bluuebunny/crossref_metadata_2025 huggingface dataset:
GAUSS-EM: Guided accumulation of ultrathin serial sections with a static magnetic field for volume electron microscopy
Authors: Fulton Kara A , Watkins Paul V , Briggman Kevin L
Published: November 2023

Serial sectioning electron microscopy of millimeter-scale 3D anatomical volumes requires the collection of thousands of ultrathin sections. Here we report a high-throughput automated approach, GAUSS-EM, utilizing a static magnetic field to collect and densely pack thousands of sections onto individual silicon wafers. The method is capable of sectioning hundreds of microns of tissue per day at section thicknesses down to 35 nm. Relative to other automated volume electron microscopy approaches, GAUSS-EM democratizes the ability to collect large 3D EM volumes because it is simple and inexpensive to implement. We present two exemplar EM volumes of a zebrafish eye and mouse olfactory bulb collected with the method.
UNSEG: unsupervised segmentation of cells and their nuclei in complex tissue samples
Authors: Kochetov Bogdan , Bell Phoenix , Garcia Paulo S. , Shalaby Akram S. , Raphael Rebecca , Raymond Benjamin , Leibowitz Brian J. , Schoedel Karen , Brand Rhonda M. , Brand Randall E. , Yu Jian , Zhang Lin , Diergaarde Brenda , Schoen Robert E. , Singhi Aatur , Uttam Shikhar
Published: November 2023

Multiplexed imaging technologies have made it possible to interrogate complex tumor microenvironments at sub-cellular resolution within their native spatial context. However, proper quantification of this complexity requires the ability to easily and accurately segment cells into their sub-cellular compartments. Within the supervised learning paradigm, deep learning based segmentation methods demonstrating human level performance have emerged. However, limited work has been done in developing such generalist methods within the label-free unsupervised context. Here we present an unsupervised segmentation (UNSEG) method that achieves deep learning level performance without requiring any training data. UNSEG leverages a Bayesian-like framework and the specificity of nucleus and cell membrane markers to construct ana posterioriprobability estimate of each pixel belonging to the nucleus, cell membrane, or background. It uses this estimate to segment each cell into its nuclear and cell-membrane compartments. We show that UNSEG is more internally consistent and better at generalizing to the complexity of tissue morphology than current deep learning methods. This allows UNSEG to unambiguously identify the cytoplasmic compartment of a cell, which we employ to demonstrate its use in an exemplar biological scenario. Within the UNSEG framework, we also introduce a new perturbed watershed algorithm capable of stably and automatically segmenting a cluster of cell nuclei into individual cell nuclei that increases the accuracy of classical watershed. Perturbed watershed can also be used as a standalone algorithm that researchers can incorporate within their supervised or unsupervised learning approaches to extend classical watershed, particularly in the multiplexed imaging context. Finally, as part of developing UNSEG, we have generated a high-quality annotated gastrointestinal tissue (GIT) dataset, which we anticipate will be useful for the broader research community. We demonstrate the efficacy of UNSEG on the GIT dataset, publicly available datasets, and on a range of practical scenarios. In these contexts, we also discuss the possibility of bias inherent in quantification of segmentation accuracy based onF1score. Segmentation, despite its long antecedents, remains a challenging problem, particularly in the context of tissue samples. UNSEG, an easy-to-use algorithm, provides an unsupervised approach to overcome this bottleneck, and as we discuss, can help improve deep learning based segmentation methods by providing a bridge between unsupervised and supervised learning paradigms.
Gait speed is a biomarker of cancer-associated cachexia decline and recovery
Authors: Roy Ishan , Binder-Markey Ben , Sychowski Danielle , Willbanks Amber , Phipps Tenisha , McAllister Donna , Bhakta Akash , Marquez Emily , D’Andrea Dominic , Franz Colin , Pichika Rajeswari , Dwinell Michael B. , Jayabalan Prakash , Lieber Richard L.
Published: November 2023

Background

Progressive functional decline is a key element of cancer-associated cachexia. No therapies have successfully translated to the clinic due to an inability to measure and improve physical function in cachectic patients. Major barriers to translating pre-clinical therapies to the clinic include lack of cancer models that accurately mimic functional decline and use of non-specific outcome measures of function, like grip strength. New approaches are needed to investigate cachexia-related function at both the basic and clinical science levels.

Methods

Survival extension studies were performed by testing multiple cell lines, dilutions, and vehicle-types in orthotopic implantation of K-rasLSL.G12D/+; Trp53R172H/+; Pdx-1-Cre (KPC) derived cells. 128 animals in this new model were then assessed for muscle wasting, inflammation, and functional decline using a battery of biochemical, physiologic, and behavioral techniques. In parallel, we analyzed a 156-subject cohort of cancer patients with a range of cachexia severity, and who required rehabilitation, to determine the relationship between gait speed via six-minute walk test (6MWT), grip strength (hGS), and functional independence measures (FIM). Cachectic patients were identified using the Weight Loss Grading Scale (WLGS), Fearon consensus criteria, and the Prognostic Nutritional Index (PNI).

Results

Using a 100-cell dose of DT10022 KPC cells, we extended the survival of the KPC orthotopic model to 8-9 weeks post-implantation compared to higher doses used (p<0.001). In this Low-dose Orthotopic (LO) model, both progressive skeletal and cardiac muscle wasting were detected in parallel to systemic inflammation; skeletal muscle atrophy at the fiber level was detected as early as 3 weeks post-implantation compared to controls (p<0.001). Gait speed in LO animals declined as early 2 week post-implantation whereas grip strength change was a late event and related to end of life. Principle component analysis (PCA) revealed distinct cachectic and non-cachectic animal populations, which we leveraged to show that gait speed decline was specific to cachexia (p<0.01) while grip strength decline was not (p=0.19). These data paralleled our observations in cancer patients with cachexia who required rehabilitation. In cachectic patients (identified by WLGS, Fearon criteria, or PNI, change in 6MWT correlated with motor FIM score changes while hGS did not (r2=0.18, p<0.001). This relationship between 6MWT and FIM in cachectic patients was further confirmed through multivariate regression (r2=0.30, p<0.001) controlling for age and cancer burden.

Conclusion

Outcome measures linked to gait are better associated with cachexia related function and preferred for future pre-clinical and clinical cachexia studies.
Convergent evolution of monocyte differentiation in adult skin instructs Langerhans cell identity
Authors: Appios Anna , Davies James , Sirvent Sofia , Henderson Stephen , Trzebanski Sébastien , Schroth Johannes , Law Morven L. , Boal Carvalho Inês , Yuan-Hao Kan Howard , Lovlekar Shreya , Major Christina , Vallejo Andres , Hall Nigel J. , Ardern-Jones Michael , Henson Sian M. , Emmerson Elaine , Jung Steffen , Polak Marta E. , Bennett Clare L.
Published: November 2023

Langerhans cells (LCs) maintain tissue and immunological homeostasis at the epidermal barrier site. They are unique among phagocytes in functioning both as embryo-derived, tissue-resident macrophages that influence skin innervation and repair, and as migrating professional antigen presenting cells, a capability classically assigned to dendritic cells (DCs). Here we report the mechanisms that determine this dual identity. Using ablation of embryo-derived LCs in murine adult skin and tracked differentiation of incoming monocyte-derived replacements, we reveal intrinsic intra-epidermal heterogeneity. We demonstrate that monocyte-dendritic cell progenitor (MDP)-derived monocytes are selected for survival in the skin environment. Within the epidermis, the hair follicle niche subsequently provides an initial site of LC commitment, likely via Notch signaling, prior to metabolic adaptation and survival of differentiated monocyte-derived LCs. In human skin, we show that embryo-derived (e)LCs in newborns retain transcriptional evidence of their macrophage origin, but this is superseded by distinct DC-like immune modules after post-natal expansion of eLCs. Thus, intrinsic and extrinsic adaptations to adult skin niches replicate conditioning of eLC at birth, permitting repair of the unique LC network.
Manual gestures facilitate learning lexical stress by modulating auditory neural responses
Authors: Zhan Tianqi , Yang Danfeng , Wu Ruoyu , Tian Xing
Published: November 2023

Gestures accompany speech and facilitate communication and learning. Most previous studies have demonstrated the effects of gestures on learning semantics, yet how gestures facilitate learning low- and intermediate-level speech features is unclear. The present study investigated the effects of manual gestures on learning lexical stress, a phonological-lexical feature that is foreign to native Mandarin speakers. Across a series of experiments, we demonstrated that the gestures with representational relations to auditory stimuli in terms of covaried amplitude modulation facilitated the learning of lexical stress for both familiar (English) and unknown (Russian) languages, but not for pseudowords that lack phonotactic properties. Interestingly, gestures with amplitude trajectory matching the stress benefited the learning of trained words, whereas gestures that only matched the timing of syllable segments but not amplitude variation generalized the learning effects to untrained stimuli. Furthermore, in the EEG experiment, we found that gesture-accompanied learning was associated with power increase and inter-trial phase coherence (ITC) decrease in the theta band at the time windows corresponding to the stress positions. These results suggest that the facilitatory effects of gestures on lexical stress learning depend on the specificity of cross-modal feature mapping at the phonological level, mediated by the neural modulation in early perceptual responses.
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

Nonsense-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

Efforts 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

Summary

Assessing 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

Lysosome-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 Statement

Better 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

Histoplasma 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.

Importance

Phagocytes 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.
Evolutionarily new genes in humans with disease phenotypes reveal functional enrichment patterns shaped by adaptive innovation and sexual selection.
Authors: Chen Jianhai , Landback Patrick , Arsala Deanna , Guzzetta Alexander , Xia Shengqian , Atlas Jared , Sosa Dylan , Zhang Yong , Cheng Jingqiu , Shen Bairong , Long Manyuan
Published: November 2023

New genes (or young genes) are genetic novelties pivotal in mammalian evolution. However, their phenotypic impacts and evolutionary patterns over time remain elusive in humans due to the technical and ethical complexities of functional studies. Integrating gene age dating with Mendelian disease phenotyping, our research shows a gradual rise in disease gene proportion as gene age increases. Logistic regression modeling indicates that this increase in older genes may be related to their longer sequence lengths and higher burdens of deleterious de novo germline variants (DNVs). We also find a steady integration of new genes with biomedical phenotypes into the human genome over macroevolutionary timescales (~0.07% per million years). Despite this stable pace, we observe distinct patterns in phenotypic enrichment, pleiotropy, and selective pressures across gene ages. Notably, young genes show significant enrichment in diseases related to the male reproductive system, indicating strong sexual selection. Young genes also exhibit disease-related functions in tissues and systems potentially linked to human phenotypic innovations, such as increased brain size, musculoskeletal phenotypes, and color vision. We further reveal a logistic growth pattern of pleiotropy over evolutionary time, indicating a diminishing marginal growth of new functions for older genes due to intensifying selective constraints over time. We propose a pleiotropy-barrier model that delineates higher potentials for phenotypic innovation in young genes compared to older genes, a process that is subject to natural selection. Our study demonstrates that evolutionarily new genes are critical in influencing human reproductive evolution and adaptive phenotypic innovations driven by sexual and natural selection, with low pleiotropy as a selective advantage.
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

The 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 summary

The 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

Diverse 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

Integrin 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

Developing 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

What 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

The 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

The 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.
Boronic acid derivative inhibits LexA mediated SOS response in Mycobacteria
Authors: Chatterjee Chitral , Mohan Gokul Raj , Hariharan V.C , Biswas Bhumika , Sundaram Vidya , Srivastava Ashutosh , Matheshwaran Saravanan
Published: November 2023

Antimicrobial resistance (AMR) properties of several pathogens pose as one of the major global concerns demanding continuous innovation in order to be controlled. The bacterial “SOS” response, regulated by LexA and RecA, contributes to AMR through advantageous mutations. Therefore, targeting LexA/RecA system with a novel inhibitor could suppress “SOS” response and may reduce AMR. However, RecA presents a challenge as a therapeutic target due to its conservation across species, including humans. Concurrently, LexA, being absent in eukaryotes, can be potentially targeted, in part, due to its association with “SOS” response and developing AMR. Our studies combining bioinformatic, biochemical, biophysical, and cell-based assays present a unique inhibitor of mycobacterial “SOS” response wherein we show that the inhibitor interacts directly with the catalytic site residues of LexA ofMycobacterium tuberculosis (Mtb), consequently hindering its cleavage, resulting in suppression of “SOS” response. We observed important genes under the “SOS” regulon to be down-regulated in the presence of the inhibitor along with a consequent reduction in the rate of mutation frequency of mycobacterial cells. In essence, this study may facilitate further research on potential LexA inhibitors, reducing mutational rates and offering promise in mitigating AMR.
GRA47 and GRA72 areToxoplasma gondiipore-forming proteins that influence small molecule permeability of the parasitophorous vacuole
Authors: Bitew Mebratu A. , Gaete Pablo S. , Swale Christopher , Maru Parag , Contreras Jorge E. , Saeij Jeroen P. J.
Published: November 2023

Toxoplasma gondii, a medically important intracellular parasite, uses GRA proteins, secreted from dense granule organelles, to mediate nutrient flux across the parasitophorous vacuole membrane (PVM). GRA17 and GRA23 are known pore-forming proteins on the PVM involved in this process, but the roles of additional proteins have remained largely uncharacterized. We recently identifiedGRA72as synthetically lethal withGRA17. DeletingGRA72produced similar phenotypes toΔgra17parasites, and computational predictions suggested it forms a pore. To understand how GRA72 functions we performed immunoprecipitation experiments and identified GRA47 as an interactor of GRA72. Deletion ofGRA47resulted in an aberrant ‘bubble vacuole’ morphology with reduced small molecule permeability, mirroring the phenotype observed inGRA17andGRA72knockouts. Structural predictions indicated that GRA47 and GRA72 form heptameric and hexameric pores, respectively, with conserved histidine residues lining the pore. Mutational analysis highlighted the critical role of these histidines for protein functionality. Validation through electrophysiology confirmed alterations in membrane conductance, corroborating their pore-forming capabilities. Furthermore, Δgra47parasites and parasites expressing GRA47 with a histidine mutation had reducedin vitroproliferation and attenuated virulence in mice. Our findings show the important roles of GRA47 and GRA72 in regulating PVM permeability, thereby expanding the repertoire of potential therapeutic targets againstToxoplasmainfections.

IMPORTANCE

Toxoplasma gondiiis a parasite that poses significant health risks to those with impaired immunity. It replicates inside host cells shielded by the parasitophorous vacuole membrane (PVM), which controls nutrient and waste exchange with the host. GRA72, previously identified as essential in the absence of the GRA17 nutrient channel, is implicated in forming an alternative nutrient channel. Here we found that GRA47 associates with GRA72 and is also important for the PVM’s permeability to small molecules. Removal of GRA47 leads to distorted vacuoles and impairs small molecule transport across the PVM, resembling the effects of GRA17 and GRA72 deletions. Structural models suggest GRA47 and GRA72 form distinct pore structures, with a pore-lining histidine critical to their function.Toxoplasmastrains lacking GRA47, or those with a histidine mutation, have impaired growth and reduced virulence in mice, highlighting these proteins as potential targets for new treatments against Toxoplasmosis.
SUMO chains depolymerization induces slender to stumpy differentiation inT. bruceibloodstream parasites
Authors: Iribarren Paula Ana , Di Marzio Lucía Ayelén , Berazategui María Agustina , Saura Andreu , Coria Lorena , Cassataro Juliana , Rojas Federico , Navarro Miguel , Alvarez Vanina Eder
Published: November 2023

Trypanosoma bruceiare extracellular protozoan parasites transmitted by tsetse flies that cause sleeping sickness in humans and nagana in cattle. Inside the mammalian host, differentiation from a bloodstream replicative slender form into a quiescent stumpy form allows the persistence of the parasite and the spread of the infection. SUMOylation is a reversible and dynamic post-translational modification of proteins that regulates diverse nuclear processes, such as DNA replication, repair and transcription. SUMO can be attached to its target proteins either as a single monomer or forming polymeric chains. We found that transgenic cell lines able to conjugate SUMO just as a monomer are attenuatedin vivo. SUMO chain mutant monomorphic parasites display relapsing and remitting waves of parasitemia, at variance with wild-type parasites that cause unremitting parasitemia and mice death. Furthermore, when mice are infected with an analogous SUMO chain mutant generated in a differentiation-competent pleomorphic background, stumpy cells can be observed at unusually low parasitemia values. Our study reveals that SUMO depolymerization could represent a coordinated signal triggered during a quiescence activation program.
USP7 Inhibition Promotes Early Osseointegration in Senile Osteoporotic Mice
Authors: Zhou F , Wang Z , Li H , Wang D , Wu Z , Bai F , Wang Q , Luo W , Zhang G , Xiong Y , Wu Y
Published: November 2023

Although elderly osteoporotic patients have similar implant survival rates compared to those of normal individuals, they require longer healing periods to achieve proper osseointegration. This may be related to chronic inflammatory responses and impaired stem cell repair functions in the osteoporotic bone microenvironment. Recently, the deubiquitinating enzyme, ubiquitin-specific peptidase 7 (USP7), was found to regulate macrophage immune response and modulate stem cell osteogenic differentiation. The selective inhibitor of USP7, P5091, has also been found to promote bone repair and homeostasis in osteoporotic conditions. However, the roles of USP7 and P5091 in osteoimmunology and dental implant osseointegration under senile osteoporotic conditions remain unclear. In this study, USP7 depletion and P5091 were showed to inhibit inflammation in senescent bone marrow derived macrophages (BMDMs) and promote osteogenic differentiation in aged BMSCs. Furthermore, mRNA-Seq revealed that USP7 depletion could enhance efferocytosis in senescent BMDMs through the EPSIN1/ low-density lipoprotein receptor-related protein 1 (LRP1) pathway and selectively induce apoptosis (senolysis) in aged BMSCs. In senile osteoporotic mice, we found that the osseointegration period was prolonged compared to young mice, and P5091 promoted the early stage of osseointegration, which may be related to macrophage efferocytosis around the implant. Collectively, this study suggests that USP7 inhibition may accelerate the osseointegration process in senile osteoporotic conditions by promoting macrophage efferocytosis and aged BMSCs apoptosis. This has implications for understanding the cellular interactions and signaling mechanisms in the peri-implant bone microenvironment under osteoporotic conditions. It may also provide clinical significance in developing new therapies to enhance osseointegration quality and shorten the edentulous period in elderly osteoporotic patients.
Mitochondria possess a large, non-selective ionic current that is enhanced during cardiac injury
Authors: Balderas Enrique , Lee Sandra H.J. , Shankar Thirupura S. , Yin Xue , Balynas Anthony M. , Kyriakopoulos Christos P. , Selzman Craig H. , Drakos Stavros G. , Chaudhuri Dipayan
Published: November 2023

Mitochondrial ion channels are essential for energy production and cell survival. To avoid depleting the electrochemical gradient used for ATP synthesis, channels so far described in the mitochondrial inner membrane open only briefly, are highly ion-selective, have restricted tissue distributions, or have small currents. Here, we identify a mitochondrial inner membrane conductance that has strikingly different behavior from previously described channels. It is expressed ubiquitously, and transports cations non-selectively, producing a large, up to nanoampere-level, current. The channel does not lead to inner membrane uncoupling during normal physiology because it only becomes active at depolarized voltages. It is inhibited by external Ca2+, corresponding to the intermembrane space, as well as amiloride. This large, ubiquitous, non-selective, amiloride-sensitive (LUNA) current appears most active when expression of the mitochondrial calcium uniporter is minimal, such as in the heart. In this organ, we find that LUNA current magnitude increases two- to threefold in multiple mouse models of injury, an effect also seen in cardiac mitochondria from human patients with heart failure with reduced ejection fraction. Taken together, these features lead us to speculate that LUNA current may arise from an essential protein that acts as a transporter under physiological conditions, but becomes a channel under conditions of mitochondrial stress and depolarization.
Transcriptional stress induces the generation of DoGs in cancer cells
Authors: Ríos Francisco , Urióstegui-Arcos Maritere , Zurita Mario
Published: November 2023

A characteristic of the cellular response to stress, is the production of RNAs generated from readthrough transcription of genes, called downstream-of-gene-(DoG) containing transcripts. Additionally, transcription inhibitor drugs are candidates for fighting cancer. In this work, we report the results of a bioinformatic analysis showing that one of the responses to transcription inhibition is the generation of DoGs in cancer cells. Although some genes that form DoGs were shared between the two cancer lines, there did not appear to be a functional correlation between them. However, our findings show that DoGs are generated as part of the cellular response to transcription inhibition like other types of cellular stress, suggesting that it may be part of the defense against transcriptional stress.
Engineering self-deliverable ribonucleoproteins for genome editing in the brain
Authors: Chen Kai , Stahl Elizabeth C. , Kang Min Hyung , Xu Bryant , Allen Ryan , Trinidad Marena , Doudna Jennifer A.
Published: November 2023

The delivery of CRISPR ribonucleoproteins (RNPs) for genome editingin vitroandin vivohas important advantages over other delivery methods, including reduced off-target and immunogenic effects1. However, effective delivery of RNPs remains challenging in certain cell types due to low efficiency and cell toxicity. To address these issues, we engineered self-deliverable RNPs that can promote efficient cellular uptake and carry out robust genome editing without the need for helper materials or biomolecules. Screening of cell-penetrating peptides (CPPs) fused to CRISPR-Cas9 protein identified potent constructs capable of efficient genome editing of neural progenitor cells. Further engineering of these fusion proteins identified a C-terminal Cas9 fusion with three copies of A22p, a peptide derived from human semaphorin-3a, that exhibited substantially improved editing efficacy compared to other constructs. We found that self-deliverable Cas9 RNPs generated robust genome edits in clinically relevant genes when injected directly into the mouse striatum. Overall, self-deliverable Cas9 proteins provide a facile and effective platform for genome editingin vitroandin vivo.