bluuebunny/crossref_metadata_2025_split huggingface dataset:
A system of reporters for comparative investigation of EJC-independent and EJC-enhanced nonsense-mediated mRNA decay
Authors: Kolakada Divya , Campbell Amy E. , Galvis Laura Baquero , Li Zhongyou , Lore Mlana , Jagannathan Sujatha 
Published: November 2023

AbstractNonsense-mediated mRNA decay (NMD) is a network of pathways that degrades transcripts that undergo premature translation termination. In mammals, NMD can be divided into the exon junction complex (EJC)-enhanced and EJC-independent branches. Fluorescence- and luminescence-based reporters have long been effective tools to investigate NMD, yet existing reporters largely focus on the EJC-enhanced pathway. Here, we present a system of reporters for comparative studies of EJC-independent and EJC-enhanced NMD. This system also enables the study of NMD-associated outcomes such as premature termination codon (PTC) readthrough and truncated protein degradation. These reporters are compatible with fluorescence or luminescence-based readouts via transient transfection or stable integration. Using this reporter system, we show that EJC-enhanced NMD RNA levels are reduced by 2- or 9-fold and protein levels are reduced by 7- or 12-fold compared to EJC-independent NMD, depending on the reporter gene used. Additionally, the extent of readthrough induced by G418 and SMG1i, alone and in combination, varies across NMD substrates. When combined, G418 and SMG1i increase readthrough product levels in an additive manner for EJC-independent reporters, while EJC-enhanced reporters show a synergistic effect. We present these reporters as a valuable toolkit to deepen our understanding of NMD and its associated mechanisms.
A Method for Sampling the Living Wood Microbiome
Authors: Arnold Wyatt , Gewirtzman Jonathan , Raymond Peter A. , Bradford Mark A. , Butler Claire , Peccia Jordan 
Published: November 2023

AbstractEfforts to characterize microbial life across diverse environments have progressed tremendously, yet the microbiome of Earth’s largest biomass reservoir—the wood of living trees—has been largely unexplored. Current understanding of the tree microbiome is largely confined to roots and leaves, with little attention given to the endophytic microbiome of wood, even though emergent studies have indicated this zone as a niche for unique taxa, of consequence for ecosystem health and global biogeochemical cycles. The lack of investigation derives partly from the physical recalcitrance of wood, which presents challenges during sampling, homogenization, and the extraction of nucleic acids. In response to these issues, we present an optimized method for processing wood for use in microbial analyses, from sampling through to downstream analyses. Using methane-cycling taxa as model endophytes, we assess losses in recovery during our method, and determine a limit-of-detection of approximately 500 cells per 100 mg of (dry) wood. For all six species evaluated—which represented several diverse taxa of hardwoods and softwoods—PCR inhibition proved minimal, and we expect this method to be applicable for a majority of tree species. The methods presented herein can facilitate future investigation into the wood microbiome and global microbial ecology of methane cycling.
Connectivity Regression
Authors: Desai Neel , Baladandayuthapani Veera , Shinohara Russell T. , Morris Jeffrey S. 
Published: November 2023

SummaryAssessing how brain functional connectivity networks vary across individuals promises to uncover important scientific questions such as patterns of healthy brain aging through the lifespan or dysconnectivity associated with disease. In this article we introduce a general regression framework, Connectivity Regression (ConnReg), for regressing subject-specific functional connectivity networks on covariates while accounting for within-network inter-edge dependence. ConnReg utilizes a multivariate generalization of Fisher’s transformation to project network objects into an alternative space where Gaussian assumptions are justified and positive semidefinite constraints are automatically satisfied. Penalized multivariate regression is fit in the transformed space to simultaneously induce sparsity in regression coefficients and in covariance elements, which capture within network inter-edge dependence. We use permutation tests to perform multiplicity-adjusted inference to identify covariates associated with connectivity, and stability selection scores to identify network edges that vary with selected covariates. Simulation studies validate the inferential properties of our proposed method and demonstrate how estimating and accounting for within-network inter-edge dependence leads to more efficient estimation, more powerful inference, and more accurate selection of covariate-dependent network edges. We apply ConnReg to the Human Connectome Project Young Adult study, revealing insights into how connectivity varies with language processing covariates and structural brain features.
Directed Evolution of Genetically Encoded LYTACs for Cell-Mediated Delivery
Authors: Yang Jonathan Lee , Yamada-Hunter Sean A. , Labanieh Louai , Sotillo Elena , Cheah Joleen S. , Roberts David S. , Mackall Crystal L. , Ting Alice Y. , Bertozzi Carolyn R. 
Published: November 2023

AbstractLysosome-targeting chimeras (LYTACs) are a promising therapeutic modality to drive the degradation of extracellular proteins. However, early versions of LYTAC contain synthetic glycopeptides that cannot be genetically encoded. Here we present our designs for a fully genetically encodable LYTAC (GELYTAC), making our tool compatible with integration into therapeutic cells for targeted delivery at diseased sites. To achieve this, we replaced the glycopeptide portion of LYTACs with the protein insulin like growth factor 2 (IGF2). After showing initial efficacy with wild type IGF2, we increased the potency of GELYTAC using directed evolution. Subsequently, we demonstrated that our engineered GELYTAC construct not only secretes from HEK293T cells but also from human primary T-cells to drive the uptake of various targets into receiver cells. Immune cells engineered to secrete GELYTAC thus represent a promising avenue for spatially-selective targeted protein degradation.Significance StatementBetter therapeutic windows can be achieved by targeting therapeutics to their desired sites of action. For protein therapeutics, this might be achieved by engineering cell therapies that home to a tissue of interest and secrete the biologic drug locally. Here, we demonstrate that human primary T cells can be engineered to produce genetically encoded lysosome targeting chimeras (GELYTACs). These GELYTACs mediate the degradation of extracellular proteins associated with cancer progression. Thus, cells engineered to produce GELYTACs represent a potential new class of cancer therapeutics.
Utilization of aHistoplasma capsulatumzinc reporter reveals the complexities of fungal sensing of metal deprivation
Authors: Blancett Logan T. , Evans Heather M. , Candor Kathleen , Buesing William R. , Landero Figueroa Julio A. , Deepe George S. 
Published: November 2023

AbstractHistoplasma capsulatumis a dimorphic fungal pathogen acquired via inhalation of soil-resident spores. Upon exposure to mammalian body temperatures, these fungal elements transform into yeasts that reside primarily within phagocytes. Macrophages (MΦ) provide a permissive environment for fungal replication until T cell-dependent immunity is engaged. MΦ activated by granulocyte-MΦ colony stimulating factor (GM-CSF) induce metallothioneins (MTs) that bind zinc (Zn) and deprive yeast cells of labile Zn, thereby disabling fungal growth. Prior work demonstrated that the high affinity zinc importer, ZRT2, was important for fungal survival in vivo. Hence, we constructed a yeast cell reporter strain that expresses green fluorescent protein (GFP) under the control of this importer. This reporter accurately responds to medium devoid of Zn. ZRT2 expression increased (∼5-fold) in GM-CSF, but not interferon-γ, stimulated MΦ. To examine the in vivo response, we infected mice with reporter yeasts and assessed ZRT2 expression at 0-, 3-, 7-, and 14-days post-infection (dpi). ZRT2 expression minimally increased at 3-dpi and peaked on 7-dpi, corresponding with onset of adaptive immunity. We discovered that the major phagocyte populations that restrict Zn to the fungus are interstitial MΦ and exudate MΦ. Neutralizing GM-CSF blunted control of infection but unexpectedly increased ZRT2 expression. This increase was dependent on another cytokine that activates MΦ to controlH. capsulatumreplication, M-CSF. These findings illustrate the reporter’s ability to sense Znin vitroandin vivoand correlate ZRT2 activity with GM-CSF and M-CSF activation of MΦ.ImportancePhagocytes use an arsenal of defenses to control replication ofHistoplasmayeasts, one of which is limitation of trace metals. On the other hand,H. capsulatumcombats metal restriction by upregulating metal importers such as the Zn importer ZRT2. This transporter contributes toH. capsulatumpathogenesis upon activation of adaptive immunity. We constructed a fluorescent ZRT2 reporter to probeH. capsulatumZn sensing during infection and exposed a role for M-CSF activation of macrophages when GM-CSF is absent. These data highlight the ways in which fungal pathogens sense metal deprivation in vivo and reveal the potential of metal-sensing reporters. The work adds a new dimension to studying how intracellular pathogens sense and respond to the changing environments of the host.
CCR5/CXCR3 antagonist TAK-779 prevents diffuse alveolar damage of the lung in murine model of the SARS-CoV-2-related acute respiratory distress syndrome
Authors: Chernov Aleksandr S. , Rodionov Maksim V. , Kazakov Vitaly A. , Ivanova Karina A. , Mesheryakov Fedor A. , Kudriaeva Anna A. , Gabibov Alexander G. , Telegin Georgii B. , Belogurov Alexey A. 
Published: November 2023

AbstractThe acute respiratory distress syndrome (ARDS) secondary to viral pneumonitis is one of the main causes of high mortality in patients with COVID-19 (novel coronavirus disease 2019) – ongoing SARS-CoV-2 infection, reached more than 0.7 billion registered cases. Recently we elaborated non- surgical and reproducible method of unilateral total diffuse alveolar damage (DAD) of the left lung in ICR mice – a publicly available imitation of the ARDS caused by SARS-CoV-2. Our data reads that two C-C chemokine receptor 5 (CCR5) ligands – macrophage inflammatory proteins (MIP) – (MIP-1α/CCL3) and (MIP-1β/CCL4) are upregulated in this DAD model up to three orders of magnitude compared to the background level. Here we showed that a nonpeptide compound TAK- 779, antagonist of CCR5/CXCR3, readily prevents DAD of the lung with a single injection of 2.5 mg/kg. Histological analysis revealed reduced peribronchial and perivascular mononuclear infiltration in the lung, and mononuclear infiltration of the wall and lumen of the alveoli in the TAK- 779-treated animals. Administration of the TAK-779 decreased 3-5-fold level of serum cytokines and chemokines in animals with DAD, including CCR5 ligands MIP-1α/β, MCP-1 and CCL5. Computed tomography revealed rapid recovery of the density and volume of the affected lung in TAK-779- treated animals. Our pre-clinical data suggest that TAK-779 is more effective than administration of dexamethasone or anti-IL6R therapeutic antibody tocilizumab, which brings novel therapeutic modality to TAK-779 and other CCR5 inhibitors recruited in ongoing clinical studies as a potential drugs for treatment of COVID19 and similar virus-induced inflammation syndromes.Abstract summaryThe pathogenesis of the SARS-CoV-2 infection is tightly linked with the cytokine storm resulting in the enormous release of cytokines and chemokines. Its clinical manifestation – the acute respiratory distress syndrome (ARDS), may be caused by self-sustaining hypersensitivity reactions leading to lung collapse even after virus clearance. Here we report that two macrophage inflammatory proteins, MIP-1α/CCL3 and MIP-1β/CCL4, seem to orchestrate mononuclear infiltration into the lungs during diffuse alveolar damage (DAD) in ICR mice – our murine model of ARDS caused by SARS-CoV-2. Inhibition of the C-C chemokine receptor 5 (CCR5) – parental receptor for MIP-1α and MIP-1β, by nonpeptide antagonist TAK-779 results in significant amelioration of DAD in terms of reduced mononuclear infiltration into the lung, suppressed cytokine storm and restored physiology of affected lung according to computed tomography data. We suggest that targeted inhibition of CCR5 should be further elucidated as safe and effective approach to overcome severe viral pneumonia in humans.
O-antigen biosynthesis mediates evolutionary trade-offs within a simple community
Authors: Spencer-Drakes Tara C.J. , Sarabia Angel , Heussler Gary , Pierce Emily C. , Morin Manon , Villareal Steven , Dutton Rachel J. 
Published: November 2023

AbstractDiverse populations of bacteriophages infect and co-evolve with their bacterial hosts. Although host recognition and infection occurs within microbiomes, the molecular mechanisms underlying host-phage interactions within a community context remain poorly studied. The biofilms (rinds) of aged cheeses contain taxonomically diverse microbial communities that follow reproducible growth patterns and can be manipulated under laboratory conditions. In this study, we use cheese as a model for studying phage-microbe interactions by identifying and characterising a tractable host-phage pair co-occurring within a model Brie community. We isolated novel bacteriophage TS33 that killsHafniasp. JB232 (hereafterHafnia), a member of the model community. TS33 is easily propagated in the lab and naturally co-occurs in the cheese with the Brie community, rendering it a prime candidate for the study of host-phage interactions. We performed growth assays of theHafnia, TS33 and the fungal community members,Geotrichum candidumandPenicillium camemberti. Employing Random Barcode Transposon Sequencing (RB-TnSeq) experiments, we identified candidate host factors that contribute to TS33 infectivity, many of which are critical to the integrity of bacterial O-antigen. Notably, disruption of these genes results in decreased susceptibility to infection by phage TS33, while simultaneously exhibiting a significant negative effect on the fitness ofHafniain the presence of the fungi. Therefore, O-antigen mutations may have pleiotropic effects on the interactions betweenHafniaand the rest of the Brie community. Ongoing and future studies aim to unearth the molecular mechanisms by which the O-antigen ofHafniamediates its interactions with its viral and fungal partners.
Axon development is regulated at genetic and proteomic interfaces between the integrin adhesome and the RPM-1 ubiquitin ligase signaling hub
Authors: Amezquita Jonathan , Desbois Muriel , Opperman Karla J. , Pak Joseph S. , Christensen Elyse L. , Nguyen Nikki T. , Diaz-Garcia Karen , Borgen Melissa A. , Grill Brock 
Published: November 2023

SUMMARYIntegrin signaling plays important roles in development and disease. An adhesion signaling network called the integrin adhesome has been principally defined using bioinformatics and proteomics. To date, the adhesome has not been studied using integrated proteomic and genetic approaches. Here, proteomic studies inC. elegansidentified physical associations between the RPM-1 ubiquitin ligase signaling hub and numerous adhesome components including Talin, Kindlin and beta-integrin.C. elegansRPM-1 is orthologous to human MYCBP2, a prominent player in nervous system development associated with a neurodevelopmental disorder. Using neuron-specific, CRISPR loss-of-function strategies, we show that core adhesome components affect axon development and interact genetically with RPM-1. Mechanistically, Talin opposes RPM-1 in a functional ‘tug-of-war’ on growth cones that is required for accurate axon termination. Thus, our findings orthogonally validate the adhesome via multi-component genetic and physical interfaces with a key neuronal signaling hub and identify new links between the adhesome and brain disorders.
Self-organised pattern formation in the developing neural tube by a temporal relay of BMP signalling
Authors: Lehr S , Brückner D B , Greunz-Schindler M , Minchington T , Merrin J , Hannezo E , Kicheva A 
Published: November 2023

AbstractDeveloping tissues interpret dynamic changes in morphogen activity to generate cell type diversity. To quantitatively study BMP signalling dynamics in the vertebrate neural tube, we developed a new ES cell differentiation system tailored for growing tissues. Differentiating cells form striking self-organised patterns of dorsal neural tube cell types driven by sequential phases of BMP signalling that are observed both in vitro and in vivo. Data-driven biophysical modelling showed that these dynamics result from coupling fast negative feedback with slow positive regulation of signalling by the specification of an endogenous BMP source. Thus, in contrast to relays that propagate morphogen signalling in space, we uncover a BMP signalling relay that operates in time. This mechanism allows rapid initial concentrationsensitive response that is robustly terminated, thereby regulating balanced sequential cell type generation. Altogether, our study provides an experimental and theoretical framework to understand how signalling dynamics are exploited in developing tissues.
Insights into vision from interpretation of a neuronal wiring diagram
Authors: Seung H. Sebastian 
Published: November 2023

AbstractWhat insects can see has been probed by over a century of behavioral experiments. Motion and color vision have also been studied through neurophysiology in insect brains. Here I study form vision by interpreting a neuronal wiring diagram of theDrosophilaoptic lobe. The Dm3 “line amacrine” cells are shown to divide into three cell types with oriented dendrites, and to be connected with three TmY cell types, also with oriented dendrites. All six cell types are predicted to respond selectively to oriented visual stimuli, with preferred orientation defined by dendrite orientation. Their receptive fields are predicted by mapping input from other cell types that chiefly convey information from single facets of the compound eye. Dm3 to Dm3 and TmY connectivity is approximated by cross-orientation inhibition and TmY to TmY connectivity by iso-orientation excitation. Both connectivity motifs were previously hypothesized for mammalian visual cortex. Two of the TmY types target a novel type of LC10 cell, which leads by multiple pathways to brain regions that support learning of visual form. Based on the spatial organization of TmY to TmY and LC10 connectivity, I conjecture that flies may see illusory contours and corners.
A short 3’UTR motif regulates gene expression in bilaterians
Authors: Eufrásio Ana , Azevedo Joana , Machado Joana , Ferreira Alexandre , Moutinho Ana , Henriques Filipe , Jesus Ana , Tavares Joana , Pereira-Castro Isabel , Teixeira Joana , Araújo Mafalda , Pinto Pedro B.P. , Bessa José , Moreira Alexandra 
Published: November 2023

AbstractThe mechanisms of gene expression regulation are essential for cell identity and function, and their disruption usually leads to human disease. The 3’ untranslated region (UTR) of mRNA contains important regulatory elements of gene expression, including upstream sequence elements (USEs) that are cis-regulatory sequences localized upstream of polyA signals (PAS). One of the best functionally characterised USEs is located in the 3’UTR of the Drosophila’s polo gene, which disruption leads to critical phenotypes in adult flies. In this work we found that the USE of the Drosophila’s polo gene (DplUSE) is also found in in the 3’UTR of vertebrate genes, including zebrafish, mouse and human genes, showing higher levels of conservation than the whole 3’UTR sequence. Using reporter assays, we show that DplUSE is able to increase gene expression in vitro in human cell lines and in vivo in zebrafish embryos. Importantly, in humans, the DplUSE containing genes are enriched for genes associated to serious diseases such as Congenital abnormalities and Malignant neoplasms, illustrating the potential of this sequence to modulate genes with relevant biological functions and related with human health. Concomitantly, when sequestering the molecular machinery that operates at the DplUSE using a dominant negative strategy, we show that this is enough to dysregulate DplUSE containing genes in human cells and disrupt proper embryo development in zebrafish. Aiming to understand the molecular mechanism operating at the DplUSE, we identified three RNA binding proteins (RBP) that specifically bind to the DplUSE in vertebrates. Importantly, one of such RBPs is PTBP1, the vertebrate orthologue of the fruit fly’s RBP Heph, that was demonstrated to be required for the DplUSE function in Drosophila. To test if PTBP1is essential for the DplUSE function, as observed in Drosophila, we depleted PTBP1 from human cells and observed a downregulation of the expression of DplUSE containing genes, demonstrating that the molecular mechanisms that operate at DplUSE are ultra-conserved. Finally, we explored if variants in DplUSE consensus could be associated to human disease. We found a reported single nucleotide polymorphism (SNP; rs3087967) that is associated with malignant tumor of colon and generates an ectopic consensus of DplUSE in the 3’ UTR of the tumorigenic POU2AF2/C11orf53 gene. We further show that this ectopic DplUSE motif causes a gain-of-function in vivo in zebrafish gut cells, suggesting its involvement in colon cancer development. These results show that a short motif present in the 3’UTR of genes from phylogenetically distant bilaterians, from fruit flies to humans, control genes’ expression through an ultra-conserved mechanism involving RBPs binding and its dysregulation might impact in human disease.
Structural and functional basis of VLDLR receptor usage by Eastern equine encephalitis virus
Authors: Adams Lucas J. , Raju Saravanan , Ma Hongming , Gilliland Theron , Reed Douglas S. , Klimstra William B. , Fremont Daved H. , Diamond Michael S. 
Published: November 2023

SUMMARYThe very low-density lipoprotein receptor (VLDLR) is comprised of eight LDLR type A (LA) domains and supports entry of distantly related Eastern equine encephalitis (EEEV) and Semliki Forest (SFV) alphaviruses. Here, by resolving multiple cryo-electron microscopy structures of EEEV-VLDLR complexes and performing mutagenesis and functional studies, we show that EEEV uses multiple sites (E1/E2 cleft and E2 A domain) to engage different LA domains simultaneously. However, no single LA domain is necessary or sufficient to support efficient EEEV infection, highlighting complexity in domain usage. Whereas all EEEV strains show conservation of two VLDLR binding sites, the EEEV PE–6 strain and other EEE complex members feature a single amino acid substitution that mediates binding of LA domains to an additional site on the E2 B domain. These structural and functional analyses informed the design of a minimal VLDLR decoy receptor that neutralizes EEEV infection and protects mice from lethal challenge.