bluuebunny/crossref_metadata_2025 huggingface dataset:
Directed evolution reveals the mechanism of HitRS signal transduction inBacillus anthracis
Authors: Pi Hualiang , Chu Michelle L. , Ivan Samuel J. , Latario Casey J. , Toth Allen M. , Carlin Sophia M. , Hillebrand Gideon H. , Lin Hannah K. , Reppart Jared D. , Stauff Devin L. , Skaar Eric P.
Published: July 2020

Bacterial twocomponentsystems (TCSs) have been studied for decades; however, most work has focused on individual domains or proteins. Systematic characterization of an entire TCS could provide a mechanistic understanding of these important signal transduction systems. Here, genetic selections were employed to dissect the molecular basis of signal transduction by the HitRS system that has been implicated in detecting cell envelope stress in the pathogenBacillus anthracis. Numerous point mutations were isolated within HitRS, 17 of which were in a 50-residue HAMP domain. Mutational analysis revealed the importance of hydrophobic interactions within the HAMP domain and highlighted its essentiality in TCS signaling. In addition, these data defined residues critical for activities intrinsic to HitRS, uncovered specific interactions among individual domains and between the two signaling proteins, and revealed that phosphotransfer is the rate-limiting step for signal transduction. This study establishes the use of unbiased genetic selections to study TCS signaling, provides a comprehensive mechanistic understanding of an entire TCS, and lays the foundation for development of novel antimicrobial therapeutics against this important infectious threat.
Gradual Compaction of the Nascent Peptide During Cotranslational Folding on the Ribosome
Authors: Liutkute Marija , Maiti Manisankar , Samatova Ekaterina , Enderlein Jörg , Rodnina Marina V.
Published: July 2020

Nascent polypeptides begin to fold in the constrained space of the ribosomal peptide exit tunnel. Here we use force profile analysis (FPA) and photo-induced energy-transfer fluorescence correlation spectroscopy (PET-FCS) to show how a small α-helical domain, the N-terminal domain of HemK, folds cotranslationally. Compaction starts vectorially as soon as the first α-helical segments are synthesized. As nascent chain grows, emerging helical segments dock onto each other and continue to rearrange at the vicinity of the ribosome. Inside or in the proximity of the ribosome, the nascent peptide undergoes structural fluctuations on the μs time scale. The fluctuations slow down as the domain moves away from the ribosome. Folding mutations have little effect on folding within the exit tunnel, but abolish the final domain stabilization. The results show the power of FPA and PET-FCS in solving the trajectory of cotranslational protein folding and in characterizing the dynamic properties of folding intermediates.
A self-regenerating synthetic cell model
Authors: Lavickova Barbora , Laohakunakorn Nadanai , Maerkl Sebastian J.
Published: July 2020

Self-regeneration is a fundamental function of all living systems. Here we demonstrate molecular self-regeneration in a synthetic cell model. By implementing a minimal transcription-translation system within microfluidic reactors, the system was able to regenerate essential protein components from DNA templates and sustained synthesis activity for over a day. By mapping genotype-phenotype landscapes combined with computational modeling we found that minimizing resource competition and optimizing resource allocation are both critically important for achieving robust system function. With this understanding, we achieved simultaneous regeneration of multiple proteins by determining the required DNA ratios necessary for sustained self-regeneration. This work introduces a conceptual and experimental framework for the development of a self-replicating synthetic cell.
Modelling the interplay between the CD4+/CD8+T-cell ratio and the expression of MHC-I in tumours
Authors: Hurry Christian John , Mozeika Alexander , Annibale Alessia
Published: July 2020

Describing the anti-tumour immune response as a series of cellular kinetic reactions from known immunological mechanisms, we create a mathematical model that shows the CD4+/CD8+T-cell ratio, T-cell infiltration and the expression of MHC-I to be interacting factors in tumour elimination. Methods from dynamical systems theory and non-equilibrium statistical mechanics are used to model the T-cell dependent anti-tumour immune response. Our model predicts a critical level of MHC-I expression which determines whether or not the tumour escapes the immune response. This critical level of MHC-I depends on the helper/cytotoxic T-cell ratio. However, our model also suggests that the immune system is robust against small changes in this ratio. We also find that T-cell infiltration and the specificity of the intra-tumour TCR repertoire will affect the critical MHC-I expression. Our work suggests that the functional form of the time evolution of MHC-I expression may explain the qualitative behaviour of tumour growth seen in patients.

Mathematics Subject Classification (2010)

MSC 37C25 · MSC 82C99 · MSC 37N25 · MSC 92B99
The role of population structure in computations through neural dynamics
Authors: Dubreuil Alexis , Valente Adrian , Beiran Manuel , Mastrogiuseppe Francesca , Ostojic Srdjan
Published: July 2020

Neural computations are currently investigated using two separate approaches: sorting neurons into functional populations, or examining the low-dimensional dynamics of collective activity. Whether and how these two aspects interact to shape computations is currently unclear. Using a novel approach to extract computational mechanisms from networks trained on neuroscience tasks, here we show that the dimensionality of the dynamics and cell-class structure play fundamentally complementary roles. While various tasks can be implemented by increasing the dimensionality in networks with fully random population structure, flexible input-output mappings instead required a non-random population structure that can be described in terms of multiple sub-populations. Our analyses revealed that such a population structure enabled flexible computations through a mechanism based on gain-controlled modulations that flexibly shape the dynamical landscape of collective dynamics. Our results lead to task-specific predictions for the structure of neural selectivity, inactivation experiments, and for the implication of different neurons in multi-tasking.
Plasmodium bergheikinesin-5 associates with the spindle apparatus during cell division and is important for efficient production of infectious sporozoites
Authors: Zeeshan Mohammad , Brady Declan , Stanway Rebecca R. , Moores Carolyn A. , Holder Anthony A. , Tewari Rita
Published: July 2020

Kinesin-5 motors play essential roles in spindle apparatus assembly during cell division, by generating forces to establish and maintain the spindle bipolarity essential for proper chromosome segregation. Kinesin-5 is largely conserved structurally and functionally in model eukaryotes, but its role is unknown in thePlasmodiumparasite, an evolutionarily divergent organism with several atypical features of both mitotic and meiotic cell division. We have investigated the function and subcellular location of kinesin-5 during cell division throughout thePlasmodium bergheilife cycle. Deletion ofkinesin-5had little visible effect at any proliferative stage except sporozoite production in oocysts, resulting in a significant decrease in the number of motile sporozoites in mosquito salivary glands, which were able to infect a new vertebrate host. Live-cell imaging showed kinesin-5-GFP located on the spindle and at spindle poles during both atypical mitosis and meiosis. Fixed-cell immunofluorescence assays revealed kinesin-5 co-localized with α-tubulin and centrin-2 and a partial overlap with kinetochore marker NDC80 during early blood stage schizogony. Dual-colour live-cell imaging showed that kinesin-5 is closely associated with NDC80 during male gametogony, but not with kinesin-8B, a marker of the basal body and axonemes of the forming flagella. Treatment of gametocytes with microtubule-specific inhibitors confirmed kinesin-5 association with nuclear spindles and not cytoplasmic axonemal microtubules. Altogether, our results demonstrate that kinesin-5 is associated with the spindle apparatus, expressed in proliferating parasite stages, and important for efficient production of infectious sporozoites.
A stutter in the coiled coil domain ofE.colico-chaperone GrpE connects structure with function
Authors: Upadhyay Tulsi , Karekar Vaibhav V , Saraogi Ishu
Published: July 2020

In bacteria, the co-chaperone GrpE acts as a nucleotide exchange factor and plays an important role in controlling the chaperone cycle of DnaK. The functional form of GrpE is an asymmetric dimer, consisting of a long non-ideal coiled-coil. During heat stress, this region partially unfolds and prevents DnaK nucleotide exchange, ultimately ceasing the chaperone cycle. In this study, we elucidate the role of thermal unfolding of the coiled-coil domain ofE. coliGrpE in regulating its co-chaperonic activity. The presence of a stutter disrupts the regular heptad arrangement typically found in an ideal coiled coil resulting in structural distortion. Introduction of hydrophobic residues at the stutter altered the structural stability of the coiled-coil. Using an in vitro FRET assay, we show for the first time that the enhanced stability of GrpE resulted in an increased affinity for DnaK. However, the mutants were defective inin vitrofunctional assays, and were unable to support bacterial growth at heat shock temperature in agrpE-deletedE. colistrain. This work provides valuable insights into the functional role of a stutter in the GrpE coiled-coil, and its role in regulating the DnaK-chaperone cycle for bacterial survival during heat stress. More generally, our findings illustrate how a sequence specific stutter in a coiled-coil domain regulates the structure function trade-off in proteins.
Alcohol Dependence Differentially Alters Action and Outcome Related Orbitofrontal Cortex Activity
Authors: Cazares Christian , Schreiner Drew C. , Gremel Christina M.
Published: July 2020

Alcohol dependence results in long-lasting deficits in decision-making and behavioral control. Neurobiological investigations have identified orbitofrontal cortex (OFC) as important for value contributions to decision-making as well as action control, and alcohol dependence induces long-lasting changes to OFC function that persist into protracted withdrawal. However, it is unclear which contributing OFC computations are disrupted in alcohol dependence. Here, we combined a well-validated mouse model of alcohol dependence with in vivo extracellular recordings during an instrumental task in which lever press duration serves as the contingency, and lever pressing is sensitive to outcome devaluation. We found prior alcohol dependence did not impair use of duration contingency control but did reduce sensitivity to outcome devaluation. Further, alcohol dependence increased OFC activity associated with lever-pressing but decreased OFC activity during outcome-related epochs. Hence, alcohol dependence induces a long-lasting disruption to OFC function such that activity associated with actions is enhanced, but OFC activity in relation to outcomes is diminished. This has important implications for hypotheses regarding compulsive and habitual phenotypes observed in addiction.
A Bayesian optimisation approach for rapidly mapping residual network function in stroke
Authors: Lorenz Romy , Johal Michelle , Dick Frederic , Hampshire Adam , Leech Robert , Geranmayeh Fatemeh
Published: July 2020

Post-stroke cognitive and linguistic impairments are debilitating conditions, with current therapies only showing small improvements. Domain-general brain networks seem to play a critical role in stroke recovery and characterising their residual function with functional neuroimaging (fMRI) has the potential to yield biomarkers capable of guiding patient-specific rehabilitation. However, this is currently challenging in patients as such detailed characterisation requires too many different cognitive tasks. Here, we use neuroadaptive Bayesian optimisation to overcome this problem, an approach combining real-time fMRI with machine-learning. By intelligently searching across many tasks, this approach rapidly maps out patient-specific profiles of residual domain-general network function. Whereas controls have highly similar profiles, patients show idiosyncratic profiles of network abnormalities that are associated with behavioural performance. This approach can be extended to diverse brain networks and combined with brain stimulation or other therapeutics, thereby opening new avenues for precision medicine targeting diverse neurological and psychiatric conditions.
The Primary Tumor Immune Microenviornment Status Predicts the Response to Immunotherapy and Overall Survival in Breast Cancer
Authors: Moorthy Arjun , Quinn Aidan
Published: July 2020

II.

The tumor immune microenvironment (TIME) of breast cancer is a known source of tumor heterogeneity and it has been increasingly recognized as having a role in the course of disease. In the present study, we used a computational approach to dissect the landscape of TIME states among TCGA breast cancer patients. Our central hypothesis is that the pre-existing TIME states represent a dimension which is informative about the prognosis and the response to immunotherapy. In order to test this hypothesis, we first classified breast cancer patients according to their primary TIME status. Next, we describe a TIME-based classification with prognostic value for overall survival among the TCGA patients. We further demonstrated that absolute quantification of mast cells, M0 macrophages, CD8 T cells and neutrophils were predictive of overall survival. In order to identify the TIME states which, predict response to immune checkpoint blockade, we performed a similar analysis of 11 different mouse models of primary invasive breast carcinoma that were subsequently treated with immune checkpoint inhibitor (ICI) therapy. These analyses revealed that the TIME content of M1 macrophages, monocytes and resting dendritic cells were predictive of sensitivity to ICI therapy. Taken together, these results indicate that (1) the landscape of human primary TIME states is diverse and can identify patients with more or less aggressive disease and (2) that pre-existing TIME states may be able to identify patients, of all molecular subtypes of breast cancer, who are good candidates for ICI therapy.
Forecasting ocean acidification impacts on kelp forest ecosystems
Authors: Schlenger Adam J. , Beas-Luna Rodrigo , Ambrose Richard F.
Published: July 2020

Ocean acidification is one the biggest threats to marine ecosystems worldwide, but its ecosystem wide responses are still poorly understood. This study integrates field and experimental data into a mass balance food web model of a temperate coastal ecosystem to determine the impacts of specific OA forcing mechanisms as well as how they interact with one another. Specifically, we forced a food web model of a kelp forest ecosystem near its southern distribution limit in the California large marine ecosystem to a 0.5 pH drop over the course of 50 years. This study utilizes a modeling approach to determine the impacts of specific OA forcing mechanisms as well as how they interact. Isolating OA impacts on growth (Production), mortality (Other Mortality), and predation interactions (Vulnerability) or combining all three mechanisms together leads to a variety of ecosystem responses, with some taxa increasing in abundance and other decreasing. Results suggest that carbonate mineralizing groups such as coralline algae, abalone, snails, and lobsters display the largest decreases in biomass while macroalgae, urchins, and some larger fish species display the largest increases. Low trophic level groups such as giant kelp and brown algae increase in biomass by 16% and 71%, respectively. Due to the diverse way in which OA stress manifests at both individual and population levels, ecosystem-level effects can vary and display nonlinear patterns. Combined OA forcing leads to initial increases in ecosystem and commercial biomasses followed by a decrease in commercial biomass below initial values over time, while ecosystem biomass remains high. Both biodiversity and average trophic level decrease over time. These projections indicate that the kelp forest community would maintain high productivity with a 0.5 drop in pH, but with a substantially different community structure characterized by lower biodiversity and relatively greater dominance by lower trophic level organisms.
Language processing in brains and deep neural networks: computational convergence and its limits
Authors: Caucheteux Charlotte , King Jean-Rémi
Published: July 2020

Abstract

Deep Learning has recently led to major advances in natural language processing. Do these models process sentences similarly to humans, and is this similarity driven by specific principles? Using a variety of artificial neural networks, trained on image classification, word embedding, or language modeling, we evaluate whether their architectural and functional properties lead them to generate activations linearly comparable to those of 102 human brains measured with functional magnetic resonance imaging (fMRI) and magnetoencephalography (MEG). We show that image, word and contextualized word embeddings separate the hierarchical levels of language processing in the brain. Critically, we compare 3,600 embeddings in their ability to linearly map onto these brain responses. The results show that (1) the position of the layer in the network and (2) the ability of the network to accurately predict words from context are the main factors responsible for the emergence of brain-like representations in artificial neural networks. Together, these results show how perceptual, lexical and compositional representations precisely unfold within each cortical region and contribute to uncovering the governing principles of language processing in brains and algorithms.
Using DNA metabarcoding to investigate diet and niche partitioning in the native European otter (Lutra lutra) and invasive American mink (Neovison vison)
Authors: Harper Lynsey R. , Watson Hayley V. , Donnelly Robert , Hampshire Richard , Sayer Carl D. , Breithaupt Thomas , Hänfling Bernd
Published: July 2020

In the UK, the native European otter (Lutra lutra) and invasive American mink (Neovison vison) have experienced concurrent declines and expansions. Currently, the otter is recovering from persecution and waterway pollution, whereas the mink is in decline due to population control and probable interspecific interaction with the otter. We explored the potential of DNA metabarcoding for investigating diet and niche partitioning between these mustelids. Otter spraints (n= 171) and mink scats (n= 19) collected from three sites (Malham Tarn, River Hull, and River Glaven) in northern and eastern England were screened for vertebrates using high-throughput sequencing. Otter diet mainly comprised aquatic fishes (81.0%) and amphibians (12.7%), whereas mink diet predominantly consisted of terrestrial birds (55.9%) and mammals (39.6%). The mink used a lower proportion (20%) of available prey (n= 40 taxa) than the otter, and low niche overlap (0.267) was observed between these mustelids. Prey taxon richness of mink scats was lower than otter spraints, and beta diversity of prey communities was driven by taxon turnover (i.e. the otter and mink consumed different prey taxa). Considering otter diet only, prey taxon richness was higher in spraints from the River Hull catchment, and beta diversity of prey communities was driven by taxon turnover (i.e. the otter consumed different prey taxa at each site). Studies using morphological faecal analysis may misidentify the predator as well as prey items. Faecal DNA metabarcoding can resolve these issues and provide more accurate and detailed dietary information. When upscaled across multiple habitat types, DNA metabarcoding should greatly improve future understanding of resource use and niche overlap between the otter and mink.
EyeLoop: An open-source, high-speed eye-tracker designed for dynamic experiments
Authors: Arvin Simon , Rasmussen Rune , Yonehara Keisuke
Published: July 2020

Eye-tracking is a method for tracking the position of the eye and size of the pupil, often employed in neuroscience laboratories and clinics. Eye-trackers are widely used, from studying brain dynamics to investigating neuropathology and disease models. Despite this broad utility, eye-trackers are expensive, hardware-intensive, and proprietary, which have limited this approach to high-resource facilities. Besides, experiments have largely been confined to static open-loop designs and post hoc analysis due to the inflexibility of current systems. Here, we developed an open-source eye-tracking system, named EyeLoop, tailored to dynamic experiments. This Python-based software easily integrates custom functions via a modular logic, tracks a multitude of eyes, including rodent, human, and non-human primate eyes, and it operates well on inexpensive consumer-grade hardware. One of the most appealing applications of EyeLoop is closed-loop experiments, in which the eyes evoke stimulus feedback, such as rapid neuronal optogenetic stimulation. By using EyeLoop, we demonstrate its utility in an open-loop, a closed-loop, and a biomedical experiment. With a remarkably low minimal hardware cost amounting to 29 USD, EyeLoop makes dynamic eye-tracking accessible to low-resource facilities, such as high schools, small laboratories, and small clinics.
Induction of C4genes evolved through changes incisallowing integration into ancestral C3gene regulatory networks
Authors: Singh Pallavi , Stevenson Sean R. , Reyna-Llorens Ivan , Reeves Gregory , Schreier Tina B. , Hibberd Julian M.
Published: July 2020

C4photosynthesis has evolved independently in over sixty lineages and in so doing repurposed existing enzymes to drive a carbon pump that limits the RuBisCO oxygenation reaction. In all cases, gene expression is modified such that C4proteins accumulate to levels matching those of the photosynthetic apparatus. To better understand this rewiring of gene expression we undertook RNA- and DNaseI-SEQ on de-etiolating seedlings of C4Gynandropsis gynandra, which is sister to C3Arabidopsis. Changes in chloroplast ultrastructure and C4gene expression were coordinated and rapid. C3photosynthesis and C4genes showed similar induction patterns, but C4genes fromG. gynandrawere more strongly induced than orthologs from Arabidopsis. A gene regulatory network predicted transcription factors operating at the top of the de-etiolation network, including those responding to light, act upstream of C4genes. Light responsive elements, especially G-, E- and GT-boxes were over-represented in accessible chromatin around C4genes. Moreover,in vivobinding of many G-, E- and GT-boxes was detected. Overall, the data support a model in which rapid and robust C4gene expression following light exposure is generated through modifications incisto allow integration into high-level transcriptional networks including those underpinned by conserved light responsive elements.
Impact of parasitic lifestyle and different types of centromere organization on chromosome and genome evolution in the plant genusCuscuta
Authors: Neumann Pavel , Oliveira Ludmila , Čížková Jana , Jang Tae-Soo , Klemme Sonja , Novák Petr , Stelmach Katarzyna , Koblížková Andrea , Doležel Jaroslav , Macas Jiří
Published: July 2020

The parasitic genusCuscuta(Convolvulaceae) is exceptional among plants with respect to centromere organization, including both monocentric and holocentric chromosomes, and substantial variation in genome size and chromosome number. We investigated 12 species representing the diversity of the genus in a phylogenetic context to reveal the molecular and evolutionary processes leading to diversification of their genomes.

We measured genome sizes and investigated karyotypes and centromere organization using molecular cytogenetic techniques. We also performed low-pass whole genome sequencing and comparative analysis of repetitive DNA composition.

A remarkable 102-fold variation in genome sizes (342–34,734 Mbp/1C) was detected for monocentricCuscutaspecies, while genomes of holocentric species were of moderate sizes (533–1,545 Mbp/1C). The genome size variation was primarily driven by the differential accumulation of repetitive sequences. The transition to holocentric chromosomes in the subgenusCuscutawas associated with loss of histone H2A phosphorylation and elimination of centromeric retrotransposons. In addition, the basic chromosome number (x) decreased from 15 to 7, presumably due to chromosome fusions.

We demonstrated that the transition to holocentricity inCuscutawas accompanied by significant changes in epigenetic marks, chromosome number and the repetitive DNA sequence composition.
Human ANKLE1 is a nuclease specific for branched DNA
Authors: Song Junfang , Freeman Alasdair D. J. , Knebel Axel , Gartner Anton , Lilley David M. J.
Published: July 2020

All physical connections between sister chromatids must be broken before cells can divide, and eukaryotic cells have evolved multiple ways in which to process branchpoints connecting DNA molecules separated both spatially and temporally. A single DNA link between chromatids has the potential to disrupt cell cycle progression and genome integrity, so it is highly likely that cells require a nuclease that can process remaining unresolved and hemi-resolved DNA junctions and other branched species at the very late stages of mitosis. We argue that ANKLE1 probably serves this function in human cells (LEM-3 inC. elegans). LEM-3 has previously been shown to be located at the cell mid-body, and we show here that human ANKLE1 is a nuclease that cleaves a range of branched DNA species. It thus has the substrate selectivity consistent with an enzyme required to process a variety of unresolved and hemi-resolved branchpoints in DNA. Our results imply that ANKLE1 acts as a catch-all enzyme of last resort that allows faithful chromosome segregation and cell division to occur.
A photosynthesis operon in the chloroplast genome drives speciation in evening primroses
Authors: Zupok Arkadiusz , Kozul Danijela , Schöttler Mark Aurel , Niehörster Julia , Garbsch Frauke , Liere Karsten , Malinova Irina , Bock Ralph , Greiner Stephan
Published: July 2020

Incompatibility between the cytoplasm and the nucleus is considered as major factor in species formation, but mechanistic understanding is poor. In evening primroses, a model plant for organelle genetics and population biology, hybrid offspring regularly displays chloroplast-nuclear incompatibility. These incompatibilities affect photosynthesis, a trait under selection in changing environments. Here we show that light-dependent misregulation of the plastidpsbBoperon (encoding core subunits of photosystem II and the cytochromeb6fcomplex), can lead to hybrid incompatibility, thus ultimately driving speciation. This misregulation results in an impaired light acclimation response in incompatible plants. Moreover, as a result of their different chloroplast genotypes, the parental lines differ in their photosynthesis performance upon exposure to different light conditions. Significantly, the incompatible chloroplast genome is naturally found in xeric habitats with high light intensities, whereas the compatible one is limited to mesic habitats. Consequently, our data raise the possibility that the hybridization barrier evolved as a result of adaptation to specific climatic conditions.
A global resource for genomic predictions of antimicrobial resistance and surveillance ofSalmonellaTyphi at Pathogenwatch
Authors: Argimón Silvia , Yeats Corin A. , Goater Richard J. , Abudahab Khalil , Taylor Benjamin , Underwood Anthony , Sánchez-Busó Leonor , Wong Vanessa K. , Dyson Zoe A. , Nair Satheesh , Park Se Eun , Marks Florian , Page Andrew J. , Keane Jacqueline A. , Baker Stephen , Holt Kathryn E. , Dougan Gordon , Aanensen David M.
Published: July 2020

Background

Microbial whole-genome sequencing (WGS) is now increasingly used to inform public health investigations of infectious disease. This approach has transformed our understanding of the global population structure ofSalmonella entericaserovar Typhi (S.Typhi), the causative agent of typhoid fever. WGS has been particularly informative for understanding the global spread of multi-drug resistant (MDR) typhoid. As WGS capacity becomes more decentralised, there is a growing opportunity for collaboration and sharing of surveillance data within and between countries to inform disease control policies. This requires freely available, community driven tools that reduce the barriers to access genomic data for public health surveillance and that deliver genomic data on a global scale.

Methods

Here we present the Pathogenwatch (https://pathogen.watch/styphi) scheme forS.Typhi, a web application enabling the rapid identification of genomic markers of antimicrobial resistance (AMR) and contextualization with public genomic data to identify high-risk clones at a population level. Data are delivered in single genome reports or in collections of genomes combined with geographic and other data using trees, maps and tables.

Results

We show that the clustering ofS.Typhi genomes in Pathogenwatch is comparable to established bioinformatics methods, and that genomic predictions of AMR are largely concordant with phenotypic drug susceptibility data. We demonstrate the public health utility of Pathogenwatch with examples selected from over 4,300 public genomes available in the application.

Conclusions

Pathogenwatch democratises genomic epidemiology ofS.Typhi by providing an intuitive entry point for the analysis of WGS and linked epidemiological data, enabling international public health monitoring of the emergence and spread of high risk clones.
A community-driven resource for genomic epidemiology and antimicrobial resistance prediction ofNeisseria gonorrhoeaeat Pathogenwatch
Authors: Sánchez-Busó Leonor , Yeats Corin A. , Taylor Benjamin , Goater Richard J. , Underwood Anthony , Abudahab Khalil , Argimón Silvia , Ma Kevin C. , Mortimer Tatum D. , Golparian Daniel , Cole Michelle J. , Grad Yonatan H. , Martin Irene , Raphael Brian H. , Shafer William M. , Spiteri Gianfranco , Town Katy , Wi Teodora , Harris Simon R. , Unemo Magnus , Aanensen David M.
Published: July 2020

Background

Antimicrobial resistant (AMR)Neisseria gonorrhoeaeis an urgent threat to public health, as strains resistant to at least one of the two last line antibiotics used in empiric therapy of gonorrhoea, ceftriaxone and azithromycin, have spread internationally. Whole genome sequencing (WGS) data can be used to identify new AMR clones, transmission networks and inform the development of point-of-care tests for antimicrobial susceptibility, novel antimicrobials and vaccines. Community driven tools that provide an easy access to and analysis of genomic and epidemiological data is the way forward for public health surveillance.

Methods

Here we present a public health focussed scheme for genomic epidemiology ofN. gonorrhoeaeat Pathogenwatch (https://pathogen.watch/ngonorrhoeae). An international advisory group of experts in epidemiology, public health, genetics and genomics ofN. gonorrhoeaewas convened to inform on the utility of current and future analytics in the platform. We implement backwards compatibility with MLST, NG-MAST and NG-STAR typing schemes as well as an exhaustive library of genetic AMR determinants linked to a genotypic prediction of resistance to eight antibiotics. A collection of over 12,000N. gonorrhoeaegenome sequences from public archives has been quality-checked, assembled and made public together with available metadata for contextualization.

Results

AMR prediction from genome data revealed specificity values over 99% for azithromycin, ciprofloxacin and ceftriaxone and sensitivity values around 99% for benzylpenicillin and tetracycline. A case study using the Pathogenwatch collection ofN. gonorrhoeaepublic genomes showed the global expansion of an azithromycin resistant lineage carrying a mosaicmtrover at least the last 10 years, emphasizing the power of Pathogenwatch to explore and evaluate genomic epidemiology questions of public health concern.

Conclusions

TheN. gonorrhoeaescheme in Pathogenwatch provides customized bioinformatic pipelines guided by expert opinion that can be adapted to public health agencies and departments with little expertise in bioinformatics and lower resourced settings with internet connection but limited computational infrastructure. The advisory group will assess and identify ongoing public health needs in the field of gonorrhoea, particularly regarding gonococcal AMR, in order to further enhance utility with modified or new analytic methods.
Hyperkalemia, not apoptosis, accurately predicts chilling injury in individual locusts
Authors: Carrington Jessica , Andersen Mads Kuhlmann , Brzezinski Kaylen , MacMillan Heath
Published: July 2020

During prolonged or severe chilling, the majority of insects accrue chilling injuries that are typically quantified by scoring neuromuscular function after rewarming. In the cold, these chill susceptible insects, like the migratory locust (Locusta migratoria) suffer a loss of ion and water balance that is hypothesized to initiate cell death. Whether apoptotic or necrotic cell death pathways are responsible for this chilling injury is unclear. Here, we use a caspase-3 specific assay to indirectly quantify apoptosis in three locust tissues (muscle, nerves, and midgut) following prolonged chilling and recovery from an injury-inducing cold exposure. Furthermore, we obtain matching measurements of injury, hemolymph [K+], and muscle caspase-3 activity in individual locusts to gain further insight into mechanistic nature of chilling injury. We hypothesized that apoptotic cell death in both muscle and nerve tissue drives motor defects following cold exposure in insects, and that there would be a strong association between cold- induced injury, hyperkalemia, and muscle caspase-3 activity. We found a significant increase in muscle caspase-3 activity, but no such increase was observed in either nervous or gut tissue from the same animals, suggesting that chill injury primarily relates to apoptotic muscle cell death. However, the levels of chilling injury measured at the whole animal level prior to tissue sampling were strongly correlated with the degree of hemolymph hyperkalemia, but not apoptosis. These results support the notion that cold-induced ion balance disruption triggers cell death but also that apoptosis is not the main cell death pathway driving injury in the cold.

Significance Statement

Temperature has profound effects on animal fitness and sets limits to animal distribution. To understand and model insect responses to climate, we need to know how temperature sets limits to their survival. There is strong evidence that a collapse of ion and water balance occurs in insects in the cold, and it is generally held that the resulting cold injury is caused by activation of programmed cell death (apoptosis). Here, we directly test this idea and show for the first time that although the loss of ion balance is a strong predictor of individual survival outcomes, apoptosis is not the primary cause of cold-induced injury.
Zinc finger protein SALL4 functions through an AT-rich motif to regulate gene expression
Authors: Kong Nikki R. , Bassal Mahmoud A. , Tan Hong Kee , Kurland Jesse V. , Yong Kol Jia , Young John J. , Yang Yang , Li Fudong , Lee Jonathan , Liu Yue , Wu Chan-Shuo , Stein Alicia , Luo Hongbo , Silberstein Leslie E. , Bulyk Martha L. , Tenen Daniel G. , Chai Li
Published: July 2020

The zinc finger transcription factor SALL4 is highly expressed in embryonic stem cells, down-regulated in most adult tissues, but reactivated in many aggressive cancers. This unique expression pattern makes SALL4 an attractive target for designing therapeutic strategies. However, whether SALL4 binds DNA directly to regulate gene expression is unclear and many of its targets in cancer cells remain elusive. Here, through an unbiased screen of protein binding microarray (PBM) and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) experiments, we identified and validated the DNA binding domain of SALL4 and its consensus binding sequence. Combined with RNA-seq analyses after SALL4 knockdown, we discovered hundreds of new SALL4 target genes that it directly regulates in aggressive liver cancer cells, including genes encoding a family of Histone 3 Lysine 9-specific Demethylases (KDMs). Taken together, these results elucidated the mechanism of SALL4 DNA binding and revealed novel pathways and molecules to target in SALL4-dependent tumors.
Map of SARS-CoV-2 spike epitopes not shielded by glycans
Authors: Sikora Mateusz , von Bülow Sören , Blanc Florian E. C. , Gecht Michael , Covino Roberto , Hummer Gerhard
Published: July 2020

The severity of the COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, calls for the urgent development of a vaccine. The primary immunological target is the SARS-CoV-2 spike (S) protein. S is exposed on the viral surface to mediate viral entry into the host cell. To identify possible antibody binding sites not shielded by glycans, we performed multi-microsecond molecular dynamics simulations of a 4.1 million atom system containing a patch of viral membrane with four full-length, fully glycosylated and palmitoylated S proteins. By mapping steric accessibility, structural rigidity, sequence conservation and generic antibody binding signatures, we recover known epitopes on S and reveal promising epitope candidates for vaccine development. We find that the extensive and inherently flexible glycan coat shields a surface area larger than expected from static structures, highlighting the importance of structural dynamics in epitope mapping.
Pex14p phosphorylation modulates import of citrate synthase 2 into peroxisomes inSaccharomyces cerevisiae
Authors: Schummer Andreas , Maier Renate , Gabay-Maskit Shiran , Hansen Tobias , Mühlhäuser Wignand W. D. , Suppanz Ida , Fadel Amir , Schuldiner Maya , Girzalsky Wolfgang , Oeljeklaus Silke , Zalckvar Einat , Erdmann Ralf , Warscheid Bettina
Published: July 2020

The peroxisomal biogenesis factor Pex14p is an essential component of the peroxisomal matrix protein import machinery. Together with Pex13p and Pex17p, it is part of the membrane-associated peroxisomal docking complex in yeast, facilitating the binding of cargo-loaded receptor proteins for translocation of cargo proteins into the peroxisome. Furthermore, Pex14p is part of peroxisomal import pores. The central role of Pex14p in peroxisomal matrix protein import processes renders it an obvious target for regulatory mechanisms such as protein phosphorylation. To explore this possibility, we examined the state of Pex14p phosphorylation inSaccharomyces cerevisiae. Phos-tag-SDS-PAGE of Pex14p affinity-purified from solubilized membranes revealed Pex14p as multi-phosphorylated protein. Using mass spectrometry, we identified 16 phosphorylation sites, with phosphorylation hot spots located in the N- and C-terminal regions of Pex14p. Analysis of phosphomimicking and nonphosphorylatable variants of Pex14p revealed a decreased import of GFP carrying a peroxisomal targeting signal type 1, indicating a functional relevance of Pex14p phosphorylation in peroxisomal matrix protein import. We show that this effect can be ascribed to the phosphomimicking mutation at serine 266 of Pex14p (Pex14p-S266D). We further screened the subcellular distribution of 23 native GFP-tagged peroxisomal matrix proteins by high-content fluorescence microscopy. Only Cit2p, the peroxisomal isoform of citrate synthase, was affected in the Pex14p-S266D mutant, showing increased cytosolic localization. Cit2p is part of the glyoxylate cycle, which is required for the production of essential carbohydrates when yeast is grown on non-fermentable carbon sources. Pex14p-S266 phosphosite mutants showed reversed growth phenotypes on oleic acid and ethanol with acetyl-CoA formed in peroxisomes and the cytosol, respectively. Our data point to the control of the peroxisomal import of Cit2p via the state of Pex14p phosphorylation at S266, which may helpS. cerevisiaecells to rapidly adjust their carbohydrate metabolism according to the nutritional conditions.
Endogenous suppression of WNT signalling in human embryonic stem cells leads to low differentiation propensity towards definitive endoderm
Authors: Dziedzicka Dominika , Tewary Mukul , Keller Alexander , Tilleman Laurentijn , Prochazka Laura , Östblom Joel , De Deckersberg Edouard Couvreu , Markouli Christina , Franck Silvie , Van Nieuwerburgh Filip , Spits Claudia , Zandstra Peter W. , Sermon Karen , Geens Mieke
Published: July 2020

Low differentiation propensity towards a targeted lineage can significantly hamper the utility of individual human pluripotent stem cell (hPSC) lines in biomedical applications. Here, we use monolayer and micropatterned cell cultures, as well as transcriptomic profiling, to investigate how variability in signalling pathway activity between human embryonic stem cell lines affects their differentiation efficiency towards definitive endoderm (DE). We show that endogenous suppression of WNT signalling in hPSCs at the onset of differentiation prevents the switch from self-renewal to DE specification. Gene expression profiling reveals that this inefficient switch is reflected inNANOGexpression dynamics. Importantly, we demonstrate that higher WNT stimulation or inhibition of the PI3K/AKT signalling can overcome the DE commitment blockage. Our findings highlight that redirection of the activity of Activin/NODAL pathway by WNT signalling towards mediating DE fate specification is a vulnerable spot, as disruption of this process can result in poor hPSC specification towards DE.
FLN-1/Filamin is required to anchor the actomyosin cytoskeleton and for global organization of sub-cellular organelles in a contractile tissue
Authors: Kelley Charlotte A. , Triplett Olivia , Mallick Samyukta , Burkewitz Kristopher , Mair William B. , Cram Erin J.
Published: July 2020

Actomyosin networks are organized in space, direction, size, and connectivity to produce coordinated contractions across cells. We use theC. elegansspermatheca, a tube composed of contractile myoepithelial cells, to study how actomyosin structures are organized. FLN-1/filamin is required for the formation and stabilization of a regular array of parallel, contractile, actomyosin fibers in this tissue. Loss offln-1results in the detachment of actin fibers from the basal surface, which then accumulate along the cell junctions and are stabilized by spectrin. In addition, actin and myosin are captured at the nucleus by the linker of nucleoskeleton and cytoskeleton complex (LINC) complex, where they form large foci. Nuclear positioning and morphology, distribution of the endoplasmic reticulum and the mitochondrial network are also disrupted. These results demonstrate that filamin is required to prevent large actin bundle formation and detachment, to prevent excess nuclear localization of actin and myosin, and to ensure correct positioning of organelles.
Cardiomyocyte-specific Srsf3 deletion reveals a mitochondrial regulatory role
Authors: Dumont Audrey-Ann , Dumont Lauralyne , Zhou Delong , Giguère Hugo , Pileggi Chantal , Harper Mary-Ellen , Blondin Denis P , Scott Michelle S , Auger-Messier Mannix
Published: July 2020

Srsf3 was recently reported as being necessary to preserve RNA stability via an mTOR mechanism in a cardiac mouse model in adulthood. Here, we demonstrate the link between Srsf3 and mitochondrial integrity in an embryonic cardiomyocyte-specific Srsf3 conditional knockout (cKO) mouse model. Fifteen-day-old Srsf3 cKO mice showed dramatically reduced (below 50%) survival and reduced left ventricular systolic performance, and histological analysis of these hearts revealed a significant increase in cardiomyocyte size, confirming the severe remodelling induced by Srsf3 deletion. RNA-seq analysis of the hearts of 5-day-old Srsf3 cKO mice revealed early changes in expression levels and alternative splicing of several transcripts related to mitochondrial integrity and oxidative phosphorylation. Likewise, the levels of several protein complexes of the electron transport chain decreased, and mitochondrial complex I-driven respiration of permeabilized cardiac muscle fibres from the left ventricle was impaired. Furthermore, transmission electron microscopy analysis showed disordered mitochondrial length and cristae structure. Together with its indispensable role in the physiological maintenance of mouse hearts, these results highlight the previously unrecognized function of Srsf3 in regulating mitochondrial integrity.
Multiple sounds degrade the frequency representation in monkey inferior colliculus
Authors: Willett Shawn M. , Groh Jennifer M.
Published: July 2020

How we distinguish multiple simultaneous stimuli is uncertain, particularly given that such stimuli sometimes recruit largely overlapping populations of neurons. One commonly proposed hypothesis is that the sharpness of tuning curves might change to limit the number of stimuli driving any given neuron when multiple stimuli are present. To test this hypothesis, we recorded the activity of neurons in the inferior colliculus while monkeys made saccades to either one or two simultaneous sounds differing in frequency and spatial location. Although monkeys easily distinguished simultaneous sounds (∼90% correct performance), the frequency selectivity of inferior colliculus neurons on dual sound trials did not improve in any obvious way. Frequency selectivity was degraded on dual sound trials compared to single sound trials: neural response functions broadened, and frequency accounted for less of the variance in firing rate. These changes in neural firing led a maximum-likelihood decoder to perform worse on dual sound trials than on single sound trials. These results fail to support the hypothesis that changes in frequency response functions serve to reduce the overlap in the representation of simultaneous sounds. Instead, these results suggest that alternative possibilities, such as recent evidence of alternations in firing rate between the rates corresponding to each of the two stimuli, offer a more promising approach.

Graphic Abstract

How sensory representations encode multiple stimuli despite coarse coding is unknown. Using a maximum likelihood decoder operating on the spike count response patterns of monkey inferior colliculus neurons, we show a marked reduction in decoding accuracy when two sounds are presented compared to one. The decoding was inferior to the behavioral performance of the animals, and thus suggests the presence of alternative coding strategies.