bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenotypically Complex Living Materials Containing Engineered Cyanobacteria
Authors: Datta Debika , Weiss Elliot L. , Wangpraseurt Daniel , Hild Erica , Chen Shaochen , Golden James W. , Golden Susan S. , Pokorski Jonathan K.
Published: January 2023

A cyanobacterial photosynthetic biocomposite material was fabricated using 3D-printing and bioengineered to produce multiple functional outputs in response to an external chemical stimulus. Our investigations show the advantages of utilizing additive manufacturing techniques in controlling the design and shape of the fabricated materials, which proved to be important for the support and growth of obligate phototrophic microorganisms within the material. As an initial proof-of-concept, a synthetic theophylline-responsive riboswitch inSynechococcus elongatusPCC 7942 was used for regulating the expression of a yellow fluorescent protein (YFP) reporter. Upon induction with theophylline, the encapsulated cells produced YFP within the hydrogel matrix. Subsequently, a strain ofS. elongatuswas engineered to produce an oxidative enzyme that is useful for bioremediation, laccase, expressed either constitutively or under the control of the riboswitch. The responsive biomaterial can decolorize a common textile dye pollutant, indigo carmine, potentially serving as a useful tool in environmental bioremediation. Finally, cells were engineered to have the capacity for inducible cell death to eliminate their presence once their activity is no longer required, which is an important function for biocontainment and minimizing unintended environmental impact. By integrating genetically engineered stimuli-responsive cyanobacteria in patterned volumetric 3D-printed designs, we demonstrate the potential of programmable photosynthetic biocomposite materials capable of producing functional outputs including, but not limited to, bioremediation.
Protein Representation Learning via Knowledge Enhanced Primary Structure Modeling
Authors: Zhou Hong-Yu , Fu Yunxiang , Zhang Zhicheng , Bian Cheng , Yu Yizhou
Published: January 2023

Abstract

Protein representation learning has primarily benefited from the remarkable development of language models (LMs). Accordingly, pre-trained protein models also suffer from a problem in LMs: a lack of factual knowledge. The recent solution models the relationships between protein and associated knowledge terms as the knowledge encoding objective. However, it fails to explore the relationships at a more granular level, i.e., the token level. To mitigate this, we propose Knowledge-exploited Auto-encoder for Protein (KeAP), which performs tokenlevel knowledge graph exploration for protein representation learning. In practice, non-masked amino acids iteratively query the associated knowledge tokens to extract and integrate helpful information for restoring masked amino acids via attention. We show that KeAP can consistently outperform the previous counterpart on 9 representative downstream applications, sometimes surpassing it by large margins. These results suggest that KeAP provides an alternative yet effective way to perform knowledge enhanced protein representation learning. Code and models are available athttps://github.com/RL4M/KeAP.
In vitroLung Epithelial Cell Model Reveals Novel Roles forPseudomonas aeruginosaSiderophores
Authors: Kang Donghoon , Xu Qi , Kirienko Natalia V.
Published: January 2023

Multidrug-resistantPseudomonas aeruginosais a common nosocomial respiratory pathogen that continues to threaten the lives of patients with mechanical ventilation in intensive care units and those with underlying comorbidities such as cystic fibrosis or chronic obstructive pulmonary disease. For over 20 years, studies have repeatedly demonstrated that the major siderophore pyoverdine is an important virulence factor forP. aeruginosain invertebrate and mammalian hostsin vivo. Despite its physiological significance, anin vitro,mammalian cell culture model to characterize the impact and molecular mechanism of pyoverdine-mediated virulence has only been developed very recently. In this study, we adapt a previously-established, murine macrophage-based model for human bronchial epithelial cells (16HBE). We demonstrate that conditioned medium fromP. aeruginosainduced rapid 16HBE cell death through the pyoverdine-dependent secretion of cytotoxic rhamnolipids. Genetic or chemical disruption of pyoverdine biosynthesis decreased rhamnolipid production and mitigated cell death. Consistent with these observations, chemical depletion of lipid factors or genetic disruption of rhamnolipid biosynthesis was sufficient to abrogate conditioned medium toxicity. Furthermore, we also examine the effects of purified pyoverdine exposure on 16HBE cells. While pyoverdine accumulated within cells, the siderophore was largely sequestered within early endosomes, showing minimal cytotoxicity. More membrane-permeable iron chelators, such as the siderophore pyochelin, decreased epithelial cell viability and upregulated several proinflammatory genes. However, pyoverdine potentiated these iron chelators in activating proinflammatory pathways. Altogether, these findings suggest that the siderophores pyoverdine and pyochelin play distinct roles in virulence during acuteP. aeruginosalung infection.
Extracting nanoscale membrane morphology from single-molecule localizations
Authors: Marin Zach , Fuentes Lukas A. , Bewersdorf Joerg , Baddeley David
Published: January 2023

Membrane surface reconstruction at the nanometer scale is required for understanding mechanisms of subcellular shape change. This historically has been the domain of electron microscopy, but extraction of surfaces from specific labels is a difficult task in this imaging modality. Existing methods for extracting surfaces from fluorescence microscopy have poor resolution or require high-quality super-resolution data that is manually cleaned and curated. Here we present NanoWrap, a new method for extracting surfaces from generalized single-molecule localization microscopy (SMLM) data. This makes it possible to study the shape of specifically-labelled membraneous structures inside of cells. We validate NanoWrap using simulations and demonstrate its reconstruction capabilities on SMLM data of the endoplasmic reticulum and mitochondria. NanoWrap is implemented in the open-source Python Microscopy Environment.

SIGNIFICANCE

We introduce a novel tool for reconstruction of subcellular membrane surfaces from single-molecule localization microscopy data and use it to visualize and quantify local shape and membrane-membrane interactions. We benchmark its performance on simulated data and demonstrate its fidelity to experimental data.
The Australasian dingo archetype:De novochromosome-length genome assembly, DNA methylome, and cranial morphology
Authors: Ballard J. William O. , Field Matt A. , Edwards Richard J. , Wilson Laura A.B. , Koungoulos Loukas G. , Rosen Benjamin D. , Chernoff Barry , Dudchenko Olga , Omer Arina , Keilwagen Jens , Skvortsova Ksenia , Bogdanovic Ozren , Chan Eva , Zammit Robert , Hayes Vanessa , Aiden Erez Lieberman
Published: January 2023

Background

One difficulty in testing the hypothesis that the Australasian dingo is a functional intermediate between wild wolves and domesticated breed dogs is that there is no reference specimen. Here we link a high-qualityde novolong read chromosomal assembly with epigenetic footprints and morphology to describe the Alpine dingo female named Cooinda. It was critical to establish an Alpine dingo reference because this ecotype occurs throughout coastal eastern Australia where the first drawings and descriptions were completed.

Findings

We generated a high-quality chromosome-level reference genome assembly (Canfam_ADS) using a combination of Pacific Bioscience, Oxford Nanopore, 10X Genomics, Bionano, and Hi-C technologies. Compared to the previously published Desert dingo assembly, there are large structural rearrangements on Chromosomes 11, 16, 25 and 26. Phylogenetic analyses of chromosomal data from Cooinda the Alpine dingo and nine previously publishedde novocanine assemblies show dingoes are monophyletic and basal to domestic dogs. Network analyses show that the mtDNA genome clusters within the southeastern lineage, as expected for an Alpine dingo. Comparison of regulatory regions identified two differentially methylated regions within glucagon receptor GCGR and histone deacetylase HDAC4 genes that are unmethylated in the Alpine dingo genome but hypermethylated in the Desert dingo. Morphological data, comprising geometric morphometric assessment of cranial morphology place dingo Cooinda within population-level variation for Alpine dingoes. Magnetic resonance imaging of brain tissue show she had a larger cranial capacity than a similar-sized domestic dog.

Conclusions

These combined data support the hypothesis that the dingo Cooinda fits the spectrum of genetic and morphological characteristics typical of the Alpine ecotype. We propose that she be considered the archetype specimen for future research investigating the evolutionary history, morphology, physiology, and ecology of dingoes. The female has been taxidermically prepared and is now at the Australian Museum, Sydney.
Bacterial mechanosensing of surface stiffness promotes signaling and growth leading to biofilm formation byPseudomonas aeruginosa
Authors: Wang Liyun , Wong Yu-Chern , Correira Joshua M. , Wancura Megan , Geiger Chris J , Webster Shanice S , Butler Benjamin J. , O’Toole George A. , Langford Richard M. , Brown Katherine A. , Dortdivanlioglu Berkin , Webb Lauren , Cosgriff-Hernandez Elizabeth , Gordon Vernita D.
Published: January 2023

The attachment of bacteria onto a surface, consequent signaling, and the accumulation and growth of the surface-bound bacterial population are key initial steps in the formation of pathogenic biofilms. While recent reports have hinted that the stiffness of a surface may affect the accumulation of bacteria on that surface, the processes that underlie bacterial perception of and response to surface stiffness are unknown. Furthermore, whether, and how, the surface stiffness impacts biofilm development, after initial accumulation, is not known. We use thin and thick hydrogels to create stiff and soft composite materials, respectively, with the same surface chemistry. Using quantitative microscopy, we find that the accumulation, motility, and growth of the opportunistic human pathogenPseudomonas aeruginosarespond to surface stiffness, and that these are linked through cyclic-di-GMP signaling that depends on surface stiffness. The mechanical cue stemming from surface stiffness is elucidated using finite-element modeling combined with experiments - adhesion to stiffer surfaces results in greater changes in mechanical stress and strain in the bacterial envelope than does adhesion to softer surfaces with identical surface chemistry. The cell-surface-exposed protein PilY1 acts as a mechanosensor, that upon surface engagement, results in higher cyclic-di-GMP levels, lower motility, and greater accumulation on stiffer surfaces. PilY1 impacts the biofilm lag phase, which is extended for bacteria attaching to stiffer surfaces. This study shows clear evidence that bacteria actively respond to different stiffness of surfaces where they adhereviaperceiving varied mechanical stress and strain upon surface engagement.

Importance

Bacteria colonize many types of biological and medical surfaces with a large range of stiffnesses. Colonization leads to the formation of biofilms, which cause costly and life-impairing chronic infections. However, whether and how bacteria can sense and respond to the mechanical cue provided by surface stiffness has remained unknown. We find that bacteria do indeed respond to surface stiffness in a way that is both consistent with expectations based on equilibrium continuum mechanics and that quantitatively impacts multiple aspects of early biofilm formation. This is a new understanding for the nascent field of bacterial mechanobiology. Furthermore, this finding suggests the possibility of a new category of approaches to hindering biofilm development by tuning the mechanical properties of biomedical surfaces.
Streptococcus pneumoniaedrives specific and lasting Natural Killer cell memory
Authors: Camarasa Tiphaine M.N. , Torné Júlia , Chevalier Christine , Rasid Orhan , Hamon Melanie Anne
Published: January 2023

NK cells are important mediators of innate immunity and play an essential role for host protection against infection, although their responses to bacteria are poorly understood. Recently NK cells were shown to display memory properties, as characterized by an epigenetic signature leading to a stronger secondary response. Although NK cell memory could be a promising mechanism to fight against infection, it has not been described upon bacterial infection. Here, we reveal that NK cells develop specific and long-term memory following sub-lethal infection with the extracellular pathogenStreptococcus pneumoniae. Memory NK cells display intrinsic sensing and response to bacteriain vitro, in a manner that is enhanced post-bacterial infection. In addition, their transfer into naïve mice confer protection from lethal infection for at least 12 weeks. Interestingly, NK cells display enhanced cytotoxic molecule production upon secondary stimulation and their protective role is dependent on Perforin and independent of IFNγ. Thus, our study identifies a new role for NK cells during bacterial infection, opening the possibility to harness innate immune memory for therapeutic purposes.
Differences in Neuropsychiatric Features in Black and White Individuals Diagnosed with Frontotemporal Degeneration
Authors: Jin Hannah , McMillan Corey T. , Yannatos Isabel , Fisher Lauren , Rhodes Emma , Jacoby Sarah F. , Irwin David J. , Massimo Lauren
Published: January 2023

Background

Neuropsychiatric symptoms are highly prevalent in frontotemporal degeneration (FTD). Prior research suggests there are disparities in the clinical presentation of dementia when comparing Black and White individuals but this has not been investigated in the context of FTD specifically. The aim of the present study is to investigate racial disparities in dementia severity, functional impairment and neuropsychiatric symptoms in individuals with a clinical diagnosis of FTD.

Methods

Using National Alzheimer’s Coordinating Center (NACC) data, we evaluated 63 Black and 2,356 White individuals living with a clinical diagnosis of FTD (behavioral variant FTD or primary progressive aphasia) and a healthy control group of 1899 Black and 9122 White individuals with normal behavior and cognition. We compared demographic characteristics, dementia severity, functional impairment and neuropsychiatric symptoms at initial NACC visit by examining differences on Clinical Dementia Rating Scale (CDR), Functional Assessment Scale (FAS) and Neuropsychiatric Inventory (NPI) using multivariable linear and logistic regression models, covarying for age at visit, disease duration, sex, and education. Models evaluating differences in neuropsychiatric symptoms additionally controlled for dementia severity.

Results

Black individuals with FTD were considerably underrepresented, comprising only 2.5% of the clincial sample. In comparison to White individuals, Black individuals with FTD had a higher degree of dementia severity on CDR (CDR sum of boxes; p=0.03 CDR global, p=.006), greater functional impairment (FAS total; p=0.01) and more delusions (p=0.01), agitation (p=0.04) and depression (p=0.03) on the NPI. White individuals with FTD were more likely to demonstrate apathy (p=0.03). Within the normal control group, Black individuals reported less depression, anxiety, agitation, and nighttime behaviors (depression; p < 0.001; anxiety; p = 0.006; agitation; p = 0.048; nighttime behaviors; p < 0.001) compared to White normal controls.

Discussion

The present study suggests there are significant disparities in dementia severity, functional impairment and neuropsychiatric presentations at initial visit between Black and White individuals with FTD. Future work must address racial disparities in FTD and their underlying social determinants as well as the lack of representation of non-White individuals in nationally representative neurodegenerative disease registries in order identify appropriate interventions.
Barriers and opportunities relating to the implementation and evaluation of a complex digital intervention in England: A case study of Greater Manchester Diabetes My Way
Authors: Goldthorpe Joanna , Allen Tommy , Rutter Martin K , Brooks Joanna
Published: January 2023

Introduction

Digital diabetes management systems have the potential to deliver cost-effective, flexible self-management support to patients with type 2 diabetes. DiabetesMyWay (DMW) is a platform for an open access website that aims to support diabetes self-management and provide patients with access to their care records. We present a case study focusing on a project involving patients across the large urban area of Greater Manchester (GM).

Methods

Interviews were undertaken with 8 members of the project team and 3 patients using the platform. Data were analysed thematically using the template analysis approach.

Results

Three themes are presented: Complex approvals, permissions and access across multiple organisations and systems; Capacity challenges in primary care settings; Ongoing support for the project. Implementation and evaluation of GMDMW involved navigating data sharing systems and regulations for complex private and public sector organisations and these impacted on the electronic data sharing necessary for the DMW platform to work as intended. Participants felt that the complexities associated with merging different systems, operated by different organisations, with different data controllers and data owners were greater than anticipated.

Discussion

The complexity, number of organisations and levels of governance involved in implementing and evaluating GMDMW were barriers to implementation and evaluation. This reflects inherent problems around mobilising innovation in the NHS. Our findings can help the development and evaluation electronic interventions to improve health to navigate this complex research landscape and ensure that patients can access the most innovative and effective ways to support their health. (245/250 words)

Author summary

Digital health interventions and Public Private Partnerships for innovation in the NHS are on the rise and in order to establish efficacy, these need to be evaluated. Often, this research and evaluation is carried out by Universities. However, challenges around navigating governance across these complex, multiple organisations exist where there is a need to share data and collaborate across systems effectively. Here, we describe the many unexpected challenges and solutions to effective data sharing reported by professionals and patients involved in the delivery and evaluation of a UK-based NHS/ private sector collaboration to provide a digital platform to support self-management of diabetes.
The effects of physiotherapy programs in COVID-19 patients during admission in the hospital
Authors: Jianramas Netchanok , Nissapatorn Veeranoot , Sivakorn Chaisith , de Lourdes Pereira Maria , Ratnarathon Anuttra (Chaovavanich) , Salesingh Chenpak , Jaiyen Eittipad , Chaiyakul Salinee , Piya-amornphan Nitita , Semphuet Thanaporn , Thiangtham Thanrada , Boontam Kornchanok , Longlalerng Khomkrip
Published: January 2023

Background and aims

Several recommendations exist regarding the role of physiotherapy programs (PTPs) in COVID-19 patients. However, none of the studies examines the frequency of bedside PTPs during admission. Thus, this study aimed to compare the different bedside PTPs frequencies on the survival rate, length of hospitalization (LoH), referrals to the intensive care unit (ICU), and in-hospital complications. The safety of patients and the physiotherapist was also investigated.

Methods

Fifty-two COVID-19 patients were equally assigned into two groups matched on gender and age (1:1 ratio). Experimental group one received 1-2 times of PTPs during hospitalization, and experimental group two received daily PTPs until hospital discharge. The primary outcomes were the survival rate, LoH, referrals to ICU, and in-hospital complications. The secondary outcomes were the adverse events for patients and the number of physiotherapists who contracted with COVID-19.

Results

Most participants were classified as having mild to moderate COVID-19 with a mean age of 45 years. There were no differences between groups in all primary outcome measures (all p > 0.05). The overall survival rate was 98%. One participant from the Ex-G2 group was referred to the ICU. Two Ex-G1 and four Ex-G2 participants had complications. There were no immediate serious adverse events found after PTPs for both groups. None of the physiotherapists tested positive for COVID-19.

Conclusion

In COVID-19 patients with mild to moderate conditions, one to two bedside PTPs were enough to achieve the same results as patients who received daily PTPs. PTPs were safe for COVID-19 patients, and physiotherapists.

CLINICAL REGISTRATION NUMBER

Thai Clinical Trials,https://www.thaiclinicaltrials.org/, TCTR20210823004.
Steamed broccoli sprouts alleviate DSS-induced inflammation and retain gut microbial biogeography in mice
Authors: Holman Johanna M. , Colucci Louisa , Baudewyns Dorien , Balkan Joe , Hunt Timothy , Hunt Benjamin , Kinney Marissa , Holcomb Lola , Chen Grace , Moses Peter L. , Mawe Gary M. , Zhang Tao , Li Yanyan , Ishaq Suzanne L.
Published: January 2023

Inflammatory Bowel Diseases (IBD) are devastating conditions of the gastrointestinal tract with limited treatments, and dietary intervention may be effective, and affordable, for managing symptoms. Glucosinolate compounds are highly concentrated in broccoli sprouts, especially glucoraphanin, and can be metabolized by certain mammalian gut bacteria into anti inflammatory isothiocyanates, such as sulforaphane. Gut microbiota exhibit biogeographic patterns, but it is unknown if colitis alters these or whether the location of glucoraphanin metabolizing bacteria affects anti-inflammatory benefits. We fed specific pathogen free C57BL/6 mice either a control diet or a 10% steamed broccoli sprout diet, and gave a three-cycle regimen of 2.5% dextran sodium sulfate (DSS) in drinking water over a 34-day experiment to simulate chronic, relapsing ulcerative colitis. We monitored body weight, fecal characteristics, lipocalin, serum cytokines, and bacterial communities from the luminal and mucosa-associated populations in the jejunum, cecum, and colon. Mice fed the broccoli sprout diet with DSS treatment performed better than mice fed the control diet with DSS, including significantly more weight gain, lower Disease Activity Indexes, lower plasma lipocalin and proinflammatory cytokines, and higher bacterial richness in all gut locations. Bacterial communities were assorted by gut location, but were more homogenous across locations in the control diet + DSS mice. Importantly, our results showed that broccoli sprout feeding abrogated the effects of DSS on gut microbiota, as bacterial richness and biogeography were similar between mice receiving broccoli sprouts with and without DSS. Collectively, these results support the protective effect of steamed broccoli sprouts against dysbiosis and colitis induced by DSS.

Importance

Evaluating bacterial communities across different locations in the gut provides a greater insight than fecal samples alone, and provides an additional metric by which to evaluate beneficial host-microbe interactions. Here, we show that 10% steamed broccoli sprouts in the diet protects mice from the negative effects of dextran sodium sulfate induced colitis, that colitis erases biogeographical patterns of bacterial communities in the gut, and that the cecum is not likely to be a significant contributor to colonic bacteria of interest in the DSS mouse model of ulcerative colitis. Mice fed the broccoli sprout diet during colitis performed better than mice fed the control diet while receiving DSS. The identification of accessible dietary components and concentrations that help maintain and correct the gut microbiome may provide universal and equitable approaches to IBD prevention and recovery, and broccoli sprouts represent a promising strategy.
Dynamic fate map of hindbrain rhombomeres in zebrafish
Authors: Kamaraj Mageshi , Savy Thierry , Desnoulez Sophie Salomé , Peyrieras Nadine , Frain Monique
Published: January 2023

Understanding of the morphogenetic processes that underlie the patterning of the vertebrate hindbrain requires establishment of rhombomere (r) cell lineages. Using long termin vivotwo-photon imaging of zebrafish transgenic lines and automated image processing tools, we provide a method to reconstruct the r2-r6 lineage trees from the onset of gastrulation through early neurulation. We provide a dynamic fate map of hindbrain patterning at single-cell resolution. We show that rhombomere progenitor domains are aligned along the anteroposterior (AP) and dorsoventral (DV) axes as early as the shield stage. Rhombomere progenitor domains show a segmental organization, parallel to the blastoderm margin that predicts the future AP order of hindbrain. The DV organization of rhombomeres is set by the segregation of the progenitors along the DV axis of the embryo. Progenitors located at the dorsal/medial part of the blastoderm form the ventral domain of the rhombomeres while the lateral progenitors constitute the dorsal part. Our study sheds light on the clonal origin of individual rhombomeres, spatial and temporal patterns of cell division and migration of rhombomere progenitors throughout the early steps of hindbrain morphogenesis.
Protein network analysis links the NSL complex to Parkinson’s disease and mitochondrial biology
Authors: Kelly Katie , Lewis Patrick A. , Plun-Favreau Helene , Manzoni Claudia
Published: January 2023

Whilst the majority of PD cases are sporadic, much of our understanding of the pathophysiological basis of disease can be traced back to the study of rare, monogenic forms of disease. In the past decade, the availability of Genome-Wide Association Studies (GWAS) has facilitated a shift in focus, toward identifying common risk variants conferring increased risk of developing PD across the population. A recent mitophagy screening assay of GWAS candidates has functionally implicated the non-specific lethal (NSL) complex in the regulation of PINK1-mitophagy. Here, a bioinformatics approach has been taken to investigate the proteome of the NSL complex, to unpick its relevance to PD progression. The mitochondrial NSL interactome has been built, mining 3 separate repositories: PINOT, HIPPIE and MIST, for curated, literature-derived protein-protein interaction (PPI) data. We built; i) the ‘mitochondrial’ interactome, applying gene-set enrichment analysis (GSEA) to explore the relevance of the NSL mitochondrial interactome to PD and, ii) the PD-oriented interactome to uncover biological pathways underpinning the NSL /PD association. In this study, we find the mitochondrial NSL interactome to be significantly enriched for the protein products of PD associated genes, including the Mendelian PD genesLRRK2andVPS35. Additionally, the PD associated interactome is enriched for mitochondrial processes;“mitochondrial cell death”,“mitochondrial protein localisation”, “membrane protein localisation”and“mitochondrial transport”. Our data points to NSL complex members OGT and WDR5 as key drivers of this increased PD association. These findings strengthen a role for mitochondrial quality control in both familial and sporadic disease.
A defined clathrin-mediated trafficking pathway regulates sFLT1/VEGFR1 secretion from endothelial cells
Authors: Kinghorn Karina , Gill Amy , Marvin Allison , Li Renee , Quigley Kaitlyn , le Noble Ferdinand , Mac Gabhann Feilim , Bautch Victoria L
Published: January 2023

FLT1/VEGFR1 negatively regulates VEGF-A signaling and is required for proper vessel morphogenesis during vascular development and vessel homeostasis. Although a soluble isoform, sFLT1, is often mis-regulated in disease and aging, how sFLT1 is trafficked and secreted from endothelial cells is not well understood. Here we define requirements for constitutive sFLT1 trafficking and secretion in endothelial cells from the Golgi to the plasma membrane, and we show that sFLT1 secretion requires clathrin at or near the Golgi. Perturbations that affect sFLT1 trafficking blunted endothelial cell secretion and promoted intracellular mis-localization in cells and zebrafish embryos. siRNA-mediated depletion of specific trafficking components revealed requirements for RAB27A, VAMP3, and STX3 for post-Golgi vesicle trafficking and sFLT1 secretion, while STX6, ARF1, and AP1 were required at the Golgi. Depletion of STX6 altered vessel sprouting in a 3D angiogenesis model, indicating that endothelial cell sFLT1 secretion is important for proper vessel sprouting. Thus, specific trafficking components provide a secretory path from the Golgi to the plasma membrane for sFLT1 in endothelial cells that utilizes a specialized clathrin-dependent intermediate, suggesting novel therapeutic targets.
The MIR-NATMAPT-AS1does not regulate Tau expression in human neurons
Authors: Policarpo Rafaela , Wolfs Leen , Martínez-Montero Saul , Vandermeulen Lina , Royaux Ines , Van Peer Gert , Mestdagh Pieter , Sierksma Annerieke , De Strooper Bart , d’Ydewalle Constantin
Published: January 2023

TheMAPTgene encodes Tau protein, a member of the large family of microtubule-associated proteins. Tau forms large insoluble aggregates that are toxic to neurons in several neurological disorders and neurofibrillary Tau tangles represent a key pathological hallmark of Alzheimer’s disease (AD) and other ‘tauopathies’1,2. Several Tau-lowering strategies are being investigated as a potential treatment for AD but the mechanisms that regulate Tau expression at the transcriptional or translational level are not well understood3,4. Recently, Simone etal.5reported the discovery of a new type of natural antisense transcripts called ‘MIR-NATs’ (referring to Mammalian-wide Interspersed Repeats – Natural Antisense Transcripts). Simone and colleagues usedMAPT-AS1, a MIR-NAT associated to theMAPTgene as an archetype of this new class of long non-coding RNAs. According to Simone etal.,MAPT-AS1represses Tau translation by competing for ribosomal RNA. We investigated the potential functions ofMAPT-AS1in neurons using the same gain- and loss-of-function experiments as described in the original report and expanded our analysis with complementary approaches. Our data do not support a role forMAPT-AS1in regulating Tau expression in human neurons and urge to cautiously interpret the data provided by Simone etal.5.
Targeted volume Correlative Light and Electron Microscopy of an environmental marine microorganism
Authors: Mocaer Karel , Mizzon Giulia , Gunkel Manuel , Halavatyi Aliaksandr , Steyer Anna , Oorschot Viola , Schorb Martin , Kieffre Charlotte Le , Yee Daniel P. , Chevalier Fabien , Gallet Benoit , Decelle Johan , Schwab Yannick , Ronchi Paolo
Published: January 2023

Photosynthetic microalgae are responsible for an important fraction of CO2fixation and O2production on Earth. Three-dimensional ultrastructural characterization of these organisms in their natural environment can contribute to a deeper understanding of their cell biology. However, the low throughput of volume electron microscopy (vEM) methods, along with the complexity and heterogeneity of environmental samples, pose great technical challenges. In the present study, we used a workflow based on a specific EM sample preparation, compatible with both light and vEM imaging in order to target one cell among a complex natural community. This method revealed the 3D subcellular landscape of a photosynthetic dinoflagellate with quantitative characterization of multiple organelles. We could show that this cell contains a single convoluted chloroplast and the arrangement of the flagellar apparatus with its associated photosensitive elements. Moreover, we observed chromatin features that could shed light on how transcriptional activity takes place in organisms where chromosomes are permanently condensed. Together with providing insights in dinoflagellates biology, this proof-of-principle study illustrates an efficient tool for the targeted ultrastructural analysis of environmental microorganisms in heterogeneous mixes.
Efficiently Quantifying DNA Methylation for Bulk- and Single-cell Bisulfite Data
Authors: Fischer Jonas , Schulz Marcel H.
Published: January 2023

Motivation

DNA CpG methylation (CpGm) has proven to be a crucial epigenetic factor in the gene regulatory system. Assessment of DNA CpG methylation values via whole-genome bisulfite sequencing (WGBS) is, however, computationally extremely demanding.

Results

We present FAst MEthylation calling (FAME), the first approach to quantify CpGm values directly from bulk or single-cell WGBS reads without intermediate output files. FAME is very fast but as accurate as standard methods, which first produce BS alignment files before computing CpGm values. We present experiments on bulk and single-cell bisulfite datasets in which we show that data analysis can be significantly sped-up and help addressing the current WGBS analysis bottleneck for large-scale datasets without compromising accuracy.

Availability

An implementation of FAME is open source and licensed under GPL-3.0 athttps://github.com/FischerJo/FAME.
Environmental and molecular regulation of asexual reproduction in the sea anemone Nematostella vectensis
Authors: Al-Shaer Layla , Leach Whitney , Baban Noor , Yagodich Mia , Gibson Mathew C. , Layden Michael J.
Published: January 2023

Cnidarians exhibit incredible reproductive diversity, with most capable of sexual and asexual reproduction. Here we investigate factors that influence asexual reproduction in the burrowing sea anemoneNematostella vectensis, which can propagate asexually by transverse fission of the body column. By altering culture conditions, we demonstrate that the presence of a burrowing substrate strongly promotes transverse fission. In addition, we show that animal size does not affect fission rates, and that the plane of fission is fixed along the oral-aboral axis of the polyp. Homeobox transcription factors and components of the TGFβ, Notch, and FGF signaling pathways are differentially expressed in polyps undergoing physal pinching suggesting they are important regulators of transverse fission. Gene ontology analyses further suggest that during transverse fission the cell cycle is suppressed and that cell adhesion and patterning mechanisms are downregulated to promote separation of the body column. Finally, we demonstrate that the rate of asexual reproduction is sensitive to population density. Collectively, these experiments provide a foundation for mechanistic studies of asexual reproduction inNematostella, with implications for understanding the reproductive and regenerative biology of other cnidarian species.
Human aneuploid cells depend on the RAF/MEK/ERK pathway for overcoming increased DNA damage
Authors: Zerbib Johanna , Ippolito Marica Rosaria , Eliezer Yonatan , De Feudis Giuseppina , Reuveni Eli , Kadmon Anouk Savir , Martin Sara , Viganò Sonia , Leor Gil , Berstler James , Laue Kathrin , Cohen-Sharir Yael , Scorzoni Simone , Vazquez Francisca , Ben-David Uri , Santaguida Stefano
Published: January 2023

Aneuploidy is a hallmark of human cancer, yet the cellular mechanisms that allow cells to cope with aneuploidy-induced cellular stresses remain largely unknown. Such coping mechanisms may present cellular vulnerabilities that can be harnessed for targeting cancer cells. Here, we induced aneuploidy in non-transformed RPE1-hTERT cells and derived multiple stable clones with various degrees of chromosome imbalances. We performed an unbiased genomic profiling of 6 isogenic clones, using whole-exome and RNA sequencing. We then functionally interrogated their cellular dependency landscapes, using genome-wide CRISPR/Cas9 screens and large-scale drug screens. We found that aneuploid clones activated the DNA damage response (DDR), and were consequently more resistant to further DNA damage induction. Interestingly, aneuploid cells also exhibited elevated RAF/MEK/ERK pathway activity, and were more sensitive to several clinically-relevant drugs targeting this pathway, and in particular to genetic and chemical CRAF inhibition. CRAF activity was functionally linked to the resistance to DNA damage induction, as CRAF inhibition sensitized aneuploid cells to DNA damage-inducing chemotherapies. The association between aneuploidy, RAF/MEK/ERK signaling, and DDR was independent of p53. The increased activity and dependency of aneuploid cells on the RAF/MEK/ERK pathway was validated in another isogenic aneuploid system, and across hundreds of human cancer cell lines, confirming their relevance to human cancer. Overall, our study provides a comprehensive resource for genetically-matched karyotypically-stable cells of various aneuploidy states, and reveals a novel therapeutically-relevant cellular dependency of aneuploid cells.
Increased RNA and protein degradation is required for counteracting transcriptional burden and proteotoxic stress in human aneuploid cells
Authors: Ippolito Marica Rosaria , Zerbib Johanna , Eliezer Yonatan , Reuveni Eli , Viganò Sonia , De Feudis Giuseppina , Kadmon Anouk Savir , Vigorito Ilaria , Martin Sara , Laue Kathrin , Cohen-Sharir Yael , Scorzoni Simone , Vazquez Francisca , Santaguida Stefano , Ben-David Uri
Published: January 2023

Aneuploidy, an abnormal chromosome composition, results in a stoichiometric imbalance of protein complexes, which jeopardizes the fitness of aneuploid cells. Aneuploid cells thus need to compensate for the imbalanced DNA levels by regulating their RNA and protein levels, a phenomenon known as dosage compensation. However, the molecular mechanisms involved in dosage compensation in human cells – and whether they can be targeted to selectively kill aneuploid cancer cells – remain unknown. Here, we addressed this question via molecular dissection of multiple diploid vs. aneuploid cell models. Using genomic and functional profiling of a novel isogenic system of RPE1-hTERT cells with various degrees of aneuploidy, we found that aneuploid cells cope with both transcriptional burden and proteotoxic stress. At the mRNA level, aneuploid cells increased RNA synthesis, but concomitantly elevated several RNA degradation pathways, in particular the nonsense-mediated decay (NMD) and the microRNA-mediated mRNA silencing pathways. Consequently, aneuploid cells were more sensitive to the genetic or chemical perturbation of several key components of these RNA degradation pathways. At the protein level, aneuploid cells experienced proteotoxic stress, resulting in reduced translation and increased protein degradation, rendering them more sensitive to proteasome inhibition. These findings were recapitulated across hundreds of human cancer cell lines and primary tumors, confirming that both non-transformed and transformed cells alter their RNA and protein metabolism in order to adapt to the aneuploid state. Our results reveal that aneuploid cells are dependent on the over- or under-activation of several nodes along the gene expression process, identifying these pathways as clinically-actionable vulnerabilities of aneuploid cells.
EomesrestrictsBrachyuryfunctions at the onset of mammalian gastrulation
Authors: Schüle Katrin M. , Weckerle Jelena , Probst Simone , Wehmeyer Alexandra E. , Zissel Lea , Schröder Chiara M. , Tekman Mehmet , Kim Gwang-Jin , Schlägl Inga-Marie , Sagar  , Arnold Sebastian J.
Published: January 2023

Mammalian specification of mesoderm and definitive endoderm (DE) is instructed by the two related Tbx transcription factors (TFs)Eomesodermin(Eomes) andBrachyurysharing partially redundant functions. Gross differences of mutant embryonic phenotypes suggest specific functions of each TF. To date, the molecular details of separated lineage-specific gene-regulation byEomesandBrachyuryremain poorly understood. Here, we combine embryonic and stem cell-based analyses to delineate the non-overlapping, lineage-specific transcriptional activities. On a genome-wide scale binding of both TFs overlaps at promoters of target genes, but shows specificity for distal enhancer regions, that is conferred by differences in Tbx DNA-binding motifs. The unique binding to enhancer sites instructs the specification of anterior mesoderm (AM) and DE byEomesand caudal mesoderm byBrachyury. Remarkably, EOMES antagonizes BRACHYURY gene-regulatory functions in co-expressing cells during early gastrulation to ensure the proper sequence of early AM and DE lineage specification followed by posterior mesoderm derivatives.

Highlights

Detailed comparative analysis of the two critical developmental regulatorsEomesandBrachyuryin mouse embryos and differentiating embryonic stem cells

Tbx factors EOMES and BRACHYURY control distinct gene programs to specify different mesoderm and endoderm subsets

Program specificity is conferred by binding to non-overlapping enhancers with distinct binding motifs

EOMES restricts the activities of BRACHYURY thus ensuring the proper sequence of mesoderm and endoderm lineage specification
Newly born mesenchymal cells disperse through a rapid mechanosensitive migration
Authors: Riddell Jon , Noureen Shahzeb Raja , Sedda Luigi , Glover James D. , Ho William K. W. , Bain Connor A. , Berbeglia Arianna , Brown Helen , Anderson Calum , Chen Yuhang , Crichton Michael L. , Yates Christian A. , Mort Richard L. , Headon Denis J.
Published: January 2023

Embryonic mesenchymal cells are dispersed within an extracellular matrix but can coalesce to form condensates with key developmental roles. Cells within condensates undergo fate and morphological changes, and induce cell fate changes in nearby epithelia to produce structures including hair follicles, feathers or intestinal villi. Here, by imaging of mouse and chicken embryonic skin, we find that mesenchymal cells undergo much of their dispersal in early interphase, in a stereotyped process of displacement driven by three hours of rapid and persistent migration, followed by a long period of low motility. The cell division plane and the elevated migration speed and persistence of newly born mesenchymal cells are mechanosensitive, aligning with tension in the tissue. This early G1 migratory behaviour disperses mesenchymal cells and allows the daughters of recent divisions to travel long distances to enter dermal condensates, demonstrating an unanticipated effect of a cell cycle sub-phase on core mesenchymal behaviour.

Highlights

After mesenchymal cell division the speed and persistence of daughter cell migration is elevated for 180 minutes

Mesenchymal cell division and migration are directed by tissue tension

Newly born mesenchymal cells are uniquely responsive to tissue strain

Newly born mesenchymal cells are preferentially recruited to dermal condensates

Increased dispersal of newly born cells enables long distance travel to dermal condensates

Graphical abstract
Backup transcription factor binding sites protect human genes from mutations in the promoter
Authors: Brown Jay C.
Published: January 2023

This study was designed to test the idea that human gene promoters have evolved to be resistant to the effects of mutations in their primary function, the control of gene expression. It is proposed that the transcription factor/transcription factor binding site (TF/TFBS) pair having the greatest effect on control of a gene is the one with the highest abundance in the promoter. Other pairs would have the same effect on gene expression and would predominate in the event of a mutation in the most abundant pair. It is expected that the overall promoter architecture proposed here will be highly resistant to mutagenic change that would otherwise affect expression of the gene. The idea was tested beginning with a database of 42 human genes highly specific for expression in brain. For each gene, information was accumulated about its expression level and about the TFBS occupancy of the five most abundant TF/TFBS pairs. Expression level was then plotted against TFBS occupancy separately for each of the five pairs, and the plots were compared with each other. The plots were found to be similar, and the results were interpreted to indicate that the TFBS occupancy ranks evolved to yield the same effect on gene expression level with multiple ranks able to function in the event of mutation in another. A similar analysis was conducted with a database of 31 human liver specific genes, and the overall result was found to be the same. Backup TFBS occupancy ranks were interpreted to be present in both brain and liver specific genes. Finally, the TFBSs in the brain specific and liver specific gene populations were compared with each other with the goal of identifying any brain selective or liver selective TFBSs. Of the 89 TFBSs in the pooled population, 58 were found only in brain specific but not liver specific genes, 8 only in liver specific but not brain specific genes and 23 were found in both brain and liver specific genes. The results were interpreted to emphasize the large number of TFBS in brain specific but not liver specific genes.
Molten globule driven and self-downmodulated phase separation of a viral factory scaffold
Authors: Salgueiro Mariano , Camporeale Gabriela , Visentin Araceli , Aran Martin , Pellizza Leonardo , Esperante Sebastián , Corbat Agustín , Grecco Hernán , Sousa Belén , Esperón Ramiro , Borkosky Silvia S. , de Prat-Gay Gonzalo
Published: January 2023

Viral factories of liquid-like nature host transcription and replication in most viruses. The syncytial respiratory virus factories include gene function proteins, brought together by the phosphoprotein (P) RNA polymerase cofactor, present across non-segmented negative stranded RNA viruses. Homotypic liquid-liquid phase separation of RSV-P is governed by an α-helical molten globule domain, and strongly self-downmodulated by adjacent sequences. Condensation of P with the nucleoprotein N is stoichiometrically tuned, defining aggregate-droplet and droplet-dissolution boundaries. Time course analysis show small N-P nuclei gradually coalescing into large granules in transfected cells. This behavior is recapitulated in infection, with small puncta evolving to large viral factories, strongly suggesting that P-N nucleation-condensation sequentially drives viral factories. Thus, the tendency of P to undergo phase separation is moderate and latent in the full-length protein but unleashed in the presence of N or when neighboring disordered sequences are deleted. This, together with its capacity to rescue nucleoprotein-RNA aggregates suggests a role as a “solvent-protein” and possible fluidity tuner of viral factories.