bluuebunny/crossref_metadata_2025_split huggingface dataset:
Automated Purification of DNA Origami with SPRI Beads
Authors: Chau Chalmers , Mohanan Gayathri , Macaulay Iain , Actis Paolo , Wälti Christoph 
Published: July 2023

AbstractDNA origami synthesis is a well-established technique and has been employed in various applications. The synthesised origami must be purified to eliminate the excess materials such as DNA oligos and other molecules. While several purification techniques are routinely used, they all have limitations, and none can be automated to simultaneously handle large numbers and quantities of samples. Here we introduce the use of solid-phase immobilisation (SPRI) beads as an easy-to-adopt, scalable, high-throughput and automation-compatible method to purify DNA origami. Not only can this method remove excess oligos and biomolecules with comparable yield to existing methods while maintaining high structural integrity of the origami, but it also allows an automated workflow to simultaneously purify large numbers of samples within a limited time. We envision that the SPRI beads purification approach will improve the scalability of DNA nanostructures synthesis both for research and commercial applications.
Fecal and skin microbiota of two rescued Mediterranean monk seal pups during rehabilitation
Authors: Dosi Aggeliki , Meziti Alexandra , Tounta Eleni , Koemtzopoulos Kimon , Komnenou Anastasia , Dendrinos Panagiotis , Kormas Konstantinos 
Published: July 2023

ABSTRACTThe role of animal host-associated microbiomes is becoming more apparent and defined for wild animals, especially for the species under conservation strategies. This study investigated the succession of fecal and skin bacterial microbiota of two rescued female Mediterranean monk seal (Monachus monachus) pups for most of their rehabilitation period. Bacterial species richness and diversity was assessed by high-throughput sequencing of nine freshly collected fecal samples and four skin swabs per individual. Both the fecal and skin microbiota highly overlapped in their containing operational taxonomic units (OTUs) and abundance patterns. The fecal microbiota was separated in two distinct periods, and was dominated by OTUs related to theShigella,Streptococcus,Enterococcus,LactobacillusandEscherichiagenera in the first period, while in the second period the dominating genera were theClostridium, Blautia, Fusobacterium, Edwardsiellaand Bacteroides. The skin microbiota was highly similar between the two individuals in each sampling and were dominated byPsychrobacter-, Elizabethkingia-andBergeyella-related OTUs. The provided antibiotic treatment along with the provided probiotics and nutritional supplements, resulted in a major turnover of the bacterial microbiota with the potentially detrimental OTUs being eliminated towards the end of the rehabilitation period, prior to the release of the pups in the wild.
Yeast-based heterologous production of the Colletochlorin family of fungal secondary metabolites
Authors: Geistodt-Kiener Aude , Totozafy Jean Chrisologue , Goff Géraldine Le , Vergne Justine , Sakai Kaori , Ouazzani Jamal , Mouille Grégory , Viaud Muriel , O’Connell Richard J. , Dallery Jean-Félix 
Published: July 2023

AbstractTranscriptomic studies have revealed that fungal pathogens of plants activate the expression of numerous biosynthetic gene clusters (BGC) exclusively when in presence of a living host plant. The identification and structural elucidation of the corresponding secondary metabolites remain challenging. Here we adapted a polycistronic vector for efficient, seamless and cost-effective cloning of biosynthetic genes using in vivo assembly (also called transformation-assisted recombination) directly in Escherichia coli followed by heterologous expression in Saccharomyces cerevisiae. Two vectors were generated with different auto-inducible yeast promoters and selection markers. The effectiveness of these vectors was validated with fluorescent proteins. As a proof-of-principle, we applied our approach to the Colletochlorin family of molecules. These polyketide secondary metabolites were known from the phytopathogenic fungus Colletotrichum higginsianum but had never been linked to their biosynthetic genes. Considering the requirement for an halogenase, and by applying comparative genomics, we identified a BGC putatively involved in the biosynthesis of Colletochlorins in C. higginsianum. Following the expression of those genes in S. cerevisiae, we could identify the presence of the precursor Orsellinic acid, Colletochlorins and their non-chlorinated counterparts, the Colletorins. In conclusion, the polycistronic vectors described herein were adapted for the host S. cerevisiae and allowed to link the Colletochlorin compound family to their corresponding biosynthetic genes. This system will now enable the production and purification of infection-specific secondary metabolites of fungal phytopathogens. More widely, this system could be applied to any fungal BGC of interest.
Machine-learning analysis of factors that shape cancer aneuploidy landscapes reveals an important role for negative selection
Authors: Jubran Juman , Slutsky Rachel , Rozenblum Nir , Rokach Lior , Ben-David Uri , Yeger-Lotem Esti 
Published: July 2023

AbstractAneuploidy, an abnormal number of chromosomes within a cell, is considered a hallmark of cancer. Patterns of aneuploidy differ across cancers, yet are similar in cancers affecting closely-related tissues. The selection pressures underlying aneuploidy patterns are not fully understood, hindering our understanding of cancer development and progression. Here, we applied interpretable machine learning (ML) methods to study tissue-selective aneuploidy patterns. We defined 20 types of features of normal and cancer tissues, and used them to model gains and losses of chromosome-arms in 24 cancer types. In order to reveal the factors that shape the tissue-specific cancer aneuploidy landscapes, we interpreted the ML models by estimating the relative contribution of each feature to the models. While confirming known drivers of positive selection, our quantitative analysis highlighted the importance of negative selection for shaping the aneuploidy landscapes of human cancer. Tumor-suppressor gene density was a better predictor of gain patterns than oncogene density, and vice-versa for loss patterns. We identified the contribution of tissue-selective features and demonstrated them experimentally for chr13q gain in colon cancer. In line with an important role for negative selection in shaping the aneuploidy landscapes, we found compensation by paralogs to be a top predictor of chromosome-arm loss prevalence, and demonstrated this relationship for one such paralog interaction. Similar factors were found to shape aneuploidy patterns in human cancer cell lines, demonstrating their relevance for aneuploidy research. Overall, our quantitative, interpretable ML models improve the understanding of the genomic properties that shape cancer aneuploidy landscapes.
Molecular insights into atypical modes of β-arrestin interaction with seven transmembrane receptors
Authors: Maharana Jagannath , Sano Fumiya K. , Sarma Parishmita , Yadav Manish K. , Duan Longhan , Stepniewski Tomasz M. , Chaturvedi Madhu , Ranjan Ashutosh , Singh Vinay , Saha Sayantan , Mahajan Gargi , Chami Mohamed , Shihoya Wataru , Selent Jana , Chung Ka Young , Banerjee Ramanuj , Nureki Osamu , Shukla Arun K. 
Published: July 2023

Abstractβ-arrestins are multifunctional proteins that are critically involved in regulating spatio-temporal aspects of GPCR signaling. The interaction of β-arrestins with GPCRs is typically conceptualized in terms of receptor activation and phosphorylation primarily in the carboxyl-terminus. Interestingly however, there are several GPCRs that harbor majority of phosphorylation sites in their 3rdintracellular loop (ICL3) instead of carboxyl-terminus but still robustly engage β-arrestins. Moreover, there are several 7TMRs that are now characterized as intrinsically-biased, β-arrestin-coupled receptors (ACRs) due to lack of functional G-protein-coupling but robust β-arrestin binding leading to functional outcomes. The molecular basis of β-arrestin interaction and activation upon binding to these types of 7TMRs is currently elusive, and it represents a major knowledge gap in our current understanding of this signaling system. Here, we present seven cryo-EM structures of β-arrestins in basal state, activated by the muscarinic M2 receptor (M2R) through its ICL3, and a β-arrestin-coupled receptor known as decoy D6 receptor (D6R). These structural snapshots combined with biochemical, cellular, and biophysical experiments including HDX-MS and MD simulation provide novel insights into the ability of β-arrestins to preferentially select specific phosphorylation patterns in the receptors, and also illuminate the structural diversity in 7TMR-β-arrestin interaction. Surprisingly, we also observe that the carboxyl-terminus of β-arrestin2 but not β-arrestin1 undergoes structural transition from a β-strand to α-helix upon activation by D6R, which may preclude the core-interaction with the activated receptor. Taken together, our study elucidates previously unappreciated aspects of 7TMR-β-arrestin interaction, and provides important mechanistic clues about how the two isoforms of β-arrestins can recognize and regulate a large repertoire of GPCRs.
Reverse engineering the anti-MUC1 hybridoma antibody 139H2 by mass spectrometry-basedde novosequencing
Authors: Peng Weiwei , Giesbers Koen C.A.P. , Šiborová Marta , Beugelink J. Wouter , Pronker Matti F. , Schulte Douwe , Hilkens John , Janssen Bert J.C. , Strijbis Karin , Snijder Joost 
Published: July 2023

AbstractMucin 1 (MUC1) is a transmembrane mucin expressed at the apical surface of epithelial cells at different mucosal surfaces including breast and intestine. In the gastrointestinal tract, MUC1 has a barrier function against bacterial invasion, but can also serve as an entry receptor for pathogenicSalmonellabacteria. Moreover, MUC1 is well known for its aberrant expression and glycosylation in adenocarcinomas The MUC1 extracellular domain contains a variable number of tandem repeats (VNTR) of 20 amino acids, which are heavilyO-linked glycosylated.. Monoclonal antibodies against the MUC1 VNTR can be powerful tools because of their multiplicity of binding and possible applications in the diagnosis and treatment of MUC1-expressing cancers. One such antibody is the hybridoma mouse monoclonal 139H2, which is also widely used as a research tool to study non-cancer MUC1. Here we report direct mass spectrometry-based sequencing of hybridoma-derived 139H2 IgG, which enabled reverse engineering of a recombinant 139H2. The performance of the reverse engineered 139H2 IgG and its Fab fragment were validated by comparison to the hybridoma-derived product in Western blot and immunofluorescence microscopy. The reverse engineering of 139H2 allowed us to characterize binding to the VNTR peptide epitope by surface plasmon resonance (SPR) and solve the crystal structure of the 139H2 Fab fragment in complex with the MUC1 VNTR peptide. These analyses reveal the molecular basis for 139H2 binding specificity to MUC1 and its tolerance toO-glycosylation of the VNTR. The available sequence of 139H2 will allow further development of MUC1-related diagnostics, targeting and treatment strategies.
Metal-induced energy transfer (MIET) imaging of cell surface engineering with multivalent DNA nanobrushes
Authors: Wang Dong-Xia , Liu Bo , Han Gui-Mei , Li Qing-Nan , Kong De-Ming , Enderlein Jörg , Chen Tao 
Published: July 2023

AbstractThe spacing between cells has a significant impact on cell-cell interactions, which are critical to the fate and function of both individual cells and multicellular organisms. However, accurately measuring the distance between cell membranes and the variations between different membranes has proven to be a challenging task. In this study, we employ metal-induced energy transfer (MIET) imaging/spectroscopy to determine and track the inter-membrane distance and variations with nanometer precision. We have developed a DNA-based molecular adhesive called the DNA nanobrush, which serves as a cellular adhesive for connecting plasma membranes of different cells. By manipulating the number of base pairs within the DNA nanobrush, we can modify various aspects of cell-cell interactions, such as adhesive directionality, distance, and forces. We demonstrate that such nanometer-level changes can be detected with the MIET imaging/spectroscopy. Moreover, we successfully employ MIET to measure distance variations between a cellular plasma membrane and a model membrane. This experiment does not only showcase the effectiveness of MIET as a powerful tool for accurately quantifying cell-cell interactions, but does also validate the potential of DNA nanobrushes as cellular adhesives. This innovative method holds significant implications for advancing the study of multicellular interactions.
Survival and Developmental Progression of Unselected Thymocytes in the Absence of the T Cell Adaptor Gads
Authors: Shalah Rose , Marzouk Manal , Hallumi Enas , Klopstock Naama , Yablonski Deborah 
Published: July 2023

AbstractThymic development is tightly regulated by TCR signaling via the immune adaptors SLP-76 and LAT. Gads bridges the TCR-induced recruitment of SLP-76 to LAT; yet is not absolutely required for thymic progression. To better identify Gads-dependent developmental transitions, we performed tamoxifen-induced ablation of Gads (GadsiKO), accompanied by expression of tdTomato, and compared the development of Gads-expressing (Tom−) and - ablated (Tom+) thymocytes within the same mouse. The frequency of GadsiKO(Tom+) thymocytes decreased at β-selection and positive selection, confirming the Gads-dependence of these junctures; nevertheless, we observed small populations of positively-selected GadsiKOSP thymocytes. Consistent with a signaling defect, expression of CD5 was strongly impaired at the β-selection checkpoint and within the DP compartment; moreover, GadsiKODP thymocytes exhibited reduced TCR-induced calcium flux. Surprisingly, MHC-non-responding (CD5−) GadsiKODP thymocytes exhibited reduced death by neglect; instead, aberrant populations of CD5−GadsiKOthymocytes progressed as far as the CD4 SP compartment, while lacking key characteristics of positively selected thymocytes. In an experimentally-induced model of death by neglect triggered by CD8 crosslinking, Gads was required for the preferential apoptosis of CD5loDP thymocytes. Our results suggest that Gads promotes passage through TCR-driven developmental checkpoints while also promoting the death by neglect of unselected thymocytes.
It’s a Trap?! Escape from an ancient, ancestral sex chromosome system and implication ofFoxl2as the putative primary sex determining gene in a lizard (Anguimorpha; Shinisauridae)
Authors: Pinto Brendan J. , Nielsen Stuart V. , Sullivan Kathryn A. , Behere Ashmika , Keating Shannon E. , van Schingen-Khan Mona , Nguyen Truong Quang , Ziegler Thomas , Pramuk Jennifer , Wilson Melissa A. , Gamble Tony 
Published: July 2023

AbstractAlthough sex determination is ubiquitous in vertebrates, mechanisms of sex determination vary from environmentally-to genetically-influenced. In vertebrates, genetic sex determination is typically accomplished with sex chromosomes. Groups like mammals maintain conserved sex chromosome systems, while sex chromosomes in most vertebrate clades aren’t conserved across similar evolutionary timescales. One group inferred to have an evolutionarily stable mode of sex determination is Anguimorpha, a clade of charismatic taxa including: monitor lizards, Gila monsters, and crocodile lizards. The common ancestor of extant anguimorphs possessed a ZW system that has been retained across the clade. However, the sex chromosome system in the endangered, monotypic family of crocodile lizards (Shinisauridae) has remained elusive. Here, we analyze genomic data to demonstrate thatShinisaurushas replaced the ancestral anguimorph ZW system on LG7 chromosome with a novel ZW system on LG3. The linkage group LG3 corresponds to chromosome 9 in chicken, and this is the first documented use of this syntenic block as a sex chromosome in amniotes. Additionally, this ∼1Mb region harbors approximately 10 genes, including a duplication of the sex-determining transcription factor,Foxl2—critical for the determination and maintenance of sexual differentiation in vertebrates, and thus a putative primary sex determining gene forShinisaurus.
Selective Inhibition of Interleukin-2 Inducible T Cell Kinase (ITK) Enhances Anti-Tumor Immunity in Association with Th1-skewing, Cytotoxic T cell Activation, and Reduced T Cell Exhaustion
Authors: Hsu Lih-Yun , Rosenbaum James T , Verner Erik , Jones William B , Hill Craig M. , Janc James W. , Buggy Joseph J. , Ding Ning , Reneau John C. , Khodadoust Michael S. , Kim Youn H. , Wilcox Ryan A. , Miller Richard A. 
Published: July 2023

ABSTRACTITK is a tyrosine kinase expressed predominantly by T lymphocytes. In mice, selective knock-out of the ITK gene produces Th1 skewing of T helper cell differentiation. We synthesized a covalent ITK inhibitor, soquelitinib, that binds ITK with greater than 100-fold selectivity compared to binding to resting lymphocyte kinase (RLK).In vitrostudies with normal or malignant T cells demonstrated that soquelitinib suppresses Th2 cytokine production preferentially with relative sparing of Th1 cytokines. Soquelitinib inhibits thein vivogrowth of several syngeneic murine tumors including those that do not express ITK. Treatment with soquelitinib leads to increased tumor infiltration of normal CD8+ cells that possess enhanced T effector function. Soquelitinib inhibited expression of T cell exhaustion markers and was able to restore T effector function to exhausted cells. Pharmacologic selective ITK inhibition may represent a novel approach to cancer immunotherapy.
Alphavirus-induced transcriptional and translational shutoffs play major roles in blocking the formation of stress granules
Authors: Palchevska Oksana , Dominguez Francisco , Frolova Elena I. , Frolov Ilya 
Published: July 2023

AbstractAlphavirus infections cause multiple alterations in the intracellular environment that can have both positive and negative effects on viral replication. The Old World alphaviruses, such as Sindbis (SINV), chikungunya (CHIKV), and Semliki Forest viruses, hinder the ability of vertebrate cells to form stress granules (SGs). Previously, this inhibitory function was attributed to the hypervariable domain (HVD) of nsP3, which sequesters the key components of SGs, G3BP1 and G3BP2, and to the nsP3 macro domain. The macro domain possesses ADP-ribosylhydrolase activity, which can diminish the ADP-ribosylation of G3BP1 during viral replication. However, our recent findings do not support the prevailing notions. We demonstrate that the interactions between SINV– or CHIKV-specific nsP3s and G3BPs, and the ADP-ribosylhydrolase activity are not major contributors to the inhibitory process, at least when nsP3 is expressed at biologically relevant levels. Instead, the primary factors responsible for suppressing SG formation are virus-induced transcriptional and translational shutoffs that rapidly develop within the first few hours post infection. Poorly replicating SINV variants carrying mutated nsP3 HVD still inhibit SG development even in the presence of NaAs. Conversely, SINV mutants lacking transcription and/or translation inhibitory functions lose their ability to inhibit SGs, despite expressing high levels of wt nsP3. Moreover, we found that stable cell lines expressing GFP-nsP3 fusions retain the capacity to form SGs when exposed to sodium arsenite. However, our results do not rule out a possibility that additional virus-induced changes in cell biology may contribute to the suppression of SG formation.ImportanceOur study highlights the mechanisms behind the cell’s resistance to SG formation after infection with Old World alphaviruses. Shortly after infection, the replication of these viruses hinders the cell’s ability to form SGs, even when exposed to chemical inducers such as sodium arsenite. This resistance is primarily attributed to virus-induced transcriptional and translational shutoffs, rather than interactions between the viral nsP3 and the key components of SGs, G3BP1/2, or the ADP-ribosylhydrolase activity of nsP3 macro domain. While interactions between G3BP and nsP3 are essential for the formation of viral replication complexes, their role in regulating SG development appears to be minimal, if any. Cells harboring replicating virus-specific RNA with modified abilities to inhibit transcription and/or translation, but encoding wt nsP3, retain the capacity for SG development. Understanding these mechanisms of regulation of SG development contributes to our knowledge of viral replication and the intricate relationships between alphaviruses and host cells.
Evaluation of AlphaFold Antibody-Antigen Modeling with Implications for Improving Predictive Accuracy
Authors: Yin Rui , Pierce Brian G. 
Published: July 2023

AbstractHigh resolution antibody-antigen structures provide critical insights into immune recognition and can inform therapeutic design. The challenges of experimental structural determination and the diversity of the immune repertoire underscore the necessity of accurate computational tools for modeling antibody-antigen complexes. Initial benchmarking showed that despite overall success in modeling protein-protein complexes, AlphaFold and AlphaFold-Multimer have limited success in modeling antibody-antigen interactions. In this study, we performed a thorough analysis of AlphaFold’s antibody-antigen modeling performance on 429 nonredundant antibody-antigen complex structures, identifying useful confidence metrics for predicting model quality, and features of complexes associated with improved modeling success. We show the importance of bound-like component modeling in complex assembly accuracy, and that the current version of AlphaFold improves near-native modeling success to over 30%, versus approximately 20% for a previous version. With this improved success, AlphaFold can generate accurate antibody-antigen models in many cases, while additional training may further improve its performance.
RegulatingPCCAgene expression by modulation of pseudoexon splicing patterns to rescue enzyme activity in propionic acidemia
Authors: Petersen Ulrika Simone Spangsberg , Dembic Maja , Martínez-Pizarro Ainhoa , Richard Eva , Holm Lise Lolle , Havelund Jesper Foged , Doktor Thomas Koed , Larsen Martin Røssel , Færgeman Nils J. , Desviat Lourdes Ruiz , Andresen Brage Storstein 
Published: July 2023

AbstractPseudoexons are nonfunctional intronic sequences that can be activated by deep intronic sequence variation. Activation increases pseudoexon inclusion in mRNA and interferes with normal gene expression. ThePCCAc.1285-1416A>G variation activates a pseudoexon and causes the severe metabolic disorder, propionic acidemia, by deficiency of the propionyl-CoA carboxylase enzyme encoded byPCCAandPCCB. We characterized this pathogenic pseudoexon activation event in detail and identified hnRNP A1 to be important for normal repression. ThePCCAc.1285-1416A>G variation disrupts an hnRNP A1-binding splicing silencer and simultaneously creates a splicing enhancer. We demonstrate that blocking this region of regulation with splice-switching antisense oligonucleotides restores normal splicing and rescues enzyme activity in patient fibroblasts and in a cellular model created by CRISPR gene editing. ThePCCApseudoexon can be exploited as a gene-regulatory switch, as healthy tissues show relatively high levels of inclusion. By blocking inclusion of the non-activated wild type pseudoexon, we increase both PCCA and PCCB protein levels, which increases the activity of the heterododecameric enzyme. Surprisingly, we can increase enzyme activity from residual levels not only in patient fibroblasts harboringPCCAmissense variants, but also those harboringPCCBmissense variants. This could be a potential treatment strategy for propionic acidemia.