bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cyclic peptide inhibitors stabilize Gq/11 heterotrimers
Authors: Mühle Jonas , Rodrigues Matthew J. , Alenfelder Judith , Jürgenliemke Lars , Guixà-González Ramon , Bacchin Arianna , Andres Fabio , Hennig Michael , Schihada Hannes , Crüsemann Max , König Gabriele M. , Kostenis Evi , Schertler Gebhard , Deupi Xavier 
Published: October 2023

ABSTRACTHeterotrimeric G proteins play a central role in cellular signaling, acting as switchable molecular regulators. Consequently, pharmacological agents to control G protein activity are of utmost importance to advance our understanding of this signal transduction system. The natural depsipeptides FR900359 (FR) and YM-254890 (YM) are two highly specific and widely used inhibitors of heterotrimeric Gq/11 proteins. These compounds have traditionally been understood to inhibit GDP dissociation by preventing the separation of the GTPase and α-helical domains of the Gα subunit. In this work, we have determined the high-resolution crystal structures of FR and YM bound to heterotrimeric G11 and used them to explain the molecular basis underlying their efficient suppression of G protein signaling. Notably, our data show that FR and YM also function as stabilizers of the interface between the Gα and Gβ subunits, acting as ‘molecular adhesives’ that stabilize the entire heterotrimer. Our results reveal unrecognized mechanistic features that explain how FR and YM effectively blunt Gq/11 signaling in living cells.
DetectEV: a functional enzymatic assays to assess integrity and bioactivity of extracellular vesicles
Authors: Adamo Giorgia , Picciotto Sabrina , Gargano Paola , Paterna Angela , Raccosta Samuele , Rao Estella , Romancino Daniele Paolo , Ghersi Giulio , Manno Mauro , Salamone Monica , Bongiovanni Antonella 
Published: October 2023

The application of extracellular vesicles (EVs) as therapeutics or nanocarriers in cell-free therapies necessitates meticulous evaluations of different features, including their identity, bioactivity, batch-to-batch reproducibility, and stability. Given the inherent heterogeneity in EV preparations, this assessment demands sensitive functional assays to provide key quality control metrics, complementing established methods to ensure that EV preparations meet the required functionality and quality standards. Here, we introduce the detectEV assay, an enzymatic-based approach for assessing EV luminal cargo bioactivity and membrane integrity. This method is fast, cost-effective, and quantifiable through enzymatic units. Utilizing microalgae-derived EVs, known as nanoalgosomes, as model systems, we optimized the assay parameters and validated its sensitivity and specificity in quantifying the enzymatic activity of esterases within the EV lumen while also evaluating EV membrane integrity. Compared to conventional methods that assess physicochemical features of EVs, our single-step analysis efficiently detects batch-to-batch variations by evaluating changes in luminal cargo bioactivity and integrity across various EV samples, including differences under distinct storage conditions and following diverse isolation and exogenous loading methods, all using small sample sizes. The detectEV assay’s application to various human-derived EV types demonstrated its versatility and potential universality. Additionally, the assay effectively predicted EV functionality, such as the antioxidant activity of different nanoalgosome batches. Our findings underscore the detectEV assay’s utility in comprehensive characterization of EV functionality and integrity, enhancing batch-to-batch reproducibility and facilitating their therapeutic applications.Graphical Abstract
Methionine oxidation of Carbohydrate-Active enZymes during white-rot wood decay
Authors: Molinelli Lise , Drula Elodie , Gaillard Jean-Charles , Navarro David , Armengaud Jean , Berrin Jean-Guy , Tron Thierry , Tarrago Lionel 
Published: October 2023

AbstractWhite-rot fungi employ secreted carbohydrate-active enzymes (CAZymes) along with reactive oxygen species (ROS), like hydrogen peroxide (H2O2), to degrade lignocellulose in wood. H2O2serves as a co-substrate for key oxidoreductases during the initial decay phase. While the degradation of lignocellulose by CAZymes is well-documented, the impact of ROS on the oxidation of the secreted proteins remains unclear and the identity of the oxidized proteins is unknown. Methionine (Met) can be oxidized to Met sulfoxide (MetO) or Met sulfone (MetO2) with potential deleterious, antioxidant, or regulatory effects. Other residues, like proline (Pro), can undergo carbonylation. Using the white-rotPycnoporus cinnabarinusgrown on aspen wood, we analyzed the Met content of the secreted proteins and their susceptibility to oxidation combining H18O with deep shotgun proteomics. Strikingly, their overall Met content was significantly lower (1.4%) compared to intracellular proteins (2.1%), a feature conserved in fungi but not in metazoans or plants. We evidenced that a catalase, widespread in white-rot fungi, protects the secreted proteins from oxidation. Our redox proteomics approach allowed identification of 49 oxidizable Met and 40 oxidizable Pro residues within few secreted proteins, mostly CAZymes. Interestingly, many of them had several oxidized residues localized in hotspots. Some Met, including those in GH7 cellobiohydrolases, were oxidized up to 47%, with a substantial percentage of sulfone (13%). These Met are conserved in fungal homologs, suggesting important functional roles. Our findings reveal that white-rot fungi safeguard their secreted proteins by minimizing their Met content and by scavenging ROS and pinpoint redox-active residues in CAZymes.ImportanceThe study of lignocellulose degradation by fungi is critical for understanding the ecological and industrial implications of wood decay. While carbohydrate-active enzymes (CAZymes) play a well-established role in lignocellulose degradation, the impact of hydrogen peroxide (H2O2) on secreted proteins remains unclear. This study aims at evaluating the effect of H2O2on secreted proteins, focusing on the oxidation of methionine (Met). Using the model white-rot modelPycnoporus cinnabarinusgrown on aspen wood, we showed that fungi protect their secreted proteins from oxidation by reducing their Met content and utilizing a secreted catalase to scavenge exogenous H2O2. The research identified key oxidizable Met within secreted CAZymes. Importantly, some Met like those of GH7 cellobiohydrolases, undergone substantial oxidation levels suggesting important roles in lignocellulose degradation. These findings highlight the adaptive mechanisms employed by white-rot fungi to safeguard their secreted proteins during wood decay and emphasize the importance of these processes in lignocellulose breakdown.
Significant roles in RNA-binding for the amino-terminal domains of Drosophila Pumilio and Nanos
Authors: Wharton Tammy H. , Marhabaie Mohammad , Wharton Robin P. 
Published: October 2023

AbstractThe Drosophila Pumilio (Pum) and Nanos (Nos) RNA-binding proteins govern abdominal segmentation in the early embryo, as well as a variety of other events during development. They bind together to a compound Nanos Response Element (NRE) present in thousands of maternal mRNAs in the ovary and embryo, includinghunchback(hb) mRNA, thereby regulating poly-adenylation, translation, and stability. Many studies support a model in which mRNA recognition and effector recruitment are achieved by distinct regions of each protein. The well-ordered Pum and Nos RNA-binding domains (RBDs) are sufficient to specifically recognize NREs; the relatively larger low-complexity N-terminal domains (NTDs) of each protein have been thought to act by recruiting mRNA regulators. Here we use yeast interaction assays to show that the NTDs also play a significant role in recognition of the NRE, acting via two mechanisms. First, the Pum and Nos NTDs interact in trans to promote assembly of the Pum/Nos/NRE ternary complex. Second, the Pum NTD acts via an unknown mechanism in cis, modifying base recognition by its RBD. These activities of the Pum NTD are important for its regulation of maternalhbmRNA in vivo.
A tuneable minimal cell membrane reveals that two lipid species suffice for life
Authors: Justice Isaac , Kiesel Petra , Safronova Nataliya , von Appen Alexander , Saenz James P. 
Published: October 2023

AbstractAll cells are encapsulated by a lipid membrane which facilitates the interaction between life and its environment. How life exploits the diverse mixtures of lipids that dictate membrane property and function has been experimentally challenging to address. We introduce an approach to tune and minimize lipidomes inMycoplasma mycoidesand the Minimal Cell (JCVI-Syn3A) revealing that a 2-component lipidome can support life. Systematically reintroducing phospholipid features demonstrated that acyl chain diversity is more critical for growth than head group diversity. By tuning lipid chirality, we explored the lipid divide between Archaea and the rest of life, showing that ancestral lipidomes could have been heterochiral. Our approach offers a tunable minimal membrane system to explore the fundamental lipidomic requirements for life, thereby extending the concept of minimal life from the genome to the lipidome.
Purine nucleosides replace cAMP in allosteric regulation of PKA in trypanosomatid pathogens
Authors: Ober Veronica , Githure George B. , Santos Yuri Volpato , Becker Sidney , Moya Gabriel , Basquin Jerôme , Schwede Frank , Lorentzen Esben , Boshart Michael 
Published: October 2023

AbstractCyclic nucleotide binding domains (CNB) confer allosteric regulation by cAMP or cGMP to many signalling proteins, including PKA and PKG. PKA of phylogenetically distantTrypanosomais the first exception as it is cyclic nucleotide-independent and responsive to nucleoside analogues (Bachmaier et al. 2019). Here we show that natural nucleosides inosine, guanosine and adenosine are nanomolar affinity CNB ligands and activators of PKA orthologs of the important tropical pathogensT. brucei,T. cruziandLeishmania.The sequence and structural determinants of binding affinity, -specificity and kinase activation of PKAR were established by structure-activity relationship (SAR) analysis, co-crystal structures and mutagenesis. Substitution of 2-3 amino acids in the binding sites is sufficient for conversion of CNB domains from nucleoside to cyclic nucleotide specificity. In addition, a trypanosomatid-specific C-terminal helix (αD) is required for high affinity binding to CNB-B. The αD helix functions as a lid of the binding site that shields ligands from solvent. Selectivity of guanosine for CNB-B and of adenosine for CNB-A results in synergistic kinase activation at low nanomolar concentration. PKA pulldown from rapid lysis establishes guanosine as the predominant ligandin vivoinT. bruceibloodstream forms, whereas guanosine and adenosine seem to synergize in the procyclic developmental stage in the insect vector. We discuss the versatile use of CNB domains in evolution and recruitment of PKA for novel nucleoside-mediated signalling.
cSTAR analysis identifies endothelial cell cycle as a key regulator of flow-dependent artery remodeling
Authors: Deng Hanqiang , Rukhlendo Oleksii S. , Joshi Divyesh , Hu Xiaoyue , Junk Philpp , Tuliakova Anna , Kholodenko Boris N. , Schwartz Martin A. 
Published: October 2023

AbstractFluid shear stress (FSS) from blood flow is sensed by vascular endothelial cells (ECs) to determine vessel stability, remodeling and susceptibility to atherosclerosis and other inflammatory diseases but the regulatory networks that govern these behaviors are only partially understood. We used cSTAR, a powerful new computational method, to define EC transcriptomic states under low shear stress (LSS) that triggers vessel inward remodeling, physiological shear stress (PSS) that stabilizes vessels, high shear stress (HSS) that triggers outward remodeling, and oscillatory shear stress (OSS) that confers disease susceptibility, all in comparison to cells under static conditions (STAT). We combined these results with the LINCS database where EC transcriptomic responses to drug treatments to define a preliminary regulatory network in which the cyclin-dependent kinases CDK1/2 play a central role in promoting vessel stability. Experimental analysis showed that PSS induced a strong late G1 cell cycle arrest in which CDK2 was activated. EC deletion of CDK2 in mice resulted in inward artery remodeling and both pulmonary and systemic hypertension. These results validate use of cSTAR to determine EC state and in vivo vessel behavior, reveal unexpected features of EC phenotype under different FSS conditions, and identify CDK2 as a key element within the EC regulatory network that governs artery remodeling.
Unraveling Evolutionary Dynamics: Insights from In Silico Experiments on Selective Mechanisms in Controlled Environments
Authors: Ledda Marco , Pluchino Alessandro , Ragusa Marco 
Published: October 2023

AbstractIn this paper, we present a series ofin silicoexperiments aimed at probing the evolutionary properties of our model. Our investigation encompasses multiple methodologies, beginning with the standard model used in population genetics for measuring natural selection. Next, we employ the Price equation, a well-established formalism known for its effectiveness in tracking how the relationship between parents and offspring evolves over time. In conclusion, we delve into the model results to explain, in the light of evolutionary theory, how the selective mechanism operates. Furthermore, the speculation about the mechanism will be hindered on the agents of the selective process. Even though natural selection can be considered as a statistical phenomenon, sprouting from the change in population frequencies, we argue that in models where there is a elevate control on the environment, it is possible to define the single element responsible for the selective pressure on theunits of selection.
Investigating a role forPUB17andPUB16in the self-incompatibility pathway in transgenicArabidopsis thaliana
Authors: Beronilla Paula K.S. , Goring Daphne R. 
Published: October 2023

AbstractIn Brassicaceae self-incompatibility (SI), self-pollen rejection is initiated by theS-haplotype specific interactions between the pollen SCR/SP11 ligand and the stigma S Receptor kinase (SRK). InBrassicaSI, a member of the Plant U-Box (PUB) E3 ubiquitin ligases, ARC1, is then activated by SRK in this stigma and cellular events downstream of this cause SI pollen rejection by inhibiting pollen hydration and pollen tube growth. During the transition to selfing,Arabidopsis thalianalost the SI components,SCR, SRK, andARC1. However, this trait can be reintroduced intoA. thalianaby adding back functional copies of these genes from closely related SI species. BothSCRandSRKare required for this, though the degree of SI pollen rejection varies between accessions, andARC1is not always needed to produce a strong SI response. ForA. thalianaC24, only transforming withA. lyrata SCRandSRKconfers a strong SI trait, and so here we investigated if ARC1-related PUBs were involved in the SI pathway. Two close ARC1 paralogs,PUB17andPUB16, were selected, and CRISPR/Cas9 technology was used to generatepub17andpub16mutations in the C24 accession. These mutants were then crossed into a transgenicA. thalianaSI-C24 line and their potential impact on SI pollen rejection was investigated. Overall, we did not observe any significant differences to implicatePUB17andPUB16functioning in the transgenicA. thalianaSI-C24 stigma to reject SI pollen.
Cell-type-resolved somatic mosaicism reveals clonal dynamics of the human forebrain
Authors: Chung Changuk , Yang Xiaoxu , Hevner Robert F. , Kennedy Katie , Vong Keng Ioi , Liu Yang , Patel Arzoo , Nedunuri Rahul , Barton Scott T. , Barrows Chelsea , Stanley Valentina , Mittal Swapnil , Breuss Martin W. , Schlachetzki Johannes C. M. , Gleeson Joseph G. 
Published: October 2023

AbstractDebate remains around anatomic origins of specific brain cell subtypes and lineage relationships within the human forebrain. Thus, direct observation in the mature human brain is critical for a complete understanding of the structural organization and cellular origins. Here, we utilize brain mosaic variation within specific cell types as distinct indicators for clonal dynamics, denoted as cell-type-specific Mosaic Variant Barcode Analysis. From four hemispheres from two different human neurotypical donors, we identified 287 and 780 mosaic variants (MVs), respectively that were used to deconvolve clonal dynamics. Clonal spread and allelic fractions within the brain reveal that local hippocampal excitatory neurons are more lineage-restricted compared with resident neocortical excitatory neurons or resident basal ganglia GABAergic inhibitory neurons. Furthermore, simultaneous genome-transcriptome analysis at both a cell-type-specific and single-cell level suggests a dorsal neocortical origin for a subgroup of DLX1+inhibitory neurons that disperse radially from an origin shared with excitatory neurons. Finally, the distribution of MVs across 17 locations within one parietal lobe reveals restrictions of clonal spread in the anterior-posterior axis precedes that of the dorsal-ventral axis for both excitatory and inhibitory neurons. Thus cell-type resolved somatic mosaicism can uncover lineage relationships governing the development of the human forebrain.
A novel glycosyltransferase organizes fungal glycogen and cell wall glucans
Authors: Loza Liza , Doering Tamara L. 
Published: October 2023

SUMMARYGlycogen is a glucose storage molecule comprised of branched α-1,4-glucan chains, best known as an energy reserve that can be broken down to fuel central metabolism. Because fungal cells have a specialized need for glucose in building cell wall glucans, we investigated whether glycogen is used for this process. For these studies we focused on the pathogenic yeastCryptococcus neoformans, which causes over 112,000 deaths per year worldwide. We identified two enzymes that influence formation of both glycogen and the cell wall: Gly-cogenin (Glg1), which initiates glycogen synthesis, and a novel protein that we callGlucanorganizingenzyme 1 (Goe1). We discovered that cells missing Glg1 lack α-1,4-glucan in their walls, indicating that this material is derived from glycogen. Without Goe1, glycogen rosettes are mislocalized and β-1,3-glucan in the cell wall is reduced. Altogether, our results provide mechanisms for a close association between glycogen and cell wall.
Cross-platform comparison of highly-sensitive immunoassays for inflammatory markers in a COVID-19 cohort1
Authors: Abe Koji , Beer Joanne C. , Nguyen Tran , Ariyapala Ishara S. , Holmes Tyson H. , Feng Wei , Zhang Bingqing , Kuo Dwight , Luo Yuling , Ma Xiao-Jun , Maecker Holden T. 
Published: October 2023

AbstractA variety of commercial platforms are available for the simultaneous detection of multiple cytokines and associated proteins, often employing antibody pairs to capture and detect target proteins. In this study, we comprehensively evaluated the performance of three distinct platforms: the fluorescent bead-based Luminex assay, the proximity extension-based Olink assay, and a novel proximity ligation assay platform known as Alamar NULISAseq. These assessments were conducted on serum samples from the NIH IMPACC study, with a focus on three essential performance metrics: detectability, correlation, and differential expression. Our results reveal several key findings. Firstly, the Alamar platform demonstrated the highest overall detectability, followed by Olink and then Luminex. Secondly, the correlation of protein measurements between the Alamar and Olink platforms tended to be stronger than the correlation of either of these platforms with Luminex. Thirdly, we observed that detectability differences across the platforms often translated to differences in differential expression findings, although high detectability did not guarantee the ability to identify meaningful biological differences. Our study provides valuable insights into the comparative performance of these assays, enhancing our understanding of their strengths and limitations when assessing complex biological samples, as exemplified by the sera from this COVID-19 cohort.
Fc-dependent functional activity of ChAdOx1-S and CoronaVac vaccine-induced antibodies to the SARS-CoV-2 spike protein
Authors: Harris Alexander W. , Kurtovic Liriye , Nogueira Jeane , Bouzas Isabel , Herbert Opi D. , Wines Bruce D. , Mark Hogarth P. , Poumbourios Pantelis , Drummer Heidi E. , Valim Clarissa , Porto Luís Cristóvão , Beeson James G. 
Published: October 2023

AbstractOngoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission and COVID-19 disease severity is influenced by immunity acquired by natural exposure and/or vaccination, whereby most vaccines are formulated on the Ancestral strain. However, population-level immunity is complicated by the emergence of variants of concern (VOCs), such as Omicron that is the dominant variant currently in circulation. Antibody Fc-dependent effector functions are being increasingly recognised as important mediators in immunity, especially against VOCs. However, induction of these functions in populations with diverse infection and/or vaccination histories, remains poorly defined. Here, we evaluated Fc-dependent functional antibodies following vaccination with two widely used vaccines: AstraZeneca (AZ; ChAdOx1-S) and Sinovac (SV). We quantified FcγR-binding and C1q-fixing antibodies against Ancestral and variant spike (S) proteins in Brazilian adults vaccinated with AZ or SV (n=222), some of which were previously exposed to SARS-CoV-2. AZ induced greater FcγR-binding responses to Ancestral S than the SV vaccine. Previously exposed individuals had significantly greater vaccine-induced responses compared to their naïve counterparts, with notably high C1q-fixation levels, irrespective of vaccine type. FcγR-binding was highest among AZ vaccinated individuals with a prior exposure, and these responses were well retained against the Omicron S protein. Overall, these findings contribute to our understanding of vaccine-induced immunity and its effectiveness against evolving variants.