bluuebunny/crossref_metadata_2025_split huggingface dataset:
A regulatory role for the unstructured C-terminal domain of the CtBP transcriptional corepressor
Authors: Raicu Ana-Maria , Suresh Megha , Arnosti David N. 
Published: May 2023

ABSTRACTThe C-terminal Binding Protein (CtBP) is a transcriptional corepressor that plays critical roles in development, tumorigenesis, and cell fate. CtBP proteins are structurally similar to alpha hydroxyacid dehydrogenases and feature a prominent intrinsically disordered region in the C-terminus. In the mammalian system, CtBP proteins lacking the C-terminal Domain (CTD) are able to function as transcriptional regulators and oligomerize, putting into question the significance of this unstructured domain for gene regulation. Yet, the presence of an unstructured CTD of ∼100 residues, including some short motifs, is conserved across Bilateria, indicating the importance of maintaining this domain over evolutionary time. To uncover the significance of the CtBP CTD, we functionally tested naturally occurring Drosophila isoforms of CtBP that possess or lack the CTD, namely CtBP(L) and CtBP(S). We used the CRISPRi system to recruit dCas9-CtBP(L) and dCas9-CtBP(S) to endogenous promoters to directly compare their transcriptional impactsin vivo. Interestingly, CtBP(S) was able to significantly repress transcription of theMpp6promoter, while CtBP(L) was much weaker, suggesting that the long CTD may modulate CtBP’s repression activity. In contrast, in cell culture, the isoforms behaved similarly on a transfectedMpp6reporter gene. The context-specific differences in activity of these two developmentally-regulated isoforms suggests that the CTD may help provide a spectrum of repression activity suitable for developmental programs.
Cytosolic iron-sulfur protein assembly system identifies clients by a C-terminal tripeptide
Authors: Marquez Melissa D. , Greth Carina , Buzuk Anastasiya , Liu Yaxi , Blinn Catharina M. , Beller Simone , Leiskau Laura , Hushka Anthony , Wu Kassandra , Nur Kübra , Netz Daili J. , Perlstein Deborah L. , Pierik Antonio J. 
Published: May 2023

AbstractThe eukaryotic cytosolic Fe-S protein assembly (CIA) machinery inserts iron-sulfur (Fe-S) clusters into cytosolic and nuclear proteins. In the final maturation step, the Fe-S cluster is transferred to the apo-proteins by the CIA-targeting complex (CTC). However, the molecular recognition determinants of client proteins are unknown. We show that a conserved [LIM]-[DES]-[WF]-COO-tripeptide present at the C-terminus of clients is necessary and sufficient for binding to the CTCin vitroand directing Fe-S cluster deliveryin vivo. Remarkably, fusion of this TCR (target complex recognition) signal enables engineering of cluster maturation on a non-native protein via recruitment of the CIA machinery. Our study significantly advances our understanding of Fe-S protein maturation and paves the way for bioengineering applications.One-Sentence SummaryA C-terminal tripeptide guides eukaryotic iron-sulfur cluster insertion into cytosolic and nuclear proteins.
Engineering Programmable Material-To-Cell Pathways Via Synthetic Notch Receptors To Spatially Control Cellular Phenotypes In Multi-Cellular Constructs
Authors: Garibyan Mher , Hoffman Tyler , Makaske Thijs , Do Stephanie , March Alexander R , Cho Nathan , Pedroncelli Nico , Lima Ricardo Espinosa , Soto Jennifer , Jackson Brooke , Khademhosseini Ali , Li Song , McCain Megan , Morsut Leonardo 
Published: May 2023

AbstractSynthetic Notch (synNotch) receptors are modular synthetic components that are genetically engineered into mammalian cells to detect signals presented by neighboring cells and respond by activating prescribed transcriptional programs. To date, synNotch has been used to program therapeutic cells and pattern morphogenesis in multicellular systems. However, cell-presented ligands have limited versatility for applications that require spatial precision, such as tissue engineering. To address this, we developed a suite of materials to activate synNotch receptors and serve as generalizable platforms for generating user-defined material-to-cell signaling pathways. First, we demonstrate that synNotch ligands, such as GFP, can be conjugated to cell- generated ECM proteins via genetic engineering of fibronectin produced by fibroblasts. We then used enzymatic or click chemistry to covalently link synNotch ligands to gelatin polymers to activate synNotch receptors in cells grown on or within a hydrogel. To achieve microscale control over synNotch activation in cell monolayers, we microcontact printed synNotch ligands onto a surface. We also patterned tissues comprising cells with up to three distinct phenotypes by engineering cells with two distinct synthetic pathways and culturing them on surfaces microfluidically patterned with two synNotch ligands.We showcase this technology by co-transdifferentiating fibroblasts into skeletal muscle or endothelial cell precursors in user-defined spatial patterns towards the engineering of muscle tissue with prescribed vascular networks. Collectively, this suite of approaches extends the synNotch toolkit and provides novel avenues for spatially controlling cellular phenotypes in mammalian multicellular systems, with many broad applications in developmental biology, synthetic morphogenesis, human tissue modeling, and regenerative medicine.
Benzylisoquinoline alkaloid production in yeast via norlaudanosoline improves selectivity and yield
Authors: Narcross Lauren , Pyne Michael E. , Kevvai Kaspar , Siu Ka-Hei , Dueber John E. , Martin Vincent J. J. 
Published: May 2023

AbstractThe benzylisoquinoline alkaloid (BIA) family of tetrahydroisoquinolines (THIQs) comprises over 2,500 members, including the pharmaceuticals morphine, codeine, and papaverine as well as the antibiotics sanguinarine and chelerythrine used in animal husbandry. Agricultural cultivation can currently supply the demand for the BIAs that accumulate in plants, but broader access to the entire BIA family would open new avenues of research and commercialization. Microbial synthesis presents an attractive option due to cheap feedstock, genetic tractability, and ease of scale-up. Previously we reported titers of the key branch-point BIA (S)-reticuline of 4.6 g/L in yeast, which was achieved through leveraging the Ehrlich pathway 2-oxoacid decarboxylase Aro10 to generate the intermediate 4-hydroxyphenylacetaldehyde (4-HPAA). Here, we establish a superior route to (S)-reticuline by switching the pathway intermediate from 4-HPAA to 3,4-dihydroxyphenylacetaldehyde (3,4-dHPAA) using human monoamine oxidase A (MAO). The resulting (S)-norlaudanosoline route to (S)-reticuline synthesis is more selective, resolving prior issues with off-pathway THIQs synthesized due to concerted enzyme promiscuity. The new pathway is also more efficient, enabling titers of 4.8 g/L (S)-reticuline while improving yields over 40%, from 17 mg/g sucrose to 24 mg/g sucrose in fed-batch fermentations. Finally, we extendde novo(S)-reticuline synthesis to dihydrosanguinarine, achieving 635 mg/L dihydrosanguinarine and sanguinarine in fed-batch fermentation the highest reported titer of these BIAs by a factor of 40.HighlightsMonoamine oxidase A (MAO) supports high-titer (S)-reticuline synthesis in yeastMAO route to (S)-reticuline improves specificity compared to Aro10 routeThis work represents a 40% increase in highest reported (S)-reticuline yield653 mg/L (dihydro-) sanguinarine was produced by extending the pathway
Plasmid-free cheater cells commonly evolve during laboratory growth
Authors: Bedore Amber M. , Waters Christopher M. 
Published: May 2023

AbstractIt has been nearly a century since the isolation and use of penicillin, heralding the discovery of a wide range of different antibiotics. In addition to clinical applications, such antibiotics have been essential laboratory tools, allowing for selection and maintenance of laboratory plasmids that encode cognate resistance genes. However, antibiotic resistance mechanisms can additionally function as public goods. For example, secretion of beta-lactamase from resistant cells, and subsequent degradation of nearby penicillin and related antibiotics, allows neighboring plasmid-free susceptible bacteria to survive antibiotic treatment. How such cooperative mechanisms impact selection of plasmids during experiments in laboratory conditions is poorly understood. Here, we show that the use of plasmid-encoded beta-lactamases leads to significant curing of plasmids in surface grown bacteria. Furthermore, such curing was also evident for aminoglycoside phosphotransferase and tetracycline antiporter resistance mechanisms. Alternatively, antibiotic selection in liquid growth led to more robust plasmid maintenance, although plasmid loss still occurred. The net outcome of such plasmid loss is the generation of a heterogenous population of plasmid-containing and plasmid-free cells, leading to experimental confounds that are not widely appreciated.ImportancePlasmids are routinely used in microbiology as readouts of cell biology or tools to manipulate cell function. Central to these studies is the assumption that all cells in an experiment contain the plasmid. Plasmid maintenance in a host cell typically depends on a plasmid-encoded antibiotic resistance marker, which provides a selective advantage when the plasmid containing cell is grown in the presence of antibiotic. Here we find that growth of plasmid-containing bacteria during laboratory conditions in the presence of three distinct antibiotic families leads to the evolution of a significant number of plasmid-free cells, which rely on the resistance mechanisms of the plasmid-containing cells for viability. This process generates a heterogenous population of plasmid-free and plasmid-containing bacteria, an outcome which could confound further experimentation.
A scalable framework for high-throughput identification of functional origins of replication in non-model bacteria
Authors: Gilbert Charlie , Brumwell Stephanie L. , Crits-Christoph Alexander , Kang Shinyoung Clair , Martin-Moldes Zaira , Alsharif Wajd , Esmurria Ariela , Nguyen Mary-Anne , Lee Henry H. , Ostrov Nili 
Published: May 2023

AbstractMicrobial genetic manipulation requires access to engineerable plasmids that can be programmed to perturb genes, pathways and genomes. The extensive repertoire of plasmids available for model microbes, such asEscherichia coli, has facilitated fundamental biology studies and synthetic biology applications. However, the scarcity of plasmids for non-model microbes hinders efforts to broaden our biological knowledge and constrains the development of biotechnological solutions. In this study, we introduce a molecular toolkit and multiplexed screen to evaluate functional plasmids in non-model microbes. We constructed a collection of genetic parts consisting of 22 origins of replication (ORIs), 20 antibiotic selectable markers, and 30 molecular barcodes, which can be assembled combinatorially to create a library of plasmids trackable by next-generation DNA sequencing. We demonstrate our approach by delivering a pooled library of 22 ORIs to 12 bacterial species including extremophiles, electroactive bacteria and bioproduction strains. We report, for the first time, DNA delivery by conjugation and functional ORIs forHalomonas alkaliphila, Halomonas neptunia,andShewanella electrodiphila. Furthermore, we expand the list of functional ORIs forDuganella zoogloeoides,Pseudomonas alcaliphila,Shewanella oneidensisandShewanella putrefaciens. This screen provides a scalable high-throughput system to rapidly build and identify functional plasmids to establish genetic tractability in non-model microbes.
Tertiary structure of single-instant RNA molecule reveals folding landscape
Authors: Liu Jianfang , McRae Ewan K.S. , Zhang Meng , Geary Cody , Andersen Ebbe Sloth , Ren Gang 
Published: May 2023

AbstractThe folding of RNA and protein molecules during their synthesis is a crucial self-assembly process that nature employs to convert genetic information into the complex molecular machinery that supports life. Misfolding events are the cause of several diseases, and the folding pathway of central biomolecules, such as the ribosome, is strictly regulated by programmed maturation processes and folding chaperones. However, the dynamic folding processes are challenging to study because current structure determination methods heavily rely on averaging, and existing computational methods do not efficiently simulate non-equilibrium dynamics. Here we utilize individual-particle cryo-electron tomography (IPET) to investigate the folding landscape of a rationally designed RNA origami 6-helix bundle that undergoes slow maturation from a “young” to “mature” conformation. By optimizing the IPET imaging and electron dose conditions, we obtain 3D reconstructions of 120 individual particles at resolutions ranging from 23-35 Å, enabling us first-time to observe individual RNA helices and tertiary structures without averaging. Statistical analysis of 120 tertiary structures confirms the two main conformations and suggests a possible folding pathway driven by helix-helix compaction. Studies of the full conformational landscape reveal both trapped states, misfolded states, intermediate states, and fully compacted states. The study provides novel insight into RNA folding pathways and paves the way for future studies of the energy landscape of molecular machines and self-assembly processes.
Application of polystyrene sulfonate (PSS) for inhibiting toxicity of ALS/FTD-linked dipeptide repeats
Authors: Bratek-Skicki Anna , Ahmed Junaid , Jonckheere Karl , Peeters Eveline , Heeren Kara , Volkov Alex , Hendrix Jelle , Batys Piotr , Bosch Ludo Van Den , Tompa Peter 
Published: May 2023

AbstractThe expansion of GGGGCC (G4C2) repeats in the noncoding region of C9orf72 is the most common genetic cause of familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeat region is translated into five different dipeptide repeats (DPRs), of which the arginine-rich DPRs (R-DPRs) poly-GR (GRn) and poly-PR (PRn) are highly neurotoxic and are probably primarily responsible for the disease. Here, we characterized the protective effect against R-DPR toxicity of polystyrene sulfonate (PSS), an FDA-approved drug applied in hyperkalemia, in biochemical, cellular, and animal models of ALS/FTD. We found that PSS, in a length-dependent manner, interacts very tightly with R-DPRs, and releases their bound RNA in R-DPR - RNA mixtures. PSS significantly influences the liquid-liquid phase separation (LLPS) of R-DPRs elicited by RNA and reduces their ensuing cell toxicity in Neuro2a cells. PSS is cell penetrable, and it is also effective in countering the toxicity of R-DPRs in zebrafish embryos. Except for the longest (n = 340) variant, PSS is toxic neither to cells nor to mice upon intracerebroventricular injection up to 1 mM concentration. Our results suggest that its polymeric nature endows PSS with an advantageous effect in C9-ALS/FTD and offers a possible remedy against this debilitating neurodegenerative disease.
Bayesian estimation of gene constraint from an evolutionary model with gene features
Authors: Zeng Tony , Spence Jeffrey P. , Mostafavi Hakhamanesh , Pritchard Jonathan K. 
Published: May 2023

AbstractMeasures of selective constraint on genes have been used for many applications including clinical interpretation of rare coding variants, disease gene discovery, and studies of genome evolution. However, widely-used metrics are severely underpowered at detecting constraint for the shortest ~25% of genes, potentially causing important pathogenic mutations to be over-looked. We developed a framework combining a population genetics model with machine learning on gene features to enable accurate inference of an interpretable constraint metric,shet. Our estimates outperform existing metrics for prioritizing genes important for cell essentiality, human disease, and other phenotypes, especially for short genes. Our new estimates of selective constraint should have wide utility for characterizing genes relevant to human disease. Finally, our inference framework, GeneBayes, provides a flexible platform that can improve estimation of many gene-level properties, such as rare variant burden or gene expression differences.
Iron Memory inE. coli
Authors: Bhattacharyya Souvik , Bhattarai Nabin , Pfannenstiel Dylan M. , Wilkins Brady , Singh Abhyudai , Harshey Rasika M. 
Published: May 2023

AbstractThe importance of memory in bacterial decision-making is relatively unexplored. We show here that a prior experience of swarming is remembered whenE. coliencounters a new surface, improving its future swarming efficiency. We conducted >10,000 single-cell swarm assays to discover that cells store memory in the form of cellular iron levels. This memory pre-exists in planktonic cells, but the act of swarming reinforces it. A cell with low iron initiates swarming early and is a better swarmer, while the opposite is true for a cell with high iron. The swarming potential of a mother cell, whether low or high, is passed down to its fourth-generation daughter cells. This memory is naturally lost by the seventh generation, but artificially manipulating iron levels allows it to persist much longer. A mathematical model with a time-delay component faithfully recreates the observed dynamic interconversions between different swarming potentials. We also demonstrate that iron memory can integrate multiple stimuli, impacting other bacterial behaviors such as biofilm formation and antibiotic tolerance.
Origin of biogeographically distinct ecotypes during laboratory evolution
Authors: Valenzuela Jacob J. , Christinal Immanuel Selva Rupa , Wilson James , Turkarslan Serdar , Ruiz Maryann , Gibbons Sean M. , Hunt Kristopher A. , Auer Manfred , Zemla Marcin , Stahl David A. , Baliga Nitin S. 
Published: May 2023

ABSTRACTResource partitioning within microbial communities is central to their incredible productivity, including over 1 gigaton of annual methane emissions through syntrophic interactions1. Here, we show how isogenic strains of a sulfate reducing bacterium (Desulfovibrio vulgaris, Dv) and a methanogen (Methanococcus maripaludis, Mm) underwent evolutionary diversification over 300-1,000 generations in a purely planktonic environmental2–4context giving rise to coexisting ecotypes that could partition resources and improve overall stability, cooperativity, and productivity in a simulated subsurface environment. We discovered that mutations in just 15 Dv and 7 Mm genes gave rise to ecotypes within each species that were spatially enriched between sediment and planktonic phases over the course of only a few generations after transferring the evolved populations to a fluidized bed reactor (FBR). While lactate utilization by Dv in the attached community was significantly greater, the resulting H2was partially consumed by low affinity hydrogenases in Mm within the same attached phase. The unutilized H2was scavenged by high affinity hydrogenases in the planktonic phase Mm, generating copious amounts of methane and higher ratio of Mm to Dv. Our findings show how a handful of mutations that arise in one environmental context can drive resource partitioning by ecologically differentiated variants in another environmental context, whose interplay synergistically improves productivity of the entire mutualistic community.
Rat microbial biogeography and age-dependent lactic acid bacteria in healthy lungs
Authors: Zhao Lan , Cunningham Christine M. , Andruska Adam M. , Schimmel Katharina , Ali Md Khadem , Kim Dongeon , Gu Shenbiao , Chang Jason L. , Spiekerkoetter Edda , Nicolls Mark R. 
Published: May 2023

AbstractThe laboratory rat emerges as a useful tool for studying the interaction between the host and its microbiome. To advance principles relevant to the human microbiome, we systematically investigated and defined a multi-tissue full lifespan microbial biogeography for healthy Fischer 344 rats. Microbial community profiling data was extracted and integrated with host transcriptomic data from the Sequencing Quality Control (SEQC) consortium. Unsupervised machine learning, Spearman’s correlation, taxonomic diversity, and abundance analyses were performed to determine and characterize the rat microbial biogeography and the identification of four inter-tissue microbial heterogeneity patterns (P1-P4). The 11 body habitats harbor a greater diversity of microbes than previously suspected. Lactic acid bacteria (LAB) abundances progressively declined in lungs from breastfeed newborn to adolescence/adult and was below detectable levels in elderly rats. LAB’s presence and levels in lungs were further evaluated by PCR in the two validation datasets. The lung, testes, thymus, kidney, adrenal, and muscle niches were found to have age-dependent alterations in microbial abundance. P1 is dominated by lung samples. P2 contains the largest sample size and is enriched for environmental species. Liver and muscle samples were mostly classified into P3. Archaea species were exclusively enriched in P4. The 357 pattern-specific microbial signatures were positively correlated with host genes in cell migration and proliferation (P1), DNA damage repair and synaptic transmissions (P2), as well as DNA transcription and cell cycle in P3. Our study established a link between metabolic properties of LAB with lung microbiota maturation and development. Breastfeeding and environmental exposure influence microbiome composition and host health and longevity. The inferred rat microbial biogeography and pattern-specific microbial signatures would be useful for microbiome therapeutic approaches to human health and good quality of life.Graphical Abstract