title: A phasevarion controls multiple virulence traits, including expression of vaccine candidates, in Streptococcus pneumoniae

abstract:
Streptococcus pneumoniae is the most common cause of bacterial illness worldwide. Current vaccines based on the polysaccharide capsule (PCV-13 and PPSV-23) are only effective against a limited number of the >100 capsular serotypes. A universal vaccine based on conserved protein antigens requires a thorough understanding of gene expression in S. pneumoniae. Restriction-Modification (R-M) systems, classically described as a defence against bacteriophage, are almost ubiquitous in the bacterial domain, and roles other than phage defence. All S. pneumoniae strains encode the SpnIII R-M system. This system contains a phase-variable methyltransferase that randomly switches specificity, and controls expression of multiple genes; a phasevarion. We aimed to determine the role of the SpnIII phasevarion during pneumococcal pathobiology and determine if phase-variation resulted in differences in expression of protein antigens that are being investigated as vaccine candidates. Using  locked S. pneumoniae strains that express a single SpnIII methyltransferase specificity, we found significant differences in clinically relevant traits, including survival in blood, and adherence to and invasion of human cells. Crucially, we also observed differences in expression of numerous proteinaceous vaccine candidates, which complicates selection of protein antigens for inclusion in a universal protein-based pneumococcal vaccine. This study will inform future vaccine design against S. pneumoniae by ensuring only stably expressed candidates are included in a rationally designed vaccine.

Significance StatementS. pneumoniae is the worlds foremost bacterial pathogen. S. pneumoniae encodes a randomly expressed epigenetic regulator, a phasevarion (phase-variable regulon), that results in random expression of multiple genes. Previous work demonstrated that the pneumococcal SpnIII phasevarion switches between six different expression states, generating six unique phenotypic variants in a pneumococcal population. Here, we show that this phasevarion generates multiple phenotypic differences relevant to pathobiology. Importantly, expression of conserved protein antigens varies with phasevarion switching. As capsule expression, a major pneumococcal virulence factor, is also controlled by the phasevarion, our work will inform the selection of the best candidates to include in a rationally designed, universal pneumococcal vaccine.


title: SARS-CoV-2 ORF6 disrupts innate immune signalling by inhibiting cellular mRNA export

abstract:
SARS-CoV-2 is a betacoronavirus and the etiological agent of COVID-19, a devastating infectious disease. Due to its far-reaching effect on human health, there is an urgent and growing need to understand the viral molecular biology of SARS-CoV-2 and its interaction with the host cell. SARS-CoV-2 encodes 9 predicted accessory proteins, which are presumed to be dispensable for in vitro replication, most likely having a role in modulating the host cell environment to aid viral replication. Here we show that the ORF6 accessory protein interacts with cellular Rae1 to inhibit cellular protein production by blocking mRNA export. We utilised cell fractionation coupled with mRNAseq to explore which cellular mRNA species are affected by ORF6 expression and show that ORF6 can inhibit the export of many mRNA including those encoding antiviral factors such as IRF1 and RIG-I. We also show that export of these mRNA is blocked in the context of SARS-CoV-2 infection. Together, our studies identify a novel mechanism by which SARS-CoV-2 can manipulate the host cell environment to supress antiviral responses, providing further understanding to the replication strategies of a virus that has caused an unprecedented global health crisis.

Author SummarySARS-CoV-2 is the virus responsible for the current COVID-19 pandemic. Coronaviruses, like SARS-CoV-2, replicate their genome in the cytoplasm of the host cell by hijacking the cellular machinery. In addition to structural proteins and viral enzymes, SARS-CoV-2 encodes 9 accessory proteins. Although these are not required for in vitro replication, they are thought to modulate the host cell environment to favour viral replication. In this work, we show that the ORF6 accessory protein can supress cellular protein production by blocking mRNA nuclear export through interacting with the cellular protein Rae1, a known mRNA export factor. We also investigated which cellular mRNAs were retained in the nucleus when ORF6 was overexpressed. Interestingly, we found that ORF6 inhibited the export of many different mRNAs, including those encoding antiviral factors, like IRF1 and RIG-I, even in the absence of stimulation by interferon. Importantly, we found that the export of these mRNAs was similarly affected in the context of SARS-CoV-2 infection. Therefore, we believe we have identified a novel mechanism that SARS-CoV-2 uses to suppress antiviral responses in order to make the cell more permissive to infection.

category: microbiology


title: Transcriptional changes in Plasmodium falciparum upon conditional knock down of mitochondrial ribosomal proteins RSM22 and L23

abstract: The mitochondrion of malaria parasites is an attractive antimalarial drug target, which require mitoribosomes to translate genes encoded in the mitochondrial (mt) DNA. Plasmodium mitoribosomes are composed of highly fragmented ribosomal RNA (rRNA) encoded in the mtDNA. All mitoribosomal proteins (MRPs) and other assembly factors are encoded in the nuclear genome. Here, we have studied one small subunit (SSU) MRP, RSM22 (Pf3D7_1027200) and one large subunit (LSU) MRP, L23 (Pf3D7_1239100) in Plasmodium falciparum. We show that both proteins localize to the mitochondrion and are essential for parasite survival. However, the parasites survive conditional knock down (KD) of PfRSM22 for two days longer than PfMRPL23 KD. RNA sequencing revealed transcriptomic changes of the nuclear and mitochondrial genomes upon KD of these MRPs. In the early phase following KD, while most mt rRNAs and transcripts of putative MRPs were downregulated in the absence of PfRSM22, several mt rRNAs and MRPs were upregulated after KD of PfMRPL23. At the late time points of KD, loss of PfRSM22 and PfMRPL23 caused defects in many essential metabolic pathways, leading to parasite death. There was a significant overlap among the mitochondrial related transcripts downregulated in the late phase of PfRSM22 and PfMRPL23 KDs. We have also identified a list of mitochondrial proteins of unknown function that are likely Plasmodium MRPs based on their structural similarity to known MRPs as well as their expression profiles in KD parasites.


title: Pectobacterium colocasium sp. nov. isolated from taro (Colocasia esculenta)

abstract:
Pectobacterium, agenus comprising gram-negative, pectinolytic phytopathogens, is responsible for economic losses in a wide host range of plants. In this study, the bacterial strains PL152T and PL155 were isolated from taro corms in Hawaii in 2018, and characterized using genomic and biochemical assays. The Next Generation Sequencing technologies, Oxford Nanopore MinION and Illumina NovaSeq, were used for whole genome sequencing of the PL152T strain. Short and long reads were assembled using the Unicycler tool accessible at the bioinformatic resource center, and PATRIC (PathoSystems Resource Integration Center) was used to generate a more accurate and reliable "hybrid" assembly. The 16S rRNA analysis of PL152T with type strains of other known Pectobacterium species showed a close relationship with P. fontis. Multi-locus sequence analysis using nine housekeeping genes (dnaA, gapA, gyrB, recA, dnaN, rpoS, mdh, rpoA and dnaK) differentiated strain PL152T from other species of Pectobacterium and formed a unique and well-defined clade. The concurrent results of average nucleotide identity (ANI) and digital DNA-DNA hybridization, with calculated values lower than 95 and 70%, respectively, supported the delineation of a novel bacterial species. Here, we proposed Pectobacterium colocasium, strain PL152T (=ICMP 24362T; LMG 32536 T) and PL155 as a novel species in the genus Pectobacterium.

1.5 RepositoriesCP091064; MZ542535 - MZ542540; OM457660


title: A bacterial pan-genome makes gene essentiality strain-dependent and evolvable

abstract: Many bacterial species are represented by a pan-genome, whose genetic repertoire far outstrips that of any single bacterial genome. Here we investigate how a bacterial pan-genome might influence gene essentiality, and whether essential genes that are initially critical for the survival of an organism can evolve to become non-essential. By using Tn-Seq, whole-genome sequencing, and RNA-Seq on a set of 36 clinical Streptococcus pneumoniae strains representative of >68% of the species pan-genome, we identify a species-wide  essentialome that can be subdivided into universal, strain-specific and accessory essential genes. By employing  forced-evolution experiments we show that specific genetic changes allow bacteria to bypass essentiality. Moreover, by untangling several genetic mechanisms we show that gene-essentiality can be highly influenced and/or dependent on: 1) the composition of the accessory genome; 2) the accumulation of toxic intermediates; 3) functional redundancy; 4) efficient recycling of critical metabolites; and 5) pathway rewiring. While this functional characterization underscores the evolvability-potential of many essential genes, we also show that genes with differential essentiality remain important antimicrobial drug target candidates, as their inactivation almost always has a severe fitness cost in vivo.


title: Comparative transcriptomics unveil distinctive metabolic pathway of phosphonate utilization by diatom Phaeodactylum tricornutum

abstract: Phosphonates are important constituents of marine organic phosphorus, however, the bioavailability and catabolism of phosphonates by eukaryotic phytoplankton remain enigmatic. Here, we use diatom Phaeodactylum tricornutum to investigate the bioavailability of phosphonates and elaborate the underlying molecular mechanism. Our results showed that 2-aminoethylphosphonic (2-AEP) can be utilized as alternative phosphorus source. Comparative transcriptomics unveil the 2-AEP utilization comprising two steps, molecular uptake through clathrin-mediated endocytosis and incorporation into the membrane phospholipids in the form of diacylglyceryl-2-AEP (DAG-2-AEP). In the global ocean, we found the prevalence of key genes responsible for vesicle formation (CLTC, AP-2) and DAG-AEP synthesis (PCYT2 and EPT1) in diatom assemblage. In accordance with the observation of elevated transcript abundance in cold waters, our culture experiments showed that cells grown in 2-AEP are more active at lower temperature. This study elucidated a distinctive mechanism of phosphonate utilization by diatom and inspected the ecological implications in adaptive mechanism.


title: N-acylethanolamine acid amide hydrolase is a novel target for drugs against SARS-CoV-2 and Zika virus

abstract:
Several compounds have been tested against SARS-CoV-2; at present, COVID-19 treatments decrease the deleterious inflammatory response and acute lung injury. However, the best therapeutic response would be expected by combining anti-inflammatory properties, while concomitantly blocking viral replication. These combined effects should drastically reduce both infection rate and severe complications induced by novel SARS-CoV-2 variants. Therefore, we explored the antiviral potency of a class of anti-inflammatory compounds that inhibit the N-Acylethanolamine acid amidase (NAAA). This enzyme catalyzes the hydrolysis of palmitoylethanolamide (PEA), a bioactive lipid that mediates anti-inflammatory and analgesic activity through the activation of peroxisome proliferator receptor- (PPAR-). Similarly, this pathway is likely to be a significant target to impede viral replication since PPAR- activation leads to dismantling of lipid droplets, where viral replication of Flaviviruses and Coronaviruses occurs.

Here, we show that either genetic or pharmacological inhibition of the NAAA enzyme leads to five-fold reduction in the replication of both SARS-CoV-2 and ZIKV in various cell lines. Once NAAA enzyme is blocked, both ZIKV and SARS CoV-2 replication decrease, which parallels a sudden five-fold decrease in virion release. These effects induced by NAAA inhibition occurs concomitantly with stimulation of autophagy during infection. Remarkably, parallel antiviral and anti-inflammatory effects of NAAA antagonism were confirmed in ex-vivo experiments, within SARS-CoV-2 infected human PBMC cells, in which both viral genomes and TNF- production drop by ~60%. It is known that macrophages contribute to viral spread, excessive inflammation and macrophage activation syndrome that NAAA inhibitors might prevent, reducing the macrophage-induced acute respiratory distress syndrome and subsequent death of COVID-19 patients.


title: Enhanced production of antiviral dsRNA in the 	Chlamydomonas reinhardtii chloroplast via a novel convergent promoter expression system

abstract: The present work presents an improvement of microalgal antiviral dsRNA production for controlling disease in shrimp aquaculture. 307 bp of sequence targeting the VP28 gene of white spot syndrome virus (WSSV) was inserted between two convergent rrnS promoters in the novel vector p2XTRBL, which was then subcloned into the transformation vector pSS116 using Golden Gate assembly. The recombinant plasmid was transformed into the Chlamydomonas reinhardtii chloroplast, and transformants selected by the restoration of photosynthesis. The presence of the cassette and homoplasmy of the algal transformants was confirmed by PCR analysis. Transcribed sense and antisense VP28-RNA were hypothesised to form an RNA duplex in the chloroplast stroma, and quantitative RT-PCR indicated that [~]100 {micro}g dsRNA was obtained per litre of transgenic microalgae culture. This accumulation of dsRNA represents a 10,000-fold increase relative to previous reports using convergent psaA promoters. Recombinant C. reinhardtii was assessed for its ability to prevent WSSV infection in shrimp larvae by direct feeding. After WSSV challenge, the survival of shrimp treated with dsRNA-expressing C. reinhardtii was significantly enhanced (95.2%) relative to the negative control without dsRNA treatment. The study suggests that this new algal production platform for dsRNA is significantly more efficient than the previous report, and it merits further scale-up and downstream processing studies.

category: molecular biology


title: Disrupted development and altered hormone signaling in male Padi2/Padi4 double knockout mice

abstract:
BackgroundPeptidylarginine deiminase enzymes (PADs) convert arginine residues to citrulline in a process called citrullination or deimination. Recently, two PADs, PAD2 and PAD4, have been linked to hormone signaling in vitro and the goal of this study was to test for links between PAD2/PAD4 and hormone signaling in vivo.

MethodsPreliminary analysis of Padi2 and Padi4 single knockout (SKO) mice did not find any overt reproductive defects and we predicted that this was likely due to genetic compensation. To test this hypothesis, we created a Padi2/Padi4 double knockout (DKO) mouse model and tested these mice for a range of reproductive defects.

ResultsControlled breeding trials found that DKO breeding pairs, particularly males, appeared to take longer to have their first litter than wild-type FVB controls (WT), and that pups and DKO male weanlings weighed significantly less than their WT counterparts. Additionally, DKO males took significantly longer than WT males to reach puberty and had lower serum testosterone levels. Furthermore, DKO males had smaller testes than WT males with increased rates of germ cell apoptosis.

ConclusionsThe Padi2/Padi4 DKO mouse model provides a new tool for investigating PAD function and outcomes from our studies provide the first in vivo evidence linking PADs with hormone signaling.


title: Validation of a colorimetric LAMP to detect Loxosceles experimental envenomation

abstract:
Diagnostic tests for brown spider accidents are unavailable and impact treatment decisions, increasing costs and patient risks. In this work, we used for the first time a fast, simple, and visual method based on the loop-mediated isothermal amplification assay (LAMP) to detect Loxosceles envenomation. Using the DNA from L. similis legs, we observed a high sensitivity using this test since as low as 0.32 pg of DNA could be detected. This pH-dependent colorimetric assay was 64 times more sensitive than PCR to detect spider DNA. The test was specific for Loxosceles once no cross-reaction was observed when testing DNA from different agents that cause similar dermonecrotic injuries. The test allowed the detection of Loxosceles intermedia DNA from hair, serum, and swab samples obtained from experimentally-envenomed rabbit within 72 h. The method sensitivity varied according to the sample and the collection time, reaching 100% sensitivity in serum and hair, respectively, 1 h and 24 h after the experimental envenomation. Due to its ease of execution, speed, sensitivity, and specificity, LAMP presents an excellent potential for identifying Loxosceles spp. envenomation. This can reduce the burden on the Health System and the morbidity for the patient by implementing the appropriate therapy immediately.In addition, this work opens up the perspective to other venomous animal accident identification using LAMP.

Highlights* Using 28S primers it was possible to identify L. similis DNA with high sensitivity;
* LAMP was 62-fold more sensitive than PCR and detected as low as 0.32 pg of DNA;
* LAMP detected L. intermedia DNA from hair, serum, and exudate from experimentally-envenomed rabbits;
* LAMP presents an excellent potential for identifying Loxosceles spp. envenomation.


title: Depletion of the RNA-binding protein PURA triggers changes in posttranscriptional gene regulation and loss of P-bodies

abstract: The RNA-binding protein PURA is implicated in the rare, monogenetic, neurodevelopmental disorder PURA Syndrome. However, very little is known about the affected molecular pathways. PURA has been reported to bind DNA and RNA and to be connected to different cellular functions. Here, we show that PURA predominantly resides in the cytoplasm, where it binds to thousands of transcripts. Many of these transcripts change abundance in response to PURA depletion, in parts reflected in altered protein expression levels. A closer inspection of the regulated proteins indicates a role of PURA in immune responses, mitochondrial function, autophagy and processing (P)-body activity. Intriguingly, reduced PURA levels lower the expression of the integral P-body component LSM14A and strongly impair P-bodies in human cells. Furthermore, PURA knockdown results in stabilization of P-body-associated transcripts, whereas other mRNAs are decreased. Hence, reduced PURA levels, as reported in patients with PURA syndrome, are associated with the formation and composition of the phase-separated RNA processing machinery. Our study on the RNA-binding protein PURA provides a blueprint for the comprehensive understanding of the tight connection between P-body-associated RNA regulation and neurodevelopmental disorders.


title: Modulation of RNA polymerase processivity affects double-strand break repair in the presence of a DNA-end binding protein

abstract: Homologous recombination is the predominant double-strand DNA break repair pathway in Escherichia coli. A recent report of non-homologous end joining involving the Ku-like Gam protein of bacteriophage Mu (MuGam) has sparked interest in understanding the functions of DNA end-binding proteins in bacteria. MuGam binds to DNA ends, but how it interferes with DNA repair or transcription in live bacteria remains elusive. In E. coli, RNA polymerase secondary channel interactors, such as the Gre factors, play a role in coordinating transcription with DNA replication and break repair. Here we show that MuGam inhibits break repair by slowing down DNA resection and impeding recombination in living cells. Loss of GreA restores break repair in the presence of MuGam by allowing for increased DNA resection due to a potential release of MuGam from the DNA. Using MuGam as a DNA break sensor, we found that breaks are generated when translation is inhibited, more so in the presence of GreA, supporting the model where transcription-translation uncoupling increases transcription/replication collisions. Significantly, this work reveals that modulation of RNA polymerase can impact DNA break repair in presence of a Ku-like protein.


title: Modulation of RNA polymerase processivity affects double-strand break repair in the presence of a DNA end-binding protein


title: Anti-inflammatory effects of WFS1 in pancreatic beta-cells

abstract: Wolfram syndrome is a rare genetic disorder characterized by juvenile-onset diabetes mellitus, optic nerve atrophy, hearing loss, diabetes insipidus, and progressive neurodegeneration. Pathogenic variants in the WFS1 gene are the main causes of Wolfram syndrome. WFS1 encodes a transmembrane protein localized to the endoplasmic reticulum (ER) and regulates the unfolded protein response (UPR). Loss of function of WFS1 leads to dysregulation of insulin production and secretion, ER calcium depletion, and cytosolic calpains activation, resulting in activation of apoptotic cascades. Although the terminal UPR has been shown to induce inflammation that accelerates {beta}-cell dysfunction and death in diabetes, the contribution of {beta}-cell inflammation to the development of diabetes in Wolfram syndrome has not been fully understood. Here we show that WFS1-deficiency enhances the gene expression of inflammatory cytokines and chemokines, leading to cytokine-induced ER-stress and cell death in pancreatic {beta}-cells. PERK and IRE1 pathways mediate high glucose-induced inflammation in a {beta}-cell model of Wolfram syndrome. M1-macrophage infiltration and hypervascularization are seen in the pancreatic islets of Wfs1 whole-body knockout mice, demonstrating that WFS1 regulates anti-inflammatory responses in {beta}-cells. Our results demonstrate that inflammation plays an essential role in the progression of {beta}-cell death and diabetes in Wolfram syndrome and reveal that the pathways involved in ER stress-mediated inflammation provide potential therapeutic targets for the treatment of Wolfram syndrome.


title: Seasonal dynamics of the wild rodent faecal virome

abstract: Viral discovery studies in wild animals often rely on cross-sectional surveys at a single time point. As a result, our understanding of the temporal stability of wild animal viromes remains poorly resolved. While studies of single host-virus systems indicate that host and environmental factors influence seasonal virus transmission dynamics, comparable insights for whole viral communities in multiple hosts are lacking. Leveraging non-invasive faecal samples from a long-term wild rodent study, we characterised viral communities of three common European rodent species (Apodemus sylvaticus, A. flavicollis, and M. glareolus) living in temperate woodland over a single year. Our findings indicate that a substantial fraction of the rodent virome is seasonally transient and associated with vertebrate or bacteria hosts. Further analyses of one of the most abundant virus families, Picornaviruses, show pronounced temporal changes in viral richness and diversity, which were associated with concurrent and up to [~]3-month lags in host density, ambient temperature, rainfall and humidity, suggesting complex feedbacks from the host and environmental factors on virus transmission and shedding in seasonal habitats. Overall, this study emphasizes the importance of understanding the seasonal dynamics of wild animal viromes in order to better predict and mitigate zoonotic risks.

category: ecology


title: Enviromic prediction is useful to define the limits of climate adaptation: A case study of common beans in Brazil

abstract:
Future environmental shifts foster plant research aiming to develop climate-smart cultivars but the past and current impacts on the environment are also a key to unraveling a major part of the phenotypic adaptation of crops. These studies may determine the most relevant environmental components of yield stability and adaptability within a breeding framework. Here, as a proof-concept study, we quantified the impacts of climate drivers in adapting common bean across Brazilian regions and seasons. We developed an  enviromic prediction approach based on Generalized Additive Models (GAM), large-scale environmental covariate data (EC), and grain yield (GY) of 18 years of a common bean breeding program. Then, we predicted the optimum limits for ECs for each production scenario. We verified the ability of GAM-based models to explain the climate driver GY variation and performed accurate predictions for diverse production scenarios (four regions, three seasons, and two grain types). Our results indicates that air temperature (maximum and minimum), accumulated solar radiation, and rainfalls are mostly associated as the main drivers of GY variation in most regions. We also observed a huge variability of the climate drivers impact for the same germplasm cultivated across different seasons for each region. Furthermore, this climate influence in common beans adaptation is more evident during the vegetative for some seasons, while more impressive for reproductive stages for other seasons. Consequently, it demands higher efforts from breeding programs in developing region- or season-specific ideotype cultivars. Enviromics prediction with GAM was useful to identify the effect of climate on critical crop stages, which indirectly might help breeders in developing climate-smart varieties. We envisage its use with research field trial data (e.g., advanced yield testing) and historical farm field yield aimed at understanding breeding gaps in developing adapted cultivars for growing scenarios.

HighlightsO_LIWe developed an  enviromic prediction approach based on Generalized Additive Models (GAM) for a large-scale environmental covariate data and grain yield
C_LIO_LIWe verified the ability of GAM-based models to explain the climate driver grain yield variation and performed accurate predictions for diverse production scenarios (four regions, three seasons, and two grain types)
C_LIO_LIClimatic limitations for cropping commun beans were identified across seasons and regions.
C_LIO_LI"Optimum" values for climate variables in different common bean regions productions were obtained .
C_LI

category: genetics


title: Strong genotype by environment interactions in the rice Global MAGIC population across seedling stage drought

abstract: Crop adaptation is required to sustainably increase the rate of yield gains to meet projected future needs under the challenging conditions of climate change and competition for resources. Future adaptation will likely need to harness both highly polygenic traits and genotype-by-environment interactions (GxE), the study of which is aided by complex recombinant populations. We used the diverse Oryza sativa Global Multiparent Advanced Generation InterCross (MAGIC) population to study the genetic architecture and contributions of seedling emergence, establishment, and development to yield components under dry-direct seeding and seedling stage drought across three growing seasons. Dry-direct seeding is an establishment practice that has the potential to reduce methane emissions, water use, and labor demands for rice farmers, but increases the possibility of early-season drought conditions. We found substantial evidence for large roles of environmental variation and GxE in controlling trait variation. Maintenance of shoot growth during seedling stage drought was positively correlated with crown root number and both directly and indirectly influenced agronomic traits. Other than the major green revolution locus sd1, most allelic effects were conditionally neutral (affecting trait values in some environments but not others) and most alleles had their strongest effect in an environmental extreme. This discovery is both a challenge and a potential opportunity: with variable genetic architecture, selection in any one environment may not result in stable trait gains across environments. However, conditionally neutral GxE is a potential route to sustainable yield stability through allele pyramiding.


title: Genome-wide association reveals host-specific genomic traits in Escherichia coli

abstract: Escherichia coli is an opportunistic pathogen that can colonize or infect various host species. There is a significant gap in our understanding to what extent genetic lineages of E. coli are adapted or restricted to specific hosts. In addition, genomic determinants underlying such host specificity are unknown.By analyzing a randomly sampled collection of 1198 whole-genome sequenced E. coli isolates from four countries (Germany, UK, Spain, and Vietnam), obtained from five host species (human, pig, cattle, chicken, and wild boar) over 16 years, from both healthy and diseased hosts, we demonstrate that certain lineages of E. coli are frequently detected in specific hosts. We report a novel nan gene cluster, designated nan-9, putatively encoding acetylesterases and determinants of uptake and metabolism of sialic acid, to be associated with the human host as identified through genome wide association studies. In silico characterization predicts nan-9 to be involved in sialic acid (Sia) metabolism. In vitro growth experiments with a representative {Delta}nan E. coli mutant strain, using sialic acids 5-N-acetyl neuraminic acid (Neu5Ac) and N-glycolyl neuraminic acid (Neu5Gc) as the sole carbon source, indicate an impaired growth behaviour compared to the wild-type. In addition, we identified several additional E. coli genes that are potentially associated with adaptation to human, cattle and chicken hosts, but not for the pig host. Collectively, this study provides an extensive overview of genetic determinants which may mediate host specificity in E. coli. Our findings should inform risk analysis and epidemiological monitoring of (antimicrobial resistant) E. coli.

category: genomics


title: The Chromosome-level Genome of Dracaena cochinchinesis Provides Insights into its Biological Features and the Mechanism of Dragon's Blood Formation

abstract:
Dracaena, a remarkably long-lived and slowly maturing species, is famous all over the world for the production of dragons blood, a precious traditional medicine used by different cultures since ancient times. However, lacking a high-quality genome,the molecular mechanisms underlying these traits are largely unknown and that greatly restricts the protection and regeneration of the rare and endangered plant resources. Here, we sequenced and assembled a chromosome-level genome of the Dracaena cochinchinensis, the first to be sequenced of Dracaena Vand. ex L. The D. cochinchinensis genome covering 1.21 Gb with a scaffold N50 of 50.06 Mb, and encodes 31,619 predicted protein-coding genes. We found D. cochinchinensis has undergone two whole genome duplications (WGDs) and two long terminal repeats (LTRs) insertion burst events. The expansion of cis-zeatin O-glucosyltransferase (cZOGT) and small auxin up-regulated RNA (SAUR) genes is account for its longevity and slow growth. In flavonoids biosynthesis pathway, transcription factors bHLH and MYB were predicted as the core regulators, and ROS as the specific signal molecule during the process of injury-induced dragons blood formation. Our study not only provides high-quality genomic knowledge of D. cochinchinensis, but also deciphered the mystery of its longevity, and preliminarily elucidated the molecular mechanism of dragons blood formation, which will facilitate the resource protection and sustainable utilization of Dracaena.

SHORT SUMMARYThis study reports a chromosome-level genome assembly for D. cochinchinensis, the first genome of Dracaena Vand. ex L. It 29 provides valuable genetic resources and creates a large scope for studying Dracaena. We found the significant expansion of genes associated with its longevity and slow growth, and genes in flavonoids biosynthesis were first completely identified. Moreover, transcription factors bHLH and MYB as the core regulator of flavonoids biosynthesis, and ROS as the specific signal molecule during the process of injury-induced dragons blood formation were also identified. These results not only deciphered the mystery of its longevity, but also elucidated the molecular mechanism of dragon[s blood formation preliminarily, which will facilitate the resource protection and sustainable utilization of Dracaena.


title: Mutagenesis at non-B DNA motifs in the human genome: a course correction

abstract: Non-B DNA structures formed by repetitive sequence motifs are known instigators of mutagenesis in experimental systems. Analyzing this phenomenon computationally in the human genome requires careful disentangling of intrinsic confounding factors, including overlapping and interrupted motifs, and recurrent sequencing errors. Accounting for these factors eliminates all signals of repeat-induced mutagenesis that extend beyond the motif boundary, and eliminates or dramatically shrinks the magnitude of mutagenesis within motifs, contradicting previous reports. Non-B DNA makes only a modest contribution to mutation in the human germline compared with other intrinsic sources of mutation. G-quadruplex motifs are potent instigators of recurrent sequencing errors, but are only mildly mutagenic. Polymerase slippage is the dominant mutagenic mechanism affecting STRs, while polymerase template-switch is exclusively responsible for mutations in palindromic repeats, which perfect and extend inverted symmetry.


title: Rtt109 promotes nucleosome replacement ahead of the replicating fork

abstract: DNA replication perturbs chromatin by triggering the eviction, replacement and incorporation of nucleosomes. How this dynamic is orchestrated in time and space is poorly understood. Here, we apply a recently established sensor for histone exchange to follow the time-resolved histone H3 exchange profile in budding yeast cells undergoing synchronous replication. We find that new histones are incorporated not only behind, but also ahead of the replication fork. We provide evidence that Rtt109, the S phase-specific acetyltransferase, stabilizes nucleosomes behind the fork, but promotes H3 replacement ahead of the fork. Unexpectedly, increased replacement ahead of the fork is independent of the primary Rtt109 acetylation target H3K56, and rather results from Rtt109 activity towards the H3 N-terminus. Our results suggest that selective incorporation of differentially modified H3s behind and ahead of the replication fork results in opposing effects on histone exchange, which may contribute to genome stability by overcoming replication-associated challenges.


title: Isocitrate dehydrogenase 3b is required for spermiogenesis but dispensable for retinal degeneration

abstract: Isocitrate dehydrogenase 3 (IDH3), a key enzyme in mitochondrial tricarboxylic acid (TCA) cycle, catalyzes the decarboxylation of isocitrate into -ketoglutarate (KG), converting NAD+ into NADH. We have found that IDH3 {beta} subunit (IDH3B) is essential for IDH3 activity in multiple tissues. Loss of Idh3b in mice causes substantial accumulation of the isocitrate and its precursors in the TCA cycle, particularly in the testes, whereas the levels of the downstream metabolites remain unchanged or slightly increased. The Idh3b-knockout (KO) mice have normal visual function without retinal degeneration. However, the male KO mice are infertile. Loss of Idh3b causes energetic deficit and disrupts the biogenesis of acrosome and flagellum, resulting in spermiogenesis arrestment in sperm cells. Together, we demonstrate IDH3B controls its substrate levels in the TCA cycle and it is required for sperm mitochondrial metabolism and spermiogenesis, highlighting the importance of the tissue-specific function of the ubiquitous TCA cycle.

category: biochemistry


title: A Multiscale study of phosphorylcholine driven cellular phenotypic targeting

abstract: Phenotypic targeting requires the ability of the drug delivery system to discriminate over cell populations expressing a particular receptor combination. Such selectivity control can be achieved using multiplexed-multivalent carriers often decorated with multiple ligands. Here, we demonstrate that the promiscuity of a single ligand can be leveraged to create multiplexed-multivalent carriers achieving phenotypic targeting. We show how the cellular uptake of poly(2-methacryloyloxyethyl phosphorylcholine)-poly(2- (diisopropylamino)ethyl methacrylate) (PMPC-PDPA) polymersomes varies depending on the receptor expression among different cells. We investigate the PMPC-PDPA polymersome insertion at the single chain/receptor level using all-atom molecular modelling. We propose a theoretical statistical mechanics-based model for polymersome-cell association that explicitly considers the interaction of the polymersome with the cell glycocalyx shedding light on its effect on the polymersome binding. We validate our model experimentally and show that the binding energy is a non-linear function, allowing us to tune interaction by varying the radius and degrees of polymerisation. Finally, we show that PMPC-PDPA polymersomes can be used to target monocytes in vivo due to their promiscuous interaction with SRB1, CD36 and CD81.


title: A catalytic mechanism for Renilla-type bioluminescence

abstract: The widely used coelenterazine-powered Renilla luciferase was discovered over 40 years ago but the oxidative mechanism by which it generates blue photons remains unclear. Here we decipher Renilla-type bioluminescence through crystallographic, spectroscopic, and computational experiments. Structures of ancestral and extant luciferases complexed with the substrate-like analogue azacoelenterazine or a reaction product were obtained, providing unprecedented snapshots of coelenterazine-to-coelenteramide oxidation. Bound coelenterazine adopts a Y-shaped conformation, enabling the deprotonated imidazopyrazinone component to attack O2 via a radical charge-transfer mechanism. A high emission intensity is secured by an aspartate from a conserved proton-relay system, which protonates the excited coelenteramide product. Another aspartate on the rim of the catalytic pocket fine-tunes the electronic state of coelenteramide and promotes the formation of the blue light-emitting phenolate anion. The results obtained also reveal structural features distinguishing flash-type from glow-type bioluminescence, providing insights that will guide the engineering of next-generation luciferase-luciferin pairs for ultrasensitive optical bioassays.


title: Membrane binding of pore-forming γ-hemolysin components studied at different lipid compositions

abstract:
Methicillin-resistant Staphylococcus aureus is is among those pathogens currently posing the highest threat to public health. Its host immune evasion strategy is mediated by pore-forming toxins (PFTs), among which the bicomponent {gamma}-hemolysin is one of the most common. The complexity of the porogenesis mechanism by {gamma}-hemolysin poses difficulties in the development of antivirulence therapies targeting PFTs from S. aureus, and sparse and apparently contrasting experimental data have been produced. Here, through a large set of molecular dynamics simulations at different levels of resolution, we investigate the first step of pore formation, and in particular the effect of membrane composition on the ability of{gamma} -hemolysin components, LukF and Hlg2, to steadily adhere to the lipid bilayer in the absence of proteinaceous receptors. Our simulations are in agreement with experimental data of {gamma}-hemolysin pore formation on model membranes, which are here explained on the basis of the bilayer properties. Our computational investigation suggests a possible rationale to explain experimental data on phospholipid binding to the LukF component, and to hypothesise a mechanism by which, on purely lipidic bilayers, the stable anchoring of LukF to the cell surface facilitates Hlg2 binding, through the exposure of its N-terminal region. We expect that further insights on the mechanism of transition between soluble and membrane bound-forms and on the role played by the lipid molecules will contribute to the design of antivirulence agents with enhanced efficacy against methicillin-resistant S. aureus infections.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/479512v1_ufig1.gif" ALT="Figure 1">
View larger version (27K):
org.highwire.dtl.DTLVardef@5f0ae5org.highwire.dtl.DTLVardef@1c46688org.highwire.dtl.DTLVardef@1277e17org.highwire.dtl.DTLVardef@1c71898_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIThe presence of cholesterol and unsaturated phospholipid tails facilitates the binding of{gamma} -hemolysin components, LukF and Hlg2, on model membranes.
C_LIO_LICoarse-grained simulations show that the two components have different absorption capabilities, with LukF undergoing the most stable binding.
C_LIO_LIThe spontaneous docking of LukF on the membrane is mediated by two distant phosphatidylcholine binding sites.
C_LI

category: biophysics


title: Matrix-driven jamming dynamics mediates transition of ovarian cancer spheroids to stable morphologies

abstract:
Metastatic progression of ovarian cancer through the peritoneal fluidic microenvironment is mediated predominantly by tumor cell clusters known as spheroids. Such spheroids also comprise the cells extracellular matrix (ECM). Spheroids with lumen, often seen in malignant ascites, are associated with a basement membrane (BM)-like coat. Herein, we combine microfluidics with high-speed time-lapse videography and imaging analysis to investigate the consequences of lumen formation and the BM coat on the architecture and integrity of ovarian cancer spheroids providing architectural robustness to the transitory ovarian cancer metastatic niche within constrained flow spaces. Upon transit through spatially constrained environments, lumen-less  moruloid spheroids deform followed by an incomplete and temporally extended ([~]4.8 s) recovery. The transit causes intercellular rearrangement during entry and shows susceptibility to cellular disintegration. On the other hand, lumen-containing  blastuloid spheroids exhibit minimal intra-spheroidal rearrangements. They exhibit a lower tendency for disintegration and quickly ([~]1.5 s) recover their morphology upon exit. Significantly, removing the blastuloid BM through collagenase treatment reverts the mechanical behaviors of these spheroids to those typifying their moruloid counterparts. Simulations using an experimentally calibrated Cellular Potts model predict the requirement of higher intercellular adhesion for blastuloid behavior, which we confirm through increased E-cadherin expression in blastuloid spheroids. Surprisingly, E-cadherin expression is dependent on the BM coat, elucidating the latters role in enhancing cell-cell adhesion. Our results show how spheroidal matrix engenders lumen formation and higher intercellular adhesion, in turn providing architectural robustness of the transitory ovarian cancer metastatic niche within constrained flow spaces.

Significance StatementInvestigating the mechanical and biophysical properties of disseminated ovarian cancer spheroids is instrumental to our understanding of their metastatic progression. We take a microfluidics-based approach to investigate the biophysical contributions of a key spheroidal constituent: basement membrane-like extracellular matrix, which is typical to hollow  blastuloid spheroids, distinguishing them from the solid  moruloid spheroids. We observe using high-speed videography, imaging analysis and multiscale computational modeling that the basement membrane renders blastuloid spheroids less deformable and resistant to disintegration than moruloid spheroids. We trace this function to jamming of cells between the blastuloid lumen and basement membrane, and high intercellular adhesion.


title: An intergeneric recombinant geminivirus causes soybean stay-green disease

abstract: Soybean is one of the most valuable legume crops in the world with high nutritional value. Recently, the outbreak of soybean stay-green syndrome has swept the soybean production in the Huang-Huai-Hai region of China, resulting in huge yield losses, which has become an epidemic and prominent problem in soybean production. However, the cause of the stay-green syndrome remains obscure. Here, we report a novel intergeneric recombinant geminivirus which causes soybean stay-green symptoms. Viral small RNA-based screening identified a new recombinant geminvirus from field soybean stay-green samples. The complete genome sequence of the virus contains 2762 nucleotide (nt) and appears to be an intergeneric recombinant virus in which protein coding for coat protein (V1) is similar to member of genus Mastrevirus, whereas proteins coding for V2, C2, C3 are most similar to those of viruses in the Maldovirus genus, and C1 and C4 are most similar to virus in genus Begomovirus. Inoculation of the infectious clone of the recombinant geminivirus through Agrobacterium rhizogenes causes typical soybean stay-green syndrome which resembles field symptoms including delayed leaf senescence, flat pods and abnormal seeds. The recombinant geminivirus can be detected in seed coat but not in cotyledon and embryo, thus failing to be transmitted by seeds. Moreover, the genome variation and epidemiological dynamic analysis were also carried out to help the continuous epidemiological surveillance of this emerging geminivirus. Collectively, this new geminivirus is tentatively named soybean stay-green associated virus (SoSGV). Our determination of the causal agent of soybean stay-green disease will bolster efforts to develop effective management strategies to control this prevalent disease in the field.


title: Multistep loading of PCNA onto DNA by RFC

abstract: The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader replication factor C (RFC), which acts at single-stranded/double-stranded DNA junctions harboring a recessed 3 end (3 ss/dsDNA junctions) and at DNA nicks. To illuminate the loading mechanism we have investigated the structure of RFC:PCNA bound to ATP{gamma}S and 3 ss/dsDNA junctions or nicked DNA using cryogenic electron microscopy. Unexpectedly, we observe open and closed PCNA conformations in the RFC:PCNA:DNA complex, revealing that PCNA can adopt an open, planar conformation that allows direct insertion of dsDNA, and indicating that PCNA ring-closure is not mechanistically coupled to ATP-hydrolysis. By resolving multiple DNA-bound states of RFC:PCNA we observe that partial melting facilitates lateral insertion into the central channel formed by RFC:PCNA. We also resolve the Rfc1 N-terminal domain and demonstrate that its single BRCT domain participates in coordinating DNA prior to insertion into the central RFC channel, which promotes PCNA loading on the lagging strand of replication forks in vitro. Combined, our data suggest a comprehensive and fundamentally revised model for the RFC-catalyzed loading of PCNA onto DNA.


title: Maternally inherited siRNAs initiate piRNA cluster formation

abstract:
PIWI-interacting RNAs (piRNAs) guide repression of transposable elements in germlines of animals. In Drosophila, piRNAs are produced from heterochromatic genomic loci, called piRNA clusters, that act as a repositories of information about genome invaders. piRNA generation by dual-strand clusters depend on the chromatin-bound Rhino-Deadlock-Cutoff (RDC) complex, which is deposited on clusters guided by piRNAs, forming a feed-forward loop in which piRNAs promote their own biogenesis. However, how piRNA clusters are formed initially, before cognate piRNAs are present, remained unknown. Here we report spontaneous de novo formation of a piRNA cluster from repetitive transgenic sequences. We show that cluster formation occurs gradually over several generations and requires continuous trans-generational transmission of small RNAs from mothers to their progeny. We discovered that maternally-supplied siRNAs are responsible for triggering de novo cluster activation in progeny. In contrast, the siRNA pathway is dispensable for cluster function after its establishment. These results revealed an unexpected cross-talk between the siRNA and piRNA pathways and suggest a mechanism for de novo formation of piRNA clusters triggered by production of siRNAs.

Highlights- A transcribed repetitive transgene is spontaneously converted into dual-strand piRNA cluster
- Establishment of piRNA cluster occurs over multiple generations and requires cytoplasmic inheritance of cognate small RNA from mothers
- Cognate siRNAs initiate the activation of piRNA cluster, but are dispensable after its establishment


title: DNA barcoding of fungal specimens using long-read high-throughput sequencing

abstract: Molecular methods are increasingly used to identify species that lack conspicuous macro- or micromorphological characters. Taxonomic and ecological research teams barcode large numbers of collected voucher specimens annually. In this study we assessed the efficiency of long-read high throughput sequencing (HTS) as opposed to the traditionally used Sanger method for taxonomic identification of multiple vouchered fungal specimens, and providing reference information about intra-individual allele polymorphism. We developed a workflow based on a test-set of 423 fungal specimens (representing 205 species), PacBio HTS method, and ribosomal rRNA operon internal transcribed spacer (ITS) and 28S rRNA gene (LSU) markers. PacBio HTS had a higher success rate than Sanger sequencing at a comparable cost. Species identification based on PacBio reads was usually straightforward, because the dominant operational taxonomic unit (OTU) typically represented the targeted organism. Unlike the Sanger method, PacBio HTS enabled detecting widespread allele polymorphism within the ITS marker in the studied specimens. We conclude that multiplex DNA barcoding of the fungal ITS and LSU markers using a PacBio HTS is a useful tool for taxonomic identification of large amounts of collected voucher specimens at competitive price. Furthermore, PacBio HTS accurately recovers various alleles, which can provide crucial information for species delimitation and population-level studies.


title: Screening for functional regulatory variants in open chromatin using GenIE-ATAC

abstract: Understanding the effects of genetic variation in gene regulatory elements is crucial to interpreting genome function. This is particularly pertinent for the hundreds of thousands of disease-associated variants identified by GWAS, which frequently sit within gene regulatory elements but whose functional effects are often unknown. Current methods are limited in their scalability and ability to assay regulatory variants in their endogenous context, independently of other tightly linked variants. Here we present a new medium-throughput screening system: genome engineering based interrogation of enhancers assay for transposase accessible chromatin (GenIE-ATAC), that measures the effect of individual variants on chromatin accessibility in their endogenous genomic and chromatin context. We employ this assay to screen for the effects of regulatory variants in human induced pluripotent stem cells, validating a subset of causal variants, and extend our software package (rgenie) to analyse these new data. We demonstrate that this methodology can be used to understand the impact of defined deletions and point mutations within transcription factor binding sites. We thus establish GenIE-ATAC as a method to screen for the effect of gene regulatory element variation, allowing identification and prioritisation of causal variants from GWAS for functional follow-up and understanding the mechanisms of regulatory element function.


title: Converging pathways found in copy number variation syndromes with high schizophrenia risk

abstract:
BackgroundSchizophrenia genetics is complex, and the contribution of common and rare variants are not fully understood. Several specific copy number variations (CNVs) confer increased risk for schizophrenia, and the study of their effects is central to molecular models of mental illness. However, these CNVs - microdeletions or -duplications - are spread across the genome and differ in number of genes affected and classes of coded proteins. This suggests that, in order to fully understand the contribution of these genetic variants to mental illness, we need to look beyond the deleted or duplicated genes, to their interaction partners and involved molecular pathways.

MethodsIn this study, we developed machine-readable interactive pathways to enable analysis of downstream effects of genes within CNV loci and identify common pathways between CNVs with high schizophrenia risk using the WikiPathways database, and schizophrenia risk gene collections from GWAS studies and a gene-disease association database.

ResultsFor CNVs that are pathogenic for schizophrenia we found overlapping pathways, including BDNF signaling, cytoskeleton, cell-cell connections, inflammation and MAPK3 signaling. Common schizophrenia risk genes identified by different studies are found in all CNV pathways but not enriched.

ConclusionsOur findings suggest that specific pathways - such as BDNF signaling - may be critical contributors to schizophrenia risk conferred by rare CNVs, and common risk variants may operate through distinct mechanisms. Our approach highlights the importance of not only investigating deleted or duplicated genes within pathogenic CNV loci, but also their direct interaction partners, which may explain pleiotropic effects of CNVs on schizophrenia risk.

category: systems biology


title: Statistical learning is not error-driven

abstract: Prediction errors have a prominent role in many forms of learning. For example, in reinforcement learning agents learn by updating the association between states and outcomes as a function of the prediction error elicited by the event. An empirical hallmark of such error-driven learning is Kamin blocking, whereby the association between a stimulus and outcome is only learnt when the outcome is not already fully predicted by another stimulus. It remains debated however to which extent error-driven computations underlie learning of automatically formed associations as in statistical learning. Here we asked whether the automatic and incidental learning of the statistical structure of the environment is error-driven, like reinforcement learning, or instead does not rely on prediction errors for learning associations. We addressed this issue in a series of Kamin blocking studies. In three consecutive experiments, we observed robust incidental statistical learning of temporal associations among pairs of images, but no evidence of blocking. Our results suggest that statistical learning is not error-driven but may rather follow the principles of basic Hebbian associative learning.

category: neuroscience


title: Transcriptional regulation and chromatin architecture maintenance are decoupled modular functions at the Sox2 locus

abstract: How distal regulatory elements control gene transcription and chromatin topology is not clearly defined, yet these processes are closely linked in lineage specification during development. Through allele-specific genome editing and chromatin interaction analyses of the Sox2 locus in mouse embryonic stem cells, we found a striking disconnection between transcriptional control and chromatin architecture. We trace nearly all Sox2 transcriptional activation to a small number of key transcription factor binding sites, whose deletions have no effect on promoter-enhancer interaction frequencies or topological domain organization. Local chromatin architecture maintenance, including at the topologically associating domain (TAD) boundary downstream of the Sox2 enhancer, is widely distributed over multiple transcription factor-bound regions and maintained in a CTCF-independent manner. Furthermore, disruption of promoter-enhancer interactions by ectopic chromatin loop formation has no effect on Sox2 expression. These findings indicate that many transcription factors are involved in modulating chromatin architecture independently of CTCF.


title: Fused in Sarcoma undergoes cold denaturation: implications on phase separation

abstract: The mediation of fused in sarcoma (FUS) protein liquid-liquid phase separation (LLPS) is generally attributed to the low-complexity and disordered domains, while the role of its folded domains remains unknown. In this work we questioned the role of the folded domains on the full-length (FL) FUS LLPS and studied the influence of several metabolites, ions and overall conditions on the LLPS process using turbidity assays, differential interference contrast microscopy and nuclear magnetic resonance spectroscopy. We demonstrate that FL FUS LLPS is highly responsive to the surrounding conditions, and that overall intrinsic disorder is crucial for LLPS. To promote such disorder, we reveal that the FUS RNA-recognition domain (RRM) and the zinc-finger motif (ZnF) undergo cold denaturation above 0{o}C, at a temperature that is determined by the conformational stability of the ZnF domain. We hypothesize that, in cold shock conditions, cold denaturation might provide a pathway that exposes additional residues to promote FUS self-assembly. Such findings mark the first evidence that FUS globular domains may have an active role in stress granule formation in cold stress.


title: Glioblastoma stem cells express non-canonical proteins and exclusive mesenchymal-like or non-mesenchymal-like protein signatures

abstract:
Glioblastomas (GBM) origin, recurrence and resistance to treatment are driven by GBM cancer stem cells (GSCs). Existing transcriptomic characterisations of GBM classify the tumours to three subtypes: classical, proneural, and mesenchymal. The comprehension of how expression patterns of the GBM subtypes are reflected at global proteome level in GSCs is limited.

To characterise protein expression in GSCs, we performed in-depth proteogenomic analysis of patient-derived GSCs by RNA-sequencing and mass-spectrometry proteomics. We identified and quantified over 10,000 proteins in two independent GSCs panels, and propose a GSC-associated proteomic signature (GSAPS) that defines two distinct morphological conditions; one defined by a set of proteins expressed in non-mesenchymal - proneural and classical - GSCs (GPC-like), and another expressed in mesenchymal GSCs (GM-like). The expression of GM-like protein set in GBM tissue was associated with hypoxia, necrosis, recurrence, and worse overall survival in GBM patients.

In a proof-of-concept proteogenomic approach, we discovered 252 non-canonical peptides expressed in GSCs, i.e., protein sequences that are variant or derive from genome regions previously considered protein-non-coding. We report new variants of the heterogeneous ribonucleoproteins (HNRNPs), which are implicated in mRNA splicing. Furthermore, we show that per-gene mRNA-protein correlations in GSCs are moderate and vary compared to GBM tissue.

category: cancer biology


title: Mate Competition Drives Aggressive Behaviour in Female Drosophila

abstract: Aggression is an adaptive set of behaviours that allows animals to compete against one another in an environment of limited resources. In Drosophila such aggressive behaviour has been extensively studied in males. Despite recent work highlighting territorial defence in females, female aggression in Drosophila is still poorly understood. Indeed, whether females compete for mating partners, as males do, has remained unknown so far. In the present work, we report that Drosophila melanogaster females reliably display aggression towards mating pairs. This aggressive behaviour is positively associated with the females mating drive and relies heavily on olfaction. Furthermore, we found that food odour in combination with OR47b-dependent fly odour sensing are required for proper expression of aggressive behaviour. Taken together, we describe a social context linked to reproduction in which Drosophila females aspiring to mate produce consistent and stereotyped displays of aggression. These findings open the door for further inquiries into the neural mechanisms that govern this behaviour.


title: Parasites make hosts more profitable but less available to predators

abstract: Parasites are omnipresent, and their eco-evolutionary significance has aroused much interest from scientists. Parasites may affect their hosts in many ways by altering host density, vulnerability to predation, and energy content, thus maximizing profitability within the optimal foraging framework. Consequently, parasites could impact predator diet and trophic links within food webs. Here, we investigate the consequences of the iridovirus Daphnia iridescent virus 1 (DIV-1) infection on the reproductive success, mortality, appearance, mobility, and biochemical composition of water fleas (Daphnia magna), a widespread freshwater crustacean. We compare search time between infected and uninfected Daphnia preyed by a common aquatic insect (Notonecta sp.) as well as the handling time and feeding preference of Notonecta sp. Our findings show that infection does not change fecundity but reduces lifespan and thereby constrains fitness. Infected Daphnia show reduced mobility and increased color reflectance in the UV and visible domains, which potentially affects their visibility and thus catchability. Infection increases body size and the amount of proteins but does not affect carbohydrate and lipid contents. Although infected Daphnia had a longer handling time, they tended to be preferred over uninfected individuals by aquatic insects. Taken together, our findings show that DIV-1 infection could make Daphnia more profitable to predators (23% energy increase), a positive effect that should be balanced with density reductions due to higher mortalities. We also highlight that exposure to infection in asymptomatic individuals leads to ecological characteristics that differ from both healthy and symptomatic infected individuals.


title: Looming stimuli reliably drive innate, but not learned, defensive responses in rats.

abstract: The ability to respond innately to stimuli such as food, water, and threat is crucial for survival as it guarantees the ability to feed, drink, and avoid danger without prior experience. These natural, unconditioned, stimuli can also drive associative learning, such that cues consistently paired with an unconditioned stimulus come to elicit a response. Threat conditioning, an extensively studied form of associative learning, typically uses painful electric shocks that mimics injury as unconditioned stimuli. Whether injury or pain is required for learning, or the threat of injury suffices, remains elusive. An approaching predator produces looming shadows and sounds. Visual looming stimuli elicit strong innate defensive responses such as escape and freezing. Here we ask whether these stimuli can also drive learned freezing or escape responses to a conditioned stimulus. We found that pairing a neutral tone with a looming stimulus failed to drive learned defensive responses, either freezing or escape, in contrast with the robust learned responses when the loom was replaced by shock. The dissociation between the capacity to drive an innate defensive response and to drive threat learning reveals new boundaries for learned defensive responses which will impact our understanding of learning processes and defensive strategies both at the mechanistic and ethological levels.

category: animal behavior and cognition


title: Ingestion of microplastic fibres, but not microplastic beads, impacts growth rates in the tropical house cricket Gryllodes sigillatus

abstract: Microplastic is a growing concern as an environmental contaminant as it is ubiquitous in our ecosystems. Microplastics are present in terrestrial environments, yet the majority of studies have focused on the adverse effects of microplastics on aquatic biota. We hypothesized that microplastic ingestion by a terrestrial insect would have localized effects on gut health and nutrient absorption, such that prolonged dietary microplastic exposure would impact growth rate and adult body size. We further hypothesized that plastic form (fibres vs. beads) would influence these effects because of the nature of gut-plastic interactions. Freshly hatched tropical house crickets (Gryllodes sigillatus) were fed a standard diet containing different concentrations of either fluorescent polyethylene microplastic beads (75-105 m), or untreated polyethylene terephthalate microfibers (<5 mm) until they died or reached adulthood (approximately 8 weeks). Weight and body length were measured weekly and microplastic ingestion was confirmed through fluorescence microscopy and visual inspection of the frass. While, to our surprise, we found no effect of polyethylene bead ingestion on growth rate or final body size of G. sigillatus, females experienced a reduction in size and weight when fed high concentrations of polyethylene terephthalate microfibers. These results suggest that high concentrations of polyethylene beads of the 100 m size range can pass through the cricket gut without a substantial negative effect on their growth and development time, but high concentrations of polyethylene terephthalate microfibers cannot. Although we report the negative effects of microplastic ingestion on the growth of G. sigillatus, it remains uncertain what threats microplastics pose to terrestrial insects.

category: physiology


title: Cerebellar and cortico-striatal-midbrain contributions to reward-cognition processes and apathy within the psychosis continuum

abstract: Negative symptoms in the psychosis continuum are linked to impairments in reward processing and cognitive function. Processes at the interface of reward processing and cognition and their relation to negative symptoms remain little studied, despite evidence suggestive of integration in mechanisms and neural circuitry. Here, we investigated brain activation during reward-dependent modulation of working memory (WM) and their relationship to negative symptoms in subclinical and early stages of the psychosis continuum. We included 27 persons with high schizotypal personality traits and 23 patients with first episode psychosis as well as 27 healthy controls. Participants underwent functional magnetic resonance imaging while performing an established 2-back WM task with two reward levels (5 CHF vs. no reward), which allowed us to assess common reward-cognition regions through whole-brain conjunction analyses and to investigate relations with clinical scores of negative symptoms. As expected for behavior, reward facilitated performance while cognitive load diminished it. At the neural level, the conjunction of high reward and high cognitive load contrasts across the psychosis continuum showed increased hemodynamic activity in the thalamus and the cerebellar vermis. During high cognitive load, more severe apathy but not diminished expression in the psychosis continuum was associated with reduced activity in right lateral orbitofrontal cortex, midbrain, posterior vermal cerebellum, caudate and lateral parietal cortex. Our results suggest that hypoactivity in the cerebellar vermis and the cortical-striatal-midbrain-circuitry in the psychosis continuum relates to apathy possibly via impaired flexible cognitive resource allocation for effective goal pursuit.


title: Developmental overproduction of cortical superficial neurons impairs adult auditory cortical processing

abstract: While evolutionary cortical expansion is thought to underlie the evolution of human cognitive capabilities, developmental expansion can lead to megalencephaly, often found in neurodevelopmental disorders such as autism spectrum disorder. Still, little is known about how the overproduction of cortical neurons during development affects cortical processing and behavior in later life. Here we show that the overproduction of cortical superficial neurons leads to hyposensitivity to auditory stimuli in mice. We took advantage of a WNT/{beta}-catenin signaling inhibitor, XAV939, to overproduce cortical superficial excitatory neurons during development. XAV939-treated adult mice exhibited a higher threshold to detect acoustic stimuli behaviorally. This behavioral deficit is associated with abnormal auditory cortical processing, such as higher delta power as well as lower spontaneous and auditory-evoked activity in putative excitatory cells. On the other hand, the auditory thalamus did not show any significant difference in neural firing between XAV939-treated and control groups. Within auditory cortical populations, functional monosynaptic connections were reduced across cell classes. A computational model predicts that the weakened excitatory-inhibitory connections lead to reduced spontaneous and evoked activity. Altogether, our results suggest that atypical auditory processing in XAV939-treated animals originates from weakened synaptic connections among cortical neurons. Although the expansion of cortical size is evolutionarily advantageous, an abnormal expansion during development can cause detrimental effects on cortical processing and perceptual behavior in adulthood.


title: Loss of SUMO-specific protease 2 causes isolated glucocorticoid deficiency by blocking zonal transdifferentiation

abstract: SUMOylation is a dynamic posttranslational modification, that provides fine-tuning of protein function involved in the cellular response to stress, differentiation, and tissue development. In the adrenal cortex, an emblematic endocrine organ that mediates adaptation to physiological demands, the SUMOylation gradient is inversely correlated with the gradient of cellular differentiation raising important questions about its role in functional zonation and the response to stress. Considering that SUMO-specific protease 2 (SENP2), a deSUMOylating enzyme, is upregulated by ACTH/PKA signalling within the zona Fasciculata (zF), we generated mice with adrenal-specific Senp2 loss to address these questions. Disruption of SENP2 activity in steroidogenic cells leads to specific hypoplasia of the zF, a blunted reponses to ACTH and isolated glucocorticoid deficiency. Mechanistically, overSUMOylation resulting from SENP2 loss shifts the balance between ACTH/PKA and WNT/{beta}-catenin signalling leading to repression of PKA activity and ectopic activation of {beta}-catenin. At the cellular level, this blocks transdifferentiation of {beta}-catenin-positive zona Glomerulosa cells into zF cells and sensitises them to premature apoptosis. Our findings indicate that the SUMO pathway is critical for adrenal homeostasis and stress responsiveness.


title: Loss of SUMO-specific protease 2 causes isolated glucocorticoid deficiency by blocking adrenal cortex zonal transdifferentiation


title: The relationship between frequency content and representational dynamics in the decoding of neurophysiological data

abstract:
Decoding of high temporal resolution, stimulus-evoked neurophysiological data is increasingly used to test theories about how the brain processes information. However, a fundamental relationship between the frequency spectra of the neural signal and the subsequent decoding accuracy timecourse is not widely recognised. We show that, in commonly used instantaneous signal decoding paradigms, each sinusoidal component of the evoked response is translated to double its original frequency in the subsequent decoding accuracy timecourses. We therefore recommend, where researchers use instantaneous signal decoding paradigms, that more aggressive low pass filtering is applied with a cut-off at one quarter of the sampling rate, to eliminate representational alias artefacts. However, this does not negate the accompanying interpretational challenges. We show that these can be resolved by decoding paradigms that utilise both a signals instantaneous magnitude and its local gradient information as features for decoding. On a publicly available MEG dataset, this results in decoding accuracy metrics that are higher, more stable over time, and free of the technical and interpretational challenges previously characterised. We anticipate that a broader awareness of these fundamental relationships will enable stronger interpretations of decoding results by linking them more clearly to the underlying signal characteristics that drive them.

HighlightsO_LIWe investigate different decoding paradigms applied to epoched data and characterise the information content available to each over time.
C_LIO_LIUnder commonly used instantaneous signal decoding paradigms, sinusoidal components of the evoked response are translated to double their original frequency in decoding accuracy metrics, presenting technical and interpretational challenges.
C_LIO_LIWhen instantaneous signal decoding is used, we recommend using low pass filters with a cut-off less than one quarter of the sampling rate to eliminate spurious representational alias artefacts.
C_LIO_LIThe interpretational issues associated with instantaneous signal decoding can be resolved with alternative paradigms such as complex spectrum decoding.
C_LIO_LIWe show that complex spectrum decoding results in decoding accuracy metrics that are higher, more stable over time, and free of representational aliasing.
C_LI


title: Conservative Significance Testing of Tripartite Interactions in Multivariate Neural Data

abstract:
An important goal in systems neuroscience is to understand the structure of neuronal interactions, frequently approached by studying functional relations between recorded neuronal signals. Commonly used pairwise metrics (e.g. correlation coefficient) offer limited insight, neither addressing the specificity of estimated neuronal interactions nor potential synergistic coupling between neuronal signals. Tripartite metrics, such as partial correlation, variance partitioning, and partial information decomposition, address these questions by disentangling functional relations into interpretable information atoms (unique, redundant and synergistic). Here, we apply these tripartite metrics to simulated neuronal recordings to investigate their sensitivity to impurities (like noise or other unexplained variance) in the data. We find that all considered metrics are accurate and specific for pure signals but experience significant bias for impure signals. We show that permutation-testing of such metrics results in high false positive rates even for small impurities and large data sizes. We present a conservative null hypothesis for significance testing of tripartite metrics, which significantly decreases false positive rate at a tolerable expense of increasing false negative rate. We hope our study raises awareness about the potential pitfalls of significance testing and of interpretation of functional relations, offering both conceptual and practical advice.

Author SummaryTripartite functional relation metrics enable the study of interesting effects in neural recordings, such as redundancy, functional connection specificity and synergistic coupling. However, common estimators of such relations are designed for pure (e.g. non-noisy) signals rare for such recordings. We study the performance of tripartite estimators using simulated impure neural signals. We demonstrate that permutation-testing is not a robust procedure for inferring ground truth interactions from studied estimators. We develop an adjusted conservative testing procedure, reducing false positive rate of studied estimators for impure data. Besides addressing significance testing, our results should aid in accurate interpretation of tripartite functional relations and functional connectivity.


title: Directional Absolute Coherence: a phase-based measure of effective connectivity for neurophysiology data

abstract:
Communication between neural structures is a topic of much clinical and scientific interest and has been linked to a variety of behavioural, cognitive, and psychiatric measures. Here, we introduce a novel effective connectivity measure, termed the directional absolute coherence (DAC). Combining aspects of magnitude squared coherence, imaginary coherence, and phase slope index, DAC provides an estimate of connectivity that is resistant to volume conduction, encapsulates the directionality of neural communication, and is bound to the interval of -1 and 1. To highlight the properties of this newly proposed method, we compare DAC to a number of established connectivity methods using data recorded from the subthalamic nucleus of patients with Parkinsons disease with deep brain stimulation electrodes. By applying a combination of real and simulated data, we demonstrate that DAC provides a reliable estimate of the magnitude and direction of connectivity, independent of the phase difference between brain signals. As such, DAC facilitates a reliable investigation of inter-regional neural communication, rendering it a valuable tool for gaining a deeper understanding of the functional architecture of the brain and its relationship to behaviour and cognition. A Python implementation of DAC is freely available at https://github.com/neurophysiological-analysis/FiNN.

Highlights- Volume conduction and limited interpretability affect many connectivity methods.
- DAC is a combined approach to overcome these pitfalls.
- DAC augments information content and interpretability in comparison to other methods.
- DAC allows for reliable estimation of effective connectivity.


title: Semantic overlap protects from interference by restoring encoding mechanisms

abstract:
Overlap between experiences can have both facilitative and detrimental effects for memory. Our aim was to establish whether overlap along one dimension (e.g. contextual, semantic, temporal) can counteract overlap-driven interference along another dimension. We hypothesized that semantic overlap facilitates episodic memory formation by modulating encoding mechanisms. We recorded scalp electroencephalography (EEG) while human participants performed a free recall task. Half of the items from late in each study list semantically overlapped with an item presented earlier in the list. We find that semantic overlap selectively improves memory and influences the neural signals engaged during the study of late list items. Relative to other recalled items, late list items that are later recalled consecutively with semantically overlapping items elicited increased high frequency activity and decreased low frequency activity, a hallmark of successful encoding. Our findings demonstrate that semantic overlap can protect from interference due to temporal overlap by modulating encoding mechanisms.

Statement of RelevancExperiences can overlap along different dimensions, including contextual, semantic, and tempo-ral. We tested the hypothesis that semantic overlap - shared meaning between experiences - may protect from interference due to temporal overlap, when experiences occur close together in time. Although evidence suggests that attention and/or encoding resources diminish across a series of study items presented in close temporal proximity, we find that semantic overlap can enable recovery of these encoding resources. Specifically, items that would typically be forgotten due to interference are better remembered and recruit distinct neural mechanisms when they share meaning with an earlier study item. These findings indicate that encoding mechanisms can be modulated by the degree of semantic overlap between two experiences. More broadly, our results suggest that experiences do not exist in isolation, rather that a prior experience can directly influence the neural mechanisms recruited to process a current experience.


title: Immunomodulatory actions of a kynurenine-derived endogenous electrophile.

abstract:
The inflammatory upregulation of kynurenine metabolism induces immunomodulatory responses via incompletely understood mechanisms. We report that increases in cellular and systemic kynurenine levels yield the electrophilic derivative kynurenine-carboxyketoalkene (Kyn-CKA), as evidenced by the accumulation of thiol-conjugates and saturated metabolites. Under physiological conditions, Kyn-CKA induces Nrf2-regulated genes and inhibits NF-{kappa}B and NLRP3-dependent pro-inflammatory signaling. Sickle Cell Disease (SCD) is a hereditary hemolytic condition characterized by basal inflammation and recurrent vaso-occlusive crises. Both a transgenic SCD murine model and SCD patients exhibit increased kynurenine synthesis and elevated Kyn-CKA metabolite levels. Plasma hemin and kynurenine concentrations are positively correlated, indicating that Kyn-CKA synthesis in SCD is upregulated during pathogenic vascular stress. Remarkably, exogenous administration of Kyn-CKA abrogated pulmonary microvasculature occlusion in SCD mice, an important factor in the development of lung injury. These findings demonstrate that the upregulation of kynurenine synthesis and its metabolism to Kyn-CKA is an adaptive response that attenuates inflammation and protects tissues.

One-Sentence SummaryKyn-CKA is a kynurenine-derived signaling mediator that transduces its immunomodulatory protective actions and attenuates vaso-occlusion in sickle cell disease.

category: immunology


title: Systematic Single Cell Pathway Analysis (SCPA) reveals novel pathways engaged during early T cell activation

abstract: Next generation sequencing technologies have revolutionized the study of T cell biology, capturing previously unrecognized diversity in cellular states and functions. Pathway analysis is a key analytical stage in the interpretation of such transcriptomic data, providing a powerful method for detecting alterations in important biological processes. Current pathway analysis tools are built on models developed for bulk-RNA sequencing, limiting their effectiveness when applied to more complex single cell RNA-sequencing (scRNA-seq) datasets. We recently developed a sensitive and distribution-free statistical framework for multisample distribution testing, which we implement here in the open-source R package Single Cell Pathway Analysis (SCPA). After demonstrating the effectiveness of SCPA over commonly used methods, we generate a scRNA-seq T cell dataset and characterize pathway activity over early cellular activation and between T cell populations. This revealed unexpected regulatory pathways in T cells, such as an intrinsic type I interferon system regulating T cell survival and a reliance on arachidonic acid metabolism throughout T cell activation. A systems level characterization of pathway activity in T cells across multiple human tissues also revealed alpha defensin expression as a hallmark of bone marrow derived T cells. Overall, our work here provides a widely applicable tool for single cell pathway analysis, and highlights unexpected regulatory mechanisms of T cells using a novel T cell dataset.


title: Spatially Resolved Transcriptomics for Evaluation of Intracranial Vessels in a Rabbit Model: Proof of Concept

abstract:
BackgroundBetter understanding of vessel biology and vascular pathophysiology is needed to improve understanding of cerebrovascular disorders. Tissue from diseased vessels can offer the best data. Rabbit models can be effective for studying intracranial vessels, filling gaps resulting from difficulties acquiring human tissue. Spatially-resolved transcriptomics (SRT) in particular hold promise for studying such models as they build on RNA sequencing methods, augmenting such data with histopathology.

MethodsRabbit brains with intact arteries were flash frozen, cryosectioned, and stained with H&E to confirm adequate inclusion of intracranial vessels before proceeding with tissue optimization and gene expression analysis using the Visium SRT platform. SRT results were analyzed with k-means clustering analysis, and differential gene expression was examined, comparing arteries to veins.

ResultsCryosections were successfully mounted on Visium proprietary slides. Quality control thresholds were met. Optimum permeabilization was determined to be 24 minutes for the tissue optimization step. In analysis of SRT data, k-means clustering distinguished vascular tissue from parenchyma. When comparing gene expression traits, the most differentially expressed genes were those found in smooth muscle cells. These genes were more commonly expressed in arteries compared to veins.

ConclusionsIntracranial vessels from model rabbits can be processed and analyzed with the Visium SRT platform. Face validity is found in the ability of SRT data to distinguish vessels from parenchymal tissue and differential expression analysis accurately distinguishing arteries from veins. SRT should be considered for future animal model investigations into cerebrovascular diseases.

category: cell biology


title: Latitudinal gradient in species diversity provides high niche opportunities for a range-expanding phytophagous insect

abstract:
O_LIWhen species undergo poleward range expansions in response to anthropogenic change, they likely encounter less diverse communities in new locations. If low diversity communities provide weak biotic interactions, such as reduced competition for resources or predation, range-expanding species may experience  high niche opportunities.
C_LIO_LIHere, we uncover if oak gall wasp communities experience a latitudinal diversity gradient (LDG) and weaker interactions at the poles that might create high niche opportunities for a range-expanding community member.
C_LIO_LIWe performed systematic surveys of oak gall wasps on a dominant oak, Quercus garryana, throughout most of its range, from northern California to Vancouver Island, British Columbia. On 540 trees at 18 sites, we identified 23 species in three guilds (leaf detachable, leaf integral, and stem galls). We performed regressions between oak gall wasp diversity, latitude, and other abiotic and habitat factors to reveal if cynipid communities follow an LDG. To uncover patterns in local interactions, we performed partial correlations on oak gall wasps co-occurring on trees within regions. Finally, we performed regressions between abundances of co-occurring gall wasps on trees to reveal potential interactions.
C_LIO_LIQ. garryana-gall wasp communities followed an LDG, with lower diversity at higher latitudes, particularly with a loss of detachable leaf gall species. Detachable leaf gall wasps, including the range-expanding species, co-occurred most on trees and had weak interactions in the northern region. Abundances of the range-expander and detachable and integral leaf galls co-occurring on trees were negatively related, suggesting antagonistic interactions. Overall, we found that LDGs create communities with weaker antagonistic interactions at the poles that might facilitate ecological release in a range-expanding community member.
C_LIO_LIHere, we uncover how regional and local scale patterns and processes create high niche opportunities for a range-expanding community member. This research provides insight into how biogeographical patterns in communities and species interactions influence the outcome of range expansions. Given the ubiquity of LDGs, these outcomes might be expected in other phytophagous insect communities.
C_LI


title: Abundance and expression of hgcAB genes and mercury availability jointly explain methylmercury formation in stratified brackish waters

abstract: Neurotoxic methylmercury (MeHg) is formed by microbial methylation of inorganic divalent Hg (HgII) and constitutes severe environmental and human health risks. The methylation is enabled by hgcA and hgcB genes, but it is not known if the associated molecular-level processes are rate-limiting or enable accurate prediction of MeHg formation in nature. In this study, we investigated the relationships between hgcA genes and MeHg across redox stratified water columns in the brackish Baltic Sea. We found that the abundance of hgcA genes and transcripts combined with the concentration of dissolved HgII-sulfide species were strong predictors of both HgII methylation rate and MeHg concentration, implying their roles as principal joint drivers of MeHg formation in these systems. In establishing relationships between hgcA genes and MeHg, we advance the fundamental understanding of mechanistic principles governing MeHg formation in nature and enable refined predictions of MeHg levels in coastal seas in response to the accelerating spread of oxygen deficient zones.


title: Landscape functional connectivity for butterflies under different scenarios of land-use, land-cover, and climate change in Australia

abstract:
Pollinating invertebrates are vital to terrestrial ecosystems but are impacted by anthropogenic habitat loss/fragmentation and climate change. Conserving and improving landscape connectivity is important to offset those threats, yet its assessment for invertebrates is lacking. In this study, we evaluated the functional connectivity between protected areas in Australia for 59 butterfly species, under present conditions and different future scenarios (for 2050 and 2090) of land-use, land-cover, and climate change. Using circuit-theory analysis, we found that functional connectivity under present conditions varies widely between species, even when their estimated geographical ranges are similar. Under future scenarios, functional connectivity is predicted to decrease overall, with negative changes worsening from 2050 to 2090, although a few species are positive exceptions. We have made our results available as spatial datasets to allow comparisons with taxa from other studies and can be used to identify priority areas for conservation in terms of establishing ecological corridors or stepping-stone habitat patches. Our study highlights the importance of considering pollinating invertebrates when seeking holistic conservation and restoration of a landscapes functional connectivity, underscoring the need to expand and promote protected areas to facilitate functional connectivity under future scenarios of global change.

Research DataThe habitat suitability maps and functional connectivity maps are made available as GeoTiff images via Figshare (10.6084/m9.figshare.19130078).


title: Resistance bioassays and allele characterisation inform Spodoptera frugiperda (Lepidoptera: Noctuidae) management and introduction pathways in Asia and Australia

abstract: The fall armyworm Spodoptera frugiperda is present in over 70 countries in Africa, Asia, and Oceania. Its rapid dispersal since 2016 since first reported in western Africa, and associated devastation to agricultural productivity, highlight the challenges posed by this pest. Currently, its management largely relies on insecticide sprays and transgenic Bacillus thuringiensis toxins, therefore understanding their responses to these agents and characteristics of any resistance genes enables adaptive strategies. In Australia, S. frugiperda was reported at the end of January 2020 in northern Queensland and by March 2020, also in northern Western Australia. We undertook bioassays on two Australian populations, one each from these initial points of establishment. To assist with resistance response interpretation, two related endemic noctuid pest species, Helicoverpa armigera and Spodoptera litura, were concurrently screened to obtained LC50 estimates for larvae against a range of insecticides. We characterised known resistance alleles from the VGSC, ACE-1, RyR, and ABCC2 genes for five populations to compared with published allele frequencies and bioassay responses from native and invasive S. frugiperda populations. An approximately 10x LC50 difference for indoxacarb was detected between Australian populations, which was approximately 28x higher than an Indian population. Characterisation of ACE-1 and VGSC alleles provided further evidence of multiple introductions in Asia, and multiple pathways involving genetically distinct individuals into Australia. Bioassay results and resistance allele patterns from invasive S. frugiperda populations therefore suggest multiple introduction events have contributed to the pests spread in the Old World, and challenge the axiom of its rapid  west-to-east spread.

category: evolutionary biology


title: Host origin is a determinant of parallel evolution between influenza virus gene segments.

abstract: Several emerging influenza viruses, including H7N9 and H5N6 viruses, trace their origins to reassortment with H9N2 viruses that contributed internal gene segments. However, the evolutionary constraints governing reassortment of H9N2 viruses remain unknown. In seasonal human influenza A viruses, gene segments evolve in parallel at both the gene and protein levels. Here, we demonstrate that parallel evolution in human H3N2 viruses differs from avian H9 viruses, with both genes and proteins of avian H9 viruses characterized by high phylogenetic divergence. Strikingly, protein trees corresponding to avian H9 polymerase subunits diverge despite known functional constraints on polymerase evolution. Gene divergence was consistent across avian H9 isolates from different continents, suggesting that parallel evolution between H9 gene segments is not dependent on regionally defined lineages. Instead, parallel evolution in H9 viruses was dependent upon host origin. Our study reveals the role of the host in parallel evolution of influenza gene segments and suggests that high reassortment potential in avian species may be a consequence of evolutionary flexibility between gene segments.


title: Genome-wide analysis and evolutionary history of the Necrosis and Ethylene-inducing peptide 1-like protein (NLP) superfamily across the Dothideomycetes class of fungi

abstract: Necrosis and Ethylene-inducing peptide 1-like proteins (NLPs) are broadly distributed across bacteria, fungi and oomycetes. Cytotoxic NLPs are usually secreted into the host apoplast where they can induce cell death and trigger plant immune responses in eudicots. To investigate the evolutionary history of the NLPs, we accessed the genomic resources of 79 species from 15 orders of Dothideomycetes. Phylogenetic approaches searched for biased patterns of NLP gene evolution and aimed to provide a phylogenetic framework for the cytotoxic activities of NLPs. Among Dothideomycetes, the NLP superfamily sizes varied, but usually contained from one to six members. Superfamily sizes were higher among pathogenic fungi, with family members that were mostly effector NLPs. Across species, members of the NLP1 family (Type I NLPs) were predominant (84%) over members of the NLP2 family (Type II NLPs). The NLP1 family split into two subfamilies (NLP1.1 and NLP1.2). The NLP1.1 subfamily was broadly distributed across Dothideomycetes. There was strong agreement between the phylogenomics of Dothideomycetes and the phylogenetic tree based on members of the NLP1 subfamilies. To a lesser extent, phylogenomics also agreed with the phylogeny based on members of the NLP2 family. While gene losses seem to have shaped the evolutionary history of NLP2 family, ancient gene duplications followed by descent with modification characterized the NLP1 family. The strongest cytotoxic activities were recorded on NLPs of the NLP1.1 subfamily, suggesting that biased NLP gene retention in this subfamily favored the cytotoxic paralogs.


title: Genome-wide profiling of histone H3K4me3 and H3K27me3 modifications in individual blastocysts by CUT&Tag without a solid support (NON-TiE-UP CUT&Tag)

abstract: Individual analysis of the epigenome of preimplantation embryos is useful for characterizing each embryo and for investigating the effects of environmental factors on their epigenome. However, it is difficult to analyze genome-wide epigenetic modifications, especially histone modifications, in a large number of single embryos due to the small number of cells and the complexity of the analysis methods. To solve this problem, we further modified the CUT&Tag method, which can analyze histone modifications in a small number of cells, such that the embryo is handled as a cell mass in the reaction solutions in the absence of the solid-phase magnetic beads that are used for antibody and enzyme reactions in the original method (NON-TiE-UP CUT&Tag; NTU-CAT). By using bovine blastocysts as a model, we showed that genome-wide profiles of representative histone modifications, H3K4me3 and H3K27me3, could be obtained by NTU-CAT that are in overall agreement with the conventional chromatin immunoprecipitation-sequencing (ChIP-seq) method, even from single embryos. However, this new approach has limitations that require attention, including false positive peaks and lower resolution for broad modifications. Despite these limitations, we conclude that NTU-CAT is a promising replacement for ChIP-seq with the great advantage of being able to analyze individual embryos.