bluuebunny/crossref_metadata_2025 huggingface dataset:
Comprehensive Unbiased Analysis of Vascular Tissue Changes in Accelerated Atherosclerosis Using High-Resolution Ultrasound combined with Photoacoustic Imaging
Authors: de Jong Alwin , Grasso Valeria , van Dijk Kayleigh , Sluiter Thijs. J. , Quax Paul. H.A. , Jose Jithin , de Vries Margreet R.
Published: November 2024

Venous bypass grafts are commonly used to circumvent complex coronary or peripheral artery occlusions. The patency rates, however, are hampered due to accelerated buildup of atherosclerotic lesions in the vein graft wall. Identification of unstable plaques is crucial to guide clinical decision making. In this study, we employ advanced high-resolution ultrasound (US) coupled with spectral photoacoustic imaging (sPAI) to enhance the accurate visualization and analysis of tissue compositionin vivo. By applying unbiased spectral analysis, we investigate the composition and plaque instability in a murine vein graft model.

Method

Male hypercholesterolemic ApoE3*Leiden mice and normocholesterolemic C57BL/6 mice underwent vein graft surgery in which a caval vein from a donor mouse was interpositioned into the arterial circulation of a recipient at the sight of the right common carotid artery. US imaging with sPAI was conducted on 7, 14, 21, and 28 days after surgery. Spectral curves from the near-infrared (NIR) I region, spanning 680 to 970nm, were extracted using a data-driven approach. Component discovery and cross-correlation analysis were performed with Matlab, and ImageJ reconstructed the components within 3D images. At the endpoint histological analysis of the vein grafts was performed.

Results

Analysis of the NIRI region revealed distinct components, with 7 and 10 components tested in the cross-correlation map. Relative abundance values identified melanin, oxidized hemoglobin, deoxygenized hemoglobin, lipids, and collagen. Lipids and collagen spectra accurately identified lipid and collagen-rich tissuesin vivo. The sPAI analysis of of the vein graft wallin vivoresulted in a 8.7% lipids in the vein graft wall compared to 1.8% lipids in the histological analysis at t=28d. For vein grafts from ApoE*3-Leiden mice no differences in the lipid positive area was observed between the sPAI analysis or histological quantification. The percentages collagen present in the vein graft walls from both strains analyzed via sPAI and histological showed comparable results at t=28d.

Conclusion

Our study demonstrates that sPAI can be utilized for compositional analysis of murine tissue in an unbiased manner. This methodology can be used to enhance our understanding of vein graft dynamics and holds promise to advance non-invasive characterization of vascular diseases to ultimately guide clinical decision making.
Inheritable cell-states shape drug-persister correlations and population dynamics in cancer cells
Authors: Iyer Anton , Granada Adrián E. , Chakrabarti Shaon
Published: November 2024

Drug tolerant persisters (DTPs) drive cancer therapy resistance by temporarily evading drug action, allowing multiple routes to eventual permanent resistance. Despite clear evidence for DTPs, the timing of their emergence, proliferative nature, and how their population dynamics arise from measured single-cell kinetics remain poorly understood. Here we use time-lapse microscopy data from two cancer cell lines, integrating single-cell and population measurements, to develop a quantitative description of drug persistence. Contrary to the expectation that increasing levels of genotoxic stress should lead to slower times to division and faster times to death, we observe minor changes in the single-cell intermitotic and death time distributions upon increasing cisplatin concentration. Yet, population decay rates increase 3-fold, suggesting a surprising independence of the overall dynamics from the measured birth and death rates. To explain this phenomenon, we argue that the observed lineage correlations and concentration-dependent decay rates imply cell-state dependent fate choices madebothpre and post-cisplatin as opposed to just post-drug birth/death rate-based competitive fate choices. We demonstrate that these cell-states, present in the ancestors of DTP and sensitive cells, exhibit no difference in cycling speed and are inherited across 2-3 cellular generations. A stochastic model implementing these rules simultaneously recapitulates the observed decay rates and cell-fate correlations, also explaining howpre-drug fate decisions are consistent with barcoding experiments where barcode diversity remains unchanged after drug administration. Our results provide a powerful perspective on drug tolerance based on general arguments, without requiring knowledge of the underlying molecular architecture of the heterogeneous cell states.
Characterisation of the historic demographic decline of the British European polecat population
Authors: Shaw R. , MacPherson J. , Kitchener A. C. , Etherington G. J. , Haerty W.
Published: November 2024

The European polecat (Mustela putorius) has a widespread distribution across many countries of mainland Europe, but is documented to be declining within these ranges. In Britain, direct persecution led to a severe decline of the polecat population during the 19thcentury. Unlike European mainland populations, it is now recovering across much of its former range. The genomic and conservation implications of such a severe demographic decline, followed by the current recovery, have still to be characterised in the European polecat in Britain. Here we carry out population-level whole-genome analyses of 65 polecats from Britain (Wales, England) and the European mainland. Welsh polecats show a unique genetic structure compared to English and European populations. We also reconstructed the demographic history of the Welsh polecat to quantify the magnitude of the bottleneck. Our analyses confirmed the drastic decline of the Welsh polecat’s effective population size, with a severe genetic bottleneck around 40 generations ago (1854-1872). We investigated whether genomic signatures reflected this demographic event and found that Welsh polecats had significantly less genetic diversity than English ones, but not European polecats. Runs of homozygosity and the genetic load present in Welsh polecat genomes also indicated recent historic inbreeding. We show that the increase of the British polecat population could be due to admixture events, but also that the Welsh polecat serves as a distinct genetic population, which could be crucial for the overall conservation of European polecats.
NANUQ+: A divide-and-conquer approach to network estimation
Authors: Allman Elizabeth S. , Baños Hector , Rhodes John A. , Wicke Kristina
Published: November 2024

Inference of a species network from genomic data remains a difficult problem, with recent progress mostly limited to the level-1 case. However, inference of theTree of Blobsof a network, showing only the network’s cut edges, can be performed for any network by TINNiK, suggesting a divide-and-conquer approach to network inference where the tree’s multifurcations are individually resolved to give more detailed structure. Here we develop a method, NANUQ+, to quickly perform such a level-1 resolution. Viewed as part of the NANUQ pipeline for fast level-1 inference, this gives tools for both understanding when the level-1 assumption is likely to be met and for exploring all highly-supported resolutions to cycles.
In a model of parasite-mediated exhaustion, stem-like CD8 T cells differentiate into an unconventional intermediate effector memory subset
Authors: Moretto Magali M. , Chen Keer , Cox Christina , Chen Jie , Khan Imtiaz A
Published: November 2024

CD8 T cell exhaustion has been reported in mice susceptible toToxoplasma gondiiinfection. While the differentiation of CD8 exhausted subsets has been extensively reported, most of these studies have been conducted in chronic viral and cancer models. During chronicT. gondiiinfection, phenotypic and transcriptomic analyses of the polyclonal antigen-specific CD8 T cell response characterize four populations based on KLRG1 and CD62L expression. Pop1 (KLRG1+CD62Llo) bears the attributes of a terminal effector subset, and pop2 (KLRG1-CD62Llo) is similar to effector memory CD8 T cells. Akin to chronic viral infection and cancer systems, pop3 (KLRG1-CD62Lhi) exhibits the characteristics of stem-like progenitor CD8 T cells (high Tcf7, Slamf6, and Cxcr5 expression), whereas pop4 (KLRG1+CD62Lhi) closely resembles a transitory subset (elevated Tbx21, low Tcf1, and Tox expression). During chronic viral infection, the stem-like progenitor CD8 T cells transition into a terminally differentiated exhausted subset via an intermediate population. However, in our system, pop3 generates pop4, which does not convert into a conventional terminally differentiated exhausted subset but instead transitions into effector pop1. Notably, during the chronic phase of the infection, pop1 cannot retain its functionality, irrespective of its origin, which may hamper its ability to control reactivation. Our observations emphasize that the differentiation of exhausted CD8 T cells in non-viral infections, like chronic toxoplasmosis, follows a different pattern than established models and highlights the need to develop new immune strategies better tailored for a broad range of pathogens.
In-silico Targeting Phosphodiesterase 5 and In-vivo Evaluation of the Aphrodisiac Effect ofAzanza garckeanaFruit
Authors: InnocentMary Ifedibaluchukwu Ejiofor , Ifeoma Ezeanya Jennifer , Ifeoma Ibe Chioma , Cyril Ogbue Onyeka , Chisom Emodi Rita , Chinwendu Agueze Jennifer , Sylvanus Ani Chibuike
Published: November 2024

Sexual dysfunction is a persistent problem affecting men, with erectile dysfunction (ED) and premature ejaculation being the most common. The current treatment involving the use of PhPDE5 inhibitors, such as sildenafil, is faced with some serious side effects. In Nigeria, there are claims thatAzanza garckeanafruit has positive effects in male sexual dysfunction. This study aims to evaluate this claim of the aphrodisiac effects ofAzanza garckeana, using computational and animal models. The 3D structures of the protein target of interest (phosphodiesterase) and 21 phytocompounds already isolated from and identified in the fruit were obtained from the Protein Data Bank and PubChem, respectively. The protein targets and the phytocompounds were prepared for molecular docking simulation using the Autodock tool. The docking was performed using Autodock-vina on the Linux platform. Phytocompounds with high specificity for the protein targets were identified, and their solubility profile was obtained. The fruits were extracted with ethanol using a Soxhlet apparatus at 60°C and concentrated with a rotary evaporator at 40°C. Phytochemical tests were conducted on the extract, and acute toxicity was evaluated using OECD 423 guidelines. The aphrodisiac effect was studied in-vivo using a rat model. Parameters such as mount and intromission frequencies, mount and ejaculation latency and testosterone levels were assessed. Three phytocompounds showed better binding affinity for phosphodiesterase 5 than the reference compounds and were also found to be a good target for the proteins of interest. The extract was found to contain steroids, flavonoids, and terpenoids. The results from the in-vivo study demonstrated significant increases (p<0.05) in mean mounting and intromission frequencies and ejaculatory latency following treatment with the extract at 500 mg/kg body weight. The study also demonstrated a significant decrease (p<0.05) in mount latency. Testosterone levels increased significantly (p<0.05) at 500mg/kg body weight. This study affirms the aphrodisiac effect ofAzanza garckeanafruit in the albino rat model and suggests that the fruit’s ethanol extract holds promise as an important phytomedicine for the development of more effective treatments for male sexual dysfunction.
Ontology-based expansion of virtual gene panels to improve diagnostic efficiency for rare genetic diseases
Authors: Shin Jaemoon , Fujiwara Toyofumi , Saitsu Hirotomo , Yamaguchi Atsuko
Published: November 2024

Background

Virtual Gene Panels (VGP) comprising disease-associated causal genes are utilized in the diagnosis of rare genetic diseases to evaluate candidate genes identified by whole-genome and whole-exome sequencing. VGPs generated by the PanelApp software were utilized in a UK 100,000 Genome Project pilot study to filter candidate genes, thus enhancing diagnostic efficiency for rare diseases. However, PanelApp also filtered out disease-causing genes in nearly 50% of the cases.

Methods

Here, we propose various methods for optimized approach to design VGPs that significantly improve the diagnostic efficiency by leveraging the hierarchical structure of the Mondo disease ontology, without excluding disease-causing genes. We also performed computational experiments on an evaluation dataset comprising 74 patients to determine the optimal VGP design method.

Results

Our results demonstrate that the proposed method can significantly enhance rare disease diagnosis efficiency by automatically identifying candidate genes. The proposed method successfully designed VGPs that improve diagnosis efficiency without excluding disease-causing genes.

Conclusion

We have developed novel methods for VGP design that leverage the hierarchical structure of the Mondo disease ontology to improve rare genetic disease diagnosis efficiency. This approach identifies candidate genes without excluding disease-causing genes, and thereby improves diagnostic efficiency.
Development and validation of an all-in-one Bat-Clade genomic sequencing and host identification protocol
Authors: de Souza Godinho Fernanda Marques , Campos Aline , Huff Rosana , Ruivo Amanda Pellenz , Bermann Thales , Bauerman Milena , dos Santos Franciellen Machado , Selayaran Taina Machado , Correa Artur Beineke , dos Santos Raissa Nunes , Roehe Paulo Michel , Wallau Gabriel da Luz , Salvato Richard Steiner
Published: November 2024

Rabies virus (RABV), a fatal zoonotic pathogen, remains a significant public health concern, with bat-maintained lineages accounting for all currently documented cases in Brazil. Despite the availability of pharmacological prophylaxis for humans and animals, the high genetic diversity of RABV in diverse natural bat hosts and continued circulation in multiple animals pose challenges for effective surveillance. Here, we developed and validated a novel, rapidly deployable amplicon-based sequencing approach for rabies virus (RABV) genomic surveillance. This “all-in-one” protocol integrates whole RABV genome sequencing with host species identification through COI gene amplification and sequencing, addressing the challenges posed by RABV’s high genetic diversity and complex transmission dynamics. We assessed the protocol’s effectiveness by sequencing 25 near-complete RABV genomes from host species across four distinct families (Bovidae, Equidae, Felidae, and Microchiroptera) obtained from the Rabies Control and Surveillance Program in Rio Grande do Sul State, Southern Brazil. The method achieved an average genome coverage of 91.4% at a minimum 5x read depth, with a mean depth coverage of 816x across sequenced genomes. The results demonstrated significant Bat-Clade sublineage diversity, which was classified using the MADDOG RABV lineage system. The protocol successfully identified three bat species (Tadarida brasiliensis, Desmodus rotundus, andMyotis nigricans) among the samples, highlighting its capability for precise host identification. This study presents a powerful tool for high-resolution evaluation of RABV genomic features and host identification, enabling more targeted public health interventions. This new approach has the potential to enhance RABV surveillance capabilities, contributing to more effective rabies control strategies within a One Health framework.
Application of ConvNeXt with Transfer Learning and Data Augmentation for Malaria Parasite Detection in Resource-Limited Settings Using Microscopic Images
Authors: Mmileng Outlwile Pako , Whata Albert , Olusanya Micheal , Mhlongo Siyabonga
Published: November 2024

Malaria is one of the most widespread and deadly diseases across the globe, especially in sub-Saharan Africa and other parts of the developing world. This is primarily because of incorrect or late diagnosis. Existing diagnostic techniques mainly depend on the microscopic identification of parasites in the blood smear stained with special dyes, which have drawbacks such as being time-consuming, depending on skilled personnel and being vulnerable to errors.

This work seeks to overcome these challenges by proposing a deep learning-based solution in the ConvNeXt architecture incorporating transfer learning and data augmentation to automate malaria parasite identification in thin blood smear images. This study’s dataset was a set of blood smear images of equal numbers of parasitised and uninfected samples drawn from a public database of malaria patients in Bangladesh. To detect malaria in the given dataset of parasitised and uninfected blood smears, the ConvNeXt models were fine-tuned. To improve the effectiveness of these models, a vast number of data augmentation strategies was used so that the models could work well in various image capture conditions and perform well even in environments with limited resources. The ConvNeXt Tiny model performed better, particularly the re-tuned version, than other models, such as Swin Tiny, ResNet18, and ResNet50, with an accuracy of 95%. On the other hand, the re-modified version of the ConvNeXt V2 Tiny model reached 98% accuracy. These findings show the potential to implement ConvNeXt-based systems in regions with scarce healthcare facilities for effective and affordable malaria diagnosis.
Up sampled PSF enables high accuracy 3D super-resolution imaging with sparse sampling rate
Authors: Chen Jianwei , Shi Wei , Feng Jianzheng , Wang Jianlin , Liu Sheng , Li Yiming
Published: November 2024

Single-molecule localization microscopy (SMLM) provides nanoscale imaging, but pixel integration of acquired SMLM images limited the choice of sample rate which restricts the information content conveyed within each image. We propose an up-sampled point spread function (PSF) inverse modeling method for large-pixel single molecule localization, enabling precise 3D super-resolution imaging with sparse sampling rate. Our approach could reduce data volume or expand the field of view by nearly an order of magnitude, while maintaining high localization accuracy, greatly improving the imaging throughput with limited pixels offered by the existing cameras.
Single-cell multiomics of pediatric BM reveals age-dependent differences in lineage differentiation linked to stromal cell heterogeneity
Authors: Hanemaaijer Evelyn S. , Müskens Konradin F. , Kal Ireen J. , te Pas Brigit M. , Fryzik Patrycja , Epskamp Nina , Balwierz Aleksandra , Candelli Tito , de Jonge Wim J. , Margaritis Thanasis , Belderbos Mirjam E.
Published: November 2024

Childhood is critical for hematopoietic development and the onset of hematologic diseases. To explore hematopoietic changes from infancy through adolescence, we generated a multi-modal single-cell atlas capturing mRNA and surface protein expression of 90.710 bone marrow (BM) cells. This includes hematopoietic stem/progenitor cells and mesenchymal stromal cells, from seven pediatric individuals and two young adults. We demonstrate that young pediatric BM is distinct from adolescents/young adults (AYA), shifting from B-lineage dominance in early childhood to myeloid and T-lineage bias in adolescence. We uncover two distinct lymphoid progenitors (LyPs) subsets regulating this shift: CD127-positive LyPs with B-lineage output, most abundant in early childhood, and CD127-negative LyPs with lymphoid and myeloid features, more common in AYAs. Age-related changes in stromal composition and signaling, mediated by IL-7 and TGF-β1, correspond with this lineage shift. This study provides an in-depth resource for understanding healthy hematologic development and potential early-life perturbations underlying pediatric hematologic diseases.
The combined use of scRNA-seq and network propagation highlights key features of pan-cancer Tumor-Infiltrating T cells
Authors: Mangelinck Adèle , Molitor Elodie , Marchiq Ibtissam , Alaoui Lamine , Bouaziz Matthieu , Andrade-Pereira Renan , Darville Hélène , Becht Etienne , Lefebvre Céline
Published: November 2024

Improving the selectivity and effectiveness of drugs represents a crucial issue for future therapeutic developments in immuno-oncology. Traditional bulk transcriptomics faces limitations in this context for the early phase of target discovery as resulting gene expression levels represent the average measure from multiple cell populations. Alternatively, single cell RNA sequencing can dive into unique cell populations transcriptome, facilitating the identification of specific targets. Here, we generated Tumor-Infiltrating regulatory T cells (TI-Tregs) and exhausted T cells (Tex) gene signatures from a single cell RNA-seq pan-cancer T cell atlas. To overcome noise and sparsity inherent to single cell transcriptomics, we then propagated the gene signatures by diffusion in a protein-protein interaction network using the Patrimony high-throughput computing platform. This methodology enabled the refining of signatures by rescoring genes based on their biological connectivity and shed light not only on processes characteristics of TI-Treg and Tex development and functions but also on their immunometabolic specificities. The combined use of single cell transcriptomics and network propagation may thus represent an innovative and effective methodology for the characterization of cell populations of interest and eventually the development of new therapeutic strategies in immuno-oncology.
Dual fragmentation via collision-induced and oxygen attachment dissociations using water and its radicals for C=C position-resolved lipidomics
Authors: Takeda Hiroaki , Okamoto Mami , Takahashi Hidenori , Buyantogtokh Bujinlkham , Kishi Noriyuki , Okano Hideyuki , Kamiguchi Hiroyuki , Tsugawa Hiroshi
Published: November 2024

Oxygen attachment dissociation (OAD) is a tandem mass spectrometry (MS/MS) technique used to annotate the positions of double bonds (C=C) in complex lipids. Although OAD has been used for untargeted lipidomics, its availability has been limited to the positive-ion mode, requiring the independent use of a collision-induced dissociation (CID) method. In this study, we demonstrated the OAD-MS/MS technique in the negative-ion mode for profiling phosphatidylserines, phosphatidylglycerols, phosphatidylinositols, and sulfatides, where the fragmentation mechanism remained consistent with that in the positive-ion mode. Furthermore, we proposed optimal conditions for the simultaneous acquisition of CID– and OAD-specific fragment ions, termed OAciD. In the collision cell for OAD, oxygen atoms and hydroxy radicals facilitate C=C position-specific fragmentation, while residual water vapor induces cleavage of low-energy covalent bonds, such as ester and peptide bonds, at higher collision energy values, preserving OAD-specific ions under high collision energy conditions. Finally, theoretical fragment ions were implemented in MS-DIAL 5 to accelerate C=C position-resolved untargeted lipidomics. The OAciD methodology was applied to lipid profiling of five marmoset brain regions: the frontal lobe, hippocampus, midbrain, cerebellum, and medulla. Region-specific marmoset lipidomes were characterized with C=C positional information, where the ratios of C=C positional isomers such as delta 9– and delta 11 of fatty acid 18:1 in phosphatidylcholine were also estimated using OAciD-MS/MS. In addition, we characterized the profiles of polyunsaturated fatty acid-containing complex lipids with C=C positional information, where lipids containing omega-3 fatty acids were enriched in the cerebellum, while those containing omega-6 fatty acids were more abundant in the hippocampus and frontal lobe.
Antigen mobility regulates the dynamics and precision of antigen capture in the B cell immune synapse
Authors: McArthur Hannah C. W. , Iliopoulou Maro , Bajur Anna T. , Spillane Katelyn M.
Published: November 2024

B cells discriminate antigens in immune synapses by capturing them from antigen-presenting cells. This discrimination relies on the application of mechanical force to B cell receptor (BCR)-antigen bonds, allowing B cells to selectively disrupt low-affinity interactions while internalizing high-affinity antigens. Using DNA-based tension sensors combined with high-resolution imaging, we demonstrate that the magnitude, location, and timing of forces within the immune synapse are influenced by the fluidity of the antigen-presenting membrane. Transitioning antigens from a high-mobility to a low-mobility substrate significantly increases the probability and speed of antigen extraction while also improving affinity discrimination. This shift in antigen mobility also reshapes the synapse architecture, altering spatial patterns of antigen uptake. Despite these adaptations, B cells maintain consistent levels of proximal and downstream signaling pathway activation regardless of antigen mobility. They also efficiently transport internalized antigens to major histocompatibility complex class II (MHCII)-positive compartments for processing. These results demonstrate that B cells mount effective responses to antigens across diverse physical environments, though the characteristics of that environment may influence the speed and accuracy of B cell adaptation during an immune response.
Fertility Preservation of Vacuum-Dried Ram Spermatozoa Stored for Four Years at Room Temperature
Authors: Moncada Margherita , Lo Sterzo Martina , Palazzese Luca , Boffa Francesca , Anzalone Debora Agata , Iuso Domenico , Czernik Marta , Gioia Luisa , Turri Federica , Mislei Beatrice , Bucci Diego , Bonnet Jacques , Colotte Marthe , Tuffet Sophie , Bochenek Michał , Loi Pasqualino
Published: November 2024

Dry storage at room temperature (RT) could simplify spermatozoa banking. Here, we explored DNA stability andin vitroandin vivodevelopment of embryos derived from vacuum-dried encapsulated (VDE) ram spermatozoa stored for four years or after accelerated aging. While some genomic damage was detected at time 0, DNA fragmentation increased from 3.32±3% (time 0) to 37.64±4% (4 years). A decrease in blastocyst rate was observed after four years of storage and 6.7 years of simulated storage (10.2% and 9%versus13.16% at time 0). Embryo quality, assessed based onCdx2and Inf-τ gene expression, declined over time. Only two of the 23 embryos transferred into synchronized ewes were implanted but were lost by day 40.

In conclusion, dry spermatozoa generated blastocysts after four years of RT storage, but their post-implantation development was impaired. Optimization of the water extraction and storage conditions could better preserve the spermatozoa’s DNA integrity, resulting in improved embryo quality, compatible with development to term.
ATGL-mediated lipolysis is essential for myocellular mitochondrial function and augments PPARδ-induced improvements in mitochondrial respiration
Authors: Gemmink Anne , van de Weijer Tineke , Schaart Gert , Grabner Gernot F. , Kornips Esther , Knoops Kèvin , Zechner Rudolf , Schweiger Martina , Hesselink Matthijs K.C.
Published: November 2024

Defects in ATGL-mediated myocellular LD lipolysis results in mitochondrial dysfunction of unknown origin, which can be rescued by PPAR agonists. Here we examine whether ATGL-mediated lipolysis is required to maintain mitochondrial network connectivity and function. Moreover, we explored if the functional implications of ATGL deficiency for mitochondrial network dynamics and function can be alleviated by promoting PPARα and/or PPARδ transcriptional activity. To this end, we cultured human primary myotubes from patients with neutral lipid storage disease with myopathy (NLSDM), a rare metabolic disorder caused by a mutation in the PNPLA2 gene. These myotubes possess dysfunctional ATGL and compromised LD lipolysis. In addition, mitochondria-LD contacts, mitochondrial network dynamics, and TMRM intensity were abrogated. Using a humanized ATGL inhibitor in myotubes cultured form healthy donors, revealed similar results. Upon stimulating PPARδ transcriptional activity, mitochondrial respiration improved by more than 50% in human primary myotubes from healthy lean individuals. This increase in respiration was dampened in myotubes with dysfunctional ATGL. Stimulation of PPARδ transcriptional activity had no effect on mitochondria-LD contacts, mitochondrial network dynamics, and TMRM intensity. Our results demonstrate that dysfunctional ATGL results in compromised mitochondrial-LD contacts and mitochondrial dynamics, and that functional ATGL is required to improve mitochondrial respiratory capacity upon stimulation of PPARδ transcriptional activity.
Model-free photon analysis of diffusion-based single-molecule FRET experiments
Authors: Terterov Ivan , Nettels Daniel , Lastiza-Male Tanya , Bartels Kim , Loew Christian , Vancraenenbroeck Rene , Carmel Itay , Rosenblum Gabriel , Hofmann Hagen
Published: November 2024

Photon-by-photon analysis tools for diffusion-based single-molecule Förster resonance energy transfer (smFRET) experiments often describe protein dynamics with Markov models. However, FRET efficiencies are only projections of the conformational space such that the measured dynamics can appear non-Markovian. Model-free methods to quantify FRET efficiency fluctuations would be desirable in this case. Here, we present such an approach. We determine FRET efficiency correlation functions free of artifacts from the finite length of photon trajectories or the diffusion of molecules through the confocal volume. We show that these functions capture the dynamics of proteins from micro-to milliseconds both in simulation and experiment, which provides a rigorous validation of current model-based analysis approaches.
In SituSingle-Cell Spatial Profiling of Matrisome Gene Expression in Glioblastoma
Authors: De Arpan , Forero Santiago A. , Sebastian Sumod , Morales John E. , De La Fuente-Granada Marisol , Huse Jason T. , Ballester Leomar Y. , Weinberg Jeffrey S. , Lang Frederick F. , Akdemir Kadir C. , McCarty Joseph H.
Published: November 2024

Glioblastoma (GBM) is the most common and aggressive primary brain cancer in adults, characterized by robust infiltrative growth and invasion within the neural microenvironment. While various stromal cells, including astrocytes, microglia, and vascular cells in the microenvironment, communicate with GBM cells to promote growth and invasion, relatively less is known about another critical component of the tumor stroma: the extracellular matrix (ECM). The matrisome comprises hundreds of ECM genes and affiliated adhesion and signaling components; however, little is understood about (i) the repertoire of matrisome genes and proteins expressed in GBM, (ii) the stromal and cancer cells of origin that express different matrisome factors, and (iii) how these components are interconnected to drive tumor malignancy. Here, we have used in situ single-cell transcriptomics to analyze the spatial expression of nearly 350 genes that constitute the human brain matrisome. Our efforts have identified several differentially expressed matrisome genes involved in ECM adhesion and signaling between cancer and stromal cells. Additionally, matrisome genes associated with low-grade glioma pathogenesis and progression to high-grade GBM have been identified and spatially mapped. Collectively, these experiments reveal the single-cell origins of differentially expressed ECM biomarkers as well as their spatial expression patterns in brain tumors. These findings may lead to new therapeutic efforts to inhibit matrisome components to benefit patients with malignant brain cancer.
Ex VivoDelivery of mRNA to Immune CellsViaa Non-Endosomal Route Obviates the Need for Nucleoside Modification
Authors: Ghoshal Bartika , Chakraborty Debajyoti , Nag Manish , Varadarajan Raghavan , Jhunjhunwala Siddharth
Published: November 2024

Base modification and the use of lipid nanoparticles (LNPs) are thought to be essential for efficientin vivodelivery and expression of mRNA. However, forex vivoimmune cell engineering, the need for either of the two is unclear. Previous reports have suggested that nucleic acids may be efficiently delivered to immune cellsex vivo,through a non-endosomal delivery route, but the need for base modification has not been determined. Herein, we demonstrate that when a non-endosomal delivery method is used, unmodified mRNA performs equally well to the commonly used base-modified mRNA, including theN1methyl pseudo-uridine modification, in terms of protein expression and inflammatory response in cells. However, if an endosomal delivery route is used, thenN1methyl pseudo-uridine modification is necessary for high expression and low inflammatory response, as demonstrated by others as well. Overall, we show that non-endosomal mRNA delivery renders nucleoside modifications non-essential, and that unmodified mRNA combined with non-endosomal delivery route may be used for efficientex vivomRNA-based engineering of immune cells.
Quantifying the nuclear localisation of fluorescently tagged proteins
Authors: Hurbain Julien , ten Wolde Pieter Rein , Swain Peter S.
Published: November 2024

Cells are highly dynamic, continually responding to intra- and extracellular signals. Monitoring and measuring the response to these signals at the single-cell level is challenging. Signal transduction is fast, but reporters for downstream gene expression are typically slow, requiring fluorescent proteins to be expressed and to mature. An alternative is to fluorescently tag and then monitor the intracellular locations of transcription factors and other effectors. These proteins move in or out of the nucleus in minutes, after upstream signalling modifies their state of phosphorylation. Although such approaches are being used increasingly, there is no consensus on how to quantify the nuclear and cytoplasmic localisation of these proteins. Using budding yeast, we developed a convolutional neural network that quantifies nuclear localisation from fluorescence and, optionally, bright-field images. Focusing on the cellular response to changing glucose, we generated ground-truth data using strains with both a transcription factor and a nuclear protein tagged with fluorescent markers. We then showed that the neural network based approach outperformed seven published methods, particularly when predicting single-cell time series, which are key to determining how cells respond and adapt. Collectively, our results are conclusive — using machine learning to automatically determine the appropriate image processing consistently outperforms ad hoc methods. Adopting such methods promises to both improve the accuracy and, with transfer learning, the consistency of single-cell analyses.
The BBS/CCT chaperonin complex ensures the localization of the adhesion G protein-coupled receptor ADGRV1 to primary cilia
Authors: Linnert Joshua , Kusuluri Deva Krupakar , Güler Baran E. , Patnaik Sarita Rani , May-Simera Helen L. , Wolfrum Uwe
Published: November 2024

Primary cilia are antenna-like sensory organelles present on almost all eukaryotic cells. Their sensory capacity relies on receptors, in particular G-protein-coupled receptors (GPCRs) which localize to the ciliary membrane. Here we show that ADGRV1, a member of the GPCR subfamily of adhesion GPCRs, is part of a large protein network, interacting with numerous proteins of a comprehensive ciliary proteome. ADGRV1 is localized to the base of prototypic primary cilia in cultured cells and the modified primary cilia of retinal photoreceptors, where it interacts with TRiC/CCT chaperonins and the Bardet Biedl syndrome (BBS) chaperonin-like proteins. Knockdown of ADGRV1, CCT2 and 3, and BBS6 result in common ciliogenesis phenotypes, namely reduced ciliated cells combined with shorter primary cilia. In addition, the localization of ADGRV1 to primary cilia depends on the activity of a co-complex of TRiC/CCT chaperonins and the BBS chaperonin-like proteins. In the absence of components of the TRiC/CCT-BBS chaperonin co-complex, ADGRV1 is depleted from the base of the primary cilium and degraded via the proteasome. Defects in the TRiC/CCT-BBS chaperonin may lead to an overload of proteasomal degradation processes and imbalanced proteostasis. Dysfunction or absence of ADGRV1 from primary cilia may underly the pathophysiology of human Usher syndrome type 2 and epilepsy caused by mutations inADGRV1.
Association of oropharyngeal cancer recurrence with tumor-intrinsic and immune-mediated sequelae of reduced genomic instability
Authors: Sannigrahi Malay K. , Raghav Lovely , Rich Dominick J. , Schrank Travis P. , Califano Joseph A. , Lukens John N. , Sun Lova , Morgan Iain M. , Cohen Roger B. , Lin Alexander , Liu Xinyi , Brown Eric J. , You Jianxin , Mirabello Lisa , Mishra Sambit K. , Shimunov David , Brody Robert M , Pearson Alexander T. , Gimotty Phyllis A. , Diab Ahmed , Jalaly Jalal B. , Basu Devraj
Published: November 2024

Background

Limited understanding of the biology predisposing certain human papillomavirus-related (HPV+) oropharyngeal squamous cell carcinomas (OPSCCs) to relapse impedes therapeutic personalization. We aimed to identify molecular traits that distinguish recurrence-prone tumors.

Methods

50 HPV+ OPSCCs that later recurred (cases) and 50 non-recurrent controls matched for stage, therapy, and smoking history were RNA-sequenced. Groups were compared by gene set enrichment analysis, and select differences were validated by immunohistochemistry. Features discriminating groups were scored in each tumor using gene set variation analysis, and scores were evaluated for recurrence prediction ability.

Results

Cases downregulated pathways linked to anti-tumor immunity (FDR-adjusted p<.05) and contained fewer tumor-infiltrating lymphocytes (p<.001), including cytotoxic T-cells (p=.005). Cases also upregulated pathways related to cell division and other aspects of tumor progression. Upregulated and downregulated pathways were respectively used to define a tumor progression score (TPS) and immune suppression score (ISS) for each tumor. Correlation between TPS and ISS (r=.603, p<.001) was potentially explained by observed upregulation of DNA repair pathways in cases, which might enhance their progression directly and by limiting cytosolic DNA-induced inflammation. Accordingly, cases contained fewer double-strand breaks based on staining for phospho-RPA32 (p=.006) and γ-H2AX (p=.005) and downregulated pro-inflammatory components of the cytoplasmic DNA sensing pathway. A combined score derived from TPS and ISS optimized recurrence prediction and stratified survival in a manner generalizable to three external cohorts.

Conclusions

We provide novel evidence that limiting genomic instability makes tumor-intrinsic and immune-mediated contributions to HPV+ OPSCC recurrence risk, opening opportunities to detect and target this treatment-resistant biology.
Meta-atlas of Juvenile and Adult Enteric Neuron scRNA-seq for Dataset Comparisons and Consensus on Transcriptomic Definitions of Enteric Neuron Subtypes
Authors: Benthal Joseph T. , May-Zhang Aaron A. , Southard-Smith E. Michelle
Published: November 2024

Background

The enteric nervous system (ENS) is a complex network of interconnected ganglia within the gastrointestinal (GI) tract. Among its diverse functions, the ENS detects bowel luminal contents and coordinates the passing of stool. ENS defects predispose to GI motility disorders. Previously, distinct enteric neuron types were cataloged by dye-filling techniques, immunohistochemistry, retrograde labeling, and electrophysiology. Recent technical advances in single cell RNA-sequencing (scRNA-seq) have enabled transcriptional profiling of hundreds to millions of individual cells from the intestine. These data allow cell types to be resolved and compared to using their transcriptional profiles (“clusters”) rather than relying on antibody labeling. As a result, greater diversity of enteric neuron types has been appreciated. Because each scRNA-seq study has relied on different methods for cell isolation and library generation, numbers of neuron clusters and cell types detected differs between analyses. Cell counts in each dataset are particularly important for characterization of rare cell types since small numbers of profiled cells may not sample rare cell types. Importantly, each dataset, depending on the isolation methods, may contain different proportions of cells that are not detected in other datasets. Aggregation of datasets can effectively increase the total number of cells being analyzed and can be helpful for confirming the presence of low-abundance neuron types that might be absent or observed infrequently in any single dataset.

Results

Here we briefly systematically review eachMus musculussingle cell or single nucleus RNA-sequencing enteric nervous system dataset. We then reprocess and computationally integrate these select independent scRNA-seq enteric neuron datasets with the aim to identify new cell types, shared marker genes across juvenile to adult ages, dataset differences, and achieve some consensus on transcriptomic definitions of enteric neuronal subtypes.

Conclusions

Data aggregation generates a consensus view of enteric neuron types and improves resolution of rare neuron classes. This meta-atlas offers a deeper understanding of enteric neuron diversity and may prove useful to investigators aiming to define alterations among enteric neurons in disease states. Future studies face the challenge of connecting these deep transcriptional profiles for enteric neurons with historical classification systems.
Source amplitude increases with body mass across avian genera
Authors: Ziegenhorn Morgan A. , Lanctot Richard B. , Brown Stephen C. , Saalfeld Sarah T. , Smith Paul A. , Lecomte Nicolas
Published: November 2024

Amplitude, or intensity, of sound is a fundamental characteristic of inter acoustic communication, with relevance in many scientific fields. The amplitude of an animal’s acoustic signal at its source (“source amplitude”) may be particularly relevant in the field of acoustic allometry, where relationships between species’ physical and acoustic features (e.g., dominant frequency) have been well-established across taxa. However, despite their potential scientific value, records and studies of source amplitudes remain remarkably scarce for avian species. Here we present novel estimates of source amplitude (range, mean, and median) for 17 species of Arctic-breeding birds, derived from measurements made in Utqiaġvik, Alaska during June 2024. We found a strong positive correlation between body mass and source amplitude in this data via a Markov Chain Monte Carlo multivariate generalized linear mixed model (MCMCglmm). Both phylogeny and individual identity were important random effects in this model. In contrast, random effects from environmental factors and measurement characteristics were minimal. Our work represents the first model describing the relationship between source amplitude and allometry across avian genera. We hope that this study will spur other investigations into avian source amplitude and its relationship to morphological and life history features for species in the Arctic and elsewhere.
High-throughput measurement of adipocyte size with open-source software using whole-slide adipose tissue images
Authors: Alan Ramalho , Marie-Frédérique Gauthier , Ina Maltais-Payette , Giada Ostinelli , Frédéric Hould , Laurent Biertho , André Tchernof
Published: November 2024

The aim of this study was to create and validate a high-throughput method based on open-source software for the measurement of adipocyte diameters in white adipose tissue histological sections. Human omental and subcutaneous adipose tissue samples collected during bariatric surgery were used to prepare hematoxylin and eosin-stained histological slides. Digital images were acquired. Adipocyte diameters were measured both manually and with an automated procedure created using ImageJ. Comparative analysis of our automated method with the manual measurement and associations of the mean adipocyte diameters with cardiometabolic markers were used to validate our method. A total of 377 adipose samples (190 participants) were included in the analysis. Pearson correlation of mean adipocyte diameters shows a strong linear relationship between methods (r=0.88, p<0.0001). The average diameter measured with the automated method was significantly smaller (8.1±5.3µm difference, p<0.0001) compared to the manual method, likely reflecting bias in selecting the cells measured with the manual approach. Pearson correlation analyses between mean omental adipocyte diameters and markers of cardiometabolic risk show that the diameters of both methods are significantly associated with the same parameters (fasting concentrations of TG, HDL-Chol, homeostasis model assessment insulin resistance, and visceral adiposity index values) with no significant differences between methods. There were also no significant differences between the manual and automated method regarding the correlations between mean subcutaneous adipocyte diameters and anthropometric or metabolic markers. In conclusion, we have created and validated a rapid automated method based on open-source software to measure adipocyte diameters from whole-slide adipose tissue images.
Epigenetic silencing and host genome evolution determine fate of viral insertions inAcanthamoeba
Authors: Blais Cédric , Colp Morgan J. , Sarre Luke A. , de Mendoza Alex , Archibald John M.
Published: November 2024

Acanthamoebais a cosmopolitan freshwater amoebae known for its association with Nucleocytoplasmic Large DNA Viruses (NCLDVs). Previous studies have shown thatAcanthamoebaspp. undergo lateral gene transfer (LGT) with NCLDVs. Here we have leveraged chromosome-scale assemblies of two strains ofAcanthamoeba castellanii, Neff and C3, to investigate the occurrence and genomic context of viral LGT inAcanthamoeba. We show that the viral ‘footprints’ in the C3 and Neff genomes are largely non-overlapping and that viral genes in Neff are found disproportionately in large sub-telomeric insertions. Multiple partial copies of these insertions are found throughout the Neff genome, but they are not expressed and do not encode functions for their own mobility. Viral regions are hyper-methylated and highly condensed, suggesting that the expression of recently acquired viral DNA is suppressed in heterochromatic regions. We propose a three-step model for the origin and fate of viral sequences inAcanthamoeba: (i) integration of DNA from giant viruses, (ii) epigenetic suppression of the viral DNAs, which allows them to persist in the genome, and (iii) deterioration of viral genomes by point mutation and intra- and inter-chromosomal recombination. Viral integrations inAcanthamoebaspp. are transient and may not have long-lasting effects on the fitness of the host amoeba. Our work strengthens a growing body of work showing widespread but transient integration of viral DNA in protists and extends the relevance of epigenetic silencing mechanisms to the evolution ofAmoebozoa. We highlight the importance of host genome dynamics for understanding the evolution of endogenized viral elements.