Hidden multivalency in phosphatase recruitment by a disordered AKAP scaffold Disordered scaffold proteins provide multivalent landing pads that, via a series of embedded Short Linear Motifs (SLiMs), bring together the components of a complex to orchestrate precise spatial and temporal regulation of cellular processes. One such protein is AKAP5, which contains SLiMs that anchor PKA and Calcineurin, and recruit substrate (the TRPV1 receptor). Calcineurin is anchored to AKAP5 by a well-characterised PxIxIT SLiM. Here we show, using a combination of biochemical and biophysical approaches, that the Calcineurin PxIxIT-binding groove also recognises several hitherto unknown lower-affinity SLiMs in addition to the PxIxIT motif. We demonstrate that the assembly is in reality a complex system with conserved SLiMs spanning a wide affinity range. The capture is analogous to that seen for many DNA-binding proteins that have a weak non-specific affinity for DNA outside the canonical binding site, but different in that it involves (i) two proteins, and (ii) hydrophobic rather than electrostatic interactions. It is also compatible with the requirement for both stable anchoring of the enzyme and responsive downstream signalling. We conclude that the AKAP5 C-terminus is enriched in lower-affinity/mini-SLiMs that, together with the canonical SLiM, maintain a structurally disordered but tightly regulated signalosome.
A Machine Learning Approach to Identify Small Molecule Inhibitors of Secondary Nucleation inalpha-Synuclein Aggregation Drug development is an increasingly active area of application of machine learning methods, due to the need to overcome the high attrition rates of conventional drug discovery pipelines. This issue is especially pressing for neurodegenerative diseases where very few disease-modifying drugs have been approved. To address this problem, we describe a machine learning approach to identify specific inhibitors of the proliferation of -synuclein aggregates through secondary nucleation, a process that has been implicated in Parkinsons disease and related synucleinopathies. We use a combination of docking simulations followed by machine learning to first identify initial hit compounds and then explore the chemical space around these compounds. Our results demonstrate that this approach leads to the identification of novel chemical matter with an improved hit rate and potency over conventional similarity search approaches.
Spatial control of cell division by GA-OsGRF7/8 module in a leaf explains the leaf length variation between cultivated and wild rice O_LICellular and genetic understanding of rice leaf size regulation is limited, despite rice being the staple food of more than half of the global population. We investigated the mechanism controlling the rice leaf length using cultivated and wild rice accessions that remarkably differed for leaf size.
C_LIO_LIComparative transcriptomics, Gibberellic Acid (GA) quantification, and leaf kinematics of the contrasting accessions suggested the involvement of GA, cell cycle, and Growth-Regulating Factors (GRFs) in the rice leaf size regulation. Zone-specific expression analysis and VIGS established the functions of specific GRFs in the process.
C_LIO_LIThe leaf length of the selected accessions was strongly correlated with GA levels. Higher GA content in wild rice accessions with longer leaves and GA-induced increase in the leaf length via an increase in cell division confirmed a GA-mediated regulation of division zone in rice. Downstream to GA, OsGRF7 and OsGRF8 function for controlling cell division to determine the rice leaf length.
C_LIO_LISpatial control of cell division to determine the division zone size mediated by GA and downstream OsGRF7 and OsGRF8 explains the leaf length differences between the cultivated and wild rice. This mechanism to control rice leaf length might have contributed to optimizing leaf size during domestication.
C_LI Toxicity Profile of Artesunate in Rats and Dogs AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSOBJECTIVESC_ST_ABSThe objectives of these studies were to investigate the toxicity, safety and toxicokinetics of single and multiple doses of artesunate for injection in rats and dogs.

METHODSSprague-Dawley rats and Beagle dogs were treated intravenously or intramuscularly for 28 consecutive days with doses of up to 30 mg/kg artesunate, evaluating toxicity, kinetics, genotoxicity, and cardiovascular and central nervous safety parameters after single and 4-week repeated administrations. Furthermore, respiratory parameters were evaluated after a single intravenous administration in rats.

RESULTSArtesunate was well tolerated with no mortality and only minor effects on clinical pathology parameters. Following repeated intramuscular administration, local reactions at the injection site became evident.

CONCLUSIONSThe results obtained in these studies support the safe use of artesunate in humans.

Subchronic data for repeated intravenous and intramuscular artesunate in animals are available in the literature for a maximum duration of 14-days. Published results of genotoxicity studies in vitro in bacteria or cells of animal or human origin and in vivo in animals are equivocal, with a couple of negative results following oral administration and positive results in human cells.

With the studies performed in rats and dogs for a treatment period of 28-days including measures of safety pharmacology, toxicokinetic evaluation and in vivo genotoxicity data, a comparison of toxicity and exposure of intravenous versus intramuscular artesunate for a prolonged treatment period is possible, delivering important information, when a decision between both routes has to be made for use in patients. Results of the peripheral blood micronucleus assay in rats were positive, suggesting genotoxic potential for the intravenous route of administration of artesunate, what is, beside patients, likewise relevant for production and health care personnel. A rationalized definition of tumor suppressor microRNAsexcludes miR-34a While several microRNAs (miRNAs) have been proposed to act as tumor suppressors, a consensual definition of tumor suppressing miRNAs is still missing. Similarly to coding genes, we propose that tumor suppressor miRNAs must show evidence of genetic or epigenetic inactivation in cancers, and exhibit an anti-tumorigenic (e.g., anti-proliferative) activity under endogenous expression levels. Here we observe that this definition excludes the most extensively studied tumor suppressor candidate miRNA, miR-34a. In analyzable cancer types, miR-34a does not appear to be down-regulated in primary tumors relatively to normal adjacent tissues. Deletion of miR-34a is occasionally found in human cancers, but it does not seem to be driven by an anti-tumorigenic activity of the miRNA, since it is not observed upon smaller, miR-34a-specific alterations. Its anti-proliferative action was observed upon large, supra-physiological transfection of synthetic miR-34a in cultured cells, and our data indicates that endogenous miR-34a levels do not have such an effect. Our results therefore argue against a general tumor suppressive function for miR-34a, providing an explanation to the lack of efficiency of synthetic miR-34a administration against solid tumors. Thermodynamic constraints on the assembly and diversity of microbial ecosystems are different near to and far from equilibrium Non-equilibrium thermodynamics has long been an area of substantial interest to ecologists because most fundamental biological processes, such as protein synthesis and respiration, are inherently energy-consuming. However, most of this interest has focused on developing coarse ecosystem-level maximisation principles, providing little insight into underlying mechanisms that lead to such emergent constraints. Microbial communities are a natural system to decipher this mechanistic basis because their interactions in the form of substrate consumption, metabolite production, and cross-feeding can be described explicitly in thermodynamic terms. Previous work has considered how thermodynamic constraints impact competition between pairs of species, but restrained from analysing how this manifests in complex dynamical systems. To address this gap, we develop a thermodynamic microbial community model with fully reversible reaction kinetics, which allows direct consideration of free-energy dissipation. This also allows species to interact via products rather than just substrates, increasing the dynamical complexity, and allowing a more nuanced classification of interaction types to emerge. Using this model, we find that community diversity increases with substrate lability, because greater free-energy availability allows for faster generation of niches. Thus, more niches are generated in the time frame of community establishment, leading to higher final species diversity. We also find that allowing species to make use of near-to-equilibrium reactions increases diversity in a low free-energy regime. In such a regime, two new thermodynamic interaction types that we identify here reach comparable strengths to the conventional (competition and facilitation) types, emphasising the key role that thermodynamics plays in community dynamics. Our results suggest that accounting for realistic thermodynamic constraints is vital for understanding the dynamics of real-world microbial communities.

Author summaryThere is a growing interest in microbial communities due to their important role in biogeochemical cycling as well as plant and animal health. Although our understanding of thermodynamic constraints on individual cells is rapidly improving, the impact of these constraints on complex microbial communities remains largely unexplored theoretically and empirically. Here, we develop a new microbial community model which allows thermodynamic efficiency and entropy production to be calculated directly. We find that availability of substrates with greater free-energy allows for a faster rate of niche generation, leading to higher final species diversity. We also show that when the free-energy availability is low, species with reactions close to thermodynamic equilibrium are favoured, leading to more diverse and efficient communities. In addition to the conventional interaction types (competition and facilitation), our model reveals the existence of two novel interaction types mediated by products rather than substrates. Though the conventional interactions are generally the strongest, the novel interaction types are significant when free-energy availability is low. Our results suggest that non-equilibrium thermodynamics need to be considered when studying microbial community dynamics. Dimerization of kringle 1 domain from hepatocyte growth factor/scatter factor provides a potent minimal MET receptor agonist. Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its cognate receptor MET play several essential roles in embryogenesis and regeneration in post-natal life of epithelial organs such as liver, kidney, lung, and pancreas, prompting a strong interest in harnessing HGF/SF-MET signalling for regeneration of epithelial organs after acute or chronic damage. The limited stability and tissue diffusion of native HGF/SF, however, which reflect the tightly controlled, local mechanism of action of the morphogen, have led to a major search for HGF/SF mimics for therapy. In this work, we describe the rational design, production and characterisation of K1K1, a novel minimal MET agonist consisting of two copies of the kringle 1 domain of HGF/SF placed in tandem. K1K1 is highly stable and displays biological activities equivalent or superior to native HGF/SF in a variety of in vitro assay systems and in a mouse model of liver disease. These data suggest that this engineered ligand may find wide applications in acute and chronic diseases of the liver and other epithelial organs dependent on MET activation. Retraction ATPase motors from three orthologous type IVa pilus systems support promiscuous retraction of the Vibrio cholerae competence pilus Bacterial surface appendages called type IVa pili (T4aP) promote diverse activities including DNA uptake, twitching motility, and virulence. These activities rely on the ability of T4aP to dynamically extend and retract from the cell surface. Dynamic extension relies on a motor ATPase commonly called PilB. Most T4aP also rely on specific motor ATPases, commonly called PilT and PilU, to dynamically and forcefully retract. Here, we systematically assess whether motor ATPases from three orthologous T4aP can functionally complement Vibrio cholerae mutants that lack their endogenous motors. We found that the PilT and PilU retraction ATPases from the three T4aP systems tested are promiscuous and promote retraction of the V. cholerae competence T4aP despite a high degree of sequence divergence. In contrast, the orthologous extension ATPases from the same T4aP systems were not able to mediate extension of the V. cholerae competence T4aP despite exhibiting a similar degree of sequence divergence. Also, we show that one of the PilT orthologs characterized does not support PilU-dependent retraction and provide some data to indicate that the C-terminus of PilT is important for PilU-dependent retraction. Together, our data suggest that retraction ATPases may have maintained a high degree of promiscuity for promoting retraction of T4aP, while extension ATPases may have evolved to become specific for their cognate systems.

IMPORTANCEOne way that bacteria interact with their environments is via hair-like appendages called type IVa pili (T4aP). These appendages dynamically extend and retract from the cell surface via the action of distinct ATPase motors. T4aP are present in diverse bacterial species. Here, we demonstrate that retraction motors from three T4aP are promiscuous, and capable of promoting retraction of a heterologous T4aP system. By contrast, the extension ATPase motors from these same T4aP systems are specific and cannot promote extension of a heterologous T4aP. Thus, these results suggest that T4aP extension may be more tightly regulated compared to T4aP retraction. Bioinformatic and mechanistic analysis of the palmerolide PKS-NRPS biosynthetic pathway from the microbiome of an Antarctic ascidian Complex interactions exist between microbiomes and their hosts. Increasingly, defensive metabolites that have been attributed to host biosynthetic capability are now being recognized as products of host-associated microbes. These unique metabolites often have bioactivity targets in human disease and can be purposed as pharmaceuticals. Polyketides are a complex family of natural products that often serve as defensive metabolites for competitive or pro-survival purposes for the producing organism, while demonstrating bioactivity in human diseases as cholesterol lowering agents, anti-infectives, and anti-tumor agents. Marine invertebrates and microbes are a rich source of polyketides. Palmerolide A, a polyketide isolated from the Antarctic ascidian Synoicum adareanum, is a vacuolar-ATPase inhibitor with potent bioactivity against melanoma cell lines. The biosynthetic gene clusters (BGCs) responsible for production of secondary metabolites are encoded in the genomes of the producers as discrete genomic elements. A candidate palmerolide BGC was identified from a S. adareanum microbiome-metagenome based on a high degree of congruence with a chemical structure-based retrobiosynthetic prediction. Protein family homology analysis, conserved domain searches, active site and motif identification were used to identify and propose the function of the [~]75 kb trans-acyltransferase (AT) polyketide synthase-non-ribosomal synthase (PKS-NRPS) domains responsible for the stepwise synthesis of palmerolide A. Though PKS systems often act in a predictable co-linear sequence, this BGC includes multiple trans-acting enzymatic domains, a non-canonical condensation termination domain, a bacterial luciferase-like monooxygenase (LLM), and is found in multiple copies within the metagenome-assembled genome (MAG). Detailed inspection of the five highly similar pal BGC copies suggests the potential for biosynthesis of other members of the palmerolide chemical family. This is the first delineation of a biosynthetic gene cluster from an Antarctic microbial species, recently proposed as Candidatus Synoicohabitans palmerolidicus. These findings have relevance for fundamental knowledge of PKS combinatorial biosynthesis and could enhance drug development efforts of palmerolide A through heterologous gene expression. Leveraging spiking deep neural networks to understand the neural mechanisms underlying selective attention Spatial attention enhances sensory processing of goal-relevant information and improves perceptual sensitivity. Yet, the specific neural mechanisms underlying the effects of spatial attention on performance are still contested. Here, we examine different attention mechanisms in spiking deep convolutional neural networks. We directly contrast effects of precision (internal noise suppression) and two different gain modulation mechanisms on performance on a visual search task with complex real-world images. Unlike standard artificial neurons, biological neurons have saturating activation functions, permitting implementation of attentional gain as gain on a neurons input or on its outgoing connection. We show that modulating the connection is most effective in selectively enhancing information processing by redistributing spiking activity, and by introducing additional task-relevant information, as shown by representational similarity analyses. Precision only produced minor attentional effects in performance. Our results, which mirror empirical findings, show that it is possible to adjudicate between attention mechanisms using more biologically realistic models and natural stimuli. A Novel Proposal for an Index for Regional Cerebral Perfusion Pressure - A Theoretical Approach Using Fluid Dynamics Cerebral blood flow (CBF) / cerebral blood volume (CBV) ratio derived by [15O] H2O/ CO2 and CO positron emission tomography (PET) examination has been used empirically as an index for cerebral perfusion pressure (CPP). However, this equation lacks a theoretical basis and furthermore, has not been confirmed to be proportionate to CPP, as direct measurement of local CPP is not practical.

We have developed a new index for CPP using the Poiseuille equation based on a simple model. Our model suggests that CBF/CBV2 is proportionate to CPP and that it is mathematically a more accurate index than CBF/CBV. The Inflammatory Role of Milk Fat Globule Epidermal Growth Factor VIII in Angiotensin II Induced Arterial Remodeling BackgroundAngiotensin II (Ang II) and milk fat globule-epidermal growth factor VIII (MFG-E8) are involved in age-associated arterial remodeling; however, the inflammatory role of MFG-E8 in Ang II associated arterial remodeling with aging remains unknown.

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

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

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


What Are the Clinical Implications?Since MFG-E8 mediates Ang II induced proinflammation in arterial wall remodeling in aging and hypertension, targeting MFG-E8 is a potential molecular approach to curb inflammatory arterial remodeling, maintaining the health of the vascular system during aging and hypertension. Thermal modulation of Zebrafish exploratory statistics reveals constraints on individual behavioral variability BackgroundVariability is a hallmark of animal behavior. It contributes to survival by endowing individuals and populations with the capacity to adapt to ever-changing environmental conditions. Intra-individual variability is thought to reflect both endogenous and exogenous modulations of the neural dynamics of the central nervous system. However, how variability is internally regulated and modulated by external cues remains elusive. Here we address this question by analyzing the statistics of spontaneous exploration of freely swimming zebrafish larvae, and by probing how these locomotor patterns are impacted when changing the water temperatures within an ethologically relevant range.

ResultsWe show that, for this simple animal model, five short-term kinematic parameters - interbout interval, turn amplitude, travelled distance, turn probability and orientational flipping rate - together control the long-term exploratory dynamics. We establish that the bath temperature consistently impacts the means of these parameters, but leave their pairwise covariance unchanged. These results indicate that the temperature merely controls the sampling statistics within a well-defined kinematic space delineated by this robust statistical structure. At a given temperature, individual animals explore the behavioral space over a timescale of tens of minutes, suggestive of a slow internal state modulation that could be externally biased through the bath temperature. By combining these various observations into a minimal stochastic model of navigation, we show that this thermal modulation of locomotor kinematics results in a thermophobic behavior, complementing direct gradient-sensing mechanisms.

ConclusionsThis study establishes the existence of a well-defined locomotor space accessible to zebrafish larvae during spontaneous exploration, and quantifies self-generated modulation of locomotor patterns. Intra-individual variability reflects a slow diffusive-like probing of this space by the animal. The bath temperature in turn restricts the sampling statistics to sub-regions, endowing the animal with basic thermophobicity. This study suggests that in Zebrafish, as well as in other ectothermic animals, ambient temperature could be used to efficiently manipulate internal states in a simple and ethological way. Synthetic periphyton as a model system to understand species dynamics in complex microbial freshwater communities Phototrophic biofilms, also known as periphyton, are microbial freshwater communities that drive crucial ecological processes in streams and lakes. Gaining a deep mechanistic understanding of the biological processes occurring in natural periphyton remains challenging due to the high complexity and variability of such communities. To address this challenge, we rationally developed a workflow to construct a synthetic community by co-culturing 26 phototrophic species (i.e., diatoms, green algae and cyanobacteria) that were inoculated in a successional sequence to create a periphytic biofilm on glass slides. We show that this community is diverse, stable and highly reproducible in terms of microbial composition, function and 3D spatial structure of the biofilm. We also demonstrate the ability to monitor microbial dynamics at the single species level during periphyton development and how their abundances are impacted by stressors such as increased temperature and a herbicide, singly and in combination. Overall, such a synthetic periphyton, grown under controlled conditions, can be used as a model system for theory testing through targeted manipulation. Seed banks alter the molecular evolutionary dynamics of Bacillus subtilis Fluctuations in the availability of resources constrains the growth and reproduction of individuals, which subsequently effects the evolution of their respective populations. Many organisms contend with such fluctuations by entering a reversible state of reduced metabolic activity, a phenomenon known as dormancy. This pool of dormant individuals (i.e., a seed bank) does not reproduce and is expected to act as an evolutionary buffer, though it is difficult to observe this effect directly over an extended evolutionary timescale. Through genetic manipulation, we analyze the molecular evolutionary dynamics of Bacillus subtilis populations in the presence and absence of a seed bank over 700 days. The ability of these bacteria to enter a dormant state increased the accumulation of genetic diversity over time and altered the trajectory of mutations, findings that were recapitulated using simulations based on a mathematical model of evolutionary dynamics. While the ability to form a seed bank did not alter the degree of negative selection, we found that it consistently altered the direction of molecular evolution across genes. Together, these results show that the ability to form a seed bank can affect the direction and rate of molecular evolution over an extended evolutionary timescale. METAbolomics data Balancing with Over-sampling Algorithms (META-BOA): an online resource for addressing class imbalance MotivationClass imbalance, or unequal sample sizes between classes, is an increasing concern in machine learning for metabolomic and lipidomic data mining, which can result in overfitting for the over-represented class. Numerous methods have been developed for handling class imbalance, but they are not readily accessible to users with limited computational experience. Moreover, there is no resource that enables users to easily evaluate the effect of different over-sampling algorithms.

ResultsMETAbolomics data Balancing with Over-sampling Algorithms (META-BOA) is a web-based application that enables users to select between four different methods for class balancing, followed by data visualization and classification of the sample to observe the augmentation effects. META-BOA outputs a newly balanced dataset, generating additional samples in the minority class, according to the users choice of Synthetic Minority Over-sampling Technique (SMOTE), Borderline-SMOTE (BSMOTE), Adaptive Synthetic (ADASYN), or Random Over-Sampling Examples (ROSE). META-BOA further displays both principal component analysis (PCA) and t-distributed stochastic neighbor embedding (t-SNE) visualization of data pre- and post-over-sampling. Random forest classification is utilized to compare sample classification in both the original and balanced datasets, enabling users to select the most appropriate method for their analyses.

Availability and implementationMETA-BOA is available at https://complimet.ca/meta-boa.

Supplementary InformationSupplementary material is available at Bioinformatics online. "Neighbourhood watch" model: embryonic epiblast cells assess positional information in relation to their neighbours In many developing and regenerating systems, tissue pattern is established through gradients of informative morphogens, but we know little about how cells interpret these. Using experimental manipulation of early chick embryos including misexpression of an inducer (VG1 or ACTIVIN) and an inhibitor (BMP4), we test two alternative models for their ability to explain how the site of primitive streak formation is positioned relative to the rest of the embryo. In one model, cells read morphogen concentrations cell-autonomously. In the other, cells sense changes in morphogen status relative to their neighbourhood. We find that only the latter model can account for the experimental results, including some counter-intuitive predictions. This mechanism (which we name "neighbourhood watch" model) illuminates the classic "French Flag Problem" and how positional information is interpreted by a sheet of cells in a large developing system.

Summary statementIn a large developing system, the chick embryo before gastrulation, cells interpret gradients of positional signals relative to their neighbours to position the primitive streak, establishing bilateral symmetry. First evidence of in vitro cytotoxic effects of marine microlitter on Merluccius merluccius and Mullus barbatus, two Mediterranean commercial fish species Marine litter is composed mainly of plastics and is recognized as a serious threats to marine ecosystems. Ecotoxicological approaches have started elucidating the potential severity of microplastics (MPs) in controlled laboratory studies with pristine materials but no information exist on marine environmental microlitter as a whole. Here, we characterized the litter in the coastal Northern Tyrrhenian sea and in the stomach of two fish species of socio-economic importance, and exposed primary cell cultures of mucosal and lymphoid organs to marine microlitter for evaluating possible cytotoxic effects. An average of 0.30 {+/-} 0.02 microlitter items m-3 was found in water samples. FT-IR analysis revealed that plastic particles, namely HDPE, polyamide and polypropylene were present in 100% and 83.3% of Merluccius merluccius and Mullus barbatus analyzed, which overall ingested 14.67 {+/-} 4.10 and 5.50 {+/-} 1.97 items/individual, respectively. Moreover, microlitter was confirmed as a vector of microorganisms. Lastly, the apical end-point of viability was found to be significantly reduced in splenic cells exposed in vitro to two microlitter conditions. Considering the role of the spleen in the mounting of adaptive immune responses, our results warrant more in-depth investigations for clarifying the actual susceptibility of these two species to anthropogenic microlitter.

HighlightsO_LI0.30 {+/-} 0.02 microlitter items m-3 were found at the surface of coastal Northern Tyrrhenian sea
C_LIO_LI14.67 {+/-} 4.10 and 5.50 {+/-} 1.97 items/individual were retrieved from the stomach of hakes and mullets
C_LIO_LIThe ingested microlitter contained plastic items
C_LIO_LIMicrolitter was validated as a carrier of bacteria, fungi and flagellates
C_LIO_LISplenic cells exposed to two microlitter conditions for 72 hours suffered cytotoxicity
C_LI FGFR2 fusion protein-driven mouse models of intrahepatic cholangiocarcinoma unveil a necessary role for Erk signaling Background and aimsAbout 15% of intrahepatic cholangiocarcinoma (iCCA) express fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs), most often in concert with mutationally inactivated TP53, CDKN2A or BAP1. FFs span residues 1-768 of FGFR2 fused to sequences encoded by any of a long list (>60) of partner genes, a configuration sufficient to ignite oncogenic FF activation. In line, FGFR-specific tyrosine kinase inhibitors (F-TKI) were shown to provide clinical benefit in FF+ iCCA, although responses were partial and/or limited by resistance mechanisms, including the FF V565F gatekeeper mutation. Herein we present an FF-driven murine iCCA model and exploit its potential for pre-clinical studies on FF therapeutic targeting.

MethodsFour iCCA FFs carrying different fusion sequences were expressed in Tp53-/- mouse liver organoids. Tumorigenic properties of genetically modified liver organoids were assessed by intrahepatic/subcutaneous transplantation in immuno-deficient mice. Cellular models derived from neoplastic lesions were exploited for pre-clinical studies.

ResultsTumors diagnosed as CCA were obtained upon transplantation of FF-expressing liver organoids. The penetrance of this tumorigenic phenotype was influenced by FF identity. Tumor organoids and 2D cell lines derived from CCA lesions were addicted to FF signaling via Ras-Erk, regardless of FF identity or presence of V565F mutation. Double blockade of FF-Ras-Erk pathway by concomitant pharmacological inhibition of FFs and Mek1/2 provided greater therapeutic efficacy than single agent F-TKI in vitro and in vivo.

ConclusionsFF-driven iCCA pathogenesis was successfully modelled in murine Tp53-/- background. This model revealed biological heterogeneity among structurally different FFs. Double blockade of FF-Erk signaling deserves consideration for improving precision-based approaches against human FF+ iCCA.

Abbreviations used in this paper: ANOVA, analysis of variance; Bap1, BRCA1-Associated-Protein 1; Cdkn2a, cyclin-dependent kinase inhibitor 2A; Cftr, cystic fibrosis transmembrane conductance regulator; Ck19, cytokeratin 19; Cyp3A, cytochrome P450, family 3, subfamily A; EGF, Epidermal growth factor; EGFR, Epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; Erk, extracellular signal-regulated kinase; FGFR2, fibroblast growth factor receptor 2; FRS2, fibroblast growth factor receptor substrate 2; GRB2, growth factor receptor-bound 2; GSEA, gene set enrichment analysis; GSVA, gene set variation analysis; H&E, hematoxylin and eosin; HepPar1, hepatocyte Paraffin 1; Hnf4-7, hepatocyte nuclear factor 4 alpha; Hprt, hypoxanthine-guanine phosphoribosyl transferase; LGR5, leucine-rich repeat-containing G-protein coupled receptor 5; NTRK, neurotrophic Tyrosine Kinase; Parp, Poly (ADP-ribose) polymerase; RECIST, response evaluation criteria in solid tumors; SHP2, Src homology phosphatase 2; Ttr, transthyretin. Polycomb-mediated repression of paternal chromosomes maintains haploid dosage in diploid embryos of Marchantia Complex mechanisms regulate gene dosage throughout eukaryotic life cycles. Mechanisms controlling gene dosage have been extensively studied in animals, however it is unknown how generalizable these mechanisms are to diverse eukaryotes. Here, we use the haploid plant Marchantia polymorpha to assess gene dosage control in its short-lived diploid embryo. We show that throughout embryogenesis, paternal chromosomes are repressed resulting in functional haploidy. The paternal genome is targeted for genomic imprinting by the Polycomb mark H3K27me3 starting at fertilization, rendering the maternal genome in control of embryogenesis. Maintaining haploid gene dosage by this new form of imprinting is essential for embryonic development. Our findings illustrate how haploid-dominant species can regulate gene dosage through paternal chromosome inactivation and initiates the exploration of the link between life cycle history and gene dosage in a broader range of organisms. Affordable Caenorhabditis elegans tracking system for classroom use For decades the nematode C. elegans has served as an outstanding research organism owing to its unsurpassed experimental amenability. This advantage has also made this tiny worm an attractive vehicle for science instruction across higher learning institutions. However, the prohibitive cost associated with the automated behavioral assessment of these animals remains an obstacle preventing their full adoption in undergraduate and high school settings. To improve this situation, we developed an inexpensive worm tracking system for use by high school interns and undergraduate students. Over the past two years this tracker has been successfully used by undergraduate students in our introductory Cell and Molecular lab (BSC220) at Illinois State University. Here we describe and demonstrate the use of our inexpensive worm tracking system. Vesalius: high-resolution in silico anatomization of Spatial Transcriptomic data using Image Analysis Characterization of tissue architecture promises to deliver insights into development, cell communication and disease. In silico spatial domain retrieval methods have been developed for spatial transcriptomics (ST) data assuming transcriptional similarity of neighboring barcodes. However, domain retrieval approaches with this assumption cannot work in complex tissues composed of multiple cell types. This task becomes especially challenging in cellular resolution ST methods. We developed Vesalius to decipher tissue anatomy from ST data by applying image processing technology. Vesalius uniquely detected territories composed of multiple cell types and successfully recovered tissue structures in high-resolution ST data including in mouse brain, embryo, liver, and colon. Utilizing this tissue architecture, Vesalius identified tissue morphology specific gene expression and regional specific gene expression changes for astrocytes, interneuron, oligodendrocytes, and entorhinal cells in the mouse brain. A liquid +TIP-network regulates microtubule dynamics through tubulin condensation Regulation of microtubule dynamics is essential for diverse cellular functions, and proteins that bind to dynamic microtubule ends can regulate network dynamics. Here we show, that two conserved microtubule end-binding proteins, CLIP-170 and EB3, undergo phase separation and form dense liquid-networks. When CLIP-170 and EB3 act together the multivalency of the network increases, which synergistically increases the amount of protein in the dense phase. In vitro and in cells these liquid networks can condense tubulin. In vitro in the presence of microtubules, EB3/CLIP-170 phase separation can co-condense tubulin all along the microtubule. At this condition microtubule growth speed increases up to two-fold and depolymerization events are strongly reduced, compared to conditions with phase separation deficient networks. Our data show that phase separated EB3/CLIP-170 networks impact microtubule growth dynamics beyond direct protein-microtubule interactions. Chimeric mRNA based COVID-19 vaccine induces protective immunity against Omicron and Delta The emerging SARS-CoV-2 variants of concern (VOCs) exhibit enhanced transmission and immune escape, reducing the efficacy and effectiveness of the two FDA-approved mRNA vaccines. Here, we explored various strategies to develop novel mRNAs vaccines to achieve safer and wider coverage of VOCs. Firstly, we constructed a cohort of mRNAs that feature a furin cleavage mutation in the spike (S) protein of predominant VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2). Not present in the mRNA vaccines currently in use, the mutation abolished the cleavage between the S1 and S2 subunits, potentially enhancing the safety profile of the immunogen. Secondly, we systematically evaluated the induction of neutralizing antibodies (nAb) in vaccinated mice, and discovered that individual VOC mRNAs elicited strong neutralizing activity in a VOC-specific manner. Thirdly, the IgG produced in mice immunized with Beta-Furin and Washington (WA)-Furin mRNAs showed potent cross-reactivity with other VOCs, which was further corroborated by challenging vaccinated mice with the live virus of VOCs. However, neither WA-Furin nor Beta-Furin mRNA elicited strong neutralizing activity against the Omicron variant. Hence, we further developed an Omicron-specific mRNA vaccine that restored protection against the original and the sublineages of Omicron variant. Finally, to broaden the protection spectrum of the new Omicron mRNA vaccine, we tested the concept of bivalent immunogen. Instead of just fusing two RBDs head-to-tail, we for the first time constructed an mRNA-based chimeric immunogen by introducing the RBD of Delta variant into the entire S antigen of Omicron. The resultant chimeric mRNA was capable of inducing potent and broadly acting nAb against Omicron (both BA.1 and BA.2) and Delta, which paves the way to develop new vaccine candidate to target emerging variants in the future. 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. Multi-omics-based label-free metabolic flux inference reveals obesity-associated dysregulatory mechanisms in liver glucose metabolism Glucose homeostasis is maintained by modulation of metabolic flux. Enzymes and metabolites regulate the involved metabolic pathways. Dysregulation of glucose homeostasis is a pathological event in obesity. Analyzing metabolic pathways and the mechanisms contributing to obesity-associated dysregulation in vivo is challenging. Here, we introduce OMELET: Omics-Based Metabolic Flux Estimation without Labeling for Extended Trans-omic Analysis. OMELET uses metabolomic, proteomic, and transcriptomic data to identify changes in metabolic flux, and to quantify contributions of metabolites, enzymes, and transcripts to the changes in metabolic flux. By evaluating the livers of fasting ob/ob mice, we found that increased metabolic flux through gluconeogenesis resulted primarily from increased transcripts, whereas that through the pyruvate cycle resulted from both increased transcripts and changes in substrates of metabolic enzymes. With OMELET, we identified mechanisms underlying the obesity-associated dysregulation of metabolic flux in liver.

HighlightsO_LIWe created OMELET to infer metabolic flux and its regulation from multi-omic data.
C_LIO_LIGluconeogenic and pyruvate cycle fluxes increased in fasting ob/ob mice.
C_LIO_LITranscripts increases mediated the increase in gluconeogenic fluxes in ob/ob mice.
C_LIO_LIIncreases in transcripts and substrates enhanced pyruvate cycle flux in ob/ob mice.
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