bluuebunny/crossref_metadata_2025 huggingface dataset:
Warming-induced range expansion effects on the diversity and composition of the gut microbiome: a case study with two butterflies
Authors: Montoya Daniel , de Jong Maaike
Published: January 2023

Species are the habitat of a diverse community of organisms, including bacteria, fungi and viruses – themicrobiome. Evidence shows that many species shift and/or expand their geographical ranges following warming climate; yet, the response of host-associated microbiome communities to species range shifts has received less attention, especially in observational studies. Here, we use two British butterfly species for which detailed long-term distributional data is available, and document for the first time a variety of effects of warming-induced range expansion on the diversity and composition of their microbiome. Our results show similar diversity and species-specific responses in the evenness of the gut microbial communities in the two butterflies. However, butterflies at the expanded ranges showed (i) a larger variability in the microbiome diversity, and (ii) a smaller core community of bacteria that is to a large extent a subset of the bacterial core community in the original range. The microbiome is responsible for many aspects of the host’s physiology and growth, and for ecosystem function, so if the changes in the gut microbial communities reported here apply to other species and taxonomic groups, the potential impact to biodiversity and functioning after range expansion could be severe.
Functional membrane microdomains and the hydroxamate siderophore transporter ATPase FhuC govern Isd-dependent heme acquisition inStaphylococcus aureus
Authors: Adolf Lea Antje , Müller-Jochim Angelika , Kricks Lara , Puls Jan-Samuel , Lopez Daniel , Grein Fabian , Heilbronner Simon
Published: January 2023

Sufficient access to transition metals such as iron is essential for bacterial proliferation and their active limitation within host tissues effectively restricts infection. To overcome iron limitation, the invasive pathogenStaphylococcus aureususes the iron-regulated surface determinant (Isd) system to acquire hemoglobin-derived heme. While heme transport over the cell wall is well understood, its transport over the membrane is hardly investigated. In this study, we show the heme-specific permease IsdF to be energized by the general ATPase FhuC. Additionally, we show that IsdF needs appropriate location within the membrane for functionality. The membrane ofS. aureuspossesses special compartments (functional membrane microdomains – FMMs) to organize membrane complexes. We show IsdF to be associated with FMMs, to directly interact with the FMM scaffolding protein flotillin A (FloA) and to co-localize with the latter on intact bacterial cells. Additionally, Isd-dependent bacterial growth required FMMs and FloA. Our study shows that Isd-dependent heme acquisition requires a highly structured cell envelope to allow coordinated transport over the cell wall and membrane and it gives the first example of a bacterial nutrient acquisition system that depends on FMMs.
Convergence of direction, location and theta in the rat anteroventral thalamic nucleus
Authors: Lomi Eleonora , Jeffery Kate J. , Mitchell Anna S.
Published: January 2023

Retrosplenial cortex (RSC) is a cortical region that computes heading direction from landmark information, but how it does this remains unknown. Recently we found that its two major subregions receive differential projections from two anteroventral thalamic (AV) subfields; dorsomedial (AVDM) and ventrolateral (AVVL). To probe the respective contributions of these inputs we recorded single neurons and local field potentials from AV in rats during foraging. We observed and characterized neurons modulated by theta oscillations, heading direction, and a conjunction of these. Unexpectedly, we also discovered place cells (neurons modulated by location). Consistent with the notion that AV contains two parallel subcircuits, there was a prevalence of non-conjunctive cells in AVDM, and of conjunctive and place neurons in AVVL. This integration of spatial and movement signals in AV is consistent with a thalamic role in multimodal integration and may be important for supplying the spatial information that modulates RSC directional responding.
Selection of Extended CRISPR RNAs with Enhanced Targeting and Specificity
Authors: Herring-Nicholas Ashley , Dimig Hillary , Roesing Miranda , Josephs Eric A.
Published: January 2023

For a CRISPR guide RNA (gRNA) with a specific target but activity at known “off-target” sequences, we present a method to screen hundreds of thousands of gRNA variants with short, randomized 5’ nucleotide extensions near its DNA-targeting segment—a modification that can increase Cas9 gene editing specificity by orders of magnitude with certain 5’- extension sequences,viasome as-yet-unknown mechanism that makesde novodesign of the extension sequence difficult to perform manually—to robustly identify extended gRNAs (x-gRNAs) that have been counter-selected against activity at those off-target sites and that exhibit significantly enhanced Cas9 specificity for their intended targets.
Chloroquine induces eryptosis inP. falciparum-infectedred blood cells and the release of extracellular vesicles with a unique protein profile
Authors: Carrera-Bravo Claudia , Zhou Tianchi , Hang Jing Wen , Modh Harshvardhan , Huang Fred , Malleret Benoit , Wacker Matthias G , Wang Jiong-Wei , Renia Laurent , Tan Kevin SW
Published: January 2023

Malaria is a vector-borne parasitic disease that affects millions worldwide. In order to reach the objective, set by the World Health Organization to decrease the cases by 2030, antimalarial drugs with novel modes of action are required. Previously, a novel mechanism of action of chloroquine (CQ) was reported involving features of programmed cell death in the parasite, mainly characterized by calcium efflux from the digestive vacuole (DV) permeabilization. Increased intracellular calcium induces the suicidal death of erythrocytes also known as eryptosis. This study aimed to identify the hallmarks of eryptosis due to calcium redistribution and the downstream cellular effects during CQ treatment in iRBCs.Plasmodium falciparum3D7 at mid-late trophozoites were used for the antimalarial drug treatment. Our results revealed increased phosphatidylserine (PS) exposure, cell shrinkage and membrane blebbing, delineating an eryptotic phenotype in the host RBC. Interestingly, the blebs on the surface of the iRBCs released to the extracellular milieu become extracellular vesicles (EVs) which are essential for intercellular communication due to their cargo of proteins, nucleic acids, lipids and metabolites. The proteomic characterization displayed 2 highly enriched protein clusters in EVs from CQ-treated iRBCs, the proteasome and ribosome. We demonstrated that this unique protein cargo is not associated with the parasite growth rate. Additionally, we found that these particular EVs might activate IFN signaling pathways mediated by IL-6 in THP-1-derived macrophages. Our findings shed new insights into a novel drug-induced cell death mechanism that targets the parasite and specific components of the infected host RBC.

IMPORTANCE

Our previous studies have shown that chloroquine (CQ) treatment in iRBCs triggersPlasmodium falciparumdigestive vacuole (DV) membrane permeabilization leading to calcium redistribution. Interestingly, increased intracellular calcium concentration is the main inducer of the suicidal death of red blood cells (RBCs) called eryptosis. The present study shed new insights into a novel CQ-induced cell death mechanism that targets the parasite and the infected host RBC by inducing key phenotypic hallmarks of eryptosis: PS exposure, cell shrinkage and membrane blebbing. Moreover, the proteomic characterization of the blebs released to the extracellular milieu also known as extracellular vesicles (EVs) revealed a cargo highly enriched in ribosomal proteins and proteasome subunits relevant for host-parasite interactions. These findings highlight CQ’s effect on calcium homeostasis disruption in infected red blood cells (iRBCs) with cellular and immunological consequences of great significance for malaria pathogenesis and potential clinical implications.
A hepatitis B virus (HBV) sequence variation graph improves sequence alignment and sample-specific consensus sequence construction for genetic analysis of HBV
Authors: Duchen Dylan , Clipman Steven , Vergara Candelaria , Thio Chloe L. , Thomas David L. , Duggal Priya , Wojcik Genevieve L.
Published: January 2023

Hepatitis B virus (HBV) remains a global public health concern, with over 250 million individuals living with chronic HBV infection (CHB) and no curative therapy currently available. Viral diversity is associated with CHB pathogenesis and immunological control of infection. Improved methods to characterize the viral genome at both the population and intra-host level could aid drug development efforts. Conventionally, HBV sequencing data are aligned to a linear reference genome and only sequences capable of aligning to the reference are captured for analysis. Reference selection has additional consequences, including sample-specific ‘consensus’ sequence construction. It remains unclear how to select a reference from available sequences and whether a single reference is sufficient for genetic analyses. Using simulated short-read sequencing data generated from full-length publicly available HBV genome sequences and HBV sequencing data from a longitudinally sampled individual with CHB, we investigate alternative graph-based alignment approaches. We demonstrate that using a phylogenetically representative ‘genome graph’ for alignment, rather than linear reference sequences, avoids issues of reference ambiguity, improves alignment, and facilitates the construction of sample-specific consensus sequences genetically similar to an individual’s infection. Graph-based methods can therefore improve efforts to characterize the genetics of viral pathogens, including HBV, and may have broad implications in host pathogen research.
The ‘phruta’ R package and ‘salphycon’ shiny app: increasing access, reproducibility, and transparency in phylogenetic analyses
Authors: Román-Palacios Cristian
Published: January 2023

Current practices for assembling phylogenetic trees often recur to sequence data stored in GenBank. However, the molecular and taxonomic make up of sequences deposited in GenBank is generally not very clear.

‘phruta’, a newly developed ‘R’ package, is designed to primarily improve access to genetic data stored in GenBank. Functions in ‘phruta’ further enable users to assemble single- and multi-gene molecular datasets, and run basic phylogenetic tasks, all within ‘R’.

The structure of the functions implemented in ‘phruta’, designed as a workflow, aim to allow users to assemble simple workflows for particular tasks, which are in turn expected to increase reproducibility of relatively simple phylogenies.

To support the use of ‘phruta’ by researchers in different fields with variable levels of coding expertise, this paper presents ‘salphycon’, a shiny web app that is expected to increase access to the fundamental functions in the ‘phruta’ ‘R’ package.
A novel role of USP7 in imparting partial EMT state in colorectal cancer through the DDX3X-β-catenin axis
Authors: Basu Bhaskar , Karmakar Subhajit , Basu Malini , Ghosh Mrinal K.
Published: January 2023

Epithelial mesenchymal transition (EMT) is a fundamental and highly regulated process that is normally observed during embryonic development and tissue repair but is deregulated during advanced cancer. Classically, through the process of EMT, cancer cells gradually transition from a predominantly epithelial phenotype to a more invasive mesenchymal phenotype. Increasing studies have, however, brought into light the existence of unique intermediary states in EMT, often referred to as partial EMT states. Through our studies we have found the deubiquitinase USP7 to be strongly associated with the development of such a partial EMT state in colon cancer cells, characterized by the acquisition of mesenchymal characteristics but without the reduction in epithelial markers. We found USP7 to be overexpressed in colon adenocarcinomas and to be closely associated with advancing tumor stage. We found that functional inhibition or knockdown of USP7 is associated with a marked reduction in mesenchymal markers and in overall migration potential of cancer cells. Starting off with a proteomics-based approach we were able to identify and later on verify the DEAD box RNA helicase DDX3X to be an interacting partner of USP7. We then went on to show that USP7, through the stabilization of DDX3X, augments Wnt/β-catenin signaling, which has previously been shown to be greatly associated with colorectal cancer cell invasiveness. Our results strongly suggest a positive role of USP7 in the development of a partial mesenchymal phenotype in colorectal cancer.
A hydrophobic funnel governs monovalent cation selectivity in the ion channel TRPM5
Authors: Ives Callum M. , Şahin Alp Tegin , Thomson Neil J. , Zachariae Ulrich
Published: January 2023

A key capability of ion channels is the facilitation of selective permeation of certain ionic species across cellular membranes at high rates. Due to their physiological significance, ion channels are of great pharmaceutical interest as drug targets. The polymodal signal-detecting Transient Receptor Potential (TRP) superfamily of ion channels form a particularly promising group of drug targets. While most members of this family permeate a broad range of cations including Ca2+, TRPM4 and TRPM5 are unique due to their strong monovalent-selectivity and impermeability for divalent cations. Here, we investigated the mechanistic basis for their unique monovalent-selectivity byin silicoelectrophysiology simulations of TRPM5. Our simulations reveal an unusual mechanism of cation selectivity, which is underpinned by the function of the central channel cavity rather than the selectivity filter. Our results suggest that a subtle hydrophobic barrier at the cavity entrance ("hydrophobic funnel") enables monovalent, but not divalent cations to pass and occupy the cavity at physiologically relevant membrane voltages. Monovalent cations then permeate efficiently by a co-operative, distant knock-on mechanism between two binding regions in the extracellular pore vestibule and the central cavity. By contrast, divalent cations do not enter or interact favourably with the channel cavity due to its raised hydrophobicity. Hydrophilic mutations in the transition zone between the selectivity filter and the central channel cavity abolish the barrier for divalent cations, enabling both monovalent and divalent cations to traverse TRPM5.
Homeodomain-interacting protein kinase maintains neuronal homeostasis during normalCaenorhabditis elegansaging and systemically regulates longevity from serotonergic and GABAergic neurons
Authors: Lazaro-Pena Maria I. , Cornwell Adam B. , Diaz-Balzac Carlos A. , Das Ritika , Ward Zachary C. , Macoretta Nicholas , Thakar Juilee , Samuelson Andrew V.
Published: January 2023

Aging and the age-associated decline of the proteome is determined in part through neuronal control of evolutionarily conserved transcriptional effectors, which safeguard homeostasis under fluctuating metabolic and stress conditions by regulating an expansive proteostatic network. We have discovered theCaenorhabditis eleganshomeodomain-interactingproteinkinase (HPK-1) acts as a key transcriptional effector to preserve neuronal integrity, function, and proteostasis during aging. Loss ofhpk-1results in drastic dysregulation in expression of neuronal genes, including genes associated with neuronal aging. During normal aginghpk-1expression increases throughout the nervous system more broadly than any other kinase. Within the aging nervous system,hpk-1induction overlaps with key longevity transcription factors, which suggestshpk-1expression mitigates natural age-associated physiological decline. Consistently, pan-neuronal overexpression ofhpk-1extends longevity, preserves proteostasis both within and outside of the nervous system, and improves stress resistance. Neuronal HPK-1 improves proteostasis through kinase activity. HPK-1 functions cell non-autonomously within serotonergic and GABAergic neurons to improve proteostasis in distal tissues by specifically regulating distinct components of the proteostatic network. Increased serotonergic HPK-1 enhances the heat shock response and survival to acute stress. In contrast, GABAergic HPK-1 induces basal autophagy and extends longevity, which requiresmxl-2(MLX),hlh-30(TFEB), anddaf-16(FOXO). Our work establisheshpk-1as a key neuronal transcriptional regulator critical for preservation of neuronal function during aging. Further, these data provide novel insight as to how the nervous system partitions acute and chronic adaptive response pathways to delay aging by maintaining organismal homeostasis.
Glycine homeostasis requires reverse SHMT flux
Authors: McBride Matthew J. , Hunter Craig J. , Rabinowitz Joshua D.
Published: January 2023

The folate-dependent enzyme serine hydroxymethyltransferase (SHMT) reversibly converts serine into glycine and a tetrahydrofolate-bound one-carbon unit. Such one-carbon unit production plays a critical role in development, the immune system, and cancer. Here we show that the whole-body SHMT flux acts to net consume rather than produce glycine. Pharmacological inhibition of whole-body SHMT1/2 and genetic knockout of liver SHMT2 elevated circulating glycine levels up to eight-fold. Stable isotope tracing revealed that the liver converts glycine to serine, which is then converted by serine dehydratase into pyruvate and burned in the tricarboxylic acid cycle. In response to diets deficient in serine and glycine, de novo biosynthetic flux was unaltered but SHMT2- and serine dehydratase-mediated catabolic flux was lower. Thus, glucose-derived serine synthesis does not respond to systemic demand. Instead, circulating serine and glycine homeostasis is maintained through variable consumption, with liver SHMT2 as a major glycine-consuming enzyme.
Plasmodium falciparumGCN5 plays a key role in regulating artemisinin resistance–related stress responses
Authors: Lucky Amuza Byaruhanga , Wang Chengqi , Shakri Ahmad Rushdi , Kalamuddin Mohammad , Chim-Ong Anongruk , Li Xiaolian , Miao Jun
Published: January 2023

Plasmodium falciparumcauses the most severe malaria and is exposed to various environmental and physiological stresses in the human host. Given that GCN5 plays a critical role in regulating stress responses in model organisms, we aimed to elucidate PfGCN5’s function in stress responses inP. falciparum. The protein level of PfGCN5 was substantially induced under three stress conditions (heat shock, low glucose starvation, and dihydroartemisinin, the active metabolite of artemisinin (ART)). With a TetR-DOZI conditional knockdown (KD) system, we successfully down-regulated PfGCN5 to ∼50% and found that KD parasites became more sensitive to all three stress conditions. Transcriptomic analysis via RNA-seq identified ∼1,000 up-and down-regulated genes in the wildtype (WT) and KD parasites under these stress conditions. Importantly, DHA induced transcriptional alteration of many genes involved in many aspects of stress responses, which were heavily shared among the altered genes under heat shock and low glucose conditions, including ART-resistance-related genes such asK13andcoronin. Based on the expression pattern between WT and KD parasites under three stress conditions, ∼300-400 genes were identified to be involved in PfGCN5-dependent, general and stress-condition-specific responses with high levels of overlaps among three stress conditions. Notably, using ring-stage survival assay (RSA), we found that KD or inhibition of PfGCN5 could sensitize the ART-resistant parasites to the DHA treatment. All these indicate that PfGCN5 is pivotal in regulating general and ART-resistance-related stress responses in malaria parasites, implicating PfGCN5 as a potential target for malaria intervention.

IMPORTANCE

Malaria leads to about half a million deaths annually and these casualties were majorly caused by the infection ofPlasmodium falciparum. This parasite strives to survive by defending against a variety of stress conditions, such as malaria cyclical fever (heat shock), starvation due to low blood sugar (glucose) levels (hypoglycemia), and drug treatment. Previous studies have revealed thatP. falciparumhas developed unique stress responses to different stresses including ART treatment, and ART-resistant parasites harbor elevated stress responses. In this study, we provide critical evidence on the role of PfGCN5, a histone modifier, and a chromatin coactivator, in regulating general and stress-specific responses in malaria parasites, indicating that PfGCN5 can be used as a potential target for anti-malaria intervention.
Data-driven Targeting of COVID-19 Vaccination Programs: An Analysis of the Evidence on Impact, Implementation, Ethics and Equity
Authors: McLaren Zoë M.
Published: January 2023

The data-driven targeting of COVID-19 vaccination programs is a major determinant of the ongoing toll of COVID-19. Targeting of access to, outreach about and incentives for vaccination can reduce total deaths by 20-50 percent relative to a first-come-first-served allocation. This piece performs a systematic review of the modeling literature on the relative benefits of targeting different groups for vaccination and evaluates the broader scholarly evidence – including analyses of real-world challenges around implementation, equity, and other ethical considerations – to guide vaccination targeting strategies. Three-quarters of the modeling studies reviewed concluded that the most effective way to save lives, reduce hospitalizations and mitigate the ongoing toll of COVID-19 is to target vaccination program resources to high-risk people directly rather than reducing transmission by targeting low-risk people. There is compelling evidence that defining vulnerability based on a combination of age, occupation, underlying medical conditions and geographic location is more effective than targeting based on age alone. Incorporating measures of economic vulnerability into the prioritization scheme not only reduces mortality but also improves equity. The data-driven targeting of COVID-19 vaccination program resources benefits everyone by efficiently mitigating the worst effects of the pandemic until the threat of COVID-19 has passed.
Fast 2-photon stimulation using holographic patterns
Authors: Brunstein Maia , Lubetzki Jules , Moutoussamy Cédric , Li Wei , Barral Jérémie
Published: January 2023

Two decades after its introduction, optogenetics – a biological technique to control the activity of neurons or other cell types with light – remains a cutting edge and promising tool to study biological processes. Its increasing usage in research varies widely from causally exploring biological mechanisms and neural computations, to neurostimulation and sensory restauration. To stimulate neurons in the brain, a variety of approaches have been developed to generate precise spatiotemporal light patterns. Yet certain constrains still exists in the current optical techniques to activate a neuronal population with both cellular resolution and millisecond precision. Here, we describe an experimental setup allowing to stimulate a few tens of neurons in a plane at sub-millisecond rates using 2-photon activation. A liquid crystal on silicon spatial light modulator (LCoS-SLM) was used to generate spatial patterns in 2 dimensions. The image of the patterns was formed on the plane of a digital micromirror device (DMD) that was used as a fast temporal modulator of each region of interest. Using fluorescent microscopy and patch-clamp recording of neurons in culture expressing the light-gated ion channels, we characterized the temporal and spatial resolution of the microscope. We described the advantages of combining the LCoS-SLM with the DMD to maximize the temporal precision, modulate the illumination amplitude, and reduce background activation. Finally, we showed that this approach can be extended to patterns in 3 dimensions. We concluded that the methodology is well suited to address important questions about the role of temporal information in neuronal coding.
Crosslinking by ZapD drives the assembly of short FtsZ filaments into toroidal structures in solution
Authors: Merino-Salomón Adrián , Schneider Jonathan , Babl Leon , Krohn Jan-Hagen , Sobrinos-Sanguino Marta , Schäfer Tillman , Luque-Ortega Juan R. , Alfonso Carlos , Jiménez Mercedes , Jasnin Marion , Schwille Petra , Rivas Germán
Published: January 2023

Bacterial cell division relies on the Z ring, a cytoskeletal structure that acts as a scaffold for the assembly of the divisome. To date, the detailed mechanisms underlying the assembly and stabilization of the Z ring remain elusive. This study highlights the role of the FtsZ-associated protein (Zap) ZapD in the assembly and stabilization of Z-ring-like structures via filament crosslinking. Using cryo-electron tomography and biochemical analysis, we show that, at equimolar concentrations of ZapD and FtsZ, ZapD induces the formation of toroidal structures composed of short, curved FtsZ filaments that are crosslinked vertically, but also laterally and diagonally. At higher concentrations of ZapD, regularly spaced ZapD dimers crosslink FtsZ filaments from above, resulting in the formation of straight bundles. Despite the simplicity of this reconstituted system, these findings provide valuable insights into the structural organization and stabilization of the Z ring by Zap proteins in bacterial cells, revealing the key role of optimal crosslinking density and geometry in enabling filament curvature and ring formation.
Genetic mapping reveals new loci and alleles for flowering time and plant height using the double round-robin population of barley
Authors: Cosenza Francesco , Shrestha Asis , Van Inghelandt Delphine , Casale Federico A. , Wu Po-Ya , Weisweiler Marius , Li Jinquan , Wespel Franziska , Stich Benjamin
Published: January 2023

Flowering time and plant height are two critical determinants of yield potential in barley (Hordeum vulgare). Although their role as key traits, a comprehensive understanding of the genetic complexity of flowering time and plant height regulation in barley is still lacking. Through a double round-robin population originated from the crossings of 23 diverse parental inbred lines, we aimed to determine the variance components in the regulation of flowering time and plant height in barley as well as identify new genetic variants by single and multi-population quantitative trait loci (QTL) analyses and allele mining. Despite similar genotypic variance, we observed higher environmental variance components for plant height than flowering time. Furthermore, we detected one new QTL for flowering time and two new QTL for plant height. Finally, we identified a new functional allelic variant of the main regulatory genePpd-H1. Our results show that the genetic architecture of flowering time and plant height might be more complex than reported earlier and that a number of undetected, small effect or low frequency, genetic variants underlie the control of these two traits.
Leftward transfer of a chemosensory polycystin initiates left-dominant calcium signaling for lateralized embryonic development
Authors: Tanaka Yosuke , Morozumi Ai , Hirokawa Nobutaka
Published: January 2023

Left-dominant [Ca2+]ielevation on the margin of ventral node furnishes the initial laterality signaling in mouse embryos. It depends on nodal flow, FGFR signaling, and PKD1L1-containing polycystin channels, of which interrelationship is still missing. Here we show that PKD1L1 protein is predominantly accumulated on the left margin of the nodal pit and serves as a chemosensory channel for Nodal-mediated [Ca2+]ielevation. PKD1L1/PKD2 overexpression augmented the Nodal sensitivity of fibroblasts. We detected PKD1L1-containing fragile meshwork of fibrous strands using KikGR-PKD1L1 knockin mice, especially when the extraembryonic membrane was preserved. The portion of meshwork bridging over nodal crown cells significantly lateralized to the left. This bridge was formed by a leftward flow of PKD1L1-containing fibrous strands, which can be suppressed by the FGFR inhibitor SU5402 that antagonized the [Ca2+]ielevation as well. These data provide evidence for a leftward transfer of chemosensory PKD1L1 polycystin channel, as a readout mechanism of nodal flow.
Reinforcement Learning informs optimal treatment strategies to limit antibiotic resistance
Authors: Weaver Davis T. , King Eshan S. , Maltas Jeff , Scott Jacob G.
Published: January 2023

Antimicrobial resistance was estimated to be associated with 4.95 million deaths worldwide in 2019. It is possible to frame the antimicrobial resistance problem as a feedback-control problem. If we could optimize this feedback-control problem and translate our findings to the clinic, we could slow, prevent or reverse the development of high-level drug resistance. Prior work on this topic has relied on systems where the exact dynamics and parameters were knowna priori. In this study, we extend this work using a reinforcement learning (RL) approach capable of learning effective drug cycling policies in a system defined by empirically measured fitness landscapes. Crucially, we show that is possible to learn effective drug cycling policies despite the problems of noisy, limited, or delayed measurement. Given access to a panel of 15β-lactam antibiotics with which to treat the simulatedE. colipopulation, we demonstrate that RL agents outperform two naive treatment paradigms at minimizing the population fitness over time. We also show that RL agents approach the performance of the optimal drug cycling policy. Even when stochastic noise is introduced to the measurements of population fitness, we show that RL agents are capable of maintaining evolving populations at lower growth rates compared to controls. We further tested our approach in arbitrary fitness landscapes of up to 1024 genotypes. We show that minimization of population fitness using drug cycles is not limited by increasing genome size. Our work represents a proof-of-concept for using AI to control complex evolutionary processes.
A paternal signal induces endosperm proliferation upon fertilization in Arabidopsis
Authors: Simonini Sara , Bencivenga Stefano , Grossniklaus Ueli
Published: January 2023

In multicellular organisms, sexual reproduction relies on the formation of highly specialized, differentiated cells, the gametes. At maturity, male and female gametes are quiescent, awaiting fertilization, with their cell cycle being arrested at a precise stage. Failure to establish quiescence leads to unwanted proliferation, abortion of the offspring, and a waste of resources. Upon fertilization, the cell cycle resumes, allowing the newly formed zygote to divide rapidly. Successful development requires that male and female gametes are in the same phase of the cell cycle. The molecular mechanisms that enforce quiescence and reinstate cell division only after fertilization occurs are poorly understood. Here, we describe a sperm-derived signal that induces proliferation of theArabidopsiscentral cell precisely upon fertilization. We show that the mature central cell is arrested in S phase, caused by the activity of the conserved RETINOBLASTOMA RELATED1 (RBR1) protein. Paternal delivery of the core cell cycle component CYCD7;1 triggers RBR1 degradation, thereby stimulating S phase progression. Absence of CYCD7;1 delays RBR1 depletion, S phase reactivation, and central cell division, whereas its constitutive expression triggers proliferation of unfertilized central cells. In summary, we show that CYCD7;1 is a paternal signal that informs the central cell that fertilization occurred, thus unlocking quiescence and ensuring that cell division initiates just at the right time to ensure functional endosperm formation.
BRCA1/BARD1 ubiquitinates PCNA in unperturbed conditions to promote replication fork stability and continuous DNA synthesis
Authors: Salas-Lloret Daniel , García-Rodríguez Néstor , Giebel Lisanne , de Ru Arnoud , van Veelen Peter A. , Huertas Pablo , Vertegaal Alfred C.O. , González-Prieto Román
Published: January 2023

Deficiencies in the BRCA1 tumor suppressor gene are the main cause of hereditary breast and ovarian cancer. BRCA1 is involved in the Homologous Recombination DNA repair pathway, and, together with BARD1, forms a heterodimer with ubiquitin E3 activity. The relevance of the BRCA1/BARD1 ubiquitin E3 activity for tumor suppression and DNA repair remains controversial and most efforts aimed to identify BRCA1/BARD1 ubiquitination substrates rely on indirect evidence. Here, we observed that the BRCA1/BARD1 ubiquitin E3 activity was not required for Homologous Recombination or resistance to Olaparib. Using TULIP2 methodology, which enables the direct identification of E3-specific ubiquitination substrates, we identified substrates for BRCA1/BARD1. We found that PCNA is ubiquitinated by BRCA1/BARD1 in unperturbed conditions independently of RAD18. PCNA ubiquitination by BRCA1/BARD1 avoids the formation of ssDNA gaps during DNA replication and promotes replication fork stability epistatically to BRCA1 S114 phosphorylation, addressing the controversy about the function of BRCA1/BARD1 E3 activity in Homologous Recombination.
A Multiplexed SEC-MS Approach to Systematically Study the Interplay Between Protein Assembly-States and Phosphorylation Events
Authors: Doron-Mandel Ella , Bokor Benjamin J. , Ma Yanzhe , Street Lena A. , Tang Lauren C. , Abdou Ahmed A. , Shah Neel H. , Rosenberger George A. , Jovanovic Marko
Published: January 2023

A protein’s molecular interactions and post-translational modifications (PTMs), such as phosphorylation, can be co-dependent and reciprocally co-regulate each other. Although this interplay is central for many biological processes, a systematic method to simultaneously study assembly-states and PTMs from the same sample is critically missing. Here, we introduce SEC-MX (Size Exclusion Chromatography fractions MultipleXed), a global quantitative method combining Size Exclusion Chromatography and PTM-enrichment for simultaneous characterization of PTMs and assembly-states. SEC-MX enhances throughput, allows phosphopeptide enrichment, and facilitates quantitative differential comparisons between biological conditions. Applying SEC-MX to HEK293 and HCT116 cells, we generated a proof-of-concept dataset mapping thousands of phosphopeptides and their assembly-states. Our analysis revealed intricate relationships between phosphorylation events and assembly-states and generated testable hypotheses for follow-up studies. Overall, we establish SEC-MX as a valuable tool for exploring protein functions and regulation beyond abundance changes.
Active Learning of the Conformational Ensemble of Proteins using Maximum Entropy VAMPNets
Authors: Kleiman Diego E. , Shukla Diwakar
Published: January 2023

Rapid computational exploration of the free energy landscape of biological molecules remains an active area of research due to the difficulty of sampling rare state transitions in Molecular Dynamics (MD) simulations. In recent years, an increasing number of studies have exploited Machine Learning (ML) models to enhance and analyze MD simulations. Notably, unsupervised models that extract kinetic information from a set of parallel trajectories have been proposed, including the variational approach for Markov processes (VAMP), VAMPNets, and time-lagged variational autoencoders (TVAE). In this work, we propose a combination of adaptive sampling with active learning of kinetic models to accelerate the discovery of the conformational landscape of biomolecules. In particular, we introduce and compare several techniques that combine kinetic models with two adaptive sampling regimes (least counts and multi-agent reinforcement learning-based adaptive sampling) to enhance the exploration of conformational ensembles without introducing biasing forces. Moreover, inspired by the active learning approach of uncertainty-based sampling, we also present MaxEnt VAMPNet. This technique consists of restarting simulations from the microstates that maximize the Shannon entropy of a VAMPNet trained to perform soft discretization of metastable states. By running simulations on two test systems, the WLALL pentapeptide and the villin headpiece subdomain, we empirically demonstrate that MaxEnt VAMPNet results in faster exploration of conformational landscapes compared to the baseline and other proposed methods.
An AlphaFold2 map of the 53BP1 pathway identifies a direct SHLD3-RIF1 interaction critical for DNA repair activity
Authors: Sifri Chérine , Hoeg Lisa , Durocher Daniel , Setiaputra Dheva
Published: January 2023

53BP1 is a chromatin-binding DNA repair protein that promotes DNA double-strand break repair through recruitment of downstream effectors including RIF1, shieldin, and CST. The structural basis of the protein-protein interactions within the 53BP1-RIF1-shieldin-CST pathway that are essential for its DNA repair activity are largely unknown. Here we used AlphaFold2-Multimer (AF2) to predict all possible pairwise combinations of proteins within this pathway and provide structural models of seven previously characterized interactions. This analysis also predicted an entirely novel binding interface between the HEAT-repeat domain of RIF1 and the eIF4E-like domain of SHLD3. Extensive interrogation of this interface through both in vitro pulldown analysis and cellular assays supports the AF2-predicted model and demonstrates that RIF1-SHLD3 binding is essential for shieldin recruitment to sites of DNA damage, and for its role in antibody class switch recombination. Direct physical interaction between RIF1 and SHLD3 is therefore essential for 53BP1-RIF1-shieldin-CST pathway activity.
pTDP-43 levels correlate with cell type specific molecular alterations in the prefrontal cortex ofC9orf72ALS/FTD patients
Authors: Wang Hsiao-Lin V. , Xiang Jian-Feng , Yuan Chenyang , Veire Austin M. , Gendron Tania F. , Murray Melissa E. , Tansey Malú G. , Hu Jian , Gearing Marla , Glass Jonathan D. , Jin Peng , Corces Victor G. , McEachin Zachary T.
Published: January 2023

Repeat expansions in theC9orf72gene are the most common genetic cause of amyotrophic lateral sclerosis and familial frontotemporal dementia (ALS/FTD). To identify molecular defects that take place in the dorsolateral frontal cortex of patients withC9orf72ALS/FTD, we compared healthy controls withC9orf72ALS/FTD donor samples staged based on the levels of cortical phosphorylated TAR DNA binding protein (pTDP-43), a neuropathological hallmark of disease progression. We identified distinct molecular changes in different cell types that take place during FTD development. Loss of neurosurveillance microglia and activation of the complement cascade take place early, when pTDP-43 aggregates are absent or very low, and become more pronounced in late stages, suggesting an initial involvement of microglia in disease progression. Reduction of layer 2-3 cortical projection neurons with high expression of CUX2/LAMP5 also occurs early, and the reduction becomes more pronounced as pTDP-43 accumulates. Several unique features were observed only in samples with high levels of pTDP-43, including global alteration of chromatin accessibility in oligodendrocytes, microglia, and astrocytes; higher ratios of premature oligodendrocytes; increased levels of the noncoding RNA NEAT1 in astrocytes and neurons, and higher amount of phosphorylated ribosomal protein S6. Our findings reveal previously unknown progressive functional changes in major cell types found in the frontal cortex ofC9orf72ALS/FTD patients that shed light on the mechanisms underlying the pathology of this disease.
Virulence and genomic diversity among clinical isolates of ST1 (BI/NAP1/027)Clostridioides difficile
Authors: Dong Qiwen , Lin Huaiying , Allen Marie-Maude , Garneau Julian R. , Sia Jonathan K. , Smith Rita C. , Haro Fidel , McMillen Tracy , Pope Rosemary L. , Metcalfe Carolyn , Burgo Victoria , Woodson Che , Dylla Nicholas , Kohout Claire , Sundararajan Anitha , Snitkin Evan S , Young Vincent B. , Fortier Louis-Charles , Kamboj Mini , Pamer Eric G.
Published: January 2023

Clostridioides difficile (C. difficile), a leading cause of nosocomial infection, produces toxins that damage the colonic epithelium and results in colitis that varies from mild to fulminant. Variation in disease severity is poorly understood and has been attributed to host factors (age, immune competence and intestinal microbiome composition) and/or virulence differences betweenC. difficilestrains, with some, such as the epidemic BI/NAP1/027 (MLST1) strain, being associated with greater virulence. We tested 23 MLST1(ST1)C. difficileclinical isolates for virulence in antibiotic-treated C57BL/6 mice. All isolates encoded a complete Tcd pathogenicity locus and achieved similar colonization densities in mice. Disease severity varied, however, with 5 isolates causing lethal infections, 16 isolates causing a range of moderate infections and 2 isolates resulting in no detectable disease. The avirulent ST1 isolates did not cause disease in highly susceptible Myd88-/-or germ-free mice. Genomic analysis of the avirulent isolates revealed a 69 base-pair deletion in the N-terminus of thecdtRgene, which encodes a response regulator for binary toxin (CDT) expression. Genetic deletion of the 69 base-paircdtRsequence in the highly virulent ST1 R20291C. difficilestrain rendered it avirulent and reduced toxin gene transcription in cecal contents. Our study demonstrates that a natural deletion withincdtRattenuates virulence in the epidemic ST1C. difficilestrain without reducing colonization and persistence in the gut. Distinguishing strains on the basis ofcdtRmay enhance the specificity of diagnostic tests forC. difficilecolitis.
Spliceosomal mutations decouple 3′ splice site fidelity from cellular fitness
Authors: Roy Kevin R. , Gabunilas Jason , Neutel Dean , Ai Michelle , Samson Joyce , Lyu Guochang , Chanfreau Guillaume F.
Published: January 2023

The fidelity of splice site selection is thought to be critical for proper gene expression and cellular fitness. In particular, proper recognition of 3′-splice site (3′SS) sequences by the spliceosome is a daunting task considering the low complexity of the 3′SS consensus sequence YAG. Here we show that inactivating the near-essential splicing factor Prp18p results in a global activation of alternative 3′SS, many of which harbor sequences that highly diverge from the YAG consensus, including some highly unusual non-AG 3′SS. We show that the role of Prp18p in 3′SS fidelity is promoted by physical interactions with the essential splicing factors Slu7p and Prp8p and synergized by the proofreading activity of the Prp22p helicase. Strikingly, structure-guided point mutations that disrupt Prp18p-Slu7p and Prp18p-Prp8p interactions mimic the loss of 3′SS fidelity without any impact on cellular growth, suggesting that accumulation of incorrectly spliced transcripts does not have a major deleterious effect on cellular viability. These results show that spliceosomes exhibit remarkably relaxed fidelity in the absence of Prp18p, and that new 3′SS sampling can be achieved genome-wide without a major negative impact on cellular fitness, a feature that could be used during evolution to explore new productive alternative splice sites.
A Single Laccase Acts as a Key Component of Environmental Sensing in a Broad Host Range Fungal Pathogen
Authors: Westrick Nathaniel M. , Dominguez Eddie G. , Hull Christina M. , Smith Damon L. , Kabbage Mehdi
Published: January 2023

Secreted laccases are important enzymes on an ecological scale for their role in mediating plant-fungal interactions, but their function in fungal pathogenesis has yet to be elucidated. Ascomycete laccases have been primarily associated with cell wall melanin deposition, and laccase mutants in ascomycete species often demonstrate reduced pigmentation. In this study, a putatively secreted laccase,Sslac2, was characterized from the broad host-range plant pathogenSclerotinia sclerotiorum, which is largely unpigmented and is not dependent on melanogenesis for plant infection. Of the seven putative laccases in theS. sclerotiorumgenome,Sslac2was the only one found to be highly upregulated during pathogenesis of soybeans and was additionally found to be induced during growth on solid surfaces. Gene knockouts ofSslac2demonstrate wide ranging developmental defects, including abolished sclerotial formation, and are functionally non-pathogenic on unwounded tissue. While these mutants demonstrated a clear radial growth defect, enhanced growth was observed in liquid culture, likely due to altered hydrophobicity and thigmotropic responsiveness.Sslac2mutants were also unable to respond to a host environment, and accordingly unable to differentiate penetration structures, respond appropriately to chemical stress, or produce the key virulence factor oxalic acid. Transmission and scanning electron microscopy of WT and mutant strains show apparent differences in extracellular matrix structure that may explain the inability of the mutant to respond to environmental cues. TargetingSslac2using host-induced gene silencing significantly improved resistance toS. sclerotiorum, suggesting that fungal laccases could be a valuable target of disease control. Collectively, we identified a laccase critical to the development and virulence of the broad host-range pathogenS. sclerotiorumand propose a potentially novel role for fungal laccases in modulating environmental sensing.
Microbiome derived acidity protects against microbial invasion inDrosophila
Authors: Barron Alexander J. , Lesperance Danielle N. A. , Doucette Jeremy , Calle Sthefany , Broderick Nichole A.
Published: January 2023

Microbial invasions underlie host-microbe interactions that result in microbial pathogenesis and probiotic colonization. While these processes are of broad interest, there are still gaps in our understanding of the barriers to entry and how some microbes overcome them. In this study, we explore the effects of the microbiome on invasions of foreign microbes inDrosophila melanogaster. We demonstrate that gut microbesLactiplantibacillus plantarumandAcetobacter tropicalisimprove survival during invasion of a lethal gut pathogen and lead to a reduction in microbial burden. Using a novel multi-organism interactions assay, we report thatL. plantaruminhibits the growth of three invasive Gram-negative bacteria, whileA. tropicalisprevents this inhibition. A series ofin vitroandin vivoexperiments revealed that inhibition byL. plantarumis linked to its ability to acidify both internal and external environments, including culture media, fly food, and the gut itself, whileA. tropicalisdiminishes the inhibition by quenching acids. We propose that acid produced by the microbiome serves as an important gatekeeper to microbial invasions, as only microbes capable of tolerating acidic environments can colonize the host. The methods described herein will add to the growing breadth of tools to study microbe-microbe interactions in broad contexts.