bluuebunny/crossref_metadata_2025 huggingface dataset:
DesiRNA: structure-based design of RNA sequences with a replica exchange Monte Carlo approach
Authors: Wirecki Tomasz K , Lach Grzegorz , Badepally Nagendar Goud , Moafinejad S Naeim , Jaryani Farhang , Klaudel Gaja , Nec Kalina , Baulin Eugene F , Bujnicki Janusz M
Published: January 2025

Designing RNA sequences that form a specific structure remains a challenge. Current computational methods often struggle with the complexity of RNA structures, especially when considering pseudoknots or restrictions related to RNA function. We developed DesiRNA, a computational tool for the design of RNA sequences based on the Replica Exchange Monte Carlo approach. It finds sequences that minimize a multiobjective scoring function, fulfill user-defined constraints and minimize the violation of restraints. DesiRNA handles pseudoknots, designs RNA–RNA complexes and sequences with alternative structures, prevents oligomerization of monomers, prevents folding into undesired structures and allows users to specify nucleotide composition preferences. In benchmarking tests, DesiRNA with a default simple scoring function solved all 100 puzzles in the Eterna100 benchmark within 24 h, outperforming all existing RNA design programs. With its ability to address complex RNA design challenges, DesiRNA holds promise for a range of applications in RNA research and therapeutic development.
The SARS-CoV-2 nucleocapsid protein interferes with the full enzymatic activation of UPF1 and its interaction with UPF2
Authors: Nuccetelli Veronica , Mghezzi-Habellah Makram , Deymier Séverine , Roisin Armelle , Gérard-Baraggia Francine , Rocchi Cecilia , Coureux Pierre-Damien , Gouet Patrice , Cimarelli Andrea , Mocquet Vincent , Fiorini Francesca
Published: January 2025

The nonsense-mediated mRNA decay (NMD) pathway triggers the degradation of defective mRNAs and governs the expression of mRNAs with specific characteristics. Current understanding indicates that NMD is often significantly suppressed during viral infections to protect the viral genome. In numerous viruses, this inhibition is achieved through direct or indirect interference with the RNA helicase UPF1, thereby promoting viral replication and enhancing pathogenesis. In this study, we employed biochemical, biophysical assays and cellular investigations to explore the interplay between UPF1 and the nucleocapsid (Np) protein of SARS-CoV-2. We evaluated their direct interaction and its impact on inhibiting cellular NMD. Furthermore, we characterized how this interaction affects UPF1’s enzymatic function. Our findings demonstrate that Np inhibits the unwinding activity of UPF1 by physically obstructing its access to structured nucleic acid substrates. Additionally, we showed that Np binds directly to UPF2, disrupting the formation of the UPF1/UPF2 complex essential for NMD progression. Intriguingly, our research also uncovered a surprising pro-viral role of UPF1 and an antiviral function of UPF2. These results unveil a novel, multi-faceted mechanism by which SARS-CoV-2 evades the host’s defenses and manipulates cellular components. This underscores the potential therapeutic strategy of targeting Np-UPF1/UPF2 interactions to treat COVID-19.
Cold suppresses virus accumulation and alters the host transcriptomic response in the turnip mosaic virus–Arabidopsis halleri system
Authors: Honjo Mie N , Emura Naoko , Kamitani Mari , Kudoh Hiroshi
Published: January 2025

Since plant viruses cause lifelong infections, virus–plant interactions are exposed to large temperature fluctuations in evergreen perennials. In such circumstances, virus–plant interactions are expected to change significantly between the warm and cold seasons. However, few studies have investigated the effects of cold temperatures on virus–plant interactions. Here, we show that in a persistent infection system of the turnip mosaic virus (TuMV)–Arabidopsis halleri, cold temperatures lead to slow viral replication/spreading within the host, attenuated host symptoms, and cold-specific transcriptomic responses. Many differentially expressed genes (DEGs) were detected between virus-inoculated and mock-inoculated plants under warm and cold conditions; however, the sets of DEGs and response timings were temperature-dependent. At cold temperatures, the expression of photosynthesis-related genes decreased in the early stages of infection. However, it recovered to the same level as that in uninfected plants in the later stages. In contrast, the transcriptomic changes under warm conditions suggest that viral infections cause auxin signaling disruption. These responses coincided with the inhibition of host growth. We identified 6 cold- and 38 warm-specific DEGs, which changed their expression in response to TuMV infection under more than half of the conditions for either cold or warm temperatures. Further validation of the putative relationships between transcriptomic and phenotypic responses of the host is required. Our findings on temperature-dependent host responses at both symptomatic and transcriptomic levels help us understand how warm and cold temperatures affect virus–plant interactions in seasonal environments.
A novel classifier of radiographic knee osteoarthritis for use on knee DXA images is predictive of joint replacement in UK Biobank
Authors: Beynon Rhona A , Saunders Fiona R , Ebsim Raja , Faber Benjamin G , Jung Mijin , Gregory Jennifer S , Lindner Claudia , Aspden Richard M , Harvey Nicholas C , Cootes Timothy , Tobias Jonathan H
Published: January 2025

Objectives

DXA scans may offer a novel means of evaluating radiographic knee OA (rKOA) in large population studies and through opportunistic screening. We aimed to develop and apply a semi-automated method for assessing rKOA using ≈20 000 knee DXA images from UK Biobank (UKB) and assess its face validity by checking for expected relationships with clinical outcomes.

Methods

Right knee DXA scans were manually annotated for osteophytes to derive corresponding grades. Joint space narrowing (JSN) grades in the medial joint compartment were determined from automatically measured minimum joint space width. Overall rKOA grade (0–4) was determined by combining osteophyte and JSN grades. Logistic regression was employed to investigate the associations of osteophyte, JSN and rKOA grades with knee pain and hospital-diagnosed KOA. Cox proportional hazards modelling was used to examine the associations of these variables with risk of subsequent total knee replacement (TKR).

Results

Of the 19 595 participants included (mean age 63.7 years), 19.5% had rKOA grade ≥1 (26.1% female, 12.5% male). Grade ≥1 osteophytes and grade ≥1 JSN were associated with knee pain, hospital-diagnosed KOA and TKR. Higher rKOA grades were linked to stronger associations with these clinical outcomes, with the most pronounced effects observed for TKR. Hazard ratios for the association of rKOA grades with TKR were 3.28, 8.75 and 28.63 for grades 1, 2 and 3–4, respectively.

Conclusions

Our DXA-derived measure of rKOA demonstrated a progressive relationship with clinical outcomes. These findings support the use of DXA for classifying rKOA in large epidemiological studies and in future population-based screening.
Hearing loss occurs prior to thrombocytopenia in both mice and humans with DFNA1
Authors: Kurasawa Shunkou , Ganaha Akira , Ayabe Shinya , Yoshiki Atsushi , Kawama Fumiya , Kitayama Shota , Tabuchi Keiji , Yamashita Kouhei , Ueyama Takehiko
Published: January 2025

DFNA1 (deafness, nonsyndromic autosomal dominant 1), initially identified as nonsyndromic sensorineural hearing loss, has been associated with an additional symptom: macrothrombocytopenia. However, the timing of the onset of hearing loss (HL) and thrombocytopenia has not been investigated, leaving it unclear which occurs earlier. Here, we generated a knock‐in (KI) DFNA1 mouse model, diaphanous‐related formin 1 (DIA1)KIΔv3/KIΔv3, in which Aequorea coerulescens green fluorescent protein (AcGFP)‐tagged human DIA1(p.R1213X) was knocked into the ATG site of Dia1. Additionally, the exon 7 of Dia1 was deleted using genome editing to knock out (KO) Dia1‐v3, a specific variant of Dia1. AcGFP‐DIA1(p.R1213X) expression and endogenous DIA1 KO were confirmed in cochleae and platelets. Hearing function in DIA1KIΔv3/KIΔv3, but not DIA1KIΔv3/+ mice, evaluated by auditory brainstem response, was significantly worse at low frequencies compared to wild‐type (WT) mice starting at 3 months of age (3M), with progressive deterioration. Using confocal microscopy and scanning electron microscopy, various stereociliary deformities were identified in the cochleae of DIA1KIΔv3/KIΔv3 mice. Platelet counts in DIA1KIΔv3/KIΔv3, but not DIA1KIΔv3/+ mice, were significantly lower than those in WT mice at 12M, but not at 6M. Furthermore, in a cohort of eight patients with DFNA1 harboring the p.R1213X mutation, HL preceded thrombocytopenia in three individuals. Thus, in both mice and humans, though HL and thrombocytopenia are progressive, HL manifests earlier than thrombocytopenia. Unlike myosin heavy chain 9 (MYH9)‐related diseases, thrombocytopenia cannot be a predictive marker for HL in DFNA1. Nevertheless, monitoring platelet counts could provide insights into the progression of the hearing impairments in patients with DFNA1.
Aerobic exercise regulates gut microbiota profiles and metabolite in the early stage of Alzheimer's disease
Authors: Wei Cuilan , Wu Xiaojing , Li Chuikun , Zhang Yeting , Yuan Qiongjia , Huang Rui
Published: January 2025

Aerobic exercise (AE) has been shown to offer significant benefits for Alzheimer's disease (AD), potentially influencing the gut microbiota. However, the impact of changes in intestinal flora in early Alzheimer's disease induced by aerobic exercise on metabolic pathways and metabolites is not well understood. In this study, 3‐month‐old APP/PS1 and C57BL/6 mice were divided into two groups each: a control group (ADC for APP/PS1 and WTC for C57BL/6) and an aerobic exercise group (ADE for APP/PS1 and WTE for C57BL/6). The exercise groups underwent a 20‐week aerobic training program on a motorized treadmill before the behavioral test (both the Morris water maze experiment (MWM) and the eight‐arm maze test). Fecal samples were collected to analyze gut microbiota profiles via 16S rRNA gene sequencing. At the same time, the metabolic pathway analysis and the detection of metabolites were carried out. At the phylum level, the ADE group exhibited a significant reduced in the relative abundance of Bacteroidetes compared to the ADC group. At the genus level, both Ileibacterium and Faecalibaculum were found to be more abundant in the ADE group than in the ADC group. Additionally, PICRUSt analysis revealed that lipid metabolism and bile acid metabolism pathways were significantly enriched in the cecal microbiota of mice in the ADE group. The metabolites detected further confirmed the changes in the metabolic pathways mentioned above. Aerobic exercise may modify gut microbiota profiles and metabolites in APP/PS1 mice, thereby potentially playing a beneficial role in delaying cognitive impairment associated with early‐stage Alzheimer's disease.
TRAF6 promotes abdominal aortic aneurysm development by activating macrophage pyroptosis via the NLRP3/Caspase1/GSDMD pathway
Authors: Cai Huoying , Li Huaming , Xiao Xiaoyong , Wang Siwen , Liu Ruiming , Qin Yuansen , Zhou Yu , Yao Chen
Published: January 2025

Abdominal aortic aneurysm represents a critical pathology of the aorta that currently lacks effective pharmacological interventions. TNF receptor‐associated factor 6 (TRAF6) has been established to be involved in cardiovascular diseases such as atherosclerosis, hypertension, and heart failure. However, its role in abdominal aortic aneurysm (AAA) remains unclear. This study aimed to explore the role of TRAF6 on AAA formation and its underlying mechanisms. Single‐cell RNA sequencing of human AAA tissues demonstrated that TRAF6 was significantly upregulated in aortic macrophages. Moreover, overexpression of TRAF6 promotes AAA formation in elastase‐induced C57BL/6 mice, while TRAF6 pharmacological inhibition could attenuate AAA development. Consistently, inhibition of TRAF6 in macrophages through in vitro methods notably limits their pyroptosis, while also diminishing proinflammatory responses in these cells. Mechanistically, TRAF6 can modulate macrophage pyroptosis through the NLRP3/Caspase1/GSDMD signaling pathway. Our study highlights the crucial role of the TRAF6/NLRP3/Caspase1/GSDMD axis in macrophage pyroptosis and AAA, offering potential biomarkers and therapeutic targets for AAA.
m6A demethylase Fto inhibited macrophage activation and glycolysis in diabetic nephropathy via m6A/Npas2/Hif‐1α axis
Authors: Zhu Sai , Jiang Ling , Liu Xinran , Chen Chaoyi , Luo Xiaomei , Jiang Shan , Yin Jiuyu , Liu Xueqi , Wu Yonggui
Published: January 2025

Macrophage infiltration and activation is a key factor in the progression of diabetic nephropathy (DN). However, aerobic glycolysis induced by m6A methylation modification plays a key role in M1‐type activation of macrophages, but the specific mechanism remains unclear in DN. In this study, the expression of m6A demethylase Fto in bone marrow derived macrophages and primary kidney macrophages from db/db mice. Loss and gain‐of‐function analysis of Fto were performed to assess the role of Fto in DN. Transcriptome and MeRIP‐seq association analysis was performed to identified the target gene was Npas2. In this study, we found that demethylase Fto exhibits low expression in type 2 DN m6A modification of Npas2 mediated by Fto regulates macrophages M1‐type activation and glucose metabolism reprogramming to participate in the process of DN. Furthermore, Fto reduces the m6A modification level of Npas2 in macrophages through a Prrc2a‐dependent mechanism, and decreasing its stability. This process mediates inflammation and glycolysis in M1 macrophages by regulating the Hif‐1α signaling pathway. Fto may act as a suppressor of M1 macrophages inflammation and glycolysis in DN through the m6A/Npas2/Hif‐1α axis. This findings providing a new basis for the prevention and treatment of DN.
Hip Joint Articulotrochanteric Distance Measurements in Children: Articulotrochanteric Distance in Children Aged 5 to 14 Years Does Not Increase With Age
Authors: Yang Di , Haiping Ouyang , Wang Zhongliang
Published: January 2025

Background:

The articulotrochanteric distance (ATD) has universally been utilized to assess greater trochanter overgrowth in pediatric orthopaedic diseases. However, its overgrowth cannot be detected in a timely manner due to the absence of a normal ATD value. This study is to determine the ATD normal value in hip radiographs of children under the age of 14 and to establish the threshold for overgrowth of the greater trochanter.

Methods:

A retrospective analysis of 1787 hip radiographs was conducted, measuring ATD, trochanter-to-trochanter distance (TTD), lesser trochanter-to-articular surface distance (LTA), epiphysis height (EH), and femoral head epiphyseal plate-to-lesser trochanter distance (ELD). The differences in TTD and ELD between adjacent age groups were denoted as ΔTTD and ΔELD, respectively. Data with normal distribution were analyzed using independent sample (tow-tailed student t test) t tests and One﻿-way ANOVA, while data with non-normal distribution were analyzed through nonparametric tests. The range defined by the 95% measurement values (mean±2SD) was considered the normal range.

Results:

Among 5-14 year-olds, the mean ATD (mean±2SD) was 23.5±10.0 mm for males and 21.2±9.6 mm (mean±2SD) for females, statistically significant differences in the ATD were observed between sexes (P<0.05), whereas no significant differences were noted among age subgroups within the same sex (P>0.05). The normal range for males was 13.5 to 33.5 mm, while for females it was 11.6 to 30.8 mm. In addition, no significant distinction (P>0.05) was noted in the EH between males and females within this age range. Furthermore, the analysis revealed no significant difference (P>0.05) between the changes in ΔTTD and ΔELD among the hips.

Conclusions:

Our findings revealed that in hip radiographs, ATD values of children aged 5 to 14 years differ significantly between males and females, although within each sex group, there was no notable variation across different age groups. We proposed that ATD <13.5 mm in males and <11.6 mm in females might indicate an overgrowth of the greater trochanter, and in such patients, prophylactic greater trochanteric epiphysiodesis may be beneficial. In addition, we believed that the growth rates of the femoral neck and greater trochanter were approximately similar.

Level of Evidence:

Level IV—diagnostic studies.
Challenges in standardizing preimplantation kidney biopsy assessments and the potential of AI-Driven solutions
Authors: Wellekens Karolien , Koshy Priyanka , Naesens Maarten
Published: January 2025

Purpose of review

This review explores the variability in preimplantation kidney biopsy processing methods, emphasizing their impact on histological interpretation and allocation decisions driven by biopsy findings. With the increasing use of artificial intelligence (AI) in digital pathology, it is timely to evaluate whether these advancements can overcome current challenges and improve organ allocation amidst a growing organ shortage.

Recent findings

Significant inconsistencies exist in biopsy methodologies, including core versus wedge sampling, frozen versus paraffin-embedded processing, and variability in pathologist expertise. These differences complicate study comparisons and limit the reproducibility of histological assessments. Emerging AI-driven tools and digital pathology show potential for standardizing assessments, enhancing reproducibility, and reducing dependence on expert pathologists. However, few studies have validated their clinical utility or demonstrated their predictive performance for long-term outcomes.

Novel AI-driven tools hold promise for improving the standardization and accuracy of preimplantation kidney biopsy assessments. However, their clinical application remains limited due to a lack of proven associations with posttransplant outcomes and insufficient evaluation of predictive performance metrics. Future research should prioritize longitudinal studies using large-scale datasets, rigorous validation, and comprehensive assessments of predictive performance for both short- and long-term outcomes to fully establish their clinical utility.
Can Sleep Predict Conversion to Mild Cognitive Impairment and Dementia? Results From the Hellenic Longitudinal Investigation of Aging and Diet Study
Authors: Bairami Styliani , Scarmeas Nikolaos , Yannakoulia Mary , Dardiotis Efthimios , Sakka Paraskevi , Hadjigeorgiou Georgios , Tsapanou Angeliki , Kosmidis Mary H.
Published: January 2025

Objective:

Sleep disturbance is considered a risk factor for cognitive decline in elderly individuals. Our aim in the current study was to investigate whether baseline sleep parameters can predict the conversion from normal cognition to mild cognitive impairment or dementia at follow-up. The Hellenic Longitudinal Investigation of Aging and Diet is a longitudinal population-based study designed to estimate the prevalence and incidence of cognitive decline and dementia in the older Greek population.

Methods:

A total of 955 cognitively normal older adults (aged ≥65 y) were drawn from the Hellenic Longitudinal Investigation of Aging and Diet study. A comprehensive neurological and neuropsychological assessment was conducted at baseline and a mean of 3.1 (SD = 0.85) years later, resulting in 160 individuals diagnosed with mild cognitive impairment and 34 with dementia at follow-up, whereas 761 remained cognitively normal.

Results:

Using Cox regression analyses, no sleep parameters increased the risk of conversion status adjusting for demographics and clinical factors. Napping, however, decreased this risk by 19.3% (P < 0.001).

Conclusions:

As several previous studies have proposed, napping constitutes a protective factor against cognitive decline. Thus, clinicians should encourage their elderly patients to adopt this healthy habit.
Hsa-MiR-483 -3p Regulates the Extracellular Matrix Proteins via TGFβ2/SMAD4 Signaling in the Glucocorticoid-responsive Human Trabecular Meshwork Cells
Authors: Haribalaganesh Ravinarayanan , Sharmila Rajendrababu , Krishnadas Ramasamy , Willoughby Colin E. , Senthilkumari Srinivasan
Published: January 2025

Purpose

To investigate the role of hsa-miR-483-3p on the regulation of extracellular matrix (ECM) in cultured human trabecular meshwork (HTM) cells with known steroid response.

Methods

Primary cultures of HTM cells with known steroid responsiveness [GC-responder (GC-R) and GC-Non-responder (GC-NR) cells] were grown on coverslip in 12-well plate until 80% confluence and treated with 100 nM dexamethasone (DEX) for 24 h and transfected with different concentrations of synthetic miRNA 483-3p mimic or inhibitor. After 24 h or 72 h post transfection, the cells were harvest for the following experiments: (i) percentage transfection efficiency (ii) RNA isolation for qPCR analysis and (iii) immunofluorescence staining, (iv) protein isolation for Western blotting respectively. All experiments were performed in triplicate with three biological samples (n□=□3).

Results

GC-R HTM cells showed significantly higher expression of SMAD4 as compared to GC-NR HTM cells. Similarly, DEX treatment up-regulated the SMAD4-dependent ECM proteins. The presence of a miR-483-3p mimic down-regulated SMAD4 expression and SMAD4-dependent ECM production in a dose-dependent manner by negatively down- regulating SMAD4/TGF-β2 signalling. The inhibition of SMAD4-dependent ECM production by miR-483-3p was more pronounced in GC-R HTM cells as compared to GC- NR cells.

Conclusion

The down-regulation in ECM production by miR-483-3p is SMAD4 dependent and may play a protective role in mitigating steroid response in GC-R HTM cells. Using miR-483-3p mimics demonstrates therapeutic potential for the management of steroid induced ocular hypertension and glaucoma.
Timelapse and volumetric imaging of mitochondrial networking using NAD(P)H autofluorescence via 2-photon microscopy
Authors: ter Hofstede Blanche , Walsh Alex J.
Published: January 2025

Significance

Mitochondria are dynamic organelles that play a key role in energy production and maintaining cellular homeostasis. The regulation of mitochondrial dynamics, involving both fission and fusion, is vital for maintaining a healthy population of mitochondria within the cell. Alterations in mitochondrial dynamics have been associated with various disease states, such as metabolic and neurodegenerative diseases and cancer.

Aim

We describe a protocol for imaging and analyzing NAD(P)H intensity to visualize the movement of mitochondria over time and in 3D to visualize the distribution of the mitochondrial network within the cell.

Approach

A multiphoton (MP) laser scanning microscope was used to image NAD(P)H autofluorescence signal of MDA-MB-231 cells at 750 nm excitation. A mitochondrial fluorescent dye, MitoSpy Orange, was used to validate the signal. Laser power, image size, dwell time, interval time, and imaging duration were optimized for timelapse and 3D imaging to minimize photodamage and maximize autofluorescence signal.

Results

The NAD(P)H signal in 2D was imaged with a frame rate of 0.4 frames per second (FPS) allowing for visualization of mitochondria movement. 3D imaging was performed with a frame rate of 0.5 FPS for a single cell with a thickness of approximately 15 microns that allowed for visualization of the mitochondria network within the cell.

Conclusions

This protocol motivates using label-free imaging techniques to study mitochondrial dynamics in a non- destructive manner, suitable for drug screening and understanding the effects of mitochondrial dynamic alterations in disease.
BIND: Large-Scale Biological Interaction Network Discovery through Knowledge Graph-Driven Machine Learning
Authors: Aamer Naafey , Asim Muhammad Nabeel , Bhatti Aamer Iqbal , Dengel Andreas
Published: January 2025

The complex interactions between biological entities provide valuable insights into fundamental life processes, which pave the way to a deeper understanding of disease mechanisms for the development of innovative therapeutic strategies. To enable large-scale predictions of biological interactions, multifarious AI-driven predictors have been developed. However, most of these are developed by leveraging information from only a limited subset of interaction types, and the broader interaction types landscape could facilitate AI algorithms to learn more informative patterns to predict unknown interactions. To address the need of a robust and precise interaction predictor, we introduce BIND (Biological Interaction Network Discovery), a predictor that leverages simultaneous learning across 10 biological entities and 30 interaction types. To develop BIND, we first evaluate 11 distinct Knowledge Graph Embedding Methods on the largest public biomedical interaction dataset namely PrimeKG. For each relation type, we extracted entity embeddings from the top 5 performing Knowledge Graph Embedding Models (KGEMs) and input them into 7 distinct machine learning classifiers. Rigorous evaluation of 1,050 predictive pipelines demonstrated that specific combinations of KGEMs and classifiers achieved F1 scores of 90% to 99% across various interaction types. Comprehensive evaluation across each relation type identified the top-performing predictive pipelines, which became the foundation of the BIND web application. To reveal the practical utility of our web application in identifying novel biological interactions, we conducted a case study on drug-phenotype interactions. The application gave 1,355 high confidence predictions, from which potential interactions were subsequently validated by scientific evidence found within the existing literature (Table 5). We believeBIND’s web application’s public accesswill serve as a valuable tool for biologists to identify unknown interactions that can be subsequently validated through wet-lab experiments.
Characterization of ECM produced by MC3T3-E1 cellular spheroids encapsulated in alginate hydrogel
Authors: Boscaro Diamante , Wahlum Lill Skovholt , Ullevålseter Marie Eline , Strand Berit Løkensgrad , Sikorski Pawel
Published: January 2025

The application of cellular spheroids in bone tissue engineering research has gained significant interest in the last decade. Compared to monolayer cell cultures, the 3D architecture allows for more physiological cell-cell and cell-extracellular matrix (ECM) interactions that make cellular spheroids a suitable model system to investigate bone ECM in vitro. The use of 3D model systems requires fine-tuning of experimental methods used to study cell morphology, ECM deposition and mineralization, and cell-ECM interactions. In this study, we use MC3T3-E1 cellular spheroids encapsulated in an alginate hydrogel to study and characterize the deposited ECM. Spheroid shape and structure were evaluated using confocal microscopy. The deposited collagenous ECM was characterized using quantitative assay and microscopy, in particular Second Harmonic Imaging Microscopy (SHIM), hydroxyproline (HYP) assay and Transmission Electron Microscopy (TEM). The use of hydrogel constructs allows easy handling and imaging of the samples and helps to preserve the spheroid’s stability by preventing cells from adhering to the culture dish surface. We use a non-modified alginate hydrogel that does not facilitate cell attachment and therefore functions as an inert encapsulating scaffold. Constructs were cultured for up to 4 weeks. SHIM, HYP assay and TEM confirmed the deposition of collagenous matrix by the spheroid, with most of it taking place between week 2 and 4. We demonstrated that alginate encapsulated bone spheroids are a promising model for studying bone ECM in vitro.
JIND-Multi: Leveraging Multiple Labeled Datasets for Automated Annotation of Single-Cell RNA and ATAC Data
Authors: Sancho Joseba , Kanhirodan Akash , Garrote Xabier , Gevaert Olivier , Hernaez Mikel , Serrano Guillermo , Ochoa Idoia
Published: January 2025

Background

The creation of single-cell atlases is essential for understanding cellular diversity and heterogeneity. However, assembling these atlases is challenging due to batch effects and the need for accurate cell annotation. Current methods for single-cell RNA and ATAC sequencing, while effective for integration, are not optimized for cell annotation. Additionally, many annotation tools rely on external databases or reference scRNA-Seq datasets, which may limit their adaptability to specific study needs, especially for rare cell-types or scATAC-Seq data.

Results

We introduce JIND-Multi, an extended version of the JIND framework, designed to transfer cell-type labels across multiple annotated datasets. JIND-Multi significantly reduces the proportion of unclassified cells in single-cell RNA sequencing (scRNA-Seq) data while maintaining the accuracy and performance of the original JIND model. Furthermore, JIND-Multi demonstrates robust and precise annotation results in its inaugural application to scATAC-Seq data, proving its versatility and effectiveness across different single-cell sequencing technologies.

Conclusions

JIND-Multi represents an improvement in cell annotation, reducing unassigned cells and offering a reliable solution for both scRNA-Seq and scATAC-Seq data. Its ability to handle multiple labeled datasets enhances the precision of annotations, making it a valuable tool for the single-cell research community. JIND-Multi is publicly available at:https://github.com/ML4BM-Lab/JIND-Multi.git.
Bidirectional communication between nucleotide and substrate binding sites in a type IV multidrug ABC transporter
Authors: Pérez Carrillo Victor Hugo , Di Cesare Margot , Rose-Sperling Dania , Javed Waqas , Neuweiler Hannes , Marcoux Julien , Orelle Cédric , Jault Jean-Michel , Hellmich Ute A.
Published: January 2025

ATP-binding cassette (ABC) transporters use ATP to transport substrates across cellular membranes. In type IV ABC transporters, including many multidrug resistance (MDR) pumps, communication between the nucleotide-binding domains (NBDs) and transmembrane domains (TMDs) occurs via intracellular coupling helices. However, the precise mechanism of interdomain crosstalk and coordination between the ATP and substrate binding sites remain unclear. Combining nuclear magnetic resonance (NMR) spectroscopy, Hydrogen-Deuterium eXchange Mass Spectrometry (HDX-MS), photo-induced electron-transfer fluorescence correlation spectroscopy (PET-FCS) and functional assays, we identified a conserved cluster of residues at the NBD/TMD interface of the bacterial MDR transporter BmrA. This cluster is crucial for transporter function and relays nucleotide and substrate binding between the two domains via coupling helix 2. Mutations impact both local and global transporter dynamics. Our findings reveal a novel interdomain communication pathway in type IV ABC transporters, shedding light on the intricate coupling mechanisms that enable the coordination of these molecular machines.
CroCoDeEL: accurate control-free detection of cross-sample contamination in metagenomic data
Authors: Goulet Lindsay , Oñate Florian Plaza , Famechon Alexandre , Quinquis Benoît , Belda Eugeni , Prifti Edi , Le Chatelier Emmanuelle , Gautreau Guillaume
Published: January 2025

Metagenomic sequencing provides profound insights into microbial communities, but it is often compromised by technical biases, including cross-sample contamination. This phenomenon arises when microbial content is inadvertently exchanged among concurrently processed samples, distorting microbial profiles and compromising the reliability of metagenomic data and downstream analyses. Existing detection methods often rely on negative controls, which are inconvenient and do not detect contamination within real samples. Meanwhile, strain-level bioinformatics approaches fail to distinguish contamination from natural strain sharing and lack sensitivity. To fill this gap, we introduce CroCoDeEL, a decision-support tool for detecting and quantifying cross-sample contamination. Leveraging linear modeling and a pre-trained supervised model, CroCoDeEL identifies specific contamination patterns in species abundance profiles. It requires no negative controls or prior knowledge of sample processing positions, offering improved accuracy and versatility. Benchmarks across three public datasets demonstrate that CroCoDeEL accurately detects contaminated samples and identifies their contamination sources, even at low rates (<0.1%), provided sufficient sequencing depth. Notably, we discovered critical contamination cases in highly cited studies, calling some of their results into question. Our findings suggest that cross-sample contamination is a widespread yet underexplored issue in metagenomics and emphasize the necessity of systematically integrating contamination detection into sequencing quality control.
Development of a High-throughput Morphological Assay for Evaluating Mesenchymal Stromal Cell-derived Extracellular Vesicle Modulation of Brain Pericyte Secretory Phenotype
Authors: Campagna Courtney E. , Larey Andrew M. , Daga Kanupriya R. , Roos Morgan , Ghosh Sneha , Grimsey Neil , Han Jin , Marklein Ross A.
Published: January 2025

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) are a promising therapeutic tool for treating many neurodegenerative diseases. Neuroinflammation plays a major role in many of these conditions through an orchestration of interdependent processes that lead to the breakdown of the blood-brain barrier (BBB), infiltration of immune cells and neuronal death. MSC-EVs have shown preliminary evidence of modulating neuroinflammation, but their mechanisms of action are still unknown. Therefore, we explored the potential of MSC-EVs in modulating brain pericytes, a cell type that plays a critical role in BBB maintenance but has not been investigated as a therapeutic target for MSC-EVs. Brain pericytes are multifaceted cells that can modulate neuroinflammation through their involvement in BBB homeostasis, as well as the innate and adaptive immune response. Pericyte morphology has been shown to change in response to inflammatory stimuliin vivo, hence, we used this behavior to develop a quantitative morphological profiling approach to assess the immunomodulatory function of MSC-EVs in a high-throughput, low-cost manner. Using this assay, we were able to demonstrate that MSC-EVs manufactured under various conditions (2D, 3D, and in response to cytokine priming) could induce distinct pericyte morphological responses indicative of changes in secretion of chemokines and cytokines relevant to neuroinflammation.
Propofol Treatment Alters the Metabolic State of MDA-MB-231 Breast Cancer Cells
Authors: Tasmi Tasbassum Ahmed , Holla Shubha , Walsh Alex J.
Published: January 2025

The mechanisms of many anesthetic drugs, such as propofol, are unknown. This study investigates the impact of propofol, a widely used intravenous anesthetic drug, on the metabolic behavior of the human triple-negative breast cancer cell line, MDA-MB 231. Utilizing fluorescence lifetime imaging microscopy (FLIM), we assessed the effect of propofol on cellular metabolism through imaging of the fluorescence lifetimes of NADH and FAD, coenzymes of metabolism reactions. Exposure to propofol-induced significant morphological and metabolic changes of the cells, including substantial cellular shrinkage, which may reveal a metabolic component to propofol’s mechanism.
DNA polymerase beta expression in head & neck cancer modulates the poly(ADP-ribose)-mediated replication checkpoint
Authors: Khan Md Maruf , Roos Wynand P. , Angajala Anusha , Gibbs Denise Y. , Liu Jeffrey C. , Ragin Camille , Sobol Robert W.
Published: January 2025

Head and Neck Squamous Cell Carcinoma (HNSCC) imposes a significant health burden, necessitating innovative therapeutic strategies to enhance treatment efficacy. Current treatments such as surgery, radiation, and chemotherapy have limited effectiveness and yield severe side effects, emphasizing the need for targeted therapies. We have focused on DNA polymerase beta (Polβ) and its roles in replication stress, cellular responses to DNA damaging therapies, and DNA damage response modifiers. Our investigations reveal a regulatory role for base excision repair (BER) proteins, including Polβ, in the cellular response to inhibitors of poly(ADP-ribose) glycohydrolase (PARG), an enzyme involved in poly(ADP-ribose) (PAR) degradation. The inhibition of PARG, in HNSCC cells, elicits replication stress. Further, this activates the PAR-induced S-phase/ATR checkpoint, leading to a block to replication, cell cycle arrest, and the onset of apoptosis. However, Polβ overexpression mitigates this response, reducing replication-stress induced PAR foci formation, suggesting a modulation of replication checkpoint activation. We found that PARG inhibitor treatment is ineffective on HNSCC cells that overexpress Polβ, implying that the PARG inhibitor-induced PAR and apoptotic response is dependent on the level of Polβ. Further, ourin vitroexperiments demonstrate that combining PARG and ATR/CHK1 inhibitors overcomes Polβ-mediated treatment resistance in HNSCC cells, producing enhanced effects as compared to the individual treatment conditions. Our findings suggest a possible treatment paradigm for HNSCC, employing ATR or CHK1 inhibitors in combination with PARG inhibitors. This strategy offers a promising path for more effective HNSCC treatments, potentially overcoming Polβ-related resistance.
SOuLMuSiC: A Computational Tool for Predicting the Impact of Mutations on Protein Solubility
Authors: Attanasio Simone , Kwasigroch Jean , Rooman Marianne , Pucci Fabrizio
Published: January 2025

Protein solubility problems arise in a wide range of applications, from antibody development to enzyme production, and are linked to several major disorders, including cataracts and Alzheimer’s diseases. To assist scientists in designing proteins with improved solubility and better understand solubility-related diseases, we introduce SOuLMuSiC, a computational tool for the fast and accurate prediction of the impact of mutations on protein solubility. Our model is based on a simple shallow artificial neural network that takes as input a series of features, including biophysical properties of wild-type and mutated residues, energetic values computed using various statistical potentials, and mutational scores derived from protein language models. SOuLMuSiC has been trained on a curated dataset of about seven hundred mutations with known solubility values, collected and manually verified from original literature. It significantly outperforms current state-of-the-art predictors in strict cross-validation and shows good performance on external datasets containing high-throughput enzyme solubility-related data as well as protein aggregation propensities. In summary, SOuLMuSiC is a valuable tool for identifying mutations that impact protein solubility, and can play a major role in the rational design of proteins with improved solubility and in understanding genetic variants’ effect. It is freely available for academic use athttp://babylone.ulb.ac.be/SoulMuSiC/.
Age and Sex-Specific Changes in Mitochondrial Quality Control in Skeletal and Cardiac Muscle
Authors: Springer-Sapp Catherine B. , Ogbara Olayinka , Da Silva Maria Canellas , Liu Yuan , Prior Steven J. , Kuzmiak-Glancy Sarah
Published: January 2025

Skeletal and cardiac muscle mitochondria exist in a dynamic reticulum that is maintained by a balance of mitochondrial biogenesis, fusion, fission, and mitophagy. This balance is crucial for adequate ATP production, and alterations in skeletal muscle mitochondria have been implicated in aging-associated declines in mitochondrial function. We sought to determine whether age and biological sex affect mitochondrial content [Complex IV (CIV)], biogenesis (PGC-1ɑ), fusion (MFN2, OPA1), fission (DRP1, FIS1), and mitophagy (Parkin, Pink1) markers in skeletal and cardiac muscle by assessing protein expression in tibialis anterior (TA) and ventricular tissue from 16 young (≤6 months) and 16 old (≥20 months) male and female Sprague-Dawley rats. In the TA, CIV expression was 40% lower in old vs. young rats (p<0.001), indicating lower mitochondrial content, and coincided with higher expression of Parkin (+4-fold, p<0.001). Further, MFN2 expression was higher (+2-fold, p<0.005) and Parkin was lower (-40%, p=0.014) in older rats. In cardiac muscle, mitochondrial content was maintained in old vs. young rats, and this occurred concomitantly with higher expression of both PGC-1ɑ and Parkin. MFN2 and OPA1 expression were also 1.2-5-fold higher in older rats (p<0.05 for all). Largely, protein expression did not differ between male and female rats, with the exception of Pink1 and FIS1 expression in the TA. Collectively, older skeletal and cardiac muscle demonstrated higher expression of fusion and mitophagy proteins, which indicates age alters the balance of biogenesis, fission, fusion, and mitophagy. This may, in turn, affect the ability to provide ATP to these metabolically active tissues.
Long-term hippocampal low-frequency stimulation alleviates focal seizures, memory deficits and synaptic pathology in epileptic mice
Authors: Kleis Piret , Paschen Enya , Djie-Maletz Andrea , Vlachos Andreas , Haas Carola A. , Häussler Ute
Published: January 2025

Background

Mesial temporal lobe epilepsy (MTLE) is a prevalent form of focal epilepsy characterized by seizures originating from the hippocampus and adjacent regions. Neurostimulation presents an alternative for surgery-ineligible patients with intractable seizures. However, conventional approaches have limited efficacy and require refinement for better seizure control. While hippocampal low-frequency stimulation (LFS) has shown promising seizure reduction in animal studies and small clinical cohorts, its mechanisms, sex-specific outcomes, and long-term effects remain unknown.

Objectives

We aimed to identify the long-term antiepileptic and cognitive outcomes and potential underlying mechanisms of hippocampal LFS in chronically epileptic male and female mice.

Methods

We used the intrahippocampal kainate mouse model replicating the features of MTLE: spontaneous seizures, hippocampal sclerosis, and memory deficits. We applied 1 Hz electrical LFS in the sclerotic hippocampus 6 hours a day, four times a week for 5 weeks and examined its effects on epileptiform activity, spatial memory, and kainate-induced pathological features at cellular and synaptic levels.

Results

Long-term hippocampal LFS consistently diminished focal seizures in epileptic male and female mice, with seizure reduction extending beyond the stimulation period. Additionally, LFS relieved spatial memory deficits and reversed pathological long-term potentiation-like changes at perforant path-dentate granule cell synapses. LFS had no significant effect on generalized convulsive seizures, anxiety-like behaviour, neurogenesis, hippocampal sclerosis, excitatory synapse marker expression, or presynaptic vesicles in perforant path fibers.

Conclusion

These findings provide clinically relevant insights into the seizure type-specific effects of hippocampal LFS, which, alongside synaptic and behavioural improvements, could contribute to enhanced seizure control and quality of life in MTLE patients.
Telomeric SUMO level influences the choices of APB formation pathways and ALT efficiency
Authors: Zhao Rongwei , Wivagg Allison , Lackner Rachel M. , Salsman Jayme , Dellaire Graham , Matunis Michael J , Chenoweth David M. , Zhao Xiaolan , Zhang Huaiying
Published: January 2025

Many cancers use an alternative lengthening of telomeres (ALT) pathway for telomere maintenance. ALT telomeric DNA synthesis occurs in ALT telomere-associated PML bodies (APBs). However, the mechanisms by which APBs form are not well understood. Here, we monitored the formation of APBs with time-lapse imaging employing CRISPR knock-in to track the promyelocytic leukemia (PML) protein at endogenous levels. We found APBs form via two pathways: telomeres recruit PML proteins to nucleate PML bodies de novo, or telomeres fuse with preformed PML bodies. Both nucleation and fusion of APBs require interactions between SUMO and SUMO interaction motifs (SIMs). Moreover, APB nucleation is associated with higher levels of SUMOs and SUMO-mediated recruitment of DNA helicase BLM, resulting in more robust telomeric DNA synthesis. Finally, further boosting SUMO levels at telomeres enhances APB nucleation, BLM enrichment, and telomeric DNA synthesis. Thus, high SUMO levels at telomeres promote APB formation via nucleation, resulting in stronger ALT activity.
SPARC is a new driver of early breast tumor progression via TGF-β -dependent mechanism
Authors: Sciacca Marianela , del Pilar Carballo María , Lacunza Ezequiel , Marino Lina , Cardozo Paola Noelia , Padilla Naira Rodríguez , López-López Tamara , Abba Martin , Courtois Laura , Bieche Ivan , Vincent-Salomon Anne , Bilbao Érica Rojas , Podsypanina Katrina , Boissan Mathieu , Chavrier Philippe , Eiján Ana María , Sáez Pablo J , Lodillinsky Catalina
Published: January 2025

Ductal carcinoma in situ (DCIS) is a pre-invasive lesion that is thought to be a precursor of invasive ductal carcinoma (IDC). The challenge lies in discriminating between DCIS progressors and DCIS non-progressors, often resulting in over- or under-treatment in many cases. Membrane type 1 (MT1)-matrix metalloproteinase (MMP) has been previously identified as an essential gene involved in DCIS progression. Here, RNA-sequencing analysis of MT1-MMPhighsubpopulation derived from invasive breast tumors in the intraductal xenograft model was compared against a dataset of human high-grade DCIS, and Secreted Protein Acidic and Cysteine Rich (SPARC) has emerged as a master candidate involved in early breast tumor progression.

We report that SPARC is up-regulated in DCIS as compared to normal breast epithelial tissues, and further increased in IDC relative to synchronous DCIS foci. We found a positive correlation between SPARC and MT1-MMP expression in DCIS lesions. At the mechanistic level, depletion of SPARC reduced MT1-MMP expression, the degradative capacity of the cells and the activation of the TGF-β signalling canonical pathway. Pharmacological inhibition of the TGF-β signalling pathway decreased SPARC and MT1-MMP at the mRNA and protein level, and concomitantly the cell degradative capacity and 3D cell migration. Strikingly, inhibition of the TGF-β signalling pathway limits the invasive transition of breast tumors in a new triple-negative mouse intraductal syngeneic xenograft model. Moreover, high SPARC expression was positively correlated with both, TGF-β and its receptor, TGFBRI, in a basal type of breast cancer collection supporting our findings. This study identifies SPARC as a new driver of early breast tumor progression via a TGF-β-dependent mechanism, suggesting TGF-β signaling pathway as a potential target for patients with high SPARC expression.