title: Dynamic profiling and functional interpretation of histone lysine crotonylation and lactylation during neural development

abstract:
BackgroundMetabolites such as crotonyl-CoA and lactyl-CoA influence gene expression through covalently modifying histones, known as histone lysine crotonylation (Kcr) and histone lysine lactylation (Kla). However, their existence patterns, dynamic changes, biological functions, as well as associations with histone lysine acetylation (Kac) and gene expression during mammalian development remain largely unknown, which imped us from understanding the epigenetic regulations of histone lysine acylations.

ResultsHistone Kcr and Kla are widely distributed and undergo global changes during neural development. By profiling genome-wide dynamics of H3K9ac, H3K9cr and H3K18la in combination with ATAC sequencing and RNA sequencing, we reveal that these histone marks cooperatively regulate chromatin state and gene expression, as well as promote transcriptome remodelling to favour cell fate transition in the developing telencephalon. Importantly, we demonstrate that global histone Kcr and Kla are not consequence of transcription and furtherly identify histone deacetylase 1-3 (HDAC1-3) as novel "erasers" of H3K18la. Taking advantage of induced differentiation system of P19 embryonal carcinoma (EC) cells, we find that a selective inhibitor of HDAC1-3, MS-275 pre-activates neural fate-related transcriptional programs via stimulating multiple histone lysine acylations simultaneously.

ConclusionsOur study uncover the interplays between histone lysine acylations to regulate gene expression and the differentiation-promoting functions of histone Kcr and Kla during development, and provide evidence that multiple histone lysine actlations synchronously alternate to orchestrate transcriptome responding under HDACs inhibition.


category: genomics


title: Universal allometry from empirical parameters

abstract: Allometric settings of population dynamics models are appealing due to their parsimonious nature and broad utility when studying system level effects. Here, we parameterise the size-scaled Rosenzweig-Macarthur ODEs to eliminate prey-mass dependency. We define the functional response term to match experiments, and examine situations where metabolic theory derivations and observation diverge. We produce dynamics consistent with observation. Our parameterisation of the Rosenzweig-Macarthur system is an accurate minimal model across 15+ orders of mass magnitude.

category: ecology


title: The evolution of size-dependent competitive interactions promotes species coexistence

abstract:
O_LITheory indicates that competing species coexist in a community when intraspecific competition is stronger than interspecific competition. When body size determines the outcome of competitive interactions between individuals, coexistence depends also on how resource use and the ability to compete for these resources change with body size. Testing coexistence theory in size-structured communities, therefore, requires disentangling the effects of size-dependent competitive abilities and niche shifts.
C_LIO_LIHere, we tested the hypothesis that the evolution of species and size-dependent competitive asymmetries increased the likelihood of coexistence between interacting species.
C_LIO_LIWe experimentally estimated the effects of size-dependent competitive interactions on somatic growth rates of two interacting fish species, Trinidadian guppies (Poecilia reticulata) and killifish (Rivulus hartii). We controlled for the effects of size-dependent changes in the niche at two competitive settings representing the early (allopatric) and late (sympatric) evolutionary stages of a killifish-guppy community. We fitted the growth data to a model that incorporates species and size-dependent competitive asymmetries to test whether changes in the competitive interactions across stages increased the likelihood of species coexistence from allopatry to sympatry.
C_LIO_LIWe found that guppies are competitively superior to killifish but were less so in sympatric populations. The decrease in the effects of interspecific competition on the fitness of killifish and increase in the interspecific effect on guppies fitness increased the likelihood that sympatric guppies and killifish will coexist. However, while the competitive asymmetries between the species changed consistently between allopatry and sympatry between drainages, the size-dependent competitive asymmetries varied between drainages.
C_LIO_LIThese results demonstrate the importance of integrating evolution and trait-based interactions into the research of how species coexistence.
C_LI


title: Taxon-specific or universal? Using target capture to study the evolutionary history of a rapid radiation

abstract: Target capture has emerged as an important tool for phylogenetics and population genetics in non-model taxa. Whereas developing taxon-specific capture probes requires sustained efforts, available universal kits may have a lower power to reconstruct relationships at shallow phylogenetic scales and within rapidly radiating clades. We present here a newly-developed target capture set for Bromeliaceae, a large and ecologically-diverse plant family with highly variable diversification rates. The set targets 1,776 coding regions, including genes putatively involved in key innovations, with the aim to empower testing of a wide range of evolutionary hypotheses. We compare the relative power of this taxon-specific set, Bromeliad1776, to the universal Angiosperms353 kit. The taxon-specific set results in higher enrichment success across the entire family, however, the overall performance of both kits to reconstruct phylogenetic trees is relatively comparable, highlighting the vast potential of universal kits for resolving evolutionary relationships. For more detailed phylogenetic or population genetic analyses, e.g. the exploration of gene tree concordance, nucleotide diversity or population structure, the taxon-specific capture set presents clear benefits. We discuss the potential lessons that this comparative study provides for future phylogenetic and population genetic investigations, in particular for the study of evolutionary radiations.

category: evolutionary biology


title: Taxon-specific or universal? Using target capture to study the evolutionary history of rapid radiations


title: A systematic review and meta-analysis of genetic parameters for complex quantitative traits in aquatic animal species

abstract: A systematic review and meta-analysis of genetic parameters underlying inheritance and complex biological relationships for quantitative traits are not available for aquatic animal species. I synthesised and conducted a comprehensive meta-analysis of the published information from 1985 to 2017 on heritability, common full-sib effects and genetic correlations for quantitative characters of biological importance (growth, carcass and flesh quality, disease resistance, deformity and reproduction) for aquaculture species. A majority of the studies (73.5%) focussed on growth related traits (body weight), followed by those on disease resistance (15.9%), whereas only a limited number of studies (10.6%) reported heritability estimates for carcass and flesh quality, deformity or reproduction characteristics. The weighted means of heritability for growth (weight, food utilisation efficiency, maturity) and carcass (fillet weight and yield) traits were moderate. Resistance against various bacteria, virus and parasites were moderately to highly heritable. Across aquatic animal species, the weighted heritability for a range of deformity measures and reproductive traits (fecundity, early survival) was low and not significantly different from zero. The common full-sibs (c2) accounted for a large proportion of total variance for body traits but it was of smaller magnitude in later phase of the growth development. The c2 effects however were not significant or in many cases they were not reported for carcass and flesh quality attributes as well as survival and deformity. The maternal genetic effects were not available for all traits studied especially for reproductive and early growth characters. Genetic correlations between body and carcass traits were high and positive, suggesting that selection for rapid growth can improve fillet weight, a carcass trait of paramount importance. Body weight, the most commonly used selection criterion in aquatic animals, showed non-significant genetic correlation with disease resistance, likely because both positive and negative genetic associations between the two types of traits. Interestingly the genetic associations between growth and reproductive performance (fecundity) and fry traits (fry weight, fry survival) were favourable. To date, there are still no published data on genetic relationships of carcass and flesh quality with disease resistance or reproductive performance in any aquaculture species. Additionally, the present study discussed new traits, including functional, immunological, behavioural and social interaction as well as uniformity that are emerging as potential selection criteria and which can be exploited in future genetic improvement programs for aquatic animals.

category: genetics


title: Personality profiling may help select better cleaner fish for sea-lice control in salmon farming

abstract: Lumpfish (Cyclopterus lumpus) are increasingly being used as cleaner fish to control parasitic sea-lice in salmon farming, but cleaning rates are very variable and not all individuals eat sea-lice, which increases the risk of emaciation and has ethical and practical implications. Selecting good cleaners is a priority to make the industry more sustainable, but there is little information on what behaviours make cleaner fish effective under a commercial setting. We examined variation in lumpfish personalities according to the five-factor personality model that takes into account differences in activity, anxiety (shelter use, thigmotaxis), aggression, sociality, and boldness (neophobia). We then quantified how variation in lumpfish personalities influenced interactions with naive Atlantic salmon (Salmo salar), without the confounding effects of variation in sea-lice loads. Variation in activity, sociality, aggression and neophobia, but not in anxiety, was repeatable, which is consistent with a heritable basis. Neophilic, non-aggressive lumpfish spent more time inspecting salmon than neophobic and aggressive individuals, but salmon fled in the presence of the most active and social individuals, suggesting there may be an optimal cleaner fish personality amenable to artificial selection. The personality screening protocols developed in this study could inform a more efficient use of cleaner fish in salmon farming and reduce the number of individuals required to control sea-lice.

category: animal behavior and cognition


title: Kmerator Suite: design of specific k-mer signatures andautomatic metadata discovery in large RNA-Seq datasets.

abstract: The huge body of publicly available RNA-seq libraries is a treasure of functional information allowing to quantify the expression of known or novel transcripts in tissues. However, transcript quantification commonly relies on alignment methods requiring a lot of computational resources and processing time, which does not scale easily to large datasets. K-mer decomposition constitutes a new way to process RNA-seq data for the identification of transcriptional signatures, as k-mers can be used to quantify accurately gene expression in a less resource-consuming way. We present the Kmerator Suite, a set of three tools designed to extract specific k-mer signatures, quantify these k-mers into RNA-seq datasets and quickly visualize large datasets characteristics. The core tool, Kmerator, produces specific k-mers for 97% of human genes, enabling the measure of gene expression with high accuracy in simulated datasets. KmerExploR, a direct application of Kmerator, uses a set of predictor genes specific k-mers to infer metadata including library protocol, sample features or contaminations from RNA-seq datasets. KmerExploR results are visualised through a user-friendly interface. Moreover, we demonstrate that the Kmerator Suite can be used for advanced queries targeting known or new biomarkers such as mutations, gene fusions or long non coding-RNAs for human health applications.

category: bioinformatics


title: On exploring effects of coevolving residues on DNA binding specificity of transcription factors

abstract: Transcription factors (TFs) regulate gene expression by specifically binding to DNA targets. Many factors have been revealed to influence TF-DNA binding specificity. Coevolution of residues in proteins occurs due to a common evolutionary history. However, it is unclear how coevolving residues in TFs contribute to DNA binding specificity. Here, we systematically analyzed TF-DNA interactions from high-throughput experiments for seven TF families, including Homeobox, HLH, bZIP_1, Ets, HMG_box, zf-C4 and Zn_clus TFs. Based on TF-DNA interactions, we detected TF subclass determining sites (TSDSs) defining the heterogeneity of DNA binding preference for each TF family. We showed that the TSDSs were more likely to be coevolving with TSDSs than with non-TSDSs, particularly for Homeobox, HLH, Ets, bZIP_1 and HMG_box TF families. Mutation of the highly coevolving residues could significantly reduce the stability of TF-DNA complex. The distant residues from the DNA interface also contributed to TF-DNA binding activity. Overall, our study gave evidence of the functional importance of coevolved residues in refining transcriptional regulation and provided clues to the application of engineered DNA-binding domains and protein.


title: Assessing and removing the effect of unwanted technical variations in microbiome data

abstract: Varying technologies and experimental approaches used in microbiome studies often lead to irreproducible results due to unwanted technical variations. Such variations, often unaccounted for and of unknown source, may interfere with true biological signals, resulting in misleading biological conclusions. In this work, we aim to characterize the major sources of technical variations in microbiome data and demonstrate how a state-of-the art approach can minimize their impact on downstream analyses. We analyzed 184 pig faecal metagenomes encompassing 21 specific combinations of deliberately introduced factors of technical and biological variations. We identify several known experimental factors, specifically storage conditions and freeze-thaw cycles, as a likely major source of unwanted variation in metagenomes. We also observed that these unwanted technical variations do not affect taxa uniformly, with freezing samples affecting taxa of class Bacteroidia the most, for example. Additionally, we benchmarked the performance of a novel batch correcting tool used in this study, RUV-III-NB (https://github.com/limfuxing/ruvIIInb/), to other popular batch correction methods, including ComBat, ComBat-seq, RUVg, and RUVs. While RUV-III-NB performed consistently robustly across our sensitivity and specificity metrics, most other methods did not remove unwanted variations optimally, with RUVg even overcorrecting and removing some of the true biological signals from the samples. Our analyses suggests that a careful consideration of possible technical confounders is critical in the experimental design of microbiome studies to ensure accurate biological reading of microbial taxa of interest, and that the inclusion of technical replicates is necessary to efficiently remove unwanted variations computationally.


title: Deep Neural Networks for Genomic Prediction Do Not Estimate Marker Effects

abstract: Genomic prediction is a promising technology for advancing both plant and animal breeding, with many different prediction models evaluated in the literature. It has been suggested that the ability of powerful nonlinear models such as deep neural networks to capture complex epistatic effects between markers offers advantages for genomic prediction. However, these methods tend not to outperform classical linear methods, leaving it an open question why this capacity to model nonlinear effects does not seem to result in better predictive capability. In this work, we propose the theory that, due to a principle called shortcut learning, deep neural networks tend to base their predictions on overall genetic relatedness, rather than on the effects of particular markers, such as epistatic effects. Using several datasets of crop plants (lentil, wheat, and Brassica carinata), we demonstrate the networks indifference to the values of the markers by showing that the same network, provided with only the locations of matches between markers for two individuals, is able to perform prediction to the same level of accuracy.


title: Decoding the effects of synonymous variants

abstract: Synonymous single nucleotide variants (sSNVs) are common in the human genome but are often overlooked. However, sSNVs can have significant biological impact and may lead to disease. Existing computational methods for evaluating the effect of sSNVs suffer from the lack of gold-standard training/evaluation data and exhibit over-reliance on sequence conservation signals. We developed synVep (synonymous Variant effect predictor), a machine learning-based method that overcomes both of these limitations. Our training data was a combination of variants reported by gnomAD (observed) and those unreported, but possible in the human genome (generated). We used positive-unlabeled learning to purify the generated variant set of any likely unobservable variants. We then trained two sequential extreme gradient boosting models to identify subsets of the remaining variants putatively enriched and depleted in effect. Our method attained 90% precision/recall on a previously unseen set of variants. Furthermore, although synVep does not explicitly use conservation, its scores correlated with evolutionary distances between orthologs in cross-species variation analysis. synVep was also able to differentiate pathogenic vs. benign variants, as well as splice-site disrupting variants (SDV) vs. non-SDVs. Thus, synVep provides an important improvement in annotation of sSNVs, allowing users to focus on variants that most likely harbor effects.


title: Validated machine learning algorithm with sub-clonal sensitivity reveals widespread pan-cancer human leukocyte antigen loss of heterozygosity

abstract: Human leukocyte antigen loss of heterozygosity (HLA LOH) allows cancer cells to escape immune recognition by deleting HLA alleles, causing the suppressed presentation of tumor neoantigens that would otherwise bind to them. Despite its importance in immunotherapy response, few methods exist to detect HLA LOH, and their accuracy is not well understood. Here, we develop DASH (Deletion of Allele-Specific HLAs), a novel machine learning-based algorithm to detect HLA LOH from paired tumor-normal sequencing data. Through validation with cell line mixtures and patient-specific digital PCR, we demonstrate increased sensitivity compared to previously published tools and pave the way for clinical utility. Using DASH on 611 patients across 15 tumor types, we found that 18% of patients had HLA LOH. Moreover, we show inflated HLA LOH rates compared to genome-wide LOH and correlations between CD274 (PD-L1) expression and MSI status, suggesting the HLA LOH is a key immune resistance strategy.


title: SC3s - efficient scaling of single cell consensus clustering to millions of cells

abstract: Technological advances have paved the way for single cell RNAseq (scRNAseq) datasets containing several million cells 1. Such large datasets require highly efficient algorithms to enable analyses at reasonable times and hardware requirements 2. A crucial step in single cell workflows is unsupervised clustering, which aims to delineate putative cell types or cell states based on transcriptional similarity 3. Here, we present a highly efficient k-means based approach, and we demonstrate that it scales linearly with the number of cells with regards to time and memory.


title: The Urtica Dioica Agglutinin Prevents Rabies Virus Infection in a Muscle Explant Model

abstract: Infection with the rabies virus (RABV) causes fatal encephalitis and paralysis in humans and animals. Post-exposure prophylaxis (PEP) consists of vaccination and the injection of anti-rabies immunoglobulins (RIGs) around the (bite) wound. This is 100% effective in preventing disease if administered in a timely manner. However, the costs, the required cold chain for storage and transport and the limited availability of RIGs makes the treatment challenging. Cheaper and easier to produce alternatives are urgently needed. To aid in the discovery and development of such alternative therapeutics, we developed a physiologically relevant infection model. Strips of freshly dissected swine skeletal muscle were placed under tension in culture medium and infected with RABV. Viral antigens were produced in the muscle explants and the virus production increased significantly over time, indicating that RABV infects and replicates in the muscle explants. Subsequently, in a search for inhibitors of RABV entry in muscle cells, we first screened a panel of 34 different lectins in a RABV / BHK-21J cell assay. The Urtica dioica (stinging nettle) Agglutinin (UDA; a N-acetyl-D-glucosamine specific agglutinin) was found to be able to completely inhibit infection of cells with the RABV (EC50 8.2 g/mL) by preventing binding of the virus to the host cell. When the infection of the muscle strips was carried out in the presence of UDA, infection of the tissue was completely prevented. We thus developed a physiologically relevant RABV muscle infection model and identified an easy to produce component that (i) may serve as a reference for further studies and (ii) holds promise as an alternative for RIGs in PEP.

category: pathology


title: Urinary Metabolomics from a Dose-Fractionated Polymyxin B Rat Model of Acute Kidney Injury

abstract:
BackgroundPolymyxin B remains an important antimicrobial against multi-drug resistant bacteria; however, kidney injury is often a treatment limiting event with kidney failure rates that range from 5-13%.

MethodsSamples were obtained from a previously conducted study of male Sprague-Dawley rats that received dose fractionated polymyxin B (12 mg/kg/day subcutaneously) once daily (QD), twice daily (BID), and thrice daily (TID) for three days. In the original study, urinary biomarkers and kidney histopathology scores were determined. Urine was sampled daily and analyzed for urinary metabolites via 1H NMR analysis.

Unsupervised Principal Components Analysis was applied for exploratory data analysis to identify trends and outliers in the spectral data. Then, supervised Orthogonal Partial Least Square Discriminant Analysis was applied to classify the samples collected in different days and identify metabolic differences during the treatment. Metabolomes were compared across study groups (i.e. those receiving QD, BID, TID, and control) using a mixed-effects models. Spearman correlation was performed for injury biomarkers and the metabolome.

ResultsA total of 27 rats contributed 77 urinary samples; n=25 rats were included that were treated with Polymyxin B and n=2 received saline. Pre-dosing samples clustered well and were characterized by higher amounts of citrate, 2-oxoglutarate, and Hippurate. On day 1 post treatment, day 1 samples showed higher taurine; day 3 samples had higher lactate, acetate and creatine. Taurine was the only metabolite significantly increased in both BID and TID compared to QD group. Taurine on day 1 correlated with increasing histopathology scores (Spearmans rho = 0.4167, P=0.038) and KIM-1 (Spearmans rho =0.4052, P=0.036); whereas KIM-1 on day one and day 3 did not reach significance with histopathology (Spearmans rho = 0.3248, P=0.11 and Spearmans rho = 0.3739, P=0.066).

ConclusionPolymyxin B causes increased amounts of urinary taurine on day 1 which then normalizes to baseline concentrations. Taurine may provide one of the earlier signals of acute kidney damage caused by polymyxin B.

category: pharmacology and toxicology


title: Endurance Exercise to Improve Physical Function in Adult and Older Mice: High Intensity Interval Training (HIIT) versus Voluntary Wheel Running (VWR)

abstract: With age comes a gradual decline in physical function and exercise capacity, concurrent with a progressive propensity for development of sarcopenia /(age-related loss of muscle mass and strength) and frailty (inability of body to thrive and maintain homeostasis). Prior research has demonstrated that exercise, while not a cure, can help mitigate sarcopenia/frailty and restore functional capacity. Reliable, validated, pre-clinical models are necessary to elucidate the underlying molecular mechanisms at the intersection of age, exercise, and functional decline. In this study, we hypothesized that endurance exercise programs mimicking typical human exercise protocols would improve physical function in both adult and older adult mice. Furthermore, our secondary hypothesis was that older mice would receive less benefit from a similar volume of exercise than adult mice. To test these hypotheses, we randomly assigned (with some selection criteria) male C57BL/6 mice either at adult ages during the study (n=24, designated 10m, aged 6 months to 10 months) or at older adult ages (n=18, designated 26m, aged 22 months to 26 months) to a 13-week program of voluntary wheel running (VWR, group termed RUN) or high intensity interval training (HIIT), with an additional 10m sedentary control (CON). The functional aptitude of each mouse was determined pre- and post-training using our composite CFAB (comprehensive functional assessment battery) scoring system consisting of voluntary wheel running (volitional exercise and activity rate), treadmill (endurance), rotarod (overall motor function), grip meter (forelimb strength), and inverted cling (whole body strength/endurance). To measure sarcopenia, we tracked body mass and body composition changes with EchoMRI, and measured muscle wet mass post-training.

category: physiology


title: PISCES: A pipeline for the Systematic, Protein Activity-based Analysis of Single Cell RNA Sequencing Data

abstract: While single-cell RNA sequencing provides a new window on physiologic and pathologic tissue biology and heterogeneity, it suffers from low signal-to-noise ratio and a high dropout rate at the individual gene level, thus challenging quantitative analyses. To address this problem, we introduce PISCES (Protein-activity Inference for Single Cell Studies), an integrated analytical framework for the protein activity-based analysis of single cell subpopulations. PISCES leverages the assembly of lineage-specific gene regulatory networks, to accurately measure activity of each protein based on the expression its transcriptional targets (regulon), using the ARACNe and metaVIPER algorithms, respectively. It implements novel analytical and visualization functions, including activity-based cluster analysis, identification of cell state repertoires, and elucidation of master regulators of cell state and cell state transitions, with full interoperability with Seurats single-cell data format. Accuracy and reproducibility assessment, via technical and biological validation assays and by assessing concordance with antibody and CITE-Seq-based measurements, show dramatic improvement in the ability to identify rare subpopulations and to assess activity of key lineage markers, compared to gene expression analysis.

category: systems biology


title: Brain-inspired Weighted Normalization for CNN Image Classification

abstract: AO_SCPLOWBSTRACTC_SCPLOWWe studied a local normalization paradigm, namely weighted normalization, that better reflects the current understanding of the brain. Specifically, the normalization weight is trainable, and has a more realistic surround pool selection. Weighted normalization outperformed other normalizations in image classification tasks on Cifar10, Imagenet and a customized textured MNIST dataset. The superior performance is more prominent when the CNN is shallow. The good performance of weighted normalization may be related to its statistical effect of gaussianizing the responses.

category: neuroscience


title: A semi-variance approach to visualising phylogenetic autocorrelation

abstract:
O_LIComparing traits across species has been a hallmark of biological research for centuries. While inter-specific comparisons can be highly informative, phylogenetic inertia can bias estimates if not properly accounted for in comparative analyses. In response, researchers typically treat phylogenetic inertia as a form of autocorrelation that can be detected, modelled, and corrected for. Despite the range of methods available for quantifying the strength of phylogenetic autocorrelation, no tools exist for visualising these autocorrelation structures.
C_LIO_LIHere we derive variogram methods suitable for phylogenic data, and show how they can be used to straightforwardly visualise phylogenetic autocorrelation. We then demonstrate their utility for three empirical examples: sexual size dimorphism (SSD) in the Musteloidea, maximum per capita rate of population growth, r, in the Carnivora, and brain size in the Artiodactyla.
C_LIO_LIWhen modelling musteloid SSD, the empirical variogram showed a tendency for the variance in SSD to stabilise over time, a characteristic feature of Ornstein-Uhlenbeck (OU) evolution. In agreement with this visual assessment, model selection identified the OU model as the best fit to the data. In contrast, the infinitely diffusive Brownian Motion (BM) model did not capture the asymptotic behaviour of the variogram and was less supported than the OU model. Phylogenetic variograms proved equally useful in understanding why an OU model was selected when modelling r in the Carnivora, and why BM was the selected evolutionary model for brain size in the Artiodactyla.
C_LIO_LIBecause the variograms of the various evolutionary processes each have different theoretical profiles, comparing fitted semi-variance functions against empirical semi-variograms can serve as a useful diagnostic tool, allowing researchers to understand why any given evolutionary model might be selected over another, which features are well captured by the model, and which are not. This allows for fitted models to be compared against the empirical variogram, facilitating model identification prior to subsequent analyses. We therefore recommend that any phylogenetic analysis begin with a non-parametric estimate of the autocorrelation structure of the data that can be visualized. The methods developed in this work are openly available in the new R package ctpm.
C_LI


title: Genetic load may increase or decrease with selfing depending upon the recombination environment

abstract: The ability of natural selection to remove deleterious mutations from a population is a function of the effective population size. Increases in selfing rate, and concomitant increases in population-level homozygosity, can increase or decrease the efficacy of selection, depending on the dominance and selection coefficients of the deleterious mutations. Most theory has focused on how (partial) selfing affects the efficacy of selection for mutations of a given dominance and fitness effect in isolation. It remains unclear how selfing affects the purging of deleterious mutations in a genome-wide context where mutations with different selection and dominance coefficients co-segregate. Here, we use computer simulations to investigate how mutation, selection and recombination interact with selfing rate to shape genome-wide patterns of genetic load. We recover various mechanisms previously described for how (partial) selfing affects the efficacy of selection against mutations of a given dominance class. However, we find that the interaction of purifying selection against mutations of different dominance classes changes with selfing rate. In particular, as outcrossing populations transition from purifying selection to pseudo-overdominance they experience a dramatic increase in the genetic load caused by additive, mildly deleterious mutations. We describe the threshold selfing rate that prevents pseudo-overdominance and decreases genetic load.


title: Golden pompano genome resource enables discovery of valuable gene determining growth traits

abstract: One important goal of fish genetic breeding is to identify valuable loci and genes that can facilitate growth and thereby productivity. Few such loci or genes have been identified in golden pompano (Trachinotus ovatus), a species of significant economic value. In this study, we produced a high-quality chromosome-level genome assembly of the golden pompano by de novo sequencing and assemblies for 2 parents and 200 F1 offspring by genome re-sequencing. We exploited these assemblies to identify loci and genes by QTL mapping, Kompetitive Allele Specific PCR (KASP) genotyping, and haplotype-based regional association analysis based on growth records of a 64 biparental and 147 individuals from a naturally occurring population. At a locus 291kb from BSNP21031, we identified a somatostatin receptor type 1-like (designated as gpsstr1) gene in which the BSNP1369 of the promoter region was highly associated with growth. Loss of sstr1a, the homolog of gpsstr1 in zebrafish, caused growth retardation. Sstr1a mediated growth via sstr2 and Wnt-gsk-3{beta} signaling pathways. Our findings provide new insights into the underlying mechanisms controlling growth. Our strategy can serve as an effective way to uncover novel genomic information and facilitate improvement of fish growth.


title: ASLPrep: A Generalizable Platform for Processing of Arterial Spin Labeled MRI and Quantification of Regional Brain Perfusion

abstract: Arterial spin labeled (ASL) magnetic resonance imaging (MRI) is the primary method for non-invasively measuring regional brain perfusion in humans. We introduce ASLPrep, a suite of software pipelines that ensure the reproducible and generalizable processing of ASL MRI data.

category: bioengineering


title: A Facile Immunopeptidomics Workflow for Capturing the HLA-I Ligandome with PEAKS XPro

abstract: Identifying antigens displayed specifically on tumour cell surfaces by human leukocyte antigen (HLA) proteins is important for the development of immunotherapies and cancer vaccines. The difficulty in capturing an HLA ligandome stems from the fact that many HLA ligands are derived from splicing events or contain mutations, hindering their identification in a standard database search. To address this challenge, we developed an immunopeptidomics workflow with PEAKS XPro that uses de novo sequencing to uncover such peptides and identifies mutations for neoantigen discovery. We demonstrate the utility of this workflow by re-analyzing HLA-I ligandome datasets and reveal a vast diversity in peptide sequences among clones derived from a colorectal cancer tumour. Over 8000 peptides predicted to bind HLA-I molecules were identified by de novo sequencing only (not found in the UniProt database) and make up over 50% of identified peptides from each sample. Lastly, tumour-specific mutations and consensus sequence motif characteristics are defined. This workflow is widely applicable to any immunopeptidomic mass spectrometry dataset and does not require custom database generation for neoantigen discovery.


title: MOFA: Multi-Objective Flux Analysis for the COBRA Toolbox

abstract:
Multi-objective Optimization (MO) is an important tool for quantitative examination of the trade-offs faced by biological organisms. Using genome-scale constraint-based models of metabolism (GSMs), Multi-Objective Flux Analysis (MOFA) allows MO analyses of trade-offs among key biological tasks. The leading software package for conducting a plethora of different types of constraint-based analyses using GSMs is the COBRA Toolbox for MATLAB. We have developed a new add-on tool for this toolbox using Normalized Normal Constraint (NNC) that performs MOFA for a number of objectives only limited by computation power (n [≤] 10). This development will facilitate MOFA analyses by COBRAs large user base and allow greater multi-faceted examination of metabolic trade-offs in complicated biological systems.

Availability and ImplementationThe MOFA software is freely available for download from https://bbs.llnl.gov under the GPL v2 license. The program runs on MATLAB with the COBRA software on Windows, Linux, and MacOS. It includes a detailed manual explaining the input and output of a simulation, a listing of the codes functions, and an example MOFA run using a well-curated GSM model of E. coli.

Contactgriesemer1@llnl.gov or navid1@llnl.gov


title: Cryptic diversity within the Poecilochirus carabi mite species complex phoretic on Nicrophorus burying beetles: phylogeny, biogeography, and host specificity

abstract: Coevolution is often considered a major driver of speciation, but evidence for this claim is not always found because diversity might be cryptic. When morphological divergence is low, molecular data are needed to uncover diversity. A taxon for which this holds true are the mites, which are known for their extensive and often cryptic diversity. We studied mites of the genus Poecilochirus that are phoretic on burying beetles (Silphidae: Nicrophorus). Poecilochirus taxonomy is poorly understood. Most studies on this genus focus on the evolutionary ecology of Poecilochirus carabi sensu lato, a complex of at least two biological species. Based on molecular data of 230 specimens from 43 locations worldwide, we identified 24 genetic clusters that may represent species. We estimate that these mites began to diversify during the Paleogene, when the clade containing P. subterraneus branched off and the remaining mites diverged into two further clades. One clade resembles P. monospinosus and P. austroasiaticus. The other clade contains 17 genetic clusters resembling P. carabi s.l.. Among these are P. carabi sensu stricto, P. necrophori, and potentially many additional cryptic species. Our analyses suggest that these clades were formed in the miocene by large-scale geographic separation. Diversification also seems to have happened on a smaller scale, potentially due to adaptation to specific hosts or local abiotic conditions, causing some clusters to specialize on certain beetle species. Our results suggest that biodiversity in this genus was generated by multiple interacting forces shaping the tangled webs of life.

category: zoology


title: Molecular basis for PICS-mediated piRNA biogenesis and cell division

abstract: By incorporating two mutually exclusive factors, PID-1 and TOST-1, C. elegans PICS complex plays important roles in piRNA biogenesis, chromosome segregation and cell division, respectively. We firstly mapped the interaction network between PICS subunits. By solving the several complex structures, including those of TOFU-6/PICS-1, ERH-2/PICS-1, and ERH-2/TOST-1, we uncover the mechanisms underlying the interactions between PICS subunits. Our biochemical experiment demonstrates that PICS exists as an octamer consisting of two copies of each subunits. Combining structural analyses with mutagenesis experiments, we identified residues of PICS subunits that are critical for maintaining intact PICS complex in vitro. Furthermore, using genetics, cell biology and imaging experiments, we found that those mutants impairing the in vitro interaction network within PICS, also lead to abnormal dysfunction PICS in vivo, including mislocalization of PICS, and reduced levels of piRNAs or abnormal chromosome segregation and cell division. Therefore, our work provides structural insights into understanding the PICS-mediated piRNA biogenesis and cell division.

category: molecular biology


title: Processing of the Ribosomal Ubiquitin-Like Fusion Protein FUBI-eS30/FAU is Required for 40S Maturation and Depends on USP36

abstract: In humans and other holozoan organisms, the ribosomal protein eS30 is synthesized as a fusion protein with the ubiquitin-like protein FUBI. However, FUBI is not part of the mature 40S ribosomal subunit and cleaved off by an as-of-yet unidentified protease. How FUBI-eS30 processing is coordinated with 40S subunit maturation is unknown. To study the mechanism and importance of FUBI-eS30 processing, we expressed non-cleavable mutants in human cells, which affected late steps of cytoplasmic 40S maturation, including the maturation of 18S rRNA and recycling of late-acting ribosome biogenesis factors. Differential affinity purification of wild-type and non-cleavable FUBI-eS30 mutants identified the deubiquitinase USP36 as a candidate FUBI-eS30 processing enzyme. Depletion of USP36 by RNAi or CRISPRi indeed impaired FUBI-eS30 processing and moreover, purified USP36 cut FUBI-eS30 in vitro. Together, these data demonstrate the functional importance of FUBI-eS30 cleavage and identify USP36 as a novel protease involved in this process.

category: cell biology


title: Morpho-functional characterization of the endo-lysosomal system by high-throughput correlative light-electron microscopy

abstract: Rab5, EEA1 and APPL1 are frequently used in fluorescence microscopy to mark early endosomes, whereas Rab7 is used as marker for late endosomes and lysosomes. However, since these proteins localize poorly in immuno-electron microscopy, systematic studies on their ultrastructural distributions are lacking. Here we address this gap by presenting a quantitative, high-throughput, on-section correlative light-electron microscopy (CLEM) approach using the sensitivity of fluorescence microscopy to infer label to hundreds of organelles classified by ultrastructure. We show that Rab5 predominantly marks small, endocytic vesicles and early endosomes. EEA1 co-localizes with Rab5 on especially early endosomes, but unexpectedly also labels Rab5-negative late endosomes and even lysosomes. APPL1 is restricted to small Rab5-positive, vesicular profiles without any visible content or ultrastructural marks. Rab7 primarily labels late endosomes and lysosomes. Our studies reveal the first ultrastructural distribution of key endosomal proteins at their endogenous levels and introduce CLEM as sensitive alternative for quantitative immuno-EM.


title: Cap-dependent translation initiation monitored in living cells

abstract: Despite extensive biochemical, genetic, and structural studies, a complete understanding of mRNA translation initiation is still lacking. Imaging methodologies able to resolve the binding dynamics of translation factors at single-cell and single-molecule resolution are necessary to fully elucidate regulation of this paramount process. We fused tags suitable for live imaging to eIF4E, eIF4G1 and 4E-BP1 without affecting their function. We combined Fluorescence Correlation Spectroscopy (FCS) and Single-Particle Tracking (SPT) to interrogate the binding dynamics of initiation factors to the 5cap. Both FCS and SPT were able to detect eIF4E:eIF4G1 binding to the mRNA in the cytoplasm of proliferating cells and neuronal processes. Upon inhibition of phosphorylation by mTOR, 4E-BP1:eIF4E complexes rapidly dissociated from the 5cap followed by eIF4G1 dissociation. Imaging of the binding dynamics of individual translation factors in living cells revealed the temporal regulation of translation at unprecedented resolution.


title: Generation of human chambered cardiac organoids from pluripotent stem cells for improved modelling of cardiovascular diseases

abstract:
Recent progress on murine and human cardiac organoids have provided understanding to the developmental processes of the heart. However, there is still an unfulfilled need for improved modelling of cardiovascular diseases using human cardiac organoids. Herein, we report successful generation of intrinsically formed human chambered cardiac organoids (CCO) and highlight its utility in modelling disease. Single cell transcriptomic profiling of CCOs showed appropriate cardiovascular cell type composition exhibiting improved maturation. Functionally, CCOs recapitulated clinical cardiac hypertrophy by exhibiting thickened chamber walls, reduced ejection fractions, increased myofibrillar disarray and tachycardia. Therefore, CCOs improve current capabilities of disease modelling as an in vitro model bridging the gap to in vivo models, with the ability to assess functional parameters that previously can only be achieved in animal systems.

One sentence summaryModelling cardiac hypertrophy using chambered cardiac organoids derived from human pluripotent stem cells.


title: Fission yeast polycystin Pkd2p promotes transition to cell growth during cytokinesis

abstract:
Polycystins are conserved mechanosensitive channels whose mutations lead to the common human renal disorder ADPKD. Previously we discovered that the plasma membrane-localized fission yeast homologue Pkd2p is an essential protein required for cytokinesis, but the mechanism remains unclear. Here, we isolated a novel temperature-sensitive mutant pkd2-B42. Among its strong growth defects, the most unique was that many mutant cells often lost significant portion of their volume in just 5 minutes followed by a gradual recovery, a process that we termed Deflation. Unlike cell lysis, deflation did not result in the plasma membrane rupture and it occurred independently from the cell cycle progression. The tip extension of pkd2-B42 cells was 80% slower than the wild type and their turgor pressure was 50% lower. Both pkd2-B42 and the other mutant pkd2-81KD partially rescued the mutants of the yeast Hippo signaling pathway Septation Initiation Network, by preventing cell lysis, enhancing septum formation, and doubling the number of Sid2/Mob1 molecules at the spindle pole bodies. We conclude that Pkd2p promotes cell size expansion during interphase by regulating turgor pressure and antagonizes SIN during cytokinesis.

Summary statementMutations of polycystins lead to human genetic disorder ADPKD. We discovered that the fission yeast homologue Pkd2p promotes the cell expansion during interphase growth and antagonizes the Hippo pathway SIN during cytokinesis.


title: Fission yeast polycystin Pkd2p promotes the cell expansion and antagonizes the Hippo pathway SIN


title: Integration of mouse ovary morphogenesis with developmental dynamics of the oviduct, ovarian ligaments, and rete ovarii.

abstract: The morphogenetic events that occur during fetal development of the mammalian ovary are crucial to the establishment of adult female fertility. While our knowledge of the cellular and molecular aspects of ovary development is increasing, the structural rearrangements that give rise to the cortex and medulla and the relation of other female reproductive tissues to the ovary have not been thoroughly investigated. In this study, we used tissue clearing and lightsheet microscopy to investigate the stepwise morphogenesis of the ovary. We found that the ovary transitioned from an elongated to a crescent-shaped structure, suggestive of a folding mechanism. As this occured, the ovarian surface epithelium wrapped from the ventral to the dorsal side of the ovary, forming the ovarian cortex and engulfing the dorsal face of the ovary to form the medulla. To identify the tissue mechanics that drive ovary folding, we investigated proximate tissues closely associated with the ovary including the Mullerian duct, the cranial suspensory ligament, and the rete ovarii. We found that relocation of the Mullerian duct to the ventral aspect of the ovary was associated with expansion of mesonephric tissue that left the ovary fully encapsulated by birth. The cranial suspensory ligament, which tethers the ovary to the body wall and to the Mullerian duct may exert mechanical tension that triggers ovarian folding. Finally, we found that the rete ovarii, a previously dismissed epithelial appendage of the ovary, significantly expanded during late gestation and may act to anchor the ovary to the mesonephros, leading to integration of extrinsic components into the ovarian medulla. This detailed atlas of ovary morphogenesis reveals novel relationships among the ovary and its surrounding tissues and paves the way towards the functional investigation of the relationship between architecture and differentiation of the mammalian ovary.

category: developmental biology


title: Impact of Type II LRRK2 inhibitors on signalling and mitophagy

abstract: Much effort has been devoted to the development of selective inhibitors of the LRRK2 as a potential treatment for LRRK2 driven Parkinsons disease. In this study we first compare the properties of Type I (GSK3357679A and MLi-2) and Type II (GZD-824, Rebastinib and Ponatinib) kinase inhibitors that bind to the closed or open conformations of the LRRK2 kinase domain, respectively. We show that Type I and Type II inhibitors suppress phosphorylation of Rab10 and Rab12, key physiological substrates of LRRK2 and also promote mitophagy, a process suppressed by LRRK2. Type II inhibitors also display higher potency towards wild type LRRK2 compared to pathogenic mutants. Unexpectedly, we find that Type II inhibitors, in contrast to Type I compounds, fail to induce dephosphorylation of a set of well-studied LRRK2 biomarker phosphorylation sites at the N-terminal region of LRRK2, including Ser935. These findings emphasize that the biomarker phosphorylation sites on LRRK2 are likely reporting on the open vs closed conformation of LRRK2 kinase and that only inhibitors which stabilize the closed conformation induce dephosphorylation of these biomarker sites. Finally, we demonstrate that the LRRK2[A2016T] mutant which is resistant to MLi-2 Type 1 inhibitor, also induces resistance to GZD-824 and Rebastinib suggesting this mutation could be exploited to distinguish off target effects of Type II inhibitors. Our observations provide a framework of knowledge to aide with the development of more selective Type II LRRK2 inhibitors.


title: Inference of Granger-causal relations in molecular systems -- a case study of the functional hierarchy among actin regulators in lamellipodia

abstract: Many cell regulatory systems implicate significant nonlinearity and redundancy among components. The regulatory network governing the formation of lamellipodial and lamellar actin structures is prototypical of such a system, containing tens of actin-nucleating and -modulating molecules with strong functional overlap. Due to instantaneous compensation, the strategy of phenotyping the system response to perturbation of individual components provides limited information on the roles the targeted component plays in the unperturbed system. Accordingly, despite the very rich data on lamellipoidial actin assembly, we have an incomplete understanding of how individual actin regulators contribute to lamellipodial dynamics. Here, we present a case study of perturbation-free reconstruction of cause-effect relations among actin regulators, applying the framework of Granger-causal inference to constitutive image fluctuations that indicate regulator recruitment and dissociation. Our analysis defines distinct active zones for actin regulators within the lamellipodia and lamella and establishes actin-dependent and actin-independent causal relations with actin filament assembly and edge motion. We demonstrate the specificity and sensitivity of the analysis and propose that edge motion is driven by assembly of two independently operating actin filament structures.


title: Multiview super-resolution microscopy

abstract: We enhance the performance of confocal microscopy over imaging scales spanning tens of nanometers to millimeters in space and milliseconds to hours in time, improving volumetric resolution more than 10-fold while simultaneously reducing phototoxicity. We achieve these gains via an integrated, four-pronged approach: 1) developing compact line-scanners that enable sensitive, rapid, diffraction-limited imaging over large areas; 2) combining line-scanning with multiview imaging, developing reconstruction algorithms that improve resolution isotropy and recover signal otherwise lost to scattering; 3) adapting techniques from structured illumination microscopy, achieving super-resolution imaging in densely labeled, thick samples; 4) synergizing deep learning with these advances, further improving imaging speed, resolution and duration. We demonstrate these capabilities on more than twenty distinct fixed and live samples, including protein distributions in single cells; nuclei and developing neurons in Caenorhabditis elegans embryos, larvae, and adults; myoblasts in Drosophila wing imaginal disks; and mouse renal, esophageal, cardiac, and brain tissues.


title: Multi-modal adaptor-clathrin contacts drive coated vesicleassembly

abstract: Clathrin-coated pits are formed by the recognition of membrane and cargo by the heterotetrameric AP2 complex and the subsequent recruitment of clathrin triskelia. A potential role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the {beta}2 subunit of AP2, and several binding sites on {beta}2 and on the clathrin heavy chain have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of {beta}2 hinge and appendage ({beta}2HA) domains. We find that the {beta}2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, {beta}2-appendage cross-links two adjacent terminal domains from different triskelia below the vertex. Functional analysis of {beta}2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the "sandwich site" on the appendage, with the appendage "platform site" having less importance. From these studies and the work of others, we propose that {beta}2-appendage binding to more than one clathrin triskelion is a key feature of the system and likely explains why clathrin assembly is driven by AP2.

category: biochemistry


title: Tumor-secreted versican co-opts myeloid IKKβ during metastasis

abstract: The mechanisms tumor cells use to hijack the immune system are largely uncharted. Here we used bioluminescent nuclear factor (NF)-{kappa}B reporter mice and macrophages to discover that metastatic tumors trigger NF-{kappa}B activation in host macrophages, dependent on mutant KRAS signaling and delivered via secretory versican. Versican activates NF-{kappa}B in tumor-associated macrophages via inhibitor of NF-{kappa}B kinase (IKK) {beta}, resulting in release of interleukin (IL)-1{beta} into the tumor microenvironment. Versican silencing in cancer cells or conditional IKK{beta} deletion in macrophages prevents myeloid NF-{kappa}B activation and metastasis. Versican is overexpressed and/or mutated in human cancers and metastatic effusions with KRAS mutations, predicts poor survival, can aid in the development of diagnostic platforms for pleural metastasis, and is druggable via toll-like receptor (TLR) 1/2 inhibition. The data indicate a cardinal role for tumor-derived versican in establishing cross-talk with macrophage IKK{beta} during metastasis and may foster the development of new therapies and diagnostic tools.

category: cancer biology


title: Circulating proteins reveal prior use of menopausal hormonal therapy and increased risk of breast cancer

abstract:
BackgroundRisk prediction is crucial for early detection and prognosis of breast cancer. Circulating plasma proteins could provide a valuable source to increase the validity of risk prediction models, however, no such markers have yet been identified for clinical use.

MethodsEDTA plasma samples from 183 breast cancer cases and 366 age-matched controls were collected prior to diagnosis from the Swedish breast cancer cohort KARMA. The samples were profiled on 700 circulating proteins using an exploratory affinity proteomics approach. Linear association analyses were performed on case-control status and a data-driven analysis strategy was applied to cluster the women on their plasma proteome profiles in an unsupervised manner. The resulting clusters were subsequently annotated for the differences in phenotypic characteristics, clinical parameters, and genetic risk.

ResultsUsing the data-driven approach we identified five clusters with distinct proteomic plasma profiles. Women in a particular sub-group (cluster 1) were significantly more likely to have used menopausal hormonal therapy (MHT), more likely to get a breast cancer diagnosis, and were older compared to the remaining clusters. The levels of circulating proteins in cluster 1 were decreased for proteins related to DNA repair and cell replication and increased for proteins related to mammographic density and female tissues. In contrast, classical dichotomous case-control analyses did not reveal any proteins significantly associated with future breast cancer.

ConclusionUsing a data-driven approach, we identified a subset of women with circulating proteins associated with previous use of MHT and risk of breast cancer. Our findings point to the potential long-lasting effects of MHT on the circulating proteome even after ending the treatment, and hence provide valuable insights concerning risk predication of breast cancer.

HighlightsO_LICurrent risk prediction models use a variety of factors to identify women at risk of developing breast cancer.
C_LIO_LIProteins circulating in blood represent an attractive but currently still underrepresented source of candidates serving as molecular risk factors.
C_LIO_LIPlasma proteomes from women participating in a prospective breast cancer cohort study were studied for proteomic risk factors related to a future breast cancer diagnosis.
C_LIO_LIUsing data-driven approaches, women with future breast cancers and previous use of menopausal hormone therapy were identified based on their circulating proteins.
C_LIO_LIMenopausal hormone therapy was found to altered the levels of the circulating proteins even years after the treatment ended.
C_LI


title: Glycerol monolaurate ameliorated intestinal barrier and immunity in broilers by regulating intestinal inflammation, antioxidant balance, and intestinal microbiota

abstract:
Extensive interactions occur between a poultry host and its gut microbiome. Glycerol monolaurate (GML) possesses a large range of antimicrobial and immunoregulatory properties. This study was conducted to investigate the impact of different doses of GML (basal diets complemented with 0, 300, 600, 900, or 1200 mg/kg GML) on growth performance, intestinal barrier, and cecal microbiota in broiler chicks. Results revealed that feed intake increased after 900 and 1200 mg/kg GML were administered during the entire 14-day experiment period. Dietary GML decreased crypt depth and increased the villus height-to-crypt depth ratio of the jejunum. In the serum and jejunum, supplementation with more than 600 mg/kg GML reduced interleukin-1{beta}, tumor necrosis factor-, and malondialdehyde levels and increased the levels of immunoglobulin G, jejunal mucin 2, total antioxidant capacity, and total superoxide dismutase. GML down-regulated jejunal interleukin-1{beta} and interferon-{gamma} expression and increased the mRNA level of zonula occludens 1 and occludin. A reduced expression of toll-like receptor 4 and a tendency of down-regulated nuclear factor kappa-B was shown in GML-treated groups. In addition, GML modulated the composition of the cecal microbiota of the broilers, improved microbial diversity, and increased the abundance of butyrate-producing bacteria. Spearmans correlation analysis revealed that the genera Barnesiella, Coprobacter, Lachnospiraceae, Faecalibacterium, Bacteroides, Odoriacter, and Parabacteroides were related to inflammation and intestinal integrity. In conclusion, GML ameliorated intestinal morphology and barrier function in broiler chicks probably by regulating intestinal immune and antioxidant balance, as well as intestinal microbiota.

IMPORTANCEAntibiotic residues and resistance issues led to the ban of antibiotic growth promoters. GML is considered an efficacious antibiotic growth promoter alternative for animal health and has the potential to become a unique fungicide owing to its established safety, antibacterial properties, and immunomodulatory capacity. Despite the potential of GML as an additive in poultry feed, little is known about the influence of GML on cecal microbiota in broilers. The significance of our research was to determine the microbial mechanism by which GML worked.


title: Syntaxin-1 is necessary for UNC5/Netrin-1-dependent macropinocytosis and chemorepulsion

abstract: Brain connectivity requires correct axonal guidance to drive axons to their appropriate targets. This process is orchestrated by guidance cues that exert attraction or repulsion to developing axons. However, the intricacies of the cellular machinery responsible for the correct response of growth cones are just being unveiled. Netrin-1 is a bifunctional molecule involved in axon pathfinding and cell migration that induces repulsion during postnatal cerebellar development. This process is mediated by Uncoordinated locomotion 5 (UNC5) receptors located on external granule layer (EGL) tracts. Here, we demonstrate that this response is characterized by enhanced membrane internalization through macropinocytosis, but not clathrin-mediated endocytosis. We show that UNC5 receptors form a protein complex with the t-SNARE syntaxin-1 (Stx1). By combining botulinum neurotoxins, a shRNA knock-down strategy and Stx1 knock-out mice, we demonstrate that this SNARE protein is required for Netrin-1-induced macropinocytosis and chemorepulsion, suggesting that Stx1 is crucial in regulating Netrin-1-mediated axonal guidance.


title: Neutrophil extracellular traps arm DC vaccination against NPM-mutant myeloproliferation

abstract:
Neutrophil extracellular traps (NET) are web-like chromatin structures composed by dsDNA and histones, decorated with anti-microbial proteins. Their interaction with dendritic cells (DC) allows DC activation and maturation toward presentation of NET-associated antigens. Differently from other types of cell death that imply protein denaturation, NETosis preserves the proteins localized onto the DNA threads for proper enzymatic activity and conformational status, including immunogenic epitopes. Besides neutrophils, leukemic cells can release extracellular traps displaying leukemia-associated antigens, prototypically mutant nucleophosmin (NPMc+) that upon mutation translocates from nucleolus to the cytoplasm localizing onto NET threads. We tested NPMc+ immunogenicity through a NET/DC vaccine to treat NPMc-driven myeloproliferation in transgenic and transplantable models. Vaccination with DC loaded with NPMc+ NET (NPMc+ NET/DC) reduced myeloproliferation in transgenic mice, favoring the development of antibodies to mutant NPMc and and the induction of a CD8+ T cell response. The efficacy of this vaccine was also tested in mixed NPMc/WT bone marrow chimeras in a competitive bone marrow transplantation setting, where the NPMc+ NET/DC vaccination impaired the expansion of NPMc+ in favor of WT myeloid compartment. NPMc+ NET/DC vaccination also achieved control of an aggressive leukemia transduced with mutant NPMc, effectively inducing an anti-leukemia CD8 T cell memory response.

eTOC SummaryNPMc is an AML-triggering mutation inducing translocation of NPM into the cytoplasm, allowing its association to NET threads. On NET, NPMc becomes immunogenic acting as an alarmin and exposing immunogenic peptides. We demonstrate that NET can be exploited as anti-tumor weapons in DC-based vaccination strategies.

category: immunology


title: DJ-1 depletion slows down immunoaging in T-cell compartments

abstract: Decline in immune function during aging increases susceptibility to different aging related diseases. However, the underlying molecular mechanisms, especially the genetic factors contributing to imbalance of naive/memory T-cell subpopulations, still remain largely elusive. Here we show that loss of DJ-1 encoded by PARK7/DJ-1, causing early-onset familial Parkinsons disease (PD), unexpectedly delayed immunoaging in both human and mice. Compared with two gender-matched unaffected sibling carriers of similar ages, the index PD patient with DJ-1 deficiency showed a decline in many critical immunoaging features, including almost doubled frequencies of non-senescent T cells. The observation of a  younger immune system in the index patient was further consolidated by the results in aged DJ-1 knockout mice. Our data from bone marrow chimera models and adoptive transfer experiments demonstrated that DJ-1 regulates several immunoaging features via hematopoietic-intrinsic and naive-CD8-intrinsic mechanisms. Our finding suggests an unrecognized critical role of DJ-1 in regulating immunoaging, discovering a potent target to interfere with immunoaging- and aging-associated diseases.


title: Repurposing of the tamoxifen metabolites to treat methicillin-resistant Staphylococcus epidermidis and vancomycin-resistant Enterococcus faecalis infections

abstract:
Repurposing drugs provides a new approach to the fight against multidrug-resistant (MDR) bacteria. We have reported that three major tamoxifen metabolites, N-desmethyltamoxifen (DTAM), 4-hydroxytamoxifen (HTAM) and endoxifen (ENDX), presented bactericidal activity against Acinetobacter baumannii and Escherichia coli. Here, we aimed to analyse the activity of a mixture of the three tamoxifen metabolites against methicillin-resistant Staphylococcus epidermidis (MRSE) and Enterococcus spp.

MRSE (n=17) and Enterococcus spp. (E. faecalis n=8, and E. faecium n=10) strains were used. MIC of the mixture of DTAM, HTAM and ENDX, and vancomycin were determined by microdilution assay. The bactericidal activity of the three metabolites together and vancomycin against MRSE (SE385 and SE742) and vancomycin-resistant E. faecalis (EVR1 and EVR2) strains was determined by time-kill curve assays. Finally, changes in membrane permeability of SE742 and EVR1 strains were analyzed using fluorescence assays.

MIC50 and MIC90 of tamoxifen metabolites were 1 mg/L for MRSE strains and 2 mg/L for Enterococcus spp. strains. In the time-killing assays, tamoxifen metabolites mixture showed bactericidal activity at 2x and 4xMIC for MRSE (SE385 and SE742) and E. faecalis (EVR1 and EVR2) strains. This antimicrobial activity of tamoxifen metabolites paralleled an increased membrane permeability of SE385 and EVR2 strains.

Altogether, these results showed that tamoxifen metabolites presented antibacterial activity against MRSE and vancomycin-resistant E. faecalis, suggesting that tamoxifen metabolites might increase the arsenal of drugs treatment against these bacterial pathogens.

category: microbiology


title: Macrophage-produced peroxynitrite induces antibiotic tolerance and supersedes intrinsic mechanisms of persister formation

abstract: Staphylococcus aureus is a leading human pathogen that frequently causes chronic and relapsing infections. Antibiotic tolerant persister cells contribute to frequent antibiotic failure in patients. Macrophages represent an important niche during S. aureus bacteremia and recent work has identified a role for oxidative burst in the formation of antibiotic tolerant S. aureus. We find that host-derived peroxynitrite, the reaction product of superoxide and nitric oxide, is the main mediator of antibiotic tolerance in macrophages. Using a collection of S. aureus clinical isolates, we find that, despite significant variation in persister formation in pure culture, all strains were similarly enriched for antibiotic tolerance following internalization by activated macrophages. Our findings suggest that host interaction strongly induces antibiotic tolerance and may negate bacterial mechanisms of persister formation, established in pure culture. These findings emphasize the importance of studying antibiotic tolerance in the context of bacterial interaction with the host suggest that modulation of the host response may represent a viable therapeutic strategy to sensitize S. aureus to antibiotics.


title: Screening of chemical libraries for new antifungal drugs against Aspergillus fumigatus reveals the potential mechanism of action of miltefosine

abstract:
Aspergillus fumigatus is an important fungal pathogen and the main etiological agent of aspergillosis, a disease characterized by a noninvasive process that can evolve to a more severe clinical manifestation called invasive pulmonary aspergillosis (IPA) in immunocompromised patients. The antifungal arsenal to threat aspergillosis is very restricted. Azoles are the main therapeutic approach to control IPA, but the emergence of azole-resistant A. fumigatus isolates has significantly increased over the last decades. Therefore, new strategies are necessary to combat aspergillosis and drug repurposing has emerged as an efficient and alternative approach for identifying new antifungal drugs. Here, we used a screening approach to analyze A. fumigatus in vitro susceptibility to 1,127 compounds. A. fumigatus was more susceptible to 10 compounds, including miltefosine, a drug that displayed fungicidal activity against A. fumigatus. By screening an A. fumigatus transcription factor null library, we identified a single mutant, which has the rmiA (resistant to miltefosine) gene deleted, conferring a phenotype of susceptibility to miltefosine. The transcriptional profiling (RNA-seq) of the wild-type and the {Delta}rmiA strains and the Chromatin Immunoprecipitation coupled to next generation sequencing (ChIP-Seq) of a RmiA-tagged strain exposed to miltefosine revealed genes of the sphingolipids pathway that are directly or indirectly regulated by RmiA. Sphingolipids analysis demonstrated that the mutant has overall decreased levels of sphingolipids when growing in the presence of miltefosine. The identification of RmiA represents the first genetic element described and characterized which plays a direct role in miltefosine response in fungi.

Author summaryThe filamentous fungus Aspergillus fumigatus causes a group of diseases named aspergillosis and their development occurs after the inhalation of conidia dispersed in the environment. Very few classes of antifungal drugs are available for aspergillosis treatment, e.g., azoles, but the emergence of global resistance to azoles in A. fumigatus clinical isolates has increased over the last decades. Repositioning or repurposing drugs already available on the market is an interesting and faster opportunity for the identification of novel antifungals agents. By using a repurposing strategy, we identified 10 different compounds that impact A. fumigatus survival. One of these compounds, miltefosine, demonstrated fungicidal activity against A. fumigatus. The mechanism of action of miltefosine is unknown and aiming to get more insights about it, we identified a transcription factor RmiA (Resistant to miltefosine) important for miltefosine resistance. Our results suggest that miltefosine plays antifungal activity against A. fumigatus interfering in the sphingolipids biosynthesis.


title: Application of transposon-insertion sequencing to determine gene essentiality in the acetogen Clostridium autoethanogenum

abstract:
The majority of the genes present in bacterial genomes remain poorly characterised with up to one third of those that are protein encoding having no definitive function. Transposon insertion sequencing represents a high-throughput technique that can help rectify this deficiency. The technology, however, can only be realistically applied to easily transformable species leaving those with low DNA-transfer rates out of reach. Here we have developed a number of approaches that overcome this barrier in the autotrophic species Clostridium autoethanogenum using a mariner-based transposon system. The inherent instability of such systems in the Escherichia coli conjugation donor due to transposition events was counteracted through the incorporation of a conditionally lethal codA marker on the plasmid backbone. Relatively low frequencies of transformation of the plasmid into C. autoethanogenum were circumvented through the use of a plasmid that is conditional for replication coupled with the routine implementation of an Illumina library preparation protocol that eliminates plasmid-based reads. A transposon library was then used to determine the essential genes needed for growth using carbon monoxide as a sole carbon and energy source.

IMPORTANCEAlthough microbial genome sequences are relatively easily determined, assigning gene function remains a bottleneck. Consequently, relatively few genes are well characterised, leaving the function of many as either hypothetical or entirely unknown. High-throughput, transposon sequencing can help remedy this deficiency, but is generally only applicable to microbes with efficient DNA-transfer procedures. These exclude many microorganisms of importance to humankind either as agents of disease or as industrial process organisms. Here we developed approaches to facilitate transposon-insertion sequencing in the acetogen Clostridium autoethanogenum, a chassis being exploited to convert single-carbon waste gases, CO and CO2, into chemicals and fuels at an industrial scale. This allowed the determination of gene essentiality under heterotrophic and autotrophic growth providing insights into the utilisation of CO as a sole carbon and energy source. The strategies implemented are translatable and will allow others to apply transposon-insertion sequencing to other microbes where DNA-transfer has until now represented a barrier to progress.


title: Proteome, bioinformatic and functional analyses reveal a distinct and conserved metabolic pathway for bile salt degradation in the Sphingomonadaceae

abstract:
Bile salts are amphiphilic steroids with a C5 carboxylic side chain with digestive functions in vertebrates. Upon excretion, they are degraded by environmental bacteria. Degradation of the bile-salt steroid skeleton resembles the well-studied pathway for other steroids like testosterone, while specific differences occur during side-chain degradation and the initiating transformations of the steroid skeleton. Of the latter, two variants via either {Delta}1,4- or {Delta}4,6-3-ketostructures of the steroid skeleton exist for 7-hydroxy bile salts. While the {Delta}1,4- variant is well-known from many model organisms, the {Delta}4,6-variant involving a 7-hydroxysteroid dehydratase as key enzyme has not been systematically studied. Here, combined proteomic, bioinformatic and functional analyses of the {Delta}4,6-variant in Sphingobium sp. strain Chol11 were performed. They revealed a degradation of the steroid rings similar to the {Delta}1,4-variant except for the elimination of the 7-OH as key difference. In contrast, differential production of the respective proteins revealed a putative gene cluster for side-chain degradation encoding a CoA-ligase, an acyl-CoA dehydrogenase, a Rieske monooxygenase, and an amidase, but lacking most canonical genes known from other steroid-degrading bacteria. Bioinformatic analyses predicted the {Delta}4,6-variant to be widespread among the Sphingomonadaceae, which was verified for three type strains which also have the predicted side-chain degradation cluster. A second amidase in the side-chain degradation gene cluster of strain Chol11 was shown to cleave conjugated bile salts while having low similarity to known bile-salt hydrolases. This study signifies members of the Sphingomonadaceae remarkably well-adapted to the utilization of bile salts via a partially distinct metabolic pathway.

ImportanceThis study highlights the biochemical diversity of bacterial degradation of steroid compounds, in particular bile salts. Furthermore, it substantiates and advances knowledge of a variant pathway for degradation of steroids by sphingomonads, a group of environmental bacteria that are well-known for their broad metabolic capabilities. Biodegradation of bile salts is a critical process due to the high input of these compounds from manure into agricultural soils and wastewater treatment plants. In addition, these results may also be relevant for the biotechnological production of bile salts or other steroid compounds with pharmaceutical functions.


title: Rapid evolution of weedy traits during sunflower de-domestication: the importance of hybridization and standing genetic variation

abstract: Hybridization between crops and their wild relatives may promote the evolution of de-domesticated (feral) weeds. Wild sunflower (Helianthus annuus L.) is typically found in ruderal environments, but crop-wild hybridization may facilitate the evolution of weedy populations. Using one crop-specific mitochondrial marker (CMS-PET1) and 14 nuclear SSR markers, we studied the origin and genetic diversity of a recently discovered weedy population of sunflower (named BRW). Then, using a resurrection approach, we tested for rapid evolution of weedy traits (seed dormancy, herbicide resistance, and competitive ability) by sampling weedy and wild populations 10 years apart (2007 and 2017). All the weedy plants present the CMS-PET1 cytotype, confirming their feral origin. At the nuclear markers, BRW showed higher genetic diversity than the cultivated lines and low differentiation with one wild population, suggesting that wild hybridization increased their genetic diversity. We found support for rapid evolution towards higher seed dormancy, but not for higher competitive ability or herbicide resistance. Our results highlight the importance of seed dormancy during the earliest stages of adaptation and show that crop-wild hybrids can evolve quickly in agricultural environments.

category: plant biology


title: Rapid evolution of seed dormancy during sunflower de-domestication


title: Comparison of the neuraminidase antigenicity in recently circulating influenza A and vaccine viruses

abstract: Neuraminidase (NA or N) antigens in circulating influenza viruses are not extensively evaluated for vaccine strain selection like hemagglutinin (HA or H) even though viral-based influenza vaccines include the recommended strain NA in varying amounts. As NA can also elicit a protective response, we assessed the antigenic similarity of the NAs from human H1N1 and H3N2 viruses that were prevalent between September 2019 to December 2020 to NAs from several recently recommended vaccine strains. To eliminate the dependence on isolates, the enzyme-linked lectin assay for analyzing NA antigenicity was performed with reverse genetic viruses carrying the same HA. Our results show that ferret antisera against NAs from the recommended H1N1 and H3N2 vaccine strains for the 2020-21 northern hemisphere influenza season recognize and inhibit the most prevalent circulating N1s and N2s, suggesting the NAs from the influenza A vaccine and circulating strains are antigenically similar. Comparisons of the recent N2s also revealed a bias in the reactivity of NA antisera from the egg and cell-based H3N2 vaccine strains due to a C-terminal substitution, indicating the C-terminus can influence N2 antigenicity and should receive consideration during the H3N2 strain selection.


title: DegP initiates regulated processing of filamentous hemagglutinin in Bordetella bronchiseptica

abstract:
Filamentous hemagglutinin (FhaB) is a critical virulence factor for both Bordetella pertussis, the causal agent of whooping cough, and the closely related species Bordetella bronchiseptica. FhaB is an adhesin, suppresses inflammatory cytokine production, and protects against phagocytic cell clearance during infection. Regulated degradation of the FhaB C-terminal prodomain is required to establish a persistent infection in mice. Two proteases, CtpA in the periplasm and SphB1 on the bacterial surface, are known to mediate FhaB processing, and we recently determined that CtpA functions before, and controls the FhaB cleavage site of, SphB1. However, the data indicate that another periplasmic protease must initiate degradation of the prodomain by removing a portion of the FhaB C terminus that inhibits CtpA-mediated degradation. Using a candidate approach, we identified DegP as the initiating protease. Deletion of degP or substitution of its predicted catalytic residue resulted in reduced creation of FHA', the main product of FhaB processing, and an accumulation of full-length FhaB in whole cell lysates. Also, FHA' was no longer released into culture supernatants in degP mutants. Alterations of the FhaB C terminus that relieve inhibition of CtpA abrogate the need for DegP, consistent with DegP functioning prior to CtpA in the processing pathway. DegP is not required for secretion of FhaB through FhaC or for adherence of the bacteria to host cells, indicating that DegP acts primarily as a protease and not a chaperone for FhaB in B. bronchiseptica. Our results highlight a role for HtrA family proteases in activation of virulence factors in pathogenic bacteria.

ImportanceTwo-partner secretion (TPS) systems are broadly distributed among Gram-negative bacteria and play important roles in bacterial pathogenesis. FhaB-FhaC is the prototypical member of the TPS family and here we identified the protease that initiates a processing cascade that controls FhaB function. Our results are significant because they provide insight into the molecular mechanism underlying the ability of Bordetella species to prevent clearance by phagocytic cells, which is critical for bacterial persistence in the lower respiratory tract. Our findings also highlight an underappreciated role for HtrA family proteases in processing specific bacterial virulence factors.


title: Plant pathogenic bacterium can rapidly evolve tolerance to an antimicrobial plant allelochemical

abstract: Crop losses to plant pathogens are a growing threat to global food security and more effective control strategies are urgently required. Biofumigation, an agricultural technique where Brassica plant tissues are mulched into soils to release antimicrobial plant allelochemicals called isothiocyanates (ITCs), has been proposed as an environmentally friendly alternative to agrochemicals. While biofumigation has been shown to suppress a range of plant pathogens, its effects on plant pathogenic bacteria remain largely unexplored. Here we used a laboratory model system to compare the efficacy of different types of ITCs against Ralstonia solanacearum plant bacterial pathogen. Additionally, we evaluated the potential for ITC-tolerance evolution under high, intermediate and low transfer frequency ITC exposure treatments. We found that allyl-ITC was the most efficient compound at suppressing R. solanacearum growth, and its efficacy was not improved when combined with other types of ITCs. Despite consistent pathogen growth suppression, ITC tolerance evolution was observed in the low transfer frequency exposure treatment, leading to cross-tolerance to ampicillin beta-lactam antibiotic. Mechanistically, tolerance was linked to insertion sequence movement at four positions in genes that were potentially associated with stress responses (H-NS histone like protein), cell growth and competitiveness (acyltransferase), iron storage ((2-Fe-2S)-binding protein) and calcium ion sequestration (calcium-binding protein). Interestingly, pathogen adaptation to the growth media also indirectly selected for increased ITC tolerance through potential adaptations linked with metabolism and antibiotic resistance (dehydrogenase-like protein) and transmembrane protein movement (Tat pathway signal protein). Together, our results suggest that R. solanacearum can rapidly evolve tolerance to allyl-ITC plant allelochemical which could constrain the long-term efficiency of biofumigation biocontrol and potentially shape pathogen evolution with plants.


title: Formylglycine-generating enzyme-like proteins constitute a novel family of widespread type VI secretion system immunity proteins

abstract:
Competition is a critical aspect of bacterial life, as it enables niche establishment and facilitates the acquisition of essential nutrients. Warfare between Gram-negative bacteria is largely mediated by the type VI secretion system (T6SS), a dynamic nanoweapon that delivers toxic effector proteins from an attacking cell to adjacent bacteria in a contact-dependent manner. Effector-encoding bacteria prevent self-intoxication and kin cell killing by the expression of immunity proteins, which prevent effector toxicity by specifically binding their cognate effector and occluding its active site. In this study, we investigate Tsi3, a previously uncharacterized T6SS immunity protein present in multiple strains of the human pathogen Acinetobacter baumannii. We show that Tsi3 is the cognate immunity protein of the antibacterial effector of unknown function Tse3. Our bioinformatic analyses indicate that Tsi3 homologs are widespread among Gram-negative bacteria, often encoded within T6SS effector-immunity modules. Surprisingly, we found that Tsi3 homologs possess a characteristic formylglycine-generating enzyme (FGE) domain, which is present in various enzymatic proteins. Our data shows that Tsi3-mediated immunity is dependent on Tse3-Tsi3 protein-protein interactions and that Tsi3 homologs from various bacteria do not protect against Tse3-dependent bacterial killing. Thus, we conclude that Tsi3 homologs are unlikely to be functional enzymes. Collectively, our work identifies FGE domain-containing proteins as important mediators of immunity against T6SS attacks and indicates that the FGE domain can be co-opted as a scaffold in multiple proteins to carry out diverse functions.

ImportanceDespite the wealth of knowledge on the diversity of biochemical activities carried out by T6SS effectors, comparably little is known about the various strategies bacteria employ to prevent susceptibility to T6SS-dependent bacterial killing. Our work establishes a novel family of T6SS immunity proteins with a characteristic FGE domain. This domain is present in enzymatic proteins with various catalytic activities. Our characterization of Tsi3 expands the known functions carried out by FGE-like proteins to include defense during T6SS-mediated bacterial warfare. Moreover, it highlights the evolution of FGE domain-containing proteins to carry out diverse biological functions.


title: Experimental Confirmation of Multiple Co-Existent DNA Secondary Structures using Low-Yield Bisulfite Sequencing

abstract: The prediction of DNA secondary structures from DNA sequences using thermodynamic models is imperfect for many biological sequences, both due to insufficient experimental data for training and to the kinetics of folding that lead to metastable structures. Here, we developed low-yield bisulfite sequencing (LYB-seq) to query the secondary structure states of cytosine (C) nucleotides in thousands of different DNA oligonucleotides on a single-molecule level. We observed that the reaction kinetics between bisulfite and C nucleotides is highly dependent on the secondary structure state of the C nucleotides, with the most accessible C nucleotides (those in small hairpin loops) reacting 70-fold faster than those in stable duplexes. Next, we developed a statistical model to evaluate the likelihood of an NGS read being consistent with a particular proposed secondary structure. By analyzing thousands of NGS reads for each DNA species, we can infer the distribution of secondary structures adopted by each species in solution. We find that 84% of 1,057 human genome subsequences studied here adopt 2 or more stable secondary structures in solution.


title: Vascular epiphytes contribute disproportionately to global centres of plant diversity

abstract:
AimVascular epiphytes are ubiquitous features of wet tropical forests where they contribute substantially to local and regional plant diversity. While some basic epiphyte distribution patterns are relatively well studied, little effort has been made to understand the drivers responsible for constraining their global distribution. This study quantifies the substantial contribution of epiphytes to global gradients and centres of vascular plant diversity and explores whether epiphytes vary from terrestrial plants in relation to contemporary and historical environmental variables.

LocationGlobal.

Time periodPresent.

Major taxa studiedVascular epiphytes.

MethodsWe integrated EpiList 1.0, a comprehensive list comprising > 30,000 vascular epiphyte species, and species distributions derived from the GIFT database to describe the global biogeography of epiphytes. We used generalized linear mixed effects models to assess the relationship between epiphytic and terrestrial plant diversity, and contemporary and historical environmental predictors.

ResultsWe find that epiphytes substantially contribute to global centres of vascular plant diversity, accounting for up to 39% of the vascular flora in the Neotropics. Epiphytes decrease in species numbers with increasing latitude at a rate three times faster than terrestrial plants, a trend that is driven mainly by the distribution of tropical forests and precipitation. Further, large regional differences emerge that are explained by several large endemic angiosperm families (e.g., Neotropical Bromeliaceae) that are absent in other tropical regions.

Main conclusionsOur results show that epiphytes are disproportionately diverse in most global centres of plant diversity and play an important role in driving the global latitudinal diversity gradient for plants. The distribution of precipitation and tropical forests emerge as major drivers of the latitudinal diversity gradient in epiphyte species richness. Finally, our findings demonstrate how epiphyte floras in different biogeographical realms are composed of different families and higher taxa revealing an important signature of historical biogeography.

category: ecology


title: A barley gene cluster for the biosynthesis of diterpenoid phytoalexins

abstract: Phytoalexins are specialized metabolites that are induced upon pathogen infection and contribute to the defense arsenal of plants. Maize and rice produce multiple diterpenoid phytoalexins and there is evidence from genomic sequences that other monocots may also produce diterpenoid phytoalexins. Here we report on the identification and characterization of a gene cluster in barley (Hordeum vulgare cv. Golden Promise) that is involved in the production of a set of labdane-related diterpenoids upon infection of roots by the fungal pathogen Bipolaris sorokiniana. The cluster is localized on chromosome 2, covers over 600 kb and comprises genes coding for a (+)-copalyl diphosphate synthase (HvCPS2), a kaurene synthase like (HvKSL4) and several cytochrome P450 oxygenases (CYPs). Expression of HvCPS2 and HvKSL4 in yeast and Nicotiana benthamiana resulted in the production of a single major product, whose structure was determined to be of the cleistanthane type and was named hordediene. Co-expression of HvCPS2, HvKSL4 and one of the CYPs from the cluster (CYP89E31) afforded two additional products, hordetriene and 11-hydroxy-hordetriene. Both of these compounds could be detected in extracts of barley roots infected by B. sorokiniana, validating the function of these genes in planta. Furthermore, diterpenoids with multiple oxidations and with molecular masses of 316, 318 and 332 were induced in infected barley roots and secreted in the medium, indicating that additional oxidases, possibly from the same genomic cluster are involved in the production of these phytoalexins. Our results provide the basis for further investigation of the role of this gene cluster in the defense of barley against pathogens and more generally in the interaction with the microbiome.


title: Design and modular assembly of synthetic intramembrane proteolysis receptors for custom gene regulation in therapeutic cells

abstract: Synthetic biology has established powerful tools to precisely control cell function. Engineering these systems to meet clinical requirements has enormous medical implications. Here, we adopted a clinically driven design process to build receptors for the autonomous control of therapeutic cells. We examined the function of key domains involved in regulated intramembrane proteolysis and showed that systematic modular engineering can generate a class of receptors we call SyNthetic Intramembrane Proteolysis Receptors (SNIPRs) that have tunable sensing and transcriptional response abilities. We demonstrate the potential transformative utility of the receptor platform by engineering human primary T cells for multi-antigen recognition and production of dosed, bioactive payloads relevant to the treatment of disease. Our design framework enables the development of fully humanized and customizable transcriptional receptors for the programming of therapeutic cells suitable for clinical translation.

category: synthetic biology


title: Bimanual digit training improves right-hand dexterity in older adults by reactivating declined ipsilateral motor-cortical inhibition

abstract: Improving deteriorated sensorimotor functions in older individuals is a social necessity in a super-aging society. Previous studies suggested that the declined interhemispheric sensorimotor inhibition observed in older adults is associated with their deteriorated hand/finger dexterity. Here, we examined whether bimanual digit exercises, which can train the interhemispheric inhibitory system, improve deteriorated hand/finger dexterity in older adults. Forty-eight healthy, right-handed, older adults (65-78 years old) were divided into two groups, i.e., the bimanual (BM) digit training and right-hand (RH) training groups, and intensive daily training was performed for 2 months. Before and after the training, we evaluated individual right hand/finger dexterity using a peg task, and the individual state of interhemispheric sensorimotor inhibition by analyzing ipsilateral sensorimotor deactivation via functional magnetic resonance imaging when participants experienced a kinesthetic illusory movement of the right-hand without performing any motor tasks. Before training, the degree of reduction/loss of ipsilateral motor-cortical deactivation was associated with dexterity deterioration. After training, the dexterity improved only in the BM group, and the dexterity improvement was correlated with reduction in ipsilateral motor-cortical activity. The capability of the brain to inhibit ipsilateral motor-cortical activity during a simple right-hand sensory-motor task is tightly related to right-hand dexterity in older adults.


title: Depression-like state induced by low-frequency repetitive transcranial magnetic stimulation to ventral medial frontal cortex in monkeys

abstract: The medial frontal cortex (MFC), especially its ventral part, has long been of great interest with respect to the pathology of mood disorders. A number of human brain imaging studies have demonstrated the abnormalities of this brain region in patients with mood disorders, however, whether it is critically involved in the pathogenesis of such disorders remains to be fully elucidated. In this study, we conducted a causal study to investigate how the suppression of neural activity in the ventral region of the MFC (vMFC) affects the behavioral and physiological states of monkeys by using repetitive transcranial magnetic stimulation (rTMS). By using low-frequency rTMS (LF-rTMS) as an inhibitory intervention, we found that LF-rTMS targeting the vMFC induced a depression-like state in monkeys, which was characterized by a reduced spontaneous behavioral activity, increased plasma cortisol level, impaired sociability, and decreased motivation level. On the other hand, no such significant changes in behavioral and physiological states were observed when targeting the other MFC regions, dorsal or posterior. We further found that the administration of an antidepressant agent, ketamine, ameliorated the abnormal behavioral and physiological states induced by the LF-rTMS intervention. These findings indicate the causal involvement of the vMFC in the regulation of mood and affect and the validity of the LF-rTMS-induced dysfunction of the vMFC as a nonhuman primate model of the depression-like state.


title: Controlling pallidal oscillations in real-time in Parkinson's disease using evoked interference deep brain stimulation (eiDBS): proof of concept in the human

abstract: Approaches to control basal ganglia neural activity in real-time are needed to clarify the causal role of 8-35 Hz ("beta band") oscillatory dynamics in the manifestation of Parkinsons disease (PD) motor signs. Here, we show that resonant beta oscillations evoked by electrical pulse with precise amplitude and timing can be used to predictably suppress or amplify spontaneous beta band activity in the internal segment of the globus pallidus (GPi) in the human. Using this approach, referred to as closed-loop evoked interference deep brain stimulation (eiDBS), we could suppress or amplify frequencyspecific (16-22 Hz) neural activity in a PD patient. Our results highlight the utility of eiDBS to characterize the role of oscillatory dynamics in PD and other brain conditions, and to develop personalized neuromodulation systems.