bluuebunny/crossref_metadata_2025_split huggingface dataset:
Semi-automated workflow for high-throughputAgrobacterium-mediated plant transformation
Authors: Annese Davide , Romani Facundo , Grandellis Carolina , Ives Lesley , Frangedakis Eftychios , Buson Felipe X. , Molloy Jennifer C. , Haseloff Jim 
Published: October 2024

ABSTRACTHigh-throughput experiments in plants are hindered by long generation times and high costs. To address these challenges, we present an optimized pipeline forAgrobacterium tumefacienstransformation and simplified a protocol to obtain stable transgenic lines of the model liverwortMarchantia polymorpha, paving the way for efficient high-throughput experiments for plant synthetic biology and other applications. Our protocol involves freeze-thawAgrobacteriumtransformation method in 6-well plates that can be adapted to robotic automation. Using the Opentrons open-source platform, we implemented a semi-automated protocol showing similar efficiency compared to manual manipulation. Additionally, we have streamlined and simplified the process of stable transformation and selection ofM. polymorpha, reducing cost, time, and manual labour without compromising transformation efficiency. The addition of sucrose in the selection media significantly enhances the production of gemmae, accelerating the generation of isogenic plants. We believe these protocols have the potential to facilitate high-throughput screenings in diverse plant species and represent a significant step towards the full automation of plant transformation pipelines. This approach allows testing ∼100 constructs per month, using conventional plant tissue culture facilities. We recently demonstrated the successful implementation of this protocol for screening hundreds of fluorescent reporters inMarchantiagemmae.
In situ crystalline structure of the human eosinophil major basic protein-1
Authors: Yang Jie E. , Mitchell Joshua M. , Bingman Craig A. , Mosher Deane F. , Wright Elizabeth R. 
Published: October 2024

AbstractEosinophils are white blood cells that participate in innate immune responses and have an essential role in the pathogenesis of inflammatory and neoplastic disorders. Upon activation, eosinophils release cytotoxic proteins such as major basic protein-1 (MBP-1) from cytoplasmic secretory granules (SGr) wherein MBP-1 is stored as nanocrystals. How the MBP-1 nanocrystalline core is formed, stabilized, and subsequently mobilized remains unknown. Here, we report thein-situstructure of crystalline MBP-1 within SGrs of human eosinophils. The structure reveals a mechanism for intragranular crystal packing and stabilization of MBP-1 via a structurally conserved loop region that is associated with calcium-dependent carbohydrate binding in other C-type lectin (CTL) proteins. Single-cell and single-SGr profiling correlating real-space three-dimensional information from cellular montage cryo-electron tomography (cryo-ET) and microcrystal electron diffraction (MicroED) data obtained from non-activated and IL33-activated eosinophils revealed activation-dependent crystal expansion and extrusion of expanded crystals from SGr. These results suggest that MBP-1 crystals play a dynamic role in the release of SGr contents. Collectively, this research demonstrates the importance ofin-situmacromolecular structure determination.
Visualization of breast cancer using contrast-enhanced optical coherence elastography based on tissue heterogeneity
Authors: Li Jiayue , Foo Ken Y. , Sanderson Rowan W. , Zilkens Renate , Hardie Mireille , Gale Laura , Yeow Yen L. , Green Celia , Abdul-Aziz Farah , Hamzah Juliana , Stephenson James , Tayaran Ammar , Cid Fernandez Jose , Jackson Lee , Chin Synn Lynn , Hamza Saud , Rijhumal Anmol , Saunders Christobel M. , Kennedy Brendan F. 
Published: October 2024

AbstractBy mapping the mechanical properties of tissue, elastography can improve identification of breast cancer. On the macro-scale, ultrasound elastography and magnetic resonance elastography have emerged as effective clinical methods for the diagnosis of tumors. On the micro-scale, optical coherence elastography (OCE) shows promise for intraoperative tumor margin assessment during breast-conserving surgery. Whilst several OCE studies have demonstrated strong potential, the mechanical models used require the assumption of uniaxial stress throughout the sample. However, breast tissue is heterogeneous and contains compressible features (e.g., ducts and blood vessels) and collagen-rich fibrotic features (e.g., stroma). This heterogeneity can invalidate the assumption of uniaxial stress and reduce the accuracy of OCE, often making it challenging to interpret images. Here, we demonstrate a new variant of OCE based on mapping the Euler angle,i.e., the angle between the principal compression and the loading axis induced by tissue heterogeneity, which removes the assumption of uniaxial deformation. This is enabled by a hybrid three-dimensional (3-D) displacement estimation method that combines phase-sensitive detection and complex cross-correlation, providing access to the 3-D displacement and 3-D strain tensor on the micro-scale. We demonstrate this new OCE technique through experiments on phantoms and 10 fresh human breast specimens. Through close correspondence with histology, our results show that mapping the Euler angle provides additional contrast to both optical coherence tomography and a current OCE technique in identifying cancer. Mapping the Euler angle in breast tissue may provide a new biomarker for intraoperative tumor margin assessment.
Quantitative Insights into Processivity of an Hsp100 Protein Disaggregase on Folded Protein Substrates
Authors: Banwait Jaskamaljot Kaur , Lucius Aaron L. 
Published: October 2024

AbstractThe Hsp100 family of proteins play important roles in maintaining protein homeostasis in cells.E. coliClpB is an Hsp100 protein that remodels misfolded proteins or aggregates. ClpB is proposed to couple the energy from ATP binding and hydrolysis to processively unfold and translocate protein substrates through its axial channel in the hexameric ring structure. However, many of the details of this reaction remain obscure. We have recently developed a transient state kinetics approach to study ClpB catalyzed protein unfolding and translocation. In this work we have used this approach to begin to examine how ATP is coupled to the protein unfolding reaction. Here we show that at saturating [ATP], ClpB induces the cooperative unfolding of a complete TitinI27 domain of 98 amino acids, which is represented by the kinetic step-size m ∼100 amino acids. This unfolding event is followed by rapid and undetected translocation up to the next folded domain. At sub-saturating [ATP], ClpB still induces cooperative unfolding of a complete TitinI27 domain but translocation becomes partially rate-limiting, which leads to an apparent reduced kinetic step-size as small as ∼ 50 amino acids. Further, we show that ClpB exhibits an unfolding processivity of P = (0.74 ± 0.06) independent of [ATP]. These findings advance our understanding of the elementary reactions catalyzed by E. coli ClpB but are broadly applicable to a variety of Hsp100 family members.Graphical Abstract
Migration strategies of a high-latitude breeding songbird (Setophaga coronata coronata) revealed using multi-sensor geolocators and stable isotopes
Authors: Szarmach Stephanie J , Beam Johanna K , Moore Mads , Van Doren Benjamin M , Brelsford Alan , Toews David P L 
Published: October 2024

ABSTRACTSeasonal migration allows animals to use habitat where conditions are unfavorable for part of the year but may constrain breeding ranges due to the costs of longer migrations as ranges expand poleward. In species with large ranges, high latitude breeding populations may employ different migratory strategies allowing them to persist far from other core non-breeding areas. The myrtle warbler (Setophaga coronata coronata) has two disjunct non-breeding ranges in North and Central America–one along the Gulf Coast and the other on the Pacific. Previous work indirectly linked birds breeding in Alaska with the Pacific non-breeding area, suggesting that high latitude populations evolved a shorter migration route. We directly tested this hypothesis using geolocators measuring both light and atmospheric pressure to track Alaskan myrtle warbler migration in fine detail and inferred non-breeding areas using hydrogen isotopes for a larger sample of birds breeding in Alaska, British Columbia, and Alberta. We found, contrary to expectations, that all geolocator-tracked birds and most birds with stable isotope data migrated to the southeastern United States, while only a small subset of birds (∼5%) likely wintered on the Pacific Coast. We additionally demonstrate the advantages of pressure geolocation for characterizing migratory behavior at a fine scale.
Sm-site containing mRNAs can accept Sm-rings and are downregulated in Spinal Muscular Atrophy
Authors: Blatnik Anton J. , Sanjeev Manu , Slivka Jacob , Pastore Benjamin , Embree Caleb M. , Tang Wen , Singh Guramrit , Burghes Arthur H. M. 
Published: October 2024

ABSTRACTSm-ring assembly is important for the biogenesis, stability, and function of uridine-rich small nuclear RNAs (U snRNAs) involved in pre-mRNA splicing and histone pre-mRNA processing. Sm-ring assembly is cytoplasmic and dependent upon the Sm-site sequence and structural motif, ATP, andSurvival motor neuron(SMN) protein complex. While RNAs other than U snRNAs were previously shown to associate with Sm proteins, whether this association follows Sm-ring assembly requirements is unknown. We systematically identified Sm-sites within the human and mouse transcriptomes and assessed whether these sites can accept Sm-rings. In addition to snRNAs, Sm-sites are highly prevalent in the 3’ untranslated regions of long messenger RNAs. RNA immunoprecipitation experiments confirm that Sm-site containing mRNAs associate with Sm proteins in the cytoplasm. In modified Sm-ring assembly assays, Sm-site containing RNAs, from either bulk polyadenylated RNAs or those transcribedin vitro, specifically associate with Sm proteins in an Sm-site and ATP-dependent manner. In cell and animal models of Spinal Muscular Atrophy (SMA), mRNAs containing Sm-sites are downregulated, suggesting reduced Sm-ring assembly on these mRNAs may contribute to SMA pathogenesis. Together, this study establishes that Sm-site containing mRNAs can accept Sm-rings and identifies a novel mechanism for Sm proteins in regulation of cytoplasmic mRNAs.GRAPHICAL ABSTRACT
Cargo adaptor identity controls the mechanism and kinetics of dynein activation
Authors: Gillies John P. , Little Saffron R. , Hancock William O. , DeSantis Morgan E. 
Published: October 2024

AbstractCytoplasmic dynein-1 (dynein), the primary retrograde motor in most eukaryotes, supports the movement of hundreds of distinct cargos, each with specific trafficking requirements. To achieve this functional diversity, dynein must bind to the multi-subunit complex dynactin and one of a family of cargo adaptors to be converted into an active, processive motor complex. Very little is known about the dynamic processes that promote the formation of this complex. To delineate the kinetic steps that lead to dynein activation, we developed a single-molecule fluorescence assay to visualize the real-time formation of dynein-dynactin-adaptor complexes in vitro. We found that dynactin and adaptors bind dynein independently rather than cooperatively. We also found that different dynein adaptors promote dynein-dynactin-adaptor assembly with dramatically different kinetics, which results in complex formation occurring via different assembly pathways. Despite differences in association rates or mechanism of assembly, all adaptors tested can generate a population of tripartite complexes that are very stable. Our work provides a model for how modulating the kinetics of dynein-dynactin-adaptor binding can be harnessed to promote differential dynein activation and reveals a new facet of the functional diversity of the dynein motor.
Gender- and Age-Based Characterization and Comparison of the Murine Primary Peritoneal Mesothelial Cell Proteome
Authors: Wang Zhikun , Liu Yueying , Safavisohi Reihaneh , Asem Marwa , Hu Daniel D. , Stack Mary Sharon , Champion Matthew M. 
Published: October 2024

ABSTRACTOrgans in the abdominal cavity are covered by a peritoneal membrane, which is comprised of a monolayer of mesothelial cells (MC). Diseases involving the peritoneal membrane include peritonitis, primary cancer (mesothelioma), and metastatic cancers (ovarian, pancreatic, colorectal). These diseases have gender- and/or age-related pathologies; however, the impact of gender and age on the peritoneal MC is not well evaluated. To address this, we identified and characterized gender- and age-related differences in the proteomes of murine primary peritoneal MC. Primary peritoneal MC were isolated from young female (FY) or male (MY) mice (3-6 months) and aged female (FA) or male (MA) mice (20-23 months), lysed, trypsin digested using S-Traps, then subjected to bottom-up proteomics using an LC-Orbitrap mass spectrometer. In each cohort, we identified >1000 protein groups. Proteins were categorized using Gene Ontology and pairwise comparisons between gender and age cohorts were conducted. This study establishes baseline information for studies on peritoneal MC in health and disease at two physiologic age/gender points. Segregation of the data by gender and age could reveal novel factors to specific disease states involving the peritoneum. [Thisin vitroprimary cell model has utility for future studies on the interaction between the mesothelium and foreign materials.]SUMMARY STATEMENTMany diseases initiate from or involve peritoneal mesothelial cells including peritonitis, primary cancer (mesothelioma) and metastatic cancers. Progression of these diseases is influenced by many host factors including gender and age; however, the influence of these factors on the peritoneal mesothelial cell proteome has not been evaluated. This study provides novel information and identifies proteins exclusive to both male and female young and aged cohorts. Given the importance of the peritoneal mesothelial cell in abdominal homeostasis, and the impact of gender and age on disease progression, these data will be key for future studies examining mesothelium in both health and disease.
Chikungunya Virus VLP Vaccine: Phase 3 Trial in Adults ≥65 Years of Age
Authors: Tindale Lauren C. , Richardson Jason S. , Anderson Debbie M. , Mendy Jason , Muhammad Sufia , Loreth Tobi , Royalty Tredo Sarah , Ramanathan Roshan , Jenkins Victoria A. , Bedell Lisa , Ajiboye Patrick ,   the EBSI-CV-317-005 Study Group
Published: October 2024

1ABSTRACTBackgroundAdults ≥65 years of age are at increased risk for atypical presentations of chikungunya disease as well as severe outcomes including death.MethodsA phase 3, randomized, double-blind, placebo-controlled, parallel-group trial was conducted in adults ≥65 years of age where participants received a single intramuscular dose of chikungunya virus (CHIKV) virus-like particle (VLP) vaccine or placebo on Day 1. Baseline and postvaccination CHIKV serum neutralizing antibody (SNA) titers (NT80) were assessed at selected timepoints. Safety was assessed through Day 183.ResultsA total of 413 participants (206 vaccine, 207 placebo) were randomized. Coprimary endpoints were met including 1) immunologic superiority of CHIKV SNA titers compared to placebo and 2) by geometric mean titer at Day 22. CHIKV VLP vaccine induced a protective seroresponse (SNA NT80≥100; considered the presumptive seroprotective antibody response), in 82% of individuals at Day 15, in 87% of individuals at Day 22, and in 76% of individuals at Day 183. There were no notable differences in AE rates between groups and most AEs were grade 1 or 2 in severity. No vaccine-related serious adverse events or deaths occurred.ConclusionsWe provide robust data from adults ≥65 years of age demonstrating that CHIKV VLP vaccine has a favorable safety profile and can provide a high rate of protection within 2 weeks postvaccination and through 6 months of follow-up. (Funded by Emergent BioSolutions Inc. and Bavarian Nordic A/S [as successor in interest to Emergent BioSolutions Inc.];ClinicalTrials.govnumber,NCT05349617).
Do self-help groups improve sexual and reproductive health and HIV outcomes among female sex workers in sub-Saharan Africa? A scoping review
Authors: Madimutsa Gracious , Machingura Fortunate , Nyamwanza Owen , Cowan Frances M , Mavhu Webster 
Published: October 2024

AbstractIntroductionSelf-help groups (SHGs) have been effective in improving the health and wellbeing of women generally but there is little evidence on whether and how they improve HIV and sexual and reproductive health (SRH) outcomes among female sex workers (FSWs), particularly in sub-Saharan Africa. This scoping review seeks to address this gap by identifying and analysing literature on SHG for FSWs in sub-Saharan Africa.Materials and methodsThis scoping review (1) identified relevant studies; (2) selected the studies; (3) charted the data; and (4) collated, summarised, and reported the results. A search strategy was developed; CINAHL, Medline and Global Health databases were searched.ResultsEleven studies were identified, two were quantitative, seven were qualitative and two were mixed methods. Studies were from seven countries in sub-Saharan Africa. The studies suggested that SHGs can improve SRH outcomes and reduce HIV vulnerabilities among FSWs by providing emotional and financial support, health education, linkage to care, and social capital (i.e., benefits derived from associations). The studies also highlighted the need for tailored interventions that address the unique needs and challenges faced by FSWs.ConclusionsThe findings of this scoping review underscore the importance of building social cohesion by incorporating SHGs into a range of HIV prevention strategies in sub-Saharan Africa. SHGs have the potential to improve SRH and HIV outcomes among FSWs. Further research is needed to explore the effectiveness of SHGs in different contexts and to identify best practices for implementing and sustaining SHGs for FSWs.
Structural basis of SIRT7 nucleosome engagement and substrate specificity
Authors: Moreno-Yruela Carlos , Ekundayo Babatunde E. , Foteva Polina N. , Calvino-Sanles Esther , Ni Dongchun , Stahlberg Henning , Fierz Beat 
Published: October 2024

AbstractChromatin-modifying enzymes selectively target distinct residues within histones to finetune gene expression profiles. SIRT7 is an NAD+-dependent histone deacylase often deregulated in cancer, which deacetylates either H3 lysine 36 (H3K36) or H3K18 with high specificity within nucleosomes. Here, we report structures of nucleosome-bound SIRT7, and uncover the structural basis of its specificity towards H3K36 and K18 deacylation, combining a mechanism-based cross-linking strategy, cryo-EM, mutagenesis and enzymatic assays. We show that the SIRT7 N-terminus represents a unique, extended nucleosome-binding domain, reaching across the nucleosomal surface to the acidic patch. The catalytic domain binds at the H3-tail exit site, engaging both DNA gyres of the nucleosome. Contacting H3K36 versus H3K18 requires a change in enzyme binding pose, and results in structural changes in both SIRT7 and the nucleosome. These structures reveal interactions critical for target lysine specificity, allowing us to engineer enzyme activity towards H3K18 or 36, and provides a basis for small molecule modulator development.
Improving HEK293-based AAV-production using GSMMs, and a multi-omics approach
Authors: Zehetner L. , Széliová D. , Kraus B. , Hernandez Bort J. A. , Zanghellini J. 
Published: October 2024

ABSTRACTHEK293 cells are a versatile cell line extensively used in the production of recombinant proteins and viral vectors, notably Adeno-associated virus (AAV) [12]. Despite their high transfection efficiency and adaptability to various culture conditions, challenges remain in achieving sufficient yields of active viral particles. This study presents a comprehensive multi-omics analysis of two HEK293 strains under good manufacturing practice conditions, focusing on the metabolic and cellular responses during AAV production. The investigation included lipidomic, exometabolomic, and transcriptomic profiling across different conditions and time points. Genome-scale metabolic models (GSMMs) were reconstructed for these strains to elucidate metabolic shifts and identify potential bottlenecks in AAV production. Notably, the study revealed significant differences between a High-producing (HP) and a Low-producing (LP) HEK293 strains, highlighting pseudohypoxia in the LP strain. Key findings include the identification of hypoxia-inducible factor 1-alpha (HIF1alpha) as a critical regulator in the LP strain, linking pseudohypoxia to poor AAV productivity. Inhibition of HIF1alpha resulted in immediate cessation of cell growth and a 2-fold increase in viral capsid production, albeit with a decreased number of viral genomes, impacting the full-to-empty particle ratio. This suggests that while HIF1alpha inhibition enhances capsid assembly, it simultaneously hampers nucleotide synthesis via the pentose phosphate pathway (PPP), necessary for genome packaging.
Mining the Proteome of Human Ovarian Cancer Extracellular Vesicles Using Thermolysin Proteolysis
Authors: Cooper Tyler T. , Lui Jaihui , Bilyk Olena , Jewer Michael ,   AOCS Study Group, Steed Helen , Fu Yangxin , Postovit Lynne-Marie , Lajoie Gilles A. 
Published: October 2024

AbstractThis study investigates the utility of Thermolysin as a proteolytic enzyme to enhance the depth and coverage of proteomic analysis in ovarian cancer (OC) extracellular vesicles (EVs). EVs were isolated from OC cell lines and ascites fluid samples from women diagnosed with high-grade serous carcinoma. Proteins were digested using Thermolysin and Trypsin/LysC, followed by label-free data-dependent acquisition (DDA) and data-independent acquisition (DIA) mass spectrometry. The proteolytic efficiency, sequence coverage, peptide complexity, and proteomic depth were compared between Thermolysin and Trypsin/LysC digests. In silico analyses predicted theoretical benchmarks of these parameters using a core set of 22 proteins, and Gene set enrichment analyses (GSEA) highlighted the biological relevance of proteins identified throughout the study. Thermolysin digestion significantly increased the complexity or of peptide pools compared to Trypsin/LysC leading to limited peptide and protein identification, albeit total sequence coverage was increased through complementation to tryptic peptides. In both cell line and ascites EVs, Thermolysin identified unique proteins not detected by Trypsin/LysC that are known drivers of metastatic solid cancers, such as Ly6E. Offline strong cation exchange (SCX) fractionation improved proteomic depth and sequence coverage obtained with Thermolysin to generate a spectral library for DIA. Our DIA analysis of Thermolysin digests revealed the presence of NODAL in OC ascites EVs, a protein associated with poor clinical prognosis, which was not detected in Trypsin/LysC digests. The importance of NODAL was cross-validated in TGCA-OV and AOCS datasets by clinical cohorts by assessing RNA levels in solid tumors or ascites fluid, respectively. Collectively, we demonstrate that Thermolysin complements traditional enzymes like Trypsin/LysC to provide a more comprehensive proteomic landscape for biomarker discovery.