Mitochondrial genomes of two parasitic Cuscuta species lack clear evidence of horizontal gene transfer and retain unusually fragmented ccmFC genes BackgroundThe intimate association between parasitic plants and their hosts favours the exchange of genetic material, potentially leading to horizontal gene transfer (HGT) between plants. With the recent publication of several parasitic plant nuclear genomes, there has been considerable focus on such non-sexual exchange of genes. To enhance the picture on HGT events in a widely distributed parasitic genus, Cuscuta (dodders), we assembled and analyzed the organellar genomes of two recently sequenced species, C. australis and C. campestris, making this the first account of complete mitochondrial genomes (mitogenomes) for this genus.

ResultsThe mitogenomes are 265,696 and 275,898 bp in length and contain a typical set of mitochondrial genes, with ten missing or pseudogenized genes often lost from angiosperm mitogenomes. Each mitogenome also possesses a structurally unusual ccmFC gene, which exhibits splitting of one exon and a shift to trans-splicing of its intron. Based on phylogenetic analysis of mitochondrial genes from across angiosperms and similarity-based searches, there is little to no indication of HGT into the Cuscuta mitogenomes. A few candidate regions for plastome-to-mitogenome transfer were identified, with one suggestive of possible HGT.

ConclusionsThe lack of HGT is surprising given examples from the nuclear genomes, and may be due in part to the relatively small size of the Cuscuta mitogenomes, limiting the capacity to integrate foreign sequences.
Enrichment analyses identify shared associations for 25 quantitative traits in over 600,000 individuals from seven diverse ancestries Since 2005, genome-wide association (GWA) datasets have been largely biased toward sampling European ancestry individuals, and recent studies have shown that GWA results estimated from self-identified European individuals are not transferable to non-European individuals due to various confounding challenges. Here, we demonstrate that enrichment analyses which aggregate SNP-level association statistics at multiple genomic scales--from genes to genomic regions and pathways--have been underutilized in the GWA era and can generate biologically interpretable hypotheses regarding the genetic basis of complex trait architecture. We illustrate examples of the robust associations generated by enrichment analyses while studying 25 continuous traits assayed in 566,786 individuals from seven diverse self-identified human ancestries in the UK Biobank and the Biobank Japan, as well as 44,348 admixed individuals from the PAGE consortium including cohorts of African-American, Hispanic and Latin American, Native Hawaiian, and American Indian/Alaska Native individuals. We identify 1,000 gene-level associations that are genome-wide significant in at least two ancestry cohorts across these 25 traits, as well as highly conserved pathway associations with triglyceride levels in European, East Asian, and Native Hawaiian cohorts.
Staphylococcus aureus-serine protease-like protein B (SplB) activates PAR2 and induces endothelial barrier dysfunction Staphylococcus aureus (S. aureus) is a major cause of life-threatening systemic infection in humans. To cause blood stream infections such as sepsis and endocarditis, the bacteria must overcome the hosts endothelial barrier. The serine protease-like proteins (Spls) of S. aureus are known to contribute to pneumonia and allergic airway inflammation in animal models, but their role in endothelial damage is unknown. Here we demonstrate that SplB induces proinflammatory cytokine release in primary human vascular endothelial cells (HUVECs) in vitro. Mechanistically, we show that SplB selectively cleaves and activates human proteinase-activated receptor-2 (PAR2), and induces biased signaling via G13, Gi1/oB (NF-k{beta}), and {beta}-arrestin-1/-2. This activation did not trigger Gq/11-mediated calcium release nor ERK phosphorylation. Inhibition of PAR2 in HUVECs reduced the SplB-mediated cytokine release. Intravital microscopy of cremaster muscles in mice demonstrated that administration of SplB causes microvascular leakage. Genetic deletion of PAR2 in mice or neutralization of SplB with a monoclonal antibody preserved the endothelial barrier. This study identifies PAR2 as a receptor and substrate for SplB and highlights its role in mediating endothelial damage.



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org.highwire.dtl.DTLVardef@16a60a2org.highwire.dtl.DTLVardef@ee4e41org.highwire.dtl.DTLVardef@153cd7borg.highwire.dtl.DTLVardef@d746eb_HPS_FORMAT_FIGEXP  M_FIG Graphical abstract C_FIG Quantifying the effect of caloric and non-caloric sweeteners in the brain response using EEG and convolutional neural network Sweetener type can influence sensory properties and consumers acceptance and preference for low-calorie products. An ideal sweetener does not exist, and each sweetener must be used in situations to which it is best suited. Aspartame and sucralose can be good substitutes for sucrose in passion fruit juice. Despite the interest in artificial sweeteners, little is known about how artificial sweeteners are processed in the human brain. Here, we evaluated brain signals of 11 healthy subjects when they tasted passion fruit juice equivalently sweetened with sucrose (9.4 g/100 g), sucralose (0.01593 g/100 g), or aspartame (0.05477 g/100 g). Electroencephalograms were recorded for two sites in the gustatory cortex (i.e., C3 and C4). Data with artifacts were disregarded, and the artifact-free data were used to feed a CNN. Our results indicated that the brain responses distinguish juice sweetened with different sweeteners with an average accuracy of 0.823.

Practical ApplicationsFinding sweeteners that best fit consumer preferences evolves understanding how the gustatory cortex processes sweeteners. Ideal equivalence will occur when the brain is no longer able to distinguish stimuli that are consciously perceived. This study presents a method of signal acquisition using a single channel and an open-source processing environment. This would allow, for example, to disregard the use of a commercial electroencephalograph and expand the studies in this area and offering to food industry additional tools in the development of products sweetened with non-caloric sweeteners. Discovery of an Antarctic ascidian-associated uncultivated Verrucomicrobia with antimelanoma palmerolide biosynthetic potential The Antarctic marine ecosystem harbors a wealth of biological and chemical innovation that has risen in concert over millennia since the isolation of the continent and formation of the Antarctic circumpolar current. Scientific inquiry into the novelty of marine natural products produced by Antarctic benthic invertebrates led to the discovery of a bioactive macrolide, palmerolide A, that has specific activity against melanoma and holds considerable promise as an anticancer therapeutic. While this compound was isolated from the Antarctic ascidian Synoicum adareanum, its biosynthesis has since been hypothesized to be microbially mediated, given structural similarities to microbially-produced hybrid non-ribosomal peptide-polyketide macrolides. Here, we describe a metagenome-enabled investigation aimed at identifying the biosynthetic gene cluster (BGC) and palmerolide A-producing organism. A 74 Kbp candidate BGC encoding the multi-modular enzymatic machinery (hybrid Type I-trans-AT polyketide synthase-non-ribosomal peptide synthetase and tailoring functional domains) was identified and found to harbor key features predicted as necessary for palmerolide A biosynthesis. Surveys of ascidian microbiome samples targeting the candidate BGC revealed a high correlation between palmerolide-gene targets and a single 16S rRNA gene variant (R=0.83 - 0.99). Through repeated rounds of metagenome sequencing followed by binning contigs into metagenome-assembled genomes, we were able to retrieve a near-complete genome (10 contigs) of the BGC-producing organism, a novel verrucomicrobium within the Opitutaceae family that we propose here as Candidatus Synoicihabitans palmerolidicus. The refined genome assembly harbors five highly similar BGC copies, along with structural and functional features that shed light on the host-associated nature of this unique bacterium.

ImportancePalmerolide A has potential as a chemotherapeutic agent to target melanoma. We interrogated the microbiome of the Antarctic ascidian, Synoicum adareanum, using a cultivation-independent high-throughput sequencing and bioinformatic strategy. The metagenome-encoded biosynthetic machinery predicted to produce palmerolide A was found to be associated with the genome of a member of the S. adareanum core microbiome. Phylogenomic analysis suggests the organism represents a new deeply-branching genus, Candidatus Synoicihabitans palmerolidicus, in the Opitutaceae family of the Verrucomicrobia phylum. The Ca. S. palmerolidicus 4.29 Mb genome encodes a repertoire of carbohydrate-utilizing and transport pathways enabling its ascidian-associated lifestyle. The palmerolide-producers genome also contains five distinct copies of the large palmerolide biosynthetic gene cluster that may provide structural complexity of palmerolide variants. A novel nongenetic murine model of hyperglycemia and hyperlipidemia-associated aggravated atherosclerosis ObjectiveAtherosclerosis, the main pathology underlying cardiovascular diseases is accelerated in diabetic patients. Genetic mouse models require breeding efforts which are time-consuming and costly. Our aim was to establish a new nongenetic model of inducible metabolic risk factors that mimics hyperlipidemia, hyperglycemia, or both and allows the detection of phenotypic differences dependent on the metabolic stressor(s).

Methods and ResultsWild-type mice were injected with gain-of-function PCSK9D377Y (proprotein convertase subtilisin/kexin type 9) mutant adeno-associated viral particles (AAV) and streptozotocin and fed either a high-fat diet (HFD) for 12 or 20 weeks or a high-cholesterol/high-fat diet (Paigen diet, PD) for 8 weeks. To evaluate atherosclerosis, two different vascular sites (aortic sinus and the truncus of the brachiocephalic artery) were examined in the mice. Combined hyperlipidemic and hyperglycemic (HGHCi) mice fed a HFD or PD displayed characteristic features of aggravated atherosclerosis when compared to hyperlipidemia (HCi HFD or PD) mice alone. Atherosclerotic plaques of HGHCi HFD animals were larger, showed a less stable phenotype (measured by the increased necrotic core area, reduced fibrous cap thickness, and less -SMA-positive area) and had more inflammation (increased plasma IL-1{beta} level, aortic pro-inflammatory gene expression, and MOMA-2-positive cells in the BCA) after 20 weeks of HFD. Differences between the HGHCi and HCi HFD models were confirmed using RNA-seq analysis of aortic tissue, revealing that significantly more genes were dysregulated in mice with combined hyperlipidemia and hyperglycemia than in the hyperlipidemia-only group. The HGHCi-associated genes were related to pathways regulating inflammation (increased Cd68, iNos, and Tnfa expression) and extracellular matrix degradation (Adamts4 and Mmp14). When comparing HFD with PD, the PD aggravated atherosclerosis to a greater extent in mice and showed plaque formation after 8 weeks. Hyperlipidemic and hyperglycemic mice fed a PD (HGHCi PD) showed less collagen (Sirius red) and increased inflammation (CD68-positive cells) within aortic plaques than hyperlipidemic mice (HCi PD). HGHCi-PD mice represent a directly inducible hyperglycemic atherosclerosis model compared with HFD-fed mice, in which atherosclerosis is severe by 8 weeks.

ConclusionWe established a nongenetically inducible mouse model allowing comparative analyses of atherosclerosis in HCi and HGHCi conditions and its modification by diet, allowing analyses of multiple metabolic hits in mice. A class of identifiable phylogenetic birth-death models In a striking result, Louca and Pennell (2020) recently proved that a large class of phylogenetic birth-death models are statistically unidentifiable from lineage-through-time (LTT) data: any pair of sufficiently smooth birth and death rate functions is "congruent" to an infinite collection of other rate functions, all of which have the same likelihood for any LTT vector of any dimension. As Louca and Pennell argue, this fact has distressing implications for the thousands of studies that have utilized birth-death models to study evolution.

In this paper, we qualify their finding by proving that an alternative and widely used class of birth-death models is indeed identifiable. Specifically, we show that piecewise constant birth-death models can, in principle, be consistently estimated and distinguished from one another, given a sufficiently large extant time tree and some knowledge of the present-day population. Subject to mild regularity conditions, we further show that any unidentifiable birth-death model class can be arbitrarily closely approximated by a class of identifiable models. The sampling requirements needed for our results to hold are explicit, and are expected to be satisfied in many contexts such as the phylodynamic analysis of a global pandemic. A robust approach to estimate relative phytoplankton cell abundance from metagenomes Phytoplankton account for >45% of global primary production, and have an enormous impact on aquatic food webs and on the entire Earth System. Their members are found among prokaryotes (cyanobacteria) and multiple eukaryotic lineages containing chloroplasts. Phytoplankton communities are generally studied by PCR amplification of bacterial (16S), nuclear (18S) or chloroplastic (16S) rRNA marker genes from DNA extracted from environmental samples. However, our appreciation of phytoplankton abundance or biomass is limited by PCR-amplification biases, rRNA gene copy number variations across taxa, and the fact that rRNA genes do not provide insights into metabolic traits such as photosynthesis. In addition, rRNA marker genes fail to capture both cyanobacteria and photosynthetic eukaryotes simultaneously. Here, we targeted the photosynthetic gene psbO from metagenomes to circumvent these limitations: the method is PCR-free, and the gene is universally and exclusively present in photosynthetic prokaryotes and eukaryotes, mainly in one copy per genome. We applied and validated this new strategy with the Tara Oceans datasets, and showed improved correlations with flow cytometry and microscopy than when based on rRNA genes. Furthermore, we revealed unexpected features of the ecology of these organisms, such as the high abundance of picocyanobacterial aggregates and symbionts in the ocean, and the decrease in relative abundance of phototrophs towards the larger size classes of marine dinoflagellates. To facilitate the incorporation of psbO in molecular-based surveys, we compiled a curated database of >18,000 unique sequences. Overall, psbO appears to be a promising new gene marker for molecular-based evaluations of entire phytoplankton communities. Scaling up to meet new challenges of brain and behaviour through deep brain electrophysiology in freely moving sheep While rodents are arguably the easiest animals to use for studying brain function, relying on them as model species for translational research comes with its own sets of limitations. Here, we propose sheep as a practical large animal species for in vivo brain function studies performed in naturalistic settings. To demonstrate their experimental usefulness, we performed proof-of-principle deep brain electrophysiological recording experiments from unrestrained sheep. Recordings were made from cortex and hippocampus both whilst sheep performed goal-directed behaviours (two-choice discrimination tasks), and across states of vigilance that included natural sleep. Hippocampal and cortical oscillatory rhythms were consistent with those seen in rodents and non-human primates, and included cortical alpha oscillations during immobility, hippocampal theta oscillations (5-6Hz) during locomotion and hippocampal sharp wave ripple oscillations ([~]150 Hz) during immobility. Moreover, we found clear examples of neurons whose activity was modulated by task, speed of locomotion, spatial position, reward and vigilance states. Recordings were conducted over a period of many months. Due to the exceptional stability of individual electrodes we were able to record from some neurons repeatedly across the span of a month. Together these experiments demonstrate that sheep are an excellent experimental animal model to use in longitudinal electrophysiological and imaging studies, particularly those requiring a large brained mammal, large scale recordings across distributed neuronal networks, experimentation outside the confounds of the traditional laboratory, or all the above concomitantly. A chemical screen based on an interruption of zebrafish gastrulation identifies the HTR2C inhibitor Pizotifen as a suppressor of EMT-mediated metastasis Metastasis is responsible for approximately 90% of cancer-associated mortality but few models exist that allow for rapid and effective screening of anti-metastasis drugs. Current mouse models of metastasis are too expensive and time consuming to use for rapid and high-throughput screening. Therefore, we created a unique screening concept utilizing conserved mechanisms between zebrafish gastrulation and cancer metastasis for identification of potential anti-metastatic drugs. We hypothesized that small chemicals that interrupt zebrafish gastrulation might also suppress metastatic progression of cancer cells and developed a phenotype-based chemical screen to test the hypothesis. The screen used epiboly, the first morphogenetic movement in gastrulation, as a marker and enabled 100 chemicals to be tested in five hours. The screen tested 1280 FDA-approved drugs and identified Pizotifen, an antagonist for serotonin receptor 2C (HTR2C) as an epiboly-interrupting drug. Pharmacologic and genetic inhibition of HTR2C suppressed metastatic progression in a mouse model. Blocking HTR2C with Pizotifen restored epithelial properties to metastatic cells through inhibition of Wnt-signaling. In contrast, HTR2C induced epithelial to mesenchymal transition (EMT) through activation of Wnt-signaling and promoted metastatic dissemination of human cancer cells in a zebrafish xenotransplantation model. Taken together, our concept offers a novel platform for discovery of anti-metastasis drugs. METHYLOME DYNAMICS UPON PROTEASOME INHIBITION BY THE PSEUDOMONAS VIRULENCE FACTOR SYRINGOLIN A DNA methylation is an important epigenetic mark required for proper gene expression and silencing of transposable elements. DNA methylation patterns can be modified by environmental factors such as pathogen infection, where modification of DNA methylation can be associated with plant resistance. To counter the plant defense pathways, pathogens produce effectors molecule, several of which act as proteasome inhibitors. Here we investigated the effect of proteasome inhibition by the bacterial virulence factor Syringolin A on genome-wide DNA methylation. We show that Syringolin A treatment results in an increase of DNA methylation at centromeric and pericentromeric regions of Arabidopsis chromosomes. We identify several CHH DMRs that are enriched in the proximity of transcriptional start sites. Syringolin A treatment does not result in significant changes in small RNA composition. However, significant changes in genome transcriptional activity can be observed, including a strong upregulation of resistance genes that are located on chromosomal arms. We hypothesize that DNA methylation changes could be linked to the upregulation of some atypical members of the de novo DNA methylation pathway: AGO3, AGO9 and DRM1. Our data suggests that modification of genome-wide DNA methylation resulting from an inhibition of the proteasome by bacterial effectors could be part of a epi-genomic arms race against pathogens. Conserved organ-specific microbial assemblages in different populations of a terrestrial crab Brachyuran crabs originated in the oceans and evolved specific morphological and physiological adaptations to live in freshwater, intertidal and even terrestrial habitats but the role of a selection mechanism involving symbiotic microorganisms long these colonization processes are not known. In this work we investigated the associated microbiota of three populations of a terrestrial brachyuran crab, Chiromantes haematocheir, to find evidence of a conserved crab-specific microbiome unrelated to the population of origin and dissimilar from environmental microbial assemblages. Bacterial 16S rRNA gene and fungal ITS sequences were obtained from selected crab organs and environmental matrices to profile microbial communities. In spite of the presence of truly marine larval stages and no gregarious behaviour, favouring microbiota exchanges, we found common, organ-specific microbiota, associated to the gut and the gills of the crabs (with more than 15% of the genera detected specifically enriched only in one organ). Our results suggest an early establishment of a new common, stable microbiota in the transition from water to land. Remarked Suppression of Aβ42 Protomer-protomer Dissociation Reaction via Pentamer Dimerization Multimeric protein complexes are molecular apparatuses to regulate biological systems and often determine their fate. Among proteins forming such molecular assemblies, amyloid proteins have drawn attention over a half-century since amyloid fibril formation of these proteins is supposed to be a common pathogenic cause for neurodegenerative diseases. This process is triggered by the accumulation of fibril-like aggregates, while the microscopic mechanisms are mostly elusive due to technical limitation of experimental methodologies in individually observing each of diverse aggregate species in the aqueous solution. We then addressed this problem by employing atomistic molecular dynamics simulations for the paradigmatic amyloid protein, amyloid-{beta} (1-42) (A{beta}42). Seven different dimeric forms of oligomeric A{beta}42 fibril-like aggregate in aqueous solution, ranging from tetramer to decamer, were considered. We found additive effects of the size of these fibril-like aggregates on their thermodynamic stability and have clarified kinetic suppression of protomer-protomer dissociation reactions at and beyond the point of pentamer dimer formation. This observation was obtained from the specific combination of the A{beta}42 protomer structure and the physicochemical condition that we here examined, while it is worthwhile to recall that several amyloid fibrils take dimeric forms of their protomers. We could thus conclude that the stable formation of fibril-like protomer dimer should be involved in a turning point where rapid growth of amyloid fibrils is triggered. Quantifying the Morphology and Mechanisms of Cancer Progression in 3D in-vitro environments: Integrating Experiments and Multiscale Models Mathematical models of cancer growth have become increasingly more accurate both in the space and time domains. However, the limited amount of data typically available has resulted in a larger number of qualitative rather than quantitative studies. In the present study, we provide an integrated experimental-computational framework for the quantification of the morphological characteristics and the mechanistic modelling of cancer progression in 3D environments. The proposed framework allows for the calibration of multiscale, spatiotemporal models of cancer growth using state-of-the-art 3D cell culture data, and their validation based on the resulting experimental morphological patterns. Its implementation enabled us to pursue two goals; first, the quantitative description of the morphology of cancer progression in 3D cultures, and second, the relation of tumour morphology with underlying biophysical mechanisms that govern cancer growth and migration. We applied this framework to the study of the spatiotemporal progression of Triple Negative Breast Cancer cells cultured in Matrigel scaffolds, and validated the hypothesis of chemotactic migration using a multiscale Keller-Segel model. The results revealed transient, non-random spatial distributions of cancer cells that consist of clustered, and dispersion patterns. The proposed model was able to describe the general characteristics of the experimental observations and suggests that cancer cells exhibited chemotactic migration and accumulation, as well as random motion during the examined time period of development. To our knowledge, this is the first time that a multiscale model is used to quantify the relationship between the spatial patterns and the underlying mechanisms of cancer growth in 3D environments. Dosage sensitivity in Pumilio1-associated diseases involves two distinct mechanisms Mutations in the RNA-binding protein (RBP) Pumilio1 (PUM1) can cause dramatically different phenotypes. Mild mutations that reduce PUM1 levels by 25% lead to a mild, adult-onset ataxia, whereas more severe mutations that reduce PUM1 levels 40-60% produce an infantile syndrome involving multiple developmental delays and seizures. Why this difference in expression should cause such different phenotypes has been unclear; known PUM1 targets are de-repressed to equal degrees in both diseases. We therefore sought to identify PUM1s protein partners in the murine brain. We identified a number of putative interactors involved in different aspects of RNA metabolism such as silencing, alternative splicing, and polyadenylation. We find that PUM1 haploinsufficiency alters the stability of several interactors and disrupts the regulation of targets of those interactors, whereas mild PUM1 loss only de-represses PUM1-specific targets. We validated these phenomena in patient-derived cell lines and show that normalizing PUM1 levels rescues the levels of interactors and their targets. We therefore propose that dosage sensitivity does not necessarily reflect a linear change in levels but can involve distinct mechanisms. Studying the interactors of RBPs in vivo will be necessary to understand their functions in neurological diseases. Establishing combination PAC-1 and TRAIL regimens for treating ovarian cancer based on patient-specific pharmacokinetic profiles using in silico clinical trials Ovarian cancer is commonly diagnosed in its late stages, and new treatment modalities are needed to improve patient outcomes and survival. We have recently established the synergistic effects of combination tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and procaspase activating compound (PAC-1) therapies in granulosa cell tumours (GCT) of the ovary, a rare form of ovarian cancer, using a mathematical model of the effects of both drugs in a GCT cell line. Here, to understand the mechanisms of combined TRAIL and PAC-1 therapy, study the viability of this treatment strategy, and accelerate preclinical translation, we leveraged our mathematical model in combination with population pharmacokinetics (PopPK) models of both TRAIL and PAC-1 to expand a realistic heterogeneous cohort of virtual patients and optimize treatment schedules. Using this approach, we investigated treatment responses in this virtual cohort and determined optimal therapeutic schedules based on patient-specific pharmacokinetic characteristics. Our results showed that schedules with high initial doses of PAC-1 were required for therapeutic efficacy. Further analysis of individualized regimens revealed two distinct groups of virtual patients within our cohort: one with high PAC-1 elimination, and one with normal PAC-1 elimination. In the high elimination group, high weekly doses of both PAC-1 and TRAIL were necessary for therapeutic efficacy, however virtual patients in this group were predicted to have a worse prognosis when compared to those in the normal elimination group. Thus, PAC-1 pharmacokinetic characteristics, particularly clearance, can be used to identify patients most likely to respond to combined PAC-1 and TRAIL therapy. This work underlines the importance of quantitative approaches in preclinical oncology. A Wnt/β-catenin signaling driven adipocyte population required for beiging and glucose homeostasis Wnt/{beta}-catenin signaling has been well established as a potent inhibitor of adipogenesis. Here, we identified a population of adipocytes that exhibit persistent activity of Wnt/{beta}-catenin signaling, as revealed by the Tcf/Lef-Gfp reporter allele, in embryonic and adult mouse fat depots, named as Wnt+ adipocytes. We showed that the {beta}-catenin-mediated signaling activation in these cells is Wnt ligand- and receptor-independent but relies on AKT/mTOR pathway and is essential for cell survival. Such adipocytes are distinct from classical ones in transcriptomic and genomic signatures and can be induced from various sources of mesenchymal stromal cells including human cells. Genetic lineage-tracing and targeted cell ablation studies revealed that these adipocytes convert into beige adipocytes directly and are also required for beige fat recruitment under thermal challenge, demonstrating both cell autonomous and non-cell autonomous roles in adaptive thermogenesis. Furthermore, mice bearing targeted ablation of these adipocytes exhibited glucose intolerance, while mice receiving exogenously supplied such cells manifested enhanced glucose utilization. Our studies uncover a unique adipocyte population in regulating beiging in adipose tissues and systemic glucose homeostasis. Binding of single-mutant epidermal growth factor (EGF) ligands alter the stability of the EGF receptor dimer and promote growth signaling The Epidermal Growth Factor Receptor (EGFR) is a membrane-anchored tyrosine kinase that is able to selectively respond to multiple extra-cellular stimuli. Previous studies have indicated that the modularity of this system is affected by ligand-induced differences in the stability of the dimerized receptor in a process known as "Biased signaling". However, this hypothesis has not been explored using single-mutant ligands thus far. Herein, we developed a new approach to identify residues responsible for functional divergence combining the conservation and co-evolution information of ortholog and paralog genes encoding the epidermal growth factor (EGF) ligand. Then, we mutated these residues and assessed the mutants effects on the receptor by employing a combination of molecular dynamics (MD) and biochemical techniques. Although the EGF mutants had comparable binding affinities to the wild type ligand for EGFR, the EGF mutants induced a different phosphorylation and cell growth pattern in multiple cell lines. The MD simulations of the EGF mutants show a long-range effect on the receptor dimer interface. For the first time in this study, a single mutation in EGF is shown to be enough to alter the activation of the pathway at the cellular level. These results also support the theory of biased signaling in the tyrosine kinase receptor system and demonstrate a promising new way to study ligand-receptor interactions. Comparison of hybrid learning and remote education in the implementation of the 'Adopt a Microorganism' methodology The Internet has changed the way teachers and students access information and build knowledge. The recent COVID-19 pandemic has created challenges for both teachers and students and a demand for new methodologies of remote learning. In the life sciences, mixing online content with practical activities represents an even greater challenge. In microbiology, the implementation of an active teaching methodology, the #Adopt project, based on the social network Facebook(R), represents an excellent option for connecting remote education with classroom activities. In 2020, the version applied in high school, "Adopt a Microorganism", was adapted to meet the demands of emergency remote education owing to the suppression of face-to-face activities caused by the pandemic. In the present study, we assessed how the change in methodology impacted the discourse richness of students from high school integrated with technical education in the Business Administration program of the Federal Institute of Sao Paulo, Sorocaba Campus. Three questionnaires related to the groups of microorganisms (Archaea, Bacteria, Virus, Fungi, and Protozoan) were applied. The students responses in the 2019 and 2020 classes were compared concerning content richness and multiplicity of concepts through the application of the Shannon diversity index, an approach that is generally used to assess biodiversity in different environments. The observed results suggest that remote learning provided students with a conceptual basis and richness of content equivalent to that achieved by students subjected to the hybrid teaching model. In conclusion, this study suggests that the #Adopt project methodology increases students discourse richness in microbiology even without face-to-face traditional classes. Missing the sweet spot: one of the two N-glycans on human Gb3/CD77 synthase is expendable N-glycosylation is a ubiquitous posttranslational modification that may influence folding, subcellular localization, secretion, solubility and oligomerization of proteins. In this study, we examined the effects of N-glycans on the activity of human Gb3/CD77 synthase, which catalyzes the synthesis of glycosphingolipids with terminal Gal1[-&gt;]4Gal (Gb3 and the P1 antigen) and Gal1[-&gt;]4GalNAc disaccharides (the NOR antigen). The human Gb3/CD77 synthase contains two occupied N-glycosylation sites at positions N121 and N203. Intriguingly, we found that while the N-glycan at N203 is essential for activity and correct subcellular localization, the N-glycan at N121 is dispensable and its absence did not reduce, but, surprisingly, even increased the activity of the enzyme. The fully N-glycosylated human Gb3/CD77 synthase and its glycoform missing the N121 glycan correctly localized in the Golgi, whereas a glycoform without the N203 site partially mislocalized in the endoplasmic reticulum. A double mutein missing both N-glycans was inactive and accumulated in the endoplasmic reticulum. Our results suggest that the decreased specific activity of human Gb3/CD77 synthase glycovariants results from their improper subcellular localization and, to a smaller degree, a decrease in enzyme solubility. Taken together, our findings show that the two N-glycans of human Gb3/CD77 synthase have opposing effects on its properties, revealing a dual nature of N-glycosylation and potentially a novel regulatory mechanism controlling the biological activity of proteins. Data-Driven Network Models for Genetic Circuits From Time-Series Data with Incomplete Measurements Synthetic gene networks are frequently conceptualized and visualized as static graphs. This view of biological programming stands in stark contrast to the transient nature of biomolecular interaction, which is frequently enacted by labile molecules that are often unmeasured. Thus, the network topology and dynamics of synthetic gene networks can be difficult to verify in vivo or in vitro, due to the presence of unmeasured biological states. Here we introduce the dynamical structure function as a new mesoscopic, data-driven class of models to describe gene networks with incomplete measurements. We introduce a network reconstruction algorithm and a code base for reconstructing the dynamical structure function from data, to enable discovery and visualization of graphical relationships in a genetic circuit diagram as time-dependent functions rather than static, unknown weights. We prove a theorem, showing that dynamical structure functions can provide a data-driven estimate of the size of crosstalk fluctuations from an idealized model. We illustrate this idea with numerical examples. Finally, we show how data-driven estimation of dynamical structure functions can explain failure modes in two experimentally implemented genetic circuits, a historical genetic circuit and a new E. coli based transcriptional event detector. Fatal COVID-19 and non-COVID-19 Acute Respiratory Distress Syndrome is Associated with Incomplete Alveolar Type 1 Epithelial Cell Differentiation from the Transitional State Without Fibrosis ARDS due to COVID-19 and other etiologies results from injury to the alveolar epithelial cell (AEC) barrier resulting in noncardiogenic pulmonary edema, which causes acute respiratory failure; clinical recovery requires epithelial regeneration. During physiologic regeneration in mice, AEC2s proliferate, exit the cell cycle, and transiently assume a transitional state before differentiating into AEC1s; persistence of the transitional state is associated with pulmonary fibrosis in humans. It is unknown whether transitional cells emerge and differentiate into AEC1s without fibrosis in human ARDS and why transitional cells differentiate into AEC1s during physiologic regeneration but persist in fibrosis. We hypothesized that incomplete but ongoing AEC1 differentiation from transitional cells without fibrosis may underlie persistent barrier permeability and fatal acute respiratory failure in ARDS. Immunostaining of postmortem ARDS lungs revealed abundant transitional cells in organized monolayers on alveolar septa without fibrosis. They were typically cuboidal or partially spread, sometimes flat, and occasionally expressed AEC1 markers. Immunostaining and/or interrogation of scRNAseq datasets revealed that transitional cells in mouse models of physiologic regeneration, ARDS, and fibrosis express markers of cell cycle exit but only in fibrosis express a specific senescence marker. Thus, in severe, fatal early ARDS, AEC1 differentiation from transitional cells is incomplete, underlying persistent barrier permeability and respiratory failure, but ongoing without fibrosis; senescence of transitional cells may be associated with pulmonary fibrosis. A novel stem cell type at the basal side of the subventricular zone maintains adult neurogenesis According to the current consensus, neural stem cells (NSCs) apically contacting the lateral ventricle generate differentiated progenitors by rare asymmetric divisions or by relocating to the basal side of the ventricular-subventricular zone V-SVZ. Both processes will then ultimately lead to the generation of adult-born olfactory bulb (OB) interneurons. In contrast to this view, we here found that adult-born OB interneurons largely derive from an additional NSC type resident in the basal V-SVZ. Despite being both capable of self-renewal and long-term quiescence, apical and basal NSCs differ in Nestin expression, primary cilia extension and frequency of cell division. The expression of Notch-related genes also differed between the two NSC groups and Notch-activation was greatest in apical NSCs. Apical downregulation of Notch-effector Hes1 decreased Notch activation while increasing proliferation across the niche and neurogenesis from apical NSCs. Underscoring their different roles in neurogenesis, lactation-dependent increase in neurogenesis was paralleled by extra activation of basal but not apical NSCs. Thus, basal NSCs support OB neurogenesis whereas apical NSCs impart Notch-mediated lateral inhibition across the V-SVZ. The Consequences of Differential Origin Licensing Dynamics in Distinct Chromatin Environments MCM complexes are loaded onto chromosomes to license DNA replication origins in G1 phase of the cell cycle, but it is not yet known how mammalian MCM complexes are adequately distributed to both euchromatin and heterochromatin. To address this question, we combined time-lapse live-cell imaging with fixed cell immunofluorescence imaging of single human cells to quantify the relative rates of MCM loading in heterochromatin and euchromatin at different times within G1. We report here that MCM loading in euchromatin is faster than in heterochromatin in very early G1, but surprisingly, heterochromatin loading accelerates relative to euchromatin loading in middle and late G1. These different loading dynamics require ORCA-dependent differences in ORC distribution during G1. A consequence of heterochromatin origin licensing dynamics is that cells experiencing a truncated G1 phase from premature cyclin E expression enter S phase with under-licensed heterochromatin, and DNA damage accumulates preferentially in heterochromatin in the subsequent S/G2 phase. Thus G1 length is critical for sufficient MCM loading, particularly in heterochromatin, to ensure complete genome duplication and to maintain genome stability. A terrain treadmill to study animal locomotion through large obstacles A major challenge to understanding locomotion in complex 3-D terrain with large obstacles is to create tools for controlled, systematic lab experiments. Existing terrain arenas only allow observations at small spatiotemporal scales ([~]10 body length, [~]10 stride cycles). Here, we create a terrain treadmill to enable high-resolution observations of animal locomotion through large obstacles over large spatiotemporal scales. An animal moves through modular obstacles on an inner sphere, while a rigidly-attached, concentric, transparent outer sphere rotated with the opposite velocity via closed-loop feedback to keep the animal on top. During sustained locomotion, a discoid cockroach moved through pillar obstacles for 25 minutes ({approx} 2500 strides) over 67 m ({approx} 1500 body lengths), and was contained within a radius of 4 cm (0.9 body length) for 83% of the duration, even at speeds of up to 10 body length/s. The treadmill enabled observation of diverse locomotor behaviors and quantification of animal-obstacle interaction.

Summary StatementA device keeps a small animal on top of a sphere while it traverses obstacles, creating a "terrain treadmill" to study locomotion over a long time and distance at high-resolution. 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.