bluuebunny/crossref_metadata_2025_split huggingface dataset:
Time-resolved transcriptomic of singleV. viniferafruits: membrane transports as switches of the double sigmoidal growth
Authors: Savoi Stefania , Shi Mengyao , Sarah Gautier , Weber Audrey , Torregrosa Laurent , Romieu Charles 
Published: September 2024

AbstractBy revealing that the grape berry loses one H+per accumulated sucrose at the inception of ripening, adopting a single fruit paradigm elucidates the fundamentals of the malate-sugar nexus, previously obscured by asynchrony in population-based models of ripening. More broadly, the development of the individual fruit was revisited from scratch to capture the simultaneous changes in gene expression and metabolic fluxes in a kinetically relevant way from flowering to overripening. Dynamics in water, tartrate, malate, hexoses, and K+fluxes obtained by combining individual single fruit growth and concentration data allowed to define eleven sub-phases in fruit development, which distributed on a rigorous curve in RNAseq PCA. WGCNA achieved unprecedented time resolutions in exploring transcript level-metabolic rate associations. A comprehensive set of membrane transporters was found specifically expressed during the first growth phase related to vacuolar over-acidification. Unlike in slightly more acidic citrus, H+V-PPase transcripts were predominantly expressed, followed by V-ATPase and PH5, clarifying the thermodynamic limit beyond which replacement by the PH1/PH5 complex turns compulsory. Puzzlingly,bona fideALMT kept a low profile at this stage, possibly replaced by a predominating uncharacterized anion channel. Then, the switch role of HT6 in sugar accumulation was confirmed, electroneutralized by malate vacuolar leakage and H+pumps activation.HighlightsTo alleviate asynchronicity biases, transcripts showing strict coincidental timing with pericarp physiological phases were disentangled on single berries, enlightening the tight multifaceted membrane developmental control of sugar and acid fluxes.
A computationally designed panel of diverse and selective peroxygenases for terpene oxyfunctionalization
Authors: Münch Judith , Soler Jordi , Gildor-Cristal Ofir , Fleishman Sarel J. , Garcia-Borràs Marc , Weissenborn Martin J. 
Published: September 2024

ABSTRACTEnzyme engineering has a critical role in the transition to economical, low-energy and environmentally friendly chemical production. Current approaches relying on costly iterations of mutation and selection are limited to reactions with a straightforward experimental readout and struggle to address mutational epistasis. We focus on unspecific peroxygenases (UPOs), prized engineering targets due to their ability to oxyfunctionalize diverse organic molecules of industrial and environmental value. To address the lack of scalable screening for UPO functions, we applied enzyme-design calculations to focus experiments. Starting from an AlphaFold2 model ofMthUPO, the automated FuncLib algorithm generated 50 diverse active-site multipoint designs—all of which were functional. Screening against nine diverse terpenes revealed large improvements and new oxyfunctionalization products, resulting in molecules of high pharmaceutical and industrial value. We rationalized observed specificity changes using AI-based docking and molecular dynamics simulations, providing molecular insights that could generalize to engineering other UPOs. Thus, computational design and modeling can dramatically accelerate the urgently needed green transition of the chemical industry.Abstract Figure
Potent and Selective SETDB1 Covalent Negative Allosteric Modulator Reduces Methyltransferase Activity in Cells
Authors: Uguen Mélanie , Shell Devan J. , Silva Madhushika , Deng Yu , Li Fengling , Szewczyk Magdalena M. , Yang Ka , Zhao Yani , Stashko Michael A. , Norris-Drouin Jacqueline L. , Waybright Jarod M. , Beldar Serap , Rectenwald Justin M. , Mordant Angie L. , Webb Thomas S. , Herring Laura E. , Arrowsmith Cheryl H. , Ackloo Suzanne , Gygi Steven P. , McGinty Robert K. , Barsyte-Lovejoy Dalia , Liu Pengda , Halabelian Levon , James Lindsey I. , Pearce Kenneth H. , Frye Stephen V. 
Published: September 2024

ABSTRACTA promising drug target, SETDB1, is a dual Kme reader and methyltransferase, which has been implicated in cancer and neurodegenerative disease progression. To help understand the role of the triple Tudor domain (3TD) of SETDB1, its Kme reader, we first identified a low micromolar small molecule ligand, UNC6535, which occupies simultaneously both the TD2 and TD3 reader binding sites. Further optimization led to the discovery of UNC10013, the first covalent 3TD ligand targeting Cys385 of SETDB1. UNC10013 is potent with a kinact/KIof 1.0 x 106M-1s-1and demonstrated proteome-wide selectivity. In cells, negative allosteric modulation of SETDB1-mediated Akt methylation was observed after treatment with UNC10013. Therefore, UNC10013 is a potent, selective and cell-active covalent ligand for the 3TD of SETDB1, demonstrating negative allosteric modulator properties and making it a promising tool to study the biological role of SETDB1 in disease progression.
What we can find in what’s left behind: DNA metabarcoding of semi-aquatic insect exuviae
Authors: Dworatzek SL , Tozer M , Austin K , Vermey A , Steinke D 
Published: September 2024

AbstractSemi-aquatic insects contribute to critical ecological functioning of freshwater habitats in both the aquatic and terrestrial phase of their life. Traditional methods of studying their emergence rely on the capture of insects as they emerge, and morphological identification with taxonomic keys. This is not only time consuming but can have large impacts on the study population, obstacles that can be removed by the use of DNA.This study investigated the potential of using exuviae collected from the water surface as DNA source. Both emergence trap samples and insect exuviae were collected from a constructed wetland and a small creek in southern Ontario. Metabarcoding provided a total of 40 samples with 254 distinct taxa from emergence traps (26 samples), and 135 from exuviae (14 samples). There were many similarities between both two sample types, especially for the rich chironomid diversity. Nonetheless, the exuviae samples were able to identify more orders containing semi-aquatic insects. Furthermore, they showed a higher level of diversity within these orders. This higher level of diversity seen in exuviae samples may be due to limitations of emergence traps, such as they only account for a small defined surface area. In contrast, exuviae are representing a much larger area and are free floating, thus collection of emerging taxa is not limited to the emerging site.We were able to show that identification of emerging aquatic insects through metabarcoding of exuviae is a useful method for the study of insect emergence.
PEG Adjustment Enables Genome Transplantation UsingMycoplasma MycoidesRecipient
Authors: Karas Bogumil J. , Moreau Nicolette G. , Venter J Craig , Smith Hamilton O , Glass John I. 
Published: September 2024

AbstractPioneering advances in synthetic biology, initiated at the J. Craig Venter Institute, have enabled the creation of the first cell driven by synthetic genome and opened a new era of creating designer microbes. This technology offers far greater potential for genetic modification than traditional genome editing techniques and holds promise for the routine creation of synthetic cells as DNA synthesis costs decrease and genetic knowledge expands. Two essential technologies underpin this achievement: the ability to clone entire genomes in host organisms, such asSaccharomyces cerevisiae, and the successful transplant of these genomes into recipient cells to generate living organisms. In all previous work the recipient cell in genome transplantation experiments has beenMycoplasma capricolum. In this study, we explored the potential of usingMycoplasma mycoidesstrains as recipient cells for genome transplantation. By increasing polyethylene glycol (PEG) concentration from 5% to 10%, we successfully transplanted variousM. mycoidesstrains using severalM. mycoidesrecipient cells. Additionally, we demonstrated the ability to transplantM. capricolumgenomes intoM. mycoidesrecipient cells; although with lower efficiency compared toM. mycoidesstrains. These findings provide a modified transplantation protocol and likely get us closer to expanding genome transplantation to other bacterial species.
Intrinsically disordered proteins can behave as different polymers across their conformational ensemble
Authors: Chakraborty Saikat , Morozova Tatiana I. , Barrat Jean-Louis 
Published: September 2024

AbstractIntrinsically disordered proteins (IDPs) are macromolecules, which in contrast to well-folded proteins, explore a large number of conformationally heterogeneous states. In this work, we investigate the conformational space of the disordered proteinβ-casein using Hamiltonian replica exchange atomistic molecular dynamics simulations in explicit water. The energy landscape contains a global minimum along with two shallow funnels. Employing static polymeric scaling laws separately for individual funnels, we find that they cannot be described by the same polymeric scaling exponent. Around the global minimum, the conformations are globular, whereas in the vicinity of local minima we recover coil-like scaling. To elucidate the implications of structural diversity on equilibrium dynamics, we initiate standard molecular dynamics simulations in the NVT ensemble with representative conformations from each funnel. Global and internal motions for different classes of trajectories show heterogeneous dynamics with globule to coil-like signatures. Thus, IDPs can behave as entirely different polymers in different regions of the conformational space.TOC Graphic
Minocycline enhances antimicrobial activity of unpolarized macrophages againstAcinetobacter baumanniiwhile reducing the inflammatory response
Authors: Guerra Alberto Daniel , Volk Cecilia F. , Peterson-Weber Alex , Peters Jason M. , Sakoulas George , Sauer John-Demian , Rose Warren E. 
Published: September 2024

ABSTRACTMinocycline activity againstAcinetobacter baumannii(AB)in vivois underestimated by standard methods of susceptibility testing. We examined pharmacologic effects of minocycline on primary immunity that may be contributing to thein vivovs.in vitrodiscrepancy of minocycline activity against AB. Minocycline MICs against 10ABstrains were compared in standard bacteriologic media (Mueller-Hinton broth, MHB) and physiologic (RPMI) media. Macrophages were pretreated with minocycline or comparator antibiotics beforeABco-culture. Macrophage cytokine production and phagocytosis ofABwere measured without and with pre-treatment with minocycline. Two to eight-fold reduction in minocycline MIC against 10ABstrains occurred in RPMI compared to MHB, which was more pronounced than other antibiotic classes. Macrophages pretreated with 1, 5, 10, 30, 50, and 100 μg/mL minocycline before bacterial co-cultures significantly decreasedABinoculum at 6 hours of co-culture in a dose-dependent manner, with no bacterial colonies observed from co-cultures with macrophages pretreated with 30 μg/mL or more of minocycline. Macrophages pretreated with minocycline for 24 hours before zymosan stimulation led to significantly higher levels of phagocytosis. Macrophages treated with minocycline for 24 hours significantly decreased production of IL-6, TNF-α, and MCP-1 in a dose dependent manner. The minocyclinein vivoefficacy may be attributed to enhanced activity in nutrient-limited, physiologic medium combined with increased macrophage phagocyte efficiency. Incorporating novel assays that recapitulate thein vivoenvironment will be important for understanding the host-pathogen-antibiotic relationship toward a goal of improved future drug discovery and overall treatment strategies againstABand other drug-resistant pathogens.
Fast hierarchical processing of orthographic and semantic parafoveal information during natural reading
Authors: Wang Lijuan , Frisson Steven , Pan Yali , Jensen Ole 
Published: September 2024

AbstractReaders extract orthographic and semantic information from parafoveal words before fixating on them. While this has to be achieved within an intersaccadic interval, the neuronal mechanisms supporting this fast parafoveal word processing within the language network remain unknown. We co-registered MEG and eye-tracking data in a natural reading paradigm to uncover the neuronal mechanisms supporting parafoveal processing. Representational similarity analysis (RSA) revealed that parafoveal orthographic neighbours (e.g., “writer” vs. “waiter”) showed higher representational similarity than non-neighbours (e.g., “writer” vs. “police”), emerging ∼68 ms after fixation onset on the preceding word (e.g., “clever”) in the visual word form area. Similarly, parafoveal semantic neighbours (e.g., “writer” vs. “author”) exhibited increased representational similarity at ∼137 ms in the left inferior frontal gyrus. Importantly, the degree of orthographic and semantic parafoveal processing predicted individual reading speed. Our findings suggest fast hierarchical processing of parafoveal words across distinct brain regions, which enhances reading efficiency.
Impact of AIM2 on HNSCC Development
Authors: Reinartz Dakota M. , Escamilla-River Vicente , Tribble Stephanie L. , Caulin Carlos , Wilson Justin E. 
Published: September 2024

AbstractHead and neck squamous cell carcinoma (HNSCC) constitutes 90% of head and neck cancers. HNSCC development is linked to chronic inflammation, while established HNSCC tumors are often immune suppressive. However, both occur through mechanisms that are not fully understood. The cytosolic double-stranded DNA sensor Absent in Melanoma 2 (AIM2) is an inflammasome forming protein that also has inflammasome-distinct roles in restricting tumorigenesis by limited PI3K signaling. Here, we used an experimental mouse model of HNSCC, involving treatment of wild type (WT) andAim2−/−mice with the carcinogen 4NQO in drinking water. Compared to WT mice, 4NQO-treatedAim2−/−mice exhibited larger tumor sizes and increased tissue dysplasia. 4NQO-treated wild type andAim2−/−mice displayed similar tongueIl6, Tnf, Il1b, Il12, and Il10expression and no consistent differences in PI3K or inflammasome activation, suggesting AIM2 may not regulate these factors during HNSCC. Instead,IfngandIrf1was elevated in 4NQO-treatedAim2−/−mice, suggesting AIM2 restricts IFNγ. In line with this, RNA-sequencing of total tongue RNA from 4NQO-treated mice revealedAim2−/−mice had enhanced expression of genes related to the MHC protein complex, cell killing, and T cell activation compared to wild type mice. In addition, we observed increased macrophage infiltration into the tongue epithelium of 4NQO-treatedAim2−/−mice. Lastly, usingAim2−/−/Rag1−/−-double deficient animals, we found that the adaptive immune compartment was necessary for the enhanced tumorigenesis during AIM2 deficiency. Taken together, these findings suggest AIM2 limits the progression of oral tumor development partially through regulating IFNγ and adaptive immune responses.
Mechanism of allosteric activation in human mitochondrial ClpP protease
Authors: Goncalves Monica M. , Uday Adwaith B. , Forrester Taylor J. B. , Currie S. Quinn W. , Kim Angelina S. , Feng Yue , Jitkova Yulia , Velyvis Algirdas , Harkness Robert W. , Kimber Matthew S. , Schimmer Aaron D. , Zeytuni Natalie , Vahidi Siavash 
Published: September 2024

AbstractHuman ClpP protease contributes to mitochondrial protein quality control by degrading misfolded proteins. ClpP is overexpressed in cancers such as acute myeloid leukemia (AML), where its inhibition leads to the accumulation of damaged respiratory chain subunits and cell death. Conversely, hyperactivating ClpP with small-molecule activators, such as the recently-discovered ONC201, disrupts mitochondrial protein degradation and impairs respiration in cancer cells. Despite its critical role in human health, the mechanism underlying the structural and functional properties of human ClpP remain elusive. Notably, human ClpP is paradoxically activated by active-site inhibitors. All available structures of human ClpP published to date are in the inactive compact or compressed states, surprisingly even when ClpP is bound to an activator molecule such as ONC201. Here, we present the first structures of human mitochondrial ClpP in the active extended state, including a pair of structures where ClpP is bound to an active-site inhibitor. We demonstrate that amino acid substitutions in the handle region (A192E and E196R) recreate a conserved salt bridge found in bacterial ClpP, stabilizing the extended active state and significantly enhancing ClpP activity. We elucidate the ClpP activation mechanism, highlighting a hormetic effect where sub-stoichiometric inhibitor binding triggers an allosteric transition that drives ClpP into its active extended state. Our findings link the conformational dynamics of ClpP to its catalytic function and provide high-resolution structures for the rational design of potent and specific ClpP inhibitors, with implications for targeting AML and other disorders with ClpP involvement.Significance statementHuman ClpP protease is essential for maintaining mitochondrial protein quality by degrading damaged proteins. In cancers like acute myeloid leukemia (AML), ClpP is overexpressed, and inhibiting it causes cancer cell death by disrupting mitochondrial function. Conversely, activating ClpP with small molecules, such as ONC201, also leads to cancer cell death by impairing mitochondrial respiration. However, the structural details of ClpP activation have been elusive. Our research presents the first structures of human ClpP in its active state, revealing a novel activation mechanism where inhibitors unexpectedly trigger activity through allosteric changes. These insights provide a foundation for designing targeted therapies for AML and other diseases where ClpP plays a crucial role.
Identification of novel toxin domains and characterization of a broadly distributed family of lipid-targeting NlpC/P60
Authors: Nicastro Gianlucca G. , Sibinelli-Sousa Stephanie , Hespanhol Julia Takuno , Santos Thomas W. C. , Munoz Joseph P. , Santos Rosangela S. , Perez-Sepulveda Blanca M. , Miyamoto Sayuri , Aravind L. , de Souza Robson F. , Bayer-Santos Ethel 
Published: September 2024

AbstractBacterial warfare is a common and ancient phenomenon in nature, where bacterial species use strategies to inhibit the growth or kill competitors. This involves the production and deployment of antibacterial toxins that disrupt essential cellular processes in target cells. Polymorphic toxins comprise a group of offensive systems with a modular structure featuring a conserved N-terminal translocation domain fused to diverse C-terminal toxin domains. The continuous arms race in which bacteria acquire new toxin and immunity proteins to promote increased adaptation to their environment is responsible for the diversification of this toxin repertoire. Here, we deployedin-silicostrategies to analyze 10,000 genomes and identify toxin domains secreted via the type VI secretion system ofSalmonella. We identified and manually curated 128 candidates, which are widespread polymorphic toxins detected in a vast array of species and linked to diverse secretion systems. In addition, 45 previously uncharacterized toxin domains were identified. STox15 was among the most frequent candidates found in the dataset and was selected for in-depth characterization. STox15 is an antibacterial effector belonging to the NlpC/P60 papain-like fold superfamily with a permuted catalytic core typical of lipid-targeting versions rather than peptidases or amidases. Biochemical analysis with recombinant protein and lipidomics of intoxicatedEscherichia colirevealed that STox15 displays phospholipase activity cleaving off acyl groups from phosphatidylglycerol and phosphatidylethanolamine.ImportanceThis work broadens our understanding of polymorphic toxin domains and provides the first direct characterization of a lipid-targeting NlpC/P60 domain in biological conflicts.
Megabase-scale loss of heterozygosity provoked by CRISPR-Cas9 DNA double-strand breaks
Authors: Regan Samantha B. , Medhi Darpan , White Travis B. , Jiang Yi-Zhen , Jia Su , Deng Qichen , Jasin Maria 
Published: September 2024

AbstractHarnessing DNA double-strand breaks (DSBs) is a powerful approach for gene editing, but it may provoke loss of heterozygosity (LOH), which predisposes to tumorigenesis. To interrogate this risk, we developed a two- color flow cytometry-based system (Flo-LOH), detecting LOH in ∼5% of cells following a DSB. After this initial increase, cells with LOH decrease due to a competitive disadvantage with parental cells, but if isolated, they stably propagate. Segmental loss from terminal deletions with de novo telomere addition and nonreciprocal translocations is observed as well as whole chromosome loss, especially following a centromeric DSB. LOH spans megabases distal from the DSB, but also frequently tens of megabases centromere-proximal. Inhibition of microhomology-mediated end joining massively increases LOH, which is synergistically increased with concomitant inhibition of canonical nonhomologous end joining. The capacity for large-scale LOH must therefore be considered when using DSB-based gene editing, especially in conjunction with end joining inhibition.
Accurate quantitation of 16S gene copies in low biomass samples post-antibiotic treatment through deep sequencing with a balanced nucleotide synthetic spike-in approach
Authors: Chang Chia-Chi , Huang Licai , Jin Yimei , Jamal Mohamed A. , Hayase Eiko , Halsey Taylor , Ortega Miriam R. , Pham Dung , Glover Israel , Flores Ivonne , McDaniel Lauren , Prasad Rishika , Peterson Christine , Jenq Robert R. 
Published: September 2024

AbstractThe microbiota significantly impacts health and treatment outcomes. While 16S rRNA gene sequencing reveals relative bacterial abundances, it does not provide absolute quantification. We developed a cost-effective solution incorporating synthetic DNA standards designed to ensure balanced nucleotide representation at each position. These standards are spiked into samples before DNA extraction, enabling simultaneous quantification of both relative and absolute bacterial abundances. We applied this method to samples collected from mice and patients, both before and after antibiotic treatment. Our approach showed a reduction in total bacterial density in mice and patients post-antibiotic treatment. This spike-in standard method can be adapted to samples with varying bacterial densities, allowing quantification of absolute taxonomical abundances without the need for an additional quantitative PCR assessment. Our approach also improves sequencing quality scores for low biomass samples.