bluuebunny/crossref_metadata_2025 huggingface dataset:
Sex-Stratified Genome-Wide Association Study of Multisite Chronic Pain in UK Biobank
Authors: Johnston Keira JA , Ward Joey , Ray Pradipta R , Adams Mark J , McIntosh Andrew M , Smith Blair H , Strawbridge Rona J , Price Theodore J , Smith Daniel J , Nicholl Barbara I , Bailey Mark E.S
Published: June 2020

Chronic pain is highly prevalent worldwide and imparts significant socioeconomic and public health burden and is more prevalent in women than in men. Factors that influence susceptibility and mechanisms of chronic pain development, are not fully understood.

To investigate sex differences in chronic pain, we carried out a sex-stratified genome-wide association study of Multisite Chronic Pain (MCP), a derived chronic pain phenotype in UK Biobank. Genetic correlations between MCP in each sex and a range of psychiatric, autoimmune, and anthropometric phenotypes were examined. The relationship between female and male MCP, and chronic widespread pain was investigated using polygenic risk scoring. Expression of sex-specific MCP-associated loci in a range of tissues was examined using GTEx, and separately in neural and non-neural human tissues with assessment for dorsal-root ganglion (DRG) enrichment. For genes enriched for neural-tissue expression the full GTEx database was queried for sex-differential gene expression in CNS regions, and for high expression in sex-specific tissues. Expression in neural mouse tissue was also examined for orthologs of these genes.

A total of 123 SNPs at five independent loci were significantly associated with MCP in men. In women, a total of 286 genome-wide significant SNPs at ten independent loci were discovered. Meta-analysis of sex-stratified GWAS output found 87 independent SNPs to be significantly associated with MCP. We found sex-specific MCP-associated genes, with 31 genes and 37 genes associated with female and male MCP respectively and one gene associated with MCP in both sexes (DCC).

We found evidence for sex-specific pleiotropy and risk for MCP was found to be associated with chronic widespread pain in a sex-differential manner. Male and female MCP were highly genetically correlated, but at rgsignificantly less than 1. All 37 male MCP-associated genes and all but one of 31 female MCP-associated genes were found to be expressed in the DRG, and many showed elevated expression in sex-specific tissues.

Overall, findings indicate sex differences in chronic pain at the SNP, gene and transcriptomic level, and highlight possible sex-specific pleiotropy for MCP. Results support the proposition of a strong nervous-system component to chronic pain in both sexes, emphasise the importance of the DRG, and indicate specific loci which may play a specialised role in nociception.
Peptide ligands of the cardiac ryanodine receptor as super-resolution imaging probes
Authors: Sheard Thomas M. D. , Howlett Luke , Kirton Hannah , Yang Zhaokang , Gurrola Georgina , Steele Derek , Jayasinghe Izzy , Valdivia Hector H. , Colyer John
Published: June 2020

To study the structural basis of pathological remodelling and altered calcium channel functional states in the heart, we sought to re-purpose high-affinity ligands of the cardiac calcium channel, the ryanodine receptor (RyR2), into super-resolution imaging probes. Imperacalcin (IpCa), a scorpion toxin peptide which induces channel sub-conduction states, and DPc10, a synthetic peptide corresponding to a sequence of the RyR2, which replicates arrhythmogenic CPVT functional changes, were used in fluorescent imaging experiments.

Isolated adult rat ventricular cardiomyocytes were saponin-permeabilised and incubated with each peptide. IpCa-A546 became sequestered into the mitochondria. This was prevented by treatment of the permeabilised cells with the ionophore FCCP, revealing a striated staining pattern in confocal imaging which had weak colocalisation with RyR2 clusters. Poor specificity (as an RyR2 imaging probe) was confirmed at higher resolution with expansion microscopy (proExM) (~70 nm).

DPc10-FITC labelled a striated pattern, which had moderate colocalisation with RyR2 cluster labelling in confocal and proExM. There was also widespread non-target labelling of the Z-discs, intercalated discs, and nuclei, which was unaffected by incubation times or 10 mM caffeine. The inactive peptide mut-DPc10-FITC (which causes no functional effects) displayed a similar labelling pattern.

Significant labelling of structures unrelated to RyR2 by both peptide conjugates makes their use as highly specific imaging probes of RyR2 in living isolated cardiomyocytes highly challenging.

We investigated the native DPc10 sequence within the RyR2 structure to understand the domain interactions and proposed mechanism of peptide binding. The native DPc10 sequence does not directly interact with another domain, and but is downstream of one such domain interface. The rabbit Arg2475 (equivalent to human Arg2474, mutated in CPVT) in the native sequence is the most accessible portion and most likely location for peptide disturbance, suggesting FITC placement does not impact peptide binding.
Ag nanoparticles-based antimicrobial polycotton fabrics to prevent the transmission and spread of SARS-CoV-2
Authors: Tremiliosi Guilherme C. , Simoes Luiz Gustavo P. , Minozzi Daniel T. , Santos Renato I. , Vilela Daiane C. B. , Durigon Edison Luiz , Machado Rafael Rahal Guaragna , Medina Douglas Sales , Ribeiro Lara Kelly , Rosa Ieda Lucia Viana , Assis Marcelo , Andrés Juan , Longo Elson , Freitas-Junior Lucio H.
Published: June 2020

Pathogens (bacteria, fungus and virus) are becoming a potential threat to the health of human beings and environment worldwide. They widely exist in the environment, with characteristics of variety, spreading quickly and easily causing adverse reactions. In this work, an Ag-based material is used to be incorporated and functionalized in polycotton fabrics using pad-dry-cure method. This composite proved to be effective for inhibiting the SARS-CoV-2 virus, decreasing the number of replicates in 99.99% after an incubation period of 2 minutes. In addition, it caused 99.99% inhibition of the pathogensS. aureus,E. coliandC. albicans, preventing cross-infections and does not cause allergies or photoirritation processes, demonstrating the safety of its use.
p53 drives premature neuronal differentiation in response to radiation-induced DNA damage during early neurogenesis
Authors: Mfossa André-Claude Mbouombouo , Verslegers Mieke , Verreet Tine , Fida Haris bin , Mysara Mohamed , Van IJcken Wilfred F.J. , De Vos Winnok H. , Moons Lieve , Baatout Sarah , Benotmane Mohammed A. , Huylebroeck Danny , Quintens Roel
Published: June 2020

p53 regulates the cellular DNA damage response (DDR). Hyperactivation of p53 during embryonic development, however, can lead to a range of developmental defects including microcephaly. Here, we induce microcephaly by acute irradiation of mouse fetuses at the onset of neurogenesis. Besides a classical DDR culminating in massive apoptosis, we observe ectopic neurons in the subventricular zone in the brains of irradiated mice, indicative of premature neuronal differentiation. A transcriptomic study indicates that p53 activates both DDR genes and differentiation-associated genes. In line with this, mice with a targeted inactivation ofTrp53in the dorsal forebrain, do not show this ectopic phenotype and partially restore brain size after irradiation. Irradiation furthermore induces an epithelial-to-mesenchymal transition-like process resembling the radiation-induced proneural-mesenchymal transition in glioma and glioma stem-like cells. Our results demonstrate a critical role for p53 beyond the DDR as a regulator of neural progenitor cell fate in response to DNA damage.
Aberrant perceptual judgements on speech-relevant acoustic features in hallucination-prone individuals
Authors: Erb Julia , Kreitewolf Jens , Pinheiro Ana P. , Obleser Jonas
Published: June 2020

Hallucinations constitute an intriguing model of how percepts are generated and how perception can fail. Here, we investigate the hypothesis that an altered perceptual weighting of the spectro-temporal modulations that characterize speech contributes to the emergence of auditory verbal hallucinations. Healthy adults (N=168) varying in their predisposition for hallucinations had to choose the ‘more speech-like’ of two presented ambiguous sound textures and give a confidence judgement. Using psychophysical reverse correlation, we quantified the contribution of different acoustic features to a listener’s perceptual decisions. Higher hallucination proneness covaried with perceptual down-weighting of speech-typical, low-frequency acoustic energy while prioritising high frequencies. Remarkably, higher confidence judgements in single trials depended not only on acoustic evidence but also on an individual’s hallucination proneness and schizotypy score. In line with an account of altered perceptual priors and differential weighting of sensory evidence, these results show that hallucination-prone individuals exhibit qualitative and quantitative changes in their perception of the modulations typical for speech.

Author summary

Hallucinations -- that is, percepts in the absence of an external stimulus -- are prevalent in psychotic disorders such as schizophrenia, but also occur in the general population. To date it is unknown whether the emergence of hallucinations is rooted in an altered perception of sounds. Fusing the psychophysical technique of reverse correlation with concepts from computational psychiatry, this research reveals alterations of sensory processing in hallucination-prone adults. We show that the higher nonclinical adults’ predisposition to hallucinations, the more they prioritise the sound features atypical for speech such as higher frequencies. At the same time, they express higher confidence in their perceptual judgements. The present approach may contribute to improving early diagnosis and prevention strategies in individuals at risk for psychosis.
Gray matter covariations and core symptoms of autism. The EU-AIMS Longitudinal European Autism Project
Authors: Mei Ting , Llera Alberto , Floris Dorothea L. , Forde Natalie J. , Tillmann Julian , Durston Sarah , Moessnang Carolin , Banaschewski Tobias , Holt Rosemary J. , Baron-Cohen Simon , Rausch Annika , Loth Eva , Dell’Acqua Flavio , Charman Tony , Murphy Declan G. M. , Ecker Christine , Beckmann Christian F. , Buitelaar Jan K. ,   the EU-AIMS LEAP group
Published: June 2020

Background

Voxel-based Morphometry (VBM) studies in Autism Spectrum Disorder (autism) have yielded diverging results. This might partly be attributed to structural alterations being associating with the combined influence of several regions rather than with a single region. Further, these structural covariation differences may relate to continuous measures of autism rather than with categorical case-control contrasts. The current study aimed to identify structural covariation alterations in autism, and assessed canonical correlations between brain covariation patterns and core autism symptoms.

Methods

We studied 347 individuals with autism and 252 typically developing individuals, aged between 6 and 30 years, who have been deeply phenotyped in the Longitudinal European Autism Project (LEAP). All participants’ VBM maps were decomposed into spatially independent components using Independent Component Analysis. A Generalized Linear Model (GLM) was used to examine case-control differences. Next, Canonical Correlation Analysis (CCA) was performed to separately explore the integrated effects between all the brain sources of gray matter variation and two sets of core autism symptoms.

Results

GLM analyses showed significant case-control differences for two independent components. The first component was primarily associated with decreased density of bilateral insula, inferior frontal gyrus, orbitofrontal cortex, and increased density of caudate nucleus in the autism group relative to typically developing individuals. The second component was related to decreased densities of the bilateral amygdala, hippocampus, and parahippocampal gyrus in the autism group relative to typically developing individuals. The CCA results showed significant correlations between components that involved variation of thalamus, putamen, precentral gyrus, frontal, parietal, and occipital lobes, and the cerebellum, and repetitive, rigid and stereotyped behaviors and abnormal sensory behaviors in autism individuals.

Limitations

Only 55.9% of the participants with autism had complete questionnaire data on continuous parent-reported symptom measures.

Conclusions

Covaried areas associated with autism diagnosis and/or symptoms are scattered across the whole brain and include the limbic system, basal ganglia, thalamus, cerebellum, precentral gyrus, and parts of the frontal, parietal, and occipital lobes. Some of these areas potentially subserve social-communicative behavior whereas others may underpin sensory processing and integration, and motor behavior.
A scalable device-less biomaterial approach for subcutaneous islet transplantation
Authors: Vlahos Alexander E. , Talior-Volodarsky Ilana , Kinney Sean M. , Sefton Michael V.
Published: June 2020

The subcutaneous space has been shown to be a suitable site for islet transplantation, however an abundance of islets is required to achieve normoglycemia, often requiring multiple donors. The loss of islets is due to the hypoxic conditions islets experience during revascularization, resulting in apoptosis. Therefore, to reduce the therapeutic dosage required to achieve normoglycemia, pre-vascularization of the subcutaneous space has been pursued. In this study, we highlight a biomaterial-based approach using a methacrylic acid copolymer coating to generate a robust pre-vascularized subcutaneous cavity for islet transplantation. We also devised a simple, but not-trivial, procedure for filling the cavity with an islet suspension in collagen. We show that the pre-vascularized site can support a marginal mass of islets to rapidly return streptozotocin-induced diabetic SCID/bg mice to normoglycemia. Furthermore, immunocompetent Sprague Daley rats remained normoglycemia for up to 70 days until they experienced graft destabilization as they outgrew their implants. This work highlights methacrylic acid-based biomaterials as a suitable pre-vascularization strategy for the subcutaneous space that is scalable and doesn’t require exogenous cells or growth factors.

In this study methacrylic acid copolymer coated tubes generated a robust vascular response in the subcutaneous space, which was critical to support islet transplantation in a streptozotocin-induced diabetic mouse model. More importantly, the subcutaneous pre-vascularization approach using this copolymer coating was scalable into a larger allogeneic rat model and returned animals to normoglycemia for up to 70 days. This platform highlights the potential of a scalable biomaterial approach for pre-vascularization of the subcutaneous space in larger animal models.
A neurogenetic mechanism of experience-dependent suppression of aggression
Authors: Ishii Kenichi , Cortese Matteo , Leng Xubo , Shokhirev Maxim N. , Asahina Kenta
Published: June 2020

Aggression is an ethologically important social behavior1but excessive aggression can be detrimental to animal fitness2,3. Social experiences among conspecific individuals reduce aggression in a wide range of animals4. However, the genetic and neural basis for the experience-dependent suppression of aggression remains largely unknown. Here we found thatnervy(nvy), aDrosophilahomolog of vertebrate myeloid translocation gene (MTG)5involved in transcriptional regulation6–8, suppresses aggression via its action in a specific subset of neurons. Loss-of-function mutation of thenvygene resulted in hyper-aggressiveness only in socially experienced flies, whereas overexpression ofnvysuppressed spontaneous aggression in socially naïve flies. The loss-of-functionnvymutant exhibited persistent aggression under various contexts in which wild-type flies transition to escape or courtship behaviors. Knockdown ofnvyin octopaminergic/tyraminergic (OA/TA) neurons increased aggression, phenocopying thenvymutation. We found that a subpopulation of OA/TA cells specifically labeled bynvyis required for the social-experience-dependent suppression of aggression. Moreover, cell-type-specific transcriptomics onnvy-expressing OA/TA neurons revealed aggression-controlling genes that are likely downstream ofnvy. Our results are the first to describe the presence of a specific neuronal subpopulation in the central brain that actively suppresses aggression in a social-experience-dependent manner, illuminating the underlying genetic mechanism.
Age and genetic background modify hybrid male sterility in house mice
Authors: Widmayer Samuel J. , Handel Mary Ann , Aylor David L.
Published: June 2020

Hybrid male sterility (HMS) contributes to reproductive isolation commonly observed among house mouse(Mus musculus)subspecies, both in the wild and in laboratory crosses. Incompatibilities involving specificPrdm9alleles and certain Chromosome (Chr) X genotypes are known determinants of fertility and HMS, and previous work in the field has demonstrated that genetic background modifies these two major loci. We constructed hybrids that have identical genotypes atPrdm9and identical X chromosomes, but differ widely across the rest of the genome. In each case, we crossed female PWK/PhJ mice representative of theM. m. musculussubspecies to males from a classical inbred strain representative ofM. m. domesticus:129S1/SvImJ, A/J, C57BL/6J, or DBA/2J. We detected three distinct trajectories of fertility among the hybrids using breeding experiments. The PWK129S1 males were always infertile. PWKDBA2 males were fertile, despite their genotypes at the major HMS loci. We also observed age-dependent changes in fertility parameters across multiple genetic backgrounds. The PWKB6 and PWKAJ males were always infertile before 15 weeks and after 35 weeks, yet some PWKB6 and PWKAJ males were fertile between fifteen and 35 weeks. This observation could resolve previous contradictory reports about the fertility of PWKB6. Taken together, these results point to multiple segregating HMS modifier alleles, some of which have age-related modes of action. The ultimate identification of these alleles and their age-related mechanisms will advance understanding both of the genetic architecture of HMS and of how reproductive barriers are maintained between house mouse subspecies.
Dynamics of a neuronal pacemaker in the weakly electric fish Apteronotus
Authors: Shifman Aaron R. , Sun Yiren , Benoit Chloé M. , Lewis John E.
Published: June 2020

The precise timing of neuronal activity is critical for normal brain function. In weakly electric fish, the medullary pacemaker network (PN) sets the timing for an oscillating electric organ discharge (EOD) used for electric sensing. This network is the most precise biological oscillator known, with sub-microsecond variation in oscillator period. The PN consists of two principle sets of neurons, pacemaker and relay cells, that are connected by gap junctions and normally fire in synchrony, one-to-one with each EOD cycle. However, the degree of gap junctional connectivity between these cells appears insufficient to provide the population averaging required for the observed temporal precision of the EOD. This has led to the hypothesis that individual cells themselves fire with high precision, but little is known about the oscillatory dynamics of these pacemaker cells. To this end, we have developed a biophysical model of a pacemaker neuron action potential based on experimental recordings. We validated the model by comparing the changes in oscillatory dynamics produced by different experimental manipulations. Our results suggest that a relatively simple model captures the complex dynamics exhibited by pacemaker cells, and that these dynamics may enhance network synchrony and precision.

Author summary

Many neural networks in the brain exhibit activity patterns which oscillate regularly in time. These oscillations, like a clock, can provide a precise sense of time, enabling drummers to maintain complex beat patterns and pets to anticipate “feeding time”. The exact mechanisms by which brain networks give rise to these biological clocks are not clear. The pacemaker network of weakly electric fish has the highest precision of all known biological clocks. In this study, we develop a detailed biophysical model of neurons in the pacemaker network. We then validate the model against experiments using a nonlinear dynamics approach. Our results show that pacemaker precision is due, at least in part, to how individual pacemaker cells generate their activity. This supports the idea that temporal precision in this network is not solely an emergent property of the network but also relies on the dynamics of individual neurons.
Capturing heterogeneous infectiousness in transmission dynamic models of tuberculosis: a compartmental modelling approach
Authors: Melsew Yayehirad A , Ragonnet Romain , Cheng Allen C , McBryde Emma S , Trauer James M
Published: June 2020

Infectiousness heterogeneity among individuals with tuberculosis (TB) is substantial and is likely to have a significant impact on the long-term dynamics of TB and the effectiveness of interventions. However, there is a gap in capturing heterogeneous infectiousness and evaluating its impact on the effectiveness of interventions.

Informed by observed distribution of secondary infections, we constructed a deterministic model of TB transmission using ordinary differential equations. The model incorporated assumption of heterogeneous infectiousness with three levels of infectivity, namely non-spreaders, low-spreaders and super-spreaders. We evaluated the effectiveness of dynamic transmission untargeted and targeted implementation of an intervention intended to represent active case finding with a point-of-care diagnostic tool. The simulated intervention detected 20% of all TB patients who would otherwise have been missed by the health system during their disease episode and was compared across four epidemiological scenarios.

Our model suggested that targeting the active case finding intervention towards super-spreaders was more effective than untargeted intervention in all setting scenarios, with more effectiveness in settings with low case detection and high transmission intensity. For instance, a targeted intervention achieved a 42.2% reduction in TB incidence, while the untargeted intervention achieved only a 20.7% reduction over 20 years, given the same number of people treated. Although the most marked impact on equilibrium TB incidence came from the rate of late reactivation, the proportion of super-spreaders and their relative infectiousness had shown substantial impact.

Targeting active case-finding interventions to highly infectious cases likely to be particularly beneficial in settings where case detection is poor. Heterogeneity-related parameters had an equivalent effect to several other parameters that have been established as being very important to TB transmission dynamics.
CRISPR-induced indels and base editing using theStaphylococcus aureusCas9 in potato
Authors: Veillet Florian , Kermarrec Marie-Paule , Chauvin Laura , Chauvin Jean-Eric , Nogué Fabien
Published: June 2020

Genome editing is now widely used in plant science for both fundamental research and molecular crop breeding. The clustered regularly interspaced short palindromic repeats (CRISPR) technology, through its precision, high efficiency and versatility, allows to edit many sites in plant genomes. This system has been highly successful to produce of knock-out mutants through the introduction of frameshift mutations due to error-prone repair pathways. Nevertheless, recent new CRISPR-based technologies such as base editing and prime editing can generate precise and on request nucleotide conversion, allowing to fine-tune protein function and generate gain-of-function mutants. However, genome editing through CRISPR systems still have some drawbacks and limitations, such as the PAM restriction and the need for more diversity in CRISPR tools to simultaneously mediate different catalytic activities. In this study, we successfully used the CRISPR-Cas9 system fromStaphylococcus aureus(SaCas9) for the introduction of frameshift mutations in the tetraploid genome of the cultivated potato (Solanum tuberosum). We also developed aS. aureus-cytosine base editor that mediate nucleotide conversions, allowing to precisely modify specific residues or regulatory elements in potato. Our proof-of-concept results in potato expand the plant dicot CRISPR toolbox for biotechnology and precision breeding applications.
Air and surface measurements of SARS-CoV-2 inside a bus during normal operation
Authors: Di Carlo Piero , Chiacchiaretta Piero , Sinjari Bruna , Aruffo Eleonora , Stuppia Liborio , De Laurenzi Vincenzo , Di Tomo Pamela , Pelusi Letizia , Potenza Francesca , Veronese Angelo , Vecchiet Jacopo , Falasca Katia , Ucciferri Claudio
Published: June 2020

Transmission pathways of SARS-CoV-2 are through aerosol, droplet and touching infected material. Indoor locations are more likely environments for the diffusion of the virus contagion among people, but direct detection of SARS-CoV-2 in air or on surfaces is quite sparse, especially regarding public transport. In fact, an important demand is to know how and if it is safe to use them. To understand the possible spreading of COVID-19 inside a city bus during normal operation and the effectiveness of the protective measures adopted for transportation, we analysed the air and the surfaces most usually touched by passengers. The measurements were carried out across the last week of the lockdown and the first week when gradually all the travel restrictions were removed.
Validation of the Social Responsiveness Scale (SRS) to screen for atypical social behaviors in juvenile macaques
Authors: Balint Z. Kovacs , Raper J. , Michopoulos V. , Howell L.H. , Gunter C. , Bachevalier J. , Sanchez M.M.
Published: June 2020

Primates form strong social bonds and depend on social relationships and networks that provide shared resources and protection critical for survival. Social deficits such as those present in autism spectrum disorder (ASD) and other psychiatric disorders hinder the individual’s functioning in communities. Given that early diagnosis and intervention can improve outcomes and trajectories of ASD, there is a great need for tools to identify early markers for screening/diagnosis, and for translational animal models to uncover biological mechanisms and develop treatments. One of the most widely used screening tools for ASD in children is the Social Responsiveness Scale (SRS), a quantitative measure used to identify individuals with atypical social behaviors. The SRS has been adapted for use in adult rhesus monkeys (Macaca mulatta) –a species very close to humans in terms of social behavior, brain anatomy/connectivity and development– but has not yet been validated or adapted for a necessary downward extension to younger ages matching those for ASD diagnosis in children. The goal of the present study was to adapt and validate the adult macaque SRS (mSRS) in juvenile macaques with age equivalent to 4-6 yr old human children. Expert primate coders modified the mSRS to adapt it to rate atypical social behaviors in juvenile macaques living in complex social groups at the Yerkes National Primate Research Center. Construct and face validity of this juvenile mSRS (jmSRS) was determined based on well-established and operationalized measures of social and non-social behaviors in this species using traditional behavioral observations. We found that the jmSRS identifies variability in social responsiveness of juvenile rhesus monkeys and shows strong construct/predictive validity, as well as sensitivity to detect atypical social behaviors in young male and female macaques across social status. Thus, the jmSRS provides a promising tool for translational research on macaque models of children social disorders.
Inferring cellular trajectories from scRNA-seq using Pseudocell Tracer
Authors: Reiman Derek , Xu Heping , Sonin Andrew , Chen Dianyu , Singh Harinder , Khan Aly A.
Published: June 2020

Single cell RNA sequencing (scRNA-seq) can be used to infer a temporal ordering of dynamic cellular states. Current methods for the inference of cellular trajectories rely on unbiased dimensionality reduction techniques. However, such biologically agnostic ordering can prove difficult for modeling complex developmental or differentiation processes. The cellular heterogeneity of dynamic biological compartments can result in sparse sampling of key intermediate cell states. This scenario is especially pronounced in dynamic immune responses of innate and adaptive immune cells. To overcome these limitations, we develop a supervised machine learning framework, called Pseudocell Tracer, which infers trajectories in pseudospace rather than in pseudotime. The method uses a supervised encoder, trained with adjacent biological information, to project scRNA-seq data into a low-dimensional cellular state space. Then a generative adversarial network (GAN) is used to simulate pesudocells at regular intervals along a virtual cell-state axis. We demonstrate the utility of Pseudocell Tracer by modeling B cells undergoing immunoglobulin class switch recombination (CSR) during a prototypic antigen-induced antibody response. Our results reveal an ordering of key transcription factors regulating CSR, including the concomitant induction ofNfkb1andStat6prior to the upregulation ofBach2expression. Furthermore, the expression dynamics of genes encoding cytokine receptors point to the existence of a regulatory mechanism that reinforces IL-4 signaling to direct CSR to the IgG1 isotype.
Epigenetic regulations follow cell cycle progression during differentiation of human pluripotent stem cells
Authors: Madrigal Pedro , Pauklin Siim , Goh Kim Jee , Grandy Rodrigo , Osnato Anna , Ortmann Daniel , Brown Stephanie , Vallier Ludovic
Published: June 2020

Most mammalian stem cells undergo cellular division during their differentiation to produce daughter cells with a new cellular identity. However, the cascade of epigenetic events and molecular mechanisms occurring between successive cell divisions upon differentiation have not yet been described in detail due to technical limitations. Here, we address this question by taking advantage of the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) reporter to develop a culture system allowing the differentiation of human Embryonic Stem Cells (hESCs) synchronised for their cell cycle. Using this approach, we have assessed the epigenome and transcriptome dynamics during the first two divisions leading to definitive endoderm. We first observed that transcription of key markers of differentiation occurs before division suggesting that differentiation is initiated during the progression of cell cycle. Furthermore, ATAC-seq shows a major decrease in chromatin accessibility after pluripotency exit indicating that the first event of differentiation is the inhibition of alternative cell fate. In addition, using digital genomic footprinting we identified novel cell cycle-specific transcription factors with regulatory potential in endoderm specification. Of particular interest, Activator protein 1 (AP-1) controlled p38/MAPK signalling seems to be necessary for blocking endoderm shifting cell fate toward mesoderm lineage. Finally, histone modifications analyses suggest a temporal order between different marks. We can also conclude that enhancers are dynamically and rapidly established / decommissioned between different cell cycle upon differentiation. Overall, these data not only reveal key the successive interplays between epigenetic modifications during differentiation but also provide a valuable resource to investigate novel mechanisms in germ layer specification.
Charge Interactions in a Highly Charge-depleted Protein
Authors: Hervø-Hansen Stefan , Højgaard Casper , Johansson Kristoffer Enøe , Wang Yong , Wahni Khadija , Young David , Messens Joris , Teilum Kaare , Lindorff-Larsen Kresten , Winther Jakob Rahr
Published: June 2020

Interactions between charged residues are difficult to study because of the complex network of interactions found in most proteins. We have designed a purposely simple system to investigate this problem by systematically introducing individual and pairs of charged and titratable residues in a protein otherwise free of such residues. We used constant pH molecular dynamics simulations, NMR spectroscopy, and thermodynamic double mutant cycles to probe the structure and energetics of the interaction between the charged residues. We found that the partial burial of surface charges contributes to a shift in pKavalue, causing an aspartate to titrate in the neutral pH range. Additionally, the interaction between pairs of residues was found to be highly context dependent, with some pairs having no apparent preferential interaction, while other pairs would engage in coupled titration forming a highly stabilized salt bridge. We find good agreement between experiments and simulations, and use the simulations to rationalize our observations and to provide a detailed mechanistic understanding of the electrostatic interactions.

Significance

Electrostatic forces are important for protein folding and are favored targets of protein engineering. However, despite the many advances in the field of protein electrostatics, the prediction of changes in protein structure and function upon introduction or removal of titratable residues is still complicated. In order to provide a basic understanding of protein electrostatics we here characterize a highly charge-depleted protein and its titratable variants by a combination of NMR spectroscopy and constant pH molecular dynamics simulations. Our investigations reveal how strongly interacting residues engaged in salt bridging, can be characterized. Furthermore, our study may also enrich and facilitate the understanding of dehydration of salt-bridges and its potential effect on protein stability.
Illuminating a Phytochrome Paradigm – a Light-Activated Phosphatase in Two-Component Signaling Uncovered
Authors: Multamäki Elina , Nanekar Rahul , Morozov Dmitry , Lievonen Topias , Golonka David , Yuan Wahlgren Weixiao , Stucki-Buchli Brigitte , Rossi Jari , Hytönen Vesa P. , Westenhoff Sebastian , Ihalainen Janne A. , Möglich Andreas , Takala Heikki
Published: June 2020

Bacterial phytochrome photoreceptors usually belong to two-component signaling systems which transmit environmental stimuli to a response regulator through a histidine kinase domain. Phytochromes switch between red light-absorbing and far-red light-absorbing states. Despite exhibiting extensive structural responses during this transition, the model bacteriophytochrome fromDeinococcus radiodurans(DrBphP) lacks detectable kinase activity. Here, we resolve this long-standing conundrum by comparatively analyzing the interactions and output activities of DrBphP and a bacteriophytochrome fromAgrobacterium fabrum(AgP1). Whereas AgP1 acts as a conventional histidine kinase, we identify DrBphP as a light-sensitive phosphatase. While AgP1 binds its cognate response regulator only transiently, DrBphP does so strongly, which is rationalized at the structural level. Our data pinpoint two key residues affecting the balance between kinase and phosphatase activities, which immediately bears on photoreception and two-component signaling. The opposing output activities in two highly similar bacteriophytochromes inform the use of light-controllable histidine kinases and phosphatases for optogenetics.
Single-cell-resolved dynamics of chromatin architecture delineate cell and regulatory states in wildtype andcloche/npas4lmutant zebrafish embryos
Authors: McGarvey Alison C. , Kopp Wolfgang , Vučićević Dubravka , Kempfer Rieke , Mattonet Kenny , Hirsekorn Antje , Bilić Ilija , Trinks Alexandra , Merks Anne Margarete , Panáková Daniela , Pombo Ana , Akalin Altuna , Junker Jan Philipp , Stainier Didier Y.R. , Garfield David , Ohler Uwe , Lacadie Scott Allen
Published: June 2020

DNA accessibility of cis regulatory elements (CREs) dictates transcriptional activity and drives cell differentiation during development. While many of the genes that regulate embryonic development have been described, the underlying CRE dynamics controlling their expression remain largely unknown. To address this, we applied single-cell combinatorial indexing ATAC-seq (sci-ATAC-seq) to whole 24 hours post fertilization (hpf) stage zebrafish embryos and developed a new computational tool, ScregSeg, that selects informative genome segments and classifies complex accessibility dynamics. We integrated the ScregSeg output with bulk measurements for histone post-translational modifications and 3D genome organization, expanding knowledge of regulatory principles between chromatin modalities. Sci-ATAC-seq profiling ofnpas4l/clochemutant embryos revealed novel cellular roles for this hemato-vascular transcriptional master regulator and suggests an intricate mechanism regulating its expression. Our work constitutes a valuable resource for future studies in developmental, molecular, and computational biology.
In situ structural analysis of SARS-CoV-2 spike reveals flexibility mediated by three hinges
Authors: Turoňová Beata , Sikora Mateusz , Schürmann Christoph , Hagen Wim J. H. , Welsch Sonja , Blanc Florian E. C. , von Bülow Sören , Gecht Michael , Bagola Katrin , Hörner Cindy , van Zandbergen Ger , Mosalaganti Shyamal , Schwarz Andre , Covino Roberto , Mühlebach Michael D. , Hummer Gerhard , Locker Jacomine Krijnse , Beck Martin
Published: June 2020

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the major focus for vaccine development. We combine cryo electron tomography, subtomogram averaging and molecular dynamics simulations to structurally analyze Sin situ. Compared to recombinant S, the viral S is more heavily glycosylated and occurs predominantly in a closed pre-fusion conformation. We show that the stalk domain of S contains three hinges that give the globular domain unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and the development of safe vaccines. The large scale tomography data set of SARS-CoV-2 used for this study is therefore sufficient to resolve structural features to below 5 Ångstrom, and is publicly available at EMPIAR-10453.
Berberine reverses multidrug resistance in Candida albicans by hijacking the drug efflux pump Mdr1p
Authors: Tong Yaojun , Sun Nuo , Wang Xiangming , Wei Qi , Zhang Yu , Huang Ren , Pu Yingying , Dai Huanqin , Ren Biao , Pei Gang , Song Fuhang , Zhu Guoliang , Wang Xinye , Xia Xuekui , Chen Xiangyin , Jiang Lan , Zhang Jingyu , Ouyang Liming , Zhang Buchang , Jiang Yuanying , Liu Xueting , Calderone Richard , Bai Fan , Zhang Lixin , Alterovitz Gil
Published: June 2020

Clinical use of antimicrobials faces great challenges from the emergence of multidrug resistant (MDR) pathogens. The overexpression of drug efflux pumps is one of the major contributors to MDR. It is considered as a promising approach to overcome MDR by reversing the function of drug efflux pumps. In the life-threatening fungal pathogenCandida albicans, the major facilitator superfamily (MFS) transporter Mdr1p can excrete many structurally unrelated antifungals, leading to multidrug resistance. Here we report a counterintuitive case of reversing multidrug resistance inC. albicansby using a natural product berberine to hijack the overexpressed Mdr1p for its own importation. Moreover, we illustrate that the imported berberine accumulates in mitochondria, and compromises the mitochondrial function by impairing mitochondrial membrane potential and mitochondrial Complex I. It results in the selective elimination of Mdr1p overexpressedC. albicanscells. Furthermore, we show that berberine treatment can prolong the mean survival time (MST) of mice with a blood-borne dissemination of Mdr1p overexpressed multidrug resistant candidiasis. This study provided a potential direction of novel anti-MDR drug discovery by screening for multidrug efflux pump converters.
Requirement for PKC Epsilon in Kras-Driven Lung Tumorigenesis
Authors: Garg Rachana , Cooke Mariana , Wang Shaofei , Benavides Fernando , Abba Martin C. , Cicchini Michelle , Feldser David M. , Kazanietz Marcelo G.
Published: June 2020

Non-small cell lung cancer (NSCLC), the most frequent subtype of lung cancer, remains a highly lethal malignancy and one of the leading causes of cancer deaths worldwide. Mutant KRAS is the prevailing oncogenic driver of lung adenocarcinoma, the most common histological form of NSCLC. In this study, we examined the role of PKCε, an oncogenic kinase highly expressed in NSCLC and other cancers, in KRAS-driven tumorigenesis. Notably, database analysis revealed an association between PKCε expression and poor outcome in lung adenocarcinoma patients specifically having KRAS mutation. By generating a PKCε-deficient, conditionally activatable allele of oncogenicKras(LSL-KrasG12D;PKCε−/−mice) we were able to demonstrate the requirement of PKCε forKras-driven lung tumorigenesisin vivo, which is consistent with the impaired transformed growth observed in PKCε-deficient KRAS-dependent NSCLC cells. Moreover, PKCε-knockout mice were found to be less susceptible to lung tumorigenesis induced by benzo[a]pyrene, a carcinogen that induces mutations inKras. Mechanistic analysis using RNA-Seq revealed little overlapping for PKCε and KRAS in the control of genes/biological pathways relevant in NSCLC, suggesting that a permissive role of PKCε inKRAS-driven lung tumorigenesis may involve non-redundant mechanisms. Our results thus highlight the relevance and potential of targeting PKCε for lung cancer therapeutics.
Modification of cell wall polysaccharide spatially controls cell division inStreptococcus mutans
Authors: Zamakhaeva Svetlana , Chaton Catherine T. , Rush Jeffrey S. , Castro Sowmya Ajay , Yarawsky Alexander E. , Herr Andrew B. , van Sorge Nina M. , Dorfmueller Helge C. , Frolenkov Gregory I. , Korotkov Konstantin V. , Korotkova Natalia
Published: June 2020

Bacterial cell division is driven by a tubulin homolog FtsZ, which assembles into the Z-ring structure leading to the recruitment of the cell division machinery. In ovoid-shaped Gram-positive bacteria, such as streptococci, MapZ guides Z-ring positioning at cell equators through an, as yet, unknown mechanism. The cell wall of the important dental pathogenStreptococcus mutansis composed of peptidoglycan decorated with SerotypecCarbohydrates (SCCs). Here, we show that an immature form of SCC, lacking the recently identified glycerol phosphate (GroP) modification, coordinates Z-ring positioning. Pulldown assays usingS. mutanscell wall combined with binding affinity analysis identified the major cell separation autolysin, AtlA, as an SCC binding protein. Importantly, AtlA binding to mature SCC is attenuated due to GroP modification. Using fluorescently-labeled AtlA, we mapped SCC distribution on the streptococcal surface to reveal that GroP-deficient immature SCCs are exclusively present at the cell poles and equators. Moreover, the equatorial GroP-deficient SCCs co-localize with MapZ throughout theS. mutanscell cycle. Consequently, in GroP-deficient mutant bacteria, proper AtlA localization is abrogated resulting in dysregulated cellular autolysis. In addition, these mutants display morphological abnormalities associated with MapZ mislocalization leading to Z-ring misplacement. Altogether, our data support a model in which GroP-deficient immature SCCs spatially coordinate the localization of AtlA and MapZ. This mechanism ensures cell separation by AtlA at poles and Z-ring alignment with the cell equator.

Graphical abstract
The structure and diversity of strain level variation in vaginal bacteria
Authors: Tortelli Brett A. , Lewis Amanda L. , Fay Justin C.
Published: June 2020

The vaginal microbiome plays an important role in human health and species of vaginal bacteria have been associated with reproductive disease. Strain level variation is also thought to be important, but the diversity, structure and evolutionary history of vaginal strains is not as well characterized. We developed and validated an approach to measure strain variation from metagenomic data based on SNPs within the core-genomes for six species of vaginal bacteria:G. vaginalis,L. crispatus,L. iners,L. jensenii,L. gasseri, andA. vaginae. Despite inhabiting the same environment, strain diversity and structure varies across species. All species exceptL. inersare characterized by multiple distinct groups of strains. Even so, strain diversity is lower in theLactobacillusspecies, consistent with a more recent colonization of the human vaginal microbiome. Both strain diversity and the frequency of multi-strain samples is related to species-level diversity of the microbiome in which they occur, suggesting similar ecological factors influencing diversity within the vaginal niche. We conclude that the structure of strain level variation provides both the motivation and means of testing whether strain level differences contribute to the function and health consequences of the vaginal microbiome.

Data Summary

All vaginal metagenomic sequence data generated for this project can be found on the Sequence Read Archive under BioProject PRJNA639592.
Phantom histories of misspecified pasts
Authors: Platt Alexander , Harris Daniel N.
Published: June 2020

Abstract

The observation that even a tiny sample of genome sequences from a natural population contains a plethora of information about the history of the population has enticed researchers to use these data to fit complex demographic histories and make detailed inference about the changes a population has experienced through time. Unfortunately, the standard assumptions required to make these inferences are often violated by natural populations in such ways as to produce specious results. This paper examines two phenomena of particular concern: when a sample is drawn from a single sub-population of a larger meta-population these models infer a spurious recent population decline, and when a genome contains loci under weak or recessive purifying selection these models infer a spurious recent population expansion.
Non-canonical activation of the ER stress sensor ATF6 byLegionella pneumophilaeffectors
Authors: Ibe Nnejiuwa U. , Mukherjee Shaeri
Published: June 2020

The intracellular bacterial pathogenLegionella pneumophila(L.p.) secretes ~330 effector proteins into the host cell to sculpt an Endoplasmic Reticulum (ER)-derived replicative niche. We previously reported fiveL.p.effectors that inhibit IRE1, a key sensor of the homeostatic unfolded protein response (UPR) pathway. In this study, we discovered a subset ofL.p.toxins that selectively activate the UPR sensor ATF6, resulting in its cleavage, nuclear translocation and target gene transcription without affecting other UPR sensors such as PERK. In a deviation from the conventional model, thisL.p. dependent activation of ATF6 does not require its transport to the Golgi or its cleavage by the S1P/S2P proteases. We believe that our findings highlight the unique regulatory control thatL.p. exerts upon the three UPR sensors and expand the repertoire of bacterial proteins that selectively perturb host homeostatic pathways.