bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid optogenetic clustering of a cytoplasmic BcLOV4 variant
Authors: Huang Zikang , Benman William , Dong Liang , Bugaj Lukasz J.
Published: September 2023

Protein clustering is a powerful form of optogenetic control, yet there is currently only one protein —Cry2—whose light-induced clustering has been harnessed for these purposes. Recently, the photoreceptor BcLOV4 was found to form protein clusters in mammalian cells in response to blue light, although clustering coincided with its translocation to the plasma membrane, potentially constraining its application as an optogenetic clustering module. Herein we identify key amino acids that couple clustering to membrane binding, allowing us to engineer a variant of BcLOV4 that clusters in the cytoplasm and does not associate with the membrane in response to blue light. This variant, BcLOVclust, clustered over many cycles with dramatically faster clustering and de-clustering kinetics compared to Cry2. The magnitude of BcLOVclust clustering could be strengthened by appending an intrinsically disordered region from the fused in sarcoma (FUS) protein, or by optimizing the fluorescent protein to which it was fused. BcLOVclust retained the temperature sensitivity of BcLOV4 such that light induced clustering was transient, and the rate of spontaneous declustering increased with temperature. At low temperatures, BcLOVclust and Cry2 could be multiplexed in the same cells, allowing light control of independent protein condensates. BcLOVclust could also be applied to control signaling proteins and stress granules in mammalian cells. Thus BcLOVclust provides an alternative to Cry2 for optogenetic clustering and a method for multiplexed clustering. While its usage is currently suited for organisms that can be cultured below ∼30 °C, a deeper understanding of BcLOVclust thermal response will further enable its use at physiological mammalian temperatures.

Graphical abstract

Highlights

Light-responsive clustering of BcLOV4 can be decoupled from its membrane association

BcLOVclust clusters in the cytoplasm with faster ON and OFF kinetics than Cry2

BcLOVclust and Cry2 can be multiplexed in the same cell

BcLOVclust can control RhoA activity and stress granule formation
Gene regulatory network that shaped the evolution of larval sensory organ in Cnidaria
Authors: Gilbert Eleanor , Craggs Jamie , Modepalli Vengamanaidu
Published: September 2023

Among non-bilaterian animals, a larval apical sensory organ with integrated neurons is only found in cnidarians. Within cnidarians, an apical organ with a ciliary tuft is mainly found in Actiniaria. Whether this apical tuft has evolved independently in Actiniaria or alternatively originated in the common ancestor of Cnidaria and Bilateria and was lost in specific groups is uncertain. We generated transcriptomes of the apical domain during the planula stage of four species representing three key groups of cnidarians:Aurelia aurita(Scyphozoa),Nematostella vectensis(Actiniaria), andAcropora millepora&Acropora tenuis(Scleractinia). We showed that the canonical genes implicated in patterning the apical domain ofNematostellaare largely absent inAurelia, indicating that scyphozoans and anthozoans do not share apical organ homology. In contrast, the apical domain of the scleractinian planula shares gene expression pattern withNematostella. By comparing the larval single-cell transcriptomes, we revealed the apical organ cell type of Scleractinia and confirmed its homology to Actiniaria. However,Fgfa2, a vital regulator of the regionalisation of theNematostellaapical organ, is absent in the scleractinian genome. Likewise, we found thatFoxJ1and 245 genes associated with cilia are exclusively expressed in theNematostellaapical domain, which is in line with the presence of ciliary apical tuft in Actiniaria and its absence in Scleractinia and Scyphozoa. Our findings suggest that the common ancestor of cnidarians and bilaterians lacked an apical organ with a ciliary tuft, and it could have evolved independently in several clades of cnidarians and bilaterians.
Strong environmental memory revealed by experimental evolution in static and fluctuating environments
Authors: Abreu Clare I. , Mathur Shaili , Petrov Dmitri A.
Published: September 2023

Evolution in a static environment, such as a laboratory setting with constant and uniform conditions, often proceeds via large-effect beneficial mutations that may become maladaptive in other environments. Conversely, natural settings require populations to endure environmental fluctuations. A sensible assumption is that the fitness of a lineage in a fluctuating environment is the time-average of its fitness over the sequence of static conditions it encounters. However, transitions between conditions may pose entirely new challenges, which could cause deviations from this time-average. To test this, we tracked hundreds of thousands of barcoded yeast lineages evolving in static and fluctuating conditions and subsequently isolated 900 mutants for pooled fitness assays in 15 environments. We find that fitness in fluctuating environments indeed often deviates from the expectation based on static components, leading to fitness non-additivity. Moreover, closer examination reveals that fitness in one component of a fluctuating environment is often strongly influenced by the previous component. We show that this environmental memory is especially common for mutants with high variance in fitness across tested environments, even if the components of the focal fluctuating environment are excluded from this variance. We employ a simple mathematical model and whole-genome sequencing to propose mechanisms underlying this effect, including lag time evolution and sensing mutations. Our results demonstrate that environmental fluctuations have large impacts on fitness and suggest that variance in static environments can explain these impacts.
Patterns of genomic variation reveal a single evolutionary origin of the wild allotetraploidMimulus sookensis
Authors: Whitener Makenzie R. , Mangelson Hayley , Sweigart Andrea L.
Published: September 2023

Polyploidy occurs across the tree of life and is especially common in plants. Because newly formed cytotypes are often incompatible with their progenitors, polyploidy is also said to trigger “instantaneous” speciation. If a polyploid can self-fertilize or reproduce asexually, it is even possible for one individual to produce an entirely new lineage. How often this extreme scenario occurs is unclear, with most studies of wild polyploids reporting multiple origins. Here, we explore the evolutionary history of the wild allotetraploidMimulus sookensis,which was formed through hybridization between self-compatible, diploid species in theMimulus guttatuscomplex. We generate a chromosome-scale reference assembly forM. sookensisand define its distinct subgenomes. Despite previous reports suggesting multiple origins of this highly selfing polyploid, we discover patterns of population genomic variation that provide unambiguous support for a single origin, which we estimate occurred ∼71,000 years ago. OneM. sookensissubgenome is clearly derived from the selferM. nasutus, which, based on organellar variation, also appears to be the maternal progenitor. The ancestor of the other subgenome is less certain, but it shares variation with bothM. decorusandM. guttatus, two outcrossing diploids that overlap broadly withM. sookensis. Whatever its precise ancestry, this study establishesM. sookensisas an example of instantaneous speciation, likely facilitated by the polyploid’s predisposition to self-fertilize. With a reference genome forM. sookensisnow available and its origin clarified, this wild tetraploid is poised to become a model for understanding the genetic and evolutionary mechanisms of polyploid persistence.
ONeSAMP 3.0: Effective Population Size via SNP Data for One Population Sample
Authors: Hong Aaron , Cheek Rebecca G. , Mukherjee Kingshuk , Yooseph Isha , Oliva Marco , Heim Mark , Funk W. Chris , Tallmon David , Boucher Christina
Published: September 2023

The genetic effective size (Ne) is arguably one of the most important characteristics of a population as it impacts the rate of loss of genetic diversity. Genetic estimators of (Ne) increasingly popular tools in population and conservation genetic studies. Yet there are very few methods that can estimate the Ne from data from a single population and without extensive information about the genetics of the population, such as a linkage map, or a reference genome of the species of interest.

We present ONeSAMP 3.0, an algorithm for estimating Ne from single nucleotide polymorphism (SNP) data collected from a single population sample using Approximate Bayesian Computation and local linear regression.

We demonstrate the utility of this approach using simulated Wright-Fisher populations, and empirical data from five endangered Channel Island fox (Urocyon littoralis) populations to evaluate the performance of ONeSAMP 3.0 compared to a commonly used Ne estimator. Our results show that ONeSAMP 3.0 is robust to the number of individual samples and number of loci included in and appears accurate even if the range of true Ne values is large.

This method is broadly applicable to natural populations and is flexible enough that future versions could easily include summary statistics appropriate for a suite of biological and sampling conditions. ONeSAMP 3.0 is publicly available under the GNU license athttps://github.com/AaronHong1024/ONeSAMP_3and also available with Bioconda (https://bioconda.github.io/index.html).
High-fidelity Image Restoration of Large 3D Electron Microscopy Volume
Authors: Kreinin Yuri , Gunn Pat , Chklovskii Dmitri , Wu Jingpeng
Published: September 2023

Volume Electron Microscopy (VEM) is an essential tool for studying biological structures. Due to the challenges of sample preparation and continuous volumetric imaging, image artifacts are almost inevitable. Such image artifacts complicate further processing both for automated computer vision methods and human experts. Unfortunately, the widely used Contrast Limited Adaptive Histogram Equalization (CLAHE) can alter the essential relative contrast information about some biological structures. We developed an image-processing pipeline to remove the artifacts and enhance the images without CLAHE. We apply our method to VEM datasets of a Microwasp head. We demonstrate that our method restores the images with high fidelity while preserving the original relative contrast. This pipeline is adaptable to other VEM datasets.
CD39 expression by regulatory T cells drives CD8+ T cell suppression during experimentalTrypanosoma cruziinfection
Authors: Araujo Furlan Cintia L. , Boccardo Santiago , Rodriguez Constanza , Robson Simon C. , Gruppi Adriana , Montes Carolina L. , Acosta Rodríguez Eva V.
Published: September 2023

An imbalance between suppressor and effector immune responses may preclude cure in chronic parasitic diseases. In the case ofTrypanosoma cruziinfection, specialized regulatory Foxp3+ T (Treg) cells suppress protective type-1 effector responses. Herein, we investigated the kinetics and underlying mechanisms behind the regulation of protective parasite-specific CD8+ T cell immunity during acuteT. cruziinfection. Using the DEREG mouse model, we found that Treg cells play a critical role during the initial stages afterT. cruziinfection, subsequently influencing CD8+ T cells. Early Treg cell depletion increased the frequencies of polyfunctional short-lived, effector T cell subsets, without affecting memory precursor cell formation or the expression of activation markers. In addition, Treg cell depletion during early infection minimally affected the antigen-presenting cell response but it boosted CD4+ T cell responses before the development of anti-parasite effector CD8+ T cell responses. Crucially, the absence of CD39 expression on Treg cells significantly bolstered effector parasite-specific CD8+ T cell responses, leading to improved parasite control duringT. cruziinfection. Our work underscores the crucial role of Treg cells in regulating protective anti-parasite immunity and provides evidence that CD39 expression by Treg cells represents a key immunomodulatory mechanism in this infection model.
Integration of Patient-Derived Organoids and Organ-on-Chip Systems: Investigating Colorectal Cancer Invasion within the Mechanical and GABAergic Tumor Microenvironment
Authors: Strelez Carly , Perez Rachel , Chlystek John S. , Cherry Christopher , Yoon Ah Young , Haliday Bethany , Shah Curran , Ghaffarian Kimya , Sun Ren X. , Jiang Hannah , Lau Roy , Schatz Aaron , Lenz Heinz-Josef , Katz Jonathan E. , Mumenthaler Shannon M.
Published: September 2023

Three-dimensional (3D) in vitro models are essential in cancer research, but they often neglect physical forces. In our study, we combined patient-derived tumor organoids with a microfluidic organ-on-chip system to investigate colorectal cancer (CRC) invasion in the tumor microenvironment (TME). This allowed us to create patient-specific tumor models and assess the impact of physical forces on cancer biology. Our findings showed that the organoid-on-chip models more closely resembled patient tumors at the transcriptional level, surpassing organoids alone. Using ’omics’ methods and live-cell imaging, we observed heightened responsiveness of KRAS mutant tumors to TME mechanical forces. These tumors also utilized the γ-aminobutyric acid (GABA) neurotransmitter as an energy source, increasing their invasiveness. This bioengineered model holds promise for advancing our understanding of cancer progression and improving CRC treatments.

Highlights

Microfluidic organ-on-chip system integrated with patient-derived CRC organoids

Physical forces influence invasion, particularly in KRAS mutant tumor cells

GABAergic signaling contributes to increased invasion within a dynamic TME

This model explores patient heterogeneity, TME interactions, and cancer progression

GRAPHICAL ABSTRACT
Acod1 Expression in Cancer Cells Promotes Immune Evasion through the Generation of Inhibitory Peptides
Authors: Schofield James H. , Longo Joseph , Sheldon Ryan D. , Albano Emma , Hawk Mark A. , Murphy Sean , Duong Loan , Rahmy Sharif , Lu Xin , Jones Russell G. , Schafer Zachary T.
Published: September 2023

Targeting PD-1 is an important component of many immune checkpoint blockade (ICB) therapeutic approaches. However, ICB is not an efficacious strategy in a variety of cancer types, in part due to immunosuppressive metabolites in the tumor microenvironment (TME). Here, we find that αPD-1-resistant cancer cells produce abundant itaconate (ITA) due to enhanced levels of aconitate decarboxylase (Acod1). Acod1 has an important role in the resistance to αPD-1, as decreasing Acod1 levels in αPD-1 resistant cancer cells can sensitize tumors to αPD-1 therapy. Mechanistically, cancer cells with high Acod1 inhibit the proliferation of naïve CD8+T cells through the secretion of inhibitory factors. Surprisingly, inhibition of CD8+T cell proliferation is not dependent on secretion of ITA, but is instead a consequence of the release of small inhibitory peptides. Our study suggests that strategies to counter the activity of Acod1 in cancer cells may sensitize tumors to ICB therapy.
Breeding for growth has resulted in increased tolerance to drought at the cost of genetic diversity in Scots pine
Authors: Gil-Muñoz Francisco , Hayatgheibi Haleh , Niemi Juha M , Östlund Lars , García-Gil M Rosario
Published: September 2023

Climate change possess a threat to forests and forestry. Drought has been identified as a one of the main issues due to its interaction with other biotic and abiotic stresses. Few studies have been done regarding breeding effect on the adaptability to climate change. After a common garden experiment with seedling families of Scots pine from northern Sweden, we have found differences in drought tolerance between families of breeding and natural origin. We performed a high throughput analysis-based phenotyping on both canopy and root traits. Root architecture traits might be related to drought tolerance and show moderate to high heritability values. The heritability of root architecture traits can be useful not only for drought but also for adaptability to other abiotic stresses. Analysis on architecture traits show that, especially on canopy-traits, families from breeding origins show less phenotypic variance than the ones from natural origins. The methodology employed to evaluate drought tolerance and plant architecture might be useful for future research and forest management focused on climate change adaptability.
Decoding DNA sequence-driven evolution of the human brain epigenome at cellular resolution
Authors: Caglayan Emre , Konopka Genevieve
Published: September 2023

DNA-based evolutionary comparisons of regulatory genomic elements enable insight into functional changes, overcoming tissue inaccessibility. Here, we harnessed adult and fetal cortex single-cell ATAC-seq datasets to uncover DNA substitutions specific to the human and human-ancestral lineages within apes. We found that fetal microglia identity is evolutionarily divergent in all lineages, whereas other cell types are conserved. Using multiomic datasets, we further identified genes linked to multiple lineage-divergent gene regulatory elements and implicated biological pathways associated with these divergent features. We also uncovered patterns of transcription factor binding site evolution across lineages and identified expansion of bHLH-PAS factor targets in human-hominin lineages, and MEF2 factor targets in the ape lineage. Finally, conserved features were more enriched in brain disease variants, whereas there was no distinct enrichment on the human lineage compared to its ancestral lineages. Our study identifies major evolutionary patterns in the human brain epigenome at cellular resolution.
Multiple parallel expansions of bilaterian-like phototransduction gene families in the eyeless Anthozoa
Authors: Hansen Stacey , Payne Meghan , McCulloch Kyle J
Published: September 2023

Opsin-mediated phototransduction cascades in photoreceptor cells are primarily responsible for light-mediated behaviors in animals. Although some visual cascades are well-studied, phototransduction mediated by non-visual opsins and in non-model animal lineages are poorly characterized. In the Cnidaria (jellyfish, corals, sea anemones etc.), the sister group to Bilateria (vertebrates, arthropods, mollusks etc.), limited evidence suggests some overlap with bilaterian phototransduction. This raises the question of whether phototransduction pathways arose a single time early in animal evolution or if light signaling cascades have evolved multiple times. These evolutionary patterns remain obscured because almost nothing is known about phototransduction in a major group within Cnidaria, the eyeless Anthozoa (corals, sea anemones, sea pens etc.). To better understand whether bilaterian-like phototransduction could be present in Anthozoa, we phylogenetically characterized 63 genes in 12 protein families known to be crucial in two types of bilaterian phototransduction in the sea anemoneNematostella vectensis. Using high quality genomic data fromN. vectensis,we took a candidate gene approach to find phototransduction genes and characterize their expression in development and regeneration. We found thatN. vectensispossesses the core suite of proteins for both r-opsin and c-opsin mediated phototransduction. In addition, several new gene subfamilies were identified, particularly in the G protein subunits and TRP channels, and many were anthozoan-specific. We identified a novel G protein α subunit family, which we call GαVI, and characterized its expression inN. vectensiswithin situhybridization. This expansion of phototransduction genes correlates with a large anthozoan-specific radiation in opsin number, suggesting possible coevolution of receptor and signaling diversity in Anthozoa. While further functional experiments on these genes are needed, our findings are in line with the hypothesis that the common ancestor of Eumetazoa had at least two related phototransduction cascades which then further diversified in each animal lineage.
Diverse signatures of convergent evolution in cacti-associated yeasts
Authors: Gonçalves Carla , Harrison Marie-Claire , Steenwyk Jacob L. , Opulente Dana A. , LaBella Abigail L. , Wolters John F. , Zhou Xiaofan , Shen Xing-Xing , Groenewald Marizeth , Hittinger Chris Todd , Rokas Antonis
Published: September 2023

Many distantly related organisms have convergently evolved traits and lifestyles that enable them to live in similar ecological environments. However, the extent of phenotypic convergence evolving through the same or distinct genetic trajectories remains an open question. Here, we leverage a comprehensive dataset of genomic and phenotypic data from 1,049 yeast species in the subphylum Saccharomycotina (Kingdom Fungi, Phylum Ascomycota) to explore signatures of convergent evolution in cactophilic yeasts, ecological specialists associated with cacti. We inferred that the ecological association of yeasts with cacti arose independently ∼17 times. Using machine-learning, we further found that cactophily can be predicted with 76% accuracy from functional genomic and phenotypic data. The most informative feature for predicting cactophily was thermotolerance, which is likely associated with duplication and altered evolutionary rates of genes impacting the cell envelope in several cactophilic lineages. We also identified horizontal gene transfer and duplication events of plant cell wall-degrading enzymes in distantly related cactophilic clades, suggesting that putatively adaptive traits evolved through disparate molecular mechanisms. Remarkably, multiple cactophilic lineages and their close relatives are emerging human opportunistic pathogens, suggesting that the cactophilic lifestyle—and perhaps more generally lifestyles favoring thermotolerance—may preadapt yeasts to cause human disease. This work underscores the potential of a multifaceted approach involving high throughput genomic and phenotypic data to shed light onto ecological adaptation and highlights how convergent evolution to wild environments could facilitate the transition to human pathogenicity.
3D-intrusions transport active surface microbial assemblages to the dark ocean
Authors: Freilich Mara A. , Poirier Camille , Dever Mathieu , Alou-Font Eva , Allen John , Cabornero Andrea , Sudek Lisa , Choi Chang Jae , Ruiz Simón , Pascual Ananda , Farrar J. Thomas , Johnston T.M. Shaun , D’Asaro Eric , Worden Alexandra Z. , Mahadevan Amala
Published: September 2023

Subtropical oceans contribute significantly to global primary production, but the fate of the picophytoplankton that dominate in these low nutrient regions is poorly understood. Working in the subtropical Mediterranean, we demonstrate that subduction of water at ocean fronts generates 3D intrusions with uncharacteristically high carbon, chlorophyll, and oxygen that extend below the sunlit photic-zone into the dark ocean. These contain “fresh” picophytoplankton assemblages that resemble the photic-zone regions where the water originated. Intrusions propagate depth-dependent seasonal variations in microbial assemblages into the ocean interior. Strikingly, the intrusions included dominant biomass contributions from non-photosynthetic bacteria and enrichment of enigmatic heterotrophic bacterial lineages. Thus, the intrusions not only deliver material that differs in composition and nutritional character from sinking detrital particles, but also drive shifts in bacterial community composition, organic matter processing, and interactions between surface and deep communities. Modeling efforts paired with global observations demonstrate that subduction can flux similar magnitudes of particulate organic carbon as sinking export, but is not accounted for in current export estimates and carbon cycle models. Intrusions formed by subduction are a particularly important mechanism for enhancing connectivity between surface and upper mesopelagic ecosystems in stratified subtropical ocean environments that are expanding due to the warming climate.
Range expansion is both slower and more variable with rapid evolution across a spatial gradient in temperature
Authors: Usui Takuji , Angert Amy L.
Published: September 2023

Rapid evolution in colonizing populations can alter our ability to predict future range expansions. Recent theory suggests that the dynamics of replicate range expansions are less variable, and hence more predictable, with increased selection at the expanding range front. Here, we test whether selection from environmental gradients across space produces more consistent range expansion speeds, using the experimental evolution of replicate duckweed populations colonizing landscapes with and without a temperature gradient. We found that range expansion across a temperature gradient was slower on average, with range-front populations displaying higher population densities, and genetic signatures and trait changes consistent with directional selection. Despite this, we found that with a spatial gradient range expansion speed became more variable and less consistent among replicates over time. Our results therefore challenge current theory, highlighting that chance can still shape the genetic response to selection to influence our ability to predict range expansion speeds.
Partitioning heritability using single-cell multi-omics identifies a novel macrophage subpopulation conveying genetic risks of coronary artery disease
Authors: Jiang Jiahao , Hiron Thomas K. , Agbaedeng Thomas , Malhotra Yashaswat , Drydale Edward , Bancroft James , Ng Esther , Reschen Michael E. , Davison Lucy J. , O’Callaghan Chris A.
Published: September 2023

Background

Coronary artery disease (CAD), the leading cause of death worldwide, is influenced by both environmental and genetic factors. While over 250 genetic risk loci have been identified through genome-wide association studies, the specific causal variants and their regulatory mechanisms are still largely unknown, particularly in disease-relevant cell types like macrophages.

Methods

We utilized single-cell RNA-seq (scRNA-seq) and single-cell multi-omics approaches in primary human monocyte-derived macrophages to explore the transcriptional regulatory network involved in a critical pathogenic event of coronary atherosclerosis—the formation of lipid-laden foam cells. Meta-analysis of scRNA-seq datasets from 26 human plaque samples was undertaken to provide a comprehensive atlas of lesional macrophages and to correlate subpopulationsin vivoandex vivo. The genetic risk levels of CAD were assessed by partitioning disease heritability across different macrophage subpopulations.

Results

We identified a novel macrophage subpopulation, termed lipid-handling macrophages, bothex vivoandin vivo, and identified associated marker genes, transcription regulators, and functional pathways. 18,782 cis-regulatory elements were identified by jointly profiling the gene expression and chromatin accessibility of >5000 macrophages. Integration with CAD GWAS data prioritized 121 CAD-related genetic variants and 56 candidate causal genes. We showed that CAD heritability was not uniformly distributed and was particularly enriched in the gene programs of lipid-handling macrophages. We investigated the cis-regulatory effect of a risk variant rs10488763 onFDX1,implicating the recruitment of AP-1 and C/EBP-beta in the causal mechanisms at this locus.

Conclusions

Our results provide genetic evidence of the divergent roles of macrophage subsets in atherogenesis and highlight lipid-handling macrophages as a key sub-population through which genetic variants actively influence disease. These findings provide an unbiased framework for functional fine-mapping of GWAS results using single-cell multi-omics and offer new insights into the genotype-environment interactions underlying atherosclerotic disease.
Identification of Genetic and Genomic Influences on Progressive Ethanol Consumption in Diversity Outbred Mice
Authors: Smith ML , Sergi Z , Mignogna KM , Rodriguez NE , Tatom Z , MacLeod L , Choi KB , Philip V , Miles MF
Published: September 2023

Genetic factors play a significant role in the risk for development of alcohol use disorder (AUD). Using 3-bottle choice intermittent access ethanol (IEA), we have employed the Diversity Outbred (DO) mouse panel as a model of alcohol use disorder in a genetically diverse population. Through use of gene expression network analysis techniques, in combination with expression quantitative trait loci (eQTL) mapping, we have completed an extensive analysis of the influence of genetic background on gene expression changes in the prefrontal cortex (PFC). This approach revealed that, in DO mice, genes whose expression was significantly disrupted by intermittent ethanol in the PFC also tended to be those whose expression correlated to intake. This finding is in contrast to previous studies of both mice and nonhuman primates. Importantly, these analyses identified genes involved in myelination in the PFC as significantly disrupted by IEA, correlated to ethanol intake, and having significant eQTLs. Genes that code for canonical components of the myelin sheath, such asMbp, also emerged as key drivers of the gene expression response to intermittent ethanol drinking. Several regulators of myelination were also key drivers of gene expression, and had significant QTLs, indicating that genetic background may play an important role in regulation of brain myelination. These findings underscore the importance of disruption of normal myelination in the PFC in response to prolonged ethanol exposure, that genetic variation plays an important role in this response, and that this interaction between genetics and myelin disruption in the presence of ethanol may underlie previously observed behavioral changes under intermittent access ethanol drinking such as escalation of consumption.
Small molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states
Authors: Riepe Celeste , Wąchalska Magda , Deol Kirandeep K. , Amaya Anais K. , Porteus Matthew H. , Olzmann James A. , Kopito Ron R.
Published: September 2023

Over 80% of people with cystic fibrosis (CF) carry the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel at the apical plasma membrane (PM) of epithelial cells. F508del impairs CFTR folding causing it to be destroyed by endoplasmic reticulum associated degradation (ERAD). Small molecule correctors, which act as pharmacological chaperones to divert CFTR-F508del from ERAD, are the primary strategy for treating CF, yet corrector development continues with only a rudimentary understanding of how ERAD targets CFTR-F508del. We conducted genome-wide CRISPR/Cas9 knockout screens to systematically identify the molecular machinery that underlies CFTR-F508del ERAD. Although the ER-resident ubiquitin ligase, RNF5 was the top E3 hit, knocking outRNF5only modestly reduced CFTR-F508del degradation. Sublibrary screens in anRNF5knockout background identified RNF185 as a redundant ligase, demonstrating that CFTR-F508del ERAD is highly buffered. Gene-drug interaction experiments demonstrated that correctors tezacaftor (VX-661) and elexacaftor (VX-445) stabilize sequential, RNF5-resistant folding states. We propose that binding of correctors to nascent CFTR-F508del alters its folding landscape by stabilizing folding states that are not substrates for RNF5-mediated ubiquitylation.

SIGNIFICANCE STATEMENT

Clinically effective small molecule cystic fibrosis (CF) correctors divert mutant CFTR molecules from ER-associated degradation (ERAD). However, the mechanisms underlying CFTR ERAD are not well-understood.

The authors used CRISPR knockout screens to identify ERAD machinery targeting CFTR-F508del and found that the pathway is highly buffered, with RNF185 serving as a redundant ubiquitin ligase for RNF5. Gene-drug interaction experiments demonstrated that correctors act synergistically by stabilizing sequential RNF5-resistant folding states.

Inhibiting proteostasis machinery is a complementary approach for enhancing current CF corrector therapies.
Simultaneous assessment of mechanical and electrical function in Langendorff-perfused ex-vivo mouse heart
Authors: Louradour Julien , Ottersberg Rahel , Segiser Adrian , Olejnik Agnieszka , Martínez-Salazar Berenice , Siegrist Mark , Egle Manuel , Barbieri Miriam , Nimani Saranda , Alerni Nicolò , Döring Yvonne , Odening Katja E. , Longnus Sarah
Published: September 2023

Background

The Langendorff-perfusedex-vivoisolated heart model has been extensively used to study cardiac function for many years. However, electrical and mechanical function are often studied separately - despite growing proof of a complex electro-mechanical interaction in cardiac physiology and pathology. Therefore, we developed an isolated mouse heart perfusion system that allows simultaneous recording of electrical and mechanical function.

Methods

Isolated mouse hearts were mounted on a Langendorff setup and electrical function was assessed via a pseudo-ECG and an octapolar catheter inserted in the right atrium and ventricle. Mechanical function was simultaneously assessed via a balloon inserted into the left ventricle coupled with pressure determination. Hearts were then submitted to an ischemia-reperfusion protocol.

Results

At baseline, heart rate, PR and QT intervals, intra-atrial and intra-ventricular conduction times, as well as ventricular effective refractory period, could be measured as parameters of cardiac electrical function. Left ventricular developed pressure (DP), left ventricular work (DP-heart rate product) and maximal velocities of contraction and relaxation were used to assess cardiac mechanical function. Cardiac arrhythmias were observed with episodes of bigeminy during which DP was significantly increased compared to that of sinus rhythm episodes. In addition, the extrasystole-triggered contraction was only 50% of that of sinus rhythm, recapitulating the “pulse deficit” phenomenon observed in bigeminy patients. After ischemia, the mechanical function significantly decreased and slowly recovered during reperfusion while most of the electrical parameters remained unchanged. Finally, the same electro-mechanical interaction during episodes of bigeminy at baseline was observed during reperfusion.

Conclusion

Our modified Langendorff setup allows simultaneous recording of electrical and mechanical function on a beat-to-beat scale and can be used to study electro-mechanical interaction in isolated mouse hearts.
Risk assessments underestimate threat of pesticides to wild bees
Authors: Shahmohamadloo René S. , Tissier Mathilde L. , Guzman Laura Melissa
Published: September 2023

Ecological risk assessments (ERA) are crucial when developing national strategies to manage adverse effects from pesticide exposure to natural populations. Yet, estimating risk with surrogate species in controlled laboratory studies jeopardizes the ERA process because natural populations exhibit intraspecific variation within and across species. Here, we investigate the extent to which the ERA process misestimates risk from pesticides on different species by conducting a meta-analysis of all records in the ECOTOX Knowledgebase for honey bees and wild bees exposed to neonicotinoids. We found the knowledgebase is largely populated by acute lethality data on the Western honey bee and exhibits within and across species variation in LD50 up to six orders of magnitude from neonicotinoid exposure. We challenge the reliability of surrogate species as predictors when extrapolating pesticide toxicity data to wild pollinators and recommend solutions to address the (a)biotic interactions occurring in nature that make such extrapolations unreliable in the ERA process.

Synopsis

Ecological risk assessments misestimate pesticide threats to pollinators sixfold by overextending acute lethality data on surrogate species to natural populations.
Brain Connectivity Changes in Meditators and Novices during Yoga Nidra: A Novel fMRI Study
Authors: Fialoke Suruchi , Tripathi Vaibhav , Thakral Sonika , Dhawan Anju , Majahan Vidur , Garg Rahul
Published: September 2023

Yoga Nidra practice aims to induce a deeply relaxed state akin to sleep while maintaining heightened awareness. Despite the growing interest in its clinical applications, a comprehensive understanding of the underlying neural correlates of the practice of Yoga Nidra (YN) remains largely unexplored. In this novel fMRI investigation, we aim to discover the differences between wakeful resting states and states attained during YN practice. The study included individuals experienced in meditation and/or yogic practices, referred to as ‘meditators’ (n=30), and novice controls (n=31). The GLM analysis, based on audio instructions, demonstrated activation related to auditory cues without concurrent Default Mode Network (DMN) deactivation. Additionally, meditators exhibited heightened bilateral thalamic activation compared to novices. DMN seed based Functional connectivity (FC) analysis revealed significant reductions in connectivity among meditators during YN as compared to controls. We did not find differences between the two groups during the pre and post resting state scans. Moreover, when DMN-FC was compared between the YN state and resting state, meditators showed distinct decoupling, whereas controls showed increased DMN-FC. Finally, meditators exhibit a remarkable correlation between reduced DMN connectivity during Yoga Nidra and self-reported hours of cumulative meditation and yoga practice. Together, these results suggest a unique neural modulation of the DMN in meditators during Yoga Nidra which results in being restful yet aware, aligned with their subjective experience of the practice. The study deepens our understanding of the neural mechanisms of Yoga Nidra, revealing distinct DMN connectivity decoupling in meditators and its relationship with meditation and yoga experience. These findings have interdisciplinary implications for neuroscience, psychology, and yogic disciplines.
A CRISPR activation screen identifies FBXO22 as an E3 ligase supporting targeted protein degradation
Authors: Basu Ananya A. , Zhang Chenlu , Riha Isabella A. , Magassa Assa , Ko Felicia , Zhang Xiaoyu
Published: September 2023

Targeted protein degradation (TPD) represents a potent chemical biology paradigm that leverages the cellular degradation machinery to pharmacologically eliminate specific proteins of interest. Although multiple E3 ligases have been discovered to facilitate TPD, there exists a compelling requirement to diversify the pool of E3 ligases available for such applications. This expansion will broaden the scope of potential protein targets, accommodating those with varying subcellular localizations and expression patterns. In this study, we describe a CRISPR-based transcriptional activation screen focused on human E3 ligases, with the goal of identifying E3 ligases that can facilitate heterobifunctional compound-mediated target degradation. This approach allows us to address the limitations associated with investigating candidate degrader molecules in specific cell lines that either lack or have low levels of the desired E3 ligases. Through this approach, we identified a candidate proteolysis-targeting chimera (PROTAC), 22-SLF, that induces the degradation of FKBP12 when the FBXO22 gene transcription is activated. 22-SLF induced the degradation of endogenous FKBP12 in a FBXO22-dependent manner across multiple cancer cell lines. Subsequent mechanistic investigations revealed that 22-SLF interacts with C227 and/or C228 in FBXO22 to achieve the target degradation. Finally, we demonstrated the versatility of FBXO22-based PROTACs by effectively degrading another endogenous protein BRD4. This study uncovers FBXO22 as an E3 ligase capable of supporting ligand-induced protein degradation through electrophilic PROTACs. The platform we have developed can readily be applied to elucidate protein degradation pathways by identifying E3 ligases that facilitate either small molecule-induced or endogenous protein degradation.
Identification of astrocyte-driven pseudolineages reveals clinical stratification and therapeutic targets in Glioblastoma
Authors: Foerster Leo Carl , Kaya Oguzhan , Wüst Valentin , Bekavac Milica , Ziegler Kevin Chris , Akcay Vuslat , Stinchcombe Nina , Gesteira Perez Noelia , Ma Xiujian , Sadik Ahmed , Le Phuong Uyen , Petrecca Kevin , Opitz Christiane , Liu Haikun , Wirtz Christian Rainer , Anders Simon , Goncalves Angela , Martin-Villalba Ana
Published: September 2023

Cancer research has predominantly targeted genetic mutations, while only recently has attention shifted to understanding tumor cell-stages. However, the key organizational principles guiding tumor dynamics towards sustainable growth remained unexplored. By analyzing tumor cell ensembles from individuals with glioblastoma through the lens of the healthy adult stem cell lineage, we identified astrocytes as central to glioblastoma progression. We found dormant tumor cells resembling astrocytes progressing to active and differentiated stages, building tumor pseudolineages that ultimately influence patient survival. These tumor stages align with specific methylomes, offering potential for patient classification. Our study identifies the Wnt antagonist SFRP1 as a missing factor in glioblastoma that plays a crucial role in the transition from quiescence to activation in the healthy lineage. Excitingly, re-introduction of SFRP1 in glioblastoma halts tumor dynamics, enhancing survival in a PDX model. This fresh view on glioblastomas underscores the importance of understanding tumor dynamics and unveils novel therapeutic avenues.
HLAfreq: Download and combine HLA allele frequency data
Authors: Wells David A. , McAuley Michael
Published: September 2023

The Allele Frequency Net Database contains publicly available data on human immune gene frequencies for different populations from a variety of sources. We introduce a python packageHLAfreqto download, combine, and analyse this data. In particular,HLAfreqcan combine multiple datasets and/or prior knowledge to improve allele frequency estimates and produce credible intervals. We demonstrate these features using real world data to visualise allele frequencies and compare populations.HLAfreqis hosted atwww.github.com/Vaccitech/HLAfreq/with documentation and detailed examples.
Bayesian model discovery for reverse-engineering biochemical networks from data
Authors: Jørgensen Andreas Christ Sølvsten , Sturrock Marc , Ghosh Atiyo , Shahrezaei Vahid
Published: September 2023

The reverse engineering of gene regulatory networks based on gene expression data is a challenging inference task. A related problem in computational systems biology lies in identifying signalling networks that perform particular functions, such as adaptation. Indeed, for many research questions, there is an ongoing search for efficient inference algorithms that can identify the simplest model among a larger set of related models. To this end, in this paper, we introduce SLInG, a Bayesian sparse likelihood-free inference method using Gibbs sampling. We demonstrate that SLInG can reverse engineer stochastic gene regulatory networks from single-cell data with high accuracy, outperforming state-of-the-art correlation-based methods. Furthermore, we show that SLInG can successfully identify signalling networks that execute adaptation. Sparse hierarchical Bayesian inference thus provides a versatile tool for model discovery in systems biology and beyond.
The Pex6 N1 domain is required for Pex15 binding and proper assembly with Pex1
Authors: Ali Bashir A. , Judy Ryan M. , Chowdhury Saikat , Jacobsen Nicole K. , Castanzo Dominic T. , Carr Kaili L. , Richardson Chris D. , Lander Gabriel C. , Martin Andreas , Gardner Brooke M.
Published: September 2023

The heterohexameric AAA-ATPase Pex1/Pex6 is essential for the formation and maintenance of peroxisomes. Pex1/Pex6, similar to other AAA-ATPases, uses the energy from ATP hydrolysis to mechanically thread substrate proteins through its central pore, thereby unfolding them. In related AAA-ATPase motors, substrates are recruited through binding to the motor’s N-terminal domains or N-terminally bound co-factors. Here we use structural and biochemical techniques to characterize the function of the N1 domain in Pex6 from budding yeast,S. cerevisiae. We found that although Pex1/ΛN1-Pex6 is an active ATPasein vitro, it does not support Pex1/Pex6 function at the peroxisomein vivo. An X-ray crystal structure of the isolated Pex6 N1 domain shows that the Pex6 N1 domain shares the same fold as the N terminal domains of PEX1, CDC48, or NSF, despite poor sequence conservation. Integrating this structure with a cryo-EM reconstruction of Pex1/Pex6, AlphaFold2 predictions, and biochemical assays shows that Pex6 N1 mediates binding to both the peroxisomal membrane tether Pex15 and an extended loop from the D2 ATPase domain of Pex1 that influences Pex1/Pex6 heterohexamer stability. Given the direct interactions with both Pex15 and the D2 ATPase domains, the Pex6 N1 domain is poised to coordinate binding of co-factors and substrates with Pex1/Pex6 ATPase activity.
The Interplay Between Sketching and Graph Generation Algorithms in Identifying Biologically Cohesive Cell-Populations in Single-Cell Data
Authors: Crawford Emma , Plotkin Alec , Ranek Jolene , Stanley Natalie
Published: September 2023

High-throughput single-cell immune profiling technologies, such as mass cytometry (CyTOF) and single-cell RNA sequencing measure the expression of multiple proteins or genes across many individual cells within a profiled sample. As it is often of interest to identify particularclustersor cell-populations driving clinical phenotypes or experimental outcomes, there is a critical need to develop automated bioinformatics approaches that can handle a large number of profiled cells. For analyzing multi-sample single-cell datasets at scale, the datasets are usually encoded as a graph, where nodes represent cells and edges imply significant between-cell similarity. As multi-sample single-cell experiments can readily result in millions of profiled cells, the construction and analysis of a graph becomes computationally prohibitive and often requires reducing the dataset size through downsampling as a pre-processing step. Here, we explore the interplay between sketching, or downsampling approaches, and the way in which the graph is constructed on the sketched data for ultimately identifying biologically-meaningful cell-populations. Our results suggest that combining a principled sketching approach with a simplek-nearest neighbor graph representation of the data can identify meaningful subsets of cells as robustly as, and sometimes better than, more sophisticated graph generation approaches. This reveals that the practical concern of downsampling or sketching a limited number of cells is a more critical pre-processing step than how the graph representation is constructed.