bluuebunny/crossref_metadata_2025 huggingface dataset:
2D representations of DNA sequence show that most transversions are misaligned nucleotides associated with replication slippage
Authors: Erives Albert J.
Published: July 2024

Homologous sequences diverge in length via insertions and deletions (indels). Consequently, evolutionary genetic analyses routinely use methods to produce gapped alignment (GA). In GA, artificial null characters (gaps) are inserted into sequences so that nucleotide characters may be placed into homological correspondence within an alignment column. However, this approach sacrifices the homological correspondence of nucleotides diverging via tandem repeats (TRs). To address this deficit, we generalize GA withmicro-paralogical gapped alignment(MPGA). While GA operates under a strict two-state homology model ofone-to-oneandone-to-none(i.e. one-to-gap) relationships, MPGA addsone-to-many,many-to-many, andmany-to-nonerelationships. This expanded, multi-state homology model is motivated by DNA replication slippage (RS). RS produces short tandem repeats, constituting interrelated micro-paralogous sequences. Together, RS and TR-associated instability have a synergistic effect in the production of indels, which generate the need for gap insertions. MPGA reduces the computational cost of determining optimal gap insertions by reducing the number of gaps required by two-dimensional (2D) representations of sequence. A 2D representation of one sequence is achieved when tandem repeats are contracted into the same columns (dimension one) by occupying multiple rows (dimension two), an internal micro-paralogical dimension. To demonstrate the benefits and challenges of 2D representation, we develop a program calledLINEUPand identify a pervasive fractal dimension in evolving sequences. We then demonstrate howLINEUP-generated 2D representations provide improved measures of substitution rates and transition-to-transversion ratios. Altogether, these results showcase significant new perspectives on basic mutational and evolutionary processes when multi-state homology models are adopted.
Deep learning guided design of dynamic proteins
Authors: Guo Amy B. , Akpinaroglu Deniz , Kelly Mark J.S. , Kortemme Tanja
Published: July 2024

Deep learning has greatly advanced design of highly stable static protein structures, but the controlled conformational dynamics that are hallmarks of natural switch-like signaling proteins have remained inaccessible tode novodesign. Here, we describe a general deep-learning-guided approach forde novodesign of dynamic changes between intra-domain geometries of proteins, similar to switch mechanisms prevalent in nature, with atom-level precision. We solve 4 structures validating the designed conformations, show microsecond transitions between them, and demonstrate that the conformational landscape can be modulated by orthosteric ligands and allosteric mutations. Physics-based simulations are in remarkable agreement with deep-learning predictions and experimental data, reveal distinct state-dependent residue interaction networks, and predict mutations that tune the designed conformational landscape. Our approach demonstrates that new modes of motion can now be realized throughde novodesign and provides a framework for constructing biology-inspired, tunable and controllable protein signaling behaviorde novo.
PINDER: The protein interaction dataset and evaluation resource
Authors: Kovtun Daniel , Akdel Mehmet , Goncearenco Alexander , Zhou Guoqing , Holt Graham , Baugher David , Lin Dejun , Adeshina Yusuf , Castiglione Thomas , Wang Xiaoyun , Marquet Céline , McPartlon Matt , Geffner Tomas , Rossi Emanuele , Corso Gabriele , Stärk Hannes , Carpenter Zachary , Kucukbenli Emine , Bronstein Michael , Naef Luca
Published: July 2024

Protein-protein interactions (PPIs) are fundamental to understanding biological processes and play a key role in therapeutic advancements. As deep-learning docking methods for PPIs gain traction, benchmarking protocols and datasets tailored for effective training and evaluation of their generalization capabilities and performance across real-world scenarios become imperative. Aiming to overcome limitations of existing approaches, we introduce PINDER, a comprehensive annotated dataset that uses structural clustering to derive non-redundant interface-based data splits and includesholo(bound),apo(unbound), and computationally predicted structures. PINDER consists of 2,319,564 dimeric PPI systems (and up to 25 million augmented PPIs) and 1,955 high-quality test PPIs with interface data leakage removed. Additionally, PINDER provides a test subset with 180 dimers for comparison to AlphaFold-Multimer without any interface leakage with respect to its training set. Unsurprisingly, the PINDER benchmark reveals that the performance of existing docking models is highly overestimated when evaluated on leaky test sets. Most importantly, by retraining DiffDock-PP on PINDER interface-clustered splits, we show that interface cluster-based sampling of the training split, along with the diverse and less leaky validation split, leads to strong generalization improvements.
Oxygen is Toxic in the Cold inC. elegans
Authors: Suraci Cameron M. , Morrison Michael L. , Roth Mark B.
Published: July 2024

Temperature and oxygen are two factors that profoundly affect survival limits of animals; too much or too little of either is lethal. However, both humans and animals can exhibit exceptional survival when oxygen and temperature are simultaneously low. To better understand this apparent synergistic interaction between oxygen and temperature, we assayed the survival ofCaenorhabditis elegansin experimental environments at different temperatures and oxygen concentrations. While nematodes cannot survive a day in room air at 2 °C (cold shock), we found that when oxygen is low, 200-fold less than room air, they can survive this temperature for 48 hours. Consistent with this, we found that worms exposed to high oxygen concentrations, 35 times greater than room air, are more sensitive to low temperature than worms in room air. These results show that normal atmospheric levels of oxygen are toxic in the cold. Using these survival assays, we found that cold acclimatization protects worms from lethal effects of high oxygen, mutations in the cold acclimatization pathway affect oxygen tolerance, and that the naturally occurring and physiologically relevant compounds glucose, manganese (II), and ascorbate improve survival limits in both low temperature and high oxygen when supplied early in life. These results show that the interdependence of temperature and oxygen on survival inC. elegansis based in part on shared physiological mechanisms involved in response to these two environmental stressors. The evolution and natural history of stress responses in animals suggest similar phenomena may function in humans.
Necrosis drives susceptibility toMycobacterium tuberculosisin PolgD257Amutator mice
Authors: Mabry CJ , Weindel CG , Stranahan LW , VanPortfliet JJ , Davis JR , Martinez EL , West AP , Patrick KL , Watson RO
Published: July 2024

The genetic and molecular determinants that underlie the heterogeneity ofMycobacterium tuberculosis(Mtb) infection outcomes in humans are poorly understood. Multiple lines of evidence demonstrate that mitochondrial dysfunction can exacerbate mycobacterial disease severity and mutations in some mitochondrial genes confer susceptibility to mycobacterial infection in humans. Here, we report that mutations in mitochondria DNA (mtDNA) polymerase gamma (POLG) potentiate susceptibility to Mtb infection in mice. PolgD257Amutator mtDNA mice fail to mount a protective innate immune response at an early infection timepoint, evidenced by high bacterial burdens, reduced M1 macrophages, and excessive neutrophil infiltration in the lungs. Immunohistochemistry reveals signs of enhanced necrosis in the lungs of Mtb-infected PolgD257Amice and PolgD257Amutator macrophages are hyper-susceptible to extrinsic triggers of necroptosisex vivo. By assigning a role for mtDNA mutations in driving necrosis during Mtb infection, this work further highlights the requirement for mitochondrial homeostasis in mounting balanced immune responses to Mtb.
The Oral Bioavailability of Lurasidone is Impacted by Changes to the Gut Microbiome: Implications for Antipsychotic Therapy
Authors: Collins Kate , Kamath Srinivas , Meola Tahlia R. , Wignall Anthony , Joyce Paul
Published: July 2024

Atypical antipsychotics are crucial for the management of schizophrenia and bipolar disorder, yet they exhibit significant pharmacokinetic variability which leads to inconsistent therapeutic responses. This study investigates the hypothesis that gut microbiome composition critically influences the oral bioavailability of lurasidone, a poorly soluble weak base antipsychotic with pH-dependent solubility. To investigate this, male Sprague-Dawley rats underwent systematic gut microbiome manipulation through pretreatment with antibiotics or prebiotics (inulin) for 14 days prior to a single oral dose of lurasidone. Pharmacokinetic analysis of collected plasma samples revealed a significant 4.3-fold increase in lurasidone bioavailability following prebiotic pretreatment, compared to a control (no pretreatment) group. Conversely, lurasidone bioavailability was highly variable in rats with a depleted microbiome (i.e., antibiotic treatment group), with 80% of animals demonstrating lower bioavailability than the control group. Characterisation of gut microbiome composition and short-chain fatty acid (SCFA) concentrations demonstrated positive correlations between lurasidone bioavailability, microbial diversity, and SCFA levels, mediated by modulation of luminal pH. Elevated SCFA levels created a favourable environment for lurasidone solubilisation by lowering intestinal pH. These findings highlight the potential for optimising antipsychotic pharmacokinetics through personalised microbiome interventions. Furthermore, the correlation between SCFAs and lurasidone bioavailability suggests their potential as biomarkers for predicting inter-patient pharmacokinetic variability, particularly for poorly soluble weak bases. Thus, new avenues are opened for developing novel co-therapies and screening tools to enhance antipsychotic pharmacokinetic performance, potentially improving treatment outcomes for patients with schizophrenia and bipolar disorder.

Graphical Abstract
PAC-MAP: Proximity Adjusted Centroid Mapping for Accurate Detection of Nuclei in Dense 3D Cell Systems
Authors: Van De Looverbosch Tim , De Beuckeleer Sarah , De Smet Frederik , Sijbers Jan , De Vos Winnok H.
Published: July 2024

Motivation

In the past decade, deep learning algorithms have surpassed the performance of many conventional image segmentation pipelines. Powerful models are now available for segmenting cells and nuclei in diverse 2D image types, but segmentation in 3D cell systems remains challenging due to the high cell density, the heterogenous resolution and contrast across the image volume, and the difficulty in generating reliable and sufficient ground truth data for model training. Reasoning that most image processing applications rely on nuclear segmentation but do not necessarily require an accurate delineation of their shapes, we implemented PAC-MAP, a 3D U-net based method that predicts the position of nuclei centroids and their proximity to other nuclei.

Results

We show that our model outperforms existing methods, predominantly by boosting recall, especially in conditions of high cell density. When trained from scratch PAC-MAP attained an average F1 score of 0.793 in dense spheroids. When pretraining using weakly supervised bulk data input and finetuning with few expert annotations the average F1 score could be significantly improved up to 0.817. We demonstrate the utility of our method for quantifying the cell content of spheroids and mapping the degree of glioblastoma multiforme infiltration in cerebral organoids.

Availability and implementation

The code is available on GitHub, athttps://github.com/DeVosLab/PAC-MAP.

Contact

Winnok H. De Vos (winnok.devos@uantwerpen.be)
Spatial ciliary signaling regulates the dorsal/ventral regionalization of human brain organoids
Authors: Shimada Issei S. , Goto Akari , Hashimoto Yutaka , Takase Hiroshi , Itoh Masayuki , Kato Yoichi
Published: July 2024

Regionalization of the brain is a fundamental question in human developmental biology. Primary cilia are known for a critical organelle for dorsal/ventral fate of brain formation in mice, but little is known about how signaling in the primary cilia regulate regionalization of the human brain. Here, we found that signaling in the primary cilia function in regionalization of the brain using brain organoids derived from human induced pluripotent stem (iPS) cells. Deletion of a ciliary GTPase,ARL13B, induced partially ventralized neural stem cells in the dorsal cortical organoids, despite using a guided dorsal cortical organoid differentiation protocol. Mechanistically,ARL13Bknockout (KO) neural stem cells decreased ciliary localization of GPR161, a negative regulator of SHH signaling in primary cilia and increased SONIC HEDGEHOG (SHH) signaling.GPR161deletion also induced ventralized neural stem cells in the dorsal cortical organoids, despite using the guided differentiation protocol.GPR161deletion increased SHH signaling mediated by decreased GLI3 repressor formation. Pharmacological treatment to increase cAMP levels rescued GLI3 repressor formation and the differentiation of dorsal neural stem cells inGPR161KO brain organoids. Importantly, elevating the amount of ciliary cAMP by optogenetics restored the generation of dorsal neural stem cells inGPR161KO brain organoids. These data indicate that spatial ciliary signaling, the ARL13B-GPR161-cAMP axis in primary cilia, is a fundamental regulator of the dorsal/ventral regionalization of the human brain.
SARS-CoV-2 B Epitope-Guided Neoantigen NanoVaccines Enhance Tumor-Specific CD4/CD8 T Cell Immunity Through B Cell Antigen Presentation
Authors: Li Chengyi , Mao Shuai , Montemayor Zera , Traore Mohamed Dit Mady , Duran Alejandra , Ke Fang , Djibo Mahamadou , Wen Hanning , Gao Wei , Sun Duxin
Published: July 2024

Current neoantigen cancer vaccines activate T cell immunity through dendritic cell /macrophage-mediated antigen presentation. It is unclear whether incorporating B cell-mediated antigen presentation into current neoantigen vaccines could enhance CD4/CD8 T cell immunity to improve their anticancer efficacy. We developed a SARS-CoV-2 B cell epitope-guided neoantigen peptide/mRNA cancer nanovaccines (BSARSTNeoAgVax) to improve anticancer efficacy by enhancing tumor-specific CD4/CD8 T cell antitumor immunity through B cell-mediated antigen presentation. BSARSTNeoAgVax crosslinked with B cell receptor, promoted SARS-CoV-2 B cell-mediated antigen presentation to tumor-specific CD4 T cells, increased tumor-specific follicular/non-follicular CD4 T cells, and enhanced B cell-dependent tumor-specific CD8 T cell immunity. BSARSTNeoAgVax achieved superior efficacy in melanoma, pancreatic, and breast cancer models compared to the current neoantigen vaccines. Our study provides a universal platform, SARS-CoV-2 B epitope-guided neoantigen nanovaccines, to improve anticancer efficacy against various cancer types by enhancing CD4/CD8 T cell antitumor immunity through viral-specific B cell-mediated antigen presentation.
The structure of a NEMO construct engineered for screening reveals novel determinants of inhibition
Authors: Kennedy Amy E. , Barczewski Adam H. , Arnoldy Christina R. , Pennington J. Pepper , Tiernan Kelly A. , Hidalgo Maria Beatriz , Reilly Caroline C. , Ragusa Michael J. , Grigoryan Gevorg , Mierke Dale F. , Pellegrini Maria M.
Published: July 2024

NEMO is an essential component in the activation of the canonical NF-κB pathway and exerts its function by recruiting the IκB kinases (IKK) to the IKK complex. Inhibition of the NEMO/IKKs interaction is an attractive therapeutic paradigm for diseases related to NF-κB mis-regulation, but a difficult endeavor because of the extensive protein-protein interface. Here we report the design and characterization of novel engineered constructs of the IKK-binding domain of NEMO, programmed to render this difficult protein domain amenable to NMR and X-ray characterization, while preserving the biological function. ZipNEMO binds IKKβ with nanomolar affinity, is amenable to heteronuclear NMR techniques and structure determination by X-ray crystallography. We show that NMR spectra of zipNEMO allow to detect inhibitor binding in solution and resonance assignment. The X-ray structure of zipNEMO highlights a novel ligand binding motif and the adaptability of the binding pocket and inspired the design of new peptide inhibitors.
Engineering the novel extremophile algaChlamydomonas pacificafor high lipid and high starch production as a path to developing commercially relevant strains
Authors: Gupta Abhishek , Dutra Molino João Vitor , Wnuk-Fink Kathryn MJ , Bruckbauer Aaron , Tessman Marissa , Kang Kalisa , Diaz Crisandra J. , Saucedo Barbara , Malik Ashleyn , Mayfield Stephen P
Published: July 2024

Microalgae offer a compelling platform for the production of commodity products, due to their superior photosynthetic efficiency, adaptability to non-arable lands and non-potable water, and their capacity to produce a versatile array of bioproducts, including biofuels and biomaterials. However, the scalability of microalgae as a bioresource has been hindered by challenges such as costly biomass production related to vulnerability to pond crashes during large-scale cultivation. This study presents a pipeline for the genetic engineering and pilot-scale production of biodiesel and thermoplastic polyurethane precursors in the extremophile speciesChlamydomonas pacifica. This extremophile microalga exhibits exceptional resilience to high pH, high salinity, and elevated temperatures. Initially, we evolved this strain to also have a high tolerance to high light intensity through mutagenesis, breeding, and selection. Subsequently, we genetically engineeredC. pacificato produce high levels of lipids and starch without compromising growth. We demonstrated the scalability of these engineered strains by cultivating them in pilot-scale raceway ponds and converting the resulting biomass into biodiesel and thermoplastic polyurethanes. This study showcases the complete cycle of transforming a newly discovered species into a commercially relevant commodity production strain. This research underscores the potential of extremophile algae, includingC. pacifica, as a key species for the burgeoning sustainable bioeconomy, offering a viable path forward in mitigating environmental challenges and supporting global bioproduct demands.

Graphical Abstract
Comparison of Methodologies for Absolute Binding Free Energy Calculations of Ligands to Intrinsically Disordered Proteins
Authors: Papadourakis Michail , Cournia Zoe , Mey Antonia S. J. S. , Michel Julien
Published: July 2024

Modulating the function of Intrinsically Disordered Proteins (IDPs) with small molecules is of considerable importance given the crucial roles of IDPs in the pathophysiology of numerous diseases. Reported binding affinities for ligands to diverse IDPs vary broadly and little is known about the detailed molecular mechanisms that underpin ligand efficacy. Molecular simulations of IDP-ligand binding mechanisms can help us understand the mode of action of small molecule inhibitors of IDP function, but it is still unclear how binding energies can be modeled rigorously for such a flexible class of proteins. Here we compare alchemical absolute binding free energy calculation (ABFE) and Markov-State Modelling (MSM) protocols to model the binding of the small molecule 10058-F4 to a disordered peptide extracted from a segment of the oncoprotein c-Myc. The ABFE results produce binding energy estimates that are sensitive to the choice of reference structure. In contrast, the MSM results produce more reproducible binding energy estimates consistent with weak mM binding affinities and transient intermolecular contacts reported in the literature.
Structural basis for allosteric regulation of the proteasome core particle
Authors: Turner Madison , Uday Adwaith B. , Velyvis Algirdas , Rennella Enrico , Zeytuni Natalie , Vahidi Siavash
Published: July 2024

Intracellular protein degradation is vital across all domains of life1. In eukaryotes, the ubiquitin proteasome system performs most non-lysosomal protein degradation and influences numerous cellular processes. Some bacteria, including the human pathogenMycobacterium tuberculosis(Mtb), encode a proteasome system that selectively degrades damaged or misfolded proteins crucial for the pathogen’s survival within host macrophages2–7. Consequently, the 20S core particle (CP), the central component of the proteasome system, has emerged as a viable target for tuberculosis treatment strategies2,8–10. Both eukaryotic andMtbproteasome systems are allosterically regulated11–13, yet the specific conformations involved have not been captured in high-resolution structures to date. Here we present the first structure ofMtb20S CP, and indeed any 20S CP, in an inactive state called 20SOFF, distinguished from the canonical active state, 20SON, by the conformation of switch helices I and II. The rearrangement of these helices collapses the S1 pocket, effectively inhibiting substrate binding. The switch helices are conserved and regulate the activity of HslV protease, the proteasome’s ancestral enzyme in bacteria, and a diverse family of serine/threonine protein phosphatases. Our results highlight the potential of harnessing allostery to develop therapeutics against the 20S CP inMtband eukaryotic systems.
Engineering microbial consortia: uptake and leakage rate differentially shape community arrangement and composition
Authors: Pignon Estelle , Holló Gábor , Steiner Théodora , van Vliet Simon , Schaerli Yolanda
Published: July 2024

Bacteria often grow as communities in intricate spatial arrangements on surfaces and interact with each other through the local exchange of diffusible molecules. Yet, our understanding of how these interactions shape the properties of the communities remains limited. Here, we study synthetic communities ofEscherichia coliamino acid auxotrophs interacting through the obligate exchange of amino acids. We genetically engineer these strains to alter their amino acid leakage and uptake abilities. We then characterise the spatial arrangement and composition of the communities when grown on a surface. By integrating experimental data with mathematical modeling, we demonstrate that amino acid uptake and leakage rates are crucial determinants of community structure. Our results show that while the spatial arrangement of the community is primarily governed by uptake rates, the community composition is predominantly influenced by leakage rates. These findings enhance our understanding of microbial community dynamics and provide a framework for predicting and engineering microbial consortia.
Prevention of ribozyme catalysis through cDNA synthesis enables accurate RT-qPCR measurements of context-dependent ribozyme activity
Authors: Alperovich Nina Y , Vasilyeva Olga B , Schaffter Samuel W
Published: July 2024

Self-cleaving ribozymes are important tools in synthetic biology, biomanufacturing, and nucleic acid therapeutics. These broad applications deploy ribozymes in many genetic and environmental contexts, which can influence activity. Thus, accurate measurements of ribozyme activity across diverse contexts are crucial for validating new ribozyme sequences and ribozyme-based biotechnologies. Ribozyme activity measurements that rely on RNA extraction, such as RNA sequencing or reverse transcription-quantitative polymerase chain reaction (RT-qPCR), are generalizable to most applications and have high sensitivity. However, the activity measurement is indirect, taking place after RNA is isolated from the environment of interest and copied to DNA. So these measurements may not accurately reflect the activity in the original context. Here we develop and validate an RT-qPCR method for measuring context-dependent ribozyme activity using a set of self-cleaving RNAs for which context-dependent ribozyme cleavage is known in vitro. We find that RNA extraction and reverse transcription conditions can induce substantial ribozyme cleavage resulting in incorrect activity measurements with RT-qPCR. To restore the accuracy of the RT-qPCR measurements, we introduce an oligonucleotide into the sample preparation workflow that inhibits ribozyme activity. We then apply our method to measure ribozyme cleavage of RNAs produced in Escherichia coli (E. coli). These results have broad implications for many ribozyme measurements and technologies.
DEVELOPMENT AND CONTENT VALIDITY OF THE HANDWASHING INDEX (HWI)
Authors: Lamptey Emmanuel , Senkyire Ephraim Kumi , Agoha Doris Selorm
Published: July 2024

Handwashing is the simplest way to stop the spread of infections and maintain health. To the best of our knowledge, there is no validated scale to measure this habit. Given the need to raise awareness and its importance, a study was undertaken to develop, test, and validate the HWI. The Physical Activity Questionnaire for Adolescents (PAQ-A) was adapted and substantially modified to draft the first version of the index (HWQ). The COnsensus-based Standards for the Selection of Health Measurement Instrument (COSMIN) guided our approach. The HWQ The questionnaire consisted of six questions with a five-point scale response for each item, ranging from the lowest [1] to the highest activity [5] as scoring criteria. We followed a four-stage procedure in validating the instrument: Stage I: items were created from the review of the relevant literature (PAQ-A, COSMIN); Stage II: the study targeted and recruited 57 health professionals from different backgrounds and vetted them as decliners, non-regulars, and regular hand washers through a scoring process.

This crucial phase ensured that the respondents had hands-on experience in handwashing in addition to their expertise. In Stage III, 10 respondents from the regular hand washers were randomly selected as expert judges. They were divided into two (2) groups (Group one and Group two) of four and six members, respectively. Further comprehensibility and comprehensiveness of the HWQ were established through a review by the four members of Group 1, and revisions were made to create HWQ-I. At stage IV, the HWQ-I was administered to the six members of the second group for ratings and content validity index calculations. The final draft of the HWI includes 4 items. Item CVI ranges between 0.8 and 1.The average scale CVI was 0.9.

The HWI demonstrated excellent content validity, showing its relevance for monitoring handwashing, and individuals can track their compliance, promoting a healthy life. We propose further studies to test the psychometric properties of the scale.
The deletion of aldehyde:ferredoxin oxidoreductase-encoding genes inClostridium ljungdahliiresults in changes in the product spectrum with various carbon sources
Authors: Baur Saskia T. , Schulz Sarah , Cluskey Joshua B. M , Velázquez Gómez José Antonio , Angenent Largus T. , Molitor Bastian
Published: July 2024

Biofuels, such as ethanol, can be produced by the microbial fermentation of waste gases that contain carbon dioxide (CO2) and carbon monoxide (CO). The acetogenic model microbeClostridium ljungdahliiconverts those substrates into acetyl-CoA with the Wood-Ljungdahl pathway. During autotrophic conditions, acetyl-CoA can be reduced further to ethanolviaacetic acid by the enzymes aldehyde:ferredoxin oxidoreductase (AOR) and alcohol dehydrogenase. Here, the genes encoding both tungsten-dependent AORs (aor1, CLJU_c20110 andaor2, CLJU_c20210) were deleted from the genome ofC. ljungdahlii. Ethanol formation was enhanced forC. ljungdahliiΔaor1with different carbon sources, that is, fructose, a mixture of hydrogen (H2) and CO2, and CO. The highest and lowest ethanol:acetic acid ratio was detected during growth with H2/CO2and CO, respectively. Oscillating patterns were observed during growth with CO, underpinning the importance of a balanced redox metabolism.
Deep Profiling of Plasma Proteoforms with Engineered Nanoparticles for Top-down Proteomics
Authors: Huang Che-Fan , Hollas Michael A. , Sanchez Aniel , Bhattacharya Mrittika , Ho Giang , Sundaresan Ambika , Caldwell Michael A. , Zhao Xiaoyan , Benz Ryan , Siddiqui Asim , Kelleher Neil L.
Published: July 2024

The dynamic range challenge for detection of proteins and their proteoforms in human plasma has been well documented. Here, we use the nanoparticle protein corona approach to enrich low-abundant proteins selectively and reproducibly from human plasma and use top-down proteomics to quantify differential enrichment for the 2841 detected proteoforms from 114 proteins. Furthermore, nanoparticle enrichment allowed top-down detection of proteoforms between ∼1 µg/mL and ∼10 pg/mL in absolute abundance, providing up to 105-fold increase in proteome depth over neat plasma in which only proteoforms from abundant proteins (>1 µg/mL) were detected. The ability to monitor medium and some low abundant proteoforms through reproducible enrichment significantly extends the applicability of proteoform research by adding depth beyond albumin, immunoglobins and apolipoproteins to uncover many involved in immunity and cell signaling. As proteoforms carry unique information content relative to peptides, this report opens the door to deeper proteoform sequencing in clinical proteomics of disease or aging cohorts.
Discovery and Engineering of Retrons for Precise Genome Editing
Authors: Buffington Jesse Dean , Kuo Hung-Che , Hu Kuang , Chang You-Chiun , Javanmardi Kamyab , Voigt Brittney , Li Yi-Ru , Little Mary E , Devanathan Sravan K , Xhemalce Blerta , GRAY RYAN , Finkelstein Ilya J
Published: July 2024

Retrons are promising gene editing tools because they can produce multi-copy single-stranded DNA in cells via self-primed reverse transcription. However, their potential for inserting genetic cargos in eukaryotes remains largely unexplored. Here we report the discovery and engineering of highly efficient retron-based gene editors for mammalian cells and vertebrates. Through bioinformatic analysis of metagenomic data and functional screening, we identified novel retron reverse transcriptases (RTs) that are highly active in mammalian cells. Rational design further improved the editing efficiency to levels comparable with conventional single-stranded oligodeoxynucleotide donors but from a genetically encoded cassette. Small molecule inhibitors of non-homologous end joining factors and Cas9-DNA repair protein fusions further increase homology-directed repair. Retron editors also exhibited robust activity with Cas12a nuclease and Cas9 nickase, expanding the genomic target scope and bypassing the need for a DNA double-stranded break. Using a rationally engineered retron editor, we incorporate a split GFP epitope tag for live cell imaging. Finally, we develop an all-RNA delivery strategy to enable DNA-free gene editing in cells and vertebrate embryos. This work establishes retron editors as a versatile and efficient tool for precise gene editing, offering new opportunities for biotechnology and biomedical research.
Mutational landscape and molecular bases of echinocandin resistance
Authors: Durand Romain , Torbey Alexandre G. , Giguere Mathieu , Pageau Alicia , Dubé Alexandre K. , Lagüe Patrick , Landry Christian R.
Published: July 2024

One of the front-line drug classes used to treat invasive fungal infections is echinocandins, which target the fungal-specific beta-glucan synthase (Fks). Treatment failure due to resistance often coincides with mutations in two protein regions known as hotspots. The biophysical bases by which such mutations confer resistance and cross-resistance among echinocandins are largely unknown. Here, we use deep-mutational scanning to quantify the resistance level of 660 mutations in the hotspots of two homologous Fks. We detail the constraints acting on drug binding and explain the resistance specificity for some mutations using the drug-protein interactions from our molecular models. Our findings will enable DNA sequence-based predictions of resistance to this important drug family and the improvement of future molecules that could overcome current resistance mutations.

One sentence summary

Disruption of specific interactions between echinocandins and key residues from their target lead to drug-specific resistance.
The N-terminal PAS domain directly regulates hERG channel gating in excised, inside-out patch-clamp fluorometry (PCF) recordings
Authors: Trudeau Matthew C.
Published: July 2024

Human ERG is a voltage-activated, K-selective channel whose physiological role is to drive action potential repolarization in cardiac myocytes. To carry out its role in the heart, hERG has specialized gating (opening and closing) transitions that are regulated by the internal N-terminal PAS and C-terminal CNBH domains. The PAS and CNBHD domains interact directly and this interaction is required for the characteristic slow deactivation (closing) of hERG channels. But it is unclear whether PAS remains globally attached or dislodges from the CNBHD during gating. Interestingly the direct PAS-CNBHD interaction can be formedin transby co-expression of the PAS domain and hERG channels with a deleted PAS domain (hERG ΔPAS) in which the PAS domain is not attached to the channel with a peptide bond.In transexpression allows us to probe the biophysical mechanism for PAS domain attachment to the rest of the channel and in a broader sense allows us to test the mechanism for intracellular domain function in an ion channel, and test whether the PAS domain detaches or remains attached to the channel during gating. We report here that in excised patches from cells containing the hERG PAS domain fused to CFP and hERG ΔPAS channels fused to Citrine that 1) regulation of deactivation (slow deactivation conveyed by the PAS domain) was similar in on-cell and excised, inside-out patch configurations, 2) that regulation of deactivation persists for the lifetime of the patch (up to 30 minutes) in excised, inside-out mode, 3) that channel activity measured by activation of the channel with voltage pulses did not alter channel deactivation and 4) dual fluorescence and ionic current measurements using patch-clamp fluorometry (PCF) showed that only membrane patches containing PAS-CFP + hERG ΔPAS-Citrine had CFP and Citrine fluorescence and slow (regulated) deactivation, whereas control patches with hERG ΔPAS -Citrine had fast (unregulated) deactivation and Citrine fluorescence (but not CFP fluorescence) and control patches from hERG PAS-CFP - injected cells had neither currents nor CFP or Citrine fluorescence. Moreover, in PCF mode, we detected FRET from PAS-CFP + hERG ΔPAS-Citrine channels. Taken together, these results suggested that PAS - CFP remained associated with hERG ΔPAS-Citrine channels after membrane excision. We interpret these results to mean that the PAS domain was not dislodged from the channel despite mechanical (excised patch) and conformational (voltage) challenges and suggests that the PAS domain remained firmly attached to the hERG channel during gating.
Identification of Juglone, a ‘first-in-class’ inhibitor of the human glutathione degrading enzyme, ChaC1, using yeast-based high throughput screens
Authors: Suyal Shradha , Choudhury Chinmayee , Kaur Deepinder , Bachhawat Anand K.
Published: July 2024

The cytosolic glutathione-degrading enzyme, ChaC1, is highly upregulated in several cancers, with the upregulation correlating to poor prognosis. The ability to inhibit ChaC1 thus becomes important in pathophysiological situations where elevated glutathione levels would be beneficial. As no inhibitors of ChaC1 are known, in this study we have focussed on this goal. We have initially taken a computational approach where a systemic structure-based virtual screening was performed. However, none of the predicted hits proved to be effective inhibitors. We also evaluated synthetic substrate analogs, but these too were not inhibitory. As both these approaches targeted the active site, we shifted to developing two high-throughput, robust, yeast-based assays that were active site independent. A small molecule compound library was screened using an automated liquid handling system using these screens. The hits were further analyzed usingin vitroassays. Among them, juglone, a naturally occurring naphthoquinone, completely inhibited ChaC1 activity with an IC50of 8.7 µM. It was also effective against the ChaC2 enzyme. Kinetic studies indicated that the inhibition was not competitive with the substrate. Juglone is known to form adducts with glutathione and is also known to selectively inhibit enzymes by covalently binding to their active site cysteine residues. However, juglone continued to inhibit a cysteine-free ChaC1 variant, indicating that it was acting through a novel mechanism. We evaluated different inhibitory mechanisms, and also analogues of juglone, and found plumbagin effective as an inhibitor. These compounds represent the ‘first-in-class’ inhibitors of the ChaC enzymes discovered using a robust yeast screen.
Structural Basis for RNA-guided DNA degradation by Cas5-HNH/Cascade complex
Authors: Liu Yanan , Wang Lin , Zhang Qian , Fu Pengyu , Zhang Lingling , Zhang Heng , Zhu Hongtao
Published: July 2024

Type I-E CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) system is the most widely investigated RNA-guided adaptive immune system in prokaryotes against the foreign genetic elements. Unlike the previously characterized Cas3 nuclease in the typical Type I-E system that exhibits uncontrolled progressive DNA cleavage, a recently discovered HNH domain linked to the Cas5 subunit functions as the nuclease for precise DNA cleavage, highlighting the potential of this new Type I-E variant system as a precise genome editing tool. Here, we present five near- atomic cryo-EM (cryo-electron microscopy) structures ofCandidatus Cloacimonetes bacteriumCas5-HNH/Cascade complex either bound or unbound to DNA. We revealed that the HNH domain extensively interacts with the adjacent subunits, including Cas6, Cas8 and Cas11. The related mutations of the crucial identified interactions can significantly weaken the performance of the enzyme. Upon the binding of DNA, the Cas-HNH/Cascade complex adopts a more compacted conformation with the subunits moving towards the center of the nuclease, thus activating the nuclease. We further identified four conserved cysteines that form a zinc-finger structure in the HNH domain and the mutations of these four cysteines can totally abolish the enzyme activity. Interestingly, we also discovered that the divalent ions such as zinc, cobalt and nickel down-regulate the enzyme performance by decreasing the stability of the Cascade complex. Together, our findings provide first structural insights into the assembly and activation of the Cas5-HNH/Cascade complex, opening new avenues for engineering this system for precise genome editing.
One-loop QCD corrections to heavy quark angular distributions in DIS
Authors: Chang Qing-Song , Zhang Guang-Peng
Published: July 2024

In this paper we calculate the fully differential cross sections for inclusive heavy quark production in deep-inelastic scattering. We construct proper projection operators to give all possible azimuthal angle distributions of the heavy quark for unpolarized and longitudinally polarized scatterings. These projection operators are expressed in terms of momenta of incoming hadron, virtual photon, and detected heavy quark. The azimuthal angle distributions are calculated to next-to-leading order of αs, i.e., O(αs2), in a unified way. Analytic expressions of the hard coefficients are given. Numerical results on future electron-ion colliders are also given. It is found that at least three azimuthal angle asymmetries can be more than 1% in typical kinematical regions of these colliders.

Published by the American Physical Society
2024
QCD equation of state and thermodynamic observables from computationally minimal Dyson-Schwinger equations
Authors: Lu Yi , Gao Fei , Liu Yu-xin , Pawlowski Jan M.
Published: July 2024

We study the QCD equation of state and other thermodynamic observables including the isentropic trajectories and the speed of sound. These observables are of eminent importance for the understanding of experimental results in heavy ion collisions and also provide a QCD input for studies of the timeline of heavy-ion-collisions with hydrodynamical simulations. They can be derived from the quark propagator whose gap equation is solved within a minimal approximation to the Dyson-Schwinger equations (DSEs) of QCD at finite temperature and density. This minimal approximation aims at a combination of computational efficiency and simplification of the truncation scheme while maintaining quantitative precision. This minimal DSE scheme is confronted and benchmarked with results for correlation functions and observables from lattice QCD at vanishing density and quantitative functional approaches at finite density.

Published by the American Physical Society
2024
Dark matter phenomenology and phase transition dynamics of the next-to-minimal composite Higgs model with a dilaton
Authors: Zhang Borui , Zhang Zhao , Cai Chengfeng , Zhang Hong-Hao
Published: July 2024

In this paper, we conduct a comprehensive study of the next-to-minimal composite Higgs model (NMCHM) extended with a dilaton field χ (denoted as NMCHMχ). A pseudo-Nambu-Goldstone boson (pNGB) η, resulting from the SO(6)→SO(5) breaking, serves as a dark matter (DM) candidate. The inclusion of the dilaton field is helpful for evading the stringent constraints from dark matter direct detection, as it allows for an accidental cancellation between the amplitudes of DM-nucleon scattering, an outcome of the mixing between the dilaton and Higgs fields. The presence of the dilaton field also enriches the phase transition patterns in the early universe. We identify two types of phase transitions: (i) a 1-step phase transition, where the chiral symmetry and electroweak symmetry breaking (EWSB) occur simultaneously, and (ii) a 2-step phase transition, where the chiral symmetry breaking transition takes place first, followed by a second phase transition corresponding to EWSB. Since the first-order phase transitions can be strong due to supercooling in our model, we also examine the stochastic background of gravitational waves generated by these phase transitions. We find that these gravitational waves hold promise for detection in future space-based gravitational wave experiments, such as LISA, Taiji, BBO, and DECIGO.

Published by the American Physical Society
2024
Impact of theoretical uncertainties on model parameter reconstruction from GW signals sourced by cosmological phase transitions
Authors: Lewicki Marek , Merchand Marco , Sagunski Laura , Schicho Philipp , Schmitt Daniel
Published: July 2024

Different computational techniques for cosmological phase transition parameters can impact the gravitational wave (GW) spectra predicted in a given particle physics model. To scrutinize the importance of this effect, we perform large-scale parameter scans of the dynamical real-singlet extended Standard Model using three perturbative approximations for the effective potential; the MS¯ and on shell schemes at leading order, and three-dimensional thermal effective theory (3D EFT) at next-to-leading order. While predictions of GW amplitudes are typically unreliable in the absence of higher-order corrections, we show that the reconstructed model parameter spaces are robust up to a few percent in uncertainty. While 3D EFT is accurate from one-loop order, theoretical uncertainties of reconstructed model parameters, using four-dimensional standard techniques, remain dominant over the experimental ones even for signals merely strong enough to claim a detection by LISA.

Published by the American Physical Society
2024
Tidal heating as a discriminator for horizons in equatorial eccentric extreme mass ratio inspirals
Authors: Datta Sayak , Brito Richard , Hughes Scott A. , Klinger Talya , Pani Paolo
Published: July 2024

Tidal heating in a binary black hole system is driven by the absorption of energy and angular momentum by the black hole’s horizon. Previous works have shown that this phenomenon becomes particularly significant during the late stages of an extreme mass ratio inspiral (EMRI) into a rapidly spinning massive black hole, a key focus for future low-frequency gravitational-wave observations by (for instance) the Laser Interferometer Space Antenna mission. Past analyses have largely focused on quasicircular inspiral geometry, with some of the most detailed studies looking at equatorial cases. Though useful for illustrating the physical principles, this limit is not very realistic astrophysically, since the population of EMRI events is expected to arise from compact objects scattered onto relativistic orbits in galactic centers through many-body events. In this work, we extend those results by studying the importance of tidal heating in equatorial EMRIs with generic eccentricities. Our results suggest that accurate modeling of tidal heating is crucial to prevent significant dephasing and systematic errors in EMRI parameter estimation. We examine a phenomenological model for EMRIs around exotic compact objects by parametrizing deviations from the black hole (BH) picture in terms of the fraction of radiation absorbed compared to the BH case. Based on a mismatch calculation, we find that reflectivities as small as |R|2∼O(10−5) are distinguishable from the BH case, irrespective of the value of the eccentricity. We stress, however, that this finding should be corroborated by future parameter estimation studies.

Published by the American Physical Society
2024
New framework to follow up candidates from continuous gravitational-wave searches
Authors: Covas P. B. , Prix R. , Martins J.
Published: July 2024

Searches for continuous gravitational waves from unknown neutron stars are limited in sensitivity due to their high computational cost. For this reason, developing new methods or improving existing ones can increase the probability of making a detection. In this paper we present a new framework that uses Markov chain Monte Carlo (MCMC) or nested sampling methods to follow up candidates of continuous gravitational-wave searches. This framework aims to go beyond the capabilities of (which is limited to the sampler), by allowing a flexible choice of sampling algorithm (using as a wrapper) and multidimensional correlated prior distributions. We show that MCMC and nested sampling methods can recover the maximum posterior point for much bigger parameter-space regions than previously thought (including for sources in binary systems), and we present tests that examine the capabilities of the new framework: a comparison between the , , and samplers, the usage of correlated priors, and its improved computational cost.

Published by the American Physical Society
2024
Critical and near-critical relaxation of holographic superfluids
Authors: Flory Mario , Grieninger Sebastian , Morales-Tejera Sergio
Published: July 2024

We investigate the relaxation of holographic superfluids after quenches, when the end state is either tuned to be exactly at the critical point, or very close to it. By solving the bulk equations of motion numerically, we demonstrate that in the former case the system exhibits a power law falloff, as well as an emergent discrete scale invariance. The latter case is in the regime dominated by critical slowing down, and we show that there is an intermediate time range before the onset of late-time exponential falloff, where the system behaves similarly to the critical point with its power law falloff. We further postulate a phenomenological Gross-Pitaevskii-like equation (corresponding to model F of Hohenberg and Halperin) that is able to make quantitative predictions for the behavior of the holographic superfluid after near-critical quenches into the superfluid and normal phase. Intriguingly, all parameters of our phenomenological equation, which describes the nonlinear time evolution, may be fixed with information from the static equilibrium solutions and linear response theory.

Published by the American Physical Society
2024