over several time points of differentiation (0, 72 hours, 7, 10 days). Aged muscle tissue was hypermethylated compared with young tissue, enriched for; ‘pathways-in-cancer’ (including; focal adhesion, MAPK signaling, PI3K-Akt-mTOR signaling, p53 signaling, Jak-STAT signaling, TGF-beta and notch signaling), ‘rap1-signaling’, ‘axon-guidance’ and ‘hippo-signalling’. Aged muscle stem cells also demonstrated a hypermethylated profile in pathways; ‘axon-guidance’, ‘adherens-junction’ and ‘calcium-signaling’, particularly at later timepoints of myotube formation, corresponding with reduced morphological differentiation and reductions in MyoD/Myogenin gene expression compared with young cells. While young cells showed little alteration in DNA methylation during differentiation, aged cells demonstrated extensive and significantly altered DNA methylation, particularly at 7 days of differentiation and most notably in the ‘focal adhesion’ and ‘PI3K-AKT signalling’ pathways. While the methylomes were vastly different between muscle tissue and isolated muscle stem cells, we identified a small number of CpG sites showing a hypermethylated state with age, in both muscle and tissue and stem cells (on genesKIF15, DYRK2, FHL2, MRPS33, ABCA17P). Most notably, differential methylation analysis of chromosomal regions identified three locations containing enrichment of 6-8 CpGs in the HOX family of genes altered with age. WithHOXD10, HOXD9, HOXD8, HOXA3, HOXC9, HOXB1, HOXB3, HOXC-AS2andHOXC10all hypermethylated in aged tissue. In aged cells the same HOX genes (and additionallyHOXC-AS3) displayed the most variable methylation at 7 days of differentiation versus young cells, withHOXD8, HOXC9, HOXB1andHOXC-AS3hypermethylated andHOXC10andHOXC-AS2hypomethylated. We also determined that there was an inverse relationship between DNA methylation and gene expression forHOXB1, HOXA3andHOXC-AS3. Finally, increased physical activity in young adults was associated with oppositely regulatingHOXB1andHOXA3methylation compared with age. Overall, we demonstrate that a considerable number of HOX genes are differentially epigenetically regulated in aged human skeletal muscle and muscle stem cells and increased physical activity may help prevent age-related epigenetic changes in these HOX genes.
Evidence for novel transient cell clusters in the neonatal mouse retina
Authors: de Montigny Jean , Krishnamoorthy Vidhyasankar , Rozenblit Fernando , Gollisch Tim , Sernagor Evelyne
Published: December 2019

Waves of spontaneous activity sweep across the neonatal mouse retinal ganglion cell (RGC) layer, driven by directly interconnected cholinergic starburst amacrine cells from postnatal day (P) 0-10, followed by waves driven by glutamatergic bipolar cells. We found transient clusters of auto-fluorescent cells in the RGC layer during the period of cholinergic waves. They appear around the optic nerve head at P2 and become gradually displaced towards the periphery between P2-8 and then they disappear. Pan-retinal multielectrode array recordings reveal that cholinergic wave origins follow a similar, non-random developmental center-to-periphery pattern. Electrical imaging unmasks hotspots of dipole electrical activity occurring in the vicinity of wave origins. We propose that these activity hotspots are the sites for wave initiation and may originate from the transient cell clusters, reminiscent of activity in transient subplate neurons in the developing cortex, suggesting a universal hyper-excitability mechanism in developing CNS networks during the critical period for brain wiring.
Cerebellum-specific deletion of the GABAAreceptor δ subunit alters anxiety-like, social and maternal behaviors without affecting motor performance
Authors: Rudolph Stephanie , Guo Chong , Pashkovski Stan , Osorno Tomas , Gillis Winthrop , Krauss Jeremy , Nyitrai Hajnalka , Flaquer Isabella , El-Rifai Mahmoud , Datta Robert Sandeep , Regehr Wade
Published: December 2019

GABAAreceptors containing the δGABAAsubunit (δGABAARs) are involved in many physiological and pathophysiological processes, such as sleep, pain, stress, anxiety-related behaviors, and postpartum depression. These extrasynaptically located, high affinity and slowly desensitizing receptors mediate tonic inhibition throughout the brain, including in granule cells (GCs) of the cerebellar input layer. However, the extent to which δGABAARs control the excitability of the cerebellar input layer and ultimately regulate behavior is unknown. We therefore deleted δGABAAsubunits specifically from GCs and determined the behavioral consequences in mice. Deletion reduced tonic inhibition and increased input layer excitability, but remarkably, did not affect either locomotion or motor learning. Unexpectedly, δGABAAdeletion heightened anxiety-like behaviors, and caused female-specific alterations in social and maternal behavior. Our findings establish that the cerebellar input layer is critical for regulating diverse behaviors that are relevant to psychiatric and neurodevelopmental disorders but were previously not associated with the cerebellum.
Trehalose Stabilizing Protein in a Water Replacement Scenario: Insights from Molecular Dynamics Simulation
Authors: Shao Qiang , Wang Jinan , Zhu Weiliang
Published: December 2019

How trehalose has exceptional property in helping biomolecules preserve their native structures remains a subject of active research. Running molecular dynamics simulations on a model protein in low-concentrated trehalose solution and pure water, respectively, the present study verifies the ability of trehalose in stabilizing protein native structure and provides a comprehensive atomic-level picture of the molecular interactions among protein, trehalose, and water in their mixed solution. Trehalose directly interacts to and meanwhile affects the interactions between the other speciesviahydrogen bonding: 1) trehalose molecules are clustered through inter-molecular hydrogen bonding interaction; 2) trehalose forms hydrogen bond with water which influences the strength of water-water hydrogen bonding network but does not impair protein-water hydrogen bonding; 3) trehalose is accessible to form hydrogen bonds towards protein and simultaneously replace water molecules around protein which reduces the hydrogen bonding possibility from water to protein, in accordance with “water replacement” scenario.
Principles for rational Cas13d guide design
Authors: Wessels Hans-Hermann , Méndez-Mancilla Alejandro , Guo Xinyi , Legut Mateusz , Daniloski Zharko , Sanjana Neville E.
Published: December 2019

Type VI CRISPR enzymes have recently been identified as programmable RNA-guided, RNA-targeting Cas proteins with nuclease activity that allow for specific and robust target gene knock-down without altering the genome. However, we currently lack information about optimal Cas13 guide RNA designs for high target RNA knock-down efficacy. To close this gap, we conducted four massively-parallel Cas13 screens targeting the mRNA of a destabilized green fluorescent protein (GFP) transgene and CD46, CD55 and CD71 cell surface proteins in human cells. In total, we measured the activity of 24,460 guide RNA including 6,469 perfect match guide RNAs and a diverse set of guide RNA variants and permutations with mismatches relative to the target sequences.

We find that guide RNAs show high diversity in knock-down efficiency driven by crRNA-specific features as well as target site context. Moreover, while single mismatches generally reduce knock-down to a modest degree, we identify a critical region spanning spacer nucleotides 15 – 21 that is largely intolerant to target site mismatches. We developed a computational model to identify guide RNAs with high knock-down efficacy. We confirmed the model’s generalizability across a large number of endogenous target mRNAs and show that Cas13 can be used in forward genetic pooled CRISPR-screens to identify essential genes. Using this model, we provide a resource of optimized Cas13 guide RNAs to target all protein-coding transcripts in the human genome, enabling transcriptome-wide forward genetic screens.
microRNA profiling of mouse cortical progenitors and neurons reveals miR-486-5p as a novel regulator of neurogenesis
Authors: Dori Martina , Cavalli Daniel , Lesche Mathias , Massalini Simone , Haj Abdullah Alieh Leila , Cardoso de Toledo Beatriz , Khudayberdiev Sharof , Schratt Gerhard , Dahl Andreas , Calegari Federico
Published: December 2019

MicroRNAs (miRNAs) are short (∼22 nt) single-stranded non-coding RNAs that regulate gene expression at the post-transcriptional level. Over the past years, many studies have extensively characterized the involvement of miRNA-mediated regulation in neurogenesis and brain development. However, a comprehensive catalog of cortical miRNAs cell-specifically expressed in progenitor types of the developing mammalian cortex is still missing. Overcoming this limitation, here we exploited a double reporter mouse line previously validated by our group to allow the identification of the transcriptional signature to neurogenic commitment and provide the field with the complete atlas of miRNAs expression in proliferating neural stem cells, neurogenic progenitors and newborn neurons during corticogenesis. By extending the currently known list of miRNAs expressed in the mouse brain by over two fold, our study highlights the power of cell type-specific analyses for the detection of transcripts that would otherwise be diluted out when studying bulk tissues. We further exploited our data by predicting putative novel miRNAs and validated the power of our approach by providing novel evidence for the involvement of miR-486 as a novel player in brain development.
Lifetime of a structure evolving by cluster aggregation and particle loss, and application to postsynaptic scaffold domains
Authors: Hakim Vincent , Ranft Jonas
Published: December 2019

The dynamics of several mesoscopic biological structures depend on the interplay of growth through the incorporation of components of different sizes laterally diffusing along the cell membrane, and loss by component turnover. In particular, a model of such an out-of-equilibrium dynamics has recently been proposed for postsynaptic scaffold domains which are key structures of neuronal synapses. It is of interest to estimate the lifetime of these mesoscopic structures, especially in the context of synapses where this time is related to memory retention. The lifetime of a structure can be very long as compared to the turnover time of its components and it can be difficult to estimate it by direct numerical simulations. Here, in the context of the model proposed for postsynaptic scaffold domains, we approximate the aggregation-turnover dynamics by a shot-noise process. This enables us to analytically compute the quasi-stationary distribution describing the sizes of the surviving structures as well as their characteristic lifetime. We show that our analytical estimate agrees with numerical simulations of a full spatial model, in a regime of parameters where a direct assessment is computationally feasible. We then use our approach to estimate the lifetime of mesoscopic structures in parameter regimes where computer simulations would be prohibitively long. For gephyrin, the scaffolding protein specific to inhibitory synapses, we estimate a lifetime longer than several months for a scaffold domain when the single gephyrin protein turnover time is about half an hour, as experimentally measured. While our focus is on postsynaptic domains, our formalism and techniques should be applicable to other biological structures that are also formed by a balance of condensation and turnover.
Time-ResNeXt for epilepsy recognition based on EEG signals
Authors: Wang shaoqiang , Wang Yifan , Wang Shudong
Published: December 2019

Objective

To automatically detect dynamic EEG signals to reduce the time cost of epilepsy diagnosis. In the signal recognition of electroencephalogram (EEG) of epilepsy, traditional machine learning and statistical methods require manual feature labeling engineering in order to show excellent results on a single data set. And the artificially selected features may carry a bias, and cannot guarantee the validity and expansibility in real-world data. In practical applications, deep learning methods can release people from feature engineering to a certain extent. As long as the focus is on the expansion of data quality and quantity, the algorithm model can learn automatically to get better improvements. In addition, the deep learning method can also extract many features that are difficult for humans to perceive, thereby making the algorithm more robust.

Method

Based on the design idea of ResNeXt deep neural network, this paper designs a Time-ResNeXt network structure suitable for time series EEG epilepsy detection to identify EEG signals.

Results

The accuracy rate of Time-ResNeXt in the detection of EEG epilepsy can reach 90.50%.

Conclusion

The Time-ResNeXt network structure produces extremely advanced performance on the benchmark dataset (Berne-Barcelona dataset), and has great potential for improving clinical practice.