Extracellular matrix and spores impart structural hierarchy and compositional heterogeneity in Bacillus subtilis biofilms Biofilms are multicellular microbial communities that encase themselves in an extracellular matrix (ECM) of secreted biopolymers and attach to surfaces and interfaces. Bacterial biofilms are detrimental in hospital and industrial settings, but they can be beneficial in agricultural contexts. An essential property of biofilms that grants them with increased survival relative to planktonic cells is phenotypic heterogeneity; the division of the biofilm population into functionally distinct subgroups of cells. Phenotypic heterogeneity in biofilms can be traced to the cellular level, however, the molecular structures and elemental distribution across whole biofilms as well as possible linkages between them remain unexplored. Mapping X-ray diffraction (XRD) across intact biofilms in time and space, we revealed the dominant structural features in Bacillus subtilis biofilms, stemming from matrix components, spores and water. By simultaneously following the X-ray fluorescence (XRF) signal of biofilms and isolated matrix components, we discovered that the ECM preferentially binds calcium ions over other metal ions, specifically, zinc, manganese and iron. These ions, remaining free to flow below macroscopic wrinkles that act as water channels, eventually accumulate and lead to sporulation. The possible link between ECM properties, regulation of metal ion distribution and sporulation across whole intact biofilms unravels the importance of molecular-level heterogeneity in shaping biofilm physiology and development.

Significance StatementBiofilms are multicellular soft microbial communities that are able to colonize synthetic surfaces as well as living organisms. To survive sudden environmental changes and efficiently share their common resources, cells in a biofilm divide into subgroups with distinct functions, leading to phenotypic heterogeneity. Here, by studying intact biofilms by synchrotron X-ray diffraction and fluorescence, we revealed correlations between biofilm macroscopic architectural heterogeneity and the spatio-temporal distribution of extracellular matrix, spores, water and metal ions. Our findings demonstrate that biofilm heterogeneity is not only affected by local genetic expression and cellular differentiation, but also by passive effects resulting from the physicochemical properties of the molecules secreted by the cells, leading to differential distribution of nutrients that propagates through macroscopic length scales.
Structure-guided synthesis of FK506 and FK520 analogs with increased selectivity exhibit in vivo therapeutic efficacy against Cryptococcus Calcineurin is an essential virulence factor that is conserved across human fungal pathogens including Cryptococcus neoformans, Aspergillus fumigatus, and Candida albicans. Although an excellent target for antifungal drug development, the serine-threonine phosphatase activity of calcineurin is conserved in mammals and inhibition of this activity results in immunosuppression. FK506 (tacrolimus) is a naturally produced macrocyclic compound that inhibits calcineurin by binding to the immunophilin FKBP12. Previously, our fungal calcineurin-FK506-FKBP12 structure-based approaches identified a non-conserved region of FKBP12 that can be exploited for fungal-specific targeting. These studies led to the design of an FK506 analog, APX879, modified at the C22 position that was less immunosuppressive yet maintained antifungal activity. We now report high resolution protein crystal structures of fungal FKBP12 and a human truncated calcineurin-FKBP12 bound to a natural FK506 analog, FK520 (ascomycin). Based on information from these structures and the success of APX879, we synthesized and screened a novel panel of C22-modified compounds derived from both FK506 and FK520. One compound, JH-FK-05 demonstrates broad-spectrum antifungal activity in vitro and is non-immunosuppressive in vivo. In murine models of pulmonary and disseminated C. neoformans infection, JH-FK-05 treatment significantly reduced fungal burden and extended animal survival alone and in combination with fluconazole. Furthermore, molecular dynamic simulations performed with JH-FK-05 binding to fungal and human FKBP12 identified additional residues outside of the C22 and C21 positions that could be modified to generate novel FK506 analogs with improved antifungal activity.

SignificanceDue to rising rates of antifungal drug resistance and a limited armamentarium of antifungal treatments, there is a paramount need for novel antifungal drugs to treat systemic fungal infections. Calcineurin has been established as an essential and conserved virulence factor in several fungi, making it an attractive antifungal target. However, due to the immunosuppressive action of calcineurin inhibitors, they have not been successfully utilized clinically for antifungal treatment in humans. Recent availability of crystal structures of fungal calcineurin bound inhibitor complexes have enabled the structure-guided design of FK506 analogs and led to a breakthrough in the development of a compound increased for fungal specificity. The development of a calcineurin inhibitor with reduced immunosuppressive activity and therapeutic antifungal activity would add a significant tool to the treatment options for these invasive fungal infections with exceedingly high rates of mortality.
Conserved organ-specific microbial assemblages in different populations of a terrestrial crab Brachyuran crabs originated in the oceans and evolved specific morphological and physiological adaptations to live in freshwater, intertidal and even terrestrial habitats but the role of a selection mechanism involving symbiotic microorganisms long these colonization processes are not known. In this work we investigated the associated microbiota of three populations of a terrestrial brachyuran crab, Chiromantes haematocheir, to find evidence of a conserved crab-specific microbiome unrelated to the population of origin and dissimilar from environmental microbial assemblages. Bacterial 16S rRNA gene and fungal ITS sequences were obtained from selected crab organs and environmental matrices to profile microbial communities. In spite of the presence of truly marine larval stages and no gregarious behaviour, favouring microbiota exchanges, we found common, organ-specific microbiota, associated to the gut and the gills of the crabs (with more than 15% of the genera detected specifically enriched only in one organ). Our results suggest an early establishment of a new common, stable microbiota in the transition from water to land. Cell Intrinsic Control of Death and Proliferation of Lymphocytes in Immune Response and Tumor Cells by pH Many biological processes are controlled by cell death and proliferation. The present study found that intracellular pH positively correlated with proliferation but negatively with apoptosis. Acid treatments lowered intracellular pH and preferentially depleted highly proliferating lymphocytes, whereas alkaline treatments increased intracellular pH and enhanced lymphocyte proliferation. Likewise, acid treatments induced apoptosis and alkaline treatments maintained proliferation of tumor cells. These findings support a unified theory of regulation of cell death and proliferation where an intracellular pH balance controls both events, and the mitochondria act as the pH-stats of the cell. Accordingly, the Warburg effect is explained as necessary for proliferating cells to have a high intracellular pH environment to accelerate proliferation and avoid low pH-induced death. PIN-FORMED1 polarity in the shoot is insensitive to the polarity of neighbouring cells In plants, epidermal cells are planar-polarized along an axis marked by the asymmetric localization patterns of several proteins including PIN-FORMED1 (PIN1), which facilitates the directional efflux of the plant hormone auxin to pattern phyllotaxis (Heisler et al., 2010; Mansfield et al., 2018; Reinhardt et al., 2003). While PIN1 polarity is known to be regulated non-cell autonomously via the MONOPTEROS (MP) transcription factor, how this occurs has not been determined (Bhatia et al., 2016). Here we use mosaic expression of the serine threonine kinase PINOID (PID) to test whether PIN1 polarizes according to the polarity of neighbouring cells. Our findings reveal that PIN1 is insensitive to the polarity of PIN1 in neighbouring cells arguing against auxin flux or extracellular auxin concentrations acting as a polarity cue, in contrast to previous model proposals (Abley et al., 2016; Stoma et al., 2008). Correlation between speed and turning naturally arises for sparsely sampled cell movements Mechanisms regulating cell movement are not fully understood. One feature of cell movement that determines how far cells displace from an initial position is persistence, the ability to perform movements in a direction similar to the previous movement direction. Persistence is thus determined by turning angles between two sequential displacements and be characterized by an average turning angle or persistence time. Recent studies found that a cells average speed and turning are negatively correlated, suggesting a fundamental cell-intrinsic program whereby cells with a lower turning ability (i.e., larger persistence time) are intrinsically faster (or faster cells turn less). By simulating correlated or persistent random walks (PRWs) using two different frameworks (one based on von Mises-Fisher (vMF) distribution and another based on Ornstein-Uhlenbeck (OU) process) we show that the negative correlation between speed and turning naturally arises when cell trajectories are sub-sampled, i.e., when the frequency of sampling is lower than frequency at which cells make movements. This effect is strongest when the sampling frequency is on the order of magnitude with the typical cell persistence time and when cells vary in persistence time. Both conditions are observed for datasets of T cell movements in vivo that we have analyzed. In simulations the correlation arises due to randomness of cell movements resulting in highly variable persistence times for individual cells that with sub-sampling leads to large variability of average cell speeds. Interestingly, previously suggested methodology of calculating displacement of cell cohorts with different speeds resulted in similar results whether or not there is a cell-intrinsic correlation between cell speed and persistence. For both vMF- and OU-based simulations of PRWs we could find parameter values (distribution of persistence times, speeds, and sampling frequency) that matched experimentally measured correlations between speed and turning for two datasets of T cell movement in vivo suggesting that such simple correlations are not fully informative on the intrinsic link between speed and persistence. Our results thus suggest that sub-sampling may contribute to (and perhaps fully explains) the observed correlation between speed and turning at least for some cell trajectory data and emphasize the role of sampling frequency in inference of critical cellular parameters of cell motility such as speeds.

Secondary AbstractMeasurement of cell movements often results in a negative correlation between average speed and average turning angle suggesting an existence of a universal, cell-intrinsic movement program. We show that such a negative correlation may arise if cells in the population differ in their ability for persistent movement when the movement data are sub-sampled. We show that sub-sampling of cell trajectories generated using two different frameworks of persistent random walk can match the experimentally observed correlation between speed and turning for T cell movements in vivo. The isoforms of pyruvate kinase act as nutrient sensors for the β-cell KATP channel Insulin release from pancreatic {beta}-cells is triggered by the metabolism-dependent closure of ATP-sensitive K+ (KATP) channels. Here, we used isoform-specific deletion of pyruvate kinase (PK) to identify the unique roles of PKm1 and PKm2 in the {beta}-cell nutrient response. In the absence of PK activity, cytosolic ATP/ADP was insufficient to close KATP channels. Both PK isoforms were found on the plasma membrane, where they endow KATP channels with nutrient sensitivity and specificity. Although PKm1 accounts for >90% of cellular activity, PKm2 controls glucosedependent KATP closure. Amino acids, which generate mitochondrial phosphoenolpyruvate (PEP) without allosterically activating PKm2, depend on PKm1 to raise ATP/ADP and close KATP channels. Shifting the {beta}-cell from PKm2 to PKm1 correspondingly increased amino acid sensitivity and boosted secretory output. In addition to these membrane depolarizing "on-switches," {beta}-cell deletion of PCK2 exposed a mitochondrial PEP-dependent "off-switch" that facilitates Ca2+ extrusion. Restricting PCK2 prolonged Ca2+ influx and increased insulin secretion. In summary, it is not the KATP channel alone, but the PK-KATP microcompartment that confers nutrient sensitivity to the {beta}-cell plasma membrane. The differential response of the PK isoforms to the glycolytic and mitochondrial sources of PEP influences {beta}-cell nutrient preference, and controls the oscillatory cycle regulating insulin secretion. Co-option of immune effectors by the hormonal signalling system triggering metamorphosis in Drosophila melanogaster Insect metamorphosis is regulated by the production, secretion and degradation of two peripheral hormones: 20-hydroxyecdysone (ecdysone) and juvenile hormone (JH). In addition to their roles in developmental regulation, increasing evidence suggests that these hormones are involved in innate immunity processes, such as phagocytosis and the induction of antimicrobial peptide (AMP) production. AMP regulation includes systemic responses as well as local responses at surface epithelia that contact with the external environment. At pupariation, Drosophila melanogaster increases dramatically the expression of three AMP genes, drosomycin (drs), drosomycin-like 2 (drsl2) and drosomycin-like 5 (drsl5). We show that the systemic action of drs at pupariation is dependent on ecdysone signalling in the fat body and operates via the ecdysone downstream target, Broad-Z4. In parallel, ecdysone also regulates local responses, specifically through the activation of drsl2 expression in the gut. Finally, we confirm the relevance of this ecdysone dependent AMP expression for the control of bacterial persistence by showing that flies lacking drs expression in the fat body have higher bacterial persistence over metamorphosis. Together, our data emphasize the importance of the association between ecdysone signalling and immunity using in vivo studies and establish a new role for ecdysone at pupariation, which impacts developmental success by regulating the immune system in a stage-dependent manner. We speculate that this co-option of immune effectors by the hormonal system may constitute a pre-emptive mechanism to control bacterial numbers in the pupa at the core of metamorphosis evolution. Non-invasive imaging of gene expression and protein secretion dynamics in living mice: identification of ectopic prothrombin expression as driver of thrombosis in cancer The topology of gene expression and protein localization is a crucial characteristic of life, where the spatiotemporal dynamic of secretory proteins instruct higher order organization, including the orchestration of developmental and adaptive programs. However tools to non-invasively interrogate the fate of secretory proteins in vivo are scarce. Here we introduce a genetic tagging strategy for in vivo imaging of the secretion and expression dynamics of secretory proteins in living animals. Applying this to a prototypical liver-derived secretory protein, we demonstrate that this approach, combined with optical in-vivo imaging, uncovers extrahepatic prothrombin expression in multiple novel anatomical sites (including testes, placenta, brain, kidney, heart and lymphatic system) and in emerging tumors, resulting in significant amounts of tumor-derived prothrombin in the blood with procoagulant properties. Syngeneic cell lines from this mouse model enable unravelling regulatory mechanisms in high resolution, and in a scalable format ex vivo. Beyond discovering new functions of proteins in a targeted manner, this model allows identifying rheostats in the cross-talk between gene expression and availability of a secretory protein. It is also a valuable resource for uncovering novel (tissue-specific) therapeutic vulnerabilities. Analysis of science journalism reveals gender and regional disparities in coverage Science journalism is a critical way in which the public can remain informed and benefit from new scientific findings. Such journalism also shapes the publics view of the current state of scientific findings and legitimizes experts. Those covering science can only cite and quote a limited number of sources. Sources may be identified by the journalists research or by recommendations by other scientists. In both cases, biases may influence who is identified and ultimately included as an expert. To analyze possible biases in science journalism, we analyzed 22,001 non-research articles published by Nature. We chose to analyze Nature non-research articles since its research articles provide a natural comparator. Our analysis considered two possible sources of disparity: gender and name origin. To explore these sources of disparity, we extracted cited authors names as well as extracted names of quoted speakers. While citations and quotations within a piece do not reflect the entire information-gathering process, they can provide insight into the demographics of visible sources. We then used the extracted names to predict gender and name origin of the cited authors and speakers.

In order to appropriately quantify the level of difference, we must identify a suitable reference set for comparison. We chose first and last authors within primary research articles in Nature and a subset of Springer Nature articles in the same time period as our comparator. In our analysis, we found a skew towards male quotation in Nature science journalism-related articles. However, quotation is trending toward equal representation at a faster rate than first and last authorship in academic publishing. Interestingly, we found that the gender disparity in Nature quotes was column-dependent, with the "Career Features" column reaching gender parity. Our name origin analysis found a significant over-representation of names with predicted Celtic/English origin and under-representation of names with a predicted East Asian origin. This finding was observed both in extracted quotes and journal citations, but dampened in citations. Unevolved proteins from modern and prebiotic amino acids manifest distinct structural profiles Natural proteins represent numerous but tiny structure/function islands in a vast ocean of possible protein sequences, most of which has not been explored by either biological evolution or research. Recent studies have suggested this uncharted sequence space possesses surprisingly high structural propensity, but development of an understanding of this phenomenon has been awaiting a systematic high-throughput approach.

Here, we designed, prepared, and characterized two combinatorial protein libraries consisting of randomized proteins, each 105 residues in length. The first library constructed proteins from the entire canonical alphabet of 20 amino acids. The second library used a subset of only 10 residues (A,S,D,G,L,I,P,T,E,V) that represent a consensus view of plausibly available amino acids through prebiotic chemistry. Our study shows that compact conformations resistant to proteolysis are (i) abundant (up to 40%) in random sequence space, (ii) independent of general Hsp70 chaperone system activity, and (iii) not granted solely by "late" and complex amino acid additions. The Hsp70 chaperone system effectively increases solubility and refoldability of the canonical alphabet but has only a minor impact on the "early" library. The early alphabet proteins are inherently more soluble and refoldable, possibly assisted by the cell-like environment in which these assays were performed.

Our work indicates that both early and modern amino acids are predisposed to supporting protein structure (either in forms of oligomers or globular/molten globule structures) and that protein structure may not be a unique outcome of evolution. Indirect restoration of ecological interactions: reintroduction of a dung-beetle associated primate increases the recruitment of large seeds The biased loss of large and medium frugivores alters seed dispersal and plant regeneration. Species reintroductions have been proposed as a strategy to reverse the consequences of species loss. However, the effects of reintroductions on ecological processes are seldom accessed, which hinders the comprehension of reintroductions potential to reestablish functioning ecosystems. In this study, we investigate the effect of howler monkey (Alouatta guariba) reintroduction on the plant regeneration of Tijuca National Park (TNP), a defaunated Atlantic Forest fragment. Howlers are folivore-frugivore primates, whose large clumped defecations attract dung beetles, which provide secondary dispersal by burying seeds present in the howlers feces. Thus, we expect that the fate of seeds dispersed by howlers will differ from those dispersed by other frugivores present in the Park. We followed the fate of seeds between 3 and 14mm in diameter in three steps of the seed dispersal loop, each one consisting of a different experiment. First, we estimated secondary seed dispersal and burial depth probabilities according to the frugivores defecation pattern; then, predation probability in different burial depths and defecation patterns; and, finally, recruitment probability in different burial depths. Considering the final result of the three experiments, the howlers reintroduction affected positively the regeneration of large seeds. The fate of 3mm seeds was little affected because they were seldom preyed upon at shallower depths anyway and could not recruit when deeply buried. On the other hand, seeds larger than 3mm reached the seedling stage more frequently when dispersed by howlers than when dispersed by other animals present in the Park. Thus, howler monkey reintroduction in defaunated areas, in addition to smaller frugivores, whose defecation patterns are less attractive for dung beetles, improves the regeneration of large seeds. We hope that this study will stimulate new howler reintroductions in defaunated areas. Vitamin C Binds to SARS Coronavirus-2 Main Protease Essential for Viral Replication There is an urgent need for anti-viral agents that treat and/or prevent Covid-19 caused by SARS-Coronavirus (CoV-2) infections. The replication of the SARS CoV-2 is dependent on the activity of two cysteine proteases, a papain-like protease, PL-pro, and the 3C-like protease known as main protease Mpro or 3CLpro. The shortest and the safest path to clinical use is the repurposing of drugs with binding affinity to PLpro or 3CLpro that have an established safety profile in humans. Several studies have reported crystal structures of SARS-CoV-2 main protease in complex with FDA approved drugs such as those used in treatment of hepatitis C. Here, we report the crystal structure of 3CLpro in complex Vitamin C (L-ascorbate) bound to the proteins active site at 2.5 [A]ngstrom resolution. The crystal structure of the Vitamin C 3CLpro complex may aid future studies on the effect of Vitamin C not only on the coronavirus main protease but on related proteases of other infectious viruses. 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. Integrated quality control of allele-specific copy numbers, mutations and tumour purity from cancer whole genome sequencing assays Cancer genomes contain thousands of somatic point mutations, chromosome copy alterations and more complex structural variants, which contribute to tumour growth and therapy response. Whole genome sequencing is a well established approach for somatic variant identification, but its broad application comes with complications, particularly in how proposed calls are quality assessed. To address this issue, we present CNAqc, a quantitative framework to quality control somatic mutations and allele-specific copy numbers, both in clonal and subclonal settings while accounting for variations in tumour purity, as commonly seen in bulk sampling. We test the model via extensive simulations, validate it using low-pass single-cell data, and apply it to 2778 single-sample PCAWG whole-genomes, 10 in-house multi-region whole-genomes and 48 TCGA whole-exomes. CNAqc is compatible with common bioinformatic pipelines and designed to support automated parameterization processes that are crucial in the era of large-scale whole genome sequencing. New Segmentation and Feature Extraction Algorithm for Classification of White Blood Cells in Peripheral Smear Images This article addresses a new method for the classification of white blood cells (WBCs) using image processing techniques and machine learning methods. The proposed method consists of three steps: detecting the nucleus and cytoplasm, extracting features, and classification. At first, a new algorithm is designed to segment the nucleus. For the cytoplasm to be detected, only a part of it located inside the convex hull of the nucleus is involved in the process. This attitude helps us overcome the difficulties of segmenting the cytoplasm. In the second phase, three shapes and four novel color features are devised and extracted. Finally, by using an SVM model, the WBCs are classified. The segmentation algorithm can detect the nucleus with a dice similarity coefficient of 0.9675. The proposed method can categorize WBCs in Raabin-WBC, LISC, and BCCD datasets with accuracies of 94.65 %, 92.21 %, and 94.20 %, respectively. It is worth mentioning that the hyperparameters of the classifier are fixed only with the Raabin-WBC dataset, and these parameters are not readjusted for LISC and BCCD datasets. The obtained results demonstrate that the proposed method is robust, fast, and accurate. Second Heart Field-derived Cells Contribute to Angiotensin II-mediated Ascending Aortopathies BackgroundThe ascending aorta is a common location for aneurysm and dissection. This aortic region is populated by a mosaic of medial and adventitial cells that are embryonically derived from either the second heart field (SHF) or the cardiac neural crest. SHF-derived cells populate areas that coincide with the spatial specificity of thoracic aortopathies. The purpose of this study was to determine whether and how SHF-derived cells contribute to ascending aortopathies.

MethodsAscending aortic pathologies were examined in patients with sporadic thoracic aortopathies and angiotensin II (AngII)-infused mice. Ascending aortas without overt pathology from AngII-infused mice were subjected to mass spectrometry assisted proteomics, and molecular features of SHF-derived cells were determined by single cell transcriptomic analyses. Genetic deletion of either low-density lipoprotein receptor-related protein 1 (Lrp1) or transforming growth factor-{beta} receptor 2 (Tgfbr2) in SHF- derived cells was conducted to examine the impact of SHF-derived cells on vascular integrity.

ResultsPathologies in human ascending aortic aneurysmal tissues were predominant in outer medial layers and adventitia. This gradient was mimicked in mouse aortas following AngII infusion that was coincident with the distribution of SHF-derived cells. Proteomics indicated that brief AngII infusion, prior to overt pathology, evoked downregulation of SMC proteins and differential expression of extracellular matrix proteins, including several LRP1 ligands. LRP1 deletion in SHF-derived cells augmented AngII-induced ascending aortic aneurysm and rupture. Single cell transcriptomic analysis revealed that brief AngII infusion decreased Lrp1 and Tgfbr2 mRNA abundance in SHF-derived cells and induced a unique fibroblast population with low abundance of Tgfbr2 mRNA. SHF-specific Tgfbr2 deletion led to embryonic lethality at E12.5 with dilatation of the outflow tract and retroperitoneal hemorrhage. Integration of proteomic and single cell transcriptomics results identified plasminogen activator inhibitor 1 (PAI1) as the most increased protein in SHF-derived SMCs and fibroblasts during AngII infusion. Immunostaining revealed a transmural gradient of PAI1 in both ascending aortas of AngII-infused mice and human ascending aneurysmal aortas that mimicked the gradient of medial and adventitial pathologies.

ConclusionSHF-derived cells exert a critical role in maintaining vascular integrity through LRP1 and TGF-{beta} signaling associated with increases of aortic PAI1.



O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/930917v6_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@185e3d9org.highwire.dtl.DTLVardef@131d702org.highwire.dtl.DTLVardef@11afa42org.highwire.dtl.DTLVardef@242786_HPS_FORMAT_FIGEXP  M_FIG C_FIG Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABS- SHF-derived SMCs and fibroblasts associate with AngII-induced aortic pathologies.
- AngII induces a distinct fibroblast sub-cluster that is less abundant for mRNAs related to major extracellular components and TGF{beta} ligands and receptors, but more abundant for proliferative genes.
- TGFBR2 deletion in SHF-derived cells are embryonic lethal with significant dilatation of the outflow tract in mice.
- SHF-specific deletion of LRP1 leads to aortic pathologies in mice, supporting the importance of SHF-derived cells in maintaining ascending aortic wall integrity.


What are the clinical implications?- Heterogeneity of the embryonic origins of SMCs and fibroblasts contributes to complex mechanisms of vasculopathy formation, which should be considered when investigating the pathogenesis of thoracic aortopathies. A phenomics approach for in vitro antiviral drug discovery BackgroundThe current COVID-19 pandemic has highlighted the need for new and fast methods to identify novel or repurposed therapeutic drugs. Here we present a method for untargeted phenotypic drug screening of virus-infected cells, combining Cell Painting with antibody-based detection of viral infection in a single assay. We designed an image analysis pipeline for segmentation and classification of virus-infected and non-infected cells, followed by extraction of morphological properties.

ResultsWe show that the methodology can successfully capture virus-induced phenotypic signatures of MRC-5 human lung fibroblasts infected with Human coronavirus 229E (CoV-229E). Moreover, we demonstrate that our method can be used in phenotypic drug screening using a panel of nine host- and virus-targeting antivirals. Treatment with effective antiviral compounds reversed the morphological profile of the host cells towards a non-infected state.

ConclusionsThe method can be used in drug discovery for morphological profiling of novel antiviral compounds on both infected and non-infected cells. Deficiency of the paternally inherited gene Magel2 alters the development of separation-induced vocalization and maternal behavior in mice The behavior of offspring results from the combined expression of maternal and paternal genes. Genomic imprinting silences some genes in a parent-of-origin specific manner, a process that, among all animals, occurs only in mammals. How genomic imprinting affects the behavior of mammalian offspring, however, remains poorly understood. Here, we studied how the loss of the paternally inherited gene Magel2 in mouse pups affects the emission of separation-induced ultrasonic vocalizations (USV). Using quantitative analysis of more than one hundred thousand USVs, we characterized the rate of vocalizations as well as their spectral features from postnatal days 6 to 12 (P6-P12), a critical phase of mouse development during which pups fully depend on the mother for survival. Our analyses show that Magel2 deficient offspring emit separation-induced vocalizations at lower rates and with altered spectral features. We also show that dams display altered behavior towards their own Magel2 deficient offspring. In a test to compare the retrieval of two pups, dams retrieve wildtype control pups first and faster than Magel2 deficient offspring. These results suggest that the loss of Magel2 impairs the expression of separation-induced vocalization in pups as well as maternal behavior, both of which support the pups growth and development. T606-phosphorylation deprives the function of Kaiso as a transcription factor and cancer repressor It is well known that Kaiso protein encoded by ZBTB33 gene is a transcription repressor and that Kaiso-P120ctn interaction increases the shift of Kaiso from the nucleus into the cytoplasm. However, the regulatory mechanisms of Kaiso compartmentalization are far from clear. Here, we reported that AKT1 could phosphorylate 606-threonine residue (T606) within the RSSTIP motif of Kaiso in the cytoplasm. The T606-phosphorylated Kaiso (pT606-Kaiso) could directly bind to 14-3-3 family proteins and the depletion of T606 phosphorylation by T606A mutation abolished most of the Kaiso-14-3-3 binding. In addition, the Kaiso-P120ctn interaction was essential for the pT606-Kaiso accumulation in the cytoplasm. Notably, enforced 14-3-3{sigma} (SFN) overexpression could increase the pT606-Kaiso accumulation in the cytoplasm and de-repress the transcription of Kaiso target gene CDH1. Decreased amounts of both pT606-Kaiso and CDH1 proteins were frequently observed in human gastric cancer tissues relative to paired normal controls. The mRNA levels of 14-3-3{sigma} and Kaiso target gene CDH1 were positively and significantly correlated with each other in bioinformatics analyses using publicly available RNA-seq datasets for human normal tissues (n=11688, r=0.60, p<0.001) in the GTEx project and for cancer cell lines (n=1156, r=0.41, p<0.001) in the CCLE project. Furthermore, Kaiso T606A mutant (unable to be phosphorylated) significantly increased the migration and invasion of cancer cells in vitro as well as boosted the growth of these cells in vivo. In conclusion, Kaiso could be phosphorylated by AKT1 at the T606 and the pT606-Kaiso accumulates in the cytoplasm through binding to 14-3-3/P120ctn that de-represses the expression of Kaiso target gene CDH1 in normal tissues. Decreased Kaiso phosphorylation may contribute to the development of gastrointestinal cancer. The status of Kaiso phosphorylation is a determinant factor for the role of Kaiso in the development of cancer. Embracing curiosity eliminates the exploration-exploitation dilemma Balancing exploration with exploitation is seen as a mathematically intractable dilemma that all animals face. In this paper, we provide an alternative view of this classic problem that does not depend on exploring to optimize for reward. We argue that the goal of exploration should be pure curiosity, or learning for learnings sake. Through theory and simulations we prove that explore-exploit problems based on this can be solved by a simple rule that yields optimal solutions: when information is more valuable than rewards, be curious, otherwise seek rewards. We show that this rule performs well and robustly under naturalistic constraints. We suggest three criteria can be used to distinguish our approach from other theories. Mouse lemur, a new primate model to investigate cardiac pacemaker activity. BackgroundGray mouse lemurs (Microcebus murinus) are one of the smallest and most ancestral primates known. Their size falls in between that of mice and rats, and their genetic proximity to humans makes them an emerging model to study age-related neurodegeneration. Since Microcebus murinus replicates similar senescence processes of humans, it constitutes a useful model also for studying cardiovascular dysfunctions. However, its cardiac physiology is unknown. Thus, we investigated sinoatrial node (SAN) pacemaker activity of mouse lemurs, presenting the first characterization of heart automaticity in nonhuman primates.

Methods and ResultsWe recorded the heart automaticity in freely moving mouse lemurs and in their SAN tissues and pacemaker myocytes. Mouse lemurs have a heart rate (HR) in between those of mice and rats and a similar generation of electrical impulses in the SAN tissue. Conversely, their ventricular depolarization is more similar to large mammals. Mouse lemur SAN myocytes express the main pacemaker currents at densities similar to mice: the hyperpolarization-activated current (If) and the L-type (ICa,L) and T-type (ICa,T) calcium currents. Moreover, mouse lemur SAN myocytes display spontaneous ryanodine receptor (RyR)-dependent Ca2+ transients. Using muscle-derived stem cells (MDSCs) from the Microcebus murinus skeletal muscle, we also differentiated pacemaker-like (PML) cells showing spontaneous automaticity and expressing markers of native SAN myocytes (HCN4 and connexin-45).

ConclusionsIn conclusion, our characterization of heart automaticity in Microcebus murinus provides new opportunities for comparative cardio-physiology studies in primates and with humans, and for pharmacologic testing of molecules that could assist in modulating age-related dysfunctions of automaticity. Localization of signaling receptors maximizes cellular informationacquisition in spatially-structured natural environments Cells in natural environments like tissue or soil sense and respond to extracellular ligands with intricately structured and non-monotonic spatial distributions, sculpted by processes such as fluid flow and substrate adhesion. In this work, we show that spatial sensing and navigation can be optimized by adapting the spatial organization of signaling pathways to the spatial structure of the environment. We develop an information-theoretic framework for computing the optimal spatial organization of a sensing system for a given signaling environment. We find that receptor localization maximizes information acquisition in simulated natural contexts, including tissue and soil. Receptor localization extends naturally to produce a dynamic protocol for continuously redistributing signaling receptors, which when implemented using simple feedback, boosts cell navigation efficiency by 30-fold. Broadly, our work shows how cells can maximize the fidelity of information transfer by adapting the spatial organization of signaling molecules to the spatial structure of the environment. Spatial phylogenetics of Japanese ferns: Patterns, processes, and implications for conservation PremiseBiodiversity is often only measured with species richness. However, this ignores evolutionary history and is not sufficient for making conservation decisions. Here, we characterize multiple facets and drivers of biodiversity to understand how these relate to bioregions and conservation status in the ferns of Japan.

MethodsWe compiled a community dataset of 1,239 20 km x 20 km grid-cells including 672 taxa based on > 300,000 specimen records. We combined this with a phylogeny and functional traits to analyze taxonomic, phylogenetic, and functional diversity, and modeled biodiversity metrics in response to environmental factors and reproductive mode. Hierarchical clustering was used to delimit bioregions. Conservation status and threats were assessed by comparing the overlap of significantly diverse grid-cells with conservation zones and range maps of native Japanese deer.

ResultsTaxonomic richness was highest at mid-latitudes. Phylogenetic and functional diversity and phylogenetic endemism were highest in small southern islands. Relative phylogenetic and functional diversity were high at high and low latitudes, and low at mid-latitudes. Grid-cells were grouped into three (phylogenetic) or four (taxonomic) major bioregions. Temperature and apomixis were identified as drivers of biodiversity patterns. Conservation status was generally high for grid-cells with significantly high biodiversity, but the threat due to herbivory by deer was greater for taxonomic richness than other metrics.

ConclusionsOur integrative approach reveals previously undetected patterns and drivers of biodiversity in the ferns of Japan. Future conservation efforts should recognize that threats can vary by biodiversity metric and consider multiple multiple metrics when establishing conservation priorities. Statistical Analysis of Three-Dimensional Chromatin Packing Domains Determined by Chromatin Scanning Transmission Electron Microscopy (ChromSTEM) Chromatin organization over a wide range of length scales plays a critical role in the regulation of transcription and deciphering the interplay of these processes requires high-resolution, three-dimensional, quantitative imaging of chromatin structure in vitro. Herein, we introduce ChromSTEM, a method that utilizes high angle annular dark-field imaging and tomography in scanning transmission electron microscopy combined with DNA-specific staining for electron microscopy. We utilized ChromSTEM to quantify chromatin structure in cultured cells and the statistical packing behavior of the chromatin polymer. Using chromatin mass and density analysis, we observed that chromatin forms spatially well-defined higher-order domains which are around 100 nm in radius, with a radially decreasing mass-density from the center to the periphery. Although the morphological properties of the domains vary within the same cell line, they seem to exhibit greater heterogeneity across cell lines, underlying a potential role of statistical chromatin packing in regulating cell-type-specific gene expression. A platform of patient-derived microtumors identifies therapeutic vulnerabilities in ovarian cancer. BackgroundIn light of the frequent development of therapeutic resistance in cancer treatment, there is a strong need for personalized model systems representing patient tumor heterogeneity, while enabling parallel drug testing and identification of appropriate treatment responses in individual patients. Using ovarian cancer as a prime example of a heterogeneous tumor disease with complex microenvironment and high recurrence rates, we developed a 3D preclinical ovarian cancer model comprised of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) for identification of treatment vulnerabilities and validation of anti-cancer drug efficacy using immunohistochemistry, immune cell phenotyping, functional assays and protein profiling analyses.

MethodsPDM and TILs were isolated from fresh primary ovarian cancer tissue specimen using mechanical disruption and limited enzymatic digestion and were subsequently cultured in suspension in defined media in the absence of serum. The heterogeneous cellular composition of isolated PDM as well as autologous TILs was analyzed by FFPE immunohistochemistry and multi-color flow cytometry, respectively. For in-depth protein profiling of PDM we established Reverse Phase Protein Array (RPPA) analyses of >110 total and phospho-proteins. Treatment efficacy in response to chemotherapeutics as well as immunotherapeutic compounds was assessed in PDM and PDM-TIL co-cultures using a functional viability assay in microplate format.

ResultsThe enzymatic digestion of primary ovarian cancer tissue and suspension culture in defined serum-free media allowed fast and efficient recovery of patient-derived microtumors (PDM). Immunohistochemical analyses demonstrated histopathological comparability of ovarian cancer PDM with corresponding patient tumor tissue. Reverse Phase Protein Array (RPPA)-based analyses of >110 total and phospho-proteins enabled the identification of patient-specific sensitivities to standard, platinum-based therapy and thereby the prediction of potential treatment-responders. Finally, combining PDM and autologous TILs for individual efficacy testing of immune checkpoint inhibitor treatment demonstrated the potential for patient-specific enhancement of cytotoxic TIL activity by this therapeutic approach.

Conclusion3D patient-derived ovarian cancer microtumors represent a preclinical, ex vivo tumor model that reflects intertumoral heterogeneity and represent the cellular complexity of individual patient tumors. Combining protein pathway analysis and anti-cancer drug efficacy testing of PDM enables drug mode-of-action analyses and therapeutic sensitivity prediction within a clinically relevant time frame after surgery. Follow-up studies in larger cohorts are currently under way to further evaluate the applicability of this platform to support clinical decision-making and personalizing cancer treatment.