bluuebunny/crossref_metadata_2025_split huggingface dataset:
Discovery of Novel Replication Proteins for Large Plasmids in Cyanobacteria and Their Potential Applications in Genetic Engineering
Authors: Ohdate Kazuma , Sakata Minori , Maeda Kaisei , Sakamaki Yutaka , Nimura-Matsune Kaori , Ohbayashi Ryudo , Hess Wolfgang R. , Watanabe Satoru 
Published: October 2023

AbstractNumerous cyanobacteria capable of oxygenic photosynthesis possess multiple large plasmids exceeding 100 kbp in size. These plasmids are believed to have distinct replication and distribution mechanisms, as they coexist within cells without causing incompatibilities between plasmids. However, information on Rep proteins necessary for plasmid replication initiation in cyanobacteria is limited.Synechocystissp. PCC 6803 hosts four large plasmids, pSYSM, pSYSX, pSYSA, and pSYSG, but Rep proteins for these plasmids, except for CyRepA1 on pSYSA, are unknown. Using Autonomous Replication sequencing (AR-seq), we identified two potential Rep genes inSynechocystis6803,slr6031andslr6090, both located on pSYSX. The corresponding Rep candidates, Slr6031 and Slr6090, share structural similarities with Rep-associated proteins of other bacteria and homologs were also identified in various cyanobacteria. We observed autonomous replication activity for Slr6031 and Slr6090 inSynechococcus elongatusPCC 7942 by fusing their genes with a construct expressing GFP and introducing them via transformation. Theslr6031/slr6090-containing plasmids exhibited lower copy numbers and instability inSynechococcus7942 cells compared to the expression vector pYS. While recombination occurred in the case ofslr6090, the engineered plasmid withslr6031coexisted with plasmids encoding CyRepA1 or Slr6090 inSynechococcus7942 cells, indicating the compatibility of Slr6031 and Slr6090 with CyRepA1. Based on these results, we designated Slr6031 and Slr6090 as CyRepX1 (CyanobacterialRep-related protein encoded on pSYSX) and CyRepX2, respectively, demonstrating that pSYSX is a plasmid with “two Reps in one plasmid”. Furthermore, we determined the copy number and stability of plasmids with cyanobacterial Reps inSynechococcus7942 andSynechocystis6803 to elucidate their potential applications. The novel properties of CyRepX1 and 2, as revealed by this study, hold promise for the development of innovative genetic engineering tools in cyanobacteria.
Optimization for High-Throughput BiFC Screening
Authors: Jia Yunlong , Reboulet Jonathan , Bleicher Françoise , Dumont Agnès , Ruscio Sylvie Di , Gillet Benjamin , Hughes Sandrine , Merabet Samir 
Published: October 2023

AbstractThe Cell-PCA screen, since its inception, has provided an efficient method for analyzing cellular interactomes and has been used in various biological studies involving proteins like MYC, PER2, and ERK. With rapid advancements in biotechnology, including tools for protein function investigation, the Cell-PCA screen remains relevant. However, despite its successful application in recent studies, there are areas for optimization to ensure its continued relevance in the face of evolving technological advancements.
An in vitro platform for quantifying cell cycle phase lengths in primary human intestinal stem cells
Authors: Cotton Michael J , Ariel Pablo , Chen Kaiwen , Walcott Vanessa A , Dixit Michelle , Breau Keith A , Hinesley Caroline M , Kedziora Kasia , Tang Cynthia Y , Zheng Anna , Magness Scott T , Burclaff Joseph 
Published: October 2023

ABSTRACTBackground and AimsThe intestinal epithelium exhibits dynamic control of cell cycle phase lengths, yet no experimental platform exists for directly analyzing cell cycle phases in living human intestinal stem cells (ISCs). Here, we develop primary human ISC lines with two different reporter constructs to provide fluorescent readouts to analyze cell cycle phases in cycling ISCs.Methods3D printing was used to construct a collagen press for making chamber slides that support primary human ISC growth and maintenance within the working distance of a confocal microscope objective. The PIP-FUCCI fluorescent cell cycle reporter and a variant with H2A-mScarlet that allows for automated tracking of cell cycle phases (PIP-H2A) were used in human ISCs along with live imaging and EdU pulsing. An analysis pipeline combining free-to-use programs and publicly available code was compiled to analyze live imaging results.ResultsChamber slides with soft collagen pressed to a thickness of 0.3 mm concurrently support ISC cycling and confocal imaging. PIP-FUCCI ISCs were found to be optimal for snapshot analysis wherein all nuclei are assigned to a cell cycle phase from a single image. PIP-H2A ISCs were better suited for live imaging since constant nuclear signal allowed for more automated analysis. CellPose2 and TrackMate were used together to track cycling cells.ConclusionsWe present two complete platforms for analyzing cell cycle phases in living primary human ISCs. The PIP-FUCCI construct allows for cell cycle phase assignment from one image of living cells, the PIP-H2A construct allows for semi-automated direct quantification of cell cycle phase lengths in human ISCs using our computational pipeline. These platforms hold great promise for future studies on how pharmaceutical agents affect the intestinal epithelium, how cell cycle is regulated in human ISCs, and more.
A Suite of Designed Protein Cages Using Machine Learning Algorithms and Protein Fragment-Based Protocols
Authors: Meador Kyle , Castells-Graells Roger , Aguirre Roman , Sawaya Michael R. , Arbing Mark A. , Sherman Trent , Senarathne Chethaka , Yeates Todd O. 
Published: October 2023

AbstractDesigned protein cages and related materials provide unique opportunities for applications in biotechnology and medicine, while methods for their creation remain challenging and unpredictable. In the present study, we apply new computational approaches to design a suite of new tetrahedrally symmetric, self-assembling protein cages. For the generation of docked poses, we emphasize a protein fragment-based approach, while forde novointerface design, a comparison of computational protocols highlights the power and increased experimental success achieved using the machine learning program ProteinMPNN. In relating information from docking and design, we observe that agreement between fragment-based sequence preferences and ProteinMPNN sequence inference correlates with experimental success. Additional insights for designing polar interactions are highlighted by experimentally testing larger and more polar interfaces. In all, using X-ray crystallography and cryo-EM, we report five structures for seven protein cages, with atomic resolution in the best case reaching 2.0 Å. We also report structures of two incompletely assembled protein cages, providing unique insights into one type of assembly failure. The new set of designed cages and their structures add substantially to the body of available protein nanoparticles, and to methodologies for their creation.
Studentsourcing - aggregating and re-using data from a practical cell biology course
Authors: Goedhart Joachim 
Published: October 2023

AbstractPractical courses mimic experimental research and may generate valuable data. Yet, data that is generated by students during a course is often lost as there is no centrally organized collection and storage of the data. The loss of data prevents its re-use. To provide access to these data, I present an approach that I call studentsourcing. It collects, aggregates and reuses data that is generated by students in a practical course on cell biology. The course runs annually, and I have recorded the data that was generated by >100 students over 3 years. Two use cases illustrate how the data can be aggregated and re-used either for the scientific record or for teaching. As the data is obtained by different students, in different groups, over different years, it is an excellent opportunity to discuss experimental design and modern data visualization methods such as the superplot. The first use case demonstrates how the data can be presented as an online, interactive dashboard, providing real-time data of the measurements. The second use case shows how central data storage provides a unique opportunity to get precise quantitative data due to the large sample size. Both use cases illustrate how data can be effectively aggregated and re-used.Abstract Figure
HSQC2STRUC: A Machine Learning Model for Protein Secondary Structure Prediction using Unassigned NMR Spectra
Authors: Dietrich Jonas , Bellstedt Peter 
Published: October 2023

AbstractDynamic changes in the secondary structure content of proteins can provide valuable insights into protein function or dysfunction. Predicting these dynamic changes is still a significant challenge but is of paramount importance for basic research as well as drug development. Here, we present a machine learning-based model that predicts the secondary structure content of proteins based on their un assigned1H,15N-HSQC NMR spectra with an RMSE of 0.11 forα-helix, 0.08 forβ-sheet and 0.12 for random coil content. Our model has been implemented into an easy-to-use and publicly available web service that estimates secondary structure content based on a provided peak list. Furthermore, a Python version is provided, ready to be integrated into Bruker’s TopSpin software or own scripts.
ReMASTER: Improved phylodynamic simulation for BEAST 2.7
Authors: Vaughan Timothy G. 
Published: October 2023

AbstractSummaryPhylodynamic models link phylogenetic trees to biologically-relevant parameters such as speciation and extinction rates (macroevolution), effective population sizes and migration rates (ecology and phylogeography), and transmission and removal/recovery rates (epidemiology) to name a few. Being able to simulate phylogenetic trees and population dynamics under these models is the basis for (a) developing and testing of phylodynamic inference algorithms, (b) performing simulation studies which quantify the biases stemming from model-misspecification, and (c) performing so-called model adequacy assessments by simulating samples from the posterior predictive distribution. Here I introduceReMASTER, a package for the BEAST 2 phylogenetic inference platform which provides a simple and efficient approach to specifying and simulating the phylogenetic trees and population dynamics arising from phylodynamic models. ReMASTER is a complete rewrite of an earlier package, MASTER, and boasts improved efficiency, ease of use, flexibility of model specification, and integration with BEAST 2.Availability and ImplementationReMASTER can be installed directly from the BEAST 2 package manager, and its documentation is available online athttps://tgvaughan.github.io/remaster. ReMASTER is free software, and is distributed under version 3 of the GNU General Public License. The Java source code for ReMASTER is available fromhttps://github.com/tgvaughan/remaster.
Intraspecific plant-soil feedbacks vary under field conditions among species in a tropical tree community
Authors: Eck Jenalle L. , Hassan Lourdes Hernández , Comita Liza S. 
Published: October 2023

AbstractPremiseSoil microbes can influence patterns of diversity in plant communities via plant-soil feedbacks. Intraspecific plant-soil feedbacks occur when plant genotype causes variation in soil microbial composition, resulting in differences in the performance of seedlings growing near their maternal plants versus seedlings growing near non-maternal conspecific plants. How commonly such intraspecific plant-soil feedbacks occur in natural plant communities is unclear, especially under variable field conditions.MethodsWe conducted an in situ experiment with four native tree species on Barro Colorado Island (BCI), Panama. Seedlings of each species were transplanted beneath their maternal tree or another conspecific tree in the BCI forest. Mortality and growth were assessed at the end of the wet season (∼4 months post-transplant) and at the end of the experiment (∼7 months post-transplant).ResultsPatterns of seedling performance varied among species and were inconsistent over time. Significant effects of field environment were detected for two of the four species: at the end of the wet season,Virola surinamensisseedlings had higher survival beneath their maternal tree than other conspecific trees, while the opposite pattern was found inOrmosia macrocalyx.However, these differences disappeared by the end of the experiment.ConclusionsOur results suggest that intraspecific plant-soil feedbacks occur inconsistently under field conditions in tropical tree species and may have a limited role in determining seedling performance in tropical tree communities. Future studies are needed to elucidate the environmental and genetic factors that determine the incidence and direction of intraspecific plant-soil feedbacks in plant communities.
HIPSD&R-seq enables scalable genomic copy number and transcriptome profiling
Authors: Lazareva Olga , Mallm Jan-Philipp , Simovic-Lorenz Milena , Philippos George , Sant Pooja , Parekh Urja , Hammann Linda , Li Albert , Yildiz Umut , Marttinen Mikael , Zaugg Judith , Noh Kyung Min , Stegle Oliver , Ernst Aurélie 
Published: October 2023

AbstractSingle-cell DNA-sequencing (scDNA-seq) enables decoding somatic cancer variation. Existing methods are hampered by low throughput or cannot be combined with transcriptome sequencing in the same cell. We propose HIPSD&R-seq (HIgh-throughPutSingle-cellDna andRna-seq), a scalable yet simple assay to profile low-coverage DNA and RNA in thousands of cells in parallel. Our approach builds on an accessible modification of the 10X Genomics platform for scATAC and multiome profiling. In applications to human cell models and primary tissue, we demonstrate the feasibility to detect rare clones and we combine the assay with combinatorial indexing to profile over 16,000 cells.
Structural Insights into the Interaction Between Adenovirus C5 Hexon and Human Lactoferrin
Authors: Dhillon Arun , Persson B. David , Volkov Alexander N. , Sülzen Hagen , Kádek Alan , Pompach Petr , Kereïche Sami , Lepšík Martin , Danskog Katarina , Uetrecht Charlotte , Arnberg Niklas , Zoll Sebastian 
Published: October 2023

ABSTRACTAdenovirus (AdV) infection of the respiratory epithelium is common, but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon’s hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development.
Bsp1, a fungal CPI motif protein, regulates actin filament capping in endocytosis and cytokinesis
Authors: Hummel Daniel R. , Hakala Markku , Toret Christopher P. , Kaksonen Marko 
Published: October 2023

AbstractThe capping of barbed filament ends is a fundamental mechanism for actin regulation. Capping protein controls filament growth and actin turnover in cells by binding to the barbed ends of the filaments with high affinity and slow off-rate. The interaction between Capping protein and actin is regulated by capping protein interaction (CPI) motif proteins. We identified a novel CPI motif protein, Bsp1, which is involved in cytokinesis and endocytosis in budding yeast. We demonstrate that Bsp1 is an actin binding protein with a high affinity for capping protein via its CPI motif. In cells, Bsp1 regulates capping protein at endocytic sites and is a major recruiter of capping protein to the cytokinetic actin ring. Lastly, we define Bsp1-related proteins as a distinct fungi-specific CPI protein group. Our results suggest that Bsp1 promotes actin filament capping by the capping protein. This study establishes Bsp1 as a new capping protein regulator and promising candidate to regulate actin networks in fungi.
G protein subunit Gγ13-mediated signaling pathway is critical to the inflammation resolution and functional recovery of severely injured lungs
Authors: Li Yi-Hong , Yang Yi-Sen , Xue Yan-Bo , Lei Hao , Zhang Sai-Sai , Qian Junbin , Yao Yushi , Zhou Ruhong , Huang Liquan 
Published: October 2023

AbstractTuft cells are a group of rare epithelial cells that can detect pathogenic microbes and parasites. Many of these cells express signaling proteins initially found in taste buds. It is, however, not well understood how these taste signaling proteins contribute to the response to the invading pathogens or to the recovery of injured tissues. In this study, we conditionally nullified the signaling G protein subunit Gγ13 and found that the number of ectopic tuft cells in the injured lung was reduced following the infection of the influenza virus H1N1. Furthermore, the infected mutant mice exhibited significantly larger areas of lung injury, increased macrophage infiltration, severer pulmonary epithelial leakage, augmented pyroptosis and cell death, greater bodyweight loss, slower recovery, worsened fibrosis and increased fatality. Our data demonstrate that the Gγ13-mediated signal transduction pathway is critical to tuft cells-mediated inflammation resolution and functional repair of the damaged lungs.To our best knowledge, it is the first report indicating subtype-specific contributions of tuft cells to the resolution and recovery.
Visualizing liquid-liquid phase transitions
Authors: Sahoo Bikash R. , Deng Xiexiong , Wong Ee Lin , Clark Nathan , Yang Harry , Subramanian Vivekanandan , Guzman Bryan B. , Harris Sarah E. , Dehury Budheswar , Miyashita Emi , Hoff J. Damon , Kocaman Vojč , Saito Hirohide , Dominguez Daniel , Plavec Janez , Bardwell James C.A. 
Published: October 2023

AbstractLiquid-liquid phase condensation governs a wide range of protein-protein and protein-RNA interactions in vivo and drives the formation of membrane-less compartments such as the nucleolus and stress granules. We have a broad overview of the importance of multivalency and protein disorder in driving liquid-liquid phase transitions. However, the large and complex nature of key proteins and RNA components involved in forming condensates such as stress granules has inhibited a detailed understanding of how condensates form and the structural interactions that take place within them. In this work, we focused on the small human SERF2 protein. We show here that SERF2 contributes to the formation of stress granules. We also show that SERF2 specifically interacts with non-canonical tetrahelical RNA structures called G-quadruplexes, structures which have previously been linked to stress granule formation. The excellent biophysical amenability of both SERF2 and RNA G4 quadruplexes has allowed us to obtain a high-resolution visualization of the multivalent protein-RNA interactions involved in liquid-liquid phase transitions. Our visualization has enabled us to characterize the role that protein disorder plays in these transitions, identify the specific contacts involved, and describe how these interactions impact the structural dynamics of the components involved in liquid-liquid phase transitions, thus enabling a detailed understanding of the structural transitions involved in early stages of ribonucleoprotein condensate formation.
Emergent Antibiotic Persistence in Cross-Feeding Bacteria in Structured Habitats
Authors: Xiong Xianyi , Othmer Hans G. , Harcombe William R. 
Published: October 2023

AbstractAntibiotic persistence allows a sub-population of bacteria to survive antibiotic-induced killing and contributes to the evolution of antibiotic resistance. Although bacteria typically live in microbial communities with complex ecological interactions, little is known about how microbial ecology affects antibiotic persistence. Here, we demonstrated that the combination of cross-feeding and community spatial structure can emergently cause high antibiotic persistence in bacteria by increasing the cell-cell heterogeneity in the metabolic state both during growth and antibiotic killing. We tracked the ampicillin-induced death on agar surfaces in a model obligate mutualism ofEscherichia coliandSalmonella enterica. We found thatE. coliformed ∼100-fold more antibiotic persisters in the cross-feeding coculture than in monoculture. This high persistence could not be explained solely by the presence ofS. enterica, the presence of cross-feeding, or the growth rate differences between the mono- and co-cultures. Time-series fluorescent microscopy revealed increased cell-cell variation inE. colilag time in the mutualistic co-culture. Furthermore, we discovered that anE. colicell can survive antibiotic killing if the nearbyS. entericacells on which it relies die first—a mechanism we termed “dynamical loss of access to nutrient.” In conclusion, we showed that the high antibiotic persistence phenotype can be an emergent phenomenon caused by a combination of cross-feeding and spatial structure. Our work highlights the importance of considering spatially-structured interactions during antibiotic treatment and to understand microbial community resilience more broadly.
Proximity proteomics reveals UCH-L1 as an NLRP3 interactor that modulates IL-1β production in human macrophages and microglia
Authors: Liang Zhu , Damianou Andreas , Vendrell Iolanda , Jenkins Edward , Lassen Frederik H. , Washer Sam J , Liu Guihai , Yi Gangshun , Lou Hantao , Cao Fangyuan , Zheng Xiaonan , Fernandes Ricardo A. , Dong Tao , Tate Edward W. , Daniel Elena Di , Kessler Benedikt M 
Published: October 2023

ABSTRACTActivation of the NACHT, LRR family pyrin domain containing 3 (NLRP3) inflammasome complex is an essential innate immune signalling mechanism. To reveal how human NLRP3 inflammasome assembly and activation are controlled, in particular by components of the ubiquitin system, proximity labelling, affinity purification and RNAi screening approaches were performed. Our study provides an intricate time-resolved molecular map of different phases of NLRP3 inflammasome activation. Also, we show that ubiquitin C-terminal hydrolase 1 (UCH-L1) interacts with the NACHT domain of NLRP3. Downregulation of UCH-L1 decreases pro-IL-1β levels. UCH-L1 chemical inhibition with small molecules interfered with NLRP3 puncta formation and ASC oligomerization, leading to altered IL-1β cleavage and secretion, particularly in microglia cells, which exhibited elevated UCH-L1 expression as compared to monocytes/macrophages. Altogether, we profiled NLRP3 inflammasome activation dynamics and highlight UCH-L1 as an important modulator of NLRP3-mediated IL-1β production, suggesting that a pharmacological inhibitor of UCH-L1 may decrease inflammation-associated pathologies.