Common physiological processes control mercury reduction during photosynthesis and fermentation Mercury (Hg) is a global pollutant and potent neurotoxin that bioaccumulates in food webs as monomethylmercury (MeHg). The production of MeHg is driven by anaerobic and Hg redox cycling pathways such as Hg reduction, which control the availability of Hg to methylators. Anaerobes play an important role in Hg reduction in methylation hotspots, yet their contributions remain underappreciated due to how challenging these pathways are to study in the absence of dedicated genetic targets and low levels of Hg0 in anoxic environments. In this study we used Hg stable isotope fractionation to explore Hg reduction during anoxygenic photosynthesis and fermentation in the model anaerobe Heliobacterium modesticaldum Ice1. We show that cells preferentially reduce lighter Hg isotopes in both metabolisms leading to mass-dependent fractionation, but mass-independent fractionation commonly induced by UV-visible light is absent. We show that isotope fractionation is affected by the interplay between pathways controlling Hg recruitment, accessibility, and availability alongside metabolic redox reactions. The combined contributions of these processes lead to isotopic enrichment during anoxygenic photosynthesis that is in between the values reported for anaerobic respiratory microbial Hg reduction and abiotic photoreduction. Isotope enrichment during fermentation is closer to what has been observed in aerobic bacteria that reduce Hg through dedicated detoxification pathways. These results demonstrate that common controls exist at the atomic level for Hg reduction during photosynthesis and fermentation in H. modesticaldum. Our work suggests that similar controls likely underpin diverse microbe-mediated Hg transformations that affect Hgs fate in oxic and anoxic habitats.

IMPORTANCEAnaerobic and photosynthetic bacteria that reduce mercury affect mercury delivery to microbes in methylation sites that drive bioaccumulation in food webs. Anaerobic mercury reduction pathways remain underappreciated in the current view of the global mercury cycle because they are challenging to study, bearing no dedicated genetic targets to establish physiological mechanisms. In this study we used stable isotopes to show that common physiological processes control mercury reduction during photosynthesis and fermentation in the model anaerobe Heliobacterium modesticaldum Ice1. The sensitivity of isotope analyses highlighted the subtle contribution of mercury uptake towards the isotope signature associated with anaerobic mercury reduction. When considered alongside the isotope signatures associated with microbial pathways for which genetic determinants have been identified, our findings underscore the narrow range of isotope enrichment that is characteristic of microbial mercury transformations. This suggests that there exist common atomic-level controls for biological mercury transformations across a broad range of geochemical conditions.
A protease-activatable luminescent biosensor andreporter cell line for authentic SARS-CoV-2 infection Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.

Author summaryTechniques for measuring infection with SARS-CoV-2 in the laboratory are laborious and time-consuming, and different laboratories use different approaches. There is therefore no generally agreed way to quantitate neutralising antibodies against SARS-CoV-2, which block infection with the virus and protect people from COVID-19. In this study, we describe a new way to measure SARS-CoV-2 infection, which is much simpler and faster than existing methods. It relies on the production of a specific protease enzyme by the virus, which is able to cleave and activate an engineered protein biosensor in infected cells. This biosensor emits light in the presence of viral infection, and the amount of light released is used as a readout for the amount of infectious SARS-CoV-2 present. The signal is very sensitive, so the number of infected cells required is very small, and the method can be scaled-up to test many samples at once. In particular, we demonstrate how it can be used to detect different variants of SARS-CoV-2, and quantitate neutralising antibodies against these viruses.
Affordable Caenorhabditis elegans tracking system for classroom use For decades the nematode C. elegans has served as an outstanding research organism owing to its unsurpassed experimental amenability. This advantage has also made this tiny worm an attractive vehicle for science instruction across higher learning institutions. However, the prohibitive cost associated with the automated behavioral assessment of these animals remains an obstacle preventing their full adoption in undergraduate and high school settings. To improve this situation, we developed an inexpensive worm tracking system for use by high school interns and undergraduate students. Over the past two years this tracker has been successfully used by undergraduate students in our introductory Cell and Molecular lab (BSC220) at Illinois State University. Here we describe and demonstrate the use of our inexpensive worm tracking system. Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads Lumen morphogenesis is key to the function of organs and results from the integration of molecular pathways and mechanical forces1-3. The mechanisms governing anisotropic lumen expansion remain elusive4-6. In contrast to epithelial cells which have simple apico-basal polarity and form tubes, hepatocytes are multi-polar and form narrow lumina that grow anisotropically between adjacent cells, collectively generating a complex 3D network of bile canaliculi (BC)7,8. Here, we studied lumen elongation and BC morphogenesis in differentiating primary mouse hepatoblasts in vitro. Remarkably, we discovered a pattern of specific extensions of the apical membrane traversing the lumen between adjacent hepatocytes and sealed by tight junctions, reminiscent of the bulkheads of boats. These structures were also present in the developing liver. A targeted screen revealed that silencing of Rab35 caused loss of the bulkheads, conversion of hepatocyte into simple epithelial polarity and formation of spherical lumina in vitro. Strikingly, we could re-engineer hepatocyte polarity and tissue morphogenesis in vivo in the embryonic liver, converting BC into simple epithelial tubes. Our results suggest that the apical bulkheads of hepatocytes are cell-intrinsic anisotropic mechanical elements that ensure stability of the elongating lumen between two cells, thus determining the structure of BC during liver tissue morphogenesis. The female pheromone (Z)-4-undecenal mediates flight attraction and courtship in Drosophila melanogaster Specific mate communication and recognition underlies reproduction and hence speciation. Mate communication evolves during adaptation to ecological niches and makes use of social signals and habitat cues.

Our study provides new insights in Drosophila melanogaster premating olfactory communication, showing that female pheromone (Z)-4-undecenal (Z4-11Al) and male pheromone cVA interact with food odour in a sex-specific manner. Furthermore, Z4-11Al, which mediates upwind flight attraction in both sexes, also elicits courtship in experienced males.

Twin variants of the olfactory receptor Or69a are co-expressed in the same olfactory sensory neurons, and feed into the same glomerulus in the antennal lobe. Z4-11Al is perceived via Or69aB, while the food odorant (R)-linalool is a main ligand for the other variant, Or69aA. That Z4-11Al mediates courtship in experienced males, not (R)- linalool, is probably due to courtship learning. Behavioural discrimination is reflected by calcium imaging of the antennal lobe, showing distinct glomerular activation patterns by these two compounds.

Male sex pheromone cVA is known to affect male and female courtship at close range, but does not elicit upwind flight attraction as a single compound, in to contrast to Z4-11Al. A blend of cVA and the food odour vinegar attracted females, while a blend of female pheromone Z4-11Al and vinegar attracted males instead.

Sex-specific upwind flight attraction to blends of food volatiles and male and female pheromone, respectively, adds a new element to Drosophila olfactory premating communication and is an unambiguous paradigm for identifying the behaviourally active components, towards a more complete concept of food-pheromone odour objects.

SummaryThe female-produced, species-specific volatile pheromone of D. melanogaster attracts both sexes from a distance, alone and in concert with food odorants, and elicits courtship in experienced males. Dynamic modulation of enhancer responsiveness by core promoter elements in living Drosophila embryo Regulatory interactions between enhancers and core promoters are fundamental for the temporal and spatial specificity of gene expression in development. The central role of core promoters is to initiate productive transcription in response to enhancers activation cues. However, it has not been systematically assessed how individual core promoter elements affect the induction of transcriptional bursting by enhancers. Here, we provide evidence that each core promoter element dynamically alters "enhancer responsiveness" by changing functional parameters of transcriptional bursting in developing Drosophila embryos. Quantitative live imaging analysis revealed that the timing and the continuity of burst induction are common regulatory steps on which core promoter elements impact. We also show that core promoters use the upstream TATA box as an amplifier of transcriptional bursting upon interaction with enhancers. Genome editing analysis of the pair-rule gene fushi tarazu further revealed that the endogenous TATA box and the downstream core promoter element are both essential for the correct expression and the function of key developmental genes in early embryos. We suggest that core promoter elements serve as a key regulatory module in converting enhancer activity into transcription dynamics during animal development. Using Hierarchical Similarity To Examine The Genetics of Behcet's disease Behcets disease (BD) is a multisystem inflammatory disease that affects patients along the historic silk road. Thus far, the pathogenesis of the disease has proved elusive due to the complex genetic interactions and unknown environmental or viral triggering factors of the disease. In this paper, we seek to clarify the genetic factors of the disease while also uncovering other diseases of interest that present with a similar genotype as BD. To do this, we employ a convergent functional genomics approach by leveraging the hierarchical similarity tool available in Geneweaver. Through our analysis, we were able to ascertain 7 BD consensus genes and 16 autoimmune diseases with genetic overlap with BD. The results of our study will inform further research into the pathogenesis of Behcets Disease. 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. Chemomechanically voxelated niches for programmable histogenesis Stem-cell derived tissue models are commonly cultured under globally-delivered stimuli that trigger histogenesis via self-organizing activity. However, the culture of such tissue models is prone to stochastic behavior, limiting the reproducibility of cellular composition and resulting in non-physiological architectures. To overcome these shortcomings, we developed a method for printing cell niche microenvironments with microstructured cues that mediate local histogenic processes, including mechanosensing and differentiation of selected cell types. Microstructured cues include independently tunable mechano-chemical properties, with conjugated peptides, proteins, and morphogens across a range of Youngs moduli. By rationally designing niches, we mediate the structure of tissues derived from stem-cell-progenitor sources, including a bone-fat assembly from stromal mesenchyme, and embryonic tissues derived from hiPSC. We show that microstructured cues can recapitulate mechano-chemical signals resembling early embryonic histogenesis. This outcome includes a role for niche mechanics in human embryonic organization, where soft niche mechanics bias markers of mesendodermal differentiation and epithelial-to-mesenchymal-transition (EMT), as well as a demonstration of a material-mediated morphogen signaling centers able to induce foci of mesenchymal and EMT differentiation. Thus, microstructured materials can mediate local histogenic processes to enhance the structure and composition of tissue models. Unsupervised clustering analysis of SARS-Cov-2population structure reveals six major subtypes atearly stage across the world Identifying the population structure of the newly emerged coronavirus SARS-CoV-2 has significant potential to inform public health management and diagnosis. As SARS-CoV-2 sequencing data accrued, grouping them into clusters is important for organizing the landscape of the population structure of the virus. Due to the limited prior information on the newly emerged coronavirus, we utilized four different clustering algorithms to group 16,873 SARS-CoV-2 strains, which automatically enables the identification of spatial structure for SARS-CoV-2. A total of six distinct genomic clusters were identified using mutation profiles as input features. Comparison of the clustering results reveals that the four algorithms produced highly consistent results, but the state-of-the-art unsupervised deep learning clustering algorithm performed best and produced the smallest intra-cluster pairwise genetic distances. The varied proportions of the six clusters within different continents revealed specific geographical distributions. In particular, our analysis found that Oceania was the only continent on which the strains were dispersively distributed into six clusters. In summary, this study provides a concrete framework for the use of clustering methods to study the global population structure of SARS-CoV-2. In addition, clustering methods can be used for future studies of variant population structures in specific regions of these fast-growing viruses. Conformational dynamics and allosteric modulation of the SARS-CoV-2 spike Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells through binding to angiotensin-converting enzyme 2 (ACE2). This interaction is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural and dynamic data have shown that S can adopt multiple conformations, which controls the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Forster resonance energy transfer (smFRET) imaging we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and the B.1 variant (D614G). We find that D614G modulates the energetics of the RBD position in a manner similar to ACE2 binding. We also find that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicate antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails. A comparison between time-constrained counts and line transects as methods to estimate butterfly diversity and monitor populations in tropical habitats Long-term species monitoring programmes have revealed catastrophic insect population declines and disruption of biological communities that are contributing to biodiversity loss. Such discoveries have been possible because of standardised methods, such as line transects, of counting butterflies and other insects. However, line transects are not feasible in many tropical and mountainous habitats, so alternative methods must be explored. To tackle this issue, we devised time-constrained (30-min) counts and compared butterfly diversity as estimated through this method with that estimated through line transects in three tropical habitats in India (evergreen forest, dry deciduous forest and an urban woodland). We tested the efficacy of the two methods to sample species richness and abundance, as well as numbers of rare, endemic and specialist butterflies. We observed greater species richness, and more species of habitat specialists and endemics per sample in time-constrained counts in evergreen forest, but not in the other two habitats. Thus, time-constrained counts were more efficient at detecting species in the species-rich evergreen habitat. Apart from this difference, the two sampling methods captured similar levels of species richness and other measures of diversity. Our study thus shows that time-constrained counts is a suitable if not a superior alternative to line transects to conduct butterfly diversity surveys and population monitoring in complex tropical landscapes. Due to methodological flexibility and simplicity, this method may be particularly useful to study the impacts of climate change, habitat fragmentation and land use practices on butterfly conservation in populous and tech-ready tropical countries using citizen science frameworks. Evidence for a hydrogen sulfide-sensing E3 ligase in yeast In yeast, control of sulfur amino acid metabolism relies upon Met4, a transcription factor which activates the expression of a network of enzymes responsible for the biosynthesis of cysteine and methionine. In times of sulfur abundance, the activity of Met4 is repressed via ubiquitination by the SCFMet30 E3 ubiquitin ligase, but the mechanism by which the F-box protein Met30 senses sulfur status to tune its E3 ligase activity remains unresolved. Herein, we show that Met30 responds to flux through the transsulfuration pathway to regulate the MET gene transcriptional program. In particular, Met30 is responsive to the biological gas hydrogen sulfide, which is sufficient to induce ubiquitination of Met4 in vivo. Additionally, we identify important cysteine residues in Met30s WD-40 repeat region that sense the availability of sulfur in the cell. Our findings reveal how SCFMet30 dynamically senses the flow of sulfur metabolites through the transsulfuration pathway to regulate synthesis of these special amino acids. 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. Intestine-enriched apolipoprotein b orthologs are required for stem cell differentiation and regeneration in planarians Lipid metabolism plays an instructive role in regulating stem cell state and differentiation. However, the roles of lipid mobilization and utilization in stem cell-driven regeneration are unclear. Planarian flatworms readily restore missing tissue due to injury-induced activation of pluripotent somatic stem cells called neoblasts. Here, we identify two intestine-enriched orthologs of apolipoprotein b, apob-1 and apob-2, which mediate transport of neutral lipid stores from the intestine to target tissues including neoblasts, and are required for tissue homeostasis and regeneration. Inhibition of apob function by RNAi causes head regression and lysis in uninjured animals, and delays body axis re-establishment and regeneration of multiple organs in amputated fragments. Furthermore, apob RNAi causes expansion of the population of differentiating neoblast progeny and dysregulates expression of genes enriched in differentiating and mature cells in eight major cell type lineages. We conclude that intestine-derived lipids serve as a source of metabolites required for neoblast differentiation. Multi-omics-based label-free metabolic flux inference reveals obesity-associated dysregulatory mechanisms in liver glucose metabolism Glucose homeostasis is maintained by modulation of metabolic flux. Enzymes and metabolites regulate the involved metabolic pathways. Dysregulation of glucose homeostasis is a pathological event in obesity. Analyzing metabolic pathways and the mechanisms contributing to obesity-associated dysregulation in vivo is challenging. Here, we introduce OMELET: Omics-Based Metabolic Flux Estimation without Labeling for Extended Trans-omic Analysis. OMELET uses metabolomic, proteomic, and transcriptomic data to identify changes in metabolic flux, and to quantify contributions of metabolites, enzymes, and transcripts to the changes in metabolic flux. By evaluating the livers of fasting ob/ob mice, we found that increased metabolic flux through gluconeogenesis resulted primarily from increased transcripts, whereas that through the pyruvate cycle resulted from both increased transcripts and changes in substrates of metabolic enzymes. With OMELET, we identified mechanisms underlying the obesity-associated dysregulation of metabolic flux in liver.

HighlightsO_LIWe created OMELET to infer metabolic flux and its regulation from multi-omic data.
C_LIO_LIGluconeogenic and pyruvate cycle fluxes increased in fasting ob/ob mice.
C_LIO_LITranscripts increases mediated the increase in gluconeogenic fluxes in ob/ob mice.
C_LIO_LIIncreases in transcripts and substrates enhanced pyruvate cycle flux in ob/ob mice.
C_LI Integrative transcriptomics reveals ectopic lipid homeostasis mechanisms in non-endocrine cells of the teleost pituitary Directing both organismal homeostasis and physiological adaptation, the pituitary is a key endocrine gland in all vertebrates. It communicates the needs of the organism to different organs by secreting hormones into the bloodstream. Here, we have used the model fish medaka to investigate the developmental dynamics in the pituitary using a comprehensive RNA-seq time series. By linking developmental expression trends to single-cell RNA-seq profiles, we show how the transcriptional activities of cell types change during sexual maturation. One of the most prominent changes is the decline of the non-endocrine folliculo-stellate cell populations, and especially of rare cells expressing genes encoding secreted lipid transport proteins. As these genes are typically associated with the liver, this reveals the existence of unexpected connections between endocrine communication, lipid homeostasis, and sexual maturation. Bacterial glycyl tRNA synthetase offers glimpses of ancestral protein topologies Aminoacyl tRNA synthetases (aaRSs) are among the proposed proteins present in the Last Universal Common Ancestor (LUCA). There are two types of glycyl tRNA synthetases (GlyRSs), from which the archaeal-eukaryal type is the one suggested to be present in LUCA. Here we solved the crystal structure of a complete bacterial glycyl tRNA synthetase (bacGlyRS) and show that indeed, bacGlyRS carries several structural signals that point it at the origin of all aaRSs. Furthermore, if bacGlyRS is ancestral, it should help to build a reliable Tree of Life (ToL). Given the modular nature of protein evolution, we used only two sub-domain segments with duplicated ancestral topologies, no detected orthologs and an assumed limited horizontal gene transfer (HGT). These motifs correspond to the non-specific RNA binding regions of contemporary bacGlyRS, archaeal CCA-adding enzyme (arch-CCAadd), and eukaryotic rRNA processing enzyme (euk-rRNA). The calculated, rooted bacterial ToL agrees with several phyla relationships unaccounted by the available trees. The Functional Nanopore Screen: A Versatile High-throughput Assay to Study and Engineer Protein Nanopores in Escherichia coli Nanopores comprise a versatile class of membrane proteins that carry out a range of key physiological functions and are increasingly developed for different biotechnological applications. Yet, a capacity to study and engineer protein nanopores by combinatorial means has so far been hampered by a lack of suitable assays that combine sufficient experimental resolution with throughput. Addressing this technological gap, the Functional Nanopore (FuN) screen now provides a quantitative and dynamic read-out of nanopore assembly and function in E. coli. The assay is based on genetically-encoded fluorescent protein sensors that resolve the nanopore-dependent influx of Ca2+ across the inner membrane of E. coli. Illustrating its versatile capacity, the FuN screen is first applied to dissect the molecular features that underlie the assembly and stability of nanopores formed by the S2168 holin. In a subsequent step, nanopores are engineered by recombining the transmembrane module of S2168 with different ring-shaped oligomeric protein structures that feature defined hexa-, hepta- and octameric geometries. Library screening highlights substantial plasticity in the ability of the S2168 transmembrane module to oligomerize in alternative geometries while the functional properties of the resultant nanopores can be fine-tuned through the identity of the connecting linkers. Overall, the FuN screen is anticipated to facilitate both fundamental studies and complex nanopore engineering endeavors with many potential applications in biomedicine, biotechnology and synthetic biology. The effect of recent habitat change on genetic diversity at putatively adaptive and neutral loci in Primula veris in semi-natural grasslands Recent habitat change in semi-natural grasslands due to a lack of management has been shown to affect the genetic diversity of grassland plants. However, it is unknown how a change in local environment affects genetic diversity at adaptive loci. We applied RADseq (restriction-site associated DNA sequencing) to extract > 3,000 SNPs across 568 individuals from 32 Estonian populations of Primula veris, a plant species common to semi-natural grasslands. We evaluated the effect of recent grassland overgrowth following management abandonment on the genetic diversity at putatively neutral and adaptive loci, which we distinguished by applying three methods, i.e., linear and categorical environmental association analyses, and an FST outlier test. For validation, we randomised the genotype to sample assignments. Effects of recent habitat change on genetic diversity differed between neutral and adaptive SNP sets. Genetic diversity assessed at putatively neutral loci was similar in open and overgrown habitats but showed a significant difference between these habitat types at putatively adaptive loci: overgrown (i.e. newly established) habitats exhibited higher genetic diversity at putatively adaptive loci than open (i.e. old) habitats, likely due to the exertion of novel selection pressures imposed by new habitat conditions. This increase in genetic diversity at putatively adaptive loci in the new environment points to currently ongoing selection processes where genetic adaptation to the old habitat is potentially lost through altered allele frequencies. Our study suggests that a recent change in local habitat conditions may not be reflected in neutral loci whereas putatively adaptive loci can inform about potential selection processes. Allopurinol Blocks The Formation And Progression Of Aortic Aneurysm In A Mouse Model Of Marfan Syndrome BackgroundThe pathogenesis and progression of aortic aneurysm in Marfan syndrome (MFS) involves dysregulated TGF-{beta} and nitric oxide signaling, altered hemodynamics, and biomechanical forces. Increasing evidence indicates that redox stress participates in MFS aortopathy, though its contribution is not well established. We reported elevated reactive oxygen species (ROS) formation and NADPH oxidase NOX4 upregulation in MFS patients and mice aortic samples. Here we address the contribution of xanthine oxidoreductase (XOR), which is transcribed as dehydrogenase (XDH) and then post-translationally modified to oxidase (XO), catabolizing mainly purines into uric acid and ROS.

Methods and ResultsWe report that in MFS patients, XOR protein levels increased in aortic samples. In MFS mice (Fbn1C1041G/+), XOR mRNA transcripts augmented in the aorta of young but not older MFS mice. Moreover, the enzymatic activity of XO form increased with respect to XDH. The administration of the XOR inhibitor allopurinol (ALO) blocked the progression of aortic root aneurysm in MFS mice. ALO was also protective when administrated before the appearance of aneurysm. MFS-induced elastic fiber fragmentation, fibrotic remodeling, nuclear translocation of pNRF2, and increased 3-nitrotyrosine levels in the aortic tunica media as well as endothelial dysfunction were all prevented by allopurinol. The large overproduction of H2O2 in MFS aorta did not occur in allopurinol-treated mice whose uric acid plasma levels remained unaltered.

ConclusionsThis study strengthens the concept that redox stress is an important determinant of aortic aneurysm formation and progression in MFS and supports a clinical trial for ALO in the pharmacological treatment of MFS aortopathy.

GRAPHICAL ABSTRACT

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/464182v3_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@eab02aorg.highwire.dtl.DTLVardef@1d3b39eorg.highwire.dtl.DTLVardef@1b54d0forg.highwire.dtl.DTLVardef@1d61ad0_HPS_FORMAT_FIGEXP  M_FIG C_FIG Default and Control networks connectivity dynamics track the stream of affect at multiple timescales In everyday life the stream of affect results from the interaction between past experiences, expectations, and the unfolding of events. How the brain represents the relationship between time and affect has been hardly explored, as it requires modeling the complexity of everyday life in the laboratory setting. Movies condense into hours a multitude of emotional responses, synchronized across subjects and characterized by temporal dynamics alike real-world experiences.

Here, we use time-varying intersubject brain synchronization and real-time behavioral reports to test whether connectivity dynamics track changes in affect during movie watching. Results show that polarity and intensity of experiences relate to connectivity of the default mode and control networks and converge in the right temporo-parietal cortex. We validate these results in two experiments including four independent samples, two movies, and alternative analysis workflows. Lastly, we reveal chronotopic connectivity maps within temporo-parietal and prefrontal cortex, where adjacent areas preferentially encode affect at specific timescales. Persistent Immune and Clotting Dysfunction Detected in Saliva and Blood Plasma after COVID-19 A growing number of studies indicate that coronavirus disease 2019 (COVID-19) is associated with inflammatory sequelae, but molecular signatures governing the normal vs. pathologic convalescence process have not been well-delineated. We characterized global immune and proteome responses in matched plasma and saliva samples obtained from COVID-19 patients collected between 4-6 weeks after initial clinical symptoms resolved. Convalescent subjects showed robust IgA and IgG responses and positive antibody correlations between matched saliva and plasma samples. However, global shotgun proteomics revealed persistent inflammatory patterns in convalescent samples including dysfunction of salivary innate immune cells and clotting factors in plasma (e.g., fibrinogen and antithrombin), with positive correlations to acute COVID-19 disease severity. Saliva samples were characterized by higher concentrations of IgA, and proteomics showed altered pathways that correlated positively with IgA levels. Our study positions saliva as a viable fluid to monitor immunity beyond plasma to document COVID-19 immune, inflammatory, and coagulation-related sequelae. First evidence of in vitro cytotoxic effects of marine microlitter on Merluccius merluccius and Mullus barbatus, two Mediterranean commercial fish species Marine litter is composed mainly of plastics and is recognized as a serious threats to marine ecosystems. Ecotoxicological approaches have started elucidating the potential severity of microplastics (MPs) in controlled laboratory studies with pristine materials but no information exist on marine environmental microlitter as a whole. Here, we characterized the litter in the coastal Northern Tyrrhenian sea and in the stomach of two fish species of socio-economic importance, and exposed primary cell cultures of mucosal and lymphoid organs to marine microlitter for evaluating possible cytotoxic effects. An average of 0.30 {+/-} 0.02 microlitter items m-3 was found in water samples. FT-IR analysis revealed that plastic particles, namely HDPE, polyamide and polypropylene were present in 100% and 83.3% of Merluccius merluccius and Mullus barbatus analyzed, which overall ingested 14.67 {+/-} 4.10 and 5.50 {+/-} 1.97 items/individual, respectively. Moreover, microlitter was confirmed as a vector of microorganisms. Lastly, the apical end-point of viability was found to be significantly reduced in splenic cells exposed in vitro to two microlitter conditions. Considering the role of the spleen in the mounting of adaptive immune responses, our results warrant more in-depth investigations for clarifying the actual susceptibility of these two species to anthropogenic microlitter.

HighlightsO_LI0.30 {+/-} 0.02 microlitter items m-3 were found at the surface of coastal Northern Tyrrhenian sea
C_LIO_LI14.67 {+/-} 4.10 and 5.50 {+/-} 1.97 items/individual were retrieved from the stomach of hakes and mullets
C_LIO_LIThe ingested microlitter contained plastic items
C_LIO_LIMicrolitter was validated as a carrier of bacteria, fungi and flagellates
C_LIO_LISplenic cells exposed to two microlitter conditions for 72 hours suffered cytotoxicity
C_LI Migrators Within Migrators: Exploring Transposable Element Landscapes, Activity, and Host Gene Associations in the Monarch Butterfly, Danaus plexippus BackgroundLepidoptera (butterflies and moths) are an important model system in ecology and evolution. A high-quality chromosomal genome assembly is available for the monarch butterfly (Danaus plexippus), but it lacks an in-depth transposable element (TE) annotation, presenting an opportunity to explore monarch TE dynamics and the impact of TEs on shaping the monarch genome.

ResultsWe find 6.21% of the monarch genome is comprised of TEs, a reduction of 6.85% compared to the original TE annotation performed on the draft genome assembly. Monarch TE content is low compared to two closely related species with available genomes, Danaus chrysippus (33.97% TE) and Danaus melanippus (11.87% TE). The biggest TE contributions to genome size in the monarch are LINEs and Penelope-like elements, and three newly identified families, r2-hero_dPle (LINE), penelope-1_dPle (Penelope-like), and hase2-1_dPle (SINE), collectively contribute 34.92% of total TE content. We find evidence of recent TE activity, with two novel Tc1 families rapidly expanding over recent timescales (tc1-1_dPle, tc1- 2_dPle). LINE fragments show signatures of genomic deletions indicating a high rate of TE turnover. We investigate associations between TEs and wing colouration and immune genes and identify a three-fold increase in TE content around immune genes compared to other host genes.

ConclusionsWe provide a detailed TE annotation and analysis for the monarch genome, revealing a considerably smaller TE contribution to genome content compared to two closely related Danaus species with available genome assemblies. We identify highly successful novel DNA TE families rapidly expanding over recent timescales, and ongoing signatures of both TE expansion and removal highlight the dynamic nature of repeat content in the monarch genome. Our findings also suggest that insect immune genes are promising candidates for future interrogation of TE-mediated host adaptation. Synthetic Cooling Agents in US-marketed E-cigarette Refill Liquids and Popular Disposable E-cigarettes: Chemical Analysis and Risk Assessment. BackgroundMenthol, through its cooling and pleasant sensory effects, facilitates smoking and tobacco product initiation, resulting in the high popularity of mint/menthol-flavored E-cigarettes. More recently, E-cigarette vendors started marketing synthetic cooling agents as additives that impart a cooling effect but lack a characteristic minty odor. Knowledge about content of synthetic coolants in US-marketed E-cigarette products and associated health risks is limited.

MethodsE-liquid vendor sites were searched with the terms "koolada", "kool/cool", "ice", or WS-3/WS-23, denoting individual cooling agents, and relevant refill E-liquids were purchased. "Ice" flavor varieties of Puffbar, the most popular disposable E-cigarette brand, were compared with non-"Ice" varieties. E-liquids were characterized, and synthetic coolants quantified using GC/MS. Margin of exposure (MOE), a risk assessment parameter, was calculated to assess the risk associated with synthetic coolant exposure from E-cigarette use.

ResultsWS-3 was detected in 24/25 refill E-liquids analyzed. All Puffbar flavor varieties contained either WS-23 (13/14) or WS-3 (5/14), in both "Ice"- and non-"Ice" flavors. Modeling consumption of WS-3 from vaped E-liquids, resulted in MOEs below the safe margin of 100 for most daily use scenarios. MOEs for WS-23 were <100 for 10/13 Puffbar flavors in all use scenarios. Puffbar power specifications are identical to Juul devices.

ConclusionsSynthetic cooling agents (WS-3/WS-23) were present in US-marketed E-cigarettes, at levels that may result in consumer exposures exceeding safety thresholds set by regulatory agencies. Synthetic coolants are not only found in mint-or menthol-flavored products, but also in fruit- and candy-flavored products, including popular disposable E-cigarette products such as Puffbar.

ImplicationsSynthetic cooling agents are widely used in "kool/cool"- and "ice"-flavored E-liquids and in E-liquids without these labels, both as a potential replacement for menthol or to add cooling  notes to non-menthol flavors. These agents may be used to bypass current and future regulatory limits on menthol content in tobacco products, and not just E-cigarettes. Since synthetic cooling agents are odorless, they may not fall under the category of "characterizing flavor", potentially circumventing regulatory measures based on this concept. Regulators need to consider the additional health risks associated with exposure to synthetic cooling agents.