bluuebunny/crossref_metadata_2025 huggingface dataset:
Predicting yield traits of individual field-grownBrassica napusplants from rosette-stage leaf gene expression
Authors: De Meyer Sam , Cruz Daniel Felipe , De Swaef Tom , Lootens Peter , De Block Jolien , Bird Kevin , Sprenger Heike , Van de Voorde Michael , Hawinkel Stijn , Van Hautegem Tom , Inzé Dirk , Nelissen Hilde , Roldán-Ruiz Isabel , Maere Steven
Published: October 2022

Background

In the plant sciences, results of laboratory studies often do not translate well to the field because lab growth conditions are very different from field conditions. To help close this lab-field gap, we developed a new strategy for studying the wiring of plant traits directly in the field, based on molecular profiling and phenotyping of individual plants of the same genetic background grown in the same field. This single-plant omics strategy leverages uncontrolled micro-environmental variation across the field and stochastic variation among the individual plants as information sources, rather than controlled perturbations. Here, we use single-plant omics on winter-typeBrassica napus(rapeseed) plants to investigate to what extent rosette-stage gene expression profiles can be linked to the early and late phenotypes of individual field-grown plants.

Results

We find that rosette leaf gene expression in autumn has substantial predictive power for both autumnal leaf phenotypes and final yield in spring. Many of the top predictor genes are linked to developmental processes known to occur in autumn in winter-typeB. napusaccessions, such as the juvenile-to-adult and vegetative-to-reproductive phase transitions, indicating that the yield potential of winter-typeB. napusis influenced by autumnal development.

Conclusions

Our results show that profiling individual plants under uncontrolled field conditions is a valid strategy for identifying genes and processes influencing crop yield in the field.
Deep Reinforcement Learning for Stabilization of Large-scale Probabilistic Boolean Networks
Authors: Moschoyiannis Sotiris , Chatzaroulas Evangelos , Sliogeris Vytenis , Wu Yuhu
Published: October 2022

Abstract

The ability to direct a Probabilistic Boolean Network (PBN) to a desired state is important to applications such as targeted therapeutics in cancer biology. Reinforcement Learning (RL) has been proposed as a framework that solves a discrete-time optimal control problem cast as a Markov Decision Process. We focus on an integrative framework powered by a model-free deep RL method that can address different flavours of the control problem (e.g., withorwithout control inputs; attractor stateora subset of the state space as the target domain). The method is agnostic to the distribution of probabilities for the next state, hence it does not use the probability transition matrix. The time complexity is onlylinearon the time steps, or interactions between the agent (deep RL) and the environment (PBN), during training. Indeed, we explore thescalabilityof the deep RL approach to (set) stabilization of large-scale PBNs and demonstrate successful control on large networks, including a metastatic melanoma PBN with200 nodes.
Bacterial aerobic methane cycling by the marine sponge-associated microbiome
Authors: Ramírez Gustavo A. , Bar-Shalom Rinat , Furlan Andrea , Romeo Roberto , Gavagnin Michelle , Calabrese Gianluca , Garber Arkadiy I. , Steindler Laura
Published: October 2022

Background

Methanotrophy by the sponge-hosted microbiome has been mainly reported in the ecological context of deep-sea hydrocarbon seep niches where methane is either produced geothermically or via anaerobic methanogenic archaea inhabiting the sulfate-depleted sediments. However, methane oxidizing bacteria from the candidate phylum Binatota have recently been described and shown to be present in oxic shallow-water marine sponges, where sources of methane remain undescribed.

Results

Here, using an integrative -omicsapproach, we provide evidence for sponge-hosted bacterial methanogenesis occurring in fully oxygenated shallow water habitats. Specifically, we suggest methanogenesis occursviaat least two independent pathways involving methylamine and methylphosphonate transformations that, concomitantly to aerobic methane production, generate bioavailable nitrogen and phosphate, respectively. Methylphosphonate may be sourced from seawater continuously filtered by the sponge host. Methylamines may also be externally sourced or, alternatively, generated by a multi-step metabolic process where carnitine, derived from sponge cell debris, is transformed to methylamine by different sponge-hosted microbial lineages. Finally, methanotrophs specialized in pigment production, affiliated to the phylum Binatota, may provide a photoprotective function, closing a previously undescribed C1-metabolic loop that involves both the sponge host and specific members of the associated microbial community.

Conclusion

Given the global distribution of this ancient animal lineage and their remarkable water filtration activity, sponge hosted methane cycling may affect methane supersaturation in oxic coastal environments. Depending on the net balance between methanogenesis and methanotrophy, sponges may serve as marine sources or sinks of this potent greenhouse gas.
Potential role of immediate early genesEgr-1, Hr-38andKakuseiin the foraging behavior and learning of in honeybees
Authors: Singh Asem Surindro , Chanu Machathoibi Takhellambam
Published: October 2022

The foraging behavior of honeybees is one of the most systematically composed behaviors among social insects which are admirable to watch. The main purpose of honeybee foraging is to collect food for their colony and since ancient days honeybee products have been used for various medicinal purposes (Singh and Takhellambam 2021). During foraging, honeybees gather information and transmit to their colony members regarding the location, distance, and profitability of forage sites with the help of unique movements called waggle dance. The capacities of honeybees time memory enable the foragers to return to a good food source in anticipation of the time of day. This highly intellectual, dynamic, and well-coordinated behavior of honeybees makes them to be one of the best choices of behavioral model to study various aspects of dynamic behaviors. As a result, vast knowledge in honeybee behavior has been accumulated and several recent studies immerge towards finding the underpinning regulatory biology of honeybee foraging behaviors. Immediate early genes (IE) genes are well documented neural markers and their promising roles in honeybee foraging have been demonstrated. Two of our recent studies showed three IE genesEgr-1, Hr38andKakuseiinvolvement during the daily foraging of honeybees. This finding has provided an avenue to further explore and identify the regulatory genes/proteins and neurons that underlie a specific behavior such as learning, memory, communication, and interaction etc. In this study we further analyze our previous published data to examine interaction of the three genes during the daily foraging of honeybees.
The role of thioredoxin proteins inMycobacterium tuberculosisprobed by proteome-wide target profiling
Authors: Sugandhi Sapna , Taunk Khushman , Jadhav Sushama , Nema Vijay , Rapole Srikanth , Mande Shekhar C.
Published: October 2022

Mycobacterium tuberculosisencounters diverse microenvironments as it attempts to establish itself within its human host. The bacterium survives oxidative assault (ROS and RNS) when it is inside the host macrophages. Redox sensory and regulation processes therefore assume significant importance, as these are essential processes forM. tuberculosisto survive under these hostile conditions. The thioredoxin system that maintains balance between the thiol/dithiol couple plays a key role in maintaining redox homeostasis inM. tuberculosis. The most explored function of the thioredoxin system is elimination of toxic molecules such as free radicals, while very little is known about its role in other metabolic processes. In the present study, we aimed to reduce the knowledge gap about the thioredoxin system inM. tuberculosis. We attempted to capture targets of all the thioredoxins (viz., TrxB and TrxC) and a thioredoxin-like protein, NrdH inM. tuberculosisunder aerobic and hypoxic conditions by performing thioredoxin trapping chromatography followed by mass spectrometry. Targets were classified using the PANTHER classification system and most enriched processes were figured out using Gene Ontology analysis. We found that TrxC captured the maximum number of targets in both the physiological conditions. Also, we suggest that the thioredoxin system might play an important role in hypoxic conditions by targeting proteins responsible to sense and maintain hypoxic conditions. Furthermore, our studies establish a link between TrxB and iron-sulfur cluster biogenesis inM. tuberculosis. Ultimately, these findings open a novel avenue to target the thioredoxin system for screening new anti-mycobacterial drug targets.

Importance

Tuberculosis (TB), an infectious disease caused by bacteriaM. tuberculosis, is the leading cause of death in the list of infectious diseases. Worldwide 1.7 billion people are estimated to be infected with TB, containing active and latent cases. An alarming situation is thatM. tuberculosishas developed resistance against one or many of the first line drugs leading to emergence of drug resistant or multidrug resistant TB. Novel drugs targeting the drug resistant bacteria is an urgent need to cure the disease. Our study provides the framework to identify new drug targets. The significance of our study is to understand the thioredoxin system in more details by identifying their target proteins, which might facilitate development of new anti-tubercular drugs.
Electrophysiological correlates of associative recognition memory for identity, spatial, and temporal relations
Authors: Hugeri Ofer , Vakil Eli , Levy Daniel A.
Published: October 2022

The formation of associative representations and their retrieval from episodic memory are vital cognitive functions. However, it is unclear to what extent retrieval of the basic component relations of episodic memory – identity, time, and space – requires different or shared brain mechanisms. In the current study, we employed EEG to track the time courses of electrophysiological correlates of retrieval processes of memory for identity relations, temporal order, and spatial configuration. Participants engaged in pair-associate learning of serially presented and spatially configured object picture pairs, followed by discrimination of identity, spatially, or temporally intact and rearranged pairs. Event-related brain potentials (ERPs) revealed distinct patterns of activity during successful retrieval of identity, spatial, and temporal relations that differed by the status of association, across the three retrieval time windows examined (300-500, 500-800, and 800-1000 ms). The identity relations condition was distinguished by a widespread greater negative-going deflection for rearranged relative to intact pairs in all three time windows. For the temporal relations condition, we observed a widespread more negative-going deflection for rearranged than intact pairs, significant in the second time window only. For the spatial relations condition, there was a widespread positive-going deflection greater for rearranged than for intact pairs, significant in the early and in middle time windows. These patterns of activity suggest that retrieval of associative memory traces for identity, spatial, and temporal relationships involve dynamically different processes, which may partially rely on different sets of basic associative mechanisms.
Systematic loss of function screens identify pathway specific functional circular RNAs
Authors: Liu Ling , Neve Matthew , Perlaza-Jimenez Laura , Hawdon Azelle , Conn Simon J. , Zenker Jennifer , Tamayo Pablo , Goodall Gregory J. , Rosenbluh Joseph
Published: October 2022

Circular RNAs (circRNAs) are covalently closed single stranded RNAs that are produced by RNA back-splicing. A small number of circRNAs have been implicated as functional, however, we still lack systematic understanding of cellular processes and signalling pathways that are regulated by circRNAs. A major gap in understanding circRNA function is the ability to define pathways that are regulated by circRNAs. Here, we generated a pooled shRNA library targeting the back-splice junction of 3,354 human circRNAs that are expressed at low to high levels in humans. We used this library for loss of function proliferation screens in a panel of 18 cancer cell lines from four tissue types that harbour mutations leading to constitutive activity of defined pathways. Using this dataset, we identify context specific and non-specific circRNAs. We validated these observations with a secondary screen and uncovered a role forcircRERE, a cell essential circRNA that regulates ferroptosis. Furthermore, we characterised the functional roles of pathway-specific circRNA,circSMAD2, a novel regulator of the WNT pathway andcircMTO1, a regulator of MAPK signalling in aPTENdependent manner. Our work sheds light on molecular pathways regulated by circRNAs and provides a catalogue of circRNAs with a measurable function in human cells.
Toehold-Mediated Strand Displacement in Random Sequence Pools
Authors: Mayer Thomas , Oesinghaus Lukas , Simmel Friedrich C.
Published: October 2022

Toehold-mediated strand displacement (TMSD) has been used extensively for molecular sensing and computing in DNA-based molecular circuits. As these circuits grow in complexity, sequence similarity between components can lead to cross-talk causing leak, altered kinetics, or even circuit failure. For small non-biological circuits, such unwanted interactions can be designed against. In environments containing a huge number of sequences, taking all possible interactions into account becomes infeasible. Therefore, a general understanding of the impact of sequence backgrounds on TMSD reactions is of great interest. Here, we investigate the impact of random DNA sequences on TMSD circuits. We begin by studying individual interfering strands and use the obtained data to build machine learning models that estimate kinetics. We then investigate the influence of pools of random strands and find that the kinetics are determined by only a small subpopulation of strongly interacting strands. Consequently, their behavior can be mimicked by a small collection of such strands. The equilibration of the circuit with the background sequences strongly influences this behavior, leading to up to one order of magnitude difference in reaction speed. Finally, we compare two established and a novel technique that speed up TMSD reactions in random sequence pools: a threeletter alphabet, protection of toeholds by intramolecular secondary structure, or by an additional blocking strand. While all of these techniques were useful, only the latter can be used without sequence constraints. We expect that our insights will be useful for the construction of TMSD circuits that are robust to molecular noise.
MAVISp: A Modular Structure-Based Framework for Protein Variant Effects
Authors: Arnaudi Matteo , Utichi Mattia , Degn Kristine , Beltrame Ludovica , Scrima Simone , Krzesińska Karolina , Sánchez-Izquierdo Besora Pablo , Meldgård Katrine , Bauer Laura , Dorčaková Terézia , Melidi Anna , Favaro Lorenzo , Kiachaki Eleni , Oswal Anu , Estad Alberte Heering , Breitenstein Joachim , Safer Jordan , Saridaki Paraskevi , Maselli Francesca , Fas Burcu Aykac , Tedeschi Guglielmo , Becker Philipp , Vinhas Jérémy , Pettenella Alberto , Sackett Peter Wad , Cava Claudia , Rohlin Anna , Nilbert Mef , Iqbal Sumaiya , Lambrughi Matteo , Tiberti Matteo , Papaleo Elena
Published: October 2022

The role of genomic variants in disease has expanded significantly with the advent of advanced sequencing techniques. The rapid increase in identified genomic variants has led to many variants being classified as Variants of Uncertain Significance or as having conflicting evidence, posing challenges for their interpretation and characterization. Additionally, current methods for predicting pathogenic variants often lack insights into the underlying molecular mechanisms. Here, we introduce MAVISp (Multi-layeredAssessment ofVarIants byStructure forproteins), a modular structural framework for variant effects, accompanied by a web server (https://services.healthtech.dtu.dk/services/MAVISp-1.0/) to enhance data accessibility, consultation, and re-usability. MAVISp currently provides data for 500 proteins, encompassing over four million variants. A team of biocurators regularly analyze and update protein entries using standardized workflows, incorporating free energy calculations or biomolecular simulations. We illustrate the utility of MAVISp through selected case studies. The framework facilitates the analysis of variant effects at the protein level and has the potential to advance the understanding and application of mutational data in disease research.
Breeding in an agricultural landscape: conservation actions increase nest survival in a ground-nesting bird
Authors: Kiss Ádám , Végvári Zsolt , Kubelka Vojtěch , Monoki Ákos , Kapocsi István , Gőri Szilvia , Székely Tamás
Published: October 2022

Agricultural intensification has affected wildlife across Europe, usually prompting steep declines and regional extinctions in farmland birds. Effective conservation activities are essential for preservation of biodiversity in agricultural landscape, but despite the efforts, the halting (or reversing) the decline of farmland species are still rare. Here we investigate a ground-nesting shorebird, the Collared Pratincole (Glareola pratincola)that has switched its habitat preferences in Central Europe in the last 20 years from alkaline grasslands to intensively managed agricultural landscapes. We show that nest success was different between three habitat types, with the highest nest success in fallow lands whereas nests in row crops showed the lowest hatching success. Nest success was also associated with timing of breeding and breeding density, since nests produced early in the breeding season and those in dense breeding sites hatched more successfully than those later in the season and low breeding density, respectively. Importantly, since 2012 direct conservation measures have been implemented that include the marking of nests and negotiating with farmers to avoid the usage of agricultural machinery around the marked areas, controlling nest predators and most recently creating suitable nesting sites and foraging areas for the Pratincoles. Due to these direct conservation actions, the probability of both nest survival increased from 0.11 in 2012 to 0.83 in year 2021, and the size of breeding population increased from 16 pairs in 2013 to 56 in 2021. Taken together, agricultural areas can continue providing important habitats for various organisms, and with targeted conservation actions we can reduce or even halt the decline of farmland species.
Glucagon-like peptide-1 receptor in the human hypothalamus is associated with body mass index and colocalizes with the anorexigenic neuropeptide nucleobindin-2/nesfatin-1
Authors: Psilopanagioti Aristea , Nikou Sofia , Logotheti Souzana , Arbi Marina , Chartoumpekis Dionysios V. , Papadaki Helen
Published: October 2022

Introduction

Glucagon-like peptide-1 (GLP-1) anorexigenic and anti-obesogenic effects are centrally mediated. Data on animals emphasize the importance of neuronal GLP-1 receptor (GLP-1R) for feeding suppression, although it is unclear whether astrocytes participate in the transduction of anorectic GLP-1R-dependent signals. In humans, the brain circuitry underlying these effects remains insufficiently investigated. GLP-1R neuroanatomic localization in human hypothalamus, a brain region with a pivotal role in energy homeostasis regulation, is essential in order to improve our understanding of GLP-1 signaling pathways and central metabolic functions. The present study aimed to explore GLP-1R protein expression in human hypothalamus and its correlation with body mass index (BMI).

Methods

Sections of hypothalamus from 28 autopsy cases, 11 with normal weight (BMI < 25 Kg/m2) and 17 with non-normal weight (BMI ≥ 25 Kg/m2), were examined using immunohistochemistry and double immunofluorescence labeling.

Results

Prominent GLP-1R immunoexpression was detected in neurons of several hypothalamic nuclei, including paraventricular, supraoptic, and infundibular nuclei, lateral hypothalamic area (LH), and basal forebrain nuclei. Interestingly, in LH, GLP-1R protein expression was significantly decreased in individuals with BMI ≥ 25 Kg/m2, compared with normal weight counterparts (p=0.03). Furthermore, GLP-1R was moderately and negatively correlated (τb=-0.347, p=0.024) with BMI levels only in the LH. GLP-1R extensively colocalized with the anorexigenic and anti-obesogenic neuropeptide nucleobindin-2/nesfatin-1, but not with the astrocytic marker glial fibrillary acidic protein (GFAP).

Conclusion

These data suggest a potential role for GLP-1R in the regulation of energy balance in human hypothalamus, possibly through interactions with nesfatin-1. In LH, an appetite- and reward-related brain region, reduced GLP-1R immunoexpression may contribute to dysregulation of homeostatic and/or hedonic feeding behavior. GLP-1R colocalization with nesfatin-1 in the basal forebrain, a cognition-related brain area, might give impetus towards elucidating additional central actions of GLP-1R.
Influence of MUC1 on trafficking of TRPV5 and TRPV6 andin vivoCa2+homeostasis
Authors: Al-bataineh Mohammad M. , Kinlough Carol L. , Marciszyn Allison , Lam Tracey , Ye Lorena , Kidd Kendrah , Maggiore Joseph C. , Poland Paul A. , Bleyer Anthony , Bain Daniel J. , Kleyman Thomas R. , Hughey Rebecca P. , Ray Evan C.
Published: October 2022

Polymorphism of the gene encoding mucin 1 (MUC1) is associated with skeletal and dental phenotypes in human genomic studies. Animals lacking MUC1 exhibit mild reduction in bone density. These phenotypes could be a consequence of modulation of bodily Ca homeostasis by MUC1, as suggested by the previous observation that MUC1 enhances cell surface expression of the Ca2+-selective channel, TRPV5 in cultured unpolarized cells. Using biotinylation of cell-surface proteins, we asked whether MUC1 influences endocytosis of TRPV5 and another Ca2+-selective TRP channel, TRPV6, in cultured polarized epithelial cells. Results indicate that MUC1 reduces endocytosis of both channels, enhancing cell surface expression. Further, mice lacking MUC1 lose apical localization of TRPV5 and TRPV6 in the renal tubular and duodenal epithelium. Females, but not males, lacking MUC1 exhibit reduced blood Ca2+. However, mice lacking MUC1 exhibited no differences in basal urinary Ca excretion or Ca retention in response to PTH receptor signaling, suggesting compensation by transport mechanisms independent of TRPV5 and TRPV6. Finally, humans with autosomal dominant tubulointerstitial kidney disease due to frame-shift mutation of MUC1 (ADTKD-MUC1) exhibit reduced plasma Ca concentrations compared to control individuals with mutations in the gene encoding uromodulin (ADTKD-UMOD), consistent with MUC1 haploinsufficiency causing reduced bodily Ca2+.
Structural basis for preservation of a subset of Topologically Associating Domains in Interphase Chromosomes upon cohesin depletion
Authors: Jeong Davin , Shi Guang , Li Xin , Thirumalai D.
Published: October 2022

Compartment formation in interphase chromosomes is a result of spatial segregation between eu- and heterochromatin on a few mega base pairs (Mbp) scale. On the sub-Mbp scales, Topologically Associating Domains (TADs) appear as interacting domains along the diagonal in the ensemble averaged Hi-C contact map. Hi-C experiments showed that most of the TADs vanish upon deleting cohesin, while the compartment structure is maintained, and perhaps even enhanced. However, closer inspection of the data reveals that a non-negligible fraction of TADs is preserved (P-TADs) after cohesin loss. Imaging experiments show that, at the single-cell level, TAD-like structures are presenteven without cohesin. To provide a structural basis for these findings, we first used polymer simulations to show that certain TADs with epigenetic switches across their boundaries survive after depletion of loops. More importantly, the three-dimensional structures show that many of the P-TADs have sharp physical boundaries. Informed by the simulations, we analyzed the Hi-C maps (with and without cohesin) in mouse liver and human colorectal carcinoma cell lines, which affirmed that epigenetic switches and physical boundaries (calculated using the predicted 3D structures using the data-driven HIPPS method that uses Hi-C as the input) explain the origin of the P-TADs. Single-cell structures display TAD-like features in the absence of cohesin that are remarkably similar to the findings in imaging experiments. Some P-TADs, with physical boundaries, are relevant to the retention of enhancer-promoter/promoter-promoter interactions. Overall, our study shows that preservation of a subset of TADs upon removing cohesin is a robust phenomenon that is valid across multiple cell lines.
Fast yet force-effective mode of supracellular collective cell migration on aligned fibers due to extracellular force transmission
Authors: Bagchi Amrit , Sarker Bapi , Zhang Jialiang , Foston Marcus , Pathak Amit
Published: October 2022

Cell collectives, like other motile entities, generate and use forces to move forward. Here, we ask whether environmental configurations alter this proportional force-speed relationship, since aligned extracellular matrix fibers are known to cause directed migration. We show that aligned fibers serve as active conduits for spatial propagation of cellular mechanotransduction through matrix exoskeleton, leading to efficient directed collective cell migration. Epithelial (MCF10A) cell clusters adhered tosoftsubstrates with aligned collagen fibers (AF) migrate faster with much lesser traction forces, compared to random fibers (RF). Fiber alignment causes higher motility waves and transmission of normal stresses deeper into cell monolayer while minimizing shear stresses and increased cell-division based fluidization. By contrast, fiber randomization induces cellular jamming due to breakage in motility waves, disrupted transmission of normal stresses, and heightened shear driven flow. Using a novel motor-clutch model, we explain that such ‘force-effective’ fast migration phenotype occurs due to rapid stabilization of contractile forces at the migrating front, enabled by higher frictional forces arising from simultaneous compressive loading of parallel fiber-substrate connections. We also model’haptotaxis’ to show that increasing ligand connectivity (but not continuity) increases migration efficiency. According to our model, increased rate of front stabilization via higher resistance to substrate deformation is sufficient to capture ‘durotaxis’. Thus, our findings reveal a new paradigm wherein the rate of leading-edge stabilization determines the efficiency of supracellular collective cell migration.
Antibody responses to Omicron BA.4/BA.5 bivalent mRNA vaccine booster shot
Authors: Wang Qian , Bowen Anthony , Valdez Riccardo , Gherasim Carmen , Gordon Aubree , Liu Lihong , Ho David D.
Published: October 2022

The SARS-CoV-2 Omicron variant and its numerous sub-lineages have exhibited a striking ability to evade humoral immune responses induced by prior vaccination or infection. The Food and Drug Administration (FDA) has recently granted Emergency Use Authorizations (EUAs) to new bivalent formulations of the original Moderna and Pfizer mRNA SARS-CoV-2 vaccines that target both the ancestral strain as well as the Omicron BA.4/BA.5 variant. Despite their widespread use as a vaccine boost, little is known about the antibody responses induced in humans. Here, we collected sera from several clinical cohorts: individuals after three or four doses of the original monovalent mRNA vaccines, individuals receiving the new bivalent vaccines as a fourth dose, and individuals with BA.4/BA.5 breakthrough infection following mRNA vaccination. Using pseudovirus neutralization assays, these sera were tested for neutralization against an ancestral SARS-CoV-2 strain, several Omicron sub-lineages, and several related sarbecoviruses. At ~3-5 weeks post booster shot, individuals who received a fourth vaccine dose with a bivalent mRNA vaccine targeting BA.4/BA.5 had similar neutralizing antibody titers as those receiving a fourth monovalent mRNA vaccine against all SARS-CoV-2 variants tested, including BA.4/BA.5. Those who received a fourth monovalent vaccine dose had a slightly higher neutralizing antibody titers than those who received the bivalent vaccine against three related sarbecoviruses: SARS-CoV, GD-Pangolin, and WIV1. When given as a fourth dose, a bivalent mRNA vaccine targeting Omicron BA.4/BA.5 and an ancestral SARS-CoV-2 strain did not induce superior neutralizing antibody responses in humans, at the time period tested, compared to the original monovalent vaccine formulation.
Visualizing the Dynamics of DNA Replication and Repair at the Single-Molecule Molecule Level
Authors: Berger Scott , Chistol Gheorghe
Published: October 2022

During cell division, the genome of each eukaryotic cell is copied by thousands of replisomes – large protein complexes consisting of several dozen proteins. Recent studies suggest that the eukaryotic replisome is much more dynamic than previously thought. To directly visualize replisome dynamics in a physiological context, we recently developed a single-molecule approach for imaging replication proteins inXenopusegg extracts. These extracts contain all the soluble nuclear proteins and faithfully recapitulate DNA replication and repairin vitro, serving as a powerful platform for studying the mechanisms of genome maintenance. Here we present detailed protocols for conducting single-molecule experiments in nuclear egg extracts and preparing key reagents. This workflow can be easily adapted to visualize the dynamics and function of other proteins implicated in DNA replication and repair.
Membrane localization accelerates association under conditions relevant to cellular signaling
Authors: Huang William Y. C. , Boxer Steven G. , Ferrell James E.
Published: October 2022

Translocation of cytoplasmic molecules to the plasma membrane is commonplace in cell signaling. Membrane localization has been hypothesized to increase intermolecular association rates; however, it has also been argued that association should be faster in the cytosol because membrane diffusion is slow. Here we directly compare an identical association reaction in solution and on supported membranes. The measured rate constants show that for 10 μm-radius spherical cell, association is 15-25-fold faster at the membrane than in the cytoplasm. The advantage is cell size-dependent, and for typical ~1 μm prokaryotic cells it should be essentially negligible. Rate enhancement is attributable to a combination of closer proximity of the signaling molecule to its targets after translocation and the higher efficiency of a two-dimensional search.
PhISCO: a simple method to infer phenotypes from protein sequences
Authors: Hernandez-Berthet Ayelén S. , Aptekmann Ariel A. , Tejero Jesús , Sánchez Ignacio E. , Noguera Martín E. , Roman Ernesto A.
Published: October 2022

Although protein sequences encode the information for folding and function, understanding their link is not an easy task. Unluckily, the prediction of how specific amino acids contribute to these features is still considerably impaired. Here, we developed PhISCO, Phenotype Inference from Sequence COmparisons, a simple algorithm that finds positions associated with any quantitative phenotype and predicts their values. From a few hundred sequences from four different protein families, we performed multiple sequence alignments and calculated per-position pairwise differences for both the sequence and the observed phenotypes. We found that from 3 to 10 positions, depending on the studied case, were enough to identify positions associated with the phenotypes and perform quantitative predictions of them. Here we show that these strong correlations can be found using individual positions while an improvement is achieved when the most correlated positions are jointly analyzed. Noteworthy, we performed phenotype predictions using a simple linear model that links per-position divergences and differences in observed phenotypes. We also show that although extremely simple, predictions are comparable to the state-of-art methodologies which, in most of the cases, are far more complex. All of the calculations are obtained at a very low information cost since the only input needed is a multiple sequence alignment of protein sequences with their associated quantitative phenotype. The diversity of the explored systems makes PhISCO a valuable tool to find sequence determinants of biological activity modulation and to predict various functional features for uncharacterized members of a protein family.
Responses of carbapenemase-producing and non-producing carbapenem-resistantPseudomonas aeruginosastrains to meropenem revealed by quantitative tandem mass spectrometry proteomics
Authors: Salvà-Serra Francisco , Jaén-Luchoro Daniel , Marathe Nachiket P. , Adlerberth Ingegerd , Moore Edward R. B. , Karlsson Roger
Published: October 2022

Pseudomonas aeruginosais an opportunistic pathogen with increasing incidence of multidrug-resistant strains, including resistance to last-resort antibiotics, such as carbapenems. Resistances are often due to complex interplays of naturally and acquired resistance mechanisms that are enhanced by its remarkably large regulatory network.

This study describes the proteomic responses of two carbapenem-resistantP. aeruginosastrains of high-risk clones ST235 and ST395 to subminimal inhibitory concentrations (sub-MICs) of meropenem by identifying differentially expressed proteins and pathways. Strain CCUG 51971, carries a VIM-4 metallo-β-lactamase or ‘classical’ carbapenemase, and strain CCUG 70744 carries no known acquired carbapenem-resistance genes and exhibits ‘non-classical’ carbapenem-resistance. Each strain was cultivated with different sub-MICs of meropenem, and analyzed, using quantitative shotgun proteomics, based on tandem mass tag (TMT) isobaric labeling followed by nano-liquid chromatography tandem-mass spectrometry.

Exposure of both strains to sub-MICs meropenem resulted in hundreds of differentially expressed proteins, including β-lactamases, proteins associated with transport, peptidoglycan metabolism, cell wall organization, and regulatory proteins. Strain CCUG 51971 showed up-regulation of intrinsic β-lactamases and VIM-4 carbapenemase, while CCUG 70744 exhibited a combination of up-regulated intrinsic β-lactamases, efflux pumps, penicillin-binding proteins and down-regulation of porins. All components of the H1 type VI secretion system were up-regulated in strain CCUG 51971. Enrichment analyses revealed multiple metabolic pathways affected in both strains.

Sub-MICs of meropenem cause marked changes in the proteomes of carbapenem-resistant strains ofP. aeruginosaexhibiting different resistance mechanisms, involving a wide range of proteins, many uncharacterized, which might play a role in the susceptibility ofP. aeruginosato meropenem.
Exploring The Behavior of Bioelectric Circuits using Evolution Heuristic Search
Authors: Hazan Hananel , Levin Michael
Published: October 2022

Characteristic spatial differences of cellular resting potential across tissues have been shown to act as instructive bioelectric prepatterns regulating embryonic and regenerative morphogenesis, as well as cancer suppression. Indeed, modulation of bioelectric patterns via specific ion channel-targeting drugs, channel misexpression, or optogenetics has been used to control growth and form in vitro, showing promise in regenerative medicine and synthetic bioengineering. Repair of defects, injury, and transformation requires quantitative understanding of bioelectric dynamics within tissues so that these can be modulated toward desired outcomes in organ patterning or the creation of entirely novel synthetic constructs. The major gap in the discovery of interventions for rational control of organ-level outcomes is the inability to predict large-scale bioelectric patterns - their emergence from symmetry breaking (given a set of channels expressed on the tissue) and their change as a function of time under specific bioelectrical interventions. It is thus essential to develop machine learning and other computational tools to help human scientists identify bioelectric states with desirable properties. Here, we tested the ability of a heuristic search algorithm to explore the parameter space of bio-electrical circuits by adjusting the parameters of simulated cells. We show that while bioelectrical space is not easy to search, it does contain parameter sets that encode rich and interesting patterning behaviors. We demonstrate proof of principle of using a computational search platform to identify circuits with desired properties, as a first step toward the design of machine learning tools for improved bioelectric control of growth and form.
The trans-regulatory landscape of gene networks in plants
Authors: Hummel Niklas F. C. , Zhou Andy , Li Baohua , Markel Kasey , Ornelas Izaiah J. , Shih Patrick M.
Published: October 2022

The effector domains of transcription factors play a key role in controlling gene expression; however, their functional nature is poorly understood, hampering our ability to explore this fundamental dimension of gene regulatory networks. To map the trans-regulatory landscape in a complex eukaryote, we systematically characterized the putative effector domains of over 400Arabidopsis thalianatranscription factors for their capacity to modulate transcription. We demonstrate that transcriptional effector activity can be integrated into gene regulatory networks capable of elucidating the functional dynamics underlying gene expression patterns. We further show how newly characterized domains can enhance genome engineering efforts and reveal how plant transcriptional activators share regulatory features conserved across distantly related eukaryotes. Our results provide a framework to systematically characterize the regulatory role of transcription factors at a genome-scale in order to understand the transcriptional wiring of biological systems.
Head-on and co-directional RNA polymerase collisions orchestrate bidirectional transcription termination
Authors: Wang Ling , Watters John W. , Ju Xiangwu , Liu Shixin
Published: October 2022

Genomic DNA is a crowded track where translocating motor proteins frequently collide. It remains unclear whether these collisions, generally thought to occur inadvertently, carry any physiological function. In this work, we developed a single-molecule assay to directly visualize the trafficking of individualE. coliRNA polymerases (RNAPs) on DNA. This assay enabled us to test the hypothesis that RNAP collisions drive bidirectional transcription termination of convergent gene pairs. We showed that the head-on collision between two converging RNAPs is necessary to prevent transcriptional readthrough, but insufficient to release the collided RNAPs from the DNA. Remarkably, co-directional collision from a trailing RNAP into the head-on collided complex dramatically increases the termination efficiency. Furthermore, a stem-loop structure formed in the nascent RNA is required for collisions to occur at well-defined positions between gene boundaries. These findings, corroborated by transcriptomic data, establish programmed RNAP collisions as an effective strategy to achieve precise gene expression and imply a broader role of genomic conflicts in cell physiology.
Molecular dynamics simulations reveal the impact of NUTD15 variants in structural conformation and dynamics
Authors: Gómez-Rubio Elena , Garcia-Marin Javier
Published: October 2022

NUDT15 or MTH2 is a member of NUDIX protein family that catalyze the hydrolysis of nucleotides and deoxynucleotides, including thioguanine analogues. NUTD15 has been reported as a DNA sanitizer in humans, and more recent studies have proved that genomic variants are related to a poor prognosis in inmoplastic and immunologic diseases with thioguanines. Despite of this, the role of NUTD15 in physiology and molecular biology is quite unclear, as well as the mechanism of action of this enzyme. The existence of clinically relevant variants has prompted the study of this enzymes, whose capacity to bound and hydrolyze thioguanine nucleotides is still poor understood. By using a combination of biomolecular modelling techniques together with molecular dynamics we have studied the monomeric wild type NUTD15, as well as two important variants R139C and R139H. Our findings reveal not only how nucleotide binding stabilizes the enzyme, but also how two loops are responsible for keeping the enzyme in a packed close conformation. Mutations in α2 helix affect a network of hydrophobic and π-interactions that are responsible of active site enclosing.
Double emulsions as a high-throughput enrichment and isolation platform for slower-growing microbes
Authors: McCully Alexandra L. , Yao McKenna Loop , Brower Kara K. , Fordyce Polly M. , Spormann Alfred M.
Published: October 2022

Our understanding ofin situmicrobial physiology is primarily based on physiological characterization of fast-growing and readily-isolatable microbes. Microbial enrichments to obtain novel isolates with slower growth rates or physiologies adapted to low nutrient environments are plagued by intrinsic biases for fastest-growing species when using standard laboratory isolation protocols. New cultivation tools to minimize these biases and enrich for less well-studied taxa are needed. In this study, we developed a high-throughput bacterial enrichment platform based on single cell encapsulation and growth within double emulsions (GrowMiDE). We showed that GrowMiDE can cultivate many different microorganisms and enrich for novel taxa that are never observed in traditional batch enrichments. For example, preventing dominance of the enrichment by fast-growing microbes due to nutrient privatization within the double emulsion droplets allowed cultivation of novelNegativicutesandMethanobacteriafrom stool samples in rich media enrichment cultures. In competition experiments between growth rate and growth yield specialist strains, GrowMiDE enrichments prevented competition for shared nutrient pools and enriched for slower-growing but more efficient strains. Finally, we demonstrated the compatibility of GrowMiDE with commercial fluorescence-activated cell sorting (FACS) to obtain isolates from GrowMiDE enrichments. Together, GrowMiDE + DE-FACS is a promising new high-throughput enrichment platform that can be easily applied to diverse microbial enrichments or screens.
Glycoengineered keratinocyte library reveals essential functions of specific glycans for all stages of HSV-1 infection
Authors: Bagdonaite Ieva , Marinova Irina N , Rudjord-Levann Asha M , Pallesen Emil MH , King-Smith Sarah , Rømer Troels B , Chen Yen-Hsi , Olofsson Sigvard , Nordén Rickard , Bergström Tomas , Dabelsteen Sally , Wandall Hans H
Published: October 2022

Viral and host glycans represent an understudied aspect of host-pathogen interactions, despite potential implications for treatment of viral infections. This is due to lack of easily accessible tools for analyzing glycan function in a meaningful context. Here we generated a glycoengineered keratinocyte library delineating human glycosylation pathways to uncover roles of specific glycans at different stages of herpes simplex virus type 1 (HSV-1) infectious cycle. We show the importance of cellular glycosaminoglycans and glycosphingolipids for HSV-1 attachment, N-glycans for entry and spread, and O-glycans for propagation. While altered virion surface structures had minimal effects on the early interactions with wild type cells, mutation of specific O-glycosylation sites affected glycoprotein surface expression and function. In conclusion, the data demonstrates the importance of specific glycans in a clinically relevant human model of HSV-1 infection and highlights the utility of genetic engineering to elucidate the roles of specific viral and cellular carbohydrate structures.
Inhibiting a mRNA motif binding protein that mediates TGF-β1 upregulation of translation attenuates pulmonary fibrosis in mice
Authors: Chen Wensheng , Pilling Darrell , Gomer Richard H.
Published: October 2022

In human lung cells, the profibrotic cytokine TGF-β1 increases sialidase 3 (NEU3) protein by increasingNEU3translation without increasing levels ofNEU3mRNA. To elucidate how TGF-β1 regulates translation, we treated human lung fibroblasts (HLF) with TGF-β1 and used proteomics and RNA-seq to determine the effect of TGF-β1 on proteins, mRNAs, and mRNA polysome/monosome ratios. We identified 181 mRNAs where TGF-β1 also increases translation to increase protein levels without significantly affecting mRNA levels. These mRNAs share a common 20 nucleotide motif. Deletion or insertion of this motif in mRNAs eliminates or induces the TGF-β1 regulation of translation. At least 5 RNA-binding proteins including DDX3 bind the RNA motif, and TGF-β1 regulates their protein levels and/or binding to the motif. Inhibiting DDX3, either by siRNA or small molecule inhibitors, reduced TGF-β1 induced NEU3 levels. In the mouse bleomycin model of pulmonary fibrosis, injections of the DDX3 inhibitor RK-33 starting 10 days after bleomycin potentiated survival and reduced lung inflammation, fibrosis, and lung tissue levels of DDX3, TGF-β1, and NEU3. Together, these results suggest that TGF-β1 regulates RNA-binding proteins that interact with a mRNA motif that is necessary and sufficient for TGF-β1 to regulate mRNA translation, and that blocking this effect can reduce fibrosis.
Spatially controlled microtubule nucleation and organization from crosslinker MAP65 condensates
Authors: Sahu Sumon , Chauhan Prashali , Lumen Ellie , Moody Kelsey , Peddireddy Karthik , Mani Nandini , Subramanian Radhika , Robertson-Anderson Rae , Wolfe Aaron J , Ross Jennifer L.
Published: October 2022

Microtubule organization in cells is essential for the internal structure and coordination of events of intracellular transport, mitosis, and cell motility. For many cell types, microtubule organization is dominated by centrosomal nucleation that use gamma-tubulin to template filaments. Yet, some cell types lack centrosomes or centrioles, such as plant cells. Instead, microtubules nucleate from regions with high concentrations of microtubule binding and nucleating proteins. A mechanism that can drive high local concentrations of nucleators is liquid-liquid phase separation of proteins with intrinsically disordered regions. Here, we report that the plant microtubule nucleator and crosslinking protein, MAP65-1, can form phase separated condensates at physiological salt and temperature without extra crowding agents. These condensates are liquid at first and can mature to gel-like phases over time and with different environmental conditions. We show that these condensates can nucleate and grow microtubule bundles that form asters, regardless of the viscoelasticity of the condensate. When gel-like droplets nucleate and grow asters from a shell of tubulin at the surface, the microtubules are able to re-fluidize the MAP65 condensate. Condensate-induced cytoskeletal formation could be a universal mechanism for organization of the microtubule and actin cytoskeletons in all cell types, especially cells without centrosomes.