bluuebunny/crossref_metadata_2025 huggingface dataset:
Fatty acid-mediated induction of CYP2E1 activity in HepaRG cells is not systematically associated with exacerbated acetaminophen cytotoxicity
Authors: Penhoat Clémence , Massart Julie , Pinon Gregory , Catheline Daniel , Rioux Vincent , Fromenty Bernard , Begriche Karima
Published: November 2024

Hepatic cytochrome P450 2E1 (CYP2E1) is thought to contribute to the pathophysiology of metabolic dysfunction-associated steatotic liver disease (MASLD) formerly known as non-alcoholic fatty liver disease (NAFLD). Indeed, increased activity of CYP2E1 in obese subjects with fatty liver may contribute to higher oxidative stress, mitochondrial dysfunction and the progression to steatohepatitis. Besides, higher CYP2E1 activity in obesity and MASLD is deemed to increase the risk of acetaminophen (APAP)-induced hepatotoxicity because this pain reliever is metabolized by CYP2E1 to N-acetyl-p-benzoquinone imine (NAPQI), a highly toxic metabolite. Although the mechanism of MASLD-associated CYP2E1 induction is still unclear, evidence suggests that hepatic CYP2E1 activity is regulated by fatty acids (FAs). In this study, we investigated the effect of 9 FAs differing by their carbon chain length and their degree of unsaturation on CYP2E1 activity in differentiated HepaRG cells. One-week incubation with palmitic acid (PA), stearic acid (SA) and linoleic acid (LA) induced CYP2E1 activity but only LA exposure induced triglyceride accumulation. APAP hepatotoxicity was then assessed in HepaRG cells cultured with or without PA, SA or LA. Acute APAP cytotoxicity was exacerbated in presence of PA or SA and this was accompanied by more severe mitochondrial dysfunction. These effects were not observed when cells were incubated with LA. Hence, FA-mediated increased CYP2E1 activity in HepaRG cells does not necessarily require steatosis. In addition, FA-mediated CYP2E1 induction does not systematically lead to higher APAP-induced cytotoxicity. By favoring triglyceride accumulation, LA might curb APAP-induced mitochondrial dysfunction and cell death.
A Quantitative Approach for Assessing Multidrug Resistance in Cancer
Authors: Ray Ashley E. , Douglass Eugene F.
Published: November 2024

Multidrug resistance (MDR) is a primary barrier to successful cancer treatment with small molecule drugs. One mechanism of resistance is through transporters which pump drug out of cancer cells before they exert any therapeutic effect. The most studied transporter is P-glycoprotein (Pgp), a member of the ATP Binding Cassette (ABC) superfamily of drug pumps. Prior research has focused on determining whether drugs are substrates of Pgp. Pgp specificity for FDA approved drugs is currently unclear due to technical variability in assays that quantify enzyme kinetics using non-cellular experimental models. Unfortunately, there is extreme variability in enzyme parameters for drugs that are characterized and gaps in literature for drugs that have not been characterized. Thus, our overall goal is to develop live cell methods to obtain quantitative scores for substrates in cells and fill gaps in the literature. Studying Pgp in the context of MDR, we improve Pgp specificity scores by leveraging new Pgp expression (cell lines, tissues) and function (drug screening) datasets. We experimentally and computationally integrate functional dataset information to better understand Pgp specificity using an approach based on underlying Michaelis-Menten enzyme kinetics. We obtain consensus scores for Pgp specificity across ∼150 FDA approved oncology drugs and validate them experimentally in a subset of 76 substrates selected to represent the spectrum of drugs for Pgp specificity. These scores can be used to calibrate clinical diagnostics (Pgp expression), and our experimental platform can be used to quantify Pgp function in clinical samples. Overall, we develop a parallel computational and experimental procedure to estimate Pgp selectivity in live cells. This procedure can be expanded to other drug transporters which contribute to MDR to further characterize this phenotype quantitatively.

Significance Statement

MDR is facilitated through the action of drug transporters which are upregulated in cancer. Even though selective inhibitors have been designed to decrease drug efflux, they failed clinically. So, current clinical practice is to select non-substrates. However, non-substrates are difficult to define. Here, we have developed a quantitative platform for characterizing MDR in cancer that factors in gene expression and enzyme kinetics. Our computational and experimental platform successfully identified the spectrum of substrates for Pgp from the FDA approved oncology drug library. This quantitative approach reflects the multigene phenotype of cancer drug resistance and can be applied to other MDR transporter genes to optimize drug selection clinically.
Staff and Service User Experiences of Specialist Open Prison Personality Disorder Services: How do Pathways Enhanced Resettlement Services Support Progression?
Authors: Mathlin Georgina , Jones Hannah , Lewis Carine , Cooper Claudia , Freestone Mark
Published: November 2024

Background

The Offender Personality Disorder (OPD) Pathway, a joint health and criminal justice initiative across England and Wales, aims to support rehabilitation of individuals with a likely diagnosis of personality disorder. Pathways Enhanced Resettlement Services (PERS) is an OPD service currently operating in five open prisons in England, which aims to support people at high risk of being returned to closed conditions or reoffending in the community after release. We aimed to understand service user and staff experiences of PERS.

Methods

We conducted semi-structured interviews with ten staff and nine service users. We then conducted a reflexive thematic analysis generating three themes.

Results

The three themes identified were: (1) “A shock to the system”, describing the challenges for service users posed by the liminal space of open prison (between higher security conditions and the community); and how in this context PERS might be viewed with suspicion but was for interviewees ultimately a space where they felt valued. (2) “We’ve got some understanding of their journey”; staff and service users described PERS staff developing more trusting relationships with service users than non-PERS staff, where service users felt understood and supported, practically and emotionally; and (3) “internal states can be real barriers to progression”; PERS staff supported service users to understand and overcome barriers, through enabling self-reflection, and tailoring support to times of greater stress, including key milestones such as parole boards or periods of trial leave.

Conclusions

Staff and service users feel PERS provides support to progress through open prison, through development of positive trusting relationships and individualised support in a challenging context where such support was not otherwise available.
Neural markers of abstinence in alcohol dependence: Insights from Reward Learning
Authors: Komarnyckyj Mica , Retzler Chris , Murphy Anna , Delis Ioannis , Fouragnan Elsa
Published: November 2024

Maladaptive reward learning and decision-making circuity are key factors in the onset and progression of alcohol use disorder and have therefore emerged as key targets for neuropsychological and pharmacological interventions. Probabilistic reversal learning studies have consistently reported impaired learning in recently detoxified alcohol dependent (AD) participants. However, the neural and behavioural changes associated with reward learning which occur throughout abstinence remain unexplored. Here, we show that AD participants, with mean abstinence of 20 months, exhibit intact behavioural performance within an electroencephalography (EEG) probabilistic reversal learning task. Reinforcement learning modelling reveals reward and punishment related learning rates and exploration rates are comparable between AD and healthy control (HC) participants, suggesting recovery of even the nuanced aspects of learning in longer term abstinence. However, EEG analysis indicates that AD, compared to HC participants, show globally elevated event-related potential (ERP) feedback related negativity (FRN) following reward valuation. Furthermore, Feedback-P3 valence prediction error signal is negatively associated with abstinence duration indicating a potential state marker of AD recovery. We then employ unsupervised machine learning (canonical polyadic tensor decomposition) to identify spatiotemporal EEG patterns of reward valuation in a purely data-driven manner. Classification analysis shows these tensor components can predict group membership with 80.4% accuracy. By probing group differences in tensor components, we discover early hyperfunctioning in centro-frontal regions linked to alcohol dependence and associated with early abstinence. The clinically meaningful EEG biomarkers presented here could guide the development of more targeted treatments and support big data approaches to objective patient monitoring.
Genetic regulation ofTERTsplicing contributes to reduced or elevated cancer risk by altering cellular longevity and replicative potential
Authors: Florez-Vargas Oscar , Ho Michelle , Hogshead Maxwell , Lee Chia-Han , Papenberg Brenen W , Forsythe Kaitlin , Jones Kristine , Luo Wen , Teshome Kedest , Blauwendraat Cornelis , Billingsley Kimberly J , Kolmogorov Mikhail , Meredith Melissa , Paten Benedict , Chari Raj , Zhang Chi , Schneekloth John S. , Machiela Mitchell J , Chanock Stephen J , Gadalla Shahinaz , Savage Sharon A , Mbulaiteye Sam M , Prokunina-Olsson Ludmila
Published: November 2024

The chromosome 5p15.33 region, which encodes telomerase reverse transcriptase (TERT), harbors multiple germline variants identified by genome-wide association studies (GWAS) as risk for some cancers but protective for others. We characterized a variable number tandem repeat withinTERTintron 6 (VNTR6-1, 38-bp repeat unit) and observed a strong association between VNTR6-1 alleles (Short: 24-27 repeats, Long: 40.5-66.5 repeats) and GWAS signals withinTERTintron 4. Specifically, VNTR6-1 fully explained the GWAS signals for rs2242652 and partially for rs10069690. VNTR6-1, rs10069690 and their haplotypes were associated with multi-cancer risk and age-related telomere shortening. Both variants reduceTERTexpression through alternative splicing and nonsense-mediated decay: rs10069690-T increases intron 4 retention and VNTR6-1-Long expands a polymorphic G quadruplex (G4, 35-113 copies) within intron 6. Treatment with G4-stabilizing ligands decreased the fraction of the functional telomerase-encodingTERTfull-length isoform, whereas CRISPR/Cas9 deletion of VNTR6-1 increased this fraction and apoptosis while reducing cell proliferation. Thus, VNTR6-1 and rs10069690 regulate the expression and splicing ofTERTtranscripts encoding both functional and nonfunctional telomerase. Altered TERT isoform ratios might modulate cellular longevity and replicative potential at homeostasis and in response to environmental factors, thus selectively contributing to the reduced or elevated cancer risk conferred by this locus.
Annotating CryoET Volumes: A Machine Learning Challenge
Authors: Peck Ariana , Yu Yue , Schwartz Jonathan , Cheng Anchi , Ermel Utz Heinrich , Kandel Saugat , Kimanius Dari , Montabana Elizabeth , Serwas Daniel , Siems Hannah , Wang Feng , Zhao Zhuowen , Zheng Shawn , Haury Matthias , Agard David , Potter Clinton , Carragher Bridget , Harrington Kyle , Paraan Mohammadreza
Published: November 2024

Cryo-electron tomography (cryoET) has emerged as a powerful structural biology tool for understanding protein complexes in their native cellular environments. Presently, 3D volumes of cellular environments can be acquired in the thousands in a few days where each volume provides a rich and complex cellular landscape. Despite numerous innovations, localizing and identifying the vast majority of protein species in these volumes remains prohibitively difficult. Machine learning based methods provide an opportunity to automate the process of labeling and annotating cryoET volumes. Due to current bottlenecks in the annotation process, and a lack of large standardized datasets, training datasets for machine learning algorithms have been scarce. Here, we present a defined “phantom” sample, along with “ground truth” annotations, that will be the basis of a machine learning challenge to bring cryoET and ML experts together and spur creativity to address this annotation problem. We have also set up a cryoET data portal that provides additional diverse sets of annotated 3D volumes from cryoET experts across the world for the machine learning challenge.
Neutralizing antibody evasion of SARS-CoV-2 JN.1 derivatives KP.3, KP.3.1.1, LB.1, and XEC
Authors: Sano Kaori , Miyakawa Kei , Kato Hideaki , Kimura Yayoi , Goto Atsushi , Ryo Akihide , Watanabe Shinji , Hasegawa Hideki
Published: November 2024

The emergence of SARS-CoV-2 variants poses ongoing challenges to vaccine efficacy. We evaluated neutralizing antibody responses against JN.1 and its derivatives (KP.3, KP.3.1.1, LB.1, and XEC) in healthcare workers who received seven doses of BNT162b2, including XBB.1.5 monovalent vaccine. In COVID-19-naïve individuals, KP.3.1.1 and LB.1 showed substantial immune escape, while previously infected individuals maintained neutralization activity against all variants. We also demonstrated that JN.1-based immunization induces robust cross-neutralizing activity against emerging variants. A single amino acid deletion at position 31 in the spike protein significantly impacted immune evasion. These findings support the potential effectiveness of JN.1-based vaccines while highlighting the need for continued surveillance and vaccine optimization.
Restoring and sustaining human postmortem retinal light responses with scalable methods for testing degenerative disease therapies
Authors: Becker Silke , Allen Jordan , Morison Zia L’Ecuyer , Saeid Sama , Koskelainen Ari , Vinberg Frans
Published: November 2024

Neuro- and retinal degenerative diseases rob millions of aging individuals of their independence. Researching these diseases in human tissue has been hindered by the immediate loss of electric activity in neurons after the circulation ceases. Recent studies indicate that limited neuronal activity can be revived postmortem, even in the retina. We capitalized on this discovery by successfully restoring and maintainingin vivo-like light responses in eyes recovered up to four hours and stored for up to 48 hours postmortem. This breakthrough significantly increases the availability of functionally viable human retinas for research. Our AI-based postmortem retinal imaging platform identifies retinal structures and allows us to compare light responses in the healthy central and peripheral retina withex vivoelectroretinography. We use this platform to measure the dark adaptation of human macular cones from controls and donors with age-related macular degeneration for the first time in the absence of the retinal pigment epithelium. We developed increased throughput technology and a model to simulate disease-associated acute ischemia-reperfusion. In this model, we demonstrate the protective or toxic effects of several drugs targeting oxidative stress or glutamate excitotoxicity.
Successful classification of clinical pediatric leukemia genetic subtypes via structural variant detection using HiFi long-read sequencing
Authors: Lansdon Lisa A. , Yoo Byunggil , Keskus Ayse , Pushel Irina , Bi Chengpeng , Ahmad Tanveer , Bryant Asher , Walter Adam , Gibson Margaret , Rindler Mary , Li Weijie , Habeebu Sultan M. , Cooley Linda D. , Herriges John , Repnikova Elena , Zhang Lei , August Keith J. , Flatt Terrie G. , Gamis Alan S. , Guest Erin M. , Hays J. Allyson , Hetherington Maxine , Lewing Karen , Pastinen Tomi , Kolmogorov Mikhail , Farooqi Midhat S.
Published: November 2024

Gene fusions are common primary drivers of pediatric leukemias and are the result of underlying structural variant (SVs). Current clinical workflows to detect such alterations rely on a multimodal approach, which often increases analysis time and overall cost of testing. In this study, we used long-read sequencing (lrSeq) as a proof-of-concept to determine whether clinically relevant (cr) SVs could be detected within a small (n = 17) pediatric leukemia cohort. We show that this methodology successfully determined all known crSVs detected through routine clinical testing. We also identified crSVs, such as an ins(11;10)(q23.3;p12p12) forming a KMT2A::MLLT10 fusion, missed by routine clinical approaches, resulting in the classification of leukemia genetic subtypes for four additional patients. This study demonstrates the diagnostic potential of lrSeq as an assay for SV detection in pediatric leukemia and supports lrSeq as a valuable tool for the accurate detection of crSVs.
MPSE provides fast, flexible, and efficient means to identify newborns who will benefit from whole genome sequencing within the first 48 hours of NICU admission
Authors: Peterson Bennet , Juarez Edwin F. , Moore Barry , Hernandez Edgar Javier , Frise Erwin , Li Jianrong , Lussier Yves , Tristani-Firouzi Martin , Reese Martin G. , Jenkins Sabrina Malone , Kingsmore Stephen F. , Bainbridge Matthew N. , Yandell Mark
Published: November 2024

Background

Identifying patients who would benefit from whole genome sequencing (WGS) is difficult and time-consuming due to complex eligibility criteria, lack of neonatologist familiarity with WGS ordering, and evolving clinical features. In previous work, we showed that MPSE, the Mendelian Phenotype Search Engine, can provide automated prioritization of probands for WGS while maintaining current diagnostic rates. MPSE is now in use in multiple hospital networks, but questions still surround how to best prioritize patients for WGS.

Methods

Here we use the clinical histories of 2,885 neonatal intensive care unit (NICU) admits from two institutions to explore further questions regarding how to best prioritize NICU admits for WGS. First, we ask if changes to the machine learning (ML) classifier and the clinical natural language processing (CNLP) tools used for generating patient phenotype descriptions might improve MPSE’s performance. Second, we explore the utility of using alternative data types as inputs to MPSE. Lastly, we conduct a longitudinal analysis of MPSE’s ability to identify probands for WGS.

Results

Eight different ML classifiers, five CNLP tools, and four previously untested alternative data types were used to train and validate MPSE models. MPSE achieved high predictive performance across multiple classifiers (max AUC=0.93), CNLP tools (max AUC=0.91), and input data types (max AUC=0.91). Longitudinal analysis of MPSE scores revealed a significant separation between cases/controls and diagnostic/non-diagnostic cases within 48 hours of NICU admission.

Conclusions

MPSE provides a highly flexible and portable framework for automated prioritization of critically ill newborns for WGS. We find that MPSE’s performance is largely agnostic with respect to CNLP tools. Moreover, structured data such as ICD codes can serve as an effective alternative input to MPSE when access to clinical notes or CNLP pipelines is problematic. Finally, MPSE can identify children most likely to benefit from WGS within 48 hours of admission to the NICU, a critical window for maximally impactful care.
Ebola Disease: Knowledge, attitude, and practices among medical students at a tertiary institution in Uganda
Authors: Gakwerere Daniel , Biryabarema Emmanuel , Namubiru Melanie , Namuyomba Marjorine , Atuhaire Bridget , Musubika Carol , Mukunya David
Published: November 2024

Background

Ebolavirus Disease (EVD) has been a public health threat since its discovery in 1976 with occasional outbreaks on the African continent. This study sought to assess the knowledge, attitudes and practices (KAP) among medical students at a tertiary university in Uganda.

Methods

We conducted a descriptive cross-sectional study using WhatsApp Messenger among students in their clinical years. A pre-validated questionnaire was adapted and modified to assess KAP towards EVD. All analyses were performed using Statistical Package for Social Sciences (SPSS) version 29.0. Bloom’s cutoff of 80% was used to assess sufficient knowledge (≥80%), good attitude (≥6.4) and good practices (≥4.8).

Results

A total of 339 participated, majority were males (n=210, 61.9%) with mean age of 24.2 years (Standard Deviation: 4.0). 77.9% (n=264) were pursuing Bachelor of Medicine and Bachelor of Surgery. Overall, 13.6% (n=46) had sufficient knowledge, 10.9% (n=37) had good attitude and 32.7% (n=111) had good practices. 54.9% (n=186) always avoided patients with signs and symptoms suggestive of EVD while 11.8% (n=40) would accept an approved ebolavirus vaccine.

Conclusion

These results revealed suboptimal EVD-related KAP among medical students. We recommend training of students on clinical presentation, transmission, treatment and prevention of EVD to effectively control future outbreaks.
Anatomical insights beyond the center edge angle in Borderline Hip Dysplasia: A Computerized tomography study
Authors: Lara Joaquín , Neira Alejandro , del Río Javier , Tomic Alexander , Garín Alan , García Nicolás , Roby Matías , De la Fuente Carlos
Published: November 2024

Background

Borderline hip dysplasia (BhD) might be associated with insufficient acetabular coverage. Thus, we investigated potential differences in acetabular anatomical measurements derived from computerized tomography (CT) that characterize BhD compared with healthy controls.

Methods

BhD patients (LCEA between 18º and 25º) and healthy controls (LCEA between 25º and 40º) underwent anteroposterior pelvic X-ray and CT to study Wiberg and Tönnis angle, extrusion and Fear index, notch width and depth, anterior and posterior wall height, anterior and posterior articular surfaces, articular circumference, the ratio between the anterior articular surface and the articular circumference, the ratio between the posterior articular surface and the articular circumference, and the ratio between the notch width and the articular circumference. An independent two-tailed t-test, U of Mann-Whitney test, and odds ratios were obtained (α = 5%).

Results

Twenty-three BhD patients aged 31.5 ± 8.3 years and LCEA 21.6° ± 4.0° and thirty-one healthy controls aged 34.1 ± 8.0 years and LCEA 33.7° ± 5.5° were included. The sensitive CT features for BhD were anterior (p< 0.001) acetabular surface, anterior (p= 0.009), anteroposterior (p= 0.008) and posterior (p< 0.008) acetabular surface ratios, and acetabular notch width (p= 0.002).

Conclusions

BhD CT characterization provides tridimensional anatomical insights beyond the LCEA. BhD insufficient acetabular coverage can be found along the superior (lower Wiberg angle, and increased Tönnis angle and extrusion index), anterior (lower anterior acetabular surface and anteroposterior acetabular surface ratio, and increased posterior acetabular ratio), and inferior (increased acetabular notch width) axis.
Machine Learning-Driven Identification of Molecular Subgroups in Medulloblastoma via Gene Expression Profiling
Authors: Hourfar Hamdam , Taklifi Pegah , Razavi Mahsa , Khorsand Babak
Published: November 2024

Background

Medulloblastoma (MB) is the most prevalent malignant brain tumor in children, characterized by substantial molecular heterogeneity across its subgroups. Accurate classification is pivotal for personalized treatment strategies and prognostic assessments.

Procedure

This study utilized machine learning (ML) techniques to analyze RNA sequencing data from 70 pediatric medulloblastoma samples. Five classifiers—K-nearest Neighbors (KNN), Decision Tree (DT), Support Vector Machine (SVM), Random Forest (RF), and Naive Bayes (NB)—were employed to predict molecular subgroups based on gene expression profiles. Feature selection identified gene subsets of varying sizes (750, 75, and 25 genes) to optimize classification accuracy.

Results

Initial analyses with the complete gene set lacked discriminative power. However, reduced feature sets significantly enhanced clustering and classification performance, particularly for Group 3 and Group 4 subgroups. The RF, KNN, and SVM classifiers consistently outperformed the DT and NB classifiers, achieving classification accuracies exceeding 90% in many scenarios, especially in Group 3 and Group 4.

Conclusions

This study highlights the efficacy of ML algorithms in classifying medulloblastoma subgroups using gene expression data. The integration of feature selection techniques substantially improves model performance, paving the way for enhanced personalized approaches in medulloblastoma management.
Efficient laminar-distributed interactions and orientation selectivity in the mouse V1 cortical column
Authors: Zou Licheng , Moreni Giulia , Pennartz Cyriel M. A. , Mejias Jorge F.
Published: November 2024

The emergence of orientation selectivity in the visual cortex is a well-known phenomenon in neuroscience, but the details of such emergence and the role of different cortical layers and cell types, particularly in rodents which lack a topographical organization of orientation-selectivity (OS) properties, are less clear. To tackle this question, we use an existing biologically detailed model of the mouse V1 cortical column, which is constrained by existing connectivity data across cortical layers and between pyramidal, PV, SST and VIP cell types. Using this model as a basis, we implemented activity-dependent structural plasticity induced by stimulation with orientated drifting gratings, leading to a good match of tuning properties of pyramidal cells with experimentally observed OS laminar distribution, their evoked firing rate and tuning width. We then employed a mean-field model to uncover the role of co-tuned subnetworks in laminar signal propagation and explain the effects of intra- and inter-laminar coupling distributions. Our plasticity-induced modified model and mean-field model were able to explain both the excitatory enhancement through co-tuned subnetworks and inter-laminar disynaptic inhibition. Overall, our work highlights the importance of the clustering of neural selectivity features for effective excitatory transmission in cortical circuits.
Repeat expansion in a Fragile X model is independent of double strand break repair mediated by Pol θ, Rad52, Rad54l or Rad54b
Authors: Hayward Bruce E , Kim Geum-Yi , Miller Carson J , McCann Cai , Lowery Megan G. , Wood Richard D. , Usdin Karen
Published: November 2024

Microsatellite instability is responsible for the human Repeat Expansion Disorders. The mutation responsible differs from classical cancer-associated microsatellite instability (MSI) in that it requires the mismatch repair proteins that normally protect against MSI. LIG4, an enzyme essential for non-homologous end-joining (NHEJ), the major pathway for double-strand break repair (DSBR) in mammalian cells, protects against expansion in mouse models. Thus, NHEJ may compete with the expansion pathway for access to a common intermediate. This raises the possibility that expansion involves an NHEJ-independent form of DSBR. Pol θ, a polymerase involved in the theta-mediated end joining (TMEJ) DSBR pathway, has been proposed to play a role in repeat expansion. Here we examine the effect of the loss of Pol θ on expansion in FXD mouse embryonic stem cells (mESCs), along with the effects of mutations inRad52,Rad54landRad54b,genes important for multiple DSBR pathways. None of these mutations significantly affected repeat expansion. These observations put major constraints on what pathways are likely to drive expansion. Together with our previous demonstration of the protective effect of nucleases like EXO1 and FAN1, and the importance of Pol β, they suggest a plausible model for late steps in the expansion process.
Improved split prime editors enable efficient in vivo genome editing
Authors: Wei Rongwei , Yu Zhenxing , Ding Lihong , Lu Zhike , Yao Keyi , Zhang Heng , Huang Binglin , He Miao , Ma Lijia
Published: November 2024

Prime editor (PE) is a precise genome-editing tool that enables all 12 possible base-to-base conversions, as well as insertions and deletions, that does not require DSBs or donor DNA. The efficient delivery of prime editors in vivo is critical for realizing the full potential of prime editing in disease modeling and therapeutic correction. Although PE has been divided into two halves and delivered using dual adeno-associated viruses (AAVs), editing efficiency at different gene loci varies among split sites, and efficient split sites within Cas9 nickase are limited. In this study, by screening multiple split sites, we demonstrated that 1115 (Asn) is an efficient split site when delivering PE by dual-AAV. Besides, we utilized a feature reported by others recently that RNase could be detached from the Cas9n and designed split sites in the first half of Cas9n. We found that split-PE-367 enabled high prime editing efficiency with Rma intein. To test the editing efficiency in vivo, dual-AAV split-ePE3-367 was packaged in AAV9 and delivered by tail vein injection in mice, achieving 24.4% precise genome editing 4 weeks after injection. Our findings establish an alternative split-PE architecture that could achieve robust gene editing efficiency, facilitating the potential utility both in model organisms and as a therapeutic modality.
Pseudomonas aeruginosaessential gene perturbations that confer vulnerability to the mammalian host environment
Authors: Prasad Neha K. , Ward Ryan D. , Yu Michelle A. , Kwon Michael S. , Banta Amy B. , Rosenberg Oren S. , Peters Jason M.
Published: November 2024

Multidrug-resistantPseudomonas aeruginosacauses highly morbid infections that are challenging to treat. While antibiotics reduce bacterial populations during infection, the host environment also plays a key role in inhibiting and eliminating pathogens. Identifying genetic targets that create vulnerabilities to the host environment may uncover strategies to synergize with nutrient limitation or inherent immune processes to clear bacterial infections. Here, we screened a partial knockdown library targetingP. aeruginosaessential and conditionally essential genes in a murine pneumonia model to identify genes with increased vulnerability in the host environment. We found that partial CRISPR interference (CRISPRi) knockdown of 178 genes showed significant fitness defects in mice relative to axenic culture. We validated two important outliers:ispD, encoding a key enzyme in isoprenoid precursor biosynthesis, andpgsA, encoding an enzyme involved in phospholipid synthesis that is strongly upregulated in human infections. Partial knockdown of both genes showed decreased virulence in a mouse survival assay but had little impact onin vitrogrowth. The use of CRISPRi screening to uncover genetic vulnerabilities represents a promising strategy to prioritize antibacterial targets that interact with the host environment.
Automatic surveillance ofEscherichia colibacteriological strains within clinical settings
Authors: Rodríguez Palomo Rafael , Guerrero-López Alejandro , Sevilla-Salcedo Carlos , Rodríguez-Temporal David , Rodríguez-Sánchez Belén , Gómez-Verdejo Vanessa
Published: November 2024

Healthcare-associated infections (HAIs) are a significant concern within hospital environments, with the World Health Organization (WHO) identifying them as a major source of bacteriological infections. HAIs affect millions of patients annually, leading to substantial morbidity and mortality. However, a significant proportion of HAIs are preventable through early detection and appropriate intervention and isolation. Traditional methods for identifying bacterial species and strains, such as antigen tests, are often time-consuming and hamper the realtime tracking of outbreaks. The matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technique has emerged as a more rapid and precise alternative, though it remains limited by manual identification processes and database constraints. Recent advancements have demonstrated the potential of combining MALDI-TOF MS data with machine learning (ML) to enhance in silico identification speed. In this paper we propose the first method for unsupervisedEscherichia colinovel strain detection through the application of a efficient PIKE tailored to strain identification, spectral clustering techniques and a one-class support vector machine (OCSVM) novelty detection model, presenting promising results.
Low dose of a non-urea selective GIRK channel activator improves hippocampal-dependent synaptic plasticity and memory disrupted by amyloid-β oligomers
Authors: Mulero-Franco Jaime , Jimenez-Herrera Raquel , Contreras Ana , Djebari Souhail , Jiménez-Díaz Lydia , Navarro-López Juan D.
Published: November 2024

Increased neural activity characterizes early Alzheimer’s disease (AD), serving as a prognostic indicator for disease progression and cognitive decline. Mechanisms that drive this hyperactivity and their behavioral effects remain mostly unrevealed, although normalizing altered excitability levels has been shown to reverse cognitive impairment in early AD, both in animals and humans. Soluble amyloid-βoligomers (oAβ)primary accumulate in limbic regions like hippocampus and induce neuronal hyperexcitability and subsequent cognitive deficits by impairing ion channel’s function. Indeed, G protein-gated inwardly rectifying K+ (GIRK) channels -that control neuronal excitability-are greatly affected and their selective pharmacological activation has already been shown very effective to counteract oAβ-induced hyperexcitability and hippocampal dysfunction. However, GIRK gain-of-function in healthy animals disrupts learning, memory and underlying synaptic plasticity, greatly limiting its therapeutic potential in preclinical asymptomatic early AD patients. Therefore, GIRK-based pharmacological treatment needs further investigation to overcome these limitations. Here we tested two doses of a novel, more potent, and neuronal selective GIRK activator, VU0810464, in healthy and early oAβ1-42-generated AD male and female mice. Both doses normalized hippocampal synaptic plasticity (long-term potentiation, LTP) and associated spatial object location memory (OLM) without sex dimorphism in AD animals. However, in healthy mice, low VU0810464 dose did not significantly alter LTP and OLM, whereas the high dose disrupted both. Our results suggest that the precise tuning of neural excitability with low dosing of VU0810464 might be a promising strategy to safely treat and prevent hippocampal overexcitation and upstreaming memory deficits in early preclinical asymptomatic phases of AD.
Effects of HA1 (a Probucol Analogue) and ApoC3 siRNA on Lipoprotein-Amyloid Metabolism, Neurovascular Integrity and Cognitive Function in db/db Mice
Authors: Sharif Arazu , Mamo John , Lam Virginie , Watts Gerald F , Luna Giuseppe , Nesbit Michael , Al-Salami Hani , Mooranian Armin , Takechi Ryu
Published: November 2024

Background and aims

Chronically elevated levels of circulating lipoprotein-amyloid-β (Aβ) are implicated in the disruption of the blood-brain barrier and the initiation of a neurodegenerative cascade leading to Alzheimer’s disease (AD). Type 2 diabetes is associated with BBB dysfunction, dyslipidaemia, and an increased risk of AD. However, alterations in triglyceride-rich lipoproteins (TRL)-Aβ homeostasis and its downstream effects on the BBB in a diabetes context remain explored. This study aimed to 1) investigate, in a preclinical model of diabetes, the hypothesis that diabetes-induced impairments in TRL-Aβ metabolism might compromise BBB integrity and exacerbate cognitive function and behavioural changes, and 2) assess the efficacy of interventions that improve TRL catabolism, including probucol, HA1, and ApoC3 siRNA, to prevent disease progression by lowering circulating TRL-Aβ levels.

Methods

Five-week-old db/db mice underwent a 9- or 23-weeks dietary interventions with probucol, a probucol prodrug HA1, or a standard diet with four-weekly injections of ApoC3 siRNA. Db/+ mice served as negative controls for each treatment duration. Blood levels of Aβ and ApoB were measured using ELISA. Immunofluorescence imaging was used to quantify enterocytic levels of Aβ and ApoB, and assess changes in neurovascular integrity (IgG, PDGFRβ, ZO1, occludin), neuroinflammation (GFAP, Iba1), and cerebral oxidative stress (8OHdG).

Results

Our results indicate that diabetes increased the abundance of plasma amyloid, specifically Aβ42, which correlated with enterocytic abundance, suggesting exaggerated postprandial excretion. Disrupted plasma amyloid homeostasis was associated with BBB breakdown, including diminished barrier function, and the loss of pericytes and astrocytes. Provision of the probucol analogue, HA1, normalised plasma and enterocytic amyloidemia concomitant with the preservation of the neurovascular junction. Treatment with ApoC3 siRNA attenuated plasma Aβ42 and modestly reduced neurovascular inflammation.

Conclusion

The findings further support the hypothesis that aberrant peripheral metabolism of lipoprotein-Aβ is associated with microvascular corruption and the development of anxiety-like behaviour. HA1 is more effective than probucol or ApoC3 siRNA in positively modulating lipoprotein-amyloid homeostasis in db/db mice and maintaining central capillary integrity.

Highlights

Enterocytic and plasma lipoprotein-Aβ levels are increased in diabetic db/db mice.

Elevated plasma lipoprotein-Aβ levels correlate with BBB breakdown and anxiety-like behavior.

HA1 lowers enterocytic and plasma Aβ levels, protects BBB, alleviates oxidative stress and anxiety.

ApoC3 siRNA lowers plasma lipoprotein-Aβ, mitigates neuroinflammation and anxiety.
A cross-species analysis of neuroanatomical covariance sex difference in humans and mice
Authors: Pham Linh , Guma Elisa , Ellegood Jacob , Lerch Jason P. , Raznahan Armin
Published: November 2024

Structural covariance in brain anatomy is thought to reflect inter-regional sharing of developmental influences - although this hypothesis has proved hard to causally test. Here, we use neuroimaging in humans and mice to study sex-differences in anatomical covariance - asking if regions that have developed shared sex differences in volume across species also show shared sex difference in volume covariance. This study design illuminates both the biology of sex-differences and theoretical models for anatomical covariance – benefitting from tests of inter-species convergence. We find that volumetric structural covariance is stronger in adult females compared to adult males for both wild-type mice and healthy human subjects: 98% of all comparisons with statistically significant covariance sex differences in mice are female-biased, while 76% of all such comparisons are female-biased in humans (q < 0.05). In both species, a region’s covariance and volumetric sex-biases have weak inverse relationships to each other: volumetrically male-biased regions contain more female-biased covariations, while volumetrically female-biased regions have more male-biased covariations (mice: r = −0.185, p = 0.002; humans: r = −0.189, p = 0.001). Our results identify a conserved tendency for females to show stronger neuroanatomical covariance than males, evident across species, which suggests that stronger structural covariance in females could be an evolutionarily conserved feature that is partially related to volumetric alterations through sex.

SIGNIFICANCE STATEMENT

Structural covariance is a potent readout of coordinated brain development, but hard to probe experimentally. Here we use sex differences as a naturally occurring test for developmental theories of structural covariance – adopting a cross-species approach for validation and translational benefit. Brain MRI reveals two conserved features of anatomical covariance across humans and mice: (i) tighter inter-regional coordination of brain development in females as evidenced by stronger volume covariance; (ii) a tendency for female-biased covariance to involve regions that are smaller in females – suggesting an unknown counterbalancing between these two distinct modes of sex-biased brain organization. These findings advance understanding of coordinated brain development and sex difference in a cross-species framework – facilitating future translational research on both topics.
Sufficiency of ITD for (biased) human auditory azimuthal perception
Authors: Azevedo Anna Carolina Pereira Fernandez Oka , Pellegrino Tomás Guimarães , Peña José Luis , Marin Bóris , Pavão Rodrigo
Published: November 2024

Experiments on human auditory perception have shown that interaural time difference (ITD) is sufficient to generate spatial percepts, even though stimuli containing only the ITD cue are perceived as being emitted from inside the head instead of from external locations at specific azimuths. These experiments are thus interpreted as “lateralization” instead of “localization” tasks. In fact, lateralized spatial perception has been quantified using tasks in which participants have to report their estimates by selecting a putative location inside the head, or matching the perceived position to sounds with a given interaural level difference. Therefore, these estimates are made with respect to internal frames of reference, but it is unclear whether these percepts have any significance for the more ecological problem of locating an external sound source.

In order to investigate the link between internalized spatial percepts and sound localization, we designed a new task in which subjects are instructed to report externalized azimuthal location for sounds containing only ITD cues. Despite the mismatch between an internalized percept having to be reported as emanating from an external location, subjects were able to estimate azimuths consistently. Furthermore, normalized estimates were indistinguishable from those obtained using traditional lateralization tasks. Our results revealed a direct relationship between perceived azimuths and ITD, which deviates from that obtained from acoustical analysis of binaural recordings, revealing estimation biases. Intriguingly, these results indicate that externalized percepts are not required for the generation of azimuthal percepts.
Structure-informed mutagenesis identifies a conserved region critical for mouse insulin receptor 5’UTR IRES function
Authors: Dahl William B. , Lan Tammy C.T. , Rouskin Silvia , Marr Michael T.
Published: November 2024

Cells under stress shift their proteome by repressing cap-dependent translation initiation. RNA elements called internal ribosome entry sites (IRES) can allow key cellular transcripts to remain efficiently translated to support an effective stress response. Well- characterized IRESes depend on RNA structures that reduce the protein requirements for translation initiation, thus circumventing translation inhibition. We have previously determined that the insulin receptor 5’ untranslated region (5’UTR) possesses a capacity for IRES activity that is conserved from insects to mammals. There are several prominent examples of viral IRES structures solved in solution; however, the RNA secondary structures of cellular IRESes remain mostly elusive, especiallyin vivo. Here we probe the secondary structure of the Insr 5’UTR IRES in tandem with two well-studied viral IRESes from Hepatitis C virus (HCV) and Encephalomyocarditis virus (EMCV) using dimethyl sulfate mutational profiling by sequencing (DMS-MaPseq) in cells andin vitro. We find that the viral IRES structures in cells are consistent with their knownin vitrostructures and that significant linearization of these well-studied IRESes occurs in the region surrounding their translation start codon in cells. Using the concurrent DMS- MaPseq probing as a constraint, we present a model of the mouse insulin receptor (Insr) 5’UTR. With this model as a guide, we employed a mutation strategy which allowed us to identify a conserved segment distal from the translation start codon as critical for Insr IRES function. This knowledge informed the design of a minimal IRES element with equivalent activity to the full-length Insr 5’UTR across translation contexts.

Background

The RNA structural requirements for cap-independent translation initiation facilitated by theMus musculusinsulin receptor (Insr) 5’ untranslated region (5’UTR) are unknown.

Results

RNA secondary structure probing of the Insr 5’UTR in cells provides a folding model used to identify elements required for cap-independent translation initiation.

Conclusion

A small region of the Insr 5’UTR distal to the translation start codon is necessary for cap-independent translation initiation and is fully sufficient in the proper structural context.
DNA replication fork speed Acts as a Pacer in Cortical Neurogenesis
Authors: Wang Jianhong , Kong Yifan , Li Xuezhuang , Xiang Kun , Tan Yulian , Shi Lei
Published: November 2024

DNA replication fork speed, which controls the rate of genome duplication, has emerged as a key regulator of cellular plasticity. However, its role in neurogenesis remains unexplored. Mini-chromosome maintenance complex (MCMs)-binding protein (MCMBP) functions as a chaperone for newly synthesized MCMs, increasing chromatin coverage to restrain fork speed. We demonstrate that selective deletion of MCMBP in neural progenitor radial glial cells (RGCs) accelerates fork speed, triggering widespread apoptosis, DNA damage, and micronuclei formation, ultimately activating p53 and causing microcephaly. Unexpectedly, concurrent deletion ofTrp53andMcmbpfurther increased fork speed, leading to extensive RGC detachment from the ventricular zone and acquisition of outer-RGC characteristics. Mechanistically, we found that the MCM3 subunit coordinates DNA and centrosome duplication, thereby mediating RGC attachment. Behavioral analysis revealed that disruption of fork speed results in anxiety-like behavior in mice. These findings unveil a previously unrecognized role for replication fork speed in neurogenesis.
ANKZF1 helps to eliminate the stress-damaged mitochondria by LC3-mediated mitophagy
Authors: Ali Mudassar , Mapa Koyeli
Published: November 2024

Mitochondria, the double membrane-bound organelles of endosymbiotic origin, are crucial centers for cellular energy production and several essential metabolic pathways. Recent studies reveal that mitochondria become dysfunctional following numerous cellular stresses, and during pathologies, demanding an extensive investigation of mitochondrial turnover mechanisms. Apart from the specific response pathways to tackle different stresses, mitophagy or degradation of mitochondria by autophagy is a critical quality control mechanism that clears irreversibly damaged mitochondria. Mitophagy is majorly executed either by receptor-mediated or PINK-Parkin-dependent pathways. Here, we show that the human orthologue of yeast Vms1, ANKZF1, participates in PINK-Parkin-mediated mitophagy. We show that ANKZF1 is extensively recruited to damaged mitochondria along with Parkin during mitochondrial proteotoxic stress induced by the expression of a single misfolded/aggregated protein or during uncoupler-induced membrane depolarization. Importantly, ANKZF1 recruitment to damaged mitochondria is significantly enhanced in the presence of Parkin, and ANKZF1 physically interacts with Parkin and LC3 during mitochondrial proteotoxic or depolarization stresses. ANKZF1 harbors six putative LC3-interacting regions (LIRs), LIR4 present at residues 333-336 is particularly important for ANKZF1-LC3 interaction. Furthermore, we show thatANKZF1knockout cells are compromised in clearing stress-damaged mitochondria by mitophagy, indicating an important role of ANKZF1 in mitochondrial turnover during stress. In summary, we show a new role of ANKZF1 in eliminating the stress-damaged mitochondria, reiterating the mito-protective role of Vms1/ANKZF1 during mitochondrial stresses.