bluuebunny/crossref_metadata_2025 huggingface dataset:
NMDAR mediated dynamic changes in m6A inversely correlates with neuronal translation
Authors: Gowda Naveen Kumar Chandappa , Nawalpuri Bharti , Ramakrishna Sarayu , Jhaveri Vishwaja , Muddashetty Ravi S
Published: June 2022

Epitranscriptome modifications are crucial in translation regulation and essential for maintaining cellular homeostasis. N6 methyladenosine (m6A) is one of the most abundant and well-conserved epitranscriptome modifications, which is known to play a pivotal role in diverse aspects of neuronal functions. However, the role of m6A modifications with respect to activity-mediated translation regulation and synaptic plasticity has not been studied. Here, we investigated the role of m6A modification in response to NMDAR stimulation. We have consistently observed that upon 5-minute NMDAR stimulation causes an increase in eEF2 phosphorylation. Correspondingly, NMDAR stimulation caused a significant increase in the m6A signal at 5 minutes time point, correlating with the global translation inhibition. The NMDAR induced increase in the m6A signal is accompanied by the redistribution of the m6A marked RNAs from translating to the non-translating pool of ribosomes. The increased m6A levels are well correlated with the reduced FTO levels observed on NMDAR stimulation. Additionally, we show that inhibition of FTO prevents NMDAR mediated changes in m6A levels. Overall, our results establish RNA-based molecular readout which corelates with the NMDAR-dependent translation regulation which helps in understanding changes in protein synthesis.
A Multitrait Locus Regulates Sarbecovirus Pathogenesis
Authors: Schäfer Alexandra , Leist Sarah R. , Gralinski Lisa E. , Martinez David R. , Winkler Emma S. , Okuda Kenichi , Hawkins Padraig E. , Gully Kendra L , Graham Rachel L. , Scobey D. Trevor , Bell Timothy A. , Hock Pablo , Shaw Ginger D. , Loome Jennifer F. , Madden Emily A. , Anderson Elizabeth , Baxter Victoria K. , Taft-Benz Sharon A. , Zweigart Mark R. , May Samantha R. , Dong Stephanie , Clark Matthew , Miller Darla R. , Lynch Rachel M , Heise Mark T. , Tisch Roland , Boucher Richard C. , Pardo Manuel de Villena Fernando , Montgomery Stephanie A. , Diamond Michael S. , Ferris Martin T. , Baric Ralph S.
Published: June 2022

Infectious diseases have shaped the human population genetic structure, and genetic variation influences the susceptibility to many viral diseases. However, a variety of challenges have made the implementation of traditional human Genome-wide Association Studies (GWAS) approaches to study these infectious outcomes challenging. In contrast, mouse models of infectious diseases provide an experimental control and precision, which facilitates analyses and mechanistic studies of the role of genetic variation on infection. Here we use a genetic mapping cross between two distinct Collaborative Cross mouse strains with respect to SARS-CoV disease outcomes. We find several loci control differential disease outcome for a variety of traits in the context of SARS-CoV infection. Importantly, we identify a locus on mouse Chromosome 9 that shows conserved synteny with a human GWAS locus for SARS-CoV-2 severe disease. We follow-up and confirm a role for this locus, and identify two candidate genes, CCR9 and CXCR6 that both play a key role in regulating the severity of SARS-CoV, SARS-CoV-2 and a distantly related bat sarbecovirus disease outcomes. As such we provide a template for using experimental mouse crosses to identify and characterize multitrait loci that regulate pathogenic infectious outcomes across species.
Computationally-guided design and selection of ribosomal active site mutants with high activity
Authors: Kofman Camila , Watkins Andrew M. , Kim Do Soon , Wooldredge Alexandra C. , Karim Ashty S. , Das Rhiju , Jewett Michael C.
Published: June 2022

Understanding how modifications to the ribosome affect function has implications for studying ribosome biogenesis, building minimal cells, and repurposing ribosomes for synthetic biology. However, efforts to design sequence-modified ribosomes have been limited because point mutations in the ribosomal RNA (rRNA), especially in the catalytic active site (peptidyl transferase center; PTC), are often functionally detrimental. Moreover, methods for directed evolution of rRNA are constrained by practical considerations (e.g., library size). Here, to address these limitations, we developed a computational rRNA design approach for screening guided libraries of mutant ribosomes. Our method includesin silicolibrary design and selection using a Rosetta stepwise Monte Carlo method (SWM), library construction andin vitrotesting, and functional characterizationin vivo. As a model, we apply our method to making modified ribosomes with mutant PTCs. We engineer ribosomes with as many as 30 mutations in their PTCs, highlighting previously unidentified epistatic interactions, and show that SWM helps identify sequences with beneficial phenotypes as compared to random library sequences. We further demonstrate that some variants improve cell growthin vivo, relative to wild type ribosomes. We anticipate that SWM design may serve as a powerful tool for high-resolution rRNA design.
RoPE: a robust profile likelihood method for differential gene expression analysis
Authors: Zhong Lehang , Strug Lisa J.
Published: June 2022

Variation in RNA-Seq data creates modeling challenges for differential gene expression (DE) analysis. Statistical approaches address conventional small sample sizes and implement empirical Bayes or non-parametric tests, but frequently produce different conclusions. Increasing sample sizes enable proposal of alternative DE paradigms. Here we develop RoPE, which uses a data-driven adjustment for variation and a robust profile likelihood ratio DE test. Simulation studies show RoPE can have improved performance over existing tools as sample size increases and has the most reliable control of error rates. Application of RoPE demonstrates that an active Pseudomonas Aeruginosa infection downregulates the SLC9A3 Cystic Fibrosis modifier gene.
Peripheral Auditory Nerve Impairment in a Mouse Model of Syndromic Autism
Authors: McChesney Nathan , Barth Jeremy L. , Rumschlag Jeffrey A. , Tan Junying , Harrington Adam J. , Noble Kenyaria V. , McClaskey Carolyn M. , Elvis Phillip , Vaena Silvia G. , Romeo Martin J. , Harris Kelly C. , Cowan Christopher W. , Lang Hainan
Published: June 2022

Dysfunction of the peripheral auditory nerve (AN) contributes to dynamic changes throughout the central auditory system, resulting in abnormal auditory processing, including hypersensitivity. Altered sound sensitivity is frequently observed in autism spectrum disorder (ASD), suggesting that AN deficits and changes in auditory information processing may contribute to ASD-associated symptoms, including social communication deficits and hyperacusis. The MEF2C transcription factor is associated with risk for several neurodevelopmental disorders, and mutations or deletions of MEF2C produce a haploinsufficiency syndrome characterized by ASD, language and cognitive deficits. A mouse model of this syndromic ASD (i.e., Mef2c+/- or Mef2c-Het) recapitulates many of the MEF2C Haploinsufficiency syndrome-linked behaviors including communication deficits. We show here that Mef2c-Het mice exhibit functional impairment of the peripheral AN and a modest reduction in hearing sensitivity. We find that MEF2C is expressed during development in multiple AN and cochlear cell types, and in Mef2c-Het mice, we observe multiple cellular and molecular alterations associated with the AN, including abnormal myelination, neuronal degeneration, neuronal mitochondria dysfunction, and increased macrophage activation and cochlear inflammation. These results reveal the importance of MEF2C function in inner ear development and function and the engagement of immune cells and other non-neuronal cells, which suggests that microglia/macrophages and other non-neuronal cells might contribute, directly or indirectly, to AN dysfunction and ASD-related phenotypes. Finally, our study establishes a comprehensive approach for characterizing AN function at the physiological, cellular, and molecular levels in mice, which can be applied to animal models with a wide range of human auditory processing impairments.

Significance Statement

This is the first report of peripheral auditory nerve (AN) impairment in a mouse model of human MEF2C haploinsufficiency syndrome that has well-characterized ASD related behaviors including communication deficits, hyperactivity, repetitive behavior, and social deficits. We identify multiple underlying cellular, sub-cellular, and molecular abnormalities that may contribute to peripheral AN impairment. Our findings also highlight the important roles of immune cells (e.g., cochlear macrophages) and other non-neuronal elements (e.g., glial cells and cells in the stria vascularis) in auditory impairment in ASD. The methodological significance of the study is the establishment of a comprehensive approach for evaluating peripheral AN function and impact of peripheral AN deficits with minimal hearing loss.
VASH1-SVBP and VASH2-SVBP generate different detyrosination profiles on microtubules
Authors: Ramirez-Rios Sacnicte , Choi Sung Ryul , Sanyal Chadni , Blum Thorsten , Bosc Christophe , Krichen Fatma , Denarier Eric , Soleilhac Jean-Marc , Blot Béatrice , Janke Carsten , Stoppin-Mellet Virginie , Magiera Maria M. , Arnal Isabelle , Steinmetz Michel O. , Moutin Marie-Jo
Published: June 2022

The detyrosination/tyrosination cycle of α-tubulin is critical for proper cell functioning. VASH1-SVBP and VASH2-SVBP are ubiquitous enzyme complexes involved in microtubule detyrosination. However, little is known about their mode of action. Here, we show in reconstituted systems and in cells that VASH1-SVBP and VASH2-SVBP drive global and local detyrosination of microtubules, respectively. We solved the cryo-electron microscopy structure of human VASH2-SVBP bound to microtubules, revealing a different microtubule-binding configuration of its central catalytic region compared to VASH1-SVBP. We further show that the divergent mode of detyrosination between the two enzymes is correlated with the microtubule-binding properties of their disordered N- and C-terminal regions. Specifically, the N-terminal region is responsible for a significantly longer residence time of VASH2-SVBP on microtubules compared to VASH1-SVBP. We suggest that this VASH domain is critical for microtubule-detachment and diffusion of VASH-SVBP enzymes on the lattice. Together, our results suggest a mechanism by which these enzymes could generate distinct microtubule subpopulations and confined areas of detyrosinated lattices to drive various microtubule-based cellular functions.

VASH1-SVBP and VASH2-SVBP produce global and local detyrosination patterns of microtubule lattices, respectively. These activities rely on the interplay between the N- and C-terminal disordered regions of the enzymes, which determine their differential molecular mechanism of action.

GRAPHICAL ABSTRACT

Schematic representation of divergent molecular mechanisms of action of VASH-SVBP detyrosination complexes.
Plant phase extraction (PPE): A novel method for enhanced discovery of RNA-binding proteome in plants
Authors: Zhang Yong , Xu Ye , Skaggs Todd H. , Ferreira Jorge F.S. , Chen Xuemei , Sandhu Devinder
Published: June 2022

RNA-binding proteins (RBPs) are versatile effectors in posttranscriptional gene regulation. Systematic profiling of RBPs in plants has been limited to proteins interacting with polyadenylated (poly(A)) RNAs due to the lack of an efficient method of isolating RBPs associated with non-poly(A) RNAs. Here we reported the establishment and application of plant phase extraction (PPE) as a novel method to comprehensively discover the RNA-binding proteome in Arabidopsis leaf tissues, leading to the isolation of 1,169 RBPs, of which 673 corroborate with previously reported RBPs and 496 are novel RBPs. PPE showed unmatched ability in capturing 374 diverse RNA-binding domains (RBDs), while 44% of the RBPs lack recognized RBDs. PPE recovered far more ribosomal and tRNA-related RBPs and metabolic enzymes with moonlighting RBP functions than any other individual report from plants, signifying its advantage in unbiasedly retrieving non-poly(A) and unconventional RBPs. We proposed that intrinsically disordered regions (IDRs) contributed to the non-classical binding of some novel RBPs, and provided the first evidence that enzymatic domains from metabolic enzymes have additional roles in RNA-binding. Taken together, our findings demonstrated that PPE is an effective approach to identifying a wide range of RBPs in complex plant tissues and may have broad biological implications.
Developing and adult reef fish show rapid, reversible light-induced plasticity in their visual system
Authors: Fogg Lily G. , Cortesi Fabio , Gache Camille , Lecchini David , Marshall N. Justin , de Busserolles Fanny
Published: June 2022

The visual capabilities of fish are optimised for their ecology and light environment over evolutionary time. Similarly, fish vision can adapt to regular changes in light conditions within their lifetime, e.g., ontogenetic or seasonal variation. However, we do not fully understand how vision responds to irregular short-term changes in the light environment, e.g., algal blooms and light pollution. In this study, we investigated the effect of short-term exposure to unnatural light conditions on opsin gene expression and retinal cell densities in larval and adult diurnal reef fish (convict surgeonfish; Acanthurus triostegus). Results revealed phenotypic plasticity in the retina across ontogeny, particularly in the larvae. The most substantial differences at both molecular and cellular levels were found under constant dim light, while constant bright light or simulated artificial light at night had a lesser effect. Under dim light, larvae and adults increased expression of the cone opsin genes, sws2a, rh2c and lws, within a few days and larvae also decreased densities of cones, inner nuclear layer cells and ganglion cells. These changes likely enhanced vision under the altered light conditions. Thus, our study suggests that plasticity mainly comes into play when conditions are extremely different to the species’ natural light environment, i.e., a diurnal fish in ‘constant night’. Finally, in a rescue experiment on adults, shifts in opsin expression were reverted within 24 hours. Overall, our study showed rapid, reversible light-induced changes in the retina of A. triostegus, demonstrating phenotypic plasticity in the visual system of a reef fish throughout life.
PRC2 clock: a universal epigenetic biomarker of aging and rejuvenation
Authors: Moqri Mahdi , Cipriano Andrea , Nachun Daniel , Murty Tara , de Sena Brandine Guilherme , Rasouli Sajede , Tarkhov Andrei , Aberg Karolina A. , van den Oord Edwin , Zhou Wanding , Smith Andrew , Mackall Crystal , Gladyshev Vadim , Horvath Steve , Snyder Michael P. , Sebastiano Vittorio
Published: June 2022

DNA methylation (DNAm) is one of the most reliable biomarkers for aging across many mammalian tissues. While the age-dependent global loss of DNAm has been well characterized, age-dependent DNAm gain is less specified. Multiple studies have demonstrated that polycomb repressive complex 2 (PRC2) targets are enriched among the CpG sites which gain methylation with age. However, a systematic whole-genome examination of all PRC2 targets in the context of aging methylome as well as whether these associations are pan-tissue or tissue-specific is lacking. Here, by analyzing DNAm data from different assays and from multiple young and old human and mouse tissues, we found that low-methylated regions (LMRs) which are highly bound by PRC2 in embryonic stem cells gain methylation with age in all examined somatic mitotic cells. We also estimated that this epigenetic change represents around 90% of the age-dependent DNAm gain genome-wide. Therefore, we propose the “PRC2 clock,” defined as the average DNAm in PRC2 LMRs, as a universal biomarker of cellular aging in somatic cells. In addition, we demonstrate the application of this biomarker in the evaluation of different anti-aging interventions, including dietary restriction and partial epigenetic reprogramming.
Temperature modifies trait-mediated infection outcomes in a Daphnia-fungal parasite system
Authors: Sun Syuan-Jyun , Dziuba Marcin K. , Jaye Riley N. , Duffy Meghan A.
Published: June 2022

One major concern related to climate change is that elevated temperatures will drive increases in parasite outbreaks. Increasing temperature is known to alter host traits and host-parasite interactions, but we know relatively little about how these are connected mechanistically – that is, about how elevated temperatures impact the relationship between epidemiologically relevant host traits and infection outcomes. Here, we used a zooplankton-fungus (Daphnia dentifera-Metschnikowia bicuspidata) disease system to investigate whether temperature interacted with host susceptibility traits in determining infection outcomes. We did this by exposing D. dentifera to M. bicuspidata or leaving them unexposed at either control (20°C) or warming temperatures (24°C) in a fully factorial design. We found that elevated temperatures altered the physical barrier and immune responses to parasites during the initial infection process, and that infected hosts at elevated temperatures suffered a greater reduction of fecundity and lifespan. Furthermore, the relationship between a key trait – gut epithelium thickness, which is a physical barrier – and the likelihood of terminal infection reversed at warmer temperatures. Together, our results highlight the complex ways that temperatures can modulate host-parasite interactions, and the importance of considering key host susceptibility traits when predicting disease dynamics in a warmer world.

This article is part of the theme issue ‘Infectious disease and evolution in a changing world’.
A Theoretical Comparison of Alternative Male Mating Strategies in Cephalopods and Fishes
Authors: Landsittel Joseph , Ermentrout G. Bard , Stiefel Klaus M.
Published: June 2022

We used computer simulations of growth, mating and death of cephalopods and fishes to explore the effect of different life-history strategies on the relative prevalence of alternative male mating strategies. Specifically, we investigated the consequences of single or multiple matings per lifetime, mating strategy switching, cannibalism, resource stochasticity, and altruism towards relatives. We found that a combination of single (semelparous) matings, cannibalism and an absence of mating strategy changes in one lifetime led to a more strictly partitioned parameter space, with a reduced region where the two mating strategies co-exist in similar numbers.

Explicitly including Hamilton’s rule in simulations of the social system of a Cichlid led to an increase of dominant males, at the expense of both sneakers and dwarf males (“super-sneakers”). Our predictions provide general bounds on the viable ratios of alternative male mating strategies with different life-histories, and under possibly rapidly changing ecological situations.
Single-cell imaging reveals non-cooperative and cooperative infection strategies ofListeria monocytogenesin macrophages
Authors: Moran Josephine , Feltham Liam , Bagnall James , Goldrick Marie , Lord Elizabeth , Nettleton Catherine , Spiller David G. , Roberts Ian , Paszek Pawel
Published: June 2022

Pathogens have developed intricate strategies to overcome the host’s innate immune responses. In this paper we use live-cell microscopy with a single bacterium resolution to follow in real time interactions between the food-borne pathogenL. monocytogenesand host macrophages, a key event controlling the infectionin vivo. We demonstrate that infection results in heterogeneous outcomes, with only a subset of bacteria able to establish a replicative invasion of macrophages. The fate of individual bacteria in the same host cell was independent from each other and non-cooperative, but a higher multiplicity of infection resulted in a reduced probability of replication. Using internalisation assays and conditional probabilities to mathematically describe the multi-stage invasion process, we demonstrate that the secreted Listeriolysin toxin (LLO) of the PrfA regulon regulates replication probability by compromising the ability to phagocytose bacteria. Using strains expressing fluorescent reporters to follow transcription of either the LLO-encodinghlyoractAgenes, we show that replicative bacteria exhibited higher PrfA regulon expression in comparison to those bacteria that did not replicate, however elevated PrfA expressionper sewas not sufficient to increase the probability of replication. Overall, this demonstrates a new role for the population-level, but not single cell PrfA-mediated cooperativity to regulate outcomes of host pathogen interactions.

Key points

L. monocytogenesinvasion of innate immune macrophages results in heterogeneous infection outcomes at the single cell level

Fate of individual bacteria in the same host cell is independent from each other and non-cooperative

Bacterial populations coordinate host cell uptake via the rate of phagocytosis to reduce internalization at high MOI

The PrfA regulon system is necessary but not sufficient forL. monocytogenesreplication, but population-level PrfA virulence regulates single cell outcome probability
A cross-disease human microglial framework identifies disease-enriched subsets and tool compounds for microglial polarization
Authors: Tuddenham John F. , Taga Mariko , Haage Verena , Roostaei Tina , White Charles , Lee Annie , Fujita Masashi , Khairallah Anthony , Green Gilad , Hyman Bradley , Frosch Matthew , Hopp Sarah , Beach Thomas G. , Corboy John , Habib Naomi , Klein Hans-Ulrich , Soni Rajesh Kumar , Teich Andrew F. , Hickman Richard A. , Alcalay Roy N. , Shneider Neil , Schneider Julie , Sims Peter A. , Bennett David A. , Olah Marta , Menon Vilas , De Jager Philip L.
Published: June 2022

Human microglia play a pivotal role in neurological diseases, but few targeted therapies that directly modulate microglial state or function exist due to an incomplete understanding of microglial heterogeneity. We use single-cell RNA sequencing to profile live human microglia from autopsies or surgical resections across diverse neurological diseases and central nervous system regions. We observe a central divide between oxidative and heterocyclic metabolism and identify subsets associated with antigen presentation, motility, and proliferation. Specific subsets are enriched in susceptibility genes for neurodegenerative diseases or the disease-associated microglial signature. We validate subtypesin situwith an RNAscope-immunofluorescence pipeline and leverage our dataset as a classification resource, finding that iPSC model systems recapitulate substantialin vivoheterogeneity. Finally, we identify and validate candidates for chemically inducing subtype-specific statesin vitro, showing that Camptothecin downregulates the transcriptional signature of disease-enriched subsets and upregulates a signature previously shown to be depleted in Alzheimer’s.
Illumination of a progressive allosteric mechanism mediating the glycine receptor activation
Authors: Shi Sophie , Lefebvre Solène N , Peverini Laurie , Cerdan Adrien , Gielen Marc , Changeux Jean-Pierre , Cecchini Marco , Corringer Pierre-Jean
Published: June 2022

Pentameric ligand-gated ion channel mediate signal transduction at chemical synapses by transiting between resting and open states upon neurotransmitter binding. Here, we investigate the gating transition of the glycine receptor fluorescently labeled at the extracellular-transmembrane interface by voltage-clamp fluorimetry (VCF). Fluorescence reports a glycine-elicited conformational transition that precedes pore opening. Low concentrations of glycine, partial agonists or specific mixtures of glycine and strychnine trigger the full fluorescence signal while weakly activating the channel. Molecular dynamic simulations of a partial agonist bound-closed Cryo-EM structure show a highly dynamic personality: a marked structural flexibility at both the extracellular-transmembrane interface and the orthosteric site, generating docking properties that recapitulate VCF data. Data thus illuminate a progressive gating transition towards activation, displaying structural plasticity with novel implication concerning the mechanism of action of allosteric effectors.
ECM-derived biophysical cues mediate interstitial flow-induced sprouting angiogenesis
Authors: Chang Chia-Wen , Shih Hsiu-Chen , Cortes-Medina Marcos , Beshay Peter E. , Avendano Alex , Seibel Alex J. , Liao Wei-Hao , Tung Yi-Chung , Song Jonathan W.
Published: June 2022

Sprouting angiogenesis is orchestrated by an intricate balance of biochemical and mechanical cues in the local microenvironment. Interstitial flow has been established as a potent regulator of angiogenesis. Similarly, extracellular matrix (ECM) physical properties, such as stiffness and microarchitecture, have also emerged as important mediators of angiogenesis. Yet, the interplay between interstitial flow and ECM physical properties in the initiation and control of angiogenesis is poorly understood. Using a 3-D microfluidic tissue analogue of angiogenic sprouting with defined interstitial flow, we found that the addition of hyaluronan (HA) to collagen-based matrices significantly enhances sprouting induced by interstitial flow compared to responses in collagen-only hydrogels. We confirmed that both the stiffness and matrix pore size of collagen-only hydrogels were increased by the addition of HA. Interestingly, interstitial flow-potentiated sprouting responses in collagen/HA matrices were not affected when functionally blocking the HA receptor CD44. In contrast, enzymatic depletion of HA in collagen/HA matrices with hyaluronidase (HAdase) resulted in decreased stiffness, pore size, and interstitial flow-mediated sprouting to the levels observed in collagen-only matrices. Taken together, these results suggest that HA enhances interstitial flow-mediated angiogenic sprouting through its alterations to collagen ECM stiffness and pore size.
The oncogenic fusion protein EWS-FLI1 promotes premature ageing of biomolecular condensates by catalyzing fibril formation
Authors: Selig Emily E. , Romero-Moreno Alma K. , Akula Shivani , Xu Xiaoping , Libich David S.
Published: June 2022

Ewing sarcoma (EwS) is an aggressive pediatric cancer of bone and soft tissue. A chromosomal translocation that joins the low-complexity domain of EWS (EWSLCD) with the DNA-binding domain of FLI1 (FLI1DBD) creates EWS-FLI1, a fusion oncoprotein essential for EwS development and accounts for 85% of all EwS cases. EWS-FLI1 acts as an aberrant transcription factor and interferes with the normal functions of nucleic acid-binding proteins via multivalent interactions and biomolecular condensation. The FLI1DBD was found to directly interact with the EWSLCD causing enhanced phase separation and induced hardening of EWSLCD condensates. Three related ETS DBDs (ERG, ETV1 and PU.1) also induced EWSLCD condensate hardening. DNA binding blocked the interaction with the EWSLCD, and NMR spectroscopy confirmed that ETS DBDs interact with EWSLCD via the DNA-binding interface. Our results provide a physical basis for the dominant-negative effect EWS-FLI1 exerts on EWS and highlight the need for further investigations of the FLI1DBD-EWSLCD interaction in vivo.
GPU-Accelerated All-atom Particle-Mesh Ewald Continuous Constant pH Molecular Dynamics in Amber
Authors: Harris Julie A. , Liu Ruibin , de Oliveira Vinicius Martins , Montelongo Erik Vaquez , Henderson Jack A. , Shen Jana
Published: June 2022

Constant pH molecular dynamics (MD) simulations sample protonation states on the fly according to the conformational environment and user specified pH condition; however, the current accuracy is limited due to the use of implicit-solvent models or a hybrid solvent scheme. Here we report the first GPU-accelerated implementation, parameterization, and validation of the all-atom continuous constant pH MD (CpHMD) method with particle-mesh Ewald (PME) electrostatics in the Amber22 pmemd. cuda engine. The titration parameters for Asp, Glu, His, Cys, and Lys were derived for the CHARMM c22 and Amber ff14sb and ff19sb force fields. We then evaluated the PME-CpHMD method using the asynchronous pH replica-exchange titration simulations with the c22 force field for six benchmark proteins, including BBL, hen egg white lysozyme (HEWL), staphylococcal nuclease (SNase), thioredoxin, ribonuclease A (RNaseA), and human muscle creatine kinase (HMCK). The root-mean-square deviation from the experimental pKa’s of Asp, Glu, His, and Cys is 0.76 pH units, and the Pearson’s correlation coefficient for the pKa shifts with respect to model values is 0.80. We demonstrated that a finite-size correction or much enlarged simulation box size can remove a systematic error of the calculated pKa’s and improve agreement with experiment. Importantly, the simulations captured the relevant biology in several challenging cases, e.g., the titration order of the catalytic dyad Glu35/Asp52 in HEWL and the coupled residues Asp19/Asp21 in SNase, the large pKa upshift of the deeply buried catalytic Asp26 in thioredoxin, and the large pKa downshift of the deeply buried catalytic Cys283 in HMCK. We anticipate that PME-CpHMD offers proper pH control to improve the accuracies of MD simulations and enables mechanistic studies of proton-coupled dynamical processes that are ubiquitous in biology but remain poorly understood due to the lack of experimental tools and limitation of current MD simulations.
Regulation of Klebsiella pneumoniae mucoidy by the bacterial tyrosine kinase Wzc
Authors: Khadka Saroj , Ring Brooke , Krzeminski Lindsey R. , Hathaway Matthew , Walker Ryan S. , Mobley Harry L.T. , Mike Laura A.
Published: June 2022

Klebsiella pneumoniae is a frequent nosocomial pathogen associated with urinary tract infections (UTI), pneumonia, and septicemia. Two lineages of K. pneumoniae have emerged that challenge the treatment of these infections. One lineage includes hypervirulent strains, causing invasive community-acquired infections, and the other encompasses carbapenem-resistant isolates. Non-mucoid, carbapenem-resistant isolates are most frequently isolated from UTI. Therefore, we hypothesized that environmental conditions may drive K. pneumoniae adaptation to the urinary tract. We found that two rmp-positive, hypervirulent strains and two rmp-negative clinical UTI isolates all suppressed mucoidy when cultured in urine compared to rich LB medium without altering capsular polysaccharide (CPS) abundance. We then performed a transposon screen to identify genetic factors through which urine suppresses mucoidy. Due to variations within transposon hits, we performed whole genome sequencing on our hits and found 100% association between secondary mutations in Wzc and increased mucoidy. Wzc is an inner membrane tyrosine kinase that regulates CPS assembly. When Wzc mutations are encoded chromosomally we observed both increased mucoidy and increased cell-free extracellular polysaccharide (EPS). However, when Wzc mutations are expressed in trans we observed increased mucoidy without impacting CPS or cell-free EPS abundance. Functionally, one periplasmic mutation increased Wzc autophosphorylation, while four active site mutations reduced Wzc autophosphorylation. These four active site mutations are also sufficient to increase mucoidy in one of the rmp-negative clinical UTI isolates without affecting EPS abundance. Combined, these data implicate mutation-driven modulation of Wzc activity as a global, rmp-independent mechanism for increasing K. pneumoniae mucoidy, without altering EPS uronic acid content. Wzc activity may represent the lynchpin that coordinates both capsule biosynthesis and mucoidy, explaining why these two phenotypes have been historically intertwined.
Biosynthetic diversification of peptaibol mediates fungus-mycohost interactions
Authors: Fan Jie , Ren Jinwei , He Ruolin , Wei Peng-Lin , Li Yuanyuan , Li Wei , Chen Dawei , Druzhinina Irina S. , Li Zhiyuan , Yin Wen-Bing
Published: June 2022

Fungi have evolved a plethora of functionally diverse secondary metabolites (SMs) to enhance their adaptation to various environments. To understand how structurally diverse metabolites contribute to fungal adaptation, we elucidate fungus-mycohost specific interactions mediated by a family of polypeptides, i.e., peptaibols. We specified that peptaibol structural diversification was attributed to the nonspecific substrate recognition by the highly conserved peptaibol synthetases (PSs) in dead wood inhabiting mycoparasitic fungi from the genus Trichoderma. Exemplified by investigation of T. hypoxylon, we characterized a library of 19 amino acid residue peptaibols, named trichohypolins, containing 42 derivatives synthesized by a single PS enzyme (NPS1Th). Elimination of trichohypolin production by the deletion of nps1Th reduced the inhibitory activities of T. hypoxylon on at least 15 saprotrophic host fungi, indicating that peptaibols are essential for interactions of Trichoderma spp. with their mycohosts. Different antagonistic effects of five trichohypolin subfractions SF1–SF5 and two pure compounds trichohypolins A (1) and B (2) on saprotrophic host fungi revealed specific activities of peptaibol derivatives in mediating fungus-mycohost interaction. Our study provides insights into the role of metabolic diversity of biosynthetic pathways in interfungal interactions.
Microstructures of Ant Chemosensory Sensilla Support a Dual Function in Detecting Both Volatile and Contact-Mediated Cues
Authors: Gellert Hannah R. , Halley Daphné C. , Sieb Zackary J. , Smith Jody C. , Pask Gregory M.
Published: June 2022

Ants and other eusocial insects emit and receive chemical signals to communicate important information within the colony. In ants, nestmate recognition, task allocation, and reproductive distribution of labor are largely mediated through the detection of cuticular hydrocarbons (CHCs) that cover the exoskeleton. With their large size and limited volatility, these CHCs are believed to be primarily detected through direct contact with the antennae during behavioral interactions. Here we use scanning electron microscopy to investigate the unique morphological features of CHC-sensitive basiconic sensilla of two ant species, the black carpenter ant Camponotus pennsylvanicus and the Indian jumping ant Harpegnathos saltator. These basiconic sensilla possess an abundance of small pores typical of most insect olfactory sensilla, but also have a large concave depression at the terminal end. Basiconic sensilla are enriched at the distal segments of the antennae in both species, further supporting their proposed role in contact chemosensation. A survey of these sensilla across other ant subfamilies shows varied microstructures at their tips, but each possess surface textures that would also increase sensory surface area. These unique ant chemosensory sensilla represent yet another example of how specialized structures have evolved to serve the functional requirements of eusocial communication.
Exploring the culturable bacterial diversity and its hydrocarbon degrading potentiality isolated from the Oxygen Minimum Zone Sediments of Bay of Bengal
Authors: Prasannakumar ChinnamaniKumar
Published: June 2022

Understanding biota distribution in oxygen minimum zone can help guide further exploration of potentially unusual habitats. The present study explores the culturable bacterial fractions in the oxygen minimum zone sediments of Bay of Bengal. The 16S rRNA gene sequences of 30 morphologically distinct bacterial colonies isolated form oxygen minimum zone of Bay of Bengal reveals 25 phylo-types, predominated by Proteobacteria (83.3%) and Actinobacteria (16.6%). Over all, Alphaproteobacteria and Gammaproteobacteria dominated the culturable fraction in this study. The overall pair-wise distances of bacterial isolates of Bay of Bengal is two times lesser when compared to overall pair-wise distance of bacterial isolates from oxygen minimum zone of Arabian Sea indicating relatively low genetic distances in Bay of Bengal. Not even 1% of bacterial cells in oxygen minimum zone of Bay of Bengal are culturable. We found that oxygen concentration alone could not be a deciding factor of culturable bacterial diversity in oxygen minimum zone. More than 50% bacterial isolates of present study is an active degraders of hydrocarbons. Higher similarity of 16S rRNA sequences produced in this study with that of previously reported efficient hydrobonoclastic bacterial isolates like Vibrio diazotrophicus, Vibrio cyclotrophicus, Pseudomonas poae, Marinobacter hydrocarbonoclasticus, Marinobacter flavimaris and Alcanivorax borkumensis further strengthens the evidence of hydrocarbon presence in Bay of Bengal sediments. This study is first of its time addresses the diversity of culturable bacterial fractions in oxygen minimum zone sediments of Bay of Bengal. Higher number of bacterial isolates from oxygen minimum zone of Bay of Bengal has carbonoclastic potentialities implying that they may play an important role in in situ hydrocarbon degradation in oxygen minimum zone of Bay of Bengal.
Dissecting the mechanisms of environment sensitivity of smart probes for quantitative assessment of membrane properties
Authors: Ragaller Franziska , Andronico Luca , Sykora Jan , Kulig Waldemar , Rog Tomasz , Urem Yagmur Balim , Amaro Mariana , Abhinav  , Danylchuk Dmytro I. , Klymchenko Andrey , Hof Martin , Vattulainen Ilpo , Sezgin Erdinc
Published: June 2022

The plasma membrane serves as a crucial platform for a multitude of cellular processes. Its collective biophysical properties are largely determined by the structural diversity of the different lipid species it accommodates. Therefore, a detailed investigation of biophysical properties of the plasma membrane is of utmost importance for a comprehensive understanding of biological processes occurring therein. During the past two decades several environment-sensitive probes have been popular tools to investigate membrane properties. Although these probes are assumed to report on membrane order in similar ways, their individual mechanisms remain to be elucidated. In this study, using model membrane systems, we studied the probes Pro12A, NR12S and NR12A in depth and examined their sensitivity to parameters with potential biological implications, such as the degree of lipid saturation, double bond position and configuration, phospholipid headgroup and cholesterol content. Applying spectral imaging together with atomistic molecular dynamics simulations and time-dependent fluorescent shift analyses, we unravelled individual sensitivities of these probes to different biophysical properties, their distinct localizations and specific relaxation processes in membranes. Overall, Pro12A, NR12S and NR12A serve together as a toolbox with a wide range of applications allowing to select the most appropriate probe for each specific research question.
CryoEM Structures of the Nitrogenase Complex During Catalytic Turnover
Authors: Rutledge Hannah L. , Cook Brian D. , Nguyen Hoang P. M. , Herzik Mark A. , Tezcan Akif
Published: June 2022

The enzyme nitrogenase couples adenosine triphosphate (ATP) hydrolysis to the multi-electron reduction of atmospheric dinitrogen into ammonia. Despite extensive research, the mechanistic details of ATP-dependent energy transduction and dinitrogen reduction by nitrogenase are not well understood, requiring new strategies to monitor its structural dynamics during catalytic action. Here we report the cryogenic electron microscopic interrogation of the nitrogenase complex under enzymatic turnover conditions, which has enabled the structural characterization of the nitrogenase reaction intermediates at high resolution for the first time. Our structures show that asymmetry governs all aspects of nitrogenase mechanism including ATP hydrolysis, protein-protein interactions, and catalysis. Furthermore, they reveal several previously unobserved, mechanistically relevant conformational changes near the catalytic iron-molybdenum cofactor that are correlated with the nucleotide-hydrolysis state of the enzyme.

One-sentence summary

High-resolution cryoEM structures of the nitrogenase complex obtained under turnover provide new mechanistic insights.
Human adenylyl cyclase 9 is auto-stimulated by its isoform-specific C-terminal domain and equipped with paradigm-switching mechanisms
Authors: Chen Zhihao , Antoni Ferenc A.
Published: June 2022

Human transmembrane adenylyl cyclase 9 (AC9) is not regulated by heterotrimeric G proteins. Key to the resistance to stimulation by Gs-coupled receptors (GsR) is auto-inhibition by the COOH-terminal domain (C2b). The present study investigated the role of the C2b domain in the regulation of cyclic AMP production by AC9 in HEK293FT cells expressing the Glosensor22F cyclic AMP-reporter protein. Surprisingly, we found C2b to be essential for sustaining the basal output of cyclic AMP by AC9. A human mutation (E326D) in the parallel coiled-coil formed by the signalling helices of AC9 dramatically increased basal activity, which was also dependent on the C2b domain. Intriguingly, the same mutation enabled stimulation of AC9 by GsRs. In summary, auto-regulation by the C2b domain of AC9 sustains its basal activity and quenches activation by GsR. Thus AC9 appears to be tailored to support constitutive activation of cyclic AMP effector systems. A switch from this paradigm to stimulation by GsRs may be occasioned by conformation changes at the coiled-coil or removal of the C2b domain.
Comprehensive functional annotation of metagenomes and microbial genomes using a deep learning-based method
Authors: Maranga Mary , Szczerbiak Pawel , Bezshapkin Valentyn , Gligorijevic Vladimir , Chandler Chris , Bonneau Richard , Xavier Ramnik J , Kosciolek Tomasz , Vatanen Tommi
Published: June 2022

Comprehensive protein function annotation is essential for understanding microbiome-related disease mechanisms in the host organisms. Still, a large portion of human gut microbial proteins lack functional annotation. Here, we have developed a new metagenome analysis workflow integrating de novo genome reconstruction, taxonomic profiling and deep learning-based functional annotations from DeepFRI. We validate DeepFRI functional annotations by comparing them to orthology-based annotations from eggNOG on a set of 1,070 infant metagenome samples from the DIABIMMUNE cohort. Using the workflow, we have generated a sequence catalogue of 1.9 million non-redundant microbial genes. The functional annotations revealed 70% concordance between GO annotations predicted by DeepFRI and eggNOG. However, DeepFRI improved the annotation coverage, with 99% of the gene catalogue obtaining GO molecular function annotations, albeit less specific compared to eggNOG. Additionally, we construct pan-genomes in a reference-free manner using high-quality metagenome assembled genomes (MAGs) and analyse the associated annotations. eggNOG annotated more genes on well-studied organisms such as Escherichia coli while DeepFRI was less sensitive to taxa. This workflow will contribute to novel understanding of the functional signature of the human gut microbiome in health and disease as well as guide future metagenomics studies.
Characterization of the continuous transcriptional heterogeneity in Wilms’ tumors using unsupervised machine learning
Authors: Trink Yaron , Urbach Achia , Dekel Benjamin , Hohenstein Peter , Goldberger Jacob , Kalisky Tomer
Published: June 2022

Wilms’ tumors are pediatric malignancies that are thought to arise from faulty kidney development. To date, the course of treatment depends on manual histopathological classification which is difficult since the tumors differ between patients in a continuous manner. Here, we used three computational approaches to characterize the continuous heterogeneity of Wilms’ tumors. We first chose a published dataset of microarray gene expression measurements from high-risk blastemal-type Wilms’ tumors. Then, we used Pareto Task Inference to show that the tumors form a triangle-shaped continuum in latent space that is bounded by three tumor archetypes with “stromal”, “epithelial”, and “blastemal” characteristics, that resemble the un-induced mesenchyme, the Cap mesenchyme, and early epithelial structures of the fetal kidney. We confirmed this by fitting a generative probabilistic “grade of membership” model whereby each tumor is represented as a unique mixture of three hidden “topics” with blastemal, stromal, and epithelial characteristics. Finally, we used cellular deconvolution to show that each tumor is composed of a unique mixture of cell populations resembling the un-induced mesenchyme, the cap mesenchyme, and the early epithelial structures of the fetal kidney. We anticipate that these methodologies will pave the way for more quantitative strategies for tumor stratification and classification.
Interspecific competition can drive the loss of conjugative plasmids from a focal species in a microbial community
Authors: Walker-Sünderhauf David , Klümper Uli , Gaze William H , Westra Edze R , van Houte Stineke
Published: June 2022

Plasmids are key disseminators of antimicrobial resistance genes and virulence factors, and it is therefore critical to predict and reduce plasmid spread within microbial communities. The cost of plasmid carriage is a key metric that can be used to predict plasmids’ ecological fate, and it is unclear whether plasmid costs are affected by growth partners in a microbial community. We carried out competition experiments and tracked plasmid maintenance using a synthetic and stable 5-species community and a broad host-range plasmid as a model. We report that both the cost of plasmid carriage and its long-term maintenance in a focal strain depended on the presence of competitors, and that these interactions were species-specific. Addition of growth partners increased the plasmid cost to a focal strain, and accordingly plasmid loss from the focal species occurred over a shorter time frame in these species combinations. We propose that the destabilising effect of interspecific competition on plasmid maintenance may be leveraged in clinical and natural environments to cure plasmids from focal strains.