bluuebunny/crossref_metadata_2025_split huggingface dataset:
RecA balances genomic stability and evolution using many successive mismatch tolerant homology tests
Authors: Prentiss Mara , Wang John , Fu Jonathan , Prévost Chantal , Godoy-Carter Veronica , Kleckner Nancy , Danilowicz Claudia 
Published: December 2022

AbstractA double-strand break (DSB) must usually be repaired with as little alteration to the genome as possible, though some rare alterations provide valuable genomic evolution. InE.coli, a DSB undergoes resection to give 3’ ssDNA tails. These invading strand tails are loaded with RecA protein and then rapidly search the genome for the corresponding (allelic) partner. Thus, a searching ssDNA/RecA filament must almost never make stable non-allelic contact; therefore, it has been puzzling that RecA forms stable products that join partially homologous sequences. Homology testing by RecA family proteins begins with an 8-bp test, followed by successive homology tests of base pair triplets. Here we introduce a highly simplified homology recognition model to highlight how mismatch sensitivity could affect non-allelic pairing in bacterial genomes. The model predicts that even if each triplet test accepts 2 mismatches, RecA can have ∼ 95% probability of establishing allelic pairing after a DSB inE. coli; however, that accuracy requires homology testing ⪆50 contiguous base pairs, consistent with the homology lengths probedin vivo. In contrast, if no mismatches are accepted testing 14 bp is sufficient, and testing more base pairs does not reduce non-allelic pairing because bacterial genomes contain long repeats.
Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging
Authors: Nikolaev Andrey , Yudenko Anna , Smolentseva Anastasia , Bogorodskiy Andrey , Tsybrov Fedor , Borshchevskiy Valentin , Bukhalovich Siarhei , Nazarenko Vera V. , Kuznetsova Elizaveta , Semenov Oleg , Remeeva Alina , Gushchin Ivan 
Published: December 2022

AbstractFlavin-binding fluorescent proteins (FbFPs) are promising genetically encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide and flavin adenine dinucleotide) are notoriously similar even between different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of twenty-two finely tuned fluorescent tags based on the thermostable LOV domain fromChloroflexus aggregans(CagFbFP). We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512 nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of FbFPs in fluorescence microscopy.Abstract Figure
Fingerprint SRS Imaging Unveils Ergosteryl Ester as a Metabolic Signature of Azole-ResistantCandida albicans
Authors: Zhang Meng , Dong Pu-Ting , Eldesouky Hassan E. , Zhan Yuewei , Lin Haonan , Wang Zian , Salama Ehab A. , Jusurf Sebastian , Zong Cheng , Chen Zhicong , Seleem Mohamed N. , Cheng Ji-Xin 
Published: December 2022

AbstractCandida albicans(C. albicans), a major fungal pathogen, causes life-threatening infections in immunocompromised individuals. Fluconazole (FLC) is recommended as first-line therapy for treatment of invasive fungal infections. Yet, the widespread use of FLC has resulted in increased antifungal resistance among different strains ofCandida, especiallyC. albicans, which is a leading source of hospital-acquired infections. Here, by hyperspectral stimulated Raman scattering (hSRS) imaging of single fungal cells in the fingerprint window and pixel-wise spectral unmixing, we report aberrant ergosteryl ester accumulation in azole-resistantC. albicanscompared to azole-susceptible species. This accumulation was a consequence ofde novolipogenesis. Lipid profiling by mass spectroscopy identified ergosterol oleate to be the major species stored in azole-resistantC. albicans. Blocking ergosterol esterification by oleate and suppressing sterol synthesis by FLC synergistically suppressed the viability ofC. albicans in vitroand limited the growth of biofilm on mouse skinin vivo. Our findings highlight a metabolic marker and a new therapeutic strategy for targeting azole-resistantC. albicansby interrupting the esterified ergosterol biosynthetic pathway.Significance StatementInvasive fungal infections and increasing antifungal resistance are emerging threats to public health with high morbidity and mortality. Despite the advances in azole resistance mechanisms, it remains unclear why some fungal species are intrinsically resistant to or easily acquire resistance to multiple antifungal drugs. Here, using fingerprint SRS microscopy, we uncovered a molecular signature, aberrant ergosteryl ester accumulation, linked to the azole resistance ofCandidaspecies. An antifungal treatment strategy combining oleate (inhibitor of ersgosteryl esterification) and azole significantly attenuates the azole resistance and the viability ofC. albicans in vitroandin vivo. Our work opens a new way to detect and treat azole-resistant fungal infections by targeting ergosterol metabolism.
The neuronal calcium sensor NCS-1 regulates the phosphorylation state and activity of the Gα chaperone and GEF Ric-8A
Authors: Muñoz-Reyes D , McClelland LJ , Arroyo-Urea S , Sánchez-Yepes S , Sabín J , Pérez-Suárez S , Menéndez M , Mansilla A , García-Nafría J , Sprang SR , Sánchez-Barrena MJ 
Published: December 2022

AbstractThe Neuronal Calcium Sensor 1 and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Gα have revealed how Ric-8A phosphorylation promotes Gα recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Gα subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted NCS-1/Ric-8A complexes to conduct a multimodal approach and determine the sequence of Ca2+signals and phosphorylation events that promote the interaction of Ric-8A with Gα. Our data show that the binding of NCS-1 and Gα to Ric-8A are mutually exclusive. Importantly, NCS-1 induces a profound structural rearrangement in Ric-8A that traps the protein in a conformational state that is inaccessible to Casein Kinase II-mediated phosphorylation, demonstrating one aspect of its negative regulation of Ric-8A-mediated G-protein signaling. Functional experiments indicate a loss of Ric-8A GEF activity towards Gα when complexed with NCS-1, and restoration of nucleotide exchange activity upon increasing Ca2+concentration. Finally, the high-resolution crystallographic data reported here that define the NCS-1/Ric-8A interface will allow the development of therapeutic synapse function regulators with improved activity and selectivity.
Structure ofToxoplasma gondiiglideosome-associated connector suggests a role as an elastic element in actomyosin force generation for gliding motility
Authors: Hung Yu-Fu , Chen Qu , Pires Isa , Rosenthal Peter B. , Kursula Inari 
Published: December 2022

AbstractToxoplasma gondiiglideosome-associated connector (GAC) is a giant armadillo-repeat protein, essential for parasite motility and conserved across Apicomplexa. It connects actin filaments to the plasma membraneviainteractions with phosphatidic acid and membrane-spanning adhesins. It is unclear how GAC contributes to gliding motility and invasion and why such a large connector is needed. We determined the crystal structure of full-lengthT. gondiiGAC at 2.3 Å resolution and explored its conformational space in solution using small-angle X-ray scattering and cryogenic electron microscopy. The crystal structure reveals a compact conformation but, in solution, GAC adopts both compact and extended forms. The PH domain stabilizes the compact form and may act as a switch triggered by membrane sensing. Based on its spring-like architecture, we suggest a role for GAC as an elastic element in actomyosin force generation during gliding motility and invasion.
Assessing Mouse Kidney Parvovirus’ Ability to Confound Research by Examining its Effects on Renally Excreted Chemotherapeutics and its Impact on Pathologic Endpoints in the Adenine Model of Chronic Kidney Disease
Authors: Ritter Amanda C , Ricart Arbona Rodolfo J , Livingston Robert S , Monette Sébastien , Lipman Neil S 
Published: December 2022

AbstractMouse kidney parvovirus (MKPV) causes inclusion body nephropathy in severely immunocompromised mice and renal interstitial inflammation in immunocompetent mice. The purpose of this 2-part study was to determine the impact that MKPV may have on preclinical models as it relates to the pharmacokinetics of chemotherapeutics as well as its impact on the adenine diet model of chronic kidney disease. To assess the impact of MKPV on pharmacokinetics of 2 renally excreted chemotherapeutics commonly used in preclinical oncology studies, methotrexate and lenalidomide, blood and urine drug concentrations were measured in MKPV-infected or uninfected immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) and immunocompetent C57BL/6NCrl (B6) female mice. Differences in plasma pharmacokinetics were observed for methotrexate, but not for lenalidomide. Differences were most profound between uninfected NSG and B6 mice. The area under the curve (AUC) of methotrexate was 1.5-fold higher in uninfected NSG mice compared to infected NSG mice, 1.9-fold higher in infected B6 mice compared to uninfected B6 mice, and 4.3-fold higher in uninfected NSG mice compared to uninfected B6 mice. Renal clearance of both drugs was not impacted by MKPV infection but was generally lower in NSG mice. To assess the impact of MKPV on the adenine diet model of chronic kidney disease, MKPV-infected and uninfected B6 female mice were fed a 0.2% adenine diet and clinical and histopathologic features of disease were assessed over 8 weeks. Infection with MKPV did not have a significant impact on serum biomarkers of renal function such as BUN, creatinine, and SDMA; urine chemistry; or hemogram. However, infection did impact select histologic outcomes. MKPV-infected mice had significantly more foci of interstitial lymphoplasmacytic infiltrates than uninfected mice after 4 and 8 weeks of diet consumption, and significantly less interstitial fibrosis at week 8. Macrophage infiltrates and renal tubular injury, assessed using various immunohistochemical stains, were similar between groups. Together, these findings indicate that MKPV infection had minimal impact on the renal excretion of 2 chemotherapeutics and serum biomarkers of renal function. However, infection significantly impacted select histologic features of renal disease in the adenine diet model. While MKPV-free mice should be used in biomedical research, it is of the utmost importance in studies evaluating renal histology as an experimental outcome.
The OSUMMER lines: a series of ultraviolet-accelerated NRAS-mutant mouse melanoma cell lines syngeneic to C57BL/6
Authors: Murphy Brandon M. , Jensen Daelin M. , Arnold Tiffany E. , Aguilar-Valenzuela Renan , Hughes Jase , Posada Valentina , Nguyen Kimberly T. , Chu Vi T. , Tsai Kenneth Y. , Burd Craig J. , Burd Christin E. 
Published: December 2022

ABSTRACTAn increasing number of cancer subtypes are treated with front-line immunotherapy. However, approaches to overcome primary and acquired resistance remain limited. Pre-clinical mouse models are often used to investigate resistance mechanisms, novel drug combinations, and delivery methods; yet most of these models lack the genetic diversity and mutational patterns observed in human tumors. Here we describe a series of thirteen C57BL/6J melanoma cell lines to address this gap in the field. The Ohio State University-Moffitt Melanoma Exposed to Radiation (OSUMMER) cell lines are derived from mice expressing endogenous, melanocyte-specific, and clinically relevantNrasdriver mutations (Q61R, Q61K, or Q61L). Exposure of these animals to a single, non-burning dose of ultraviolet B accelerates the onset of spontaneous melanomas with mutational patterns akin to human disease. Furthermore, in vivo irradiation selects against potent tumor antigens, which could prevent the outgrowth of syngeneic cell transfers. Each OSUMMER cell line possesses distinct in vitro growth properties, trametinib sensitivity, mutational signatures, and predicted antigenicity. Analysis of OSUMMER allografts shows a correlation between strong, predicted antigenicity and poor tumor outgrowth. These data suggest that the OSUMMER lines will be a valuable tool for modeling the heterogeneous responses of human melanomas to targeted and immune-based therapies.SIGNIFICANCENRAS-activating mutations are the second most common genetic driver event in cutaneous melanoma, occurring in 15% to 25% of cases. With few therapeutic options beyond immunotherapy, patients with NRAS-mutant melanoma have a poorer prognosis. Pre-clinical mouse models that mimic the high mutational burden of human NRAS-mutant melanomas are lacking in the field. Here, we describe a series of NRAS-mutant melanoma cell lines, derived from ultraviolet (UV)-induced, spontaneous tumors. These lines permit the study of targeted, NRAS mutant-specific, immune, and combination therapies in C57BL/6J mice. With the release of this resource, we hope to catalyze new therapeutic approaches for NRAS-mutant melanoma.
Vimo: Visual Analysis of Neuronal Connectivity Motifs
Authors: Troidl Jakob , Warchol Simon , Choi Jinhan , Matelsky Jordan , Dhanyasi Nagaraju , Wang Xueying , Wester Brock , Wei Donglai , Lichtman Jeff W. , Pfister Hanspeter , Beyer Johanna 
Published: December 2022

AbstractRecent advances in high-resolution connectomics provide researchers with access to accurate petascale reconstructions of neuronal circuits and brain networks for the first time. Neuroscientists are analyzing these networks to better understand information processing in the brain. In particular, scientists are interested in identifying specific small network motifs, i.e., repeating subgraphs of the larger brain network that are believed to be neuronal building blocks. Although such motifs are typically small (e.g., 2 - 6 neurons), the vast data sizes and intricate data complexity present significant challenges to the search and analysis process. To analyze these motifs, it is crucial to review instances of a motif in the brain network and then map the graph structure to detailed 3D reconstructions of the involved neurons and synapses. We presentVimo, an interactive visual approach to analyze neuronal motifs and motif chains in large brain networks. Experts can sketch network motifs intuitively in a visual interface and specify structural properties of the involved neurons and synapses to query large connectomics datasets. Motif instances (MIs) can be explored in high-resolution 3D renderings. To simplify the analysis of MIs, we designed a continuous focus&context metaphor inspired by visual abstractions. This allows users to transition from a highly-detailed rendering of the anatomical structure to views that emphasize the underlying motif structure and synaptic connectivity. Furthermore,Vimosupports the identification of motif chains where a motif is used repeatedly (e.g., 2 - 4 times) to form a larger network structure. We evaluateVimoin a user study and an in-depth case study with seven domain experts on motifs in a large connectome of the fruit fly, including more than 21,000 neurons and 20 million synapses. We find thatVimoenables hypothesis generation and confirmation through fast analysis iterations and connectivity highlighting.
Anin vivoplatform for rebuilding functional neocortical tissue
Authors: Quezada Alexandra , Ward Claire , Bader Edward R. , Zolotavin Pavlo , Altun Esra , Hong Sarah , Killian Nathan , Xie Chong , Batista-Brito Renata , Hébert Jean M. 
Published: December 2022

AbstractRecent progress in cortical stem cell transplantation has demonstrated its potential to repair the brain. However, current transplant models have yet to demonstrate that the circuitry of transplant-derived neurons can encode useful function to the host. This is likely due to missing cell types within the grafts, abnormal proportions of cell types, abnormal cytoarchitecture, and inefficient vascularization. Here, we devised a transplant platform for testing neocortical tissue prototypes. Dissociated mouse embryonic telencephalic cells in a liquid scaffold were transplanted into aspiration-lesioned adult mouse cortices. The donor neuronal precursors differentiated into upper and deep layer neurons that exhibited synaptic puncta, projected outside of the graft to appropriate brain areas, became electrophysiologically active within one month post-transplant, and responded to visual stimuli. Interneurons and oligodendrocytes were present at normal densities in grafts. Grafts became fully vascularized by 1-week post-transplant and vessels in grafts were perfused with blood. With this paradigm, we could also organize cells into layers. Overall, we have provided proof of concept for anin vivoplatform that can be used for developing and testing neocortical-like tissue prototypes.
Cauliflower mosaic virusdisease spectrum uncovers novel susceptibility factor NCED9 inArabidopsis thaliana
Authors: Hoffmann Gesa , Shukla Aayushi , López-González Silvia , Hafrén Anders 
Published: December 2022

AbstractViruses are intimately linked with their hosts and especially dependent on gene-for-gene interactions to establish successful infections. The genotype of their hosts thus has a strong influence on the outcome virus disease. On the host side, defence mechanisms like tolerance and resistance can occur within the same species leading to differing virus accumulation in relation to symptomology and plant fitness. The identification of novel resistance genes and susceptibility factors against viruses is an important part in understanding viral pathogenesis and securing food production. The model plantArabidopsis thalianadisplays a wide symptom spectrum in response to RNA virus infections and unbiased genome-wide association studies have proven a powerful tool to identify novel disease-genes. In this study we infected natural accessions ofArabidopsis thalianawith the pararetrovirusCauliflower mosaic virusto study the phenotypic variations between accessions and their correlation with virus accumulation. Through genome-wide association mapping of viral accumulation differences, we identified several susceptibility factors for CaMV, the strongest of which was the abscisic acid synthesis geneNCED9. Further experiments confirmed the importance of abscisic acid homeostasis and its disruption for CaMV disease.
Upregulated extracellular matrix-related genes and impaired synaptic activity in dopaminergic and hippocampal neurons derived from Parkinson’s disease patients withPINK1andPARK2mutations
Authors: Tripathi Utkarsh , Rosh Idan , Ezer Ran Ben , Nayak Ritu , Choudhary Ashwani , Djamus Jose , Manole Andreea , Haulden Henry , Gage Fred H. , Stern Shani 
Published: December 2022

AbstractParkinson’s disease (PD) is the second most prevalent neurodegenerative disease. Primary symptoms of PD arise with the loss of dopaminergic (DA) neurons in the Substantia Nigra Pars Compacta, but it affects the hippocampus and cortex also, usually in its later stage. Approximately 15% of PD cases familial with a genetic mutation. Two of the most associated genes with autosomal recessive (AR) early-onset familial PD arePINK1 and PARK2. There is a need for in-vitro studies of these genetic mutations in order to understand the neurophysiological changes in patients’ neurons that may contribute to neurodegeneration. In this work, we generated and differentiated DA and hippocampal neurons from iPSCs derived from two patients with a double mutation in theirPINK1 and PARK2(one homozygous and one heterozygous) genes and assessed their neurophysiology compared to two healthy controls. We showed that the synaptic activity of PD neurons generated from patients with thePINK1 and PARK2mutations is impaired in the hippocampus and dopaminergic neurons. Mutant dopaminergic neurons had enhanced excitatory post-synaptic activity. In addition, DA neurons with the homozygous mutation ofPINK1exhibited more pronounced electrophysiological differences compared to the control neurons. Signaling network analysis of RNA sequencing results revealed that Focal adhesion and ECM receptor pathway were the top 2 upregulated pathways in the mutant PD neurons. These phenotypes are reversed to PD phenotypes of other mutations, suggesting that the interaction of the two mutations may yield different mechanisms of PD.
May the force be with you: the role of hyper-mechanostability of the bone sialoprotein binding protein during early stages of Staphylococci infections
Authors: Gomes Priscila S. F. C. , Forrester Meredith , Pace Margaret , Gomes Diego E. B. , Bernardi Rafael C. 
Published: December 2022

AbstractThe bone sialoprotein-binding protein (Bbp) is a mechanoactive MSCRAMM protein expressed on the surface ofStaphylococcus aureusthat mediates adherence of the bacterium to fibrinogen-α(Fgα), a component of the bone and dentine extracellular matrix of the host cell. Mechanoactive proteins like Bbp have key roles in several physiological and pathological processes. Particularly, the Bbp:Fgαinteraction is important in the formation of biofilms, an important virulence factor of pathogenic bacteria. Here, we investigated the mechanostability of the Bbp:Fgαcomplex usingin silicosingle-molecule force spectroscopy (SMFS), in an approach that combines results from all-atom and coarse-grained steered molecular dynamics (SMD) simulations. Our results show that Bbp is the most mechanostable MSCRAMM investigated thus far, reaching rupture forces beyond the 2 nN range in typical experimental SMFS pulling rates. Our results show that high force-loads, which are common during initial stages of bacterial infection, stabilize the interconnection between the protein’s amino acids, making the protein more “rigid”. Our results offer new insights that are crucial on the development of novel anti-adhesion strategies.
Monomer and dimer structures of cytochromebo3ubiquinol oxidase fromEscherichia coli
Authors: Guo Yirui , Karimullina Elina , Emde Tabitha , Otwinowski Zbyszek , Borek Dominika , Savchenko Alexei 
Published: December 2022

AbstractTheE. colicytochromebo3ubiquinol oxidase is a four-subunit heme-copper oxidase that serves as a proton pump in theE. coliaerobic respiratory chain. Despite many mechanistic studies on this protein, it is unclear whether this ubiquinol oxidase functions as a monomer, or as a dimer in a manner similar to its eukaryotic counterparts – the mitochondrial electron transport complexes. In this study, we determined the monomeric and dimeric structures of theE. colicytochromebo3ubiquinol oxidase reconstituted in amphipol by cryogenic electron microscopy single particle reconstruction (cryo-EM SPR) to a resolution of 3.15 Å and 3.46 Å, respectively. We have discovered that the protein can form a dimer in C2 symmetry, with the dimerization interface maintained by interactions between the subunit II of one monomer and the subunit IV of the other monomer. Moreover, the dimerization does not induce significant structural changes in each monomer, except the movement of a loop in subunit IV (residues 67–74).