Wolbachia load variation in Drosophila is more likely caused by drift than by host genetic factors Symbiosis is a continuum of long-term interactions ranging from mutualism to parasitism, according to the balance between costs and benefits for the protagonists. The density of endosymbionts is, in both cases, a key factor that determines both the transmission of symbionts and the host extended phenotype, and is thus tightly regulated within hosts. However, the evolutionary and molecular mechanisms underlying bacterial density regulation are currently poorly understood. In this context, the symbiosis between the fruit fly and its intracellular bacteria Wolbachia (wMelPop strain) is particularly interesting to study. Although vertically transmitted, the symbiont is pathogenic, and a positive correlation between virulence and wMelPop density is observed. In addition, the number of repeats of a bacterial genomic region -Octomom-is positively correlated with Wolbachia density, underlying a potential genetic mechanism that controls bacterial density. Interestingly, the number of repeats varies between host individuals, but most likely also within them. Such genetic heterogeneity within the host could promote conflicts between bacteria themselves and with the host, notably by increasing within-host competition between symbiont genotypes through a process analogous to the tragedy of the commons. To characterize the determinisms at play in the regulation of bacterial density, we first introgressed wMelPop in different genetic backgrounds of D. melanogaster, and found different density levels and Octomom copy numbers in each host lineage. To determine whether such variations reflect a host genetic determinism on density regulation through Octomom copy number selection, we replicated the introgressions and performed reciprocal crosses on the two Drosophila populations with the most extreme density levels. In both experiments, we detected an absence of directionality in the patterns of infection, associated with a strong instability of these patterns across generations. Given that bacterial density was highly correlated with Octomom copy numbers in all experiments, these results rather suggest a strong influence of drift and a random increase in the frequency of certain bacterial variants. We then discuss how drift, both on the symbiont population during transmission and on the host population, could limit the efficiency of selection in such a symbiotic system, and the consequences of drift on the regulation of density and composition of bacterial populations.
PRDM9 losses in vertebrates are coupled to those of paralogs ZCWPW1 and ZCWPW2 In most mammals and likely throughout vertebrates, the gene PRDM9 specifies the locations of meiotic double strand breaks; in mice and humans at least, it also aids in their repair. For both roles, many of the molecular partners remain unknown. Here, we take a phylogenetic approach to identify genes that may be interacting with PRDM9, by leveraging the fact that PRDM9 arose before the origin of vertebrates, but was lost many times, either partially or entirely--and with it, its role in recombination. As a first step, we characterize PRDM9 domain composition across 446 vertebrate species, inferring at least thirteen independent losses. We then use the interdigitation of PRDM9 orthologs across vertebrates to test whether it co-evolved with any of 241 candidate genes co-expressed with PRDM9 in mice or associated with recombination phenotypes in mammals. Accounting for the phylogenetic relationship among species, we find two genes whose presence and absence is unexpectedly coincident with that of PRDM9: ZCWPW1, which was recently shown to facilitate double strand break repair, and its paralog ZCWPW2, as well as more tentative evidence for TEX15 and FBXO47. ZCWPW2 is expected to be recruited to sites of PRDM9 binding; its tight coevolution with PRDM9 across vertebrates suggests that it is a key interactor, with a role either in recruiting the recombination machinery or in double strand break repair.

Author SummaryOur understanding of meiotic recombination in mammals has seen great progress over the past 15 years, spurred in part by the convergence of lines of evidence from molecular biology, statistical genetics and evolutionary biology. We now know that in most mammals and likely in many vertebrates, the gene PRDM9 specifies the location of meiotic double strand breaks and that in mice and humans at least, it also aids in their repair. For both roles, however, many of the molecular partners remain unknown. To search for these, we take a phylogenetic approach, leveraging the fact that the complete PRDM9 has been lost at least thirteen times in vertebrates and thus that its presence is interdigitated across species. By this approach, we identify two genes whose presence or absence across vertebrates is coupled to the presence or absence of PRDM9, ZCWPW1 and ZCWPW2, as well as two genes for which the evidence is weaker, TEX15 and FBXO47. ZCWPW1 was recently shown to be recruited to sites of PRDM9 binding and to aid in the repair of double strand breaks. ZCWPW2 is likely recruited to sites of PRDM9 binding as well; its tight coevolution with PRDM9 across vertebrates suggests that it plays an important role either in double strand break formation, potentially as the missing link that recruits the recombination machinery to sites of PRDM9 binding, or in double strand break repair.
Social behavioral profiling by unsupervised deep learning reveals a stimulative effect of dopamine D3 agonists on zebrafish sociality It has been a major challenge to systematically evaluate and compare how pharmacological perturbations influence social behavioral outcomes. Although some pharmacological agents are known to alter social behavior, precise description and quantification of such effects have proven difficult. The complexity of brain functions regulating sociality makes it challenging to predict drug effects on social behavior without testing in live animals, and most existing behavioral assays are low-throughput and provide only unidimensional readouts of social function. To achieve richer characterization of drug effects on sociality, we developed a scalable social behavioral assay for zebrafish named ZeChat based on unsupervised deep learning. High-dimensional and dynamic social behavioral phenotypes are automatically classified using this method. By screening a neuroactive compound library, we found that different classes of chemicals evoke distinct patterns of social behavioral fingerprints. By examining these patterns, we discovered that dopamine D3 agonists possess a social stimulative effect on zebrafish. The D3 agonists pramipexole, piribedil, and 7-hydroxy-DPAT-HBr rescued social deficits in a valproic acid-induced zebrafish autism model. The ZeChat platform provides a promising approach for dissecting the pharmacology of social behavior and discovering novel social-modulatory compounds. Human 14-3-3 proteins site-selectively bind the mutational hotspot region of SARS-CoV-2 nucleoprotein modulating its phosphoregulation Phosphorylated within its Ser/Arg-rich region, the SARS-CoV-2 nucleoprotein (N) recruits the phosphopeptide-binding human 14-3-3 proteins that play a well-recognized role in replication of many viruses. Here we use genetic code expansion to demonstrate that phosphorylation of SARS-CoV-2 N at either of two pseudo-repeats centered at Ser197 and Thr205 is sufficient for 14-3-3 binding. According to fluorescence anisotropy, the pT205-motif, present in SARS-CoV-2 but not in SARS-CoV, is preferred over the pS197-motif by all seven human 14-3-3 isoforms, which display unforeseen pT205/pS197 binding selectivity hierarchy. Crystal structures demonstrate that pS197 and pT205 are mutually exclusive 14-3-3-binding sites, whereas SAXS and biochemical data indicate 14-3-3 binding occludes the Ser/Arg-rich region, inhibiting its dephosphorylation. This Ser/Arg-rich region of N is highly prone to mutations, as exemplified by the Omicron and Delta variants, with our data suggesting how the strength of its 14-3-3 binding can be linked with the replicative fitness of the virus. 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. The Inflammatory Role of Milk Fat Globule Epidermal Growth Factor VIII in Angiotensin II Induced Arterial Remodeling BackgroundAngiotensin II (Ang II) and milk fat globule-epidermal growth factor VIII (MFG-E8) are involved in age-associated arterial remodeling; however, the inflammatory role of MFG-E8 in Ang II associated arterial remodeling with aging remains unknown.

Methods and ResultsIn this study, 30-week-old MFG-E8 knock out (KO) and age-matched wild-type (WT) mice were infused with Ang II or saline. After infusion the with Ang II, the aortic molecular, cellular, and structural remodeling were observed in mice and compared to those infused with saline, but these effects were dependent on the expression of MFG-E8: (1) In the WT mice, Ang II infusion substantially increased intimal-medial thickness, elastic lamina degradation, collagen deposition, and the proliferation of VSMCs; in contrast, in the KO mice, these effects were significantly reduced; (2) In the WT mice, Ang II treatment significantly increased the activation and expression of MMP2, TGF-{beta}1, and its downstream signaling molecule p-SMAD2, and collagen type I production, however, in the KO mice, these molecular effects were significantly reduced; (3) In the WT mice, Ang II treatment increased inflammatory p-NF-{kappa}B p65, MCP1, TNF-, ICAM1, and VCAM1 molecular expression, while conversely, in the KO mice, no significant inflammatory changes were found; (4) Importantly, compared to untreated control mice with a wide range of age from 4-96 weeks, Ang II infused "younger" mice produced an "older" arterial inflammatory phenotype, which was alleviated by MFG-E8 deficiency.

ConclusionsMFG-E8 mediates Ang II associated arterial inflammatory remodeling. Targeting MFG-E8 is a novel molecular approach to curb adverse arterial remodeling during aging and hypertension.

CLINICAL PERSPECTIVEO_ST_ABSWhat Is New?C_ST_ABS* Both Ang II and MFG-E8 increases are involved in proinflammatory arterial remodeling mediating the molecular, cellular and tissue events in aging and hypertension.
* MFG-E8 is essential for Ang II induced and age-associated adverse arterial remodeling via the increase of proinflammation, intimal medial thickening, elastin fragmentation, collagen deposition, and VSMC proliferation.


What Are the Clinical Implications?Since MFG-E8 mediates Ang II induced proinflammation in arterial wall remodeling in aging and hypertension, targeting MFG-E8 is a potential molecular approach to curb inflammatory arterial remodeling, maintaining the health of the vascular system during aging and hypertension. Pathways of savannization in a mesic African savanna-forest mosaic O_LIFires in savannas limit tree cover, thereby promoting flammable grass accumulation and fuelling further frequent fires. Meanwhile, forests and thickets form dense canopies that reduce C4-grass fuel loads and creating a humid microclimate, thereby excluding fires under typical climatic conditions.
C_LIO_LIHowever, extreme fires occasionally burn into these closed-canopy systems. Although these rare fires cause substantial tree mortality and can make repeat fires more likely, the long-term consequences of an extreme fire for closed canopy vegetation structure and potential to convert to savanna (hereafter "savannization") remain largely unknown.
C_LIO_LIHere, we analysed whether an extreme fire could, alone, alter species composition, vegetation structure, and fire regimes of closed-canopy ecosystems in an intact savanna-forest-thicket mosaic, or whether successive fires after an initial extreme fire were necessary to trigger a biome transition between from forest to savanna.
C_LIO_LIWe found that forests that only burned once recovered, whereas those that burned again following an initial extreme fire transitioned from closed-canopy forests towards open, grassy savannas.
C_LIO_LIWhile thickets had less tree mortality in fires than forests, repeat fires nonetheless precipitated a transition towards savannas.
C_LIO_LIColonization of the savanna tree community lagged behind the grass community, but also began to transition.
C_LI

SynthesisOur results suggest that rare extreme fires, followed by repeated burning can indeed result in savannization in places where savanna and forest represent alternative stable states. N-glycan chitobiose core biosynthesis by Agl24 strengthens the hypothesis of an archaeal origin of the eukaryal N-glycosylation Protein N-glycosylation is the most common posttranslational modifications found in all three domains of life. The crenarchaeal N-glycosylation begins with the synthesis of a lipid-linked chitobiose core structure, identical to that in eukaryotes. Here, we report the identification of a thermostable archaeal beta-1,4-N-acetylglucosaminyltransferase, named archaeal glycosylation enzyme 24 (Agl24), responsible for the synthesis of the N-glycan chitobiose core. Biochemical characterization confirmed the function as an inverting {beta}-D-GlcNAc-(1[-&gt;]4)--D-GlcNAc-diphosphodolichol glycosyltransferase. Substitution of a conserved histidine residue, found also in the eukaryotic and bacterial homologs, demonstrated its functional importance for Agl24. Furthermore, bioinformatics and structural modeling revealed strong similarities between Agl24 and both the eukaryotic Alg14/13 and a distant relation to the bacterial MurG, which catalyze the identical or a similar process, respectively. Our data, complemented by phylogenetic analysis of Alg13 and Alg14, revealed similar sequences in Asgardarchaeota, further supporting the hypothesis that the Alg13/14 homologs in eukaryotes have been acquired during eukaryogenesis.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/427365v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@13d1ffdorg.highwire.dtl.DTLVardef@1ff48d0org.highwire.dtl.DTLVardef@1056e75org.highwire.dtl.DTLVardef@196ff58_HPS_FORMAT_FIGEXP  M_FIG C_FIG HighlightsO_LIFirst identification and characterization of a thermostable {beta}-D-GlcNAc-(1[-&gt;]4)--D-GlcNAc-diphosphodolichol glycosyltransferase (GT family 28) in Archaea.
C_LIO_LIA highly conserved histidine, within a GGH motif in Agl24, Alg14, and MurG, is essential for function of Agl24.
C_LIO_LIAgl24-like homologs are broadly distributed among Archaea.
C_LIO_LIThe eukaryotic Alg13 and Alg14 are closely related to the Asgard homologs, suggesting their acquisition during eukaryogenesis.
C_LI Preprints in motion: tracking changes between preprint posting and journal publication during a pandemic Amidst the COVID-19 pandemic, preprints in the biomedical sciences are being posted and accessed at unprecedented rates, drawing widespread attention from the general public, press and policymakers for the first time. This phenomenon has sharpened longstanding questions about the reliability of information shared prior to journal peer review. Does the information shared in preprints typically withstand the scrutiny of peer review, or are conclusions likely to change in the version of record? We assessed preprints from bioRxiv and medRxiv that had been posted and subsequently published in a journal through 30th April 2020, representing the initial phase of the pandemic response. We utilised a combination of automatic and manual annotations to quantify how an article changed between the preprinted and published version. We found that the total number of figure panels and tables changed little between preprint and published articles. Moreover, the conclusions of 7.2% of non-COVID-19-related and 17.2% of COVID-19-related abstracts undergo a discrete change by the time of publication, but the majority of these changes do not qualitatively change the conclusions of the paper. Identifying and correcting for misspecifications in GWAS summary statistics and polygenic scores Publicly available genome-wide association studies (GWAS) summary statistics exhibit uneven quality, which can impact the validity of follow-up analyses. First, we present an overview of possible misspecifications that come with GWAS summary statistics. Then, in both simulations and real data analyses, we show that additional information such as imputation INFO scores, allele frequencies, and per-variant sample sizes in GWAS summary statistics can be used to detect possible issues and correct for misspecifications in the GWAS summary statistics. One important motivation for us is to improve the predictive performance of polygenic scores built from these summary statistics. Unfortunately, due to the lack of reporting standards for GWAS summary statistics, this additional information is not systematically reported. We also show that using well-matched LD references can improve model fit and translate into more accurate prediction. Finally, we discuss how to make polygenic score methods such as lassosum and LDpred2 more robust to these misspecifications to improve their predictive power. Robust and Remote Measurement of Heart Rate Based on A Surveillance Camera Arrhythmia is a marked symptom of many cardiovascular diseases (CVDs). The accurate and timely detection of heart rate can greatly reduce the harm of arrhythmia to people. However, it is still a challenge to robustly and remotely measure heart rate in daily life due to the changing environmental conditions during measurement, such as the varying light intensity, the movement of people, and the uncertain distance between the sensor to people. In this study, we propose a method to accurately measure human heart rate within a distance of 4.5 meters under different light intensities by simply using a surveillance camera. After a 20-second color video of a persons hand is captured by the camera, a method based on the Fast Fourier Transform (FFT) algorithm is designed to extract the blood volume pulse wave to calculate the heart rate. According to the comparison between the real heart rate and results measured by electrocardiography (ECG), the proposed method achieves an accuracy of 95.8% when the measurement is performed within a distance of 4.5 meters and 90% when within 5.0 meters. Our experiments show that when the illuminance varies between 100-1000 lux (lighting level indoor), we still get the correct results. Our experiments also demonstrate that the proposed method accurately obtain heart rate even when the light intensity is below 32 lux (300-500 lux in a workplace environment). The methods strong adaptability to changing environmental conditions makes it applicable to many scenarios, such as homes of the elderly, classrooms, and other public spaces. PIN-FORMED1 polarity in the shoot is insensitive to the polarity of neighbouring cells In plants, epidermal cells are planar-polarized along an axis marked by the asymmetric localization patterns of several proteins including PIN-FORMED1 (PIN1), which facilitates the directional efflux of the plant hormone auxin to pattern phyllotaxis (Heisler et al., 2010; Mansfield et al., 2018; Reinhardt et al., 2003). While PIN1 polarity is known to be regulated non-cell autonomously via the MONOPTEROS (MP) transcription factor, how this occurs has not been determined (Bhatia et al., 2016). Here we use mosaic expression of the serine threonine kinase PINOID (PID) to test whether PIN1 polarizes according to the polarity of neighbouring cells. Our findings reveal that PIN1 is insensitive to the polarity of PIN1 in neighbouring cells arguing against auxin flux or extracellular auxin concentrations acting as a polarity cue, in contrast to previous model proposals (Abley et al., 2016; Stoma et al., 2008). High temporal resolution systems profiling reveals distinct patterns of interferon response after Covid-19 mRNA vaccination and SARS-CoV2 infection Knowledge of the mechanisms underpinning the development of protective immunity conferred by mRNA vaccines is fragmentary. Here we investigated responses to COVID-19 mRNA vaccination via ultra-low-volume sampling and high-temporal-resolution transcriptome profiling (23 subjects across 22 timepoints, and with 117 COVID-19 patients used as comparators). There were marked differences in the timing and amplitude of the responses to the priming and booster doses. Notably, we identified two distinct interferon signatures. The first signature (A28/S1) was robustly induced both post-prime and post-boost and in both cases correlated with the subsequent development of antibody responses. In contrast, the second interferon signature (A28/S2) was robustly induced only post-boost, where it coincided with a transient inflammation peak. In COVID19 patients, a distinct phenotype dominated by A28/S2 was associated with longer duration of intensive care. In summary, high-temporal-resolution transcriptomic permitted the identification of post- vaccination phenotypes that are determinants of the course of COVID-19 disease. Synthetic Nanobody-Functionalized Nanoparticles for Accelerated Development of Rapid, Accessible Detection of Viral Antigens Successful control of emerging infectious diseases requires accelerated development of fast, affordable, and accessible assays to be widely implemented at a high frequency. Here we present a generalizable assay platform, nanobody-functionalized nanoparticles for rapid, electronic detection (Nano2RED), demonstrated in the detection of Ebola and COVID-19 antigens. To efficiently generate high-quality affinity reagents, synthetic nanobody co-binders and mono-binders with high affinity, specificity, and stability were selected by phage display screening of a vastly diverse, rationally randomized combinatorial library, bacterially expressed and site-specifically conjugated to gold nanoparticles (AuNPs) as multivalent in-solution sensors. Without requiring fluorescent labelling, washing, or enzymatic amplification, these AuNPs reliably transduce antigen binding signals upon mixing into physical AuNP aggregation and sedimentation processes, displaying antigen-dependent optical extinction readily detectable by spectrometry or simple electronic circuitry. With nanobodies against an Ebola virus secreted glycoprotein (sGP) and a SARS-CoV-2 spike protein receptor binding domain (RBD) as targets, Nano2RED showed a high sensitivity (limit of detection of [~]10 pg/mL for sGP and [~]40 pg/mL for RBD in diluted human serum), a high specificity, and a large dynamic range ([~]7 logs). Unlike conventional assays where slow mass transport for surface binding limits the assay time, Nano2RED features fast antigen diffusion at micrometer scale, and can be accelerated to deliver results within a few minutes. The rapid detection, low material cost (estimated < $0.01 per test), inexpensive and portable readout system (< $5 and < 100 cm3), and digital data output, make Nano2RED particularly suitable for screening of patient samples with simplified operation and accelerated data transmission. Our method is widely applicable for prototyping diagnostic assays for other antigens from new emerging viruses. Modeling microRNA-driven post-transcriptional regulation by using exon-intron split analysis (EISA) in pigs The contribution of microRNAs (miRNAs) to mRNA regulation has often been explored by post hoc selection of downregulated genes and determining whether they harbor binding sites for miRNAs of interest. This approach, however, does not discriminate whether these mRNAs are also downregulated at the transcriptional level. Here, we have characterized the transcriptional and post-transcriptional changes of mRNA expression in two porcine tissues: gluteus medius muscle of fasted and fed Duroc gilts and adipose tissue of lean and obese Duroc-Gottingen minipigs. Exon-intron split analysis (EISA) of RNA-seq data allowed us to identify downregulated mRNAs with high post-transcriptional signals in fed or obese states, and we assessed whether they harbor binding sites for upregulated miRNAs in any of these two physiological states. We found 26 downregulated mRNAs with high post-transcriptional signals in the muscle of fed gilts and 21 of these were predicted targets of upregulated miRNAs also in the fed state. For adipose tissue, 44 downregulated mRNAs in obese minipigs displayed high post-transcriptional signals, and 25 of these were predicted targets of miRNAs upregulated in the obese state. These results suggest that the contribution of miRNAs to mRNA repression is more prominent in the skeletal muscle system. Finally, we identified several genes that may play relevant roles in the energy homeostasis of the pig skeletal muscle (DKK2 and PDK4) and adipose (SESN3 and ESRRG) tissues. By differentiating transcriptional from post-transcriptional changes in mRNA expression, EISA provides a valuable view about the regulation of gene expression, complementary to canonical differential expression analyses. Frequency modulated timer regulates mammalian hibernation Mammalian hibernators decrease basal metabolism and body temperature (Tb) to minimize energy expenditure in harsh seasons. During hibernation, Tb drops to low temperature (<10 {degrees}C) and remains constant for days, known as deep torpor in small mammalian hibernators. Spontaneous interbout arousals interrupt torpor bouts, when Tb recovers to euthermic state ~37 {degrees}C. Torpor-interbout arousal event repeats during several months of hibernation. However, little is known about mechanisms governing Tb fluctuation across torpor-interbout arousal cycles during hibernation. Recent improvement in data-logging techniques enables us to monitor Tb for more than hundred days with high precision, opening up new avenues for quantitative analysis to address the principle governing Tb fluctuation. Here, we analyzed Tb fluctuation across torpor-interbout arousal cycle of Syrian hamster, which can hibernate in response to chronic cold and short photoperiod under a laboratory condition, using generalized harmonic analysis and discovered a model with frequency modulation quantitatively reproducing Tb fluctuation. This analysis also identified an unexpectedly longer period of 120-430 days as the period that modulates another period of several days, generating Tb fluctuation for Syrian hamster. We propose that concerted action of two endogenous periods governs torpor-interbout arousal cycles during hibernation. Bulk-assembly of monodisperse coacervates and giant unilamellar vesicles with programmable hierarchical complexity Compartmentalization is an essential step for the emergence of protocellular life on the early earth and the endeavor to bottom-up construct synthetic cells in the lab. Among manifold natural strategies for compartmentalization, assembly of lipid vesicles or coacervate droplets with resemblance to cell envelope or cytoplasm have emerged as two dominant paradigms. However, the spontaneous assembly of lipid vesicles or coacervates generally results in polydisperse microcompartments and lacks the ability to efficiently integrate functional building blocks towards defined complexity, which give rise to the difficulty to achieve the order and complexity towards the scope of that of life via spontaneous assembly. Herein, we show that the interplay of coacervates and colloidal particles provides a bulk-assembly approach to form monodisperse coacervates and giant unilamellar vesicles (GUVs) with programmable hierarchical complexity. The hierarchical complexity of the synthetic cell microcompartments can be delicately engineered from the control of the spatial or temporal organization of components in entity unit to manifold forms of interconnected synthetic cell consortia, achieving a compartment hierarchy analogous to the organelle-cell-tissue structure. The increase in complexity gives rise to emergent properties, for instance, collective morphology evolution of coacervate assemblages, gated permeability of GUVs without sophisticated protein machinery, and remarkable structural and functional stability of GUVs at extreme conditions. This work paves an unprecedented step to form monodisperse, hierarchical coacervates and GUVs via bulk-assembly, which provides a prebiotically plausible approach for the proper integration of inanimate matter towards the order and complexity of life and monodisperse, hierarchical microdroplets for follow-up scientific and technological applications. Localization of signaling receptors maximizes cellular informationacquisition in spatially-structured natural environments Cells in natural environments like tissue or soil sense and respond to extracellular ligands with intricately structured and non-monotonic spatial distributions, sculpted by processes such as fluid flow and substrate adhesion. In this work, we show that spatial sensing and navigation can be optimized by adapting the spatial organization of signaling pathways to the spatial structure of the environment. We develop an information-theoretic framework for computing the optimal spatial organization of a sensing system for a given signaling environment. We find that receptor localization maximizes information acquisition in simulated natural contexts, including tissue and soil. Receptor localization extends naturally to produce a dynamic protocol for continuously redistributing signaling receptors, which when implemented using simple feedback, boosts cell navigation efficiency by 30-fold. Broadly, our work shows how cells can maximize the fidelity of information transfer by adapting the spatial organization of signaling molecules to the spatial structure of the environment. Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging Biocompatible fluorescent reporters with spectral properties spanning the entire visible spectrum are indispensable tools for imaging the biochemistry of living cells and organisms in real time. Here, we present the engineering of a fluorescent chemogenetic reporter with tunable optical and spectral properties. A collection of live-cell compatible fluorogenic chromophores with various electronic properties enables to generate bimolecular fluorescent assemblies that cover the visible spectrum from blue to red using a single protein tag engineered and optimized by directed evolution and rational design. We showed that the ability to tune the fluorescence color and properties through simple molecular modulation provides an unprecedent experimental versatility for imaging proteins in live cells, including delicate cultured hippocampal neurons, and in multicellular organisms. The ability to tune the spectral properties and fluorescence performance enables to match the spectral specifications and requirements of the most advanced imaging techniques, and allowed us to achieve efficient stimulated emission depletion (STED) nanoscopy of fusion proteins in live cells and live primary cultured neurons. Antennapedia and optix regulate metallic silver wing scale development and cell shape in Bicyclus anynana butterflies Butterfly wing scale cells can develop very intricate cuticular nanostructures that interact with light to produce structural colors including silvery hues, but the genetic basis of such nanostructures is mostly unexplored. Here, we first identified that optix is necessary for metallic scale development in the butterfly Bicyclus anynana. We then characterized different subtypes of wildtype metallic silver scales and addressed the function of five genes - apterous A, Ultrabithorax, doublesex, Antennapedia, and optix - in the differentiation of silver scales, at a single-cell resolution, by leveraging crispants that exhibited either ectopic gains or losses of silver scales. Wildtype silver scales were generally rounded and had low amounts of pigmentation, exhibiting a common ultrastructural modification for metallic broadband reflectance, i.e., an undulatory air layer enclosed by an upper and lower lamina. Our results indicated that the varying air layer thickness was the important parameter of the bilaminate scale for producing a broadband reflectance across visible wavelengths. While a single lamina of the appropriate thickness could also produce broadband colors, the bilaminate structure is advantageous as it increases the overall reflectivity. Crispant brown scales differed from wildtype silver scales via the loss of the continuous upper lamina, increased lower lamina thickness, and increased pigmentation. The reverse was seen when brown scales became silver. We identified Antennapedia and optix as high-level regulators in the network differentiating different silver scale subtypes and determining overall cell shape in both sexes. In addition, Antp exhibits a novel, post-embryonic role in the determination of ridge and crossrib orientation. Nucleus size and its effect on the chromatin structure in living cells. DNA-architectural proteins play a major role in organization of chromosomal DNA in living cells by packaging it into chromatin, whose spatial conformation is determined by an intricate interplay between the DNA-binding properties of architectural proteins and physical constraints applied to the DNA by a tight nuclear space. Yet, the exact effects of the cell nucleus size on DNA-protein interactions and chromatin structure currently remain obscure. Furthermore, there is even no clear understanding of molecular mechanisms responsible for the nucleus size regulation in living cells. To find answers to these questions, we developed a general theoretical framework based on a combination of polymer field theory and transfer-matrix calculations, which showed that the nucleus size is mainly determined by the difference between the surface tensions of the nuclear envelope and the endoplasmic reticulum membrane as well as the osmotic pressure exerted by cytosolic macromolecules on the nucleus. In addition, the model demonstrated that the cell nucleus functions as a piezoelectric element, changing its electrostatic potential in a size-dependent manner. This effect has been found to have a profound impact on stability of nucleosomes, revealing a previously unknown link between the nucleus size and chromatin structure. Overall, our study provides new insights into the molecular mechanisms responsible for regulation of the cell nucleus size, as well as the potential role of nuclear organization in shaping the cell response to environmental cues.

SIGNIFICANCE STATEMENTThe cell nucleus plays a central role in the life of eukaryotic cells, providing the highest level of control of intracellular processes. Depending on the stage of the cell cycle and / or surrounding environment, the size of the cell nucleus may undergo changes that are believed to cause chromatin reorganization, affecting gene transcription. At present, however, there is no clear understanding of the molecular mechanisms that may be responsible for such regulation, whose exact effect on chromatin structure remains unclear. In this study, by developing an advanced computational approach, we explore these issues from a physical perspective, revealing previously unknown mechanisms contributing to organization of the cell nucleus and chromatin. Assessing sequence-based protein-protein interaction predictors for use in therapeutic peptide engineering Engineering peptides to achieve a desired therapeutic effect through the inhibition of a specific target activity or protein interaction is a non-trivial task. Few of the existing in silico peptide design algorithms generate target-specific peptides. Instead, many methods produce peptides that achieve a desired effect through an unknown mechanism. In contrast with resource-intensive high-throughput experiments, in silico screening is a cost-effective alternative that can prune the space of candidates when engineering target-specific peptides. Using a set of FDA-approved peptides we curated specifically for this task, we assess the applicability of several sequence-based protein-protein interaction predictors as a screening tool within the context of peptide therapeutic engineering. We show that similarity-based protein-protein interaction predictors are more suitable for this purpose than the state-of-the-art deep learning methods publicly available at the time of writing. We also show that this approach is mostly useful when designing new peptides against targets for which naturally-occurring interactors are already known, and that deploying it for de novo peptide engineering tasks may require gathering additional target-specific training data. Taken together, this work offers evidence that supports the use of similarity-based protein-protein interaction predictors for peptide therapeutic engineering, especially peptide analogs. 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. Integrative analysis reveals aged clonal B cells, microenvironment and c-Myc activation in the origin of age-related lymphoma While cancer is an age-related disease, most studies focus on genetically engineered younger mouse models. Here, we uncover how cancer develops as a consequence of the naturally aged immune system in mice. B-cell lymphoma frequently occurs in aged mice and is associated with increased cell size, splenomegaly, and a novel clonal B-cell population. Age-emergent B cells clonally expand outside of germinal centers driven by somatic mutations, activated c-Myc and hypermethylated promoters, and both genetically and epigenetically recapitulate human follicular and diffuse-large B-cell lymphomas. Mechanistically, mouse cancerous B cells originate from age-associated B cells, which are atypical memory B cells. Age-associated B cells secrete a spectrum of proinflammatory cytokines and activate paracrinally the expression of c-Myc in surrounding B cells. Although clonal B cells are a product of an aging microenvironment, they evolve being self-sufficient and support malignancy when transferred into young mice. Inhibition of mTOR and c-Myc attenuates premalignant changes in B cells during aging and emerges as a therapeutic strategy to delay the onset of age-related lymphoma. Together, we uncover how aging generates cancerous B cells, characterize a model that captures the origin of spontaneous cancer during aging and identify interventions that may postpone age-associated lymphoma. Kaposi's sarcoma-associated herpesvirus (KSHV) utilizes the NDP52/CALCOCO2 selective autophagy receptor to disassemble processing bodies Kaposis sarcoma-associated herpesvirus (KSHV) causes the inflammatory and angiogenic endothelial cell neoplasm, Kaposis sarcoma (KS). We previously demonstrated that expression of the KSHV protein, Kaposin B (KapB), promoted inflammation via the disassembly of cytoplasmic ribonucleoprotein granules called processing bodies (PBs). Processing bodies (PBs) modify gene expression by silencing or degrading labile messenger RNAs (mRNAs) including many transcripts that encode inflammatory or angiogenic proteins that are associated with KS disease. Although our work implicated PB disassembly as one of the causes of inflammation during KSHV infection, the precise mechanism used by KapB to elicit PB disassembly was unclear. Here we reveal a new connection between the degradative process of autophagy and PB disassembly. We show that both latent KSHV infection and KapB expression enhanced autophagic flux via the phosphorylation of the autophagy regulatory protein, Beclin 1. KapB was necessary for this effect, as infection with a recombinant virus that does not express the KapB protein did not induce Beclin 1 phosphorylation or autophagic flux. Moreover, we reveal that PB disassembly mediated by KSHV or KapB depended on canonical autophagy genes and the selective autophagy receptor NDP52/CALCOCO2 and that the PB scaffolding protein, Pat1b, co-immunoprecipitated with NDP52. These studies reveal a new role for autophagy and the selective autophagy receptor NDP52 in promoting PB turnover and the concomitant synthesis of inflammatory molecules during KSHV infection.

Author SummaryKaposis sarcoma-associated herpesvirus (KSHV) is the causative agent of the inflammatory, endothelial cell cancer, Kaposis Sarcoma (KS). KSHV induces a pro-tumourigenic inflammatory environment which aids in the establishment and maintenance of the KS lesion. Processing bodies (PBs) are cellular structures that dampen inflammatory gene expression by suppression or decay of their cognate RNA molecules. We previously showed that the viral protein KapB caused PB disappearance during KSHV infection, identifying a new pathway used by KSHV to elicit inflammation. Now, we show that KSHV and KapB hijack the cellular degradative process of autophagy to promote PB disassembly and increase inflammatory gene expression. This places autophagy as central to the regulation of inflammation by KSHV and illustrates another remarkable strategy used by these viruses to create a tumourigenic microenvironment. NOMIS: Quantifying morphometric deviations from normality over the lifetime of the adult human brain We present NOMIS (https://github.com/medicslab/NOMIS), a comprehensive open MRI tool to assess morphometric deviation from normality in the adult human brain. Based on MR anatomical images from 6,909 cognitively healthy individuals aged 18-100 years, we modeled 1,344 measures computed using the open access FreeSurfer pipeline, considering account personal characteristics (age, sex, intracranial volume) and image quality (resolution, contrast-to-noise ratio and surface reconstruction defect holes), and providing expected values for any new individual. Then, for each measure, the NOMIS tool was built to generate Z-score effect sizes denoting the extent of deviation from the normative sample. Depending on the user need, NOMIS offers four versions of Z-score adjusted on different sets of variables. While all versions consider head size and image quality, they can also incorporate age and/or sex, thereby facilitating multi-site neuromorphometric research across adulthood.