bluuebunny/crossref_metadata_2025 huggingface dataset:
Spatiotemporal Determination of Metabolite Activities in Multiple Corneal Epithelium Barriers on a Chip
Authors: Abdalkader Rodi , Chaleckis Romanas , Wheelock Craig E. , Kamei Ken-ichiro
Published: January 2021

The corneal epithelial barrier maintains the metabolic activities of the ocular surface by regulating membrane transporters and metabolic enzymes responsible for the homeostasis of the eye as well as the pharmacokinetic behavior of drugs. Despite its importance, no established biomimetic in vitro methods are available to perform the spatiotemporal investigation of corneal metabolism and determine the transportation of endogenous and exogenous molecules. This study introduces multiple corneal epithelium barriers on a chip, namely, Cornea-Chip, which enables the spatiotemporal collection as well as analysis of micro-scaled extracellular metabolites from both the apical and basolateral sides of the barriers. Longitudinal samples collected during 48 h period were analyzed using untargeted liquid chromatography–mass spectrometry metabolomics method, and 104 metabolites were annotated. The shifts in extracellular metabolites and quantitative analysis of the mRNA associated with membrane transporters could allow the investigation of the correlation between the expression of and the secretion and transportation of metabolites across the polarized corneal epithelial barrier. The Cornea-Chip might provide a non-invasive, simple, and effectively informative method to determine pharmacokinetics and pharmacodynamics as well as to discover novel biomarkers for drug toxicological and safety tests as an alternative to animal experiments.
UCsim2: 2D Structured Illumination Microscopy using UC2
Authors: Wang Haoran , Lachmann René , Marsikova Barbora , Heintzmann Rainer , Diederich Benedict
Published: January 2021

State-of-the-art microscopy techniques enable the imaging of sub-diffraction barrier biological structures at the price of high-costs or lacking transparency. We try to reduce some of these barriers by presenting a super-resolution upgrade to our recently presented open-source optical toolbox UC2. Our new injection moulded parts allow larger builds with higher precision. The 4× lower manufacturing tolerance compared to 3D printing makes assemblies more reproducible. By adding consumer-grade available open-source hardware such as digital mirror devices (DMD) and laser projectors we demonstrate a compact 3D multimodal setup that combines image scanning microscopy (ISM) and structured illumination microscopy (SIM). We demonstrate a gain in resolution and optical sectioning using the two different modes compared to the widefield limit by imaging Alexa Fluor 647- and SiR-stained HeLa cells. We compare different objective lenses and by sharing the designs and manuals of our setup, we make super-resolution imaging available to everyone.
LuxT controls specific quorum-sensing-regulated behaviors inVibrionaceaespp. via repression ofqrr1, encoding a small regulatory RNA
Authors: Eickhoff Michaela J. , Fei Chenyi , Huang Xiuliang , Bassler Bonnie L.
Published: January 2021

Quorum sensing (QS) is a process of chemical communication bacteria use to transition between individual and collective behaviors. QS depends on the production, release, and synchronous response to signaling molecules called autoinducers (AIs). The marine bacteriumVibrio harveyimonitors AIs using a signal transduction pathway that relies on five small regulatory RNAs (called Qrr1-5) that post-transcriptionally control target genes. Curiously, the small RNAs function redundantly making it difficult to understand the necessity for five of them. Here, we identify LuxT as a transcriptional repressor ofqrr1. LuxT does not regulateqrr2-5, demonstrating thatqrrgenes can be independently controlled to drive unique downstream QS gene expression patterns. LuxT reinforces its control over the same genes it regulates indirectly via repression ofqrr1, through a second transcriptional control mechanism. Genes dually regulated by LuxT specify public goods including an aerolysin-type pore-forming toxin. Phylogenetic analyses reveal that LuxT is conserved amongVibrionaceaeand sequence comparisons predict that LuxT repressesqrr1 in additional species. The present findings reveal that redundant QS regulatory RNAs can carry out both shared and unique functions to endow bacteria with plasticity in their output behaviors.

Author Summary

Bacteria communicate and count their cell numbers using a process called quorum sensing (QS). In response to changes in cell density, QS bacteria alternate between acting as individuals and participating in collective behaviors.Vibrio harveyiis used as a model organism to understand QS-mediated communication. Five small RNAs lie at the heart of theV. harveyiQS system, and they regulate the target genes that underlie the QS response. The small RNAs function redundantly making it difficult to understand whyV. harveyirequires five of them. Here, we discover a regulator, called LuxT, that exclusively represses the gene encoding one of the QS small RNAs. LuxT regulation of one QS small RNA enables unique control of a specific subset of QS target genes. LuxT is broadly conserved amongVibrionaceae. Our findings show how redundant regulatory components can possess both common and unique roles that provide bacteria with plasticity in their behaviors.
Fatty acid oxidation participates of the survival to starvation, cell cycle progression and differentiation in the insect stages ofTrypanosoma cruzi
Authors: Souza Rodolpho Ornitz Oliveira , Damasceno Flávia Silva , Marsiccobetre Sabrina , Biran Marc , Murata Gilson , Curi Rui , Bringaud Frédéric , Silber Ariel Mariano
Published: January 2021

During its complex life cycle,Trypanosoma cruzicolonizes different niches in its insect and mammalian hosts. This characteristic determined the types of parasites that adapted to face challenging environmental cues. The primary environmental challenge, particularly in the insect stages, is poor nutrient availability. TheseT. cruzistages could be exposed to fatty acids originating from the degradation of the perimicrovillar membrane. In this study, we revisit the metabolic fate of fatty acid breakdown inT. cruzi. Herein, we show that during parasite proliferation, the glucose concentration in the medium can regulate the fatty acid metabolism. At the stationary phase, the parasites fully oxidize fatty acids. [U-14C]-palmitate can be taken up from the medium, leading to CO2production via beta-oxidation. Lastly, we also show that fatty acids are degraded through beta-oxidation. Additionally, through beta-oxidation, electrons are fed directly to oxidative phosphorylation, and acetyl-CoA is supplied to the tricarboxylic acid cycle, which can be used to feed other anabolic pathways such as thede novobiosynthesis of fatty acids.

Author Summary

Trypanosoma cruziis a protist parasite with a life cycle involving two types of hosts, a vertebrate one (which includes humans, causing Chagas disease) and an invertebrate one (kissing bugs, which vectorize the infection among mammals). In both hosts, the parasite faces environmental challenges such as sudden changes in the metabolic composition of the medium in which they develop, severe starvation, osmotic stress and redox imbalance, among others. Because kissing bugs feed infrequently in nature, an intriguing aspect ofT. cruzibiology (it exclusively inhabits the digestive tube of these insects) is how they subsist during long periods of starvation. In this work, we show that this parasite performs a metabolic switch from glucose consumption to lipid oxidation, and it is able to consume lipids and the lipid-derived fatty acids from both internal origins as well as externally supplied compounds. When fatty acid oxidation is chemically inhibited by etomoxir, a very well-known drug that inhibits the translocation of fatty acids into the mitochondria, the proliferative insect stage of the parasites has dramatically diminished survival under severe metabolic stress and its differentiation into its infective forms is impaired. Our findings place fatty acids in the centre of the scene regarding their extraordinary resistance to nutrient-depleted environments.
Role of extracellular mycobacteria in blood-retinal barrier invasion in a zebrafish model of ocular TB
Authors: Damera Santhosh Kumar , Panigrahi Ranjan K. , Mitra Sanchita , Basu Soumyava
Published: January 2021

Intraocular inflammation following mycobacterial dissemination to the eye is common in tuberculosis (TB)-endemic countries. However, the early host-pathogen interactions during ocular dissemination are unknown. In this study, we investigate the early events during mycobacterial invasion of the blood-retinal barriers (BRB) with fluorescent taggedMycobacterium marinum(Mm), host macrophages and retinal vasculature in a zebrafish model of ocular TB. We found thatMminvaded the vascular endothelium either in extracellular or intracellular (inside phagocytes) state, typically 3-4 days post-injection (dpi). ExtracellularMmare phagocytosed in the retinal tissue, and progress to form a compact granuloma around 6 dpi. IntracellularMmcrossing the BRB are likely to be less virulent, and either persist inside solitary macrophages (most cases), or progress to loosely arranged granuloma (rare). The early interactions between mycobacteria and host immune cells can thus determine the course of disease during mycobacterial dissemination to the eye.
Evolution in salmon life-history induced by direct and indirect effects of fishing
Authors: Czorlich Y. , Aykanat T. , Erkinaro J. , Orell P. , Primmer CR.
Published: January 2021

Understanding the drivers of evolution is a fundamental aim in biology. However, identifying the evolutionary impacts of human activities, both direct and indirect, is challenging because of lack of temporal data and limited knowledge of the genetic basis of most traits1. Atlantic salmon is a species exposed to intense anthropogenic pressures during its anadromous life cycle2. Previous research has shown that salmon age at maturity has evolved towards earlier maturation over the last 40 years, with an 18% decrease3in the allele associated with late maturation at the large-effectvgll3locus4; but the drivers of this change remain unknown. Here, we link genetic and phenotypic changes in a large Atlantic salmon population with salmon prey species biomass in the Barents Sea, temperature, and fishing effort in order to identify drivers of age at maturity evolution. We show that age at maturity evolution is associated with two different types of fisheries induced evolution acting in opposing directions: an indirect effect linked with commercial harvest of a salmon prey species (capelin) at sea (selection against late maturation), and a direct effect due to temporal changes in net fishing pressure in the river (surprisingly, selection against early maturation). Although the potential for direct and indirect evolutionary effects of fishing have been acknowledged, empirical evidence for induced changes at the genetic level has been lacking5. As capelin are primarily harvested to produce fish meal and oil for aquaculture6, we hereby identify an indirect path by which Atlantic salmon aquaculture may negatively affect wild populations.
Genetic influences on brain activation and large-scale functional connectivity during nociceptive processing: a twin study
Authors: Kastrati Gránit , Rosén Jörgen , Thompson William H. , Chen Xu , Larsson Henrik , Nichols Thomas E. , Tracey Irene , Fransson Peter , Åhs Fredrik , Jensen Karin B.
Published: January 2021

Nociceptive processing in the human brain is a signal that enables harm avoidance, with large interindividual variance. The relative contributions of genes and environment to the neural structures that support nociception have not been studied in twins previously. Here, we employed a classic twin-design to determine brain structures influenced by additive genetics. We found genetic influences on nociceptive processing in the midcingulate cortex, bilateral posterior insulae and thalamus. In addition to brain activations, we found genetic contributions to large-scale functional connectivity during nociceptive processing. We conclude that additive genetics influence specific aspects of nociceptive processing, which improves our understanding of human nociceptive processing.
compaRe, an ultra-fast and robust suite for multiparametric screening, identifies phenotypic drug responses in acute myeloid leukemia
Authors: Hajkarim Morteza Chalabi , Karjalainen Ella , Osipovitch Mikhail , Dimopoulos Konstantinos , Gordon Sandra Leigh , Ambri Francesca , Rasmussen Kasper Dindler , Grønbæk Kirsten , Helin Kristian , Wennerberg Krister , Won Kyoung Jae
Published: January 2021

Multiparametric phenotypic screening of cells, for example assessing their responses to small molecules or knockdown/knockout of specific genes, is a powerful approach to understanding cellular systems and identifying potential new therapeutic strategies. However, automated tools for analyzing similarities and differences between a large number of tested conditions have not been readily available. Methods designed for clustering cells cannot identify differences between samples effectively. We introducecompaRefor ultra-fast and robust analysis of multiparametric high-throughput screening. Applying a mass-aware gridding algorithm using hypercubes,compaReperforms automatic and effective similarity comparison for hundreds to thousands of tests and provides information about the treatment effect. Particularly for screening data,compaReis equipped with modules to remove various sources of bias.

Benchmarking tests show thatcompaRecan circumvent batch effects and perform a similarity analysis substantially faster than conventional analysis tools. ApplyingcompaReto high-throughput flow cytometry screening data, we were able to distinguish subtle phenotypic drug responses in a human sample and a genetically engineered mouse model with acute myeloid leukemia (AML).compaRerevealed groups of drugs with similar responses even though their mechanisms are distinct from each other. In another screening,compaReeffectively circumvented batch effects and grouped samples from AML and myelodysplastic syndrome (MDS) patients using clinical flow cytometry data.
An optimized retroviral toolbox for overexpression and genetic perturbation of primary lymphocytes
Authors: van der Donk Lieve EH , van der Spek Jet , van Duivenvoorde Tom , ten Brink Marieke S , Geijtenbeek Teunis BH , Kuijl Coenraad P , van Heijst Jeroen WJ , Ates Louis S
Published: January 2021

Genetic manipulation of primary lymphocytes is crucial for both clinical purposes and fundamental research. Despite their broad use, we encountered a paucity of data on systematic comparison and optimization of retroviral vectors, the workhorses of genetic modification of primary lymphocytes. Here, we report the construction and validation of a versatile range of retroviral expression vectors. These vectors can be used for the knockdown or overexpression of genes of interest in primary human and murine lymphocytes, in combination with a wide choice of selection and reporter strategies. By streamlining the vector backbone and insert design, these publicly available vectors allow easy interchangeability of the independent building blocks, such as different promoters, fluorescent proteins, surface markers and antibiotic resistance cassettes. We validated these vectors and tested the optimal promoters for in vitro and in vivo overexpression and knockdown of the murine T cell antigen receptor. By publicly sharing these vectors and the data on their optimization, we aim to facilitate genetic modification of primary lymphocytes for researchers entering this field.
Measuring arterial pulsatility with Dynamic Inflow MAgnitude Contrast (DIMAC)
Authors: Whittaker Joseph R. , Fasano Fabrizio , Venzi Marcello , Liebig Patrick , Gallichan Daniel , Murphy Kevin
Published: January 2021

The pulsatility of blood flow through cerebral arteries is clinically important, as it is intrinsically associated with cerebrovascular health. In this study we outline a new MRI approach to measuring the real-time pulsatile flow in cerebral arteries, which is based on the inflow phenomenon associated with fast gradient-recalled-echo acquisitions. Unlike traditional phase-contrast techniques, this new method, which we dubDynamicInflowMagnitudeContrast (DIMAC), does not require velocity-encoding gradients as sensitivity to flow velocity is derived purely from the inflow effect. We achieved this using a highly accelerated single slice EPI acquisition with a very short TR (15 ms) and a 90° flip angle, thus maximizing inflow contrast. We simulate the spoiled GRE signal in the presence of large arteries and perform a sensitivity analysis to demonstrate that in the regime of high inflow contrast it shows much greater sensitivity to flow velocity over blood volume changes. We support this theoretical prediction within-vivodata collected in two separate experiments designed to demonstrate the utility of the DIMAC signal contrast. We perform a hypercapnia challenge experiment in order to experimentally modulate arterial tone within subjects, and thus modulate the arterial pulsatile flow waveform. We also perform a thigh-cuff release challenge, designed to induce a transient drop in blood pressure, and demonstrate that the continuous DIMAC signal captures the complex transient change in the pulsatile and non-pulsatile components of flow. In summary, this study proposes a new role for a well-established source of MR image contrast and demonstrates its potential for measuring both steady-state and dynamic changes in arterial tone.

Highlights

We present a novel method for measuring pulsatility of cerebral arteries.

The inflow effect on fast GRE imaging can be exploited to yield a flow velocity dependent signal.

We measure pulsatile flow through cerebral arteries dynamically on a beat-to-beat basis.

We use physiological challenges to demonstrate sensitivity to dynamic and steady-state changes in vascular tone.
Understanding the holobiont: crosstalk between gut microbiota and mitochondria during endurance
Authors: Mach Núria , Moroldo Marco , Rau Andrea , Lecardonnel Jérôme , Moyec Laurence Le , Robert Céline , Barrey Eric
Published: January 2021

Endurance exercise has a dramatic impact on the functionality of mitochondria and on the composition of the intestinal microbiome, but the mechanisms regulating the crosstalk between these two components are still largely unknown. Here, we sampled 20 elite horses before and after an endurance race and used blood transcriptome, blood metabolome and fecal microbiome to describe the gut-mitochondria crosstalk. A subset of mitochondria-related differentially expressed genes involved in pathways such as energy metabolism, oxidative stress and inflammation was discovered and then shown to be associated with butyrate-producing bacteria of theLachnospiraceaefamily, especiallyEubacterium. The mechanisms involved were not fully understood, but through the action of their metabolites likely acted onPPARγ,theFRX-CREBaxis and their downstream targets to delay the onset of hypoglycemia, inflammation and extend running time. Our results also suggested that circulating free fatty acids may act not merely as fuel but drive mitochondrial inflammatory responses triggered by the translocation of gut bacterial polysaccharides following endurance. Targeting the gut-mitochondria axis therefore appears to be a potential strategy to enhance athletic performance.
Joint development recovery on resumption of embryonic movement following paralysis
Authors: Rolfe Rebecca A. , O’Callaghan David Scanlon , Murphy Paula
Published: January 2021

Fetal activityin uterois a normal part of pregnancy and reduced or absent movement can lead to long-term skeletal defects such as Fetal Akinesia Deformation Sequence (FADS), joint dysplasia and arthrogryposis. A variety of animal models with decreased or absent embryonic movements show a consistent set of developmental defects providing insight into the aetiology of congenital skeletal abnormalities. At developing joints defects include reduced joint interzones with frequent fusion of cartilaginous skeletal rudiments across the joint. At the spine defects include shortening and a spectrum of curvature deformations. An important question, with relevance to possible therapeutic interventions for human conditions, is the capacity for recovery with resumption of movement following short term immobilisation. Here we use the well-established chick model to compare the effects of sustained immobilisation from embryonic day (E) 4-10 to two different recovery scenarios: (i) natural recovery from E6 until E10 and (ii) the addition of hyperactive movement stimulation during the recovery period. We demonstrate partial recovery of movement and partial recovery of joint development under both recovery conditions, but no improvement in spine defects. The joints examined (elbow, hip and knee) showed better recovery in hindlimb than forelimb, with hyperactive mobility leading to greater recovery in the knee and hip. The hip joint showed the best recovery with improved rudiment separation, tissue organisation and commencement of cavitation. This work demonstrates that movement post paralysis can partially-recover specific aspects of joint development which could inform therapeutic approaches to ameliorate the effects of human fetal immobility.

Summary Statement

The study reveals that embryonic movement post paralysis can partially-recover specific aspects of joint development, which could inform therapeutic approaches to ameliorate the effects of restricted fetal movementin utero.
Humanization of antibodies using a machine learning approach on large-scale repertoire data
Authors: Chin Mark , Marks Claire , Deane Charlotte M.
Published: January 2021

Monoclonal antibody therapeutics are often produced from non-human sources (typically murine), and can therefore generate immunogenic responses in humans. Humanization procedures aim to produce antibody therapeutics that do not elicit an immune response and are safe for human use, without impacting efficacy. Humanization is normally carried out in a largely trial-and-error experimental process. We have built machine learning classifiers that can discriminate between human and non-human antibody variable domain sequences using the large amount of repertoire data now available. Our classifiers consistently outperform existing best-in-class models, and our output scores exhibit a negative relationship with the experimental immunogenicity of existing antibody therapeutics. We used our classifiers to develop a novel, computational humanization tool, Hu-mAb, that suggests mutations to an input sequence to reduce its immunogenicity. For a set of existing therapeutics with known precursor sequences, the mutations suggested by Hu-mAb show significant overlap with those deduced experimentally. Hu-mAb is therefore an effective replacement for trial- and-error humanization experiments, producing similar results in a fraction of the time. Hu-mAb is freely available to use at opig.stats.ox.ac.uk/webapps/humab.
Targeting conserved sequences circumvents the evolution of resistance in a viral gene drive against human cytomegalovirus
Authors: Walter Marius , Perrone Rosalba , Verdin Eric
Published: January 2021

Gene drives are genetic systems designed to efficiently spread a modification through a population. They have been designed almost exclusively in eukaryotic species, and especially in insects. We recently developed a CRISPR-based gene drive system in herpesviruses that relies on similar mechanisms and could efficiently spread into a population of wildtype viruses. A common consequence of gene drives in insects is the appearance and selection of drive-resistant sequences that are no longer recognized by CRISPR-Cas9. Here, we analyze in cell culture experiments the evolution of resistance in a viral gene drive against human cytomegalovirus. We report that after an initial invasion of the wildtype population, a drive-resistant population is positively selected over time and outcompetes gene drive viruses. However, we show that targeting evolutionary conserved sequences ensures that drive-resistant viruses acquire long-lasting mutations and are durably attenuated. As a consequence, and even though engineered viruses don’t stably persist in the viral population, remaining viruses have a replication defect, leading to a long-term reduction of viral levels. This marks an important step toward developing effective gene drives in herpesviruses, especially for therapeutic applications.
The neural basis of effort valuation: A meta-analysis of functional magnetic resonance imaging studies
Authors: Lopez-Gamundi Paula , Yao Yuan-Wei , Chong Trevor T-J. , Heekeren Hauke R. , Herrero Ernest Mas , Pallares Josep Marco
Published: January 2021

Choosing how much effort to expend is a critical for everyday decisions. While effort-based decision-making is altered in common psychopathologies and many neuroimaging studies have been conducted to examine how effort is valued, it remains unclear where the brain processes effort-related costs and integrates them with rewards. Using meta-analyses of combined maps and coordinates of functional magnetic resonance imaging (fMRI) studies (total N = 22), we showed that raw effort demands consistently activated the pre-supplementary motor area (pre-SMA). In contrast, the net value of effortful reward consistently activated regions, such as the ventromedial prefrontal cortex (vmPFC) and ventral striatum (VS), that have been previously implicated in value integration in other cost domains. The opposite activation patterns of the pre-SMA and vmPFC imply a double dissociation of these two regions, in which the pre-SMA is involved in pure effort cost representation and the vmPFC in net value integration. These findings advance our understanding of the neural basis of effort-related valuation and reveal potential brain targets to treat motivation-related disorders.
Human gut bacteria produce TH17-modulating bile acid metabolites
Authors: Paik Donggi , Yao Lina , Zhang Yancong , Bae Sena , D’Agostino Gabriel D. , Kim Eunha , Franzosa Eric A. , Avila-Pacheco Julian , Bisanz Jordan E. , Rakowski Christopher K. , Vlamakis Hera , Xavier Ramnik J. , Turnbaugh Peter J. , Longman Randy S. , Krout Michael R. , Clish Clary B. , Huttenhower Curtis , Huh Jun R. , Devlin A. Sloan
Published: January 2021

The microbiota plays a pivotal role in gut immune homeostasis. Bacteria influence the development and function of host immune cells, including T helper cells expressing interleukin-17a (TH17 cells). We previously reported that the bile acid metabolite 3-oxolithocholic acid (3-oxoLCA) inhibits TH17 cell differentiation1. While it was suggested that gut-residing bacteria produce 3-oxoLCA, the identity of such bacteria was unknown. Furthermore, it was not clear whether 3-oxoLCA and other immunomodulatory bile acids are associated with gut inflammatory pathologies in humans. Using a high-throughput screen, we identified human gut bacteria and corresponding enzymes that convert the secondary bile acid lithocholic acid into 3-oxoLCA as well as the abundant gut metabolite isolithocholic acid (isoLCA). Like 3-oxoLCA, isoLCA suppressed TH17 differentiation by inhibiting RORγt (retinoic acid receptor-related orphan nuclear receptor γt), a key TH17 cell-promoting transcription factor. Levels of both 3-oxoLCA and isoLCA and the 3α-hydroxysteroid dehydrogenase (3α-HSDH) genes required for their biosynthesis were significantly reduced in patients with inflammatory bowel diseases (IBD). Moreover, levels of these bile acids were inversely correlated with expression of TH17 cell-associated genes. Overall, our data suggest that bacterially produced TH17 cell-inhibitory bile acids may reduce the risk of autoimmune and inflammatory disorders such as IBD.
Selective modulation of population dynamics during neuroprosthetic skill learning
Authors: Zippi Ellen L. , You Albert K. , Ganguly Karunesh , Carmena Jose M.
Published: January 2021

Learning to control a brain-machine interface (BMI) is associated with the emergence of coordinated neural dynamics in populations of neurons whose activity serves as direct input to the BMI decoder (direct subpopulation). While previous work shows differential modification of firing rate modulation in this population relative to a population whose activity was not directly input to the BMI decoder (indirect subpopulation), little is known about how learning-rated changes in cortical population dynamics within these groups compare. To investigate this, we monitored both direct and indirect subpopulations as two macaque monkeys learned to control a BMI. We found that while the combined population increased coordinated neural dynamics, this coordination was primarily driven by changes in the direct subpopulation while the indirect subpopulation remained relatively stable. These findings indicate that motor cortex refines cortical dynamics throughout the entire network during learning, with a more pronounced effect in ensembles causally linked to behavior.
Strong and tunable anti-CRISPR/Cas9 activity of AcrIIA4 in plants
Authors: Calvache Camilo , Vazquez-Vilar Marta , Selma Sara , Uranga Mireia , Daròs José Antonio , Orzáez Diego
Published: January 2021

This study describes the strong anti-CRISPR activity of the bacterial AcrIIA4 protein inNicotiana benthamiana, a model plant used as molecular farming platform. The results demonstrate that AcrIIA4 abolishes site-directed mutagenesis in leaves when transiently co-expressed with CRISPR/Cas9. We also show that AcrIIA4 represses CRISPR/dCas9-based transcriptional activation (CRISPRa) of both reporter and endogenous genes in a highly efficient, dose-dependent manner. Furthermore, the fusion of an auxin degron to AcrIIA4 results in auxin-regulated activation of a downstream reporter gene. The strong anti-Cas9 activity of AcrIIA4 reported here opens new possibilities for customized control of gene editing and gene expression in plants.
Low light intensity delays vegetative phase change inArabidopsis thaliana
Authors: Xu Mingli , Hu Tieqiang , Poethig R. Scott
Published: January 2021

Plants that develop under low intensity light (LL) often display a phenotype known as the “shade tolerance syndrome (STS)”. This syndrome is similar to the phenotype of plants in the juvenile phase of shoot development, but the basis for this similarity is unknown. We tested the hypothesis that the STS is regulated by the same mechanism that regulates the juvenile vegetative phase by examining the effect of LL on rosette development inArabidopsis thaliana. We found that LL prolonged the juvenile vegetative phase and that this was associated with an increase the expression of the master regulators of vegetative phase change, miR156 and miR157, and a decrease in the expression of their SPL targets. Exogenous sucrose partially corrected the effect of LL on seedling development and miR156 expression. Our results suggest that the response of Arabidopsis to LL is mediated by an increase in miR156/miR157 expression and by factors that repress SPL gene expression independently of miR156/miR157, and is caused in part by a decrease in carbohydrate production. The effect of LL on vegetative phase change does not require the photoreceptors and transcription factors responsible for the shade avoidance syndrome, implying that light intensity and light quality regulate rosette development by different pathways.
Proton-gated coincidence detection is a common feature of GPCR signaling
Authors: Kapolka Nicholas J. , Rowe Jacob B. , Taghon Geoffrey J. , Morgan William M. , O’Shea Corin R. , Isom Daniel G.
Published: January 2021

The evolutionary expansion of G protein-coupled receptors (GPCRs) has produced a rich diversity of transmembrane sensors for many physical and chemical signals. In humans alone, over 800 GPCRs detect stimuli such as light, hormones, and metabolites to guide cellular decision making primarily using intracellular G protein signaling networks. This diversity is further enriched by GPCRs that function as molecular logic gates capable of discerning multiple inputs to transduce cues encoded in complex, context-dependent signals. Here, we show that many GPCRs are switch-like Boolean-gated coincidence detectors that link proton (H+) binding to GPCR signaling. Using a panel of 28 receptors, covering 280 individual GPCR-Gα coupling combinations, we show that H+gating both positively and negatively modulates and controls GPCR signaling. Notably, these observations extend to all modes of GPCR pharmacology including ligand efficacy, potency, and cooperativity. Additionally, we show that GPCR antagonism and constitutive activity are regulated by H+gating and report the discovery of a new acid sensor, the adenosine A2a receptor (ADORA2A), which can be activated solely by acidic pH. Together, these findings establish a new paradigm for GPCR biology and pharmacology in acidified microenvironments such as endosomes, synapses, tumors, and ischemic vasculature.
Systematically quantifying morphological features reveals constraints on organoid phenotypes
Authors: Beck Lauren E. , Lee Jasmine , Coté Christopher , Dunagin Margaret C. , Lukonin Ilya , Salla Nikkita , Chang Marcello K. , Hughes Alex J. , Mornin Joseph D. , Gartner Zev J. , Liberali Prisca , Raj Arjun
Published: January 2021

Organoids recapitulate complex 3D organ structures and represent a unique opportunity to probe the principles of self-organization. While we can alter an organoid’s morphology by manipulating the culture conditions, the morphology of an organoid often resembles that of its original organ, suggesting that organoid morphologies are governed by a set of tissue-specific constraints. Here, we establish a framework to identify constraints on an organoid’s morphological features by quantifying them from microscopy images of organoids exposed to a range of perturbations. We apply this framework to Madin-Darby Canine Kidney cysts and show that they obey a number of constraints taking the form of scaling relationships or caps on certain parameters. For example, we found that the number, but not size, of cells increases with increasing cyst size. We also find that these constraints vary with cyst age and can be altered by varying the culture conditions. We observed similar sets of constraints in intestinal organoids. This quantitative framework for identifying constraints on organoid morphologies may inform future efforts to engineer organoids.
Speech-like sounds dominate the human infant vocal landscape
Authors: Oller D. Kimbrough , Ramsay Gordon , Bene Edina , Long Helen L. , Griebel Ulrike
Published: January 2021

Early human infant vocalization is viewed as forming not only a critical foundation for vocal learning of language, but also a crucial realm of communication affecting emotional and social development. Although speech-like sounds are rare or absent in other ape infants, they share distress sounds (shrieks and cries) and laughter with humans, forming a potential basis for especially informative cross-species comparisons as well as potential insights regarding usage and learning of vocal sounds. A fundamental need to make such comparisons possible is empirical research to document frequency of occurrence of vocalizations of various types in natural environments.

The present work focuses on laughter in the human infant, a topic that has been viewed by many as a key factor in social development for humans and other apes. Yet we know of no research quantifying frequency of occurrence of human infant laughter in natural environments across the first year. In the past two decades it has been shown that the predominant vocalizations of the human infant are “protophones”, the precursor sounds to speech. Longitudinal research has indicated unambiguously that protophones outnumber cries by a factor of at least five based on data from random-sampling of all-day recordings across the whole first year. The present work expands on the prior reports by reporting data showing that human infant laughter occurs even more rarely than cry in all-day recordings. Yet laughter is clearly a salient and important aspect of social development. We reason about the dominance of protophones in the infant vocal landscape in light of their role in illuminating human vocal learning and the origin of language.
Variance misperception under skewed empirical noise statistics explains overconfidence in the visual periphery
Authors: Winter Charles J. , Peters Megan A. K.
Published: January 2021

Perceptual confidence typically corresponds to accuracy. However, observers can be overconfident relative to accuracy, termed ‘subjective inflation’. Inflation is stronger in the visual periphery relative to central vision, especially under conditions of peripheral inattention. Previous literature suggests inflation stems from errors in estimating noise, i.e. ‘variance misperception’. However, despite previous Bayesian hypotheses about metacognitive noise estimation, no work has systematically explored how noise estimation may critically depend on empirical noise statistics which may differ across the visual field, with central noise distributed symmetrically but peripheral noise positively skewed. Here we examined central and peripheral vision predictions from five Bayesian-inspired noise-estimation algorithms under varying usage of noise priors, including effects of attention. Models that failed to optimally estimate noise exhibited peripheral inflation, but only models that explicitly used peripheral noise priors -- but used them incorrectly -- showed increasing peripheral inflation under increasing peripheral inattention. Further, only one model successfully captured previous empirical results which showed a selective increase in confidence in incorrect responses under performance reductions due to inattention accompanied by no change in confidence in correct responses; this was the model that implemented Bayesian estimation of peripheral noise, but using an (incorrect) symmetric rather than the correct positively skewed peripheral noise prior. Our findings explain peripheral inflation, especially under inattention, and suggest future experiments that might reveal the noise expectations used by the visual metacognitive system.

Significance

Perceptual confidence can dissociate from accuracy in peripheral visual perception, a phenomenon known as peripheral inflation. No previous model has considered how this phenomenon may arise from metacognitive noise estimation which depends on empirical noise statistics. Here we simulate Bayesian-inspired noise estimation algorithms to show that the system’s erroneous beliefs about distributions of noise in the visual periphery can explain the occurrence of peripheral inflation, including how inflation varies with attentional manipulations in surprising ways. Our results explain why peripheral inflation occurs by positing a new Bayesian metacognitive noise estimation mechanism, paving the way for future psychophysical studies.
Synaptojanin1 deficiency upregulates basal level autophagosome formation in astrocytes
Authors: Pan Ping-Yue , Zhu Justin , Rizvi Asma , Zhu Xinyu , Tanaka Hikari , Dreyfus Cheryl F.
Published: January 2021

Macroautophagy (hereafter, autophagy) dysregulation is implicated in multiple neurological disorders. While the autophagy pathways are heavily investigated in heterologous cells and neurons, how autophagy is regulated in the astrocyte, the most abundant cell type in the mammalian brain, is less understood. Here we report that Synaptojanin1 (Synj1), a neuron enriched lipid phosphatase, is expressed in low levels in astrocytes and represses autophagy at the basal level. Synj1 is encoded by theSynj1gene, whose missense mutations are linked to Parkinsonism with seizure. While the best-known role of Synj1 is to facilitate synaptic vesicle recycling, recent studies suggest that Synj1 also regulates autophagy. Our previous study using theSynj1haploinsufficient (Synj1+/−) mouse demonstrated thatSynj1deficiency was associated with an age-dependent autophagy impairment in multiple brain regions. We now use cultured astrocytes fromSynj1deficient mice to investigate its role in astrocyte autophagy. We demonstrate that Synj1 deficient astrocytes exhibit increased LC3 puncta, which is more pronounced when lysosomal acidification is blocked. The increased autophagosome formation is accompanied by reduced autophagy substrate, p62, but an insensitivity to starvation induced autophagy clearance. Moreover, we show, for the first time, that the Parkinsonism associated R839C mutation impacts astrocyte autophagy. The profound impact of this mutation on Synj1’s phosphatase functions results in elevated basal level autophagosome formation and clearance that mimicsSynj1deletion. We find that energy sensing molecules, including mTOR and AMPK, are altered inSynj1deficient astrocytes, which may contribute to the enhanced basal level autophagy.
The collapse of genetic incompatibilities in hybridizing populations
Authors: Xiong Tianzhu , Mallet James
Published: January 2021

Diverging species are often genetically incompatible upon hybridization. Such incompatibilities are considered important in keeping the integrity of species from the disruption of hybrids. However, recent empirical work has shown that not all incompatibilities are gene-flow-proof, and they can collapse due to continuing hybridization. Counterintuitively, many studies found that incompatible alleles are already segregating within species, whereas they should go extinct quickly in a randomly mating population. Due to the complexity of multilocus epistasis, few general principles explain behaviors of incompatibilities under gene flow both within and between species. In the current work, we argue that the redundancy of genetic mechanisms can robustly determine the dynamics of intrinsic incompatibilities under gene flow. While higher genetic redundancy decreases the stability of incompatibilities during hybridization, it also increases the tolerance of incompatibility polymorphism within each species. We treat two general classes of incompatibilities. In the redundant class, similar to the classical Dobzhansky-Muller system, the collapse is continuous and eventually approaches quasi-neutral polymorphism between broadly-sympatric species, often as a result of isolation-by-distance. In the non-redundant class, analogous to the shifting-balance process, incompatibilities collapse abruptly with spatial traveling waves. We obtained simulated and analytical results for several incompatibility models to demonstrate the differences between the two classes. As both redundant and non-redundant genetic mechanisms of incompatibilities are common, the proposed conceptual framework may help understand the abundance of incompatibilities in natural populations.
Explosive diversification following continental colonizations by canids
Authors: Porto Lucas M. V. , Etienne Rampal S. , Maestri Renan
Published: January 2021

Colonization of a new environment may trigger an explosive radiation process, defined as an accelerated accumulation of species in a short period of time. However, how often colonization events trigger explosive radiations is still an open question. We studied the worldwide dispersal of the subfamily Caninae, to investigate whether the invasion of new continents resulted in explosive radiations. We used a combination of phylogenetic analyses and ancestral area reconstructions to estimate ancestral ranges of 56 extant and extinct species of Caninae, as well as variation in speciation and extinction rates through time and across clades. Our findings indicate that canids experienced an explosive radiation event when lineages were able to cross the Bering Strait and the Isthmus of Panama to reach Eurasia and South America, respectively, around 11 million years ago. This large number of species arising in a short period of time suggests that canids experienced ecological opportunity events within the new areas, implying that the differences in the ecological settings between continents may be responsible for the variation in clade dynamics. We argue that interaction with other carnivores probably also affected the diversification dynamics of canids.
Anti-stress effects of the glucagon-like peptide-1 receptor agonist liraglutide in zebrafish
Authors: Bertelli Pablo R. , Mocelin Ricieri , Marcon Matheus , Sachett Adrieli , Gomez Rosane , Rosa Adriane R. , Herrmann Ana P. , Piato Angelo
Published: January 2021

Stress-related disorders are extremely harmful and cause significant impacts on the individual and society. Despite the limited evidence regarding glucagon-like peptide-1 receptor (GLP-1R) and mental disorders, a few clinical and preclinical studies suggest that modulating this system could improve symptoms of stress-related disorders. This study aimed to investigate the effects of liraglutide, a GLP-1R agonist, on neurobehavioral phenotypes and brain oxidative status in adult zebrafish. Acute liraglutide promoted anxiolytic-like effects in the light/dark test, while chronic treatment blocked the impact of unpredictable chronic stress on behavioral and physiological parameters. Taken together, our study demonstrates that liraglutide is active on zebrafish brain and may counteract some of the effects induced by stress. More studies are warranted to further elucidate the potential of GLP-1R agonists for the management of brain disorders.
In vivoCGG repeat RNA binding protein capture identifies RAN translation modifiers and suppressors of repeat toxicity
Authors: Malik Indranil , Tseng Yi-Ju , Wright Shannon E. , Zheng Kristina , Ramaiyer Prithika , Green Katelyn M. , Todd Peter K.
Published: January 2021

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a transcribed CGG repeat expansion in the 5’ UTR ofFMR1. Expanded CGG repeat RNAs both sequester RNA-binding proteins (RBPs) into nuclear foci and undergo repeat-associated non-AUG (RAN) translation into toxic homopolymeric peptides. RBPs that interact with CGG repeats may play a pivotal role in foci formation and/or RAN translation. Here we employed a CGG repeat RNA-tagging system to capture and identify CGG repeat binding RBPsin vivounder different cellular conditions. We found that several SR (serine/arginine-rich domain) proteins interact with CGG repeat RNAs basally and under cellular stress. These same proteins strongly modify toxicity in a Drosophila model of FXTAS, improving eye degeneration and survival. Furthermore, genetic or pharmacological targeting of the serine/arginine protein kinases (SRPKs) suppresses RAN translation in cellular reporters and toxicity in fly models of FXTAS and C9orf72 ALS/FTD. Finally, pharmacological targeting of SRPK1 supressed CGG repeat toxicity and enhanced survival in rodent neurons. Taken together, these findings demonstrate roles for CGG repeat RNA binding proteins in both RAN translation and repeat toxicity and suggest SRPK inhibition may serve as a possible therapeutic strategy in repeat expansion disorders.