bluuebunny/crossref_metadata_2025 huggingface dataset:
The ultrastructural nature of human oocytes’ cytoplasmatic abnormalities and the role of cytoskeleton dysfunction
Authors: Tatíčková Martina , Trebichalská Zuzana , Kyjovská Drahomíra , Otevřel Pavel , Kloudová Soňa , Holubcová Zuzana
Published: April 2023

Background

Egg quality is a limiting factor of female fertility and assisted reproductive technology (ART) success. Oocytes recovered from hyperstimulated ovaries often display morphological anomalies suspected to compromise their fertilization and developmental potential. Knowledge of (ab)normal oocyte’s intracellular organization is vital to establish reliable criteria for morphological evaluation of oocytes intended for in vitro fertilization (IVF).

Methods

Transmission electron microscopy (TEM) was used to investigate the fine morphology of 22 dysmorphic IVF eggs exhibiting different types of cytoplasmic irregularities, namely (1) refractile bodies, (2) centrally-located cytoplasmic granularity (CLCG), (3) smooth endoplasmic reticulum (SER) disc, and (4) vacuoles. The cytoskeleton targeting compounds were employed to address the causative mechanism behind the anomalous cytoplasmic architecture observed in abnormal egg samples. A total of 133 immature oocytes were exposed to chemical inhibitors/control conditions, and their morphology was examined by fluorescent and electron microscopy.

Results

TEM exposed the structural basis of the common oocyte aberrations and revealed that the underlying cause of two of the studied morphotypes was excessive organelle clustering. Inhibition experiments showed that disruption of actin, not microtubules, allows inordinate aggregation of subcellular structures resembling the ultrastructural pattern seen in morphologically abnormal eggs retrieved in IVF cycles. These results imply that actin serves as a regulator of organelle distribution during human oocyte maturation.

Conclusions

The ultrastructural analogy between dysmorphic eggs and oocytes, in which actin network integrity was perturbed, suggests that malfunction of the actin cytoskeleton might be implicated in generating common cytoplasmic aberrations. Knowledge of human oocytes’ inner workings and the origin of morphological abnormalities is a step forward to more objective egg quality assessment in clinical practice.
The genome ofTalinum fruticosum
Authors: Brilhaus Dominik , Denton Alisandra K. , Maleckova Eva , Reichel-Deland Vanessa , Weber Andreas P. M.
Published: April 2023

Research on crassulacean acid metabolism (CAM) has in recent years focused on obligate CAM species, such asKalanchoë fedtschenkoiand pineapple (Ananas comosus). To fully understand the plasticity of the CAM pathway, its evolutionary trajectory and regulation, genomic resources of additional species, including facultative CAM species are desirable. To this end, we sequenced the genome and full-length transcripts (Iso-Seq) of the facultative CAM dicotTalinum fruticosum.

The provided resources may aid in CAM engineering as an approach to improving crop water-use efficiency.
Nociception in chicken embryos, Part III: Analysis of movements before and after application of a noxious stimulus
Authors: Süß Stephanie C. , Werner Julia , Saller Anna M. , Weiss Larissa , Reiser Judith , Ondracek Janie M. , Zablotski Yury , Kollmansperger Sandra , Anders Malte , Schusser Benjamin , Fenzl Thomas , Baumgartner Christine
Published: April 2023

Many potentially noxious interventions are performed on chicken embryos in research and in the poultry industry. It is therefore in the interest of animal welfare to define the point at which a chicken embryo is capable of nociception. The present part III of a comprehensive study examined the movements of developing chicken embryos with the aim of identifying behavioral responses to aww noxious stimulus. For this purpose, a noxious mechanical stimulus and a control stimulus were applied. The recorded movements of the embryos were evaluated using the markerless pose estimation software DeepLabCut and manual observations. After the application of the noxious stimulus, a significant increase in beak movement was identified in 15- to 18-day-old embryos. In younger embryos, no behavioral changes related to the noxious stimulus were observed. The results indicate that noxious stimuli at the beak base evoke a nocifensive reaction in chicken embryos starting at embryonic day 15.
Gaze-centered gating and reactivation of value encoding in orbitofrontal cortex
Authors: Ferro Demetrio , Cash-Padgett Tyler , Wang Maya Zhe , Hayden Benjamin , Moreno-Bote Rubén
Published: April 2023

During economic choice, we often consider options in alternation, until we commit to one. Nonetheless, neuroeconomics typically ignores the dynamic aspects of deliberation. We trained macaques to perform a value-based decision-making task where two risky offers were presented in sequence at different locations of the visual field, each followed by a delay epoch where offers were invisible. Subjects looked at the offers in sequence, as expected. Surprisingly, during the delay epochs, we found that subjects still tend to look at empty locations where the visual offers were formerly presented; and, moreover, longer fixation to given empty location increases the probability of choosing the associated offer, even after controlling for the offer values. We show that activity in orbitofrontal cortex (OFC) reflects the value of the gazed offer, but also the value of the offer associated to the gazed spatial location, even if it is not the most recently viewed. This reactivation reflects a reevaluation process, as fluctuations in neural spiking during offer stimuli presentation and delays correlate with upcoming choice. Our results suggest that look-at-nothing gazing triggers the reactivation of a previously seen offer for further reevaluation, revealing novel aspects of deliberation.
Biophysical characterization and analysis of a mesophilic chorismate mutase fromB. pumilus
Authors: Wilkins Ryan Scott , Lund Bjarte Aarmo , Isaksen Geir Villy , Åqvist Johan , Brandsdal Bjørn Olav
Published: April 2023

Chorismate mutases have extensively been used as computational benchmarking systems for enzyme catalysis, yet the roles entropy and enthalpy play in catalysis are still not fully understood. Thus, it is important to better understand these enzymes for potential research or industrial applications. Here, we report the first crystal structure and kinetic characterization of a chorismate mutase fromBacillus pumilus. This enzyme exhibits a high degree of similarity to a known mesophilic chorismate mutase fromBacillus subtilis. Using this crystal structure, we further employ EVB/MD simulations to construct Arrhenius plots, allowing us to extract thermodynamic activation parameters. Overall, this study provides new insights into the structural and functional features of theB. pumiluschorismate mutase and highlights its potential as a valuable enzyme for biocatalytic and biotechnological applications.
Establishment of a screening platform based on human coronavirus OC43 for the identification of microbial natural products with antiviral activity
Authors: Martínez-Arribas Blanca , Annang Frederick , Díaz-González Rosario , Pérez-Moreno Guiomar , Martín Jesús , Mackenzie Thomas A. , Castillo Francisco , Reyes Fernando , Genilloud Olga , Ruiz-Pérez Luis Miguel , Vicente Francisca , Ramos María C. , González-Pacanowska Dolores
Published: April 2023

Human coronaviruses (HCoVs) cause respiratory tract infections and are of great importance due to the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Human betacoronavirus OC43 (HCoV-OC43) is an adequate surrogate for SARS-CoV-2 because it infects the human respiratory system, presents a comparable biology, and is transmitted in a similar way. Its use is advantageous since it only requires biosafety level (BSL)-2 infrastructure which minimizes costs and biosafety associated limitations. In this report, we describe a high-throughput screening (HTS) platform to identify compounds that inhibit the propagation of HCoV-OC43. Optimization of assays based on inhibition of the cytopathic effect and virus immunodetection with a specific antibody, has provided a robust methodology for the screening of a selection of microbial natural product extracts from the Fundación MEDINA collection. Using this approach, a subset of 1280 extracts has been explored. Of these, upon hit confirmation and early LC-MS dereplication, 10 extracts were identified that contain potential new compounds. In addition, we report on the novel antiviral activity of some previously described natural products whose presence in bioactive extracts was confirmed by LC/MS analysis.

IMPORTANCE

The COVID-19 pandemic has revealed the lack of effective treatments against betacoronaviruses and the urgent need for new broad-spectrum antivirals. Natural products are a valuable source of bioactive compounds with pharmaceutical potential that may lead to the discovery of new antiviral agents. Specifically, compared to conventional synthetic molecules, microbial natural extracts possess a unique and vast chemical diversity and are amenable to large-scale production. The implementation of a high-throughput screening platform using the betacoronavirus OC43 in a human cell line infection model has provided proof of concept of the approach and has allowed for the rapid and efficient evaluation of 1280 microbial extracts. The identification of several active compounds validates the potential of the platform for the search for new compounds with antiviral capacity.
replicAnt: A pipeline for generating annotated images of animals in complex environments using Unreal Engine
Authors: Plum Fabian , Bulla René , Beck Hendrik K. , Imirzian Natalie , Labonte David
Published: April 2023

Deep learning-based computer vision methods are transforming animal behavioural research. Transfer learning has enabled work in non-model species, but still requires hand-annotation of example footage, and is only performant in well-defined conditions. To overcome these limitations, here we presentreplicAnt, a configurable pipeline implemented in Unreal Engine 5 and Python, designed to generate large and variable training datasets on consumer-grade hardware.replicAntplaces 3D animal models into complex, procedurally generated environments, from which automatically annotated images can be exported. We demonstrate that synthetic data generated withreplicAntcan significantly reduce the hand-annotation required to achieve benchmark performance in common applications such as animal detection, tracking, pose-estimation, and semantic segmentation. We also show that it increases the subject-specificity and domain-invariance of the trained networks, thereby conferring robustness. In some applications,replicAntmay even remove the need for hand-annotation altogether. It thus represents a significant step towards porting deep learning-based computer vision tools to the field.
Camera trap methods and drone thermal surveillance provide reliable, comparable density estimates of large, free-ranging ungulates
Authors: Baldwin Robert W. , Beaver Jared T. , Messinger Max , Muday Jeffrey , Windsor Matt , Larsen Gregory D. , Silman Miles , Anderson T. Michael
Published: April 2023

Camera traps and drone surveys both leverage advancing technologies to study dynamic wildlife populations with little disturbance. Both techniques entail strengths and weaknesses, and common camera trap methods can be confounded by unrealistic assumptions and prerequisite conditions. We compared three methods to estimate the population density of white-tailed deer (Odocoileus virgnianus) at a section of Pilot Mountain State Park, NC, USA: (1) camera trapping using mark-resight ratios or (2) N-mixture modelling, and (3) aerial thermal videography from a drone platform. All three methods yielded similar density estimates, suggesting that they converged on an accurate estimate. We also included environmental covariates in N-mixture modeling to explore spatial habitat use, and we fit models for each season to understand temporal changes in population density. Deer occurred in greater densities on warmer, south-facing slopes in the fall and winter, and on cooler north-facing slopes and in areas with flatter terrain in the summer. Seasonal density estimates over two years suggested an annual cycle of higher densities in fall and winter than in summer, suggesting that the region may function as a refuge during the hunting season.

Simple Summary

Wildlife researchers and managers can choose from several techniques to estimate the number of animals in a population. Camera traps and drones are increasingly common and cost-effective options that can estimate the number and density of wildlife. We compared three simple methods that are used to estimate the number and density of deer in Pilot Mountain State Park: (1) mark-resighting and (2) N-mixture modeling, both using camera trap data, and (3) extrapolating transect counts from thermal videos collected from an aerial drone. We found that all three methods provided similar estimates of the population’s density, with complementary strengths and weaknesses: drone surveys collected data quickly and precisely, mark-resight ratios provided estimates of different demographic groups, and N-mixture modeling revealed changes in density across different habitat qualities and seasons.
A core NRF2 gene set defined through comprehensive transcriptomic analysis predicts selective drug resistance and poor multi-cancer prognosis
Authors: Luo George , Kumar Harshita , Aldridge Kristin , Rieger Stevie , Jiang Ethan , Chan Ernest R. , Soliman Ahmed , Mahdi Haider , Letterio John J.
Published: April 2023

The NRF2-KEAP1 pathway plays an important role in the cellular response to oxidative stress but may also contribute to metabolic changes and drug resistance in cancer. We investigated the activation of NRF2 in human cancers and fibroblast cells through KEAP1 inhibition and cancer associated KEAP1/NRF2 mutations. We define a core set of 14 upregulated NRF2 target genes from seven RNA-Sequencing databases that we generated and analyzed, which we validated this gene set through analyses of published databases and gene sets. An NRF2 activity score based on expression of these core target genes correlates with resistance to drugs such as PX-12 and necrosulfonamide but not to paclitaxel or bardoxolone methyl. We validated these findings and also found NRF2 activation led to radioresistance in cancer cell lines. Finally, our NRF2 score is prognostic for cancer survival and validated in additional independent cohorts for novel cancers types not associated with NRF2-KEAP1 mutations. These analyses define a core NRF2 gene set that is robust, versatile, and useful as a NRF2 biomarker and for predicting drug resistance and cancer prognosis.
Unsaturated fatty acid synthesis is associated with poor prognosis and differentially regulated byMYCNand tumor suppressor microRNAs in neuroblastoma
Authors: Sheeter Dennis A. , Garza Secilia , Park Hui Gyu , Badi Niharika R. , Espinosa Erika C. , Kothapalli Kumar , Brenna J. Thomas , Powers John T.
Published: April 2023

MYCNamplification and disruption of tumor suppressor microRNA (TSmiR) function are central drivers of poor outcomes in neuroblastoma (NB). MYC, MYCN, and TSmiRs regulate glucose metabolism; however, their role in unsaturated fatty acid synthesis (UFAS) remains poorly understood. Here we show thatde novoand UFAS pathway genesFASN,ELOVL6,SCD,FADS2, andFADS1are upregulated in high-risk NB and are associated with poor prognosis. RNA-Seq analysis of eight human NB cell lines revealed parallel UFAS gene expression patterns. Consistent with this, we found that NB-related TSmiRs were predicted to extensively target these genes. In addition, we observed that both MYC and MYCN upregulated UFAS pathway genes while suppressing TSmiR host gene expression, thereby creating a possible UFAS regulatory network betweenMYCNand TSmiRs in NB. Furthermore, NB cells are high in omega 9 (ω9) unsaturated fatty acids that can be synthesizedde novoand low in both ω6 and ω3, providing a plausible means for NB to limit cell-autonomous immune stimulation and reactive oxygen species (ROS)-driven apoptosis from ω6 and ω3 unsaturated fatty acid derivatives, respectively. We propose a model in which the UFAS pathway, through novel regulation byMYCNand TSmiRs, plays a key role in neuroblastoma pathology with implications for otherMYC-driven cancers.
Transcript Assembly and Annotations: Bias and Adjustment
Authors: Zhang Qimin , Shao Mingfu
Published: April 2023

Motivation

Transcript annotations play a critical role in gene expression analysis as they serve as a reference for quantifying isoform-level expression. The two main sources of annotations are RefSeq and Ensembl/GENCODE, but discrepancies between their methodologies and information resources can lead to significant differences. It has been demonstrated that the choice of annotation can have a significant impact on gene expression analysis. Furthermore, transcript assembly is closely linked to annotations, as assembling large-scale available RNA-seq data is an effective data-driven way to construct annotations, and annotations are often served as benchmarks to evaluate the accuracy of assembly methods. However, the influence of different annotations on transcript assembly is not yet fully understood.

Results

We investigate the impact of annotations on transcript assembly. We observe that conflicting conclusions can arise when evaluating assemblers with different annotations. To understand this striking phenomenon, we compare the structural similarity of annotations at various levels and find that the primary structural difference across annotations occurs at the intron-chain level. Next, we examine the biotypes of annotated and assembled transcripts and uncover a significant bias towards annotating and assembling transcripts with intron retentions, which explains above the contradictory conclusions. We develop a standalone tool, available athttps://github.com/Shao-Group/irtool, that can be combined with an assembler to generate an assembly without intron retentions. We evaluate the performance of such a pipeline and offer guidance to select appropriate assembling tools for different application scenarios.
Hold out the genome: A roadmap to solving the cis-regulatory code
Authors: de Boer Carl G. , Taipale Jussi
Published: April 2023

Gene expression is regulated by transcription factors that work together to read cis-regulatory DNA sequences. The “cis-regulatory code” - the rules that cells use to determine when, where, and how much genes should be expressed - has proven to be exceedingly complex, but recent advances in the scale and resolution of functional genomics assays and Machine Learning have enabled significant progress towards deciphering this code. However, we will likely never solve the cis-regulatory code if we restrict ourselves to models trained only on genomic sequences; regions of homology can easily lead to overestimation of predictive performance, and there is insufficient sequence diversity in our genomes to learn all relevant parameters. Fortunately, randomly synthesized DNA sequences enable us to test a far larger sequence space than exists in our genomes in each experiment, and designed DNA sequences enable a targeted query of the sequence space to maximally improve the models. Since cells use the same biochemical principles to interpret DNA regardless of its source, models that are trained on these synthetic data can predict genomic activity, often better than genome-trained models. Here, we provide an outlook on the field, and propose a roadmap towards solving the cis-regulatory code by training models exclusively on non-genomic DNA sequences, and using genomic sequences solely for evaluating the resulting models.
Extreme structural heterogeneity rewires glioblastoma chromosomes to sustain patient-specific transcriptional programs
Authors: Xie Ting , Danieli-Mackay Adi , Buccarelli Mariachiara , Barbieri Mariano , Papadionysiou Ioanna , Giorgio D’Alessandris Q. , Übelmesser Nadine , Vinchure Omkar Suhas , Lauretti Liverana , Fotia Giorgio , Wang Xiaotao , Ricci-Vitiani Lucia , Gopalakrishnan Jay , Pallini Roberto , Papantonis Argyris
Published: April 2023

Glioblastoma multiforme (GBM) encompasses brain malignancies marked by phenotypic and transcriptional heterogeneity thought to render these tumors aggressive, resistant to therapy, and inevitably recurrent. However, little is known about how the spatial organization of GBM genomes underlies this heterogeneity and its effects. Here, we compiled a cohort of 28 patient-derived glioblastoma stem cell-like lines (GSCs) known to reflect the properties of their tumor-of-origin; six of these were primary-relapse tumor pairs from the same patient. We generated and analyzed kbp-resolution chromosome conformation capture (Hi-C) data from all GSCs to systematically map >3,100 standalone and complex structural variants (SVs) and the >6,300 neoloops arising as a result. By combining Hi-C, histone modification, and gene expression data with chromatin folding simulations, we explain how the pervasive, uneven, and idiosyncratic occurrence of neoloops sustains tumor-specific transcriptional programs via the formation of new enhancer-promoter contacts. We also show how even moderately recurrent neoloops can help us infer patient-specific vulnerabilities. Together, our data provide a resource for dissecting GBM biology and heterogeneity, as well as for informing therapeutic approaches.
Influence of Alzheimer’s disease related neuropathology on local microenvironment gene expression in the human inferior temporal cortex
Authors: Kwon Sang Ho , Parthiban Sowmya , Tippani Madhavi , Divecha Heena R. , Eagles Nicholas J. , Lobana Jashandeep S. , Williams Stephen R. , Mak Michelle , Bharadwaj Rahul A. , Kleinman Joel E. , Hyde Thomas M. , Page Stephanie C. , Hicks Stephanie C. , Martinowich Keri , Maynard Kristen R. , Collado-Torres Leonardo
Published: April 2023

Neuropathological lesions in the brains of individuals affected with neurodegenerative disorders are hypothesized to trigger molecular and cellular processes that disturb homeostasis of local microenvironments. Here, we applied the 10x Genomics Visium Spatial Proteogenomics (Visium-SPG) platform, which measures spatial gene expression coupled with immunofluorescence protein co-detection, in post-mortem human brain tissue from individuals with late-stage Alzheimer’s disease (AD) to investigate changes in spatial gene expression with respect to amyloid-β (Aβ) and hyperphosphorylated tau (pTau) pathology. We identified Aβ-associated transcriptomic signatures in the human inferior temporal cortex (ITC) during late-stage AD, which we further investigated at cellular resolution with combined immunofluorescence and single molecule fluorescent in situ hybridization (smFISH) co-detection technology. We present a workflow for analysis of Visium-SPG data and demonstrate the power of multi-omic profiling to identify spatially-localized changes in molecular dynamics that are linked to pathology in human brain disease. We provide the scientific community with web-based, interactive resources to access the datasets of the spatially resolved AD-related transcriptomes athttps://research.libd.org/Visium_SPG_AD/.
Actin polymerization drives lumen formation in a human epiblast model
Authors: Indana Dhiraj , Zakharov Andrei , Lim Youngbin , Dunn Alexander R. , Bhutani Nidhi , Shenoy Vivek B. , Chaudhuri Ovijit
Published: April 2023

Lumens or fluid-filled cavities are a ubiquitous feature of mammals and are often evolutionarily linked to the origin of body-plan complexity. Post-implantation, the pluripotent epiblast in a human embryo forms a central lumen, paving the way for gastrulation. While osmotic pressure gradients drive lumen formation in many developmental contexts, mechanisms of human epiblast lumenogenesis are unknown. Here, we study lumenogenesis in a pluripotent-stem-cell-based model of the epiblast using engineered hydrogels that model the confinement faced by the epiblast in the blastocyst. Actin polymerization into a dense mesh-like network at the apical surface generates forces to drive early lumen expansion, as leaky junctions prevent osmotic pressure gradients. Theoretical modeling reveals that apical actin polymerization into a stiff network drives lumen opening, but predicts that a switch to pressure driven lumen growth at larger lumen sizes is required to avoid buckling of the cell layer. Consistent with this prediction, once the lumen reaches a radius of around 12 μm, tight junctions mature, and osmotic pressure gradients develop to drive further lumen growth. Human epiblasts show a transcriptional signature of actin polymerization during early lumenogenesis. Thus, actin polymerization drives lumen opening in the human epiblast, and may serve as a general mechanism of lumenogenesis.
Simultaneous tracking of near-isogenic bacterial strains in syntheticArabidopsismicrobiota by chromosomally-integrated barcodes
Authors: Ordon Jana , Thouin Julien , Nakano Ryohei Thomas , Ma Ka-Wai , Zhang Pengfan , Huettel Bruno , Garrido-Oter Ruben , Schulze-Lefert Paul
Published: April 2023

DNA amplicon-based microbiota profiling currently relies on polymorphisms in microbial marker genes to estimate species diversity and abundance. However, this technique cannot resolve genetic differences among microbial individuals of the same species. We report here the development of modular bacterial tags (MoBacTags) encoding DNA barcodes. These tags facilitate tracking of near-isogenic bacterial commensals in synthetic communities (SynComs), which allow assessment of the contributions of individual bacterial genes to root microbiota establishment inArabidopsis thaliana. Chromosomally-integrated DNA barcodes are co-amplified with endogenous marker genes of the community by integrating corresponding primer binding sites into the barcode. We generated MoBacTag-labeled strains of wild-typePseudomonas capeferrumWCS358 and of pqqF and cyoB mutants with known defects in gluconic acid-mediated host immunosuppression and validated reduced root colonization of both mutants in a 15-member synthetic microbiota. We detected a reduced SynCom load on roots in the presence of the WCS358:pqqF mutant, but not WCS358:cyoB, revealing distinctpqqFandcyoBactivities in a community context. Using MoBacTags, we also show that WCS358 pqqF mutant-specific colonization and community establishment is nottrans-complemented by wild-type WCS358. Given that gluconic acid production inP. capeferrumis indirectly abolished in the pqqF mutant by disruption of pyrroloquinoline quinone (PQQ) biosynthesis, we propose that drastic changes in the root-associated community result from depletion of the cofactor PQQ, which might serve as a common good during root microbiota establishment. Our proof-of-principle experiments illustrate how MoBacTags can be applied to assess scaling of individual bacterial genetic determinants in the plant microbiota.
Antagonistic nanobodies reveal mechanism of GSDMD pore formation and unexpected therapeutic potential
Authors: Schiffelers Lisa D.J. , Normann Sabine , Binder Sophie C. , Hagelauer Elena , Kopp Anja , Alon Assaf , Geyer Matthias , Ploegh Hidde L. , Schmidt Florian I.
Published: April 2023

Activation of various inflammasomes converges on the cleavage of gasdermin D (GSDMD) by pro-inflammatory caspases, followed by oligomerization of the N-terminal domain (GSDMDNT) and the assembly of pores penetrating target membranes. Yet, it remained unclear what triggers the conformational changes that allow membrane insertion, as methods to study pore formation in living cells were limited. We raised nanobodies specific for human GSDMD and found two nanobodies that prevent pyroptosis and IL-1β release when expressed in the cytosol of human macrophages. Nanobody binding to GSDMDNTblocked its oligomerization, while inflammasome assembly and GSDMD processing itself were not affected. The nanobody-stabilized monomers of GSDMDNTpartitioned into the plasma membrane, suggesting that pore formation is initiated by insertion of monomers, followed by oligomerization in the target membrane. When GSDMD pore formation was inhibited, cells still underwent caspase-1-dependent apoptosis, likely due to the substantially augmented caspase-1 activity. This hints at a novel layer of regulation of caspase-1 activity by GSDMD pores. Moreover, we revealed the unexpected therapeutic potential of antagonistic GSDMD nanobodies, as recombinant nanobodies added to the medium prevented cell death by pyroptosis, likely by entering through GSDMD pores and curtailing the assembly of additional pores. GSDMD nanobodies may thus be suitable to treat the ever-growing list of diseases caused by activation of the (non-) canonical inflammasomes.
Assessment of the Glycan-Binding Profile ofPseudomonas aeruginosaPAO1
Authors: Sanchez Hector , O’Toole George A. , Berwin Brent
Published: April 2023

Pseudomonas aeruginosais an opportunistic pathogen that can establish acute and chronic infections in individuals that lack fully functional innate immunity. In particular, phagocytosis by neutrophils and macrophages is a key mechanism that modulates host control and clearance ofP. aeruginosa. Individuals with neutropenia or cystic fibrosis are highly susceptible toP. aeruginosainfection thus underscoring the importance of the host innate immune response. Cell-to-cell contact between host innate immune cells and the pathogen, a first step in phagocytic uptake, is facilitated by simple and complex glycan structures present at the host cell surface. We have previously shown that endogenous polyanionic N-linked glycans localized to the cell surface of phagocytes mediate binding and subsequent phagocytosis ofP. aeruginosa. However, the suite of glycans thatP. aeruginosabinds to on host phagocytic cells remains poorly characterized. Here we demonstrate, with the use of exogenous N-linked glycans and a glycan array, thatP. aeruginosaPAO1 preferentially attaches to a subset of glycans, including a bias towards monosaccharide versus more complex glycan structures. Consistent with these findings, we were able to competitively inhibit bacterial adherence and uptake by the addition of exogenous N-linked mono- and di-saccharide glycans. We discuss of findings in the context of previous reports ofP. aeruginosaglycan binding.

IMPORTANCE

P. aeruginosabinds to a variety of glycans as part of its interaction with host cells, and a number ofP. aeruginosa-encoded receptors and target ligands have been described that allow this microbe to bind to such glycans. Here we extend this work by studying the glycans used byP. aeruginosaPAO1 to bind to phagocytic cells and by using a glycan array to characterize the suite of such molecules that could facilitate host cell-binding by this microbe. This study provides an increased understanding of the glycans bound byP. aeruginosa, and furthermore, provides a useful dataset for future studies ofP. aeruginosa-glycan interactions.
Feature-Based 3D+t Descriptors of Hyperactivated Human Sperm Beat Patterns
Authors: Hernández Haydee O. , Montoya Fernando , Hernández-Herrera Paul , Díaz-Guerrero Dan S. , Olveres Jimena , Darszon Alberto , Escalante-Ramírez Boris , Corkidi Gabriel
Published: April 2023

The flagellar movement of the mammalian sperm is essential for male fertility as it enables this cell to reach and fertilize an egg. In the female reproductive tract, human spermatozoa undergo a process called capacitation which promotes changes in their motility. Only those spermatozoa that change to hyperactivated (HA) motility are capable of fertilizing the egg; this type of motility is characterized by asymmetric flagellar bends of greater amplitude and lower frequency. Historically, clinical fertilization studies have used two-dimensional analysis to classify sperm motility, although, sperm motility is three-dimensional (3D). Recent studies have described several 3D beating features of sperm flagella, including curvature, torsion, and asymmetries. However, the 3D motility pattern of hyperactivated spermatozoa has not yet been characterized. One of the main difficulties in classifying these patterns in 3D is the lack of a ground-truth reference, as it can be difficult to visually assess differences in flagellar beat patterns. Additionally, only about 10 − 20% of sperm that have been induced to capacitate are truly capacitated (i.e., hyperactivated). In this work, we used an image acquisition system that can acquire, segment, and track sperm flagella in 3D+t. We developed a feature-based vector that describes the spatio-temporal flagellar sperm motility patterns by an envelope of ellipses. Our results demonstrate that the proposed descriptors can effectively be used to distinguish between hyperactivated and nonhyperactivated spermatozoa, providing a tool to characterize the 3D sperm flagellar beat motility patterns without prior training or supervision. We demonstrated the effectiveness of the descriptors by applying them to a dataset of human sperm cells and showing that they can accurately classify the motility patterns of the sperm cells. This work is potentially useful for assessing male fertility or for diagnosing.
Efficient protein tagging andcis-regulatory element engineering via precise and directional oligonucleotide-based targeted insertion in plants
Authors: Kumar Jitesh , Char Si Nian , Weiss Trevor , Liu Hua , Liu Bo , Yang Bing , Zhang Feng
Published: April 2023

Efficient and precise targeted insertion holds great promise but remains challenging in plant genome editing. An efficient NHEJ-mediated targeted insertion method was recently developed by combining CRISPR-Cas9 with phosphorothioate modified double-stranded oligodeoxynucleotides (dsODNs). Yet this approach often led to imprecise insertions with no control over the insertion direction. In this study, we first quantitatively compared the impact of the chemical protection on efficiency of targeted insertion. With the observation that CRISPR-SpCas9 could frequently induce staggered cleavages with 5′ 1-nucleotide overhangs, we then evaluated the impact of the donor end structures on the direction and preciseness of targeted insertions. Our study demonstrated that the chemically protected dsODNs with 5′ 1-nt overhangs could significantly improve the precision and direction control of target insertions in all tested CRIPSR targeting sites. Lastly, we applied this method to endogenous gene tagging inSetaria viridis, andcis-regulatory element engineering for disease resistance in rice. Two distinct TAL effector binding elements were directionally inserted into the promoter region of a recessive rice bacterial blight resistance gene at up to 24.4% efficiency. The resulting rice lines with heritable insertions exhibited strong resistance to the infection ofXanthomonas oryzaepv.oryzaepathogen in an inducible and strain-specific manner.
Stitching flexible electronics into the brain
Authors: Lee Jung Min , Lin Dingchang , Pyo Young-Woo , Kim Ha-Reem , Park Hong-Gyu , Lieber Charles M.
Published: April 2023

Understanding complex neuronal networks requires monitoring long-term neuronal activity in various regions of the brain. Significant progress has been made in multi-site implantations of well-designed probes, such as multi-site implantation of Si-based and polymer-based probes. However, these multi-probe strategies have been limited by the sizes and weights of interfaces to the multiple probes and the inability to track the activity of the same neurons and changes in neuronal activity over longer time periods. Here, we report a long single flexible probe that can be implanted by stitching into multiple regions of the mouse brain and subsequently transmit chronically-stable neuronal signals from the multiple sites via a single low-mass interface. We implanted the probe at four different sites using a glass capillary needle or two sites using an ultrathin metal needle. In-vitro tests in brain-mimicking hydrogel showed that multi-site probe implantations achieved a high connection yield of >86%. In-vivo histological images at each site of probes, implanted by stitching using either glass capillary or ultrathin metal insertion needles exhibit seamless tissue-probe interfaces with negligible chronic immune response. In addition, electrophysiology studies demonstrated the ability to track single neuron activities at every injection site with chronic stability over at least one month. Notably, the measured spike amplitudes and signal-to-noise ratios at different implantation sites showed no statistically significant differences. Multi-site stitching implantation of flexible electronics in the brain opens up new opportunities for both fundamental neuroscience research and electrotherapeutic applications.
High-resolution structure of a mercury cross-linked ZIP metal transporter reveals delicate motions and metal relay for regulated zinc transport
Authors: Zhang Tuo , Zhang Yao , Sui Dexin , Hu Jian
Published: April 2023

Zrt-/Irt-like protein (ZIP) divalent metal transporters play a central role in maintaining trace element homeostasis. The prototypical ZIP fromBordetella bronchiseptica(BbZIP) is an elevator-type transporter, but the dynamic motions and detailed transport mechanism remain to be elucidated. Here, we report a high-resolution crystal structure of a mercury-crosslinked BbZIP variant at 1.95 Å, revealing an upward rotation of the transport domain in the new inward-facing conformation and a water-filled metal release channel that is divided into two parallel pathways by the previously disordered cytoplasmic loop. Mutagenesis and transport assays indicated that the newly identified high-affinity metal binding site in the primary pathway acts as a “metal sink” to reduce the transport rate. The discovery of a hinge motion around an extracellular axis allowed us to propose a sequential hinge-elevator-hinge movement of the transport domain to achieve alternating access. These findings provide key insights into the transport mechanisms and activity regulation.
Genetic basis of aposematic coloration in a mimetic radiation of poison frogs
Authors: Linderoth Tyler , Aguilar-Gómez Diana , White Emily , Twomey Evan , Stuckert Adam , Bi Ke , Ko Amy , Graham Natalie , Rocha Joana L. , Chang Jason , MacManes Matthew D. , Summers Kyle , Nielsen Rasmus
Published: April 2023

The evolution of mimicry in a single species or population has rippling inter and intraspecific effects across ecological communities, providing a fascinating mechanism of phenotypic diversification. In this study we present the first identification of genes underlying Müllerian mimicry in a vertebrate, the Peruvian mimic poison frog,Ranitomeya imitator. We sequenced 124R. imitatorexomes and discovered loci with both strong divergence between different mimetic morphs and phenotypic associations within an intraspecific admixture zone, implicatingmc1r,asip,bsn,retsat, andkrt8.2in the evolution of mimetic color phenotypes. We confirmed these associations for most candidate genes through linkage mapping in a lab-reared pedigree. We also sequenced transcriptomes from the model species, allowing tests for introgression and revealing that the mimetic resemblance betweenR. imitatorand the models evolved independently. Selection analyses of the candidate genes show that the mimicry phenotypes likely have evolved through selective sweeps acting on polygenic variation. Our results suggest that the evolutionary origins and molecular mechanisms underlying mimicry phenotypes in vertebrates may be radically different from those previously documented in invertebrates such as the iconicHeliconiusbutterfly mimicry complex.

One Sentence Summary

Müllerian mimicry evolved through independent selective sweeps on color and pattern loci in the mimic poison frog.
Circadian redox rhythm gates immune-induced cell death distinctly from the genetic clock
Authors: Karapetyan Sargis , Mwimba Musoki , Dong Xinnian
Published: April 2023

Organisms use circadian clocks to synchronize physiological processes to anticipate the Earth’s day-night cycles and regulate responses to environmental stresses to gain competitive advantage1. While divergent genetic clocks have been studied extensively in bacteria, fungi, plants, and animals, a conserved circadian redox rhythm has only recently been reported and hypothesized to be a more ancient clock2, 3. However, it is controversial whether the redox rhythm serves as an independent clock and controls specific biological processes4. Here, we uncovered the coexistence of redox and genetic rhythms with distinct period lengths and transcriptional targets through concurrent metabolic and transcriptional time-course measurements in anArabidopsislong-period clock mutant5. Analysis of the target genes indicated regulation of the immune-induced programmed cell death (PCD) by the redox rhythm. Moreover, this time-of-day-sensitive PCD was eliminated by redox perturbation and by blocking the signalling pathway of the plant defence hormones jasmonic acid/ethylene, while remaining intact in a genetic-clock-impaired line. We demonstrate that compared to robust genetic clocks, the more sensitive circadian redox rhythm serves as a signalling hub in regulating incidental energy-intensive processes, such as immune-induced PCD6, to provide organisms a flexible strategy to prevent metabolic overload caused by stress, a unique role for the redox oscillator.
Multidimensional analysis of matched primary and recurrent glioblastoma identifies Fcγ receptors upregulation on microglia as a contributor of tumor recurrence
Authors: Shekarian Tala , Hogan Sabrina , Martins Tomás A. , Schmassmann Philip , Gerber Alexandra , Roux Julien , Kaymak Deniz , Durano Célia , Burger Bettina , Matter Matthias , Ritz Marie-Françoise , Hutter Gregor
Published: April 2023

Background: Glioblastoma (GBM) is a lethal brain tumor without effective treatment options. The aim of this study was to characterize longitudinal tumor immune microenvironment (iTME) changes in order to find potential actionable targets to prevent GBM-induced immune evasion mechanisms.

Methods: This study included 15 patient-matched treatment-naïve WHO grade 4 primary (pGBM) and recurrent (rGBM) tumors. RNA and proteins extracted from fresh frozen tumor samples from matched pGBM and rGBM were profiled via transcriptomics and proteomics, respectively. A tissue microarray containing paired formalin-fixed paraffin-embedded tumor samples was processed for spatial transcriptomics analysis.

Results: Differentially expressed genes and proteins between pGBM and rGBM were involved in pathways responsible for synapse development and myelination which have been shown to play a role in GBM recurrence. By categorizing patients into short and long time-to-relapse (STTR vs LTTR), we identified genes positively or negatively associated with TTR. Expression of Fcγ receptors and complement system genes such asFCGR1A(CD64),FCGR3AandC3in rGBM samples were negatively correlated with TTR, whereas expression ofDNMT1/3A, andSMARCA4, involved in DNA methylation, were positively correlated with TTR. Spatial transcriptomic analysis of the tumor cell compartment showed enrichment of oligodendrocytes in rGBM, whereas the myeloid cell compartment switched from quiescent to activated microglia, was enriched in B and T cells, specifically in rGBM with STTR.

Conclusions:Our results uncover a role for CD64-expressing activated microglia in GBM recurrence and suggest that interfering with these cells may represent a therapeutic option for hindering GBM relapse.

Key points:

Transcriptomic and proteomic differences exist between patient-paired primary and recurrent GBM tumors

High expression of Fcy receptors genes on activated microglia at tumor recurrence is associated with shorter time to relapse.

Importance of this study:

In glioblastoma (GBM), the tumor recurs in almost all cases after standard treatment such as surgery and chemo-radiotherapy. In this study, we longitudinally evaluated the immune- and neoplastic compartments using transcriptomic, proteomic, and spatial transcriptomics in patient-matched treatment-naive and recurrent tumor samples. By correlating gene expression with time-to-relapse, we identified a geneset associated with treatment resistance and faster tumor recurrence. Moreover, this study highlighted the plasticity of the myeloid compartment during disease progression and an unfavorable role of activated microglia in tumor recurrence.
Translating deep learning to neuroprosthetic control
Authors: Deo Darrel R. , Willett Francis R. , Avansino Donald T. , Hochberg Leigh R. , Henderson Jaimie M. , Shenoy Krishna V.
Published: April 2023

Advances in deep learning have given rise to neural network models of the relationship between movement and brain activity that appear to far outperform prior approaches. Brain-computer interfaces (BCIs) that enable people with paralysis to control external devices, such as robotic arms or computer cursors, might stand to benefit greatly from these advances. We tested recurrent neural networks (RNNs) on a challenging nonlinear BCI problem: decoding continuous bimanual movement of two computer cursors. Surprisingly, we found that although RNNs appeared to perform well in offline settings, they did so by overfitting to the temporal structure of the training data and failed to generalize to real-time neuroprosthetic control. In response, we developed a method that alters the temporal structure of the training data by dilating/compressing it in time and re-ordering it, which we show helps RNNs successfully generalize to the online setting. With this method, we demonstrate that a person with paralysis can control two computer cursors simultaneously, far outperforming standard linear methods. Our results provide evidence that preventing models from overfitting to temporal structure in training data may, in principle, aid in translating deep learning advances to the BCI setting, unlocking improved performance for challenging applications.
TopoDoE: A Design of Experiment strategy for selection and refinement in ensembles of executable Gene Regulatory Networks
Authors: Bouvier Matteo , Zreika Souad , Vallin Elodie , Fourneaux Camille , Giraud-Gonin Sandrine , Bonnaffoux Arnaud , Gandrillon Olivier
Published: April 2023

Background

Inference of Gene Regulatory Networks (GRNs) is a difficult and long-standing question in Systems Biology. Numerous approaches have been proposed with the latest methods exploring the richness of single-cell data. One of the current difficulties lies in the fact that many methods of GRN inference do not result in one proposed GRN but in a collection of plausible networks that need to be further refined. In this work, we present a Design of Experiment strategy to use as a second stage after the inference process. It is specifically fitted for identifying the next most informative experiment to perform for deciding between multiple network topologies, in the case where proposed GRNs are executable models. This strategy first performs a topological analysis to reduce the number of perturbations that need to be tested, then predicts the outcome of the retained perturbations by simulation of the GRNs and finally compares predictions with novel experimental data.

Results

We apply this method to the results of our divide-and-conquer algorithm called WASABI, adapt its gene expression model to produce perturbations and compare our predictions with experimental results. We show that our networks were able to producein silicopredictions on the outcome of a gene knock-out, which were qualitatively validated for 48 out of 49 genes. Finally, we eliminate as many as two thirds of the candidate networks for which we could identify an incorrect topology, thus greatly improving the accuracy of our predictions.

Conclusion

These results both confirm the inference accuracy of WASABI and show how executable gene expression models can be leveraged to further refine the topology of inferred GRNs. We hope this strategy will help systems biologists further explore their data and encourage the development of more executable GRN models.