bluuebunny/crossref_metadata_2025_split huggingface dataset:
Arrayedin vivobarcoding for multiplexed sequence verification of plasmid DNA and demultiplexing of pooled libraries
Authors: Li Weiyi , Miller Darach , Liu Xianan , Tosi Lorenzo , Chkaiban Lamia , Mei Han , Hung Po-Hsiang , Parekkadan Biju , Sherlock Gavin , Levy Sasha F 
Published: October 2023

AbstractSequence verification of plasmid DNA is critical for many cloning and molecular biology workflows. To leverage high-throughput sequencing, several methods have been developed that add a unique DNA barcode to individual samples prior to pooling and sequencing. However, these methods require an individual plasmid extraction and/orin vitrobarcoding reaction for each sample processed, limiting throughput and adding cost. Here, we develop an arrayedin vivoplasmid barcoding platform that enables pooled plasmid extraction and library preparation for Oxford Nanopore sequencing. This method has a high accuracy and recovery rate, and greatly increases throughput and reduces cost relative to other plasmid barcoding methods or Sanger sequencing. We usein vivobarcoding to sequence verify >45,000 plasmids and show that the method can be used to transform error-containing dispersed plasmid pools into sequence-perfect arrays or well-balanced pools.In vivobarcoding does not require any specialized equipment beyond a low-overhead Oxford Nanopore sequencer, enabling most labs to flexibly process hundreds to thousands of plasmids in parallel.
“Fluorescent minihelix assay for translation quantification in high-throughput cell-free systems”
Authors: Willi Jessica A , Jewett Michael C 
Published: October 2023

AbstractWe report the use of an optimized tetra-cysteine minihelix both as a fusion protein and as a standalone reporter in vitro with the Flash dye to study cell-free protein expression dynamics and engineered ribosome activity. The fluorescent product can be detected and quantified via its characteristic emission spectrum in a standard 96/384-well plate reader, RT-qPCR system, or gel electrophoresis. The fluorescent reporter helix is short enough to be encoded on a primer pair and can tag any protein of interest via PCR. Both tagged protein or standalone reporter can be detected in real time during and in terminal cell-free expression reactions, or in gel, without the need for staining. The fluorescent signal is stable and linearly correlates with protein concentration, thus is suitable for product quantification. Finally, we demonstrate how this reporter can be used for future efforts in engineering in vitro translation systems.Graphical abstract
The H3.3 K36M oncohistone disrupts the establishment of epigenetic memory through loss of DNA methylation
Authors: Sinha Joydeb , Nickels Jan F. , Thurm Abby R. , Ludwig Connor H. , Archibald Bella N. , Hinks Michaela M. , Wan Jun , Fang Dong , Bintu Lacramioara 
Published: October 2023

SummaryHistone H3.3 is frequently mutated in cancers, with the lysine 36 to methionine mutation (K36M) being a hallmark of chondroblastomas. While it is known that H3.3K36M changes the cellular epigenetic landscape, it remains unclear how it affects the dynamics of gene expression. Here, we use a synthetic reporter to measure the effect of H3.3K36M on silencing and epigenetic memory after recruitment of KRAB: a member of the largest class of human repressors, commonly used in synthetic biology, and associated with H3K9me3. We find that H3.3K36M, which decreases H3K36 methylation, leads to a decrease in epigenetic memory and promoter methylation weeks after KRAB release. We propose a new model for establishment and maintenance of epigenetic memory, where H3K36 methylation is necessary to convert H3K9me3 domains into DNA methylation for stable epigenetic memory. Our quantitative model can inform oncogenic mechanisms and guide development of epigenetic editing tools.
NMR analysis of the homodimeric structure of a D-Ala-D-Ala metallopeptidase, VanX, from vancomycin-resistant bacteria
Authors: Konuma Tsuyoshi , Takai Tomoyo , Tsuchiya Chieko , Nishida Masayuki , Hashiba Miyu , Yamada Yudai , Oe Ayaka , Shirai Haruka , Nagadoi Aritaka , Chikaishi Eriko , Akagi Ken-ichi , Akashi Satoko , Yamazaki Toshio , Akutsu Hideo , Ikegami Takahisa 
Published: October 2023

AbstractBacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin-resistant enterococci was a tremendous shock considering that vancomycin is the last resort for controlling methicillin-resistantStaphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, which is typically considered too large for NMR studies, we successfully assigned the main chain using an amino acid-selective unlabeling method. Because we found that the Zn2+-coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by mutating the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR.
Refining the transcriptional landscapes for distinct clades of virulent phages infectingpseudomonas aeruginosa
Authors: Putzeys Leena , Wicke Laura , Boon Maarten , Noort Vera van , Vogel Jörg , Lavigne Rob 
Published: October 2023

AbstractThe introduction of high-throughput sequencing has resulted in a surge of available bacteriophage genomes, unveiling their tremendous genomic diversity. However, our current understanding of the complex transcriptional mechanisms that dictate their gene expression during infection is limited to a handful of model phages. Here, we applied ONT-cappable-seq to reveal the transcriptional architecture of six different clades of virulent phages infectingPseudomonas aeruginosa. This long-read microbial transcriptomics approach is tailored to globally map transcription start and termination sites, transcription units and putative RNA-based regulators on dense phage genomes. Specifically, the full-length transcriptomes of LUZ19, LUZ24, 14-1, YuA, PAK_P3 and giant phage phiKZ during early, middle and late infection were collectively charted. Beyond pinpointing traditional promoter and terminator elements and transcription units, these transcriptional profiles provide insights in transcriptional attenuation and splicing events and allow straightforward validation of Group I intron activity. In addition, ONT-cappable-seq data can guide genome-wide discovery of novel regulatory element candidates, including non-coding RNAs and riboswitches. This work substantially expands the number of annotated phage-encoded transcriptional elements identified to date, shedding light on the intricate and diverse gene expression regulation mechanisms inPseudomonasphages, which can ultimately be sourced as tools for biotechnological applications in phage and bacterial engineering.
The V-ATPase-ATG16L1 axis recruits LRRK2 to facilitate lysosomal stress responses
Authors: Eguchi Tomoya , Sakurai Maria , Wang Yingxue , Saito Chieko , Yoshii Gen , Wileman Thomas , Mizushima Noboru , Kuwahara Tomoki , Iwatsubo Takeshi 
Published: October 2023

AbstractLeucine-rich repeat kinase 2 (LRRK2), a Rab kinase associated with Parkinson’s disease and several inflammatory diseases, has been shown to localize to stressed lysosomes and get activated to regulate lysosomal homeostasis. However, the mechanisms of LRRK2 recruitment and activation have not been well understood. Here we found that the ATG8 conjugation system regulates the recruitment of LRRK2 as well as LC3 onto single membranes of stressed lysosomes/phagosomes. This recruitment did not require FIP200-containing autophagy initiation complex, nor did it occur on double-membrane autophagosomes, suggesting independence from canonical autophagy. Consistently, LRRK2 recruitment was regulated by the V-ATPase-ATG16L1 axis, which requires the WD40 domain of ATG16L1 and specifically mediates ATG8 lipidation on single membranes. This mechanism was also responsible for the lysosomal stress-induced activation of LRRK2 and the resultant regulation of lysosomal secretion and enlargement. These results indicate that the V-ATPase-ATG16L1 axis serves a novel non-autophagic role in the maintenance of lysosomal homeostasis by recruiting LRRK2.
High-speed imaging of giant unilamellar vesicle formation in cDICE
Authors: Van de Cauter Lori , Jawale Yash K. , Tam Daniel , Baldauf Lucia , van Buren Lennard , Koenderink Gijsje H. , Dogterom Marileen , Ganzinger Kristina A. 
Published: October 2023

AbstractGiant unilamellar vesicles (GUVs) are widely used asin vitromodel membranes in biophysics and as cell-sized containers in synthetic biology. Despite their ubiquitous use, there is no one-size-fits-all method for their production. Numerous methods have been developed to meet the demanding requirements of reproducibility, reliability, and high yield, while simultaneously achieving robust encapsulation. Emulsion-based methods are often praised for their apparent simplicity and good yields; hence, methods like continuous droplet interface crossing encapsulation (cDICE) that make use of this principle, have gained popularity. However, the underlying physical principles governing the formation of GUVs in cDICE and related methods remain poorly understood. To this end, we have developed a high-speed microscopy setup that allows us to visualize GUV formation in real-time. Our experiments reveal a complex droplet formation process occurring at the capillary orifice, generating both larger droplets and, likely, GUV-sized satellite droplets. According to existing theoretical models, the oil-water interface should allow for crossing of all droplets, but based on our observations and theoretical modelling of the fluid dynamics within the system, we find a size-selective crossing of GUV-sized droplets only. Finally, we demonstrate that proteins in the inner solution affect GUV formation by increasing the viscosity and altering lipid adsorption kinetics. These results will not only contribute to a better understanding of GUV formation processes in cDICE, but ultimately also aid the development of more reliable and efficient methods for GUV production.Graphical abstractWe developed a high-speed microscopy setup to study giant unilamellar vesicle formation in cDICE, revealing a complex droplet formation process occurring at the capillary orifice and size-selectivity at the oil-water interface.
Unlocking the Mycorrhizal Nitrogen Pathway Puzzle: Metabolic Modelling and multi-omics unveil Pyrimidines’ Role in Maize Nutrition via Arbuscular Mycorrhizal Fungi Amidst Nitrogen Scarcity
Authors: Decouard Bérengère , Chowdhury Niaz Bahar , Saou Aurélien , Rigault Martine , Quilleré Isabelle , Sapir Thomas , Marmagne Anne , le Roux Christine Paysant , Launay-Avon Alexandra , Guerard Florence , Mauve Caroline , Gakière Bertrand , Lévy-Leduc Céline , Barbillon Pierre , Courty Pierre-Emmanuel , Wipf Daniel , Hirel Bertrand , Saha Rajib , Dellagi Alia 
Published: October 2023

AbstractMaize is currently the most productive cereal crop in the world (www.faostat.org). Maize can form a symbiotic relationship with the Arbuscular Mycorrhizal Fungus (AMF),Rhizophagus irregularis. In this relationship, the fungus provides the plant with additional water and mineral nutrients, while the plant supplies carbon compounds to the fungus. Little is known about the N metabolism disruption during symbiosis in both partners. To address this issue, two genetically distant maize lines were studied in terms of physiological and molecular responses to AMF inoculation by dual RNA-seq, metabolomics and phenotyping. Interestingly, the beneficial effects of the AMF were observed mainly under conditions of limited N fertilization. Under such conditions, the AMF helped maintain plant biomass production. The availability of nitrogen was found to be a crucial factor influencing all the traits studied showing that the level of N supply plays a pivotal role in determining how maize plants interact with the AMF. Despite the two maize lines showing different transcriptomic and metabolomic responses toR. irregularis, their agro-physiological traits remained similar. Both the plant and fungal transcriptomes were more significantly influenced by the level of N nutrition rather than the specific maize genotype. This suggests that N availability has a more profound impact on gene expression in both organisms than the genetic makeup of the maize plant. To understand the metabolic implications of this symbiotic relationship, we integrated transcriptomic data into our recently built multi-organ Genome-scale metabolic model (GSM) called iZMA6517. Remarkably, this modelling approach was supported by metabolomics profiling, in particular increased leaf pyrimidine levels in response to AMF inoculation under limiting N supply. Consistently, fungal genes involved in pyrimidine de novo synthesis and salvage were found to be expressed in symbiotic roots. Our work highlights nucleotide and ureides metabolism as previously unrecognized factors contributing to the symbiotic N nutrition facilitated byR. irregularis, thereby enhancing maize growth. This study demonstrates the effectiveness of integrating multi-omics approaches with mathematical modelling to uncover novel metabolic mechanisms associated with AM symbiosis, without a priori.
A singleMicrocoleusspecies causes benthic cyanotoxic blooms worldwide
Authors: Junier Pilar , Cailleau Guillaume , Fatton Mathilda , Udriet Pauline , Hashmi Isha , Bregnard Danae , Corona-Ramirez Andrea , di Francesco Eva , Kuhn Thierry , Mangia Naïma , Zhioua Sami , Hunkeler Daniel , Bindschedler Saskia , Sieber Simon , Gonzalez Diego 
Published: October 2023

AbstractRecently, proliferations of benthic cyanobacteria producing derivatives of anatoxin-a have been reported in rivers all over the world. In three river systems, in New Zealand, the USA, and Canada, a cohesive cluster ofMicrocoleusstrains was responsible for toxin production. Here, we document a similar toxigenic event that occurred at the mouth of the river Areuse in lake Neuchâtel (Switzerland) and caused the death of several dogs. Using 16S RNA-based community analysis, we show that riverine benthic communities are dominated by Oscillatoriales and especially byMicrocoleusstrains. We correlate the detection of one sequence variant with the presence of anatoxin-a derivatives and use metagenomics to assemble a complete circular genome of the strain. The strain is distinct from the ones isolated in New Zealand, the USA, and Canada, but belongs to the same species; it shares significant traits with them, in particular a relatively small genome and incomplete vitamin biosynthetic pathways. Overall, our results suggest that the major anatoxin-a-associated benthic proliferations worldwide can be traced back to a single ubiquitous species,Microcoleus anatoxicus,rather than to a diversity of cyanobacterial lineages. We recommend that this species be monitored internationally in order to help predict and mitigate similar cyanotoxic events.
PARP10 is Critical for Stress Granule Initiation
Authors: Jayabalan Aravinth Kumar , Bhambhani Krishna , L Leung Anthony K 
Published: October 2023

ABSTRACTStress granules (SGs) are cytoplasmic biomolecular condensates enriched with RNA, translation factors, and other proteins. They form in response to stress and are implicated in various diseased states including viral infection, tumorigenesis, and neurodegeneration. Understanding the mechanism of SG assembly, particularly its initiation, offers potential therapeutic avenues. Although ADP-ribosylation plays a key role in SG assembly, and one of its key forms—poly(ADP-ribose) or PAR—is critical for recruiting proteins to SGs, the specific enzyme responsible remains unidentified. Here, we systematically knock down the human ADP-ribosyltransferase family and identify PARP10 as pivotal for SG assembly. Live-cell imaging reveals PARP10’s crucial role in regulating initial assembly kinetics. Further, we pinpoint the core SG component, G3BP1, as a PARP10 substrate and find that PARP10 regulates SG assembly driven by both G3BP1 and its modeled mechanism. Intriguingly, while PARP10 only adds a single ADP-ribose unit to proteins, G3BP1 is PARylated, suggesting its potential role as a scaffold for protein recruitment. PARP10 knockdown alters the SG core composition, notably decreasing translation factor presence. Based on our findings, we propose a model in which ADP-ribosylation acts as a rate-limiting step, initiating the formation of this RNA-enriched condensate.HIGHLIGHTSPARP10 plays a crucial role in the initial SG assembly kinetics.The core SG component G3BP1 is a substrate of PARP10.PARP10 is required for SG assembly mediated by G3BP1 or its synthetic mimic.PARP10 knockdown reduces the levels of translation factors within the SG core.
Structure of the endosomal CORVET tethering complex
Authors: Shvarev Dmitry , König Caroline , Susan Nicole , Langemeyer Lars , Walter Stefan , Perz Angela , Fröhlich Florian , Ungermann Christian , Moeller Arne 
Published: October 2023

AbstractCells depend on their endolysosomal system for nutrient uptake and downregulation of plasma membrane proteins. These processes rely on endosomal maturation, which requires multiple membrane fusion steps. Early endosome fusion is promoted by the Rab5 GTPase and its effector, the hexameric CORVET tethering complex, which is homologous to the lysosomal HOPS. How these related complexes recognize their specific target membranes remains entirely elusive. Here, we solved the structure of CORVET by cryo-electron microscopy and revealed its minimal requirements for membrane tethering. As expected, the core of CORVET and HOPS resembles each other. However, the function-defining subunits show marked structural differences. Notably, we discovered that unlike HOPS, CORVET depends not only on Rab5 but also on phosphatidylinositol-3-phosphate (PI3P) and membrane packaging defects for tethering, implying that an organelle-specific membrane code enables fusion. Our data suggest that both shape and membrane interactions of CORVET and HOPS are conserved in metazoans, thus providing a paradigm how tethering complexes function.One-Sentence SummaryUsing cryo-EM, we solved the structure of the CORVET tethering complex and explained how it promotes endosome fusion.
Cnot3is required for male germ cell development and spermatogonial stem cell maintenance
Authors: Chen Qing , Malki Safia , Xu Xiaojiang , Bennett Brian , Lackford Brad L. , Kirsanov Oleksandr , Geyer Christopher B. , Hu Guang 
Published: October 2023

SummaryThe foundation of spermatogenesis and lifelong fertility is provided by spermatogonial stem cells (SSCs). SSCs divide asymmetrically to either replenish their numbers (self-renewal) or produce undifferentiated progenitors that proliferate before committing to differentiation. However, regulatory mechanisms governing SSC maintenance are poorly understood. Here, we show that the CCR4-NOT mRNA deadenylase complex subunit CNOT3 plays a critical role in maintaining spermatogonial populations in mice.Cnot3is highly expressed in undifferentiated spermatogonia, and its deletion in spermatogonia resulted in germ cell loss and infertility. Single cell analyses revealed thatCnot3deletion led to the de-repression of transcripts encoding factors involved in spermatogonial differentiation, including those in the glutathione redox pathway that are critical for SSC maintenance. Together, our study reveals that CNOT3 – likely via the CCR4-NOT complex – actively degrades transcripts encoding differentiation factors to sustain the spermatogonial pool and ensure the progression of spermatogenesis, highlighting the importance of CCR4-NOT-mediated post-transcriptional gene regulation during male germ cell development.HighlightsCnot3is predominantly expressed in undifferentiated spermatogonia, and its expression decreases in later stages.Conditional deletion ofCnot3leads to total germ cell loss and male infertility.CNOT3 represses transcripts encoding factors involved in spermatogonial differentiation.CNOT3 protects spermatogonial stem cells maintenance via GSH/redox pathway.Graphical AbstractCnot3 promotes SSC self-renewal by balancing ROS production via the regulation of GSH production
Cytotoxic rhamnolipid micelles drive acute virulence inPseudomonas aeruginosa
Authors: Xu Qi , Kang Donghoon , Meyer Matthew D. , Pennington Christopher L. , Gopal Citrupa , Schertzer Jeffrey W. , Kirienko Natalia V. 
Published: October 2023

AbstractPseudomonas aeruginosais an opportunistic human pathogen that has developed multi- or even pan-drug resistance towards most frontline and last resort antibiotics, leading to increasing infections and deaths among hospitalized patients, especially those with compromised immune systems. Further complicating treatment,P. aeruginosaproduces numerous virulence factors that contribute to host tissue damage and immune evasion, promoting bacterial colonization and pathogenesis. In this study, we demonstrate the importance of rhamnolipid production in host-pathogen interactions. Secreted rhamnolipids form micelles that exhibited highly acute toxicity towards murine macrophages, rupturing the plasma membrane and causing organellar membrane damage within minutes of exposure. While rhamnolipid micelles (RMs) were particularly toxic to macrophages, they also caused membrane damage in human lung epithelial cells, red blood cells, Gram-positive bacteria, and even non-cellular models like giant plasma membrane vesicles. Most importantly, rhamnolipid production strongly correlated toP. aeruginosavirulence against murine macrophages in various panels of clinical isolates. Altogether, our findings suggest that rhamnolipid micelles are highly cytotoxic virulence factors that drive acute cellular damage and immune evasion duringP. aeruginosainfections.
The ribonucleoprotein-mediated CRISPR–Cas9 system induces recurrent Aire gene mutations in contrast to the nickase expression vector in murinein vitroorin vivomodels
Authors: Tanaka Pedro P. , Monteiro Cíntia J. , Duarte Max J. , Oliveira Ernna D. , Monteleone-Cassiano Ana C. , Mascarenhas Romário S. , Machado Mayara C. Vieira , Matos Adriana A. , Brito Letícia A. , Oliveira Alina O. , Cunha Thiago M. , Donadi Eduardo A. , Passos Geraldo A. 
Published: October 2023

AbstractAlthoughin vitromTEC cultures can help determine the role of the autoimmune regulator (Aire) gene, the mechanisms of Aire mutations were identified using Aire mutant mice. Nevertheless, long-term cultures of mTECs from mice are difficult to establish. To overcome this, we used a CRISPR–Cas9 system to edit Aire in a murine mTEC linein vitroand mouse embryos. Two ribonucleoprotein (RNP) complexes were designed to separately target Aire exons 6 and 8. NHEJ-derived indels or HDR-derived mutations were obtained. Recurrent NHEJ-derived mutations were observed among editions, one in exon 6 (del 3554G) and the other in exon 8 (del 5676_5677TG), i.e., the exon 6 mutation was kept in an mTEC clone editedin vitroandin vivoin a mouse, and the exon 8 mutation was kept in mTECsin vitro. None of the mutations obtained with the nickase system were recurrent, indicating the participation of the RNP complex.