title: Structural and functional analysis of the human Cone-rod homeobox transcription factor

abstract: The cone-rod homeobox (CRX) protein is a critical K50 homeodomain transcription factor responsible for the differentiation and maintenance of photoreceptor neurons in the vertebrate retina. Mutant alleles in the human gene encoding CRX result in a variety of distinct blinding retinopathies, including retinitis pigmentosa, cone-rod dystrophy, and Leber congenital amaurosis. Despite the success of using in vitro biochemistry, animal models, and genomics approaches to study this clinically relevant transcription factor over the past 24 years since its initial characterization, there are no high-resolution structures in the published literature for the CRX protein. In this study, we use bioinformatic approaches and small-angle x-ray scattering (SAXS) structural analysis to further understand the biochemical complexity of the human CRX homeodomain (CRX-HD). We find that the CRX-HD is a compact, globular monomer in solution that can specifically bind functional cis-regulatory elements encoded upstream of retina -specific genes. This study presents the first structural analysis of CRX, paving the way for a new approach to studying the biochemistry of this protein and its disease-causing mutant protein variants.


title: The transcriptomic signature of physiological trade-offs caused by larval overcrowding in Drosophila melanogaster

abstract: Intraspecific competition at the larval stage is an important ecological factor affecting life-history, adaptation and evolutionary trajectory in holometabolous insects. However, the molecular pathways and physiological trade-offs underpinning these ecological processes are poorly characterised. We reared Drosophila melanogaster at three egg densities (5, 60 and 300 eggs/ml) and sequenced the transcriptomes of pooled third-instar larvae. We also examined emergence time, egg-to-adult viability, adult mass and adult sex-ratio at each density. Medium crowding had minor detrimental effects on adult phenotypes compared to low density and yielded 24 differentially expressed genes (DEGs) including several chitinase enzymes. In contrast, high crowding had substantial detrimental effects on adult phenotypes and yielded 2107 DEGs. Among these, upregulated gene sets were enriched in sugar, steroid and amino acid metabolism as well as DNA replication pathways, whereas downregulated gene sets were enriched in ABC transporters, Taurine, Toll/Imd signalling and P450 xenobiotics metabolism pathways. Overall, our findings show that larval overcrowding has a large consistent effect on several molecular pathways (i.e., core responses) with few pathways displaying density-specific regulation (i.e., idiosyncratic responses). This provides important insights into how holometabolous insects respond to intraspecific competition during development.

category: ecology


title: Dominance of coniferous and broadleaved trees drives bacterial associations with boreal feather mosses

abstract: The composition of ecologically important moss-associated bacterial communities seems to be mainly driven by host species, but may also be shaped by environmental conditions related with tree-canopy dominance. The moss phyllosphere has been studied in coniferous forests while broadleaf forests remain understudied. To determine if host species or environmental conditions defined by tree-canopy dominance drives the bacterial diversity in the moss phyllosphere, we used 16S rRNA gene amplicon sequencing to quantify changes in bacterial communities as a function of host species (Pleurozium schreberi and Ptilium crista-castrensis) and forest type (coniferous black spruce versus deciduous broadleaf trembling aspen) in eastern Canada. Forest type, not host species, was the main factor affecting moss phyllosphere bacterial community composition, though the interaction of both variables was significant. Bacterial -diversity was highest in spruce forests, while there was greater turnover ({beta}- diversity) and higher {gamma}-diversity in aspen forests. Unexpectedly, Cyanobacteria were much more relatively abundant in aspen than in spruce forests, with the bacterial family Nostocaceae (Cyanobacteria) differing the most between both forest types. Our results suggest that the increasing change in dominance from coniferous to broadleaf trees due to natural and anthropic disturbances is likely to affect the composition of moss-associated bacteria in boreal forests.

category: microbiology


title: Glucocorticoid receptor dysregulation underlies 5-HT2A receptor-dependent synaptic and behavioral deficits in a mouse neurodevelopmental disorder model

abstract: Prenatal environmental insults increase the risk of neurodevelopmental psychiatric conditions in the offspring. Structural modifications of dendritic spines are central to brain development and plasticity. Using maternal immune activation (MIA) as a rodent model of prenatal environmental insult, previous results have reported dendritic structural deficits in the frontal cortex. However, very little is known about the molecular mechanism underlying MIA-induced synaptic structural alterations in the offspring. Using prenatal (E12.5) injection with poly-(I:C) as a mouse MIA model, we show that upregulation of the serotonin 5-HT2A receptor (5-HT2AR) is at least in part responsible for some of the effects of prenatal insults on frontal cortex dendritic spine structure and sensorimotor gating processes. Mechanistically, we report that this upregulation of frontal cortex 5-HT2AR expression is associated with MIA-induced reduction of nuclear translocation of the glucocorticoid receptor (GR) and, consequently, a decrease in the enrichment of GR at the 5-HT2AR promoter. The translational significance of these preclinical findings is supported by data in postmortem human brain samples suggesting dysregulated nuclear GR translocation in frontal cortex of schizophrenia subjects. Repeated (twice a day for 4 days) corticosterone administration augmented frontal cortex 5-HT2AR expression and reduced GR binding to the 5-HT2AR promoter. However, virally (AAV)-mediated augmentation of GR function reduced frontal cortex 5-HT2AR expression and improved sensorimotor gating processes via 5-HT2AR. Together, these data support a negative regulatory relationship between GR signaling and 5-HT2AR expression in mouse frontal cortex that may carry implications for the pathophysiology underlying 5-HT2AR dysregulation in neurodevelopmental psychiatric disorders.

category: neuroscience


title: Interspecies transfer of syntenic RAMOSA1 orthologs and promoter cis sequences impacts maize inflorescence architecture

abstract: Grass inflorescences support floral structures that each bear a single grain, where variation in branch architecture directly impacts yield. The maize RAMOSA1 (ZmRA1) transcription factor acts as a key regulator of inflorescence development by imposing branch meristem determinacy. Here, we show RA1 transcripts accumulate in boundary domains adjacent to spikelet meristems in Sorghum bicolor (Sb) and Setaria viridis (Sv) inflorescences similar as in the developing maize tassel and ear. To evaluate functional conservation of syntenic RA1 orthologs and promoter cis sequences in maize, sorghum and setaria, we utilized interspecies gene transfer and assayed genetic complementation in a common inbred background by quantifying recovery of normal branching in highly ramified ra1-R mutants. A ZmRA1 transgene that includes endogenous upstream and downstream flanking sequences recovered normal tassel and ear branching in ra1-R. Interspecies expression of two transgene variants of the SbRA1 locus, modeled as the entire endogenous tandem duplication or just the non-frameshifted downstream copy, complemented ra1-R branching defects and induced novel fasciation and branch patterns. The SvRA1 locus lacks conserved, upstream noncoding cis sequences found in maize and sorghum; interspecies expression of an SvRA1 transgene did not or only partially recovered normal inflorescence forms. Driving expression of the SvRA1 coding region by the ZmRA1 upstream region, however, recovered normal inflorescence morphology in ra1-R. These data leveraging interspecies gene transfer suggest that cis-encoded temporal regulation of RA1 expression is a key factor in modulating branch meristem determinacy that ultimately impacts grass inflorescence architecture.

category: plant biology


title: WhyD tailors surface polymers to prevent bacteriolysis and direct cell elongation in Streptococcus pneumoniae

abstract: Penicillin and related antibiotics disrupt cell wall synthesis in bacteria and induce lysis by misactivating cell wall hydrolases called autolysins. Despite the clinical importance of this phenomenon, little is known about the factors that control autolysins and how penicillins subvert this regulation to kill cells. In the pathogen Streptococcus pneumoniae (Sp), LytA is the major autolysin responsible for penicillin-induced bacteriolysis. We recently discovered that penicillin treatment of Sp causes a dramatic shift in surface polymer biogenesis in which cell wall-anchored teichoic acids (WTAs) increase in abundance at the expense of lipid-linked lipoteichoic acids. Because LytA binds to these polymers, this change recruits the enzyme to its substrate where it cleaves the cell wall and elicits lysis. In this report, we identify WhyD (SPD_0880) as a new factor that controls the level of WTAs in Sp cells to prevent LytA misactivation and lysis. We show that WhyD is a WTA hydrolase that restricts the WTA content of the wall to areas adjacent to active PG synthesis. Our results support a model in which the WTA tailoring activity of WhyD directs PG remodeling activity required for proper cell elongation in addition to preventing autolysis by LytA.


title: High seroprevalence of SARS-CoV-2 in white-tailed deer (Odocoileus virginianus) at one of three captive cervid facilities in Texas

abstract:
Free-ranging white-tailed deer (Odocoileus virginanus) across the United States are increasingly recognized as involved in SARS-CoV-2 transmission cycles. Through a cross-sectional study of 80 deer at three captive cervid facilities in central and southern Texas, we provide evidence of 34 of 36 (94.4%) white-tailed deer at a single captive cervid facility seropositive for SARS-CoV-2 by neutralization assay (PRNT90), with endpoint titers as high as 1280. In contrast, all tested white-tailed deer and axis deer (Axis axis) at two other captive cervid facilities were seronegative, and SARS-CoV-2 RNA was not detected in respiratory swabs from deer at any of the three facilities. These data support transmission among captive deer that cannot be explained by human contact for each infected animal, as only a subset of the seropositive does had direct human contact. The facility seroprevalence was more than double of that reported from wild deer, suggesting that the confined environment may facilitate transmission. Further exploration of captive cervids and other managed animals for their role in the epizootiology of SARS-CoV-2 is critical for understanding impacts on animal health and the potential for spillback transmission to humans or other animal taxa.

ImportanceAs SARS-CoV-2 vaccine coverge of the human population increases and variants of concern continue to emerge, identification of the epidemiologic importance of animal virus reservoirs is critical. We found that nearly all (94.4%) of the captive white-tailed deer at a cervid facility in central Texas had neutralizing antibodies for SARS-CoV-2. This seroprevalence is over double than that which has been reported from free-ranging deer from other regions of the US. Horizontal transmission among deer may be facilitated in confinement. Tracking new infections among wild and confined deer is critical for understanding the importance of animal reservoirs for both veterinary and human health.

category: zoology


title: Toxin-producing endosymbionts shield pathogenic fungus against micropredators

abstract:
The phytopathogenic fungus Rhizopus microsporus harbours a bacterial endosymbiont (Mycetohabitans rhizoxinica) for the production of the toxin rhizoxin, the causative agent of rice seedling blight. This toxinogenic bacterial-fungal alliance is, however, not restricted to the plant disease, but has been detected in numerous environmental isolates from geographically distinct sites covering all five continents. Yet, the ecological role of rhizoxin beyond rice seedling blight has been unknown.

Here we show that rhizoxin serves the fungal host in fending off protozoan and metazoan predators. Fluorescence microscopy and co-culture experiments with the fungivorous amoeba Protostelium aurantium revealed that ingestion of R. microsporus spores is toxic to P. aurantium. This amoebicidal effect is caused by the bacterial rhizoxin congener rhizoxin S2, which is also lethal towards the model nematode Caenorhabditis elegans. By combining stereomicroscopy, automated image analyses, and quantification of nematode movement we show that the fungivorous nematode Aphelenchus avenae actively feeds on R. microsporus that is lacking endosymbionts, while worms co-incubated with symbiotic R. microsporus are significantly less lively.

This work uncovers an unexpected ecological role of rhizoxin as shield against micropredators. This finding suggests that predators may function an evolutionary driving force to maintain toxin-producing endosymbionts in non-pathogenic fungi.


title: Dynamic Turnover of PI4P and PI(4,5)P2 under Hypoxia Controls Electrostatic Plasma Membrane Targeting

abstract: Phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-biphosphate (PIP2) are key phosphoinositides that determine the identity of the plasma membrane (PM) and regulate numerous key biological events there. To date, mechanisms regulating the homeostasis and dynamic turnover of PM PI4P and PIP2 in response to various physiological conditions and stresses remain to be fully elucidated. Here we report that hypoxia in Drosophila induces acute and reversible depletion of PM PI4P and PIP2 that severely disrupts the electrostatic PM targeting of multiple polybasic polarity proteins. Genetically encoded ATP sensors confirmed that hypoxia induces acute and reversible reduction of cellular ATP levels which showed a strong real-time correlation with the levels of PM PI4P and PIP2 in cultured cells. By combining genetic manipulations with quantitative imaging assays we showed that PI4KIII, as well as Rbo/EFR3 and TTC7 that are essential for targeting PI4KIII to PM, are required for maintaining the homeostasis and dynamic turnover of PM PI4P and PIP2 under normoxia and hypoxia. Our results revealed that in cells challenged by energetic stresses triggered by hypoxia, ATP inhibition and possibly ischemia, dramatic turnover of PM PI4P and PIP2 could have profound impact on many cellular processes including electrostatic PM targeting of numerous polybasic proteins.

category: cell biology


title: Dissecting conformational rearrangements and allosteric modulation in metabotropic glutamate receptor activation

abstract: Selective allosteric modulators bear great potential to fine-tune neurotransmitter-induced brain receptor responses. Promising targets are metabotropic glutamate (mGlu) receptors, which are associated to different brain diseases. These multidomain class C GPCRs experience concerted structural rearrangements and rely on allosteric modulation of agonist action to be fully activated. Here we establish live cell compatible fluorescence labeling of mGlu2 by click chemistry through genetic code expansion. Using lanthanide resonance energy transfer, we establish multiple FRET sensors to monitor ligand effects on conformational changes in mGlu2 extracellular domain and subsequently dissect the underlying conformational states by smFRET. Using three distinct FRET sensors, we demonstrate that mGlu activation relies on a ligand-induced sampling of three conformational states. Orthosteric agonists act by promoting the closure of the mGlu2 ligand binding domains, leading to an equilibrium between an inactive intermediate and the active state. Allosteric modulator further push this equilibrium toward the active state, promoting and stabilizing the relative reorientation of the mGlu protomers. These results underline the complex and dynamic nature of such type of neuroreceptors, pointing out that ligands fine-tune activation by differentially acting on the equilibria between multiple states.

category: biophysics


title: The scaffolding protein Flot2 regulates cytoneme-based transport of Wnt3 in gastric cancer

abstract: The Wnt/{beta}-catenin signalling pathway regulates multiple cellular processes during development and many diseases, including cell proliferation, migration, and differentiation. Despite their hydrophobic nature, Wnt proteins exert their function over long distances to induce paracrine signalling. Recent studies have identified several factors involved in Wnt secretion, however, our understanding of how Wnt ligands are transported between cells to interact with their cognate receptors is still debated. Here, we demonstrate that gastric cancer cells utilise cytonemes to transport Wnt3 intercellularly to promote proliferation. Furthermore, we identify the membrane-bound scaffolding protein Flotillin-2 (Flot2), frequently overexpressed in gastric cancer, as a regulator of these cytonemes. Together with the Wnt co-receptor and cytoneme initiator Ror2, Flot2 determines the number and length of Wnt3 cytonemes in gastric cancer. Finally, we show that Flot2 is necessary for Wnt8a cytonemes during zebrafish embryogenesis, suggesting a conserved mechanism for Flot2-mediated Wnt transport on cytonemes in development and disease.

category: cancer biology


title: Exploration of Methanomethylophilus alvus pyrrolysyl-tRNA synthetase activity in yeast

abstract:
Archaeal pyrrolysyl-tRNA synthetases (PylRSs) have been used to genetically encode over 200 distinct noncanonical amino acids (ncAAs) in proteins in E. coli and mammalian cells. This vastly expands the range of chemical functionality accessible within proteins produced in these organisms. Despite these clear successes, explorations of PylRS function in yeast remains limited. In this work, we demonstrate that the Methanomethylophilus alvus PylRS (MaPylRS) and its cognate tRNACUA support the incorporation of ncAAs into proteins produced in S. cerevisiae using stop codon suppression methodologies. Additionally, we prepared three MaPylRS mutants originally engineered in E. coli and determined that all three were translationally active with one or more ncAAs, although with low efficiencies of ncAA incorporation in comparison to the parent MaPylRS. Alongside MaPylRS variants, we evaluated the translational activity of previously reported Methanosarcina mazei, Methanosarcina barkeri, and chimeric M. mazei and M. barkeri PylRSs. Using the yeast strain RJY100, and pairing these aaRSs with the M. barkeri tRNACUA, we did not observe any detectable stop codon suppression activity under the same conditions that produced moderately efficient ncAA incorporation with MaPylRS. The addition of MaPylRS to the orthogonal translation machinery toolkit in yeast potentially opens the door to hundreds of ncAAs that have not previously been genetically encodable using other aminoacyl-tRNA synthetase/tRNA pairs. Extending the scope of ncAA incorporation in yeast could powerfully advance chemical and biological research for applications ranging from basic biological discovery to enzyme engineering and therapeutic protein lead discovery.

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category: synthetic biology


title: A Proposal for Finite But Unbounded Human Lifetimes

abstract: Close analysis of an extensive data set combined with independent evidence prompts our proposal to view human lifetimes as individually finite but collectively unbounded. We formulate a model incorporating this idea whose predictions agree very well with the observed data. In the model, human lifetimes are theoretically unbounded, but the probability of an individual living to an extreme age is negligible, so lifetimes are effectively limited. Our model incorporates a mortality hazard rate plateau and a late-life mortality deceleration effect in conjunction with a newly observed advanced age mortality acceleration. This reconciles many previously observed effects. The model is temporally stable: consistent with observation, parameters do not change over time. As an application, assuming no major medical advances, we predict the emergence of many individuals living past 120, but due to accelerating mortality find it unlikely that any will subsequently survive to an age of 125.

category: systems biology


title: Defining the dynamics of naive CD4 and CD8 T cells across the mouse lifespan

abstract: Naive CD4 and CD8 T cells are cornerstones of adaptive immunity, but the dynamics of their establishment early in life and how their kinetics change as they mature following release from the thymus are poorly understood. Further, due to the diverse signals implicated in naive T cell survival, it has been a long-held and conceptually attractive view that they are sustained by active homeostatic control as thymic activity wanes. Here we employ multiple experimental systems to identify a unified model of naive CD4 and CD8 T cell population dynamics across the mouse lifespan. We infer that both subsets divide rarely and progressively increase their survival capacity with cell age. Strikingly, this simple model captures naive CD4 T cell dynamics throughout life. In contrast, we find that newly generated naive CD8 T cells are lost more rapidly during the first 3-4 weeks of life, likely due to increased recruitment into memory. We find no evidence for elevated division rates in neonates, or for feedback regulation of naive T cell numbers at any age. We show how confronting mathematical models with diverse datasets can reveal a quantitative and remarkably simple picture of naive T cell dynamics from birth into old age.

category: immunology


title: Towards a unified model of naive T cell dynamics across the lifespan


title: Orally administered antigen can reduce or exacerbate pathology in an animal model of inflammatory arthritis dependent upon the timing of administration

abstract:
Currently, treatments for rheumatoid arthritis (RA) are focussed on treatment of disease symptoms rather than addressing the cause of disease, which could lead to remission and cure. Central to disease development is the induction of autoimmunity through a breach of self-tolerance. There is considerable research in RA focussed on antigens and approaches to re-establish antigen specific tolerance. A crucial step in this research is to employ appropriate animal models to test prospective antigen specific immunotherapies, preferably in the context of joint inflammation.

In this short communication, we use our previously developed model of antigen specific inflammatory arthritis in which OVA-specific TcR tg T cells drive breach of tolerance to endogenous antigens to determine the impact that the timing of therapy administration has upon disease progression. Using antigen feeding to induce tolerance we demonstrate that administration prior to articular challenge results in a reduced disease score as evidenced by pathology and serum antibody responses. By contrast, feeding antigen after articular challenge had the opposite effect and resulted in the exacerbation of pathology.

Although preliminary, these data suggest that the timing of antigen administration may be key to the success of tolerogenic immunotherapies. This has important implications for the timing of potential tolerogenic therapies in patients.


title: Analysis of historical selection in winter wheat

abstract:
Winter wheat is a major crop with a rich selection history in the modern era of crop breeding. Genetic gains across economically important traits like yield have been well characterized and are the major force driving its production. Winter wheat is also an excellent model for analyzing historical genetic selection. As a proof of concept, we analyze two major collections of winter wheat varieties that were bred in western Europe from 1916 to 2010, namely the Triticeae Genome (TG) and WAGTAIL panels, which include 333 and 403 varieties respectively. We develop and apply a selection mapping approach, Regression of Alleles on Years (RALLY), in these panels, as well as in simulated populations. RALLY maps loci under sustained historical selection by using a simple logistic model to regress allele counts on years of variety release. To control for drift-induced allele frequency change, we develop a hybrid approach of genomic control and delta control. Within the TG panel, we identify 22 significant RALLY quantitative selection loci (QSLs) and estimate the local heritabilities for 12 traits across these QSLs. By correlating predicted marker effects with RALLY regression estimates, we show that alleles whose frequencies have increased over time are heavily biased towards conferring positive yield effect, but negative effects in flowering time, lodging, plant height and grain protein content. Altogether, our results (1) demonstrate the use of RALLY to identify selected genomic regions while controlling for drift, and (2) reveal key patterns in the historical selection in winter wheat and guide its future breeding.

Key MessageModelling of the distribution of allele frequency over year of variety release identifies major loci involved in historical breeding of winter wheat.

category: genetics


title: Transcription shapes 3D chromatin organization by interacting with loop-extruding cohesin complexes

abstract: Cohesin organizes mammalian interphase chromosomes by reeling chromatin fibers into dynamic loops (Banigan and Mirny, 2020; Davidson et al., 2019; Kim et al., 2019; Yatskevich et al., 2019). "Loop extrusion" is obstructed when cohesin encounters a properly oriented CTCF protein (Busslinger et al., 2017; de Wit et al., 2015; Fudenberg et al., 2016; Nora et al., 2017; Sanborn et al., 2015; Wutz et al., 2017), and recent work indicates that other factors, such as the replicative helicase MCM (Dequeker et al., 2020), can also act as barriers to loop extrusion. It has been proposed that transcription relocalizes (Busslinger et al., 2017; Glynn et al., 2004; Lengronne et al., 2004) or interferes with cohesin (Heinz et al., 2018; Jeppsson et al., 2020; Valton et al., 2021; S. Zhang et al., 2021), and that active transcription start sites function as cohesin loading sites (Busslinger et al., 2017; Kagey et al., 2010; Zhu et al., 2021; Zuin et al., 2014), but how these effects, and transcription in general, shape chromatin is unknown. To determine whether transcription can modulate loop extrusion, we studied cells in which the primary extrusion barriers could be removed by CTCF depletion and cohesins residence time and abundance on chromatin could be increased by Wapl knockout. We found evidence that transcription directly interacts with loop extrusion through a novel "moving barrier" mechanism, but not by loading cohesin at active promoters. Hi-C experiments showed intricate, cohesin-dependent genomic contact patterns near actively transcribed genes, and in CTCF-Wapl double knockout (DKO) cells (Busslinger et al., 2017), genomic contacts were enriched between sites of transcription-driven cohesin localization ("cohesin islands"). Similar patterns also emerged in polymer simulations in which transcribing RNA polymerases (RNAPs) acted as "moving barriers" by impeding, slowing, or pushing loop-extruding cohesins. The model predicts that cohesin does not load preferentially at promoters and instead accumulates at TSSs due to the barrier function of RNAPs. We tested this prediction by new ChIP-seq experiments, which revealed that the "cohesin loader" Nipbl (Ciosk et al., 2000) co-localizes with cohesin, but, unlike in previous reports (Busslinger et al., 2017; Kagey et al., 2010; Zhu et al., 2021; Zuin et al., 2014), Nipbl did not accumulate at active promoters. We propose that RNAP acts as a new type of barrier to loop extrusion that, unlike CTCF, is not stationary in its precise genomic position, but is itself dynamically translocating and relocalizes cohesin along DNA. In this way, loop extrusion could enable translocating RNAPs to maintain contacts with distal regulatory elements, allowing transcriptional activity to shape genomic functional organization.

category: genomics


title: Toxoflavin secreted by Pseudomonas alcaliphila inhibits growth of Legionella pneumophila and its host Vermamoeba vermiformis

abstract: Legionella pneumophila is a natural inhabitant of water systems. From there, it can be transmitted to humans by aerosolization resulting in severe pneumonia. Most large outbreaks are caused by cooling towers contaminated with L. pneumophila. The resident microbiota of the cooling tower is a key determinant for the colonization and growth of L. pneumophila. The genus Pseudomonas correlates negatively with the presence of L. pneumophila, but it is not clear which species is responsible. Therefore, we identified the Pseudomonas species inhabiting 14 cooling towers using a Pseudomonas-specific 16S rRNA amplicon sequencing strategy. Cooling towers free of L. pneumophila contained a high relative abundance of members from the Pseudomonas alcaliphila/oleovorans phylogenetic cluster. In vitro, P. alcaliphila JCM 10630 inhibited the growth of L. pneumophila on agar plates. Analysis of the P. alcaliphila genome revealed the presence of a genes cluster predicted to produce toxoflavin. L. pneumophila growth was inhibited by pure toxoflavin and by extract from P. alcaliphila culture found to contain toxoflavin by LC-ESI-MS. In addition, toxoflavin inhibits growth of Vermameoba vermiformis, a host cell of L. pneumophila. Our study indicates that P. alcaliphila may be important to restrict growth of L. pneumophila in water systems through the production of toxoflavin. A sufficiently high concentration is likely not achieved in the bulk water but might have a local inhibitory effect such as in biofilm.


title: Estimating Pan evolutionary history from nucleotide site patterns

abstract: Introgression appears increasingly ubiquitous in the evolutionary history of various taxa, including humans. However, accurately estimating introgression is difficult, particularly when 1) there are many parameters, 2) multiple models fit the data well, and 3) parameters are not simultaneously estimated. Here, we use the software Legofit to investigate the evolutionary history of bonobos (Pan paniscus) and chimpanzees (P. troglodytes) using whole genome sequences. This approach 1) ignores within-population variation, reducing the number of parameters requiring estimation, 2) allows for model selection, and 3) simultaneously estimates all parameters. We tabulated site patterns from the autosomes of 71 bonobos and chimpanzees representing all five extant Pan lineages. We then compared previously proposed demographic models and estimated parameters using a deterministic approach. We further considered sex bias in Pan evolutionary history by analyzing the site patterns from the X chromosome. Introgression from bonobos into the ancestor of eastern and central chimpanzees and from western into eastern chimpanzees best explained the autosomal site patterns. This second event was substantial with an estimated 0.21 admixture proportion. Estimates of effective population size and most divergence dates are consistent with previous findings; however, we observe a deeper divergence within chimpanzees at 987 ka. Finally, we identify male-biased reproduction in Pan evolutionary history and suggest that western to eastern chimpanzee introgression was driven by western males mating with eastern females.


title: Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Tumor

abstract:
Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of mitochondrial TCA cycle enzyme, succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect in PC tumors, triggers dysregulation of Ca2+ homeostasis, and aberrantly activates calpain and the protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a cascade of phospho- signaling where GSK3 inhibition inactivates energy sensing by AMP-kinase through dephosphorylation of the AMP-kinase {gamma} subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC tumor formation. A novel Cdk5 inhibitor, MRT3-007, reversed this phospho-cascade, invoking an anti- Warburg effect, cell cycle arrest, and senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important novel mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers.

HighlightsO_LILoss of SDHB function in pheochromocytoma causes Ca2+ dysregulation, calpain activation, and aberrant activation of the protein kinase Cdk5.
C_LIO_LIHyperactive Cdk5 deregulates a GSK3/PRKAG2/AMPK signaling cascade.
C_LIO_LIp25 overexpression and consequent aberrant Cdk5 activity in chromaffin cells causes pheochromocytoma.
C_LIO_LIInhibition of Cdk5 activates the PRKAG2/AMPK/p53 signaling to rescue cell senescence and block PC tumor progression.
C_LI

Graphical Abstract

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title: Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Cancer


title: Characterization of CAP1 and ECA4 adaptors participating in clathrin-mediated endocytosis

abstract: Formation of endomembrane vesicles is crucial in all eukaryotic cells and relies on vesicle coats such as clathrin. Clathrin-coated vesicles form at the plasma membrane and the trans-Golgi Network. They contain adaptor proteins, which serve as binding bridges between clathrin, vesicle membranes, and cargoes. A large family of monomeric ANTH/ENTH/VHS adaptors is present in A. thaliana. Here, we characterize two homologous ANTH-type clathrin adaptors, CAP1 and ECA4, in clathrin-mediated endocytosis (CME). CAP1 and ECA4 are recruited to sites at the PM identified as clathrin-coated pits (CCPs), where they occasionally exhibit early bursts of high recruitment. Subcellular binding preferences of N- and C-terminal fluorescent protein fusions of CAP1 identified a functional adaptin-binding motif in the unstructured tails of CAP1 and ECA4. In turn, no function can be ascribed to a double serine phosphorylation site conserved in these proteins. Double knockout mutants do not exhibit deficiencies in general development or CME, but a contribution of CAP1 and ECA4 to these processes is revealed in crosses into sensitized endocytic mutant backgrounds. Overall, our study documents a contribution of CAP1 and ECA4 to CME in A. thaliana and opens questions about functional redundancy among non-homologous vesicle coat components.


title: Developmental patterning function of GNOM ARF-GEF mediated from the plasma membrane

abstract: The GNOM (GN) Guanine nucleotide Exchange Factor for ARF small GTPases (ARF-GEF) is among the best studied trafficking regulators in plants, playing crucial and unique developmental roles in patterning and polarity. The current models place GN at the Golgi apparatus (GA), where it mediates secretion/recycling, and at the plasma membrane (PM) presumably contributing to clathrin-mediated endocytosis (CME). The mechanistic basis of the developmental function of GN, distinct from the other ARF-GEFs including its homologue GNOM-LIKE1 (GNL1), remains elusive. Insights from this study redefine the current notions of GN function. We show that GN, but not GNL1, localizes to the PM at long-lived structures distinct from clathrin-coated pits, while CME and secretion proceed normally in gn knockouts. The functional GN mutant variant GNfewerroots, absent from the GA, suggests that PM is the major place of GN action responsible for its developmental function. Following inhibition by Brefeldin A, GN, but not GNL1, relocates to the PM likely on exocytic vesicles, suggesting selective molecular associations. A study of GN-GNL1 chimeric ARF-GEFs indicate that all GN domains contribute to the specific GN function in a partially redundant manner. Together, this study offers significant steps towards the elucidation of the mechanism underlying unique cellular and development functions of GN.


title: SH3Ps recruit auxilin-like vesicle uncoating factors into clathrin-mediated endocytosis

abstract: Clathrin-mediated endocytosis (CME) is an essential process of cellular cargo uptake operating in all eukaryotes. In animal and yeast, CME involves BAR-SH3 domain proteins, endophilins and amphiphysins, which function at the conclusion of CME to recruit factors for vesicle scission and uncoating. Arabidopsis thaliana contains BAR-SH3 domain proteins SH3P1-3, but their role is poorly understood. We identify SH3P1-3 as functional homologues of endophilin/amphiphysin. SH3P1-3 bind to discrete foci at the plasma membrane (PM), and colocalization indicates late recruitment of SH3P2 to a subset of clathrin-coated pits. PM recruitment pattern of SH3P2 is nearly identical to its interactor, a putative vesicle uncoating factor AUXILIN-LIKE1, and SH3P1-3 are required for most of AUXILIN-LIKE1 PM binding. This indicates a plant-specific modification of CME, where BAR-SH3 proteins recruit auxilin-like uncoating factors, rather than the uncoating phosphatases synaptojanins. Furthermore, we identify an unexpected redundancy between SH3P1-3 and a plant-specific endocytic adaptor, TPLATE complex, showing a contribution of SH3P1-3 to gross CME.


title: Resource allocation to cell envelopes and the scaling of bacterial growth rate

abstract:
1Although various empirical studies have reported a positive correlation between the specific growth rate and cell size across bacteria, it is currently unclear what causes this relationship. We conjecture that such scaling occurs because smaller cells have a larger surface-to-volume ratio and thus have to allocate a greater fraction of the total resources to the production of the cell envelope, leaving fewer resources for other biosynthetic processes. To test this theory, we developed a coarse-grained model of bacterial physiology composed of the proteome that converts nutrients into biomass, with the cell envelope acting as a resource sink. Assuming resources are partitioned to maximize the growth rate, the model yields expected scalings. Namely, the growth rate and ribosomal mass fraction scale negatively, while the mass fraction of envelope-producing enzymes scales positively with surface-to-volume. These relationships are compatible with growth measurements and quantitative proteomics data reported in the literature.

2 Author summaryIt is widely known that smaller eukaryotes tend to grow faster. However, this trend does not hold in bacteria, where small-celled species grow slower. We propose that small bacteria - compared to their larger counterparts - have to invest a greater fraction of the total resources to cell envelope owing to their large surface-to-volume ratio, leaving fewer resources to internal biosynthetic processes that build the cell. By representing the cell as being composed of proteins (that convert nutrients into biomass), and cell envelope, we find that cells with large surface-to-volume ratio grow more slowly because they have to invest resources in the production of the cell envelope and the enzyme machinery that builds this structure, thus leaving fewer resources to ribosomes that replicate the cell. These predictions are corroborated by comparison with growth rate data across more than 200 bacterial species.


title: Metabolic phenotyping of marine heterotrophs on refactored media reveals diverse metabolic adaptations and lifestyle strategies

abstract:
Microbial communities, through their metabolism, drive carbon cycling in marine environments. These complex communities are composed of many different microorganisms including heterotrophic bacteria, each with its own nutritional needs and metabolic capabilities. Yet, models of ecosystem processes typically treat heterotrophic bacteria as a "black box", which does not resolve metabolic heterogeneity nor address ecologically important processes such as the successive modification of different types of organic matter. Here we directly address the heterogeneity of metabolism by characterizing the carbon source utilization preferences of 63 heterotrophic bacteria representative of several major marine clades. By systematically growing these bacteria on 10 media containing specific subsets of carbon sources found in marine biomass, we obtained a phenotypic fingerprint that we used to explore the relationship between metabolic preferences and phylogenetic or genomic features. At the class level, these bacteria display broadly conserved patterns of preference for different carbon sources. Despite these broad taxonomic trends, growth profiles correlate poorly with phylogenetic distance or genome-wide gene content. However, metabolic preferences are strongly predicted by a handful of key enzymes that preferentially belong to a few enriched metabolic pathways, such as those involved in glyoxylate metabolism and biofilm formation. We find that enriched pathways point to enzymes directly involved in the metabolism of the corresponding carbon source and suggest potential associations between metabolic preferences and other ecologically-relevant traits. The availability of systematic phenotypes across multiple synthetic media constitutes a valuable resource for future quantitative modeling efforts and systematic studies of inter-species interactions.

ImportanceHalf of the Earths annual primary production is carried out by phytoplankton in the surface ocean. However, this metabolic activity is heavily impacted by heterotrophic bacteria, which dominate the transformation of organic matter released from phytoplankton. Here, we characterize the diversity of metabolic preferences across many representative heterotrophs by systematically growing them on different fractions of dissolved organic carbon (DOC). Our analysis suggests that different clades of bacteria have substantially distinct preferences for specific carbon sources, in a way that cannot be simply mapped onto phylogeny. These preferences are associated with the presence of specific genes and pathways, reflecting an association between metabolic capabilities and ecological lifestyles. In addition to helping understand the importance of heterotrophs under different conditions, the phenotypic fingerprint we obtained can help build higher resolution quantitative models of global microbial activity and biogeochemical cycles in the oceans.


title: HIF-1α Contributes to the Progression of Chronic Obstructive Pulmonary Disease

abstract:
BackgroundHypoxia-inducible factor-1 (HIF-1) plays an important regulatory role in inflammatory and hypoxic diseases. Higher HIF-1 level was found in lungs of chronic obstructive pulmonary disease (COPD) patients, however, its role in cigarette smoke (CS)-induced COPD has not been fully studied. Digoxin has been showed to inhibit HIF-1 translation and block HIF-1 activity and thus is often used as the HIF-1 inhibitor. Therefore, in the present study, we chose digoxin as the inhibitor to investigate whether HIF-1 contributes to the progression of COPD and possible mechanism.

MethodsCS-exposed mice were intragastrically treated with different doses of digoxin, and COPD-associated phenotypes such as pathological changes in lungs, inflammation, lung function and mucus secretion in airways were evaluated. Meanwhile, CSE-treated A549 cells were administrated with digoxin or S7959. Moreover, EMT-associated markers together with HIF-1\TGF-{beta}1\Smad3 signaling pathway were detected both in vivo and in vitro.

ResultsThe level of HIF-1 was significantly increased in lungs of COPD mice and CSE-exposed A549 cells, which was markedly suppressed by digoxin. Moreover, digoxin inhibited CS-induced inflammatory responses, lung function decline, and mucus hyper-secretion in COPD mouse model. In in vitro studies, digoxin decreased CSE-induced pro-inflammatory cytokine release. Importantly, CS-induced or CSE-induced EMT and up-regulation of HIF-1/TGF-{beta}1/Smad pathway was inhibited by digoxin. Additionally, S7959 mitigated CSE-induced EMT in A549 cells.

ConclusionsDigoxin can protect CS-induced COPD and prevent CS-induced EMT possibly through HIF-1/TGF-{beta}1/Smad3 signaling pathway. This study suggests HIF1- could be a potential intervention target for COPD prevention and treatment, especially for EMT in CS-induced COPD.

category: molecular biology


title: The Cryo-EM Structures of two Amphibian Antimicrobial Cross-β Amyloid Fibrils

abstract: The amyloid-antimicrobial link hypothesis is based on antimicrobial properties found in human amyloids involved in neurodegenerative and systemic diseases, along with amyloidal structural properties found in antimicrobial peptides (AMPs) across kingdoms of life. Supporting this hypothesis, we here determined the fibril structure of two AMPs from amphibians, uperin 3.5 and aurein 3.3, by cryogenic electron microscopy (cryo-EM), revealing amyloid cross-{beta} fibrils of mated {beta}-sheets at atomic resolution. Uperin 3.5 displayed substantial polymorphism with a protofilament of two mated {beta}-sheets. The determined structure was a polymorph showing a 3-blade symmetrical propeller of nine peptides per fibril layer including tight {beta}-sheet interfaces. This cross-{beta} cryo-EM structure complements the cross- fibril conformation previously determined by a crystal structure, substantiating a secondary structure switch mechanism of uperin 3.5. The aurein 3.3 arrangement consisted of six peptides per fibril layer, all showing kinked {beta}-sheets allowing a rounded compactness of the fibril. The kinked {beta}-sheets are similar to LARKS (Low-complexity, Amyloid-like, Reversible, Kinked segments) found in human functional amyloids. The amyloidal properties of antimicrobial peptides shed light on a mechanism of regulation of animicrobial activity involving self-assembly and fibril morphological variations. Moreover, the known endurance of amyloid structures can provide a template for the design of sturdy antimicrobials.


title: RAB18 is a key regulator of GalNAc conjugated siRNA induced silencing in Hep3B cells

abstract: Small interfering RNAs (siRNA) therapeutics have developed rapidly in recent years, despite the challenges associated with delivery of large, highly charged nucleic acids. Delivery of siRNA therapeutics to the liver has been established, with conjugation of siRNA to N-acetylgalactosamine (GalNAc) providing durable gene knockdown in hepatocytes following subcutaneous injection. GalNAc binds the asialoglycoprotein receptor (ASGPR) that is highly expressed on hepatocytes and exploits this scavenger receptor to deliver siRNA across the plasma membrane by endocytosis. However, siRNA needs to access the RNA-induced silencing complex (RISC) in the cytoplasm to provide effective gene knockdown and the entire siRNA delivery process is very inefficient, likely due to steps required for endosomal escape, intracellular trafficking, and stability of siRNA. To reveal the cellular factors limiting delivery of siRNA therapeutics, we performed a pooled, genome wide knockout screen based on delivery of GalNAc conjugated siRNA targeting the HPRT1 gene in the human hepatocellular carcinoma line Hep3B. Our primary pooled genome wide knockout screen identified candidate genes that when knocked out significantly enhanced siRNA efficacy in Hep3B cells. Follow-up studies indicate that knockout of one gene in particular, RAB18, improved siRNA efficacy.


title: BML-257, a small molecule that protects against drug induced liver injury in zebrafish

abstract: The use of many essential drugs is restricted due to their deleterious effects on the liver. Molecules that can prevent or protect the liver from drug induced liver injury (DILI) would be valuable in such situations. We used hepatocyte-specific expression of bacterial nitroreductase in zebrafish to cause temporally controlled liver damage. This transgenic line was used to run a whole organism based chemical screen in zebrafish larvae. In this screen we identified BML-257, a potent small molecule AKT inhibitor, that protected the liver against metronidazole-induced liver injury. BML-257 also showed potent prophylactic and pro-regenerative activity in this liver damage model. BML-257 also showed remarkable protective action in two independent toxicological models of liver injury caused by acetaminophen and Isoniazid. This suggests that BML-257 may have the potential to protect against multiple kinds of drug induced liver injury.


title: NOX4 inhibition promotes the remodeling of dystrophic muscle

abstract:
The muscular dystrophies (MDs) are genetic muscle diseases that result in progressive muscle degeneration followed by the fibrotic replacement of affected muscles as regenerative processes fail. Therapeutics that specifically address the fibrosis and failed regeneration associated with MDs represent a major unmet clinical need for MD patients, particularly those with advanced stage disease progression. The current study investigates targeting NAD(P)H oxidase (NOX) 4 as a potential strategy to reduce fibrosis and promote regeneration in disease-burdened muscle that models Duchenne muscular dystrophy (DMD). NOX4 is elevated in the muscles of dystrophic mice and DMD patients, localizing primarily to interstitial cells located between muscle fibers. Genetic and pharmacological targeting of NOX4 significantly reduces fibrosis in dystrophic respiratory and limb muscles. Mechanistically, NOX4 targeting decreases the number of fibrosis-depositing cells (myofibroblasts) and restores the number of muscle-specific stem cells (satellite cells) to their physiological niche, thereby, rejuvenating muscle regeneration. Furthermore, acute inhibition of NOX4 is sufficient to induce apoptotic clearing of myofibroblasts within dystrophic muscle. These data indicate that targeting NOX4 is an effective strategy to promote the beneficial remodeling of disease-burdened muscle representative of DMD and, potentially, other MDs and muscle pathologies.

SIGNIFICANCE STATEMENTMuscular dystrophies are progressive muscle diseases. Therapeutics capable of combating the severe fibrosis that replaces functional muscle in these devastating diseases is a major unmet clinical need, particularly for the treatment of older patients. The current work reveals that targeting NOX4 in dystrophic muscle promotes the beneficial remodeling of disease-burdened musculature. This is achieved by the removal of disease-causing cells, known as myofibroblasts, which results in reduced muscle fibrosis and rejuvenation of muscle regeneration. NOX4-targeting strategies, therefore, represent remodeling therapeutics capable of improving muscle disease caused by muscular dystrophy, and, likely, other muscle pathologies.


title: Free diffusion of PI(4,5)P2 in the plasma membrane in the presence of high density effector protein complexes

abstract: The lipid phosphatidyl-D-myo-inositol-4,5-bisphosphate [PI(4,5)P2] is a master regulator of plasma membrane (PM) function. It engages effector proteins that regulate diverse traffic, transport, signaling and cytoskeletal processes that define PM structure and function. How a single class of lipid molecules independently regulate so many parallel processes remains an open question. We tested the hypothesis that spatially segregated pools of PI(4,5)P2 are associated with, and thus reserved for regulation of, different functional complexes in the PM. The mobility of PI(4,5)P2 in the membrane was measured using lipid biosensors by single particle tracking photoactivation localization microscopy (sptPALM). We found that PI(4,5)P2, and several other classes of inner PM lipids, diffuse rapidly at approximately 0.3 {micro}m2/s with largely Brownian motion, although they spend approximately a third of their time diffusing much more slowly. Surprisingly, areas of the PM occupied by PI(4,5)P2-dependent complexes, such endoplasmic-reticulum:PM contact sites, clathrin-coated structures, and several actin cytoskeletal elements including focal adhesions, did not cause a change in PI(4,5)P2 lateral mobility. Only the spectrin and septin cytoskeletons were observed to produce a slowing of PI(4,5)P2 diffusion. We conclude that even structures with high densities of PI(4,5)P2-engaging effector proteins, such as clathrin coated pits and focal adhesions, do not corral free PI(4,5)P2, questioning a role for spatially segregated PI(4,5)P2 pools in organizing and regulating parallel PM functions.


title: Purification of synchronized E. coli transcription elongation complexes by reversible immobilization on magnetic beads

abstract: Synchronized transcription elongation complexes (TECs) are a fundamental tool for in vitro studies of transcription and RNA folding. Transcription elongation can be synchronized by omitting one or more NTPs from an in vitro transcription reaction so that RNA polymerase can only transcribe to the first occurrence of the omitted nucleotide(s) in the coding DNA strand. This approach was developed over four decades ago and has been applied extensively in biochemical investigations of RNA polymerase enzymes, but has not been optimized for RNA-centric assays. In this work, we describe the development of a system for isolating synchronized TECs from an in vitro transcription reaction. Our approach uses a custom 5 leader sequence, called C3-SC1, to reversibly capture synchronized TECs on magnetic beads. We first show that complexes isolated by this procedure, called C3-SC1TECs, are >95% pure, >98% active, highly synchronous (94% of complexes chase in <15s upon addition of saturating NTPs), and compatible with solid-phase transcription; the yield of this purification is [~]8%. We then show that C3-SC1TECs perturb, but do not interfere with, the function of ZTP-sensing and ppGpp-sensing transcriptional riboswitches. For both riboswitches, transcription using C3-SC1TECs improved the efficiency of transcription termination in the absence of ligand but did not inhibit ligand-induced transcription antitermination. Given these properties, C3-SC1TECs will likely be useful for developing biochemical and biophysical RNA assays that require high-performance, quantitative bacterial in vitro transcription.

category: biochemistry


title: Active site geometry stabilization of a presenilin homolog by the lipid bilayer promotes intramembrane proteolysis

abstract: Cleavage of membrane proteins in the lipid bilayer by intramembrane proteases is crucial for health and disease. Although different lipid environments can potently modulate their activity, how this is linked to their structural dynamics is unclear. Here we show that the carboxy-peptidase-like activity of the archaeal intramembrane protease PSH, a homolog of the Alzheime[r]s disease-associated presenilin/{gamma}-secretase is impaired in micelles and promoted in a lipid bilayer. Comparative molecular dynamics simulations revealed that important elements for substrate binding such as transmembrane domain 6a of PSH are more labile in micelles and stabilized in the lipid bilayer. Moreover, consistent with an enhanced interaction of PSH with a transition- state analog inhibitor, the bilayer promoted the formation of the enzymes catalytic active site geometry. Our data indicate that the lipid environment of an intramembrane protease plays a critical role in structural stabilization and active site arrangement of the enzyme-substrate complex thereby promoting intramembrane proteolysis.


title: Membrane tension propagation couples axon growth and collateral branching

abstract: Neuronal axons must navigate a mechanically heterogeneous environment to reach their targets, but the biophysical mechanisms coupling mechanosensation, growth, and branching are not fully understood. Here, we show that local changes in membrane tension propagate along axons at approximately 20 {micro}m/s, more than 1000-fold faster than in other non-motile cells. This rapid and long-range mechanical signaling mediates bidirectional competition between axonal branch initiation and growth cone extension. Our data suggest a mechanism by which mechanical cues at one part of a growing axon can affect growth dynamics remotely.


title: Trade-Offs Between Hepatic Host Defense and Metabolic Programs Underlie Sex-Biased Diseases

abstract: Current concepts in evolutionary medicine propose that trade-offs and mismatches with a shifting environment increase disease risk. While biological sex also impacts disease prevalence, contributions of environmental pressures to sex-biased diseases remain unexplored. Here, we show that sex-dependent hepatic programs confer a robust (~300%) survival advantage for male mice during lethal bacterial infection. The transcription factor BCL6, which masculinizes hepatic gene expression at puberty, is essential for this advantage. However, protection by BCL6 comes at a cost following dietary excess, resulting in overt fatty liver and glucose intolerance in males. Deleting hepatic BCL6 reverses these phenotypes but markedly lowers male fitness during infection, thus establishing a sex-dependent tradeoff between host defense and metabolic systems. We suggest that these tradeoffs, coupled with current environmental pressures, drive metabolic disease in males.

category: physiology


title: Interactions between influenza A virus nucleoprotein and gene segment UTRs facilitate selective modulation of viral gene expression

abstract:
The influenza A virus (IAV) genome is divided into eight negative-sense, single-stranded RNA segments. Each segment exhibits a unique level and temporal pattern of expression, however the exact mechanisms underlying the patterns of individual gene segment expression are poorly understood. We previously demonstrated that a single substitution in the viral nucleoprotein (NP:F346S) selectively modulates neuraminidase (NA) gene segment expression while leaving other segments largely unaffected. Given what is currently known about NP function, there is no obvious explanation for how changes in NP can selectively modulate the replication of individual gene segments. We found that the specificity of this effect for the NA segment is virus strain specific and depends on the UTR sequences of the NA segment. While the NP:F346S substitution did not significantly alter the RNA binding or oligomerization activities of NP in vitro, it specifically decreased the ability of NP to promote NA segment vRNA synthesis. In addition to NP residue F346, we identified two other adjacent aromatic residues in NP (Y385 & F479) capable of similarly regulating NA gene segment expression, suggesting a larger role for this domain in gene-segment specific regulation. Our findings reveal a new role for NP in selective regulation of viral gene segment replication and demonstrate how the expression patterns of individual viral gene segments can be modulated during adaptation to new host environments.

Author summaryInfluenza A virus (IAV) is a respiratory pathogen that remains a significant source of morbidity and mortality. Escape from host immunity or emergence into new host species often requires mutations that modulate the functional activities of the viral glycoproteins hemagglutinin (HA) and neuraminidase (NA) which are responsible for virus attachment to and release from host cells, respectively. Maintaining the functional balance between the activities of HA and NA is required for fitness across multiple host systems. Thus, selective modulation of viral gene expression patterns may be a key determinant of viral immune escape and cross-species transmission potential. We identified a novel mechanism by which the viral nucleoprotein (NP) gene can selectively modulate NA segment replication and gene expression through interactions with the segment UTR. Our work highlights an unexpected role for NP in selective regulation of expression from the individual IAV gene segments.


title: Antibiotic action revealed by real-time imaging of the mycobacterial membrane

abstract: The current understanding of mycobacterial cell envelope remodeling in response to antibiotics is limited. Chemical tools that report on phenotypic changes with minimal cell wall perturbation are critical to understanding such time-dependent processes. We employed a fluorogenic chemical probe to image how antibiotics perturb mycobacterial cell envelope assembly in real-time. Time-lapse microscopy revealed that differential antibiotic treatment elicited unique cellular phenotypes, providing a platform for simultaneously monitoring cell envelope construction and remodeling responses. Our data show that rifampicin, which does not directly inhibit cell wall biosynthesis, affords a readily detected mycomembrane phenotype. The fluorogenic probe revealed the production of extracellular vesicles in response to antibiotics, and analyses of these vesicles indicate that antibiotic treatment elicits the release of agents that attenuate macrophage activation.


title: Increased resolution in the face of conflict: phylogenomics of the Neotropical bellflowers (Campanulaceae: Lobelioideae), a rapid plant radiation

abstract:
Background and AimsThe centropogonid clade (Lobelioideae: Campanulaceae) is an Andean-centered rapid radiation characterized by repeated convergent evolution of morphological traits, including fruit type and pollination syndromes. While previous studies have resolved relationships of lineages with fleshy fruits into subclades, relationships among capsular species remain unresolved, particularly along the phylogenetic backbone. This lack of resolution has impeded reclassification of non-monophyletic genera, whose current taxonomy relies heavily on traits that have evolved multiple times within the clade.

MethodsTargeted sequence capture using a probeset recently developed for the centropogonid clade was used to obtain phylogenomic data from DNA extracted from both silica-dried and herbarium leaf tissue. These data were used to infer relationships among species using concatenated and partitioned species tree methods, as well as to quantify gene tree discordance.

Key ResultsWhile silica-dried leaf tissue resulted in generally more and longer sequence data, the inclusion of herbarium samples improved phylogenetic reconstruction. Relationships among baccate lineages are similar to those inferred by previous studies, though they differ within and among capsular lineages. We improve resolution of Siphocampylus, which forms ten groups of closely related species to which we provide informal names that largely do not correspond to current infrageneric taxonomy. Two subclades of Siphocampylus and two individual species are rogue taxa whose placement differs widely across analyses. Gene tree discordance is high.

ConclusionsThe first phylogenomic study of the centropogonid clade considerably improves our understanding of relationships in this rapid radiation. Differences across analyses and the possibility of additional lineage discoveries still hamper a solid and stable reclassification. Rapid morphological innovation corresponds with a high degree of phylogenomic complexity, including cytonuclear discordance, nuclear gene tree conflict, and well-supported differences between analyses based on different nuclear loci. Taken together, these results point to a potential role of hemiplasy underlying repeated convergent evolution. This hallmark of rapid radiations is likely present in many other species-rich Andean plant radiations.

category: evolutionary biology


title: Transcriptome-based phylogeny of the semi-aquatic bugs (Hemiptera: Heteroptera: Gerromorpha) reveals patterns of lineage expansion in a series of new adaptive zones

abstract: Key innovations enable access to new adaptive zones and are often linked to increased species diversification. As such, they have attracted much attention, yet their concrete consequences on the subsequent evolutionary trajectory and diversification of the bearing lineages remain unclear. The monophyletic group of water striders and relatives (Hemiptera: Heteroptera: Gerromorpha) represent a group of insects that transited to live on the water-air interface and diversified to occupy ponds, puddles, streams, mangroves and even oceans. This lineage offers an excellent model to study the patterns and processes underlying species diversification following the conquest of new adaptive zones. However, such studies require a reliable and comprehensive phylogeny of the infraorder. Based on whole transcriptomic datasets of 97 species and fossil records, we reconstructed a new phylogeny of the Gerromorpha that resolved inconsistencies and uncovered strong support for previously unknown relationships between some important taxa. We then used this phylogeny to reconstruct the ancestral state of a set of adaptations associated with water surface invasion (fluid locomotion, dispersal and transition to saline waters) and sexual dimorphism. Our results uncovered important patterns and dynamics of phenotypic evolution revealing how the initial event of water surface invasion enabled multiple subsequent transitions to new adaptive zones, representing distinct niches of water surfaces, and further diversification of the group. This phylogeny and the associated transcriptomic datasets constitute highly valuable resources, making Gerromorpha an attractive model lineage to study phenotypic evolution.


title: A Saturated Map of Common Genetic Variants Associated with Human Height from 5.4 Million Individuals of Diverse Ancestries

abstract: Common SNPs are predicted to collectively explain 40-50% of phenotypic variation in human height, but identifying the specific variants and associated regions requires huge sample sizes. Here we show, using GWAS data from 5.4 million individuals of diverse ancestries, that 12,111 independent SNPs that are significantly associated with height account for nearly all of the common SNP-based heritability. These SNPs are clustered within 7,209 non-overlapping genomic segments with a median size of ~90 kb, covering ~21% of the genome. The density of independent associations varies across the genome and the regions of elevated density are enriched for biologically relevant genes. In out-of-sample estimation and prediction, the 12,111 SNPs account for 40% of phenotypic variance in European ancestry populations but only ~10%-20% in other ancestries. Effect sizes, associated regions, and gene prioritization are similar across ancestries, indicating that reduced prediction accuracy is likely explained by linkage disequilibrium and allele frequency differences within associated regions. Finally, we show that the relevant biological pathways are detectable with smaller sample sizes than needed to implicate causal genes and variants. Overall, this study, the largest GWAS to date, provides an unprecedented saturated map of specific genomic regions containing the vast majority of common height-associated variants.


title: Molecular basis of SARS-CoV-2 Omicron variant receptor engagement and antibody evasion and neutralization

abstract: The SARS-CoV-2 Omicron variant exhibits striking immune evasion and is spreading globally at an unprecedented speed. Understanding the underlying structural basis of the high transmissibility and greatly enhanced immune evasion of Omicron is of high importance. Here through cryo-EM analysis, we present both the closed and open states of the Omicron spike, which appear more compact than the counterparts of the G614 strain, potentially related to the Omicron substitution induced enhanced protomer-protomer and S1-S2 interactions. The closed state showing dominant population may indicate a conformational masking mechanism of immune evasion for Omicron spike. Moreover, we capture two states for the Omicron S/ACE2 complex with S binding one or two ACE2s, revealing that the substitutions on the Omicron RBM result in new salt bridges/H-bonds and more favorable electrostatic surface properties, together strengthened interaction with ACE2, in line with the higher ACE2 affinity of the Omicron relative to the G614 strain. Furthermore, we determine cryo-EM structures of the Omicron S/S3H3 Fab, an antibody able to cross-neutralize major variants of concern including Omicron, elucidating the structural basis for S3H3-mediated broad-spectrum neutralization. Our findings shed new lights on the high transmissibility and immune evasion of the Omicron variant and may also inform design of broadly effective vaccines against emerging variants.


title: Potent protease inhibitors of deadly lagoviruses: rabbit hemorrhagic disease virus and European brown hare syndrome virus

abstract: Rabbit hemorrhagic disease (RHD) and European brown hare syndrome (EBHS) are highly contagious diseases caused by lagoviruses in the Caliciviridae family and mainly affect rabbits and hares, respectively. These infectious diseases are associated with high mortality and a serious threat to domesticated and wild rabbits and hares, including endangered species such as Riparian brush rabbits. In the US, only isolated cases of RHD had been reported until Spring 2020. However, RHD caused by RHD type 2 virus (RHDV2) was unexpectedly reported in April 2020 in New Mexico and has subsequently spread to several US states infecting wild rabbits and hares, making it highly likely that RHD will become endemic in the US. Vaccines are available for RHD, however, there is no specific treatment for these diseases. RHDV and EBHSV encode a 3C-like protease (3CLpro), which is essential for virus replication and a promising target for antiviral drug development. We have previously generated focused small molecule libraries of 3CLpro inhibitors and demonstrated the in vitro potency and in vivo efficacy of some protease inhibitors against viruses that encode 3CLpro including caliciviruses and coronaviruses. Here we established the enzyme and cell-based assays for these uncultivable viruses to determine the in vitro activity of 3CLpro inhibitors, including GC376, a protease inhibitor being developed for feline infectious peritonitis, and identified potent inhibitors of RHDV1 and 2 and EBHSV. In addition, structure-activity relationship study and homology modelling of the 3CLpros and inhibitors revealed that lagoviruses share similar structural requirements for 3CLpro inhibition with other caliciviruses.


title: Bacterial lifestyle switch in response to algal metabolites

abstract:
Unicellular algae, termed phytoplankton, greatly impact the marine environment by serving as the basis of marine food webs and by playing central roles in biogeochemical cycling of elements. The interactions between phytoplankton and heterotrophic bacteria affect the fitness of both partners. It is becoming increasingly recognized that metabolic exchange determines the nature of such interactions, but the underlying molecular mechanisms remain underexplored. Here, we investigated the molecular and metabolic basis for the bacterial lifestyle switch, from coexistence to pathogenicity, in Sulfitobacter D7 during its interaction with Emiliania huxleyi, a cosmopolitan bloom-forming phytoplankter. To unravel the bacterial lifestyle switch, we profiled bacterial transcriptomes in response to infochemicals derived from algae in exponential and stationary growth, which induced the Sulfitobacter D7 coexistence and pathogenicity lifestyles, respectively. We found that algal dimethylsulfoniopropionate (DMSP) was a pivotal signaling molecule that mediated the transition between the lifestyles. However, the coexisting and pathogenic lifestyles were evident only in the presence of additional algal metabolites. In the pathogenic mode, Sulfitobacter D7 upregulated flagellar motility and many transport systems, presumably to maximize assimilation of E. huxleyi-derived metabolites released by algal cells upon cell death. Specifically, we discovered that algae-produced benzoate promoted the growth of Sulfitobacter D7, and negated the DMSP-inducing lifestyle switch to pathogenicity, demonstrating that benzoate is important for maintaining the coexistence of algae and bacteria. We propose that bacteria can sense the physiological status of the algal host through changes in the metabolic composition, which will determine the bacterial lifestyle during the interactions.

Significance StatementMicroorganisms in the marine environment play crucial roles in the regulation of Earths climate and elemental cycling. Understanding microbial interactions and the metabolic exchange that drives them is necessary for disentangling the complexity of the marine ecosystem. Here we demonstrate how the opportunistic pathogen Sulfitobacter D7 switches its lifestyle from coexistence to pathogenicity in response to metabolites released by Emiliania huxleyi, a bloom-forming unicellular alga. By mapping bacterial transcriptional profiles, we show that the algal metabolite dimethylsulfoniopropionate (DMSP), an important signaling molecule in the marine environment, is essential for the bacterial lifestyle switch. However, the activity of DMSP depended on additional algal signals. This work emphasizes how metabolic crosstalk can influence the nature and fate of microbial interactions, which have cascading effects on large-scale oceanic processes.


title: An NMR fingerprint matching approach for the identification and structural re-evaluation of Pseudomonas lipopeptides.

abstract:
Cyclic lipopeptides (CLiPs) are secondary metabolites secreted by a range of bacterial phyla. CLiPs from Pseudomonas in particular display diverse structural variations in terms of the number of amino acid residues, macrocycle size, amino acid identity and stereochemistry (e.g. D- vs. L-amino acids). Reports detailing the discovery of novel or already characterized CLiPs from new sources appear regularly in literature. Increasingly however, the lack of detailed characterization threatens to cause considerable confusion, especially if configurational heterogeneity is present for one or more amino acids. Using Pseudomonas CLiPs from the Bananamide, Orfamide and Xantholysin groups as test cases, we demonstrate and validate that 1H and 13C NMR chemical shifts of CLiPs are sufficiently sensitive to differentiate between possible diastereomers of a particular sequence even when they only differ in a single D/L configuration. Rapid screening, involving simple comparison of the NMR fingerprint of a newly isolated CLiP with that of a reference CLiP of known stereochemistry, can then be applied to resolve dead-ends in configurational characterization and avoid the much more cumbersome chemical characterization protocols. Even when the stereochemistry of a particular reference CLiP remains to be established, NMR fingerprinting still allows verifying whether a CLiP from a novel source is already present in the reference collection, thus contributing to dereplication. To benefit research involving CLiPs, we have made a publicly available  knowledge base at https://www.rhizoclip.be, where we present an overview of published NMR fingerprint data of characterized CLiPs, together with literature data on the originally determined structures.

Significance StatementPseudomonas CLiPs, are ubiquitous specialized metabolites, impacting the producers lifestyle and interactions with the (a)biotic environment. Consequently, they generate interest for agricultural and clinical applications. Establishing structure-activity relationships as premise to their development is hindered because full structural characterization including stereochemistry requires labor-intensive analyses, without guarantee for success. Moreover, increasing use of superficial comparison with previously characterized CLiPs introduces or propagates erroneous attributions, clouding further scientific progress. We provide a generally applicable characterization methodology for structural comparison of newly isolated CLiPs to reference compounds with (un)known stereochemistry based on NMR fingerprints. The reference compound database available for the wide scientific community promises to facilitate structural assessment and dereplication of newly isolated CLiPs, and to support genome mining for novel CLiPs.


title: Visual threats reduce blood-feeding and trigger escape responses in Aedes aegypti mosquitoes.

abstract:
The diurnal mosquitoes Aedes aegypti are vectors of several arboviruses, including dengue, yellow fever, and Zika viruses. To find a host to feed on, they rely on the sophisticated integration of olfactory, visual, thermal, and gustatory cues reluctantly emitted by the hosts. If detected by their target, this latter may display defensive behaviors that mosquitoes need to be able to detect and escape. In humans, a typical response is a swat of the hand, which generates both mechanical and visual perturbations aimed at a mosquito. While the neuro-sensory mechanisms underlying the approach to the host have been the focus of numerous studies, the cues used by mosquitoes to detect and identify a potential threat remain largely understudied. In particular, the role of vision in mediating mosquitoes ability to escape defensive hosts has yet to be analyzed. Here, we used programmable visual displays to generate expanding objects sharing characteristics with the visual component of an approaching hand and quantified the behavioral response of female mosquitoes. Results show that Ae. aegypti is capable of using visual information to decide whether to feed on an artificial host mimic. Stimulations delivered in a LED flight arena further reveal that landed females Ae. aegypti display a stereotypical escape strategy by taking off at an angle that is a function of the distance and direction of stimulus introduction. Altogether, this study demonstrates mosquitoes can use isolated visual cues to detect and avoid a potential threat.

Summary StatementIn isolation, visual stimuli programmed to mimic a human swat prevent mosquitoes from blood-feeding by triggering take-offs and escape responses.

category: animal behavior and cognition


title: HDX-MS performed on BtuB in E. coli outer membranes delineates the luminal domain's allostery and unfolding upon B12 and TonB binding

abstract:
To import large metabolites across the outer membrane of Gram-negative bacteria, TonB dependent transporters (TBDTs) undergo significant conformational change. After substrate binding in BtuB, the E. coli vitamin B12 TBDT, TonB binds and couples BtuB to the inner membrane proton motive force that powers transport (1). But, the role of TonB in rearranging the plug domain to form a putative pore remains enigmatic. Some studies focus on force-mediated unfolding (2) while others propose force-independent pore formation (3) by TonB binding leading to breakage of a salt bridge termed the "Ionic Lock". Our hydrogen exchange/mass spectrometry measurements in E. coli outer membranes find that the region surrounding the Ionic Lock, far from the B12 site, is fully destabilized upon substrate binding. A comparison of the exchange between the B12 bound and the B12&TonB bound complexes indicates that B12 binding is sufficient to unfold the Ionic Lock region with the subsequent binding of a TonB fragment having much weaker effects. TonB binding accelerates exchange in the third substrate binding loop, but pore formation does not obviously occur in this or any region. This study provides a detailed structural and energetic description of the early stages of B12 passage that provides support both for and against current models of the transport process.

Significance StatementTonB dependent transporters such as BtuB are found in the outer membranes of Gram-negative bacteria. They import scarce nutrients essential for growth, such as B12, the substrate of BtuB. Many transport steps remain enigmatic. Recent studies have emphasized force-mediated unfolding or the breakage of the "Ionic Lock", a moiety far from the B12 binding site. A strong dependence on the membrane environment has been noted. Accordingly, we measured hydrogen exchange on BtuB still embedded in native outer membranes and found that B12 binding is sufficient to break the Ionic Lock. The amino terminus then extends into the periplasm to bind TonB. But we find no evidence of pore formation, which likely requires energy transduction from the inner membrane by TonB.


title: Intravenous gene transfer throughout the brain of infant Old World primates using AAV

abstract: Adeno-associated viruses (AAVs) can enable robust and safe gene delivery to the mammalian central nervous system (CNS). While the scientific community has developed numerous neurotropic AAV variants for systemic gene-transfer to the rodent brain, there are few AAVs that efficiently access the CNS of higher order primates. We describe here AAV.CAP-Mac, an engineered AAV variant that enables systemic, brain-wide gene delivery in infants of two Old World primate species--the rhesus macaque (Macaca mulatta) and the green monkey (Chlorocebus sabaeus). We identified CAP-Mac using a multi-species selection strategy, initially screening our library in the adult common marmoset (Callithrix jacchus) and narrowing our pool of test-variants for another round of selection in infant macaques. In individual characterization, CAP-Mac robustly transduces human neurons in vitro and Old World primate neurons in vivo, where it targets all lobes of cortex, the cerebellum, and multiple subcortical regions of disease relevance. We use CAP-Mac for Brainbow-like multicolor labeling of macaque neurons throughout the brain, enabling morphological reconstruction of both medium spiny neurons and cortical pyramidal cells. Because of its broad distribution throughout the brain and high neuronal efficiency in infant Old World primates compared to AAV9, CAP-Mac shows promise for researchers and clinicians alike to unlock novel, noninvasive access to the brain for efficient gene transfer.

category: bioengineering


title: Lipid nanoparticles delivering constitutively active STING mRNA as a novel anti-cancer therapeutic approach

abstract: Treating immunosuppressive tumors represents a major challenge in cancer therapies. Activation of STING signaling has shown remarkable potential to invigorate the immunologically  cold tumor microenvironment (TME). However, we and others have shown that STING is silenced in many cancers, including pancreatic ductal adenocarcinoma (PDAC) and Merkel cell carcinoma (MCC), both of which are associated with an immune-dampened TME. In this study, we applied mRNA lipid nanoparticles (LNP) to deliver a permanently active gain-of-function STINGR284S mutant into PDAC and MCC cells. Expression of STINGR284S induces cytokines and chemokines crucial for promoting intratumoral infiltration of CD8+ T cells and, importantly, also leads to robust cancer cell death while avoiding T cell entry and toxicity. Our studies demonstrated that mRNA-LNP delivery of STINGR284S could be explored as a novel therapeutic tool to reactivate antitumor response in an array of STING-deficient cancers while overcoming the toxicity and limitations of conventional STING agonists.


title: Oncogenic role of a developmentally regulated NTRK2 splice variant

abstract: Temporally-regulated alternative splicing choices are vital for proper development yet the wrong splice choice may be detrimental. Here we highlight a novel role for the neurotrophin receptor splice variant TrkB.T1 in neurodevelopment, embryogenesis, transformation, and oncogenesis across multiple tumor types in both humans and mice. TrkB.T1 is the predominant NTRK2 isoform across embryonic organogenesis and forced over-expression of this embryonic pattern causes multiple solid and nonsolid tumors in mice in the context of tumor suppressor loss. TrkB.T1 also emerges the predominant NTRK isoform expressed in a wide range of adult and pediatric tumors, including those harboring TRK fusions. Affinity purification-mass spectrometry (AP-MS) proteomic analysis reveals TrkB.T1 has distinct interactors with known developmental and oncogenic signaling pathways such as Wnt, TGF-{beta}, Hedgehog, and Ras. From alterations in splicing factors to changes in gene expression, the discovery of isoform specific oncogenes with embryonic ancestry has the potential to shape the way we think about developmental systems and oncology.


title: Cut-and-Paste DNA Insertion with Engineered Type V-K CRISPR-associated Transposases

abstract: CRISPR-associated transposases (CASTs) enable recombination-independent, multi-kilobase DNA insertions at RNA-programmed genomic locations. Type V-K CASTs offer distinct technological advantages over type I CASTs given their smaller coding size, fewer components, and unidirectional insertions. However, the utility of type V-K CASTs is hindered by a replicative transposition mechanism that results in a mixture of desired simple cargo insertions and undesired plasmid co-integrate products. Here, we overcome this limitation by engineering new CASTs with dramatically improved product purity. To do so, we compensate for the absence of the TnsA subunit in multiple type V-K CASTs by engineering a Homing Endonuclease-assisted Large-sequence Integrating CAST compleX, or HELIX system. HELIX utilizes a nicking homing endonuclease (nHE) fused to TnsB to restore the 5 " nicking capability needed for dual-nicking of the DNA donor. By leveraging distinct features of both type V-K and type I systems, HELIX enables cut-and-paste DNA insertion with up to 99.3% simple insertion product purity, while retaining robust integration efficiencies on genomic targets. Furthermore, we demonstrate the versatility of this approach by generating HELIX systems for other CAST orthologs. We also establish the feasibility of creating a minimal, 3-component HELIX, simplifying the number of proteins that must be expressed. Together, HELIX streamlines and improves the application of CRISPR-based transposition technologies, eliminating barriers for efficient and specific RNA-guided DNA insertions.


title: Targeting Host PIM Protein Kinases Reduces Mayaro Virus Replication

abstract: Mayaro virus (MAYV) manipulates cell machinery to successfully replicate. Thus, identifying host proteins implicated in MAYV replication, represents an opportunity to discover potential antiviral targets. PIM kinases are enzymes that regulate essential cell functions and also appear to be critical factors in the replication of certain viruses. In this study we explored the consequences of PIM kinase inhibition in the replication of MAYV and other arboviruses. Cytopathic effects or viral titers in samples from MAYV-, Chikungunya-, Una-or Zika-infected cells treated with PIM kinase inhibitors were evaluated using an inverted microscope or plaque-forming assays. The expression of viral proteins E1 and nsP1 in MAYV-infected cells was assessed using an immunofluorescence confocal microscope or Western blot. Our results revealed that PIM kinase inhibition partially prevented MAYV-induced cell damage and also promoted a decrease in viral titers for MAYV, UNAV and ZIKV. The inhibitory effect of PIM kinase blocking was observed for each of the MAYV strains tested and also occurred as late as 8 hpi. Finally, PIM kinase inhibition suppressed the expression of MAYV E1 and nsP1 proteins. Taken together, these findings suggest that PIM kinases could represent an antiviral target for MAYV and other arboviruses.

category: microbiology


title: Polymer-directed inhibition of reversible to irreversible attachment prevents Pseudomonas aeruginosa biofilm formation

abstract:
Non-toxic, biocompatible materials that inhibit bacterial biofilm formation on implanted medical devices and so prevent infection are urgently required. Weakly amphiphilic acrylate polymers with rigid hydrocarbon pendant groups resist bacterial biofilm formation in vitro and in vivo but the biological mechanism involved is not known. By comparing biofilm formation on polymers with the same acrylate backbone but with different pendant groups, we show that poly(ethylene glycol dicyclopentenyl ether acrylate; pEGdPEA) but not neopentyl glycol propoxylate diacrylate (pNGPDA) inhibited the transition from reversible to irreversible attachment. By using single-cell tracking algorithms and controlled flow microscopy we observed that fewer Pseudomonas aeruginosa PAO1 cells accumulated on pEGdPEA compared with pNGPDA. Bacteria reaching the pEGdPEA surface exhibited shorter residence times and greater asymmetric division with more cells departing from the surface post-cell division, characteristic of reversible attachment. Migrating cells on pEGdPEA deposited fewer exopolysaccharide trails and were unable top adhere strongly. Discrimination between the polymers required type IV pili and flagella. On pEGdPEA, the lack of accumulation of cyclic diguanylate or expression of sadB were consistent with the failure to transit from reversible to irreversible attachment. Constitutive expression of sadB increased surface adhesion sufficient to enable P. aeruginosa to form biofilms in a Mot flagellar stator dependent manner. These findings were extendable to other biofilm resistant acrylates highlighting their unique ability to inhibit reversible to irreversible attachment as a mechanism for preventing biofilm-associated infections.

SignificanceBacteria readily attach to surfaces forming biofilms. These are commonly associated with medical device-associated infections and highly refractory to antibiotics. Biocompatible, weakly amphiphilic acrylate polymers with large hydrophobic pendant groups that inhibit biofilm formation and can prevent such infections have been described. However, the biological mechanism involved is not understood. By comparing a biofilm-inhibiting with a biofilm-supporting acrylate, we showed that Pseudomonas aeruginosa PAO1 cells responded differentially to the two polymers and were unable to accumulate and adhere strongly, activate cyclic diguanylate signalling or transit from reversible to irreversible attachment on the inhibitory polymer. Constitutive expression of sadB increased surface adhesion sufficient to enable P. aeruginosa to form biofilms in a flagellar stator dependent manner overcoming the biofilm inhibitory properties of the polymer.


title: A single amino acid substitution in PB1 of pandemic H1N1 with A/Ann Arbor/6/60 master donor virus mutations as a novel live-attenuated influenza virus vaccine

abstract:
Influenza A viruses (IAV) remain emerging threats to human public health. Live-attenuated influenza vaccines (LAIV) are one of the most effective prophylactic options to prevent disease caused by influenza infections. However, licensed LAIV remain restricted for use in 2- to 49-year old healthy and non-pregnant people. Therefore, development of LAIV with increased safety, immunogenicity, and protective efficacy is highly desired. The United States (U.S.) licensed LAIV is based on the master donor virus (MDV) A/Ann Arbor/6/60 H2N2 backbone, which was generated by adaptation of the virus to growth at low temperatures. Introducing the genetic signature of the U.S. MDV into the backbone of other IAV strains resulted in varying levels of attenuation. While the U.S. MDV mutations conferred an attenuated phenotype to other IAV strains, the same amino acid changes did not significantly attenuate the pandemic A/California/04/09 H1N1 (pH1N1) strain. To attenuate pH1N1, we replaced the conserved leucine at position 319 with glutamine (L319Q) in PB1 and analyzed the in vitro and in vivo properties of pH1N1 viruses containing either PB1 L319Q alone or in combination with the U.S. MDV mutations using two animal models of influenza infection and transmission, ferrets and guinea pigs. Our results demonstrated that L319Q substitution in the pH1N1 PB1 alone or in combination with the mutations of the U.S. MDV resulted in reduced pathogenicity (ferrets) and transmission (guinea pigs), and an enhanced temperature sensitive phenotype. These results demonstrate the feasibility of generating an attenuated MDV based on the backbone of a contemporary pH1N1 IAV strain.

IMPORTANCEVaccination represents the most effective strategy to reduce the impact of seasonal IAV infections. Although LAIV are superior in inducing protection and sterilizing immunity, they are not recommended for many individuals who are at high risk for severe disease. Thus, development of safer and more effective LAIV are needed. A concern with the current MDV used to generate the U.S. licensed LAIV is that it is based on a virus isolated in 1960. Moreover, mutations that confer the temperature sensitive, cold-adapted, and attenuated phenotype of the U.S. MDV resulted in low level of attenuation in the contemporary pandemic A/California/04/09 H1N1 (pH1N1). Here, we show that introduction of PB1 L319Q substitution, alone or in combination with the U.S. MDV mutations, resulted in pH1N1 attenuation. These findings support the development of a novel LAIV MDV based on a contemporary pH1N1 strain as a medical countermeasure against currently circulating H1N1 IAV.


title: Identification of domestic cat hepadnavirus from a cat blood sample in Japan

abstract: The hepatitis B virus (Hepadnaviridae) induces chronic hepatitis and hepatic cancer in humans. A novel domestic cat hepadnavirus (DCH) was recently identified in several countries, however, the DCH infection status of cats in Japan is unknown. Therefore, we investigated the DCH infection rate of 139 cat samples collected in Japan. We identified one positive blood sample (0.78%) from a 17-year-old female cat with chronically elevated alanine aminotransferase. Phylogenetic analysis demonstrated that the DCH strain identified in this study is genetically distinct from strains in other countries. Further investigations are required to elucidate the evolution of DCH and the impact of DCH infection on hepatic diseases in domestic cats.


title: Liquid-liquid phase separation of the chromosomal passenger complex drives parallel bundling of midzone microtubules

abstract: The spindle midzone is a dynamic structure that forms during anaphase, mediates chromosome segregation, and provides a signaling platform to position the cleavage furrow. The spindle midzone comprises two antiparallel bundles of microtubules (MTs) but the process of their formation is poorly understood. Here, we show that the Chromosomal Passenger Complex (CPC) undergoes liquid-liquid phase separation (LLPS) to generate parallel MT bundles in vitro when incubated with free tubulin and GTP. MT bundles emerge from CPC droplets with protruding minus-ends that then grow into long, tapered MT structures. During this growth, the CPC in condensates apparently reorganize to coat and bundle the resulting MT structures. CPC mutants attenuated for LLPS or MT binding prevented the generation of parallel MT bundles in vitro and reduced the number of MTs present at spindle midzones in HeLa cells. Our data uncovers a kinase-independent function of the CPC and provides models for how cells generate parallel-bundled MT structures that are important for the assembly of the mitotic spindle.


title: Isolation and Characterization of Extracellular Vesicles from the Fungal Phytopathogen Colletotrichum higginsianum

abstract: Fungal phytopathogens secrete extracellular vesicles (EVs) associated with enzymes and phytotoxic metabolites. While these vesicles are thought to promote infection, defining the true contents and functions of fungal EVs, as well as suitable protein markers, is an ongoing process. To expand our understanding of fungal EVs and their possible roles during infection, we purified EVs from the hemibiotrophic phytopathogen Colletotrichum higginsianum, the causative agent of anthracnose disease in multiple plant species, including Arabidopsis thaliana. EVs were purified in large numbers from the supernatant of protoplasts but not the supernatant of intact mycelial cultures. We purified two separate populations of EVs, each associated with over 700 detected proteins, including proteins involved in vesicle transport, cell wall biogenesis and the synthesis of secondary metabolites. We selected two SNARE proteins (Snc1 and Sso2) and one 14-3-3 protein (Bmh1) as potential EV markers and generated transgenic lines expressing fluorescent fusions. Each marker was confirmed to be protected inside EVs. Fluorescence microscopy was used to examine the localization of each marker during infection on Arabidopsis leaves. These findings further our understanding of EVs in fungal phytopathogens and will help build an experimental system to study EV inter-kingdom communication between plants and fungi.