bluuebunny/crossref_metadata_2025_split huggingface dataset:
Optical characterization of molecular interaction strength in protein condensates
Authors: Beck Timon , van der Linden Lize-Mari , Borcherds Wade M. , Kim Kyoohyun , Schlüßler Raimund , Müller Paul , Franzmann Titus , Möckel Conrad , Goswami Ruchi , Leaver Mark , Mittag Tanja , Alberti Simon , Guck Jochen 
Published: March 2024

AbstractBiomolecular condensates have been identified as a ubiquitous means of intracellular organization, exhibiting very diverse material properties. However, techniques to characterize these material properties and their underlying molecular interactions are scarce. Here, we introduce two optical techniques – Brillouin microscopy and quantitative phase imaging (QPI) – to address this scarcity. We establish Brillouin shift and linewidth as measures for average molecular interaction and dissipation strength, respectively, and we used QPI to obtain the protein concentration within the condensates. We monitored the response of condensates formed by FUS and by the low-complexity domain of hnRNPA1 (A1-LCD) to altering temperature and ion concentration. Conditions favoring phase separation increased Brillouin shift, linewidth, and protein concentration. In comparison to solidification by chemical crosslinking, the ion-dependent aging of FUS condensates had a small effect on the molecular interaction strength inside. Finally, we investigated how sequence variations of A1-LCD, that change the driving force for phase separation, alter the physical properties of the respective condensates. Our results provide a new experimental perspective on the material properties of protein condensates. Robust and quantitative experimental approaches such as the presented ones will be crucial for understanding how the physical properties of biological condensates determine their function and dysfunction.
In search of visual opsin ofLissachatina fulica:in silicoprediction
Authors: Dominova Irina N. , Fedotov Daniil A. , Shirina Svetlana , Zikin Vladimir , Zhukov Valerii V. 
Published: March 2024

AbstractHere we present a complex study of possible visual opsin ofL. fulica. 8 candidate sequences were chosen for visual opsin protein and only one (Afu005002) was confirmed as a possible candidate after domain search, structural (structures were stimulated with AlphaFold2) and BLAST analysis. In order to evaluate spectroscopic properties of the Afu005002 protein, we have computed excitation energy and intensity of the main absorption band using QM/MM methodology at the ADC(2) level of theory. The theoretically predicted position of the main absorption maxima shows a good agreement with available experimental data.
Antigen-driven CD8+T cell clonal expansion is a prominent feature of MASH in humans and mice
Authors: Burtis Abbigayl E.C. , DeNicola Destiny M.C. , Ferguson Megan E. , Santos Radleigh G. , Pinilla Clemencia , Kriss Michael S. , Orlicky David J. , Tamburini Beth A. Jirón , Gillen Austin E. , Burchill Matthew A. 
Published: March 2024

AbstractBackground and AimsChronic liver disease due to metabolic dysfunction-associated steatohepatitis (MASH) is a rapidly increasing global epidemic. MASH progression is a consequence of the complex interplay between inflammatory insults and dysregulated hepatic immune responses. T lymphocytes have been shown to accumulate in the liver during MASH, but the cause and consequence of T cell accumulation in the liver remain unclear. Our study aimed to define the phenotype and T cell receptor diversity of T cells from human cirrhotic livers and an animal model of MASH to begin resolving their function in disease.Approach and ResultsIn these studies, we evaluated differences in T cell phenotype in the context of liver disease we isolated liver resident T cell populations from individuals with cirrhosis and a murine model of MASH. Using both 5’ single cell sequencing and flow cytometry we defined the phenotype and T cell receptor repertoire of liver resident T cells during health and disease.ConclusionsMASH-induced cirrhosis and diet-induced MASH in mice resulted in the accumulation of activated and clonally expanded T cells in the liver. The clonally expanded T cells in the liver expressed markers of chronic antigenic stimulation, includingPD1,TIGITandTOX. Overall, this study establishes for the first time that T cells undergo antigen-dependent clonal expansion and functional differentiation during the progression of MASH. These studies could lead to the identification of potential antigenic targets that drive T cell activation, clonal expansion, and recruitment to the liver during MASH.
Probing chromatin accessibility with small molecule DNA intercalation and nanopore sequencing
Authors: Bai Gali , Dhillon Namrita , Felton Colette , Meissner Brett , Saint-John Brandon , Shelansky Robert , Meyerson Elliot , Hrabeta-Robinson Eva , Hodjat Babak , Boeger Hinrich , Brooks Angela N. 
Published: March 2024

ABSTRACTGenome-wide identification of chromatin organization and structure has been generally probed by measuring accessibility of the underlying DNA to nucleases or methyltransferases. These methods either only observe the positioning of a single nucleosome or rely on large enzymes to modify or cleave the DNA. We developed adduct sequencing (Add-seq), a method to probe chromatin accessibility by treating chromatin with the small molecule angelicin, which preferentially intercalates into DNA not bound to core nucleosomes. We show that Nanopore sequencing of the angelicin-modified DNA is possible and allows visualization and analysis of long single molecules with distinct chromatin structure. The angelicin modification can be detected from the Nanopore current signal data using a neural network model trained on unmodified and modified chromatin-free DNA. Applying Add-seq toSaccharomyces cerevisiaenuclei, we identified expected patterns of accessibility around annotated gene loci in yeast. We also identify individual clusters of single molecule reads displaying different chromatin structure at specific yeast loci, which demonstrates heterogeneity in the chromatin structure of the yeast population. Thus, using Add-seq, we are able to profile DNA accessibility in the yeast genome across long molecules.GRAPHICAL ABSTRACT
Higher prevalence of sacbrood virus in highbush blueberry pollination units
Authors: McAfee Alison , French Sarah K. , Tsvetkov Nadejda , Higo Heather , Common Julia , Pernal Stephen F. , Giovenazzo Pierre , Hoover Shelley E. , Guzman-Novoa Ernesto , Currie Robert W , Veiga Patricia Wolf , Conflitti Ida M. , Pepinelli Mateus , Tran Lan , Zayed Amro , Guarna M. Marta , Foster Leonard J. 
Published: March 2024

AbstractHighbush blueberry pollination depends on managed honey bees (Apis mellifera) for adequate fruit set; however, beekeepers have raised concerns about poor health of colonies after pollinating this crop. Postulated causes include agrochemical exposure, nutritional deficits, and interactions with parasites and pathogens, particularlyMelisococcus plutonius(the causal agent of European foulbrood disease), but other pathogens could be involved. To broadly investigate common honey bee pathogens in relation to blueberry pollination, we sampled adult honey bees from colonies at time points corresponding to before (t1), during (t2), at the end (t3), and after (t4) highbush blueberry pollination in British Columbia (BC), Canada, across two years (2020 and 2021). Nine viruses as well asM. plutonius,Vairimorpha ceranaeandV. apis(formerlyNosema ceranaeandN. apis) were detected by PCR and microscopy and compared among colonies located near and far from blueberry fields. We found a significant interactive effect of time and blueberry proximity on the multivariate pathogen community, mainly due to differences at t4 (corresponding to roughly six weeks after the beginning of the pollination period). Post-hoc comparisons of pathogens in near and far groups at t4 showed that detections of sacbrood virus (SBV), which was significantly higher in the exposed group, was the primary driver. The association of SBV with highbush blueberry pollination may be contributing to the health decline that beekeepers observe after pollinating this crop, likely in combination with other factors.
Structure ofAquifex aeolicusLumazine Synthase by Cryo-Electron Microscopy to 1.42Å Resolution
Authors: Savva Christos G. , Sobhy Mohamed A. , De Biasio Alfredo , Hamdan Samir M. 
Published: March 2024

AbstractSingle particle Cryo-Electron microscopy (Cryo-EM) has become an essential structural determination technique with recent hardware developments making it possible to reach atomic resolution at which individual atoms, including hydrogen atoms, can be resolved. Thus Cryo-EM allows not only unprecedented detail regarding the structural architecture of complexes but also a better understanding surrounding their chemical states. In this study we used the enzyme involved in the penultimate step of riboflavin biosynthesis as a test specimen to benchmark a recently installed microscope and determine if other protein complexes could reach a resolution of 1.5Å or better which so far has only been achieved for the iron carrier ferritin. Using state of the art microscope and detector hardware as well as the latest software techniques to overcome microscope and sample limitations, a 1.42Å map ofAquifex aeolicuslumazine synthase (AaLS) was obtained from a 48-hour microscope session. In addition to water molecules and ligands involved in AaLS function, we can observe positive density for ∼50% of hydrogen atoms. A small improvement in resolution was achieved by Ewald sphere correction which was expected to limit the resolution to ∼1.5Å for a molecule of this diameter. Our study confirms that other protein complexes can be solved to near-atomic resolution. Future improvements in specimen preparation and protein complex stabilization may allow more flexible macromolecules to reach this level of resolution and should become a priority of study in the field.
VASP phase separation with priming proteins of fast endophilin mediated endocytosis modulates actin polymerization
Authors: Narayan Karthik B. , James Honey Priya , Cope Jonathan , Mondal Samsuzzoha , Baeyens Laura , Milano Francesco , Zheng Jason , Krause Matthias , Baumgart Tobias 
Published: March 2024

AbstractActin polymerization is essential in several clathrin-independent endocytic pathways including fast endophilin mediated endocytosis (FEME), however the actin machinery involved in FEME has been elusive. Here, we show that the actin polymerase VASP colocalizes and interacts directly with the FEME priming complex. We identify endophilin (EDP) as a VASP binding partner and establish novel non-canonical interactions between EDP and the EVH1 and EVH2 domains of VASP. The major FEME regulators EDP and lamellipodin (LPD) interact multivalently with VASP undergoing liquid-liquid phase separation both in solution and on lipid membranes. We show that priming complex mimicking condensates localise actin polymerization, with LPD-VASP promoting and EDP antagonising actin assembly, suggesting a novel role for EDP during the priming step of FEME. Finally, we show that LPD and EDP recruits and clusters VASP on lipid membranes mimicking the plasma membrane’s inner leaflet to locally assemble actin filaments. Our results support a model where actin polymerization in FEME is spatiotemporally initiated by the depletion of EDP, mediated by receptor activation.TeaserLiquid-like condensates formed by actin polymerases and endocytic proteins, localise and regulate actin polymerization.
A TLR4 ligand-based adjuvant for promoting the immunogenicity of Typhoid subunit vaccines
Authors: Alugupalli Kishore R. 
Published: March 2024

ABSTRACTNone of the typhoid Vi Polysaccharide (ViPS) subunit vaccines incorporate adjuvants, and the immunogenicity of ViPS vaccines (e.g. Typbar TCV®and Typhim Vi®) is in part due to associated TLR4 ligands such as endotoxin present in these vaccines. Since endotoxin content in vaccines is variable and kept very low due to inherent toxicity, I hypothesized that incorporating a defined amount of a non-toxic TLR4-ligand such as monophosphoryl lipid A in ViPS vaccines would improve their immunogenicity. To test this hypothesis, I developed an monophosphoryl lipid A-based adjuvant formulation named Turbo. Admixing Turbo with Typbar TCV®(ViPS-conjugated to tetanus toxoid) increased the levels of anti-ViPS IgM, IgG1, IgG2b, IgG2a/c and IgG3 in inbred and outbred mice. In infant mice, a single immunization with Turbo adjuvanted Typbar TCV®, resulted in a significantly increased and durable IgG response, and improved the control of bacterial burden compared to mice immunized without Turbo. Similarly, when adjuvanted with Turbo, the antibody response and control of bacteremia were also improved in mice immunized with Typhim Vi®, an unconjugated vaccine. The immunogenicity of unconjugated ViPS is inefficient in young mice and is lost in adult mice when immunostimulatory ligands in ViPS are removed. Nevertheless, when adjuvanted with Turbo, poorly immunogenic ViPS induced a robust IgG response in young and adult mice, and this was observed even under antigen-limiting conditions. These data suggest that incorporation of Turbo as an adjuvant will make typhoid vaccines more immunogenic regardless of their intrinsic immunogenicity or conjugation status and maximize the efficacy across all ages.
Exploring variations in lipids among drug-resistant and sensitiveMycobacterium tuberculosisby Thin layer chromatography and mass spectrometry
Authors: Kumar Kavitha , Giribhattanavar Prashant , Sagar B. K. Chandrasekhar , Patil Shripad A. 
Published: March 2024

AbstractMycobacterium tuberculosis (M.tb)lipids are important in the host–pathogen interplay, variation in the lipids organization of cell wall can act as an adaptive response. Specific cell wall structures can possibly result in suboptimal intracellular concentrations of anti-TB drugs, which favors the acquisition of drug resistance. Therefore, lipids fromM.tb(drug resistant and sensitive) were analyzed by 2D-thin layer chromatography and mass spectrometry. GraphPad Prism was used to perform Mann Whitney-U test to determine the statistical significance. Difference observed for total lipid content among different resistant isolates was insignificant. However, increase in phospholipids was identified in multi-drug resistant (MDR) isolate compared to sensitive isolate. Isoniazid, streptomycin-isoniazid, and isoniazid-ethambutol resistant isolates showed increased alpha-mycolic acids. MDR isolate showed a marginal decrease in alpha- and keto-form. Mycolipenic acid was seen only in sensitive isolate, and mycosanoic acids were observed in all the resistant isolates. Among the resistant isolates, there was an insignificant increase in the total phthiocerol dimycocerosates and sulfolipids. Drug resistance was associated with compositional imbalance of lipids. However, investigations to determine whether the changes notices are induced by the drugs is to be explored, which could give an insight into the drug resistant organisms pathogenesis.
Functional characterization of fatty acid synthase inHermetia illucens
Authors: Jiang Yuguo , Xue Wei , Peng Zhidan , Hou Peng , Kou Zongqing , Chen Bihui , Lu Huimeng , Huang Yongping 
Published: March 2024

AbstractFatty acid synthase (FAS) is a key role inde novolipogenesis (DNL) in most lives. In insects, type I FAS functions in homodimer, activated by a phosphopantetheine transferase (PPT), tode novoproduce essential saturated fatty acid for cellular processes. In a nova insect speciesHermetia illucens(Diptera: Stratiomyidae), different from most animals and insects, lauric acid (LA) accumulates to a noticeable level, near content of that in coconut oil or palm kernel oil. However, the origin of LA, fromde novobiosynthesis or as degraded products from long chain fatty acid, was still ambiguous. Here, in this study, we mined aFASgenes (HiOGS16234) fromH. illucensgenome and initially characterized its abilities of LA production in a free-fatty-acid producible yeast. After clear the yeast metabolites background of fatty acid by deletion ofScFAS1andScELO1sequentially, which responsible for fatty acid genesis and elongation respectively, functions of HiOGS16234 fromH. illucenswasin vitroelucidated by analyzing its product portfolio. Through protein domain swap in HiOGS16234, we found the ketoacyl synthase domain and thioesterases domain were responsible for the different product portfolio of HiOGS16234, compared with DmFASN1. Throughin vitroassay in yeast, the origin of LA in BSF was firstly elucidated at molecular level, which support DNL origin hypothesis in BSF.
Activity of zebrafish THAP9 transposase and zebrafish P element-like transposons
Authors: Kutnowski Nitzan , Ghanim George E. , Lee Yeon , Rio Donald C. 
Published: March 2024

ABSTRACTTransposable elements are mobile DNA segments that are found ubiquitously across the three domains of life. One family of transposons, called P elements, were discovered in the fruit flyDrosophila melanogaster. Since their discovery, P element transposase-homologous genes (called THAP-domain containing 9 or THAP9) have been discovered in other animal genomes. Here, we show that the zebrafish (Danio rerio) genome contains both an active THAP9 transposase (zfTHAP9) and mobile P-like transposable elements (calledPdre). zfTHAP9 transposase can excise one of its own elements (Pdre2) andDrosophilaP elements.DrosophilaP element transposase (DmTNP) is also able to excise the zebrafishPdre2 element, even though it’s distinct from theDrosophilaP element. However, zfTHAP9 cannot transposePdre2 orDrosophilaP elements, indicating partial transposase activity. Characterization of the N-terminal THAP DNA binding domain of zfTHAP9 shows distinct DNA binding site preferences from DmTNP and mutation of the zfTHAP9, based on known mutations in DmTNP, generated a hyperactive protein,. These results define an active vertebrate THAP9 transposase that can act on the endogenous zebrafishPdreandDrosophilaP elements.
All eukaryotic SMC proteins induce a twist of -0.6 at each DNA-loop-extrusion step
Authors: Janissen Richard , Barth Roman , Davidson Iain F. , Taschner Michael , Gruber Stephan , Peters Jan-Michael , Dekker Cees 
Published: March 2024

ABSTRACTEukaryotes carry three types of Structural Maintenance of Chromosomes (SMC) protein complexes, condensin, cohesin, and SMC5/6, which are ATP-dependent motor proteins that remodel the genome via DNA loop extrusion. SMCs modulate DNA supercoiling, but it has remained incompletely understood how this is achieved. Here we present a single-molecule magnetic tweezers assay that directly measures how much twist is induced by an individual SMC in each loop-extrusion step. We demonstrate that all three SMC complexes induce the same large negative twist (i.e., a linking number change ΔLk of -0.6 at each loop-extrusion step) into the extruded loop, independent of step size. Using ATP-hydrolysis mutants and non-hydrolysable ATP analogues, we find that ATP binding is the twist-inducing event during the ATPase cycle, which coincides with the force-generating loop-extrusion step. The fact that all three eukaryotic SMC proteins induce the same amount of twist indicates a common DNA-loop-extrusion mechanism among these SMC complexes.
Upstream open reading frames may contain hundreds of novel human exons
Authors: Ji Hyun Joo , Salzberg Steven L 
Published: March 2024

AbstractSeveral recent studies have presented evidence that the human gene catalogue should be expanded to include thousands of short open reading frames (ORFs) appearing upstream or downstream of existing protein-coding genes, each of which would comprise an additional bicistronic transcript in humans. Here we explore an alternative hypothesis that would explain the translational and evolutionary evidence for these upstream ORFs without the need to create novel genes or bicistronic transcripts. We examined 2,199 upstream ORFs that have been proposed as high-quality candidates for novel genes, to determine if they could instead represent protein-coding exons that can be added to existing genes. We checked for the conservation of these ORFs in four recently sequenced, high-quality human genomes, and found a large majority (87.8%) to be conserved in all four as expected. We then looked for splicing evidence that would connect each upstream ORF to the downstream protein-coding gene at the same locus, thus creating a novel splicing variant using the upstream ORF as its first exon. These protein coding exon candidates were further evaluated using protein structure predictions of the protein sequences that included the proposed new exons. We determined that 582 out of 2,199 upstream ORFs have strong evidence that they can form protein coding exons that are part of an existing gene, and that the resulting protein is predicted to have similar or better structural quality than the currently annotated isoform.Author SummaryWe analyzed over 2000 human sequences that have been proposed to represent novel protein-coding genes, and that reside just upstream of known genes. These “upstream ORFs” (uORFs) would represent a surprisingly large addition to the human gene catalogue, which after decades of refinement now contains just under 20,000 protein-coding genes. They would also create over 2000 new bicistronic genes, which number only 10 in current human annotation databases. We hypothesized that rather than novel genes, these sequences might instead represent novel exons that can be spliced into existing protein-coding genes, creating new isoforms of those genes. Using a combination of transcriptional evidence and computational predictions, we show that at least 582 of the previously-described uORFs can be used to create novel protein-coding exons, generating new transcripts and new protein isoforms, but not requiring the addition of entirely new genes to the human gene catalogue. We also demonstrate that the predicted three-dimensional structure of some of the new protein isoforms hints at new or improved functions for existing proteins.