bluuebunny/crossref_metadata_2025_split huggingface dataset:
optiPRM: A targeted immunopeptidomics LC-MS workflow with ultra-high sensitivity for the detection of mutation-derived tumor neoepitopes from limited input material
Authors: Salek Mogjiborahman , Förster Jonas D. , Becker Jonas P. , Meyer Marten , Charoentong Pornpimol , Lyu Yanhong , Lindner Katharina , Lotsch Catharina , Volkmar Michael , Momburg Frank , Poschke Isabel , Fröhling Stefan , Schmitz Marc , Offringa Rienk , Platten Michael , Jäger Dirk , Zörnig Inka , Riemer Angelika B. 
Published: August 2023

ABSTRACTPersonalized cancer immunotherapies such as vaccines and T cell receptor (TCR)-transgenic T cells rely on the presentation of tumor-specific peptides by human leukocyte antigen (HLA) class I molecules to cytotoxic T cells. Such neoepitopes can for example arise from somatic mutations and their identification is crucial for the rational design of new therapeutic interventions. For their detection by liquid chromatography mass spectrometry (LC-MS), we have developed a parameter optimization workflow to tune targeted assays for maximum detection sensitivity on a per peptide basis, termed optiPRM. Optimization of collision energy using optiPRM allows for improved detection of low abundant peptides that are very hard to detect using standard parameters. Applying this to immunopeptidomics, we detected a neoepitope in a patient-derived xenograft (PDX) from as little as 2.5×106cells input. Application of the workflow on small patient tumor samples allowed for the detection of five mutation-derived neoepitopes in three patients. One neoepitope was confirmed to be recognized by patient T cells. In conclusion, we here present optiPRM, a targeted MS workflow reaching ultra-high sensitivity by per peptide parameter optimization, which allowed for the identification of actionable neoepitopes from sample sizes usually available in the clinic.
Self-supervised learning for characterising histomorphological diversity and spatial RNA expression prediction across 23 human tissue types
Authors: Cisternino Francesco , Ometto Sara , Chatterjee Soumick , Giacopuzzi Edoardo , Levine Adam P. , Glastonbury Craig A. 
Published: August 2023

AbstractAs vast histological archives are digitised, there is a pressing need to be able to associate specific tissue substructures and incident pathology to disease outcomes without arduous annotation. Such automation provides an opportunity to learn fundamental biology about how tissue structure and function varies in a population. Recently, self-supervised learning has proven competitive to supervised machine learning approaches in classification, segmentation and representation learning. Here, we leverage self-supervised learning to generate histology feature representations using 1.7M images across 23 healthy tissues in 838 donors from GTEx. Using these representations, we demonstrate we can automatically segment tissues into their constituent tissue substructures and pathology proportions, and surpass the performance of conventionally used pre-trained models. We observe striking population variability in canonical tissue substructures, highlight examples of missing pathological diagnoses, incorrect assignment of target tissue and cross-tissue contamination. We demonstrate that this variability in tissue composition leads to a likely overestimation of eQTL tissue sharing and drives dramatic differential gene expression changes. We use derived tissue substructures to detect 284 tissue substructures and pathology specific eQTLs. As our derived histology representations are rich morphological descriptors of the underlying tissue, we introduce a multiple instance learning model that can predict and spatially localise individual RNA expression levels directly from histology to specific substructures and pathological features. We validate our RNA spatial predictions with matched ground truth immunohistochemistry (IHC) for several well characterised marker genes, recapitulating their known spatial specificity. Finally, we derive a gene expression spatial enrichment metric, allowing us to detect genes specifically expressed within sites of pathology (e.g. arterial calcification). Together, these results demonstrate the power of self-supervised machine learning when applied to vast histological datasets to allow researchers to pose and answer questions about tissue pathology, its spatial organisation and the interplay between morphological tissue variability and gene expression.
Testing a non-destructive assay to trackPlasmodiumsporozoites in mosquitoes over time
Authors: Oke Catherine E. , Reece Sarah E. , Schneider Petra 
Published: August 2023

AbstractBackgroundThe extrinsic incubation period (EIP), defined as the time it takes for malaria parasites in a mosquito to become infectious to a vertebrate host, is one of the most influential parameters for malaria transmission but remains poorly understood. The EIP is usually estimated by quantifying salivary gland sporozoites in subsets of mosquitoes, which requires terminal sampling. However, assays that allow repeated sampling of individual mosquitoes over time could provide better resolution of the EIP.MethodsWe tested a non-destructive assay to quantify sporozoites of two rodent malaria species,Plasmodium chabaudiandPlasmodium berghei, expelled throughout 24hr windows, from sugar-feeding substrates using quantitative PCR.ResultsThe assay can quantify sporozoites from sugar-feeding substrates, but the prevalence of parasite positive substrates is low. Multiple methods to increase the detection of expelled parasites (running additional technical replicates; using groups rather than individual mosquitoes) did not increase the detection rate, suggesting that expulsion of sporozoites is variable and infrequent.ConclusionsWe reveal successful detection of expelled sporozoites from sugar-feeding substrates. However, investigations of the biological causes underlying the low detection rate of sporozoites (e.g. mosquito feeding behaviour, frequency of sporozoite expulsion, or sporozoite clumping) are needed to maximise the utility of using non-destructive assays to quantify sporozoite dynamics. Increasing detection rates will facilitate the detailed investigation on infection dynamics within mosquitoes, which is necessary to explainPlasmodium’shighly variable EIP and improve understanding of malaria transmission dynamics.
A single-dose intramuscular immunization of pigs with lipid-nanoparticle DNA vaccines based on the hemagglutinin antigen confers complete protection against challenge infection with the homologous influenza virus strain
Authors: Nguyen The Nhu , Kumari Sushmita , Sillman Sarah , Chaudhari Jayeshbhai , Lai Danh Cong , Vu Hiep L.X. 
Published: August 2023

AbstractInfluenza A virus of swine (IAV-S) is highly prevalent and causes significant economic losses to swine producers. Due to the highly variable and rapidly evolving nature of the virus, it is critical to develop a safe and versatile vaccine platform that allows frequent updates of the vaccine immunogens to cope with the emergence of new viral strains. The main objective of this study was to assess the feasibility of using lipid nanoparticles (LNPs) as a nanocarrier to deliver DNA plasmid encoding the viral hemagglutinin (HA) gene in pigs. Intramuscular administration of a single dose of the LNP-DNA vaccines resulted in robust systemic and mucosal in pigs. Importantly, the vaccinated pigs were fully protected against challenge infection with the homologous IAV-S strain, with only one out of 12 vaccinated pigs shedding a low amount of viral genomic RNA in its nasal cavity. No gross or microscopic lesions were observed in the lungs of the vaccinated pigs at necropsy. Thus, the LNP-DNA vaccines are highly effective in protecting pigs against the homologous IAV-S strain and can serve as a promising platform for the rapid development of IAV-S vaccines.ImportanceInfluenza A virus of swine (IAV-S) is a significant pathogen of swine. The virus also poses a great public health concern due to its zoonotic potential. Although whole-inactivated virus (WIV) vaccines are available to control IAV-S, their heterologous efficacy is limited due to the substantial genetic/antigenic variation of the viral genome. This study provides compelling evidence demonstrating that lipid nanoparticle encapsulated DNA (LNP-DNA) vaccines induce robust systemic and mucosal immunity and complete protection of pigs against challenge infection with the homologous IAV-S strain. Importantly, the LNP-DNA vaccine approach meets the regulatory requirements to serve as an adaptable platform that can be frequently updated to match the emergence of new IAV-S variants. Thus, this LNP-DNA vaccine strategy exhibits great potential in effectively mitigating the economic impact of IAV-S on the swine industry and the associated public health threat.
Design of Cytotoxic T Cell Epitopes by Machine Learning of Human Degrons
Authors: Truex Nicholas L. , Mohapatra Somesh , Melo Mariane , Rodriguez Jacob , Li Na , Abraham Wuhbet , Sementa Deborah , Touti Faycal , Keskin Derin B. , Wu Catherine J. , Irvine Darrell J. , Gómez-Bombarelli Rafael , Pentelute Bradley L. 
Published: August 2023

AbstractAntigen processing is critical for producing epitope peptides that are presented by HLA molecules for T cell recognition. Therapeutic vaccines aim to harness these epitopes for priming cytotoxic T cell responses against cancer and pathogens, but insufficient processing often reduces vaccine efficacy through limiting the quantity of epitopes released. Here, we set out to improve antigen processing by harnessing protein degradation signals called degrons from the ubiquitin-proteasome system. We used machine learning to generate a computational model that ascribes a proteasomal degradation score between 0 and 100. Epitope peptides with varying degron activities were synthesized and translocated into cells using nontoxic anthrax proteins: protective antigen (PA) and the N-terminus of lethal factor (LFN). Immunogenicity studies revealed epitope sequences with a low score (<25) show pronounced T-cell activation but epitope sequences with a higher score (>75) provide limited activation. This work sheds light on the sequence–activity relationships between proteasomal degradation and epitope immunogenicity, through conserving the epitope region but varying the flanking sequence. We anticipate that future efforts to incorporate proteasomal degradation signals into vaccine designs will lead to enhanced cytotoxic T cell priming by vaccine therapeutics in clinical settings.
Mycobacterium tuberculosisresponse to cholesterol is integrated with environmental pH and potassium levels via a lipid utilization regulator
Authors: Chen Yue , MacGilvary Nathan J. , Tan Shumin 
Published: August 2023

SUMMARYHow bacterial response to environmental cues and nutritional sources may be integrated in enabling host colonization is poorly understood. Exploiting a reporter-based screen, we discovered that overexpression ofMycobacterium tuberculosis(Mtb) lipid utilization regulators altered Mtb acidic pH response dampening by low environmental potassium (K+). Transcriptional analyses unveiled amplification of Mtb response to acidic pH in the presence of cholesterol, a major carbon source for Mtb during infection, and vice versa. Strikingly, deletion of the putative lipid regulatormce3Rresulted in loss of augmentation of (i) cholesterol response at acidic pH, and (ii) low [K+] response by cholesterol, with minimal effect on Mtb response to each signal individually. Finally, the Δmce3Rmutant was attenuated for colonization in a murine model that recapitulates lesions with lipid-rich foamy macrophages. These findings reveal critical coordination between bacterial response to environmental and nutritional cues, and establish Mce3R as a crucial integrator of this process.
N4-Hydroxycytidine/Molnupiravir Inhibits RNA-Virus Induced Encephalitis by Producing Mutated Viruses with Reduced Fitness
Authors: Ojha Durbadal , Hill Collin S. , Zhou Shuntai , Evans Alyssa B. , Leung Jacqueline M. , Lewis Christine Schneider , Amblard Franck , Schinazi Raymond F. , Baric Ralph S. , Peterson Karin E. , Swanstrom Ronald 
Published: August 2023

AbstractA diverse group of RNA viruses including Rabies, Polio, La Crosse, West Nile, Zika, Nipah, Eastern and Western equine encephalitis, Venezuelan equine encephalitis, Japanese encephalitis, and tick-borne encephalitis viruses have the ability to gain access to and replicate in the central nervous system (CNS), causing severe neurological disease. Current treatment for these patients is generally limited to supportive care. To address the need for a generalizable antiviral, we utilized a strategy of mutagenesis to limit virus replication. We evaluated ribavirin (RBV), favipiravir (FAV) andN4-hydroxycytidine (NHC) against La Crosse virus (LACV) which is the primary cause of pediatric arboviral encephalitis cases in North America. NHC was more potent than RBV or FAV in neuronal cells. Oral administration of molnupiravir (MOV), the 5’-isobutyryl prodrug of NHC, decreased neurological disease development by 32% following intraperitoneal (IP) infection of LACV. MOV also reduced disease by 23% when virus was administered intranasally (IN). NHC and MOV produced less fit viruses by incorporating predominantly G-to-A or C-to-U mutations. Furthermore, NHC also inhibited two other orthobunyaviruses, Jamestown Canyon virus and Cache Valley virus. Collectively, these studies indicate that NHC/MOV has therapeutic potential to inhibit virus replication and subsequent neurological disease caused by this neurotropic RNA virus.
Genome-wide assays to characterize rAAV integration into human genomic DNA in vivo
Authors: Prout Jaime , Von Stetina Jessica , Cerqueira Gustavo , Chasse Madison , Chen Huei-Mei , Pla Andrew , Resendes Rachel , Sookiasian Danielle , Tazearslan Cagdas , Wright Jason , Thompson John F. 
Published: August 2023

AbstractAdeno-associated viral (AAV) vectors are used to treat genetic diseases, expressing therapeutic genes from both extrachromosomal episomes and payloads that integrate into the host genome. Assays were developed to evaluate HR-mediated on-target integration and the potential occurrence of off-target integration. While many studies have addressed elements of these processes, proper characterization requires long-read sequencing to ensure that integrated viral DNA is examined and not the more prevalent episomes. We used Oxford Nanopore to characterize integrated DNA and scan the whole genome for off-target integrations. These assays were applied to cell-based and in vivo models to study vectors that correct phenylketonuria (PKU), caused by loss of phenylalanine hydroxylase (PAH). Administration of the human-specific vector in a humanized-liver mouse xenograft model resulted in stable, nuclease-free integration intoPAH. Because detection of rare integration events in a much larger pool of episomal DNA is subject to artifacts, careful assay validation was required. A long-read, genome-wide assay capable of detecting on- and off-target vector integrations showed no evidence of off-target integration. Artifactual false positive events were below the limit of blank. These data support rAAV as an investigational therapeutic for genetic diseases and reinforce the need for characterization of integration assays to avoid artifacts.
DNA polymerase diversity reveals multiple incursions of Polintons during nematode evolution
Authors: Jeong Dae-Eun , Sundrani Sameer , Hall Richard Nelson , Krupovic Mart , Koonin Eugene V. , Fire Andrew Z. 
Published: August 2023

AbstractPolintons are dsDNA, virus-like self-synthesizing transposons widely found in eukaryotic genomes. Recent metagenomic discoveries of Polinton-like viruses are consistent with the hypothesis that Polintons invade eukaryotic host genomes through infectious viral particles. Nematode genomes contain multiple copies of Polintons and provide an opportunity to explore the natural distribution and evolution of Polintons during this process. We performed an extensive search of Polintons across nematode genomes, identifying multiple full-length Polinton copies in several species. We provide evidence of both ancient Polinton integrations and recent mobility in strains of the same nematode species. In addition to the major nematode Polinton family, we identified a group of Polintons that are overall closely related to the major family, but encode a distinct protein-primed B family DNA polymerase (pPolB) that is related to homologs from a different group of Polintons present outside of theNematoda. Phylogenetic analyses on the pPolBs support the evolutionary scenarios in which these extrinsic pPolBs that seem to derive from Polinton families present in oomycetes and molluscs replaced the canonical pPolB in subsets of Polintons found in terrestrial and marine nematodes, respectively, suggesting inter-phylum horizontal gene transfers. The pPolBs of the terrestrial nematode and oomycete Polintons share a unique feature, an insertion of a HNH nuclease domain, whereas the pPolBs in the marine nematode Polintons share an insertion of a VSR nuclease domain with marine mollusc pPolBs. We hypothesize that horizontal gene transfer occurs among Polintons from widely different but cohabiting hosts.
cGAS/STING and NLRP3 cooperatively activate CD8+ T cell-mediated anti-tumor immunity in colorectal cancer
Authors: Mowat Courtney , Schiller Daniel , Baker Kristi 
Published: August 2023

AbstractColorectal cancer (CRC) is a highly prevalent and deadly disease that is largely refractory to immunotherapy. The only CRC subset that responds to these therapies is characterized by prevalent microsatellite instability (MSI), extensive CD8+ T cell infiltration and high expression of innate immune signaling pathways. Endogenous activation of the cGAS/STING pathway is essential for the CD8+ T cell antitumor response in MSI CRCs, suggesting that activating it in other CRCs could boost immunotherapy response rates. We show that cGAS/STING signaling can be enhanced by costimulation of the NLRP3 inflammasome and that dual stimulation increases CD8+ T cell-mediated antitumor immunity in both MSI and non-MSI CRCs. The ability of NLRP3 to boost cGAS/STING signaling was specific and did not occur with activation of other innate immune pathways such as AIM2 or TLRs. Cooperativity between cGAS/STING and NLRP3 proceeded via a positive feedback loop that was inflammasome-independent and required early crosstalk between the signaling mediators and regulation of their gene expression. Notably, increased cGAS/STING signaling enhanced CD8+ T cell activation when in conjunction with anti-PD1 immunotherapy, suggesting that signaling via NLRP3 could further boost this response and render otherwise resistant CRC susceptible to immunotherapy.SignificanceInnate immune signaling pathways cooperatively regulate CD8+ T cell-mediated antitumor immunity in both hot and cold tumors. In addition to serving as predictive biomarkers, these pathways can be therapeutically targeted to increase response rates to immunotherapy while minimizing undesirable adverse events.
De novodesign of buttressed loops for sculpting protein functions
Authors: Jiang Hanlun , Jude Kevin M. , Wu Kejia , Fallas Jorge , Ueda George , Brunette TJ , Hicks Derrick , Pyles Harley , Yang Aerin , Carter Lauren , Lamb Mila , Li Xinting , Levine Paul M. , Stewart Lance , Garcia K. Christopher , Baker David 
Published: August 2023

AbstractIn natural proteins, structured loops play central roles in molecular recognition, signal transduction and enzyme catalysis. However, because of the intrinsic flexibility and irregularity of loop regions, organizing multiple structured loops at protein functional sites has been very difficult to achieve byde novoprotein design. Here we describe a solution to this problem that generates structured loops buttressed by extensive hydrogen bonding interactions with two neighboring loops and with secondary structure elements. We use this approach to design tandem repeat proteins with buttressed loops ranging from 9 to 14 residues in length. Experimental characterization shows the designs are folded and monodisperse, highly soluble, and thermally stable. Crystal structures are in close agreement with the computational design models, with the loops structured and buttressed by their neighbors as designed. We demonstrate the functionality afforded by loop buttressing by designing and characterizing binders for extended peptides in which the loops form one side of an extended binding pocket. The ability to design multiple structured loops should contribute quite generally to efforts to design new protein functions.
Modeling membrane geometries implicitly in Rosetta
Authors: Woods Hope M. , Leman Julia Koehler , Meiler Jens 
Published: August 2023

AbstractInteractions between membrane proteins (MPs) and lipid bilayers are critical for many cellular functions. In the Rosetta molecular modeling suite, the implicit membrane energy function is based on a “slab” model, which represent the membrane as a flat bilayer. However, in nature membranes often have a curvature that is important for function and/or stability. Even more prevalent, in structural biology research MPs are reconstituted in model membrane systems such as micelles, bicelles, nanodiscs, or liposomes. Thus, we have modified the existing membrane energy potentials within the RosettaMP framework to allow users to model MPs in different membrane geometries. We show that these modifications can be utilized in core applications within Rosetta such as structure refinement, protein-protein docking, and protein design. For MPs structures found in curved membranes, refining these structures in curved, implicit membranes produces higher quality models with structures closer to experimentally determined structures. For MP systems embedded in multiple membranes, representing both membranes results in more favorable scores compared to only representing one of the membranes. Modeling MPs in geometries mimicking the membrane model system used in structure determination can improve model quality and model discrimination.
A pectin-binding peptide with a structural and signaling role in the assembly of the plant cell wall
Authors: Schoenaers Sébastjen , Lee Hyun Kyung , Gonneau Martine , Faucher Elvina , Levasseur Thomas , Akary Elodie , Claeijs Naomi , Moussu Steven , Broyart Caroline , Balcerowicz Daria , AbdElgawad Hamada , Bassi Andrea , Damineli Daniel Santa Cruz , Costa Alex , Feijó José A , Moreau Celine , Bonnin Estelle , Cathala Bernard , Santiago Julia , Höfte Herman , Vissenberg Kris 
Published: August 2023

SummaryPressurized cells with strong walls make up the hydrostatic skeleton of plants. Assembly and expansion of such stressed walls depend on a family of secreted RAPID ALKALINIZATION FACTOR (RALF) peptides which, curiously, bind both a membrane receptor complex and wall-localized LEUCINE-RICH REPEAT EXTENSINs (LRXs) in a mutually exclusive way. Here we show that, in root hairs, the RALF22 peptide has a dual structural and signaling role in cell expansion. Together with LRX1, it directs the compaction of charged pectin polymers at the root hair tip into periodic circumferential rings. Free RALF22 induces the formation of a complex with LORELEI-LIKE-GPI-ANCHORED PROTEIN 1 (LLG1) and FERONIA (FER), triggering adaptive cellular responses. These findings show how a peptide simultaneously functions as a structural component organizing cell wall architecture and as a signaling molecule that regulates this process. This mechanism may also underlie wall assembly and expansion in other plant cell types.