title: In-scanner head motion and structural covariance networks

abstract: In-scanner head motion systematically reduces estimated regional gray matter volumes obtained from structural brain MRI. Here, we investigate how head motion affects structural covariance networks that are derived from regional gray matter volumetric estimates. We acquired motion-affected and motion-free whole brain T1-weighted MRI in 29 healthy adult subjects and estimated relative regional gray matter volumes using a voxel-based morphometry approach. Structural covariance network analyses were undertaken while systematically increasing the number of motion-affected scans included in the analysis. We demonstrate that the standard deviation in regional gray matter estimates increases as the number of motion-affected scans increases. This subsequently increases pair-wise correlations between regions, a key determinant for construction of structural covariance networks. We further demonstrate that head motion systematically alters graph theoretic metrics derived from these networks. Our findings suggest that in-scanner head motion is a source of error that violates the assumption that structural covariance networks reflect neuroanatomical connectivity between brain regions. Results of structural covariance studies should be interpreted with caution, particularly when subject groups are likely to move their heads in the scanner.


title: Genomic imprinting drives eusociality

abstract: The origin of eusociality, altruistically foregoing personal reproduction to help others, has been a long-standing paradox ever since Darwin. Most eusocial insects and rodents likely evolved from subsocial precursors, in which older offspring "helpers" contribute to the development of younger siblings without a permanent sterile caste. The driving mechanism for the transition from subsociality (with helpers) to eusociality (with lifelong sterile workers) remains an enigma because individuals in subsocial groups are subject to direct natural selection rather than kin selection. Our genomic imprinting theory demonstrates that natural selection generates eusociality in subsocial groups when parental reproductive capacity is linked to a delay in the sexual development of offspring due to sex-antagonistic action of transgenerational epigenetic marks. Focusing on termites, our theory provides the missing evolutionary link to explain the evolution of eusociality from their subsocial wood-feeding cockroach ancestors, and provides a novel framework for understanding the origin of eusociality.

category: evolutionary biology


title: Gene Family Complexity and Expression Divergence as a Mechanism of Adaptation In Coral

abstract: Gene family complexity and its influence on expression dynamics has long been theorized to be an important source of adaptation in natural systems through providing novel genetic material and influencing gene dosage. There is now growing empirical support for this theory; however, this process has only been demonstrated in a limited number of systems typically using recently diverged species or populations. In particular, examples of how this process operates in basal animals with deeper species splits has not been well explored. To address this issue, we investigated the evolution of gene family complexity in five species of common Caribbean coral. We demonstrate widespread divergence in gene repertoires owing to slow rates of gene turnover occurring along deep species splits. The resulting differences in gene family complexity involve numerous biologic processes, shedding light on to the selective forces that have influenced the evolution of each species. By coupling these findings with gene expression data, we show that increased gene family complexity promotes increased expression divergence between species, indicating an interplay between gene family complexity and expression divergence. Finally, we show that immune genes are evolving particularly fast demonstrating the importance of interactions with other organisms in the evolutionary history of Caribbean corals. Overall, these findings provide support for gene copy number change as an important evolutionary force in Caribbean corals, which may influence their ability to persist in a rapidly changing environment.

category: genomics


title: Distortion Discovery: A Framework to Model, Spot and Explain Tumor Heterogeneity and Mitigate its Negative Impact on Cancer Risk Assessment

abstract: In a complex system of inter-genome interactions, false negatives remain an overwhelming problem when using omics data for disease risk prediction. This is especially clear when dealing with complex diseases like cancer in which the infiltration of stromal and immune cells into the tumor tissue can affect the degree of its tumor purity and hence its cancer signal. Previous work was done to estimate the degree of cancer purity in a tissue. In this work, we introduce a data and biomarker selection independent, information theoretic, approach to tackle this problem. We model distortion as a source of false negatives and introduce a mechanism to detect and remove its impact on the accuracy of disease risk prediction.

category: bioinformatics


title: SPIRE, Surface Projection Image Recognition Environment for bicontinuous phases: application for plastid cubic membranes

abstract: Bicontinuous membranes in cell organelles epitomise natures ability to create complex functional nanostructures. Like their synthetic counterparts, these membranes are characterised by continuous membrane sheets draped onto topologically complex saddle-shaped surfaces with a periodic network-like structure. In cell organelles, their structure sizes around 50-500 nm and fluid nature make Transmission Electron Microscopy (TEM) the analysis method of choice to decipher nanostructural features. Here we present a tool to identify bicontinuous structures from TEM sections by comparison to mathematical "nodal surface" models, including the hexagonal lonsdaleite geometry. Our approach, following pioneering work by Deng and Mieczkowski (1998), creates synthetic TEM images of known bicontinuous geometries for interactive structure identification. We apply the method to the inner membrane network in plant cell chloroplast precursors and achieve a robust identification of the bicontinuous diamond surface as the dominant geometry in several plant species. This represents an important step in understanding their as yet elusive structure-function relationship.

category: cell biology


title: Development of a 3D atlas of the embryonic pancreas for topological and quantitative analysis of heterologous cell interactions

abstract: Generating comprehensive image maps, while preserving spatial 3D context, is essential to quantitatively assess and locate specific cellular features and cell-cell interactions during organ development. Despite the recent advances in 3D imaging approaches, our current knowledge of the spatial organization of distinct cell types in the embryonic pancreatic tissue is still largely based on 2D histological sections. Here, we present a light-sheet fluorescence microscopy approach to image the pancreas in 3D and map tissue interactions at key development time points in the mouse embryo. We used transgenic mouse models and antibodies to visualize the three main cellular components within the developing pancreas, including epithelial, mesenchymal and endothelial cell populations. We demonstrated the utility of the approach by providing volumetric data, 3D distribution of distinct progenitor populations and quantification of relative cellular abundance within the tissue. Lastly, our image data were combined in an open source online repository (referred to as Pancreas Embryonic Cell Atlas). This image dataset will serve the scientific community by enabling further investigation on pancreas organogenesis but also for devising strategies for the in vitro generation of transplantable pancreatic tissue for regenerative therapies.

category: developmental biology


title: A re-analysis of the data in Sharkey et al.'s (2021) minimalist revision reveals that BINs do not deserve names, but BOLD Systems needs a stronger commitment to open science

abstract: Halting biodiversity decline is one of the most critical challenges for humanity, but monitoring biodiversity is hampered by taxonomic impediments. One impediment is the large number of undescribed species (here called "dark taxon impediment") while another is caused by the large number of superficial species descriptions which can only be resolved by consulting type specimens ("superficial description impediment"). Recently, Sharkey et al. (2021) proposed to address the dark taxon impediment for Costa Rican braconid wasps by describing 403 species based on barcode clusters ("BINs") computed by BOLD Systems. More than 99% of the BINs (387 of 390) are converted into species by assigning binominal names (e.g., BIN "BOLD:ACM9419" becomes Bracon federicomatarritai) and adding a minimal diagnosis (usually consisting only of a consensus barcode). We here show that many of Sharkey et al.s species are unstable when the underlying data are analyzed using different species delimitation algorithms. Add the insufficiently informative diagnoses, and many of these species will become the next "superficial description impediment" for braconid taxonomy because they will have to be tested and redescribed after obtaining sufficient evidence for confidently delimiting species. We furthermore show that Sharkey et al.s approach of using consensus barcodes as diagnoses is not functional because it cannot be consistently applied. Lastly, we reiterate that COI alone is not suitable for delimiting and describing species and voice concerns over Sharkey et al.s uncritical use of BINs because they are calculated by a proprietary algorithm (RESL) that uses a mixture of public and private data. We urge authors, reviewers, and editors to maintain high standards in taxonomy by only publishing new species that are rigorously delimited with open-access tools and supported by publicly available evidence.


title: The YPhi Motif Defines the Structure-Activity Relationships of Human 20S Proteasome Activators

abstract: The 20S proteasome (20S) facilitates turnover of most eukaryotic proteins. Substrate entry into the 20S first requires opening of gating loops through binding of HbYX motifs that are present at the C-termini of certain proteasome activators (PAs). The HbYX motif has been predominantly characterized in the archaeal 20S, whereas little is known about the sequence preferences of the human 20S (h20S). Here, we synthesized and screened [~]120 HbYX-like peptides, revealing unexpected differences from the archaeal system and defining the h20S recognition sequence as the Y-F/Y (Y{Phi}) motif. To gain further insight, we created a functional chimera of the optimized sequence, NLSYYT, fused to the model activator, PA26E102A.A cryo-EM structure of PA26E102A-h20S identified key interactions, including non-canonical contacts and gate-opening mechanisms. Finally, we demonstrated that the Y{Phi} sequence preferences are tuned by valency, allowing multivalent PAs to sample greater sequence space. These results expand the model for termini-mediated gating and provide a template for the design of h20S activators.

category: biochemistry


title: Modeling intracranial electrodes

abstract:
BackgroundIntracranial electrodes are implanted in patients with drug-resistant epilepsy as part of their pre-surgical evaluation. This allows investigation of normal and pathological brain functions with excellent spatial and temporal resolution. The spatial resolution relies on methods that precisely localize the implanted electrodes in the cerebral cortex, which is critical for drawing valid anatomical inferences about brain function.

Multiple methods have been developed to localize implanted electrodes, mainly relying on pre-implantation MRI and post-implantation CT images. However, there is no standard approach to quantify the performance of these methods systematically.

The purpose of our work is to model intracranial electrodes to simulate realistic implantation scenarios, thereby providing methods to optimize localization algorithm performance.

ResultsWe implemented novel methods to model the coordinates of implanted grids, strips, and depth electrodes, as well as the CT artifacts produced by these.

We successfully modeled a large number of realistic implantation "scenarios", including different sizes, inter-electrode distances, and brain areas. In total, more than 3300 grids and strips were fitted over the brain surface, and more than 850 depth electrode arrays penetrating the cortical tissue were modeled. More than 37000 simulations of electrode array CT artifacts were performed in these "scenarios", mimicking the intensity profile and orientation of real artifactual voxels. Realistic artifacts were simulated by introducing different noise levels, as well as overlapping electrodes.

ConclusionsWe successfully developed the first platform to model implanted intracranial grids, strips, and depth electrodes and realistically simulate CT artifacts and noise.

These methods set the basis for developing more complex models, while simulations allow the performance evaluation of electrode localization techniques systematically.

The methods described in this article, and the results obtained from the simulations, are freely available via open repositories. A graphical user interface implementation is also accessible via the open-source iElectrodes toolbox.


title: Expression of a CO2-permeable aquaporin enhances mesophyll conductance in the C4 species Setaria viridis

abstract: A fundamental limitation of photosynthetic carbon fixation is the availability of CO4. In C4 plants, primary carboxylation occurs in mesophyll cytosol, and little is known about the role of CO2 diffusion in facilitating C4 photosynthesis. We have examined the expression, localization, and functional role of selected plasma membrane intrinsic aquaporins (PIPs) from Setaria italica (foxtail millet) and discovered that SiPIP2;7 is CO2-permeable. When ectopically expressed in mesophyll cells of S. viridis (green foxtail), SiPIP2;7 was localized to the plasma membrane and caused no marked changes in leaf biochemistry. Gas-exchange and C18O16O discrimination measurements revealed that targeted expression of SiPIP2;7 enhanced the conductance to CO2 diffusion from the intercellular airspace to the mesophyll cytosol. Our results demonstrate that mesophyll conductance limits C4 photosynthesis at low pCO2 and that SiPIP2;7 is a functional CO2 permeable aquaporin that can improve CO2 diffusion at the airspace/mesophyll interface and enhance C4 photosynthesis.

category: plant biology


title: Sensory processing dysregulations as reliable translational biomarkers in SYNGAP1 haploinsufficiency

abstract: Amongst the numerous genes associated with intellectual disability, SYNGAP1 stands out for its frequency and penetrance of loss-of-function variants found in patients, as well as the wide range of co-morbid disorders associated with its mutation. Most studies exploring the pathophysiological alterations caused by Syngap1 haploinsufficiency in mouse models have focused on cognitive problems and epilepsy, however whether and to what extent sensory perception and processing are altered by Syngap1 haploinsufficiency is less clear. By performing EEG recordings in awake mice, we identified specific alterations in multiple aspects of auditory and visual processing, including increased baseline gamma oscillation power, increased theta/gamma phase amplitude coupling following stimulus presentation and abnormal neural entrainment in response to different sensory modality-specific frequencies. We also report lack of habituation to repetitive auditory stimuli and abnormal deviant sound detection. Interestingly, we found that most of these alterations are present in human patients as well, thus making them strong candidates as translational biomarkers of sensory-processing alterations associated with SYNGAP1/Syngap1 haploinsufficiency.

category: neuroscience


title: Systematic morphological and morphometric analysis of identified olfactory receptor neurons in Drosophila melanogaster

abstract: The biophysical properties of sensory neurons are influenced by their morphometric and morphological features, whose precise measurements require high-quality volume electron microscopy (EM). However, systematic surveys of these nanoscale characteristics for identified neurons are scarce. Here, we characterize the morphology of Drosophila olfactory receptor neurons (ORNs) across the majority of genetically identified sensory hairs. By analyzing serial block-face electron microscopy (SBEM) images of cryofixed antennal tissues, we compile an extensive morphometric dataset based on 122 reconstructed 3D models of 33 identified ORN types. In addition, we observe multiple novel features--including extracellular vacuoles within sensillum lumen, intricate dendritic branching, mitochondria enrichment in select ORNs, novel sensillum types, and empty sensilla containing no neurons--which raise new questions pertinent to cell biology and sensory neurobiology. Our systematic survey is critical for future investigations into how the size and shape of sensory neurons influence their responses, sensitivity and circuit function.


title: Judgments of agency are affected by sensory noise without recruiting metacognitive processing

abstract: Judgments of agency, our sense of control over our actions and the environment, often occur in noisy conditions. We examined the computations underlying judgments of agency, in particular under the influence of sensory noise. Building on previous literature, we studied whether judgments of agency incorporate uncertainty in the same way that confidence judgments do, which would imply that the former share computational mechanisms with metacognitive judgments. In two tasks, participants rated agency, or confidence in a decision about their agency, over a virtual hand that tracked their movements, either synchronously or with a delay and either under high or low noise. We compared the predictions of two computational models to participants ratings and found that agency ratings, unlike confidence, were best explained by a model involving no estimates of sensory noise. We propose that agency judgments reflect first-order measures of the internal signal, without involving metacognitive computations, challenging the assumed link between the two cognitive processes.


title: Gonadal hormones and sex chromosome complement differentially contribute to ethanol intake, preference, and relapse-like behavior in Four Core Genotypes mice

abstract: Alcohol use and high-risk alcohol drinking behaviors among women are rapidly rising. In rodent models, females typically consume more ethanol (EtOH) than males. Here, we used the Four Core Genotypes (FCG) mouse model to investigate the influence of gonadal hormones and sex chromosome complement on EtOH drinking behaviors. FCG mice were given access to escalating concentrations of EtOH in a two-bottle, 24-h continuous access drinking paradigm to assess consumption and preference. Relapse-like behavior was measured by assessing escalated intake following repeated cycles of deprivation and re-exposure. Twenty-four hour EtOH consumption was greater in mice with ovaries (Sry-), relative to those with testes, and in mice with the XX chromosome complement, relative to those with XY sex chromosomes. EtOH preference was higher in XX vs. XY mice. Escalated intake following repeated cycles of deprivation and re-exposure emerged only in XX mice (vs. XY). Mice with ovaries (Sry-FCG mice and C57Bl/6J females) were also found to consume more water than mice with testes. These results demonstrate that aspects of EtOH drinking behavior may be independently regulated by sex hormones and chromosomes and inform our understanding of the neurobiological mechanisms which contribute to EtOH dependence in male and female mice. Future investigation of the contribution of sex chromosomes to EtOH drinking behaviors is warranted.


title: Prostacyclin as a Negative Regulator of Angiogenesis in the Neurovasculature

abstract: In this study, multiple measures of angiogenic processes were assessed in murine brain endothelial (bEnd.3) cells after exposure to the stable prostacyclin analog, iloprost. Additionally, changes in the {gamma}-secretase enzyme were evaluated after activation of prostacyclin signaling using PGI2 overexpressing mouse brain tissue and immunohistology studies in bEnd.3 cells. A three-dimensional assay of tube formation revealed that iloprost inhibits normal formation by significantly reduced tube lengths and vessel mesh area. The iloprost-mediated inhibition of tube-like structures was ameliorated by a specific IP-receptor antagonist, CAY10449. Reductions in wound healing were observed with iloprost application in a dose-dependent manner and this effect was reversed using CAY10449. Iloprost did not exhibit anti-proliferative effects in the bEnd.3 cells. When subjected to a Transwell assay to evaluate changes in trans-epithelial electrical resistance (TEER), bEnd.3 cells displayed reduced TEER values in the presence of iloprost an effect that lasted over prolonged periods (24 hours). Again, CAY10449 was able to reverse iloprost-mediated reductions in TEER value. Surprisingly, the adenylyl cyclase activator, forskolin, produced higher TEER values in the bEnd.3 cells over the same time. The TEER results suggest that iloprost may not activating the Gs protein of the IP receptor to increase cAMP levels given by the opposing results seen with iloprost and forskolin. In terms of {gamma}-secretase expression, PGI2 overexpression in mice increased the expression of the APH-1 subunit in the hippocampus and cortex. In bEnd.3 cells, iloprost application slightly increased APH-1 subunit expression measured by western blot and interrupted the colocalization of Presenilin 1 and APH-1 subunits using immunohistochemistry. The results suggest that prostacyclin signaling within bEnd.3 cells is anti-angiogenic and further downstream events have effects on the expression and most likely the activity of the A{beta} cleaving enzyme, {gamma}-secretase.


title: Regulation of layered T cell tolerance mechanisms by the NR4A family is essential to preserve immune homeostasis and suppress autoimmunity

abstract: The NR4A family of orphan nuclear receptors (Nr4a1-3) plays redundant roles upstream of Foxp3 to establish and maintain Treg identity; deletion of multiple family members in the thymus results in Treg deficiency and a severe inflammatory disease. Consequently, it has been challenging to isolate the functions of this family in other immune cells. Here we take advantage of a competitive bone marrow chimera strategy, coupled with conditional genetic tools, to rescue Treg homeostasis and unmask such functions. Unexpectedly, chimeras harboring Nr4a1-/- Nr4a3-/- (DKO) bone marrow develop autoantibodies and a systemic inflammatory disease despite a replete Treg compartment of largely wild-type origin. This disease differs qualitatively from that seen with Treg-deficiency and is B cell-extrinsic. Negative selection of DKO thymocytes is profoundly impaired in a cell-intrinsic manner. Consistent with escape of self-reactive T cells into the periphery, DKO T cells with functional and phenotypic features of anergy accumulate in chimeric mice. Despite this, DKO T cells exhibit enhanced IL-2 production, implying a cell-intrinsic role for the NR4A family in peripheral T cell tolerance. These studies reveal roles for the NR4A family in multiple layered T cell tolerance mechanisms and demonstrate that each is essential to preserve immune homeostasis.

category: immunology


title: Transplantation of high fat fed mouse microbiota into zebrafish embryos identifies pathobiont species

abstract: Gut dysbiosis is an important modifier of pathologies including cardiovascular disease but our understanding of the role of individual microbes is limited. Here, we have used transplantation of mouse microbiota into microbiota-deficient zebrafish larvae to study the interaction between members of a mammalian high fat diet-associated gut microbiota with a lipid rich diet challenge in a tractable model species. We find zebrafish larvae are more susceptible to hyperlipidaemia when exposed to the mouse high fat-diet-associated microbiota and that this effect can be driven by two individual bacterial species fractionated from the mouse high fat-diet-associated microbiota. We find Stenotrophomonas maltophilia increases the hyperlipidaemic potential of chicken egg yolk to zebrafish larvae independent of direct interaction between S. maltophilia and the zebrafish host. Colonisation by live, or exposure to heat-killed, Enterococcus faecalis accelerates hyperlipidaemia via host Myd88 signalling. The hyperlipidaemic effect is replicated by exposure to the Gram positive Toll-like receptor agonists peptidoglycan and lipoteichoic acid in a MyD88-dependent manner. In this work, we demonstrate the applicability of zebrafish as a tractable host for the identification of gut microbes that can induce conditional host phenotypes via microbiota transplantation and subsequent challenge with a high fat diet.

category: microbiology


title: Transplantation of high fat fed mouse microbiota into zebrafish larvae identifies MyD88-dependent acceleration of hyperlipidaemia by Gram positive cell wall components


title: Efficacy of oseltamivir treatment in influenza virus infected obese mice

abstract:
Obesity has been epidemiologically and empirically linked with more severe disease upon influenza infection. To ameliorate severe disease, treatment with antivirals, such as the neuraminidase inhibitor oseltamivir, are suggested to begin within days of infection, especially in hosts at higher risk for poor outcomes. However, this treatment is often poorly effective and can generate resistance variants within the treated host. Here, we hypothesized that oseltamivir treatment would not be effective in genetically obese mice and would generate a more diverse and drug resistant viral population. We demonstrated that oseltamivir treatment does not improve viral clearance in obese mice. While no traditional variants associated with oseltamivir resistance emerged, we did note that drug treatment failed to quench the viral population and did lead to phenotypic drug resistance in vitro. Mechanistically, we demonstrate the blunted interferon response in obese hosts may be contributing to treatment failure, as type I interferon receptor deficient mice also fail to clear influenza virus infection upon oseltamivir administration. Together, these studies suggest that the unique pathogenesis and immune responses in obese mice could have implications for pharmaceutical interventions and the within-host dynamics of the influenza virus population.

IMPORTANCEInfluenza virus infections, while typically resolving within days to weeks, can turn critical especially in high-risk populations. Prompt antiviral administration is crucial to mitigating these severe sequalae, yet concerns remain if antiviral treatment is effective in hosts with obesity. Here, we show that oseltamivir does not improve viral clearance in genetically obese or type I IFN receptor-deficient mice and increases the genetic entropy of the within-host viral population. This suggests a blunted immune response may impair oseltamivir efficacy and render a host more susceptible to severe disease. This study furthers our understanding of oseltamivir treatment dynamics both systemically and in the lungs of obese mice, as well as the consequences of oseltamivir treatment for the within-host emergence of drug-resistant variants.


title: Evaluation of the virucidal efficacy of Klaran UVC LEDs against surface dried norovirus

abstract: Human norovirus (HuNoV) is a highly contagious pathogenic virus that is transmitted through contaminated food, water, high-touch surfaces and aerosols. Globally, there are an estimated 685 million infections annually due to norovirus, among them 200 million children under the age of 5, causing approximately 50,000 child deaths per year and costing an estimated $60 billion annually in healthcare. In the USA, HuNoV is responsible for 19-21 million illnesses, with an average of 570-800 deaths per year. HuNoV is especially pernicious because it requires less than 100 viral particles to cause an infection, and there are few effective disinfectants. It is believed that Ultraviolet Subtype C (UVC) irradiation might prove to be an effective disinfectant. This study seeks to determine the inactivation profile of UVC against norovirus using a Klaran UVC Light-emitting diode (LED) array product number KL265-50V-SM-WD, emitting radiation at 269 nm peak wavelength and a measured fluence of 1.25 mW/cm2 at a 7 cm source-surface distance. Since the HuNoV cannot currently be propagated in cell cultures, the study utilized feline calicivirus (FCV), a recommended surrogate as challenge organism. The test followed Modified ASTM E2197. Assessment of virus inactivation was performed using plaque assay method, with Crystal Violet as a staining agent to enhance plaque visualization. Within 18 seconds of exposure time at a UVC irradiance of 1.25mW/cm2 and a dose of 22.5 mJ/cm2, the study obtained 99.9 % virus reduction (3 log reduction value). These results demonstrate that Klaran UVC-LED array (KL265-50V-SM-WD) can provide effective inactivation of HuNoV.


title: Integration of embryo-endosperm interaction into a holistic and dynamic picture of seed development using a rice mutant with notched-belly grains

abstract: The interaction between the embryo and endosperm affects seed development, an essential process in yield formation in crops such as rice. Signals that communicate between embryo and endosperm are largely unknown. Here we use the notched-belly (NB) mutant with impaired communication between embryo and endosperm to evaluate 1) the impact of embryo on developmental staging of the endosperm; 2) signaling pathways emanating from the embryo that regulate endosperm development. Hierachical clustering of mRNA datasets from embryo and endosperm samples collected through development in NB and wild type showed a delaying effect of the embryo on the developmental transition of the endosperm by extending the middle stage. K-means clustering further identified coexpression modules of gene sets specific for embryo and endosperm development. Combined gene expression and biochemical analysis showed that T6P-SnRK1, gibberellin and auxin signalling from the embryo regulate endosperm developmental transition. The data enable us to propose a new seed developmental staging system for rice and the most detailed signature of rice grain formation to date, that will direct genetic strategies for rice yield improvement.


title: Bacteriophages encoding human immune evasion factors adapt to livestock-associated MRSA through rounds of integration and excision

abstract: In recent years there has been an increase in human infections with methicillin-resistant Staphylococcus aureus (MRSA) originating from livestock and strains carrying bacterial viruses of the Sa3int-family have disseminated into the community. Sa3int phages express immune evasion factors and are common in human staphylococcal strains. As the bacterial attachment site (attB) for Sa3int phages is mutated in livestock-associated strains, the integration frequency is low and a key question is how the phages are established. Here we show that Sa3int phages adapt to alternative bacterial integration sites by mutating the phage attachment sequence, attP, leading to enhanced integration at these sites. Using a model strain carrying the mutated attBLA of livestock-associated strains we find that once established, the Sa3int phage, {Phi}13 is inducible with release of heterogenous phage populations carrying mutations in attP that in part increase homology to alternative integration sites or attBLA. Compared to the original phage, the adaptive mutations increase phage integration in new rounds of infection. Also, Sa3int phages induced from livestock-associated outbreak strains reveal mutated attP sequences. We suspect that promiscuity of the phage-encoded recombinase allows this adaptation and propose it may explain how phages mediate "host jumps" that are regularly observed for staphylococcal lineages.


title: PLURIPOTENT STEM CELL-DERIVED CARDIOVASCULAR PROGENITORS DIFFFERENTIATED ON LAMININ 221 REGENERATE AND IMPROVE FUNCTION OF INFARCTED SWINE HEARTS

abstract:
BackgroundIschemic heart disease is a huge global burden where patients often have irreversibly damaged heart muscle. State-of-the-art technology using stem cell-derived products for cellular therapy could potentially replace damaged heart muscle for regenerative cardiology.

Methods and ResultsPluripotent human embryonic stem cells (hESCs) were differentiated on a laminin LN521+221 matrix to cardiovascular progenitors (CVPs). Global transcriptome analyses at multiple time points by single-cell RNA-sequencing demonstrated high reproducibility (R2 > 0.95) between two hESCs lines. We identified several CVP signature genes as quality batch control parameters which are highly specific to our CVPs as compared to canonical cardiac progenitor genes. A total of 200 million CVPs were injected into the infarcted region caused by permanent ligation of the coronary arteries of 10 immunosuppressed pigs and maintained for 4- and 12-weeks post transplantation. The transplanted cells engrafted and proliferated in the infarcted area as indicated by IVIS imaging, histology staining and spatial transcriptomic analysis. Spatial transcriptomic analysis at 1 week following transplantation showed that the infarcted region expressed human genes in the same area as immunohistology sections. Heart function was analyzed by magnetic resonance imaging (MRI) and computerized tomography (CT). Functional studies revealed overall improvement in left ventricular ejection fraction by 21.35 {+/-} 3.3 %, which was accompanied by significant improvements in ventricular wall thickness and wall motion, as well as a reduction in infarction size after CVP transplantation as compared to medium control pigs (P < 0.05). Immunohistology analysis revealed maturation of the CVPs to cardiomyocytes (CMs) where the human grafts aligned with host tissue forming end-to-end connections typical for heart muscle. Electrophysiology analyses revealed electric continuity between injected and host tissue CMs. Episodes of ventricular tachyarrhythmia (VT) over a period of 25 days developed in four pigs, one pig had persistent VT, while the rest remained in normal sinus rhythm. All ten pigs survived the experiment without any VT-related death.

ConclusionsWe report a highly reproducible, chemically defined and fully humanized differentiation method of hESCs for the generation of potent CVPs. This method may pave the way for lasting stem cell therapy of myocardial infarction (MI) in humans.

Clinical PerspectiveO_ST_ABSWhat is New?C_ST_ABSO_LIWe present a highly reproducible, chemically defined and fully humanized laminin-based differentiation method for generation of large amounts of cardiovascular progenitors (CVP); 20 million cells in a 10 cm2 culture dish which were used for a preclinical study in pigs.
C_LIO_LITransplantation of the CVPs into the myocardial infarcted pig hearts yields maturation of the progenitor cells to cardiomyocytes (CMs) and improved cardiac function (21.35 {+/-} 3.3 % LVEF improvement) using only 200 million CVPs.
C_LIO_LITemporary episodes of ventricular arrhythmia (50%) were observed after CVP transplantation. No fatal ventricular arrhythmia occurred.
C_LI

What are the clinical implications?O_LIOur laminin-based approach generated potent CVPs in vivo and largely restored function of the damaged heart.
C_LIO_LICardiovascular progenitors may provide a new and safe therapeutic strategy for myocardial infarction.
C_LIO_LIThe results may have a significant impact on regenerative cardiology.
C_LI


title: ZAF -- An Open Source Fully Automated Feeder for Aquatic Facilities

abstract: In the past few decades, aquatic animals have become popular model organisms in biology, spurring a growing need for establishing aquatic facilities. Zebrafish are widely studied and relatively easy to culture using commercial systems. However, a challenging aspect of maintaining aquatic facilities is animal feeding, which is both time- and resource-consuming. We have developed an open-source fully automatic daily feeding system, Zebrafish Automatic Feeder (ZAF). ZAF is reliable, provides a standardized amount of food to every tank, is cost-efficient and easy to build. The advanced version, ZAF+, allows for the precise control of food distribution as a function of fish density per tank, and has a user-friendly interface. Both ZAF and ZAF+ are adaptable to any laboratory environment and facilitate the implementation of aquatic colonies. Here we provide all blueprints and instructions for building the mechanics, electronics, fluidics, as well as to setup the control software and its user-friendly graphical interface. Importantly, the design is modular and can be scaled to meet different user needs. Furthermore, our results show that ZAF and ZAF+ do not adversely affect zebrafish culture, enabling fully automatic feeding for any aquatic facility.


title: ZAF -- The First Open Source Fully Automated Feeder for Aquatic Facilities


title: Identification and correction of phase switches with Hi-C data in the Nanopore and HiFi chromosome-scale assemblies of the dikaryotic leaf rust fungus Puccinia triticina

abstract:
BackgroundMost animals and plants have more than one set of chromosomes and package these haplotypes into a single nucleus within each cell. In contrast, many fungal species carry multiple haploid nuclei per cell. Rust fungi are such species with two nuclei (karyons) that contain a full set of haploid chromosomes each. The physical separation of haplotypes in dikaryons means that, unlike in diploids, Hi-C chromatin contacts between haplotypes are false positive signals.

ResultsWe generate the first chromosome-scale, fully-phased assembly for the dikaryotic leaf rust fungus Puccinia triticina and compare Nanopore MinION and PacBio HiFi sequence-based assemblies. We show that false positive Hi-C contacts between haplotypes are predominantly caused by phase switches rather than by collapsed regions or Hi-C read mis-mappings. We introduce a method for phasing of dikaryotic genomes into the two haplotypes using Hi-C contact graphs, including a phase switch correction step. In the HiFi assembly, relatively few phase switches occur, and these are predominantly located at haplotig boundaries and can be readily corrected. In contrast, phase switches are widespread throughout the Nanopore assembly. We show that haploid genome read coverage of 30-40 times using HiFi sequencing is required for phasing of the leaf rust genome (~0.7% heterozygosity) and that HiFi sequencing resolves genomic regions with low heterozygosity that are otherwise collapsed in the Nanopore assembly.

ConclusionsThis first Hi-C based phasing pipeline for dikaryons and comparison of long-read sequencing technologies will inform future genome assembly and haplotype phasing projects in other non-haploid organisms.


title: Ground tissue circuitry regulates organ complexity in cereal roots

abstract:
Most plant roots have multiple cortex layers that make up the bulk of the organ and play key roles in physiology, such as flood tolerance and symbiosis. However, little is known about the formation of cortical layers outside of the highly reduced anatomy of the model Arabidopsis. Here we use single-cell RNAseq to rapidly generate a cell resolution map of the maize root, revealing an alternative configuration of the tissue formative SHORT-ROOT (SHR) signaling pathway adjacent to an expanded cortex. We show that maize SHR protein is hypermobile, moving at least eight cell layers into the cortex. Higher-order SHR mutants in both maize and Setaria have reduced numbers of cortical layers, showing that the SHR pathway controls expansion of cortical tissue in grasses that sets up anatomical complexity and a host of key traits.

One sentence summarySingle-cell RNA-seq maps the maize root transcriptome uncovering a mechanism that regulates cortex layer number.


title: No reproductive benefits of dear enemy recognition in a territorial songbird

abstract: Territorial animals often exhibit the dear enemy effect, in which individuals respond less aggressively to neighbours than to other individuals. The dear enemy effect is hypothesized to be adaptive by reducing unnecessary aggressive interactions with individuals that are not a threat to territory ownership. A key prediction of this hypothesis, that individual fitness will be affected by variation in the speed and extent to which individuals reduce their aggression towards neighbours relative to strangers, has never been tested. We used a series of song playbacks to measure the change in response of male great tits on their breeding territories to a simulated establishment of a neighbour on an adjacent territory. Males reduced their approach to the speaker and sang fewer songs on later repetitions of the playback trials, consistent with a dear enemy effect through habituation learning. However, not all males discriminated between the neighbour and stranger playbacks at the end of the series of trials, and there was evidence that individuals consistently differed from one another in performing this discrimination. We monitored nests and analysed offspring paternity to determine male reproductive success. Unexpectedly, individuals that exhibited dear enemy behaviour towards the simulated neighbour did not have higher reproductive success, and in fact one measure, total offspring biomass, was lower for individuals that showed the dear enemy effect. Although the general capability to recognize neighbours is most likely adaptive, it seems that individuals who decrease their responsiveness to familiar neighbours too quickly may gain no advantage or even be at a disadvantage.

category: animal behavior and cognition


title: Automating UbiFast for High-throughput and Multiplexed Ubiquitin Enrichment

abstract: Robust methods for deep-scale enrichment and site-specific identification of ubiquitylation sites is necessary for characterizing the myriad roles of protein ubiquitylation. To this end we previously developed UbiFast, a sensitive method for highly multiplexed ubiquitylation profiling where K-{varepsilon}-GG peptides are enriched with anti-K-{varepsilon}-GG antibody and labeled on-antibody with isobaric labeling reagents for sample multiplexing. Here, we present robotic automation of the UbiFast method using a magnetic bead-conjugated K-{varepsilon}-GG antibody (mK-{varepsilon}-GG) and a magnetic particle processor. We report the identification of [~]20,000 ubiquitylation sites from a TMT10-plex with 500 g input per sample processed in [~]2 hours. Automation of the UbiFast method greatly increased the number of identified and quantified ubiquitylation sites, improved reproducibility and significantly reduced processing time. The workflow enables processing of up to 96 samples in a single day making it suitable to study ubiquitylation in large sample sets. Here we demonstrate the applicability of this method to profile small amounts of tissue using breast cancer patient-derived xenograft (PDX) tissue samples.


title: Mechanical Metric for Skeletal Biomechanics Derived from Spectral Analysis of Stiffness matrix

abstract: A new metric for the quantitative and qualitative evaluation of bone stiffness is introduced. It is based on the spectral decomposition of stiffness matrix computed with finite element method. The here proposed metric is defined as an amplitude rescaled eigenvalues of stiffness matrix. The metric contains unique information on the principal stiffness of bone and reflects both bone shape and material properties. The metric was compared with anthropometrical measures and was tested for sex sensitivity on pelvis bone. Further, the smallest stiffness of pelvis was computed under a certain loading condition and analyzed with respect to sex and direction. The metric complements anthropometrical measures and provides a unique information about the smallest bone stiffness independent from the loading configuration and can be easily computed by state-of-the-art subject specified finite element algorithms.


title: AGENT: the Arabidopsis Gene Regulatory Network Tool for Exploring and Analyzing GRNs

abstract: Gene regulatory networks (GRNs) are complex networks that capture multi-level regulatory events between one or more regulatory macromolecules, such as transcription factors (TFs), and their target genes. Advancements in screening technologies such as enhanced yeast-one-hybrid screens have allowed for high throughput determination of GRNs. However, visualization of GRNs in Arabidopsis has been limited to ad hoc networks and are not interactive. Here, we describe the Arabidopsis GEne Network Tool (AGENT) that houses curated GRNs and provides tools to visualize and explore them. AGENT features include expression overlays, subnetwork motif scanning, and network analysis. We show how to use AGENTs multiple built-in tools to identify key genes that are involved in flowering and seed development along with identifying temporal multi-TF control of a key transporter in nitrate signaling. AGENT can be accessed at https://bar.utoronto.ca/AGENT.


title: The molecular structure and surface accumulation dynamics of hyaluronan at the water/air interface

abstract: Hyaluronan is a biopolymer that is essential for many biological processes in the human body, like the regulation of tissue lubrication and inflammatory responses. Here we study the behavior of hyaluronan at aqueous surfaces using heterodyne-detected vibrational sum-frequency generation spectroscopy (HD-VSFG). We find that high-molecular weight hyaluronan (>1 MDa) does not come to the surface, even hours after addition of the polymer to the aqueous solution. In contrast, low-molecular weight hyaluronan (~150 kDa) gradually covers the water-air interface within hours, leading to a negatively charged surface and a reorientation of the interfacial water molecules. This strong dependence on the polymer molecular weight can be explainend from entanglements of the hyaluronan polymers. We also find that the migration kinetics of hyaluronan in aqueous media shows an anomalous dependence on the pH of the solution, which can be explained from the interplay of hydrogen-bonding and electrostatic interactions of the hyaluronan polymers.

category: biophysics


title: Antitumor activity of a lectibody targeting cancer-associated high-mannose glycans

abstract: Aberrant protein glycosylation is a hallmark of cancer, but few drugs targeting cancer glycobiomarkers are currently available. Here, we show that a "lectibody" consisting of the high-mannose glycan-binding lectin Avaren and human IgG1 Fc (AvFc) selectively recognizes a range of cell lines derived from lung, breast, colon and blood cancers at nanomolar concentrations. AvFcs binding to the non-small cell lung cancer (NSCLC) cell lines A549 and H460 was characterized in detail. Co-immunoprecipitation proteomics analysis revealed that epidermal growth factor receptor (EGFR) and insulin-like growth factor 1 receptor (IGF1R) are among the lectibodys common targets in these cells. AvFc blocked the activation of EGFR and IGF1R by their respective ligands in A549 cells and inhibited the migration of A549 and H460 cells upon stimulation with EGF and IGF1. Furthermore, AvFc induced potent Fc-mediated cytotoxic effects and significantly retarded A549 and H460 tumor growth in SCID mice. Immunohistochemistry analysis of primary lung tissues from NSCLC patients demonstrated that AvFc preferentially binds to tumors over adjacent non-tumor tissues. Our findings provide evidence that increased abundance of high-mannose glycans in the glycocalyx of cancer cells can be a druggable target, and AvFc may provide a new tool to probe and target this tumor-associated glycobiomarker.

category: cancer biology


title: Phosphorylation of the M1 muscarinic acetylcholine receptor mediates protection in neurodegenerative disease

abstract: There are currently no treatments that can slow the progression of neurodegenerative diseases such as Alzheimers disease (AD). There is, however, a growing body of evidence that activation of the M1 muscarinic acetylcholine receptor (M1-receptor) can not only restore memory loss in AD patients, but in preclinical animal models can also slow neurodegenerative disease progression. The generation of an effective medicine targeting the M1-receptor has however been severely hampered by associated cholinergic adverse responses. By using genetically engineered mouse models that express a G protein-biased M1-receptor, we recently established that M1-receptor mediated adverse responses can be minimised by ensuring activating ligands maintain receptor phosphorylation/arrestin-dependent signalling. Here, we use these same genetic models in concert with murine prion disease, a terminal neurodegenerative disease showing key hallmarks of AD, to establish that phosphorylation/arrestin-dependent signalling delivers neuroprotection that both extends normal animal behaviour and prolongs the life span of prion diseased mice. Our data point to an important neuroprotective property inherent to the M1-receptor and indicate that next generation M1-receptor ligands designed to drive receptor phosphorylation/arrestin-dependent signalling would potentially show low adverse responses whilst delivering neuroprotection that will slow disease progression.

category: pharmacology and toxicology


title: Tissue-specific metabolic reprogramming during wound induced de novo organ formation in tomato hypocotyl explants

abstract:
O_LIPlants have remarkable regenerative capacity, which allows them to survive tissue damaging after biotic and abiotic stress. Some of the key transcription factors and the hormone crosstalk involved in wound-induced organ regeneration have been extensively studied in the model plant Arabidopsis thaliana. However, little is known about the role of metabolism in wound-induced organ regeneration.
C_LIO_LIHere, we performed detailed transcriptome analysis and targeted metabolomics approach during de novo organ formation in tomato hypocotyl explants and found tissue-specific metabolic differences and divergent developmental pathways after wounding.
C_LIO_LIOur results indicate that callus growth in the apical region of the hypocotyl depends on a specific metabolic switch involving the upregulation of the photorespiratory pathway and the differential regulation of photosynthesis-related genes and of the gluconeogenesis pathway.
C_LIO_LIThe endogenous pattern of ROS accumulation in the apical and basal region of the hypocotyl during the time-course were dynamically regulated, and contributed to tissue-specific wound-induced regeneration.
C_LIO_LIOur findings provide a useful resource for further investigation on the molecular mechanisms involved in wound-induced organ formation in a crop species such as tomato.
C_LI

One-sentence SummaryMetabolic switch during wound-induced regeneration


title: Reconstitution of prenyltransferase activity on nanodiscs by components of the rubber synthesis machinery of the Para rubber tree and guayule

abstract: Prenyltransferases mediate the biosynthesis of various types of polyisoprene compound in living organisms. Natural rubber (NR) of the Para rubber tree (Hevea brasiliensis) is synthesized as a result of prenyltransferase activity, with the proteins HRT1, HRT2, and HRBP having been identified as candidate components of the rubber biosynthetic machinery. To clarify the contribution of these proteins to prenyltransferase activity, we established a cell-free translation system for nanodisc-based protein reconstitution and measured the enzyme activity of the protein-nanodisc complexes. Cell-free synthesis of HRT1, HRT2, and HRBP in the presence of asolectin nanodiscs revealed that all three proteins were membrane associated. A complex of HRT1 and HRBP formed as a result of co-expression of the two proteins in the presence of nanodiscs manifested marked polyisoprene synthesis activity, whereas neither HRT1, HRT2, or HRBP alone nor a complex of HRT2 and HRBP exhibited such activity. Similar analysis of guayule (Parthenium argentatum) proteins revealed that three HRT1 homologs (CPT1-3) manifested prenyltransferease activity only if co-expressed with the homolog of HRBP (CBP). Our results thus indicate that the core prenyltransferase of the rubber biosynthetic machinery of both the Para rubber tree and guayule is formed by the assembly of heterologous subunits (HRT1 and HRBP in the former species).


title: Using Stakeholder Insights to Enhance Engagement in PhD Professional Development

abstract: There is increasing awareness of the need for predoctoral and postdoctoral professional development and career guidance, however many academic institutions are only beginning to build out these functional roles. As a graduate career educator, accessing the vast silos and resources at a university and with industrial partners can be daunting, yet collaborative endeavors and network development both on and off campus are crucial to the success of any career and professional development office. To better inform and direct the efforts of graduate career offices, forty-five stakeholders external and internal to academic institutions were identified and interviewed to gather and categorize perspectives on topics critical to career and professional development offices. Using a stakeholder network visualization tool developed by the authors, stakeholder engagement can be rapidly assessed to ascertain areas where offices have strong connections and other areas where additional efforts could be directed to enhance engagement. General themes from interviews with internal and external stakeholders are discussed to provide graduate career educators with various stakeholder subgroup perspectives to help prepare for successful interactions. Benefits include increased engagement and opportunities to collaborate, as well as the opportunity to build or expand graduate career development offices.

category: scientific communication and education


title: Identification that ADAM17 mediates proteolytic maturation of calcium channel auxiliary α2δ subunits, and enables calcium current enhancement

abstract: The auxiliary 2{delta} subunits of voltage-gated calcium (CaV) channels are key to augmenting expression and function of CaV1 and CaV2 channels, and are also important drug targets in several therapeutic areas, including neuropathic pain. The 2{delta} proteins are translated as pre-proteins encoding both 2 and {delta}, and post-translationally proteolysed into 2 and {delta} subunits, which remain associated as a complex. In this study we have identified ADAM17 as a key protease involved in proteolytic processing of pro-2{delta}-1 and 2{delta}-3 subunits. We provide three lines of evidence: firstly, proteolytic cleavage is inhibited by chemical inhibitors of particular metalloproteases, including ADAM17. Secondly, proteolytic cleavage of both 2{delta}-1 and 2{delta}-3 is markedly reduced in cell lines by knockout of ADAM17 but not ADAM10. Thirdly, proteolytic cleavage is reduced by the N-terminal active domain of TIMP-3 (N-TIMP-3), which selectively inhibits ADAM17. We have found previously that proteolytic cleavage into mature 2{delta} is essential for the enhancement of CaV function, and in agreement, knockout of ADAM17 inhibited the ability of 2{delta}-1 to enhance both CaV2.2 and CaV1.2 calcium currents. Thus, our study identifies ADAM17 as a key protease required for proteolytic maturation of 2{delta}-1 and 2{delta}-3, and thus a potential drug target in neuropathic pain.


title: Evaluation of data-driven MEG analysis approaches for the extraction of fMRI resting state networks

abstract:
The electrophysiological basis of resting state networks (RSN) is still under debate. In particular, no principled mechanism has been determined that is capable of explaining all RSN equally well. While magnetoencephalography (MEG) and electroencephalography (EEG) are the methods of choice to determine the electrophysiological basis of RSN, no standard analysis pipeline of RSN yet exists. In this paper, we compare the two main existing data-driven analysis strategies for extracting resting state networks from MEG data. The first approach extracts RSN through an independent component analysis (ICA) of the Hilbert envelope in different frequency bands. The second approach uses phase -amplitude coupling to determine the RSN. To evaluate the performance of these approaches, we compare the MEG-RSN to the functional magnetic resonance imaging (fMRI)-RSN from the same subjects.

Overall, it was possible to extract the canonical fMRI RSN with MEG. The approach based on phase-amplitude coupling yielded the best correspondence to the fMRI-RSN. The Hilbert envelope-ICA produced different dominant frequency-bands underlying RSN for different ICA runs, suggesting the absence of a single dominant frequency underlying the RSN. Our results also suggest that individual RSN are not characterized by one single dominant frequency. Instead, the resting state networks seem to be based on a combination of the delta/theta phase and gamma amplitude.


title: Sex differences in behaviour and molecular pathology in the 5XFAD model

abstract:
BackgroundThe prevalence of Alzheimers Disease (AD) is greater in women compared to men, but the reasons for this remain unknown. This sex difference has been widely neglected in experimental studies using transgenic mouse models of AD.

ObjectiveHere, we studied behaviour and molecular pathology of 5-month-old 5XFAD mice, which express mutated human amyloid precursor protein and presenilin-1 on a C57BL/6J background, vs. their wild-type littermate controls, to compared both sex- and genotype-dependent differences.

MethodsA novel behavioural paradigm was utilised (OF-NO-SI), comprising activity measures (Open Field, OF) arena, followed by Novel Object exploration (NO) and Social Interaction (SI) of a sex-matched conspecific. Each segment consisted of two repeated trials to assess between-trial habituation. Subsequently, brain pathology (amyloid load, stress response and inflammation markers, synaptic integrity, trophic support) was assessed using qPCR and Western blotting.

ResultsFemale 5XFAD mice had higher levels of human APP and beta-amyloid (A{beta}) and heightened inflammation vs males. These markers correlated with hyperactivity observed in both sexes, yet only female 5XFAD mice presented with deficits in object and social exploration. Male animals had higher expression of stress markers and neurotrophic factors irrespective of genotype, this correlated with cognitive performance.

ConclusionThe impact of sex on AD-relevant phenotypes is in line with human data and emphasises the necessity of appropriate study design and reporting. Differential molecular profiles observed in male vs. female mice offer insights into possible protective mechanisms, and hence treatment strategies.


title: A Single Cell Atlas of Spared Tissue Below a Spinal Cord Injury Reveals Cellular Mechanisms of Repair

abstract: After spinal cord injury (SCI), the "spared" tissue below the lesion contains undamaged cells that could support or augment recovery, but targeting these cells requires a clearer understanding of their injury responses and capacity for repair. Here, we used single nucleus sequencing to profile how each cell type in the lumbar spinal cord changes over time after a thoracic injury. We present an atlas of these dynamic responses and explore two unexpected findings. Amongst neurons, rare cell types expressed a molecular signature of regeneration and amongst microglia, we identified a population of "trauma associated microglia" (TAM). These TAM cells were present in the white matter near degenerating axons and expressed the trophic factors Igf1 and Spp1(OPN). Viral over-expression of Igf1 and Spp1(OPN) expanded the TAM population and promoted the clearance of myelin debris. These findings expose endogenous mechanisms of repair in spared neural tissue, uncovering potential candidates for targeted therapy.


title: Citrullination of glucokinase linked to autoimmune diabetes

abstract: Inflammation, including reactive oxygen species and inflammatory cytokines in tissue microenvironments amplify the appearance of various post-translational modifications (PTMs) of self-proteins. Previously, a number of PTMs have been identified as autoimmune biomarkers in the initiation and progression of Type 1 diabetes (T1D). Herein, we have identified the citrullination of glucokinase (GK) as a result of inflammation, triggering autoimmunity and affecting its biological functions. Glucokinase is predominantly expressed in hepatocytes to regulate glycogen synthesis, and in pancreatic beta cells, where it acts as a glucose sensor to initiate glycolysis and insulin signaling. Herein, we demonstrate that glucokinase is citrullinated in inflamed non-obese diabetic (NOD) islets as well as in human GK. Autoantibodies against both native and citrullinated GK arise in both spontaneous human T1D and murine models. Likewise, autoreactive CD4+ T cells to both native and citrullinated glucokinase epitopes are present in the circulation of T1D patients. Finally, citrullination alters GK biologic activity and suppresses glucose-stimulated insulin secretion. Our studies define glucokinase as a T1D biomarker, providing new insights into altering glucose metabolism, creating neoautoantigens, and better define the broad impact of PTMs on the tissue pathology of T1D.


title: An Aerobic Anoxygenic Phototrophic Bacterium Fixes CO2 via the Calvin-Benson-Bassham Cycle

abstract: Aerobic anoxygenic phototrophic bacteria (AAnPB) are photoheterotrophs, which use light as auxiliary energy and require organic carbon (OC) for growth. Herein, we report the unusual strain B3, which is a true AAnPB because it requires oxygen for growth, harbours genes for cbb3- and bd-type cytochromes and acsF, and produces bacteriochlorophyll. The B3 genome encodes the complete metabolic pathways for AAnPB light utilization, CO2 fixation via Calvin-Benson-Bassham (CBB) cycle and oxidation of sulfite and H2, and the transcriptome indicated that all components of these pathways were fully transcribed. Expression of the marker genes related to photosynthesis, including pufM for light harnessing and rbcL for CO2 fixation, and the activity of RubisCO, the key enzyme in the Calvin-Benson-Bassham (CBB) cycle, increased in response to decreased OC supply. Large amounts of cell biomass were obtained in liquid BG11 medium under illumination. The strain thus likely photoautotrophically grows using sulfite or H2 as an electron donor. Similar GC contents between photosynthesis, the CBB cycle and 16S rRNA genes and the consistency of their phylogenetic topologies implied that light harnessing and carbon fixation genes evolved vertically from an anaerobic phototrophic ancestor of Rhodospirillaceae in Alphaproteobacteria. In conclusion, strain B3 represents a novel AAnPB characterized by photoautotrophy using the CBB cycle. This kind of AAnPB may be ecologically significant in the global carbon cycle.


title: Biofilm hydrophobicity in environmental isolates of Bacillus subtilis

abstract:
Biofilms are communities of bacteria that are attached to a surface and surrounded by an extracellular matrix. The extracellular matrix protects the community from stressors in the environment, making biofilms robust. The Gram-positive soil bacterium Bacillus subtilis, particularly the isolate NCIB 3610, is widely used as a model for studying biofilm formation. B. subtilis NCIB 3610 forms colony biofilms that are architecturally complex and highly hydrophobic. The hydrophobicity is linked, in part, to the localisation of the protein BslA at the surface of the biofilm, which provides the community with increased resistance to biocides. As most of our knowledge about B. subtilis biofilm formation comes from one isolate, it is unclear if biofilm hydrophobicity is a widely distributed feature of the species. To address this knowledge gap, we collated a library of B. subtilis soil isolates and acquired their whole genome sequences. We used our new isolates to examine biofilm hydrophobicity and found that, although BslA is encoded and produced by all isolates in our collection, hydrophobicity is not a universal feature of B. subtilis colony biofilms. To test whether the matrix exopolymer poly {gamma}-glutamic acid could be masking hydrophobicity in our hydrophilic isolates, we constructed deletion mutants and found, contrary to our hypothesis, that the presence of poly {gamma}-glutamic acid was not the reason behind the observed hydrophilicity. This study highlights the natural variation in the properties of biofilms formed by different isolates and the importance of using a more diverse range of isolates as representatives of a species.

RepositoriesRaw sequence reads and annotated assemblies have been submitted to the European Nucleotide Archive under accession PRJEB43128.


title: Virtual partition digital PCR for high precision chromosomal counting applications

abstract: Digital PCR (dPCR) is the gold standard analytical platform for rapid high precision quantification of genomic fragments. However, current dPCR assays are generally limited to monitoring 1-2 analytes per sample, thereby limiting the platforms ability to address some clinical applications that require the simultaneous monitoring of 20 - 50 analytes per sample. Here we present Virtual Partition dPCR (VPdPCR), a novel analysis methodology enabling the detection of 10 or more target regions per color channel using conventional dPCR hardware and workflow. Furthermore, VPdPCR enables dPCR instruments to overcome upper quantitation limits caused by partitioning error. While traditional dPCR analysis establishes a single threshold to separate negative and positive partitions, VPdPCR establishes multiple thresholds to identify the number of unique targets present in each positive droplet based on fluorescent intensity. Each physical partition is then divided into a series of virtual partitions, and the resulting increase in partition count substantially decreases partitioning error. We present both a theoretical analysis of the advantages of VPdPCR and an experimental demonstration in the form of a 20-plex assay for non-invasive fetal aneuploidy testing. This demonstration assay - tested on 432 samples contrived from sheared cell-line DNA at multiple input concentrations and simulated fractions of euploid or trisomy-21 "fetal" DNA - is analyzed using both traditional dPCR thresholding and VPdPCR. VPdPCR analysis significantly lowers variance of chromosome ratio across replicates and increases the accuracy of trisomy identification when compared to traditional dPCR, yielding >98% single-well sensitivity and specificity. VPdPCR has substantial promise for increasing the utility of dPCR in applications requiring ultra-high-precision quantitation.

category: molecular biology


title: Single-cell resolution of MET and EMT programs during zebrafish fin regeneration

abstract: Zebrafish regenerate fin rays following amputation through epimorphic regeneration, a process that has been proposed to involve the epithelial-to-mesenchymal transition (EMT). We performed single-cell RNA-sequencing (scRNA-seq) to elucidate osteoblastic transcriptional programs during zebrafish caudal fin regeneration. We show that osteoprogenitors are enriched with components associated with EMT and its reverse, mesenchymal-to-epithelial transition (MET), and provide evidence that the EMT markers cdh11 and twist2 are co-expressed in dedifferentiating cells at the amputation stump at 1 dpa, and in differentiating osteoblastic cells in the regenerate, the latter of which are enriched in EMT signatures. We also show that esrp1, a regulator of alternative splicing in epithelial cells that is associated with MET, is expressed in a subset of osteoprogenitors during outgrowth. This study provides a single cell resource for the study of osteoblastic cells during zebrafish fin regeneration, and supports the contribution of MET- and EMT-associated components to this process.


title: Temporal single-cell transcriptomes of zebrafish spinal cord pMN progenitors reveal distinct neuronal and glial progenitor populations

abstract:
Ventral spinal cord progenitor cells, which express the basic helix loop helix transcription factor Olig2, sequentially produce motor neurons and oligodendrocyte precursor cells (OPCs). Following specification some OPCs differentiate as myelinating oligodendrocytes while others persist as OPCs. Though a considerable amount of work has described the molecular profiles that define motor neurons, OPCs, and oligodendrocytes, less is known about the progenitors that produce them. To identify the developmental origins and transcriptional profiles of motor neurons and OPCs, we performed single-cell RNA sequencing on isolated pMN cells from embryonic zebrafish trunk tissue at stages that encompassed motor neurogenesis, OPC specification, and initiation of oligodendrocyte differentiation. Downstream analyses revealed two distinct pMN progenitor populations: one that appears to produce neurons and one that appears to produce OPCs. This latter population, called Pre-OPCs, is marked by expression of GS Homeobox 2 (gsx2), a gene that encodes a homeobox transcription factor. Using fluorescent in situ hybridizations, we identified gsx2-expressing Pre-OPCs in the spinal cord prior to expression of canonical OPC marker genes. Our data therefore reveal heterogeneous gene expression profiles among pMN progenitors, supporting prior fate mapping evidence.

HighlightsO_LISingle-cell RNA sequencing reveals the developmental trajectories of neurons and glia that arise from spinal cord pMN progenitor cells in zebrafish embryos
C_LIO_LITranscriptionally distinct subpopulations of pMN progenitors are the apparent sources of neurons or oligodendrocytes, consistent with fate mapping data
C_LIO_LIgsx2 expression marks pMN progenitors that produce oligodendrocyte lineage cells
C_LI


title: Disentangling how multiple traits drive 2 strain frequencies in SIS dynamics with coinfection

abstract: A general theory for competitive dynamics among many strains at the epidemiological level is required to understand polymorphisms in virulence, transmissibility, antibiotic resistance and other biological traits of infectious agents. Mathematical coinfection models have addressed specific systems, focusing on the criteria leading to stable coexistence or competitive exclusion, however, due to their complexity and nonlinearity, analytical solutions in coinfection models remain rare. Here we study a 2-strain SIS compartmental model with co-infection/co-colonization, incorporating multiple fitness dimensions under the same framework: variation in transmissibility, duration of carriage, pairwise susceptibilities to coinfection, coinfection duration, and transmission priority effects from mixed coinfection. Taking advantage of a singular perturbation approach, under the assumption of strain similarity, we expose how strain dynamics on a slow timescale are explicitly governed by a replicator equation which encapsulates all traits and their interplay. This allows us to predict explicitly not only the final epidemiological outcome of a given 2-player competition, but moreover, their entire frequency dynamics as a direct function of their relative variation and of strain-transcending global parameters. Based on mutual invasion fitnesses, we analyze and report rigorous results on transition phenomena in the 2-strain system, strongly mediated via coinfection prevalence. We show that coinfection is not always a promoter of coexistence; instead, its effect to favour or prevent polymorphism is non-monotonic and depends on the type and level of phenotypic differentiation between strains. This framework offers a deeper analytical understanding of 2-strain competitive games in coinfection, with theoretical and practical applications in epidemiology, ecology and evolution.

category: ecology


title: Climate trends and behavior of an avian forest specialist in central Amazonia indicate thermal stress during the dry season

abstract: Rainforest loss threatens terrestrial insectivorous birds throughout the worlds tropics. Recent evidence shows these birds to be declining in undisturbed Amazonian rainforest, possibly due to climate change. Here, we first addressed whether Amazonian terrestrial insectivores were exposed to climate change using 38 years of climate data. We found that climate has changed in central Amazonia, especially in the dry season, which was [~]1.3{degrees}C hotter and 21% drier in 2019 than in 1981. Second, to test whether birds actively avoided hot and dry conditions, we used field sensors to identify ambient extremes and prospective microclimate refugia within undisturbed rainforest from 2017 - 2019. Simultaneously, we examined how tagged Black-faced Anthrushes (Formicarius analis) used this space. We collected 1.4 million field measurements quantifying ambient conditions in the forest understory, including along elevation gradients. For 11 birds, we obtained GPS locations to test whether birds adjusted their shelter use (n = 2,724) or elevation (n = 640) across seasonal and daily cycles. For four additional birds, we collected >180,000 light and temperature readings to assess exposure. Field measurements in the modern landscape revealed that temperature was higher in the dry season and highest on plateaus. Thus, low-lying areas were relatively buffered, providing microclimate refugia during hot afternoons in the dry season. At those times, birds entered shelters and shifted downslope, reducing their thermal exposure by 50%. Because climate change intensifies the hot, dry conditions that antthrushes avoid, our results are consistent with the hypothesis that climate change lowers habitat quality for terrestrial insectivores. This sensitivity may be related to their declines within  undisturbed Amazonian rainforest.


title: The Taxus genome provides insights into paclitaxel biosynthesis

abstract:
The ancient gymnosperm genus Taxus is the exclusive source of the anticancer drug paclitaxel, yet no reference genome sequences are available for comprehensively elucidating the paclitaxel biosynthesis pathway. We have completed a chromosome-level genome of Taxus chinensis var. mairei with a total length of 10.23 Gb. Taxus shared an ancestral whole-genome duplication with the coniferophyte lineage and underwent distinct transposon evolution. We discovered a unique physical and functional grouping of CYP725As (cytochrome P450) in the Taxus genome for paclitaxel biosynthesis. We also identified a gene cluster in the taxadiene biosynthesis, which was mainly formed by gene duplications. This study will facilitate the elucidation of paclitaxel biosynthesis and unleash the biotechnological potential of Taxus.

One Sentence SummaryA chromosome-level genome assembly of Taxus chinensis var. mairei uncovers its unique genome evolution process and genetic architectures for the paclitaxel biosynthesis pathway.


title: Interpreting Neural Networks for Biological Sequences by Learning Stochastic Masks

abstract: Sequence-based neural networks can learn to make accurate predictions from large biological datasets, but model interpretation remains challenging. Many existing feature attribution methods are optimized for continuous rather than discrete input patterns and assess individual feature importance in isolation, making them ill-suited for interpreting non-linear interactions in molecular sequences. Building on work in computer vision and natural language processing, we developed an approach based on deep generative modeling - Scrambler networks - wherein the most salient sequence positions are identified with learned input masks. Scramblers learn to generate Position-Specific Scoring Matrices (PSSMs) where unimportant nucleotides or residues are  scrambled by raising their entropy. We apply Scramblers to interpret the effects of genetic variants, uncover non-linear interactions between cis-regulatory elements, explain binding specificity for protein-protein interactions, and identify structural determinants of de novo designed proteins. We show that interpretation based on a generative model allows for efficient attribution across large datasets and results in high-quality explanations, often outperforming state-of-the-art methods.


title: scvi-tools: a library for deep probabilistic analysis of single-cell omics data

abstract: AO_SCPLOWBSTRACTC_SCPLOWProbabilistic models have provided the underpinnings for state-of-the-art performance in many single-cell omics data analysis tasks, including dimensionality reduction, clustering, differential expression, annotation, removal of unwanted variation, and integration across modalities. Many of the models being deployed are amenable to scalable stochastic inference techniques, and accordingly they are able to process single-cell datasets of realistic and growing sizes. However, the community-wide adoption of probabilistic approaches is hindered by a fractured software ecosystem resulting in an array of packages with distinct, and often complex interfaces. To address this issue, we developed scvi-tools (https://scvi-tools.org), a Python package that implements a variety of leading probabilistic methods. These methods, which cover many fundamental analysis tasks, are accessible through a standardized, easy-to-use interface with direct links to Scanpy, Seurat, and Bioconductor workflows. By standardizing the implementations, we were able to develop and reuse novel functionalities across different models, such as support for complex study designs through nonlinear removal of unwanted variation due to multiple covariates and reference-query integration via scArches. The extensible software building blocks that underlie scvi-tools also enable a developer environment in which new probabilistic models for single cell omics can be efficiently developed, benchmarked, and deployed. We demonstrate this through a code-efficient reimplementation of Stereoscope for deconvolution of spatial transcriptomics profiles. By catering to both the end user and developer audiences, we expect scvi-tools to become an essential software dependency and serve to formulate a community standard for probabilistic modeling of single cell omics.


title: Cost-effective mapping of genetic interactions in mammalian cells

abstract: Comprehensive maps of genetic interactions in mammalian cells are daunting to construct because of the large number of potential interactions, ~ 2 x 108 for protein coding genes. We previously used co-inheritance of distant genes from published radiation hybrid (RH) datasets to identify genetic interactions. However, it was necessary to combine six legacy datasets from four species to obtain adequate statistical power. Mapping resolution was also limited by the low density PCR genotyping. Here, we employ shallow sequencing of nascent human RH clones as an economical approach to constructing interaction maps. In this initial study, 15 clones were analyzed, enabling construction of a network with 225 genes and 2359 interactions (FDR < 0.05). Despite its small size, the network showed significant overlap with the previous RH network and with a protein-protein interaction network. Consumables were [lsim] $50 per clone, showing that affordable, high quality genetic interaction maps are feasible in mammalian cells.


title: An enveloped virus-like particle vaccine expressing a stabilized prefusion form of the SARS-CoV-2 spike protein elicits potent immunity after a single dose.

abstract:
Development of efficacious single dose vaccines would substantially aid efforts to stop the uncontrolled spread of the COVID-19 pandemic. We evaluated enveloped virus-like particles (eVLPs) expressing various forms of the SARS-CoV-2 spike protein and several adjuvants in an effort to identify a COVID-19 vaccine candidate efficacious after a single dose. The eVLPs expressing a modified prefusion form of SARS-CoV-2 spike protein were selected as they induced the highest antibody binding titers and neutralizing activity after a single injection in mice. Formulation of SARS-CoV-2 S eVLPs with aluminum phosphate resulted in balanced induction of IgG2 and IgG1 isotypes and antibody binding and neutralization titers were undiminished for more than 3 months after a single immunization. A single dose of this candidate, VBI-2902a (prefusion S eVLPs formulated with aluminum phosphate), protected Syrian golden hamsters from challenge with SARS-CoV-2 and supports the on-going clinical evaluation of VBI-2902a as a potential single dose vaccine against COVID-19.

HighlightsO_LIVBI-2902a is a VLP-based vaccine candidate against SARS-COV-2
C_LIO_LIVBI-2902a contains VLPs pseudotyped with a modified prefusion SARS-COV-2 S in Alum.
C_LIO_LIVBI-2902a induces robust neutralization antibody response against SARS-COV-2 S
C_LIO_LIVBI-2902a protects hamsters from SARS-CoV-2 induced lung inflammation
C_LIO_LIA single dose of VBI-2902a provides protective benefit in hamsters
C_LI


title: Allelic variation in Class I HLA determines pre-existing memory responses to SARS-CoV-2 that shape the CD8+ T cell repertoire upon viral exposure

abstract:
Effective presentation of antigens by HLA class I molecules to CD8+ T cells is required for viral elimination and generation of long-term immunological memory. In this study, we applied a single-cell, multi-omic technology to generate the first unified ex vivo characterization of the CD8+ T cell response to SARS-CoV-2 across 4 major HLA class I alleles. We found that HLA genotype conditions key features of epitope specificity, TCR /{beta} sequence diversity, and the utilization of pre-existing SARS-CoV-2 reactive memory T cell pools. Single-cell transcriptomics revealed functionally diverse T cell phenotypes of SARS-CoV-2-reactive T cells, associated with both disease stage and epitope specificity. Our results show that HLA variations influence pre-existing immunity to SARS-CoV-2 and shape the immune repertoire upon subsequent viral exposure.

One-Sentence SummaryWe perform a unified, multi-omic characterization of the CD8+ T cell response to SARS-CoV-2, revealing pre-existing immunity conditioned by HLA genotype.


title: Protracted yet coordinated differentiation of long-lived SARS-CoV-2-specific CD8+ T cells during COVID-19 convalescence

abstract: CD8+ T cells are important antiviral effectors that can potentiate long-lived immunity against COVID-19, but a detailed characterization of these cells has been hampered by technical challenges. We screened 21 well-characterized, longitudinally-sampled convalescent donors that recovered from mild COVID-19 against a collection of SARS-CoV-2 tetramers, and identified one participant with an immunodominant response against Nuc322-331, a peptide that is conserved in all the SARS-CoV-2 variants-of-concern reported to date. We conducted 38- parameter CyTOF phenotyping on tetramer-identified Nuc322-331-specific CD8+ T cells, and on CD4+ and CD8+ T cells recognizing the entire nucleocapsid and spike proteins from SARS- CoV-2, and took 32 serological measurements on longitudinal specimens from this participant. We discovered a coordination of the Nuc322-331-specific CD8+ T response with both the CD4+ T cell and antibody pillars of adaptive immunity. Nuc322-331-specific CD8+ T cells were predominantly central memory T cells, but continually evolved over a [~]6-month period of convalescence. We observed a slow and progressive decrease in the activation state and polyfunctionality of the Nuc322-331-specific CD8+ T cells, accompanied by an increase in their lymph-node homing and homeostatic proliferation potential. These results suggest that following a typical case of mild COVID-19, SARS-CoV-2-specific CD8+ T cells not only persist but continuously differentiate in a coordinated fashion well into convalescence, into a state characteristic of long-lived, self-renewing memory.


title: Phylogenetic analysis of SARS-CoV-2 lineage development across the first and second waves in Eastern Germany, 2020

abstract: SARS-CoV-2 lineages prevalent in the first and second waves in Eastern Germany were different, with many new variants including four predominant lineages in the second wave, having been introduced into Eastern Germany between August to October 2020, indicating the major cause of the second wave was the introduction of new variants.


title: The natural stilbenoid (-)-hopeaphenol inhibits cellular entry of SARS-CoV-2 USA-WA1/2020, B.1.1.7 and B.1.351 variants

abstract:
Antivirals are urgently needed to combat the global SARS-CoV-2/COVID-19 pandemic, supplement existing vaccine efforts, and target emerging SARS-CoV-2 variants of concern. Small molecules that interfere with binding of the viral spike receptor binding domain (RBD) to the host ACE2 receptor may be effective inhibitors of SARS-CoV-2 cell entry. Here we screened 512 pure compounds derived from natural products using a high-throughput RBD/ACE2 binding assay and identified (-)-hopeaphenol, a resveratrol tetramer, in addition to vatalbinoside A and vaticanol B, as potent and selective inhibitors of RBD/ACE2 binding and viral entry. For example, (-)-hopeaphenol disrupted RBD/ACE2 binding with a 50% inhibitory concentration (IC50) of 0.11 M in contrast to an IC50 of 28.3 M against the unrelated host ligand/receptor binding pair PD-1/PD-L1 (selectivity index = 257.3). When assessed against the USA-WA1/2020 variant, (-)-hopeaphenol also inhibited entry of a VSV{Delta}G-GFP reporter pseudovirus expressing SARS-CoV-2 spike into ACE2-expressing Vero-E6 cells and in vitro replication of infectious virus in cytopathic effect assays (IC50 = 10.2 M) without cytotoxicity. Notably, (-)- hopeaphenol also inhibited two emerging variants of concern originating from the United Kingdom (B.1.1.7) and South Africa (B.1.351) in both cytopathic effect and spike-containing pseudovirus assays with similar (B.1.1.7) or improved (B.1.351) efficacies over the USA- WA1/2020 variant. These results identify (-)-hopeaphenol and related stilbenoid analogues as potent and selective inhibitors of viral entry across multiple SARS-CoV-2 variants including those with increased infectivity and/or reduced susceptibility to existing vaccines.

ImportanceSARS-CoV-2 antivirals are needed to supplement existing vaccine efforts and target emerging viral variants with increased infectivity or reduced susceptibility to existing vaccines. Here we show that (-)-hopeaphenol, a naturally-occurring stilbenoid compound, in addition to its analogues vatalbinoside A and vaticanol B, inhibit SARS-CoV-2 by blocking the interaction of the viral spike protein with the cellular ACE2 entry receptor. Importantly, in addition to inhibiting the early USA-WA1/2020 SARS-CoV-2 variant, hopeaphenol also inhibits emerging variants of concern including B.1.1.7 ("United Kingdom variant") and B.1.351 ("South Africa variant"), with improved efficacy against B.1.351. (-)-Hopeaphenol therefore represents a new antiviral lead against infection from multiple SARS-CoV-2 variants.


title: Molecular interactions of the M and E integral membrane proteins of SARS-CoV-2

abstract: Specific lipid-protein interactions are key for cellular processes, and even more so for the replication of pathogens. The COVID-19 pandemic has drastically changed our lives and cause the death of nearly three million people worldwide, as of this writing. SARS-CoV-2 is the virus that causes the disease and has been at the center of scientific research over the past year. Most of the research on the virus is focused on key players during its initial attack and entry into the cellular host; namely the S protein, its glycan shield, and its interactions with the ACE2 receptors of human cells. As cases continue to raise around the globe, and new mutants are identified, there is an urgent need to understand the mechanisms of this virus during different stages of its life cycle. Here, we consider two integral membrane proteins of SARS-CoV-2 known to be important for viral assembly and infectivity. We have used microsecond-long all-atom molecular dynamics to examine the lipid-protein and protein-protein interactions of the membrane (M) and envelope (E) structural proteins of SARS-CoV-2 in a complex membrane model. We contrast the two proposed protein complexes for each of these proteins, and quantify their effect on their local lipid environment. This ongoing work also aims to provide molecular-level understanding of the mechanisms of action of this virus to possibly aid in the design of novel treatments.


title: Possible link between higher transmissibility of B.1.617 and B.1.1.7 variants of SARS-CoV-2 and increased structural stability of its spike protein and hACE2 affinity

abstract: The Severe Acute syndrome corona Virus 2 (SARS-CoV-2) outbreak in December 2019 has caused a global pandemic. The rapid mutation rate in the virus has caused alarming situations worldwide and is being attributed to the false negativity in RT-PCR tests, which also might lead to inefficacy of the available drugs. It has also increased the chances of reinfection and immune escape. We have performed Molecular Dynamic simulations of three different Spike-ACE2 complexes, namely Wildtype (WT), B.1.1.7 variant (N501Y Spike mutant) and B.1.617 variant (L452R, E484Q Spike mutant) and compared their dynamics, binding energy and molecular interactions. Our result shows that mutation has caused the increase in the binding energy between the Spike and hACE2. In the case of B.1.617 variant, the mutations at L452R and E484Q increased the stability and intra-chain interactions in the Spike protein, which may change the interaction ability of human antibodies to this Spike variant. Further, we found that the B.1.1.7 variant had increased hydrogen interaction with LYS353 of hACE2 and more binding affinity in comparison to WT. The current study provides the biophysical basis for understanding the molecular mechanism and rationale behind the increase in the transmissivity and infectivity of the mutants compared to wild-type SARS-CoV-2.


title: Loss of Smad4 promotes aggressive lung cancer metastasis by de-repression ofPAK3 via miRNA regulation

abstract: Over 85% of lung cancer patients harbor overt or subclinical metastases at diagnosis, and therefore most patients die of progressive metastatic disease despite aggressive local and systemic therapies. Somatic mutations in the Smad4 gene have been found in non-small-cell lung cancer, but the underlying mechanism by which Smad4 loss-of-function (LOF) accelerates lung cancer metastasis is yet to be elucidated. Here, we generated a highly aggressive lung cancer mouse model bearing conditional KrasG12D, p53fl/fl LOF and/or Smad4 fl/fl LOF mutations. The Smad4fl/fl; p53 fl/fl; KrasG12D (SPK) mutant mice manifested a much higher incidence of tumor metastases than the p53 fl/fl; KrasG12D (PK) mice. Molecularly, PAK3 was identified as a novel downstream effector of Smad4, mediating metastatic signal transduction via the PAK3-JNK-Jun pathway. Upregulation of PAK3 by Smad4 LOF in SPK mice was achieved by attenuating Smad4-dependent transcription of miR-495 and miR-543. These microRNAs (miRNAs) directly bind to the PAK3 3UTR for blockade of PAK3 production, ultimately regulating lung cancer metastasis. An inverse correlation between Smad4 and PAK3 pathway components suggests clinical use of Smad4 LOF as a potential marker for prognosis in human lung cancer. Our study highlights the Smad4-PAK3 regulation as a point of potential therapy in metastatic lung cancer.


title: The JAK2-STAT3 pathway controls a beneficial proteostasis response of reactive astrocytes in Huntington disease

abstract:
Huntingtons disease is a fatal neurodegenerative disease characterized by striatal neurodegeneration, aggregation of mutant Huntingtin and the presence of reactive astrocytes. Astrocytes are important partners for neurons and engage in a specific reactive response in Huntingtons disease that involves morphological, molecular and functional changes. How reactive astrocytes contribute to Huntingtons disease is still an open question, especially because their reactive state is poorly reproduced in experimental mouse models.

Here, we show that the JAK2-STAT3 pathway, a central cascade controlling astrocyte reactive response, is activated in the putamen of Huntingtons disease patients. Selective activation of this cascade in astrocytes through viral gene transfer reduces the number and size of mutant Huntingtin aggregates in neurons and improves neuronal defects in two complementary mouse models of Huntingtons disease. It also reduces striatal atrophy and increases glutamate levels, two central clinical outcomes measured by non-invasive magnetic resonance imaging. Moreover, astrocyte-specific transcriptomic analysis shows that activation of the JAK2-STAT3 pathway in astrocytes coordinates a transcriptional program that increases their intrinsic proteolytic capacity, through the lysosomal and ubiquitin-proteasome degradation systems. This pathway also enhances their production and exosomal release of the co-chaperone DNAJB1, which contributes to mutant Huntingtin clearance in neurons.

Together, our results show that the JAK2-STAT3 pathway controls a beneficial proteostasis response in reactive astrocytes in Huntingtons disease, which involves bi-directional signalling with neurons to reduce mutant Huntingtin aggregation, eventually improving disease outcomes.


title: Reactive astrocytes promote proteostasis in Huntington disease through the JAK2-STAT3 pathway


title: Distinct bioenergetic features of human invariant natural killer T (iNKT) cells enable retained functions in nutrient-deprived states

abstract: Invariant natural killer T (iNKT) cells comprise a unique subset of lymphocytes that are primed for activation and possess innate NK-like functional features. Currently, iNKT cell-based immunotherapies remain in early clinical stages, and little is known about the ability of these cells to survive and retain effector functions within the solid tumor microenvironment (TME) long-term. In conventional T cells (TCONV), cellular metabolism is linked to effector functions and their ability to adapt to the nutrient-poor TME. In contrast, the bioenergetic requirements of iNKT cells - particularly those of human iNKT cells - at baseline and upon stimulation are not well understood; neither is how these requirements affect cytokine production or anti-tumor effector functions. We find that unlike TCONV, human iNKT cells are not dependent upon glucose or glutamine for cytokine production and cytotoxicity upon stimulation with anti-CD3 and anti-CD28. Additionally, transcriptional profiling revealed that stimulated human iNKT cells are less glycolytic than TCONV and display higher expression of fatty acid oxidation (FAO) and adenosine monophosphate-activated protein kinase (AMPK) pathway genes. Furthermore, stimulated iNKT cells displayed higher mitochondrial mass and membrane potential relative to TCONV. Real-time Seahorse metabolic flux analysis revealed that stimulated human iNKT cells utilize fatty acids as substrates for oxidation more than stimulated TCONV. Together, our data suggest that human iNKT cells possess different bioenergetic requirements from TCONV and display a more memory-like metabolic program relative to effector TCONV. Importantly, iNKT cell-based immunotherapeutic strategies could co-opt such unique features of iNKT cells to improve their efficacy and longevity of anti-tumor responses.


title: NEU1 and NEU3 enzymes alter CD22 organization on B cells

abstract: The B cell membrane expresses sialic acid binding Immunoglobulin-like lectins, also called Siglecs, that are important for modulating immune response. Siglecs have interactions with sialoglycoproteins found on the same membrane (cis ligands) that result in homotypic and heterotypic receptor clusters. The regulation and organization of these clusters, and their effect on cell activation, is not clearly understood. We investigated the role of human neuraminidase enzymes, NEU1 and NEU3, on the clustering of CD22 on B cells using confocal microscopy. We observed that native NEU1 and NEU3 activity influence the cluster size of CD22. Using single-particle tracking, we observed that NEU3 activity increased the lateral mobility of CD22, which was in contrast to the effect of exogenous bacterial NEU enzymes. Moreover, we show that native NEU1 and NEU3 activity influenced cellular Ca2+levels, supporting a role for these enzymes in regulating B cell activation. Our results establish a role for native NEU activity in modulating CD22 organization and function on B cells.


title: High-Yield Monocyte, Macrophage, and Dendritic Cell Differentiation from Induced Pluripotent Stem Cells

abstract: Differentiation of induced pluripotent stem cells (iPSC) into monocytes, monocyte-derived macrophages (MDM), and monocyte-derived dendritic cells (moDC) represents a powerful tool for studying human innate immunology and developing novel iPSC-derived immune therapies. Challenges include inefficiencies in iPSC-derived cell cultures, labor-intensive culture conditions, low purity of desired cell types, and feeder cell requirements. Here, a highly efficient method for differentiating monocytes, MDMs, and moDCs that overcomes these challenges is described. The process utilizes commercially-available materials to derive CD34+ progenitor cells that are apically released from a hemogenic endothelium. Subsequently, the hemogenic endothelium gives rise to highly pure (>95%), CD34-CD14+ monocytes in 19-23 days and yields 13.5-fold more monocytes by day 35 when compared to previous methods. These iPSC-monocytes are analogous to human blood-derived monocytes and readily differentiate into MDM and moDC. The efficient workflow and increase in monocyte output heightens feasibility for high throughput studies and enables clinical-scale iPSC-derived manufacturing processes.