bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthetic dosage-compensating miRNA circuits for quantitative gene therapy
Authors: Flynn Michael James , Mayfield Acacia M. , Du Rongrong , Gradinaru Viviana , Elowitz Michael B
Published: March 2024

A longstanding challenge in gene therapy is expressing a dosage-sensitive gene within a tight therapeutic window. For example, loss of MECP2 function causes Rett syndrome, while its duplication causes MECP2 duplication syndrome. Viral gene delivery methods generate variable numbers of gene copies in individual cells, creating a need for gene dosage-invariant expression systems. Here, we introduce a compact miRNA-based, incoherent feed-forward loop circuit that achieves precise control of Mecp2 expression in cells and brains, and improves outcomes in an AAV-based mouse model of Rett syndrome gene therapy. Single molecule analysis of endogenous and ectopic Mecp2 mRNA revealed precise, sustained expression across a broad range of gene dosages. Delivered systemically in a brain-targeting AAV capsid, the circuit strongly suppressed Rett behavioral symptoms for over 24 weeks, outperforming an unregulated gene therapy. These results demonstrate that synthetic miRNA-based regulatory circuits can enable precise in vivo expression to improve the safety and efficacy of gene therapy.
Electrochemical Biosensor Using Polymer Nanocomposites for G6PD Deficiency Detection
Authors: Salas Ronald Rastre , Paula Marcos Marques da Silva , Romaguera Barcelay Yonny , Brito Walter Ricardo
Published: March 2024

Electrochemical biosensors based on the combination of DHP, OG, AuNPs, and G6PD, used to determine the enzymatic activity of G6PD, showed optimal current intensity values close to pH 7.8 at room temperature. Square wave voltammetry was optimized and used to evaluate the responses of the biosensors during the enzymatic reaction and construct the analytical curves. The best results were found under the following conditions: 30 minutes of incubation, in the range of NAD+concentrations from 0.75 to 6 μmol L−1; obtaining coefficient of determination (R2) values between 98 and 99% about the analytical curves. The biosensors maintained their stability after five days of storage. The calculated LOD and LOQ limits showed good sensitivity of the method, achieving results in the order of 10-6mol L−1.
Rare Copy Number Variant analysis in case-control studies using SNP Array Data: a scalable and automated data analysis pipeline
Authors: Artaza Haydee , Lavrichenko Ksenia , Wolff Anette S.B. , Røyrvik Ellen C. , Vaudel Marc , Johansson Stefan
Published: March 2024

Background

Rare copy number variants (CNVs) significantly influence the human genome and may contribute to disease susceptibility. High-throughput SNP genotyping platforms provide data that can be used for CNV detection, but it requires the complex pipelining of bioinformatic tools. Here, we propose a flexible bioinformatic pipeline for rare CNV analysis from human SNP array data.

Results

The pipeline performs two major tasks: (1) CNV detection and quality control, and (2) rare CNV analysis. It is implemented in Snakemake following a rule-based structure that enables automation and scalability while maintaining flexibility.

Conclusions

Our pipeline automates the detection and analysis of rare CNVs. It implements a rigorous CNV quality control, assesses the frequencies of these rare CNVs in patients versus controls, and evaluates the impact of CNVs on specific genes or pathways. We hence aim to provide an efficient yet flexible bioinformatic framework to investigate rare CNVs in biomedical research.
The molecular basis of pine wilt disease resistance inPinus massoniana
Authors: Wang Jinjin , Cai Shouping , Zeng Liqiong , Tong Jie , Hang Danling , Zhang Xinliang , Fang Yu , Su Shunde , Su Jun
Published: March 2024

Pine wilt disease (PWD), caused by the pine wood nematode (PWN)Bursaphelenchus xylophilus, results in significant economic and ecological damage toPinusforests and plantations worldwide.Pinus massonianais the primary host of PWD in southern China, but its response to the PWN remains largely unstudied. Previously, we observed PWD in aP. massoniananursery that contained over 71 commonly used cultivars. Through field phenotyping, we identified two groups of PWN resistant cultivars. Resistant cultivars (RC) exhibited very low PWN carrying amounts (PCA) and had relatively low mortality, and tolerant cultivars (TC) had high PCA but low mortality. In this study, we confirmed via PWN inoculation assays that the resistant and tolerant cultivars had lower mortality rates, 10% and 11%, respectively, than other cultivars (which had a mortality rate of 83.3%). The RC had a PCA that was significantly lower than that of other cultivars, while the TC exhibited a higher PCA. To explore the molecular mechanisms underlying the response ofP. massonianato PWN, the transcriptome and metabolome of the above cultivars were profiled by high throughput sequencing. As no reference genome is available forP. massoniana, we generated a new full-length transcriptome library with iso-seq. Using the transcriptome and metabolome from differentP. massonianacultivars inoculated with PWN, we found three major PWD resistance strategies. 1) The common response strategy involved three important molecular pathways. First, synthesis of indole-3-acetic acid (IAA) and abcisic acid (ABA) were suppressed during PWN invasion, thus suppressing the synthesis of polysaccharides (especially myo-inositol and trehalose) and (-)-riboflavin through ABC transporters. Second, by inhibiting aspartic acid, the synthesis of arginine and proline through APS5 (aspartate aminotransferase 5) were suppressed. Third, by reducing cysteine, GERD (germacrene D synthase) was up-regulated, and sesquiterpenoid and triterpenoid metabolites were accumulated to resist PWN invasion. 2) The most effective resistance strategy involved the accumulation of reactive oxygen species (ROS), which enhanced jasmonic acid (JA) accumulation and highly induced the expression of chitinase, thus improving resistance to PWN. 3) The tolerance strategy involved the induction of phosphatidylcholine, which promoted flavonoid and anthocyanin synthesis via LPIN (phosphatidate phosphatase), LOX2S (lipoxygenase), and LOX1_5 (linoleate 9S-lipoxygenase), thus significantly inhibiting the pathogenicity of the PWN but not the PCA. These results illustrate the molecular mechanisms by whichP. massonianacultivars resist or tolerate PWN and are of great significance in the prevention and control of PWD.
Protein nanoparticle vaccines induce potent neutralizing antibody responses against MERS-CoV
Authors: Chao Cara W. , Sprouse Kaitlin R. , Miranda Marcos C. , Catanzaro Nicholas J. , Hubbard Miranda L. , Addetia Amin , Stewart Cameron , Brown Jack T. , Dosey Annie , Valdez Adian , Ravichandran Rashmi , Hendricks Grace G. , Ahlrichs Maggie , Dobbins Craig , Hand Alexis , Treichel Catherine , Willoughby Isabelle , Walls Alexandra C. , McGuire Andrew T. , Leaf Elizabeth M. , Baric Ralph S. , Schäfer Alexandra , Veesler David , King Neil P.
Published: March 2024

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic betacoronavirus that causes severe and often lethal respiratory illness in humans. The MERS-CoV spike (S) protein is the viral fusogen and the target of neutralizing antibodies, and has therefore been the focus of vaccine design efforts. Currently there are no licensed vaccines against MERS-CoV and only a few candidates have advanced to Phase I clinical trials. Here we developed MERS-CoV vaccines utilizing a computationally designed protein nanoparticle platform that has generated safe and immunogenic vaccines against various enveloped viruses, including a licensed vaccine for SARS-CoV-2. Two-component protein nanoparticles displaying MERS-CoV S-derived antigens induced robust neutralizing antibody responses and protected mice against challenge with mouse-adapted MERS-CoV. Electron microscopy polyclonal epitope mapping and serum competition assays revealed the specificities of the dominant antibody responses elicited by immunogens displaying the prefusion-stabilized S-2P trimer, receptor binding domain (RBD), or N-terminal domain (NTD). An RBD nanoparticle vaccine elicited antibodies targeting multiple non-overlapping epitopes in the RBD, whereas anti-NTD antibodies elicited by the S-2P– and NTD-based immunogens converged on a single antigenic site. Our findings demonstrate the potential of two-component nanoparticle vaccine candidates for MERS-CoV and suggest that this platform technology could be broadly applicable to betacoronavirus vaccine development.
Exploiting WEE1 kinase activity as FUS::DDIT3-dependent therapeutic vulnerability in myxoid liposarcoma
Authors: Heinst Lorena , Lee Kwang Seok , Berthold Ruth , Isfort Ilka , Wosnig Svenja , Kuntze Anna , Hafner Susanne , Altvater Bianca , Rössig Claudia , Åman Pierre , Wardelmann Eva , Scholl Claudia , Hartmann Wolfgang , Fröhling Stefan , Trautmann Marcel
Published: March 2024

The pathognomonic FUS::DDIT3 fusion protein drives myxoid liposarcoma (MLS) tumorigenesis via aberrant transcriptional activation of oncogenic signaling. Since FUS::DDIT3 has so far not been pharmacologically tractable to selectively target MLS cells, this study investigated the functional role of the cell cycle regulator WEE1 as novel FUS::DDIT3-dependent therapeutic vulnerability in MLS. Here we demonstrate that enhanced WEE1 pathway activity represents a hallmark of FUS::DDIT3-expressing cell lines as well as MLS tissue specimens and that WEE1 is required for MLS cellular survivalin vitroandin vivo. Pharmacologic inhibition of WEE1 activity results in DNA damage accumulation and cell cycle progression forcing cells to undergo apoptotic cell death. In addition, our results uncover FUS::DDIT3-dependent WEE1 expression as an oncogenic survival mechanism to tolerate high proliferation and resulting replication stress in MLS. Fusion protein-driven G1/S cell cycle checkpoint deregulation via overactive Cyclin E/CDK2 complexes thereby contributes to enhanced WEE1 inhibitor sensitivity in MLS. These findings identify WEE1-mediated replication stress tolerance as molecular vulnerability in FUS::DDIT3-driven MLS tumorigenesis that could represent a novel target for therapeutic intervention.
Viewing stomata in action: Autonomousin plantaimaging of individual stomatal movement
Authors: van den Berg Tomas E. , Sanders Remco G. P. , Kaiser Elias , Schmitz Jurriaan
Published: March 2024

Stomata regulate plant gas exchange under changing environments, but observations of the dynamics of single stomatain plantaare sparse. We developed a compact microscope system that can measure the kinetics of tens of stomatain plantasimultaneously, with sub-minute time resolution. Dark field imaging with green light was used to create 3D stacks from which 2D surface projection were constructed to resolve stomatal apertures within the field of view.

Stomatal dynamics ofChrysanthemum morifolium (Chrysanthemum)andZea Mays(Maize) under dynamically changing light intensity were categorized, and a kinetic model was fitted to the data for quantitative comparison. In addition, we also resolved dynamics of the surface position of the leaf, related to dynamics of leaf thickness or bending.

Maize stomata oscillated frequently between open and closed states under constant growth light and these oscillating stomata responded faster to changes in light than non-oscillating stomata at the same aperture.

The slow closure of Chrysanthemum stomata reduced water use efficiency (WUE). Over 50% showed delayed or partial closure, leading to unnecessarily large apertures after reduced light. Stomata with larger apertures had more lag and similar closure speeds compared to those with smaller apertures and lag, further reducing WUE. In contrast, maize stomata with larger apertures closed faster, with no lag.

In conclusion, our new system enables fine mapping of the heterogeneity of movement in neighboring stomata, providing new insights on the relations between stomatal dimensions, relative position and aperture changes under fluctuating light intensity.
Muscle fiber Myc is dispensable for muscle growth and forced expression severely perturbs homeostasis
Authors: Ham Daniel J. , Semeraro Michelangelo , Berger Bianca M. , Lin Shuo , Maino Eleonora , Oliveri Filippo , Rüegg Markus A.
Published: March 2024

The oncogenic transcription factor Myc stimulates many growth processes including cell cycle progression and ribosome biogenesis. Myc expression is low in adult skeletal muscle, but is upregulated upon growth stimuli. Furthermore, muscle fiber Myc overexpression recapitulates many aspects of growth-related gene expression, suggesting Myc may mediate pro-growth responses to anabolic stimuli, such as exercise. Here, we tested this hypothesis by examining mouse models in which Myc was specifically eliminated or overexpressed in skeletal muscle fibers or muscle stem cells (MuSC). While muscle fiber Myc expression increased during muscle growth and Myc expression in MuSCs was required for successful muscle regeneration, muscle fiber Myc expression was dispensable for post-natal, mechanical overload or PKB/Akt-induced muscle growth in mice. Similarly, constitutive Myc expression did not promote skeletal muscle hypertrophy, but instead impaired muscle fiber structure and function within days. These data question the role of Myc in skeletal muscle growth.
Bayesian semantic surprise based on different types of regularities predicts the N400 and P600 brain signals
Authors: Hodapp Alice , Lindborg Alma , Rabovsky Milena
Published: March 2024

The brain’s remarkable ability to extract patterns from sequences of events has been demonstrated across cognitive domains and is a central assumption of predictive processing theories. While predictions shape language processing at the level of meaning, little is known about the underlying learning mechanism. Here, we investigated how continuous statistical inference in a semantic sequence influences the neural response. 60 participants were presented with a semantic oddball-like roving paradigm, consisting of sequences of nouns from different semantic categories. Unknown to the participants, the overall sequence contained an additional manipulation of transition probability between categories. Two Bayesian sequential learner models that captured different aspects of probabilistic learning were used to derive theoretical surprise levels for each trial and investigate online probabilistic semantic learning. The N400 ERP component was primarily modulated by increased probability with repeated exposure to the categories throughout the experiment, which essentially represents repetition suppression. This N400 repetition suppression likely prevented sizeable influences of more complex predictions such as those based on transition probability, as any incoming information was already continuously active in semantic memory. In contrast, the P600 was associated with semantic surprise in a transition probability model over recent observations, possibly indicating a working memory update in response to violations of these conditional dependencies. The results support probabilistic predictive processing of semantic information and demonstrate that continuous update of distinct statistics differentially influences language related ERPs.
N-acetylcysteine counteracts immune dysfunction and autistic-related behaviors in theShank3bmouse model of Autism Spectrum Disorders
Authors: Pangrazzi Luca , Cerilli Enrica , Balasco Luigi , Dall’O’ Ginevra Matilde , Chelini Gabriele , Pastore Anna , Weinberger Birgit , Bozzi Yuri
Published: March 2024

Autism Spectrum Disorder (ASD) includes a range of neurodevelopmental disabilities characterized by social interaction deficits, communication impairments, and repetitive behaviors. Previous studies have shown that pro-inflammatory conditions play a key role in ASD. Here we reported that increased levels of molecules related to inflammation are present in the cerebellum and peripheral blood (PB) of mice lacking Shank3b, established model of syndromic ASD. In parallel, immune dysfunction was documented in the bone marrow (BM) and spleen of mutant mice. N-acetylcysteine (NAC) treatment rescued inflammation in the cerebellum and PB, as well as impaired production of pro-inflammatory molecules in the BM and spleen. In addition, social impairment was counteracted in NAC-treated Shank3b-/-animals. Taken together, our study further confirms the key role of cerebellar inflammation in the establishment of ASD-related behaviors. Furthermore, our findings underscore the importance of considering ASD as a systemic disorder.

Our findings therefore suggest that the interplay between oxidative stress and inflammation may support ASD-related behaviors in mice.
CDK4is co-amplified with eitherTP53promoter gene fusions orMDM2through distinct mechanisms in osteosarcoma
Authors: Saba Karim H , Difilippo Valeria , Styring Emelie , Nilsson Jenny , Magnusson Linda , Bos Hilda van den , Spierings Diana C. J. , Foijer Floris , Nathrath Michaela , Haglund de Flon Felix , Baumhoer Daniel , Nord Karolin H
Published: March 2024

Amplification of theMDM2andCDK4genes on chromosome 12 is commonly associated with low-grade osteosarcomas. In this study, we conducted high-resolution genomic and transcriptomic analyses on 33 samples from 25 osteosarcomas, encompassing both high- and low-grade cases withMDM2and/orCDK4amplification. We identified four major subgroups: (i) low-grade osteosarcoma with chromosome 12 amplicons as the sole acquired alteration, (ii) high- and low-grade tumours withCDK4andMDM2amplification along with few changes affecting other chromosomes, (iii) high-grade osteosarcomas with heavily rearranged genomes including eitherCDK4andMDM2amplification or (iv)CDK4amplification andTP53structural alterations. The amplicons involvingMDM2exhibited signs of an initial chromothripsis event affecting chromosome 12. In contrast, there was no indication of a chromothripsis event on chromosome 12 inTP53-rearranged cases. Instead, the initial disruption of theTP53locus resulted in breakage and repair processes that co-amplified theCDK4locus. Furthermore, our investigation revealed recurring promoter swapping events that involved the regulatory regions of theFRS2,PLEKHA5, andTP53genes. These events led to the ectopic expression of partner genes, with theELF1gene being upregulated by theFRS2andTP53promoter regions, respectively, in two distinct cases.
GEGA (Gallus Enriched Gene Annotation): an online tool providing genomics and functional information across 47 tissues for a chicken gene-enriched atlas gathering Ensembl & Refseq genome annotations
Authors: Degalez Fabien , Bardou Philippe , Lagarrigue Sandrine
Published: March 2024

GEGA is a user-friendly tool to navigate through different genomics and functional information related to an enriched gene atlas in chicken that unifies the gene catalogues from the two reference databases, NCBI-RefSeq & EMBL-Ensembl/GENCODE, and four other additional rich resources as FAANG and NONCODE. Using the latest GRCg7b genome assembly, GEGA offers a total of 78,323 genes, including 24,102 protein-coding genes (PCGs) and 44,428 long non-coding RNAs (lncRNAs), greatly enhancing the number of genes provided by each resource separately. But GEGA is more than just a gene database. It offers a range of features that allow to go deeper into the functional aspects of these genes,e.g., by exploring their expression and co-expression profiles across 47 tissues from 36 datasets and 1400 samples, by discovering tissue-specific variations and their expression as a function of sex or age, by extracting their orthologous genes or their configuration related to the genomics closest gene. For the communities interested in one specific gene, a list of genes or a QTL region in chicken, GEGA’s user-friendly interface enables efficient gene analysis, easy downloading of results and a multitude of graphical representations, from genomic information to detailed visualization of expression levels.

GRAPHICAL ABSTRACT
Adoptive transfer of CD49a+Tissue resident memory cells reverses pulmonary fibrosis in mice
Authors: Collins Samuel L , Chan-Li Yee , Shenderov Kevin , Gillich Astrid , Nelson Andrew M. , Loube Jeffrey M. , Mitzner Wayne A , Powell Jonathan D. , Horton Maureen R.
Published: March 2024

Pulmonary fibrosis is a devastating disease with no effective treatments to cure, stop or reverse the unremitting, fatal fibrosis. A critical barrier to treating this disease is the lack of understanding of the pathways leading to fibrosis as well as those regulating the resolution of fibrosis. Fibrosis is the pathologic side of normal tissue repair that results when the normal wound healing programs go awry. Successful resolution of tissue injury requires several highly coordinated pathways, and this research focuses on the interplay between these overlapping pathways: immune effectors, inflammatory mediators and fibroproliferation in the resolution of fibrosis. Previously we have successfully prevented, mitigated, and even reversed established fibrosis using vaccinia vaccination immunotherapy in two models of murine lung fibrosis. The mechanism by which vaccinia reverses fibrosis is by vaccine induced lung specific Th1 skewed tissue resident memory (TRMs) in the lung. In this study, we isolated a population of vaccine induced TRMs - CD49a+CD4+T cells - that are both necessary and sufficient to reverse established pulmonary fibrosis. Using adoptive cellular therapy, we demonstrate that intratracheal administration of CD49a+CD4+TRMs into established fibrosis, reverses the fibrosis histologically, by promoting a decrease in collagen, and functionally, by improving lung function, without the need for vaccination. Furthermore, co-culture ofin vitroderived CD49+CD4+human TRMs with human fibroblasts from individuals with idiopathic pulmonary fibrosis (IPF) results in the down regulation of IPF fibroblast collagen production. Lastly, we demonstrate in human IPF lung histologic samples that CD49a+CD4+TRMs, which can down regulate human IPF fibroblast function, fail to increase in the IPF lungs, thus potentially failing to promote resolution. Thus, we define a novel unappreciated role for tissue resident memory T cells in regulating established lung fibrosis to promote resolution of fibrosis and re-establish lung homeostasis. We demonstrate that immunotherapy, in the form of adoptive transfer of CD49a+CD4+TRMs into the lungs of mice with established fibrosis, not only stops progression of the fibrosis but more importantly reverses the fibrosis. These studies provide the insight and preclinical rationale for a novel paradigm shifting approach of using cellular immunotherapy to treat lung fibrosis.
Distractor-response binding influences visual search
Authors: Allenmark Fredrik , Yu Hao , Müller Hermann J. , Shi Zhuanghua , Frings Christian
Published: March 2024

Intertrial priming effects in visual search and action control suggest the involvement of binding and retrieval processes. However, the role of distractor-response binding (DRB) in visual search has been largely overlooked, and the specific processing stage within the functional architecture of attentional guidance where the DRB occurs remains unclear. To address these gaps, we implemented two search tasks, where participants responded based on a separate feature from the one defining the target. We kept the target dimension consistent across trials while varying the color and shape of the distractor. Moreover, we either repeated or randomized the target position in different sessions. Our results revealed a pronounced response priming, a difference between trials where the response changed vs. repeated: they were stronger when distractor features or the target position were repeated than they varied. Furthermore, the distractor feature priming, a difference between the distractor features repetition and switch, was contingent on the target position, suggesting that DRB likely operates at late stages of target identification and response selection. These insights affirm the presence of DRB during visual search and support the framework of binding and retrieval in action control as a basis for observed intertrial priming effects related to distractor features.

Public significance statement

This study investigated inter-trial effects within visual search tasks and uncovered significant evidence for the role of distractor-response binding. This phenomenon involves linking a response in a given trial to the perceptual features (e.g. color and shape) of non-target items. Crucially, the study revealed that this distractor-response binding effect depends on whether the target location is repeated nearly repeated, suggesting that the retrieval of a previous response occurs at the later stages of target identification or response selection, even though non-target features likely are identified at an earlier stage.
Information is asymmetry: spatial relations were encoded by asymmetric mnemonic manifolds
Authors: Jiang Longsheng , Zhu Yanlin , Liu Jia
Published: March 2024

Substantial evidence suggests that working memory (WM) leverages relational representations to provide flexible support for cognitive functions, a capacity likely derived from the dynamic nature of neural codes in WM. However, how these dynamic codes represent and maintain relations remains unclear. Here, we examined the transformation of neural geometries in the dorsal prefrontal cortex of monkeys performing a visuospatial delayed-match/nonmatch task, where the monkeys were instructed to hold the spatial location of a white square in WM to match it with the spatial location of a subsequent square. We found that the sensory manifold during the square’s presence and the mnemonic manifold after the square’s offset both aligned with the stimulus manifold. However, significant differences emerged between the sensory and mnemonic manifolds, exhibiting little correlation in their neural geometries. Further analysis on the dynamic transformation from the sensory to mnemonic manifold revealed a process of expansion followed by flattening: the asymmetric sensory manifold first expanded into a symmetric neural geometry immediately after the square’s onset offset, which then gradually flattened along dimensions different from those initially expanded, culminating in an asymmetric mnemonic manifold. This dynamic process of reconstruction not only remained its faithfulness to the stimulus geometry but also gained the flexibility to meet task demands. In sum, this transformation from asymmetry to symmetry and back to asymmetry in neural geometry precisely illustrates the dynamics of memory reconstruction, shedding lights on the subjective nature of WM that generates both accurate and illusory representation of the world we lived in.
idh-1neomorphic mutation confers sensitivity to vitamin B12 via increased dependency on one-carbon metabolism inCaenorhabditis elegans
Authors: Ponomarova Olga , Starbard Alyxandra N. , Belfi Alexandra , Anderson Amanda V. , Sundaram Meera V. , Walhout Albertha J.M.
Published: March 2024

The isocitrate dehydrogenase neomorphic mutation (idh-1neo) generates increased levels of cellular D-2-hydroxyglutarate (D-2HG), a proposed oncometabolite. However, the physiological effects of increased D-2HG and whether additional metabolic changes occur in the presence of anidh-1neomutation are not well understood. We created aC. elegansmodel to study the effects of theidh-1neomutation in a whole animal. Comparing the phenotypes exhibited by theidh-1neotoΔdhgd-1(D-2HG dehydrogenase) mutant animals, which also accumulate D-2HG, we identified a specific vitamin B12 diet-dependent vulnerability inidh-1neomutant animals that leads to increased embryonic lethality. Through a genetic screen we found that impairment of the glycine cleavage system, which generates one-carbon donor units, exacerbates this phenotype. Additionally, supplementation with an alternate source of one-carbon donors suppresses the lethal phenotype. Our results indicate that theidh-1neomutation imposes a heightened dependency on the one-carbon pool and provides a further understanding how this oncogenic mutation rewires cellular metabolism.
Spotlight on 10x Visium: a multi-sample protocol comparison of spatial technologies
Authors: Du Mei R. M. , Wang Changqing , Law Charity W. , Amann-Zalcenstein Daniela , Anttila Casey J. A. , Ling Ling , Hickey Peter F. , Sargeant Callum J. , Chen Yunshun , Ioannidis Lisa J. , Rajasekhar Pradeep , Yip Raymond K. H. , Rogers Kelly L. , Hansen Diana S. , Bowden Rory , Ritchie Matthew E.
Published: March 2024

Background

Spatial transcriptomics allows gene expression to be measured within complex tissue contexts. Among the array of spatial capture technologies available is 10x Genomics’ Visium platform, a popular method which enables transcriptomewide profiling of tissue sections. Visium offers a range of sample handling and library construction methods which introduces a need for benchmarking to compare data quality and assess how well the technology can recover expected tissue features and biological signatures.

Results

Here we presentSpatialBench, a unique reference dataset generated from spleen tissue of mice responding to malaria infection spanning several tissue preparation protocols (both fresh frozen and FFPE samples, with and without CytAssist tissue placement). We noted better quality control metrics in reference samples prepared using probe-based capture methods, particularly those processed with CytAssist, validating the improvement in data quality produced with the platform. Our analysis of replicate samples extends to explore spatially variable gene detection, the outcomes of clustering and cell deconvolution using matched single-cell RNA-sequencing data and publicly available reference data to identify cell types and tissue regions expected in the spleen. Multi-sample differential expression analysis recovered known gene signatures related to biological sex or gene knockout.

Conclusions

We framed a comprehensive multi-sample analysis workflow that allowed us to generate consistent results both within and between different subsets of replicate samples, enabling broader comparisons and interpretations to be made at the group-level. OurSpatialBenchdataset, analysis, and workflow can serve as a practical guide for Visium users and may prove valuable in other benchmarking studies.
Global Rearrangement of Degree Centrality Reflects Cognitive Impairment and Fatigue in Multiple Sclerosis
Authors: Hok Pavel , Thai Quang Thong , Rehák Bučková Barbora , Domin Martin , Řasová Kamila , Tintěra Jaroslav , Lotze Martin , Grothe Matthias , Hlinka Jaroslav
Published: March 2024

Abstract

Background and Objectives

The aim of this secondary data analysis was to determine whether multiple sclerosis (MS) is associated with changes in global degree rank order disruption index (kD), a graph theory-based functional connectivity measure representing shift in overall distribution of nodal degree centrality. Additionally, we tested the relationship betweenkDand MS symptoms (cognitive and motor impairment, fatigue, and global disability).

Methods

GlobalkDwas computed in a pre-existing cross-sectional fMRI dataset and compared between patients with MS (PwMS) and healthy controls (HCs). Group differentiation was tested against other known biomarkers in MS (regional degree centrality, structural MRI with volumetry, diffusion-weighted imaging, lesion mapping) using receiver operating characteristic and logistic regression analysis. Associations betweenkDand cognitive processing speed (Symbol Digit Modalities Test), fatigue (Fatigue Scale for Motor and Cognitive Functions), gait (Timed Up and Go Test), and disability (Expanded Disability Status Scale [EDSS]) were evaluated using Spearman correlation coefficient and ordinal regression adjusted for structural imaging, age, sex, and disease duration.

Results

Analysis included 56 PwMS and 58 HCs (35/27 women, median age 45.1/40.5 years). GlobalkDwas lower in PwMS (median −0.30, inter-quartile range [IQR] 0.55) than in HCs (median −0.06, IQR 0.54;p= 0.009, Mann-Whitney U test).kDyielded acceptable differentiation between groups (area under curve 0.64), but did not improve such differentiation on top of structural imaging. BothkDand regional degree in medial prefrontal cortex (MPFC) were correlated with cognitive decline (kD: Spearman’sρ= 0.32,p= 0.019; MPFC:ρ= −0.45,p= 0.001,n= 55), whilekDwas also correlated with fatigue (ρ= −0.34,p= 0.010,n= 56), but not with EDSS (ρ= −0.06, p = 0.674,n= 56) or gait (ρ= −0.18,p= 0.211,n= 52).kDsignificantly explained cognitive impairment (χ2= 4.49,p= 0.034) and fatigue (χ2= 7.18,p= 0.007).

Discussion

Our data provide evidence thatkDis a potential biomarker of cognitive decline and fatigue. Further cross-validations are required to assess its generalizability.
Role of non-coding RNA hsa_circ_0001495 in 16HBE cellular inflammation induced by PM2.5and O3combined exposure
Authors: Wang HongJie , Tan Yi , Li CaiXia , Jin WenJia , Yu Ying , Mu Xuan , Peng XiaoWu
Published: March 2024

Background

PM2.5and O3are the main air pollutants in China, and inflammation of the respiratory system is one of their main toxic effects. Cyclic RNAs are involved in many pathophysiological processes, but their relationship to the combined exposure to PM2.5and O3has not yet been investigated.

Objective

To elucidate the biological function played by hsa_circ_0001495 in the induction of 16HBE cellular inflammation by combined exposure to PM2.5and O3.

Method

Detection of cell survival after 24h exposure of 16HBE cells to a combination of PM2.5and O3by CCK8. RT-qPCR and ELISA were used to detect inflammatory factors in 16HBE cells after co-exposing to PM2.5and O3. CircRNA was screened using high throughput sequencing and bioinformatics analysis approaches. RNaseR experiments were carried out to verify the circular RNA properties of the circRNAs. Cytoplasmic-nuclear subcellular localisation assays and fish assays were used to verify the distribution of circRNAs in the nucleus versus the cytoplasm of the cell. To validate functions related with circRNA,RT-qPCR and ELISA were employed.

Result

Combined exposure to PM2.5and O3resulted in decreased cell viability.Combined exposure to PM2.5and O3resulted in 16HBE inflammation. High throughput sequencing and RT-qPCR results showed that the expression of hsa_circ_0001495 was significantly downregulated in 16HBE exposed to PM2.5and O3in combination. Hsa_circ_0001495 is not easily digested by RNaseR enzymes and has the properties of a circular RNA. Hsa_circ_0001495 is expressed in the cytoplasm as well as in the nucleus, but its distribution is predominantly in the cytoplasm.

Conclusion

In 16HBE cells, combined exposure to PM2.5and O3can induce an inflammatory response.hsa_circ_0001495 plays an inhibitory role in the inflammatory response of 16HBE cells that can be induced by combined exposure to PM2.5and O3.
Open-Source Tools to Analyze Temporal and Spatial Properties of Local Field Potentials
Authors: Barrett Geoffrey M. , Vajram Srujan , Shetler Oliver , Aoun Andrew , Hussaini S. Abid
Published: March 2024

Analysis of local field potentials (LFPs) is important for understanding how ensemble neurons function as a network in a specific region of the brain. Despite the availability of tools for analyzing LFP data, there are some missing features such as analysis of high frequency oscillations (HFOs) and spatial properties. In addition, accessibility of most tools is restricted due to closed source code and/or high costs. To overcome these issues, we have developed two freely available tools that make temporal and spatial analysis of LFP data easily accessible. The first tool, hfoGUI (High Frequency Oscillation Graphical User Interface), allows temporal analysis of LFP data and scoring of HFOs such as ripples and fast ripples which are important in understanding memory function and neurological disorders. To complement the temporal analysis tool, a second tool, SSM (Spatial Spectral Mapper), focuses on the spatial analysis of LFP data. The SSM tool maps the spectral power of LFPs as a function of subject’s position in a given environment allowing investigation of spatial properties of LFP signal. Both hfoGUI and SSM are open-source tools that have unique features not offered by any currently available tools, and allow visualization and spatio-temporal analysis of LFP data.
Transcriptomic and epigenomic consequences of heterozygous loss of function mutations inAKAP11, the first large-effect shared risk gene for bipolar disorder and schizophrenia
Authors: Farhangdoost Nargess , Liao Calwing , Liu Yumin , Alda Martin , Dion Patrick A. , Rouleau Guy A. , Khayachi Anouar , Chaumette Boris
Published: March 2024

The gene A-kinase anchoring protein 11 (AKAP11) recently emerged as a shared risk factor between bipolar disorder and schizophrenia, driven by large-effect loss-of-function (LoF) variants. Recent research has uncovered the neurophysiological characteristics and synapse proteomics profile ofAkap11-mutant mouse models. Considering the role of AKAP11 in binding cAMP-dependent protein kinase A (PKA) and mediating phosphorylation of numerous substrates, such as transcription factors and epigenetic regulators, and given that chromatin alterations have been implicated in the brains of patients with bipolar disorder and schizophrenia, it is crucial to uncover the transcriptomic and chromatin dysregulations following the heterozygous knockout ofAKAP11, particularly in human neurons. In this study, we use genome-wide approaches to investigate such aberrations in human induced pluripotent stem cell (iPSC)-derived neurons. We show the impact of heterozygousAKAP11LoF mutations on the gene expression landscape and profile the methylomic and acetylomic modifications. Altogether we highlight the involvement of aberrant activity of intergenic and intronic enhancers, which are enriched in PBX homeobox 2 (PBX2) and Nuclear Factor-1 (NF1) known binding motifs, respectively, in transcription dysregulations of genes functioning as DNA-binding transcription factors, actin and cytoskeleton regulators, and cytokine receptors, as well as genes involved in G-protein-coupled receptors (GPCRs) binding and signaling. A better understanding of the dysregulations resulting from haploinsufficiency inAKAP11improves our knowledge of the biological roots and pathophysiology of BD and SCZ, paving the way for better therapeutic approaches.
Polymorphic positions 349 and 725 of the autoimmunity-protective allotype 10 of ER aminopeptidase 1 are key in determining its unique enzymatic properties
Authors: Georgaki Galateia , Mpakali Anastasia , Trakada Myrto , Papakyriakou Athanasios , Stratikos Efstratios
Published: March 2024

ER aminopeptidase 1 (ERAP1) is a polymorphic intracellular aminopeptidase with key roles in antigen presentation and adaptive immune responses. ERAP1 allotype 10 is highly protective towards developing some forms of autoimmunity and displays unusual functional properties, including very low activity versus some substrates. To understand the molecular mechanisms that underlie the biology of allotype 10 we studied its enzymatic and biophysical properties focusing on its unique polymorphisms V349M and Q725R. Compared to ancestral allotype 1, allotype 10 is much less effective in trimming small substrates but presents allosteric kinetics that ameliorate activity differences at high substrate concentrations. Furthermore, it is inhibited by a transition-state analogue via a non-competitive mechanism and is much less responsive to an allosteric small-molecule modulator. It also presents opposite enthalpy, entropy and heat capacity of activation compared to allotype 1 and its catalytic rate is highly dependent on viscosity. Polymorphisms V349M and Q725R significantly contribute to the lower enzymatic activity of allotype 10 for small substrates, especially at high substrate concentrations, influence the cooperation between the regulatory and active sites and regulate viscosity dependence, likely by limiting product release. Overall, our results suggest that allotype 10 is not just an inactive variant of ERAP1 but rather carries distinct enzymatic properties that largely stem from changes at positions 349 and 725. These changes affect kinetic and thermodynamic parameters that likely control rate-limiting steps in the catalytic cycle, resulting in an enzyme optimized for sparing small substrates and contributing to the homeostasis of antigenic epitopes in the ER.
Amyloid Beta Oligomers Accelerate ATP-Dependent Phase Separation of miRNA-Bound Ago2 to RNA Processing Bodiesin vitro
Authors: Ray Sritama , Roychowdhury Sumangal , Chakraborty Yogaditya , Banerjee Saikat , Chattopadhyay Krishnananda , Mukherjee Kamalika , Bhattacharyya Suvendra N.
Published: March 2024

Phase separation to insoluble membrane-less organelles is a major way of activity regulation of specific proteins in eukaryotic cells. miRNA-repressed mRNAs and Ago proteins are known to be localized to RNA-processing bodies, the subcellular structures which are formed due to assembly of several RNA binding and regulatory proteins in eukaryotic cells. Ago2 is the most important miRNA binding protein that by forming complex with miRNA binds to mRNAs having cognate miRNA binding sites and represses protein synthesis in mammalian cells. Factors which control compartmentalization of Ago2 and miRNA-repressed mRNAs to RNA processing bodies are largely unknown. We have adopted a detergent permeabilized cell-based assay system to follow the phase separation of exogenously added Ago2 to RNA processing bodiesin vitro. The Ago2 phase separation process is ATP dependent and is influenced by osmolarity and salt concentration of the reaction buffer. miRNA binding of Ago2 is essential for its targeting to RNA processing bodies and the compartmentalization process gets retarded by miRNA binding “sponge” protein HuR. This assay system found to be useful in identification of amyloid beta oligomers as miRNA-activity modulators which repress miRNA activity by enhancing Ago2-miRNP targeting to RNA processing bodies.

Graphical Abstract

miRNA bound Ago2 gets phase separatedin vitroto RNA processing bodies (PBs) in detergent permeabilized mammalian cells.

Phase separation of Ago2 to PBs is controlled by presence of ATP and RNA.

Amyloid beta oligomers retard dynamics of Ago2 bodies to inhibit miRNA function and enhance PB targeting of Ago2 miRNPs.

microRNA binding protein HuR can rescue Ago2 miRNP from PBs and inverse the effect of amyloid beta oligomers.
The T4bSS ofLegionellafeatures a two-step secretion pathway with an inner membrane intermediate for secretion of transmembrane effectors
Authors: Malmsheimer Silke , Grin Iwan , Bohn Erwin , Franz-Wachtel Mirita , Macek Boris , Sahr Tobias , Smollich Fabian , Chetrit David , Meir Amit , Roy Craig , Buchrieser Carmen , Wagner Samuel
Published: March 2024

To promote intracellular survival and infection,Legionella spp.translocate hundreds of effector proteins into eukaryotic host cells using a type IV b protein secretion system (T4bSS). T4bSS are well known to translocate soluble as well as transmembrane domain-containing effector proteins (TMD-effectors) but the mechanisms of secretion are still poorly understood. Herein we investigated the secretion of hydrophobic TMD-effectors, of which about 80 were previously reported to be encoded byL. pneumophila. A proteomic analysis of fractionated membranes revealed that TMD-effectors are targeted to and inserted into the bacterial inner membranes ofL. pneumophilaindependent of the presence of a functional T4bSS. While the T4bSS chaperones IcmS and IcmW were critical for secretion of all tested TMD-effectors, they did not influence inner membrane targeting of these proteins. As for soluble effector proteins, translocation of TMD-effectors into host cells depended on a C-terminal secretion signal and this signal needed to be presented towards the cytoplasmic side of the inner membrane. A different secretion behavior of TMD- and soluble effectors and the need for small periplasmic loops within TMD-effectors provided strong evidence that TMD-effectors are secreted in a two-step secretion process: Initially, an inner membrane intermediate is formed, that is extracted towards the cytoplasmic side, possibly by the help of the type IV coupling protein complex and subsequently secreted into eukaryotic host cells by the T4bSS core complex. Overall, our study highlights the amazing versatility of T4bSS to secrete soluble and TMD-effectors from different subcellular locations of the bacterial cell.
Neurogenomic diversity enhances collective antipredator performance inDrosophila
Authors: Sato Daiki X. , Takahashi Yuma
Published: March 2024

Collective behavior is a unique social behavior that plays crucial roles in detecting and avoiding predators. Despite a long history of research on the ecological significance, its neural and genetic underpinnings remain elusive. Here we focus on the mesmerizing nature that visual cues from surrounding conspecifics alleviate the fear response to threatening stimuli inDrosophila melanogaster. A large-scale behavioral experiment and genome-wide association analysis utilizing 104 strains with known genomes uncovered the genetic foundation of the emergent behavioral properties of flies. We found genes involved in visual neuron development associated with visual response to conspecifics, and the functional assay confirmed the regulatory significance of lamina neurons. Furthermore, behavioral synchronization combined with interindividual heterogeneity in freezing drove nonadditive, synergistic changes in group performance for predatory avoidance. Our novel approach termed genome-wide higher-level association study (GHAS) identified loci whose within-group genetic diversity potentially contributes to such an emergent effect. Population genetic analysis revealed that selective pressure may favor increased responsiveness to conspecifics, indicating that by-productive genomic diversity within the group leads to a collective phenomenon. This work opens up a new avenue to understand the genomics underpinning the group-level phenotypes and offers an evolutionary perspective on the mechanism of collective behavior.
Distributed encoding of hippocampal information in mossy cells
Authors: Ouchi Ayako , Toyoizumi Taro , Ikegaya Yuji
Published: March 2024

In neural information processing, the nervous system transmits neuronal activity across layers of neural circuits, occasionally passing through small layers composed only of sparse neurons. Hippocampal hilar mossy cells (MCs) constitute such a typical bottleneck layer.In vivo/vitropatch-clamp recordings revealed that MCs were reliably depolarized in response to sharp-wave ripples (SWRs), synchronous neuronal events transmitted from the CA3 region to the dentate gyrus via the MC layer. Machine-learning algorithms predicted the waveforms of SWRs in the CA3 region, based on the MC depolarization waveforms, suggesting that CA3 neural information is indeed transmitted to the MC layer. However, the prediction accuracy varied;i.e., a particular MC showed a more robust association with a particular SWR cluster, and the SWR cluster associated with one MC rarely overlapped with the SWR clusters associated with other MCs. Thus, CA3 network activity is distributed across MC ensembles with pseudo-orthogonal neural representations, allowing the small MC layer to effectively compress hippocampal information.