title: Cytoplasmic Colocalization of Granulins and TDP-43 Prion-like Domain Involves Electrostatically Driven Complex Coacervation Tuned by the Redox State of Cysteines.

abstract: Cytoplasmic inclusions containing aberrant proteolytic fragments of TDP-43 are associated with frontotemporal lobar degeneration (FTLD) and other related pathologies. In FTLD, TDP-43 is translocated into the cytoplasm and proteolytically cleaved to generate a prion-like domain (PrLD) containing C-terminal fragments (C25 and C35) that form toxic inclusions. Under stress, TDP-43 partitions into membraneless organelles called stress granules (SGs) by coacervating with RNA and other proteins. We were interested in understanding if and how cysteine-rich granulins (GRNs 1-7), which are the proteolytic products of a genetic risk factor in FTLD called progranulin, interact with TDP-43. We show that extracellular GRNs internalize and colocalize with PrLD as puncta in the cytoplasm of neuroblastoma cells but show no presence in SGs. In addition, we show GRNs and PrLD coacervate to undergo liquid-liquid phase separation (LLPS) or form gel- or solid-like aggregates. Identification of the sequence determinants within GRNs for the observed phase transitions reveal the negative charges to be the drivers of LLPS modulated by the positive charges and the redox state of cysteines. Furthermore, RNA and GRNs compete and expunge one another from PrLD condensates, providing a basis for GRNs absence in SGs. Together, the results illustrate the potential mechanisms by which extracellular GRNs, formed during chronic inflammatory conditions, could internalize, and modulate cytoplasmic TDP-43 inclusions in proteinopathies.


title: Cytoplasmic Colocalization of Granulins and TDP-43 Prion-like Domain Involves Electrostatically Driven Coacervation Tuned by the Redox State of Cysteines.

category: biophysics


title: Intraspecific diversity observed in the wild tomato species Solanum chilense in initial immune responses towards a glucan elicitor.

abstract: Natural plant populations are highly polymorphic and often show intraspecific quantitative, variation in resistance properties against pathogens. The activation of the underlying defence responses can depend on the perception of conserved pathogen-associated molecular patterns (PAMPs). To dissect and understand such variation, we evaluated the diversity of responses induced by laminarin (representing a general glucan elicitor of plant immune responses) in the wild tomato species Solanum chilense. We confirm considerable overlap of the plants global transcriptional responses to laminarin and to the oomycete pathogen Phytophthora infestans. We further measured key components of basal defence responses such as reactive oxygen species (ROS) production and levels of diverse phytohormones and their derivatives upon elicitation with laminarin in 83 plants originating from nine natural populations of S. chilense from distinct geographic origin. We found high diversity in these components at basal and elicitor-induced levels. We generated generalised linear mixed models (GLMMs) with these components to explain the observed resistance phenotype against P. infestans in the plants and found that additive effects of multiple components best explain resistance at the species level. For individual components, we observed the strongest positive correlation between the resistance phenotype and ethylene (ET) production upon laminarin elicitation. The strength of this correlation differed between individual populations. Chemical inhibition of ET production in individuals from a population, in which ET production was associated with resistance, provoked more severe disease symptoms. Our findings reveal high diversity in the strength of induced defence responses within a species and in the basal levels of stress-related phytohormones. We show the involvement of multiple components with a quantitatively different contribution of individual components to resistance in geographically separated populations of S. chilense against P. infestans.

category: plant biology


title: Clathrin-independent endocytic retrieval of SV proteins mediated by the clathrin adaptor AP-2 at mammalian central synapses

abstract: Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Conflicting models have been proposed regarding the mechanisms of SV endocytosis, most notably clathrin/ AP-2-mediated endocytosis and clathrin-independent ultrafast endocytosis. Partitioning between these pathways has been suggested to be controlled by temperature and stimulus paradigm. We report on the comprehensive survey of six major SV proteins to show that SV endocytosis in hippocampal neurons at physiological temperature occurs independent of clathrin while the endocytic retrieval of a subset of SV proteins including the vesicular transporters for glutamate and GABA depend on sorting by the clathrin adaptor AP-2. Our findings highlight a clathrin-independent role of the clathrin adaptor AP-2 in the endocytic retrieval of select SV cargos from the presynaptic cell surface and suggest a unified model for the endocytosis of SV membranes at mammalian central synapses.

category: neuroscience


title: Mutations in PIKFYVE cause autosomal dominant congenital cataract

abstract: Congenital cataract, an ocular disease predominantly occurring within the first decade of life, is one of the leading causes of blindness in children. Through whole exome sequencing of a Chinese family with congenital cataract, we identified a disease-causing mutation (p.G1943E) in PIKFYVE, which affecting the PIP kinase domain of the PIKfyve protein. We demonstrated that heterozygous/homozygous disruption of PIKfyve kinase domain, instead of overexpression of PIKFYVEG1943E in zebrafish mimicked the cataract defect in human patients, suggesting that haploinsufficiency, rather than dominant-negative inhibition of PIKfyve activity caused the disease. Phenotypical analysis of pikfyve zebrafish mutants revealed that loss of Pikfyve caused aberrant vacuolation (accumulation of Rab7+Lc3+ amphisomes) in lens cells, which was significantly alleviated by treatment with the V-ATPase inhibitor bafilomycin A1 (Baf-A1). Collectively, we identified PIKFYVE as a novel causative gene for congenital cataract and demonstrated the potential application of Baf-A1 in treatment of congenital cataract.

category: genomics


title: Large structural variations in the haplotype-resolved African cassava genome.

abstract:
Cassava (Manihot esculenta Crantz, 2n=36) is a global food security crop. Cassava has a highly heterozygous genome, high genetic load, and genotype-dependent asynchronous flowering. It is typically propagated by stem cuttings and any genetic variation between haplotypes, including large structural variations, is preserved by such clonal propagation. Traditional genome assembly approaches generate a collapsed haplotype representation of the genome. In highly heterozygous plants, this results in artifacts and an oversimplification of heterozygous regions. We used a combination of Pacific Biosciences (PacBio), Illumina, and Hi-C to resolve each haplotype of the genome of a farmer-preferred cassava line, TME7 (Oko-iyawo). PacBio reads were assembled using the FALCON suite. Phase switch errors were corrected using FALCON-Phase and Hi-C read data. The ultra-long-range information from Hi-C sequencing was also used for scaffolding. Comparison of the two phases revealed more than 5,000 large haplotype-specific structural variants affecting over 8 Mb, including insertions and deletions spanning thousands of base pairs. The potential of these variants to affect allele specific expression was further explored. RNA-seq data from 11 different tissue types were mapped against the scaffolded haploid assembly and gene expression data are incorporated into our existing easy-to-use web-based interface to facilitate use by the broader plant science community. These two assemblies provide an excellent means to study the effects of heterozygosity, haplotype-specific structural variation, gene hemizygosity, and allele specific gene expression contributing to important agricultural traits and further our understanding of the genetics and domestication of cassava.

Significance statementThe cassava varieties grown by subsistence farmers in Africa largely differ from the inbred reference genome due to their highly heterozygous nature. We used multiple sequencing technologies to assemble and resolve both haplotypes in TME7, a farmer-preferred cassava line, enabling us to study the considerable haplotypic structural variation in this line.


title: An acetyl-click screening platform identifies a small molecule inhibitor of Histone Acetyltransferase 1 (HAT1) with anti-tumor activity

abstract: HAT1 is a central regulator of chromatin synthesis that acetylates nascent histone H3:H4 tetramers in the cytoplasm. It may have a role in cancer metabolism by linking cytoplasmic production of acetyl-CoA to nuclear acetyl flux. This is because the HAT1 di-acetylation mark is not propagated in chromatin and instead is de-acetylated after nascent histone insertion into chromatin. Thus, HAT1 likely provides a nuclear source of free acetate that may be recycled to acetyl-CoA for nuclear acetylation reactions. Correspondingly, suppression of HAT1 protein expression impairs tumor growth. To ascertain whether targeting HAT1 is a viable anti-cancer treatment strategy we sought to identify small molecule inhibitors of HAT1. We developed a high-throughput HAT1 acetyl-click assay to facilitate drug discovery and enzymology. Screening of small molecules computationally predicted to bind the active site led to the discovery of multiple riboflavin analogs that inhibited HAT1 enzymatic activity by competing with acetyl-CoA binding. These hits were refined by synthesis and testing over 70 analogs, which yielded structure-activity relationships. The isoalloxazine core was required for enzymatic inhibition, whereas modifications of the ribityl sidechain improved enzymatic potency and cellular growth suppression. These efforts resulted in a lead compound (JG-2016) that suppressed growth of human cancer cells lines in vitro and impaired tumor growth in vivo. This is the first report of a small molecule inhibitor of the HAT1 enzyme complex and represents a step towards targeting this pathway for cancer therapy.

category: biochemistry


title: Performance characteristics of next-generation sequencing for antimicrobial resistance gene detection in genomes and metagenomes

abstract: Short-read sequencing provides a culture-independent method for the detection of antimicrobial resistance (AMR) genes from single bacterial genomes and metagenomic samples. However, the performance characteristics of these approaches have not been systematically characterized. We compared assembly- and read-based approaches to determine sensitivity, positive predictive value, and sequencing limits of detection required for AMR gene detection in an Escherichia coli ST38 isolate spiked into a synthetic microbial community at varying abundances. Using an assembly-based method the limit of detection was 15X genome coverage. We are confident in AMR gene detection at target relative abundances of 100% to 1%, where a target abundance of 1% would require assembly of approximately 30 million reads to achieve 15X target coverage. Recent studies assessing AMR gene content in metagenomic samples may be inadequately sequenced to achieve high sensitivity. Our study informs future sequencing projects and analytical strategies for genomic and metagenomic AMR gene detection.

category: bioinformatics


title: Plant inositol-phosphate-glycans and fucosylated xyloglucan oligosaccharide are accumulated upon Arabidopsis thaliana/ Botrytis cinerea infection

abstract: In mammals, insulin is involved in controlling blood glucose levels and its role in modulating immunity is being more and more documented. This hormone promotes the release of inositolphosphate glycans (IPG) which act as mediators. In plants, one IG has already been identified in plant culture cells (Smith and Fry, 1999; Smith et al., 1999) but, to our knowledge, no IPG have been yet identified. Here, we discovered 7 IPG that are accumulated upon Arabidopsis thaliana-Botrytis cinerea interaction, concomitantly with oligogalacturonides and a fucosylated xyloglucan oligosaccharide. Further structural characterization showed that they come from the hydrolysis of polar heads of Serie A to H glycosylinositol phosphorylceramides presumably via a phospholipase C activity. Taken together with the emerging role of insulin as immune regulator, these results question the role of IPG as damage associated molecular pattern both in animal and plant kingdoms.


title: Plant inositol-phosphate-glycans and a fucosylated xyloglucan oligosaccharide are accumulated upon Arabidopsis thaliana/ Botrytis cinerea infection


title: Knocking out AtCALS7 disturbs photoassimilate distribution and weakens defence against phytoplasma infection

abstract:
Callose deposition around sieve pores, under control of callose synthase 7 (AtCALS7), has been interpreted as a mechanical response to limit pathogen spread in phytoplasma-infected plants. Wild-type and Atcals7ko mutants were therefore employed to unveil the mode of involvement of CALS7 in the plants response to phytoplasma infection. The fresh weights of healthy and CY-(Chrysanthemum Yellows) phytoplasma-infected Arabidopsis wild-type and mutant plants indicated two superimposed effects of the absence of CALS7: a partial impairment of photo-assimilate transport and a stimulated phytoplasma proliferation as illustrated by a significantly increased phytoplasma titre in Atcal7ko mutants. Further studies solely dealt with the effects of CALS7 absence on phytoplasma growth. Phytoplasma infection affected sieve-element substructure to a larger extent in mutants than in wild-type plants, which was also true for the levels of some free carbohydrates. Moreover, infection induced a similar upregulation of gene expression of enzymes involved in sucrose cleavage (AtSUS5, AtSUS6) and transmembrane transport (AtSWEET11) in mutants and wild-type plants, but an increased gene expression of carbohydrate transmembrane transporters (AtSWEET12, AtSTP13, AtSUC3) in infected mutants only. It remains still unclear how the absence of AtCALS7 leads to gene upregulation and how an increased intercellular mobility of carbohydrates and possibly effectors contributes to a higher susceptibility. It is also unclear if modified sieve-pore structures in mutants allow a better spread of phytoplasmas giving rise to higher titre.

Author SummaryPhytoplasma infections are one of the most limiting factors for production of important crops all over the world. Phytoplasma disease epidemics can be handled mainly by insect-vector control using insecticides. Basic information about plant-phytoplasma interactions are still limited, nevertheless it is necessary to design new management and breeding strategies aimed to obtain more tolerant or resistant cultivars. Phytoplasmas are obligate intracellular parasites restricted to the phloem sieve tubes. Callose deposition at the sieve plates has been described since the  70s as a mechanical defence process to limit pathogen spread by occluding sieve pores. Studies reported 40 years later demonstrated that callose at the sieve pores is also involved in sieve-pore development and function and, hence, in mass-flow regulation, carbohydrate metabolism and distribution, and plant growth. Here, we reported on the role(s) of sieve-element callose in phytoplasma-infected Arabidopsis, using a mutant lacking AtCALS7, the enzyme responsible for callose synthesis in the sieve elements. The results indicate that loss of AtCAL7 appears to confer increased susceptibility to phytoplasma infection, due to alterations in expression of genes involved in sugar metabolism and membrane transport. In the long run, the identification of plant resistance or susceptibility traits against phytoplasmas will allow a complete re-organization of chemical control strategies, with obvious opportunities of reducing insecticide burden.


title: Increased susceptibility to Chrysanthemum Yellows phytoplasma infection in Atcals7 ko plants is accompanied by enhanced expression of carbohydrate transporters.


title: CaProDH2-mediated modulation of proline metabolism confers tolerance to Ascochyta in chickpea under drought

abstract: Drought and leaf blight caused by the fungus Ascochyta rabiei often co-occur in chickpea (Cicer arietinum)-producing areas. While the responses of chickpea to either drought or A. rabiei infection have been extensively studied, their combined effect on plant defense mechanisms is unknown. Fine modulation of stress-induced signaling pathways under combined stress is an important stress adaptation mechanism that warrants a better understanding. Here we show that drought facilitates resistance against A. rabiei infection in chickpea. The analysis of proline levels and gene expression profiling of its biosynthetic pathway under combined drought and A. rabiei infection revealed the gene encoding proline dehydrogenase (CaProDH2) as a strong candidate conferring resistance to A. rabiei infection. Transcript levels of CaProDH2, pyrroline-5-carboxylate (P5C) quantification, and measurement of mitochondrial reactive oxygen species (ROS) production showed that fine modulation of the proline-P5C cycle determines the observed resistance. In addition, CaProDH2-silenced plants lost basal resistance to A. rabiei infection induced by drought, while overexpression of the gene conferred higher resistance to the fungus. We suggest that the drought-induced accumulation of proline in the cytosol helps maintain cell turgor and raises mitochondrial P5C contents by a CaProDH2-mediated step, which results in ROS production that boosts plant defense responses and confers resistance to A. rabiei infection. Our findings indicate that manipulating the proline-P5C pathway may be a possible strategy for improving stress tolerance in plants suffering from combined drought and A. rabiei infection.


title: Successional stages in infant gut microbiota maturation

abstract:
BackgroundDisturbances in the primary colonization of the infant gut can result in life-long consequences and have been associated with a range of host conditions. Although early life factors have been shown to affect the infant gut microbiota development, our current understanding of the human gut colonization in early life remains limited.

To gain more insights in the unique dynamics of this rapidly evolving ecosystem, we investigated the microbiota over the first year of life in eight densely sampled infants (total number of samples, n=303). To evaluate gut microbiota maturation transition towards an adult configuration, we compared the microbiome composition of the infants to the Flemish Gut Flora Project population (n=1,106).

ResultsWe observed the infant gut microbiota to mature through three distinct, conserved stages of ecosystem development. Across these successional gut microbiota maturation stages, genus predominance was observed to shift from Escherichia over Bifidobacterium to Bacteroides. Both disease and antibiotic treatment were observed to be associated occasionally with gut microbiota maturation stage regression, a transient setback in microbiota maturation dynamics. Although the studied microbiota trajectories evolved to more adult-like constellations, microbiome community typing against the background of the Flemish Gut Flora Project (FGFP) cohort clustered all infant samples within the (in adults) potentially dysbiotic Bact2 enterotype.

ConclusionWe confirmed similarities between infant gut microbial colonization and adult dysbiosis. A profound knowledge about the primary gut colonization process in infants might provide crucial insights into how the secondary colonization of a dysbiotic adult gut can be redirected.

category: microbiology


title: Environmental RNAi-based reverse genetics in the predatory mite Neoseiulus californicus: towards improved methods of biological control

abstract:
The predatory mite Neoseiulus californicus (McGregor) (Mesostigmata: Phytoseiidae) has been commercialized by manufacturers in the pest control industry and is used worldwide as a natural enemy of spider mites. However, because its genome has not been sequenced, reverse genetics techniques that could be used to analyze gene function have not been established. Here we partially sequenced the gene that encodes the vacuolar-type H+-ATPase (V-ATPase), an ATP-dependent proton pump, in N. californicus (NcVATPase) and then conducted a functional analysis using environmental RNA interference (eRNAi) by orally administering sequence-specific exogenous dsRNA (dsRNA-NcVATPase) to larvae and adult females. The larvae treated with dsRNA-NcVATPase took longer to develop and had lower survivorship, fecundity, and offspring viability at the adult stage than those treated with a control dsRNA. Adult females treated with dsRNA-NcVATPase showed significant reductions in survival, fecundity, and prey consumption, and their endogenous gene expression level of NcVATPase was reduced by approximately 65% compared with the control. Our findings suggest that the NcVATPase gene, silencing of which inhibits feeding and reproduction, is an excellent biomarker for investigating the eRNAi mechanism in N. californicus. The highly efficient experimental system of eRNAi established in this study paves the way for applied research using eRNAi to enhance the predatory ability of N. californicus.

Key messageO_LIEnvironmental RNAi-inducing double-stranded RNAs have the potential to improve biological control as well as biopesticide applications.
C_LIO_LIWe investigated the efficacy of eRNAi against the predatory mite Neoseiulus californicus, a major natural enemy of spider mites.
C_LIO_LIOral administration of dsRNA targeting NcVATPase decreased the gene expression level, developmental time, survival, fecundity, and prey consumption.
C_LIO_LINeoseiulus californicus, which was found to have the high eRNAi effects, can be used as a model for the study on eRNAi-mediated improvement of biological control.
C_LI

category: molecular biology


title: Testing methods for quantifying Monte Carlo variation for categorical variables in Probabilistic Genotyping

abstract:
Two methods for applying a lower bound to the variation induced by the Monte Carlo effect are trialled. One of these is implemented in the widely used probabilistic genotyping system, STRmix. Neither approach is giving the desired 99% coverage. In some cases the coverage is much lower than the desired 99%. The discrepancy (i.e. the distance between the LR corresponding to the desired coverage and the LR observed coverage at 99%) is not large. For example, the discrepancy of 0.23 for approach 1 suggests the lower bounds should be moved downwards by a factor of 1.7 to achieve the desired 99% coverage.

Although less effective than desired these methods provide a layer of conservatism that is additional to the other layers. These other layers are from factors such as the conservatism within the sub-population model, the choice of conservative measures of co-ancestry, the consideration of relatives within the population and the resampling method used for allele probabilities, all of which tend to understate the strength of the findings.

HighlightsO_LITwo methods for quantifying Monte Carlo variability are tested,
C_LIO_LIBoth give less than the desired 99% coverage,
C_LIO_LIThe magnitude of possible discrepancy is small,
C_LIO_LIFor example an LR of 4.3 x 1011 could be reported as 1.8 x 1012
C_LIO_LIAn LR of 18 could be reported as 22.
C_LI


title: The WZA: A window-based method for characterizing genotype-environment association

abstract: Genotype environment association (GEA) studies have the potential to elucidate the genetic basis of local adaptation in natural populations. Specifically, GEA approaches look for a correlation between allele frequencies and putatively selective features of the environment. Genetic markers with extreme evidence of correlation with the environment are presumed to be tagging the location of alleles that contribute to local adaptation. In this study, we propose a new method for GEA studies called the weighted-Z analysis (WZA) that combines information from closely linked sites into analysis windows in a way that was inspired by methods for calculating FST. We analyze simulations modelling local adaptation to heterogeneous environments either using a GEA method that controls for population structure or an uncorrected approach. In the majority of cases we tested, the WZA either outperformed single-SNP based approaches or performed similarly. The WZA outperformed individual SNP approaches when the measured environment is not perfectly correlated with the true selection pressure or when a small number of individuals or demes was sampled. We apply the WZA to previously published data from lodgepole pine and identified candidate loci that were not found in the original study.

category: evolutionary biology


title: A wAlbB Wolbachia transinfection displays stable phenotypic effects across divergent Aedes aegypti mosquito backgrounds

abstract: Aedes mosquitoes harboring intracellular Wolbachia bacteria are being released in arbovirus and mosquito control programs. With releases taking place around the world, understanding the contribution of host variation to Wolbachia phenotype is crucial. We generated a Wolbachia transinfection (wAlbBQ) in Aedes aegypti and performed backcrossing to introduce the infection into Australian or Malaysian nuclear backgrounds. Whole Wolbachia genome sequencing shows that the wAlbBQ transinfection is near-identical to the reference wAlbB genome, suggesting few changes since the infection was first introduced to Ae. aegypti over 15 years ago. However, these sequences were distinct from other available wAlbB genome sequences, highlighting the potential diversity of wAlbB in natural Ae. albopictus populations. Phenotypic comparisons demonstrate effects of wAlbB infection on egg hatch and nuclear background on fecundity and body size, but no interactions between wAlbB infection and nuclear background for any trait. The wAlbB infection was stable at high temperatures and showed perfect maternal transmission and cytoplasmic incompatibility regardless of host background. Our results demonstrate the stability of wAlbB across host backgrounds and point to its long-term effectiveness for controlling arbovirus transmission and mosquito populations.


title: DatumKB: A Database of Biological Experimental Results

abstract:
1.DatumKB is a freely accessible database of experimental results involving the function and regulation of human proteins in cultured cells. The results are manually curated from biological research literature using a shorthand language and stored as datums. Datums were originally designed to be used as evidence for rules in a Pathway Logic model of intracellular signal transduction (STM8, http://pl.csl.sri.com/online.html) (1). They are independent units that can be understood by biologists, traced back to their source, and have enough structure to be interrogated computationally. The information is expressed using a controlled vocabulary with links to well known databases such as HUGO Gene Nomenclature Committee (HGNC), UniProt, PubChem, and Cellosaurus. DatumKB can be searched using a query interface and the results can be downloaded in the original datum format, a simplified text format, or a JSON file. Links to detailed documentation of datum structure and a tutorial for the search engine are provided.

Database URLhttps://datum.csl.sri.com

category: cell biology


title: RNAseq analysis reveals dynamic metaboloepigenetic profiles of human, mouse and bovine pre-implantation embryos

abstract: Metaboloepigenetic regulation (metabolites regulating the cellular epigenome inducing long-term changes), has been reported in stem cells, germ cells and tumor cells. Embryonic metaboloepigenetics, however, have just begun to be described. Here we analyzed RNAseq data to characterize the metaboloepigenetic profiles of human, mouse and bovine pre-implantation embryos. In embryos, metaboloepigenetic reprogramming is species specific, varies with the developmental stage and is disrupted with in vitro culture. Although the idea that the in vitro environment may influence embryo development is not new, there has been little progress on improving pregnancy rates after more than 43 years using in vitro fertilization. Hence, the present data on embryonic metaboloepigenetic will contribute to understanding how the in vitro manipulation affects the metaboloepigenetic status of early embryos, which can be used to establish culture strategies aimed at improving the in vitro environment and, consequently, pregnancy rates and offspring health.

category: developmental biology


title: MassGenie: a transformer-based deep learning method for identifying small molecules from their mass spectra

abstract: The  inverse problem of mass spectrometric molecular identification ( given a mass spectrum, calculate the molecule whence it came) is largely unsolved, and is especially acute in metabolomics where many small molecules remain unidentified. This is largely because the number of experimentally available electrospray mass spectra of small molecules is quite limited. However, the forward problem ( calculate a small molecules likely fragmentation and hence at least some of its mass spectrum from its structure alone) is much more tractable, because the strengths of different chemical bonds are roughly known. This kind of molecular identification problem may be cast as a language translation problem in which the source language is a list of high-resolution mass spectral peaks and the  translation a representation (for instance in SMILES) of the molecule. It is thus suitable for attack using the deep neural networks known as transformers. We here present MassGenie, a method that uses a transformer-based deep neural network, trained on ~6 million chemical structures with augmented SMILES encoding and their paired molecular fragments as generated in silico, explicitly including the protonated molecular ion. This architecture (containing some 400 million elements) is used to predict the structure of a molecule from the various fragments that may be expected to be observed when some of its bonds are broken. Despite being given essentially no detailed nor explicit rules about molecular fragmentation methods, isotope patterns, rearrangements, neutral losses, and the like, MassGenie learns the effective properties of the mass spectral fragment and valency space, and can generate candidate molecular structures that are very close or identical to those of the  true molecules. We also use VAE-Sim, a previously published variational autoencoder, to generate candidate molecules that are  similar to the top hit. In addition to using the  top hits directly, we can produce a rank order of these by  round-tripping candidate molecules and comparing them with the true molecules, where known. As a proof of principle, we confine ourselves to positive electrospray mass spectra from molecules with a molecular mass of 500Da or lower. The transformer method, applied here for the first time to mass spectral interpretation, works extremely effectively both for mass spectra generated in silico and on experimentally obtained mass spectra from pure compounds. The ability to create and to  learn millions of fragmentation patterns in silico, and therefrom generate candidate structures (that do not have to be in existing libraries) directly, thus opens up entirely the field of de novo small molecule structure prediction from experimental mass spectra.


title: FRET and LRET Biosensors for Cell-based Imaging and Screening of Rac1 Activation

abstract: Rac1 is a key regulator of several cell signaling pathways and dysregulated Rac1 activation has been implicated in cancer. Genetically encoded Forster resonance energy transfer (FRET) biosensors with enhanced dynamic range enabled live cell fluorescence imaging of Rac1 activity and a cell lysate-based assay of Rac1 inhibition in 96-well plates. We prepared HEK293 cell lines that stably expressed polypeptides with a general domain sequence (N- to C-terminus) of 1) FRET acceptor; 2) Rac/Cdc42 binding domain of human p21 protein kinase A (residues 68-150); 3) a linker domain; 4) FRET donor; and 5) full-length Rac1. Activated Rac1 binds to the protein kinase A domain, bringing donors and acceptors close together to increase FRET. We evaluated the effects on FRET signal dynamic range of alpha helical linkers comprised of alternating repeats of roughly four glutamate and four arginine or lysine residues. So-called ER/K linkers had limited effects on conventional FRET biosensors that incorporated the fluorescent protein (FP) pairs mCerulean/YPet, or mTFP1(cp227)/mVenus(cp229). Fluorometric measurements of cells that co-expresssed biosensors with positive (TIAM1) or negative (RhoGDI) Rac1 regulators revealed significant dynamic range enhancement in only one FP construct (mCerulean/YPet with 20 nm ER/K linker) relative to an analogous structure that incorporated an unstructured linker. We transfected this construct into a cell line that stably expressed a rapamycin-inducible c-Src analog (RapR-Src) and observed activation of Rac1 at protruding edges following rapamycin stimulation. In cells that expressed lanthanide-based FRET (LRET biosensors) that incorporated a luminescent terbium complex donor and GFP fluorescent acceptor, time-gated luminescence (TGL) measurements showed substantial gains in dynamic range that increased with linker length (up to 1200%). We robustly detected small molecule Rac1 inhibition following lysis of LRET biosensor-expressing cells grown directly in 96-well plates. The results herein highlight the potential of FRET and LRET biosensors with ER/K linkers for cell-based imaging and screening of protein activities.


title: Arrival and proliferation of the invasive seaweed Rugulopteryx okamurae in NE Atlantic islands

abstract: The present study reports the recent occurrence and expansion of Rugulopteryx okamurae in the Azores archipelago (NE Atlantic). Morphological and molecular characters confirmed the species identification. Quick surveys around the island of Sao Miguel showed that it has successfully colonized the island and is quickly expanding. In some locations, R. okamurae is currently the dominant organism smothering all other benthic biota and posing a serious threat to the benthic ecosystems across the region. The species first record dates from 2019 near the main harbour of the island, suggesting that its introduction was driven by human-assisted transport, via boat ballast waters or adhered to ship hulls and likely originating from the Mediterranean populations that have been proliferating in recent years across the Strait of Gibraltar.

category: ecology


title: Dynamical transitions of the actomyosin cortex can trigger single cell morphogenesis

abstract: Morphogenetic changes driven by actomyosin contractile forces are well-characterized at the tissue level. At the single cell level, shape changes steered by actomyosin contractile forces include mitotic rounding and cytokinetic furrow ingression. In some cases, more complex shape transitions associated with spatial patterning of the cortex were observed. The actomyosin cortex was widely studied using active gel frameworks, and stabilized contractile instabilities were shown to generate patterns, but whether complex shapes can emerge from these cortical patterns is not established. Here we show that complex morphogenetic changes at the single cell level can accompany cortical patterns, using a minimal active gel model. For sufficiently low membranecortex drag, an initially homogeneous cortex spontaneously develops stripes associated with stable furrows, similar to furrowing observed in cells. Our work suggests that controlled cortical instability can trigger morphogenesis at the cellular level.


title: Diffuse ions coordinate dynamics in a ribonucleoprotein assembly

abstract:
Proper ionic concentrations are required for the functional dynamics of RNA and ribonucleoprotein (RNP) assemblies. While experimental and computational techniques have provided many insights into the properties of chelated ions, less is known about the energetic contributions of diffuse ions to large-scale conformational rearrangements. To address this, we present a model that is designed to quantify the influence of diffuse monovalent and divalent ions on the dynamics of biomolecular assemblies. This model employs all-atom (non-H) resolution and explicit ions, where effective potentials account for hydration effects. We first show that the model accurately predicts the number of excess Mg2+ ions for prototypical RNA systems, at a level comparable to modern coarse-grained models. We then apply the model to a complete ribosome and show how the balance between diffuse Mg2+ and K+ ions can control the dynamics of tRNA molecules during translation. The model predicts differential effects of diffuse ions on the free-energy barrier associated with tRNA entry and the energy of tRNA binding to the ribosome. Together, this analysis reveals the direct impact of diffuse ions on the dynamics of an RNP assembly.

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title: Progression and Resolution of SARS-1 CoV-2 Infection in Golden Syrian Hamsters

abstract: To catalyze SARS-CoV-2 research including development of novel interventive and preventive strategies, we characterized progression of disease in depth in a robust COVID-19 animal model. In this model, male and female golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 USA-WA1/2020. Groups of inoculated and mock-inoculated uninfected control animals were euthanized at day 2, 4, 7, 14, and 28 days post-inoculation to track multiple clinical, pathology, virology, and immunology outcomes. SARS-CoV-2-inoculated animals consistently lost body weight during the first week of infection, had higher lung weights at terminal timepoints, and developed lung consolidation per histopathology and quantitative image analysis measurements. High levels of infectious virus and viral RNA were reliably present in the respiratory tract at days 2 and 4 post-inoculation, corresponding with widespread necrosis and inflammation. At day 7, when infectious virus was rare, interstitial and alveolar macrophage infiltrates and marked reparative epithelial responses (type II hyperplasia) dominated in the lung. These lesions resolved over time, with only residual epithelial repair evident by day 28 post-inoculation. The use of quantitative approaches to measure cellular and morphologic alterations in the lung provides valuable outcome measures for developing therapeutic and preventive interventions for COVID-19 using the hamster COVID-19 model.

category: pathology


title: Woman Authorship in Pre-print Versus Peer-Reviewed Oral Health-Related Publications: A Two-Year Observational Study

abstract:
ObjectivesWomen in oral health science face similar societal issues and challenges as those in other STEMM careers, and gender disparities continue to exist as evidenced by fewer women represented as first and last authors in scientific publications. Pre-prints may serve as a conduit to immediately disseminating ones work, bypassing the arduous peer review process and its associated inherent biases. Therefore, the purpose of this study was to 1] compare the gender of first and last authors in pre-print versus peer reviewed publications, 2] examine the composition of first and last author pairs as stratified by publication type, and 3] examine the correlation between woman authorship and institutional geographic location and publication metrics stratified by publication type.

MethodsThe keyword "oral health" was used to search for publications in BioRxiv and Pubmed in the years 2018 and 2019. Gender of first and last authors were determined, and its frequency was considered as the primary outcome. Additionally, the geographic location of the authors associated institution and publication metrics measured by Altmetrics score were extracted. Data was descriptively summarized by frequencies and percentages. Chi-square analysis was conducted for categorical variables which included the relationship between gender and publication type as well as gender and region of authors associated institution. Binomial regression analysis was conducted to analyze the relationship between gender and Altmetrics.

ResultsWoman first authors comprised 40.3% of pre-prints and 64.5% of peer reviewed publications [p<0.05]. Woman last authors comprised 31.3% of pre-prints and 61.5% of peer reviewed publications [p<0.05]. When analyzing the relationships between first and last author, the Man-Man pairing represented 47.7% of the pre-print publications and the Woman-Woman pairing comprised a majority of the of the peer review publications at 47.5%. All results were statistically significant with a p-value <0.05. No significant correlation was found between region of institution or Altmetrics and gender of first or last authors [p>0.05].

ConclusionFor the first time in oral health science, it was found that women show higher representation as first and last author positions in peer reviewed publications versus pre-prints.

category: scientific communication and education


title: Dynamic rewiring of biological activity across genotype and lineage revealed by context-dependent functional interactions

abstract: Coessentiality networks derived from CRISPR screens in cell lines provide a powerful framework for identifying functional modules in the cell and for inferring the role of uncharacterized genes. However, these networks integrate signal across all underlying data, and can mask strong interactions that occur in only a subset of the cell lines analyzed. Here we decipher dynamic functional interactions by identifying significant cellular contexts, primarily by oncogenic mutation, lineage, and tumor type, and discovering coessentiality relationships that depend on these contexts. We recapitulate well-known gene-context interactions such as oncogene-mutation, paralog buffering, and tissue-specific essential genes, show how mutation rewires known signal transduction pathways, including RAS/RAF and IGF1R-PIK3CA, and illustrate the implications for drug targeting. We further demonstrate how context-dependent functional interactions can elucidate lineage-specific gene function, as illustrated by the maturation of proreceptors IGF1R and MET by proteases FURIN and CPD. This approach advances our understanding of context-dependent interactions and how they can be gleaned from these data. We provide an online resource to explore these context-dependent interactions at diffnet.hart-lab.org.

category: systems biology


title: Use of viral motif mimicry improves the proteome-wide discovery of human linear motifs

abstract: Linear motifs have an integral role in dynamic cell functions including cell signalling, the cell cycle and others. However, due to their small size, low complexity, degenerate nature, and frequent mutations, identifying novel functional motifs is a challenging task. Viral proteins rely extensively on the molecular mimicry of cellular linear motifs for modifying cell signalling and other processes in ways that favour viral infection. This study aims to discover human linear motifs convergently evolved also in disordered regions of viral proteins, under the hypothesis that these will result in enrichment in functional motif instances. We systematically apply computational motif prediction, combined with implementation of several functional and structural filters to the most recent publicly available human-viral and human-human protein interaction network. By limiting the search space to the sequences of viral proteins, we observed an increase in the sensitivity of motif prediction, as well as improved enrichment in known instances compared to the same analysis using only human protein interactions. We identified > 8,400 motif instances at various confidence levels, 105 of which were supported by all functional and structural filters applied. Overall, we provide a pipeline to improve the identification of functional linear motifs from interactomics datasets and a comprehensive catalogue of putative human motifs that can contribute to our understanding of the human domain-linear motif code and the mechanisms of viral interference with this.


title: A unique cerebellar pattern of microglia activation in a mousemodel of encephalopathy of prematurity

abstract: Preterm infants often show pathologies of the cerebellum, which are associated with impaired motor performance, lower IQ and poor language skills at school ages. Because 1 in 10 babies is born preterm cerebellar injury is a significant clinical problem. The causes of cerebellar damage are yet to be fully explained. Herein, we tested the hypothesis that perinatal inflammatory stimuli may play a key role in cerebellar injury of preterm infants. We undertook our studies in an established mouse model of inflammation-induced encephalopathy of prematurity driven by systemic administration of the prototypic pro-inflammatory cytokine interleukin-1{beta} (IL-1{beta}). Inflammation is induced between postnatal day (P) 1 to day 5, timing equivalent to the last trimester for brain development in humans the period of vulnerability to preterm birth related brain injury. We investigated acute and long-term consequences for the cerebellum on brain volume expansion, oligodendroglial maturation, myelin levels and the microglial transcriptome. Perinatal inflammation induced global mouse brain volume reductions, including specific grey and white matter volume reductions in cerebellar lobules I and II (5% FDR) in IL-1{beta} versus control treated mice from P15 onwards. Oligodendroglia damage preceded the MRI-detectable volume changes, as evidenced by a reduced proliferation of OLIG2+ cells at P10 and reduced levels of the myelin proteins MOG, MBP and MAG at P10 and P15. Increased density of Iba1+ cerebellar microglia was observed at P5 and P45, with evidence for increased microglial proliferation at P5 and P10. Comparison of the transcriptome of microglia isolated from P5 cerebelli and cerebrum revealed significant enrichment of pro-inflammatory markers in microglia from both regions, but in the cerebellum microglia displayed a unique type I interferon signalling dysregulation. Collectively, these data suggest that in our model that systemic inflammation causes chronic activation of microglia and maldevelopment of cerebellum that includes myelin deficits which is driven in the cerebellum by type I interferon signalling. Future protective strategies for preterm infants should consider sustained type I interferon signalling driven cerebellar inflammation as an important target.


title: Gestationally-Dependent Immune Organization at the Maternal-Fetal Interface

abstract:
The immune system and placenta have a dynamic relationship across gestation to accommodate fetal growth and development. High-resolution characterization of this maternal- fetal interface is necessary to better understand the immunology of pregnancy and its complications. We developed a single-cell framework to simultaneously immuno-phenotype circulating, endovascular, and tissue-resident cells at the maternal-fetal interface throughout gestation, discriminating maternal and fetal contributions. Our data reveal distinct immune profiles across the endovascular and tissue compartments with tractable dynamics throughout gestation that respond to a systemic immune challenge in a gestationally-dependent manner. We uncover that mononuclear phagocytes and neutrophils drive the temporal immune composition of the placenta with remarkably diverse populations, including PD-L1-expressing subsets having compartmental and early gestational bias. Our approach and accompanying datasets provide a resource for additional investigations into gestational immunology and evoke a more significant role for the innate immune system in establishing the microenvironment of early pregnancy.

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category: immunology


title: New intranasal and injectable gene therapy for healthy life extension

abstract: As the global elderly population grows, it is socioeconomically and medically critical to have diverse and effective means of mitigating the impact of aging on human health. Previous studies showed that adenovirus-associated virus (AAV) vector induced overexpression of certain proteins can suppress or reverse the effects of aging in animal models. Here, we sought to determine whether the high-capacity cytomegalovirus vector can be an effective and safe gene delivery method for two such-protective factors: telomerase reverse transcriptase (TERT) and follistatin (FST). We found that the mouse cytomegalovirus (MCMV) carrying exogenous TERT or FST (MCMVTERT or MCMVFST) extended median lifespan by 41.4% and 32.5%, respectively. This is the first report of CMV being used successfully as both an intranasal and injectable gene therapy system to extend longevity. Treatment significantly improved glucose tolerance, physical performance, and prevented loss of body mass and alopecia. Telomere shortening seen with aging was ameliorated by TERT, and mitochondrial structure deterioration was halted in both treatments. Intranasal and injectable preparations performed equally well in safely and efficiently delivering gene therapy to multiple organs, with long-lasting benefits and without carcinogenicity or unwanted side effects. Translating this research to humans could have significant benefits associated with increased health span.


title: Host-specific epibiomes of distinct Acropora cervicornis genotypes persist after field transplantation

abstract: Microbiome studies across taxa have established the influence of host genotype on microbial recruitment and maintenance. However, research exploring host-specific epibionts in scleractinian corals is scant and the influence of intraspecific differences across environments remains unclear. Here, we studied the epibiome of ten Acropora cervicornis genotypes to investigate the relative roles of host genotype and environment in structuring the epibiome. Coral mucus was sampled in a common garden nursery from replicate ramets of distinct genotypes (T0). Coral fragment replicates (n=3) of each genotype were then transplanted to nine different field sites in the Lower Florida Keys and mucus was again sampled one year later from surviving ramets (T12). 16S rRNA amplicon sequencing was used to assess microbial composition, richness, and beta-diversity. The most abundant and consistent amplicon sequencing variants (ASVs) in all samples belonged to Fokiniaceae (MD3-55 genus) and Cyanobacteria (Synechococccus). The abundances of these bacterial taxa varied consistently between genotypes whereas neither the composition nor taxonomic abundance were significantly different among field sites. Interestingly, several high MD3-55 hosting genotypes showed rapid diversification and an increase in MD3-55 following transplantation. Overall, our results indicate healthy A. cervicornis genotypes retain distinct epibiome signatures through time, suggesting a strong host component. Lastly, our results show that differences in MD3-55 abundances can be consistently detected in the epibiome of distinct host-genotypes of A. cervicornis. As this organism (sensu Aquarickettsia rohweri) has been implicated as a marker of disease resistance, this finding reinforces the potential use of microbial indicators in reef restoration efforts via non-invasive surface/mucus sampling.


title: Enforced specialization fosters mutual cheating and not division of labour in the bacterium Pseudomonas aeruginosa

abstract: A common way for bacteria to cooperate is via the secretion of beneficial public goods (proteases, siderophores, biosurfactants) that can be shared among individuals in a group. Bacteria often simultaneously deploy multiple public goods with complementary functions. This raises the question whether natural selection could favour division of labour where subpopulations or species specialise in the production of a single public good, whilst sharing the complementary goods at the group level. Here we use an experimental system, where we genetically enforce specialization in the bacterium Pseudomonas aeruginosa with regard to the production of its two siderophores, pyochelin and pyoverdine, and explore the conditions under which specialization can lead to division of labour. When growing pyochelin and pyoverdine specialists at different mixing ratios in various iron limited environments, we found that specialists could only successfully complement each other in environments with moderate iron limitation and grow as good as the generalist wildtype but not better. Under more stringent iron limitation, the dynamics in specialist communities was characterized by mutual cheating and with higher proportions of pyochelin producers greatly compromising group productivity. Nonetheless, specialist communities remained stable through negative frequency-dependent selection. Our work shows that specialization in a bacterial community can be spurred by mutual cheating and does not necessarily result in beneficial division of labour. We propose that natural selection might favour fine-tuned regulatory mechanisms in generalists over division of labour because the former enables generalists to remain flexible and adequately adjust public good investments in fluctuating environments.


title: Genomic Analyses of the Metastasis-derived LNCaP, VCaP, and PC3-AR Prostate Cancer Cell Lines

abstract: The lymph node metastasis-derived LNCaP, the bone metastasis-derived PC3 (skull), and VCaP (vertebral) cell lines are widely used preclinical models of human prostate cancer (CaP) and have been described in >19,000 publications. Here, we report on short-read whole-genome sequencing and genomic analyses of LNCaP, VCaP, and PC3 cells stably transduced with WT AR (PC3-AR). LNCaP cells are composed of multiple subpopulations, which results in non-integral copy number states and a high mutational load when the data is analyzed in bulk. All three cell lines contain pathogenic mutations and homozygous deletions in genes involved in DNA mismatch repair, along with deleterious mutations in cellcycle, Wnt signaling, and other cellular processes. Furthermore, LNCaP cells contain a missense mutation in a well-known CaP hotspot of TP53, whereas both PC3-AR and VCaP have truncating mutations in TP53 and do not express p53 protein. In addition, we detect the signatures of chromothripsis of the q arms of chromosome 5 in both PC3-AR and VCaP cells, strengthening the association of TP53 inactivation with chromothripsis reported in other systems. Our work provides a resource for genetic, genomic, and biological studies employing these commonly-used prostate cancer cell lines.


title: Polyunsaturated fatty acid structure determines the strength of potentiation of Acid-sensing ion channels

abstract:
Acid-sensing ion channels (ASICs) are sensitized to activation by inflammatory mediators like the polyunsaturated fatty acid (PUFA) arachidonic acid (AA). Previous work has shown that AA can potentiate ASIC currents at subsaturating proton concentrations, but the structural mechanisms of this change in gating are not understood. Here we show that PUFAs cause multiple gating changes in ASIC3 including shifting the pH dependence of activation, slowing the rate of desensitization, and increasing the current even at a saturating pH. The impact on gating depends on the nature of both the head and tail of the lipid with the head group structure primarily determining the magnitude of the effect on the channel. An N-acyl amino acid (NAAA), arachidonoyl glycine (AG), is such a strong regulator that it can act as a ligand at neutral pH. Mutation of an arginine in the outer segment of TM1 (R64) eliminated the effect of docosahexaenoic acid (DHA) even at high concentrations suggesting a potential interaction site for the lipid on the channel. Our results suggest a model where PUFAs bind to ASICs both via their tail group as well as an electrostatic interaction between the negatively charged PUFA head group and the positively charged arginine side chain. These data provide the first look at the structural features of lipids that are important for modulating ASICs and suggest a potential binding site for PUFAs on the channel.

Short SummaryArachidonic acid (AA) is a known modulator of ASICs. Bankston and Klipp examine the structural requirements for ASIC modulation by a number of lipids related to AA. Negatively charged head groups are stronger potentiators and may interact with an arginine in TM1 near the outer leaflet of the plasma membrane.


title: Structural determinants of Acid-sensing ion channel potentiation by single chain lipids


title: The Mixed Blessing of AMPK Signaling in Cancer Treatments

abstract: Nutrient acquisition and metabolism pathways are altered in cancer cells to meet bioenergetic and biosynthetic demands. A major regulator of cellular metabolism and energy homeostasis, in normal and cancer cells, is AMP-activated protein kinase (AMPK). AMPK influences cell growth via its modulation of the mechanistic target of Rapamycin (mTOR) pathway, specifically, by inhibiting mTOR complex mTORC1, which facilitates cell proliferation, and by activating mTORC2 and cell survival. Given its conflicting roles, the effects of AMPK activation in cancer can be counter-intuitive. Prior to the establishment of cancer, AMPK acts as a tumor suppressor. However, following the onset of cancer, AMPK has been shown to either suppress or promote cancer, depending on cell type or state. To unravel the controversial roles of AMPK in cancer, we developed a computational model to simulate the effects of pharmacological maneuvers that target key metabolic signalling nodes, with specific focus on AMPK, mTORC, and their modulators. Model simulations clarify the competing effects and the roles of key metabolic signalling pathways in tumorigenesis, which may yield insights on innovative therapeutic strategies.


title: Cellular intelligence: dynamic specialization through non-equilibrium multi-scale compartmentalization

abstract:
Intelligence is usually associated with the ability to perceive, retain and use information to adapt to changes in ones environment. In this context, systems of living cells can be thought of as intelligent entities. Here, we show that the concepts of non-equilibrium tuning and compartmentalization are sufficient to model manifestations of cellular intelligence such as specialization, division, fusion and communication using the language of operads. We implement our framework as an unsupervised learning algorithm, IO_SCPLOWNTC_SCPLOWCO_SCPLOWYTC_SCPLOW, which we show is able to memorize, organize and abstract reference machine-learning datasets through generative and self-supervised tasks. Overall, our learning framework captures emergent properties programmed in living systems, and provides a powerful new approach for data mining.

Structured abstractO_ST_ABSBackgroundC_ST_ABSAlthough intelligence has been given many definitions, we can associate it with the ability to perceive, retain, and use information to adapt to changes in ones environment. In this context, systems of living cells can be thought of as intelligent entities. While one can reasonably describe their adaptive abilities within the realm of homeostatic mechanisms, it is challenging to comprehend the principles governing their metabolic intelligence. In each organism, cells have indeed developed as many ways to adapt as there are cell types, and elucidating the impetus of their evolutionary behaviors could be the key to understanding life processes and likely diseases.

AdvancesThe goal of this article is to propose principles for understanding cellular intelligence. Specifically, we show that the concepts of non-equilibrium tuning and compartmentalization are enough to recover cellular adaptive behaviors such as specialization, division, fusion, and communication. Our model has the advantage to encompass all scales of life, from organelles to organisms through systems of organs and cell assemblies. We achieve this flexibility using the language of operads, which provides an elegant framework for reasoning about nested systems and, as an emergent behavior, non-equilibrium compartmentalization.

To demonstrate the validity and the practical utility of our model, we implement it in the form of an unsupervised learning algorithm, IO_SCPLOWNTC_SCPLOWCO_SCPLOWYTC_SCPLOW, and apply it to reference machine learning datasets through generative and self-supervised tasks. We find that IO_SCPLOWNTC_SCPLOWCO_SCPLOWYTC_SCPLOWs interpretability, plasticity and accuracy surpass that of a wide range of machine learning algorithms, thus providing a powerful approach for data mining.

OutlookOur results indicate that the nested hierarchical language of operads captures the emergent properties of programmed cellular metabolism in the development of living systems, and provide a new biologically-inspired, yet practical and lightweight, computational paradigm for memorizing, organizing and abstracting datasets.


title: A lineage tree-based hidden Markov model to quantify cellular heterogeneity and plasticity

abstract:
Cell plasticity, or the ability of cells within a population to reversibly alter their phenotype, is an important feature of tissue homeostasis during processes such as wound healing and cancer. Plasticity operates alongside other sources of cell-to-cell heterogeneity such as genetic mutations and variation in signaling. Ultimately these processes prevent most cancer therapies from being curative. The predominant methods of quantifying tumor-drug response operate on snapshot population-level measurements and therefore lack evolutionary dynamics, which are particularly critical for dynamic processes such as plasticity. Here we apply a tree-based adaptation of a hidden Markov model (tHMM) that employs single cell lineages as input to learn the characteristic patterns of single cell heterogeneity and state transitions in an unsupervised fashion. This model enables single cell classification based on the phenotype of individual cells and their relatives for improved specificity in pinpointing the structure and dynamics of variability in drug response. Integrating this model with a modular interface for defining observed phenotypes allows the model to easily be adapted to any phenotype measured in single cells. To benchmark our model, we paired cell fate with either cell lifetimes or individual cell cycle phase lengths (G1 and S/G2) as our observed phenotypes on synthetic data and demonstrated that the model successfully classifies cells within experimentally tractable dataset sizes. As an application, we analyzed experimental measurements of cell fate and phase duration in cancer cell populations treated with chemotherapies to determine the number of distinct subpopulations. In total, this tHMM framework allows for the flexible classification of single cell heterogeneity across lineages.

Summary pointsO_LIA lineage tree-based hidden Markov model (tHMM) quantifies cell-to-cell variability and dynamic population changes.
C_LIO_LIA modular interface to define phenotypes allows the model to work with any single cell phenotypic measurements.
C_LIO_LIThe model accurately classifies cells within experimentally tractable dataset sizes.
C_LIO_LIClassifying cells based on their phenotypic heterogeneity and relationships uncovers dynamics hidden at the population level.
C_LI

Author SummaryHeterogeneous traits, such as resistance or susceptibility to a drug, can be correlated in related cells because of shared or inherited factors. These acquired traits may be the result of the microenvironment, epigenetics, or mutations. Using cells relationships we capture these dynamic transitions between different cell states and arrive at a more accurate quantification of cell heterogeneity within tumor populations compared to clustering such as K-means that ignores any relationship between the individuals. Our computational approach employing a modified hidden Markov model provides greater specificity by identifying intratumor heterogeneity on a single cell level based on lineage histories and can identify dynamic changes in population structure upon treatment.


title: Assessing the influence of local neural activity on global connectivity fluctuations: Application to human intracranial EEG during a cognitive task

abstract: Cognitive-relevant information is processed by different brain areas that cooperate to eventually produce a response. The relationship between local activity and global brain states during such processes, however, remains for the most part unexplored. To address this question, we designed a simple face-recognition task performed in drug-resistant epileptic patients with intracranial EEG. Based on our observations, we developed a novel analytical framework (named "local-global" framework) to statistically correlate the brain activity in every recorded gray-matter region with the widespread connectivity functions as proxy to assess the level of influence of local neural activations into the brains global state during cognition. The application of our analysis to the data from two subjects was able to detect the local activity in task-relevant brain areas including the primary visual and motor cortices. Despite substantial differences in the recorded regions of each subject, the connectivity functions consistently showed a significant global desynchronization occurring a few hundred milliseconds after the stimulus onset. In this context, the local-global framework revealed that the reported desynchronization was better explained by the local activity of brain areas involved in face information processing, providing evidence that the global measures might be a novel signature of functional brain activity reorganization taking place when a stimulus is processed in a task context.


title: On the neuronal dynamics of aesthetic experience: Evidence from electroencephalographic oscillatory dynamics

abstract: Aesthetic experiences have an influence on many aspects of life. Interest in the neural basis of aesthetic experiences has grown rapidly in the past decade, and fMRI studies have identified several brain systems supporting aesthetic experiences. Work on the rapid neuronal dynamics of aesthetic experience, however, is relatively scarce. The present study adds to this field by investigating the experience of being aesthetically moved by means of ERP and time-frequency analysis. Participants EEG was recorded while they viewed a diverse set of artworks and evaluated the extent to which these artworks moved them. Results show that being aesthetically moved is associated with a sustained increase in gamma activity over centroparietal regions. Also, alpha power over right frontocentral regions was reduced in high and low moving images, compared to artworks given intermediate ratings. We interpret the gamma effect as an indication for sustained savoring processes for aesthetically moving artworks compared to aesthetically less moving artworks. The alpha effect is interpreted as an indication of increased attention for aesthetically salient images. In contrast to previous works, we observed no significant effects in any of the established ERP components, but we did observe effects at latencies longer than 1 s. We conclude that EEG time-frequency analysis provides useful information on the neuronal dynamics of aesthetic experience.


title: Towards the Interpretability of Deep Learning Models for Human Neuroimaging

abstract: Brain-age (BA) estimates based on deep learning are increasingly used as neuroimaging biomarker for brain health; however, the underlying neural features have remained unclear. We combined ensembles of convolutional neural networks with Layer-wise Relevance Propagation (LRP) to detect which brain features contribute to BA. Trained on magnetic resonance imaging (MRI) data of a population-based study (n=2637, 18-82 years), our models estimated age accurately based on single and multiple modalities, regionally restricted and whole-brain images (mean absolute errors 3.37-3.86 years). We find that BA estimates capture aging at both small and large-scale changes, revealing gross enlargements of ventricles and subarachnoid spaces, as well as lesions, iron accumulations and atrophies that appear throughout the brain. Divergence from expected aging reflected cardiovascular risk factors and accelerated aging was more pronounced in the frontal lobe. Applying LRP, our study demonstrates how superior deep learning models detect brain-aging in healthy and at-risk individuals throughout adulthood.


title: Room-temperature Storage of Lyophilized Engineered Bacteria using Tardigrade Intrinsically Disordered Proteins

abstract: Tardigrades, which live in transiently wet environments such as moss, are well-known for their extreme resistance to desiccation. Tardigrade intrinsically disordered proteins (TDPs) have been reported to also protect bacteria and yeast under desiccation [4, 5, 32]. In this study, we utilized lyophilization to achieve room-temperature storage of engineered bacteria. By using TDPs, engineered bacteria are protected under lyophilization and their original functions are preserved [12, 17, 18]. This study shows that TDPs can be expressed in the Escherichia coli (E. coli) BL21 and DH5, and bacteria treated with Cytosolic-abundant heat soluble protein (CAHS) 106094 displayed the highest survival rate after lyophilization [16, 41, 44]. Moreover, this study shows that the co-expression of TDPs can improve the preservation of bacteria and maintain high survival rates after prolonged room temperature storage. Additionally, the TDPs can be expressed using different vectors, which means that they can be used in different types of engineered bacteria. This study offers a new storage method that not only improves the storage of biological material for industrial and daily usage, but also for future iGEM (International Genetically Engineered Machine Competition) teams to store and use their engineered bacteria in different applications.

category: synthetic biology


title: Oxytocin receptors influence the development and maintenance of social behavior in zebrafish (Danio rerio).

abstract: Zebrafish are highly social teleost fish and an excellent model to study social behavior. The neuropeptide Oxytocin is associated different social behaviors as well as disorders resulting in social impairment like autism spectrum disorder. However, how Oxytocin receptor signaling affects the development and expression kinetics of social behavior is not known. In this study we investigated the role of the two oxytocin receptors, Oxtr and Oxtrl, in the development and maintenance of social preference and shoaling behavior in 2- to 8-week-old zebrafish. Using CRISPR/Cas9 mediated oxtr and oxtrl knock-out fish, we found that the development of social preference is accelerated if one of the Oxytocin receptors is knocked-out and that the knock-out fish reach significantly higher levels of social preference. Moreover, oxtr -/- fish showed impairments in the maintenance of social preference. Social isolation prior to testing led to impaired maintenance of social preference in both wild-type and oxtr and oxtrl knock-out fish. Knocking-out one of the Oxytocin receptors also led to increased group spacing and reduced polarization in a 20-fish shoal at 8 weeks post fertilization, but not at 4. These results show that the development and maintenance of social behavior is influenced by the Oxytocin receptors and that the effects are not just pro- or antisocial, but dependent on both the age and social context of the fish.


title: Age-related change in adult chimpanzee social network integration

abstract:
BackgroundSocial isolation is a key risk factor for the onset and progression of age-related disease and mortality in humans, yet older people commonly have narrowing social networks. Few models explain why human networks shrink with age, despite the risk that small networks and isolation pose. We evaluate models grounded in a life history perspective by studying social aging in wild chimpanzees, which are long-lived and show physical decline with age.

MethodologyWe applied social network analysis to examine age-related changes in social integration in a 7+ year mixed-longitudinal dataset comprised of 38 wild adult chimpanzees (22 F, 16 M) in the Kanyawara community in the Kibale National Park, Uganda. Metrics of social integration included social attractivity and overt effort (directed degree and strength), gregariousness (undirected strength), social roles (betweenness and local transitivity), and embeddedness (eigenvector centrality) in grooming and spatial association networks.

ResultsMales reduced overt social effort yet increased in attractivity, roles in cliques, and embeddedness. Females were overall less integrated than males, and their decreased integration with age suggested social avoidance. Effects of age were largely independent of rank. Both sexes maintained highly repeatable inter-individual differences in several aspects of integration, particularly among mixed-sex partners.

Conclusions and implicationsAs in humans, chimpanzees experience age-related declines in social effort. However, important facets of integration aged more similarly to humans in non-industrialized vs. industrialized societies, suggesting an evolutionary social mismatch between conserved declines in effort and dynamics of industrialized society. Lastly, individual and sex differences have the potential to be important mediators of successful social aging in chimpanzees, as in humans.

category: animal behavior and cognition


title: OSCAR: a framework to identify and quantify cells in densely packed three-dimensional biological samples

abstract: We have developed an Object Segmentation, Counter and Analysis Resource (OSCAR) that is designed specifically to quantify densely packed biological samples with reduced signal-to-background ratio. OSCAR uses as input three dimensional images reconstructed from confocal 2D sections stained with dies such as nuclear marker and immunofluorescence labeling against specific antibodies to distinguish the cell types of interest. Taking advantage of a combination of arithmetic, geometric and statistical algorithms, OSCAR is able to reconstruct the objects in the 3D space bypassing segmentation errors due to the typical reduced signal to noise ration of biological tissues imaged in toto. When applied to the zebrafish developing retina, OSCAR is able to locate and identify the fate of each nuclei as a cycling progenitor or a terminally differentiated cell, providing a quantitative characterization of the dynamics of the developing vertebrate retina in space and time with unprecedented accuracy.


title: Dopamine reports reward prediction errors, but does not update policy, during inference-guided choice

abstract: Dopamine is thought to carry reward prediction errors (RPEs), which update values and hence modify future behaviour. However, updating values is not always the most efficient way of adapting to change. If previously encountered situations will be revisited in future, inferring that the state of the world has changed allows prior experience to be reused when situations are reencountered. To probe dopamines involvement in such inference-based behavioural flexibility, we measured and manipulated dopamine while mice solved a sequential decision task using state inference. Dopamine was strongly influenced by the value of states and actions, consistent with RPE signalling, using value information that respected task structure. However, though dopamine responded strongly to rewards, stimulating dopamine at the time of trial outcome had no effect on subsequent choice. Therefore, when inference guides choice, rewards have a dopamine-independent influence on policy through the information they carry about the worlds state.


title: Temporal derivative computation in the dorsal raphe network revealed by an experimentally-driven augmented integrate-and-fire modeling framework

abstract: By means of an expansive innervation, the relatively few phylogenetically-old serotonin (5-HT) neurons of the dorsal raphe nucleus (DRN) are positioned to enact coordinated modulation of circuits distributed across the entire brain in order to adaptively regulate behavior. In turn, the activity of the DRN is driven by a broad set of excitatory inputs, yet the resulting network computations that naturally emerge from the excitability and connectivity features of the various cellular elements of the DRN are still unknown. To gain insight into these computations, we developed a flexible experimental and computational framework based on a combination of automatic characterization and network simulations of augmented generalized integrate-and-fire (aGIF) single-cell models. This approach enabled the examination of causal relationships between specific excitability features and identified population computations. We found that feedforward inhibition of 5-HT neurons by heterogeneous DRN somatostatin (SOM) neurons implemented divisive inhibition, while endocannabinoid-mediated modulation of excitatory drive to the DRN increased the gain of 5-HT output. The most striking computation that arose from this work was the ability of 5-HT output to linearly encode the derivative of the excitatory inputs to the DRN. This network computation primarily emerged from the prominent adaptation mechanisms found in 5-HT neurons, including a previously undescribed dynamic threshold. This novel computation in the DRN provides a potential mechanism underlying some of the functions recently ascribed to 5-HT in the context of reinforcement learning.


title: Ipsilesional motor cortex activation with high-force unimanual handgrip contractions of the less-affected limb in participants with stroke

abstract: Stroke is a leading cause of severe disability that often presents with unilateral motor impairment. Conventional rehabilitation approaches focus on motor practice of the affected limb and aim to suppress brain activity in the contralesional hemisphere to facilitate ipsilesional hemispheric neuroplasticity subserving motor recovery. Previous research has also demonstrated that exercise of the less-affected limb can promote motor recovery of the affected limb through the interlimb transfer of the trained motor task, termed cross-education. One of the leading theories for cross-education proposes that the interlimb transfer manifests from ipsilateral cortical activity during unimanual motor tasks, and that this ipsilateral cortical activity results in motor related neuroplasticity giving rise to contralateral improvements in motor performance. Conversely, exercise of the less-affected limb promotes contralesional brain activity which is typically viewed as contraindicated in stroke recovery due to the interhemispheric inhibitory influence onto the ipsilesional hemisphere. High-force unimanual handgrip contractions are known to increase ipsilateral brain activation in control participants, but it remains to be determined if this would be observed in participants with stroke. Therefore, this study aimed to determine how parametric increases in handgrip force during repeated contractions with the less-affected limb impacts brain activity bilaterally in participants with stroke and in a cohort of neurologically intact controls. In this study, higher force contractions were found to increase brain activation in the ipsilesional/ipsilateral hemisphere in both groups (p = .002), but no between group differences were observed. These data suggest that high-force exercise with the less-affected limb may promote ipsilesional cortical plasticity to promote motor recovery of the affected-limb in participants with stroke.


title: Molecular evolution of the anti-CRISPR family AcrIF7 reveals conservation despite mutation tolerance

abstract: Anti-CRISPRs are proteins capable of blocking CRISPR-Cas systems and typically encoded in mobile genetic elements. Since their discovery, numerous anti-CRISPR families have been identified. However, little is known about the distribution and sequence diversity of members within a family, nor how these traits influence the anti-CRISPRs function and evolution. Here we use AcrIF7 to explore the dissemination and molecular evolution of an anti-CRISPR family. We uncovered five sub-clusters and prevalent anti-CRISPR variants within the group. Remarkably, AcrIF7 homologs display high similarity despite their broad geographical, ecological and temporal distribution. Although mainly associated with Pseudomonas aeruginosa, AcrIF7 was identified in distinct genetic backgrounds indicating horizontal dissemination, primarily by phages. Using mutagenesis, we recreated variation observed in databases but also extended the sequence diversity of the group. Characterisation of the variants identified residues key for the anti-CRISPR function and other contributing to its mutational tolerance. Moreover, molecular docking revealed that variants with affected function lose key interactions with its CRISPR-Cas target. Analysis of publicly available data and the generated variants suggests that the dominant AcrIF7 variant corresponds to the minimal and optimal anti-CRISPR selected in the family. Our study provides a blueprint to investigate the molecular evolution of anti-CRISPR families.


title: Distribution, molecular evolution, and mutational scanning of the anti-CRISPR family AcrIF7 reveal conservation, key residues for the protein function and a putative alternative binding mode to Cas8f


title: Distribution and molecular evolution of the anti-CRISPR family AcrIF7


title: Characterization of an operon required for growth on cellobiose in Clostridioides difficile

abstract: Cellobiose metabolism is linked to the virulence properties in numerous bacterial pathogens. Here, we characterized a putative cellobiose PTS operon of Clostridiodes difficile to investigate the role of cellobiose metabolism in C. difficile pathogenesis. Our gene knockout experiments demonstrated that the putative cellobiose operon enables uptake of cellobiose into C. difficile and allows growth when cellobiose is provided as the sole carbon source in minimal medium. Additionally, using reporter gene fusion assays and DNA pull-down experiments, we show that its transcription is regulated by CelR, a novel transcriptional repressor protein, which directly binds to the upstream region of the cellobiose operon to control its expression. We have also identified cellobiose metabolism to play a significant role in C. difficile physiology as observed by the reduction of sporulation efficiency when cellobiose uptake was compromised in the mutant strain. In corroboration to in vitro study findings, our in vivo hamster challenge experiment showed a significant reduction of pathogenicity by the cellobiose mutant strain in both the primary and the recurrent infection model- substantiating the role of cellobiose metabolism in C. difficile pathogenesis.


title: Longitudinal accumulation of in vivo and in vitro-grown Treponema pallidum subsp. pallidum TprK variants in the presence and absence of immune pressure

abstract:
Immune evasion by Treponema pallidum subspecies pallidum (T. pallidum) has been attributed to antigenic variation of its putative outer-membrane protein TprK. In TprK, amino acid diversity is confined to seven variable (V) regions, and generation of sequence diversity within the V regions occurs via a non-reciprocal segmental gene conversion mechanism where donor cassettes recombine into the tprK expression site. Although previous studies have shown the significant role of immune selection in driving accumulation of TprK variants, the contribution of baseline gene conversion activity to variant diversity is less clear. Here, combining longitudinal tprK deep sequencing of near clonal Chicago C from immunocompetent and immunosuppressed rabbits along with the newly developed in vitro cultivation system for T. pallidum, we directly characterized TprK alleles in the presence and absence of immune selection. Our data confirm significantly greater sequence diversity over time within the V6 region during syphilis infection in immunocompetent rabbits compared to immunosuppressed rabbits, consistent with previous studies on the role of TprK in evasion of the host immune response. Compared to strains grown in immunocompetent rabbits, strains passaged in vitro displayed low level changes in allele frequencies of TprK variable region sequences similar to that of strains passaged in immunosuppressed rabbits. Notably, we found significantly increased rates of V6 allele generation relative to other variable regions in in vitro cultivated T, pallidum strains, illustrating that the diversity within these hypervariable regions occurs in the complete absence of immune selection. Together, our results demonstrate antigenic variation in T. pallidum can be studied in vitro and occurs even in the complete absence of immune pressure, allowing the T. pallidum population to continuously evade the immune system of the infected host.

Author SummarySyphilis continues to be a disease of global and public health concern, even though the infection can be easily diagnosed and effectively treated with penicillin. Although infected individuals often develop a strong immunity to the pathogen, repeated infection with the syphilis agent, Treponema pallidum subspecies pallidum (T. pallidum), is possible. Several studies point at antigenic variation of the T. pallidum TprK protein as the mechanism responsible for evasion of the immunity that develops during infection, pathogen persistence, and re-infection. Past studies have highlighted the importance of immune clearance of dominant variants that, in turn, allows less represented variants to emerge. The contribution of an immunity-independent baseline generation of variability in the tprK gene is less clear. Here, we used deep sequencing to profile tprK variants using a laboratory-isolated T. pallidum strain nearly isogenic for tprK that was propagated over time in vitro, where no immune pressure is exerted on the pathogen, as well as in samples obtained from immunosuppressed and immunocompetent rabbits infected with the same strain. We confirmed that tprK accumulates significantly more diversity under immune pressure, and demonstrated a low but discernible basal rate of gene conversion in complete absence of immune pressure.


title: Resolving the structure of phage-bacteria interactions in the context of natural diversity

abstract: Microbial communities are shaped by viral predators1. Yet, resolving which viruses (phages) and bacteria are interacting is a major challenge in the context of natural levels of microbial diversity2. Thus, fundamental features of how phage-bacteria interactions are structured and evolve in "the wild" remain poorly resolved3, 4. Here we use large-scale isolation of environmental marine Vibrio bacteria and their phages to obtain quantitative estimates of strain-level phage predator loads, and use all-by-all host range assays to discover how phage and host genomic diversity shape interactions. We show that killing in environmental interaction networks is sparse - with phage predator loads low for most bacterial strains and phages host-strain-specific in their killing. Paradoxically, we also find that although overlap in killing is generally rare between phages, recombination is common. Together, these results indicate that the number of hosts that phages infect is often larger than the number that they kill and suggest that recombination during cryptic co-infections is an important mode of phage evolution in microbial communities. In the development of phages for bioengineering and therapeutics it will be important to consider that nucleic acids of introduced phages may spread into local phage populations through recombination, and that the likelihood of transfer is not predictable based on killing host range.


title: Candida albicans Isolates 529L and CHN1 Exhibit Stable Colonization of the Murine Gastrointestinal Tract

abstract:
Candida albicans is a pathobiont that colonizes multiple niches in the body including the gastrointestinal (GI) tract, but is also responsible for both mucosal and systemic infections. Despite its prevalence as a human commensal, the murine GI tract is generally refractory to colonization with the C. albicans reference isolate SC5314. Here, we identify two C. albicans isolates, 529L and CHN1, that stably colonize the murine GI tract in three different animal facilities under conditions where SC5314 is lost from this niche. Analysis of the bacterial microbiota did not show notable differences between mice colonized with the three C. albicans strains. We compared the genotypes and phenotypes of these three strains and identified thousands of SNPs and multiple phenotypic differences, including their ability to grow and filament in response to nutritional cues. Despite striking filamentation differences under laboratory conditions, however, analysis of cell morphology in the GI tract revealed that the three isolates exhibited similar filamentation properties in this in vivo niche. Notably, we found that SC5314 is more sensitive to the antimicrobial peptide CRAMP, and the use of CRAMP-deficient mice increased the ability of SC5314 to colonize the GI tract relative to CHN1 and 529L. These studies provide new insights into how strain-specific differences impact C. albicans traits in the host and advance CHN1 and 529L as relevant strains to study C. albicans pathobiology in its natural host niche.

IMPORTANCEUnderstanding how fungi colonize the GI tract is increasingly recognized as highly relevant to human health. The animal models used to study Candida albicans commensalism commonly rely on altering the host microbiome (via antibiotic treatment or defined diets) to establish successful GI colonization by the C. albicans reference isolate SC5314. Here, we characterize two C. albicans isolates that can colonize the murine GI tract without antibiotic treatment and can therefore be used as tools for studying fungal commensalism. Importantly, experiments were replicated in three different animal facilities and utilized three different mouse strains. Differential colonization between fungal isolates was not associated with alterations in the bacterial microbiome but rather with distinct responses to CRAMP, a host antimicrobial peptide. This work emphasizes the importance of C. albicans intra-species variation as well as host anti-microbial defense mechanisms in defining commensal interactions.


title: Evaluation of Host Defense Peptide (CaD23)-Antibiotic Interaction and Mechanism of Action: Insights from Experimental and Molecular Dynamics Simulations Studies

abstract:
Background/aimHost defense peptides (HDPs) have the potential to provide a novel solution to antimicrobial resistance (AMR) in view of their unique and broad-spectrum antimicrobial activities. We had recently developed a novel hybrid HDP based on LL-37 and human beta-defensin-2, named CaD23, which was shown to exhibit good in vivo antimicrobial efficacy against Staphylococcus aureus in a bacterial keratitis murine model. This study aimed to examine the potential CaD23-antibiotic synergism and to evaluate the underlying mechanism of action of CaD23.

MethodsAntimicrobial efficacy was determined using minimum inhibitory concentration (MIC) assay with broth microdilution method. Peptide-antibiotic interaction was evaluated against S. aureus, methicillin-resistant S. aureus (MRSA), and Pseudomonas aeruginosa using established checkerboard assay and time-kill kinetics assay. Fractional inhibitory concentration index (FICI) was calculated and interpreted as synergistic (FICI<0.5), additive (FICI between 0.5-1.0), indifferent (FICI between >1.0 and [≤]4), or antagonistic (FICI>4). SYTOX green uptake assay was performed to determine the membrane-permeabilising action of CaD23. Molecular dynamics (MD) simulations were performed to evaluate the interaction of CaD23 with bacterial and mammalian mimetic membranes.

ResultsCaD23-amikacin and CaD23-levofloxacin combination treatment exhibited a strong additive effect against S. aureus SH1000 (FICI=0.56) and MRSA43300 (FICI=0.56) but a borderline additive-to-indifferent effect against P. aeruginosa (FIC=1.0-2.0). CaD23 (at 25 g/ml; 2x MIC) was able to achieve complete killing of S. aureus within 30 mins. When used at sub-MIC concentration (3.1 g/ml; 0.25x MIC), it was able to expedite the antimicrobial action of amikacin against S. aureus by 50%. The rapid antimicrobial action of CaD23 was attributed to the underlying membrane-permeabilising mechanism of action, evidenced by the SYTOX green uptake assay and MD simulations studies. MD simulations revealed that cationicity, alpha-helicity, amphiphilicity and hydrophobicity (related to the Trp residue at C-terminal) play important roles in the antimicrobial action of CaD23.

ConclusionsCaD23 is a novel membrane-active synthetic HDP that can enhance and expedite the antimicrobial action of antibiotics against Gram-positive bacteria when used in combination. MD simulation serves as a useful tool in dissecting the mechanism of action and guiding the design and optimisation of HDPs.


title: Microbiota responses to different prebiotics are conserved within individuals and associated with habitual fiber intake

abstract: Background: Short-chain fatty acids (SCFAs) derived from gut bacteria are associated with protective roles in diseases ranging from obesity to colorectal cancers. Intake of microbially accessible dietary fibers (prebiotics) lead to varying effects on SCFA production in human studies, and gut microbial responses to nutritional interventions vary by individual. It is therefore possible that prebiotic therapies will require customizing to individuals. Results: Here, we explored prebiotic personalization by conducting a three-way crossover study of three prebiotic treatments in healthy adults. We found that within individuals, metabolic responses were correlated across the three prebiotics. Individual identity, rather than prebiotic choice, was also the major determinant of SCFA response. Across individuals, prebiotic response was inversely related to basal fecal SCFA concentration, which, in turn, was associated with habitual fiber intake. Experimental measures of gut microbial SCFA production for each participant also negatively correlated with fiber consumption, supporting a model in which individuals' gut microbiota are limited in their overall capacity to produce fecal SCFAs from fiber. Conclusions: Our findings support developing personalized prebiotic regimens that focus on selecting individuals who stand to benefit, and that such individuals are likely to be deficient in fiber intake.


title: Anaerobic conditions unmask antimicrobial resistance

abstract: The antibiotic formulary is threatened by high rates of antimicrobial resistance (AMR) among enteropathogens. Enteric bacteria often undergo anaerobic respiration within the gastrointestinal tract, yet little is known about how anaerobic conditions influence AMR. The facultative enteropathogen Vibrio cholerae was chosen as a model to address this knowledge gap. V. cholerae isolates were tested for resistance under aerobic and anaerobic conditions against clinically relevant antibiotics. Using conventional breakpoints established for aerobic conditions, the odds of classifying isolates as resistant increased over 10 times for ciprofloxacin and 100 times for azithromycin under anaerobic conditions compared to aerobic conditions; for doxycycline, nearly all isolates remained sensitive. Genome-wide association studies found associations between genetic elements and AMR phenotypes that varied by oxygen exposure and antibiotic concentrations; AMR phenotypes were more heritable - and more AMR genes were discovered - under anaerobic conditions. These findings challenge the paradigm of testing facultative enteropathogens for AMR under aerobic conditions alone.


title: Anaerobic conditions unmask antimicrobial resistance in Vibrio cholerae


title: Identification and characterisation of splicing regulators in Toxoplasma gondii

abstract: The splicing of mRNA constitutes a major source of co- and post-transcriptional regulation in metazoans. In particular, members of the serine/arginine (SR) protein family are essential splicing factors that are implicated in the regulation of gene expression and RNA metabolism. However, very little is known about these proteins in apicomplexans, a phylum that includes some of the most important global parasites. In this study, we investigated the suite of three uncharacterised SR proteins in Toxoplasma gondii and show that all three are found localised to nuclear speckles. We show, by genetic ablation, that TgSR1 is particularly important for T. gondii growth. Using RNA-seq, we also characterised the global gene expression and splicing regulation of these proteins. We find that the SR proteins regulate several types of alternative splicing of distinct but overlapping subsets of transcripts, as well as impacting transcript abundance. Most of the alternative splicing events are non-productive intron retention events that do not appear to affect transcript abundance. The splicing sites of the impacted transcripts are enriched in characteristic SR binding motifs. We also identified and conditionally knocked down two putative kinases of SR proteins. The kinases are localised to nuclear speckles and are essential to parasite survival. Their perturbation resulted in widespread changes to splicing, but the affected transcripts did not mirror the patterns seen in knockouts of individual SRs, suggesting an absence of a simple relationship between SRs and these putative kinase regulators. Overall, this study reveals a complex system of splicing factors and kinases that post-transcriptionally regulate gene expression in T. gondii.