Collective polymerase dynamics emerge from DNA supercoiling during transcription All biological processes ultimately come from physical interactions. The mechanical properties of DNA play a critical role in transcription. RNA polymerase can over or under twist DNA (referred to as DNA supercoiling) when it moves along a gene resulting in mechanical stresses in DNA that impact its own motion and that of other polymerases. For example, when enough supercoiling accumulates, an isolated polymerase halts and transcription stops. DNA supercoiling can also mediate non-local interactions between polymerases that shape gene expression fluctuations. Here, we construct a comprehensive model of transcription that captures how RNA polymerase motion changes the degree of DNA supercoiling which in turn feeds back into the rate at which polymerases are recruited and move along the DNA. Surprisingly, our model predicts that a group of three or more polymerases move together at a constant velocity and sustain their motion (forming what we call a polymeton) whereas one or two polymerases would have halted. We further show that accounting for the impact of DNA supercoiling on both RNA polymerase recruitment and velocity recapitulates empirical observations of gene expression fluctuations. Finally, we propose a mechanical toggle switch whereby interactions between genes are mediated by DNA twisting as opposed to proteins. Understanding the mechanical regulation of gene expression provides new insights into how endogenous genes can interact and informs the design of new forms of engineered interactions.

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Gradient-mixing LEGO robots for purifying DNA origami nanostructures of multiple components by rate-zonal centrifugation DNA origami purification is critical in emerging applications of functionalized DNA nanostructures from basic fundamental biophysics, nanorobots to therapeutics. Advances in DNA origami purification have led to the establishment of rate-zonal centrifugation (RZC) as a scalable, high-yield, and contamination-free approach to purifying DNA origami nanostructures. In RZC purification, a linear density gradient is created using viscous agents, such as glycerol and sucrose, to separate molecules based on their mass and shape during high-rpm centrifugation. However, current methods for creating density gradients are typically time-consuming because of their reliance on slow passive diffusion. Here, we built a LEGO gradient mixer to rapidly create a quasi-continuous density gradient with minimal layering of concentrations using simple rotational motion. We found that rotating two layers of different concentrations at an angle can reduce the diffusion time from a few hours to mere minutes. The instrument needed to perform the movement can be constructed from low-cost components, such as Arduino and LEGO Mindstorms pieces, and has comparable efficacy to commercial gradient mixers currently available. Our results demonstrate that the creation of a linear density gradient can be achieved with minimal labor, time, and cost with this machine. With the recent advances in DNA origami production, we anticipate our findings to further improve the viability of scaling up DNA origami purification in grams quantities. Our simple process enables automated large-scale purification of functionalized DNA origami more feasible in resource-constrained settings.
S-acylation stabilizes ligand-induced receptor kinase complex formation during plant pattern-triggered immune signalling. Plant receptor kinases are key transducers of physical extracellular stimuli, such as the presence of beneficial or pathogenic microbes or secreted signalling molecules. Receptor kinases are regulated by numerous post-translational modifications. Here, using the bacterial flagellin perceiving receptor kinase FLS2, we show that S-acylation at an evolutionarily conserved cysteine is crucial for function. S-acylation involves the addition of long-chain fatty acids to cysteine residues within proteins, altering their biophysical properties and behaviour within the membrane environment. We observe S-acylation of FLS2 at C-terminal kinase domain cysteine residues within minutes of treatment with flg22 ligand, and in a BAK1 co-receptor dependent manner. We demonstrate that S-acylation is essential for FLS2-mediated immune signalling, including anti-bacterial immunity. Similarly, mutating the corresponding conserved cysteine residue in the immune receptor kinase EFR supressed elf18 mediated signalling. Biochemical analysis of unstimulated and activated FLS2 containing complexes using detergents and native membrane DIBMA nanodiscs indicates that S-acylation assists the stabilization of activated receptor kinase complexes withing the membrane environment to increase signalling efficiency. Increased biting rate and decreased Wolbachia density in irradiated mosquitoes BackgroundReleasing considerable numbers of radiation-sterilized males is a promising strategy to suppress mosquito vectors. However, releases may also include small percentages of biting females which translate to large numbers when releases are large. Currently, the effects of irradiation on the host-seeking and host-biting behaviors have not been exhaustively investigated. Information is also lacking regarding the effects of sterilizing treatment on the endosymbiotic bacterium Wolbachia, which is known to affect the vector competence of infected mosquitos.

MethodsTo ascertain the effects of irradiation on females, the pupae of two Aedes albopictus strains, differing in their natural or artificial Wolbachia infection type, and Ae. aegypti--which is not infected by Wolbachia--were treated with various doses of X-rays and monitored for key fitness parameters and biting behavior over a period of two weeks. The effect of radiation on Wolbachia was investigated by qPCR and FISH analysis.

ResultsPartial Ae. albopictus female sterility was achieved at 28 Gy but the number of weekly bites more than doubled compared to that of the controls. Radiation doses of 35 and 45 Gy completely inhibited progeny production but did not significantly affect the survival or flight ability of Ae. albopictus females and caused a tripling of the number of bites per female per week (compared to untreated controls). These results were also confirmed in Ae. aegypti after treatment at 50 Gy. Wolbachia density decreased significantly in 45-Gray-irradiated females, with the greatest decreases in the early irradiation group (26{+/-}2-hour-old pupae). Wolbachia density also decreased as adults aged. This trend was confirmed in ovaries but not in extra-ovarian tissues. FISH analysis showed a strongly reduced Wolbachia-specific fluorescence in the ovaries of 13{+/-}1-day-old females.

ConclusionsThese results suggest that, under SIT programs, the vector capacity of a target population could increase with the frequency of the irradiated females co-released with the sterile males due to an increased biting rate. In the context of a successful suppression, the related safety issues could be generally negligible, but they should be conservatively evaluated when large scale programs relying on imperfect sexing and high overflooding release ratios are run for long time in areas endemic for arboviral diseases. Also, the effects of irradiation on the vector competence deserve further investigation. Crowdsourced identification of multi-target kinase inhibitors for RET- and TAU- based disease: the Multi-Targeting Drug DREAM Challenge A continuing challenge in modern medicine is the identification of safer and more efficacious drugs. Precision therapeutics, which have one molecular target, have been long promised to be safer and more effective than traditional therapies. This approach has proven to be challenging for multiple reasons including lack of efficacy, rapidly acquired drug resistance, and narrow patient eligibility criteria. An alternative approach is the development of drugs that address the overall disease network by targeting multiple biological targets ( polypharmacology). Rational development of these molecules will require improved methods for predicting single chemical structures that target multiple drug targets. To address this need, we developed the Multi-Targeting Drug DREAM Challenge, in which we challenged participants to predict single chemical entities that target pro-targets but avoid anti-targets for two unrelated diseases: RET-based tumors and a common form of inherited Tauopathy. Here, we report the results of this DREAM Challenge and the development of two neural network-based machine learning approaches that were applied to the challenge of rational polypharmacology. Together, these platforms provide a potentially useful first step towards developing lead therapeutic compounds that address disease complexity through rational polypharmacology.

Author SummaryMany modern drugs are developed with the goal of modulating a single cellular pathway or target. However, many drugs are, in fact,  dirty; they target multiple cellular pathways or targets. This phenomenon is known as multi-targeting or polypharmacology. While some strive to develop  cleaner therapeutics that eliminate secondary targets, recent work has shown that multi-targeting therapeutics have key advantages for a variety of diseases. However, while multi-targeting drugs that affect a precisely-defined profile of targets may be more effective, it is difficult to computationally predict which molecules have desirable target profiles. Here, we report the results of a competitive crowdsourcing project (the Multi-Targeting Drug DREAM Challenge), where we challenged participants to predict chemicals that have desired target profiles for cancer and neurodegenerative disease. Genome editing excisase origins illuminated by somatic genome of Blepharisma Massive DNA excision occurs regularly in ciliates, ubiquitous microbial eukaryotes with somatic and germline nuclei in the same cell. Tens of thousands of internally eliminated sequences (IESs) scattered throughout a copy of the ciliate germline genome are deleted during development of the streamlined somatic genome. Blepharisma represents one of the two earliest diverging ciliate classes, and, unusually, has dual pathways of somatic nuclear development, making it ideal for investigating the functioning and evolution of these processes. Here, we report the somatic genome assembly of Blepharisma stoltei strain ATCC 30299 (41 Mb), arranged as numerous alternative telomere-capped minichromosomes. This genome encodes eight PiggyBac transposase homologs liberated from transposons. All are subject to purifying selection, but just one, the putative IES excisase, has a complete catalytic triad. We propose PiggyBac homologs were ancestral excisases that enabled evolution of extensive, natural genome editing. Sexual selection for males with beneficial mutations Sexual selection is the process by which traits providing a mating advantage are favoured. Theoretical treatments of the evolution of sex by sexual selection propose that it operates by reducing the load of deleterious mutations. Here, we postulate instead that sexual selection primarily acts through females preferentially mating with males carrying beneficial mutations. We used simulation and analytical modelling to investigate the evolutionary dynamics of beneficial mutations in the presence of sexual selection. We found that female choice for males with beneficial mutations had a much greater impact on genetic quality than choice for males with low mutational load. We also relaxed the typical assumption of a fixed mutation rate. For deleterious mutations, mutation rate should always be minimized, but when rare beneficial mutations can occur, female choice for males with those rare beneficial mutations could overcome a decline in average fitness and allow an increase in mutation rate. We propose that sexual selection for beneficial mutations could overcome the  two-fold cost of sex much more readily than choice for males with low mutational load and may therefore be a more powerful explanation for the prevalence of sexual reproduction than the existing theory. If sexual selection results in higher fitness at higher mutation rates and if the variability produced by mutation itself promotes sexual selection, then a feedback loop between these two factors could have had a decisive role in driving adaptation. A map of binary SARS-CoV-2 protein interactions implicates host immune regulation and ubiquitination Key steps in viral propagation, immune suppression, and pathology are mediated by direct, binary, physical interactions between viral and host proteins. To understand the biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we generated an unbiased systematic map of binary interactions between viral and host proteins, complementing previous co-complex association maps by conveying more direct mechanistic understanding and potentially enabling targeted disruption of direct interactions. To this end, we deployed two parallel strategies, identifying 205 virus-host and 27 intraviral binary interactions amongst 171 host and 19 viral proteins, and confirming high quality of these interactions via a calibrated orthogonal assay. Host proteins interacting with SARS-CoV-2 proteins are enriched in various cellular processes, including immune signaling and inflammation, protein ubiquitination, and membrane trafficking. Specific subnetworks provide new hypotheses related to viral modulation of host protein homeostasis and T-cell regulation. The binary virus-host protein interactions we identified can now be prioritized as targets for therapeutic intervention. More generally, we provide a resource of systematic maps describing which SARS-CoV-2 and human proteins interact directly. A novel two-generation approach to the population dynamics of gregarious parasitoids Parasitoids, as important natural enemies, help maintain balance in natural ecosystems. Their population dynamics is generally predicted from the number of individuals. Here, using gregarious parasitoids as models, we show that this traditional approach omits one important parameter: the mothers manipulation of offspring fertility due to the clutch size-body size- fertility correlation. As a result of this correlation, when females deliberately adjust the number of offspring laid in a host, they determine not only the number but also the body sizes and reproductive potentials of those offspring. For the first time in the model species Anaphes flavipes, we determined the parasitoids offspring fertility from clutch size. Using this, we experimentally clarified the advantage of specific clutch size combinations and we show that identical fertility in the F1 generation can lead to distinctly different fertility values in the F2 generation. Even with the same number of hosts, lower fertility in the F1 generation can cause higher fertility in the F2 generation. Based on these results, we propose a novel two-generation approach which includes the clutch size-body size-fertility correlation. Our novel approach provides a new perspective for determining the individual fitness levels of gregarious parasitoids with new options for the modelling of parasitoid population dynamics. Joint representations of color and form in mouse visual cortex described by random pooling from rods and cones Spatial transitions in color can aid any visual perception task, and its neural representation - the "integration of color and form" - is thought to begin at primary visual cortex (V1). An integration of color and form is untested in mouse V1, yet studies show that the ventral retina provides the necessary substrate from green-sensitive rods and UV-sensitive cones. Here, we used two-photon imaging in V1 to measure spatial frequency (SF) tuning along four axes of rod and cone contrast space, including luminance and color. We first reveal that V1s sensitivity to color is similar to luminance, yet average SF tuning is significantly shifted lowpass for color. Next, guided by linear models, we used SF tuning along all four color axes to estimate the proportion of neurons that fall into classic models of color opponency - "single-", "double-", and "non-opponent". Few neurons (~6%) fit criteria for double-opponency, which are uniquely tuned for chromatic borders. Most of the population can be described as a unimodal distribution ranging from strongly single-opponent to non-opponent. Consistent with recent studies of the rodent and primate retina, our V1 data is well-described by a simple model in which ON and OFF channels to V1 sample the photoreceptor mosaic randomly. Snrpb, the CCMS gene, is required in neural crest cells for proper splicing of genes essential for craniofacial morphogenesis Heterozygous mutations in SNRPB, an essential core component of the five small ribonucleoprotein particles of the spliceosome, are responsible for Cerebrocostomandibular Syndrome (CCMS). We show that Snrpb heterozygous embryos arrest shortly after implantation. Additionally, heterozygous deletion of Snrpb in the developing brain and neural crest cells models craniofacial malformations found in CCMS, and results in death shortly after birth. RNAseq analysis of mutant heads prior to morphological defects revealed increased exon-skipping and intron-retention in association with increased 5 splice site strength. We found increased exon-skipping in negative regulators of the P53-pathway, increased levels of nuclear P53, P53-target genes. However, removing TrP53 in Snrpb heterozygous mutant neural crest cells did not completely rescue craniofacial development. We also found a small but significant increase in exon-skipping of several transcripts required for head and midface development, including Smad2 and Rere. Furthermore, mutant embryos exhibited ectopic or missing expression of Fgf8 and Shh, which are required to coordinate face and brain development. Thus, we propose that mis-splicing of transcripts that regulate P53-activity and craniofacial-specific genes both contribute to craniofacial malformations. Reconstitution of minimal motility system based on Spiroplasma swimming by expressing two bacterial actins in synthetic minimal bacterium Motility is one of the most important features of life, but its evolutionary origin remains unknown. In this study, we focused on Spiroplasma, commensal, or parasitic bacteria. They swim by switching the helicity of a ribbon-like cytoskeleton that comprises six proteins, each of which evolved from a nucleosidase and bacterial actin called MreB. We expressed these proteins in a synthetic, non-motile minimal bacterium, JCVI-syn3.0B, whose reduced genome was computer-designed and chemically synthesized. The synthetic bacterium exhibited swimming motility with features characteristic of Spiroplasma swimming. Moreover, some combinations of the two proteins produced a helical cell shape and swimming, suggesting that the swimming originated from the differentiation and coupling of bacterial actins, and we also obtained a minimal system for motility of the synthetic bacterium.

One-Sentence SummaryThe minimal system comprised two bacterial actins that provided cell helicity and swimming to the synthetic minimal bacterium. Computational analysis of B cell receptor repertoires in COVID-19 patients using deep embedded representations of protein sequences Analyzing B cell receptor (BCR) repertoires is immensely useful in evaluating ones immunological status. Conventionally, repertoire analysis methods have focused on comprehensive assessments of clonal compositions, including V(D)J segment usage, nucleotide insertions/deletions, and amino acid distributions. Here, we introduce a novel computational approach that applies deep-learning-based protein embedding techniques to analyze BCR repertoires. By selecting the most frequently occurring BCR sequences in a given repertoire and computing the sum of the vector representations of these sequences, we represent an entire repertoire as a 100-dimensional vector and eventually as a single data point in vector space. We demonstrate that this new approach enables us to not only accurately cluster BCR repertoires of coronavirus disease 2019 (COVID-19) patients and healthy subjects but also efficiently track minute changes in immune status over time as patients undergo treatment. Furthermore, using the distributed representations, we successfully trained an XGBoost classification model that achieved a mean accuracy rate of over 87% given a repertoire of CDR3 sequences. The potential of dynamic physiological traits in young tomato plants to predict field-yield performance To address the challenge of predicting tomato yields in the field, we used whole-plant functional phenotyping to evaluate water relations under well-irrigated and drought conditions. The genotypes tested are known to exhibit variability in their yields in wet and dry fields. The examined lines included two lines with recessive mutations that affect carotenoid biosynthesis, zeta z2083 and tangerine t3406, both isogenic to the processing tomato variety M82. The two mutant lines were reciprocally grafted onto M82, and multiple physiological characteristics were measured continuously, before, during and after drought treatment in the greenhouse. A comparative analysis of greenhouse and field yields showed that the whole-canopy stomatal conductance (gsc) in the morning and cumulative transpiration (CT) were strongly correlated with field measurements of total yield (TY: r2 = 0.9 and 0.77, respectively) and plant vegetative weight (PW: r2 = 0.6 and 0.94, respectively). Furthermore, the minimum CT during drought and the rate of recovery when irrigation was resumed were both found to predict resilience. BRCA2 BRC missense variants disrupt RAD51-dependent DNA repair Pathogenic mutations in the BRCA2 tumor suppressor gene predispose to breast, ovarian, pancreatic, prostate, and other cancers. BRCA2 maintains genome stability through homology-directed repair (HDR) of DNA double-strand breaks (DSBs) and replication fork protection. Nonsense or frameshift mutations leading to truncation of the BRCA2 protein are typically considered pathogenic, however, missense mutations resulting in single amino acid substitutions can be challenging to functionally interpret. The majority of missense mutations in BRCA2 have been classified as Variants of Uncertain Significance (VUS) with unknown functional consequences. In this study, we identified three BRCA2 VUS located within the BRC repeat region to determine their impact on canonical HDR and fork protection functions. We provide evidence that S1221P and T1980I, which map to conserved residues in the BRC2 and BRC7 repeats, compromise the cellular response to chemotherapeutics and ionizing radiation, and display deficits in fork protection. We further demonstrate biochemically that S1221P and T1980I disrupt RAD51 binding and diminish the ability of BRCA2 to stabilize RAD51-ssDNA complexes. The third variant, T1346I, located within the spacer region between BRC2 and BRC3 repeats, is fully functional. We conclude that T1346I is a benign allele whereas S1221P and T1980I are hypomorphic disrupting the ability of BRCA2 to fully engage and stabilize RAD51 nucleoprotein filaments. Our results underscore the importance of correctly classifying BRCA2 VUS as pathogenic variants can impact both future cancer risk and guide therapy selection during cancer treatment.

Graphical Abstract

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org.highwire.dtl.DTLVardef@1e5e3c6org.highwire.dtl.DTLVardef@1cd0e60org.highwire.dtl.DTLVardef@16adf4corg.highwire.dtl.DTLVardef@16a0089_HPS_FORMAT_FIGEXP  M_FIG C_FIG 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. The Rab GTPase activating protein TBC-2 regulates endosomal localization of DAF-16 FOXO and lifespan FOXO transcription factors have been shown to regulate longevity in model organisms and are associated with longevity in humans. To gain insight into how FOXO functions to increase lifespan, we examined the subcellular localization of DAF-16 in C. elegans. We show that DAF-16 is localized to endosomes and that this endosomal localization is increased by the insulin-IGF signaling (IIS) pathway. Endosomal localization of DAF-16 is modulated by endosomal trafficking proteins. Disruption of the Rab GTPase activating protein TBC-2 increases endosomal localization of DAF-16, while inhibition of TBC-2 targets, RAB-5 or RAB-7 GTPases, decreases endosomal localization of DAF-16. Importantly, the amount of DAF-16 that is localized to endosomes has functional consequences as increasing endosomal localization through mutations in tbc-2 reduced the lifespan of long-lived daf-2 IGFR mutants, depleted their fat stores, and DAF-16 target gene expression. Overall, this work identifies endosomal localization as a mechanism regulating DAF-16 FOXO, which is important for its functions in metabolism and aging. 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. Immune complex solubility and size govern individual Fc-gamma receptor responses: Novel biomarkers for autoimmune diseases Fc-gamma receptor (Fc{gamma}R) activation by soluble IgG immune complexes (sICs) represents a major mechanism of inflammation in certain autoimmune diseases such as systemic lupus erythematosus (SLE). A robust and scalable test system allowing for the detection and quantification of sIC bioactivity is missing. Previously described Fc{gamma}R interaction assays are limited to certain Fc{gamma}Rs, lack scalability and flexibility, are not indicative of receptor activation or lack sensitivity towards sIC size. We developed a comprehensive reporter cell panel detecting individual activation of Fc{gamma}Rs from humans and the mouse. The reporter cell lines were integrated into an assay format that provides flexible read-outs enabling the quantification of sIC reactivity via ELISA or a fast detection using flow cytometry. This identified Fc{gamma}RIIA(H) and Fc{gamma}RIIIA as the most sIC-sensitive Fc{gamma}Rs in our test system. Applying the assay we demonstrate that sICs versus immobilized ICs are fundamentally different Fc{gamma}R-ligands with regard to Fc{gamma}R preference and signal strength. Reaching a detection limit in the very low nanomolar range, the assay proved also to be sensitive to sIC stoichiometry and size enabling for the first time a complete reproduction of the Heidelberger-Kendall precipitation curve in terms of immune receptor activation. Analyzing sera from SLE patients and mouse models of lupus and arthritis proved that sIC-dependent Fc{gamma}R activation has predictive capabilities regarding severity of SLE disease. The new methodology provides a sensitive, scalable and comprehensive tool to evaluate the size, amount and bioactivity of sICs in all settings.

One Sentence SummaryIn this study we established a comprehensive Fc{gamma}R reporter cell assay enabling the detection and quantification of soluble immune complexes generated in experimental and clinical settings. Creatine transporter deficient rat model show motor dysfunction, cerebellar alterations and muscle creatine deficiency without muscle atrophy Creatine (Cr) is a nitrogenous organic acid and plays roles as fast phosphate energy buffer to replenish ATP, osmolyte, antioxidant, neuromodulator, and as a compound with anabolic and ergogenic properties in muscle. Cr is taken from the diet or endogenously synthetized by the enzymes AGAT and GAMT, and specifically taken up by the transporter SLC6A8. Loss-of-function mutations in the genes encoding for the enzymes or the transporter cause Cerebral Creatine Deficiency Syndromes (CCDS). CCDS are characterized by brain Cr deficiency, intellectual disability with severe speech delay, behavioral troubles, epilepsy and motor dysfunction. Among CCDS, the X-linked Cr transporter deficiency (CTD) is the most prevalent with no efficient treatment so far. Different animal models of CTD show reduced brain Cr levels, cognitive deficiencies and together they cover other traits similar to those of patients. However, motor function was poorly explored in CTD models and some controversies in the phenotype exist in comparison with CTD patients. Our recently described Slc6a8Y389C knock-in (KI) rat model of CTD showed mild impaired motor function, morphological alterations in cerebellum, reduced muscular mass, Cr deficiency and increased guanidinoacetate content in muscle, although no consistent signs of muscle atrophy. Our results indicate that such motor dysfunction co-occurred with both nervous and muscle dysfunction, suggesting that muscle strength and performance as well as neuronal connectivity might be affected by this Cr deficiency in muscle and brain. A novel method for producing functionalized vesicles that efficiently deliver oligonucleotides in vitro in cancer cells and in vivo in mice Nano-based delivery systems have enhanced our ability to administer and target drugs and macromolecules to their targets. Oligonucleotide drugs have great therapeutic potential but often have off-target effects and stability issues. Therefore, they are often encapsulated in vesicles with targeting ligands such as antibodies (Ab) to deliver their cargo. Herein, we describe a novel, scalable and straightforward approach to producing functionalized vesicles called the "Functionalized Lipid Insertion Method," which differs from the older "Detergent-Dialysis Method." The latter method required excess detergent and extensive dialysis over many hours to produce the functionalized vesicles. With our method, only the functionalized lipid is detergent-solubilized during the engineering of the vesicle. The approach reduces the dialysis time, keeps the vesicles intact while orienting the targeting moieties of the functionalized lipid toward the outside of the vesicle. Pilot in vitro and in vivo experiments was performed to show the feasibility of our method. Dynamic light scattering (DLS) experiments suggested that the original vesicular structure was relatively unperturbed, and the functionalized lipid was inserted externally. Our approach efficiently delivered oligonucleotides and affected the function of liver cancer HepG2 cells. Furthermore, functionalized vesicles achieved targeted delivery of oligonucleotides in mice without inducing a significant innate immune response. The industrial and therapeutic significance and implications of functionalized vesicles produced by our method are also discussed. Additional experiments and analyses are recommended to bring out the full potential of this molecular delivery technology. A Bayesian model selection approach to mediation analysis Mediation analysis is a powerful tool for discovery of causal relationships. We describe a Bayesian model selection approach to mediation analysis that is implemented in our bmediatR software. Using simulations, we show that bmediatR performs as well or better than established methods including the Sobel test, while allowing greater flexibility in both model specification and in the types of inference that are possible. We applied bmediatR to genetic data from mice and human cell lines to demonstrate its ability to derive biologically meaningful findings. The Bayesian model selection framework is extensible to support a wide variety of mediation models. H3K36 methylation and DNA-binding both promote Ioc4 recruitment and Isw1b remodeller function The Isw1b chromatin-remodelling complex is specifically recruited to gene bodies to help retain pre-existing histones during transcription by RNA polymerase II. Recruitment is dependent on H3K36 methylation and the Isw1b subunit Ioc4, which contains an N-terminal PWWP domain. Here, we present the crystal structure of the Ioc4-PWWP domain, including a detailed functional characterization of the domain on its own as well as in the context of full-length Ioc4 and the Isw1b remodeller. The Ioc4-PWWP domain preferentially binds H3K36me3-containing nucleosomes. Its ability to bind DNA is required for nucleosome binding. It is also furthered by the unique insertion motif present in Ioc4-PWWP. The ability to bind H3K36me3 and DNA promote the interaction of full-length Ioc4 with nucleosomes in vitro and they are necessary for its recruitment to gene bodies in vivo. Furthermore, a fully functional Ioc4-PWWP domain promotes efficient remodelling by Isw1b and the maintenance of ordered chromatin in vivo, thereby preventing the production of non-coding RNAs. Groping in the fog: soaring migrants exhibit wider scatter in flight directions and respond differently to wind under low visibility conditions Atmospheric conditions are known to affect flight propensity, behaviour during flight, and migration route in birds. Yet, the effects of fog have only rarely been studied although they could disrupt orientation and hamper flight. Fog could limit the visibility of migrating birds such that they might not be able to detect landmarks that guide them during their journey. Soaring migrants modulate their flight speed and direction in relation to the wind vector to optimize the cost of transport. Consequently, landmark-based orientation, as well as adjustments of flight speed and direction in relation to wind conditions, could be jeopardized when flying in fog. Using a radar system operated in a migration bottleneck (Strait of Messina, Italy), we studied the behaviour of soaring birds under variable wind and fog conditions over two consecutive springs (2016 and 2017), discovering that migrating birds exhibited a wider scatter of flight directions and responded differently to wind under fog conditions. Birds flying through fog deviated more from the mean migration direction and increased their speed with increasing crosswinds. In addition, airspeed and groundspeed increased in the direction of the crosswind, causing the individuals to drift laterally. Our findings represent the first quantitative empirical evidence of flight behaviour changes when birds migrate through fog and explain why low visibility conditions could risk their migration journey. From adverse to beneficial - contrasting dietary effects of freshwater mixotrophs on zooplankton O_LIThe importance of mixotrophic algae as key bacterivores in microbial food webs is increasingly acknowledged, but their effects on the next trophic level remain poorly understood. Their high stoichiometric food quality is contrasted by anti-grazing strategies.
C_LIO_LIWe tested the quality of freshwater mixotrophs as prey for zooplankton, using four non-colonial chrysophyte species and a cryptophyte as a high quality reference food. We (1) analyzed the stoichiometric and biochemical (fatty acid) composition of the mixotrophs, and (2) quantified their dietary effects on Daphnia longispina survival.
C_LIO_LISurvival of D. longispina significantly depended on the identity of species provided as food, ranging from higher to lower as compared to starvation. This was not reflected in differences in cellular stoichiometry or fatty acid profiles of the mixotrophs. We suggest that toxicity may be the driver for the observed differences.
C_LIO_LIGeneralization of the dietary effects of mixotrophic chrysophytes does not appear straightforward. Besides fundamental species-specific differences, potential toxic effects may vary depending on environmental cues or physiological strategies. Notably in our study, Ochromonas tuberculata, a species previously reported to be deleterious turned out to be a beneficial food source, in terms of enabling high survival of D. longispina.
C_LIO_LIWe challenge the generality of the assumption that chrysophytes are of low value as food for zooplankton. We recommend that future studies test how environmental conditions and physiological strategies shape the quality of mixotrophs as food for consumers at higher trophic levels, specifically focusing on effects of dietary toxicity.
C_LI 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.