bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multifocal Ectopic Purkinje Premature Contractions due to neutralization of anSCN5Anegative charge: structural insights into the gating pore hypothesis
Authors: Glazer Andrew M. , Yang Tao , Li Bian , Page Dana , Fouda Mohamed , Wada Yuko , Lancaster Megan C. , O’Neill Matthew J. , Muhammad Ayesha , Gao Xiaozhi , Ackerman Michael J. , Sanatani Shubhayan , Ruben Peter C. , Roden Dan M. 
Published: February 2024

AbstractBackgroundWe identified a novelSCN5Avariant, E171Q, in a neonate with very frequent ectopy and reduced ejection fraction which normalized after arrhythmia suppression by flecainide. This clinical picture is consistent with multifocal ectopic Purkinje-related premature contractions (MEPPC). Most previous reports of MEPPC have implicatedSCN5Avariants such as R222Q that neutralize positive charges in the S4 voltage sensor helix of the channel protein NaV1.5 and generate a gating pore current.Methods and ResultsE171 is a highly conserved negatively-charged residue located in the S2 transmembrane helix of NaV1.5 domain I. E171 is a key component of the Gating Charge Transfer Center, a region thought to be critical for normal movement of the S4 voltage sensor helix. We used heterologous expression, CRISPR-edited induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), and molecular dynamics simulations to demonstrate that E171Q generates a gating pore current, which was suppressed by a low concentration of flecainide (IC50 = 0.71±0.07 µM). R222Q shifts voltage dependence of activation and inactivation in a negative direction but we observed positive shifts with E171Q. E171Q iPSC-CMs demonstrated abnormal spontaneous activity and prolonged action potentials. Molecular dynamics simulations revealed that both R222Q and E171Q proteins generate a water-filled permeation pathway that underlies generation of the gating pore current.ConclusionPreviously identified MEPPC-associated variants that create gating pore currents are located in positively-charged residues in the S4 voltage sensor and generate negative shifts in the voltage dependence of activation and inactivation. We demonstrate that neutralizing a negatively charged S2 helix residue in the Gating Charge Transfer Center generates positive shifts but also create a gating pore pathway. These findings implicate the gating pore pathway as the primary functional and structural determinant of MEPPC and widen the spectrum of variants that are associated with gating pore-related disease in voltage-gated ion channels.
A SENSORIZED 3D-PRINTED KNEE TEST RIG FOR EXPERIMENTAL VALIDATION OF PATELLAR TRACKING AND CONTACT SIMULATION AFTER TOTAL KNEE REPLACEMENT
Authors: Michaud Florian , Mouzo Francisco , Dopico Daniel , Cuadrado Javier 
Published: February 2024

AbstractTotal knee arthroplasty aims to relieve pain and restore function in the affected joint through artificial implants. Despite advances in design and surgical techniques, there are complications associated with surgery, especially concerning the patella. The use of musculoskeletal models in orthopedic surgery allows for objective prediction of postoperative function and optimization of results for each patient. To ensure that simulations are trustworthy and can be used for predictive purposes, comparing simulation results with experimental data is crucial. Although progress has been made in obtaining 3D bone geometry and estimating contact forces, validation of these predictions has been limited due to the lack of direct in vivo measurements and the economic and ethical constraints associated with available alternatives. In this study, an existing commercial surgical training station was transformed into a sensorized test bench. The use of 3D-printed models and sensors allowed low-cost and reproducible experimental validation of computer simulation (patellar movement and forces) while avoiding ethical issues.
Vitamin D is Glucoprotective in Aging Males but Not Females
Authors: Ginnard Olivia Z.B. , Morales Maria , Youn Ji Youn , Xu Yong , Sisley Stephanie R. 
Published: February 2024

AbstractType 2 diabetes is strongly linked to vitamin D deficiency in older adults. However, there is a discrepancy between clinical trials in adults on the efficacy of vitamin D treatment in prediabetes and diabetes. In addition, human data indicates there may be sexual dimorphism in the effect of vitamin D deficiency on dysglycemia that is more pronounced in men. These incongruities may be due to our limited understanding of the underlying mechanisms of vitamin D in glucose homeostasis among its vast target tissues across the body. Furthermore, vitamin D deficiency is diagnosed by low levels of a storage form of vitamin D, which may not be an accurate indicator of vitamin D status in these individuals. Thus, measuring expression levels of vitamin D receptor (VDR)-target genes across tissues involved in glucose regulation and vitamin D pathways may be a more promising marker of vitamin D status. Here we describe the sex-specific physiological effects of vitamin D supplementation in an aged, non-obese mouse model on glucose homeostasis and tissue-specific gene regulation.
Analog epigenetic memory revealed by targeted chromatin editing
Authors: Palacios Sebastian , Bruno Simone , Weiss Ron , Salibi Elia , Kane Andrew , Ilia Katherine , Del Vecchio Domitilla 
Published: February 2024

SummaryChemical modifications to histones and DNA play a crucial role in the regulation of transcription and in the maintenance of chromatin states that are not permissive to gene expression [1–3]. However, the landscape of gene expression states that these modifications stably maintain remains uncharted. Here, we show that gene expression can be memorized at a wide range of levels thus implementing analog epigenetic memory. Mechanistically, we find that DNA methylation serves a primary role in maintaining memory across cell divisions while histone modifications only follow DNA methylation to regulate gene expression. Employing targeted epigenetic editing and time-course analysis, we analyzed the temporal stability of gene expression and DNA methylation post removal of epigenetic effectors. We found that the grade of DNA methylation in the gene’s promoter, defined as the mean fraction of methylated CpGs, remains stable over time and inversely correlates with gene expression level. By contrast, Histone 3 lysine 9 trimethylation (H3K9me3) could not persist after removal of its writer in the absence of DNA methylation. These experimental findings, combined with our chromatin modification model, indicate that the absence of positive feedback mechanisms around DNA methylation - unlike those found in histone modifications - enable the temporal stability of the DNA methylation grade, which leads to analog memory. These results expand current knowledge on how epigenetic memory is achieved in natural systems. Moreover, we anticipate that analog memory through graded DNA methylation will enable to program mammalian cells with fine-grained information storage. This capability will significantly enhance the sophistication of engineered cell functionality in applications including tissue engineering, organoids, and cell therapies.
Cancer-Specific Alterations in Nuclear Matrix Proteins Determined by Multi-omics Analyses of Ductal Carcinomain Situ
Authors: Almutairy Ali , Alhamed Abdullah , Grant Stephen G. , Sarachine Falso Miranda J. , Day Billy W. , Simmons Colton R. , Latimer Jean J. 
Published: February 2024

AbstractBreast cancer (BC) is the most common cancer affecting women in the United States. Ductal carcinomain situ(DCIS) is the earliest identifiable pre-invasive BC lesion. Estimates show that 14 to 50% of DCIS cases progress to invasive BC. Our objective was to identify nuclear matrix proteins (NMP) with specifically altered expression in DCIS and later stages of BC compared to non-diseased breast reduction mammoplasty and a contralateral breast explant using mass spectrometry and RNA sequencing to accurately identify aggressive DCIS. Sixty NMPs were significantly differentially expressed between the DCIS and non-diseased breast epithelium in an isogenic contralateral pair of patient-derived extended explants. Ten of the sixty showed significant mRNA expression level differences that matched the protein expression. These 10 proteins were similarly expressed in non-diseased breast reduction cells. Three NMPs (RPL7A, RPL11, RPL31) were significantly upregulated in DCIS and all other BC stages compared to the matching contralateral breast culture and an unrelated non-diseased breast reduction culture. RNA sequencing analyses showed that these three genes were upregulated increasingly with BC progression. Finally, we identified three NMPs (AHNAK, CDC37 and DNAJB1) that were significantly downregulated in DCIS and all other BC stages compared to the isogenically matched contralateral culture and the non-diseased breast reduction culture using both proteomics and RNA sequencing techniques.
Deciphering the function of intrinsic and genomics-driven epigenetic heterogeneity in head and neck cancer progression with single-nucleus CUT&RUN
Authors: Womersley Howard J. , Muliaditan Daniel , DasGupta Ramanuj , Cheow Lih Feng 
Published: February 2024

AbstractInterrogating regulatory epigenetic alterations during tumor progression at the resolution of single cells has remained an understudied area of research. Here we developedahighly sensitive single-nucleus CUT&RUN (snCUT&RUN) assay to profile histone modifications in isogenic primary, metastatic, and cisplatin-resistant head and neck squamous cell carcinoma (HNSCC) patient-derived tumor cell lines. We find that the epigenome can be involved in diverse modes to contribute towards HNSCC progression. First, we demonstrate that gene expression changes during HNSCC progression can be co-modulated by alterations in both copy number and chromatin activity, driving epigenetic rewiring of cell-states. Furthermore, intratumour epigenetic heterogeneity (ITeH) may predispose sub-clonal populations within the primary tumour to adapt to selective pressures and foster the acquisition of malignant characteristics. In conclusion, snCUT&RUN serves as a valuable addition to the existing toolkit of single-cell epigenomic assays and can be used to dissect the functionality of the epigenome during cancer progression.
Myosin-19 and Miro regulate mitochondria-endoplasmatic reticulum contacts and mitochondria inner membrane architecture
Authors: Attia Aya , Majstrowicz Katarzyna , Shembekar Samruddhi , Honnert Ulrike , Nikolaus Petra , Lohman Birgit , Bähler Martin 
Published: February 2024

AbstractMitochondrial dynamics is important for cellular health and includes morphology, fusion, fission, vesicle formation, transport and contact formation with other organelles. Myosin XIX (Myo19) is an actin-based motor which competes with TRAK1/2 adaptors of microtubule-based motors for binding to the outer mitochondrial membrane receptors Mitochondrial Rho GTPases 1/2 (Miro). Currently, it is poorly understood how Myo19 contributes to mitochondrial dynamics. Here, we report on a Myo19-deficient mouse model and the ultrastructure of the mitochondria from cells of Myo19-deficient mice and HEK cells, Miro-deficient HEK cells and TRAK1-deficient HAP1 cells. Myo19-deficient mitochondria in kidney, skeletal and cardiac muscle cells, MEFs and HEK cells have morphological alterations in the inner mitochondrial membrane with reduced numbers of malformed cristae. In addition, mitochondria in Myo19-deficient cells showed fewer ER-mitochondria contact sites (ERMCS). In accordance with the ultrastructural observations, Myo19-deficient MEFs had lower oxygen consumption rates and a reduced abundance of OXPHOS supercomplexes. The simultaneous loss of Miro1 and Miro 2 led to a comparable mitochondria phenotype and reduced ERMCS as observed upon loss of Myo19. However, the loss of TRAK1 caused only a reduction in the number of cristae, but not ERMCS. These results demonstrate that both actin- and microtubule-based motors regulate cristae formation, but only Myo19 and its membrane receptor Miro regulate ERMCS.
E93 controls adult differentiation by repressingbroadinDrosophila
Authors: Cruz Josefa , Ureña Enric , Franch-Marro Xavier , Martín David 
Published: February 2024

ABSTRACTInDrosophila melanogaster, successful development relies on the precise coordination of both spatial and temporal regulatory axes. The temporal axis governs stage-specific identity and developmental transitions through a number of genes, collectively forming the Metamorphic Gene Network. Among these, Ecdysone inducible protein 93F (E93) serves as the critical determinant for adult specification, but its mechanism of action remains unclear. Here, we found that, rather than acting as an instructive signal, E93 promotes adult differentiation through the repression of the pupal specifierbroad (br). In the absence of E93, sustained high levels of Br during the pupal stage strongly represses pupal-specific enhancers that are essential for the terminal differentiation of the wing. We also show that Br represses the pupal-enhancers during the larval and prepupal stages preventing the premature implementation of the adult genetic program, and that it also dampens the activity of larval enhancers during the latter stages of larval development. This mechanism of action seems to be a derived feature acquired in Diptera, as in the coleopteranTribolium castaneum, repression ofbrby E93 is not sufficient to allow adult differentiation. In summary, our study elucidates the crucial role of the intricate interplay between E93 and Br as the governing mechanism in the process of terminal differentiation inDrosophila. This discovery holds significant implications for advancing our understanding of the evolution of insect metamorphosis.
In vitro activity of cefiderocol against carbapenemase-producing and meropenem-nonsusceptible gram-negative bacteria collected in the Japan Antimicrobial Resistant Bacterial Surveillance (JARBS-GNR)
Authors: Kayama Shizuo , Kawakami Sayoko , Kondo Kohei , Kitamura Norikazu , Yu Liansheng , Hayashi Wataru , Yahara Koji , Sugawara Yo , Sugai Motoyuki 
Published: February 2024

AbstractThe treatment options available for infections caused by multidrug-resistant gram-negative pathogens are often limited. Cefiderocol (CFDC) is a novel siderophore cephalosporin that exhibits activity against multidrug-resistant gram-negative pathogens. Several studies have reported the in vitro activity of CFDC using clinical isolates from Europe, the United States, and China. However, no large-scale studies on the in vitro activities of CFDC have been conducted using all isolates with available genomic backgrounds based on whole-genome sequencing (WGS). We evaluated the antimicrobial activities of CFDC, ceftolozane/tazobactam (CTLZ/TAZ), imipenem-relebactam (IPM/REL), and ceftazidime/avibactam (CAZ/AVI) against carbapenemase-producing Enterobacterales, carbapenemase-non-producing meropenem-nonsusceptible Enterobacterales, and carbapenemase-producing non-fermentative bacteria. We selected 603 isolates (528 Enterobacterales, 18Pseudomonas aeruginosa, and 57Acinetobacterspp.) from the recent surveillance of clinical isolates in Japan using WGS data. Among these, 97.7% (300/307 strains) of carbapenemase-producing Enterobacterales, 100% (18/18 strains) of carbapenemase-producingP. aeruginosa, and 91.2% (52/57 strains) of carbapenemase-producingAcinetobacterspp. were susceptible to CFDC, showing better antimicrobial activity than the other antimicrobial agents evaluated in this study. In addition, CFDC was highly effective against class A, B, and D β-lactamase harboring isolates when compared to the other antimicrobial agents in this study. While β-lactam antibiotics were essentially ineffective against CFDC-resistant Enterobacterales, minocycline was the most effective, and gentamicin and amikacin were also effective. This is the first large-scale study to systematically demonstrate the efficacy of CFDC using carbapenemase-producing strains with transparent genomic backgrounds.
The NAPstar family of NADP redox state sensors highlights glutathione as the primary mediator of anti-oxidative electron flux
Authors: Scherschel Marie , Niemeier Jan-Ole , Jacobs Lianne J.H.C. , Hoffmann Markus , Diederich Anika , Bell Christopher , Höhne Pascal , Raetz Sonja , Kroll Johanna B. , Steinbeck Janina , Lichtenauer Sophie , Multhoff Jan , Zimmermann Jannik , Sadhanasatish Tanmay , Rothemann R. Alexander , Grashoff Carsten , Messens Joris , Ampofo Emmanuel , Laschke Matthias , Riemer Jan , Roma Leticia Prates , Schwarzländer Markus , Morgan Bruce 
Published: February 2024

AbstractThe NADPH/NADP+redox couple is central to metabolism and redox signalling. NADP redox state is differentially regulated by distinct enzymatic machineries at the subcellular compartment level. Nonetheless, a detailed understanding of subcellular NADP redox dynamics is limited by the availability of appropriate tools. Here, we introduce NAPstars, a family of genetically encoded, fluorescent protein-based NADP redox state biosensors. NAPstars offer real-time, specific, pH-resistant measurements, across a broad-range of NADP redox states, with subcellular resolution. We establish NAPstar measurements in yeast, plants and mammalian cell models, revealing a conserved robustness of cytosolic NADP redox homeostasis. NAPstars uncovered NADP redox oscillations linked to the cell cycle in yeast and illumination- and hypoxia-dependent NADP redox changes in plant leaves. By selectively impairing the glutathione and thioredoxin anti-oxidative pathways under acute oxidative challenge, NAPstars demonstrated an unexpected role for the glutathione system as the primary mediator of anti-oxidative electron flux that is conserved across eukaryotic kingdoms.
R-Loop Functions inBrca1-Associated Mammary Tumorigenesis
Authors: Chiang Huai-Chin , Qi Leilei , Mitra Payal , Hu Yanfen , Li Rong 
Published: February 2024

AbstractExcessive R-loops, a DNA-RNA hybrid structure, are associated with genome instability andBRCA1mutation-related breast cancer. Yet the causality of R-loops in tumorigenesis remains unclear. Here we show that R-loop removal byRnaseh1overexpression (Rh1-OE) inBrca1-knockout (BKO) mouse mammary epithelium exacerbates DNA replication stress without affecting homology-directed DNA repair. R-loop removal also diminishes luminal progenitors, the cell of origin for estrogen receptor α (ERα)-negative BKO tumors. However, R-loop reduction does not dampen spontaneous BKO tumor incidence. Rather, it gives rise to a significant percentage of ERα-expressing BKO tumors. Thus, R-loops reshape mammary tumor subtype rather than promoting tumorigenesis.
Expanding the molecular grammar of polar residues and arginine in FUS prion-like domain phase separation and aggregation
Authors: Wake Noah , Weng Shuo-Lin , Zheng Tongyin , Wang Szu-Huan , Kirilenko Valentin , Mittal Jeetain , Fawzi Nicolas L 
Published: February 2024

AbstractA molecular grammar governing low-complexity prion-like domains phase separation (PS) has been proposed based on mutagenesis experiments that identified tyrosine and arginine as primary drivers of phase separation via aromatic-aromatic and aromatic-arginine interactions. Here we show that additional residues make direct favorable contacts that contribute to phase separation, highlighting the need to account for these contributions in PS theories and models. We find that tyrosine and arginine make important contacts beyond only tyrosine-tyrosine and tyrosine-arginine, including arginine-arginine contacts. Among polar residues, glutamine in particular contributes to phase separation with sequence/position-specificity, making contacts with both tyrosine and arginine as well as other residues, both before phase separation and in condensed phases. For glycine, its flexibility, not its small solvation volume, favors phase separation by allowing favorable contacts between other residues and inhibits the liquid-to-solid (LST) transition. Polar residue types also make sequence-specific contributions to aggregation that go beyond simple rules, which for serine positions is linked to formation of an amyloid-core structure by the FUS low-complexity domain. Hence, here we propose a revised molecular grammar expanding the role of arginine and polar residues in prion-like domain protein phase separation and aggregation.
Breaking Barriers: pCF10 Type 4 Secretion System relies on a self-regulating muramidase to modulate the cell wall
Authors: Sun Wei-Sheng , Torrens Gabriel , Beek Josy ter , Cava Felipe , Berntsson Ronnie P-A 
Published: February 2024

AbstractConjugative Type 4 Secretion Systems (T4SS) are a main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. To deliver the DNA substrate to recipient cells, it must cross the cell envelopes of both donor and recipient mating bacteria. In the T4SS from the enterococcal conjugative plasmid pCF10, PrgK is known to be the active cell wall degrading enzyme. It has 3 predicted extracellular hydrolase domains, LytM, SLT and CHAP. Here, we report the structure of the LytM domain, and show that its active site is degenerate and lacks the active site metal. Further, we show that only the predicted SLT domain is functional, and that it unexpectedly has a muramidase instead of a lytic transglycosylase activity. While we did not observe any peptidoglycan hydrolytic activity for the LytM or CHAP domain, we found that these domains downregulated the SLT muramidase activity. The CHAP domain was also found to be involved in PrgK dimer formation. Further, we show that PrgK interacts with PrgL, which likely targets PrgK to the rest of the T4SS. The presented data provides important information towards understanding the function of Gram-positive T4SSs.
The co-chaperone DNAJA2 buffers proteasomal degradation of cytosolic proteins with missense mutations
Authors: Baker Heather A , Bernardini Jonathan P , Csizmók Veronika , Madero Angel , Kamat Shriya , Eng Hailey , Lacoste Jessica , Yeung Faith Au , Comyn Sophie , Calabrese Gaetano , Raught Brian , Taipale Mikko , Mayor Thibault 
Published: February 2024

AbstractMutations can result in the loss of a protein’s native function due to protein misfolding, which is generally handled by an intricate protein quality control network. To better understand the triaging mechanisms of misfolded cytosolic proteins, we screened a human mutation library to identify a panel of unstable mutations. The degradation of these mutated cytosolic proteins is largely dependent on the ubiquitin proteasome system. Using BioID proximity labelling, we found that the co-chaperones DNAJA1 and DNAJA2 are key interactors of one of the mutated proteins. Notably, the absence of DNAJA2 increases the turnover of the mutant protein but not of the wild-type protein. Our work indicates that missense mutations in cytosolic proteins can promote interactions with molecular chaperones that normally do not occur. Assessment of the broader panel of cytosolic mutant proteins shows that the co-chaperone DNAJA2 exhibits three distinct behaviours: acting to stabilize solely the mutant, both the wild-type and mutant proteins, or being dispensable. Our work illustrates how distinct elements of the protein homeostasis network are utilized in the presence of a cytosolic misfolded protein.Summary StatementWe identified a panel of cytosolic mutant proteins degraded by the proteasome. DNAJA2 is often required to prevent mutant protein turnover, even if it is sometimes dispensable for the wild-type protein.