Using machine learning to design adeno-associated virus capsids with high likelihood of viral assembly We study the application of machine learning in designing adeno-associated virus (AAV) capsid sequences with high likelihood of viral assembly, i.e. capsid viability. Specifically, we design and implement Origami, a model-based optimization algorithm, to identify highly viable capsid sequences within the vast space of 2033 possibilities. Our evaluation shows that Origami performs well in terms of optimality and diversity of model-designed sequences. Moreover, these sequences are ranked according to their viability score. This helps designing experiments given budget constraint.
Characterization of DNA extrusion scenarios underlying aggregate Hi-C patterns by a new computational framework 1Aggregate Hi-C maps centered on several genetic elements show significant DNA extrusion features. However, these features are missing from the component Hi-C images. To resolve this discrepancy, we present a new computational framework that reconstructs DNA extrusion scenarios (DESs) and explains the enhancement of DNA extrusion features by aggregation. We showed that the reconstructed DES is essential to understand the site interactions of the relevant genetic elements. For example, CCCTC-binding factor (CTCF) binding elements (CBE) with a specific motif direction are only affected by DNA extrusion activities initiated from one side, manifested as a directional bias. About half of promoters search for enhancers in a specific direction, as indicated by the corresponding DES. Moreover, since DNA extrusion does not pause at promoter or enhancer elements, direct promoter-enhancer contacts are not long-lasting; rather, promoters and enhancers are brought in proximity by continuous, dynamic DNA extrusion.
Transcriptomic profiling of myeloid cells in Alzheimer's Disease brain illustrates heterogeneity of microglia endolysosomal subtypes Microglia-mediated neuroinflammation contributes to disease progression in Alzheimers Disease (AD). Microglia demonstrate heterogeneous states with proposed beneficial, harmful, and disease-specific subtypes. Defining the spectrum of microglia phenotypes is crucially important to the design of neuroinflammation-modulating therapies. We performed single-nucleus RNA-seq on over 120,000 microglia nuclei isolated from dorsolateral prefrontal cortices collected from 12 AD and 10 controls. We detected several previously unrecognized microglia transcriptomic sub-populations, including three with predominant endolysosomal gene expression patterns and heterogeneous metabolic and inflammatory signatures. One endoloysomal subtype is more abundant in AD individuals and uniquely enriched for transcripts involved in nucleic acid detection and interferon signaling. AD individuals also exhibited increased expression of unfolded protein response and inflammatory genes in homeostatic microglia subpopulations. These results highlight the heterogenous nature of the microglia response to AD pathology and inform efforts to target specific subtypes of microglia in the development of novel AD therapies. Can double PPO mutations exist in the same allele and are such mutants functional? BackgroundResistance to PPO-inhibiting herbicides is primarily endowed by target site mutations at the PPO2 gene that compromise binding of the herbicide to the catalytic domain. In Amaranthus ssp. PPO2, the most prevalent target mutations are deletion of the G210 codon and the R128G and G339A substitutions. These mutations strongly affect the dynamic of the PPO2 binding pocket resulting in reduced affinity with the ligand. Here we investigated the likelihood of co-occurrence of the most widespread target site mutations in the same ppo2 allele.

ResultsPlants carrying R128G+/+ {Delta}G210+/-, where + indicates presence of the mutation, were crossed with each other. The ppo2 of the offspring was subjected to pyrosequencing and E. coli- based Sanger sequencing to determine mutation frequencies and allele co-occurrence. The data show that R128G {Delta}G210 can occur only in one allele; the second allele carries only one mutation. Double mutation in both alleles is less likely because of significant loss of enzyme activity. The segregation of offspring populations derived from a cross between heterozygous plants carrying {Delta}G210 G399A also showed no co-occurrence in the same allele. The offspring exhibited the expected mutation distribution patterns with few exceptions.

ConclusionsHomozygous double-mutants are not physiologically viable. Double-mutant plants can only exist in a heterozygous state. Alternatively, if two mutations are detected in one plant, each mutation would occur in a separate allele.

NomenclaturePalmer amaranth, Amaranthus palmeri S. Wats.; protoporphyrinogen oxidase, PPO; tall waterhemp, Amaranthus tuberculatus (Moq.) J.D.Sauer Positive supercoiling favors transcription elongation through lac repressor-mediated DNA loops Some proteins, like the lac repressor (LacI), mediate long-range loops that alter DNA topology and create torsional barriers. During transcription, RNA polymerase generates supercoiling that may facilitate passage through such barriers. We monitored E. coli RNA polymerase progress along templates in conditions that prevented, or favored, LacI-mediated DNA looping. Tethered particle motion measurements revealed that RNA polymerase paused longer at unlooped LacI obstacles or those barring entry to a loop than those barring exit from the loop. Enhanced dissociation of a LacI roadblock by the positive supercoiling generated ahead of a transcribing RNA polymerase within a torsion-constrained DNA loop may be responsible. In support of this, RNA polymerase transcribed a looped segment more slowly than an unlooped segment and paused more briefly at LacI obstacles on positively supercoiled templates, especially under increased tension (torque). Positive supercoiling propagating ahead of polymerase appears to facilitate elongation along topologically-complex, protein-coated templates. Combined MEK and STAT3 inhibition reprograms stromal inflammation to overcome immunotherapy resistance in pancreatic cancer Pancreatic ductal adenocarcinoma (PDAC) is characterized by immune exclusion, pro-inflammatory polarization of cancer-associated fibroblasts (CAF), and resistance to immune checkpoint inhibition (ICI). We have previously demonstrated that reciprocally activated RAS/MEK/ERK and JAK/STAT3 pathways mediate therapeutic resistance, while combined MEK and STAT3 inhibition (MEKi+STAT3i) overcomes such resistance in preclinical models. We now show that combined MEKi+STAT3i not only alters stromal architecture but also uncovers stromal plasticity by revealing a substantial attenuation of Il6/Cxcl1-expressing secretory and Lrrc15-expressing myofibroblastic CAF phenotypes with a concomitant enrichment of Ly6a/Cd34-expressing CAF phenotypes exhibiting mesenchymal progenitor-like properties via single-cell RNA sequencing in Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox(PKT) mice. This remodeling of CAF heterogeneity is associated with reprogramming of immunosuppressive myeloid populations and enhanced trafficking of CD8+ T-cells which exhibit a distinct effector transcriptional program. These MEKi+STAT3i-mediated repercussions are in part CAF-dependent, since CRISPR/Cas9 genetic silencing of CAF-restricted Mek1/Stat3 mitigates inflammatory CAF polarization and myeloid infiltration in vivo. Addition of MEKi+STAT3i to PD-1 blockade overcomes ICI resistance by significantly augmenting anti-tumor responses and dramatically improving survival in PKT mice compared with anti-PD-1 monotherapy. The addition of MEKi+STAT3i to PD-1 blockade not only augments the recruitment of activated and memory T-cell populations, but also improves their degranulating capacity and functional cytotoxicity compared to PD-1 blockade alone. Importantly, treatment of a patient with chemotherapy-refractory metastatic PDAC with MEKi (Trametinib), STAT3i (Ruxolitinib), and PD-1 inhibitor (Nivolumab) was well-tolerated and yielded clinical benefit. These data uncover a novel paradigm in which combined MEKi+STAT3i reprograms stromal inflammation and immune tolerance to overcome immunotherapy resistance in PDAC. Population differences in the length and early-life dynamics of telomeres among European pied flycatchers Telomere length and shortening rate are increasingly used as biomarkers for long-term costs in ecological and evolutionary studies because of their relationships with survival and fitness. Both early-life conditions and growth, and later-life stressors can create variation in telomere shortening rate. Studies on between- population telomere length and dynamics are scarce, despite the expectation that populations exposed to varying environmental constraints would present divergent telomere length patterns. The pied flycatcher (Ficedula hypoleuca) is a passerine bird breeding across Eurasia (from Spain to western Siberia) and migrating through the Iberian Peninsula to spend the non-breeding period in sub-Saharan Africa. Thus, different populations show marked differences in migration distance. We studied the large-scale variation of telomere length and early-life dynamics in the pied flycatcher by comparing six European populations across a north- south gradient (Finland, Estonia, England, and Spain) predicting negative effect of migration distance on adult telomere length, and of nestling growth on nestling telomere dynamics. There were clear population differences in telomere length, with English birds from mid-latitudes having the longest telomeres. Telomere length did not thus show consistent latitudinal variation and was not linearly linked to differences in migration distance. Early-life telomere shortening rate tended to vary between populations. Fast growth was associated with shorter telomeres in the early life, but faster nestling growth affected telomeres more negatively in northern than southern populations. While the sources of between-population differences in telomere-related biology remain to be more intensively studied, our study illustrates the need to expand telomere studies at the between-population level. Gene loss during a transition to multicellularity Multicellular evolution is a major transition associated with momentous diversification of multiple lineages and increased developmental complexity. The volvocine algae comprise a valuable system for the study of this transition, as they span from unicellular to undifferentiated and differentiated multicellular morphologies despite their genomes being highly similar, suggesting multicellular evolution requires few genetic changes to undergo dramatic shifts in developmental complexity. Here, the evolutionary dynamics of six volvocine genomes were examined, where a gradual loss of genes was observed in parallel to the co-option of a few key genes. Protein complexes in the six species exhibited a high degree of novel interactions, suggesting that gene loss plays a role in evolutionary novelty. This finding was supported by gene network modeling, where gene loss outpaces gene gain in generating novel stable network states. These results suggest developmental complexity may be driven by gene loss, and not just by gene gain.

Significance StatementIncreased developmental complexity is thought to evolve mainly through genetic innovation and co-option. Comparison of the genomes of the closely related volvocines revealed that despite large changes in developmental complexity their genomes are exhibiting significant gene loss. Further, a burst of gene loss is shown to occur at the transition to undifferentiated multicellularity by gene inactivation and decay. This likely results in changes to protein-protein interactions within the cell, suggesting evolutionary novelty does not always require gene gain. Using empirical and modelling approaches we demonstrate gene loss can more easily produce protein network novelty than gene gain. We propose that gene loss can be a driver of biological innovation, allowing for reconfiguration of gene networks and differential use of existing functional repertoires. Phylogenomic evidence for the Origin of Obligately Anaerobic Anammox Bacteria around the Great Oxidation Event The anaerobic ammonium oxidation (anammox) bacteria could transform ammonium and nitrite to dinitrogen gas, and this obligate anaerobic process accounts for up to half of the global nitrogen loss in surface environments. Yet its origin and evolution, which may give important insights into the biogeochemistry of early Earth, remains enigmatic. Here, we performed comprehensive phylogenomic analysis and showed a single origin of anammox bacteria within the phylum Planctomycetes. After accommodating the uncertainties and factors influencing time estimates, which includes implementing both a traditional cyanobacteria-based and a recently developed mitochondria-based approach, we estimated that anammox bacteria originated at early Proterozoic and most likely around the so-called Great Oxidation Event (GOE; 2.32 to 2.5 billion years ago [Ga]) which fundamentally changed global biogeochemical cycles. We further showed that during the origin of anammox bacteria, genes involved in oxidative stress, bioenergetics and anammox granules formation were recruited, which might have contributed to their survival on an increasingly oxic Earth. Our findings suggest the rising levels of atmospheric oxygen, which made nitrite increasingly available, was a potential driving force for the emergence of anammox bacteria. This is one of the first studies that link the GOE to the evolution of obligate anaerobic bacteria. Second Heart Field-derived Cells Contribute to Angiotensin II-mediated Ascending Aortopathies BackgroundThe ascending aorta is a common location for aneurysm and dissection. This aortic region is populated by a mosaic of medial and adventitial cells that are embryonically derived from either the second heart field (SHF) or the cardiac neural crest. SHF-derived cells populate areas that coincide with the spatial specificity of thoracic aortopathies. The purpose of this study was to determine whether and how SHF-derived cells contribute to ascending aortopathies.

MethodsAscending aortic pathologies were examined in patients with sporadic thoracic aortopathies and angiotensin II (AngII)-infused mice. Ascending aortas without overt pathology from AngII-infused mice were subjected to mass spectrometry assisted proteomics, and molecular features of SHF-derived cells were determined by single cell transcriptomic analyses. Genetic deletion of either low-density lipoprotein receptor-related protein 1 (Lrp1) or transforming growth factor-{beta} receptor 2 (Tgfbr2) in SHF- derived cells was conducted to examine the impact of SHF-derived cells on vascular integrity.

ResultsPathologies in human ascending aortic aneurysmal tissues were predominant in outer medial layers and adventitia. This gradient was mimicked in mouse aortas following AngII infusion that was coincident with the distribution of SHF-derived cells. Proteomics indicated that brief AngII infusion, prior to overt pathology, evoked downregulation of SMC proteins and differential expression of extracellular matrix proteins, including several LRP1 ligands. LRP1 deletion in SHF-derived cells augmented AngII-induced ascending aortic aneurysm and rupture. Single cell transcriptomic analysis revealed that brief AngII infusion decreased Lrp1 and Tgfbr2 mRNA abundance in SHF-derived cells and induced a unique fibroblast population with low abundance of Tgfbr2 mRNA. SHF-specific Tgfbr2 deletion led to embryonic lethality at E12.5 with dilatation of the outflow tract and retroperitoneal hemorrhage. Integration of proteomic and single cell transcriptomics results identified plasminogen activator inhibitor 1 (PAI1) as the most increased protein in SHF-derived SMCs and fibroblasts during AngII infusion. Immunostaining revealed a transmural gradient of PAI1 in both ascending aortas of AngII-infused mice and human ascending aneurysmal aortas that mimicked the gradient of medial and adventitial pathologies.

ConclusionSHF-derived cells exert a critical role in maintaining vascular integrity through LRP1 and TGF-{beta} signaling associated with increases of aortic PAI1.



O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/930917v6_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@185e3d9org.highwire.dtl.DTLVardef@131d702org.highwire.dtl.DTLVardef@11afa42org.highwire.dtl.DTLVardef@242786_HPS_FORMAT_FIGEXP  M_FIG C_FIG Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABS- SHF-derived SMCs and fibroblasts associate with AngII-induced aortic pathologies.
- AngII induces a distinct fibroblast sub-cluster that is less abundant for mRNAs related to major extracellular components and TGF{beta} ligands and receptors, but more abundant for proliferative genes.
- TGFBR2 deletion in SHF-derived cells are embryonic lethal with significant dilatation of the outflow tract in mice.
- SHF-specific deletion of LRP1 leads to aortic pathologies in mice, supporting the importance of SHF-derived cells in maintaining ascending aortic wall integrity.


What are the clinical implications?- Heterogeneity of the embryonic origins of SMCs and fibroblasts contributes to complex mechanisms of vasculopathy formation, which should be considered when investigating the pathogenesis of thoracic aortopathies. Nuclear NAD+-biosynthetic enzyme NMNAT1 facilitates survival of developing retinal neurons Despite mounting evidence that the mammalian retina is exceptionally reliant on proper NAD+ homeostasis for health and function, the specific roles of subcellular NAD+ pools in retinal development, maintenance, and disease remain obscure. Here, we show that deletion of the nuclear-localized NAD+ synthase nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1) in the developing murine retina causes early and severe degeneration of photoreceptors and select inner retinal neurons via multiple distinct cell death pathways. This severe phenotype is associated with disruptions to retinal central carbon metabolism, purine nucleotide synthesis, and amino acid pathways. Furthermore, large-scale transcriptomics reveals dysregulation of a collection of photoreceptor and synapse-specific genes in NMNAT1 knockout retinas prior to detectable morphological or metabolic alterations. Collectively, our study reveals previously unrecognized complexity in NMNAT1-associated retinal degeneration and suggests a yet-undescribed role for NMNAT1 in gene regulation during photoreceptor terminal differentiation. A coupled mechano-biochemical framework for root meristem morphogenesis Plants grow roots to adjust their bodies to dynamic changes in the surrounding environment. How do plant roots emerge from the stem cell reservoir during embryogenesis is however poorly understood. Here, we present a bottom-up strategy to address this challenge by combining empirical observations with advanced computer modeling techniques. We demonstrate that the anisotropy of root growth results from differential growth rates of adjacent tissues, whereas the root meristem development incorporates a multi-level feedback loop between complex transport network of phytohormone auxin, auxin-dependent cell growth and cytoskeleton rearrangements. In silico model predictions are in close agreement with in vivo patterns of anisotropic growth, auxin distribution, and cell polarity, as well as several root phenotypes caused by chemical, mechanical, or genetic perturbations. Our findings reveal a minimal set of design principles connecting tissue mechanics, cell anisotropy, and directional transport that are sufficient for self-organization of the root meristem shape. A mobile auxin signal transported through immobile cells orchestrates polarity and growth mechanics to instruct the morphogenesis of an independent organ. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Multi-omics investigation of tacrolimus nephrotoxicity BackgroundTacrolimus, an immunosuppressive drug prescribed to a majority of transplanted patients is nephrotoxic, through still unclear mechanisms. This study aims to evaluate the impact of tacrolimus on a lineage of proximal tubular cells using a multi-omics approach.

MethodsLLC-PK1 cells were exposed to 5 M of tacrolimus for 24h. Intracellular proteins and metabolites, and extracellular metabolites were extracted and analysed by LC-MS/MS. The transcriptional expression of the dysregulated proteins PCK-1, FBP1 and FBP2 was measured using RT-qPCR.

ResultsIn our cell model, tacrolimus impacted different metabolic pathways including those of arginine (e.g., citrulline, ornithine) (p < 0.0001), amino acids (e.g., valine, isoleucine, aspartic acid) (p < 0.0001) and pyrimidine (p<0.01). In addition, it induced oxidative stress (p < 0.01) as shown by a decrease in total cell glutathione quantity. It impacted cell energy through an increase in Krebs cycle intermediates (e.g., citrate, aconitate, fumarate) (p < 0.01) and down-regulation of PCK-1 (p < 0.05) and FPB1 (p < 0.01), which are key enzymes in gluconeogenesis. Apart from glucose synthesis, gluconeogenesis is an important process in kidney mediated acid-base balance control.

ConclusionThe variations found using this multi-omics approach clearly point towards a dysregulation of energy production in epithelial cells of the renal tubule, and potentially of their functions, that may be implicated in tacrolimus nephrotoxicity in the clinics. Naming Human Diseases: Ethical Principles of Curating Exclusive Substitute for Inopportune Nosology BackgroundIn the medical sphere, understanding naming conventions strengthen the integrity of naming human diseases remains nominal rather than substantial yet. Since the current nosology-based standard for human diseases could not offer a one-size-fits-all corrective mechanism, many idiomatic but flawed names frequently appear in scientific literature and news outlets at the cost of sociocultural impacts.

ObjectiveWe attempt to examine the ethical oversights of current naming practices and propose heuristic rationales and approaches to determine a pithy name instead of an inopportune nosology.

MethodsFirst, we examined the compiled global online news volumes and emotional tones on some inopportune nosology like German measles, Middle Eastern Respiratory Syndrome, Spanish flu, Hong Kong flu, and Huntingtons disease in the wake of COVID-19. Second, we prototypically scrutinize the lexical dynamics and pathological differentials of German measles and common synonyms by leveraging the capacity of the Google Books Ngram Corpus. Third, we demonstrated the empirical approaches to curate an exclusive substitute for an anachronistic nosology German measles based on deep learning models and post-hoc explanations.

ResultsThe infodemiological study shows that the public informed the offensive names with extremely negative tones in textual and visual narratives. The findings of the historiographical study indicate that many synonyms of German measles did not survive, while German measles became an anachronistic usage, and rubella has taken the dominant place since 1994. The PubMedBERT model could identify rubella as a potential substitution for German measles with the highest semantic similarity. The results of the semantic drift experiments further indicate that rubella tends to survive during the ebb and flow of semantic drift.

ConclusionsOur findings indicate that the nosological evolution of anachronistic names could result in sociocultural impacts without a corrective mechanism. To mitigate such impacts, we introduce some ethical principles for formulating an improved naming scheme. Based on deep learning models and post-hoc explanations, our illustrated experiments could provide hallmark references to the remedial mechanism of naming practices and pertinent credit allocations. Phagocytic Uptake of Polymeric Particles by Immune cells Under Flow Conditions Particles injected intravenously are thought to be cleared by macrophages residing in the liver and spleen, but they also encounter circulating immune cells. It remains to be established if the circulating cells can take up particles while flowing, and if the uptake capacity is similar under static and flow conditions. Here, we use an in vitro peristaltic pump setup that mimics pulsatile blood flow to determine if immune cells take up particles under constant fluidic flow. We use polystyrene particles of varying sizes as the model of a polymeric particle for these studies. Our results show that the immune cells do phagocytose under flow conditions. We demonstrate that cell lines representing myeloid cells, primary human neutrophils and monocytes take up sub-micrometer-sized particles at similar or better rates under flow compared to static conditions. Experiments with whole human blood show that even under the crowding effects of red blood cells, neutrophils and monocytes take up particles while flowing. Together, these data suggest that circulating immune cells are likely to phagocytose intravenously injected particulates, which has implications for design of particles to evade or target these cells. Comprehensive O-GlcNAc glycoproteomics on NOTCH1 EGF repeats implicated unique Lewis X epitopes in mammals The O-GlcNAc modification of Notch receptors regulates Notch ligand interactions in a manner distinct from other forms of O-glycans on epidermal growth factor-like (EGF) repeats of Notch receptors. Although many proteins, besides Notch receptors, are expected to be O-GlcNAcylated by EGF domain-specific O-GlcNAc transferase (EOGT), only a small number of proteins have been reported to be modified in vivo, and elongated O-GlcNAc glycans have not been extensively explored. To extend our view of the specificity and variety of the glycan modification, we conducted a comprehensive analysis of O-GlcNAc glycans on NOTCH1 in mammals. Mass spectrometric analysis of NOTCH1 fragments expressed in HEK293T cells revealed that several EGF domains with putative O-GlcNAcylation sites were hardly modified with O-GlcNAc. Although amino acid residues before the modification site are preferentially occupied with aromatic residues, Phe and Tyr are preferable to Trp for the apparent modification with O-GlcNAc. Furthermore, a minor form of fucosylated O-GlcNAc glycans was detected in a subset of EGF domains. Fucosylation of O-GlcNAc glycans was enhanced by FUT1, FUT2, or FUT9 expression. The FUT9-dependent Lewis X epitope was confirmed by immunoblotting using an anti-Lewis X antibody. As expected from the similarity in the glycan structures, the Lexis X antigen was detected on O-fucose glycans. Our results refined the putative consensus sequence for the EOGT-dependent extracellular O-GlcNAc modification in mammals and revealed the structural diversity of functional Notch O-glycans. Assigning metabolic rate measurements to torpor and euthermy in heterothermic endotherms: 'torpor', a new package for R. Torpor is a state of controlled reduction of metabolic rate (M) in endotherms. Assigning measurements of M to torpor or euthermy can be challenging, especially when the difference between euthermic M and torpid M is small, in species defending a high minimal body temperature in torpor, in thermolabile species, and slightly below the thermoneutral zone (TNZ). Here, we propose a novel method for distinguishing torpor from euthermy. We use the variation in M measured during euthermic rest and torpor at varying ambient temperatures (Ta) to objectively estimate the lower critical temperature (Tlc) of the TNZ and to assign measurements to torpor, euthermic rest or rest within TNZ. In addition, this method allows the prediction of M during euthermic rest and torpor at varying Ta, including resting M within the TNZ. The present method has shown highly satisfactory results using 28 published sets of metabolic data obtained by respirometry on 26 species of mammals. Ultimately, this novel method aims to facilitate analysis of respirometry data in heterothermic endotherms. Finally, the development of the associated R-package (torpor) will enable widespread use of the method amongst biologists.

Summary statementThe presented method and its associated R-package (torpor) enable the assignment of metabolic rate measurements to torpor or euthermy, ultimately improving the standardization of respirometry analyses in heterotherms. The Rab GTPase activating protein TBC-2 regulates endosomal localization of DAF-16 FOXO and lifespan FOXO transcription factors have been shown to regulate longevity in model organisms and are associated with longevity in humans. To gain insight into how FOXO functions to increase lifespan, we examined the subcellular localization of DAF-16 in C. elegans. We show that DAF-16 is localized to endosomes and that this endosomal localization is increased by the insulin-IGF signaling (IIS) pathway. Endosomal localization of DAF-16 is modulated by endosomal trafficking proteins. Disruption of the Rab GTPase activating protein TBC-2 increases endosomal localization of DAF-16, while inhibition of TBC-2 targets, RAB-5 or RAB-7 GTPases, decreases endosomal localization of DAF-16. Importantly, the amount of DAF-16 that is localized to endosomes has functional consequences as increasing endosomal localization through mutations in tbc-2 reduced the lifespan of long-lived daf-2 IGFR mutants, depleted their fat stores, and DAF-16 target gene expression. Overall, this work identifies endosomal localization as a mechanism regulating DAF-16 FOXO, which is important for its functions in metabolism and aging. Activation of the type 3 secretion system of enteropathogenic E. coli leads to remodeling of its membrane composition and function The cell envelope of gram-negative bacteria is a complex structure, essential for bacterial survival and for resistance to many antibiotics. Channels that cross the bacterial envelope and the host cell membrane form secretion systems that are activated upon attachment to host, enabling bacteria to inject effector molecules into the host cell, required for bacterial-host interaction. The type III secretion system (T3SS) is critical for the virulence of several pathogenic bacteria, including enteropathogenic E. coli (EPEC). The EPEC T3SS activation is associated with repression of carbon storage regulator (CsrA), resulting in gene expression remodeling, which is known to affect EPEC central carbon metabolism and contributes to the adaptation to a cell-adherent lifestyle in a poorly understood manner. We reasoned that the changes in bacterial envelope upon attachment to host and the activation of a secretion system may involve a modification of the lipid composition of bacterial envelope. Accordingly, we performed a lipidomics analysis on mutant strains that simulate T3SS activation. We saw a shift in glycerophospholipid metabolism towards the formation of lysophospholipids, attributed to corresponding upregulation of the phospholipase pldA and the acyltransferase ygiH upon T3SS activation in EPEC. We also detected a shift from menaquinones and ubiquinones to undecaprenyl lipids, concomitant to abnormal synthesis of O-antigen. The remodeling of lipid metabolism is mediated by CsrA and associated with increased bacteria cell size and Zeta potential, and a corresponding alteration in EPEC permeability to vancomycin, increasing the sensitivity of T3SS-activated strains and of adherent wild type EPEC to the antibiotic.

ImportanceThe characterization of EPEC membrane lipid metabolism upon attachment to host is an important step towards a better understanding the shift of EPEC, a notable human pathogen, from a planktonic to adherent life style. It may also apply to other pathogenic bacteria that use this secretion system. We predict that upon attachment to host cells the lipid remodeling upon T3SS activation contributes to bacterial fitness and promotes host colonization, and show that it is associated with increased cell permeability and higher sensitivity to vancomycin.

To the best of our knowledge, this is the first demonstration of a bacterial lipid remodeling due to activation of a secretion system. Consumer risk perception towards pesticide stained tomatoes in Uganda. BackgroundTomatoes are consumeddaily. Unfortunately, abuse of pesticides application by vegetable growers in Uganda increases risks of exposing consumers through the pesticide residues, as it may be above European Union Maximum Residue Limits (used as a standard in Uganda). This study is aimed to determine consumer attitudes and risk perception towards pesticide stained tomatoes available on the Ugandan local markets.

MethodsA cross-sectional study sampled 468 household consumers in four districts one from each region of Uganda. In each district, 60 household members from three randomly selected Sub Counties were interviewed. In addition, in each district 9 tomato handlers (3 tomato farmers, 3 tomato retailers and 3 tomato wholesalers) participated in Focus Group Discussion (FGDs) perdistrict. Collected data was entered into MS-Excel 13 and exported into STATA SE 14.0 for cleaning and analysis at below a 5% level of significance and 95% Confidence Intervals (CI). Proportion of risk perceptions and attitudes were computed and presented as percentages while factors associated with risk perception were determined using Fisher exact test. Qualitative data was analyzed using deductive and inductive approaches under thematic content analysis.

ResultsMore than half, 54.2% (253/468), of the respondents were females, mean age was 37 years (SD=13.13, ranging from 18 to 88 years). Half of the respondents, 50.9% (238/467), were farmers by occupation and 40.3% (188/468) had completed upper primary education. Only 5.0% (20/396) of consumers reported a high risk perception towards tomatoes stained with pesticide residues, the rest, 95.0% (376/396), were buying pesticide stained tomatoes despite their awareness about the possible health effects. The main reason for buying the pesticide stained tomatoes was that a majority, 59.0% (230/390), lacked an alternative to stained tomatoes. However, consumers generally had a negative attitude towards pesticide stained tomatoes, with 67.0% (313/468) of the consumers disagreeing to a statement that tomatoes sold on the market are safe. Consumer risk perception was significantly associated with consumer awareness about residues in the tomatoes; where the proportino of consumers who were aware of the risk of pesticide stained tomatoes were 42.8 times more likely not to buy stained tomatoes compared to the proportion of those who were not aware. OR, 42.8 (95% CI: 10.76-170.28). However, level of education P(0.975), gender P(0.581) and age-group P(0.680) were not associated with consumer risk perception after Fisher-Exact tests analysis. (95% CI and 5% level of significance).

ConclusionConsumer risk perception on pesticide stained tomatoes among Ugandan consumers ranked low with a majority of consumers buying tomatoes stained with pesticide residuesdue to lack of an alternative, except for a few who had a high risk perception about the pesticide health effects. Topaz1, an essential gene for murine spermatogenesis, down-regulates the expression of numerous testis-specific long non-coding RNAs. Spermatogenesis involves coordinated processes, including meiosis, to produce functional gametes. We previously reported Topaz1 as a germ cell-specific gene highly conserved in vertebrates. Topaz1 knockout males are sterile with testes that lack haploid germ cells because of meiotic arrest after prophase I. To better characterize Topaz1-/- testes, we used RNA-sequencing analyses at two different developmental stages (P16 and P18). The absence of TOPAZ1 disturbed the expression of genes involved in microtubule and/or cilium mobility, which was consistent with testicular histology showing the disruption of microtubules and centrosomes. Moreover, a quarter of P18 dysregulated genes are long non-coding RNAs (lncRNAs), and three of them are testis-specific and located in spermatocytes, their expression starting between P11 and P15. The suppression of one of them, 4939463O16Rik, did not alter fertility although sperm parameters were disturbed and sperm concentration fell. The transcriptome of P18-4939463O16Rik-/- testes was altered and the molecular pathways affected included microtubule-based processes, the regulation of cilium movement and spermatogenesis. The absence of TOPAZ1 protein or 4930463O16Rik produced the same enrichment clusters in mutant testes despite a contrasted phenotype on male fertility. In conclusion, TOPAZ1 appeared to stabilize the expression of numerous lncRNAs. Its suppression is not essential for fertility but required during the terminal differentiation of male gametes.

Author SummaryThe Topaz1 gene was initially characterized during the initiation of meiosis in the sheep fetal ovary. In order to determine its function, a KO of the murine gene was performed. In this species, only males were sterile and spermatogenesis was blocked before the first meiotic division. Here, we show that cytoskeletal elements are markedly disturbed in mutant testes, indicating that these elements play an important function in spermatogenesis. While the mitotic spindle of spermatogonia was normal, the meiotic spindle of spermatocytes was hemi-spindle-shaped and the homologous chromosome pairs could position themselves on the equatorial plate. In addition, lncRNAs account for 25% of genes whose expression in testes varies significantly in the absence of Topaz1. This suggests a key role for these factors in spermatogenesis. Largely testis-specific, they may be involved in spermatogenesis and play a more or less critical role in mouse fertility, which probably also depends on their redundancies. Common anti-haemostatic medications increase the severity of uropathogenic Escherichia coli sepsis Uropathogenic Escherichia coli (UPEC) causes urinary tract infections that can result in sepsis. The haemostatic system is protective in the pyelonephritis stage of ascending UPEC infection, but the role of the haemostatic system has not been investigated during sepsis. Here we utilize a zebrafish-UPEC systemic infection model to visualize infection-induced coagulation and examine the effects of commonly prescribed anti-haemostatic medications on the infection severity. Treatment of systemically infected zebrafish with warfarin, aspirin, or ticagrelor reduced host survival, while stabilization of clots with aminocaproic acid increased host survival. Anti-haemostatic drug treatment increased UPEC burden. Our findings provide evidence that commonly prescribed anti-haemostatic medications may worsen the outcome of severe UPEC infection. Trait-based responses to land use and canopy dynamics modify long-term diversity changes in forest understories AimLand use is the foremost cause of global biodiversity decline, but species do not respond equally to land-use practices. Instead, it is suggested that responses vary with species traits, but long-term data on the trait-mediated effects of land-use on communities is scarce. Here we study how forest understory communities have been affected by two land-use practices during 4-5 decades, and whether changes in plant diversity are related to changes in functional composition.

LocationFinland

Time period1968-2019

Major taxa studiedVascular plants

MethodsWe resurveyed 245 vegetation plots in boreal herb-rich forest understories, and used hierarchical Bayesian linear models to relate changes in diversity, species composition, average plant size, and leaf economic traits to reindeer abundance, forest management intensity, and changes in climate, canopy cover and composition.

ResultsIntensively managed forests decreased in species richness and had increased turnover, but management did not affect functional composition. Increased reindeer densities corresponded with increased leaf dry matter content, evenness and diversity, and decreased height and specific leaf area. Successional development in the canopy was associated with increased specific leaf area and decreased leaf dry matter content and height in the understory over the study period. Effects of reindeer abundance and canopy density on diversity were partially mediated by vegetation height, which had a negative relationship with evenness. Observed changes in climate had no discernible effect on any variable.

Main conclusionsFunctional traits are useful in connecting vegetation changes to the mechanisms that drive them, and provide unique information compared to turnover and diversity metrics. These trait-dependent selection effects could inform whether species benefit or suffer from land use changes and explain observed biodiversity changes under global change. A network approach to genetic circuit designs As genetic circuits become more sophisticated, the size and complexity of data about their designs increases. This data captured goes beyond monolithic genetic sequences and towards circuit modularity and functional details, which are beneficial for analyzing circuit performance and establishing design automation techniques. However, the accessibility, visualisation and usability of design data (and metadata) have received relatively little attention to date. Here, we present a method to turn circuit designs into networks and showcase its potential to enhance the utility of design data. Since networks are dynamic structures, initial graphs can be interactively shaped into sub-networks of relevant information based on requirements such as abstraction, hierarchy and protein interactions. Additionally, several visual changes can be applied, such as colouring or clustering nodes based on types (e.g., genes or promoters), resulting in easier comprehension from a user perspective. This approach allows circuit designs to be coupled to other networks, such as metabolic pathways or implementation protocols captured in graph-like formats. Therefore, we advocate using networks to structure, access and improve synthetic biology information. Illuminating the plant transposon insertion landscape in real time using Cas9-targeted Nanopore sequencing and a novel pipeline Transposable elements (TEs) are a major source of genomic novelty, contributing to plant adaptation, speciation and new cultivar production. The often large, complex genomes of plants make identifying TE insertions from short reads challenging, while whole-genome sequencing remains expensive. In addition, rare somatic insertions reflecting mobilome dynamic are difficult to track using short reads. To overcome these challenges, we applied the Cas9-targeted Nanopore sequencing (CANS) approach and a novel pipeline, NanoCasTE, to trace both genetically inherited and somatic TE insertions in plants. We performed CANS of EVD retrotransposon in wild-type Arabidopsis thaliana and obtained up to 40x coverage of the targets after only a few hours of sequencing on a MinION sequencer. To estimate the ability to detect new TE and T-DNA insertions, we exploited the A. thaliana ddm1 mutant, which has elevated TE activity. Using CANS, we detect the hemizygous T-DNA insertion site and genetically inherited EVD insertions. CANS also detected >800 somatic TE insertions allowing monitoring of TE insertions in real time. By this, we built a high-density physical map of EVD insertion preferences in the A. thaliana ddm1 and found that EVD insertions are biased toward pericentromeric regions in ddm1 that differ from wild-type A. thaliana ecotypes suggesting a pivotal role of DNA methylation in the shaping of EVD insertion preferences. Using CANS, we were able to detect transposition events of ONSEN transposon family that occurred in response to heat stress and found hotspots of ONSEN insertions. Our results indicate that CANS and NanoCasTE are effective tools for the detection of TEIs in real time, opening an avenue for obtaining a deeper understanding of mobilome organization in response to stress and in different genetic backgrounds. The ability of CANS to detect genetically inherited TEIs and T-DNA integration sites make it useful for screening insertion mutants and transgenic plants.