bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid DNA unwinding accelerates genome editing by engineered CRISPR-Cas9
Authors: Eggers Amy R. , Chen Kai , Soczek Katarzyna M. , Tuck Owen T. , Doherty Erin E. , Thornton Brittney W. , Xu Bryant , Trinidad Marena I. , Doudna Jennifer A. 
Published: December 2023

SUMMARYThermostable CRISPR-Cas9 enzymes could improve genome editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold higher genome editing levels. Cryo-EM structures of the wildtype and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome editing activity.
Effective seed sterilization methods require optimization across maize genotypes
Authors: Parnell J. Jacob , Pal Gaurav , Awan Ayesha , Vintila Simina , Houdinet Gabriella , Hawkes Christine V. , Balint-Kurti Peter J. , Wagner Maggie R. , Kleiner Manuel 
Published: December 2023

AbstractStudies of plant-microbe interactions using synthetic microbial communities (SynComs) often require the removal of seed-associated microbes by seed sterilization prior to inoculation to provide gnotobiotic growth conditions. A diversity of seed sterilization protocols have been developed in the past and have been used on different plant species with various amounts of validation. From these studies it has become clear that each plant species requires its own optimized sterilization protocol. It has, however, so far not been tested if the same protocol works equally well for different varieties and seed sources of one plant species. We evaluated six seed sterilization protocols on two different varieties (Sugar Bun & B73) of maize. All unsterilized maize seeds showed fungal growth upon germination on filter paper, highlighting the need for a sterilization protocol. A short sterilization protocol with hypochlorite and ethanol was sufficient to prevent fungal growth on Sugar Bun germinants, however a longer protocol with heat treatment and germination in fungicide was needed to obtain clean B73 germinants. This difference may have arisen from the effect of either genotype or seed source. We then tested the protocol that performed best for B73 on three additional maize genotypes from four sources. Seed germination rates and fungal contamination levels varied widely by genotype and geographic source of seeds. Our study shows that consideration of both variety and seed source is important when optimizing sterilization protocols and highlights the importance of including seed source information in plant-microbe interaction studies that use sterilized seeds.
An optimized method for gene knockdown in differentiating human and mouse adipocyte cultures
Authors: Chua Ruiming , Ghosh Sujoy 
Published: December 2023

AbstractAdipocyte cultures are a mainstay of metabolic disease research, yet loss-of-function studies in differentiating adipocytes is complicated by the refractoriness of lipid-containing adipocytes to standard siRNA transfections. Alternative methods, such as electroporation or adenovirus/lentivirus-based delivery systems are complex, expensive and often accompanied with unacceptable levels of cell death. To address this problem, we have tested two commercially available siRNA delivery systems in this study using a multi-parameter optimization approach. Our results identified a uniform siRNA transfection protocol that can be applied to human and mouse adipocyte cultures throughout the time course of differentiation, beginning with pre-differentiated cells and continuing up to lipid-accumulated differentiated adipocytes. Our findings allow for efficient transfection of human and mouse adipocyte cultures using standard and readily available methodologies, and should help significantly expand the scope of gene manipulation studies in these cell types.
A versatile and upgraded version of the LundTax classification algorithm applied to independent cohorts
Authors: Aramendía Cotillas Elena , Bernardo Carina , Veerla Srinivas , Liedberg Fredrik , Sjodahl Gottfrid , Eriksson Pontus 
Published: December 2023

Stratification of cancer into biologically and molecularly similar subgroups is a cornerstone of precision medicine and transcriptomic profiling has revealed that urothelial carcinoma (UC) is a heterogeneous disease with several distinct molecular subtypes. The Lund Taxonomy classification system for urothelial carcinoma aims to be applicable across the whole disease spectrum including both non-muscle invasive and invasive bladder cancer. For a classification system to be useful it is of critical importance that it can be applied robustly and reproducibly to new samples. Many transcriptomic methods used for subtype classification are affected by the choice of expression platform, data preprocessing, cohort composition, and tumor purity. The application of a subtype classification system across studies therefore comes with a degree of uncertainty regarding whether the predictions in a new cohort accurately recapitulate the originally intended stratification. Currently, only limited data has been published evaluating the transferability and applicability of existing stratification systems and their respective classification-algorithms to external datasets. In the present investigation we develop a single sample classifier based on in-house microarray and RNA-sequencing data intended to be broadly applicable across datasets, studies, and tumor stages. We evaluate the performance of the proposed method and the Lund Taxonomical classification across 10 published bladder cancer cohorts (n=2560 cases) by examining the expression of characteristic subtype associated gene signatures, and whether complementary data such as mutations, clinical outcomes, response, or variant histologies are captured by our classification. Effects of varying sample purity on the classification results were also evaluated by generating low-purity versions of samples in silico. We show that the classifier is robustly applicable across different gene expression profiling platforms and preprocessing methods, and less sensitive to variations in sample purity.The classifier is available as the ‘LundTaxonomy2023Classifier’ R package onGitHub.
A site-specific analysis of the ADP-Ribosylome unveils Homogeneous DNA Damage-Induced Serine ADP-Ribosylation across wild-type and BRCA-mutant Breast Cancer cell lines
Authors: Anagho Holda A. , Mullari Meeli , Prósz Aurél G. , Buch-Larsen Sara C. , Locard-Paulet Marie , Szallasi Zoltan , Nielsen Michael L. 
Published: December 2023

SummaryADP-ribosylation (ADPr) signaling plays a crucial role in the DNA damage response. Inhibitors against the main enzyme catalyzing ADPr after DNA damage – PARP1 – are used as targeted therapies against breast cancers with BRCA1/2 mutations. However, development of resistance to PARP inhibitors (PARPi) is a major obstacle in treating patients. To better understand the role of ADPr in PARPi sensitivity, we used Liquid Chromatography-Mass Spectrometry (LC-MS) for systems level analysis of the ADP-ribosylome in six breast cancer cell lines exhibiting different PARPi sensitivities. We identified 1,632 sites on 777 proteins across all cell lines, primarily on serine residues, with site-specific overlap of targeted residues across DNA damage-related proteins across all cell lines, demonstrating high conservation of serine ADPr signaling networks upon DNA damage. We furthermore observed site-specific differences in ADPr intensities in PARPi-sensitive BRCA mutants, and unique ADPr sites in PARPi-resistant BRCA mutant cells, which we notably show to have low PARG levels and longer ADPr chains on PARP1.
Parallel Molecular Data Storage by Printing Epigenetic Bits on DNA
Authors: Zhang Cheng , Wu Ranfeng , Sun Fajia , Lin Yisheng , Zhang Yizheng , Liang Yuan , Chen Yiming , Teng Jiongjiong , Song Zichen , Liu Na , Ouyang Qi , Qian Long , Yan Hao 
Published: December 2023

SummaryDNA storage has shown potential to transcend current silicon-based data storage technologies in storage density, lifetime and energy consumption. However, writing large-scale data directly into DNA sequences byde novosynthesis remains uneconomical in time and cost. Inspired by the natural genomic modifications, in this work, we report an alternative, parallel strategy that enables the writing of arbitrary data on DNA using premade nucleic acids. With epigenetic modifications as information bits, our system employed DNA self-assembly guided enzymatic methylation to perform movable-type printing on universal DNA templates. By programming with a finite set of 700 DNA movable types and 5 templates, we achieved the synthesis-free writing of ∼270000 bits on an automated platform with 350 bits written per reaction. The data encoded in complex epigenetic patterns were retrieved high-throughput by nanopore sequencing, and algorithms were developed to finely resolve 240 modification patterns per sequencing reaction. Our framework presents a new modality of DNA-based data storage that is parallel, programmable, stable and scalable. Such a modality opens up avenues towards practical data storage and dual-mode data functions in biomolecular systems.
Claudin-4 polymerizes after the incorporation of just two extracellular claudin-3 residues
Authors: van der Veen Rozemarijn E. , Piontek Jörg , Bieck Marie , Saiti Arbesa , Gonschior Hannes , Lehmann Martin 
Published: December 2023

AbstractTight junctions play a pivotal role in the functional integrity of the human body by forming barriers crucial for tissue compartmentalization and protecting the body from external threats. Essential components of tight junctions are the transmembrane claudin proteins, which can polymerize into tight junction strands and meshworks. This study delves into the structural determinants of claudin polymerization, utilizing the close homology yet strong difference in polymerization capacity between claudin-3 and claudin-4. Through a combination of sequence alignment and structural modeling, critical residues in the second extracellular segment are pinpointed. Molecular dynamics simulations provide insights into the interactions of and the conformational changes induced by the identified extracellular segment 2 residues, shedding light on the intricacies of claudin polymerization. Live-STED imaging demonstrates that introduction of these residues from claudin-3 into claudin-4 significantly enhances polymerization in non-epithelial cells. In tight junction-deficient epithelial cells, mutated claudin-4 not only influences tight junction morphology but also partially restores barrier function. Understanding the structural basis of claudin polymerization is of paramount importance, as it offers insights into the dynamic nature of tight junctions. This knowledge could be applied to targeted therapeutic interventions, offering insight to repair or prevent barrier defects associated with pathological conditions, or introduce temporary barrier openings during drug delivery.
Population-scale skeletal muscle single-nucleus multi-omic profiling reveals extensive context specific genetic regulation
Authors: Varshney Arushi , Manickam Nandini , Orchard Peter , Tovar Adelaide , Ventresca Christa , Zhang Zhenhao , Feng Fan , Mears Joseph , Erdos Michael R , Narisu Narisu , Nishino Kirsten , Rai Vivek , Stringham Heather M , Jackson Anne U , Tamsen Tricia , Gao Chao , Yang Mao , Koues Olivia I , Welch Joshua D , Burant Charles F , Williams L Keoki , Jenkinson Chris , DeFronzo Ralph A , Norton Luke , Saramies Jouko , Lakka Timo A , Laakso Markku , Tuomilehto Jaakko , Mohlke Karen L , Kitzman Jacob O , Koistinen Heikki A , Liu Jie , Boehnke Michael , Collins Francis S , Scott Laura J , Parker Stephen C J 
Published: December 2023

AbstractSkeletal muscle, the largest human organ by weight, is relevant in several polygenic metabolic traits and diseases including type 2 diabetes (T2D). Identifying genetic mechanisms underlying these traits requires pinpointing cell types, regulatory elements, target genes, and causal variants. Here, we use genetic multiplexing to generate population-scale single nucleus (sn) chromatin accessibility (snATAC-seq) and transcriptome (snRNA-seq) maps across 287 frozen human skeletal muscle biopsies representing nearly half a million nuclei. We identify 13 cell types and integrate genetic variation to discover>7,000 expression quantitative trait loci (eQTL) and>100,000 chromatin accessibility QTLs (caQTL) across cell types. Learning patterns of e/caQTL sharing across cell types increased precision of effect estimates. We identify high-resolution cell-states and context-specific e/caQTL with significant genotype by context interaction. We identify nearly 2,000 eGenes colocalized with caQTL and construct causal directional maps for chromatin accessibility and gene expression. Almost 3,500 genome-wide association study (GWAS) signals across 38 relevant traits colocalize with sn-e/caQTL, most in a cell-specific manner. These signals typically colocalize with caQTL and not eQTL, highlighting the importance of population-scale chromatin profiling for GWAS functional studies. Finally, our GWAS-caQTL colocalization data reveal distinct cell-specific regulatory paradigms. Our results illuminate the genetic regulatory architecture of human skeletal muscle at high resolution epigenomic, transcriptomic, and cell-state scales and serve as a template for population-scale multi-omic mapping in complex tissues and traits.
An Integrated Optogenetic and Bioelectronic Platform for Regulating Cardiomyocyte Function
Authors: Bolonduro Olurotimi A. , Chen Zijing , Lai Yan-Ru , Cote Megan , Rao Akshita A. , Liu Haitao , Tzanakakis Emmanuel S. , Timko Brian P. 
Published: December 2023

AbstractWe report an integrated optogenetic and bioelectronic platform for stable and long-term modulation and monitoring of cardiomyocyte function in vitro. Optogenetic inputs were achieved through expression of a photoactivatable adenylyl cyclase (bPAC), that when activated by blue light caused a dose-dependent and time-limited increase in autonomous cardiomyocyte beat rate. Bioelectronic readouts were achieved through an integrated planar multi-electrode array (MEA) that provided real-time readouts of electrophysiological activity from 32 spatially-distinct locations. Irradiation at 27 μW/mm2resulted in a ca. 14% increase in beat rate within 20-25 minutes, which remained stable for at least 2 hours. The beating rate could be cycled through repeated “on” and “off” states, and its magnitude was a monotonic function of irradiation intensity. Our integrated platform opens new avenues in bioelectronic medicine, including closedloop feedback systems, with potential applications for cardiac regulation including arrhythmia diagnosis and intervention.TeaserA system that integrates optogenetic stimulation and bioelectronic recording capabilities allows for on-demand regulation of cardiac cell function.
Impact of climate change and oligotrophication on quality and quantity of lake primary production: A case study in Lake Biwa
Authors: Kazama Takehiro , Hayakawa Kazuhide , Nagata Takamaru , Shimotori Koichi , Imai Akio 
Published: December 2023

AbstractGlobal climate change and anthropogenic oligotrophication are expected to reshape the dynamics of primary production (PP) in aquatic ecosystems; however, few studies have explored their long-term effects. In theory, the PP of phytoplankton in Lake Biwa may decline over decades due to warming, heightened stratification, and anthropogenic oligotrophication. Furthermore, the PP of large phytoplankton, which are inedible to zooplankton, along with biomass-specific productivity (PBc), could decrease. In this study, data from 1976–2021 and active fluorometry measurements taken in 2020 and 2021 were evaluated. Quantitatively, the temporal dynamics of mean seasonal PP during 1971–2021 were assessed according to the carbon fixation rate to investigate relationships among environmental factors. Qualitatively, phytoplankton biomass, PP, and PBc were measured in two size fractions [edible (S) or inedible (L) for zooplankton] in 2020 and 2021, and the L:S balance for these three measures was compared between 1992 (low-temperature/high-nutrient conditions) and 2020–2021 (high-temperature/low-nutrient conditions) to assess seasonal dynamics. The results indicated that climate change and anthropogenic oligotrophication over the past 50 years have diminished Lake Biwa’s PP since the 1990s, impacting the phenology of PP dynamics. However, the L:S balance in PP and PBc has exhibited minimal change since 1992. These findings suggest that, although climate change and oligotrophication may reduce overall PP, they do not markedly alter the inedible/edible phytoplankton balance in terms of PP and PBc. Instead, as total PP declines, the production of small edible phytoplankton decreases proportionally, potentially affecting trophic transfer efficiency and material cycling in Lake Biwa.
Encompassing view of spatial and single-cell RNA-seq renews the role of the microvasculature in human atherosclerosis
Authors: Bleckwehl Tore , Maryam Sidrah , Babler Anne , Nyberg Michael , Bosteen Markus , Halder Maurice , Pyke Charles , Hvid Henning , Voetmann Louise Marie , Sluimer Judith C. , Das Vivek , Baumgart Simon , Kramann Rafael , Hayat Sikander 
Published: December 2023

AbstractAtherosclerosis is a pervasive contributor to cardiovascular diseases including ischemic heart disease and stroke. Despite the advance and success of effective lipid lowering-therapies and hypertensive agents, the residual risk of an atherosclerotic event remains high and improving disease understanding and development of novel therapeutic strategies has proven to be challenging. This is largely due to the complexity of atherosclerosis with a spatial interplay of multiple cell types within the vascular wall. Here, we generated an integrative high-resolution map of human atherosclerotic plaques by combining single-cell RNA-seq from multiple studies and novel spatial transcriptomics data from 12 human specimens to gain insights into disease mechanisms. Comparative analyses revealed cell-type and atherosclerosis-specific expression changes and associated alterations in cell-cell communication. We highlight the possible recruitment of lymphocytes via different endothelial cells of the vasa vasorum, the migration of vascular smooth muscle cells towards the lumen to become fibromyocytes, and cell-cell communication in the plaque, indicating an intricate cellular interplay within the adventitia and the subendothelial space in human atherosclerosis.
Pneumococcal Extracellular Vesicles Mediate Horizontal Gene Transfer via the Transformation Machinery
Authors: Lass Sarah Werner , Camphire Shaw , Smith Bailey E. , Eutsey Rory A. , Prentice Jojo A. , Yerneni Saigopalakrishna S. , Arun Ashni , Bridges Andrew A. , Rosch Jason W. , Conway James F. , Campbell Phil , Hiller N. Luisa 
Published: December 2023

AbstractBacterial cells secrete extracellular vesicles (EVs), the function of which is a matter of intense investigation. Here, we show that the EVs secreted by the human pathogenStreptococcus pneumoniae(pneumococcus) are associated with bacterial DNA on their surface and can deliver this DNA to the transformation machinery of competent cells. These findings suggest that EVs contribute to gene transfer in Gram-positive bacteria, and in doing so, may promote the spread of drug resistance genes in the population.SignificanceThis work extends our understanding of horizontal gene transfer and the roles of extracellular vesicles in pneumococcus. This bacterium serves as the model for transformation, a process by which bacteria can take up naked DNA from the environment. Here we show that extracellular vesicles secreted by the pneumococcus have DNA on their surface, and that this DNA can be imported by the transformation machinery facilitating gene transfer. Understanding EV-mediated gene transfer may provide new avenues to manage the spread of antibiotic drug resistance.
Impact of Cell Shape on Mitotic Spindle Positioning Forces
Authors: Xie Jing , Najafi Javad , Nommick Aude , Lederer Luc , Salle Jeremy , Dmitrieff Serge , Minc Nicolas 
Published: December 2023

ABSTRACTCell geometry is a key parameter for the regulation of mitotic spindle positioning during early embryo development and tissue morphogenesis. To date, however, we still lack an understanding for how intracellular forces that position, orient or hold mitotic spindles depend on cell geometry. Here, we usedin vivomagnetic tweezers to directly measure the forces that maintain the mitotic spindle in the center of sea urchin cells that adopt different shapes during early embryo development. We found that spindles are held by viscoelastic forces that progressively increase in amplitude as cells become more elongated during early development. By coupling direct cell shape manipulations andin vivoforce measurements, we establish how spindle associated forces increase in dose dependence with cell shape anisotropy. Cytoplasm flow analysis and hydrodynamic simulations suggest that this geometry-dependent mechanical enhancement results from a stronger hydrodynamic coupling between the spindle and cell boundaries, which dampens cytoplasm flows and spindle mobility as cells become more elongated. These findings establish how cell shape affects spindle associated forces, and suggest a novel mechanism for shape-sensing and division positioning mediated by intracellular hydrodynamics with functional implications for early embryo morphogenesis.
Darkness inhibits autokinase activity of bacterial bathy phytochromes
Authors: Huber Christina , Strack Merle , Schultheiß Isabel , Pielage Julia , Mechler Xenia , Hornbogen Justin , Diller Rolf , Frankenberg-Dinkel Nicole 
Published: December 2023

AbstractBathy phytochromes are a subclass of bacterial biliprotein photoreceptors that carry a biliverdin IXα chromophore. In contrast to prototypical phytochromes that adopt a Pr ground state, the Pfr-form is the thermally stable ground state of bathy phytochromes. Although the photobiology of bacterial phytochromes has been extensively studied since their discovery in the late 1990s, our understanding of the signal transduction process to the connected transmitter domains, which are often histidine kinases, remains insufficient. Initiated by the analysis of the bathy phytochromePaBphP fromPseudomonas aeruginosa, we performed a systematic analysis of five different bathy phytochromes with the aim to derive a general statement on the correlation of photostate and autokinase output. While all proteins adopt different Pr/Pfr-fractions in response to red, blue, and far-red light, only darkness leads to a pure or highly-enriched Pfr-form, directly correlated with the lowest level of autokinase activity. Using this information, we developed a method to quantitatively correlate the autokinase activity of phytochrome samples with well-defined stationary Pr/Pfr-fractions. We demonstrate that the off-state of the phytochromes is the Pfr-form and that different Pr/Pfr-fractions enable the organisms to fine-tune their kinase output in response to a certain light environment. Furthermore, the output response is regulated by the rate of dark reversion, which differs significantly from 5 seconds to 50 minutes half-life. Overall, our study indicates that bathy phytochromes function as sensors of light and darkness, rather than red and far-red light, as originally postulated.