bluuebunny/crossref_metadata_2025_split huggingface dataset:
Human xenobiotic metabolism proteins have full-length and split homologs in the gut microbiome
Authors: Rendina Matthew , Turnbaugh Peter J. , Bradley Patrick H. 
Published: November 2024

AbstractXenobiotics, including pharmaceutical drugs, can be metabolized by both host and microbiota, in some cases by homologous enzymes. We conducted a systematic search for all human proteins with gut microbial homologs. Because gene fusion and fission can obscure homology detection, we built a pipeline to identify not only full-length homologs, but also cases where microbial homologs were split across multiple adjacent genes in the same neighborhood or operon (“split homologs”). We found that human proteins with full-length gut microbial homologs disproportionately participate in xenobiotic metabolism. While this included many different enzyme classes, short-chain and aldo-keto reductases were the most frequently detected, especially in prevalent gut microbes, while cytochrome P450 homologs were largely restricted to lower-prevalence facultative anaerobes. In contrast, human proteins with split homologs tended to play roles in central metabolism, especially of nucleobase-containing compounds. We identify twelve specific drugs that gut microbial split homologs may metabolize; two of these, 6-mercaptopurine by xanthine dehydrogenase (XDH) and 5-fluorouracil by dihydropyrimidine dehydrogenase (DPYD), have been recently confirmed in mouse models. This work provides a comprehensive map of homology between the human and gut microbial proteomes, indicates which human xenobiotic enzyme classes are most likely to be shared by gut microorganisms, and finally demonstrates that split homology may be an underappreciated explanation for microbial contributions to drug metabolism.Article SummaryWe develop a pipeline to systematically find human proteins with gut microbial homologs, including those split across multiple microbial genes (e.g., operons). This reveals thousands of proteins with full-length gut homologs, especially reductases and hydrolases that metabolize xenobiotics. Nearly two dozen split homologs are also observed for central metabolic enzymes, many of which can transform substrate analogs; in two cases, previous studies verify that microbial split homologs enable the expected drug to be metabolizedin vivo. These results, which we provide as a resource, map out homology and shed light on parallel drug metabolism between host and microbiome.
Efficient and Versatile Rapeseed Transformation for New Breeding Technologies
Authors: Ille Kea , Melzer Siegbert 
Published: November 2024

SummaryMany gene functions are widely studied and understood in Arabidopsis; however, the lack of efficient transformation systems often limits the application and verification of this knowledge in crop plants.Brassica napusL., a member of the Brassicaceae family, is usually transformed byAgrobacterium-mediated hypocotyl transformation, but not all growth types are equally amenable to transformation. In particular, winter-type rapeseed, which requires vernalization to initiate flowering, is recalcitrant toin vitroregeneration and transformation. The analysis of gene functions in rapeseed is further complicated by the allotetraploid nature of its genome and the genome triplication within theBrassicagenus, which has led to the presence of a large number of gene homologs for each Arabidopsis ortholog. We have established a transformation method that facilitates the regeneration of winter-type rapeseed by using theWUSCHELgene fromBeta vulgaris. This allowed us to efficiently transform winter-type and spring-type rapeseed in small-scale experiments. As proof of principle, we targetedBna.CLV3andBna.SPL9/15with CRISPR/Cas9 and showed that entire gene families are effectively edited using this transformation protocol. This allowed us to simultaneously study many redundantly acting homologous genes in rapeseed. We observed mutant phenotypes forBna.CLV3andBna.SPL9/15in primary transformants, indicating that biallelic knockouts were obtained for up to eight genes. This allowed an initial phenotypic characterization to be performed already a few months after starting the experiment.
An mRNA Display Approach for Covalent Targeting of aStaphylococcus aureusVirulence Factor
Authors: Wang Sijie , Woods Emily C. , Jo Jeyun , Zhu Jiyun , Hansel-Harris Althea , Holcomb Matthew , Pedowitz Nichole J. , Upadhyay Tulsi , Bennett John , Fellner Matthias , Park Ki Wan , Zhang Anna , Valdez Tulio A. , Forli Stefano , Chan Alix I , Cunningham Christian N. , Bogyo Matthew 
Published: November 2024

ABSTRACTStaphylococcus aureus (S. aureus) is an opportunistic human pathogen that causes over one million deaths around the world each year. We recently identified a family of serine hydrolases termed fluorophosphonate binding hydrolases (Fphs) that play important roles in lipid metabolism and colonization of a host. Because many of these enzymes are only expressed inStaphylococcusbacteria, they are valuable targets for diagnostics and therapeutics. Here we developed and screened highly diverse cyclic peptide libraries using mRNA display with a genetically encoded oxadiazolone (Ox) electrophile that was previously shown to potently and covalently inhibit multiple Fph enzymes. By performing multiple rounds of counter selections with WT and catalytic dead FphB, we were able to tune the selectivity of the resulting selected cyclic peptides containing the Ox residue towards the desired target. From our mRNA display hits, we developed potent and selective fluorescent probes that label the active site of FphB at single digit nanomolar concentrations in liveS. aureusbacteria. Taken together, this work demonstrates the potential of using direct genetically encoded electrophiles for mRNA display of covalent binding ligands and identifies potent new probes for FphB that have the potential to be used for diagnostic and therapeutic applications.
A Single Chimeric Spike Antigen Induces Pan-Sarbecovirus Immunity
Authors: Counoupas Claudio , Pino Paco , Armitano Joshua , Johansen Matt D , Smith Lachlan J , Chan Elizabeth , Ashley Caroline , Estapé Eva , Troyon Jean , Alca Sibel , Miem-czyk Stefan , Hansbro Nicole G. , Gabriella Scandurra , Britton Warwick J. , Courant Thomas , Dubois Patrice M. , Collin Nicolas , Mohan V Krishna , Hansbro Philip M , Wurm Maria J , Wurm Florian M. , Steain Megan , Triccas James A. 
Published: November 2024

AbstractNext-generation vaccines are required to address the evolving nature of SARS-CoV-2 and to protect against emerging pandemic threats from other coronaviruses. These vaccines should aim to elicit broad-protection, provide long-lasting immunity and facilitate equitable access for all populations. In this study, a panel of chimeric, full-length spike antigens were developed that incorporate mutations from previous, circulating and predicted SARS-CoV-2 variants. The lead candidate (CoVEXS5) was obtained from a high-yield production process in stable CHO cells with purity of >95%, long-term stability and elicitation of broadly cross-reactive neutralising antibodies when delivered to mice in a squalene emulsion adjuvant (Sepivac SWE™). In both mice and hamsters, CoVEXS5 immunisation reduced clinical disease signs, lung inflammation and organ viral titres after SARS-CoV-2 infection, including challenge with the highly immunoevasive Omicron XBB.1.5 subvariant. In mice previously primed with a licenced protein vaccine (NVX-CoV2373), CoVEXS5 could boost T cell immunity, as well as neutralising antibodies levels against viruses from three sarbecoviruses clades. The breadth of sarbecovirus cross-reactivity elicited by CoVEXS5 exceeded that observed after boosting with the NVX-CoV2373 vaccine. These findings highlight the potential of a chimeric spike antigen, formulated in an open-access adjuvant, as a next-generation vaccine candidate to enhance cross-protection against emerging sarbecoviruses in vaccinated populations globally.
Predictors of Response and Rational Combinations for the Novel MCL-1 Inhibitor MIK665 in Acute Myeloid Leukemia
Authors: Saad Joseph , Newman Rhiannon , Khabusheva Elmira , Aakko Sofia , Durand Eric , Tambe Mahesh , Kuusanmäki Heikki , Parsons Alun , Miettinen Juho J. , Javarappa Komal Kumar , Ikonen Nemo , Kontro Mika , Porkka Kimmo , Maacke Heiko , Woo Janghee , Halilovic Ensar , Heckman Caroline A. 
Published: November 2024

AbstractDespite promising anti-leukemic activity of MCL-1 inhibitors in preclinical studies of acute myeloid leukemia (AML), their progress through clinical evaluation has in part been challenged by limited knowledge of patient subgroups suitable for treatment. To stratify patients with AML for MCL-1 inhibitor-based treatment, we evaluated the sensitivity of 42 primary AML samples to MCL-1 inhibitor MIK665 (S64315) and contrasted their molecular profiles. We observed that MIK665 sensitive samples had a more differentiated phenotype, whereas resistant samples displayed higher levels of ABCB1 (MDR1) and the anti-apoptotic protein BCL-XL. Further evaluation revealed thatABCB1expression has good predictive performance in identifying MIK665 primary resistant samples. To induce sensitivity, we treated MIK665-resistant samples with ABCB1 inhibitor elacridar, BCL-XL inhibitor A1331852, or BCL-2 inhibitor venetoclax in combination with MIK665. While combinations with elacridar and A1331852 were not effective, the combination of MIK665 and venetoclax effectively eliminated AML blasts compared to either of the agents alone. Additionally, the combination restored sensitivity of samples with primary venetoclax resistance. Overall, this study indicates that elevatedABCB1expression is a potential predictor of resistance to MIK665 in AML, and that a combination of MIK665 with venetoclax may be effective for overcoming resistance to either MCL-1 or BCL-2 inhibition.
Autophagic stress activates distinct compensatory secretory pathways in neurons
Authors: Palumbos Sierra D. , Popolow Jacob , Goldsmith Juliet , Holzbaur Erika L.F. 
Published: November 2024

ABSTRACTAutophagic dysfunction is a hallmark of neurodegenerative disease, leaving neurons vulnerable to the accumulation of damaged organelles and proteins. However, the late onset of diseases suggests that compensatory quality control mechanisms may be engaged to delay the deleterious effects induced by compromised autophagy. Neurons expressing common familial Parkinson’s disease (PD)-associated mutations in LRRK2 kinase exhibit defective autophagy. Here, we demonstrate that both primary murine neurons and human iPSC-derived neurons harboring pathogenic LRRK2 upregulate the secretion of extracellular vesicles. We used unbiased proteomics to characterize the secretome of LRRK2G2019Sneurons and found that autophagic cargos including mitochondrial proteins were enriched. Based on these observations, we hypothesized that autophagosomes are rerouted toward secretion when cell-autonomous degradation is compromised, likely to mediate clearance of undegraded cellular waste. Immunoblotting confirmed the release of autophagic cargos and immunocytochemistry demonstrated that secretory autophagy was upregulated in LRRK2G2019Sneurons. We also found that LRRK2G2019Sneurons upregulate the release of exosomes containing miRNAs. Live-cell imaging confirmed that this upregulation of exosomal release was dependent on hyperactive LRRK2 activity, while pharmacological experiments indicate that this release staves off apoptosis. Finally, we show that markers of both vesicle populations are upregulated in plasma from mice expressing pathogenic LRRK2. In sum, we find that neurons expressing pathogenic LRRK2 upregulate the compensatory release of secreted autophagosomes and exosomes, to mediate waste disposal and transcellular communication, respectively. We propose that this increased secretion contributes to the maintenance of cellular homeostasis, delaying neurodegenerative disease progression over the short term while potentially contributing to increased neuroinflammation over the longer term.SIGNIFICANCEA hallmark feature of many neurodegenerative diseases is autophagy dysfunction, resulting in the accumulation of damaged proteins and organelles that is detrimental to neuronal health. The late onset of neurodegenerative diseases, however, suggests alternative quality control mechanisms may delay neuronal degeneration. Here, we demonstrate that neurons expressing a Parkinson’s Disease-causing mutation upregulate the release of two extracellular vesicle populations. First, we observe the increased expulsion of secreted autophagosomes to mediate cellular waste disposal. Second, we observe the increased release of exosomes, likely to facilitate transcellular communication. Thus, we propose that increases in secretory autophagy and exosome release are a homeostatic response in neurons undergoing chronic stress.
Creating overlapping genes by alternate-frame insertion
Authors: Leonard Sean P. , Halvorsen Tiffany , Lim Bentley , Park Dan M. , Jiao Yongqin , Yung Mimi , Ricci Dante 
Published: November 2024

AbstractOverlapping genes–wherein two different proteins are translated from alternative frames of the same DNA sequence–provide a means to stabilize an engineered gene by directly linking its evolutionary fate with that of an overlapped gene. However, creating overlapping gene pairs is challenging as it requires redesign of both protein products to accommodate overlap constraints. Here, we present a new “overlapping,alternate-frame insertion” (OAFI) method for creating overlapping genes byinsertionof an “inner” gene, encoded in an alternate frame, into a flexible region of an “outer” gene. Using OAFI, we create new overlapping gene pairs of bacterial toxins within an antibiotic resistance gene. We show that both the inner and outer genes retain functionality despite redesign, with translation of the inner gene is influenced by its overlap position in the outer gene. Additionally, we show that selection for the outer gene alters the permitted inactivating mutations in the inner gene and that overlapping toxins can restrict horizontal gene transfer of the antibiotic resistance gene. Overall, OAFI offers a versatile tool for synthetic biology, expanding the applications of overlapping genes in gene stabilization and biocontainment.
GCfix: A Fast and Accurate Fragment Length-Specific Method for Correcting GC Bias in Cell-Free DNA
Authors: Rahman Chowdhury Rafeed , Poh Zhong Wee , Skanderup Anders Jacobsen , Wong Limsoon 
Published: November 2024

AbstractMotivationCell-free DNA (cfDNA) analysis has wide-ranging clinical applications due to its non-invasive nature. However, cfDNA fragmentomics and copy number analysis can be complicated by GC bias. There is a lack of GC correction software based on rigorous cfDNA GC bias analysis. Furthermore, there is no standardized metric for comparing GC bias correction methods across large sample sets, nor a rigorous experiment setup to demonstrate their effectiveness on cfDNA data at various coverage levels.ResultsWe present GCfix, a method for robust GC bias correction in cfDNA data across diverse coverages. Developed following an in-depth analysis of cfDNA GC bias at the region and fragment length levels, GCfix is both fast and accurate. It works on all reference genomes and generates correction factors, tagged BAM files, and corrected coverage tracks. We also introduce two orthogonal performance metrics for (1) comparing the fragment count density distribution of GC content between expected and corrected samples, and (2) evaluating coverage profile improvement post-correction. GCfix outperforms existing cfDNA GC bias correction methods on these metrics.AvailabilityGCfix software and code for reproducing the figures are publicly accessible on GitHub:https://github.com/Rafeed-bot/GCfix_Software.
Upscaling production of immunogenic poliovirus virus-like particles inPichia Pastorisby controlled fermentation
Authors: Sherry Lee , Grehan Keith , Bahar Mohammad W , Swanson Jessica J , Fox Helen , Matthews Sue , Carlyle Sarah , Qin Ling , Porta Claudine , Wilkinson Steven , Robb Suzanne , Clark Naomi , Liddell John , Fry Elizabeth E , Stuart David I , Macadam Andrew J , Rowlands David J , Stonehouse Nicola J 
Published: November 2024

AbstractThe success of the poliovirus (PV) vaccines has enabled the near-eradication of wild PV, however, their continued use post-eradication poses a significant threat to maintaining a polio-free world. Recombinant virus-like particles (VLPs) that lack the viral genome remove this risk. Here, we demonstrate the production of PV VLPs for all three serotypes by controlled fermentation usingPichia pastoris. We determined the cryo-EM structure of a new PV-2 mutant, termed SC5a, in comparison to PV-2 SC6b VLPs described previously and investigated the immunogenicity of PV-2 SC5a VLPs. Finally, a trivalent immunogenicity trial using bioreactor-derived VLPs of all three serotypes in the presence of Alhydrogel adjuvant, showed that these VLPs outperform the current IPV vaccine in the standard vaccine potency assay, offering the potential for dose-sparing. Overall, these results provide further evidence that yeast-produced VLPs have the potential to be a next-generation polio vaccine in a post-eradication world.
Phenotypic plasticity in bacterial elongation among closely related species
Authors: Delaby Marie , Yang Liu , Jacq Maxime , Gallagher Kelley A , Kysela David T , Hughes Velocity , Pulido Francisco , Veyrier Frederic J. , VanNieuwenhze Michael S. , Brun Yves V. 
Published: November 2024

ABSTRACTCell elongation is a fundamental component of the bacterial cell cycle and has been studied over many decades, in part owing to its mechanisms being a target of numerous antibiotic classes. While several distinct modes of cell elongation have been described, these studies have largely relied on a handful of model bacterial species. Therefore, we have a limited view of the diversity of cell elongation approaches that are employed by bacteria, and how these vary in response to evolutionary and environmental influences. Here, by employing fluorescent D-amino acids (FDAAs) to track the spatiotemporal dynamics of elongation, we reveal previously unsuspected diversity of elongation modes among closely related species of theCaulobacteraceae, with species-specific combinations of dispersed, midcell and polar elongation that can be either unidirectional or bidirectional. Using genetic, cell biology, and phylogenetic approaches, we demonstrate that evolution of unidirectional-midcell elongation is accompanied by changes in the localization pattern of the peptidoglycan synthase PBP2 and infer that elongation complexes display a high degree of phenotypic plasticity, both among theCaulobacteraceaeand more widely among the Alphaproteobacteria. Demonstration that even closely related bacterial species employ highly distinct mechanisms of cell elongation reshapes our understanding of the evolution and regulation of bacterial cell growth, with broad implications for bacterial morphology, adaptation, and antibiotic resistance.
Comparative Aerosol Persistence of HypermucoidKlebsiella pneumoniae
Authors: Chirichella Nicole , Beddingfield Brandon J. , Redmann Rachel , Norton Elizabeth B. , Kolls Jay K. , Roy Chad J. 
Published: November 2024

AbstractKlebsiella pneumoniaeis becoming an increasing cause for concern due to antibiotic resistance and emerging hypervirulent strains. It results in a wide variety of infections, most commonly pneumonia. Immunocompromised individuals and those needing ventilators are among the most at-risk for infection and systemic spread. It is crucial to further understand the behavior ofK. pneumoniaeto prevent future infections and severe illness. In the past, little research has examinedK. pneumoniaein the air to determine whether aerosol transmission is a realistic infection pathway. Therefore, we observed survival of K. pneumoniae suspensions using a 10.7 L custom-built rotating chamber, characterizing laboratory and clinical strains in separate aerosol events. Airborne bacterial suspensions were assessed by culture and quantitative polymerase chain reaction (qPCR). The K2 laboratory strain remained viable until yielding no colony forming units (CFU) at 16 hours. K1 clinical strain did not become unculturable until 32 hours. For each strain, qPCR data showed Ct values slightly increased as time in drum also increased. Our data suggestsK. pneumoniaecan survive after multiple hours in the air, indicating aerosol transmission ofK. pneumoniaeis a possible infection pathway.
Decoding protein dynamicity in DNA ligase activity through deep learning-based structural ensembles
Authors: Mishra Nitesh , Callaghan Sean , Briney Bryan 
Published: November 2024

ABSTRACTNumerous proteins perform their functions by transitioning between various structures. Understanding the conformational ensembles associated with these states is essential for uncovering crucial mechanistic aspects that regulate protein function. In this study, we utilized AlphaFold3 (AF3) to investigate the structural dynamics and mechanisms of enzymes involved in DNA homeostasis, using NAD-dependent Taq ligases and human DNA Ligase 1 as a case example. Modifying the input parameters for AF3 yielded detailed conformational states of a DNA-binding enzyme, thereby offering enhanced mechanistic insights. We applied AF3 to model the various stages of thermophilic Taq DNA ligase activity, from its ground state to substrate-bound complexes, revealing significant mobility in the N-terminal adenylation and C-terminal BRCT domains. These detailed structural ensembles provided novel insights into the enzyme’s behavior during DNA repair, underscoring the potential of AF3 in elucidating mechanistic details critical for therapeutic and biotechnological targeting. Extending this approach to human LIG1, we examined its end-joining activity on double-strand breaks (DSBs) with short 3’ and 5’ overhangs. In alignment with published experimental data, AF3 conformational ensembles indicated LIG1 has lower catalytic efficiency for 5’ overhangs due to suboptimal DNA positioning within the catalytic site, demonstrating AF3’s capability to capture subtle yet functionally significant conformational differences by generating conformational ensembles capturing greater structural variance.
Rab40 GTPases regulate AMBRA1-mediated transcription and cell migration
Authors: Sampath Revathi , Vaeth Katherine , Mikalayeva Valeryia , Skeberdis Vytenis Arvydas , Prekeris Rytis , Han Ke-Jun 
Published: November 2024

ABSTRACTThe Rab40 subfamily are unique small monomeric GTPases that form CRL5-based ubiquitin E3 ligase complex and regulate ubiquitylation of specific target proteins. Recent studies have shown that Rab40s play an important role in regulating cell migration, but the underlying mechanisms of Rab40/CRL5 complex function are still not fully understood. In this study we identified AMBRA1 as a novel binding partner of Rab40 GTPases and showed that this interaction mediates a bi-directional crosstalk between CRL4 and CRL5 E3 ligases. Importantly, we found that Rab40/CRL5 ubiquitylates AMBRA1, which does not result in AMBRA1 degradation, but instead it seems to induce AMBRA1-dependent regulation of gene transcription. The global transcriptional profiles identified by RNA-seq showed that AMBRA1 regulates transcription of genes related to cell adhesion and migration. Additionally, we have shown that AMBRA1-dependent transcription regulation does not require the enzymatic activity of AMBRA1/CRL4, and that Rab40-induced AMBRA1 ubiquitylation leads to dissociation of AMBRA1/CRL4 complex. Taken together, our findings reveal a novel function of Rab40/CRL5 complex as an important regulator for AMBRA1-dependent transcription of genes involved in cell migration.