bluuebunny/crossref_metadata_2025 huggingface dataset:
Commensal Microbiota Regulate Renal Gene Expression
Authors: Moore Brittni N. , Pluznick Jennifer L.
Published: December 2022

The gut microbiome impacts host gene expression not only in the colon, but also at distal sites including liver, white adipose tissue, and spleen. The gut microbiome also influences the kidney and is associated with renal diseases and pathologies; however, a role for the gut microbiome to modulate renal gene expression has not been examined. To determine if microbes modulate renal gene expression, we used whole-organ RNA sequencing (RNA-Seq) to compare gene expression in C57Bl/6 mice that are germ-free (lacking gut microbiota) versus conventionalized (with gut microbiota). 16S sequencing showed that males and females were similarly conventionalized, although Verrucomicrobia was higher in male mice. We find that renal gene expression is differentially regulated in the presence versus absence of microbiota, and that these changes are largely sex-specific. Although microbes also influence gene expression in the liver and large intestine, most differentially expressed genes (DEGs) in the kidney are not similarly regulated in the liver or large intestine. This demonstrates that the influence of the gut microbiota on gene expression is tissue specific. However, a minority of genes (n=4 in males, n=6 in females) were similarly regulated in all three tissues examined, including genes associated with circadian rhythm (Per1in males andPer2in females) and metal binding (Mt1 and Mt2in both males and females). Finally, using a previously published single cell RNA-Seq (scRNA-Seq) dataset, we assigned a subset of DEGs to specific kidney cell types, revealing clustering of DEGs by cell type and/or sex.

NEW & NOTEWORTHY

It is unknown whether the microbiome influences host gene expression in the kidney. Here, we utilize an unbiased, bulk RNA-Seq approach to compare gene expression in the kidneys of male and female mice with or without gut microbiota, as well as in liver and large intestine. This report demonstrates that renal gene expression is modulated by the microbiome in a sex- and tissue-specific manner.
Taxonomy of interventions at academic institutions to improve research quality
Authors: Davidson Alexandra R , Barbour Ginny , Nakagawa Shinichi , Holcombe Alex O. , Fidler Fiona , Glasziou Paul P
Published: December 2022

Research institutions and researchers have become increasingly concerned about poor research reproducibility and replicability, and research waste more broadly. Research institutions play an important role and understanding their intervention options is important. This review aims to identify and classify possible interventions to improve research quality, reduce waste, and improve reproducibility and replicability within research-performing institutions.

Taxonomy development steps: 1) use of an exemplar paper of journal-level research quality improvement interventions, 2) 2-stage search in PubMed using seed and exemplar articles, and forward and backward citation searching to identify articles evaluating or describing research quality improvement, 3) elicited draft taxonomy feedback from researchers at an open-sciences conference workshop, and 4) cycles of revisions from the research team.

The search identified 11 peer-reviewed articles on relevant interventions. Overall, 93 interventions were identified from peer-review literature and researcher reporting. Interventions covered before, during, and after study conduct research stages and whole of institution. Types of intervention included: Tools, Education & Training, Incentives, Modelling & Mentoring, Review & Feedback, Expert involvement, and Policies & Procedures. Identified areas for research institutions to focus on to improve research quality and for further research includes improving incentives to implement quality research practices, evaluating current interventions, encourage no- or low-cost/high-benefit interventions, examine institution research culture, and encourage mentor-mentee relationships.
Multiomic Profiling Reveals Metabolic Alterations Mediating Aberrant Platelet Activity and Inflammation in Myeloproliferative Neoplasms
Authors: He Fan , Laranjeira Angelo B. A. , Kong Tim , Liu Alice , Ashworth Katrina J. , Lasky Nina M. , Fisher Daniel A. C. , Cox Maggie J. , Fulbright Mary C. , Heck Lilian A. , Yu LaYow , Sykes Stephen M. , D’Alessandro Angelo , Di Paola Jorge , Oh Stephen T.
Published: December 2022

Platelets from patients with myeloproliferative neoplasms (MPNs) exhibit a hyperreactive phenotype. Here, we found elevated P-selectin exposure and platelet-leukocyte aggregates indicating activation of platelets from essential thrombocythemia (ET) patients. Single cell RNA-seq analysis of primary samples revealed significant enrichment of transcripts related to platelet activation, mTOR and oxidative phosphorylation (OXPHOS) in ET patient platelets. These observations were validated via proteomic profiling. Platelet metabolomics revealed distinct metabolic phenotypes consisting of elevated ATP generation, accompanied by increases in the levels of multiple intermediates of the tricarboxylic acid (TCA) cycle, but lower alpha-ketoglutarate (α-KG) in MPN patients. Inhibition of PI3K/AKT/mTOR signaling significantly reduced metabolic responses and hyperreactivity in MPN patient platelets, while α-KG supplementation markedly reduced oxygen consumption and ATP generation.Ex vivoincubation of platelets from both MPN patients andJak2 V617Fmice with α-KG significantly reduced platelet activation responses. Oral α-KG supplementation ofJak2 V617Fmice decreased splenomegaly and reduced hematocrit, monocyte and platelet counts. Finally, α-KG incubation significantly decreased proinflammatory cytokine secretion from MPN CD14+ monocytes. Our results reveal a previously unrecognized metabolic disorder in conjunction with aberrant PI3K/AKT/mTOR signaling, contributing to platelet hyperreactivity in MPN patients.
Lipid remodeling by hypoxia aggravates migratory potential in pancreatic cancer while maintaining membrane homeostasis
Authors: Agarwala Prema Kumari , Nie Shuai , Reid Gavin E. , Kapoor Shobhna
Published: December 2022

Membranes are crucial cell components underlying optimal cellular functioning under diverse conditions including cancer. The membrane physiology requires acute maintenance of biophysical properties and a regulation of cellular lipidome. Homeostatic adaptation of membranes to temperature, pressure and anti-cancer drugs is a well-recognized. However, how the same is regulated under the influence of oxygen deprivation in pancreatic cancers-highly hypoxic cancer- is not known. Here, we report robust lipidomic remodelling in response to HIF-1α induction in pancreatic cancer cells and significant accumulation of lipid droplets. The lipidome rewiring span changes across various lipid classes, levels of unsaturation and acyl chain lengths. Interestingly, despite extensive lipidome alteration, cellular membrane homeostatic response ensures no major modulation of membrane biophysical properties underlying enhanced migratory potential. The correlation of lipidome changes, with pathway analysis and proteomics provide the basis for mutually exclusive regulation of lipidome and membrane properties. These findings help to understand the hypoxic regulation of pancreatic membrane homeostasis.
Maternal Mental Health and Engagement in Developmental Care Activities with Preterm Infants in the NICU
Authors: Dubner Sarah E. , Morales Maya Chan , Marchman Virginia A. , Shaw Richard J. , Travis Katherine E. , Scala Melissa
Published: December 2022

Objective

To examine associations between maternal mental health and involvement in developmental care in the NICU.

Study Design

Mothers of infants born at <32 weeks gestation (n=55) were screened for anxiety, depression, and post traumatic stress disorder two weeks after admission. Mothers’ frequency, rate, and duration of developmental care activities were documented in the electronic health record. Group differences between unscreened mothers, and mothers with and without elevated screener scores and developmental care measures were assessed retrospectively.

Results

35% of screened mothers scored above the cutoff for clinical concern on ≥1 measure. No significant group differences were identified for rates, frequency, or amount of overall developmental care, kangaroo care, or holding.

Conclusion

Maternal mental health ratings did not relate to developmental care. Maternal developmental care engagement may not indicate mental health status. Universal screening for psychological distress is required to accurately detect symptoms in mothers of hospitalized preterm infants.
The Changing Global Epidemiology of Re-emerging Human Monkeypox Virus Infection: A Systematic Review
Authors: Sham Sunder , Sapna FNU , Anjali FNU , Kumar Sanjay , Podder Vivek , Jaladi Soumya , Bendari Ahmed , Al-Refai Reham , Baloch Manal Mohammad , Abdelwahed Mohammed , Kiran FNU , Laharwani Hansini
Published: December 2022

Background

Human monkeypox (MPXV) virus infection, an emerging zoonotic disease caused by an orthopoxvirus, leads to smallpox-like disease. Human MPXV infection was first reported in 1970 in the Democratic Republic of Congo (DRC). Before April 2022, MPXV cases were endemic and seldom reported outside African regions; but recent global outbreaks of MPXV are concerning. We aimed to investigate the epidemiology of emerging human monkeypox virus infection including the number of suspected, confirmed, and fatal cases as well as risk factors for contracting monkeypox infection.

Design

We performed a systematic review of peer-reviewed literature by following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline. An electronic database search (PubMed, online Willey library, science direct) was undertaken. For monkeypox related studies, we included 25 peer-reviewed articles from 2018 and 2022 and data were extracted to inform current evidence on the cases and public health risk factors for developing infection, and public health advice.

Results

Our reports show a rapid rise of MPVX cases in highly endemic African regions after the 1970s, spread to other countries, and increased median age from young children to young adults. Cessation of smallpox vaccination might be one of the factors responsible for the findings. Till 2022 genomic sequences of ten MPXV strains, associated with the recent countrywide outbreak, have been determined. While West African Clade is mostly implicated in the recent viral surge, data were insufficient to determine which mutation contributed to increased transmissibility. In DRC, sleeping on the floor was significantly (odds ratio [OR] 6.1, 95% confidence interval [CI] 1.2-31.6) associated with contracting MPVXwhile eating or processing animal foods was not a significant risk factor. In the United States, cleaning cages, bedding sick animals (OR 5.3, 95% CI 1.4-20.7), or touching infected animals (OR 4.0, 95% CI 1.2-13.4), daily sick animal exposure (OR 4.0, 95% CI 1.2-13.4) were associated with contracting MPVX infection.

Conclusion

Recent global outbreaks, the rising incidence in young adults and endemic zones might result from smallpox vaccine cessation. Increased risk with sick animal exposure or sleeping on the floor suggests high infectivity from animal excretions. Increasing awareness, strict surveillance, and contact tracing can contain global outbreaks. Ring vaccination approach to exposed people can also be a strategy. Future studies should investigate to determine measures for rapid laboratory diagnosis, maintaining lab safety, and also transmissibility.
User and Usability Testing of a web-basedGBAandLRRK2genetics education tool
Authors: Han Noah , Paul Rachel A. , Bardakjian Tanya , Kargilis Daniel , Bradbury Angela R. , Chen-Plotkin Alice , Tropea Thomas F.
Published: December 2022

Background

Genetic testing is essential to identify research participants for clinical trials enrolling people with Parkinson’s disease (PD) carrying a variant in theGBAorLRRK2genes. Limited availability of professionals trained in genetic counseling is a major barrier to increased testing.

Objective

As an alternative to pre-test genetic counseling, we developed a web-based genetics education tool focused onGBAandLRRK2testing for PD called the Interactive Multimedia Approach to Genetic counseling to INform and Educate in PD (IMAGINE-PD) and conducted user testing and usability testing.

Methods

Genetic counselors and PD and neurogenetics subject matter experts developed content for IMAGINE-PD, specifically focused onGBAandLRRK2genetic testing. User testing to review content was conducted with thirteen patients with PD and eleven movement disorder specialists (MDS). Twelve PD patients were enrolled in usability testing of a high-fidelity prototype in accordance with national guidelines. Qualitative data analysis was used in both phases.

Results

Qualitative analysis conducted by three evaluators identified themes emerging from MDS and PD patient feedback in user testing in three content areas: content, function, and appearance. Similarly, qualitative analysis of usability testing feedback identified additional themes in these three content areas. Refinements were made to IMAGINE-PD based on consensus recommendation by evaluators.

Conclusions

User testing for content review and usability testing have informed refinements to IMAGINE-PD to develop this alternative to standard pre-test genetic counseling. Comparison of this stakeholder-informed intervention to standard telegenetic counseling approaches is ongoing.
Molecular determinants of inhibition of UCP1-mediated respiratory uncoupling
Authors: Gagelin Antoine , Largeau Corentin , Masscheleyn Sandrine , Piel Mathilde S. , Calderon-Mora Daniel , Bouillaud Frédéric , Hénin Jérôme , Miroux Bruno
Published: December 2022

Brown adipose tissue expresses uncoupling protein 1 (UCP1), a mitochondrial transporter that uncouples respiration from ATP synthesis and dissipates energy as heat, making it a target for treating obesity and related metabolic disorders. Here, we combine molecular dynamics simulations with mitochondrial respiration assays to investigate how purine nucleotides inhibit respiration uncoupling by UCP1. Simulations predict that GDP binds UCP1 in the common substrate binding site in an upright orientation, where the base moiety interacts with a pair of charged residues (R92/E191) that are specifically conserved in the subfamily of UCPs. E191, among others, interacts with purine but not pyrimidine bases, suggesting a rationale for nucleotide specificity in UCP1 inhibition. We also identify a triplet of uncharged residues involved in hydrophobic contacts with GDP. Site-directed mutagenesis of either I187 or W281 to alanine increases lauric acid-induced uncoupling activity of UCP1 and partially suppresses inhibition of UCP1 activity by GDP in yeast spheroplasts. The triple mutant (F88, I187, W281) to alanine is overactivated by lauric acid even in a high concentration of purine nucleotides. Variants at these positions may help increase energy expenditure in a cellular and therapeutic context.
Novel porcine model reveals two distinct LGR5 cell types during lung development and homeostasis
Authors: Polkoff Kathryn M. , Lampe Ross , Gupta Nithin K. , Murphy Yanet , Chung Jaewook , Carter Amber , Simon Jeremy M. , Gleason Katherine , Moatti Adele , Murthy Preetish K. , Edwards Laura , Greenbaum Alon , Tata Aleksandra , Tata Purushothama Rao , Piedrahita Jorge A.
Published: December 2022

Cells expressing LGR5 play a pivotal role in homeostasis, repair, and regeneration in multiple organs including skin and gastrointestinal tract, yet little is known about their role in the lung. Findings from mice, a widely used animal model, suggest that lung LGR5 expression differs from that of humans. In this work, using a new transgenic pig model, we identify two main populations of LGR5+cells in the lung that are conserved in human, but not mouse lungs. Using RNA sequencing, 3D imaging and organoid models, we determine that in the fetal lung, epithelial LGR5 expression is transient in a subpopulation of SOX9+/ETV+/SFTPC+progenitor lung tip cells. In contrast, epithelial LGR5 expression is absent from postnatal lung, but is reactivated in bronchioalveolar organoids derived from basal airway cells. We also describe a separate population of mesenchymal LGR5+cells that surrounds developing and mature airways, lies adjacent to airway basal cells, and is closely associated with nerve fibers. Transcriptionally, mesenchymal LGR5+cells include a subset of peribronchial fibroblasts (PBF) that express unique patterns ofSHH, FGF, WNTandTGF-β signaling pathway genes. These results support distinct roles for LGR5+cells in the lung and describe a physiologically relevant animal model for further studies on the function of these cells in repair and regeneration.
METTL3 drives telomere targeting of TERRA lncRNA through m6A-dependent R-loop formation: a therapeutic target for ALT-positive neuroblastoma
Authors: Vaid Roshan , Thombare Ketan , Mendez Akram , Burgos-Panadero Rebeca , Djos Anna , Jachimowicz Daniel , Lundberg Kristina Ihrmark , Bartenhagen Christoph , Kumar Navinder , Tümmler Conny , Sihlbom Carina , Fransson Susanne , Johnsen John Inge , Kogner Per , Martinsson Tommy , Fischer Matthias , Mondal Tanmoy
Published: December 2022

Telomerase-negative tumors maintain telomere length by alternative lengthening of telomeres (ALT), but the underlying mechanism behind ALT remains poorly understood. A proportion of aggressive neuroblastoma (NB), particularly relapsed tumors, are positive for ALT (ALT+), suggesting that a better dissection of the ALT mechanism could lead to novel therapeutic opportunities. TERRA, a long non-coding RNA (lncRNA) derived from telomere ends, localizes to telomeres in a R-loop-dependent manner and plays a crucial role in telomere maintenance. Here we present evidence that RNA modification at theN6position of internal adenosine (m6A) in TERRA by the methyltransferase METTL3 is essential for telomere maintenance in ALT+ cells, and the loss of TERRA m6A/METTL3 results in telomere damage. We observed that m6A modification is abundant in R-loop enriched TERRA, and the m6A-mediated recruitment of hnRNPA2B1 to TERRA is critical for R-loop formation. Our findings suggest that m6A drives telomere targeting of TERRA via R-loops, and this m6A-mediated R-loop formation could be a widespread mechanism employed by other chromatin-interacting lncRNAs. Furthermore, treatment of ALT+ NB cells with a METTL3 inhibitor resulted in compromised telomere targeting of TERRA and accumulation of DNA damage at telomeres, indicating that METTL3 inhibition may represent a therapeutic approach for ALT+ NB.
Random genetic drift sets an upper limit on mRNA splicing accuracy in metazoans
Authors: Bénitière Florian , Necsulea Anamaria , Duret Laurent
Published: December 2022

Most eukaryotic genes undergo alternative splicing (AS), but the overall functional significance of this process remains a controversial issue. It has been noticed that the complexity of organisms (assayed by the number of distinct cell types) correlates positively with their genome-wide AS rate. This has been interpreted as evidence that AS plays an important role in adaptive evolution by increasing the functional repertoires of genomes. However, this observation also fits with a totally opposite interpretation: given that ‘complex’ organisms tend to have small effective population sizes (Ne), they are expected to be more affected by genetic drift, and hence more prone to accumulate deleterious mutations that decrease splicing accuracy. Thus, according to this “drift barrier” theory, the elevated AS rate in complex organisms might simply result from a higher splicing error rate. To test this hypothesis, we analyzed 3,496 transcriptome sequencing samples to quantify AS in 53 metazoan species spanning a wide range ofNevalues. Our results show a negative correlation betweenNeproxies and the genome-wide AS rates among species, consistent with the drift barrier hypothesis. This pattern is dominated by low abundance isoforms, which represent the vast majority of the splice variant repertoire. We show that these low abundance isoforms are depleted in functional AS events, and most likely correspond to errors. Conversely, the AS rate of abundant isoforms, which are relatively enriched in functional AS events, tends to be lower in more complex species. All these observations are consistent with the hypothesis that variation in AS rates across metazoans reflects the limits set by drift on the capacity of selection to prevent gene expression errors.
Positions of pivot points in quadrupedal locomotion: limbs and trunk global control in four different dog breeds
Authors: Andrada Emanuel , Hildebrandt Gregor , Witte Hartmut , Fischer Martin S.
Published: December 2022

Dogs (Canis familiaris) prefer the walk at lower speeds and the more economical trot at speeds ranging from 0.5 Fr up to 3 Fr. Important works were carried out to understand these gaits at the levels of center of mass, joint mechanics, and muscular control. However, less is known about the global control goals for limbs and overall locomotion, and of whether these global control goals are gait or breed specific. For walk and trot, we analyzed dog global dynamics based on motion capture and single leg kinetic data recorded from treadmill locomotion of French Bulldog (N = 4), Whippet (N = 5), Malinois (N = 4) and Beagle (N = 5). Dogs displayed two virtual pivot points (VPP) during walk and trot each. One resembles control of both thoracic (fore) limbs and is roughly located above and caudally to the scapular pivot, while the second is located roughly above and cranially to the hip and mirrors the control of the pelvic (hind-) limbs. The positions of VPPs and the patterns of the legs‘ axial and tangential functions were gait and breed related. However, breed related changes were mainly exposed by the French Bulldog. The position of VPPs relative to the proximal pivots explains the propulsive and breaking forces observed in quadrupedal locomotion and may help to reduce limb work. In combination with former work, from the present study the VPP template emerges as the expression of a simple and general global control rule for both bipeds and quadrupeds.
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

A 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

Flavin-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

Candida 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 Statement

Invasive 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

The 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

Toxoplasma 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

Mouse 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

An 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.

SIGNIFICANCE

NRAS-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

Recent 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

Recent 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

Viruses 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

Parkinson’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

The 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

TheE. 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).
Pooled genome-wide CRISPR activation screening for rapamycin resistance genes inDrosophilacells
Authors: Xia Baolong , Viswanatha Raghuvir , Hu Yanhui , Mohr Stephanie E. , Perrimon Norbert
Published: December 2022

Loss-of-function and gain-of-function genetic perturbations provide valuable insights into gene function. InDrosophilacells, while genome-wide loss-of-function screens have been extensively used to reveal mechanisms of a variety of biological processes, approaches for performing genome-wide gain-of-function screens are still lacking. Here, we describe a pooled CRISPR activation (CRISPRa) screening platform inDrosophilacells and apply this method to both focused and genome-wide screens to identify rapamycin resistance genes. The screens identified three genes as novel rapamycin resistance genes: a member of SLC16 family of monocarboxylate transporters (CG8468), a member of the lipocalin protein family (CG5399), and a zinc finger C2H2 transcription factor (CG9932). Mechanistically, we demonstrate thatCG5399overexpression activates the RTK-Akt-mTOR signaling pathway and that activation of InR byCG5399requires cholesterol and clathrin-coated pits at the cell membrane. This study establishes a novel platform for functional genetic studies inDrosophilacells.