Cell surface hydrophobicity determines aggregate assembly type in Pseudomonas aeruginosa Bacteria live in spatially organized aggregates during chronic infections, where they adapt to the host environment, evade immune responses and resist therapeutic interventions. Although it is known that environmental factors such as polymers influence bacterial aggregation, it is not clear how bacterial adaptation during chronic infection impacts the formation and spatial organization of aggregates in the presence of polymers. Here we show that in an in vitro model of cystic fibrosis (CF) containing the polymers eDNA and mucin, O-specific antigen is a major factor in determining the formation of two distinct aggregate assembly types of Pseudomonas aeruginosa due to alterations in cell surface hydrophobicity. Our findings suggest that during chronic infection, interplay between cell surface properties and polymers in the environment may influence the formation and structure of bacterial aggregates, which would shed new light on the fitness costs and benefits of O-antigen production in environments such as CF lungs.

ImportanceDuring chronic infection, several factors contribute to the biogeography of microbial communities. Heterogeneous populations of Pseudomonas aeruginosa form aggregates in cystic fibrosis airways, however, the impact of this population heterogeneity on spatial organization and aggregate assembly is not well understood. In this study we found that changes in O-specific antigen determine the spatial organization of P. aeruginosa cells by altering the relative cell surface hydrophobicity. This finding suggests a role for O-antigen in regulating P. aeruginosa aggregate size and shape in cystic fibrosis airways.
Perturbation of alphavirus and flavivirus infectivity by components of the bacterial cell wall The impact of the host microbiota on arbovirus infections is currently not well understood. Arboviruses are viruses transmitted through the bites of infected arthropods, predominantly mosquitoes or ticks. The first site of arbovirus inoculation is the biting site in the host skin, which is colonized by a complex microbial community that could possibly influence arbovirus infection. We demonstrated that pre-incubation of arboviruses with certain components of the bacterial cell wall, including lipopolysaccharides (LPS) of some Gram-negative bacteria and lipoteichoic acids or peptidoglycan of certain Gram-positive bacteria, significantly reduced arbovirus infectivity in vitro. This inhibitory effect was observed for arboviruses of different virus families, including chikungunya virus of the Alphavirus genus and Zika virus of the Flavivirus genus, showing that this is a broad phenomenon. A modest inhibitory effect was observed following incubation with a panel of heat-inactivated bacteria, including bacteria residing on the skin. No viral inhibition was observed after pre-incubation of cells with LPS. Furthermore, a virucidal effect of LPS on viral particles was noticed by electron microscopy. Therefore, the main inhibitory mechanism seems to be due to a direct effect on the virus particles. Together, these results suggest that bacteria are able to decrease the infectivity of alphaviruses and flaviviruses.

ImportanceDuring the past decades the world has experienced a vast increase in epidemics of alphavirus and flavivirus infections. These viruses can cause severe diseases such as hemorrhagic fever, encephalitis and arthritis. Several alpha- and flaviviruses, such as chikungunya virus, Zika virus and dengue virus, are significant global health threats because of their high disease burden, their widespread (re-)emergence and the lack of (good) anti-arboviral strategies. Despite the clear health burden, alphavirus and flavivirus infection and disease are not fully understood. A knowledge gap in the interplay between the host and the arbovirus is the potential interaction with host skin bacteria. Therefore, we studied the effect of (skin) bacteria and bacterial cell wall components on alphavirus and flavivirus infectivity in cell culture. Our results show that certain bacterial cell wall components markedly reduced viral infectivity by directly interacting with the virus particle.
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. An Extended Motif in the SARS-CoV-2 Spike Modulates Binding and Release of Host Coatomer in Retrograde Trafficking {beta}-Coronaviruses such as SARS-CoV-2 hijack coatomer protein-I (COPI) for spike protein retrograde trafficking to the progeny assembly site in endoplasmic reticulum-Golgi intermediate compartment (ERGIC). However, limited residue-level details are available into how the spike interacts with COPI. Here we identify an extended COPI binding motif in the spike that encompasses the canonical K-x-H dibasic sequence. This motif demonstrates selectivity for COPI subunit. Guided by an in silico analysis of dibasic motifs in the human proteome, we employ mutagenesis and binding assays to show that the spike motif terminal residues are critical modulators of complex dissociation, which is essential for spike release in ERGIC. COPI residues critical for spike motif binding are elucidated by mutagenesis and crystallography and found to be conserved in the zoonotic reservoirs, bats, pangolins, camels, and in humans. Collectively, our investigation on the spike motif identifies key COPI binding determinants with implications for retrograde trafficking. A human monoclonal antibody bivalently binding two different epitopes in streptococcal M protein protects against infection Group A streptococci have evolved multiple strategies to evade human antibodies, making it challenging to create effective vaccines or antibody treatments. Here, we have generated antibodies derived from the memory B cells of an individual who had successfully cleared a group A streptococcal infection. The antibodies bind with high affinity in the central region of the surface-bound M protein. Such antibodies are typically non-opsonic. However, one antibody could effectively promote vital immune functions, including phagocytosis and in vivo protection. Remarkably, this antibody primarily interacts through a bivalent dual-Fab cis mode, where the Fabs bind to two distinct epitopes in the M protein. The dual-Fab cis binding phenomenon is conserved across different groups of M types. In contrast, other antibodies binding with normal single-Fab mode to the same region can not bypass the M proteins virulent effects. A broadly binding, protective monoclonal antibody could be a candidate for anti-streptococcal therapy. Our findings highlight the concept of dual-Fab cis binding as a means to access conserved, and normally non-opsonic regions, for protective antibody targeting. Agrobacterium mediated transformation and deciphering SNPs in TaLr67 gene homeologs for gene editing in wheat. Diseases adversely affect grain yield of crop plants. Leaf rust is a major disease of wheat. As race-specific resistance breaks down, introduction of newer sources of resistance often from older accessions is necessary. Linkage drag from the donor accessions adversely impacts grain yield. As a result, CIMMYT breeding effort has shifted to using durable resistance, also referred to as race non-specific host resistance, which allows slow rusting but maintains grain yield. One of the key genes for durable resistance is Lr67 and the resistant form of this gene is absent in CIMMYT elite lines. Hence, we have initiated efforts to convert the susceptible copy of Lr67 into its resistant form directly in these elite lines using gene editing. This would eliminate backcrossing and thus save time as well as eliminate linkage drag that would accompany the resistant copy of the gene if it were to be introgressed from an older accession. As first steps, we have isolated and sequenced the genomic copies from each of the A, B, and D genome of the Lr67 gene from three elite lines and an experimental line. Identification of more than 50 single nucleotide polymorphisms (SNPs) in the open reading frames (ORF) among these lines would be useful in designing the guide RNA molecules with precision. Further, we have streamlined genetic transformation of these elite lines, a prerequisite step for gene editing. Polycystins are upregulated in fibrotic kidneys and promote renal fibrosis Autosomal polycystic kidney disease (ADPKD) is caused by mutations in PKD1or PKD2 gene, which encodes polycystin-1 or polycystin-2 respectively. Paradoxically, polycystins are up-regulated in human ADPKD kidneys and overexpression of polycystin-1 even induced renal cyst formation in mice, suggesting that up-regulation of polycystin is pathogenic. In this study, we determined the role of polycystin in renal fibrosis and explored its underlying mechanisms. We found that the expression of polycystin-1 or polycystin-2 was positively correlated with the expression of collagen-I in the kidney of three different fibrotic mouse models. Pharmaceutical inhibition or genetic knockout of polycystin-2 reduced the expression of pro-fibrotic markers in fibrotic kidneys of two animal models. Moreover, conditional knockout of Pkd1 gene attenuated renal fibrosis in two mouse models. Enhancer of Zeste homolog 2 (EZH2) is a methyltransferase which plays important role in renal fibrosis. We found that EZH2 was positively correlated with the expression of polycystins in fibrotic kidneys. Conditional knockout of the Ezh2 attenuated the anti-fibrotic responses induced by Pkd1 deletion in fibrotic kidneys.

In conclusion, polycystins are up-regulated in injured kidneys and polycystins promote renal fibrosis through up-regulation of EZH2 in fibrotic kidneys, suggesting that renal fibrosis in polycystic kidneys is not the direct effect of PKD gene mutations.

SignificanceMutations of polycystins cause autosomal polycystic kidney disease (ADPKD) which is associated with increased renal fibrosis in cystic kidneys. However, the role of polycystin in renal fibrosis remains unclear. Here, we showed that the expression of polycystins is positively correlated with the development of renal fibrosis in injured kidneys. Pharmaceutical inhibition or genetic deletion of polycystin-2 attenuated renal fibrosis in fibrotic kidneys. Moreover, genetic deletion of polycystin-1 inhibited renal fibrosis in two different animal models. Finally, we showed that ablation of the methyltransferase EZH2 mitigated the anti-fibrotic effect of polycystin-1 deletion in fibrotic kidneys. We conclude that polycystin promotes through EZH2 and renal fibrosis in cystic kidney is not the direct effect of PKD gene mutations. Implicit Laplacian of Enhanced Edge: An Unguided Algorithm for Accurate and Automated Quantitative Analysis of Cytoskeletal Images The eukaryotic cytoskeleton plays essential roles in cell signaling and trafficking, which is broadly associated with immunity and diseases of human and plants. To date, most analyses aiming at defining the temporal and spatial dynamics of the cytoskeleton have relied on qualitative and quantitative analysis of fluorescence images to describe cytoskeletal function. While state-of-the-art, these approaches have limitations: the diverse shape and brightness of the cytoskeleton cause considerable inaccuracy in both human-driven and automated approaches, and the widely adopted image projection process (3D to 2D) leads to bias and information loss. Here, we describe the development and application of Implicit Laplacian of Enhanced Edge (ILEE), an unguided approach that uses a 2D/3D-compatible local thresholding algorithm for the quantitative evaluation of cytoskeletal status and organization at high performance. Using ILEE, we constructed a Python library to enable automated cytoskeletal image analysis, providing numerous biologically-interpretable indices measuring the density, bundling, severing, branching, and directionality of the cytoskeleton. The data presented herein demonstrate that ILEE resolves the defects of classic cytoskeleton analysis approaches, enables the measurement of novel cytoskeletal features, and yields quantitatively descriptive data with superior accuracy, stability, and robustness. We released the ILEE algorithm as an open-source library and further developed a Google Colab interface as a community resource. Modulation of P2X4 pore closure by magnesium,potassium, and ATP The P2X4 receptor plays a prominent role in cellular responses to extracellular ATP. Through classical all-atom molecular dynamics (MD) simulations totaling 24 {micro}s we have investigated how metal-complexed ATP stabilizes the channels open state and prevents its closing. We have identified two metal-binding sites, magnesium (Mg2+) and potassium (K+), one at the intersection of the three subunits in the ectodomain (MBS1) and the second one near the ATP binding site (MBS2), similar to those characterized in Gulf coast P2X. Our data indicate that when Mg2+ and K+ ions are complexed with ATP, the channel is locked into an open state. Interestingly, irrespective of the number of bound ATP molecules, Mg2+ ions bound to the MBS2 resisted collapsing of the open state protein to a closed state by stabilizing the ATP-protein interactions. However, when Mg2+ in the MBS2 was replaced with K+ ions, as might be expected when in equilibrium with an extracellular solution, the interactions between the subunits were weakened and we found evidence of pore collapse. This collapse was apparent when fewer than two ATP were bound to MBS2 in the presence of K+. Therefore, the different capacities of common cations to stabilize the channel may underlie a mechanism governing P2X4 channel gating in physiological systems. This study provides structural insights into the differential modulation of ATP activation of P2X4 by Mg2+ and K+. Diverse classes of constraints enable broader applicability of a linear programming-based dynamic metabolic modeling framework Current metabolic modeling tools suffer from a variety of limitations, from scalability to simplifying assumptions, that preclude their use in many applications. We recently created a modeling framework, LK-DFBA, that addresses a key gap: capturing metabolite dynamics and regulation while retaining a potentially scalable linear programming structure. Key to this frameworks success are the linear kinetics and regulatory constraints imposed on the system. However, while the linearity of these constraints reduces computational complexity, it may not accurately capture the behavior of many biochemical systems. Here, we developed three new classes of LK-DFBA constraints to better model interactions between metabolites and the reactions they regulate. We tested these new approaches on several synthetic and biological systems, and also performed the first-ever comparison of LK-DFBA predictions to experimental data. We found that no single constraint approach was optimal across all systems examined, and systems with the same topological structure but different parameters were often best modeled by different types of constraints. However, we did find that the optimal constraint approach was generally robust to local perturbations of the system, indicating that just a single wild-type dataset could allow identification of the ideal constraint for a given system. These results suggest that the availability of multiple constraint approaches will allow LK-DFBA to model a wider range of metabolic systems. 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. Synthetic periphyton as a model system to understand species dynamics in complex microbial freshwater communities Phototrophic biofilms, also known as periphyton, are microbial freshwater communities that drive crucial ecological processes in streams and lakes. Gaining a deep mechanistic understanding of the biological processes occurring in natural periphyton remains challenging due to the high complexity and variability of such communities. To address this challenge, we rationally developed a workflow to construct a synthetic community by co-culturing 26 phototrophic species (i.e., diatoms, green algae and cyanobacteria) that were inoculated in a successional sequence to create a periphytic biofilm on glass slides. We show that this community is diverse, stable and highly reproducible in terms of microbial composition, function and 3D spatial structure of the biofilm. We also demonstrate the ability to monitor microbial dynamics at the single species level during periphyton development and how their abundances are impacted by stressors such as increased temperature and a herbicide, singly and in combination. Overall, such a synthetic periphyton, grown under controlled conditions, can be used as a model system for theory testing through targeted manipulation. Environment dependent benefits of inter-individual variation in honey bee recruitment Inter-individual differences in behaviour within the members of a social group can affect the groups productivity. In eusocial insects, individual differences amongst workers in a colony play a central role division of labour and task allocation. Extensive empirical and theoretical work has highlighted variation in response thresholds as a proximate mechanism underlying individual behavioural differences and hence division of labour. However, other response parameters, like response probability and intensity, can affect these differences. In this study, we first extended a previously published agent-based model on honey bee foraging to understand the relative importance of response (dance) probability and response (dance) intensity in the task of recruitment. Comparing variation obtained from the simulations with previously published empirical data, we found that response intensity plays a more important role than probability in producing consistent inter-individual differences in recruitment behaviour. We then explored the benefits provided by this individual variation in recruitment behaviour to the colonys collective foraging effort under different environmental conditions. Our results revealed that individual variation leads to a greater energetic yield per forager, but only when food is abundant. Our study highlights the need to consider all response parameters while studying division of labour and adds to the growing body of evidence linking individual variation in behavioural responses to the success of social groups. Apolipoprotein M attenuates anthracycline cardiotoxicity and lysosomal injury ObjectivesDetermine the role of apolipoprotein M (ApoM) in anthracycline (Dox) cardiotoxicity.

BackgroundApoM binds the cardioprotective sphingolipid sphingosine-1-phosphate (S1P). Circulating ApoM is inversely associated with mortality in human heart failure (HF).

MethodsIn the Penn HF Study (PHFS), we tested the relationship between ApoM and mortality in a subset with anthracycline-induced cardiomyopathy. We measured ApoM in humans and mice treated with Dox and utilized hepatic ApoM transgenic (ApomTG), ApoM knockout (ApomKO), ApoM knock-in mice with impaired S1P binding, and S1P receptor 3 (S1PR3) knockout mice in Dox cardiotoxicity. We assayed autophagy in left ventricular tissue from anthracycline-induced HF patients versus donor controls.

ResultsApoM was inversely associated with mortality in PHFS, and Dox reduced circulating ApoM in mice and breast cancer patients. ApomTG mice were protected from Dox-induced cardiac dysfunction and loss of left ventricular mass. ApomTG attenuated Dox-induced impairment in autophagic flux in vivo and accumulation of insoluble p62, which was also observed in the myocardium of patients with anthracycline-induced HF. In vehicle-treated mice, ApoM negatively regulated transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis. The effect of ApoM on TFEB required both S1P binding and S1PR3. In the presence of Dox, ApoM preserved TFEB and cardiomyocyte lysosomal abundance assessed as lysosomal associated membrane protein 1 positive structures in vivo, while S1P mimetic pretreatment of cardiomyocytes prevented Dox-induced changes in lysosomal pH.

ConclusionsApoM attenuates Dox cardiotoxicity via the autophagy-lysosome pathway. The association between ApoM and reduced mortality may be explained by its role in sustaining autophagy.

HighlightsO_LICirculating ApoM is inversely associated with survival in human anthracycline-induced cardiomyopathy
C_LIO_LIAnthracycline treatment reduces circulating ApoM in humans and mice
C_LIO_LIIncreasing ApoM attenuates doxorubicin cardiotoxicity, lysosomal injury and preserves myocardial autophagic flux, but does not impact doxorubicin anti-neoplastic efficacy
C_LIO_LIAutophagic impairment is characteristic of human anthracycline cardiomyopathy
C_LI An IPI based immune prognostic model for diffuse large B-cell lymphoma BackgroundInternational Prognostic Index (IPI) was widely used to better discriminate prognosis of patients with diffuse large B-cell lymphoma (DLBCL). However, there is a significant need to identify novel valuable biomarkers in the context of targeted therapies, such as immune checkpoint blockade (ICB) therapy.

MethodsGene expression data and clinical information of DLBCL were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. 371 immune-related hub genes in DLBCL patients with different IPI levels were identified by weighted gene co-expression network analysis (WGCNA), and 8 genes were selected to construct an IPI-based immune prognostic model (IPI-IPM). Afterward, the genetic, somatic mutational and molecular profiles of IPI-IPM subgroups were analyzed, as well as the potential clinical response of ICB in different IPI-IPM subgroups.

ResultsThe IPI-IPM was constructed based on the expression of CMBL, TLCD3B, SYNDIG1, ESM1, EPHA3, HUNK, PTX3 and IL12A, where high-risk patients had shorter overall survival (OS) than low-risk patients, consistent with the results in the GEO cohorts. The comprehensive results showed that high IPI-IPM risk scores were correlated with immune-related signaling pathways, high KMT2D and CD79B mutation rates, as well as up-regulation of inhibitory immune checkpoints including PD-L1, BTLA and SIGLEC7, indicating more potential response to ICB therapy.

ConclusionThe IPI-IPM has independent prognostic significance for DLBCL patients, which provides an immunological perspective to elucidate the mechanisms on tumor progression, also sheds a light on developing immunotherapy for DLBCL. 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. Liver Endothelium Promotes HER3-mediated Cell Survival in KRAS Wild-type and Mutant Colorectal Cancer Cells We previously showed that liver endothelial cells (ECs) secreted soluble factors in a paracrine fashion and activated human epidermal growth factor receptor 3 (HER3, also known as ERBB3) for promoting colorectal cancer (CRC) growth and chemoresistance. However, RAS proteins play a critical role in receptor tyrosine kinase signaling pathways, and KRAS mutations mediate CRC resistance to therapies targeting EGFR, another HER protein. Therefore, the role of KRAS mutation status in EC-induced HER3 activation and CRC survival was investigated as it has therapeutic implications. We used CRC cell lines and patient-derived xenografts harboring KRAS wild-type or mutant genes and demonstrated that liver EC-secreted factors promoted HER3-mediated CRC cell growth independent of KRAS mutation status. Also, blocking HER3 in CRC cells by siRNAs or a HER3 antibody seribantumab blocked EC-induced CRC survival. Our findings highlight the potential of utilizing HER3 targeted therapies for treating patients with mCRC independent of RAS mutational status. Climate change and chill accumulation: implications for tree fruit production in cold-winter regions Winter chill accumulation is critical for the productivity and profitability of perennial tree fruit systems. Several studies have quantified the impacts of global warming on chill accumulation in the warmer production regions of the world, where insufficient chill events occur and their frequency is increasing. In contrast, we focus on a region with relatively cold winters-the Pacific Northwest United States (PNW)-where insufficient chill events are currently absent, and quantify the potential for introduction of these risks under climate change. Results identified spatial variation within the PNW, with chill accumulation projected to increase in some areas but decrease in others. There was also spatiotemporal variation in the driving factors of changes to chill accumulation. Even with decreases in chill accumulations, there are likely minimal issues with insufficient chill accumulation. However, delayed chill accumulation in combination with advances in the onset of heat accumulation can potentially shift the region from one where spring phenology is primarily forcing-driven to one where the dynamic interplay between chilling and forcing processes become important. These interactions might create production risks for varieties with high chill requirements, post mid-21st-century under high emissions scenarios. Future work should focus on understanding, modeling, and projecting responses across these overlapping chilling and forcing processes. Additionally, given significant spatial differences across a relatively small geographic range, it is also critical to understand and model these dynamics at a local landscape resolution for regions such as the PNW. Extracellular Matrix Hydrogels Promote Expression of Muscle-Tendon Junction Proteins Muscle and tendon injuries are prevalent and range from minor sprains and strains to traumatic, debilitating injuries. However, the interactions between these tissues during injury and recovery remain unclear. Three-dimensional tissue models that incorporate both tissues and a physiologically relevant junction between muscle and tendon may help understand how the two tissues interact. Here, we use tissue specific extracellular matrix (ECM) derived from muscle and tendon to determine how cells of each tissue interact with the microenvironment of the opposite tissue resulting in junction specific features. ECM materials were derived from the Achilles tendon and gastrocnemius muscle, decellularized, and processed to form tissue specific pre-hydrogel digests. ECM materials were unique in respect to protein composition and included many types of ECM proteins, not just collagens. After digestion and gelation, ECM hydrogels had similar complex viscosities which were less than type I collagen hydrogels at the same concentration. C2C12 myoblasts and tendon fibroblasts were cultured in tissuespecific ECM conditioned media or encapsulated in tissue-specific ECM hydrogels to determine cell-matrix interactions and the effects on a muscle-tendon junction marker, paxillin. ECM conditioned media had only a minor effect on upregulation of paxillin in cells cultured in monolayer. However, cells cultured within ECM hydrogels had 50-70% higher paxillin expression than cells cultured in type I collagen hydrogels. Contraction of the ECM hydrogels varied by the type of ECM used. Subsequent experiments with varying density of type I collagen (and thus contraction) showed no correlation between paxillin expression and the amount of gel contraction, suggesting that a constituent of the ECM was the driver of paxillin expression in the ECM hydrogels. In addition, the extracellular matrix protein type XXII collagen had similar expression patterns as paxillin, with smaller effect sizes. Using tissue specific ECM allowed for the de-construction of the cell-matrix interactions similar to muscletendon junctions to study the expression of MTJ specific proteins.

Impact StatementThe muscle-tendon junction is an important feature of muscle-tendon units; however, despite crosstalk between the two tissue types, it is overlooked in current research. Deconstructing the cell-matrix interactions will provide the opportunity to study significant junction specific features and markers that should be included in tissue models of the muscletendon unit, while gaining a deeper understanding of the natural junction. This research aims to inform future methods to engineer a more relevant multi-tissue platform to study the muscletendon unit. Sufu regulation of Hedgehog signaling in P19 cells is required for proper astrocyte differentiation Hedgehog signaling is essential for vertebrate development, however, less is known about the negative regulators that influence this pathway during the differentiation of cell fates. Using the mouse P19 embryonal carcinoma cell model, Suppressor of Fused (SUFU), a negative regulator of the Hedgehog pathway, was investigated during retinoic acid-induced neural differentiation. We found Hedgehog signaling was activated in the early phase of neural differentiation but was inactive during terminal differentiation of neurons and astrocytes. This early activation was required for neural differentiation however, it alone was not sufficient to induce neural lineages. SUFU, which regulates signaling at the level of GLI, remained relatively unchanged during the differentiation process, but its loss through CRISPR-Cas9 gene editing resulted in ectopic expression of Hedgehog target genes. Interestingly, these SUFU-deficient cells were unable to differentiate in the absence of retinoic acid, but when differentiated using retinoic acid, they showed delayed and decreased astrocyte differentiation; neuron differentiation was unaffected. Ectopic activation of Hh target genes in SUFU-deficient cells remained throughout retinoic acid-induced differentiation and this phenotype was accompanied by the loss of GLI3, despite the presence of the Gli3 message. Thus, the study indicates the proper timing and proportion of astrocyte differentiation requires SUFU, and its normal regulation of GLI3 to maintain Hh signaling in an inactive state. Laminar Mechanisms of Saccadic Suppression in Primate Visual Cortex Saccades are a ubiquitous and crucial component of our visual system, allowing for the efficient deployment of the fovea and its accompanying neural resources. Initiation of a saccade is known to cause saccadic suppression, a temporary reduction in visual sensitivity1, 2 and visual cortical firing rates3-6. While saccadic suppression has been well characterized at the level of perception and single neurons, relatively little is known about the visual cortical networks governing this phenomenon. Here we examine the effects of saccadic suppression on distinct neural subpopulations within visual area V4. We find cortical layer- and cell type-specific differences in the magnitude and timing of peri-saccadic modulation. Neurons in the input layer show changes in firing rate and inter-neuronal correlations prior to saccade onset, indicating that this layer receives information about impending saccades. Putative inhibitory interneurons in the input layer elevate their firing rate during saccades, suggesting they play a role in suppressing the activity of other cortical subpopulations. A computational model of this circuit recapitulates our empirical observations and demonstrates that an input layer-targeting pathway can initiate saccadic suppression by enhancing local inhibitory activity. Collectively, our results provide a mechanistic understanding of how eye movement signaling interacts with cortical circuitry to enforce visual stability. Penalized Logistic Regression Analysis for Genetic Association Studies of Binary Phenotypes IntroductionIncreasingly, logistic regression methods for genetic association studies of binary phenotypes must be able to accommodate data sparsity, which arises from unbalanced case-control ratios and/or rare genetic variants. Sparseness leads to maximum likelihood estimators (MLEs) of log-OR parameters that are biased away from their null value of zero and tests with inflated type 1 errors. Different penalized-likelihood methods have been developed to mitigate sparse-data bias. We study penalized logistic regression using a class of log-F priors indexed by a shrinkage parameter m to shrink the biased MLE towards zero.

MethodsWe propose a two-step approach to the analysis of a genetic association study: first, a set of variants that show evidence of association with the trait is used to estimate m; and second, the estimated m is used for log-F -penalized logistic regression analyses of all variants using data augmentation with standard software. Our estimate of m is the maximizer of a marginal likelihood obtained by integrating the latent log-ORs out of the joint distribution of the parameters and observed data. We consider two approximate approaches to maximizing the marginal likelihood: (i) a Monte Carlo EM algorithm (MCEM) and (ii) a Laplace approximation (LA) to each integral, followed by derivative-free optimization of the approximation.

ResultsWe evaluate the statistical properties of our proposed two-step method and compared its performance to other shrinkage methods by a simulation study. Our simulation studies suggest that the proposed log-F -penalized approach has lower bias and mean squared error than other methods considered. We also illustrate the approach on data from a study of genetic associations with "super senior" cases and middle aged controls.

Discussion/ConclusionWe have proposed a method for single rare variant analysis with binary phenotypes by logistic regression penalized by log-F priors. Our method has the advantage of being easily extended to correct for confounding due to population structure and genetic relatedness through a data augmentation approach. Ubiquity and origins of structural maintenance of chromosomes (SMC) proteins in eukaryotes Structural maintenance of chromosomes (SMC) protein complexes are common in Bacteria, Archaea, and Eukaryota. SMC proteins, together with the proteins related to SMC (SMC-related proteins), constitute a superfamily of ATPases. Bacteria/Archaea and Eukaryotes are distinctive from one another in terms of the repertory of SMC proteins. A single type of SMC protein is dimerized in the bacterial and archaeal complexes, whereas eukaryotes possess six distinct SMC subfamilies (SMC1-6), constituting three heterodimeric complexes, namely cohesin, condensin, and SMC5/6 complex. Thus, to bridge the homodimeric SMC complexes in Bacteria and Archaea to the heterodimeric SMC complexes in Eukaryota, we need to invoke multiple duplications of an SMC gene followed by functional divergence. However, to our knowledge, the evolution of the SMC proteins in Eukaryota had not been examined for more than a decade. In this study, we reexamined the ubiquity of SMC1-6 in phylogenetically diverse eukaryotes that cover the major eukaryotic taxonomic groups recognized to date and provide two novel insights into the SMC evolution in eukaryotes. First, multiple secondary losses of SMC5 and SMC6 occurred in the eukaryotic evolution. Second, the SMC proteins constituting cohesin and condensin (i.e., SMC1-4), and SMC5 and SMC6 were derived from closely related but distinct ancestral proteins. Based on the above-mentioned findings, we discuss how SMC1-6 have diverged from the archaeal homologs. The physics of liquid-to-solid transitions in multi-domain protein condensates Many RNA-binding proteins (RBPs) that assemble into membraneless organelles, have a common architecture including disordered prion-like domain (PLD) and folded RNA-binding domain (RBD). An enrichment of PLD within the condensed phase gives rise to formation, on longer time scales, amyloid-like fibrils (aging). In this study, we employ coarse-grained Langevin dynamics simulations to explore the physical basis for the structural diversity in condensed phases of multi-domain RBPs. We discovered a highly cooperative first order transition between disordered structures and an ordered phase whereby chains of PLD organize in fibrils with high nematic orientational order. An interplay between homo-domain (PLD-PLD) and hetero-domain (PLD-RBD) interactions results in variety of structures with distinct spatial architectures. Interestingly, the different structural phases also exhibit vastly different intra-cluster dynamics of proteins, with diffusion coefficients 5 (disordered structures) to 50 times (ordered structures) lower than that of the dilute phase. Cooperativity of this liquid-solid transition makes fibril formation highly malleable to mutations or post-translational modifications. Our results provide a mechanistic understanding of how multi-domain RBPs could form assemblies with distinct structural and material properties.

Significance StatementAssembly of proteins and nucleic acids into dense, liquid-like pockets is associated with several key functions including stress response, gene-regulation, DNA-repair and RNA processing. Several RNA binding proteins such as FUS are known to form liquid-like condensates that progressively harden into more dynamically, solid-like structures, a phenomenon that gets accelerated by disease mutations. In this study, we discover the mechanistic origins of this transition and show that small mutational or posttranslational modifications could result in sharp disorder-order transitions that could characterize accelerated liquid-solid transition in disease mutants. Evaluation of critical data processing steps for reliable prediction of gene co-expression from large collections of RNA-seq data MotivationGene co-expression analysis is an attractive tool for leveraging enormous amounts of public RNA-seq datasets for the prediction of gene functions and regulatory mechanisms. However, the optimal data processing steps for the accurate prediction of gene co-expression from such large datasets remain unclear. Especially the importance of batch effect correction is understudied.

ResultsWe processed RNA-seq data of 68 human and 76 mouse cell types and tissues using 50 different workflows into 7,200 genome-wide gene co-expression networks. We then conducted a systematic analysis of the factors that result in high-quality co-expression predictions, focusing on normalization, batch effect correction, and measure of correlation. We confirmed the key importance of high sample counts for high-quality predictions. However, choosing a suitable normalization approach and applying batch effect correction can further improve the quality of co-expression estimates, equivalent to a >80% and >40% increase in samples. In larger datasets, batch effect removal was equivalent to a more than doubling of the sample size. Finally, Pearson correlation appears more suitable than Spearman correlation, except for smaller datasets.

ConclusionA key point for accurate prediction of gene co-expression is the collection of many samples. However, paying attention to data normalization, batch effects, and the measure of correlation can significantly improve the quality of co-expression estimates. Development of a yeast-based assay for bioavailable phosphorus Preventing eutrophication of inland freshwater ecosystems requires quantifying the phosphorus (P) content of the streams and rivers that feed them. Typical methods for measuring P assess soluble reactive P (SRP) or total P (TP) and require expensive analytical techniques that produce hazardous waste. Here we present a novel method for measuring the more relevant bioavailable P (BAP); this assay utilizes the growth of familiar bakers yeast, avoids production of hazardous waste, and reduces cost relative to measurements of SRP and TP. The yeast BAP (yBAP) assay takes advantage of the observation that yeast density at saturating growth increases linearly with provided P. We show that this relationship can be used to measure P in freshwater in concentration ranges relevant to eutrophication. In addition, we measured yBAP in water containing known amounts of fertilizer and in samples from agricultural waterways. We observed that the majority of yBAP values were between those obtained from standard SRP and TP measurements, demonstrating that the assay is compatible with real-world settings. The cost-effective and nonhazardous nature of the yeast-based assay suggests that it could have utility in a range of settings, offering added insight to identify water systems at risk of eutrophication from excess phosphorus.