Fractal polymer with loops recapitulates key features of chromosome organization Chromosomes are exceedingly long polymers compacted in a cell nucleus. While it has long been suggested that mammalian chromosomes during interphase are folded into loops, experimental detection of such loops has remained a daunting task. The most comprehensive experimental information about chromosome spatial organization is provided by Hi-C experiments that measure the frequency of contacts between all chromosomal positions. The lack of a tractable physical model of a polymer folded into loops limits our ability to interpret experimental data. It thus remains unknown how to obtain accurate and quantitative information about the nature of chromosomal looping from Hi-C. Here, we introduce a model of a polymer with random loops, solve it analytically and extend it by simulations for real chains. Remarkably, our model faithfully reproduces complex shapes of experimental contact probability curves universal among mammalian cells. Furthermore, our model allows one to estimate sizes of randomly positioned chromosomal loops from experimental data. We also show that excluded volume in real chains can induce osmotic and topological repulsion between loops, stiffening interphase chromosomes. Thus, our new framework allows interpretation of experimental data and suggests that interphase chromosomes are crumpled polymers further folded into a sequence of randomly positioned loops.
Orientation dependent proton transverse relaxation in the human brain white matter: The magic angle effect on a cylindrical helix PurposeTo overcome some limitations of prior orientation-dependent proton transverse relaxation formalisms in white matter (WM) with a novel framework based on the generalized magic angle effect function.

MethodsA cylindrical helix model was developed embracing anisotropic rotational and translational diffusion of restricted molecules in human brain WM, with the former characterized by an axially symmetric system. Transverse relaxation rates R2 and [Formula] were divided into isotropic [Formula] and anisotropic parts, [Formula], with  denoting an open angle and{varepsilon} 0 an orientation ({Phi}) offset from DTI-derived primary diffusivity direction. The proposed framework (Fit A) was compared with prior models without{varepsilon} 0 on previously published water and methylene proton transverse relaxation rates from developing, healthy, and pathological WM at 3T. Goodness of fit was represented by root-mean-square error (RMSE). F-test and linear correlation were used with statistical significance set to P [&le;] 0.05.

ResultsFit A significantly (P<0.01) outperformed prior models as demonstrated by reduced RMSEs, e.g., 0.349 vs. 0.724 in myelin water. Fitted{varepsilon} 0 was in good agreement with calculated{varepsilon} 0 from directional diffusivities. Compared with those from healthy adult, the fitted [Formula], and  from neonates were substantially reduced but{varepsilon} 0 increased, consistent with incomplete myelination. Significant positive [Formula] and negative ( and [Formula]) correlations were found with aging (demyelination) in elderly.

ConclusionThe developed framework can better characterize orientation dependences from a wide range of proton transverse relaxation measurements in human brain WM, shedding new light on myelin microstructural alterations at the molecular level.
Inter-microscope comparability of dental microwear texture data obtained from different optical profilometers. Dental microwear texture analysis has become a well-established approach for dietary inference and reconstruction of mammals and other tetrapods, both extant and extinct. As the amount of available data grows immensely, researchers could benefit from combining data gathered by others to perform meta-analyses. However, different devices used to capture three-dimensional surface scans for DMTA are known to produce variation even when measuring the same surface. Here we compare DMTA data of 36 guinea pigs that received different diets in a controlled feeding experiment, measured on a confocal disc-scanning and a confocal laser-scanning microscope. We are testing different pre-analysis filtering protocols to mitigate differences. We find inter-microscopes and filter-related differences for the majority of analysed 41 DMTA parameters. Certain microscope-specific filter routines resulted in less differences than other pre-analysis protocols. We further identify DMTA parameters which were stable regardless of microscope or pre-analysis treatment. Overall, the results obtained on both microscopes show the same dietary differentiation between guinea pig feeding groups, which supports that DMTA is a suitable method to obtain repeatable, objective dietary inferences. We finally propose a roadmap to enhance data exchange and inter-lab comparability and collaboration in the future. Dynamic modulation of enhancer responsiveness by core promoter elements in living Drosophila embryo Regulatory interactions between enhancers and core promoters are fundamental for the temporal and spatial specificity of gene expression in development. The central role of core promoters is to initiate productive transcription in response to enhancers activation cues. However, it has not been systematically assessed how individual core promoter elements affect the induction of transcriptional bursting by enhancers. Here, we provide evidence that each core promoter element dynamically alters "enhancer responsiveness" by changing functional parameters of transcriptional bursting in developing Drosophila embryos. Quantitative live imaging analysis revealed that the timing and the continuity of burst induction are common regulatory steps on which core promoter elements impact. We also show that core promoters use the upstream TATA box as an amplifier of transcriptional bursting upon interaction with enhancers. Genome editing analysis of the pair-rule gene fushi tarazu further revealed that the endogenous TATA box and the downstream core promoter element are both essential for the correct expression and the function of key developmental genes in early embryos. We suggest that core promoter elements serve as a key regulatory module in converting enhancer activity into transcription dynamics during animal development. The RAG1 Ubiquitin Ligase Domain Enhances Assembly and Selection of T Cell Receptor Genes to Restrain the Autoimmune Hazard of Generating T Cell Receptor Diversity RAG1/RAG2 (RAG) endonuclease-mediated assembly of diverse lymphocyte antigen receptor genes by V(D)J recombination is critical for the development and immune function of T and B cells. However, this process creates highly self-reactive cells that must be properly selected to suppress autoimmunity. The RAG1 protein contains a ubiquitin ligase domain that stabilizes RAG1 and stimulates RAG endonuclease activity in vitro. We report that mice with a mutation that inactivates the RAG1 ubiquitin ligase in vitro exhibit modestly reduced thymic cellularity, decreased assembly and altered repertoires of T cell receptor (TCR) {beta} and  genes in thymocytes, and impaired thymocyte developmental transitions that require the assembly of TCR{beta} or  genes and signaling by their proteins. These RAG1 mutant mice also exhibit less efficient positive selection and superantigen-mediated negative selection of conventional {beta} T cells, 2) impaired differentiation of iNKT lineage {beta} T cells, and 3) CD4+ {beta} T cells with elevated autoimmune potential. Our findings demonstrate that the RAG1 ubiquitin ligase domain functions in vivo to stimulate the assembly and selection of TCR{beta} and TCR genes, thereby establishing replete diversity of {beta} TCRs and {beta} T cell lineages while restraining the inherent autoimmune hazard of generating diverse antigen specificities. Morphological aspects of opossum (Didelphis aurita) pancreas during intramarsupial development Functionalized nanostructured systems can be used for imaging and drug delivery for anti-tumor therapy, including breast tumors. This is a more efficient approach that offers reduced systemic side effects compared to conventional diagnostic and chemotherapy methods. Multifunctional nanoparticles are potential tools in the diagnosis, location tracing and kill tumor cells through a less invasive manner. Functionalized phosphate-based nanoparticles are capable of encapsulating, or may be associated, with fluorescent probes. In this study, we synthesize a nanoparticle phosphate-based composite (NPC) and functionalize it with poly-ethylene glycol (PEG), hyaluronic acid (HA), the fluorescent probe rhodamin 6G (R6G) and the antimitotic doxorubicin (DOX). We focused on targeting human breast cancer cells reporting the biological effects of functionalized NPC on them. NPC and NPC formulations containing PEG, HA, and R6G did not cause cell viability reduction on MCF-7 and MDA-MB-231 cell lines. The cellular internalization of NPC was quantified by real-time in vitro observation, and confirmed by electron microscopy techniques. Intracellular NPC distribution is detected in the cytoplasm and nucleus of tumor cells by confocal fluorescent images. The percent association of doxorubicin to NPC matrix was approximately 18% and NPC formulations associated with doxorubicin led to a significant reduction in cell viability in MDA-MB-231 and MCF-7 cells. This data suggest the potential use of NPC as a non-cytotoxic platform for association with functional ligands to selective targeting breast cancer cells. NPC use can be also explored in drug delivery to cancer cells. GCN5L1 promotes diastolic dysfunction by inhibiting cardiac pyruvate oxidation Left ventricular diastolic dysfunction is a structural and functional condition that precedes the development of heart failure with preserved ejection fraction (HFpEF). The etiology of diastolic dysfunction includes alterations in fuel substrate metabolism that negatively impact cardiac bioenergetics, and may precipitate the eventual transition to heart failure. To date, the molecular mechanisms that regulate early changes in fuel metabolism leading to diastolic dysfunction remain unclear. In this report, we use a diet-induced obesity model and quantitative acetylproteomics in aged mice to show that inhibitory lysine acetylation of the pyruvate dehydrogenase (PDH) complex promotes energetic deficits and diastolic dysfunction in mouse hearts. Cardiomyocyte-specific deletion of the mitochondrial lysine acetylation regulatory protein GCN5L1 prevented hyperacetylation of the PDH complex subunit PDHA1, allowing aged obese mice to continue using pyruvate as a bioenergetic substrate in the heart. Our findings suggest that changes in mitochondrial protein lysine acetylation represent a key metabolic component of diastolic dysfunction that precedes the development of heart failure. Modeling miRNA-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. Artificial seawater based long-term culture of colonial ascidians Tunicates are highly diverse marine invertebrate filter-feeders that are vertebrates closest relatives. These organisms, despite a drastically different body plan during their adulthood, have a tissue complexity related to that of vertebrates. Ascidians, which compose most of the Tunicata, are benthic sessile hermaphrodites that reproduce sexually through a motile tadpole larval stage. Over half of the known ascidians species are able to reproduce asexually by budding, typically leading to the formation of colonies where animals, called zooids, are interconnected through an external vascular system. In addition, colonial ascidians are established models for important biological processes including allorecognition, immunobiology, aging, angiogenesis and whole-body regeneration. However, the current paucity in breeding infrastructures limits the study of these animals to coastal regions.

To promote a wider scientific spreading and popularity of colonial ascidians, we have developed a flexible recirculating husbandry setup for their long-term in-lab culture. Our system is inspired both by the flow-through aquariums used by coastal ascidian labs, as well as by the recirculating in-lab systems used for zebrafish research. Our hybrid system thus combines colony breeding, water filtering and food culturing in a semi-automated system where specimens develop on hanging microscopy glass slides. Temperature, light/dark cycles, flow speed and feeding rates can be controlled independently in four different breeding environments to provide room for species-specific optimization as well as for running experiments. This setup is complemented with a quarantine for the acclimatization of wild isolates.

Herein we present our success in breeding Botrylloides diegensis, a species of colonial ascidians, for more than 3 years in recirculating artificial seawater over 600 km away from their natural habitat. We show that colonies adapt well to in-lab culturing provided that a suitable marine microbiome is present, and that a specific strain can be isolated, propagated and efficiently used for research over prolonged periods of time. The flexible and modular structure of our system can be scaled and adapted to the needs of specific species, such as Botryllus schlosseri, as well as of particular laboratory spaces. Overall, we show that Botrylloides diegensis can be proficiently bred in-land and suggest that our results can be extended to other species of colonial ascidians to promote research on these fascinating animals.

HighlightsO_LIFirst artificial seawater based recirculating aquaculture for colonial ascidians
C_LIO_LIOver 3 years of continuous breeding
C_LIO_LISemi-automated setup with minimized maintenance
C_LIO_LIGood biomass production for strain propagation
C_LIO_LI4 different culture conditions for optimized breeding for species of interest
C_LI Design, Mutate, Screen: High-throughput creation of genetic clocks with different period-amplitude characteristics Directed evolution has become an invaluable tool in protein engineering and has also greatly influenced the construction of synthetic gene circuits. The ability to generate diversity at precise targets for directed evolution approaches has improved vastly, allowing researchers to create large, specific mutant libraries with relative ease. Screening approaches for large mutant libraries have similarly come a long way, especially when the desired behavior can easily be tested for with static, single time-point assays. For more complex gene circuits with dynamic phenotypes that change over time, directed evolution approaches to controlling and tuning circuit behavior have been hindered by the lack of sufficiently high-throughput screening methods to isolate variants with desired characteristics. Here we utilize directed mutagenesis and multiplexed microfluidics to develop a workflow for creating, screening and tuning dynamic gene circuits that operate at the population level. Specifically, we create a mutant library of an existing oscillator, the synchronized lysis circuit, and tune its dynamics while uncovering principles regarding its behavior. Lastly, we utilize this directed evolution workflow to construct a new synchronized genetic oscillator that exhibits robust dynamics over long time scales. The "dark matter" of protein variants carries a distinct DNA signature and predicts damaging variant effects AO_SCPLOWBSTRACTC_SCPLOWSignatures of DNA motifs associated with distinct mutagenic exposures have been defined for somatic variants, but little is known about the consequences different mutational processes pose to the cell, especially how mutagens exert damage on specific proteins and their three-dimensional structures. Here we identify a DNA mutational signature which corresponds to damaging protein variants. We show that this mutational signature is under-sampled in sequencing data from tumour cohorts, constituting the "dark matter" of the mutational landscape which could only be accessed using deep mutational scanning (DMS) data. By training a set of gradient boosting classifiers, we illustrate that DMS data from only a handful ({approx} 10) of experiments can accurately predict variant impact, and that DNA mutational signatures embed information about the protein-level impact of variants. We bridge the gap between DNA sequence variations and protein-level consequences, discuss the significance of this signature in informing protein design and molecular principles of protein stability, and clarify the relationship between disease association and the true impact mutations bring to protein function. Extreme parallel evolution of flagellar motility facilitated by silent mutations Mutational hotspots can determine evolutionary outcomes and make evolution repeatable. Hotspots are products of multiple evolutionary forces including mutation rate heterogeneity, but this variable is often hard to identify. In this work we reveal that a powerfully deterministic genetic hotspot can be built and broken by a handful of silent mutations. We observed this when studying homologous immotile variants of the bacteria Pseudomonas fluorescens, AR2 and Pf0-2x. AR2 resurrects motility through highly repeatable de novo mutation of the same nucleotide in >95% lines in minimal media (ntrB A289C). Pf0-2x, however, evolves via a number of mutations meaning the two strains diverge significantly during adaptation. We determined that this evolutionary disparity was owed to just 6 synonymous variations within the ntrB locus, which we demonstrated by swapping the sites and observing that we were able to both break (>95% to 0% in AR2) and build (0% to 80% in Pf0-2x) a powerfully deterministic mutational hotspot. Our work reveals a fundamental role for silent genetic variation in determining adaptive outcomes. Heterogeneous recruitment abilities to RNA polymerases generate nonlinear scaling of gene expression with cell volume While most genes expression levels are proportional to cell volumes, some genes exhibit nonlinear scaling between their expression levels and cell volume. Therefore, their mRNA and protein concentrations change as the cell volume increases, which often have crucial biological functions such as cell-cycle regulation. However, the biophysical mechanism underlying the nonlinear scaling between gene expression and cell volume is still unclear. In this work, we show that the nonlinear scaling is a direct consequence of the heterogeneous recruitment abilities of promoters to RNA polymerases based on a gene expression model at the whole-cell level. Those genes with weaker (stronger) recruitment abilities than the average ability spontaneously exhibit superlinear (sublinear) scaling with cell volume. Analysis of the promoter sequences and the nonlinear scaling of Saccharomyces cerevisiaes mRNA levels shows that motifs associated with transcription regulation are indeed enriched in genes exhibiting nonlinear scaling, in concert with our model. Combined epigenetic and metabolic inhibition blocks platinum-induced ovarian cancer stem cell enrichment High grade serous ovarian cancer (HGSOC) is the most common and aggressive type of ovarian cancer. Platinum resistance is a common occurrence in HGSOC and a main cause of tumor relapse resulting in high patient mortality rates. Recurrent OC is enriched in aldehyde dehydrogenase (ALDH)+ ovarian cancer stem cells (OCSCs), which are resistant to platinum agents. We demonstrated that acute platinum treatment induced a DNA damage-dependent decrease in BRCA1 levels. In a parallel response associated with G2/M arrest, platinum treatment also induced an increase in expression of NAMPT, the rate limiting regulator of NAD+ production from the salvage pathway, and levels of NAD+, the cofactor required for ALDH activity. Concurrent inhibition of DNA methyltransferases (DNMTs) and NAMPT synergistically abrogated the platinum-induced increase in OCSCs. Combining pharmacological inhibitors of DNMT and NAMPT with carboplatin reduced tumorigenesis and OCSC percentage in vivo. We conclude that both epigenetic and metabolic alterations lead to platinum induced OCSC enrichment, providing preclinical evidence that in the neoadjuvant setting, combining DNMT and NAMPT inhibitors with platinum has the potential to reduce OC recurrence and avert the development of platinum resistance. Balancing sensitivity and specificity in preclinical research The success of scientific discovery in preclinical drug development is based on the different roles of exploration and confirmation. Exploration requires sensitivity while confirmation needs to be specific. Based on published empirical data, we simulated two preclinical research trajectories to examine their effectiveness to detect true effects while keeping animal numbers low. We compare a conventional p-value based approach with a method based on an a priori determined smallest effect size of interest (SESOI). Using a SESOI increases transition rates from exploration to confirmation and results in higher detection rates across the trajectory. Specificity in the SESOI trajectory increases if the prior probability of true effects is low. We conclude that employing a SESOI is superior to a p-value based approach. Based on our findings, we propose a reconsideration of planning and conducting preclinical experiments, especially when the prior probability of true hypotheses is low. A coupled mechano-biochemical framework for root meristem morphogenesis Plants grow roots to adjust their bodies to dynamic changes in the surrounding environment. How do plant roots emerge from the stem cell reservoir during embryogenesis is however poorly understood. Here, we present a bottom-up strategy to address this challenge by combining empirical observations with advanced computer modeling techniques. We demonstrate that the anisotropy of root growth results from differential growth rates of adjacent tissues, whereas the root meristem development incorporates a multi-level feedback loop between complex transport network of phytohormone auxin, auxin-dependent cell growth and cytoskeleton rearrangements. In silico model predictions are in close agreement with in vivo patterns of anisotropic growth, auxin distribution, and cell polarity, as well as several root phenotypes caused by chemical, mechanical, or genetic perturbations. Our findings reveal a minimal set of design principles connecting tissue mechanics, cell anisotropy, and directional transport that are sufficient for self-organization of the root meristem shape. A mobile auxin signal transported through immobile cells orchestrates polarity and growth mechanics to instruct the morphogenesis of an independent organ. Phagocytic Uptake of Polymeric Particles by Immune cells Under Flow Conditions Particles injected intravenously are thought to be cleared by macrophages residing in the liver and spleen, but they also encounter circulating immune cells. It remains to be established if the circulating cells can take up particles while flowing, and if the uptake capacity is similar under static and flow conditions. Here, we use an in vitro peristaltic pump setup that mimics pulsatile blood flow to determine if immune cells take up particles under constant fluidic flow. We use polystyrene particles of varying sizes as the model of a polymeric particle for these studies. Our results show that the immune cells do phagocytose under flow conditions. We demonstrate that cell lines representing myeloid cells, primary human neutrophils and monocytes take up sub-micrometer-sized particles at similar or better rates under flow compared to static conditions. Experiments with whole human blood show that even under the crowding effects of red blood cells, neutrophils and monocytes take up particles while flowing. Together, these data suggest that circulating immune cells are likely to phagocytose intravenously injected particulates, which has implications for design of particles to evade or target these cells. Do males know? Evidence-driven rainmaking mating strategy by male bonobos to meet the fertile window of females Female bonobos exhibit prolonged receptivity, one suggested function of which is to lower male mating competition. However, it is questionable whether easier access to receptive females can reduce male-male competition, given the exclusive nature of male reproductive success. We tested whether males could determine the fertile phase of females. We found that ovulation probability predicted male mating effort. High-ranking males copulated with females with higher fertility, and male-male agonistic interactions increased when there were fertile females in the party. When there were multiple females with maximal swelling, males concentrated their mating effort on females with an older infant whose maximal swelling started earlier, and they continued mating efforts until detumescence (rainmaking). These findings suggest that male bonobos distinguish between fertile and non-fertile phases of females and that having more receptive females in the party does not reduce male-male competition for fertile females.

TeaserMales use the rainmakers rule to meet the periovulatory phase of the female bonobo for better reproductive success. Bursting emerges from the complementary roles of neurons in a four-cell network Reciprocally inhibitory modules that form half-center oscillators require mechanisms for escaping or being released from inhibition. The central pattern generator (CPG) underlying swimming by the nudibranch mollusc, Dendronotus iris, is composed of only four neurons that are organized into two competing modules of a half center oscillator. In this system, bursting activity in left-right alternation is not driven by any of the neurons but is an emergent property of the network as a whole. We found that the unique synaptic actions and membrane properties of the two neurons in each module (Si2 and Si3) play complementary roles in generating stable bursting in this network oscillator. Only Si2 evokes fast and strong inhibition of its contralateral counterpart, the termination of which initiates post-inhibitory rebound in the Si3 of that module. Only Si3 is responsible for the rebound excitation because it has a hyperpolarization-activated inward current. Within each module, the synaptic actions and membrane properties of the two neurons complement each other: Si3 excites Si2, which then feeds back slow inhibition to Si3, terminating the burst. Using dynamic clamp, we showed that the magnitude of the slow inhibition sets the period of the oscillator. Thus, the synaptic actions of Si2 provide the hyperpolarization needed for the other module to rebound stably, whereas the membrane properties of Si3 in each module cause it to rebound first and excite Si2 and maintain the burst until the cycle repeats and the other module becomes active. Missing the sweet spot: one of the two N-glycans on human Gb3/CD77 synthase is expendable N-glycosylation is a ubiquitous posttranslational modification that may influence folding, subcellular localization, secretion, solubility and oligomerization of proteins. In this study, we examined the effects of N-glycans on the activity of human Gb3/CD77 synthase, which catalyzes the synthesis of glycosphingolipids with terminal Gal1[-&gt;]4Gal (Gb3 and the P1 antigen) and Gal1[-&gt;]4GalNAc disaccharides (the NOR antigen). The human Gb3/CD77 synthase contains two occupied N-glycosylation sites at positions N121 and N203. Intriguingly, we found that while the N-glycan at N203 is essential for activity and correct subcellular localization, the N-glycan at N121 is dispensable and its absence did not reduce, but, surprisingly, even increased the activity of the enzyme. The fully N-glycosylated human Gb3/CD77 synthase and its glycoform missing the N121 glycan correctly localized in the Golgi, whereas a glycoform without the N203 site partially mislocalized in the endoplasmic reticulum. A double mutein missing both N-glycans was inactive and accumulated in the endoplasmic reticulum. Our results suggest that the decreased specific activity of human Gb3/CD77 synthase glycovariants results from their improper subcellular localization and, to a smaller degree, a decrease in enzyme solubility. Taken together, our findings show that the two N-glycans of human Gb3/CD77 synthase have opposing effects on its properties, revealing a dual nature of N-glycosylation and potentially a novel regulatory mechanism controlling the biological activity of proteins. 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. Single-Cell Multiomics Reveals Distinct Cell States at the Top of the Human Hematopoietic Hierarchy The advent of single cell (Sc) genomics has challenged the dogma of haematopoiesis as a tree-like structure of stepwise lineage commitment through distinct and increasingly restricted progenitor populations. Instead, analysis of ScRNA-seq has proposed that the earliest events in human hematopoietic stem cell (HSC) differentiation are characterized by only subtle molecular changes, with hematopoietic stem and progenitor cells (HSPCs) existing as a continuum of low-primed cell-states that gradually transition into a specific lineage (CLOUD-HSPCs). Here, we combine ScRNA-seq, ScATAC-seq and cell surface proteomics to dissect the heterogeneity of CLOUD-HSPCs at different stages of human life. Within CLOUD-HSPCs, pseudotime ordering of both mRNA and chromatin data revealed a bifurcation of megakaryocyte/erythroid and lympho/myeloid trajectories immediately downstream a subpopulation with an HSC-specific enhancer signature. Importantly, both HSCs and lineage-restricted progenitor populations could be prospectively isolated based on correlation of their molecular signatures with CD35 and CD11A expression, respectively. Moreover, we describe the changes that occur in this heterogeneity as hematopoiesis develops from neonatal to aged bone marrow, including an increase of HSCs and depletion of lympho-myeloid biased MPPs. Thus, this study dissects the heterogeneity of human CLOUD-HSPCs revealing distinct HSPC-states of relevance in homeostatic settings such as ageing. Predictive uncertainty in mechanistic models of cellular processes calibrated to experimental data Mathematical models are often used to explore network-driven cellular processes from a systems perspective. However, a dearth of quantitative data suitable for model calibration leads to models with parameter unidentifiability and questionable predictive power. Here we introduce a Bayesian and Machine-Learning based Measurement Model approach to explore how quantitative and non-quantitative data constrain models of apoptosis execution within a missing data context. We find two orders of magnitude more ordinal (e.g. immunoblot) data are necessary to achieve accuracy comparable to quantitative (e.g. fluorescence) data. Notably, ordinal and nominal (e.g. immunostain) non-quantitative data synergize to reduce model uncertainty and improve accuracy. Further, model prediction accuracy and certainty strongly depend on rigorous data-driven formulations of the measurement, and the size and make-up of the datasets. Finally, we demonstrate the potential of a data-driven Measurement Model approach to identify model features that could lead to informative experimental measurements and improve model predictive power. A new method for the sampling and preservation of placental specimens in low-resource settings for the identification of P. falciparum and analysis of nucleic acids Collection, preservation, and shipment of histological specimens in low-resource settings is challenging. We present a novel method that achieved excellent preservation of placental specimens from rural Mali by using formalin fixation, ethanol dehydration, and long-term storage in a solar-powered freezer. Sample preservation success was 92%, permitting evaluation of current and past malaria infection, anemia, placental maturity, and inflammation. Using RNAscope(R) hybridization we were able to visualize cell-specific gene expression patterns in the formalin-fixed paraffin-embedded (FFPE) specimens. Additionally, our method entailed mirrored sampling from the two cut faces of a cotyledon, one for the FFPE workflows and the other for storage in RNAlater and RNA-seq. Improving human coronavirus OC43 (HCoV-OC43) research results comparability in studies using HCoV-OC43 as a surrogate for SARS-CoV-2 The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has renewed interest in human coronaviruses that cause the common cold, particularly as research with them at biosafety level (BSL)-2 avoids the added costs and biosafety concerns that accompany work with SARS-COV-2, BSL-3 research. One of these, human coronavirus OC43 (HCoV-OC43), is a well-matched surrogate for SARS-CoV-2 because it is also a Betacoronavirus, targets the human respiratory system, is transmitted via respiratory aerosols and droplets and is relatively resistant to disinfectants. Unfortunately, growth of HCoV-OC43 in the recommended human colon cancer (HRT-18) cells does not produce obvious cytopathic effect (CPE) and its titration in these cells requires expensive antibody-based detection. Consequently, multiple quantification approaches for HCoV-OC43 using alternative cell lines exist, which complicates comparison of research results. Hence, we investigated the basic growth parameters of HCoV-OC43 infection in three of these cell lines (HRT-18, human lung fibroblasts (MRC-5) and African green monkey kidney (Vero E6) cells) including the differential development of cytopathic effect (CPE) and explored reducing the cost, time and complexity of antibody-based detection assay. Multi-step growth curves were conducted in each cell type in triplicate at a multiplicity of infection of 0.1 with daily sampling for seven days. Samples were quantified by tissue culture infectious dose50(TCID50)/ml or plaque assay (cell line dependent) and additionally analyzed on the Sartorius Virus Counter 3100 (VC), which uses flow virometry to count the total number of intact virus particles in a sample. We improved the reproducibility of a previously described antibody-based detection based TCID50 assay by identifying commercial sources for antibodies, decreasing antibody concentrations and simplifying the detection process. The growth curves demonstrated that HCoV-O43 grown in MRC-5 cells reached a peak titer of [~]107 plaque forming units/ml at two days post infection (dpi). In contrast, HCoV-OC43 grown on HRT-18 cells required six days to reach a peak titer of [~]106.5 TCID50/ml. HCoV-OC43 produced CPE in Vero E6 cells but these growth curve samples failed to produce CPE in a plaque assay after four days. Analysis of the VC data in combination with plaque and TCID50 assays together revealed that the defective:infectious virion ratio of MRC-5 propagated HCoV-OC43 was less than 3:1 for 1-6 dpi while HCoV-OC43 propagated in HRT-18 cells varied from 41:1 at 1 dpi, to 329:4 at 4 dpi to 94:1 at 7 dpi. These results should enable better comparison of extant HCoV-OC43 study results and prompt further standardization efforts. Medium-throughput zebrafish optogenetic platform identifies deficits in subsequent neural activity following brief early exposure to cannabidiol and Δ-9-tetrahydrocannabinol In the light of legislative changes and the widespread use of cannabis as a recreational and medicinal drug, delayed effects of cannabis upon brief exposure during embryonic development are of high interest as early pregnancies often go undetected. Here, zebrafish embryos were exposed to cannabidiol (CBD) and {Delta}-9-tetrahydrocannabinol l (THC) until the end of gastrulation (1-10 hours post-fertilization) and analyzed later in development (4-5 days post-fertilization). In order to measure neural activity, we implemented CaMPARI (Calcium-Modulated Photoactivatable Ratiometric Integrator) and optimized the protocol for a 96-well format complemented by locomotor analysis. Our results revealed that neural activity was decreased by CBD more than THC. At higher doses, both cannabinoids could dramatically reduce neural activity and locomotor activity. Interestingly, the decrease was more pronounced when CBD and THC were combined. At the receptor level, CBD-mediated reduction of locomotor activity was partially prevented using cannabinoid type 1 and 2 receptor inhibitors. Overall, we report that CBD toxicity occurs via two cannabinoid receptors and is synergistically enhanced by THC exposure to negatively impact neural activity late in larval development. Future studies are warranted to reveal other cannabinoids and receptors involved in this pathway to understand the subsequent health implications of cannabis consumption on fetal development.