Biofilm self-patterning: mechanical forces drive a reorientation cascade In growing active matter systems, a large collection of engineered or living autonomous units metabolize free energy and create order at different length scales as they proliferate and migrate collectively. One such example is bacterial biofilms, which are surface-attached aggregates of bacterial cells embedded in an extracellular matrix. However, how bacterial growth coordinates with cell-surface interactions to create distinctive, long-range order in biofilms remains elusive. Here we report a collective cell reorientation cascade in growing Vibrio cholerae biofilms, leading to a differentially ordered, spatiotemporally coupled core-rim structure reminiscent of a blooming aster. Cell verticalization in the core generates differential growth that drives radial alignment of the cells in the rim, while the radially aligned rim in turn generates compressive stresses that expand the verticalized core. Such self-patterning disappears in adhesion-less mutants but can be restored through opto-manipulation of growth. Agent-based simulations and two-phase active nematic modeling reveal the strong interdependence of the driving forces for the differential ordering. Our findings provide insight into the collective cell patterning in bacterial communities and engineering of phenotypes and functions of living active matter.
Nucleus size and its effect on the chromatin structure in living cells. DNA-architectural proteins play a major role in organization of chromosomal DNA in living cells by packaging it into chromatin, whose spatial conformation is determined by an intricate interplay between the DNA-binding properties of architectural proteins and physical constraints applied to the DNA by a tight nuclear space. Yet, the exact effects of the cell nucleus size on DNA-protein interactions and chromatin structure currently remain obscure. Furthermore, there is even no clear understanding of molecular mechanisms responsible for the nucleus size regulation in living cells. To find answers to these questions, we developed a general theoretical framework based on a combination of polymer field theory and transfer-matrix calculations, which showed that the nucleus size is mainly determined by the difference between the surface tensions of the nuclear envelope and the endoplasmic reticulum membrane as well as the osmotic pressure exerted by cytosolic macromolecules on the nucleus. In addition, the model demonstrated that the cell nucleus functions as a piezoelectric element, changing its electrostatic potential in a size-dependent manner. This effect has been found to have a profound impact on stability of nucleosomes, revealing a previously unknown link between the nucleus size and chromatin structure. Overall, our study provides new insights into the molecular mechanisms responsible for regulation of the cell nucleus size, as well as the potential role of nuclear organization in shaping the cell response to environmental cues.

SIGNIFICANCE STATEMENTThe cell nucleus plays a central role in the life of eukaryotic cells, providing the highest level of control of intracellular processes. Depending on the stage of the cell cycle and / or surrounding environment, the size of the cell nucleus may undergo changes that are believed to cause chromatin reorganization, affecting gene transcription. At present, however, there is no clear understanding of the molecular mechanisms that may be responsible for such regulation, whose exact effect on chromatin structure remains unclear. In this study, by developing an advanced computational approach, we explore these issues from a physical perspective, revealing previously unknown mechanisms contributing to organization of the cell nucleus and chromatin.
Elucidation of global and local genomic epidemiology of Salmonella enterica  Enteritidis through multilevel genome typing Salmonella enterica serovar Enteritidis is a major cause of foodborne Salmonella infections and outbreaks in humans. Effective surveillance and timely outbreak detection are essential for public health control. Multilevel genome typing (MGT) with multiple levels of resolution has been previously demonstrated as a promising tool for this purpose. In this study, we developed MGT with nine levels for S. Enteritidis and characterised the genomic epidemiology of S. Enteritidis in detail. We examined 26,670 publicly available S. Enteritidis genome sequences from isolates spanning 101 years from 86 countries to reveal their spatial and temporal distributions. Using the lower resolution MGT levels, globally prevalent and regionally restricted sequence types (STs) were identified; avian associated MGT4-STs were found that were common in human cases in the USA were identified; temporal trends were observed in the UK with MGT5-STs from 2014 to 2018, revealing both long lived endemic STs and the rapid expansion of new STs. Using MGT3 to MGT6, we identified MDR associated STs at various MGT levels, which improves precision of detection and global tracking of MDR clones. We also found that the majority of the global S. Enteritidis population fell within two predominant lineages, which had significantly different propensity of causing large scale outbreaks. An online open MGT database has been established for unified international surveillance of S. Enteritidis. We demonstrated that MGT provides a flexible and high-resolution genome typing tool for S. Enteritidis surveillance and outbreak detection.

Impact statementSalmonella enterica serovar Enteritidis is a common foodborne pathogen that can cause large outbreaks. Surveillance and high-resolution typing are essential for outbreak prevention and control. Genome sequencing offers unprecedented power for these purposes and a standardised method or platform for the interpretation, comparison and communication of genomic typing data is highly desirable. In this work, we developed a genomic typing scheme called Multilevel Genome Typing (MGT) for S. Enteritidis. We analysed 26,670 publicly available genomes of S. Enteritidis using MGT. We characterised the geographic and temporal distribution of S. Enteritidis MGT types as well as their association with multidrug resistance (MDR) and virulence genes. A publicly available MGT database for S. Enteritidis was established, which has the potential facilitate the unified global public health surveillance for this pathogen.

Data SummaryO_LIThe MGT database for S. Enteritidis is available at https://mgtdb.unsw.edu.au/enteritidis/.
C_LIO_LIAll accession numbers of the public available genomes were available in the MGT database and Data Set S1, Tab 1. And there were no newly sequenced data in this study.
C_LIO_LISupplementary material: Supplementary Fig. S1 to S7, supplementary methods and supporting results about the evaluation of potential repeat sequencing bias.
C_LIO_LIData Set S1: Supporting tables of the main results.
C_LIO_LIData Set S2. Supporting tables of the repeat sequencing bias evaluation by removing the potential repeat sequencing isolates. Note outbreak isolates may also be removed.
C_LI Circulating immune complexes drive immunopathology in COVID-19 A dysregulated immune response with high levels of SARS-CoV-2 specific IgG antibodies characterizes patients with severe or critical COVID-19. Although a robust IgG response is traditionally considered to be protective, excessive triggering of activating Fc-gamma-receptors (Fc{gamma}Rs) could be detrimental and cause immunopathology. Here, we document that patients who develop soluble circulating IgG immune complexes (sICs) during infection are subject to enhanced immunopathology driven by Fc{gamma}R activation. Utilizing cell-based reporter systems we provide evidence that sICs are predominantly formed prior to a specific humoral response against SARS-CoV-2. sIC formation, together with increased afucosylation of SARS-CoV-2 specific IgG eventually leads to an enhanced CD16 (Fc{gamma}RIII) activation of immune cells reaching activation levels comparable active systemic lupus erythematosus (SLE) disease. Our data suggest a vicious cycle of escalating immunopathology driven by an early formation of sICs in predisposed patients. These findings reconcile the seemingly paradoxical findings of high antiviral IgG responses and systemic immune dysregulation in severe COVID-19.

Clinical implicationsThe identification of sICs as drivers of an escalating immunopathology in predisposed patients opens new avenues regarding intervention strategies to alleviate critical COVID-19 progression.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/449893v4_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@b4616corg.highwire.dtl.DTLVardef@682d1aorg.highwire.dtl.DTLVardef@16946cborg.highwire.dtl.DTLVardef@a6ef7d_HPS_FORMAT_FIGEXP  M_FIG C_FIG A vicious cycle of immunopathology in COVID-19 patients is driven by soluble multimeric immune complexes (sICs). SARS-CoV-2 infection triggers sIC formation in prone individuals. Activation of Fc{gamma}RIII/CD16 expressing immune cells by sICs precedes a humoral response to SARS-CoV2 infection. sICs and infection add to IgG afucosylation, further enhancing Fc{gamma}RIII/CD16 activation by opsonized targets. High inflammation induces further sIC mediated immune cell activation ultimately leading to an escalating immunopathology. 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. 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. Coupled myovascular expansion directs cardiac growth and regeneration BackgroundHeart regeneration requires multiple cell types to enable cardiomyocyte (CM) proliferation. How these cells interact to create growth niches is unclear. Here we profile proliferation kinetics of cardiac endothelial cells (CECs) and CMs in the neonatal mouse heart and find that they are spatiotemporally coupled.

Methods and ResultsWe show that coupled myovascular expansion during cardiac growth or regeneration is dependent upon VEGF-VEGFR2 signaling, as genetic deletion of Vegfr2 from CECs or inhibition of VEGFA abrogates both CEC and CM proliferation. Repair of cryoinjury displays poor spatial coupling of CEC and CM proliferation. Boosting CEC density after cryoinjury with virus encoding Vegfa enhances regeneration. Using Mendelian randomization, we demonstrate that circulating VEGFA levels are positively linked with human myocardial mass, suggesting that Vegfa can stimulate human cardiac growth.

ConclusionsOur work demonstrates the importance of coupled CEC and CM expansion and reveals a myovascular niche that may be therapeutically targeted for heart regeneration. Design of Specific Primer Sets for the Detection of B.1.1.7, B.1.351, P.1, B.1.617.2 and B.1.1.519 Variants of SARS-CoV-2 using Artificial Intelligence As the COVID-19 pandemic continues, new SARS-CoV-2 variants with potentially dangerous features have been identified by the scientific community. Variant B.1.1.7 lineage clade GR from Global Initiative on Sharing All Influenza Data (GISAID) was first detected in the UK, and it appears to possess an increased transmissibility. At the same time, South African authorities reported variant B.1.351, that shares several mutations with B.1.1.7, and might also present high transmissibility. Earlier this year, a variant labelled P.1 with 17 non-synonymous mutations was detected in Brazil. Recently the World Health Organization has raised concern for the variants B.1.617.2 mainly detected in India but now exported worldwide. It is paramount to rapidly develop specific molecular tests to uniquely identify new variants. Using a completely automated pipeline built around deep learning and evolutionary algorithms techniques, we designed primer sets specific to variants B.1.1.7, B.1.351, P.1 and respectively. Starting from sequences openly available in the GISAID repository, our pipeline was able to deliver the primer sets for each variant. In-silico tests show that the sequences in the primer sets present high accuracy and are based on 2 mutations or more. In addition, we present an analysis of key mutations for SARS-CoV-2 variants. Finally, we tested the designed primers for B.1.1.7 using RT-PCR. The presented methodology can be exploited to swiftly obtain primer sets for each new variant, that can later be a part of a multiplexed approach for the initial diagnosis of COVID-19 patients. The impact of housing conditions on mesenchymal stromal/stem cell populations differ between adipose tissue and skeletal muscle BackgroundIn pigs, the ratio between lean mass and fat mass in the carcass determines production efficiency and is strongly influenced by the number and size of cells in tissues. During growth, the increase in the number of cells results from the recruitment of different populations of multipotent mesenchymal stromal/stem cells (MSCs) residing in the tissues. We hypothesized that the impact of hygiene of housing conditions during growth on the proportions of MSCs in adipose tissue and skeletal muscle may differ between pigs with different residual feed intake (RFI), a measure of feed efficiency.

MethodsAt the age of 12 weeks, Large White pigs from two lines divergently selected for low and high RFI were housed in two contrasting hygiene conditions (good vs poor). After six weeks, pigs were slaughtered (n = 30; 5-9/group). Samples of subcutaneous adipose tissue and longissimus skeletal muscle were collected, and cells from the stromal vascular fraction (SVF), which includes mesenchymal stromal/stem cell populations, were isolated from each tissue. Adipose and muscle cell populations from the SVF were phenotyped by flow cytometry using antibodies that targeted different cell surface markers (CD45 to separate hematopoietic cells from MSCs; CD34, CD38, CD56 and CD140a to identify MSC populations with adipogenic and/or myogenic potential).

ResultsAdipose tissue and muscle shared some common MSC populations although MSC diversity was higher in muscle than in adipose tissue. In muscle, the CD45-CD56+CD34-CD140a+ and CD45-CD56+CD34+CD140a+ cell populations were abundant. Of these two cell populations, only the proportions of CD45-CD56+CD34+CD140a+ cells increased (P < 0.05) in pigs housed in poor hygiene as compared with pigs in good hygiene conditions. For the CD45-CD56-CD34- cell population, present in low proportion, there was an interaction between hygiene condition and genetic line (P < 0.05) with a decrease in low RFI pigs housed in poor hygiene conditions. In adipose tissue, the two abundant MSC populations were CD45-CD56-CD34- and CD45-CD56+CD34-. The proportion of CD45-CD56-CD34- cells increased (P < 0.05) whereas the proportion of CD45-CD56+CD34- tended to decrease (P < 0.1) in pigs housed in poor conditions. This study shows that the proportions of some MSC populations were affected by hygiene of housing conditions in a tissue-dependent manner in pigs of both RFI lines. It suggests that MSCs may play a significant role in adipose tissue and skeletal muscle homeostasis and may influence later growth and body composition in growing animals. Fine-scale adaptations to environmental variation and growth strategies drive phyllosphere Methylobacterium diversity. Methylobacterium is a prevalent bacterial genus of the phyllosphere. Despite its ubiquity, little is known about the extent to which its diversity reflects neutral processes like migration and drift, versus environmental filtering of life history strategies and adaptations. In two temperate forests, we investigated how phylogenetic diversity within Methylobacterium was structured by biogeography, seasonality, and growth strategies. Using deep, culture-independent barcoded marker gene sequencing coupled with culture-based approaches, we uncovered a considerable diversity of Methylobacterium in the phyllosphere. We cultured different subsets of Methylobacterium lineages depending upon the temperature of isolation and growth (20 {degrees}C or 30 {degrees}C), suggesting long-term adaptation to temperature. To a lesser extent than temperature adaptation, Methylobacterium diversity was also structured across large (>100km; between forests) and small geographical scales (<1.2km within forests), among host tree species, and was dynamic over seasons. By measuring growth of 79 isolates at different temperature treatments, we observed contrasting growth performances, with strong lineage- and season-dependent variations in growth strategies. Finally, we documented a progressive replacement of lineages with a high-yield growth strategy typical of cooperative, structured communities, in favor of those characterized by rapid growth, resulting in convergence and homogenization of community structure at the end of the growing season. Together our results show how Methylobacterium is phylogenetically structured into lineages with distinct growth strategies, which helps explain their differential abundance across regions, host tree species, and time. This works paves the way for further investigation of adaptive strategies and traits within a ubiquitous phyllosphere genus.

ImportanceMethylobacterium is a bacterial group tied to plants. Despite its ubiquity and importance to their hosts, little is known about the processes driving Methylobacterium community dynamics. By combining traditional culture-dependent and independent (metabarcoding) approaches, we monitored Methylobacterium diversity in two temperate forests over a growing season. On the surface of tree leaves, we discovered remarkably diverse and dynamic Methylobacterium communities over short temporal (from June to October) and spatial scales (within 1.2 km). Because we cultured different subsets of Methylobacterium diversity depending on the temperature of incubation, we suspected that these dynamics partly reflected climatic adaptation. By culturing strains in lab conditions mimicking seasonal variations, we found that diversity and environmental variations were indeed good predictors of Methylobacterium growth performances. Our findings suggest that Methylobacterium community dynamics at the surface of tree leaves results from the succession of strains with contrasted growth strategies in response to environmental variations. Pathways of savannization in a mesic African savanna-forest mosaic following an extreme fire O_LIFires in savannas limit tree cover, thereby promoting flammable grass accumulation and fuelling further frequent fires. Meanwhile, forests and thickets form dense canopies that reduce C4-grass fuel loads and creating a humid microclimate, thereby excluding fires under typical climatic conditions.
C_LIO_LIHowever, extreme fires occasionally burn into these closed-canopy systems. Although these rare fires cause substantial tree mortality and can make repeat fires more likely, the long-term consequences of an extreme fire for closed canopy vegetation structure and potential to convert to savanna (hereafter "savannization") remain largely unknown.
C_LIO_LIHere, we analysed whether an extreme fire could, alone, alter species composition, vegetation structure, and fire regimes of closed-canopy ecosystems in an intact savanna-forest-thicket mosaic, or whether successive fires after an initial extreme fire were necessary to trigger a biome transition between from forest to savanna.
C_LIO_LIWe found that forests that only burned once recovered, whereas those that burned again following an initial extreme fire transitioned from closed-canopy forests towards open, grassy savannas.
C_LIO_LIWhile thickets had less tree mortality in fires than forests, repeat fires nonetheless precipitated a transition towards savannas.
C_LIO_LIColonization of the savanna tree community lagged behind the grass community, but also began to transition.
C_LI

SynthesisOur results suggest that rare extreme fires, followed by repeated burning can indeed result in savannization in places where savanna and forest represent alternative stable states. Juvenile Gouldian Finches (Erythrura gouldiae) form sibling sub-groups during social integration The formation of social relationships in complex groups is critical in shaping patterns of social organization and behavioral development. In many birds, young individuals remain dependent on their parents for extended periods but must abruptly transition to navigating interactions in the wider group after independence. While lack of social relationships during this period is detrimental in the development of later social skills, little is known about the social relationships juveniles form after independence in many bird species. In this study we describe patterns of social interactions in juvenile Gouldian Finches (Erythrura gouldiae) during transitions from family groups into flocks of unfamiliar individuals. A group of 20 juveniles from 4 families were introduced into two flocks. After introductions juveniles showed a gradient of approach rates with most approaches directed towards siblings, followed by juvenile peers, adult females, and lastly adult males. Significant preferences for siblings resulted in the emergence of sibling subgroups within the larger social network. This active self-assortment of siblings suggests that sibling sub-groups are an important bridge linking social connections within the family and the wider group. Such findings suggests that sibling relationships have a critical role in the socialization after independence, as well as structuring the social organization of Gouldian finch flocks. Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using electron cryo-microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using HDX-MS, we observed dynamic behavior of the major five-fold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.

TeaserCryo-EM and HDX-MS analysis of an encapsulin nanocompartment shows that the pores at the five-fold icosahedral vertex of the shell are flexible. Data-Driven Network Models for Genetic Circuits From Time-Series Data with Incomplete Measurements Synthetic gene networks are frequently conceptualized and visualized as static graphs. This view of biological programming stands in stark contrast to the transient nature of biomolecular interaction, which is frequently enacted by labile molecules that are often unmeasured. Thus, the network topology and dynamics of synthetic gene networks can be difficult to verify in vivo or in vitro, due to the presence of unmeasured biological states. Here we introduce the dynamical structure function as a new mesoscopic, data-driven class of models to describe gene networks with incomplete measurements. We introduce a network reconstruction algorithm and a code base for reconstructing the dynamical structure function from data, to enable discovery and visualization of graphical relationships in a genetic circuit diagram as time-dependent functions rather than static, unknown weights. We prove a theorem, showing that dynamical structure functions can provide a data-driven estimate of the size of crosstalk fluctuations from an idealized model. We illustrate this idea with numerical examples. Finally, we show how data-driven estimation of dynamical structure functions can explain failure modes in two experimentally implemented genetic circuits, a historical genetic circuit and a new E. coli based transcriptional event detector. Optical regulation of endogenous RhoA reveals selection of cellular responses by signal amplitude How protein signaling networks respond to different input strengths is an important but poorly understood problem in cell biology. For example, the small GTPase RhoA regulates both focal adhesion (FA) growth or disassembly, but whether RhoA serves as a switch selecting between cellular outcomes, or if outcomes are simply modulated by additional factors in the cell, is not clear. Here, we develop a photoswitchable RhoA guanine exchange factor, psRhoGEF, to precisely control endogenous RhoA activity. We also develop a FRET-based biosensor to allow visualization of RhoA activity together with psRhoGEF control. Using these new optical tools, we discover that low levels of RhoA activation preferentially induce FA disassembly in a Src-dependent manner, while high levels induce both FA growth and disassembly in a ROCK-dependent manner. Thus, rheostatic control of RhoA activation with photoswitchable RhoGEF reveals that cells can use signal amplitude to produce multiple responses to a single biochemical signal. First detection of herpesvirus and prevalence of mycoplasma infection in free-ranging Hermann s tortoises (Testudo hermanni), and in potential pet vectors AO_SCPLOWBSTRACTC_SCPLOWTwo types of pathogens cause highly contagious upper respiratory tract diseases (URTD) in Chelonians: testudinid herpesviruses (TeHV) and a mycoplasma (Mycoplasma agassizii). In captivity, these infections are frequent and can provoke outbreaks. Pet trade generates international flow of tortoises, often without sanitary checking; individuals intentionally or accidentally released in the wild may spread pathogens. A better understanding of the transmission of infectious agents from captivity to wild tortoises is needed. Many exotic individuals have been introduced in populations of the endangered western Hermanns tortoise (Testudo hermanni hermanni), notably spur-thighed tortoises (Testudo graeca). We assessed the presence of TeHV and mycoplasma in native western Hermanns tortoises and in potential pet vectors in south-eastern France. Using a large sample (N=572 tortoises), this study revealed, by PCR, the worrying presence of herpesvirus in 7 free-ranging individuals (3 sub-populations). Additionally, Mycoplasma agassizii was detected, by PCR, in 15 of the 18 populations sampled with a frequency ranging from 3.4% (1 of 29 tortoises) to 25% (3 of 12 tortoises). Exotic spur-thighed tortoises showed high frequency of mycoplasma infection in captivity (18.2%) and in individuals (50%) found in native Hermanns tortoise sub-populations, suggesting that this species could be a significant vector. The paucity of information of TeHV on European tortoise URTD in natural settings, especially in combination with mycoplasma, prompts for further studies. Indeed, sick tortoises remain concealed and may not be easily detected in the field. Our results indicate a good health for most infected tortoise but it should be screened in the field as well as in captivity. The diverse effects of phenotypic dominance on hybrid fitness When divergent populations interbreed, their alleles are brought together in hybrids. These hybrids may express novel phenotypes, not previously exposed to selection. In the initial F1 cross, most divergent alleles are present as heterozygotes. Therefore, F1 fitness can be influenced by dominance effects that first appear together in the hybrids, and so could not have been selected to function well together. We present a systematic study of these F1 dominance effects by introducing variable phenotypic dominance into Fishers geometric model. We show that dominance often reduces hybrid fitness, which can lead to patterns of optimal outbreeding and a steady decline in F1 fitness at high levels of divergence. We also show that "lucky" beneficial effects sometimes arise by chance, which might be especially important when hybrids can access novel environments. We then explore the interaction of phenotypic dominance with uniparental inheritance, showing that dominance can lead to violations of Haldanes Rule (reduced fitness of the heterogametic sex) while strengthening Darwins Corollary (fitness differences between cross directions). Taken together, our results show that dominance could play an important role in the outcomes of hybridisation after secondary contact, and thus to the maintenance or collapse of isolating barriers. Nevertheless, the telltale signs of dominance are relatively few and subtle. Results also suggest that dominance effects are smaller than the cost of segregation variance, implying that simple additive models may still give good predictions for later-generation recombinant hybrids, even when dominance qualitatively alters outcomes for the F1. Abscisic Acid Drives Stomatal Closure through Increases in Hydrogen Peroxide in Distinct Subcellular Compartments Including Mitochondria Abscisic acid (ABA) closes stomata to minimize transpiration and gas exchange in response to drought. ABA signaling increases accumulation of reactive oxygen species (ROS) in guard cells to drive stomatal closure. We used chemical dyes and genetically-encoded biosensors to identify the subcellular localizations where ABA increases hydrogen peroxide (H2O2) in guard cells. The general redox sensor, dichlorofluorescein (DCF), revealed ABA-driven ROS increases in distinct subcellular locations including chloroplasts, cytosol, and nuclei. ABA also increased fluorescence of both DCF and an H2O2-selective probe, Peroxy Orange 1, in mitochondria, which colocalize with Mitotracker Red. Whole cell and mitochondrial localized ABA-increased ROS signals were lost in an ABA-insensitive quintuple ABA receptor mutant and accentuated in an ABA-hypersensitive mutant. The ABA-induced ROS accumulation in mitochondria was lost in guard cells of mutants with defects in genes encoding H2O2 elevating respiratory burst oxidase homolog (RBOH) enzymes and reduced by treatment with the RBOH inhibitor VAS2870. ABA treatment of guard cells transformed with the genetically-encoded H2O2 reporter targeted to the cytoplasm, roGFP2-Orp1, and the mitochondrial targeted, mt-roGFP2-Orp1, revealed significant ABA-induced H2O2 increases. Furthermore, H2O2 treatment is sufficient to close stomata. These results reveal that ABA elevates H2O2 accumulation in guard cell cytosol and mitochondria to drive stomatal closure.

One sentence summaryGenetically encoded biosensors and chemical probes revealed ABA-dependent increases in hydrogen peroxide, a reactive oxygen species with signaling activity, in guard cell cytoplasm and mitochondria. Citation needed?Wikipedia bibliometrics during the first waveof the COVID-19 pandemic. BackgroundWith the COVID-19 pandemics outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an "infodemic", ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedias COVID-19-related articles before and during the pandemics first wave (January-May 2020).

ResultsWe found that coronavirus-related articles referenced trusted media sources and high-quality academic research. Moreover, despite a surge in COVID-19 preprints, Wikipedia had a clear preference for open-access studies published in respected journals and made little use of preprints. Building a timeline of English COVID-19 articles from 2001-2020 revealed a nuanced trade-off between quality and timeliness. It further showed how preexisting articles on key topics related to the virus created a framework for integrating new knowledge. Supported by a rigid sourcing policy, this "scientific infrastructure" facilitated contextualization and regulated the influx of new information. Lastly, we constructed a network of DOI-Wikipedia articles, which showed the shifting landscape of pandemic-related knowledge on Wikipedia and how academic citations create a web of shared knowledge supporting topics like COVID-19 vaccine development.

ConclusionsUnderstanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its "citizen encyclopedists", it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts. Induced Cognitive Impairments Reversed by Grafts of Neural Precursors: a Longitudinal Study in a Macaque Model of Parkinson's Disease Parkinsons disease (PD) evolves over an extended and variable period in humans; several years prior to the onset of classical motor symptoms, cognitive deficits as well as sleep and biological rhythm disorders develop and worsen with disease progression, significantly impacting the quality of life of patients. The gold standard MPTP macaque model of PD recapitulates the progression of motor and non-motor symptoms over contracted periods of time.

Here, this multidisciplinary and multiparametric study follows, in five animals, the steady progression of motor and non-motor symptoms and describes their reversal following bilateral grafts of neural precursors in diverse functional domains of the basal ganglia.

Results show unprecedented recovery from cognitive symptoms in addition to a strong clinical motor recuperation. Both motor and cognitive recovery and partial circadian rhythm recovery correlate with the degree of graft integration into the host environment as well as with in-vivo levels of striatal dopaminergic innervation and function.

Given inter-individuality of disease progression and recovery the present study underlines the importance of longitudinal multidisciplinary assessments in view of clinical translation and provides empirical evidence that integration of neural precursors following transplantation efficiently restores function at multiple levels in parkinsonian non-human primates.

One Sentence SummaryEmpirical evidence that cell therapy efficiently reverts cognitive and clinical motor symptoms in the non-human primate model of Parkinsons disease. Meiotic DSB-independent role of protein phosphatase 4 in Hop1 assembly to promote meiotic chromosome axis formation in budding yeast Dynamic changes in chromosomal structure that occur during meiotic prophase play an important role in the progression of meiosis. Among these structures, meiosis-specific chromosomal axis-loop structures are important as a scaffold for integrated control between the meiotic recombination reaction and the associated checkpoint system to ensure accurate chromosome segregation. However, the molecular mechanism of the initial step of chromosome axis-loop construction is not well understood. Here, we showed that in budding yeast, protein phosphatase 4 (PP4) that primarily counteracts Mec1/Tel1 phosphorylation is required to promote the assembly of chromosomal axis components Hop1 and Red1 onto meiotic chromatin via interaction with Hop1. PP4, in contrast, did not affect Rec8 assembly. Notably, unlike the previously known function of PP4, this novel function of PP4 was independent of meiotic DSB-dependent Tel1/Mec1 kinase activities. The defect in Hop1/Red1 assembly in the absence of PP4 function was not suppressed by dysfunction of Pch2, which removes Hop1 protein from the chromosome axis. This suggests that PP4 is required for the initial step of chromatin loading of Hop1 rather than stabilization of Hop1 on axes. These results indicate that phosphorylation/dephosphorylation-mediated regulation of Hop1 recruitment onto chromatin occurs during chromosome axis construction before meiotic double-strand break formation. 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. Revisiting regulatory decoherence and phenotypic integration: accounting for temporal bias in co-expression analyses Environment can alter the degree of phenotypic variation and covariation, potentially influencing evolutionary trajectories. However, environment-driven changes in phenotypic variation remain understudied. In an effort to exploit the abundance of RNASequencing data now available, an increasing number of ecological studies rely on population-level correlation to characterize the plastic response of the entire transcriptome and to identify environmentally responsive molecular pathways. These studies are fundamentally interested in identifying groups of genes that respond in concert to environmental shifts. We show that population-level differential co-expression exhibits biases when capturing changes of regulatory activity and strength in rice plants responding to elevated temperature. One possible cause of this bias is regulatory saturation, the observation that detectable co-variance between a regulator and its target may be low as their transcript abundances are induced. This phenomenon appears to be particularly acute for rapid-onset environmental stressors. However, our results suggest that temporal correlations may be a reliable means to detect transient regulatory activity following rapid onset environmental perturbations such as temperature stress. Such temporal bias is likely to confound the studies of phenotypic integration, where high-order organismal traits are hypothesized to be more integrated with strong correlation under stressful conditions, while recent transcriptome studies exhibited weaker coexpression between genes under stressful conditions. Collectively, our results point to the need to account for the nuances of molecular interactions and the possibly confounding effects that these can introduce into conventional approaches to study transcriptome datasets. RadGenNets: Deep Learning-Based Radiogenomics Model For Gene Mutation Prediction In Lung Cancer AO_SCPLOWBSTRACTC_SCPLOWIn this paper, we present our methodology that can be used for predicting gene mutation in patients with non-small cell lung cancer (NSCLC). There are three major types of gene mutations that a NSCLC patients gene structure can change to: epidermal growth factor receptor (EGFR), Kirsten rat sarcoma virus (KRAS), and Anaplastic lymphoma kinase (ALK). We worked with the clinical and genomics data for each patient as well CT scans. We preprocessed all of the data and then built a novel pipeline to integrate both the image and tabular data. We built a novel pipeline that used a fusion of Convolutional Neural Networks and Dense Neural Networks. Also, using a search approach, we pick an ensemble of deep learning models to classify the separate gene mutations. These models include EfficientNets, SENet, and ResNeXt WSL, among others. Our model achieved a high area under curve (AUC) score of 94% in detecting gene mutation. Cell-type specific circadian bioluminescence rhythms in Dbp reporter mice Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein (Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre-recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vivo and ex vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei ex vivo. In vivo studies show Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;DbpKI/+ "liver reporter" mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies confirm the previously demonstrated circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues. Designing single-cell experiments to harvest fluctuation information while rejecting measurement noise Measurement error is a complicating factor that reduces or distorts the information contained in any experiment. This problem becomes even more serious in the context of experiments that are specifically designed to quantify single-cell gene expression heterogeneity, where important RNA and protein copy numbers are themselves subject to the inherently random fluctuations of biochemical reactions. It is not clear how measurement noise should be managed, in addition to other experiment design variables such as sampling size and frequency, to ensure that collected data will provide useful insights on gene expression mechanisms of interest. To address these fundamental single-cell experiment design challenges, we propose a computational framework that makes explicit use of measurement error modeling and Fisher Information Matrix-based criteria to decide between experimental methods. This unified approach not only quantifies how different noise characteristics affect uncertainty in parameter estimation, but also enables a systematic approach to design hybrid experiments that combine different measurement modalities.