bluuebunny/crossref_metadata_2025_split huggingface dataset:
Additive Manufacturing Leveraged Microfluidic Setup for Sample to Answer Colorimetric Detection of Pathogens
Authors: Yedire Sripadh Guptha , Hosseini Imman Isaac , Shieh Hamed , Jahromi Arash Khorrami , AbdelFatah Tamer , Jalali Mahsa , Mahshid Sara 
Published: August 2023

AbstractColorimetric readout for the detection of infectious diseases is gaining traction at the point of care/need owing to its ease of analysis and interpretation, and integration potential with highly specific Loop-mediated amplification (LAMP) assays. However, coupling colorimetric readout with LAMP is rife with challenges including, rapidity, inter-user variability, colorimetric signal quantification, and user involvement in sequential steps of the LAMP assay, hindering its application. To address these challenges, for the first time, we propose a remotely smartphone-operated automated setup consisting of (i) an additively manufactured microfluidic cartridge, (ii) a portable reflected-light imaging setup with controlled epi-illumination (PRICE) module, and (iii) a control and data analysis module. The microfluidic cartridge facilitates sample collection, lysis, mixing of amplification reagents stored on-chip, and subsequent isothermal heating for initiation of amplification in a novel way by employing tunable elastomeric chambers and auxiliary components (heaters and linear actuators). PRICE offers a new imaging setup that captures the colorimetric change of the amplification media over a plasmonic nanostructured substrate in a controlled and noise-free environment for rapid minute-scale nucleic acid detection. The control and data analysis module employs microprocessors to automate cartridge operation in tandem with the imaging module. The different device components were characterized individually and finally, as a proof of concept, SARS-CoV-2 wild-type RNA was detected with a turnaround time of 13 minutes, showing the device’s clinical feasibility. The suggested automated device can be adopted in future iterations for other detection and molecular assays that require sequential fluid handling steps.
Spontaneous Mutation in2310061I04RikResults in Reduced Expression of Mitochondrial Genes and Impaired Brain Myelination
Authors: Tsitsikov Erdyni N. , Phan Khanh P. , Liu Yufeng , Tsytsykova Alla V. , Paterno Rosalia , Sherry David M. , Johnson Anthony C. , Dunn Ian F. 
Published: August 2023

SummaryHere, we describe a spontaneous mouse mutant with a deletion in a predicted gene2310061I04Rik(Rik) of unknown function located on chromosome 17. A 59 base pair long deletion occurred in the first intron of theRikgene and disrupted its expression.Riknullmice were born healthy and appeared anatomically normal up to two weeks of age. After that, these mice showed inhibited growth, ataxic gait, and died shortly after postnatal day 24 (P24). Transcriptome analysis at P14 and P23 revealed significantly reduced expression of mitochondrial genes inRiknullbrains compared to wild type controls includingmt-Nd4,mt-Cytb,mt-Nd2,mt-Co1,mt-Atp6,and others. Similarly, genes specific for myelinating oligodendrocytes also showed reduced expression in P23Riknullbrains compared to controls. Histological examination of anterior thalamic nuclei demonstrated decreased myelination of anteroventral nuclei but not of anterodorsal nuclei in P23Riknullmice. Myelination of the anterior commissure was also impaired and displayed extensive vacuolation. Consistent with these findings, immunohistochemistry showed reduced expression of Opalin, a glycoprotein expressed in differentiated oligodendrocytes. Taken together, these results suggest that RIK is important for oligodendrocyte maturation, and myelination in the developing brain.
One-Tip enables comprehensive proteome coverage in minimal cells and single zygotes
Authors: Ye Zilu , Sabatier Pierre , Martin-Gonzalez Javier , Eguchi Akihiro , Bekker-Jensen Dorte B. , Bache Nicolai , Olsen Jesper V. 
Published: August 2023

AbstractWe present One-Tip, a lossless proteomics methodology that seamlessly combines swift, one-pot sample preparation with narrow-window data-independent acquisition mass spectrometric analysis. With simplest sample processing, One-Tip reproducibly identifies > 9,000 proteins from ∼1000 cells and ∼ 6,000 proteins in a single mouse zygote with a throughput of 40 samples-per-day. This easy-to-use method expands capabilities of proteomics research by enabling greater depth, scalability and throughput covering low to high input samples.
DNA end sensing and cleavage by the Shedu anti-phage defense system
Authors: Loeff Luuk , Walter Alexander , Rosalen Gian Tizio , Jinek Martin 
Published: August 2023

AbstractThe detection of molecular patterns associated with invading pathogens is a hallmark of innate immune systems. Prokaryotes deploy sophisticated host-defense mechanisms in innate anti-phage immunity. Shedu is a single-component defense system comprising a putative nuclease SduA. Here we report cryo-EM structures of apo- and dsDNA-bound tetrameric SduA assemblies, revealing that the N-terminal domains of SduA form a clamp that recognizes free DNA ends. End binding positions the DNA over the PD/ExK nuclease domain, resulting in dsDNA nicking at a fixed distance from the 5’ end. The end-directed DNA nicking activity of Shedu prevents propagation of linear DNAin vivo. Finally, we show that phages escape Shedu immunity by suppressing their recombination-dependent DNA replication pathway. Taken together, these results define the antiviral mechanism of Shedu systems, underlining the paradigm that recognition of pathogen-specific nucleic acid structures is a conserved feature of innate immunity across all domains of life.
Sphingolipid metabolism is spatially regulated in the developing embryo bySOXEgenes
Authors: Piacentino Michael L. , Fasse Aria J. , Camacho-Avila Alexis , Grabylnikov Ilya , Bronner Marianne E. 
Published: August 2023

AbstractDuring epithelial-to-mesenchymal transition (EMT), significant rearrangements occur in plasma membrane protein and lipid content that are important for membrane function and acquisition of cell motility. To gain insight into how neural crest cells regulate their lipid content at the transcriptional level during EMT, here we identify critical enhancer sequences that regulate the expression ofSMPD3, a gene responsible for sphingomyelin hydrolysis to produce ceramide, which is necessary for neural crest EMT. We uncovered three enhancer regions within the first intron of theSMPD3locus that drive reporter expression in distinct spatial and temporal domains, together collectively recapitulating the expression domains of endogenousSMPD3within the ectodermal lineages. We further dissected one enhancer that is specifically active in the migrating neural crest. By mutating putative transcriptional input sites or knocking down upstream regulators, we find that the SoxE-family transcription factors Sox9 and Sox10 regulate the expression ofSMPD3in migrating neural crest cells. Together these results shed light on how core components of developmental gene regulatory networks interact with metabolic effector genes to control changes in membrane lipid content.HighlightsSMPD3is expressed in the neural tube, neural crest, and notochord during early developmentSMPD3expression is regulated by at least three intronic enhancersSox10 and its binding sites are required for expression by a migratory neural crest-specificSMPD3enhancerSox10 is a positive regulator of endogenousSMPD3expression during neural crest migration
Metal Ions Guide the Production of Silkworm Fibers
Authors: Brookstein Ori , Shimoni Eyal , Eliaz Dror , Kaplan-Ashiri Ifat , Carmel Itay , Shimanovich Ulyana 
Published: August 2023

AbstractSilk fibers’ unique mechanical properties have made them a desirable material for various applications, from medical to optical materials and even in sensing. Yet, to date, no synthetic method has come close to reproducing this remarkably strong biomaterial due to the complexity and insufficient understanding of the mechanism of silk fiber formation. While ions are known to play a key role in the production of natural silk fiber, how they do so has thus far eluded discovery. Here we report that a broad composition of metal ions guides structural transformations in the silk fibroin protein inside the silkworm silk gland. By using a combination of cryo-electron microscopy techniques coupled with elemental analysis, we followed the changes in the composition and spatial localization of metal ions inside the silk gland. We observed that ions are homogenously dispersed during the initial stages of silk secretion and storage inside the silk gland, but once the fibers are spun, the ions delocalize from the silk fibroin fiber core to the sericin coating gum layer. This shift in ion localization is accompanied by the alignment of protein chains and an increase in silk feedstock viscosity inside the silk gland - changes that make the protein more sensitive to shear and enable the initiation of the liquid-to-solid transition in the silk. Moreover, the selective doping of the spun silk fibers with metal ions modifies their mechanical performance. These findings highlight the importance and the dynamic role of metal ions in the evolution of silk fibers’ mechanical properties, enhance our understanding of the mechanism of silk fiber formation, and lay the foundations for developing new concepts in biomaterial design.
The PP1 phosphatase exhibits pleiotropic roles controlling both the tachyzoite cell cycle and amylopectin-steady state levels inToxoplasma gondii
Authors: Khelifa Asma Sarah , Boissavy Tom , Mouveaux Thomas , Araujo Silva Tatiana , Chhuon Cerina , Attias Marcia , Guerrera Ida Chiara , De Souza Wanderley , Dauvillee David , Roger Emmanuel , Gissot Mathieu 
Published: August 2023

ABSTRACTVirulence of apicomplexan parasites is based on their ability to divide rapidly to produce significant biomass. The regulation of their cell-cycle is therefore key to their pathogenesis. Phosphorylation is a crucial post-transcriptional modification that regulates many aspects of the eucaryotic cell cycle. The phosphatase PP1 is known to play a major role in the phosphorylation balance in eukaryotes. We explored the role of TgPP1 during the cell cycle of the tachyzoite form of the apicomplexan parasiteToxoplasma gondii. Using a conditional mutant strain, we show that TgPP1 regulates many aspects of the cell cycle including the proper assembly of the daughter cells inner membrane complex (IMC), the segregation of organelles and nuclear division. Unexpectedly, depletion of TgPP1 also results in the accumulation of amylopectin, a storage polysaccharide that is normally found in the latent bradyzoite form of the parasite. Using transcriptomics and phosphoproteomics, we show that TgPP1 mainly acts through post-transcriptional mechanisms by dephosphorylating target proteins including IMC proteins. TgPP1 also dephosphorylate a protein bearing a starch binding domain. Mutagenesis analysis reveals that the targeted phospho-sites are linked to the ability of the parasite to regulate amylopectin steady-state levels. Therefore, we show that TgPP1 has pleiotropic roles during the tachyzoite cell cycle regulation, but also regulates amylopectin accumulation.
Nucleolar stress caused by arginine-rich peptides triggers a ribosomopathy and accelerates ageing in mice
Authors: Sirozh Oleksandra , Jung Bomi , Sanchez-Burgos Laura , Ventoso Ivan , Lafarga Vanesa , Fernández-Capetillo Oscar 
Published: August 2023

SummaryNucleolar stress (NS) has been associated to several age-related diseases such as cancer or neurodegeneration. To investigate the mechanisms of toxicity triggered by NS, we here used (PR)n arginine-rich peptides that are found in patients of some neurodegenerative diseases. Although these peptides accumulate at nucleoli and generate NS, how this translates into cellular toxicity is poorly understood. We here reveal that whereas (PR)n expression leads to an overall decrease in protein abundance, this occurs concomitant with an accumulation of free ribosomal (r) proteins in the cytoplasm, a hallmark of ribosomopathies. Conversely, cells with acquired resistance to (PR)n peptides present global downregulation of r-proteins and low levels of mTOR signaling. In mice, systemic expression of (PR)97drives widespread NS and accelerated ageing, which is associated to an increased expression of r-proteins and mTOR hyperactivation. Furthermore, the reduced lifespan of (PR)97-expressing mice was alleviated by the mTOR inhibitor rapamycin. Importantly, we show that the generalised accumulation of free r-proteins is a common outcome in response to chemical or genetic perturbations that trigger NS, such as Actinomycin D, TIF-IA depletion, or the expression of mutant HMGB1 variants recently associated to rare human diseases. Together, our study presentsin vivoevidence supporting the role of NS as a driver of ageing, and provides a general framework to explain the toxicity caused by NS in mammalian cells.
Non-homologous end joining shapes the genomic rearrangement landscape of chromothripsis from mitotic errors
Authors: Hu Qing , Espejo Valle-Inclan Jose , Dahiya Rashmi , Guyer Alison , Mazzagatti Alice , Maurais Elizabeth G. , Engel Justin L. , Cortés-Ciriano Isidro , Ly Peter 
Published: August 2023

ABSTRACTErrors in mitosis can generate micronuclei that entrap mis-segregated chromosomes, which are susceptible to catastrophic fragmentation through a process termed chromothripsis. The reassembly of fragmented chromosomes by error-prone DNA double-strand break (DSB) repair generates a spectrum of simple and complex genomic rearrangements that are associated with human cancers and disorders. How specific DSB repair pathways recognize and process these lesions remains poorly understood. Here we used CRISPR/Cas9 to systematically inactivate distinct DSB processing or repair pathways and interrogated the rearrangement landscape of fragmented chromosomes from micronuclei. Deletion of canonical non-homologous end joining (NHEJ) components, including DNA-PKcs, LIG4, and XLF, substantially reduced the formation of complex rearrangements and shifted the rearrangement landscape toward simple alterations without the characteristic patterns of cancer-associated chromothripsis. Following reincorporation into the nucleus, fragmented chromosomes localize within micronuclei bodies (MN bodies) and undergo successful ligation by NHEJ within a single cell cycle. In the absence of NHEJ, chromosome fragments were rarely engaged by polymerase theta-mediated alternative end-joining or recombination-based mechanisms, resulting in delayed repair kinetics and persistent 53BP1-labeled MN bodies in the interphase nucleus. Prolonged DNA damage signaling from unrepaired fragments ultimately triggered cell cycle arrest. Thus, we provide evidence supporting NHEJ as the exclusive DSB repair pathway generating complex rearrangements following chromothripsis from mitotic errors.
Spatiotemporal optical control of Gαq-PLCβ interactions
Authors: Ubeysinghe Sithurandi , Kankanamge Dinesh , Thotamune Waruna , Wijayaratna Dhanushan , Mohan Thomas M. , Karunarathne Ajith 
Published: August 2023

AbstractCells experience time-varying and spatially heterogeneous chemokine signals in vivo, activating cell surface proteins, including G protein-coupled receptors (GPCRs). The Gαq pathway activation by GPCRs is a major signaling axis with a broad physiological and pathological significance. Compared to other Gα members, GαqGTP activates many crucial effectors, including PLCβ (Phospholipase Cβ) and Rho GEFs (Rho guanine nucleotide exchange factors). PLCβ regulates many key processes, such as hematopoiesis, synaptogenesis, and cell cycle, and is therefore implicated in terminal - debilitating diseases, including cancer, epilepsy, Huntington’s Disease, and Alzheimer’s Disease. However, due to a lack of genetic and pharmacological tools, examining how the dynamic regulation of PLCβ signaling controls cellular physiology has been difficult. Since activated PLCβ induces several abrupt cellular changes, including cell morphology, examining how the other pathways downstream of Gq-GPCRs contribute to the overall signaling has also been difficult. Here we show the engineering, validation, and application of a highly selective and efficient optogenetic inhibitor (Opto-dHTH) to completely disrupt GαqGTP-PLCβ interactions reversibly in user-defined cellular-subcellular regions on optical command. Using this newly gained PLCβ signaling control, our data indicate that the molecular competition between RhoGEFs and PLCβ for GαqGTP determines the potency of Gq-GPCR-governed directional cell migration.
Mesoscopic Oblique Plane Microscopy via Light-sheet Mirroring
Authors: Daetwyler Stephan , Chang Bo-Jui , Chen Bingying , Zhou Felix , Fiolka Reto 
Published: August 2023

AbstractUnderstanding the intricate interplay and inter-connectivity of biological processes across an entire organism is important in various fields of biology, including cardiovascular research, neuroscience, and developmental biology. Here, we present a mesoscopic oblique plane microscope (OPM) that enables whole organism imaging with high speed and subcellular resolution. A microprism underneath the sample enhances the axial resolution and optical sectioning through total internal reflection of the light-sheet. Through rapid refocusing of the light-sheet, the imaging depth is extended up to threefold while keeping the axial resolution constant. Using low magnification objectives with a large field of view, we realize mesoscopic imaging over a volume of 3.7×1.5×1 mm3with ∼2.3 microns lateral and ∼9.2 microns axial resolution. Applying the mesoscopic OPM, we demonstratein vivoandin totowhole organism imaging of the zebrafish vasculature and its endothelial nuclei, and blood flow dynamics at 12 Hz acquisition rate, resulting in a quantitative map of blood flow across the entire organism.
Pretrainable Geometric Graph Neural Network for Antibody Affinity Maturation
Authors: Cai Huiyu , Zhang Zuobai , Wang Mingkai , Zhong Bozitao , Li Quanxiao , Zhong Yuxuan , Wu Yanling , Ying Tianlei , Tang Jian 
Published: August 2023

AbstractIncreasing the binding affinity of an antibody to its target antigen is a crucial task in antibody therapeutics development. This paper presents a pretrainable geometric graph neural network, GearBind, and explores its potential inin silicoaffinity maturation. Leveraging multi-relational graph construction, multi-level geometric message passing and contrastive pretraining on mass-scale, unlabeled protein structural data, GearBind outperforms previous state-of-the-art approaches on SKEMPI and an independent test set. A powerful ensemble model based on GearBind is then derived and used to successfully enhance the binding of two antibodies with distinct formats and target antigens. ELISA EC50values of the designed antibody mutants are decreased by up to 17 fold, andKDvalues by up to 6.1 fold. These promising results underscore the utility of geometric deep learning and effective pretraining in macromolecule interaction modeling tasks.
Bioluminescence Assay of Lysine Deacylase Sirtuin Activity
Authors: Van Scoyk Alexandria N. , Antelope Orlando , Franzini Anca , Ayer Donald E. , Peterson Randall T. , Pomicter Anthony D. , Owen Shawn C. , Deininger Michael W. 
Published: August 2023

ABSTRACTLysine acylation can direct protein function, localization, and interactions. Sirtuins deacylate lysine towards maintaining cellular homeostasis, and their aberrant expression contributes to the pathogenesis of multiple pathological conditions, including cancer. Measuring sirtuins’ activity is essential to exploring their potential as therapeutic targets, but accurate quantification is challenging. We developed ‘SIRTify’, a high-sensitivity assay for measuring sirtuin activityin vitroandin vivo. SIRTifyis based on a split-version of the NanoLuc® luciferase consisting of a truncated, catalytically inactive N-terminal moiety (LgBiT) that complements with a high-affinity C-terminal peptide (p86) to form active luciferase. Acylation of two lysines within p86 disrupts binding to LgBiT and abates luminescence. Deacylation by sirtuins reestablishes p86 and restores binding, generating a luminescence signal proportional to sirtuin activity. Measurements accurately reflect reported sirtuin specificity for lysine acylations and confirm the effects of sirtuin modulators. SIRTifyeffectively quantifies lysine deacylation dynamics and may be adaptable to monitoring additional post-translational modifications.
Aging and senescence-associated analysis of the aged kidney glomerulus highlights the role of mesangial cells in renal aging
Authors: Korin Ben , Avraham Shimrit , McKay Andrew , Durinck Steffen , Moncada Reuben , Menon Hari , Darmanis Spyros , Liang Yuxin , Modrusan Zora , Webster Joshua D. , Shaw Andrey S 
Published: August 2023

AbstractMost causes of chronic kidney disease begin with injury to the glomerulus and involve progressive loss of kidney function. The glomerulus is a capillary bed where blood filtration to produce urine in the kidney occurs. During aging, there is progressive loss of glomeruli and filtration capacity of the kidney because podocytes, the glomerular epithelial cell, are lost with aging and after injury. Podocytes cannot divide and therefore cannot be replaced. Our histological analysis confirmed the presence of glomerulosclerosis, generalized interstitial fibrosis and glomerular hypertrophy in the aged mouse kidney. One barrier to studies of glomeruli is their low frequency in the kidney, less than 1.5% of the cells, and as such, they are often underrepresented in whole kidney analyses. To address this challenge, we used both bulk and single cell RNA sequencing (scRNA-Seq) to characterize purified glomeruli from young and aged mice. Aged glomeruli displayed increased inflammation and expressed a variety of injury and senescence-associated markers, most notably in mesangial cells and macrophages. This increased expression of senescence markers in mesangial cells of aged kidneys suggests a potential cellular target to address age-related renal dysfunction and chronic kidney disease (CKD), which represent a tremendous unmet medical need.
Flow cytometry methods for targeted isolation of ctenophore cells
Authors: Dieter Abigail C , True Aliyah BK , Gilbertson Emily , Snyder Grace , Lacy-Hulbert Adam , Traylor-Knowles Nikki , Browne William E , Vandepas Lauren E 
Published: August 2023

AbstractCell suspension fluidics, such as flow cytometry (FCS) and fluorescence-activated cell sorting (FACS), facilitates the identification and precise separation of individual cells based on phenotype. Since its introduction, flow cytometry has been used to analyze cell types and cellular processes in diverse non-vertebrate taxa, including cnidarians, molluscs, and arthropods. Ctenophores, which diverged very early from the metazoan stem lineage, have emerged as an informative clade for the study of metazoan cell type evolution. We present standardized methodologies for flow cytometry-mediated identification and analyses of cells from the model ctenophoreMnemiopsis leidyithat can also be applied to isolate targeted cell populations. Here we focus on the identification and isolation of ctenophore phagocytes. Implementing flow cytometry methods in ctenophores allows for fine scale analyses of fundamental cellular processes conserved broadly across animals, as well as potentially revealing novel cellular phenotypes and behaviors restricted to the ctenophore lineage.