bluuebunny/crossref_metadata_2025 huggingface dataset:
Genomic rearrangements have consequences for introgression breeding as revealed by genome assemblies of wild and cultivated lentil species
Authors: Ramsay Larissa , Koh Chu Shin , Kagale Sateesh , Gao Dongying , Kaur Sukhjiwan , Haile Teketel , Gela Tadesse S. , Chen Li-An , Cao Zhe , Konkin David J. , Toegelová Helena , Doležel Jaroslav , Rosen Benjamin D. , Stonehouse Robert , Humann Jodi L. , Main Dorrie , Coyne Clarice J. , McGee Rebecca J. , Cook Douglas R. , Varma Penmetsa R. , Vandenberg Albert , Chan Crystal , Banniza Sabine , Edwards David , Bayer Philipp E. , Batley Jacqueline , Udupa Sripada M. , Bett Kirstin E.
Published: July 2021

Understanding the genomic relationship between wild and cultivated genomes would facilitate access to the untapped variability found in crop wild relatives. We developed genome assemblies of a cultivated lentil (Lens culinaris) as well as a wild relative (L. ervoides). Comparative analyses revealed large-scale structural rearrangements and additional repetitive DNA in the cultivated genome, resulting in regions of reduced recombination, segregation distortion and permanent heterozygosity in the offspring of a cross between the two species. These novel findings provide plant breeders with better insight into how best to approach accessing the novel variability available in wild relatives.
Trait-trait relationships and tradeoffs vary with genome size in prokaryotes
Authors: Beier Sara , Werner Johannes , Bouvier Thierry , Mouquet Nicolas , Violle Cyrille
Published: July 2021

We report genomic traits that have been associated with the life history of prokaryotes and highlight conflicting findings concerning earlier observed trait correlations and tradeoffs. In order to address possible explanations for these contradictions we examined trait-trait variations of 11 genomic traits from ~ 18,000 sequenced genomes. The studied trait-trait variations suggested: (i) the predominance of two resistance and resilience-related orthogonal axes and (ii) at least in free living species with large effective population sizes whose evolution is little affected by genetic drift an overlap between a resilience axis and an axis of resource usage efficiency. These findings imply that resistance associated traits of prokaryotes are globally decoupled from resilience related traits and in the case of free-living communities also from resource use efficiencies associated traits. However, further inspection of pairwise scatterplots showed that resistance and resilience traits tended to be positively related for genomes up to roughly five million base pairs and negatively for larger genomes. This in turn may preclude a globally consistent assignment of prokaryote genomic traits to the competitor - stress-tolerator - ruderal (CSR) schema that sorts species depending on their location along disturbance and productivity gradients into three ecological strategies and may serve as an explanation for conflicting findings from earlier studies. All reviewed genomic traits featured significant phylogenetic signals and we propose that our trait table can be applied to extrapolate genomic traits from taxonomic marker genes. This will enable to empirically evaluate the assembly of these genomic traits in prokaryotic communities from different habitats and under different productivity and disturbance scenarios as predicted via the resistance-resilience framework formulated here.
Determination of the Two-Component Systems regulatory network reveals core and accessory regulations across Pseudomonas aeruginosa lineages
Authors: Trouillon Julian , Imbert Lionel , Villard Anne-Marie , Vernet Thierry , Attrée Ina , Elsen Sylvie
Published: July 2021

Pseudomonas aeruginosa possesses one of the most complex bacterial regulatory networks, which largely contributes to its success as a human opportunistic pathogen. However, most of its transcription factors (TFs) are still uncharacterized and the potential intra-species variability in regulatory networks has been mostly ignored so far. Here, to provide a first global view of the two-component systems (TCSs) regulatory network in P. aeruginosa, we produced and purified all DNA-binding TCS response regulators (RRs) and used DAP-seq to map the genome-wide binding sites of these 55 TFs across the three major P. aeruginosa lineages. The resulting networks encompass about 40% of all genes in each strain and contain numerous new important regulatory interactions across most major physiological processes, including virulence and antibiotic resistance. Strikingly, the comparison between the three representative strains shows that about half of the detected targets are specific to only one or two of the tested strains, revealing a previously unknown large functional diversity of TFs within a single species. Three main mechanisms were found to drive this diversity, including differences in accessory genome content, as exemplified by the strain-specific plasmid in the IHMA87 outlier strain which harbors numerous binding sites of chromosomally-encoded RRs. Additionally, most RRs display potential auto-regulation or RR-RR cross-regulation, bringing to light the vast complexity of this network. Overall, we provide the first complete delineation of the TCS regulatory network in P. aeruginosa that will represent an important resource for future studies on this pathogen.
Malaria parasite evades mosquito immunity by glutaminyl cyclase mediated protein modification
Authors: Kolli Surendra Kumar , Molina-Cruz Alvaro , Araki Tamasa , van Geurten Fiona J.A. , Ramesar Jai , Chevalley-Maurel Severine , Kroeze Hans J , Bezemer Sacha , de Korne Clarize , Withers Roxanne , Raytselis Nadia , El Hebieshy Angela F. , Kim Robert Q , Child Matthew , Kakuta Soichiro , Hisaeda Hajime , Kobayashi Hirotaka , Annoura Takeshi , Hensbergen Paul J. , Franke-Fayard Blandine M. , Barillas-Mury Carolina , Scheeren Ferenc A. , Janse Chris J.
Published: July 2021

Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of Plasmodium QC. We show that Plasmodium sporozoites of QC-null mutants are recognized by the mosquito immune system and melanized when they reach the hemocoel. Sporozoite numbers in salivary glands are also reduced in mosquitoes infected with QC-null or QC catalytically-dead mutants. This phenotype can be rescued by genetic complementation or by disrupting mosquito hemocytes or melanization immune responses. Mutation of a single QC-target glutamine of the major sporozoite surface protein (CSP) also results in immune recognition of sporozoites. These findings reveal QC-mediated post-translational modification of surface proteins as a major mechanism of mosquito immune evasion by Plasmodium sporozoites.
The human neuronal receptor NgR1 bridges reovirus capsid proteins to initiate infection
Authors: Sutherland Danica M. , Strebl Michael , Koehler Melanie , Welsh Olivia L. , Yu Xinzhe , Hu Liya , dos Santos Natividade Rita , Knowlton Jonathan J. , Taylor Gwen M. , Moreno Rodolfo A. , Wörz Patrick , Lonergan Zachary R. , Aravamudhan Pavithra , Guzman-Cardozo Camila , Alsteens David , Wang Zhao , Prasad B. V. V. , Stehle Thilo , Dermody Terence S.
Published: July 2021

Human Nogo-66 receptor 1 (NgR1) is a receptor for mammalian orthoreoviruses (reoviruses), but the mechanism of virus-receptor engagement is unknown. NgR1 binds a variety of structurally dissimilar ligands in the adult central nervous system (CNS) to inhibit axon outgrowth. Disruption of ligand binding to NgR1 and subsequent signaling can improve neuron regrowth, making NgR1 an important therapeutic target for diverse conditions such as spinal crush injuries and Alzheimer disease. To elucidate how NgR1 mediates cell binding and entry of reovirus, we defined the affinity of interaction between virus and receptor, determined the structure of the virus-receptor complex, and identified residues in the receptor required for virus binding and infection. These studies revealed that NgR1 sequences in a central concave region of the molecule establish a bridge between two copies of the viral capsid protein, σ3. This unusual binding interface produces high-avidity interactions between virus and receptor and likely primes early entry steps. NgR1 sequences engaged by reovirus also are required for NgR1 binding to ligands expressed by neurons and oligodendrocytes. These studies redefine models of reovirus cell-attachment and highlight the evolution of viruses to engage multiple receptors using distinct capsid components.
Regulation of neuronal progenitor delamination by dynein-driven post-Golgi apical transport
Authors: Brault J.B. , Bardin S. , Lampic M. , Carpentieri J.A. , Coquand L. , Penisson M. , Lachuer Hugo , Victoria G.S. , Baloul S. , Boncompain G. , Miserey-Lenkei S. , Fraisier V. , Francis F. , Perez F. , Goud B. , Baffet A. D.
Published: July 2021

Radial glial (RG) cells are the neural stem cells of the developing neocortex. Apical RG (aRG) cells can delaminate to generate basal RG (bRG) cells, a cell type associated with human brain expansion. Here, we report that this delamination is regulated by the post-Golgi secretory pathway. Using in situ subcellular live imaging, we show that post-Golgi transport of RAB6+ vesicles occurs toward the minus ends of microtubules and depends on dynein. We demonstrate that the apical determinant Crumbs3 (CRB3) is also transported by dynein. Double knockout of RAB6A/A’ and RAB6B impairs apical localization of CRB3, and induces a retraction of aRG cell apical process, leading to delamination and ectopic division. These defects are phenocopied by knock-out of the dynein activator LIS1. Overall, our results identify a RAB6-dynein-LIS1 complex for Golgi to apical surface transport in aRG cells, and highlights the role of this pathway in the maintenance of neuroepithelial integrity.
Mechanosensitivity of nucleocytoplasmic transport
Authors: Andreu Ion , Granero-Moya Ignasi , Chahare Nimesh R. , Clein Kessem , Jordàn Marc Molina , Beedle Amy E. M. , Elosegui-Artola Alberto , Trepat Xavier , Raveh Barak , Roca-Cusachs Pere
Published: July 2021

Mechanical force controls fundamental cellular processes in health and disease, and increasing evidence shows that the nucleus both experiences and senses applied forces. Here we show that nuclear forces differentially control passive and facilitated nucleocytoplasmic transport, setting the rules for the mechanosensitivity of shuttling proteins. We demonstrate that nuclear force increases permeability across nuclear pore complexes, with a dependence on molecular weight that is stronger for passive than facilitated diffusion. Due to this differential effect, force leads to the translocation into or out of the nucleus of cargoes within a given range of molecular weight and affinity for nuclear transport receptors. Further, we show that the mechanosensitivity of several transcriptional regulators can be both explained by this mechanism, and engineered exogenously by introducing appropriate nuclear localization signals. Our work sets a novel framework to understand mechanically induced signalling, with potential general applicability across signalling pathways and pathophysiological scenarios.

One sentence summary

Force application to the nucleus leads to nuclear accumulation of proteins by differentially affecting passive versus facilitated nucleocytoplasmic transport.
DNA mismatch and damage detection using a FRET-based assay for monitoring the loading of multiple MutS sliding clamps
Authors: Kunetsky Vladislav , Storozhuk Olha , Brouwer Gwendolyn , Laffeber Charlie , Dillingham Mark S. , Lebbink Joyce , Friedhoff Peter
Published: July 2021

We developed a sensitive, homogeneous fluorescence assay for the detection of DNA mismatches and DNA damage based on the mismatch repair (MMR) protein MutS. The assay is based on Förster resonance energy transfer (FRET) between SYBR Green I (SG), non-covalently bound to DNA, and Alexa Fluor 647 (AF647) conjugated to MutS. In contrast to previous assays using only the mismatch binding activity of MutS, we exploited the ATP-dependent loading of multiple MutS sliding clamps provoked by mismatch/damage to the DNA, which increases the overall sensitivity of the assay. The assay was validated using a well-characterized 3 kb circular DNA containing a single G/T mismatch. We also demonstrate that treatment of long (multiple kb) DNA with various chemical or physical agents including non-denaturing bisulfite conversion of cytosine to uracil, cisplatin modification or ultraviolet light (UVC) results in changes in the DNA that can be detected by the FRET-based MutS biosensor.
The desmin mutation R349P increases contractility and fragility of stem cell-generated muscle micro-tissues
Authors: Spörrer Marina , Kah Delf , Gerum Richard C. , Reischl Barbara , Huraskin Danyil , Dessalles Claire A. , Schneider Werner , Goldmann Wolfgang H. , Herrmann Harald , Thievessen Ingo , Clemen Christoph S. , Friedrich Oliver , Hashemolhosseini Said , Schröder Rolf , Fabry Ben
Published: July 2021

Desminopathies comprise hereditary myopathies and cardiomyopathies caused by mutations in the intermediate filament protein desmin that lead to severe and often lethal degeneration of striated muscle tissue. Animal and single cell studies hinted that this degeneration process is associated with massive ultrastructural defects correlating with increased susceptibility of the muscle to acute mechanical stress. The underlying mechanism of mechanical susceptibility, and how muscle degeneration develops over time, however, has remained elusive. Here, we investigated the effect of a desmin mutation on the formation, differentiation, and contractile function of in vitro-engineered three-dimensional micro-tissues grown from muscle stem cells (satellite cells) isolated from heterozygous R349P desmin knock-in mice. Micro-tissues grown from desmin-mutated cells exhibited spontaneous unsynchronized contractions, higher contractile forces in response to electrical stimulation, and faster force recovery compared to tissues grown from wild-type cells. Within one week of culture, the majority of R349P desmin-mutated tissues disintegrated, whereas wild-type tissues remained intact over at least three weeks. Moreover, under tetanic stimulation lasting less than five seconds, desmin-mutated tissues partially or completely ruptured, whereas wild-type tissues did not display signs of damage. Our results demonstrate that the progressive degeneration of desmin-mutated micro-tissues is closely linked to extracellular matrix fiber breakage associated with increased contractile forces and unevenly distributed tensile stress. This suggests that the age-related degeneration of skeletal and cardiac muscle in patients suffering from desminopathies may be similarly exacerbated by mechanical damage from high-intensity muscle contractions. We conclude that micro-tissues may provide a valuable tool for studying the organization of myocytes and the pathogenic mechanisms of myopathies.
Sulfated host glycan recognition by carbohydrate sulfatases of the human gut microbiota
Authors: Luis Ana , Baslé Arnaud , Byrne Dominic P , Wright Gareth SA , London James , Chunsheng Jin , Karlsson Niclas , Hansson Gunnar , Eyers Patrick , Czjzek Mirjam , Barbeyron Tristan , Yates Edwin A , Martens Eric C. , Cartmell Alan
Published: July 2021

The vast microbial community that resides in the human colon, termed the human gut microbiota, performs important roles in maintaining host health. Sulfated host glycans comprise both a major nutrient source and important colonisation factors for this community. Carbohydrate sulfatases remove sulfate groups from glycans and are essential in many bacteria for the utilisation of sulfated host glycans. Additionally, carbohydrate sulfatases are also implicated in numerous host diseases, but remain some of the most understudied carbohydrate active enzymes to date, especially at the structural and molecular level. In this work, we analyse 7 carbohydrate sulfatases, spanning 4 subfamilies, from the human gut symbiont Bacteroides thetaiotaomicron, a major utiliser of sulfated host glycans, correlating structural and functional data with phylogenetic and environmental analyses. Together, these data begin to fill the knowledge gaps in how carbohydrate sulfatases orchestrate sulfated glycan metabolism within their environment.
Blood flow coordinates collective endothelial cell migration during vascular plexus formation and promotes angiogenic sprout regression via vegfr3/flt4
Authors: Chen Yan , Jiang Zhen , Fisher Katherine H. , Kim Hyejeong R. , Evans Paul C. , Wilkinson Robert N.
Published: July 2021

Nascent vascular networks adapt to the increasing metabolic demands of growing tissues by expanding via angiogenesis. As vascular networks expand, blood vessels remodel, progressively refining vascular connectivity to generate a more haemodynamically efficient network. This process is driven by interplay between endothelial cell (EC) signalling and blood flow. While much is known about angiogenesis, considerably less is understood of the mechanisms underlying vessel remodelling by blood flow. Here we employ the zebrafish sub-intestinal venous plexus (SIVP) to characterise the mechanisms underlying blood flow-dependent remodelling. Using live imaging to track ECs we show that blood flow controls SIVP remodelling by coordinating collective migration of ECs within the developing plexus. Blood flow opposes continuous ventral EC migration within the SIVP and is required for regression of angiogenic sprouts to support plexus growth. Sprout regression occurs by coordinated polarisation and migration of ECs from non-perfused leading sprouts, which migrate in opposition to blood flow and incorporate into the SIV. Sprout regression is compatible with low blood flow and is dependent upon vegfr3/flt4 function under these conditions. Blood flow limits expansive venous remodelling promoted by vegfr3/flt4. Collectively, these studies reveal how blood flow sculpts a developing vascular plexus by coordinating EC migration and balancing vascular remodelling via vegfr3/flt4.
Fecal Microbiota Transplantation Drives Colonic Expression of Immune Activation Genes in a Mouse Model of Antibiotic Use
Authors: Moreau G. Brett , Ozbek Hale , Kumar Pankaj , Frisbee Alyse , Leslie Jhansi , Petri William A.
Published: July 2021

Clostridioides difficile infection (CDI) is the leading hospital acquired infection in North America. While the standard treatment for CDI remains antibiotics, fecal microbiota transplantation (FMT) has gained attention as an effective therapy to prevent relapse. Previous work has focused on colonization resistance mounted against C. difficile by FMT-delivered commensals, but the effects of FMT on the gut mucosal immune response are poorly understood. Better understanding of the molecular mechanisms driven by FMT would allow for more targeted therapy against CDI. To address this important gap in knowledge, microbial community structure and host gene expression were assessed after FMT in a mouse model of antibiotic use. Administration of FMT led to a significant increase in microbial diversity and partial restoration of community structure within 48 hours of treatment. RNA sequencing of cecal tissue identified large changes in gene expression between FMT recipient and vehicle control groups. Strikingly, genes upregulated after FMT treatment were enriched in immune activation pathways, many of which were associated with pro-inflammatory immune responses. FMT also upregulated several genes associated with type 2 immunity while repressing several associated with type 3 immunity, trends that are associated with improved response to CDI. These results highlight the interplay between the intestinal microbiota and host transcriptome and identify pathways of interest for exploring the role of FMT on treatment of recurring CDI.
Adult fibroblasts use aggresomes only in distinct cell-states
Authors: Morrow Christopher S. , Arndt Zachary P. , Peng Bo , Zewdie Eden Y. , Benayoun Bérénice A. , Moore Darcie L.
Published: July 2021

The aggresome is a protein turnover system in which proteins are trafficked along microtubules to the centrosome for degradation. Despite extensive focus on aggresomes in immortalized cell lines, it remains unclear if the aggresome is conserved in all primary cells and all cell-states. Here we examined the aggresome in primary adult mouse dermal fibroblasts shifted into four distinct cell-states. We found that in response to proteasome inhibition, quiescent and immortalized fibroblasts formed aggresomes whereas proliferating and senescent fibroblasts did not. Using this model, we generated a resource to better understand the aggresome, providing a characterization of the proteostasis networks in which the aggresome is used and transcriptomic features associated with the presence or absence of aggresome formation. Using this resource, we validate a previously reported role for p38 MAPK signaling in aggresome formation and identify TAK1 as a novel driver of aggresome formation upstream of p38 MAPKs. Together, our data demonstrate that the aggresome is a non-universal protein degradation system which can be used cell-state specifically and provide a resource for better understanding aggresome formation and function.
Knowledge Graph-based Recommendation Framework Identifies Novel Drivers of Resistance in EGFR mutant Non-small Cell Lung Cancer
Authors: Gogleva Anna , Polychronopoulos Dimitris , Pfeifer Matthias , Poroshin Vladimir , Ughetto Michaël , Sidders Benjamin , Dry Jonathan R. , Ahdesmäki Miika , McDermott Ultan , Papa Eliseo , Bulusu Krishna
Published: July 2021

Resistance to EGFR inhibitors (EGFRi) presents a major obstacle in treating non-small cell lung cancer (NSCLC). One of the most exciting new ways to find potential resistance markers involves running functional genetic screens, such as CRISPR, followed by manual triage of significantly enriched genes. This triage process to identify ‘high value’ hits resulting from the CRISPR screen involves significant manual curation that requires specialized knowledge and can take even experts several months to comprehensively complete.

To find key drivers of resistance faster we built a hybrid recommendation system on top of a heterogeneous biomedical knowledge graph integrating preclinical, clinical, and literature evidence. Genes were ranked based on trade-offs between diverse types of evidence linking them to potential mechanisms of EGFRi resistance. This unbiased approach identified 36 resistance markers from >3,000 genes, reducing hit identification time from months to minutes. In addition to reproducing known resistance markers, our method identified novel resistance mechanisms that we prospectively validated.
Trade-offs between cost of ingestion and rate of intake drive defensive toxin use
Authors: Douglas Tyler E. , Beskid Sofia G. , Gernand Callie E. , Nirtaut Brianna E. , Tamsil Kristen E. , Fitch Richard W. , Tarvin Rebecca D.
Published: July 2021

Animals that ingest toxins can become unpalatable and even toxic to predators and parasites through toxin sequestration. Because most animals rapidly eliminate toxins to survive their ingestion, it is unclear how populations transition from susceptibility and toxin elimination to tolerance and accumulation as chemical defense emerges. Studies of chemical defense have generally focused on species with active toxin sequestration and target-site insensitivity mutations or toxin-binding proteins that permit survival without necessitating toxin elimination. Here, we investigate whether animals that presumably rely on toxin elimination for survival can utilize ingested toxins for defense. We use the A4 and A3 Drosophila melanogaster fly strains from the Drosophila Synthetic Population Resource (DSPR), which respectively possess elevated and reduced metabolic nicotine resistance amongst DSPR fly lines. We find that ingesting nicotine increased A4 but not A3 fly survival against Leptopilina heterotoma wasp parasitism.Further, we find that despite possessing genetic variants that enhance toxin elimination, A4 flies accrued more nicotine than A3 individuals likely by consuming more media. Our results suggest that enhanced toxin metabolism can allow for greater toxin intake by offsetting the cost of toxin ingestion. Passive toxin accumulation that accompanies increased toxin intake may underlie the early origins of chemical defense.
In vitro and in vivo analysis of microvesicle-mediated metastasis using a bright, red-shifted bioluminescent reporter protein of extracellular vesicles
Authors: Zarea Ahmed A. , Perez Gloria I. , Broadbent David , Dolgikh Benedikt , Bernard Matthew P. , Withrow Alicia , McGill Amelia , Toomajian Victoria , Thampy Lukose K. , Harkema Jack , Walker Joel R. , Kirkland Thormas A. , Bachmann Michael H. , Schmidt Jens , Kanada Masamitsu
Published: July 2021

Cancer cells produce heterogeneous extracellular vesicles (EVs) as mediators of intercellular communication. Our study focused on a novel method to image EV subtypes and their biodistribution in vivo. Regardless of injection routes, we established that reporter EVs isolated from murine mammary carcinoma cells expressing PalmReNL, which utilizes bioluminescence resonance energy transfer (BRET), localized to the lungs. This new EV reporter allowed highly sensitive EV tracking in vitro and in vivo and enabled us to begin studies to understand the commonalities and functional differences of the EV subtypes. We demonstrated the early appearance of metastatic foci in the lungs of mammary tumor-bearing mice following multiple injections of the microvesicle (MV)-enriched fraction derived from mammary carcinoma cells. In addition, the results we present here show that tumor cell-derived MVs act on distant tissues through upregulating LC3 expression within the lung.
IFITM3 regulates virus-induced inflammatory cytokine production by titrating Nogo-B orchestration of TLR responses
Authors: Clement M. , Forbester J.L. , Marsden M. , Sabberwal P. , Wellington D. , Dimonte S. , Clare S. , Harcourt K. , Yin Z. , Nobre L. , Antrobus R , Jin B. , Chen M. , Makvandi-Nejad S. , Lindborg J.A , Strittmatter S.M. , Weekes M.P. , Stanton R.J. , Dong T. , Humphreys I.R.
Published: July 2021

Interferon induced transmembrane protein 3 (IFITM3) is an important viral restriction factor in viral pathogenesis that also exhibits poorly understood immune regulatory functions. Here, using human and mouse models, we demonstrate that IFITM3 regulates MyD88-dependent TLR-mediated cytokine production following dendritic cell exposure to cytomegalovirus (CMV), and this process limits viral pathogenesisin vivo. IFITM3 also restricted pro-inflammatory (IL-6) cytokine production in response to influenza. IFITM3 bound to and promoted ubiquitination and proteasomal degradation of the reticulon 4 isoform Nogo-B. We reveal that Nogo-B mediates TLR-dependent pro-inflammatory cytokine production and promotes viral pathogenesisin vivo, and this process involved alteration of TLR dynamics. The anti-inflammatory function of IFITM3 was intrinsically linked to its ability to regulate Nogo-B. Thus, we uncover Nogo-B as an unappreciated driver of viral pathogenesis and highlight a novel immune regulatory pathway where IFITM3 fine-tunes TLR responsiveness of myeloid cells to viral stimulation.
Combinatorial drug-microenvironment interaction mapping reveals cell-extrinsic drug resistance mechanisms and clinically relevant patient subgroups in CLL
Authors: Bruch Peter-Martin , Giles Holly A. R. , Kolb Carolin , Herbst Sophie A. , Becirovic Tina , Roider Tobias , Lu Junyan , Scheinost Sebastian , Wagner Lena , Huellein Jennifer , Berest Ivan , Kriegsmann Mark , Kriegsmann Katharina , Zgorzelski Christiane , Dreger Peter , Zaugg Judith B. , Müller-Tidow Carsten , Zenz Thorsten , Huber Wolfgang , Dietrich Sascha
Published: July 2021

The tumour microenvironment and genetic alterations collectively influence drug efficacy in cancer, but current evidence is limited to small scale studies and systematic analyses are lacking. We chose Chronic Lymphocytic Leukaemia (CLL), the most common leukaemia in adults, as a model disease to study this complex interplay systematically. We performed a combinatorial assay using 12 drugs individually co-applied with each of 17 microenvironmental stimuli in 192 primary CLL samples, generating a comprehensive map of drug-microenvironment interactions in CLL. This data was combined with whole-exome sequencing, DNA-methylation, RNA-sequencing and copy number variant annotation. Our assay identified four distinct CLL subgroups that differed in their responses to the panel of microenvironmental stimuli. These subgroups were characterized by distinct clinical outcomes independently of known prognostic markers. We investigated the effect of CLL- specific recurrent genetic alterations on microenvironmental responses and identified trisomy 12 as an amplifier of multiple microenvironmental stimuli. We further quantified the impact of microenvironmental stimuli on drug response, confirmed known interactions such as Interleukin (IL) 4 mediated resistance to B cell receptor (BCR) inhibitors, and identified new interactions such as Interferon-γ induced resistance to BCR inhibitors. Finally, we identified interactions which were limited to genetic subgroups. Resistance to chemotherapeutics, such as Fludarabine, induced by Toll-Like Receptor (TLR) agonists could be observed in IGHV unmutated patient samples and IGHV mutated samples with trisomy 12. In-vivo relevance was investigated in CLL-infiltrated lymph nodes, which showed increased IL4 and TLR signalling activity compared to healthy samples (p<0.001). High IL4 activity in lymph nodes correlated with faster disease progression (p=0.038).

We provide a publicly available resource (www.dietrichlab.de/CLL_Microenvironment/) which uncovers tumour cell extrinsic influences on drug response and disease progression in CLL, and how these interactions are modulated by cell intrinsic molecular features.
A BioBricks® toolbox for multiplexed metabolic engineering of central carbon metabolism in the tetracenomycin pathway
Authors: Nguyen Jennifer T. , Riebschleger Kennedy K. , Brown Katelyn V. , Gorgijevska Nina M. , Nybo S. Eric
Published: July 2021

The tetracenomycins are aromatic anticancer polyketides that inhibit peptide translation via binding to the large ribosomal subunit. Here, we expressed the elloramycin biosynthetic gene cluster in the heterologous host Streptomyces coelicolor M1146 to facilitate the downstream production of tetracenomycin analogs. We developed a BioBricks® genetic toolbox of genetic parts for substrate precursor engineering in S. coelicolor M1146::cos16F4iE. We cloned a series of integrating vectors based on the VWB, TG1, and SV1 integrase systems to interrogate gene expression in the chromosome. We genetically engineered three separate genetic constructs to modulate tetracenomycin biosynthesis: 1) the vhb hemoglobin from obligate aerobe Vitreoscilla stercoraria to improve oxygen utilization; (2) the accA2BE acetyl-CoA carboxylase to enhance condensation of malonyl-CoA; (3) lastly, the sco6196 acyltransferase, which is a “metabolic regulatory switch” responsible for mobilizing triacylglycerols to β-oxidation machinery for acetyl-CoA. In addition, we engineered the tcmO 8-O-methyltransferase and newly identified tcmD 12-O-methyltransferase from Amycolatopsis sp. A23 to generate tetracenomycins C and X. We also co-expressed the tcmO methyltransferase with oxygenase urdE to generate the analog 6-hydroxy-tetracenomycin C. Altogether, this system is compatible with the BioBricks® [RFC 10] cloning standard for the co-expression of multiple gene sets for metabolic engineering of Streptomyces coelicolor M1146::cos16F4iE.
Chemically-induced epimutagenesis allows bypassing reproductive barriers in hybrid seeds
Authors: Huc Jonathan , Dziasek Katarzyna , Pachamuthu Kannan , Woh Tristan , Köhler Claudia , Borges Filipe
Published: July 2021

The “triploid block” prevents interploidy hybridizations in flowering plants, and is characterized by failure in endosperm development, arrest in embryogenesis, and seed collapse. Many genetic components of triploid seed lethality have been successfully identified in the model plant Arabidopsis thaliana, most notably the paternally expressed imprinted genes (PEGs) that are up-regulated in the tetraploid endosperm with paternal excess. Previous studies have shown that the paternal epigenome is a key determinant of the triploid block response, as the loss of DNA methylation in diploid pollen suppresses the triploid block almost completely. Here, we demonstrate that triploid seed collapse is bypassed in Arabidopsis plants treated with the DNA methyltransferase inhibitor 5-Azacytidine during seed germination and early growth. We have identified strong suppressor lines showing stable transgenerational inheritance of hypomethylation in CG context, as well as normalized expression of PEGs in triploid seeds. Importantly, differentially methylated loci segregate in the progeny of “epimutagenized” plants, which may allow the identification of epialleles involved in the triploid block response in future studies. Finally, we demonstrate that chemically-induced epimutagenesis allows bypassing hybridization barriers in crosses between different Capsella species, thus potentially emerging as a novel strategy for producing triploids and interspecific hybrids with high agronomical interest.

One sentence summary

Genome-wide loss of DNA methylation induced by 5-Azacytidine allows bypassing interploidy and interspecific hybridization barriers in Arabidopsis and Capsella.
Opponent control of behavior by dorsomedial striatal pathways depends on task demands and internal state
Authors: Bolkan Scott S. , Stone Iris R. , Pinto Lucas , Ashwood Zoe C. , Iravedra Garcia Jorge M. , Herman Alison L. , Singh Priyanka , Bandi Akhil , Cox Julia , Zimmerman Christopher A. , Cho Jounhong Ryan , Engelhard Ben , Pillow Jonathan W. , Witten Ilana B.
Published: July 2021

A classic view of the striatum holds that activity in direct and indirect pathways oppositely modulates motor output. Whether this involves direct control of movement, or reflects a cognitive process underlying movement, remains unresolved. Here we find that strong, opponent control of behavior by the two pathways of the dorsomedial striatum (DMS) depends on the cognitive requirements of a task. Furthermore, a latent state model (a hidden markov model with generalized linear model observations) reveals that—even within a single task—the contribution of the two pathways to behavior is state-dependent. Specifically, the two pathways have large contributions in one of two states associated with a strategy of evidence accumulation, compared to a state associated with a strategy of repeating previous choices. Thus, both the demands imposed by a task, as well as the internal state of mice when performing a task, determine whether DMS pathways provide strong and opponent control of behavior.
Genomic and local microenvironment effects shaping epithelial-to-mesenchymal trajectories in cancer
Authors: Tagliazucchi Guidantonio Malagoli , Wiecek Anna J , Withnell Eloise , Secrier Maria
Published: July 2021

The epithelial to mesenchymal transition (EMT) is a key cellular process underlying cancer progression, with multiple intermediate states whose molecular hallmarks remain poorly characterized. To fill this gap, we explored EMT trajectories in 7,180 tumours of epithelial origin and identified three macro-states with prognostic and therapeutic value, attributable to epithelial, hybrid E/M and mesenchymal phenotypes. We show that the hybrid state is remarkably stable and linked with increased aneuploidy and APOBEC mutagenesis. We further employed spatial transcriptomics and single cell datasets to show that local effects impact EMT transformation through the establishment of distinct interaction patterns with cytotoxic, NK cells and fibroblasts in the tumour microenvironment. Additionally, we provide an extensive catalogue of genomic events underlying distinct evolutionary constraints on EMT transformation. This study sheds light on the aetiology of distinct stages along the EMT trajectory, and highlights broader genomic and environmental hallmarks shaping the mesenchymal transformation of primary tumours.
Individual differences in members of Actinobacteria, Bacteroidetes, and Firmicutes is associated with resistance or vulnerability to addiction-like behaviors in heterogeneous stock rats
Authors: Simpson S. , de Guglielmo G. , Brennan M. , Maturin L. , Peters G. , Jia H. , Wellmeyer E. , Andrews S. , Woods L. Solberg , Palmer A. A. , George O.
Published: July 2021

An emerging element in psychiatry is the gut-brain-axis, the bi-directional communication pathways between the gut microbiome and the brain. A prominent hypothesis, mostly based on preclinical studies, is that individual differences in the gut microbiome composition and drug-induced dysbiosis may be associated with vulnerability to psychiatric disorders including substance use disorder. However, most studies used small sample size, ignored individual differences, or used animal models with limited relevance to addiction. Here, we test the hypothesis that pre-existing microbiome composition and drug-induced changes in microbiome composition can predict addiction-like behaviors using an advanced animal model of extended access to cocaine self-administration in a large cohort of heterogenous stock (HS) rats. Adult male and female HS rats were allowed to self-administer cocaine under short (2h/day) and long access (6h/day) for ~7 weeks under various schedule of reinforcement to identify individuals that are resistant or vulnerable to addiction-like behaviors and fecal samples were collected before the first session and after the last session to assess differences in the microbiome composition. Linear discriminant analysis (LDA) identified sex-dependent and sex-independent differences at the phylum, order, and species level that are differentially abundant in resistant vs. vulnerable individuals, including high level of actinobacteria both before the first exposure to cocaine and after 7 weeks of cocaine self-administration in resistant animals. Predictions of functional gene content using PICRUSt revealed differential regulation of short-chain fatty acid processing in the vulnerable group after self-administration. These results identify microbiome constituents as well as metabolic pathways that are associated with resistance or vulnerability to addiction-like behaviors in rats. Identification of microbes and tangential metabolic pathways involved in cocaine resilience/vulnerability may represent an innovative strategy for the development of novel biomarkers and medication for the treatment of cocaine use disorder.
Glutamatergic dysfunction leads to a hyper-dopaminergic phenotype through deficits in short-term habituation: a mechanism for aberrant salience
Authors: Panayi Marios C , Boerner Thomas , Jahans-Price Thomas , Huber Anna , Sprengel Rolf , Gilmour Gary , Sanderson David J , Harrison Paul J , Walton Mark E , Bannerman David M
Published: July 2021

Psychosis in disorders like schizophrenia is commonly associated with aberrant salience and elevated striatal dopamine. However, the underlying cause(s) of this hyper-dopaminergic state remain elusive. Various lines of evidence point to glutamatergic dysfunction and impairments in synaptic plasticity in the aetiology of schizophrenia, including deficits associated with the GluA1 AMPAR subunit. GluA1 knockout (Gria1-/-) mice provide a model of impaired synaptic plasticity in schizophrenia and exhibit a selective deficit in a form of short-term memory which underlies short-term habituation. As such, these mice are unable to reduce attention to recently presented stimuli. In this study we used fast-scan cyclic voltammetry to measure phasic dopamine responses in the nucleus accumbens ofGria1-/-mice to determine whether this behavioral phenotype might be a key driver of a hyper-dopaminergic state. There was no effect of GluA1 deletion on electrically-evoked dopamine responses in anaesthetized mice, demonstrating normal endogenous release properties of dopamine neurons inGria1-/-mice. Furthermore, dopamine signals were initially similar inGria1-/-mice compared to controls in response to both sucrose rewards and neutral light stimuli. They were also equally sensitive to changes in the magnitude of delivered rewards. In contrast, however, these stimulus-evoked dopamine signals failed to habituate with repeated presentations inGria1-/-mice, resulting in a task-relevant, hyper-dopaminergic phenotype. Thus, here we show that GluA1 dysfunction, resulting in impaired short-term habituation, is a key driver of enhanced striatal dopamine responses, which may be an important contributor to aberrant salience and psychosis in psychiatric disorders like schizophrenia.
FTO suppresses STAT3 activation and modulates proinflammatory interferon-stimulated gene expression
Authors: McFadden Michael J. , Sacco Matthew T. , Murphy Kristen A. , Park Moonhee , Gokhale Nandan S. , Somfleth Kim Y. , Horner Stacy M.
Published: July 2021

Signaling initiated by type I interferon (IFN) results in the induction of hundreds of IFN-stimulated genes (ISGs). The type I IFN response is important for antiviral restriction, but aberrant activation of this response can lead to inflammation and autoimmunity. Regulation of this response is incompletely understood. We previously reported that the mRNA modification m6A and its deposition enzymes, METTL3 and METTL14 (METTL3/14), promote the type I IFN response by directly modifying the mRNA of a subset of ISGs to enhance their translation. Here, we determined the role of the RNA demethylase FTO in the type I IFN response. FTO, which can remove either m6A or the cap-adjacent m6Am RNA modifications, has previously been associated with obesity and body mass index, type 2 diabetes, cardiovascular disease, and inflammation. We found that FTO suppresses the transcription of a distinct set of ISGs, including many known pro-inflammatory genes, and that this regulation is not through the actions of FTO on m6Am. Further, we found that depletion of FTO led to activation of STAT3, a transcription factor that mediates responses to various cytokines, but whose role in the type I IFN response is not well understood. This activation of STAT3 increased the expression of a subset of ISGs. Importantly, this increased ISG induction resulting from FTO depletion was partially ablated by depletion of STAT3. Together, these results reveal that FTO negatively regulates STAT3-mediated signaling that induces proinflammatory ISGs during the IFN response, highlighting an important role for FTO in suppression of inflammatory genes.
SRS-FISH: High-Throughput Platform Linking Microbiome Function to Identity at the Single Cell Level
Authors: Ge Xiaowei , Pereira Fátima C. , Mitteregger Matthias , Berry David , Zhang Meng , Wagner Michael , Cheng Ji-Xin
Published: July 2021

One of the biggest challenges in microbiome research in environmental and medical samples is to better understand functional properties of microbial community members at a single cell level. Single cell isotope probing has become a key tool for this purpose, but the currently applied detection methods for measuring isotope incorporation into single cells do not allow high-throughput analyses. Here, we report on the development of an imaging-based approach termed stimulated Raman scattering - two-photon fluorescence in situ hybridization (SRS-FISH) for high-throughput structure-function analyses of microbial communities with single cell resolution. SRS-FISH has an imaging speed of 10 to 100 milliseconds per cell, which is two to three orders of magnitude faster than spontaneous Raman-FISH. Using this technique, we delineated metabolic responses of thirty thousand individual cells to various mucosal sugars in the human gut microbiome via incorporation of deuterium from heavy water as an activity marker. Application of SRS-FISH to investigate the utilization of host-derived nutrients by two major human gut microbiome taxa revealed that response to mucosal sugars tends to be dominated by Bacteroidales, with an unexpected finding that Clostridia can outperform Bacteroidales at foraging fucose.
Golden Gate Assembly of Aerobic and Anaerobic Microbial Bioreporters
Authors: Hinz Aaron J. , Stenzler Benjamin , Poulain Alexandre J.
Published: July 2021

Microbial bioreporters provide direct insight into cellular processes by producing a quantifiable signal dictated by reporter gene expression. The core of a bioreporter is a genetic circuit in which a reporter gene (or operon) is fused to promoter and regulatory sequences that govern its expression. In this study, we develop a system for constructing novelEscherichia colibioreporters based on Golden Gate assembly, a synthetic biology approach for the rapid and seamless fusion of DNA fragments. Gene circuits are generated by fusing promoter and reporter sequences encoding yellow fluorescent protein, mCherry, bacterial luciferase, and an anaerobically active flavin-based fluorescent protein. We address a barrier to the implementation of Golden Gate assembly by designing a series of compatible destination vectors that can accommodate the assemblies. We validate the approach by measuring the activity of constitutive bioreporters and mercury and arsenic biosensors in quantitative exposure assays. We also demonstrate anaerobic quantification of mercury and arsenic in biosensors that produce flavin-based fluorescent protein, highlighting the expanding range of redox conditions that can be examined by microbial bioreporters.

IMPORTANCE

Microbial bioreporters are versatile genetic tools with wide-ranging applications, particularly in the field of environmental toxicology. For example, biosensors that produce a signal output in the presence of a specific analyte offer less costly alternatives to analytical methods for the detection of environmental toxins such as mercury and arsenic. Biosensors of specific toxins can also be used to test hypotheses regarding mechanisms of uptake, toxicity, and biotransformation. In this study, we develop an assembly platform that uses a synthetic biology technique to streamline construction of novelEscherichia colibioreporters that produce fluorescent or luminescent signals. We validate the approach by synthesizing and testing an array of bioreporters, including arsenic and mercury biosensors, that produce signal outputs in environments ranging from aerobic to highly reduced anaerobic growth conditions.