bluuebunny/crossref_metadata_2025_split huggingface dataset:
3D-printing fabrication of microwave-microfluidic device for droplets network formation and characterisation
Authors: Silver Kai , Li Jin , Dimitriou Pantelitsa , Kallnik Colin , Porch Adrian , Barrow David 
Published: April 2024

AbstractMicrowave-microfluidic devices (MMDs) have emerged as precision tools for the rapid, accurate, sensitive, and non-invasive characterisation of low-volume liquids. However, the fabrication of MMDs remains a significant challenge due to the complexities associated with integrating fluidic ducts and electronic components. Herein, we present a versatile and economical 3D-printing approach for MMD fabrication, using liquid metal as an electrical conductor. Cyclic olefin copolymer, polylactic acid and polypropylene were identified as potential printable dielectric materials for MMD fabrication. 3D-printed cyclic olefin copolymer substrates exhibited the lowest loss tangent of 0.002 at 2.7GHz, making it an ideal material for high frequency engineering. Liquid metal, specifically gallium indium eutectic, was injected into the printed ducts to form conductive microwave structures. Exemplar MMDs were fabricated to integrate split-ring type microwave resonators and droplet-forming fluidic junctions. These devices were applied in the formation and characterisation of water-in-oil emulsions for constructing definable lipid-segregated droplet interface bilayer (DIB) networks. This work not only indicates the feasibility of using 3D-printing for rapid prototyping of customised MMDs but also demonstrates the potential of MMDs as a new research tool for biochemistry and synthetic biology.
RGF1 controls PLT2 protein stability through ROS-dependent regulation of a cysteine residue in root meristem development
Authors: Hsiao Yu-Chun , Shiue Shiau-Yu , Yen Ming-Ren , Lai Joon-Keat , Yamada Masashi 
Published: April 2024

AbstractThe protein concentration gradients of the master regulators of the root meristem, named the PLETHORA proteins, modulate the root meristem size. Root meristem growth factor 1 (RGF1) peptide extends the PLETHORA2 (PLT2) protein gradients by altering reactive oxygen species (ROS) distributions. However, the underlying mechanism through which the ROS alterations regulate PLT2 remains unknown. Here, we demonstrate that the 212thcysteine of the PLT2 protein plays a pivotal role in modulating PLT2 stability through the ROS altered by RGF1. The substitution of the 212thcysteine of PLT2 with serine (PLT2C212S) enhanced the PLT2 protein stability upon RGF1 and resulted in robust resistance to ROS relative to the native PLT2. Accordingly, PLT2C212Smodulated expressions of certain specific root development-related genes to a greater extent than native PLT2. In summary, these findings show that the PLT2 concentration gradient formation through ROS, modulated by RGF1, is dependent on a mechanism involving the 212thcysteine of PLT2.
Natural variation in salt-induced changes in root:shoot ratio reveals SR3G as a negative regulator of root suberization and salt resilience in Arabidopsis
Authors: Ishka Maryam Rahmati , Sussman Hayley , Hu Yunfei , Alqahtani Mashael Daghash , Craft Eric , Sicat Ronell , Wang Minmin , Yu Li’ang , Ait-Haddou Rashid , Li Bo , Drakakaki Georgia , Nelson Andrew , Pineros Miguel , Korte Arthur , Jaremko Lukasz , Testerink Christa , Tester Mark , Julkowska Magdalena M. 
Published: April 2024

AbstractSoil salinity is one of the major threats to agricultural productivity worldwide. Salt stress exposure alters root and shoot growth rates, thereby affecting overall plant performance. While past studies have extensively documented the effect of salt stress on root elongation and shoot development separately, here we take an innovative approach by examining the coordination of root and shoot growth under salt stress conditions. Utilizing a newly developed tool for quantifying the root:shoot ratio in agar-grown Arabidopsis seedlings, we found that salt stress results in a loss of coordination between root and shoot growth rates. We identify a specific gene cluster encoding domain-of-unknown-function 247 (DUF247), and characterize one of these genes asSaltRoot:shootRatioRegulatorGene (SR3G). Further analysis elucidates the role of SR3G as a negative regulator of salt stress tolerance, revealing its function in regulating shoot growth, root suberization, and sodium accumulation. We further characterize thatSR3Gexpression is modulated byWRKY75transcription factor, known as a positive regulator of salt stress tolerance. Finally, we show that the salt stress sensitivity ofwrky75mutant is completely diminished when it is combined withsr3gmutation. Together, our results demonstrate that utilizing root:shoot ratio as an architectural feature leads to the discovery of new stress resilience gene. The study’s innovative approach and findings not only contribute to our understanding of plant stress tolerance mechanisms but also open new avenues for genetic and agronomic strategies to enhance crop environmental resilience.
Quantification of tissue creatine content using capillary electrophoresis
Authors: Choi Y. Jane , Bautista Angelo P , Terrill Jessica R , Pillow J. Jane , Arthur Peter G 
Published: April 2024

AbstractCreatine plays a fundamental role in cellular energy homeostasis. The current protocol describes an alternative method for creatine quantification in biological tissue samples using capillary electrophoresis, with high separation efficiency that enables rapid analysis with low sample volumes. The protocol involves homogenization of snap-frozen tissue in phosphate buffer, followed by electrophoresis through a bare-fused capillary (75 µm internal diameter) and measurement at 200 nm on the Agilent 7100 CE system. Under the optimised conditions, there was excellent linearity in creatine standards between 6.3 – 100 µM. The overall intra-assay variability for concentrations between 6.3 – 100 µM was 1.5 %, and the inter-assay variability was 6.4 %, with a limit of detection at 6 nmol/mg protein. The protocol was further benchmarked against a commercially available enzyme assay kit using lung samples from lambs that received continuous creatine or saline supplementation. There was good agreement between the two methods (mean difference = 0.42 [-0.26-1.1] nmol/mg protein). Importantly, capillary electrophoresis enables reliable detection of creatine in biological samples from just ∼1.5 mg of wet-weight lung tissue. Capillary electrophoresis enables rapid (<10 minutes) and highly efficient analysis of tissue samples and avoids challenges faced with traditional enzymatic assays. The current protocol was developed and optimised with ovine lung tissue, but it can be easily adapted to analyse various tissue types. For tissues with higher baseline creatine content, such as the skeletal muscles or brain, <1 mg wet weight tissue would be sufficient to detect creatine using capillary electrophoresis.
Artificial Intelligence-Enabled AlphaFold II Pipeline Guides Functional Fluorescence Labeling of Tubulin Across Species
Authors: Xu Kaiming , Li Zhiyuan , Mao Linfan , Guo Zhengyang , Chen Zhe , Chai Yongping , Xie Chao , Yang Xuerui , Na Jie , Li Wei , Ou Guangshuo 
Published: April 2024

AbstractDynamic properties are essential for microtubule (MT) physiology. Current techniques forin vivoimaging of MTs present intrinsic limitations in elucidating the isotype-specific nuances of tubulins, which contribute to their versatile functions. Harnessing the power of AlphaFold II pipeline, we engineered a strategy for the minimally invasive fluorescence labeling of endogenous tubulin isotypes or those harboring missense mutations. We demonstrated that a specifically designed 16-amino acid linker, coupled with sfGFP11 from the split-sfGFP system and integration into the H1-S2 loop of tubulin, facilitated tubulin labeling without compromising MT dynamics, embryonic development, or ciliogenesis inC. elegans. Extending this technique to human cells and murine oocytes, we visualized MTs with the minimal background fluorescence and a pathogenic tubulin isoform with fidelity. The utility of our approach across biological contexts and species set an additional paradigm for studying tubulin dynamics and functional specificity, with implications for understanding tubulin-related diseases known as tubulinopathies.
Two-ended recombination at a Flp-nickase-broken replication fork
Authors: Elango Rajula , Nilavar Namrata , Li Andrew G. , Duffey Erin E. , Jiang Yuning , Nguyen Daniel , Abakir Abdulkadir , Willis Nicholas A. , Houseley Jonathan , Scully Ralph 
Published: April 2024

SummaryCollision of a replication fork with a DNA nick is thought to generate a one-ended break, fostering genomic instability. Collision of the opposing converging fork with the nick could, in principle, form a second DNA end, enabling conservative repair by homologous recombination (HR). To study mechanisms of nickase-induced HR, we developed the Flp recombinase “step arrest” nickase in mammalian cells. Flp-nickase-induced HR entails two-ended, BRCA2/RAD51-dependent short tract gene conversion (STGC), BRCA2/RAD51-independent long tract gene conversion, and discoordinated two-ended invasions. HR induced by a replication-independent break and by the Flp-nickase differ in their dependence onBRCA1. To determine the origin of the second DNA end during Flp-nickase-induced STGC, we blocked the opposing fork using a site-specific Tus/Terreplication fork barrier. Flp-nickase-induced STGC remained robust and two-ended. Thus, collision of a single replication fork with a Flp-nick can trigger two-ended HR, possibly reflecting replicative bypass of lagging strand nicks. This response may limit genomic instability during replication of a nicked DNA template.
Efficient Generation of Human Pluripotent Stem Cells from Frozen Cord Tissue via Chemical Reprogramming
Authors: Nansubuga Coneria , Mahnke Donna K. , Li Siqi , Multerer Abigail , Minx Jake , Miller Bradley , Shen Mengcheng , Beyer Andreas , Han Lu , Lincoln Joy , Liu Chun 
Published: April 2024

ABSTRACTChemical reprogramming presents an innovative approach for generating induced pluripotent stem cells (iPSCs), bypassing the genetic instability and safe concern associated with viral vector approach. We describe a novel, efficient chemical method for reprogramming human umbilical cord tissue-derived mesenchymal stem cells (MSCs) into induced pluripotent stem cells (iPSCs). Compared to previous sources like adipose tissue and skin, frozen umbilical cord tissue offers an abundant, non-invasive, long-term storable, and ethically sound cell source. Our findings not only showcase the feasibility and safety of utilizing chemical reprogramming on cells from frozen umbilical cords but also underscore its potential in regenerative medicine, especially for developing safer and more effective therapies for cardiovascular diseases.
Profiling microRNA expression during senescence and aging: mining for a diagnostic tool of senescent-cell burden
Authors: Weigl Moritz , Krammer Teresa L. , Pultar Marianne , Wieser Matthias , Chaib Selim , Suda Masayoshi , Diendorfer Andreas , Khamina-Kotisch Kseniya , Giorgadze Nino , Pirtskhalava Tamar , Johnson Kurt O , Inman Christina L. , Xue Ailing , Lämmermann Ingo , Meixner Barbara , Wang Lichao , Xu Ming , Grillari Regina , Ogrodnik Mikolaj , Tchkonia Tamar , Hackl Matthias , Kirkland James L , Grillari Johannes 
Published: April 2024

AbstractIn the last decade cellular senescence, a hallmark of aging, has come into focus for pharmacologically targeting aging processes. Senolytics are one of these interventive strategies that have advanced into clinical trials, creating an unmet need for minimally invasive biomarkers of senescent cell load to identify patients at need for senotherapy. We created a landscape of miRNA and mRNA expression in five human cell types induced to senescencein-vitroand provide proof-of-principle evidence that miRNA expression can track senescence burden dynamicallyin-vivousing transgenic p21highsenescent cell clearance in HFD fed mice. Finally, we profiled miRNA expression in seven different tissues, total plasma, and plasma derived EVs of young and 25 months old mice. In a systematic analysis, we identified 22 candidate senomiRs with potential to serve as circulating biomarkers of senescence not only in rodents, but also in upcoming human clinical senolytic trials.
Ergothioneine boosts mitochondrial respiration and exercise performance via direct activation of MPST
Authors: Sprenger Hans-Georg , Mittenbühler Melanie J. , Sun Yizhi , Van Vranken Jonathan G. , Schindler Sebastian , Jayaraj Abhilash , Khetarpal Sumeet A. , Vargas-Castillo Ariana , Puszynska Anna M. , Spinelli Jessica B. , Armani Andrea , Kunchok Tenzin , Ryback Birgitta , Seo Hyuk-Soo , Song Kijun , Sebastian Luke , O’Young Coby , Braithwaite Chelsea , Dhe-Paganon Sirano , Burger Nils , Mills Evanna L. , Gygi Steven P. , Arthanari Haribabu , Chouchani Edward T. , Sabatini David M. , Spiegelman Bruce M. 
Published: April 2024

AbstractErgothioneine (EGT) is a diet-derived, atypical amino acid that accumulates to high levels in human tissues. Reduced EGT levels have been linked to age-related disorders, including neurodegenerative and cardiovascular diseases, while EGT supplementation is protective in a broad range of disease and aging models in mice. Despite these promising data, the direct and physiologically relevant molecular target of EGT has remained elusive. Here we use a systematic approach to identify how mitochondria remodel their metabolome in response to exercise training. From this data, we find that EGT accumulates in muscle mitochondria upon exercise training. Proteome-wide thermal stability studies identify 3-mercaptopyruvate sulfurtransferase (MPST) as a direct molecular target of EGT; EGT binds to and activates MPST, thereby boosting mitochondrial respiration and exercise training performance in mice. Together, these data identify the first physiologically relevant EGT target and establish the EGT-MPST axis as a molecular mechanism for regulating mitochondrial function and exercise performance.
Delivering large genes using adeno-associated virus and the CRE-lox DNA recombination system
Authors: Datta Poppy , Rhee Kun Do , Staudt Rylee J , Thompson Jacob M , Hsu Ying , Hassan Salma , Drack Arlene V. , Seo Seongjin 
Published: April 2024

AbstractAdeno-associated virus (AAV) is a safe and efficient gene delivery vehicle for gene therapies. However, its relatively small packaging capacity limits its use as a gene transfer vector. Here, we describe a strategy to deliver large genes that exceed the AAV’s packaging capacity using up to four AAV vectors and the CRE-lox DNA recombination system. We devised novel lox sites by combining non-compatible and reaction equilibrium-modifying lox site variants. These lox sites facilitate sequence-specific and near-unidirectional recombination of AAV vector genomes, enabling efficient reconstitution of up to 16 kb of therapeutic genes in a pre-determined configuration. Using this strategy, we have developed AAV gene therapy vectors to deliverIFT140,PCDH15,CEP290, andCDH23and demonstrate efficient production of full-length proteins in cultured mammalian cells and mouse retinas. Notably, this approach significantly surpasses the trans-splicing and split-intein-based reconstitution methods in efficiency, requiring lower doses, minimizing or eliminating the production of truncated protein products, and offering flexibility in selecting splitting positions. The CRE-lox approach described here provides a simple and effective platform for producing AAV gene therapy vectors beyond AAV’s packaging capacity.
Microbial Metabolite Trimethylamine N-Oxide PromotesCampylobacter jejuniInfection by Escalating Intestinal Inflammation, Epithelial Damage, and Barrier Disruption
Authors: Smith Caitlyn B. , Gao Angela , Bravo Paloma , Alam Ashfaqul 
Published: April 2024

ABSTRACTThe interactions betweenCampylobacter jejuni, a critical foodborne cause of gastroenteritis, and the intestinal microbiota during infection are not completely understood. The crosstalk betweenC. jejuniand its host is impacted by the gut microbiota through mechanisms of competitive exclusion, microbial metabolites, or immune response. To investigate the role of gut microbiota onC. jejunipathogenesis, we examined campylobacteriosis in the IL10KO mouse model, which was characterized by an increase in the relative abundance of intestinal proteobacteria,E. coli, and inflammatory cytokines duringC. jejuniinfection. We also found a significantly increased abundance of microbial metabolite Trimethylamine N-Oxide (TMAO) in the colonic lumens of IL10KO mice. We further investigated the effects of TMAO onC. jejunipathogenesis. We determined thatC. jejunisenses TMAO as a chemoattractant and the administration of TMAO promotesC. jejuniinvasion into Caco-2 monolayers. TMAO also increased the transmigration ofC. jejuniacross polarized monolayers of Caco-2 cells, decreased TEER, and increasedC. jejuni-mediated intestinal barrier damage. Interestingly, TMAO treatment and presence duringC. jejuniinfection of Caco-2 cells synergistically caused an increased inflammatory cytokine expression, specifically IL-1β and IL-8. These results establish thatC. jejuniutilizes microbial metabolite TMAO for increased virulence during infection.
Four enzymes control natural variation in the steroid core ofErysimumcardenolides
Authors: Younkin Gordon C. , Alani Martin L. , Züst Tobias , Jander Georg 
Published: April 2024

ABSTRACTPlants commonly produce families of structurally related metabolites with similar defensive functions. This apparent redundancy raises the question of underlying molecular mechanisms and adaptive benefits of such chemical variation. Cardenolides, a class defensive compounds found in the wallflower genusErysimum(L., Brassicaceae) and scattered across other plant families, show substantial structural variation, with glycosylation and hydroxylation being common modifications of a steroid core, which itself may vary in terms of stereochemistry and saturation. Through a combination of chemical mutagenesis and analysis of gene coexpression networks, we identified four enzymes involved in cardenolide biosynthesis inErysimumthat work together to determine stereochemistry at carbon 5 of the steroid core: Ec3βHSD, a 3β-hydroxysteroid dehydrogenase, Ec3KSI, a ketosteroid isomerase, EcP5βR2, a progesterone 5β-reductase, and EcDET2, a steroid 5α-reductase. We biochemically characterized the activity of these enzymesin vitroand generated CRISPR/Cas9 knockout lines to confirm activityin vivo. Cardenolide biosynthesis was not eliminated in any of the knockouts. Instead, mutant plants accumulated cardenolides with altered saturation and stereochemistry of the steroid core. Furthermore, we found variation in carbon 5 configuration among the cardenolides of 44 species ofErysimum, where the occurrence of some 5β-cardenolides is associated with the expression and sequence of P5βR2. This may have allowedErysimumspecies to fine-tune their defensive profiles to target specific herbivore populations over the course of evolution.SIGNIFICANCE STATEMENTPlants use an array of toxic compounds to defend themselves from attack against insects and other herbivores. One mechanism through which plants may evolve more toxic compounds is through modifications to the structure of compounds they already produce. In this study, we show how plants in the wallflower genusErysimumuse four enzymes to fine-tune the structure of toxic metabolites called cardenolides. Natural variation in the sequence and expression of a single enzyme called progesterone 5β-reductase 2 partly explains the variation in cardenolides observed across theErysimumgenus. These alterations to cardenolide structure over the course of evolution suggests that there may be context-dependent benefits toErysimumto invest in one cardenolide variant over another.
Genome editing in almond: A CRISPR-based approach through hairy root transformation
Authors: Jedličková Veronika , Štefková Marie , Sánchez López Juan Francisco , Grimplet Jérôme , Rubio Cabetas María José , Robert Hélène S. 
Published: April 2024

AbstractClustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) technology has revolutionized genome manipulation for crop enhancement, providing a powerful toolkit. However, the tissue culture and plant regeneration steps that are critical to the CRISPR/Cas editing framework are often challenging, especially in some woody plant species that exhibit substantial resistance to these procedures. To address this, we have developed an injection-based protocol for inducing hairy roots in almond (Prunus dulcis, syn.Prunus amygdalus), a species known for its recalcitrance to conventional transformation methods. Notably, the hairy root induction method also proved effective in almond x peach hybrids. To evaluate its utility for gene functional analysis, we combined the hairy root transformation system with CRISPR/Cas9 gene editing technology, targeting two transcription factor genes (ERF74andGAI). Our efforts resulted in transformants with target knock-out, suggesting the potential of this genetic transformation technology as a valuable tool for future routine gene function studies in almond.
Uptake of small extracellular vesicles by recipient cells is facilitated by paracrine adhesion signaling
Authors: Hirosawa Koichiro M. , Sato Yusuke , Kasai Rinshi S. , Yamaguchi Eriko , Komura Naoko , Ando Hiromune , Hoshino Ayuko , Yokota Yasunari , Suzuki Kenichi G. N. 
Published: April 2024

AbstractSmall extracellular vesicles (sEVs) play crucial roles in intercellular communication. However, the internalization of individual sEVs by recipient cells has not yet been directly observed. Here, we examined these mechanisms using state-of-the-art imaging techniques. Single-molecule imaging showed that tumor-derived sEVs can be classified into several subtypes. Simultaneous single-sEV particle tracking and observation of super-resolution movies of membrane invaginations in living cells revealed that all sEV subtypes were internalized via clathrin-independent endocytosis mediated by galectin-3 and LAMP-2C, while some subtypes that recruited raft markers were internalized through caveolae. Integrin β1 and talin-1 accumulated in recipient cell plasma membranes beneath all sEV subtypes. Paracrine, but not autocrine, sEV binding triggered Ca2+mobilization induced by the activation of Src family kinases and PLCγ. Subsequent Ca2+-induced activation of calcineurin–dynamin promoted sEV internalization, leading to the recycling pathway. Thus, we have clarified the detailed mechanisms of sEV internalization driven by paracrine adhesion signaling.
Strong-field processes induced by an ultrashort linearly polarized pulse with two carrier frequencies
Authors: Habibović D. , Milošević D. B. 
Published: April 2024

We perform a detailed semianalytical study of the characteristics of strong-field processes induced by an ultrashort linearly polarized pulse with two carrier frequencies. We show that the photoelectron spectra depend to a great extent on the absolute phases of the field components and on the duration of the pulse. By using the saddle-point method, we show that this dependence can be exploited to control the electron dynamics in the laser field. Besides the photoelectron spectra, we also investigate the spectra of high-order harmonics. Again, we find a strong dependence on the absolute phases and the duration of the pulse. We present the spectra using a false color scale in the absolute phase-harmonic energy plane, and show that particular regions in this plane can be assessed using the simple man's model founded on the classical solution of the Newton equation of motion. Finally, we investigate the symmetry properties of the photoelectron and high-order harmonic spectra with respect to the transformation which includes the change of the absolute phase, and compare them with those valid for a long driving pulse with a flat envelope.

Published by the American Physical Society
2024