Optimism and pessimism in optimised replay The replay of task-relevant trajectories is known to contribute to memory consolidation and improved task performance. A wide variety of experimental data show that the content of replayed sequences is highly specific and can be modulated by reward as well as other prominent task variables. However, the rules governing the choice of sequences to be replayed still remain poorly understood. One recent theoretical suggestion is that the prioritization of replay experiences in decision-making problems is based on their effect on the choice of action. We show that this implies that subjects should replay sub-optimal actions that they dysfunctionally choose rather than optimal ones, when, by being forgetful, they experience large amounts of uncertainty in their internal models of the world. We use this to account for recent experimental data demonstrating exactly pessimal replay, fitting model parameters to the individual subjects choices.
White matter connections of high-level visual areas predict cytoarchitecture better than category-selectivity in childhood, but not adulthood Ventral temporal cortex (VTC) consists of high-level visual regions that are arranged in consistent anatomical locations across individuals. This consistency has led to several hypotheses about the factors that constrain the functional organization of VTC. A prevailing theory is that white matter connections influence the organization of VTC, however, the nature of this constraint is unclear. Here, we test two hypotheses: (1) white matter tracts are specific for each category or (2) white matter tracts are specific to cytoarchitectonic areas of VTC. To test these hypotheses, we used diffusion magnetic resonance imaging (dMRI) to identify white matter tracts and functional magnetic resonance imaging (fMRI) to identify category-selective regions in VTC in children and adults. We find that in childhood, white matter connections are linked to cytoarchitecture rather than category-selectivity. In adulthood, however, white matter connections are linked to both cytoarchitecture and category-selectivity. These results suggest a rethinking of the view that category-selective regions in VTC have category-specific white matter connections early in development. Instead, these findings suggest that the neural hardware underlying the processing of categorical stimuli may be more domain-general than previously thought, particularly in childhood.
Data-Driven Network Models for Genetic Circuits From Time-Series Data with Incomplete Measurements Synthetic gene networks are frequently conceptualized and visualized as static graphs. This view of biological programming stands in stark contrast to the transient nature of biomolecular interaction, which is frequently enacted by labile molecules that are often unmeasured. Thus, the network topology and dynamics of synthetic gene networks can be difficult to verify in vivo or in vitro, due to the presence of unmeasured biological states. Here we introduce the dynamical structure function as a new mesoscopic, data-driven class of models to describe gene networks with incomplete measurements. We introduce a network reconstruction algorithm and a code base for reconstructing the dynamical structure function from data, to enable discovery and visualization of graphical relationships in a genetic circuit diagram as time-dependent functions rather than static, unknown weights. We prove a theorem, showing that dynamical structure functions can provide a data-driven estimate of the size of crosstalk fluctuations from an idealized model. We illustrate this idea with numerical examples. Finally, we show how data-driven estimation of dynamical structure functions can explain failure modes in two experimentally implemented genetic circuits, a historical genetic circuit and a new E. coli based transcriptional event detector. Classification of daily crop phenology in PhenoCams using deep learning and hidden markov models Near surface cameras, such as those in the PhenoCam network, are a common source of ground truth data in modelling and remote sensing studies. Despite having locations across numerous agricultural sites, few studies have used near surface cameras to track the unique phenology of croplands. Due to management activities, crops do not have a natural vegetation cycle which many phenological extraction methods are based on. For example, a field may experience abrupt changes due to harvesting and tillage throughout the year. A single camera can also record several different plants due to crop rotations, fallow fields, and cover crops. Current methods to estimate phenology metrics from image time series compress all image information into a relative greenness metric, which discards a large amount of contextual information. This can include the type of crop present, whether snow or water is present on the field, the crop phenology, or whether a field lacking green plants consists of bare soil, fully senesced plants, or plant residue. Here we developed a modelling workflow to create a daily time series of crop type and phenology, while also accounting for other factors such as obstructed images and snow covered fields. We used a mainstream deep learning image classification model, VGG16. Deep learning classification models do not have a temporal component, so to account for temporal correlation among images our workflow incorporates a hidden markov model in the post-processing. The initial image classification model had out of sample F1 scores of 0.83-0.85, which improved to 0.86-0.91 after all post-processing steps. The resulting time series show the progression of crops from emergence to harvest, and can serve as a daily, local scale dataset of field states and phenological stages for agricultural research. C. elegans PEZO-1 is a Mechanosensitive Ion Channel Involved in Food Sensation PIEZO channels are force sensors essential for physiological processes, including baroreception and proprioception. The Caenorhabditis elegans genome encodes an ortholog gene of the Piezo family, pezo-1, which is expressed in several tissues, including the pharynx. This myogenic pump is an essential component of the C. elegans alimentary canal, whose contraction and relaxation are modulated by mechanical stimulation elicited by food content. Whether pezo-1 encodes a mechanosensitive ion channel and contributes to pharyngeal function remain unknown. Here, we leverage genome editing, genetics, microfluidics, and electropharyngeogram recording to establish that pezo-1 is expressed in the pharynx, including in a proprioceptive-like neuron, and regulates pharyngeal function. Knockout (KO) and gain-of-function (GOF) mutants reveal that pezo-1 is involved in fine-tuning pharyngeal pumping frequency, sensing osmolarity, and food mechanical properties. Using pressure-clamp experiments in primary C. elegans embryo cultures, we determine that pezo-1 KO cells do not display mechanosensitive currents, whereas cells expressing wild-type or GOF PEZO-1 exhibit mechanosensitivity. Moreover, infecting the Spodoptera frugiperda cell line with a baculovirus containing the G-isoform of pezo-1 (among the longest isoforms) demonstrates that pezo-1 encodes a mechanosensitive channel. Our findings reveal that pezo-1 is a mechanosensitive ion channel that regulates food sensation in worms. Consumer risk perception towards pesticide stained tomatoes in Uganda. BackgroundTomatoes are consumeddaily. Unfortunately, abuse of pesticides application by vegetable growers in Uganda increases risks of exposing consumers through the pesticide residues, as it may be above European Union Maximum Residue Limits (used as a standard in Uganda). This study is aimed to determine consumer attitudes and risk perception towards pesticide stained tomatoes available on the Ugandan local markets.

MethodsA cross-sectional study sampled 468 household consumers in four districts one from each region of Uganda. In each district, 60 household members from three randomly selected Sub Counties were interviewed. In addition, in each district 9 tomato handlers (3 tomato farmers, 3 tomato retailers and 3 tomato wholesalers) participated in Focus Group Discussion (FGDs) perdistrict. Collected data was entered into MS-Excel 13 and exported into STATA SE 14.0 for cleaning and analysis at below a 5% level of significance and 95% Confidence Intervals (CI). Proportion of risk perceptions and attitudes were computed and presented as percentages while factors associated with risk perception were determined using Fisher exact test. Qualitative data was analyzed using deductive and inductive approaches under thematic content analysis.

ResultsMore than half, 54.2% (253/468), of the respondents were females, mean age was 37 years (SD=13.13, ranging from 18 to 88 years). Half of the respondents, 50.9% (238/467), were farmers by occupation and 40.3% (188/468) had completed upper primary education. Only 5.0% (20/396) of consumers reported a high risk perception towards tomatoes stained with pesticide residues, the rest, 95.0% (376/396), were buying pesticide stained tomatoes despite their awareness about the possible health effects. The main reason for buying the pesticide stained tomatoes was that a majority, 59.0% (230/390), lacked an alternative to stained tomatoes. However, consumers generally had a negative attitude towards pesticide stained tomatoes, with 67.0% (313/468) of the consumers disagreeing to a statement that tomatoes sold on the market are safe. Consumer risk perception was significantly associated with consumer awareness about residues in the tomatoes; where the proportino of consumers who were aware of the risk of pesticide stained tomatoes were 42.8 times more likely not to buy stained tomatoes compared to the proportion of those who were not aware. OR, 42.8 (95% CI: 10.76-170.28). However, level of education P(0.975), gender P(0.581) and age-group P(0.680) were not associated with consumer risk perception after Fisher-Exact tests analysis. (95% CI and 5% level of significance).

ConclusionConsumer risk perception on pesticide stained tomatoes among Ugandan consumers ranked low with a majority of consumers buying tomatoes stained with pesticide residuesdue to lack of an alternative, except for a few who had a high risk perception about the pesticide health effects. Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin inflammation ObjectiveUltraviolet B (UVB) is an important trigger of skin inflammation and lupus with leukocyte recruitment to inflamed skin. We recently reported the involvement of neutrophil NETosis in UVB-induced skin inflammation, and that NETotic nuclear envelope rupture is driven by PKC-mediated nuclear lamin B disassembly. To address the role of Actin cytoskeleton in NETosis, we investigated the effects of Rho kinase (ROCK) and its downstream actomyosin cytoskeletal networks on PKC nuclear translocation and NET formation, as well as their involvement in UVB-induced skin inflammation.

MethodsWe studied the dynamic changes of ROCK and actomyosin cytoskeletal networks during NETosis induction and their involvement in PKC nuclear translocation. Using mice with hematopoietic-specific ROCK1 deficiency, we investigated the effects of ROCK1 deficiency on NETosis, and its involvement in UVB-induced skin inflammation.

ResultsOur time course studies demonstrated the dynamic changes of actin polymerization and ROCK activation, support the role of actin cytoskeleton in nuclear translocation of cytosolic PKC in early stage of NETosis induction. Inhibition of actin polymerization or key molecules of the ROCK/MLCK/myosin pathway decreased PKC nuclear translocation and NET formation. Genetic deficiency of ROCK1, inhibited NETosis ex vivo and in vivo, decreased extracellular display of NET-associated IL-17A, TNF, IFN{gamma}, and IFN in inflamed skin, which were correlated with the ameliorated skin inflammation in UVB-irradiated mice with hematopoietic-specific ROCK1 deficiency.

ConclusionsROCK regulated NETosis through modulation of PKC nuclear translocation via actomyosin cytoskeletal networks in neutrophils. ROCK1 deficiency ameliorated UVB- induced skin inflammation by attenuation of NETosis and NET-associated cytokines. Somatic uniparental disomy mitigates the most damaging EFL1 allele combination in Shwachman-Diamond syndrome Shwachman-Diamond syndrome (SDS; OMIM: #260400) is caused by variants in SBDS (Shwachman-Bodian-Diamond syndrome gene), which encodes a protein that plays an important role in ribosome assembly. Recent reports suggest that recessive variants in EFL1 are also responsible for SDS. However, the precise genetic mechanism that leads to EFL1-induced SDS remains incompletely understood. Here we present three unrelated Korean SDS patients that carry biallelic pathogenic variants in EFL1 with biased allele frequencies, resulting from a bone marrow-specific somatic uniparental disomy (UPD) in chromosome 15. The recombination events generated cells that were homozygous for the relatively milder variant, allowing for the evasion of catastrophic physiological consequences. Still, the milder EFL1 variant was solely able to impair 80S ribosome assembly and induce SDS features in cell line, zebrafish, and mouse models. The loss of EFL1 resulted in a pronounced inhibition of terminal oligo-pyrimidine element-containing ribosomal protein transcript 80S assembly. Therefore, we propose a more accurate pathogenesis mechanism of EFL1 dysfunction that eventually leads to aberrant translational control and ribosomopathy. A tool for assessing alignment of biomedical data repositories with open, FAIR, citation and trustworthy principles Increasing attention is being paid to the operation of biomedical data repositories in light of efforts to improve how scientific data is handled and made available for the long term. Multiple groups have produced recommendations for functions that biomedical repositories should support, with many using requirements of the FAIR data principles as guidelines. However, FAIR is but one set of principles that has arisen out of the open science community. They are joined by principles governing open science, data citation and trustworthiness, all of which are important aspects for biomedical data repositories to support. Together, these define a framework for data repositories that we call OFCT: Open, FAIR, Citable and Trustworthy. Here we developed an instrument using the open source PolicyModels toolkit that attempts to operationalize key aspects of OFCT principles and piloted the instrument by evaluating eight biomedical community repositories listed by the NIDDK Information Network (dkNET.org). Repositories included both specialist repositories that focused on a particular data type or domain, in this case diabetes and metabolomics, and generalist repositories that accept all data types and domains. The goal of this work was both to obtain a sense of how much the design of current biomedical data repositories align with these principles and to augment the dkNET listing with additional information that may be important to investigators trying to choose a repository, e.g., does the repository fully support data citation? The evaluation was performed from March to November 2020 through inspection of documentation and interaction with the sites by the authors. Overall, although there was little explicit acknowledgement of any of the OFCT principles in our sample, the majority of repositories provided at least some support for their tenets. Resolving when (and where) the Thylacine went extinct Like the Dodo and Passenger Pigeon before it, the predatory marsupial Thylacine (Thylacinus cynocephalus), or  Tasmanian tiger, has become an iconic symbol of human-caused extinction. The last captive animal died in 1936, but even today reports of the Thylacines possible ongoing survival in remote regions of Tasmania are newsworthy and capture the publics imagination. Extirpated from mainland Australia in the mid-Holocene, the large island of Tasmania became the species final stronghold. Following European settlement in the 1800s, the Thylacine was heavily persecuted and pushed to the margins of its range, although many sightings were reported thereafter--even well beyond the 1930s. To gain a new depth of insight into the extinction of the Thylacine, we assembled an exhaustive database of 1,237 observational records from Tasmania (from 1910 onwards), quantified their uncertainty, and charted the patterns these revealed. We also developed a new method to visualize the species 20th-century spatio-temporal dynamics, to map potential post-bounty refugia and pinpoint the most-likely location of the final persisting subpopulation. A direct reading of the high-quality records (confirmed kills and captures, in combination with sightings by past Thylacine hunters and trappers, wildlife professionals and experienced bushmen) implies a most-likely extinction date within four decades following the last capture (i.e., 1940s to 1970s). However, uncertainty modelling of the entire sighting record, where each observation is assigned a probability and the whole dataset is then subject to a sensitivity analysis, suggests that extinction might have been as recent as the late 1980s to early 2000s, with a small chance of persistence in the remote south-western wilderness areas. Beyond the intrinsically fascinating problem of reconstructing the final fate of the Thylacine, the new spatio-temporal mapping of extirpation developed herein would also be useful for conservation prioritization and search efforts for other rare taxa of uncertain status. Decreased cell stiffness facilitates detachment and migration of breast cancer cells in 3D collagen matrices: An exploratory study BackgroundTumour-cell detachment is a critical early event in the metastatic cascade. However, the role of the cells mechanical properties in detachment and migration is not well understood. This exploratory study aimed to assess how intracellular stiffness changes these processes.

MethodsMDA-MB-231 cells were embedded as 10,000-cell spheroids in 2 and 4 mg/ml collagen matrices. Intracellular stiffness was assessed with mitochondria tracking microrheology of cells that migrated distances equivalent to four and six times the cell diameter (dC) from the spheroid and compared to cells at the spheroid surface (0dC), representing medium, high and no migration, respectively.

Results and discussionThe mitochondrial mean square displacement and intracellular stiffness decreased during detachment and migration for both collagen concentrations (i.e. rigidities). The mean square displacement of 4dC and 6dC cells was similar, whereas cell stiffness was lower for 4dC than for 6dC cells. With increasing matrix rigidity, the intracellular stiffness decreased for 0dC cells and did not change for 4dC and 6dC cells. It is proposed that decreased cell stiffness drives detachment and migration and increased matrix rigidity physically hinders migration, and cells need to become softer or remodel the environment to migrate. The independence of the stiffness of migrated cells from matrix rigidity suggests that cells remodel their environment through matrix proteins cleavage to migrate.

ConclusionsThe study revealed the collective effects of enhanced migratory conditions and increased matrix rigidity on the mechanical properties of the cells. The expression of matrix metalloproteinases and transforming growth factor {beta} and the role of cell volume on detachment and migration in matrices with varying pore sizes are proposed targets for further studies on metastatic cancer cells. Linking plankton size spectra and community composition to carbon export and its efficiency The magnitude and efficiency of particulate carbon export from the ocean surface depends not only on net primary production (NPP) but also on how carbon is consumed, respired, and repackaged by organisms. We contend that several of these processes can be captured by the size spectrum of the plankton community. However, most global models have relatively simple food-web structures that are unable to generate plankton sizespectra. Moreover, the life-cycles of multicellular zooplankton are typically not resolved, restricting the ability of models to represent time-lags that are known to impact carbon export and its efficiency (pe-ratio). Here, we use a global mechanistic size-spectrum model of the marine plankton community to investigate how particulate export and pe-ratio relate to the community size spectrum, community composition, and time-lags between predators and prey. The model generates emergent food-webs with associated size distributions for organisms and detrital particles. To resolve time-lags between phytoplankton and zooplankton, we implement the life-cycle of multicellular zooplankton (here represented by copepods). The simulation successfully captures observed patterns in biomass and energy fluxes across regions. We find that carbon export correlates best with copepod biomass and trophic level, whereas the pe-ratio correlates best with the exponent of the size spectrum and sea surface temperature (SST). Community metrics performed better than NPP or SST for both deep export and pe-ratio. Time-lags between phytoplankton and copepods did not strongly affect export or pe-ratio. We conclude by discussing how can we reconcile size-spectrum theory with field sampling.

Plain Language SummaryPlankton are tiny but extremely abundant aquatic organisms. Plankton lock CO2 away from the atmosphere as they sink to the deep ocean, where carbon can be stored for hundreds of years. However, how much carbon is locked away and for how long depends on how organisms eat, defecate, and respire. We argue that these processes are reflected in the size composition of the plankton community. The size composition shows a clear relationship between the number of organisms and their body-size. The steepness of this "size-abundance relationship" describes the balance between small vs. large organisms, and has been argued to reflect how energy is transferred from small to large organisms. Since large organisms create fast-sinking particles, the size-abundance relationship could be used to estimate how much carbon is being stored in the deep ocean. Here we use a computer simulation of the global plankton community to investigate how the removal of carbon relates to the plankton community and the steepness of the sizeabundance relationship. The model successfully captures patterns observed in nature. We found that the size-abundance relationship, together with the quantity of large zooplankton better explained carbon export than other measures typically used, such as photosynthesis and temperature.

Key PointsO_LIWe use a global mechanistic size-spectrum model to investigate the relation between particulate export and plankton community metrics.
C_LIO_LIWe find a good correlation between export efficiency and the exponent of the size spectrum.
C_LIO_LITotal carbon export correlated well with copepod biomass and trophic level of active copepods in the model.
C_LI MS2AI: Automated repurposing of public peptide LC-MS data for machine learning applications MotivationLiquid-chromatography mass-spectrometry (LC-MS) is the established standard for analyzing the proteome in biological samples by identification and quantification of thousands of proteins. Machine learning (ML) promises to considerably improve the analysis of the resulting data, however, there is yet to be any tool that mediates the path from raw data to modern ML applications. More specifically, ML applications are currently hampered by three major limitations: (1) absence of balanced training data with large sample size; (2) unclear definition of sufficiently information-rich data representations for e.g. peptide identification; (3) lack of benchmarking of ML methods on specific LC-MS problems.

ResultsWe created the MS2AI pipeline that automates the process of gathering vast quantities of mass spectrometry (MS) data for large scale ML applications. The software retrieves raw data from either in-house sources or from the proteomics identifications database, PRIDE. Subsequently, the raw data is stored in a standardized format amenable for ML encompassing MS1/MS2 spectra and peptide identifications. This tool bridges the gap between MS and AI, and to this effect we also present an ML application in the form of a convolutional neural network for the identification of oxidized peptides.

AvailabilityAn open source implementation of the software can be found freely available for non-commercial use at https://gitlab.com/roettgerlab/ms2ai.

Contactveits@bmb.sdu.dk

Supplementary informationSupplementary data are available at Bioinformatics online. Fast ATP-dependent Subunit Rotation in Reconstituted FoF1-ATP Synthase Trapped in Solution FoF1-ATP synthases are ubiquitous membrane-bound, rotary motor enzymes that can catalyze ATP synthesis and hydrolysis. Their enzyme kinetics are controlled by internal subunit rotation, by substrate and product concentrations, by mechanical inhibitory mechanisms, but also by the electrochemical potential of protons across the membrane. Single-molecule Forster resonance energy transfer (smFRET) has been used to detect subunit rotation within FoF1-ATP synthases embedded in freely diffusing liposomes. We now report that kinetic monitoring of functional rotation can be prolonged from milliseconds to seconds by utilizing an Anti-Brownian electrokinetic trap (ABEL trap). These extended observation times allowed us to observe fluctuating rates of functional rotation for individual FoF1-liposomes in solution. Broad distributions of ATP-dependent catalytic rates were revealed. The buildup of an electrochemical potential of protons was confirmed to limit the maximum rate of ATP hydrolysis. In the presence of ionophores or uncouplers, the fastest subunit rotation speeds measured in single reconstituted FoF1-ATP synthases were 180 full rounds per second. This was much faster than measured by biochemical ensemble averaging, but not as fast as the maximum rotational speed reported previously for isolated single F1 complexes uncoupled from the membrane-embedded Fo complex. Further application of ABEL trap measurements should help resolve the mechanistic causes of such fluctuating rates of subunit rotation.

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org.highwire.dtl.DTLVardef@1bd800org.highwire.dtl.DTLVardef@228aeorg.highwire.dtl.DTLVardef@1bce39forg.highwire.dtl.DTLVardef@3f01a_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOFigure 1.C_FLOATNO C_FIG Define protein variant functions with high-complexity mutagenesis libraries and enhanced mutation detection software ASMv1.0 Open reading frame (ORF) variant libraries have advanced our ability to query the functions of a large number of variants of a protein simultaneously in a single experiment. Variant libraries targeting full-length ORFs typically consists of all possible single-amino-acid substitutions and a stop codon at each amino-acid position. Because a variant differs from the template ORF by a single codon variation, variant quantification presents the most profound challenge to this technology. Efforts such as dividing a library into sub-libraries for direct sequencing, or tag-directed subassembly are practical only for small ORFs. Our approach, however, features an enhanced variant-calling tool that processes massive shotgun sequencing data, allowing one to screen a single variant library for ORFs sized up to [~]3600 bases. The variant-calling tool detects variants reliably, and also presents variant annotations in datafiles enabling analyses that have reshaped our strategies governing library design, screen deconvolution, sequencing and sequencing data analysis. 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. Dynamics of SARS-CoV-2 mutations reveals regional-specificity and similar trends of N501 and High-Frequency mutation N501Y in different levels of control measures Coronavirus disease 2019 (COVID-19) is a contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This disease has spread globally, causing more than 161.5 million cases and 3.3 million deaths to date. Surveillance and monitoring of new mutations in the virus genome are crucial to our understanding of the adaptation of SARS-CoV-2. Moreover, how the temporal dynamics of these mutations is influenced by control measures and non-pharmaceutical interventions (NPIs) is poorly understood. Using 1 058 020 SARS-CoV-2 from sequenced COVID-19 cases from 98 countries (totaling 714 country-month combinations), we perform a normalization by COVID-19 cases to calculate the relative frequency of SARS-CoV-2 mutations and explore their dynamics over time. We found 115 mutations estimated to be present in more than 3 % of global COVID-19 cases and determined three types of mutation dynamics: High-Frequency, Medium-Frequency, and Low-Frequency. Classification of mutations based on temporal dynamics enable us to examine viral adaptation and evaluate the effects of implemented control measures in virus evolution during the pandemic. We showed that Medium-Frequency mutations are characterized by high prevalence in specific regions and/or in constant competition with other mutations in several regions. Finally, taking N501Y mutation as representative of High-Frequency mutations, we showed that level of control measure stringency negatively correlates with the effective reproduction number of SARS-CoV-2 with High-Frequency or not-High-Frequency and both follows similar trends in different levels of stringency. Multispecific DARPin(R) therapeutics demonstrate very high potency against SARS-CoV-2 variants in vitro SARS-CoV-2 has infected millions of people globally and continues to undergo evolution. Emerging variants can be partially resistant to vaccine induced immunity and therapeutic antibodies, emphasizing the urgent need for accessible, broad-spectrum therapeutics. Here, we report a comprehensive study of ensovibep, the first trispecific clinical DARPin candidate, that can simultaneously engage all three units of the spike protein trimer to potently inhibit ACE2 interaction, as revealed by structural analyses. The cooperative binding of the individual modules enables ensovibep to retain inhibitory potency against all frequent SARS-CoV-2 variants, including Omicron BA.1 and BA.2, as of February 2022. Moreover, viral passaging experiments show that ensovibep, when used as a single agent, can prevent development of escape mutations comparably to a cocktail of monoclonal antibodies (mAb). Finally, we demonstrate that the very high in vitro antiviral potency also translates into significant therapeutic protection and reduction of pathogenesis in Roborovski dwarf hamsters infected with either the SARS-CoV-2 wild-type or the Alpha variant. In this model, ensovibep prevents fatality and provides substantial protection equivalent to the standard of care mAb cocktail. These results support further clinical evaluation and indicate that ensovibep could be a valuable alternative to mAb cocktails and other treatments for COVID-19. 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. Production of Offspring from Azoospermic Mice with Meiotic Failure: Precise Biparental Meiosis within Halved Oocytes While the large volume of mammalian oocytes is necessary for embryo development, it can lead to error-prone chromosomal segregation during meiosis. Conversely, we hypothesized that smaller oocytes would have a great unidentified potential to stabilize unstable meiosis and improve the development of the resultant embryos. Here, we show that reducing ooplasmic volume can rescue highly error-prone fertilization using primary spermatocytes by preventing segregation errors of chromosomes during biparental meiosis. High-resolution live-imaging analysis revealed that erroneous chromosome segregation occurred in most (90%) spermatocyte-injected oocytes of normal size, but could be ameliorated to 40% in halved oocytes. The birth rate improved remarkably from 1% to 19% (P < 0.0001). Importantly, this technique enabled the production of offspring from azoospermic mice with spermatocyte arrest caused by STX2 deficiency, an azoospermia factor also found in humans. Thus, contrary to popular opinion, oocytes inherently have a strong potential for precise meiotic divisions, which can be evoked by reduction of the ooplasmic volume. Their potential might help rescue cases of untreatable human azoospermia with spermatocyte arrest. The pH Dependence of Niclosamide Solubility, Dissolution, and Morphology Motivates Potentially Universal Mucin-Penetrating Nasal and Throat Sprays for COVID19, its Contagious Variants, and Other Respiratory Viral Infections MotivationWith the coronavirus pandemic still raging, prophylactic nasal and early treatment throat sprays could help prevent infection and reduce viral load. Niclosamide has the potential to treat a broad range of viral infections if local bioavailability is optimized as mucin-penetrating solutions as opposed to more traditional microparticle-based sprays that cannot penetrate the mucin.

ExperimentalpH-dependence of supernatant concentrations and dissolution rates of niclosamide were measured in buffered solutions by Nanodrop-UV/Vis-spectroscopy for niclosamide from different suppliers, as precipitated material, and as cosolvates. Data was compared to predictions from Henderson Hasselbalch and precipitation pH models. Optimal microscopy was used to observe the morphologies of precipitated and converted niclosamide.

ResultsSupernatant-concentrations of niclosamide increased with increasing pH, from 1.77uM at pH 3.66 to 30uM at pH 8, and more rapidly from 90uM at pH8.5 to 300uM at pH 9.1, reaching 641uM at pH 9.5. Logarithmic rates for dissolution increased by [~]3x for pHs 8.62 to 9.44. However, when precipitated from supersaturated solution, niclosamide equilibrated to much lower final supernatant concentrations, reflective of more stable polymorphs at each pH that were also apparent for niclosamide from other suppliers and cosolvates.

ConclusionsGiven niclosamides activity against COVID19, its more contagious variants, and other respiratory viral infections, these niclosamide solutions, that put the virus in lockdown, could represent universal prophylactic nasal and early treatment throat sprays. As solutions they would be the simplest and potentially most effective formulations from both an efficacy standpoint as well as manufacturing and distribution, with no cold chain. They now just need testing. ATP-binding cassette protein ABCF1 couples gene transcription with maintenance of genome integrity in embryonic stem cells OCT4 and SOX2 confer pluripotency by recruiting coactivators to activate stem cell-specific gene expression programs. However, the composition of coactivator complexes and their roles in maintaining stem cell fidelity remain unclear. Here we report the identification of ATP-binding cassette subfamily F member 1 (ABCF1) as a critical coactivator for OCT4/SOX2. ABCF1 is required for pluripotency gene expression and stem cell self-renewal. ABCF1 binds co-dependent coactivators XPC and DKC1 via its intrinsically disordered region and stimulates transcription by linking SOX2 to the transcription machinery. Furthermore, in response to pathogen infection and DNA damage, ABCF1 binds intracellular DNAs accumulated in cells, concomitant with loss of SOX2 interaction and pluripotency gene transcription. This results in spontaneous differentiation of compromised stem cells and elimination from the self-renewing population. Thus, ABCF1 directly couples pluripotency gene transcription with sensing aberrant DNAs and acts as a checkpoint for self-renewal to safeguard stem cell fidelity and genome integrity. Hybridization alters the shape of the genotypic fitness landscape, increasing access to novel fitness peaks during adaptive radiation Estimating the complex relationship between fitness and genotype or phenotype (i.e. the adaptive landscape) is one of the central goals of evolutionary biology. However, adaptive walks connecting genotypes to organismal fitness, speciation, and novel ecological niches are still poorly understood and processes for surmounting fitness valleys remain controversial. One outstanding system for addressing these connections is a recent adaptive radiation of ecologically and morphologically novel pupfishes (a generalist, molluscivore, and scale-eater) endemic to San Salvador Island, Bahamas. We leveraged whole-genome sequencing of 139 hybrids from two independent field fitness experiments to identify the genomic basis of fitness, estimate genotypic fitness networks, and measure the accessibility of adaptive walks on the fitness landscape. We identified 132 SNPs that were significantly associated with fitness in field enclosures. Six out of the 13 regions most strongly associated with fitness contained differentially expressed genes and fixed SNPs between trophic specialists; one gene (mettl21e) was also misexpressed in lab-reared hybrids, suggesting a potential intrinsic genetic incompatibility. We then constructed genotypic fitness networks from adaptive alleles and show that scale-eating specialists are the most isolated of the three species on these networks. Intriguingly, introgressed and de novo variants reduced fitness landscape ruggedness as compared to standing variation, increasing the accessibility of genotypic fitness paths from generalist to specialists. Our results suggest that adaptive introgression and de novo mutations alter the shape of the fitness landscape, providing key connections in adaptive walks circumventing fitness valleys and triggering the evolution of novelty during adaptive radiation. Computation of the mitochondrial age distribution along the axon length We describe a compartmental model of mitochondrial transport in axons, which we apply to compute mitochondrial age at different distances from the soma. The model predicts that the average mitochondrial age at the tip of an axon with a length of 1 cm is approximately 22 hours. The mitochondria are youngest closest to the soma and their age scales approximately linearly with distance from the soma. To the best of the authors knowledge, this is the first attempt to predict the spatial distribution of mitochondrial age within an axon. A sensitivity study of the mean age of mitochondria to various model parameters is also presented. Global analysis of human-to-mouse contact-dependent intercellular mRNA and lncRNA transfer in cell culture Full-length mRNAs can transfer between adjacent mammalian cells via direct cell-to-cell connections called tunneling nanotubes (TNTs). However, the extent of mRNA transfer at the transcriptome-wide level (the transferome) is unknown. Here, we analyzed whole transcriptome mRNA and lncRNA transfer between heterogeneous human-mouse cell populations in in vitro co-culture using RNA-sequencing. Our data indicate that mRNA transfer is non-selective, prevalent across the human transcriptome, and that the amount of transfer to mouse embryonic fibroblasts (MEFs) strongly correlates with the endogenous level of gene expression in donor human breast cancer cells (MCF7). These results were validated by both quantitative RT-PCR and in situ hybridization, and analysis shows that typically <1% of endogenous mRNAs and lncRNAs undergo transfer. Non-selective expression-dependent RNA transfer was further validated using synthetic RNA reporters. Notably, significant differential changes in the native MEF transcriptome were observed in response to co-culture, including the upregulation of multiple cancer and cancer-associated fibroblast-related genes and pathways. Together, these results lead us to suggest that TNT-mediated RNA transfer could be a phenomenon of physiological importance under both normal and pathogenic conditions.

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org.highwire.dtl.DTLVardef@13a7176org.highwire.dtl.DTLVardef@1ef1c75org.highwire.dtl.DTLVardef@1355eeforg.highwire.dtl.DTLVardef@ebc275_HPS_FORMAT_FIGEXP  M_FIG C_FIG Universal COVID-19 vaccine with updated spike antigen confers full protection against all SARS-CoV-2 variants of concern. Current first-generation COVID-19 vaccines are based on prototypic spike sequences from ancestral 2019 SARS-CoV-2 strains. However, the ongoing pandemic is fueled by variants of concern (VOC) that threaten to escape vaccine-mediated protection. Here we show in a stringent hamster model that immunization using prototypic spike expressed from a potent YF17D viral vector (1) provides vigorous protection against infection with ancestral virus (B lineage) and VOC Alpha (B.1.1.7), however, is insufficient to provide maximum protection against the Beta (B.1.351) variant. To improve vaccine efficacy, we created a revised vaccine candidate that carries an evolved spike antigen. Vaccination of hamsters with this updated vaccine candidate provides full protection against intranasal challenge with all four VOCs Alpha, Beta, Gamma (P.1) and Delta (B.1.617.2) resulting in complete elimination of infectious virus from the lungs and a marked improvement in lung pathology. Vaccinated hamsters did also no longer transmit the Delta variant to non-vaccinated sentinels. Hamsters immunized with our modified vaccine candidate also mounted marked neutralizing antibody responses against the recently emerged Omicron (B.1.1.529) variant, whereas the old vaccine employing prototypic spike failed to induce immunity to this antigenically distant virus. Overall, our data indicate that current first-generation COVID-19 vaccines need to be urgently updated to cover newly emerging VOCs to maintain vaccine efficacy and to impede virus spread at the community level.

Significance StatementSARS-CoV-2 keeps mutating rapidly, and the ongoing COVID-19 pandemic is fueled by new variants escaping immunity induced by current first-generation vaccines. There is hence an urgent need for universal vaccines that cover variants of concern (VOC). In this paper we show that an adapted version of our vaccine candidate YF-S0* provides full protection from infection, virus transmission and disease by VOCs Alpha, Beta, Gamma and Delta, and also results in markedly increased levels of neutralizing antibodies against recently emerged Omicron VOC in a stringent hamster model. Our findings underline the necessity to update COVID-19 vaccines to curb the pandemic, providing experimental proof on how to maintain vaccine efficacy in view of an evolving SARS-CoV-2 diversity.