bluuebunny/crossref_metadata_2025 huggingface dataset:
Predicting hERG repolarization power at 37°C from recordings at room temperature
Authors: Oliveira-Mendes Barbara B.R. , Alameh Malak , Montnach Jérôme , Ollivier Béatrice , Gibaud Solène , Feliciangeli Sylvain , Lesage Florian , Charpentier Flavien , Loussouarn Gildas , De Waard Michel , Baró Isabelle
Published: April 2023

Mutations in theKCNH2gene cause long or short QT syndromes (LQTS or SQTS) predisposing to life-threatening arrhythmias.KCNH2encodes for the voltage-gated K+channel hERG involved in the late repolarization phase of the cardiac action potential (AP). For the last decades, sequencingKCNH2has provided a plethora of variants associated or not with clear pathological cardiac phenotypes. Identifying pathogenic or likely pathogenic variants from the benign ones would provide useful information to clarify the genetic background of LQTS patients and relatives, and to stratify the risk of adverse events. In face of a wide spectrum of hERG biophysical defects, we looked for a way to summarize the net loss or gain of function in a unique index. In a previous work, we defined as the repolarization power the time integral of the K+currents developed during an AP clamp. Here, with the aim of accelerating the functional characterization of hERG variants using automated patch-clamp, we adapted the AP-clamp protocol to establish, at room temperature, at which the recording success rate is high, a repolarization power index, as reliable and informative as the one measured at physiological temperature. We also illustrate that the repolarization power determined at room temperature is predictive of the repolarization power at physiological temperature for 2 pathogenic hERG variants with different biophysical dysfunctions.
RBP-J regulates homeostasis and function of circulating Ly6Clomonocytes
Authors: Kou Tiantian , Kang Lan , Zhang Bin , Li Jiaqi , Zhao Baohong , Zeng Wenwen , Hu Xiaoyu
Published: April 2023

Notch-RBP-J signaling plays an essential role in maintenance of myeloid homeostasis. However, its role in monocyte cell fate decisions is not fully understood. Here we showed that conditional deletion of transcription factor RBP-J in myeloid cells resulted in marked accumulation of blood Ly6Clomonocytes that highly expressed chemokine receptor CCR2. Bone marrow transplantation and parabiosis experiments revealed a cell intrinsic requirement of RBP-J for controlling blood Ly6CloCCR2himonocytes. RBP-J-deficient Ly6Clomonocytes exhibited enhanced capacity competing with wildtype counterparts in blood circulation. In accordance with alterations of circulating monocytes, RBP-J deficiency led to markedly increased population of lung tissues with Ly6Clomonocytes and CD16.2+interstitial macrophages. Furthermore, RBP-J deficiency-associated phenotypes could be genetically corrected by further deletingCcr2in myeloid cells. These results demonstrate that RBP-J functions as a crucial regulator of blood Ly6Clomonocytes and thus derived lung-resident myeloid populations, at least in part through regulation of CCR2.
Nanomolar interactions of alpha-synuclein fibrils to tau determined by FCS
Authors: Ramirez Jennifer , Brackhahn Emily Ann Elizabeth , Petersson E. James , Rhoades Elizabeth
Published: April 2023

Age-related neurodegenerative disorders like Alzheimer’s disease (AD) and Parkinson’s disease (PD) are characterized by deposits of protein aggregates, or amyloid, in various regions of the brain. Traditionally, aggregation of a single protein was observed to be correlated with these different pathologies: tau in AD and α-synuclein (αS) in PD. However, there is increasing evidence that the pathologies of these two diseases overlap, and the individual proteins may promote each other’s aggregation. Both tau and αS are intrinsically disordered proteins (IDPs), lacking stable secondary and tertiary structure under physiological conditions. In this study we used a combination of biochemical and biophysical techniques to interrogate the interaction of tau with both soluble and fibrillar αS. Fluorescence correlation spectroscopy (FCS) was used to assess the interactions of specific domains of fluorescently labeled tau with full length and C-terminally truncated αS in both monomeric and fibrillar forms. We found that full-length tau as well as individual tau domains interact with monomer αS weakly, but this interaction is much more pronounced with αS seeds. This interaction does not impact tau aggregation or fibril formation. These findings provide insight into the nature of interactions between tau and αS as well as the domains responsible.
Characterizing the monomer-dimer equilibrium of UbcH8/Ube2L6: A combined SAXS and NMR study
Authors: Kahraman Kerem , Robson Scott A. , Göcenler Oktay , Yenici Cansu M. , Tozkoparan Cansu D. , Klein Jennifer M. , Dötsch Volker , Elgin Emine Sonay , Haas Arthur L. , Ziarek Joshua J. , Dağ Çağdaş
Published: April 2023

Interferon-stimulated gene-15 (ISG15) is an interferon-induced protein with two ubiquitin-like (Ubl) domains linked by a short peptide chain, and the conjugated protein of the ISGylation system. Similar to ubiquitin and other Ubls, ISG15 is ligated to its target proteins through a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8, and HERC5, respectively. Ube2L6/UbcH8 plays a literal central role in ISGylation, underscoring it as an important drug target for boosting innate antiviral immunity. Depending on the type of conjugated protein and the ultimate target protein, E2 enzymes have been shown to function as monomers, dimers, or both. UbcH8 has been crystalized in both monomeric and dimeric forms, but the functional state is unclear. Here, we used a combined approach of small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy to characterize UbcH8’s oligomeric state in solution. SAXS revealed a dimeric UbcH8 structure that could be dissociated when fused N-terminally to glutathione S-transferase. NMR spectroscopy validated the presence of a concentration-dependent monomer-dimer equilibrium and suggested a backside dimerization interface. Chemical shift perturbation and peak intensity analysis further suggest dimer-induced conformational dynamics at E1 and E3 interfaces - providing hypotheses for the protein’s functional mechanisms. Our study highlights the power of combining NMR and SAXS techniques in providing structural information about proteins in solution.
Zn2+is Essential for Ca2+Oscillations in Mouse Eggs
Authors: Akizawa Hiroki , Lopes Emily , Fissore Rafael A.
Published: April 2023

Changes in the intracellular concentration of free calcium (Ca2+) underpin egg activation and initiation of development in animals and plants. In mammals, the Ca2+release is periodical, known as Ca2+oscillations, and mediated by the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1). Another divalent cation, zinc (Zn2+), increases exponentially during oocyte maturation and is vital for meiotic transitions, arrests, and polyspermy prevention. It is unknown if these pivotal cations interplay during fertilization. Here, using mouse eggs, we showed that basal concentrations of labile Zn2+are indispensable for sperm-initiated Ca2+oscillations because Zn2+-deficient conditions induced by cell-permeable chelators abrogated Ca2+responses evoked by fertilization and other physiological and pharmacological agonists. We also found that chemically- or genetically generated eggs with lower levels of labile Zn2+displayed reduced IP3R1 sensitivity and diminished ER Ca2+leak despite the stable content of the stores and IP3R1 mass. Resupplying Zn2+restarted Ca2+oscillations, but excessive Zn2+prevented and terminated them, hindering IP3R1 responsiveness. The findings suggest that a window of Zn2+concentrations is required for Ca2+responses and IP3R1 function in eggs, ensuring optimal response to fertilization and egg activation.
Volatile profiling distinguishesStreptococcus pyogenesfrom other respiratory streptococcal species
Authors: Berna Amalia Z. , Merriman Joseph A. , Mellet Leah , Parchment Danealle K. , Caparon Michael G. , John Audrey R. Odom
Published: April 2023

Sore throat is one of the most common complaints encountered in the ambulatory clinical setting. Rapid, culture-independent diagnostic techniques that do not rely on pharyngeal swabs would be highly valuable as a point-of-care strategy to guide outpatient antibiotic treatment. Despite the promise of this approach, efforts to detect volatiles during oropharyngeal infection have yet been limited. In our research study, we sought to evaluate for specific bacterial volatile organic compounds (VOC) biomarkers in isolated culturesin vitro, in order to establish proof-of-concept prior to initial clinical studies of breath biomarkers. A particular challenge for diagnosis of pharyngitis due toStreptococcus pyogenesis the likelihood that many metabolites may be shared byS. pyogenesand other related oropharyngeal colonizing bacterial species. Therefore, we evaluated whether sufficient metabolic differences are present that distinguish the volatile metabolome of Group A streptococci from other streptococcal species that also colonize the respiratory mucosa, such asS. pneumoniaeandS. intermedius. In this work, we identify candidate biomarkers that distinguishS. pyogenesfrom other species, and establish highly produced VOCs that indicate presence ofS. pyogenes in vitro, supporting future breath-based diagnostic testing for streptococcal pharyngitis.

IMPORTANCE

Acute pharyngitis accounts for approximately 15 million ambulatory care visits in the USA. The most common and important bacterial cause of pharyngitis isStreptococcus pyogenesis, accounting for 15% to 30% of pediatric pharyngitis. Distinguishing between bacterial and viral pharyngitis is key to management in US practice. Culture of a specimen obtained by throat swab is the standard laboratory procedure for the microbiologic confirmation of pharyngitis, however this method is time consuming which delays appropriate treatment. If left untreated,S. pyogenespharyngitis may lead to local and distant complications. In this study, we characterized the volatile metabolomes ofS. pyogenesand other related oropharyngeal colonizing bacterial species. We identify candidate biomarkers that distinguishS. pyogenesfrom other species and provides evidence to support future breath-based diagnostic testing for streptococcal pharyngitis.
Impact of inflammatory preconditioning on murine microglial proteome response induced by focal ischemic brain injury
Authors: Helbing Dario Lucas , Haas Fabienne , Cirri Emilio , Rahnis Norman , Dau Therese Thuy Dung , Sacramento Erika Kelmer , Oraha Nova , Böhm Leopold , Morrison Helen , Bauer Reinhard
Published: April 2023

Preconditioning with LPS induces neuroprotection against subsequent cerebral ischemic injury, mainly involving innate immune pathways. Microglia are CNS-resident immune cells that respond early to danger signals through memory-like differential reprogramming. However, the cell-specific molecular mechanisms underlying preconditioning are not fully understood. To elucidate the distinct molecular mechanisms of preconditioning on microglia, we compared these cell-specific proteomic profiles in response to LPS preconditioning and without preconditioning and subsequent transient focal brain ischemia and reperfusion, – using an established mouse model of transient focal brain ischemia and reperfusion. A proteomic workflow, based on isolated microglia obtained from mouse brains by cell sorting and coupled to mass spectrometry for identification and quantification, was applied. Our data confirm that LPS preconditioning induces marked neuroprotection, as indicated by a significant reduction in brain infarct volume. The established brain cell separation method was suitable for obtaining an enriched microglial cell fraction for valid proteomic analysis. The results show a significant impact of LPS preconditioning on microglial proteome patterns by type I interferons, presumably driven by the interferon cluster regulator proteins Stat1/2.
Linear and Neural Network Models for Predicting N-glycosylation in Chinese Hamster Ovary Cells Based on B4GALT Levels
Authors: Seber Pedro , Braatz Richard D.
Published: April 2023

Glycosylation is an essential modification to proteins that has positive effects, such as improving the half-life of antibodies, and negative effects, such as promoting cancers. Despite the importance of glycosylation, predictive models have been lacking. This article constructs linear and neural network models for the prediction of the distribution of glycans on N-glycosylation sites. The models are trained on data containing normalized B4GALT levels in Chinese Hamster Ovary cells. The ANN models achieve a median prediction error of 1.39%, which is 10-fold smaller than for previously published models, and a narrow error distribution. We also discuss issues with other models reported in the literature. We provide all of the software used in this work, allowing other researchers to reproduce the work and reuse or improve the code in future endeavors.
Evidence of a pan-tissue decline in stemness during human aging
Authors: dos Santos Gabriel Arantes , Vega Magdaleno Gustavo Daniel , de Magalhães João Pedro
Published: April 2023

Despite their biological importance, the role of stem cells in human aging remains to be elucidated. In this work, we applied a machine learning methodology to GTEx transcriptome data and assigned stemness scores to 17,382 healthy samples from 30 human tissues aged between 20 and 79 years. We found that ∼60% of the studied tissues present a significant negative correlation between the subject’s age and stemness score. The only significant exception to this pattern was the uterus, where we observed an increased stemness with age. Moreover, we observed a global trend of positive correlations between cell proliferation and stemness. When analyzing the tissues individually, we found that ∼50% of human tissues present a positive correlation between stemness and proliferation and 20% a negative correlation. Furthermore, all our analyses show negative correlations between stemness and cellular senescence, with significant results in ∼80% of the tissues analyzed. Finally, we also observed a trend that hematopoietic stem cells derived from old patients might have more stemness. In short, we assigned stemness scores to human samples and show evidence of a pan-tissue loss of stemness during human aging, which adds weight to the idea that stem cell deterioration contributes to human ageing.
The Power of Two: integrating deep diffusion models and variational autoencoders for single-cell transcriptomics analysis
Authors: Sadria Mehrshad , Layton Anita
Published: April 2023

Discovering a lower-dimensional embedding of single-cell data can greatly improve downstream analysis. The embedding should encapsulate both the high-level semantics and low-level variations in order to be meaningful and interpretable. Although current generative models have been used to learn such a low-dimensional representation, they have several limitations. Here, we introduce scVAEDer, a scalable deep-learning model that combines the power of variational autoencoders and deep diffusion models to learn a meaningful representation which can capture both global semantics and local variations in the data. By using the learned embedding, we show that scVAEDer can generate novel scRNA-seq data, predict the effect of the perturbation on various cell types, identify changes in gene expression during dedifferentiation, and detect master regulators in a biological process.
Sequential chromogenic immunohistochemistry: spatial analysis of lymph nodes identifies contact interactions between plasmacytoid dendritic cells and plasmablasts
Authors: Claudio N.M. , Nguyen M.T. , Wanner A.A. , Pucci F.
Published: April 2023

Recent clinical observations highlight the importance of the spatial organization of immune cells into lymphoid structures for the success of cancer immunotherapy and patient survival. Sequential chromogenic immunohistochemistry (scIHC) supports the analysis of multiple biomarkers on a single tissue section thus providing unique information about relative location of cell types and assessment of disease states. Unfortunately, widespread implementation of scIHC is limited by lack of a standardized, rigorous guide to the development of customized biomarker panels and by the need for user-friendly analysis pipelines able to streamline the extraction of meaningful data. Here, we examine major steps from classical IHC protocols and highlight the impact they have on the scIHC procedure. We report practical examples and illustrations of the most common complications that can arise during the setup of a new biomarker panel and how to avoid them. We described in detail how to prevent and detect cross- reactivity between secondary reagents and carry over between detection antibodies. We developed a novel analysis pipeline based on non-rigid tissue deformation correction, Cellpose-inspired automated cell segmentation and computational network masking of low-quality data. The resulting biomarker panel and pipeline was used to study regional lymph nodes from head and neck cancer patients. We identified contact interactions between plasmablasts and plasmacytoid dendritic cellsin vivo. Given that TLR receptors, which are highly expressed in plasmacytoid dendritic cells play a key role in vaccine efficacy, the significance of this cell-cell interaction decisively warrants further studies. In conclusion, this work streamlines the development of novel biomarker panels for scIHC, which will ultimately improve our understanding of immune responses in cancer.
Acute myeloid leukemia stratifies as two clinically relevant sphingolipidomic subtypes
Authors: Paudel B. Bishal , Tan Su-Fern , Fox Todd E. , Ung Johnson , Shaw Jeremy , Dunton Wendy , Lee Irene , Sharma Arati , Viny Aaron D. , Barth Brian M. , Tallman Martin S. , Cabot Myles , Garrett-Bakelman Francine E. , Levine Ross L. , Kester Mark , Claxton David , Feith David J. , Janes Kevin A. , Loughran Thomas P.
Published: April 2023

Acute myeloid leukemia (AML) is an aggressive disease with complex and heterogeneous biology. Although several genomic classifications have been proposed, there is a growing interest in going beyond genomics to stratify AML. In this study, we profile the sphingolipid family of bioactive molecules in 213 primary AML samples and 30 common human AML cell lines. Using an integrative approach, we identify two distinct sphingolipid subtypes in AML characterized by a reciprocal abundance of hexosylceramide (Hex) and sphingomyelin (SM) species. The two Hex-SM clusters organize diverse samples more robustly than known AML driver mutations and are coupled to latent transcriptional states. Using transcriptomic data, we develop a machine-learning classifier to infer the Hex-SM status of AML cases in TCGA and BeatAML clinical repositories. The analyses show that the sphingolipid subtype with deficient Hex and abundant SM is enriched for leukemic stemness transcriptional programs and comprises an unappreciated high-risk subgroup with poor clinical outcomes. Our sphingolipid-focused examination of AML identifies patients least likely to benefit from standard of care and raises the possibility that sphingolipidomic interventions could switch the subtype of AML patients who otherwise lack targetable alternatives.

Key Points

1.Sphingolipidomics separates acute myeloid leukemia (AML) patients and cell lines into two subtypes.

2.The subtype with low hexosylceramide and high sphingomyelin defines a new high-risk subtype with poor clinical outcomes.
Weakly supervised contrastive learning infers molecular subtypes and recurrence of breast cancer from unannotated pathology images
Authors: Liu Hui , Zhang Yang , Zhu Aichun , Sun Zhiqiang , Luo Judong
Published: April 2023

The deep learning-powered computational pathology has led to sig-nificant improvements in the speed and precise of tumor diagnosis,, while also exhibiting substantial potential to infer genetic mutations and gene expression levels. However,current studies remain limited in predicting molecular subtypes and recurrence risk in breast cancer. In this paper, we proposed a weakly supervised contrastive learning framework to address this challenge. Our framework first performed contrastive learning pretraining on large-scale unlabeled patches tiled from whole slide images (WSIs) to extract patch-level features. The gated attention mechanism was leveraged to aggregate patch-level features to produce slide feature that was then applied to various downstream tasks. To confirm the effectiveness of the proposed method, we have conducted extensive experiments on four independent cohorts of breast cancer. For gene expression prediction task, rather than one model per gene, we adopted multitask learning to infer the expression levels of 21 recurrence-related genes, and achieved remarkable performance and generalizability that were validated on an external cohort. Particularly, the predictive power to infer molecular subtypes and recurrence events was strongly validated by cross-cohort experiments. In addition, the learned patch-level attention scores enabled us to generate heatmaps that were highly consistent with pathologist annotations and spatial transcriptomic data. These findings demonstrated that our model effectively established the high-order genotype-phenotype associations, thereby enhances the potential of digital pathology in clinical applications.
Biotic Assessment of Crowdsourced Data Defines Four Ecoregions in Thar: A Novel Approach for Citizen Engagement in Conservation
Authors: Mukherjee Manasi , Paul Angshuman , Mukerji Mitali
Published: April 2023

Distinct ecosystems have representative biota that can allow delineation of ecoregions. Despite being one of the most populated desert ecosystems in the world with extreme climatic conditions, Thar has been classified as a single ecoregion by WWF. The present study aimed to test this on the basis of biotic assessment using a citizen science-based crowdsourced data from ebird (492 species of birds through 50,000 checklists of 4000 birders). Unsupervised clustering (k=4) and mathematical validation using cosine similarity revealed the presence of four distinct ecoregionsEastern Thar (ET), Western Thar (WT), Transitional Zone (TZ) and Cultivated Zone (CZ) in Thar. Most strikingly, these ecoregions when overlaid on geographical regions of Thar show that the CZ ecoregion was split between three distant and distinct geographic regions. Further spatial diversity estimates show CZ had the leastαdiversity (273) and highestβdiversity (1.8) indicating the least similarity between the districts that comprised this ecoregion. The results suggest CZ as an evolving ecoregion or a consequence of habitat fragmentation due to anthropogenic effects. The presence of least number of exclusive species in CZ (0.2%) and TZ (0.8%) including the near-threatened species likeLaticilla burnesii(Rufous-vented grass babbler) andPelecanus philippensis(Spot-billed pelican) and an endangered speciesAthene blewitti(Forest Owlet) highlights the need for restoration of these threatened biota and ecoregions. This study for the first time proposes crowd-sourced bird data as an important biota-based tool to understand (a) established ecoregions in an ecosystem, (b) anthropogenic effects of agri-farmlands and (c) relation between geographic regions and biota.
Surveillance of the Impact of Antimicrobial Resistant infections in Immunosuppressed Children’s therapy: A systematic review
Authors: Domo Danielle , Woogeng Ivo Ngundu
Published: April 2023

Background

Antimicrobial resistance (AMR) is a global problem. Resistant bacteria, for many reasons, do not cease to emerge and re-emerge. The impact of AMR on patient therapy is not scares in literature to date, but there is still much to do, mainly in pediatric settings. It is vital to assess the necessity of observing the impact of such infections on the clinical care trends to which some kids may already be subject, strengthening, improving, and, where necessary, implementing new age policies and regulations that may help contain the spread of AMR.

Method

We performed a yearlong review of literature on antimicrobial resistance in paediatrics immunosuppressed patients until September 2022. We draw-up a protocol of the review, to which we adhered rigorously, following the prescribed including and excluding criteria.

Results

From the 110 articles finally selected following the PRISMA workflow diagram, from which 29% of them were in majority randomized controlled trials studies, the remaining selection ranged from case controls to cohort studies, systematic reviews, controlled before and after reports trials, matched case-control and placebo reports trials and few not clearly reported research article types (15%).

Conclusion

The process yielded to the confirmation that there are literally good evidences of the serious lethal impact of resistant microbiological infections among immunocompromised children in and out of hospitals’ settings. What lacks the most are practical evidences of such damages both to the patient and to the public health sector, which can be conquer though, through well-programmed cohort–based studies.
Ultra-fast genotyping of SNPs and short indels using GPU acceleration
Authors: Henriksen Jørgen Wictor , Rand Knut Dagestad , Sandve Geir Kjetil , Grytten Ivar
Published: April 2023

As decreasing DNA sequencing costs leads to a steadily increasing rate of generated data, the development of efficient algorithms for processing of the sequence data is increasingly important to reduce costs and energy consumption. Recent work have shown that genotyping can be done efficiently and accurately using alignment-free methods that are based on analyzing kmers from sequenced reads. In particular, we have recently presented the KAGE genotyper, which uses an efficient pangenome representation of known individuals in a population to further increase accuracy and efficiency. While existing genotypers like KAGE use the Central Processing Unit (CPU) to count and analyze kmers, the Graphical Processing Unit (GPU) has shown promising for reducing runtime for similar problems.

We here present GKAGE, a new and improved version of KAGE that utilizes the GPU to further increase the computational efficiency. This is done by counting and analyzing large amounts of kmers in the many parallel cores of a GPU. We show that GKAGE is, on hardware of comparable cost, able to genotype an individual up to an order of magnitude faster than KAGE while producing the same output, which makes it by far the fastest genotyper available today. GKAGE can run on consumer-grade GPUs, and enables genotyping of a human sample in only a matter of minutes without the need for expensive high-performance computers. GKAGE is open source and available athttps://github.com/kage-genotyper/kage.
Integrative identification of non-coding regulatory regions driving metastatic prostate cancer
Authors: Woo Brian J , Moussavi-Baygi Ruhollah , Karner Heather , Karimzadeh Mehran , Garcia Kristle , Joshi Tanvi , Yin Keyi , Navickas Albertas , Gilbert Luke A. , Wang Bo , Asgharian Hosseinali , Feng Felix Y. , Goodarzi Hani
Published: April 2023

Large-scale sequencing efforts of thousands of tumor samples have been undertaken to understand the mutational landscape of the coding genome. However, the vast majority of germline and somatic variants occur within non-coding portions of the genome. These genomic regions do not directly encode for specific proteins, but can play key roles in cancer progression, for example by driving aberrant gene expression control. Here, we designed an integrative computational and experimental framework to identify recurrently mutated non-coding regulatory regions that drive tumor progression. Application of this approach to whole-genome sequencing (WGS) data from a large cohort of metastatic castration-resistant prostate cancer (mCRPC) revealed a large set of recurrently mutated regions. We used (i)in silicoprioritization of functional non-coding mutations, (ii) massively parallel reporter assays, and (iii)in vivoCRISPR-interference (CRISPRi) screens in xenografted mice to systematically identify and validate driver regulatory regions that drive mCRPC. We discovered that one of these enhancer regions, GH22I030351, acts on a bidirectional promoter to simultaneously modulate expression of U2-associated splicing factor SF3A1 and chromosomal protein CCDC157. We found that both SF3A1 and CCDC157 are promoters of tumor growth in xenograft models of prostate cancer. We nominated a number of transcription factors, including SOX6, to be responsible for higher expression of SF3A1 and CCDC157. Collectively, we have established and confirmed an integrative computational and experimental approach that enables the systematic detection of non-coding regulatory regions that drive the progression of human cancers.
Electrostatic interactions control the adsorption of extracellular vesicles onto supported lipid bilayers
Authors: Ridolfi Andrea , Cardellini Jacopo , Gashi Fatlinda , van Herwijnen Martijn J.C. , Trulsson Martin , Campos-Terán José , Wauben Marca H. M. , Berti Debora , Nylander Tommy , Stenhammar Joakim
Published: April 2023

Communication between cells located in different parts of an organism is often mediated by membrane-enveloped nanoparticles, such as extracellular vesicles (EVs). EV binding and cell uptake mechanisms depend on the heterogeneous composition of the EV membrane. From a colloidal perspective, the EV membrane interacts with other biological interfaces via both specific and non-specific interactions, where the latter include long-ranged electrostatic and van der Waals forces, and short-ranged repulsive “steric-hydration” forces. While electrostatic forces are generally exploited in most EV immobilization protocols, the roles played by various colloidal forces in controlling EV adsorption on surfaces have not yet been thoroughly addressed. In the present work, we study the interaction and adsorption of EVs with supported lipid bilayers (SLBs) carrying different surface charge densities. By probing the EV-SLB interaction using quartz crystal microbalance with dissipation monitoring (QCM-D) and confocal laser scanning microscopy (CLSM), we demonstrate that EV adsorption onto lipid membranes can be controlled by varying the strength of electrostatic forces. We then model the observed phenomena within the framework of nonlinear Poisson-Boltzmann theory. Modelling results confirm the experimental observations and highlight the crucial role played by attractive electrostatics in EV adsorption onto lipid membranes. Our results provide new fundamental insights into EV-membrane interactions and could be useful for developing novel EV separation and immobilization strategies.
Recursive self-embedded vocal motifs in wild orangutans
Authors: Lameira Adriano R. , Hardus Madeleine E. , Ravignani Andrea , Raimondi Teresa , Gamba Marco
Published: April 2023

Recursive procedures that allow placing a vocal signal inside another of similar kind provide a neuro-computational blueprint for syntax and phonology in spoken language and human song. There are, however, no known vocal sequences among nonhuman primates arranged in self-embedded patterns that evince vocal recursion or potential insipient or evolutionary transitional forms thereof, suggesting a neuro-cognitive transformation exclusive to humans. Here, we uncover that wild flanged male orangutan long calls feature rhythmically isochronous call sequences nested within isochronous call sequences, consistent with two hierarchical strata. Remarkably, three temporally and acoustically distinct call rhythms in the lower stratum were not related to the overarching rhythm at the higher stratum by any low multiples, which suggests that these recursive structures were neither the result of parallel non-hierarchical procedures or anatomical artifacts of bodily constrains or resonances. Findings represent a case of temporally recursive hominid vocal combinatorics in the absence syntax, semantics, phonology or music. Second-order combinatorics, ‘sequences within sequences’, involving hierarchically organized and cyclically structured vocal sounds in ancient hominids may have preluded the evolution of recursion in modern language-able humans.
A humanin vitroneuronal model for studying homeostatic plasticity at the network level
Authors: Yuan Xiuming , Puvogel Sofía , van Rhijn Jon-Ruben , Esteve-Codina Anna , Meijer Mandy , Rouschop Simon , van Hugte Eline J.H. , Oudakker Astrid , Schoenmaker Chantal , Schubert Dirk , Franke Barbara , Nadif Kasri Nael
Published: April 2023

Mechanisms that underlie homeostatic plasticity have been extensively investigated at single-cell levels in animal models, but are less well understood at the network level. Here, we used microelectrode arrays to characterize neuronal networks following induction of homeostatic plasticity in human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons co-cultured with rat astrocytes. Chronic suppression of neuronal activity through tetrodotoxin (TTX) elicited a time-dependent network re-arrangement. Increased expression of AMPA receptors and the elongation of axon initial segments were associated with increased network excitability following TTX treatment. Transcriptomic profiling of TTX-treated neurons revealed up-regulated genes related to extracellular matrix organization, while down-regulated genes related to cell communication; also astrocytic gene expression was found altered. Overall, our study shows that hiPSC-derived neuronal networks provide a reliablein vitroplatform to measure and characterize homeostatic plasticity at network and single-cell level; this platform can be extended to investigate altered homeostatic plasticity in brain disorders.
What AlphaFold tells us about cohesin’s retention on and release from chromosomes
Authors: Nasmyth Kim A. , Lee Byung-Gil , Roig Maurici B. , Löwe Jan
Published: April 2023

Cohesin is a trimeric complex containing a pair of SMC proteins (Smc1 and Smc3) whose ATPase domains at the end of long coiled coils (CC) are interconnected by Scc1. During interphase, it organizes chromosomal DNA topology by extruding loops in a manner dependent on Scc1’s association with two large hook shaped proteins called SA (yeast: Scc3) and Nipbl (Scc2). The latter’s replacement by Pds5 recruits Wapl, which induces release from chromatin via a process requiring dissociation of Scc1’s N-terminal domain (NTD) from Smc3. If blocked by Esco (Eco)-mediated Smc3 acetylation, cohesin containing Pds5 merely maintains pre-existing loops, but a third fate occurs during DNA replication, when Pds5-containing cohesin associates with Sororin and forms structures that hold sister DNAs together. How Wapl induces and Sororin blocks release has hitherto remained mysterious. In the twenty years since their discovery, not a single testable hypothesis has been proposed as to their role. Here, AlphaFold 2 (AF) three-dimensional protein structure predictions lead us to propose formation of a quarternary complex between Wapl, SA, Pds5, and Scc1’s NTD, in which the latter is juxtaposed with (and subsequently sequestered by) a highly conserved cleft within Wapl’s C-terminal domain (CTD). AF also reveals how Scc1’s dissociation from Smc3 arises from a distortion of Smc3’s CC induced by engagement of SMC ATPase domains, how Esco acetyl transferases are recruited to Smc3 by Pds5, and how Sororin prevents release by binding to the Smc3/Scc1 interface. Our hypotheses explain the phenotypes of numerous existing mutations and are highly testable.
Reversal of cell, circuit and seizure phenotypes in a mouse model ofDNM1epileptic encephalopathy
Authors: Bonnycastle K. , Dobson K.L. , Blumrich E-M. , Gajbhiye A. , Davenport E.C. , Pronot M. , Steinruecke M. , Trost M. , Gonzalez-Sulser A. , Cousin M.A.
Published: April 2023

Pathogenic heterozygous missense mutations in theDNM1gene result in a novel form of epileptic encephalopathy.DNM1encodes for the large GTPase dynamin-1, an enzyme with an obligatory role in the endocytosis of synaptic vesicles (SVs) at mammalian nerve terminals. PathogenicDNM1mutations cluster within regions required for its essential GTPase activity, implicating disruption of this enzyme activity as being central to epileptic encephalopathy. We reveal that the most prevalent pathogenic mutation ofDNM1, R237W, disrupts dynamin-1 enzyme activity and SV endocytosis when overexpressed in central neurons. To determine how this dominant-negative heterozygous mutant impacted cell, circuit and behaviour when expressed from its endogenous locus, we generated a mouse carrying the R237W mutation. Neurons isolated from heterozygous mice displayed dysfunctional SV endocytosis, which translated into altered excitatory neurotransmission and seizure-like phenotypes. Importantly, these phenotypes were corrected at the cell, circuit andin vivolevel by the drug, BMS-204352, which accelerates SV endocytosis. This study therefore provides the first direct link between dysfunctional SV endocytosis and epilepsy, and importantly reveals that SV endocytosis is a viable therapeutic route for monogenic intractable epilepsies.
In vivostructure probing of RNA inArchaea: Novel insights into the ribosome structure ofMethanosarcina acetivorans
Authors: Williams Allison M. , Jolley Elizabeth A. , Santiago-Martínez Michel Geovanni , Chan Cheong Xin , Gutell Robin R. , Ferry James G. , Bevilacqua Philip C.
Published: April 2023

Structure probing combined with next-generation sequencing (NGS) has provided novel insights into RNA structure-function relationships. To date such studies have focused largely on bacteria and eukaryotes, with little attention given to the third domain of life, archaea. Furthermore, functional RNAs have not been extensively studied in archaea, leaving open questions about RNA structure and function within this domain of life. With archaeal species being diverse and having many similarities to both bacteria and eukaryotes, the archaea domain has the potential to be an evolutionary bridge. In this study, we introduce a method for probing RNA structurein vivoin the archaea domain of life. We investigated the structure of ribosomal RNA (rRNA) fromMethanosarcina acetivorans, a well-studied anaerobic archaeal species, grown with either methanol or acetate. After probing the RNAin vivowith dimethyl sulfate (DMS), Structure-seq2 libraries were generated, sequenced, and analyzed. We mapped the reactivity of DMS onto the secondary structure of the ribosome, which we determined independently with comparative analysis, and confirmed the accuracy of DMS probing inM. acetivorans. Accessibility of the rRNA to DMS in the two carbon sources was found to be quite similar, although some differences were found. Overall, this study establishes the Structure-seq2 pipeline in the archaea domain of life and informs about ribosomal structure withinM. acetivorans.
MicroRNA-Mediated Obstruction of Stem-loop Alternative Splicing (MIMOSAS): a global mechanism for the regulation of alternative splicing
Authors: Ruan Kai , Perez German Farinas , Liu Jiaqi , Kubat Miroslav , Hofacker Ivo , Wuchty Stefan , Zhai R. Grace
Published: April 2023

While RNA secondary structures are critical to regulate alternative splicing of long-range pre-mRNA, the factors that modulate RNA structure and interfere with the recognition of the splice sites are largely unknown. Previously, we identified a small, non-coding microRNA that sufficiently affects stable stem structure formation ofNmnatpre-mRNA to regulate the outcomes of alternative splicing. However, the fundamental question remains whether such microRNA-mediated interference with RNA secondary structures is a global molecular mechanism for regulating mRNA splicing. We designed and refined a bioinformatic pipeline to predict candidate microRNAs that potentially interfere with pre-mRNA stem-loop structures, and experimentally verified splicing predictions of three different long-range pre-mRNAs in theDrosophilamodel system. Specifically, we observed that microRNAs can either disrupt or stabilize stem-loop structures to influence splicing outcomes. Our study suggests that MicroRNA-Mediated Obstruction of Stem-loop Alternative Splicing (MIMOSAS) is a novel regulatory mechanism for the transcriptome-wide regulation of alternative splicing, increases the repertoire of microRNA function and further indicates cellular complexity of post-transcriptional regulation.

One-Sentence Summary

MicroRNA-Mediated Obstruction of Stem-loop Alternative Splicing (MIMOSAS) is a novel regulatory mechanism for the transcriptome-wide regulation of alternative splicing.
Nociception in chicken embryos, Part I: Analysis of cardiovascular responses to a mechanical noxious stimulus
Authors: Weiss Larissa , Saller Anna M. , Werner Julia , Süß Stephanie C. , Reiser Judith , Kollmansperger Sandra , Anders Malte , Fenzl Thomas , Schusser Benjamin , Baumgartner Christine
Published: April 2023

While it is assumed that chicken embryos acquire the ability for nociception during the developmental period in the egg, an exact time point has not yet been specified. This study aimed to determine the onset of nociception during embryonic development in chicken. Changes in blood pressure and heart rate (HR) in response to a mechanical noxious stimulus at the base of the beak versus a light touch on the beak in chicken embryos between embryonic days (EDs) 7 and 18 were examined. Mean arterial pressure (MAP) was the most sensitive parameter for assessing cardiovascular responses. Significant changes in MAP in response to a noxious stimulus were detected in embryos at ED16 to ED18, while significant changes in HR were observed on ED17 and ED18. Infiltration anesthesia with the local anesthetic lidocaine significantly reduced reactions in MAP on ED18, so the cardiovascular changes can be assumed to be nociceptive responses.
A data-driven approach for enhancing forest productivity by accounting for indirect genetic effects
Authors: Ferreira Filipe M. , Chaves Saulo F. S. , Bhering Leonardo L. , Alves Rodrigo S. , Takahashi Elizabete K. , Resende Marcos D. V. , Souza João E. , Gezan Salvador A. , Viana José M. S. , Fernandes Samuel B. , Dias Kaio O. G.
Published: April 2023

Maintaining the past decades current genetic gains for tree species is a challenging task for foresters and tree breeders due to biotic and abiotic factors. Planting a mixture of genotypes or clonal composites can be an alternative to increase the phytosanitary security and yield of forest plantations. These clonal composites are more complex than monocultures due to inter-genotypic competition and indirect genetic effects that can affect the total heritable variation. This study aims to understand how indirect genetic effects can impact the response to selection and how the stand composition can be used to explore these effects and enhance forest yield. We used a clonally trial ofEucalyptus urophylla×Eucalyptus grandishybrids in a randomized complete block design with 24 replications, containing a single tree per plot evaluated for mean annual increment at 3 and 6 years. We focus on partitioning the genetic variation of trees into direct and indirect genetic effects based on competition intensity factors. We identified clones as aggressive, homeostatic, and sensitive based on the magnitude of indirect genetic effects. By accounting for indirect genetic effects, for mean annual increment, the total heritability decreased 39 and 44% for 3 and 6 years, respectively. We proposed a workflow that uses the direct and indirect genetic effect to predict the mean value of clonal composite combinations and to select the one with highest yield. Our methodology accounted for spatial variability and interplot competition that can contribute to the total heritable variance and response to selection in forest trials. Based on the models evaluated, the clones are easily classified according to their deviation from the indirect genetic effects mean. Also, we extract useful information to predict different clonal composites compositions, their expected average performance, and define the best recommended combination to be planted in large scale.
Visualization oftranshomophilic interaction of clustered protocadherin in neurons
Authors: Hoshino Natsumi , Kanadome Takashi , Itoh Mizuho , Kaneko Ryosuke , Inoue Yukiko U. , Inoue Takayoshi , Hirabayashi Takahiro , Watanabe Masahiko , Matsuda Tomoki , Nagai Takeharu , Tarusawa Etsuko , Yagi Takeshi
Published: April 2023

Clustered protocadherin (Pcdh) functions as a cell recognition molecule through the homophilic interaction in CNS. However, its interactions have yet not been visualized in neurons. We previously reported PcdhγB2-FRET probes to be applicable only for cell lines. Herein, we newly designed PcdhγB2-FRET probes by fusing FRET donor and acceptor fluorescent proteins to a single PcdhγB2 molecule and succeeded in visualizing PcdhγB2 homophilic interaction in cultured hippocampal neurons. The γB2-FRET probe localized in the soma and neurites, and FRET signals were observed at contact sites between neurites and eliminated by EGTA addition. Live imaging revealed that the FRET-negative γB2 signals were rapidly moving along neurites and soma, whereas the FRET-positive signals remained in place. We observed that the γB2 proteins at synapses rarely interact homophilically. The γB2-FRET probe would allow us to elucidate the function of the homophilic interaction and the cell recognition mechanism.

Significance Statement

We visualize the Pcdh homophilic interaction using a novel FRET-based probe, and reveal that the homophilically interacting Pcdh proteins are found at contact sites between the neurites and roots of neurites from the soma, and are stable at a location. Additionally, in neurons, Pcdh proteins are located at synapses but rarely interact homophilically.
ENOD93 interacts with cytochrome c oxidase altering respiratory ATP production and root growth in plants
Authors: Lee Chun Pong , Le Xuyen H. , Gawryluk Ryan M.R. , Casaretto José A. , Rothstein Steven J. , Millar A. Harvey
Published: April 2023

The early nodulin 93 (ENOD93) gene family in plants can regulate biological nitrogen fixation in legumes and nitrogen use efficiency in cereals but its molecular function is unknown. We show profile hidden Markov models define ENOD93 as a distant homolog of the N-terminal domain of RESPIRATORY SUPERCOMPLEX FACTOR 2 (RCF2). RCF2 is reported to regulate cytochrome oxidase (CIV) influencing the generation of a mitochondria proton motive force in yeast. Knockout ofenod93in Arabidopsis leads to a short root phenotype. ENOD93 is associated with a protein complex the size of CIV in isolated mitochondria but neither CIV abundance nor its activity in ruptured organelles changed inenod93. However, a progressive loss of ADP-dependent respiration rate was observed inenod93mitochondria which could be fully recovered in complemented lines. Mitochondrial membrane potential was higher inenod93but ATP synthesis and ADP depletion rates progressively decreased. Respiration rate of wholeenod93seedlings was elevated and root ADP content was nearly double that in WT without a change in ATP content. These altered energetic states correlated with elevated respiratory substrate levels in roots ofenod93compared to WT and complemented lines. Overexpression of ENOD93 lowered ATP content in roots and increased the abundance of a range of amino acids in both roots and leaves. We propose that two previously unconnected gene families in plants, ENOD93 and HYPOXIA INDUCED GENE DOMAIN, are the functional equivalent of yeast RCF2 but have remained undiscovered in many eukaryotic lineages because they are encoded in two separate genes.

Highlight significance

We identify the enigmatic early nodulin ENOD93 gene family as the plant homolog of the N-terminal regulatory domain of the yeast RESPIRATORY SUPERCOMPLEX 2 (RCF2) of the mitochondrial oxidative phosphorylation system and provide biochemical and physiological evidence of its role in plant ATP production, broadly explaining the role of ENOD93 in plants.