bluuebunny/crossref_metadata_2025 huggingface dataset:
High fidelity sensory-evoked responses in neocortex after intravenous injection of genetically encoded calcium sensors
Authors: Leikvoll Austin , Kara Prakash
Published: March 2023

Two-photon imaging of genetically-encoded calcium indicators (GECIs) has traditionally relied on intracranial injections of adeno-associated virus (AAV) or transgenic animals to achieve expression. Intracranial injections require an invasive surgery and result in a relatively small volume of tissue labeling. Transgenic animals, although they can have brain-wide GECI expression, often express GECIs in only a small subset of neurons, may have abnormal behavioral phenotypes, and are currently limited to older generations of GECIs. Inspired by recent developments in the synthesis of AAVs that readily cross the blood brain barrier, we tested whether an alternative strategy of intravenously injecting AAV-PhP.eB is suitable for two-photon calcium imaging of neurons over many months after injection. We injected young (postnatal day 23 to 31) C57BL/6J mice with AAV-PhP.eB-Synapsin-jGCaMP7s via the retro-orbital sinus. After allowing 5 to 34 weeks for expression, we performed conventional and widefield two-photon imaging of layers 2/3, 4 and 5 of the primary visual cortex. We found reproducible trial-by-trial neural responses and tuning properties consistent with known feature selectivity in the visual cortex. Thus, intravenous injection of AAV-PhP.eB does not interfere with the normal processing in neural circuits.In vivoand histological images show no nuclear expression of jGCaMP7s for at least 34 weeks post-injection.
Diet-Induced Glial Insulin Resistance Impairs The Clearance Of Neuronal Debris
Authors: Alassaf Mroj , Rajan Akhila
Published: March 2023

Obesity significantly increases the risk of developing neurodegenerative disorders, yet the precise mechanisms underlying this connection remain unclear. Defects in glial phagocytic function are a key feature of neurodegenerative disorders, as delayed clearance of neuronal debris can result in inflammation, neuronal death, and poor nervous system recovery. Mounting evidence indicates that glial function can affect feeding behavior, weight, and systemic metabolism, suggesting that diet may play a role in regulating glial function. While it is appreciated that glial cells are insulin sensitive, whether obesogenic diets can induce glial insulin resistance and thereby impair glial phagocytic function remains unknown. Here, using aDrosophilamodel, we show that a chronic obesogenic diet induces glial insulin resistance and impairs the clearance of neuronal debris. Specifically, obesogenic diet exposure downregulates the basal and injury-induced expression of the glia-associated phagocytic receptor, Draper. Constitutive activation of systemic insulin release fromDrosophilaInsulin-producing cells (IPCs) mimics the effect of diet-induced obesity on glial draper expression. In contrast, genetically attenuating systemic insulin release from the IPCs rescues diet-induced glial insulin resistance and draper expression. Significantly, we show that genetically stimulating Phosphoinositide 3-kinase (PI3K), a downstream effector of Insulin receptor signaling, rescues HSD-induced glial defects. Hence, we establish that obesogenic diets impair glial phagocytic function and delays the clearance of neuronal debris.
Sex differences in neural representations of social and nonsocial reward in the medial prefrontal cortex
Authors: Isaac Jennifer , Karkare Sonia , Balasubramanian Hymavathy , Schappaugh Nicholas , Javier Jarildy , Rashid Maha , Murugan Malavika
Published: March 2023

The reinforcing nature of social interactions is necessary for the maintenance of appropriate social behavior. However, the neural substrates underlying social reward processing and how they might differ based on the sex and internal state of the animal remains unknown. It is also unclear whether these neural substrates are shared with those involved in nonsocial rewarding processing. We developed a fully automated, novel two choice (social-sucrose) operant assay in which mice choose between social and nonsocial rewards to directly compare the reward-related behaviors associated with two competing stimuli. We performed cellular resolution calcium imaging of medial prefrontal cortex (mPFC) neurons in male and female mice across varying states of water restriction and social isolation. We found that mPFC neurons maintain largely non-overlapping, flexible representations of social and nonsocial reward that vary with internal state in a sex-dependent manner. Additionally, optogenetic manipulation of mPFC activity during the reward period of the assay disrupted reward-seeking behavior across male and female mice. Thus, using a novel operant assay, we have identified sex-dependent, non-overlapping neural representations of social and nonsocial reward in the mPFC that vary with internal state and that are essential for appropriate reward-seeking behavior.
Icariin prevents depression-like behaviors in chronic unpredictable mild stress-induced rats through Bax/cytoplasm C/caspase-3 axis to alleviate neuronal apoptosis
Authors: Wu Xiao , Zhang Xiaona , Sun Lulu , Lu Xiaomin , Shen Cunsi
Published: March 2023

Major depressive disorder (MDD) affects approximately 16% of the global population. Our previous study has demonstrated that icariin (ICA) exhibits anti-depressant activity by increasing the expression of Brain Derived Neurotrophic Factor (BDNF) in a rat model of chronic unpredictable mild stress (CUMS). In this study, we investigated whether and how ICA can prevent CUMS-induced depression-like behaviors in rats by modulating hippocampus neuronal apoptosis. Forty male rats were randomly divided into control, CUMS, CUMS-fluoxetine (Flx) (10 mg/kg), and CUMS-ICA (20 mg/kg) groups. Behavior tests including sucrose preference test (SPT), open field test (OFT), elevated plus-maze (EPM), and forced swimming tests (FST) were performed. The Nissl staining and TUNNEL assay were used to determine neuronal apoptosis. Subsequently, expression of glucocorticoid receptor (GR), Bcl-2, cytochrome C, caspase-3 and Bax in the hippocampus were tested by western blot. Our results show that a chronic administration of ICA (20 mg/kg) can prevent CUMS-induced depressant-like behaviors in male model rats. Additionally, ICA significantly inhibited mitochondrial translocation of GR, reduced mitochondrial outer membrane permeabilization (MOMP) to suppress the release of cytochrome C, and then inhibit the activation of caspase-3. In conclusion, our research provides new evidence to understand the anti-depressant activity of ICA, which relates to its inhibition of neuronal apoptosis in hippocampus through mitochondrial apoptotic pathway.
Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the earlyDrosophilaembryo
Authors: Ho Emily K. , Oatman Harrison R. , McFann Sarah E. , Yang Liu , Johnson Heath E. , Shvartsman Stanislav Y. , Toettcher Jared E.
Published: March 2023

Positional information in developing tissues often takes the form of stripes of gene expression that mark the boundaries of a particular cell type or morphogenetic process. How stripes form is still in many cases poorly understood. Here we use optogenetics and live-cell biosensors to investigate one such pattern: the posterior stripe ofbrachyenteron (byn)expression in the earlyDrosophilaembryo. Thisbynstripe depends on interpretation of an upstream signal – a gradient of ERK kinase activity – and the expression of two target genestailless (tll)andhuckebein (hkb)that exert antagonistic control overbyn. We find that high or low doses of ERK signaling produce either transient or sustainedbynexpression, respectively. These ERK stimuli also regulatetllandhkbexpression with distinct dynamics:tlltranscription is rapidly induced under both low and high stimuli, whereashkbtranscription converts graded ERK inputs into an output switch with a variable time delay. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop architecture, which is sufficient to explain transient or sustainedbyndynamics and adds temporal complexity to the steady-state model ofbynstripe formation. We further show that an all-or-none stimulus can be ‘blurred’ through intracellular diffusion to non-locally produce a stripe ofbyngene expression. Overall, our study provides a blueprint for using optogenetic inputs to dissect developmental signal interpretation in space and time.
Microbiota Succession Influences Nematode Physiology in a Beetle Microcosm Ecosystem
Authors: Han Ziduan , Sommer Ralf J. , Lo Wen-Sui
Published: March 2023

Research during the last decade has generated a substantial understanding of the role of microbiota in animal development, metabolism and immunity, including humans. However, many organismal interactions involve microbial successions, such as in animal decay but also human health and disease. The complexity of most microbiota makes it difficult to obtain insight into such microbial successions, resulting in a limited understanding of microbiota for ecosystem functioning. One potential, relatively simple, model system for the analysis of microbial successions is insect decay in soil ecosystems, a highly abundant process that has however, not been investigated in detail. For example, microbiota and nematodes are the two most abundant groups of organisms in soil systems, but their interplay and successions during the decomposition of insects are currently unknown. Here, we use a semi-artificial decaying rose chafer grub microcosm to study the reciprocal interactions between microbiota and nematodes through metagenomics and transcriptomic studies. We show that the controlled addition of nematodes to beetle grub carcasses will strongly influence the microbial succession and result in a massive increase in microbial diversity. Nematodes select microbes of high nutritional value for consumption, thereby influencing the composition of microbiota on the decaying insect. Reciprocally, the activity of nematode metabolic pathways strongly responds to their microbial diet and affects fat metabolism and the formation of dauer larvae, the nematode dispersal stage. These results indicate the importance of microbial successions and their reciprocal interactions with nematodes for insect decay in soil ecosystems.
Dysregulation of Prefrontal Oligodendrocyte Lineage Cells Across Mouse Models of Adversity and Human Major Depressive Disorder
Authors: Sharma Sumeet , Ma Wenjing , Ressler Kerry J. , Anderson Thea , Li Dan. C. , Jin Peng , Gourley Shannon L. , Qin Zhaohui
Published: March 2023

Animal models of adversity have yielded few molecular mechanisms that translate to human stress-related diseases like major depressive disorder (MDD). We congruently analyze publicly available bulk-tissue transcriptomic data from prefrontal cortex (PFC) in multiple mouse models of adversity and in MDD. We apply strategies, to quantify cell-type specific enrichment from bulk-tissue transcriptomics, utilizing reference single cell RNA sequencing datasets. These analyses reveal conserved patterns of oligodendrocyte (OL) dysregulation across animal experiments, including susceptibility to social defeat, acute cocaine withdrawal, chronic unpredictable stress, early life stress, and adolescent social isolation. Using unbiased methodologies, we further identify a dysregulation of layer 6 neurons that associate with deficits in goal-directed behavior after social isolation. Human post-mortem brains with MDD show similar OL transcriptome changes in Brodmann Areas 8/9 in both male and female patients. This work assesses cell type involvement in an unbiased manner from differential expression analyses across animal models of adversity and human MDD and finds a common signature of OL dysfunction in the frontal cortex.
CXCR4 blockade alleviates pulmonary and cardiac outcomes in early COPD
Authors: Dupin Isabelle , Henrot Pauline , Maurat Elise , Abohalaka Reshed , Chaigne Sébastien , El Hamrani Dounia , Eyraud Edmée , Prevel Renaud , Esteves Pauline , Campagnac Maryline , Dubreuil Marielle , Cardouat Guillaume , Bouchet Clément , Ousova Olga , Dupuy Jean-William , Trian Thomas , Thumerel Matthieu , Bégueret Hugues , Girodet Pierre-Olivier , Marthan Roger , Zysman Maeva , Freund-Michel Véronique , Berger Patrick
Published: March 2023

Chronic obstructive pulmonary disease (COPD) is a prevalent respiratory disease lacking effective treatment. Focusing on early COPD should help to discover disease modifying therapies. We aimed to examine the role of the CXCL12/CXCR4 axis in early COPD from both human samples and murine models. Blood samples and lung tissues of early COPD patients and controls were obtained in order to analyse CXCL12 and CXCR4 levels. To generate an early COPD model, ten-week-old male C57BL/6J mice were exposed to cigarette smoke (CS) for 10 weeks and intranasal instillations of polyinosinic–polycytidylic acid (poly(I:C)) for the last 5 weeks to mimic exacerbations. CXCR4 expressing cells number was increased in the blood of patients with COPD, as well as in the blood of exposed mice. Lung CXCL12 expression was higher in both early COPD patients and exposed mice. Exposed mice presented mild airway obstruction, peri-bronchial fibrosis and right heart thickening. The density of fibrocytes expressing CXCR4 was increased in the bronchial submucosa of these mice. Conditional inactivation of CXCR4 at adult stage as well as pharmacological inhibition of CXCR4 with plerixafor injections improved lung function, reduced inflammation, and protected against CS and poly-(I:C)-induced airway and cardiac remodeling. CXCR4-/-and plerixafor-treated mice also had less CXCR4-expressing circulating cells and a lower density of peri-bronchial fibrocytes. We demonstrate that targeting CXCR4 has beneficial effects in an animal model of early COPD and provide a framework to translate these preclinical findings to clinical settings in a drug repurposing approach.

Clinical relevance

We demonstrate that CXCL12/CXCR4 axis plays an important role in the pathogenesis of early COPD. Inhibition of this axis improves lung function and cardiac tissue remodeling, supporting the future use of CXCR4 inhibitors to slow down the progression of the disease.
Molecular cascades and cell-type specific signatures in ASD revealed by single cell genomics
Authors: Wamsley Brie , Bicks Lucy , Cheng Yuyan , Kawaguchi Riki , Quintero Diana , Grundman Jennifer , Liu Jianyin , Xiao Shaohua , Hawken Natalie , Margolis Michael , Mazariegos Samantha , Geschwind Daniel H
Published: March 2023

Understanding how genetic variation exerts its effects on the human brain in health and disease has been greatly informed by functional genomic characterization. Studies over the last decade have demonstrated robust evidence of convergent transcriptional and epigenetic profiles in post-mortem cerebral cortex from individuals with Autism Spectrum Disorder (ASD). Here, we perform deep single nuclear (sn) RNAseq to elucidate changes in cell composition, cellular transcriptomes and putative candidate drivers associated with ASD, which we corroborate using snATAC-seq and spatial profiling. We find changes in cell state composition representing transitions from homeostatic to reactive profiles in microglia and astrocytes, a pattern extending to oligodendrocytes and blood brain barrier cells. We identify profound changes in differential expression involving thousands of genes across neuronal and glial subtypes, of which a substantial portion can be accounted for by specific transcription factor networks that are significantly enriched in common and rare genetic risk for ASD. These data, which are available as part of the PsychENCODE consortium, provide robust causal anchors and resultant molecular phenotypes for understanding ASD changes in human brain.
An adaptive behavioral control motif mediated by cortical axo-axonic inhibition
Authors: Jung Kanghoon , Chang Minhyeok , Steinecke André , Berke Benjamin , Choi Youngjin , Oisi Yasuhiro , Fitzpatrick David , Taniguchi Hiroki , Kwon Hyung-Bae
Published: March 2023

Neural circuits are reorganized with specificity during learning. Genetically-defined subgroups of inhibitory interneurons are thought to play distinct roles in learning, but heterogeneity within these subgroups has limited our understanding of the scope and nature of their specific contributions to learning. Here we reveal that the chandelier cell (ChC), an interneuron type that specializes in inhibiting the axon-initial segment (AIS) of pyramidal neurons, establishes cortical microcircuits for organizing neural coding through selective axo-axonic synaptic plasticity. We find that organized motor control is mediated by enhanced population coding of direction-tuned premotor neurons, whose tuning is refined through suppression of irrelevant neuronal activity. ChCs are required for learning-dependent refinements via providing selective inhibitory control over pyramidal neurons rather than global suppression. Quantitative analysis on structural plasticity of axo-axonic synapses revealed that ChCs redistributed inhibitory weights to individual pyramidal neurons during learning. These results demonstrate an adaptive logic of the inhibitory circuit motif responsible for organizing distributed neural representations. Thus, ChCs permit efficient cortical computation in a target cell specific manner, which highlights the significance of interneuron diversity.
Ketogenic diet prevents sucrose withdrawal-induced anxiety-like behaviour in male mice
Authors: Kumar Mohit , Lal Roshan , Bhatt Babita , Bishnoi Mahendra
Published: March 2023

Sugar bingeing has been shown to induce withdrawal effects when sugar is no longer available in the diet. In this study, we investigated the sex differences in sugar-addiction-like behaviour and the effects of the ketogenic diet on these outcomes. We used the two-bottle sucrose choice paradigm as a pre-clinical sucrose overeating and withdrawal model. Female mice consumed more sucrose than males when given free access to water and 10% sucrose for four weeks. One week of sucrose withdrawal after four weeks of consecutive sucrose overeating showed anxiety-like behaviour in male mice. However, the ketogenic diet did not affect sucrose overconsumption in males and females but prevented the sucrose withdrawal-induced anxiety-like behaviour in male mice with a concomitant increase incFosmRNA in the prefrontal cortex. These findings provide evidence of sex differences in sucrose addiction-like behaviour and also indicate that a ketogenic diet may prevent sucrose withdrawal-induced anxiety-like behaviour in males. However, further research is needed to explore the underlying molecular mechanisms driving sex differences in sugar-addiction-like behaviour and the anxiolytic effects of the ketogenic diet.
Uptake of environmental DNA inBacillus subtilisoccurs all over the cell surface through a dynamic pilus structure
Authors: Kilb Alexandra , Burghard-Schrod Marie , Holtrup Sven , Graumann Peter L.
Published: March 2023

At the transition to stationary phase, a subpopulation ofBacillus subtiliscells can enter the developmental state of competence, where DNA is taken up through the cell envelope, is processed to single stranded DNA, which is incorporated into the genome if sufficient homology between sequences exists. We show here that the initial step of transport across the cell wall occurs via a true pilus structure, with an average length of about 500 nm, which assembles at various places on the cell surface. Once assembled, the pilus remains at one position and can be retracted in a time frame of seconds. The major pilin, ComGC, was studied at a single molecule level in live cells. ComGC was found in two distinct populations, one that would correspond to ComGC freely diffusing throughout the cell membrane, and one that is relatively stationary, likely reflecting pilus-incorporated molecules. The ratio of 65% diffusing and 35% stationary ComGC changed towards more stationary molecules upon addition of external DNA, while the number of pili in the population did not strongly increase. These findings suggest that the pilus assembles stochastically, but engages more pilin monomers from the membrane fraction in the presence of transport substrate. Our data reveal that localized transport of DNA into the cytosol at a single cell pole is preceded by localization throughout the periplasm and efficient uptake through the entire cell surface by a dynamic pilus.

Importance

Horizontal gene transfer (HGT) is important for bacterial evolution and the adaption to new environments. Through HGT, genes can be transferred between bacteria, which can lead to antibiotic resistance development, especially critical with regards to pathogenic bacterial species. One mechanism of HGT is natural competence, through which bacteria can take up DNA under natural conditions and incorporate it into their chromosomes. For DNA uptake, a complex will be formed that is conserved among Gram-positive and Gram-negative bacteria, in spite of different envelope architectures. We show that inBacillus subtilis, where the part of the uptake complex spanning the cell membrane localizes to a single cell pole, transport through the cell wall is mediated by a visible pilus structure that can extend and retract to pull DNA into the periplasm. Thus, DNA uptake from the environment is not limited to polar sites, but is channelled into the cytosol at the pole. Single molecule dynamics of ComGC revealed a static, likely pilus-bound, and a mobile fraction of ComGC within the cell membrane. Addition of environmental DNA increased the static fraction of molecules, showing that pilus dynamics respond to DNA binding.
Mitochondrial morphology dynamics and ROS regulate apical polarity and differentiation inDrosophilafollicle cells
Authors: Uttekar Bhavin , Tomer Darshika , Rikhy Richa
Published: March 2023

Mitochondrial morphology dynamics regulate signaling pathways during epithelial cell formation and differentiation. The mitochondrial fission protein Drp1 affects the appropriate activation of EGFR and Notch signaling-driven differentiation of posterior follicle cells inDrosophilaoogenesis. The mechanisms by which Drp1 regulates epithelial polarity during differentiation are not known. In this study, we show that Drp1 depleted follicle cells are constricted in early stages and present in multiple layers at later stages with decreased levels of apical polarity protein aPKC. This defect is suppressed by additional depletion of mitochondrial fusion protein Opa1. Opa1 depletion leads to mitochondrial fragmentation and increased reactive oxygen species (ROS) in follicle cells. We find that increasing ROS by depleting the ROS scavengers, mitochondrial SOD2, and catalase also leads to mitochondrial fragmentation. Further, the loss of Opa1, SOD2, and catalase partially restores the defects in epithelial polarity and aPKC along with EGFR and Notch signaling in Drp1 depleted follicle cells. Our results show a crucial interaction between mitochondrial morphology, ROS generation, and epithelial cell polarity formation during the differentiation of follicle epithelial cells inDrosophilaoogenesis.

Summary statement

Mitochondrial fission protein Drp1 regulates epithelial follicle cell differentiation inDrosophilaoogenesis. Increasing ROS and mitochondrial fragmentation suppresses the defects in epithelial polarity, and differentiation in Drp1 depleted follicle cells.
YAP signaling regulates the cellular uptake and therapeutic effect of nanoparticles
Authors: Cassani Marco , Fernandes Soraia , Oliver-De La Cruz Jorge , Durikova Helena , Vrbsky Jan , Patočka Marek , Hegrova Veronika , Klimovic Simon , Pribyl Jan , Debellis Doriana , Skladal Petr , Cavalieri Francesca , Caruso Frank , Forte Giancarlo
Published: March 2023

Interactions between living cells and nanoparticles have been extensively studied to enhance the delivery of therapeutics. Nanoparticles size, shape, stiffness and surface charge have been regarded as the main features able to control the fate of cell-nanoparticle interactions. However, the clinical translation of nanotherapies has so far been limited, and there is a need to better understand the biology of cell-nanoparticle interactions. This study investigated the role of cellular mechanosensitive components in cell-nanoparticle interactions. We demonstrate that the genetic and pharmacologic inhibition of yes-associated protein (YAP), a key component of cancer cell mechanosensing apparatus and Hippo pathway effector, improves nanoparticle internalization in triple-negative breast cancer cells regardless of nanoparticle properties or substrate characteristics. This process occurs through YAP-dependent regulation of endocytic pathways, cell mechanics, and membrane organization. Hence, we propose targeting YAP may sensitize triple negative breast cancer cells to chemotherapy and increase the selectivity of nanotherapy.
Olfactory search with finite-state controllers
Authors: Verano Kyrell Vann , Panizon Emanuele , Celani Antonio
Published: March 2023

Long-range olfactory search is an extremely difficult task in view of the sparsity of odor signals that are available to the searcher and the complex encoding of the information about the source location. Current algorithmic approaches typically require a continuous memory space, sometimes of large dimensionality, which may hamper their optimization and often obscure their interpretation. Here, we show how finite-state controllers with a small set of discrete memory states are expressive enough to display rich, time-extended behavioral modules that resemble the ones observed in living organisms. Finite-state controllers optimized for olfactory search have an immediate interpretation in terms of approximate clocks and coarse-grained spatial maps, suggesting connections with neural models of search behavior.
Cell Type-specific Hypothalamic Pathways to Brainstem Drive Context-dependent Strategies in Response to Stressors
Authors: Ahmadlou Mehran , Giannouli Maria , van Vierbergen Jacqueline F. M. , van Leeuwen Tom , Bloem Wouter , Houba Janou H. W. , Yasamin Shirazi Maryam , Cazemier J. Leonie , Haak Robin , Dubey Mohit , de Winter Fred , Heimel J. Alexander
Published: March 2023

Adaptive behavioral responses to stressors are critical for survival. However, which brain areas orchestrate switching the appropriate stress responses to distinct contexts is an open question. This study aimed to identify the cell type-specific brain circuitry governing the selection of distinct behavioral strategies in response to stressors. Through novel mouse behavior paradigms, we observed distinct stressor-evoked behaviors in two psycho-spatially distinct contexts, characterized by stressors inside or outside the safe zone. The identification of brain regions activated in both conditions revealed the involvement of the dorsomedial hypothalamus (DMH). Further investigation using optogenetics, chemogenetics, and photometry uncovered that glutamatergic projections from the DMH to periaqueductal gray (PAG) mediated responses to inside-stressors, while GABAergic projections, particularly from tachykinin1-expressing neurons, played a crucial role in coping with outside-stressors. These findings elucidate the role of cell type-specific circuitry from the DMH to the PAG in shaping behavioral strategies in response to stressors. These findings have the potential to advance our understanding of fundamental neurobiological processes and inform the development of novel approaches for managing context-dependent and anxiety-associated pathological conditions such as agoraphobia and claustrophobia.
VirRep: accurate identification of viral genomes from human gut metagenomic data via a hybrid language representation learning framework
Authors: Dong Yanqi , Chen Wei-Hua , Zhao Xing-Ming
Published: March 2023

Accurate identification of viral genomes from metagenomic data provides a broad avenue for studying viruses in the human gut. Here, we introduce VirRep, a novel virus identification method based on a hybrid language representation learning framework. VirRep employs a context-aware encoder and a composition-focused encoder to incorporate the learned knowledge and known biological insights to better describe the source of a DNA sequence. We benchmarked VirRep on multiple human gut virome datasets under different conditions and demonstrated significant superiority than state-of-the-art methods and even combinations of them. A comprehensive validation has also been conducted on real human gut metagenomes to show the great utility of VirRep in identifying high-quality viral genomes that are missed by other methods.
De novo Design of a Polycarbonate Hydrolase
Authors: Holst Laura H. , Madsen Niklas G. , Toftgaard Freja T. , Roenne Freja , Moise Ioana-Malina , Petersen Evamaria I. , Fojan Peter
Published: March 2023

Enzymatic degradation of plastics is currently limited to the use of engineered natural enzymes. As of yet, all engineering approaches applied to plastic degrading enzymes retain the natural α/β-fold. While mutations can be used to increase thermostability, an inherent maximum likely exists for the α/β-fold. It is thus of interest to introduce catalytic activity toward plastics in a different protein fold to escape the sequence space of plastic degrading enzymes. Here, a method for designing highly thermostable enzymes that can degrade plastics is described. This has been used to design an enzyme that can catalyze the hydrolysis of polycarbonate, which no known natural enzymes can degrade. Rosetta enzyme design is used to introduce a catalytic triad into a set of thermostable scaffolds. Through computational evaluation, a potential PCase was selected and produced recombinantly in E. coli. CD spectroscopy suggests that the design has a melting temperature of >95°C. Activity towards a commercially used polycarbonate (Makrolon 2808) was confirmed using AFM, which showed that a PCase had been designed successfully.
Perinatal Opioid Exposure Leads to Decreased Social Play in Adolescent Male and Female Rats: Potential Role of Oxytocin Signaling in Brain Regions Associated with Social Reward
Authors: Harder Hannah J. , Searles Christopher T. , Vogt Meghan E. , Murphy Anne Z.
Published: March 2023

Over the last two decades, the number of infants exposed to opioidsin uterohas quadrupled in the United States, with some states reporting rates as high as 55 infants per 1000 births. Clinical studies report that children previously exposed to opioids during gestation show significant deficits in social behavior, including an inability to form friendships or other social relationships. To date, the neural mechanisms whereby developmental opioid exposure disrupts social behavior remain unknown. Using a novel paradigm of perinatal opioid administration, we tested the hypothesis that chronic opioid exposure during critical developmental periods would disrupt juvenile play. As oxytocin is a major regulator of sociability, the impact of perinatal morphine exposure on oxytocin peptide and receptor expression was also examined. Juvenile play was assessed in vehicle- or morphine-exposed male and female rats at P25, P35, and P45. Classical features of juvenile play were measured, including time spent engaged in social play, time not in contact, number of pins, and number of nape attacks. We report that morphine-exposed females spend less time engaged in play behavior than control males and females, with a corresponding increase in time spent alone. Morphine-exposed females also initiated fewer pins and nape attacks. Oxytocin receptor binding was reduced in morphine-exposed females in the nucleus accumbens, a brain region critical for social reward. Together, these data suggest that females exposed to morphine during critical developmental periods are less motivated to participate in social play, potentially due to alterations in oxytocin-mediated reward signaling.
A computational framework for characterizing normative development of structural brain connectivity in the perinatal stage
Authors: Wu Yihan , Gholipour Ali , Vasung Lana , Karimi Davood
Published: March 2023

Quantitative assessment of the brain’s structural connectivity in the perinatal stage is useful for studying normal and abnormal neurodevelopment. However, estimation of the structural connectome from diffusion MRI data involves a series of complex and ill-posed computations. For the perinatal period, this analysis is further challenged by the rapid brain development and difficulties of imaging subjects at this stage. These factors, along with high inter-subject variability, have made it difficult to chart the normative development of the structural connectome. Hence, there is a lack of baseline trends in connectivity metrics that can be used as reliable references for assessing normal and abnormal brain development at this critical stage. In this paper we propose a computational framework, based on spatio-temporal atlases, for determining such baselines. We apply the framework on data from 169 subjects between 33 and 45 postmenstrual weeks. We show that this framework can unveil clear and strong trends in the development of structural connectivity in the perinatal stage. Some of our interesting findings include that connection weighting based on neurite density produces more consistent trends and that the trends in global efficiency, local efficiency, and characteristic path length are more consistent than in other metrics.
RAB18 confers protection to lipid droplets from lysolipophagy and counters cellular steatosis
Authors: Rieck Adrian , Guenther Georgia , Hengstler Jan , Vartak Nachiket
Published: March 2023

Lipid droplet (LD) enlargement is the most conspicuous cellular phenotype of steatotic liver diseases such as NAFLD and NASH. The small GTPase RAB18 localizes to lipid droplets yet its effects on LD dynamics are unknown. We show that RAB18 localizes to LDs dynamically via the acylation cycle in an activity dependent manner. Removal of RAB18 from LDs through acylation cycle inhibition of RNAi-mediated downregulation leads to an increase in LD size, that can be prevented by inhibition of autophagy. We propose a model where RAB18 on LDs decreases the susceptibility of LDs to autophagy by lysosomes, allowing them to exist longer in cells and enabling an even distribution of exogenous lipid load. In the absence of RAB18, fewer LDs survive autophagic removal, and enlarge disproportionately as they absorb the incumbent exogenous lipid load on cells, leading to cellular steatosis. Inhibition of autophagy prevented cellular steatosis in primary human hepatocytes. Finally, mice treated with the autophagy inhibitor chloroquine were resistant to the development of liver steatosis when on a steatogenic diet. The modulation of RAB18 activity and autophagy inhibition represents a promising approach to prevent steatotic liver disease in the clinical setting.
A Novel ARNT-Dependent HIF-2α Signaling as a Protective Mechanism for Cardiac Microvascular Barrier Integrity and Heart Function Post-Myocardial Infarction
Authors: Ullah Karim , Ai Lizhuo , Li Yan , Liu Lifeng , Zhang Qin , Pan Kaichao , Humayun Zainab , Piao Lin , Sitikov Albert , Su Qiaozhu , Zhao Qiong , Sharp Willard , Fang Yun , Wu David , Liao James K. , Wu Rongxue
Published: March 2023

Myocardial infarction (MI) significantly compromises the integrity of the cardiac microvascular endothelial barrier, leading to enhanced leakage and inflammation that contribute to the progression of heart failure. While HIF2α is highly expressed in cardiac endothelial cells (ECs) under hypoxic conditions, its role in regulating microvascular endothelial barrier function during MI is not well understood. In this study, we utilized mice with a cardiac-specific deletion of HIF2α, generated through an inducible Cre (Cdh5Cre-ERT2) recombinase system. These mice exhibited no apparent phenotype under normal conditions. However, following left anterior descending (LAD) artery ligation-induced MI, they showed increased mortality associated with enhanced cardiac vascular leakage, inflammation, worsened cardiac function, and exacerbated heart remodeling. These outcomes suggest a protective role for endothelial HIF2α in response to cardiac ischemia. Parallel investigations in human cardiac microvascular endothelial cells (CMVECs) revealed that loss of ecHif2α led to diminished endothelial barrier function, characterized by reduced tight-junction protein levels and increased cell death, along with elevated expression of IL6 and other inflammatory markers. These effects were substantially reversed by overexpressing ARNT, a critical dimerization partner for HIF2α during hypoxia. Additionally, ARNT deletion also led to increased CMVEC permeability. Interestingly, ARNT, rather than HIF2α itself, directly binds to the IL6 promoter to suppress IL6 expression. Our findings demonstrate the critical role of endothelial HIF2α in response to MI and identify the HIF2α/ARNT axis as a transcriptional repressor, offering novel insights for developing therapeutic strategies against heart failure following MI.
Cortical thickness and sub-cortical volumes in post-H1N1 narcolepsy type 1: A brain-wide MRI case-control study
Authors: Juvodden Hilde T. , Alnæs Dag , Agartz Ingrid , Andreassen Ole A. , Server Andres , Thorsby Per M. , Westlye Lars T. , Knudsen-Heier Stine
Published: March 2023

Objective

There was an increased incidence of Narcolepsy type 1(NT1) after the H1N1-mass vaccination in 2009/2010 which has been associated with the Pandemrix®-vaccine. We performed the first case-control comparison of MRI-based global and sub-cortical volume and cortical thickness in post-H1N1(largely Pandemrix®-vaccinated) NT1 patients compared with healthy controls.

Methods

We included 54 post-H1N1 NT1 patients (51 with confirmed hypocretin-deficiency; 48 H1N1-vaccinated with Pandemrix®; 39 females, mean age 21.8 ± 11.0 years) and 114 healthy controls (77 females, mean age 23.2 ± 9.0 years). 3T MRI brain scans were obtained, and the T1-weighted MRI data were processed using FreeSurfer. Group differences among three global and 10 sub-cortical volume measures and 34 cortical thickness measures for bilateral brain regions were tested using general linear models with permutation testing. We corrected for multiple testing with the Benjamini-Hochberg procedure with the false discovery rate at 5%.

Results

Patients had significantly thinner brain cortex bilaterally in the temporal poles (Cohen’s d=0.68,p=0.00080), entorhinal cortex (d=0.60,p=0.0018) and superior temporal gyrus (d=0.60,p=0.0020) compared to healthy controls. The analysis revealed no significant group differences for sub-cortical volumes.

Conclusions

Post-H1N1(largely Pandemrix®-vaccinated) NT1 patients have significantly thinner cortex in temporal brain regions compared to controls. We speculate that this effect can be partly attributed to the hypothalamic neuronal change in NT1, including loss of function of the widely projecting hypocretin-producing neurons and secondary effects of the abnormal sleep-wake pattern in NT1. Alternatively, the findings could be specific for post-H1N1 (largely Pandemrix®-vaccinated) NT1 patients.
In silicoagent-based modeling approach to characterize multiplein vitrotuberculosis infection models
Authors: Petrucciani Alexa , Hoerter Alexis , Kotze Leigh , Plessis Nelita Du , Pienaar Elsje
Published: March 2023

In vitromodels ofMycobacterium tuberculosis (Mtb)infection are a valuable tool to examine host-pathogen interactions and screen drugs. With the development of more complexin vitromodels, there is a need for tools to help analyze and integrate data from these models. We introduce an agent-based model (ABM) representation of the interactions between immune cells and bacteria in anin vitrosetting. Thisin silicomodel was used to independently simulate both traditional and spheroid cell culture models by changing the movement rules and initial spatial layout of the cells. These two setups were calibrated to published experimental data in a paired manner, by using the same parameters in both simulations. Within the calibrated set, heterogeneous outputs are seen for outputs of interest including bacterial count and T cell infiltration into the macrophage core of the spheroid. The simulations are also able to predict many outputs with high time resolution, including spatial structure. The structure of a single spheroid can be followed across the time course of the simulation, allowing the relationship between cell localization and immune activation to be explored. Uncertainty analyses are performed for both model setups using latin hypercube sampling and partial rank correlation coefficients to allow for easier comparison, which can provide insight into ideal use cases for the independent setups. Future model iterations can be guided by the limitations of the current model, specifically which parts of the output space were harder to reach. This ABM can be used to represent morein vitro Mtbinfection models due to its flexible structure, providing a powerful analysis tool that can be used in tandem with experiments.

Author Summary

Tuberculosis is an infectious disease that causes over 1.4 million deaths every year. During infection, immune cells surround the bacteria forming structures called granulomas in the lungs. New laboratory models generate spheroids that aim to recreate these structures to help understand infection and find new ways to treat tuberculosis. Computational modeling is used to compare these newer spheroid models to traditional models, which don’t recreate the structure of the cell clusters. After calibration to data from laboratory experiments to ensure that the computational model can represent both systems, the structures were characterized over time. The traditional and spheroid model were also compared by looking at how model inputs impact outputs, allowing users to figure out when one model should be used over the other. This computational tool can be used to help integrate data from different laboratory models, generate hypothesis to be tested in laboratory models, and predict pathways to be targeted by drugs.
Voltage-dependent volume regulation controls epithelial cell extrusion and morphology
Authors: Mitchell Saranne J. , Pardo-Pastor Carlos , Zangle Thomas A. , Rosenblatt Jody
Published: March 2023

Epithelial cells work collectively to provide a protective barrier, yet also turn over rapidly by cell death and division. If the number of dying cells does not match those dividing, the barrier would vanish, or tumors can form. Mechanical forces and thestretch-activated ionchannel (SAC) Piezo1 link both processes; stretch promotes cell division and crowding triggers cell death by initiating live cell extrusion1,2. However, it was not clear how particular cells within a crowded region are selected for extrusion. Here, we show that individual cells transiently shrink via water loss before they extrude. Artificially inducing cell shrinkage by increasing extracellular osmolarity is sufficient to induce cell extrusion. Pre-extrusion cell shrinkage requires the voltage-gated potassium channels Kv1.1 and Kv1.2 and the chloride channel SWELL1, upstream of Piezo1. Activation of these voltage-gated channels requires the mechano-sensitive Epithelial Sodium Channel, ENaC, acting as the earliest crowd-sensing step. Imaging with a voltage dye indicated that epithelial cells lose membrane potential as they become crowded and smaller, yet those selected for extrusion are markedly more depolarized than their neighbours. Loss of any of these channels in crowded conditions causes epithelial buckling, highlighting an important role for voltage and water regulation in controlling epithelial shape as well as extrusion. Thus, ENaC causes cells with similar membrane potentials to slowly shrink with compression but those with reduced membrane potentials to be eliminated by extrusion, suggesting a chief driver of cell death stems from insufficient energy to maintain cell membrane potential.
Discovery of DTX3L inhibitors through a homogeneous FRET-based assay that monitors formation and removal of poly-ubiquitin chains
Authors: Vela-Rodríguez Carlos , Scarpulla Ilaria , Ashok Yashwanth , Lehtiö Lari
Published: March 2023

Ubiquitination is a complex and reversible protein post-translational modification in which the subsequent action of enzymes belonging to three different families, broadly referred to as E1, E2 and E3, results in the covalent linking of ubiquitin to a target protein. While this linkage is canonically an isopeptide bond between the C-terminus of ubiquitin and the lysine residue of the target protein, Ser, Thr, and Tyr can also be susceptible to ubiquitination through an oxyester bond. Once ubiquitinated, multiple units of ubiquitin can be attached to the initial ubiquitin thus extending it to a chain of ubiquitins. Ubiquitination regulates multiple cellular processes, but it is best known as a modification that targets proteins for proteasomal degradation following the formation poly-ubiquitin chains linked through lysine 48 or 63 of ubiquitin. Dysregulation of ubiquitination has been associated with multiple types of cancer and efforts have been carried out to develop technologies that lead to the identification of inhibitors of the enzymes involved in the ubiquitination cascade. Herein, we present the development of a FRET-based assay that allows us to monitor auto-ubiquitination of DTX3L, a RING-type E3 ubiquitin ligase. Our method shows a robust signal window with a robust average Z’ factor of 0.76. From a validatory screening experiment we have identified the first molecules that inhibit DTX3L with potencies in the low micromolar range. Additionally, we have expanded the system to study deubiquitinases such as USP28 that lead to reduction of FRET due to hydrolysis of fluorescent poly-Ub chains.
Structural basis of Cas12a R-loop propagation on pathway to DNA cleavage
Authors: Strohkendl Isabel , Moy Catherine , Nguyen Alexander-Hoi , Russell Rick , Taylor David W.
Published: March 2023

Cas12a is a CRISPR RNA-guided nuclease that engages target DNA through protein-DNA and RNA-DNA contacts. Initial PAM binding by Cas12a leads to formation of a 20bp R-loop between the complementary crRNA guide and target strand. Following specificity-determining R-loop formation, both DNA strands undergo RuvC-mediated cleavage. Current structures of Cas12a bound to its target only show the R-loop after formation, leaving an important gap in knowledge as to how Cas12a accommodates the extending R-loop and coordinates R-loop formation with nuclease activation. Here, we use cryo-EM to capture a series of kinetically trapped Cas12a R-loop intermediates and observe how Cas12a delivers each DNA strand into the RuvC active site. We show that Cas12a first interrogates target DNA via a 5bp seed, followed by dramatic Rec domain conformational flexibility to accommodate R-loop extension. Only during formation of the final R-loop base pairs do the Rec and BH domains engage in the majority of contacts with the R-loop. R-loop completion leads the nontarget strand to displace the RuvC lid and kink into the active site via a base stacking interaction. Following nontarget strand cleavage, we observe substantial Rec2 and Nuc domain dynamics as the TS is brought to the RuvC active site. Our kinetics-guided structural snapshots provide a comprehensive model describing Cas12a DNA targeting and highlight mechanistic differences between Cas12a and Cas9.