bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA METHYLATION PROFILING OF A CNIDARIAN-ALGAL SYMBIOSIS USING NANOPORE SEQUENCING
Authors: Dimond James L. , Nguyen Nhung , Roberts Steven B.
Published: February 2021

Symbiosis with protists is common among cnidarians such as corals and sea anemones, and is associated with homeostatic and phenotypic changes in the host that could have epigenetic underpinnings, such as methylation of CpG dinucleotides. We leveraged the sensitivity to base modifications of nanopore sequencing to probe the effect of symbiosis with the chlorophyteElliptochloris marinaon methylation in the sea anemoneAnthopleura elegantissima. We first validated the approach by comparison of nanopore-derived methylation levels with CpG depletion analysis of a published transcriptome, finding that high methylation levels are associated with CpG depletion as expected. Next, using reads generated exclusively from aposymbiotic anemones, a largely complete draft genome comprising 243 Mb was assembled. Reads from aposymbiotic and symbiotic sea anemones were then mapped to this genome and assessed for methylation using the program Nanopolish, which detects signal disruptions from base modifications as they pass through the nanopore. Based on assessment of 452,841 CpGs for which there was adequate read coverage (approximately 8% of the CpGs in the genome), symbiosis withE. marinawas, surprisingly, associated with only subtle changes in the host methylome. However, we did identify one extended genomic region with consistently higher methylation among symbiotic individuals. The region was associated with a DNA polymerase zeta that is noted for its role in translesion synthesis, which opens interesting questions about the biology of this symbiosis. Our study highlights the power and relative simplicity of nanopore sequencing for studies of nucleic acid base modifications in non-model species.
The impact of distributional assumptions in gene-set and pathway analysis: how far can it go wrong?
Authors: Ho Chi-Hsuan , Huang Yu-Jyun , Lai Ying-Ju , Mukherjee Rajarshi , Hsiao Chuhsing Kate
Published: February 2021

Gene-set analysis (GSA) has been one of the standard procedures for exploring potential biological functions when a group of differentially expressed genes have been derived. The development of its methodology has been an active research topic in recent decades. Many GSA methods, when newly proposed, rely on simulation studies to evaluate their performance with a common implicit assumption that the multivariate expression values are normally distributed. The validity of this assumption has been disputed in several studies but no systematic analysis has been carried out to assess the influence of this distributional assumption. Our goal in this study is not to propose a new GSA method but to first examine if the multi-dimensional gene expression data in gene sets follow a multivariate normal distribution (MVN). Six statistical methods in three categories of MVN tests were considered and applied to a total of twenty-two datasets of expression data from studies involving tumor and normal tissues, with ten signaling pathways chosen as the gene sets. Second, we evaluated the influence of non-normality on the performance of current GSA tools, including parametric and non-parametric methods. Specifically, the scenario of mixture distributions representing the case of different tumor subtypes was considered. Our first finding suggests that the MVN assumption should be carefully dealt with. It does not hold true in many applications tested here. The second investigation of the GSA tools demonstrates that the non-normality does affect the performance of these GSA methods, especially when subtypes exist. We conclude that the use of the inherent multivariate normality assumption should be assessed with care in evaluating new GSA tools, since this MVN assumption cannot be guaranteed and this assumption affects strongly the performance of GSA methods. If a newly proposed GSA method is to be evaluated, we recommend the incorporation of multivariate non-normal distributions or sampling from large databases if available.
Role of transmembrane spanning domain 1 in cystic fibrosis transmembrane conductance regulator folding
Authors: Patrick Anna E. , Millen Linda , Thomas Philip J.
Published: February 2021

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein that disrupt its folding pathway. The most common mutation causing CF is a deletion of phenylalanine at position 508 (ΔF508). CFTR contains five domains that each form cotranslational structures that interact with other domains as they are produced and folded. CFTR is comprised of two transmembrane spanning domains (TMDs), two nucleotide binding domains (NBDs) and a unique regulatory region (R). The first domain translated, TMD1, forms interdomain interactions with the other domains in CFTR. In TMD1, long intracellular loops extend into the cytoplasm and interact with both NBDs via coupling helices and with TMD2 via transmembrane spans (TMs). We examined mutations in TMD1 to determine the impact on individual domain and multidomain constructs. We found that mutations in a TM span or in the cytosolic ICLs interfere with specific steps in the hierarchical folding of CFTR. TM1 CF-causing mutants, G85E and G91R, directly affect TMD1, whereas most ICL1 and ICL2 mutant effects become apparent in the presence of TMD2. A single mutant in ICL2 worsened CFTR trafficking in the presence of NBD2, supporting its role in the ICL2-NBD2 interface. Mutation of hydrophobic residues in ICL coupling helices tended to increased levels of pre-TMD2 biogenic intermediates but caused ER accumulation in the presence of TMD2. This suggests a tradeoff between transient stability during translation and final structure. NBD2 increased the efficiency of mutant trafficking from the ER, consistent with stabilization of the full-length constructs. While the G85E and G91R mutants in TM1 have immediately detectable effects, most of the studied mutant effects and the ΔF508 mutant are apparent after production of TMD2, supporting this intermediate as a major point of recognition by protein quality control.
Protein lactylation induced by neural excitation
Authors: Hagihara Hideo , Shoji Hirotaka , Otabi Hikari , Toyoda Atsushi , Katoh Kaoru , Namihira Masakazu , Miyakawa Tsuyoshi
Published: February 2021

Lactate is known to have diverse roles in the brain at the molecular and behavioral levels under both physiological and pathophysiological conditions, such as learning and memory and regulation of mood. Recently, a novel post-translational modification called lysine lactylation has been found in histone H3 of mouse macrophages, and the lactylation levels paralleled the intracellular lactate levels1. However, it is unknown whether lysine lactylation occurs in brain cells, and if it does, whether lactylation is induced by the stimuli that accompany changes in lactate levels. Herein, we reveal that lysine lactylation in brain cells is regulated by systemic changes in lactate levels, neural excitation, and behaviorally relevant stimuli. Lysine lactylation levels were increased by lactate treatment and by high-potassium-induced depolarization in cultured primary neurons; these increases were attenuated by pharmacological inhibition of monocarboxylate transporter 2 and lactate dehydrogenase, respectively, suggesting that both cell-autonomous and non-cell-autonomous neuronal mechanisms are involved in overall lysine lactylation.In vivo, electroconvulsive stimulation increased lysine lactylation levels in the prefrontal cortices of mice, and its levels were positively correlated with the expression levels of the neuronal activity marker c-Fos on an individual cell basis. In the social defeat stress model of depression in which brain lactate levels increase, lactylation levels were increased in the prefrontal cortices of the defeated mice, which was accompanied by increased c-Fos expression, decreased social behaviors, and increased anxiety-like behaviors, suggesting that stress-induced neuronal excitation may induce lysine lactylation, thereby affecting mood-related behaviors. Further, we identified 63 candidate lysine-lactylated proteins in the mouse cortex and found that lactylation levels in histone H1 increased in response to defeat stress. This study may open up an avenue for exploration of a novel role of neuronal activity-induced lactate mediated by protein lactylation in the brain.
Sorghum pan-genome explores the functional utility to accelerate the genetic gain
Authors: Ruperao Pradeep , Thirunavukkarasu Nepolean , Gandham Prasad , S. Sivasubramani , M Govindaraj , Nebie Baloua , Manyasa Eric , Gupta Rajeev , Das Roma Rani , Gandhi Harish , Edwards David , Deshpande Santosh P. , Rathore Abhishek
Published: February 2021

Sorghum (Sorghum bicolorL.) is one of the most important food crops in the arid and rainfed production ecologies. It is a part of resilient farming and is projected as a smart crop to overcome the food and nutritional challenges in the developing world. The development and characterisation of the sorghum pan-genome will provide insight into genome diversity and functionality, supporting sorghum improvement. We built a sorghum pan-genome using reference genomes as well as 354 genetically diverse sorghum accessions belonging to different races. We explored the structural and functional characteristics of the pan-genome and explain its utility in supporting genetic gain. The newly-developed pan-genome has a total of 35,719 genes, a core genome of 16,821 genes and an average of 32,795 genes in each cultivar. The variable genes are enriched with environment responsive genes and classify the sorghum accessions according to their race. We show that 53% of genes display presence-absence variation, and some of these variable genes are predicted to be functionally associated with drought traits. Using more than two million SNPs from the pan-genome, association analysis identified 398 SNPs significantly associated with important agronomic traits, of which, 92 were in genes. Drought gene expression analysis identified 1,788 genes that are functionally linked to different conditions, of which 79 were absent from the reference genome assembly. This study provides comprehensive genomic diversity resources in sorghum which can be used in genome assisted crop improvement.
Resolving phylogenetic and biochemical barriers to functional expression of heterologous iron-sulphur cluster enzymes
Authors: Shomar Helena , Garcia Pierre Simon , Fernández-Fueyo Elena , D’Angelo Francesca , Pelosse Martin , Manuel Rita Rebelo , Büke Ferhat , Liu Siyi , van den Broek Niels , Duraffourg Nicolas , de Ram Carol , Pabst Martin , Gribaldo Simonetta , Py Beatrice , de Choudens Sandrine Ollagnier , Bokinsky Gregory , Barras Frédéric
Published: February 2021

Many of the most promising applications of synthetic biology, including engineering of microbes for renewable chemical production, relies upon the ability of genetically-tractable hosts to express heterologous enzymes from foreign species. While countless methods for facilitating heterologous enzyme expression have been developed, comparable tools for facilitating heterologous enzyme activity are generally lacking. Such tools are needed to fully exploit the biosynthetic potential of the natural world. Here, using the model bacterium Escherichia coli, we investigate why iron-sulphur (Fe-S) enzymes are often inactive when heterologously expressed. By applying a simple growth complementation assay with collections of Fe-S enzyme orthologs from a wide range of prokaryotic diversity, we uncover a striking correlation between phylogenetic distance and probability of functional expression. Moreover, co-expression of a heterologous Fe-S biogenesis pathway increases the phylogenetic range of orthologs that can be functionally expressed. On the other hand, we find that heterologous Fe-S enzymes that require specific electron carrier proteins within their natural host are rarely functionally expressed unless their specific reducing partners are identified and co-expressed. We demonstrate in vitro that such selectivity in part derives from a need for low-potential electron donors. Our results clarify how phylogenetic distance and electron transfer biochemistry each separately impact functional heterologous expression and provide insight into how these barriers can be overcome for successful microbial engineering involving Fe-S enzymes.
Angiotensin-II modulates GABAergic neurotransmission in the mouse substantia nigra
Authors: Singh Maibam R. , Vigh Jozsef , Amberg Gregory C.
Published: February 2021

GABAergic projections neurons of the substantia nigra reticulata (SNr), through an extensive network of dendritic arbors and axon collaterals, provide robust inhibitory input to neighboring dopaminergic neurons in the substantia nigra compacta (SNc). Angiotensin-II (Ang-II) receptor signaling increases SNc dopaminergic neuronal sensitivity to insult, thus rendering these cells susceptible to dysfunction and destruction. However, the mechanisms by which Ang-II regulates SNc dopaminergic neuronal activity are unclear. Given the complex relationship between SN dopaminergic and GABAergic neurons, we hypothesized that Ang-II could regulate SNc dopaminergic neuronal activity directly and indirectly by modulating SNr GABAergic neurotransmission. Herein, using transgenic mice, slice electrophysiology, and optogenetics, we provide evidence of an AT1 receptor-mediated signaling mechanism in SNr GABAergic neurons where Ang-II suppresses electrically-evoked neuronal output by facilitating postsynaptic GABAA receptors and prolonging the action potential duration. Unexpectedly, Ang-II had no discernable effects on the electrical properties of SNc dopaminergic neurons. Also, and indicating a nonlinear relationship between electrical activity and neuronal output, following phasic photoactivation of SNr GABAergic neurons, Ang-II paradoxically enhanced the feedforward inhibitory input to SNc dopaminergic neurons. In sum, our observations describe an increasingly complex and heterogeneous response of the SN to Ang-II by revealing cell-specific responses and nonlinear effects on intranigral GABAergic neurotransmission. Our data further implicate the renin-angiotensin-system as a functionally relevant neuromodulator in the basal ganglia, thus underscoring a need for additional inquiry.

SIGNIFICANCE STATEMENT

Angiotensin II (Ang-II) promotes dopamine release in the striatum and, in the substantia nigra compacta (SNc), exacerbates dopaminergic cell loss in animal models of Parkinson’s disease. Despite a potential association with Parkinson’s disease, the effects of Ang-II on neuronal activity in the basal ganglia is unknown. Here we describe a novel AT1 receptor-dependent signaling mechanism in GABAergic projection neurons of the SN reticulata (SNr), a major inhibitory regulator of SNc dopaminergic neurons. Specifically, Ang-II suppresses SNr GABAergic neuronal activity, subsequently altering GABAergic modulation of SNc dopaminergic neurons in a nonlinear fashion. Altogether, our data provide the first indication of Ang-II-dependent modulation of GABAergic neurotransmission in the SN, which could impact output from the basal ganglia in health and disease.
Perturbations in 3D genome organization can promote acquired drug resistance
Authors: Manjón Anna G , Hupkes Daan Peric , Liu Ning Qing , Friskes Anoek , Joosten Stacey , Teunissen Hans , Aarts Marleen , Prekovic Stefan , Zwart Wilbert , de Wit Elzo , van Steensel Bas , Medema René H
Published: February 2021

Acquired drug resistance is a major problem in the treatment of cancer. hTERT-immortalized, untransformed RPE-1 (RPE) cells can acquire resistance to taxol by derepressing the ABCB1 gene, encoding for the multidrug transporter P-gP. Here we have investigated how the ABCB1 gene is derepressed. We show that activation of the ABCB1 gene is associated with reduced DNA methylation, reduced H3K9 trimethylation and increased H3K27 acetylation at the ABCB1 promoter. In addition, we find that the ABCB1 locus has moved away from the nuclear lamina in the taxol-resistant cells. This raises the question which of these alterations were causal to derepression. Directly modifying DNA methylation or H3K27 methylation had neither significant effect on ABCB1 expression, nor did it promote drug resistance. In contrast, the disruption of Lamin B Receptor (LBR), a component of the nuclear lamina involved in genome organization, did promote the acquisition of a taxol-resistant phenotype in a subset of cells. Using CRISPRa-mediated gene activation, we could further substantiate a model in which disruption of lamina association renders the ABCB1 gene permissive to derepression. Based on these data we propose a model in which nuclear lamina dissociation of a repressed gene allows for its activation, implying that deregulation of the 3D genome topology could play an important role in tumor evolution and the acquisition of drug resistance.
Endothelial cell senescence exacerbates pulmonary hypertension through Notch-mediated juxtacrine signaling
Authors: Ramadhiani Risa , Ikeda Koji , Miyagawa Kazuya , Ryanto Gusty Rizky Teguh , Tamada Naoki , Suzuki Yoko , Hirata Ken-ichi , Emoto Noriaki
Published: February 2021

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by pathological pulmonary artery remodeling. Endothelial cells (EC) injury including DNA damage is critically involved in the vascular remodeling in PAH, and persistent injury leads to cellular senescence in ECs. Here, we show that EC senescence exacerbates pulmonary hypertension through Notch-mediated juxtacrine signaling. EC-specific progeroid mice that we recently generated showed exacerbated pulmonary hypertension after chronic hypoxia exposure, accompanied by the enhanced pulmonary arterial smooth muscle cells (PASMCs) proliferation in the distal pulmonary arteries. Mechanistically, we identified that senescent ECs highly expressed Notch ligands, and thus activated Notch signaling in PASMCs, leading to enhanced PASMCs proliferation and migration capacities. Consistently, pharmacological inhibition of Notch signaling attenuated the effects of senescent ECs on SMCs functions in vitro, and on the pulmonary hypertension in EC-specific progeroid mice in vivo. These data establish EC senescence as a crucial disease-modifying facor in PAH.
Establishing regulated unconventional secretion for production of heterologous proteins
Authors: Hussnaetter Kai P. , Philipp Magnus , Müntjes Kira , Feldbrügge Michael , Schipper Kerstin
Published: February 2021

Heterologous protein production is a highly demanded biotechnological process. Secretion of the product to the culture broth is advantageous because it drastically reduces downstream pro-cessing costs. We exploit unconventional secretion for heterologous protein expression in the fungal model microorganism Ustilago maydis. Proteins of interest are fused to carrier chitinase Cts1 for export via the fragmentation zone of dividing yeast cells in a lock-type mechanism. Here, we are first to develop regulatory systems for unconventional protein secretion. This enables uncoupling the accumulation of biomass and protein synthesis of a product of choice from its export. Regulation was successfully established at two different levels using transcriptional and post-translational induction strategies. As a proof-of-principle, we applied autoinduction based on transcriptional regulation for the production and secretion of functional anti-Gfp nanobodies. The presented developments comprise tailored solutions for differentially prized products and thus constitute another important step towards a competitive protein production platform.
ChIPflow: from raw data to epigenomic dynamics
Authors: Daunesse Maëlle , Legendre Rachel , Varet Hugo , Pain Adrien , Chica Claudia
Published: February 2021

We present ChIPflow, a Snakemake-based pipeline for epigenomic data from the raw fastq files to the differential analysis. It can be applied to any chromatin factor, e.g. histone modification or transcription factor, which can be profiled with ChIP-seq. ChIPflow streamlines critical steps like the quality assessment of the immunoprecipitation using cross-correlation and the replicate comparison for both narrow and broad peaks. For the differential analysis ChIPflow provides linear and nonlinear methods for normalisation between samples as well as conservative and stringent models for estimating the variance and testing the significance of the observed binding/marking differences.

ChIPflow can process in parallel multiple chromatin factors with different experimental designs, number of biological replicates and/or conditions. It also facilitates the specific parametrisation of each dataset allowing both narrow or broad peak calling, as well as comparisons between the conditions using multiple statistical settings. Finally, complete reports are produced at the end of the bioinformatic and the statistical part of the analysis, which facilitate the data quality control and the interpretation of the results.

We explored the discriminative power of the statistical settings for the differential analysis, using a published dataset of three histone marks (H3K4me3, H3K27ac and H3K4me1) and two transcription factors (Oct4 and Klf4) profiled with ChIP-seq in two biological conditions (shControl and shUbc9). We show that distinct results are obtained depending on the sources of ChIP-seq variability and the dynamics of the chromatin factor under study. We propose that ChIPflow can be used to measure the richness of the epigenomic landscape underlying a biological process by identifying diverse regulatory regimes and the associated genes sets.
Transmission of pre-XDR and XDR-TB in the Mumbai Metropolitan Region, India
Authors: Dreyer Viola , Mandal Ayan , Dev Prachi , Merker Matthias , Barilar Ivan , Utpatel Christian , Nilgiriwala Kayzad , Rodrigues Camilla , Crook Derrick W. ,   the CRyPTIC Consortium, Mistry Nerges , Niemann Stefan
Published: February 2021

Multidrug-resistant (MDR) and extensively drug resistant (XDR)Mycobacterium tuberculosiscomplex (MTBC) strains are a great challenge for tuberculosis (TB) control in India. Still, factors driving the MDR/XDR epidemic in India are not well defined.

To address this, whole genome sequencing (WGS) data from 1 852 MTBC strains obtained from patients from a tertiary care hospital laboratory in Mumbai were used for phylogenetic strain classification, resistance prediction, and cluster analysis (12 allele distance threshold). Factors associated with pre-XDR/XDR-TB were defined by odds ratios and a multivariate logistic regression model.

Overall, 1 017 MTBC strains were MDR, out of which 57.8 % (n=591) were pre-XDR, and 17.9 % (n=183) were XDR. Lineage 2 (L2) strains represented 41.7 % of the MDR, 77.2 % of the pre-XDR, and 86.3 % of the XDR strains, and were significantly associated with pre-XDR/XDR-TB (P < 0.001). Cluster rates were high among MDR (78 %) and pre-XDR/XDR (85 %) strains with three dominant L2 strain clusters (Cl 1-3) representing half of the pre-XDR and two thirds of the XDR-TB cases. Cl 1 strains accounted for 52.5 % of the XDR MTBC strains. Transmission could be confirmed by identical mutation patterns of particular pre-XDR/XDR strains.

As a conclusion high rates of pre-XDR/XDR strains among MDR-TB patients require rapid changes in treatment and control strategies. Transmission of particular pre-XDR/XDR L2 strains is the main driver of the pre-XDR/XDR-TB epidemic. Accordingly, control of the epidemic in the region requires measures with stopping transmission especially of pre-XDR/XDR L2 strains.
A variant selection framework for genome graphs
Authors: Jain Chirag , Tavakoli Neda , Aluru Srinivas
Published: February 2021

Motivation

Variation graph representations are projected to either replace or supplement conventional single genome references due to their ability to capture population genetic diversity and reduce reference bias. Vast catalogues of genetic variants for many species now exist, and it is natural to ask which among these are crucial to circumvent reference bias during read mapping.

Results

In this work, we propose a novel mathematical framework for variant selection, by casting it in terms of minimizing variation graph size subject to preserving paths of lengthαwith at mostδdifferences. This framework leads to a rich set of problems based on the types of variants (SNPs, indels), and whether the goal is to minimize the number of positions at which variants are listed or to minimize the total number of variants listed. We classify the computational complexity of these problems and provide efficient algorithms along with their software implementation when feasible. We empirically evaluate the magnitude of graph reduction achieved in human chromosome variation graphs using multipleαandδparameter values corresponding to short and long-read resequencing characteristics. When our algorithm is run with parameter settings amenable to long-read mapping (α= 10 kbp,δ= 1000), 99.99% SNPs and 73% indel structural variants can be safely excluded from human chromosome 1 variation graph. The graph size reduction can benefit downstream pan-genome analysis.

Implementation

https://github.com/at-cg/VF

Contact

chirag@iisc.ac.in,neda.tavakoli@gatech.edu,aluru@cc.gatech.edu
Evidence that standard metabolic rate and risk-taking to breathe air are linked to boldness, activity level and exploratory behaviour in a catfish
Authors: McKenzie David J. , Belão Thiago C. , Killen Shaun S. , Blasco Felipe R. , Svendsen Morten B.S. , Rantin F. Tadeu
Published: February 2021

We used an air-breathing catfish,Clarias gariepinus,to investigate the hypothesis that individual variation in metabolic rate, and the propensity to take risks to obtain a resource (oxygen from air), would be correlated with behavioural tendencies such as boldness, activity level and exploratory behaviour. The standard metabolic rate (SMR) of 58 juvenile catfish was positively correlated with their rates of aerial respiration in daylight when surfacing was inherently risky. SMR was positively correlated with boldness measured in two contexts, namely the time-lag to resume air-breathing in a potentially dangerous environment (T-res, measured in a respirometer), and the timelag to enter the centre of a novel environment (T-centre, measured in an open field test (OFT)). These two measures of boldness were very highly correlated. Thus, these data support the hypothesis that high SMR and an associated tendency to take risks to acquire resources are linked to increased boldness in animals. Individual SMR was positively correlated with the proportion of time the fish spent moving in the OFT, but was negatively correlated with movement speed. The data confirmed previous observations that these catfish may exhibit a bimodal distribution of T-res phenotypes, whereby individuals either resumed air-breathing relatively rapidly (< 85 min, bold n = 26) or more slowly (> 115 min, shy n = 31) after a startle stimulus. Bold T-res phenotypes had significantly higher SMR than shy; breathed more air during the day, and showed greater boldness but less activity and exploration in the OFT. No parallel bold/shy dichotomy was observed, however, in any measure of boldness in the OFT. Therefore, the data support propositions regarding how SMR and risk-taking should relate to boldness, but provide mixed results about how SMR relates to activity and exploration, and whether bold/shy is a dichotomy or spectrum.
Pluripotent stem cell SOX9 and INS reporters facilitate differentiation into insulin-producing cells
Authors: Dettmer Rabea , Niwolik Isabell , Mehmeti Ilir , Jörns Anne , Naujok Ortwin
Published: February 2021

Differentiation of human pluripotent stem cells into insulin-producing stem cell-derived beta cells harbors great potential for research and therapy of diabetes. The SOX9 gene plays a crucial role during development of the pancreas and particularly in the development of insulin-producing cells as SOX9+ cells form the source for NEUROG3+ endocrine progenitor cells. For the purpose of easy monitoring of differentiation efficiencies into pancreatic progenitors and insulin-producing cells, we generated new reporter lines by knocking in a P2A-H-2Kk-F2A-GFP2 reporter genes into theSOX9locus and a P2A-mCherry reporter gene into theINSlocus mediated by CRISPR/CAS9-technology. The knock-ins enable co-expression of the endogenous genes and reporter genes, report the endogenous gene expression and enable the purification of pancreatic progenitors and insulin-producing cells using FACS or MACS. Using these cell lines we established a new differentiation protocol geared towards SOX9+ cells to efficiently drive human pluripotent stem cells into glucose-responsive beta cells.
“A feed-forward Ca2+-dependent mechanism boosting glycolysis and OXPHOS by activating Aralar-malate-aspartate shuttle, upon neuronal stimulation”
Authors: Pérez-Liébana Irene , Juaristi Inés , González-Sánchez Paloma , González-Moreno Luis , Rial Eduardo , Podunavac Maša , Zakarian Armen , Molgó Jordi , Pardo Beatriz , Satrústegui Jorgina , del Arco Araceli
Published: February 2021

Calcium is an important second messenger regulating a bioenergetic response to the workloads triggered by neuronal activation. In cortical neurons using glucose as only fuel, activation by NMDA, which elicits a strong workload dependent on Na+entry, stimulates glucose uptake, glycolysis, pyruvate and lactate production, and OXPHOS in a Ca2+-dependent way. We find that Ca2+-upregulation of glycolysis, pyruvate levels and respiration, but not glucose uptake, all depend on Aralar/AGC1/Slc25a12, the Ca2+regulated mitochondrial aspartate-glutamate carrier, component of the malate-aspartate shuttle (MAS). Ca2+-activation of MAS increases pyruvate production, which directly fuels workload-stimulated respiration. Also it stimulates glycolysis. MCU silencing had no effect indicating that none of these processes required mitochondrial Ca2+. The neuronal respiratory response to carbachol was also dependent on Aralar, but not on MCU. We also find that cortical neurons are endowed with a constitutive ER-to-mitochondria Ca2+flow maintaining basal cell bioenergetics in which Ryanodine receptors, RyR2, rather than InsP3R, are responsible for Ca2+release, and in which MCU does not participate. The results reveal that in neurons using glucose MCU does not participate in OXPHOS regulation under basal or stimulated conditions, while Aralar-MAS appears as the major Ca2+-dependent pathway tuning simultaneously glycolysis and OXPHOS to neuronal activation.
Latent gammaherpesvirus exacerbates arthritis and requires age-associated B cells
Authors: Mouat Isobel C. , Morse Zach J. , Shanina Iryna , Brown Kelly L. , Horwitz Marc S.
Published: February 2021

Epstein-Barr virus (EBV) infection is associated with rheumatoid arthritis (RA) in adults, though the nature of the relationship remains unknown. Herein, we examine the contribution of viral infection to the severity of arthritis in mice. We provide the first evidence that latent gammaherpesvirus infection enhances clinical arthritis, modeling EBV’s role in RA. Mice latently infected with a murine analog of EBV, gammaherpesvirus 68 (γHV68), develop more severe collagen-induced arthritis and a Th1-skewed immune profile reminiscent of human disease. We demonstrate that disease enhancement requires viral latency and is not due to active virus stimulation of the immune response. Age-associated B cells (ABCs) are associated with several human autoimmune diseases, including arthritis, though their contribution to disease is not well understood. Using ABC knockout mice, we provide the first evidence that ABCs are mechanistically required for viral enhancement of disease, thereby establishing that latent gammaherpesvirus infection stimulates ABCs to provoke arthritis.

Conflict of interest statement

The authors have declared that no conflict of interest exists.
Sleeping at the Switch
Authors: Bouchard Maude , Lina Jean-Marc , Gaudreault Pierre-Olivier , Lafrenière Alexandre , Dubé Jonathan , Gosselin Nadia , Carrier Julie
Published: February 2021

Sleep slow waves are studied for their role in brain plasticity, homeostatic regulation and their changes during aging. Here, we address the possibility that two types of slow waves co-exist in humans. Thirty young and 29 older adults underwent a night of polysomnographic recordings. Using theTransition frequency, slow waves with a slow transition (slow switchers) and with a fast transition (fast switchers) were discovered. Slow switchers had a high EEG connectivity along their depolarization transition while fast switchers had a lower connectivity dynamic and dissipated faster during the night. Aging was associated with lower temporal dissipation of sleep pressure in slow and fast switchers and lower EEG connectivity at the microscale of the oscillations, suggesting a decreased flexibility in the connectivity network of older individuals. Our findings show that two different types of slow waves with possible distinct underlying functions, coexist in the slow wave spectrum.
AN INULIN-TYPE FRUCTAN ENRICHED EXCLUSIVE ENTERAL NUTRITION FORMULA SUPPRESSES COLITIS THROUGH GUT MICROBIOME MODULATION AND PROMOTING EXPANSION OF ANTI-INFLAMMATORY T CELL SUBSETS
Authors: Healey Genelle R. , Tsai Kevin , Lisko Daniel J. , Cook Laura , Vallance Bruce A. , Jacobson Kevan
Published: February 2021

Background & Aims

Exclusive enteral nutrition (EEN) is used to treat pediatric Crohn’s disease (CD), but therapeutic benefits are not long lasting. Due to reported lower efficacy EEN is not routinely used to treat pediatric ulcerative colitis (UC). Inulin-type fructans (IN) beneficially modulate the gut microbiome and promote expansion of anti-inflammatory immune cells. We hypothesized that enriching EEN with IN (EENIN) would enhance treatment efficacy. To test this, we examined the effects of EEN-IN on colitis development, the gut microbiome and CD4+T cells using an adoptive T cell transfer model of colitis.

Methods

TCR-ß deficient mice were randomized to one of four groups: 1) Control, 2) Chow, 3) EEN and 4) EEN-IN, and naïve CD4+T cells were adoptively transferred into groups 2-4, after which mice were monitored for 5-weeks prior to experimental endpoint.

Results

Mice fed EEN-IN showed greater colitis protection, with colonic shortening, goblet cell and crypt density loss reduced over that of EEN fed mice and reduced disease activity and immune cell infiltration compared to chow fed mice, and less crypt hyperplasia and higher survival compared to both groups. EENIN mice maintained colonic mucus layer thickness and had increased levels of Foxp3+IL-10+and Rorγt+IL- 22+and reduced levels of Tbet+IFNγ+and Tbet+TNF+CD4+T cells. EEN-IN also lead to higher butyrate,Bifidobacteriumspp. andBacteroidesspp. concentrations.

Conclusion

The EEN-IN group showed reduced colitis development as compared to the chow and EEN groups. This highlights the potential benefits of EEN-IN as a novel induction therapy for pediatric CD and UC patients.

Synopsis

We demonstrated that inulin-type fructan enriched exclusive enteral nutrition formula reduced colitis development likely due to butyrate-dependent pathways that helped preserve the mucus layer and promote an anti-inflammatory intestinal environment via expansion of regulatory T cells.
Dynamic modulation of spleen germinal center reactions by gut bacteria duringPlasmodiuminfection
Authors: Mandal Rabindra K. , Denny Joshua E. , Namazzi Ruth , Opoka Robert O. , Datta Dibyadyuti , John Chandy C. , Schmidt Nathan W.
Published: February 2021

Gut microbiota educate the local and distal immune system in early life to imprint long-term immunological outcomes while maintaining the capacity to dynamically modulate the local mucosal immune system throughout life. It is unknown if gut microbiota provide signals that dynamically regulate distal immune responses following an extra-gastrointestinal infection. Using the murine model of malaria, we show that existing spleen germinal center reactions are malleable to dynamic cues provided by gut bacteria that impact parasite burden. Gut bacteria composition was also shown to correlate with the severity of malaria in humans. Whereas antibiotic-induced changes in gut bacteria has been associated with immunopathology or impairment of immunity, our data demonstrate antibiotic-induced changes in gut bacteria can enhance humoral immunity toPlasmodium. This effect is not universal, but depends on baseline gut bacteria composition. These data demonstrate the dynamic communications that exist between gut bacteria and the systemic immune system as well as the plasticity of an ongoing humoral immune response.

The study by Mandal R, et al. provides new insight into the dynamic communications that exist between gut bacteria, the systemic immune system and the plasticity of spleen germinal center reactions duringPlasmodiuminfection.
Deficits of Hippocampal RNA Editing and Social Interaction Resulting from Prenatal Stress are Mitigated by Clozapine
Authors: Bristow Greg C. , Dong Erbo , Nwabuisi-Heath Evelyn , Gentile Saverio , Guidotti Alessandro , Sodhi Monsheel
Published: February 2021

Background

Neurodevelopmental deficits resulting from prenatal stress are associated with neurological disorders that include deficits of social behavior, such as schizophrenia1and autism2–7. Studies of human brain and animal models indicate that an epitranscriptomic process known as ‘RNA editing’ contributes to the pathophysiology of these disorders, which occur more frequently in males than in females8–20. RNA editing plays an important role in brain development through its modification of excitatory and inhibitory neurotransmission21.

Methods

We exposed pregnant mice to restraint stress three times daily during gestational weeks 2 and 3. We treated the adult male offspring with haloperidol (1mg/kg), clozapine (5mg/kg) or saline twice daily for 5 days. Subsequently we measured social interaction behavior (SI) and locomotor activity, followed by next-generation sequencing analyses of hippocampal RNA editing.

Results

Mice exposed to PRS exhibited reduced SI, which correlated with hippocampal RNA editing of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA2, GluA3 and GluA4, the potassium channel Kv1.1, the calcium channel subunit Cav1.3, calcium-dependent secretion activator (CAPS-1) and the calcium-dependent cell adhesion protein, cadherin 22 (CDH22). Treatment with clozapine, but not haloperidol, normalized SI behavior, and selectively reduced the deficits in GluA2 RNA editing in PRS mice.

Conclusions

RNA editing may contribute to impaired hippocampal function after exposure to PRS. The efficacy of clozapine in improving SI behavior may include indirect stimulation of GluA2 RNA editing in the hippocampus. Although these data are from male mice and not humans, the results suggest a new molecular pathway by which PRS leads to life-long impairments of hippocampal function.
The anterior cingulate cortex and its role in controlling contextual fear memory to predatory threats
Authors: de Lima Miguel Antonio Xavier , Baldo Marcus Vinicius C. , Oliveira Fernando A. , Canteras Newton Sabino
Published: February 2021

Predator exposure is a life-threatening experience and elicits learned fear responses to the context in which the predator was encountered. The anterior cingulate area (ACA) occupies a pivotal position in a cortical network responsive to predatory threats, and it exerts a critical role in processing fear memory. Ours results revealed that the ACA is involved in both the acquisition and expression of contextual fear to predatory threat. Overall, the ACA can provide predictive relationships between the context and the predator threat and influences fear memory acquisition through projections to the basolateral amygdala and perirhinal region and the expression of contextual fear through projections to the dorsolateral periaqueductal gray. Our results expand previous studies based on classical fear conditioning and open interesting perspectives for understanding how the ACA is involved in processing contextual fear memory to ethologic threatening conditions that entrain specific medial hypothalamic fear circuits (i.e., predator- and conspecific-responsive circuits).
Mesophyll cells mediate systemic reactive oxygen signaling during wounding or heat stress
Authors: Zandalinas Sara I. , Mittler Ron
Published: February 2021

Sensing of heat, high light (HL), or mechanical injury by a single leaf of a plant results in the activation of different systemic signals that reach systemic tissues within minutes and trigger systemic acquired acclimation (SAA) or systemic wound responses (SWRs), resulting in a heightened state of stress readiness of the entire plant. Among the different signals associated with rapid systemic responses to stress in plants are electric, calcium and reactive oxygen species (ROS) waves. These signals propagate from the stressed or injured leaf to the rest of the plant through the plant vascular bundles, and trigger SWRs and SAA in systemic tissues. However, whether they can propagate through other cell types, and whether or not they are interlinked, remain open questions. Here we report that in response to wounding or heat stress (HS), but not HL stress, the ROS wave can propagate through mesophyll cells ofArabidopsis thaliana. Moreover, we show that propagation of the ROS wave through mesophyll cells during these stresses is sufficient to restore SWR and SAA transcript accumulation in systemic leaves, as well as SAA to HS (but not HL). We further show that propagation of the ROS wave through mesophyll cells could contribute to systemic signal integration during HL&HS stress combination. Our findings reveal that the ROS wave can propagate through tissues other than the vascular bundles of plants, and that different stresses can trigger different types of systemic signals that propagate through different cell layers and induce stress-specific systemic responses.

One-sentence summary

In addition to vascular bundles, mesophyll cells can mediate the ROS wave during systemic responses to wounding or heat stress in Arabidopsis.
SpatialDWLS: accurate deconvolution of spatial transcriptomic data
Authors: Dong Rui , Yuan Guo-Cheng
Published: February 2021

Recent development of spatial transcriptomic technologies has made it possible to systematically characterize cellular heterogeneity while preserving spatial information, which greatly enables the investigation of structural organization of a tissue and its impact on modulating cellular behavior. On the other hand, the technology often does not have sufficient resolution to distinguish neighboring cells which may belong to different cell types, therefore it is difficult to identify cell-type distribution directly from the data. To overcome this challenge, we have developed a computational method, called spatialDWLS, to quantitatively estimate the cell-type composition at each spatial location. We benchmarked the performance of spatialDWLS by comparing with a number of existing deconvolution methods using both real and simulated datasets, and we found that spatialDWLS outperformed the other methods in terms of accuracy and speed. By applying spatialDWLS to analyze a human developmental heart dataset, we observed striking spatial-temporal changes of cell-type composition which becomes increasing spatially coherent during development. As such, spatialDWLS provides a valuable computational tool for faithfully extracting biological information from spatial transcriptomic data.
Brain2Pix: Fully convolutional naturalistic video reconstruction from brain activity
Authors: Le Lynn , Ambrogioni Luca , Seeliger Katja , Güçlütürk Yağmur , van Gerven Marcel , Güçlü Umut
Published: February 2021

Reconstructing complex and dynamic visual perception from brain activity remains a major challenge in machine learning applications to neuroscience. Here we present a new method for reconstructing naturalistic images and videos from very large single-participant functional magnetic resonance imaging data that leverages the recent success of image-to-image transformation networks. This is achieved by exploiting spatial information obtained from retinotopic mappings across the visual system. More specifically, we first determine what position each voxel in a particular region of interest would represent in the visual field based on its corresponding receptive field location. Then, the 2D image representation of the brain activity on the visual field is passed to a fully convolutional image-to-image network trained to recover the original stimuli using VGG feature loss with an adversarial regularizer. In our experiments, we show that our method offers a significant improvement over existing video reconstruction techniques.
Cardiac fibroblast GSK-3α mediates adverse myocardial fibrosis via IL-11 and ERK pathway
Authors: Umbarkar Prachi , Tousif Sultan , Singh Anand P. , Anderson Joshua C. , Zhang Qinkun , Lal Hind
Published: February 2021

Background

Heart failure is the leading cause of mortality, morbidity, and healthcare expenditures worldwide. Numerous studies have implicated Glycogen Synthase Kinase-3 (GSK-3) as a promising therapeutic target for cardiovascular diseases. GSK-3 isoforms appear to play overlapping, unique, and even opposing functions in the heart. Recently our group has identified cardiac fibroblast (CF) GSK-3β as a negative regulator of fibrotic remodeling in the ischemic heart. However, the role of CF-GSK-3α in myocardial fibrosis is unknown.

Methods and Results

Herein, we employed two entirely novel conditional fibroblast-specific and tamoxifen-inducible mouse models to define the role of CF-GSK-3α in fibroblast activation and myocardial fibrosis. Specifically, GSK-3α was deleted from cardiac fibroblasts or myofibroblasts with tamoxifen-inducible Tcf21- or periostin-promoter-driven Cre recombinase. At 2 months of age, WT and KO mice were subjected to cardiac injury, and heart functions were monitored by serial echocardiography. Histological analysis and morphometric studies were performed at 8 weeks post-injury. In both settings, GSK-3α deletion restricted fibrotic remodeling and improved cardiac function. To investigate underlying mechanisms, we examined the effect of GSK-3α deletion on myofibroblast transformation and pro-fibrotic TGFβ1-SMAD3 signalingin vitro. A significant reduction in cell migration, collagen gel contraction, and α-SMA expression in TGFβ1 treated GSK-3α KO MEFs confirmed that GSK-3α is required for myofibroblast transformation. Surprisingly, GSK-3α deletion did not affect SMAD3 activation, indicating the pro-fibrotic role of GSK-3α is SMAD3 independent. To further delineate the underlying mechanism, total proteins were isolated from CFs of WT and KO animals at 4 weeks post-injury, and kinome profiling was performed by utilizing PamStation®12 high throughput microarray platform. The kinome analysis identified the downregulation of RAF family kinase activity in GSK3α-KO-CFs. Moreover, mapping of significantly altered kinases against literature annotated interactions generated ERK-centric networks. Importantly, flow cytometric analysis of CFs confirmed a significant decrease in pERK levels in KO mice. Additionally, ourin vitrostudies demonstrated that GSK-3α deletion prevented TGFβ1 induced ERK activation thereby validating our findings from kinome analysis. Interestingly, IL-11, a fibroblast specific downstream effector of TGFβ1, was very low in GSK-3α KO MEFs as compared to WT and ERK inhibition further reduced IL-11 expression in them. All these results indicate that GSK-3α mediates pro-fibrotic response in the injured heart through IL-11 and ERK pathway.

Conclusion

CF-GSK-3α plays a causal role in myocardial fibrosis that could be therapeutically targeted for future clinical applications.