bluuebunny/crossref_metadata_2025 huggingface dataset:
zol & fai: large-scale targeted detection and evolutionary investigation of gene clusters
Authors: Salamzade Rauf , Tran Patricia Q , Martin Cody , Manson Abigail L , Gilmore Michael S , Earl Ashlee M , Anantharaman Karthik , Kalan Lindsay
Published: June 2023

Many universally and conditionally important genes are genomically aggregated within clusters. Here, we introduce fai and zol, which together enable large-scale comparative analysis of different types of gene clusters and mobile-genetic elements (MGEs), such as biosynthetic gene clusters (BGCs) or viruses. Fundamentally, they overcome a current bottleneck to reliably perform comprehensive orthology inference at large scale across broad taxonomic contexts and thousands of genomes. First, fai allows the identification of orthologous instances of a query gene cluster of interest amongst a database of target genomes. Subsequently, zol enables reliable, context-specific inference of ortholog groups for individual protein-encoding genes across gene cluster instances. In addition, zol performs functional annotation and computes a variety of evolutionary statistics for each inferred ortholog group. Importantly, in comparison to tools for visual exploration of homologous relationships between gene clusters, zol can scale to thousands of gene cluster instances and produce detailed reports that are easy to digest. To showcase fai and zol, we apply them for: (i) longitudinal tracking of a virus in metagenomes, (ii) discovering novel population-level genetic insights of two common BGCs in the fungal speciesAspergillus flavus, and (iii) uncovering large-scale evolutionary trends of a virulence-associated gene cluster across thousands of genomes from a diverse bacterial genus.
A 3Din vitrocortical tissue model based on dense collagen to study the effects of gamma radiation on neuronal function
Authors: Lojek Neal M. , Williams Victoria A. , Rogers Andrew M. , Sajo Erno , Black Bryan J. , Ghezzi Chiara E.
Published: June 2023

Studies on gamma radiation-induced injury have long been focused on hematopoietic, gastrointestinal, and cardiovascular systems, yet little is known about the effects of gamma radiation on the function of human cortical tissue. The challenge in studying radiation-induced cortical injury is, in part, due to a lack of human tissue models and physiologically relevant readouts. Here, we have developed a physiologically relevant 3D collagen-based cortical tissue model (CTM) for studying the functional response of human iPSC derived neurons and astrocytes to a sub-lethal radiation exposure (5 Gy). We quantified cytotoxicity, DNA damage, morphology, and extracellular electrophysiology. We reported that 5 Gy exposure significantly increased cytotoxicity, DNA damage, and astrocyte reactivity while significantly decreased neurite length and neuronal network activity. Additionally, we found that clinically deployed radioprotectant amifostine ameliorated the DNA damage, cytotoxicity, and astrocyte reactivity. The CTM provides a critical experimental platform to understand cell-level mechanisms by which GR affects human cortical tissue and to screen prospective radioprotectant compounds.
Calling Cards: a customizable platform to longitudinally record protein-DNA interactions over time in cells and tissues
Authors: Yen Allen , Mateusiak Chase , Sarafinovska Simona , Gachechiladze Mariam A. , Guo Juanru , Chen Xuhua , Moudgil Arnav , Cammack Alexander J. , Hoisington-Lopez Jessica , Crosby MariaLynn , Brent Michael R. , Mitra Robi D. , Dougherty Joseph D.
Published: June 2023

Calling Cards is a platform technology to record a cumulative history of transient protein-DNA interactions in the genome of genetically targeted cell types. The record of these interactions is recovered by next generation sequencing. Compared to other genomic assays, whose readout provides a snapshot at the time of harvest, Calling Cards enables correlation of historical molecular states to eventual outcomes or phenotypes. To achieve this, Calling Cards uses the piggyBac transposase to insert self-reporting transposon (SRT) “Calling Cards” into the genome, leaving permanent marks at interaction sites. Calling Cards can be deployed in a variety ofin vitroandin vivobiological systems to study gene regulatory networks involved in development, aging, and disease. Out of the box, it assesses enhancer usage but can be adapted to profile specific transcription factor binding with custom transcription factor (TF)-piggyBac fusion proteins. The Calling Cards workflow has five main stages: delivery of Calling Card reagents, sample preparation, library preparation, sequencing, and data analysis. Here, we first present a comprehensive guide for experimental design, reagent selection, and optional customization of the platform to study additional TFs. Then, we provide an updated protocol for the five steps, using reagents that improve throughput and decrease costs, including an overview of a newly deployed computational pipeline. This protocol is designed for users with basic molecular biology experience to process samples into sequencing libraries in 1-2 days. Familiarity with bioinformatic analysis and command line tools is required to set up the pipeline in a high-performance computing environment and to conduct downstream analyses.

Basic Protocol 1: Preparation and delivery of Calling Cards reagents

Basic Protocol 2: Sample preparation

Basic Protocol 3: Sequencing library preparation

Basic Protocol 4: Library pooling and sequencing

Basic Protocol 5: Data analysis
Identification of dialects and individuals of globally threatened Yellow Cardinals using neural networks
Authors: Bocaccio Hernan , Domínguez Marisol , Mahler Bettina , Reboreda Juan Carlos , Mindlin Gabriel
Published: June 2023

Audio-based analysis of bird songs has proven to be a valuable practice for the growth of knowledge in the fields of ethology and ecology. In recent years, machine learning techniques applied to audio field recordings of bird calls have yielded successful results in studying population distributions and identification of individuals for their monitoring in a variety of bird species. This offers promising possibilities in the study of social behavior, biodiversity, and conservation strategies for birds. In this work, we trained deep learning models, directly from the sonograms of audio field recordings, to investigate the statistical properties of vocalizations in the endangered Yellow Cardinal. To our knowledge, this is the first time that this approach has been applied to this threatened species. Our results indicate the presence of vocal signatures that reflect similarities in songs of individuals that inhabit the same region, determining dialects, but which also show differences between individuals. These differences can be exploited by a deep learning classifier to discriminate the bird identities through their songs. Models trained with data labeled by regions showed a good performance in the recognition of dialects with a mean accuracy of (0.84 +/-0.04) significantly higher than accuracy obtained by chance. Precision and recall values also reflected the classifier’s ability to find alike vocal patterns in the songs of neighboring individuals. Models trained with data labeled by individuals showed an accuracy of (0.63 +/-0.03) significantly higher than that obtained by chance. However, the individual discrimination model showed greater confusion with neighboring individuals. This reflects a hierarchical structure in the characteristics of the Yellow Cardinal’s vocalization, where the intra-individual variability is lower than the inter-individual variability, but it is even lower than the variability obtained when individuals inhabit different regions, providing evidence of the existence of dialects. This reinforces the results of previous works but also offers an automated method for characterizing cultural units within the species. Along with genetic data, this method could help better define management units, thereby benefiting the success of reintroduction programmes of recovered individuals of Yellow Cardinal. Moreover, the novelty of individual discrimination using neural networks for the Yellow Cardinal, which has limited data availability, shows promise for non-invasive acoustic monitoring strategies with potentially relevant implications for its conservation.
In silicodiscovery of the myxosortases that process MYXO-CTERM and three novel prokaryotic C-terminal protein-sorting signals that share invariant Cys residues
Authors: Haft Daniel H.
Published: June 2023

The LPXTG protein-sorting signal, found in surface proteins of various Gram-positive pathogens, was the founding member of a growing panel of prokaryotic small C-terminal sorting domains. Sortase A (SrtA) cleaves LPXTG, exosortases (XrtA and XrtB) cleave the PEP-CTERM sorting signal, archaeosortase A (ArtA) cleaves PGF-CTERM, and rhombosortase (RrtA) cleaves GlyGly-CTERM domains. Four sorting signal domains without previously known processing proteases are the MYXO-CTERM, JDVT-CTERM, Synerg-CTERM, and CGP-CTERM domains. These exhibit the standard tripartite architecture of short signature motif, then a hydrophobic transmembrane segment, then an Arg-rich cluster. Each has an invariant cysteine in its signature motif. Computational evidence strongly suggests that each of these four Cys-containing sorting signals is processed, at least in part, by a cognate family of glutamic-type intramembrane endopeptidases, related to eukaryotic type II CAAX-processing protease Rce1. For the MYXO-CTERM sorting signals of different lineages, their sorting enzymes, called myxosortases, include MrtX (MXAN_2755 inMyxococcus xanthus), MrtC, and MrtP, all with radically different N-terminal domains but with a conserved core. Predicted cognate sorting enzymes were identified also for JDVT-CTERM (MrtJ), Synerg-CTERM (MrtS), and CGP-CTERM (MrtA). This work establishes a major new family of protein-sorting housekeeping endopeptidases contributing to surface attachment of proteins in prokaryotes.

Importance

Homologs of the eukaryotic type II CAAX-box protease Rce1, a membrane-embedded endopeptidase found in yeast and human ER and involved in sorting proteins to their proper cellular locations, are abundant in prokaryotes but are not well understood there. This bioinformatics paper identifies several subgroups of the family as cognate endopeptidases for four protein-sorting signals processed by previously unknown machinery. Sorting signals with newly identified processing enzymes include three novel ones, but also MYXO-CTERM, which had been the focus of previous experimental work in the model fruiting and gliding bacteriumMyxococcus xanthus. The new findings will substantially improve our understanding of Cys-containing C-terminal protein-sorting signals and of protein trafficking generally in bacteria and archaea.
Per-ischemic changes in penumbral blood supply and its microscopic distribution
Authors: Iversen Nina K. , Jimenéz Eugenio Gutierréz , Rasmussen Peter Mondrup , Angelys Hugo , Mikkelsen Irene Klærke , Hollyer Tristan R. , Østergaard Leif
Published: June 2023

Acute ischemic stroke (AIS) is a frequent cause of death and adult disability. AIS patient management targets theischemic penumbra: Hypoperfused, electrically silent brain tissue, which can be salvaged by restoring blood flow during the first, critical hours after symptom onset. Neuroimaging studies in AIS patients suggest that penumbral tissue is characterized not only by hypoperfusion, but also by microvascular flow disturbances that strongly affect tissue outcome. Here, we demonstrate that microvascular flows become increasingly chaotic in the ischemic penumbra in the hours after middle cerebral artery occlusion in a rat model of AIS. Biophysical models suggest that these disturbances are accompanied by increasing hypoxia in the absence of blood flow changes. Unlike findings in severe ischemia, pericyte constrictions do not appear to occlude penumbral capillaries. We propose that microvascular flow disturbances represent a critical feature of penumbral tissue, and a potential target for neuroprotective therapy after AIS.
KV7 but not dual SK/IK channel openers inhibit the activation of colonic afferents by noxious stimuli
Authors: Bhebhe Charity N , Higham James P , Gupta Rohit A , Raine Tim , Bulmer David C
Published: June 2023

In numerous subtypes of central and peripheral neurons, small and intermediate conductance Ca2+-activated K+(SK and IK, respectively) channels are important regulators of neuronal excitability. Transcripts encoding SK channel subunits, as well as the closely related IK subunit, are co-expressed in the soma of colonic afferent neurons with receptors for the algogenic mediators adenosine triphosphate (ATP) and bradykinin, P2X3and B2, highlighting the potential utility of these channels as drug targets for the treatment of abdominal pain in gastrointestinal diseases such as irritable bowel syndrome. Despite this, pre-treatment with the dual SK/IK channel opener SKA-31 had no effect on the colonic afferent response to ATP, bradykinin or noxious ramp distention of the colon. Inhibition of SK or IK channels with apamin or TRAM-34, respectively, yielded no change in spontaneous baseline afferent activity, indicating these channels are not tonically active. In contrast to its lack of effect in electrophysiological experiments, comparable concentrations of SKA-31 abolished ongoing peristaltic activity in the colonex vivo. Treatment with the KV7 channel opener retigabine blunted the colonic afferent response to all applied stimuli. Our data therefore highlight the potential utility of KV7, but not SK/IK, channel openers as analgesic agents for the treatment of abdominal pain.
Alarm barks of male vervet monkeys as displays of male quality
Authors: Schad Lukas , Dongre Pooja , van de Waal Erica , Fischer Julia
Published: June 2023

Research on the vocal behaviour of non-human primates is often motivated by a desire to understand the origins of semantic communication, which led to a partial separation of this research from ecological-evolutionary approaches. To bridge this gap, we returned to the textbook example of semantic communication in animals, the vervet monkey,Chlorocebus pygerythrus, alarm call system, and investigated whether male alarm barks fulfil a dual function of alarming and indicating male quality. Barks are loud calls, produced by adult males, in response to large carnivores. However, since barks occasionally occur in agonistic interactions, we investigated whether barks may also indicate male quality. We recorded natural barking events over 23 months, sampling individual male participation from 45 individuals in six free-ranging groups at the Mawana Game Reserve, KwaZulu-Natal, South Africa. We hypothesized that barking frequency is under intra-sexual selection and predicted that barking frequency would increase with male rank and the degree of male-male competition. We found that the highest-ranking males were more likely to produce barks than lower-ranking males and that the number of daily barking events increased during the mating season. We advocate studying primate communication in its evolutionary context to achieve a comprehensive understanding of call “meaning”.
Distinctive Whole-brain Cell Types Predict Tissue Damage Patterns in Thirteen Neurodegenerative Conditions
Authors: Pak Veronika , Adewale Quadri , Bzdok Danilo , Dadar Mahsa , Zeighami Yashar , Iturria-Medina Yasser
Published: June 2023

For over a century, brain research narrative has mainly centered on neuron cells. Accordingly, most neurodegenerative studies focus on neuronal dysfunction and their selective vulnerability, while we lack comprehensive analyses of other major cell types’ contribution. By unifying spatial gene expression, structural MRI, and cell deconvolution, here we describe how the human brain distribution of canonical cell types extensively predicts tissue damage in thirteen neurodegenerative conditions, including early-and late-onset Alzheimer’s disease, Parkinson’s disease, dementia with Lewy bodies, amyotrophic lateral sclerosis, mutations in presenilin-1, and three clinical variants of frontotemporal lobar degeneration (behavioural variant, semantic and non-fluent primary progressive aphasia) along with associated 3-repeat and 4-repeat tauopathies and TDP43 proteinopathies types A and C. We reconstructed comprehensive whole-brain reference maps of cellular abundance for six major cell types and identified characteristic axes of spatial overlapping with atrophy. Our results support the strong mediating role of non-neuronal cells, primarily microglia and astrocytes, in spatial vulnerability to tissue loss in neurodegeneration, with distinct and shared across-disorders pathomechanisms. These observations provide critical insights into the multicellular pathophysiology underlying spatiotemporal advance in neurodegeneration. Notably, they also emphasize the need to exceed the current neuro-centric view of brain diseases, supporting the imperative for cell-specific therapeutic targets in neurodegeneration.
Astrocyte store-operated calcium entry is required for centrally mediated neuropathic pain
Authors: Prokhorenko Mariya A. , Smyth Jeremy T.
Published: June 2023

Central sensitization is a critical step in chronic neuropathic pain formation following acute nerve injury. Central sensitization is defined by nociceptive and somatosensory circuitry changes in the spinal cord leading to dysfunction of antinociceptive gamma-aminobutyric acid (GABA)ergic cells (Li et al., 2019), amplification of ascending nociceptive signals, and hypersensitivity (Woolf, 2011). Astrocytes are key mediators of the neurocircuitry changes that underlie central sensitization and neuropathic pain, and astrocytes respond to and regulate neuronal function through complex Ca2+signaling mechanisms. Clear definition of the astrocyte Ca2+signaling mechanisms involved in central sensitization may lead to new therapeutic targets for treatment of chronic neuropathic pain, as well as enhance our understanding of the complex central nervous system (CNS) adaptions that occur following nerve injury. Ca2+release from astrocyte endoplasmic reticulum (ER) Ca2+stores via the inositol 1,4,5-trisphosphate receptor (IP3R) is required for centrally mediated neuropathic pain (Kim et al, 2016); however recent evidence suggests the involvement of additional astrocyte Ca2+signaling mechanisms. We therefore investigated the role of astrocyte store-operated Ca2+entry (SOCE), which mediates Ca2+influx in response to ER Ca2+store depletion. Using an adultDrosophila melanogastermodel of central sensitization based on thermal allodynia in response to leg amputation nerve injury (Khuong et al., 2019), we show that astrocytes exhibit SOCE-dependent Ca2+signaling events three to four days following nerve injury. Astrocyte-specific suppression of Stim and Orai, the key mediators of SOCE Ca2+influx, completely inhibited the development of thermal allodynia seven days following injury, and also inhibited the loss of ventral nerve cord (VNC) GABAergic neurons that is required for central sensitization in flies. We lastly show that constitutive SOCE in astrocytes results in thermal allodynia even in the absence of nerve injury. Our results collectively demonstrate that astrocyte SOCE is necessary and sufficient for central sensitization and development of hypersensitivity inDrosophila, adding key new understanding to the astrocyte Ca2+signaling mechanisms involved in chronic pain.
Punishment resistance for cocaine is associated with inflexible habits in rats
Authors: Jones Bradley O. , Paladino Morgan S. , Cruz Adelis M. , Spencer Haley F. , Kahanek Payton L. , Scarborough Lauren N. , Georges Sandra F. , Smith Rachel J.
Published: June 2023

Addiction is characterized by continued drug use despite negative consequences. In an animal model, a subset of rats continues to self-administer cocaine despite footshock consequences, showing punishment resistance. We sought to test the hypothesis that punishment resistance arises from failure to exert goal-directed control over habitual cocaine seeking. While habits are not inherently permanent or maladaptive, continued use of habits under conditions that should encourage goal-directed control makes them maladaptive and inflexible. We trained male and female Sprague Dawley rats on a seeking-taking chained schedule of cocaine self-administration (2 h/day). We then exposed them to 4 days of punishment testing, in which footshock (0.4 mA, 0.3 s) was delivered randomly on one-third of trials, immediately following completion of seeking and prior to extension of the taking lever. Before and after punishment testing (4 days pre-punishment and ≥4 days post-punishment), we assessed whether cocaine seeking was goal-directed or habitual using outcome devaluation via cocaine satiety. We found that punishment resistance was associated with continued use of habits, whereas punishment sensitivity was associated with increased goal-directed control. Although punishment resistance was not predicted by habitual responding pre-punishment, it was associated with habitual responding post-punishment. In parallel studies of food self-administration, we similarly observed that punishment resistance was associated with habitual responding post-punishment but not pre-punishment. These findings indicate that punishment resistance is related to habits that have become inflexible and persist under conditions that should encourage a transition to goal-directed behavior.
Tapered Drug delivery, Optical stimulation, and Electrophysiology (T-DOpE) probes reveal the importance of cannabinoid signaling in hippocampal CA1 oscillations in behaving mice
Authors: Kim Jongwoon , Huang Hengji , Gilbert Earl , Arndt Kaiser , English Daniel Fine , Jia Xiaoting
Published: June 2023

Understanding the neural basis of behavior requires monitoring and manipulating combinations of physiological elements and their interactions in behaving animals. Here we developed a thermal tapering process (TTP) which enables the fabrication of novel, low-cost, flexible probes that combine ultrafine features of dense electrodes, optical waveguides, and microfluidic channels. Furthermore, we developed a semi-automated backend connection allowing scalable assembly of the probes. We demonstrate that our T-DOpE (TaperedDrug delivery,Optical stimulation, andElectrophysiology) probe achieves in a single neuron-scale device (1) high-fidelity electrophysiological recording (2) focal drug delivery and (3) optical stimulation. With a tapered geometry, the device tip can be minimized (as small as 50 μm) to ensure minimal tissue damage while the backend is ~20 times larger allowing for direct integration with industrial-scale connectorization. Acute and chronic implantation of the probes in mouse hippocampus CA1 revealed canonical neuronal activity at the level of local field potentials and spiking. Taking advantage of the triple-functionality of the T-DOpE probe, we monitored local field potentials with simultaneous manipulation of endogenous type 1 cannabinoid receptors (CB1R; via microfluidic agonist delivery) and CA1 pyramidal cell membrane potential (optogenetic activation). Electro-pharmacological experiments revealed that focal infusion of CB1R agonist CP-55,940 in dorsal CA1 downregulated theta and sharp wave-ripple oscillations. Furthermore, using the full electro-pharmacological-optical feature set of the T-DOpE probe we found that CB1R activation reduces sharp wave-ripples (SPW-Rs) by impairing the innate SPW-R-generating ability of the CA1 circuit.
A Cost-Effective CRISPR/Cas9-Based Method for Sequencing the Functional Antibody Kappa Chain cDNA from Hybridomas Expressing Aberrant Kappa Chain mRNAs
Authors: Ahnoud Armin , He Chong , Strainic Michael , Zhai Wenwu
Published: June 2023

One prerequisite for generating recombinant antibodies for therapeutic and non-therapeutic purposes from hybridoma clones is the reliable, efficient, and cost-effective sequencing of the hybridoma clone that produces the desired antibody. However, many hybridoma fusion partners produce aberrant endogenous mRNA transcripts most of which resemble kappa chains. These aberrant kappa chain mRNAs can interfere with or even prevent the determination of the functional murine antibody kappa chain cDNA sequences during the PCR amplification step. In this paper, we report the development of a rapid and cost-effective CRISPR/Cas9 based method to eliminate the aberrant endogenous sequence. We have demonstrated the effectiveness of this method by significantly reducing the number of an endogenous aberrant kappa chain transcript known as the aberrant SP2/0 kappa chain. This transcript is produced by the commonly used fusion partner known as SP2/0 which is a myeloma-derived cell line. Here, we first cloned and sequenced two hybridoma clones using this method (clones A1E7 and A9E11). Our results showed a 24 to 25 percent reduction of the aberrant chain sequences (method 1) from the sequencing results. We then optimized this method (method 2) and used it to clone and sequence a third hybridoma clone (clone 262). This optimized method allowed us to achieve an 88% reduction of the aberrant kappa chain sequences.
Unifying sparse coding, predictive coding, and divisive normalization
Authors: Lian Yanbo , Burkitt Anthony N.
Published: June 2023

Sparse coding, predictive coding and divisive normalization have each been found to be principles that underlie the function of neural circuits in many parts of the brain, supported by substantial experimental evidence. However, the connections between these related principles are still poorly understood. In this paper, we show how sparse coding, predictive coding, and divisive normalization can be described within a unified framework, and illustrate this explicitly within the context of a two-layer neural model. This two-layer model is constructed in a way that implements sparse coding with a network structure that is constructed by implementing predictive coding. The results demonstrate how a homeostatic function that regulates neural responses in the model can shape the nonlinearity of neural responses in a way that replicates different forms of divisive normalization. The equivalence of the model to divisive normalization in a single-neuron case is shown analytically. Simulations show that the model can learn simple cells in the primary visual cortex with the property of contrast saturation, which has previously been explained by divisive normalization. In summary, the study demonstrates that the three principles of sparse coding, predictive coding, and divisive normalization can be unified to provide a learning framework based on biophysical properties, such as Hebbian learning and homeostasis, and this framework incorporates both learning and more diverse response nonlinearities observed experimentally. This framework has the potential to also be used to explain how the brain learns to integrate input from different sensory modalities.
An exosome-based gene delivery platform for cell-specific CRISPR/Cas9 genome editing
Authors: Dubey Sunil , Chen Zhe , Talis Austin , Molotkov Andrei , Ali Alessandra , Mintz Akiva , Momen-Heravi Fatemeh
Published: June 2023

Exosomes are naturally occurring vesicles that have the potential to be manipulated to become promising drug delivery vehicles for on-demand in vitro and in vivo gene editing. Here, we developed the modular safeEXO platform, a prototype exosome delivery vehicle that is mostly devoid of endogenous RNA and can efficaciously deliver RNA and ribonucleoprotein (RNP) complexes to their intended intracellular targets manifested by downstream biologic activity. We also successfully engineered producer cells to produce safeEXO vehicles that contain endogenous Cas9 (safeEXO-CAS) to effectively deliver efficient ribonucleoprotein (RNP)-mediated CRISPR genome editing machinery to organs or diseased cells in vitro and in vivo. We confirmed that safeEXO-CAS exosomes could co-deliver ssDNA, sgRNA and siRNA, and efficaciously mediate gene insertion in a dose-dependent manner. We demonstrated the potential to target safeEXO-CAS exosomes by engineering exosomes to express a tissue-specific moiety, integrin alpha-6 (safeEXO-CAS-ITGA6), which increased their uptake to lung epithelial cells in vitro and in vivo. We tested the ability of safeEXO-CAS-ITGA6 loaded with EMX1 sgRNAs to induce lung-targeted editing in mice, which demonstrated significant gene editing in the lungs with no signs of morbidity or detectable changes in immune cell populations. Our results demonstrate that our modular safeEXO platform represents a targetable, safe and efficacious vehicle to deliver nucleic acid-based therapeutics that successfully reach their intracellular targets. Furthermore, safeEXO producer cells can be genetically manipulated to produce safeEXO vehicles containing CRISPR machinery for more efficient RNP-mediated genome editing. This platform has the potential to improve current therapies and increase the landscape of treatment for various human diseases using RNAi and CRISPR approaches.
Pentose Phosphate Pathway Inhibition activates Macrophages towards phagocytic Lymphoma Cell Clearance
Authors: Beielstein Anna C. , Izquierdo Elena , Blakemore Stuart , Nickel Nadine , Michalik Michael , Chawan Samruddhi , Brinker Reinhild , Bartel Hans-Henrik , Vorholt Daniela , Nolte Janica L. , Linke Rebecca , Costa Picossi Carolina Raissa , Sáiz Jorge , Picard Felix , Florin Alexandra , Meinel Jörn , Büttner Reinhard , Villaseñor Alma , Winkels Holger , Hallek Michael , Krüger Marcus , Barbas Coral , Pallasch Christian P.
Published: June 2023

Macrophages in the B-cell lymphoma microenvironment represent a functional node in progression and therapeutic response. We assessed metabolic regulation of macrophages in the context of therapeutic antibody-mediated phagocytosis. Pentose phosphate pathway (PPP) inhibition by specific compounds and shRNA targeting induced increased phagocytic lymphoma cell clearance.

Moreover, macrophages provided decreased support for survival of lymphoma cells. PPP inhibition induced metabolic activation, cytoskeletal re-modelling and pro-inflammatory polarization of macrophages. A link between PPP and immune regulation was identified as mechanism of macrophage repolarization. Inhibition of the PPP causes suppression of glycogen synthesis and subsequent modulation of the immune modulatory UDPG-Stat1-Irg1-Itaconate axis. PPP inhibition rewired macrophage maturation and activationin vivo. Addition of the PPP inhibitor S3 to antibody therapy achieved significantly prolonged overall survival in an aggressive B-cell lymphoma mouse model.

We hypothesize the PPP as key regulator and targetable modulator of macrophage activity in lymphoma to improve efficacy of immunotherapies.

Highlights

Macrophage-mediated lymphoma cell phagocytosis is increased by pentose phosphate pathway (PPP) inhibition as an immune regulatory switch for macrophage function and polarization

PPP inhibition is linked to decreased glycogen synthesis and subsequent modulation of the UDPG-Stat1-Irg1-Itaconate axis

PPP inhibition is tolerablein vivoand facilitates therapeutic targeting of B-cell lymphoma
Molecular mechanisms reconstruction from single-cell multi-omics data with HuMMuS
Authors: Trimbour Remi , Deutschmann Ina Maria , Cantini Laura
Published: June 2023

The molecular identity of a cell results from a complex interplay between heterogeneous molecular layers. Recent advances in single-cell sequencing technologies have opened the possibility to measure such molecular layers of regulation.

Here, we present HuMMuS, a new method for inferring regulatory mechanisms from single-cell multi-omics data. Differently from the state-of-the-art, HuMMuS captures cooperation between biological macromolecules and can easily include additional layers of molecular regulation.

We benchmarked HuMMuS with respect to the state-of-the-art on both paired and unpaired multi-omics datasets. Our results proved the improvements provided by HuMMus in terms of TF targets, TF binding motifs and regulatory regions prediction. Finally, once applied to snmC-seq, scATAC-seq and scRNA-seq data from mouse brain cortex, HuMMuS enabled to accurately cluster scRNA profiles and to identify potential driver TFs.
IRF2BP2 counteracts the ATF7/JDP2 AP-1 heterodimer to prevent inflammatory overactivation in acute myeloid leukemia (AML) cells
Authors: Fischer Sabrina , Weber Lisa M. , Stielow Bastian , Frech Miriam , Simon Clara , Könnecke Julie , Forné Ignasi , Nist Andrea , Bauer Uta Maria , Stiewe Thorsten , Neubauer Andreas , Liefke Robert
Published: June 2023

Acute myeloid leukemia (AML) is a hematological malignancy characterized by abnormal proliferation and accumulation of immature myeloid cells in the bone marrow. Inflammation plays a crucial role in AML progression, but excessive activation of cell-intrinsic inflammatory pathways can also trigger cell death. IRF2BP2 is a chromatin regulator implicated in AML pathogenesis, although its precise role in this disease is not fully understood. In this study, we demonstrate that IRF2BP2 interacts with the AP-1 heterodimer ATF7/JDP2, which is involved in activating inflammatory pathways in AML cells. We show that IRF2BP2 is recruited by the ATF7/JDP2 dimer to chromatin and counteracts its gene-activating function. Loss of IRF2BP2 leads to overactivation of inflammatory pathways, resulting in strongly reduced proliferation. Our research indicates that a precise equilibrium between activating and repressive transcriptional mechanisms creates a pro-oncogenic inflammatory environment in AML cells. The ATF7/JDP2-IRF2BP2 regulatory axis is likely a key regulator of this process and may therefore represent a promising therapeutic vulnerability for AML. Thus, our study provides new insights into the molecular mechanisms underlying AML pathogenesis and identifies a potential therapeutic target for AML treatment.
Touch sensation requires the mechanically-gated ion channel Elkin1
Authors: Chakrabarti Sampurna , Klich Jasmin D. , Khallaf Mohammed A. , Sánchez-Carranza Oscar , Baran Zuzanna M. , Rossi Alice , Huang Angela Tzu-Lun , Pohl Tobias , Fleischer Raluca , Fürst Carina , Hammes Annette , Bégay Valérie , Hörnberg Hanna , Poole Kate , Lewin Gary R.
Published: June 2023

The slightest touch to the skin initiates tactile perception that is almost immediate1. The extraordinary speed of touch perception is enabled by mechanically-activated ion channels, the opening of which excites the endings of sensory neurons innervating the skin to initiate sensation. Here we identify a new mechanically-activated ion channel, Elkin12, that, when ablated in mice, leads to a profound behavioural touch insensitivity. Touch insensitivity inElkin1-/-mice was caused by a loss of mechanically-activated currents (MA-currents) in around half of all sensory neurons that are activated by light touch (low threshold mechanoreceptors, LTMRs). Reintroduction of Elkin1 into sensory neurons fromElkin1-/-mice acutely restored MA-currents. Piezo23–6is an established mechanosensitive ion channel required for touch sensation. In mice genetic ablation ofPiezo2renders many, but not all, LTMRs insensitive to mechanical force4,5,7. Here we show that Elkin1 underpins PIEZO2-independent touch sensation. Additionally, we find that Elkin1 is present in many nociceptive sensory neurons which detect potentially damaging and painful mechanical force. These nociceptors depend onElkin1for effectively communicating information on sustained noxious mechanical forces. We further identified molecular and functional interactions between the known mechanotransduction protein Stoml38,9and Elkin1 ion channels. Our data identify Elkin1 as a novel core component of touch transduction in mammals. The specific sensory deficits exhibited byElkin1-/-mice make Elkin1 a highly desirable target that could be harnessed to treat somatic sensory disorders including pain.
Blueprinting expandable nanomaterials with standardized protein building blocks
Authors: Huddy Timothy F. , Hsia Yang , Kibler Ryan D. , Xu Jinwei , Bethel Neville , Nagarajan Deepesh , Redler Rachel , Leung Philip J. Y. , Courbet Alexis , Yang Erin C. , Bera Asim K. , Coudray Nicolas , Calise S. John , Davila-Hernandez Fatima A. , Weidle Connor , Han Hannah L. , Li Zhe , McHugh Ryan , Reggiano Gabriella , Kang Alex , Sankaran Banumathi , Dickinson Miles S. , Coventry Brian , Brunette TJ , Liu Yulai , Dauparas Justas , Borst Andrew J. , Ekiert Damian , Kollman Justin M. , Bhabha Gira , Baker David
Published: June 2023

A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies in comparison has been much more complex, largely due to the irregular shapes of protein structures1. Here we describe extendable linear, curved, and angled protein building blocks, as well as inter-block interactions that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight “train track” assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not been previously possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank 3D canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to “back of an envelope” architectural blueprints.
The involvement of the human prefrontal cortex in the emergence of visual awareness
Authors: Fang Zepeng , Dang Yuanyuan , Ling Zhipei , Han Yongzheng , Zhao Hulin , Xu Xin , Zhang Mingsha
Published: June 2023

Exploring the neural mechanisms of awareness is a fundamental task of cognitive neuroscience. There is an ongoing dispute regarding the role of the prefrontal cortex (PFC) in the emergence of awareness, which is partially raised by the confound between report- and awareness-related activity. To address this problem, we designed a visual awareness task that can minimize report-related motor confounding. Our results show that saccadic latency is significantly shorter in the aware trials than in the unaware trials. Local field potential (LFP) data from 6 patients consistently show early (200-300 ms) awareness-related activity in the PFC, including event-related potential and high-gamma activity. Moreover, the awareness state can be reliably decoded by the neural activity in the PFC since the early stage, and the neural pattern is dynamically changed rather than being stable during the representation of awareness. Furthermore, the enhancement of dynamic functional connectivity, through the phase modulation at low frequency, between the PFC and other brain regions in the early stage of the awareness trials may explain the mechanism of conscious access. These results indicate that the PFC is critically involved in the emergence of awareness.
Spatial Mapping of Mobile Genetic Elements and their Cognate Hosts in Complex Microbiomes
Authors: Grodner Benjamin , Shi Hao , Farchione Owen , Vill Albert C. , Ntekas Ioannis , Diebold Peter J. , Zipfel Warren R. , Brito Ilana L. , De Vlaminck Iwijn
Published: June 2023

The frequent exchange of mobile genetic elements (MGEs) between bacteria accelerates the spread of functional traits, including antimicrobial resistance, within the human microbiome. Yet, progress in understanding these intricate processes has been hindered by the lack of tools to map the spatial spread of MGEs in complex microbial communities, and to associate MGEs to their bacterial hosts. To overcome this challenge, we present an imaging approach that pairs single molecule DNA Fluorescence In Situ Hybridization (FISH) with multiplexed ribosomal RNA FISH, thereby enabling the simultaneous visualization of both MGEs and host bacterial taxa. We used this methodology to spatially map bacteriophage and antimicrobial resistance (AMR) plasmids in human oral biofilms, and we studied the heterogeneity in their spatial distributions and demonstrated the ability to identify their host taxa. Our data revealed distinct clusters of both AMR plasmids and prophage, coinciding with densely packed regions of host bacteria in the biofilm. These results suggest the existence of specialized niches that maintain MGEs within the community, possibly acting as local hotspots for horizontal gene transfer. The methods introduced here can help advance the study of MGE ecology and address pressing questions regarding antimicrobial resistance and phage therapy.
Detecting genetic effects on phenotype variability to capture gene-by-environment interactions: a systematic method comparison
Authors: Zhang Xiaopu , Bell Jordana T.
Published: June 2023

Phenotypic variability has been widely observed across organisms and traits, including in humans. Both gene-gene and gene-environment interactions can lead to an increase in phenotypic variability. Therefore, detecting the underlying genetic variants, or variance Quantitative Trait Loci (vQTLs), can provide novel insights into complex traits. Established approaches to detect vQTLs apply different methodologies from variance-only approaches to mean-variance joint tests, but a comprehensive comparison of these methods is lacking. Here, we review available methods to detect vQTLs in humans, carry out a simulation study to assess their performance under different biological scenarios of gene-environment interactions, and apply the optimal approaches for vQTL identification to gene expression data. We find that the squared residual value linear model (SVLM) method is optimal when the interacting exposure is discrete, and both SVLM and the deviation regression model (DRM) perform well when the interacting exposure is continuous. Additionally, a larger sample size, smaller minor allele frequency, and more balanced sample distribution in different exposure categories increase power of SVLM and DRM. Our results highlight vQTL detection methods that perform optimally under realistic simulation settings and show that their relative performance depends on the type of exposure in the interaction model underlying the vQTL.

Author Summary

Genetic background can influence organismal response to changes in the environment, including through effects of gene-environment (GxE) interactions. GxE interactions form a fundamental component of complex phenotypes and disease. The presence of GxE interactions leads to increases in phenotypic variability, where individuals of a certain genotype can display more divergent phenotypes. Therefore, identifying genetic variation that can alter phenotypic variance is an alternative approach to identifying genetic variants that underlie GxE effects. Here, we conduct a systematic assessment of methods that detect genetic variants linked to phenotypic variance under several GxE scenarios. We identify the most optimal approaches for different environmental exposure settings and apply these to validate previously detected GxE signals. We also estimate power of these methods to detect GxE effects. Our results help guide experimental design for future studies aiming to identify genetic variant impacts on phenotypic variance, and their result interpretation in context of gene-environment interactions in complex traits.
CNP blocks mitochondrial depolarization and inhibits SARS-CoV-2 replicationin vitroandin vivo
Authors: Logue James , Melville Victoria M. , Ardanuy Jeremy , Frieman Matthew B.
Published: June 2023

The COVID-19 pandemic has claimed over 6.5 million lives worldwide and continues to have lasting impacts on the world’s healthcare and economic systems. Several approved and emergency authorized therapeutics that inhibit early stages of the virus replication cycle have been developed however, effective late-stage therapeutical targets have yet to be identified. To that end, our lab identified that 2’,3’ cyclic-nucleotide 3’-phosphodiesterase (CNP) inhibits SARS-CoV-2 virion assembly. We show that CNP inhibits the generation of new SARS-CoV-2 virions, reducing intracellular titers without inhibiting viral structural protein translation. Additionally, we show that targeting of CNP to mitochondria is necessary for inhibition, blocking mitochondrial depolarization and implicating CNP’s proposed role as an inhibitor of the mitochondrial permeabilization transition pore (mPTP) as the mechanism of virion assembly inhibition. We also demonstrate that an adenovirus expressing virus expressing both human ACE2 and CNP inhibits SARS-CoV-2 titers to undetectable levels in lungs of mice. Collectively, this work shows the potential of CNP to be a new SARS-CoV-2 antiviral target.

Author Summary

Upon entry into a cell, viruses manipulate the cellular environment for the benefit of their replication. We have found that upon infection, SARS-CoV-2 induces the mitochondria to release reactive oxygen species (ROS) into the cytoplasm which benefits the replication of the virus. We identified a phosphodiesterase, called CNP, that blocks this release via inhibition of an inducible pore called the mitochondrial permeability transition pore or mPTP. We also found that a drug targeting this pore inhibits SARS-CoV-2 replication. We test the function of CNP in vivo and find that if we overexpress CNP in mouse lungs, we inhibit SARS-CoV-2 replication. Together this demonstrates a key function of the mitochondria on SARS-CoV-2 replication and that antithetically ROS release enhances viral replication. We propose this is common across coronaviruses and potentially other viruses identifying a novel target for future therapies.
Metric information in cognitive maps: Euclidean embedding of non-Euclidean environments
Authors: Baumann Tristan , Mallot Hanspeter A
Published: June 2023

The structure of the internal representation of surrounding space, the so-calledcognitive map, has long been debated. A Euclidean metric map is the most straight-forward hypothesis, but human navigation has been shown to systematically deviate from the Euclidean ground truth. Vector navigation based on non-metric models can better explain the observed behavior, but also discards useful geometric properties such as fast shortcut estimation and cue integration.

Here, we propose another alternative, a Euclidean metric map that is systematically distorted to account for the observed behavior. The map is found by embedding the non-metric model, a labeled graph, into 2D Euclidean coordinates. We compared these two models using human data from Warren et al. (2017), where participants had to navigate and learn a non-Euclidean maze (i.e., with Wormholes) and perform direct shortcuts between different locations. Even though the Euclidean embedding cannot correctly represent the non-Euclidean environment, both models predicted the data equally well. We argue that the so embedded graph naturally arises from integrating the local position information into a metric framework, which makes the model more powerful and robust than the non-metric alternative. It may therefore be a better model for the human cognitive map.