Clarification of human blood ILC subtype interrelatedness and discovery of amphiregulin production by human NK cells shed light on HIV-1 pathogenesis Human blood innate lymphoid cells (ILCs), which include ILCs and natural killer (NK) cells, derive from a common CD117+ILC precursor (ILCP). Yet, the relationship among the ILC subsets remains unclear. Bulk and single cell RNA-Seq and ATAC-Seq showed that blood ILC subsets cluster into ILC2s, ILCPs, a mixed cluster of CD56dim and CD56- NK cells, and a separate cluster of CD56hiNK cells that share features with both ILCs and CD56dimNK cells. In surprising contrast to mice, tissue repair protein amphiregulin was produced by human NK cells, with higher levels in CD56hiNK cells than in ILCs. Amphiregulin production by human NK cells was promoted by TCF7/WNT signaling and inhibited by TGFB1, a cytokine elevated in people living with HIV-1. Knockout of RUNX3, a WNT antagonist downstream of TGFB1, increased amphiregulin production in human NK cells. CD4+T cell depletion in people living with HIV-1, or from PBMCs in tissue culture, was associated with expansion of metabolically inert, nonfunctional CD56-NK cells. Experiments in tissue culture and in humanized mice revealed that CD56-NK cells are derived from CD56dimNK cells, and that CD4+T cell-derived IL-2 stimulates MTOR activity in CD56dimNK cells to prevent this transition. These findings clarify how ILC subsets are related to each other and provide insight into how HIV-1 infection disrupts ILC homeostasis and contributes to pathology.
Antibody-induced glomerulonephritis is amplified by RTEC-intrinsic IL-17 signaling and restrained by IL-17-mediated induction of the endoribonuclease Regnase-1 (Zc3h12a) Antibody-mediated glomerulonephritis (AGN) is a clinical manifestation of many autoimmune kidney diseases for which few effective treatments exist. Chronic inflammatory circuits in renal glomerular and tubular cells lead to tissue damage in AGN. These cells are targeted by the cytokine IL-17, which has recently been shown to be a central driver of the pathogenesis of AGN. However, surprisingly little is known about the regulation of pathogenic IL-17 signaling in the kidney. Here, using a well characterized mouse model of AGN, we show that IL-17 signaling in renal tubular epithelial cells (RTECs) is necessary for AGN development. We also show that Regnase-1, an RNA binding protein with endoribonuclease activity, is a negative regulator of IL-17 signaling in RTECs. Accordingly, mice with a selective Regnase-1 deficiency in RTECs exhibited exacerbated kidney dysfunction in AGN. Mechanistically, Regnase-1 inhibits IL-17-driven expression of the transcription factor I{kappa}B{xi} and consequently its downstream gene targets including Il6 and Lcn2. Moreover, deletion of Regnase-1 in human RTECs reduced inflammatory gene expression in an I{kappa}B{xi}-dependent manner. Overall, these data identify an IL-17-driven inflammatory circuit in RTECs during AGN that is constrained by Regnase-1.
Nanoscale ligand density modulates gap junction intercellular communication of cell condensates during chondrogenesis To unveil the influence of cell-matrix adhesions in the establishment of gap junction intercellular communication (GJIC) during cell condensation in chondrogenesis.

Materials & MethodsPreviously developed nanopatterns of the cell adhesive ligand arginine-glycine-aspartic acid (RGD) were used as cell culture substrates to control cell adhesion at the nanoscale. We conducted in vitro chondrogenesis of mesenchymal stem cells on the nanopatterns. We evaluated cohesion and GJIC in cell condensates.

ResultsMechanical stability and GJIC are enhanced by a nanopattern configuration in which 90% of the surface area presents adhesion sites separated less than 70 nm, thus providing an onset for cell signaling.

ConclusionCell-matrix adhesions regulate GJIC of mesenchymal cell condensates during in vitro chondrogenesis from a threshold configuration at the nanoscale. Royal knifefish generate powerful suction feeding through large neurocranial elevation and high epaxial muscle power Suction feeding in ray-finned fishes involves powerful buccal cavity expansion to accelerate water and food into the mouth. Previous XROMM studies in largemouth bass (Micropterus salmoides), bluegill sunfish (Lepomis macrochirus), and channel catfish (Ictalurus punctatus) have shown that more than 90% of suction power in high performance strikes comes from the axial musculature. Thus, the shape of the axial muscles and skeleton may impact suction feeding mechanics. Royal knifefish (Chitala blanci) have an unusual postcranial morphology, with a ventrally flexed vertebral column and relatively large mass of epaxial muscle. Based on their body shape, we hypothesized that royal knifefish would generate high power strikes by utilizing large neurocranial elevation, vertebral column extension, and epaxial shortening. As predicted, C. blanci generated high suction expansion power compared to the other three species studied to date (up to 160 W), which was achieved by increasing both the rate of volume change and the intraoral subambient pressure. The large epaxial muscle (25% of body mass) shortened at high velocities to produce large neurocranial elevation and vertebral extension (up to 41 deg, combined), as well as high muscle mass-specific power (up to 800 W kg-1). For the highest power strikes, axial muscles generated 95% of the power, and 64% of the axial muscle mass consisted of the epaxial muscles. The epaxial-dominated suction expansion of royal knifefish supports our hypothesis that postcranial morphology may be a strong predictor of suction feeding biomechanics.

SUMMARY STATEMENTRoyal knifefish rely on their distinct postcranial morphology--with a curved vertebral column and large dorsal body muscles--to produce large neurocranial elevation and powerful suction feeding. Reconstitution of minimal motility system based on Spiroplasma swimming by expressing two bacterial actins in synthetic minimal bacterium Motility is one of the most important features of life, but its evolutionary origin remains unknown. In this study, we focused on Spiroplasma, commensal, or parasitic bacteria. They swim by switching the helicity of a ribbon-like cytoskeleton that comprises six proteins, each of which evolved from a nucleosidase and bacterial actin called MreB. We expressed these proteins in a synthetic, non-motile minimal bacterium, JCVI-syn3.0B, whose reduced genome was computer-designed and chemically synthesized. The synthetic bacterium exhibited swimming motility with features characteristic of Spiroplasma swimming. Moreover, some combinations of the two proteins produced a helical cell shape and swimming, suggesting that the swimming originated from the differentiation and coupling of bacterial actins, and we also obtained a minimal system for motility of the synthetic bacterium.

One-Sentence SummaryThe minimal system comprised two bacterial actins that provided cell helicity and swimming to the synthetic minimal bacterium. On-pathway multivalency of a broadly neutralizing evolution drug candidate A promising approach to prevent antibiotic resistance is design of multivalent antibiotics that constrain bacterial evolvability by blocking multiple escape routes. Previously we discovered that an "evolution drug" lead designed to broadly neutralize bacterial dihydrofolate reductase (DHFR) and its trimethoprim escape variants is multivalent in E. coli cells. In this work we identified the second target of this new lead. We analysed antibiotic induced global metabolic perturbations and deployed metabolic-network analysis and structural similarity search of the putative targets and narrowed down the search to a few enzymes. Further, combining in vivo and in vitro analyses, we identified HPPK (folK), an essential protein in bacterial folate-metabolism, as the non-DHFR target of the lead. We observed that compounds with interaction specificity against WT do not develop multivalency against related proteins. This multivalent-design strategy is effective in blocking the emergence of drug-resistant phenotypes.

HighlightsO_LIAn evolution drug lead designed to block bacterial evolvability manifested behaviours suggesting an intra-cellular multivalent activity.
C_LIO_LIA multi-layered metabolomic and structure guided approach unravelled the second intra-cellular target of the evolution-drug lead, showing folate-pathway inhibitions at two metabolic points.
C_LIO_LIThe on-pathway anti-folate activity is exhibited by inhibiting two essential proteins viz. DHFR and its upstream protein HPPK.
C_LIO_LIMultivalent drug development schemes present a promising anti-evolution strategy to address the problem of "drug-resistance" by inhibiting more than one metabolic protein.
C_LI Shedding Light on the Circadian Clock of the Threespine Stickleback The circadian clock is an internal timekeeping system shared by most organisms, and knowledge about its functional importance and evolution in natural environments is still needed. Here, we investigated the circadian clock of wild-caught threespine sticklebacks (Gasterosteus aculeatus) at the behavioural and molecular levels. While their behaviour, ecology, and evolution are well studied, information on their circadian rhythms are scarce. We quantified the daily locomotor activity rhythm under a light-dark cycle (LD) and under constant darkness (DD). Under LD, all fish exhibited significant daily rhythmicity, while under DD, only 18% of individuals remained rhythmic. This interindividual variation suggests that the circadian clock controls activity only in certain individuals. Moreover, under LD, some fish were almost exclusively nocturnal, while others were active around the clock. Furthermore, the most nocturnal fish were also the least active. These results suggest that light masks activity (i.e. suppresses activity without entraining the internal clock) more strongly in some individuals than others. Finally, we quantified the expression of five clock genes in the brain of sticklebacks under DD using qPCR. We did not detect circadian rhythmicity, which could either indicate that the clock molecular oscillator is highly light-dependent, or that there was an oscillation but that we were unable to detect it. Overall, our study suggests that a strong circadian control on behavioural rhythms may not necessarily be advantageous in a natural population of sticklebacks and that the daily phase of activity varies greatly between individuals because of a differential masking effect of light.

Summary statementWe found that in wild-caught threespine sticklebacks, the circadian clock does not control locomotor activity in most, but not all, individuals. Sticklebacks are mostly nocturnal, although interindividual variation exists. Glucose dynamics during ozone exposure measured using radiotelemetry: Stress drivers BackgroundStress-related neurobehavioral and metabolic disorders are associated with altered circulating adrenal-derived hormones and hyperglycemia. Temporal assessment of glucose and these hormones is critical for insights on an individuals health.

ObjectivesHere we used implantable-telemetry in rats to assess real-time changes in circulating glucose during and after exposure to the air pollutant ozone, and link responses to circulating neuroendocrine stress and metabolic hormones. We also compared rodent glucose and corticosterone (cortisol in humans) responses to humans exposed to ozone.

MethodsFirst, using a cross-over design, we monitored glucose levels during single or repeated ozone exposures (0.0, 0.2, 0.4 and 0.8-ppm) and non-exposure periods in male Wistar-Kyoto-rats implanted with glucose-telemeters. A second cohort of un-implanted rats was exposed to ozone (0.0, 0.4 or 0.8-ppm) for 30-min, 1-hour, 2-hour, or 4-hour with hormones measured immediately after exposure. Then we assessed glucose metabolism in sham and adrenalectomized rats with or without pharmacological interventions of adrenergic and glucocorticoid receptors. Finally, we assessed glucose and cortisol in serum samples from a clinical study involving exposure of human volunteers to air or 0.3 ppm ozone.

ResultsOzone (0.8-ppm) caused hyperglycemia and hypothermia beginning 90-min into exposure, with reversal of effects 4-6 hours post-exposure. Glucose monitoring during four daily 4-hour ozone exposures revealed duration of hyperglycemia, adaptation, and diurnal variations. Ozone-induced hyperglycemia was preceded by increased adrenocorticotropic hormone, corticosterone, and epinephrine, but depletion of thyroid-stimulating, prolactin, and luteinizing hormones. Hyperglycemia was inhibited in rats that were adrenalectomized and/or treated with glucocorticoid inhibitor. The depletion of cortisol was dampened in humans exposed to ozone during intermittent exercise.

DiscussionWe demonstrate for the first time the temporality of neuroendocrine-stress-mediated biological sequalae responsible for ozone-induced metabolic dysfunction as exposure occurs. Real-time glucose monitoring with stress hormones assessment may be useful in identifying interactions among pollutants and stress-related illnesses. Usutu virus escapes langerin-induced restriction to productively infect human Langerhans cells, unlike West Nile virus. Usutu virus (USUV) and West Nile virus (WNV) are phylogenetically close emerging arboviruses transmitted by mosquitoes, and constitute a global public health threat. Since USUV and WNV enter the body through the skin, the first immune cells they encounter are skin-resident dendritic cells, the most peripheral outpost of immune defense. This unique network is composed of Langerhans cells (LCs) and dermal DCs, which reside in the epidermis and the dermis, respectively.

Using human skin explants, we show that while both viruses can replicate in keratinocytes, they can also infect resident DCs with distinct tropism, since WNV preferentially infects DCs in the dermis, whereas USUV has a greater propensity to infect LCs. Using both purified human epidermal LCs (eLCs) and monocyte derived LCs (MoLCs), we confirm that LCs sustain a faster and more efficient replication of USUV compared with WNV and that this correlates with a more intense innate immune response to USUV compared with WNV. Next, we show that ectopic expression of the LC-specific C-type lectin receptor (CLR), langerin, in HEK293T cells allows WNV and USUV to bind and enter, but supports the subsequent replication of USUV only. Conversely, blocking or silencing langerin in MoLCs or eLCs made them resistant to USUV infection, thus demonstrating that USUV uses langerin to enter and replicate in LCs. Altogether, our results demonstrate that LCs constitute privileged target cells for USUV in human skin, because langerin favors its entry and replication. Intriguingly, this suggests that USUV efficiently escapes the antiviral functions of langerin, which normally safeguards LCs from most viral infections. Resetting of the 24-nt siRNA landscape in rice zygotes BackgroundThe zygote, a totipotent stem cell, constitutes a critical stage of the life cycle of sexually reproducing organisms. It is produced by the fusion of two differentiated cells -- the egg and sperm, which in plants have radically different siRNA transcriptomes from each other and from multicellular embryos. Due to technical challenges, the epigenetic changes that accompany the zygotic transition are poorly understood.

ResultsHere, we characterized the small RNA transcriptome of rice zygotes. We found widespread redistribution of 24-nt siRNAs relative to gametes, including absence of sperm signature siRNAs, reduction at egg signature siRNA loci, and upregulation at seedling signature siRNA loci. Loci with reduced siRNAs in zygote relative to egg were gene-distal and heterochromatic, while loci with increased siRNAs relative to egg had a similar genomic distribution to canonical siRNA loci. Although both egg and zygote siRNA loci had higher mCHH level in wildtype than in drm2 embryo, zygote but not egg siRNA loci were associated with hypermethylation in mature embryo. A small fraction of siRNA loci (~1%) called siren loci accounted for 60% of all siRNAs within zygote siRNA loci, that likely arose from maternal carryover as they had similarly abundant siRNAs in egg; these siren loci were not associated with embryo hypermethylation.

ConclusionsTaken together, our results indicate re-distribution of siRNAs in rice zygotes towards the canonical vegetative profile, that are consistent with the initiation of resetting of the gametic epigenome before the first embryonic division. Analyses of symbiotic bacterial communities in the plant pest Bemisia tabaci reveal high prevalence of Candidatus Hemipteriphilus asiaticus on the African continent AO_SCPLOWBSTRACTC_SCPLOWMicrobial symbionts are widespread in insects and some of them have been associated to adaptive changes. Primary symbionts (P-symbionts) have a nutritional role that allows their hosts to feed on unbalanced diets (plant sap, wood, blood). Most of them have undergone genome reduction, but their genomes still retain genes involved in pathways that are necessary to synthesize the nutrients that their hosts need. However, in some P-symbionts, essential pathways are incomplete and secondary symbionts (S-symbionts) are required to complete parts of their degenerated functions. The P-symbiont of the phloem sap-feeder Bemisia tabaci, Candidatus Portiera aleyrodidarium, lacks genes involved in the synthesis of vitamins, cofactors, and also of some essential amino-acids. Seven S-symbionts have been detected in the B. tabaci species complex. Phenotypic and genomic analyses have revealed various effects, from reproductive manipulation to fitness benefits, notably some of them have complementary metabolic capabilities to Candidatus Portiera aleyrodidarium, suggesting that their presence may be obligatory. In order to get the full picture of the symbiotic community of this pest, we investigated, through metabarcoding approaches, the symbiont content of individuals from Burkina Faso, a West African country where B. tabaci induces severe crop damage. While no new putative B. tabaci S-symbiont was identified, Candidatus Hemipteriphilus asiaticus, a symbiont only described in B. tabaci populations from Asia, was detected for the first time on this continent. Phylogenetic analyses however reveal that it is a different strain than the reference found in Asia. Specific diagnostic PCRs showed a high prevalence of these S-symbionts and especially of Candidatus Hemipteriphilus asiaticus in different genetic groups. These results suggest that Candidatus Hemipteriphilus asiaticus may affect the biology of B. tabaci and provide fitness advantage in some B. tabaci populations. Citation needed?Wikipedia bibliometrics during the first waveof the COVID-19 pandemic. BackgroundWith the COVID-19 pandemics outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an "infodemic", ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedias COVID-19-related articles before and during the pandemics first wave (January-May 2020).

ResultsWe found that coronavirus-related articles referenced trusted media sources and high-quality academic research. Moreover, despite a surge in COVID-19 preprints, Wikipedia had a clear preference for open-access studies published in respected journals and made little use of preprints. Building a timeline of English COVID-19 articles from 2001-2020 revealed a nuanced trade-off between quality and timeliness. It further showed how preexisting articles on key topics related to the virus created a framework for integrating new knowledge. Supported by a rigid sourcing policy, this "scientific infrastructure" facilitated contextualization and regulated the influx of new information. Lastly, we constructed a network of DOI-Wikipedia articles, which showed the shifting landscape of pandemic-related knowledge on Wikipedia and how academic citations create a web of shared knowledge supporting topics like COVID-19 vaccine development.

ConclusionsUnderstanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its "citizen encyclopedists", it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts. Global connectivity fingerprints predict the domain generality of multiple-demand regions A set of distributed cognitive control networks are known to contribute to diverse cognitive demands, yet it is unclear how these networks gain this domain-general capacity. We hypothesized that this capacity is largely due to the particular organization of the human brains intrinsic network architecture. Specifically, we tested the possibility that each brain regions domain generality is reflected in its level of global (hub-like) intrinsic connectivity, as well as its particular global connectivity pattern (connectivity fingerprint). Consistent with prior work, we found that cognitive control networks exhibited domain generality, as they represented diverse task context information covering sensory, motor response, and logic rule domains. Supporting our hypothesis, we found that the level of global intrinsic connectivity (estimated with resting-state fMRI) was correlated with domain generality during tasks. Further, using a novel information fingerprint mapping approach, we found that each cognitive control regions unique rule response profile (information fingerprint) could be predicted based on its unique intrinsic connectivity fingerprint and the information content in non-cognitive control regions. Together these results suggest that the human brains intrinsic network architecture supports its ability to represent diverse cognitive task information, largely via the location of multiple-demand regions within the brains global network organization. Synergy of chemotherapy and macrophage depletion leads to B cell-mediated T cell memory activation and durable triple negative breast cancer regression Immunosuppressive elements within the tumor microenvironment such as Tumor Associated Macrophages (TAMs) can present a barrier to successful anti-tumor responses by cytolytic T cells. We employed preclinical syngeneic p53 null mouse models of triple negative breast cancer (TNBC) to develop a treatment regimen that harnessed the immunostimulatory effects of low-dose cyclophosphamide coupled with the pharmacologic inhibition of TAMs using either a small molecule CSF1R inhibitor or an anti-CSF1R antibody. This therapeutic combination was used to successfully treat several highly aggressive TNBC murine mammary tumors and lung metastasis. Using this regimen and single cell RNA sequencing we characterized tumor infiltrating lymphocytes (TILs) including helper T cells and antigen-presenting B cells that were highly enriched in good responders to combination therapy. Using high dimensional imaging techniques, we identified the close spatial localization of B220+ CD86+ activated B cells and CD4+ T cells in tertiary lymphoid structures that were present up to 6 weeks post-treatment in one model that also exhibited long-term tumor regression post-treatment. We also characterized the transcriptional and metabolic heterogeneity of TAMS in these two closely related claudin-low/mesenchymal subtype tumor models with differential treatment responses. A murine TAM signature derived from the T12 model is highly expressed and conserved in human claudin-low breast cancers, and high expression of the T12 signature correlated with reduced overall survival. This T12 tumor TAM signature may help identify human claudin-low breast cancer patients that will benefit from the combination of cyclophosphamide and anti-CSF1R therapy. These studies illustrate the complexity of the tumor immune microenvironment and highlight different immune responses that result from rationale combinations of immunotherapy.

SignificanceA treatment regimen that harnessed the immunostimulatory effects of cyclophosphamide coupled with the inhibition of CSF1R was used to successfully treat several highly aggressive claudin-low TNBC murine mammary tumors and lung metastasis. Mitochondrial F1FO ATP synthase determines the local proton motive force in cristae tips The classical view of oxidative phosphorylation is that a proton motive force PMF generated by the respiratory chain complexes fuels ATP synthesis. Under glycolytic conditions, ATP synthase in its reverse mode also can contribute to the PMF. Here, we dissected the two functions of ATP synthase and the role of its inhibitory factor 1 (IF1) under different metabolic conditions in detail. pH profiles of mitochondrial sub-compartments were recorded with high spatial resolution in live mammalian cells by positioning a pH-sensor directly at F1 and FO of ATP synthase, complex IV and in the matrix. Our results clearly show that ATP synthase activity is substantially controlling the PMF and that IF1 is essential under OXPHOS conditions to prevent reverse ATP synthase activity due to an almost negligible {Delta}pH.

GRAPHICAL ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/430746v2_ufig1.gif" ALT="Figure 1">
View larger version (50K):
org.highwire.dtl.DTLVardef@1a975eforg.highwire.dtl.DTLVardef@7ddb49org.highwire.dtl.DTLVardef@c7a128org.highwire.dtl.DTLVardef@2559e8_HPS_FORMAT_FIGEXP  M_FIG C_FIG HIGHLIGHTSO_LIThe {Delta}pH along and across the inner mitochondrial membrane is not homogeneous
C_LIO_LIThe proton motive force at cristae tips is controlled by F1 FO ATP synthase
C_LIO_LIUnder OXPHOS conditions, the pH difference between FO and F1 of active ATP synthase is almost negligible (1.2 proton vs. 1 proton equivalent)
C_LIO_LIIF1 is required to prevent the onset of ATP hydrolysis under OXPHOS conditions
C_LI A Novel Proposal for an Index for Regional Cerebral Perfusion Pressure - A Theoretical Approach Using Fluid Dynamics Cerebral blood flow (CBF) / cerebral blood volume (CBV) ratio derived by [15O] H2O/ CO2 and CO positron emission tomography (PET) examination has been used empirically as an index for cerebral perfusion pressure (CPP). However, this equation lacks a theoretical basis and furthermore, has not been confirmed to be proportionate to CPP, as direct measurement of local CPP is not practical.

We have developed a new index for CPP using the Poiseuille equation based on a simple model. Our model suggests that CBF/CBV2 is proportionate to CPP and that it is mathematically a more accurate index than CBF/CBV. Reuse of a wing venation gene-regulatory network in patterning the eyespot rings of butterflies Novel organismal traits might reuse ancestral gene-regulatory networks (GRNs) in their development, but data supporting this mechanism are still sparse. Here we propose that a conserved venation gene network, used in patterning insect wings, has been recruited to pattern the sharp and distinct rings of color in butterfly eyespot development. This network involves Decapentaplegic (Dpp), acting as a central morphogen, activating optix and spalt at different concentration thresholds. It also involves Spalt cross-regulating optix resulting in the formation of a sharp boundary between the two genes. First, using in situ hybridizations, we show that dpp is expressed in the eyespot centers during the early pupal stage and that CRISPR-Cas9 embryonic knockouts of dpp, optix, and spalt result in the loss of eyespots, loss of the orange ring, and loss of the black scales, respectively. Furthermore, using RNAi during the pupal stage, we show that dpp down-regulation results in the complete or partial loss of eyespots. Using CRISPR disruptions of optix or spalt, followed by immunostainings, we show that Spalt represses optix in the central region of the eyespot, limiting optix expression to a more peripheral ring. We further illustrate that optix disruptions alter scale pigmentation and scale ultrastructure as they transform orange to brown scales. Altogether, the regulatory interactions between dpp, spalt and optix mimic those found in earlier larval developmental stages, in the anterior compartment of an insects wing, where these genes play a role in venation patterning. The network similarities suggest that the venation GRN was co-opted to aid in the differentiation of the eyespot rings. 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 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. Brominated Lipid Probes Expose Structural Asymmetries in Constricted Membranes Lipids in biological membranes are thought to be functionally organized, but few experimental tools can probe nanoscale membrane structure. Using brominated lipids as contrast probes for cryo-EM and a model ESCRT-III membrane remodeling system, we observed leaflet-level and protein-localized lipid structural patterns within highly constricted and thinned membrane nanotubes. These nanotubes differed markedly from protein-free, flat bilayers in leaflet thickness, lipid diffusion rates, and lipid compositional and conformational asymmetries. Simulations and cryo-EM imaging of brominated SDPC showed how a pair of phenylalanine residues scored the outer leaflet with a helical hydrophobic defect where polyunsaturated docosahexaenoyl (DHA) tails accumulated at the bilayer surface. Combining cryo-EM of halogenated lipids with molecular dynamics thus enables new characterizations of the composition and structure of membranes on molecular length scales.

One-Sentence SummaryCryo-EM imaging of halogenated lipids and MD simulations provide molecular-scale insight into constricted bilayer structure. cinaR: A comprehensive R package for the differential analyses and functional interpretation of ATAC-seq data SummaryATAC-seq is a frequently used assay to study chromatin accessibility levels. Differential chromatin accessibility analyses between biological groups and functional interpretation of these differential regions are essential in ATAC-seq data analyses. Although distinct methods and analyses pipelines are developed for this purpose, a stand-alone R package that combines state-of-the art differential and functional enrichment analyses pipelines is missing. To fill this gap, we developed cinaR (Chromatin Analyses in R), which is a single wrapper function and provides users with various data analyses and visualization options, including functional enrichment analyses with gene sets curated from multiple sources.

Availability and implementationcinaR is an R/CRAN package which is under GPL-3 License and its source code is freely accessible at https://CRAN.R-project.org/package=cinaR.

Gene sets are available at https://CRAN.R-project.org/package=cinaRgenesets.

Bone marrow ATAC-seq data is available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE165120

Contactonur.karakaslar@jax.org or duygu.ucar@jax.org Fatal neuroinvasion and SARS-CoV-2 tropism in K18-hACE2 mice is partially independent on hACE2 expression Animal models recapitulating distinctive features of severe COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. The precise mechanisms of lethality in this mouse model remain unclear. Here, we evaluated the spatiotemporal dynamics of SARS-CoV-2 infection for up to 14 days post-infection. Despite infection and moderate pneumonia, rapid clinical decline or death of mice was invariably associated with viral neuroinvasion and direct neuronal injury (including brain and spinal neurons). Neuroinvasion was observed as early as 4 dpi, with virus initially restricted to the olfactory bulb supporting axonal transport via the olfactory neuroepithelium as the earliest portal of entry. No evidence of viremia was detected suggesting neuroinvasion occurs independently of entry across the blood brain barrier. SARS-CoV-2 tropism was not restricted to ACE2-expressing cells (e.g., AT1 pneumocytes), and some ACE2-positive lineages were not associated with the presence of viral antigen (e.g., bronchiolar epithelium and brain capillaries). Detectable ACE2 expression was not observed in neurons, supporting overexpression of ACE2 in the nasal passages and neuroepithelium as more likely determinants of neuroinvasion in the K18-hACE2 model. Although our work incites caution in the utility of the K18-hACE2 model to study global aspects of SARS-CoV-2 pathogenesis, it underscores this model as a unique platform for exploring the mechanisms of SARS-CoV-2 neuropathogenesis that may have clinical relevance acknowledging the growing body of evidence that suggests COVID-19 may result in long-standing neurologic consequences.

IMPORTANCECOVID-19 is predominantly a respiratory disease caused by SARS-CoV-2 that has infected more than 191 million people with over 4 million fatalities (2021-07-20). The development of animal models recapitulating distinctive features of severe COVID-19 is critical to enhancing our understanding of SARS-CoV-2 pathogenesis and in the evaluation of vaccine and therapeutic efficacy. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. Here, we show lethality of this model is invariably associated with viral neuroinvasion linked with viral replication and assembly. Importantly, pneumonia albeit invariably present was generally moderate with the absence of culturable infectious virus at peak neuroinvasion. The dynamics of viral neuroinvasion and pneumonia were only partially dependent on hACE2. Overall, this study provides an in-depth sequential characterization of the K18-hACE2 model following SARS-CoV-2 infection, highlighting its significance to further study the mechanisms of SARS-CoV-2 neuropathogenesis. RNA polymerase II is recruited to DNA double-strand breaks for dilncRNA transcription in Drosophila DNA double-strand breaks are among the most toxic lesions that can occur in a genome and their faithful repair is thus of great importance. Recent findings have uncovered local transcription that initiates at the break and forms a non-coding transcript, called damage-induced long non-coding RNA (dilncRNA), which helps to coordinate the DNA transactions necessary for repair. We provide nascent RNA sequencing-based evidence that RNA polymerase II transcribes the dilncRNA in Drosophila and that this is more efficient for DNA breaks in an intron-containing gene, consistent with the higher damage-induced siRNA levels downstream of an intron. The spliceosome thus stimulates recruitment of RNA polymerase II to the break, rather than merely promoting the annealing of sense and antisense RNA to form the siRNA precursor. In contrast, RNA polymerase III nascent RNA libraries did not contain reads corresponding to the cleaved loci and selective inhibition of RNA polymerase III did not reduce the yield of damage-induced siRNAs. Finally, the damage-induced siRNA density was unchanged downstream of a T8 sequence, which terminates RNA polymerase III transcription. We thus found no evidence for a participation of RNA polymerase III in dilncRNA transcription in cultured Drosophila cells. Understanding the mechanism of monoADP ribosylation in OsSRT1 and its linkage to the DNA repair system under stress conditions The role of sirtuins in plants are slowly unraveling. There are only reports of H3K9Ac deacetylation by OsSRT1. Here our studies shade light on its dual enzyme capability with preference for mono ADP ribosylation over deacetylation. OsSRT1 can specifically transfer the single ADP ribose group on its substrates in an enzymatic manner. This mono ADPr effect is not well known in plants, more so for deacetylases. The products of this reaction (NAM and ADP ribose) have immense negative effect on this enzyme suggesting a tighter regulation. Resveratrol, a natural plant polyphenol proves to be a strong activator of this enzyme at 150 M concentration. Under different abiotic stress conditions, we could link this ADP ribosylase activity to the DNA repair pathway by activating the enzyme PARP1. Metal stress in plants also influences these enzyme activities.

HighlightsO_LIOsSRT1 can transfer a single moiety of ADP-ribose on itself as well as other nuclear proteins like histones H3 and H2A.
C_LIO_LINAM, ADP-ribose and certain metal ions negatively regulate this ADP-ribose transfer.
C_LIO_LIADPr of OsPARP1 and OsPARP2 links OsSRT1 to DNA damage repair pathways.
C_LIO_LIOsSRT1 positively regulates the activity of OsPARP1 by ADP ribosylating it.
C_LIO_LIOn plants exposure to H2O2 (oxidative stress) and Arsenic toxicity, there is a link between the increased activity of the players of DNA damage repair system and overexpression of OsSRT1.
C_LI Fixed or random? On the reliability of mixed-effects models for a small number of levels in grouping variables O_LIBiological data are often intrinsically hierarchical. Due to their ability to account for such dependencies, mixed-effects models have become a common analysis technique in ecology and evolution. While many questions around their theoretical foundations and practical applications are solved, one fundamental question is still highly debated: When facing a low number of levels should we model a grouping (blocking, clustering) variable as a random or fixed effect? In such situation, the variance of the random effect is imprecise, but whether this affects the statistical properties of the population effect is unclear.
C_LIO_LIHere, we analyzed the consequences of including a grouping variable as fixed or random effect in the correctly specified and other possible miss-specified models (too simple or too complex models) for data with small number of levels (2 - 8). For all these options, we calculated type I error rates and power. Moreover, we show how these statistical properties change with the study design.
C_LIO_LIWe found that the model choice does not influence the statistical properties of the population effect when the effect is the same at all levels However, if an ecological effect differs among levels, using a random slope and intercept model, and switching to a fixed-effect model only in case of a singular fit, avoids overconfidence in the results. Additionally, power and type I error are strongly influenced by the number of and difference between levels.
C_LIO_LIWe conclude that inferring the correct random effect structure is of high importance to get correct statistical properties. When in doubt, we recommend starting with the simpler model and using model diagnostics to identify missing components. When having identified the correct structure, we encourage to start with a mixed-effects model independent of the number of levels and switch to a fixed-effect model only in case of a singular fit. With these recommendations, we allow for more informative choices about study design and data analysis and thus make ecological inference with mixed-effects models more robust for small number of levels.
C_LI Single cell profiling of functionally cured Chronic Hepatitis B patients reveals the emergence of activated innate and an altered adaptive immune response in the intra-hepatic environment Hepatitis B surface antigen (HBsAg) loss or functional cure (FC), is considered the desirable therapeutic outcome for chronic hepatitis B (CHB) patients. However, the immuno-pathological biomarkers and underlying mechanisms remain unclear. Here we present a comprehensive single cell-transcriptomic atlas together with immune-phenotyping of disease-associated cell states (DACS) isolated from intra-hepatic tissue and matched PBMCs of either CHB or FC patients. We find that the intra-hepatic environment displays specific cell identities and molecular signatures that are distinct from PBMCs. FC is associated with emergence of an altered adaptive immune response marked by CD4 cytotoxic T lymphocytes (CD4-CTLs), and an activated innate response represented by liver-resident natural killer (LR-NK) cells. Overall, these findings provide novel insights into immuno-pathological cell states associated with FC that could serve as prognostic biomarkers.