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The adaptive immune system protects against infection via selection of specific antigen receptors on B-cells and T-cells. We studied the prevalent CD8 ‘killer’ T-cell response mounted against SARS-CoV-2 Spike269-277 epitope YLQPRTFLL via the most frequent Human Leukocyte Antigen (HLA) class I worldwide, HLA A*02. The widespread Spike P272L mutation has arisen in at least 14 different SARS-CoV-2 lineages to date, including in lineages identified as variants of concern. P272L was common in the B.1.177 lineage associated with establishing the ‘second wave’ in Europe. The large CD8 T-cell response seen across a cohort of HLA A*02+ convalescent patients, comprising of over 120 different TCRs, failed to respond to the P272L. Sizable populations (0.01%-0.2%) of total CD8 T-cells from individuals vaccinated against SARS-CoV-2 stained with HLA A*02-YLQPRTFLL multimers but failed to bind to the P272L reagent. Viral escape at prevalent T-cell epitopes restricted by high frequency HLA may be particularly problematic when vaccine immunity is focussed on a single protein such as SARS-CoV-2 Spike and provides a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlights the urgent need for monitoring T-cell escape in new SARS-CoV-2 variants.New in Version 2Updated as P272L now seen in 14 different SARS-CoV-2 lineages including the B.1.1.7/Alpha (UK variant) lineageAtomic structures of HLA A*02-YLQPRTFLL and HLA A*02-YLQLRTFLL addedPierre Rizkallah added as an author and author list changed to reflect the contributions of Aaron Wall and Anna Fuller to the newly added datasets

Leaf mold caused by the fungus Cladosporium fulvum (syn. Passalora fulva) has been a serious disease in greenhouse tomatoes in Cuba, but less damage is seen in outdoor grown plants. This study was conducted to identify races of the causal agent of tomato leaf mold. A total of 36 single conidial isolates obtained from different provinces in Cuba and the reference strain race 0 were inoculated on a differential set of tomato cultivars carrying different Cf resistance genes. Further, a polymerase chain reaction (PCR) was developed using Avr9 gene-specific primers. As result, all isolates could overcome the Cf-9 resistance gene only, but not resistance genes Cf-2, Cf-4 and Cf-5 and must therefore be coined race 9. Race identity was also confirmed by PCR analysis, which showed that the Avr9 gene was not amplified in any of 36 single conidial isolates studied proving that they all lack this gene. This is the first report of the occurrence of race 9 of C. fulvum in Cuba. Pyramiding other Cf genes in hybrid of tomato may provide a more durable level of resistance to C. fulvum.

Immunocompromised patients are highly susceptible to invasive aspergillosis. Herein, we identified a homozygous deletion mutation (507 del C) resulting in a frameshift (N170I) and early stop codon in the fungal binding Dectin-2 receptor, in an immunocompromised patient. The mutated form of Dectin-2 was weakly expressed, did not form clusters at/near the cell surface and was functionally defective. Peripheral blood mononuclear cells from this patient were unable to mount a cytokine (tumor necrosis factor, interleukin 6) response to Aspergillus fumigatus, and this first identified Dectin-2–deficient patient died of complications of invasive aspergillosis., We identified a Dectin-2 N170I mutation in an immunocompromised patient who died of complications of invasive aspergillosis. This mutation results in an early stop codon and poor receptor expression and renders Dectin-2 functionally defective.

Cellular homeostasis plays a critical role in how an organism will develop and age. Disruption of this fragile equilibrium is often associated with health degradation and ultimately, death. Reactive oxygen species (ROS) have been closely associated with health decline and neurological disorders, such as Alzheimer’s disease or Parkinson’s disease. ROS were first identified as by-products of the cellular activity, mainly mitochondrial respiration, and their high reactivity is linked to a disruption of macromolecules such as proteins, lipids and DNA. More recent research suggests more complex function of ROS, reaching far beyond the cellular dysfunction. ROS are active actors in most of the signaling cascades involved in cell development, proliferation and survival, constituting important second messengers. In the brain, their impact on neurons and astrocytes has been associated with synaptic plasticity and neuron survival. This review provides an overview of ROS function in cell signaling in the context of aging and degeneration in the brain and guarding the fragile balance between health and disease.

The evolution of DNA methylome and methylation intra-tumor heterogeneity (ITH) during early carcinogenesis of lung adenocarcinoma has not been systematically studied. We perform reduced representation bisulfite sequencing of invasive lung adenocarcinoma and its precursors, atypical adenomatous hyperplasia, adenocarcinoma in situ and minimally invasive adenocarcinoma. We observe gradual increase of methylation aberrations and significantly higher level of methylation ITH in later-stage lesions. The phylogenetic patterns inferred from methylation aberrations resemble those based on somatic mutations suggesting parallel methylation and genetic evolution. De-convolution reveal higher ratio of T regulatory cells (Tregs) versus CD8 + T cells in later-stage diseases, implying progressive immunosuppression with neoplastic progression. Furthermore, increased global hypomethylation is associated with higher mutation burden, copy number variation burden and AI burden as well as higher Treg/CD8 ratio, highlighting the potential impact of methylation on chromosomal instability, mutagenesis and tumor immune microenvironment during early carcinogenesis of lung adenocarcinomas., It is known that invasive lung adenocarcinomas evolve from pre-cancerous dysplastic lesions. In this study, the authors show that evolution of pre-cancerous lesions is accompanied by DNA methylation alterations, and that global hypomethylation correlates with immune infiltration, mutational burden and copy number alterations.

Rationale: The BMPs (bone morphogenetic proteins) are essential morphogens in angiogenesis and vascular development. Disruption of BMP signaling can trigger cardiovascular diseases, such as arteriovenous malformations. Objective: A computational model predicted that BMP4 and BMP9 and their inhibitors MGP (matrix gamma-carboxyglutamic acid [Gla] protein) and CV2 (crossveinless-2) would form a regulatory system consisting of negative feedback loops with time delays and that BMP9 would trigger oscillatory expression of the 2 inhibitors. The goal was to investigate this regulatory system in endothelial differentiation and vascular growth. Methods and Results: Oscillations in the expression of MGP and CV2 were detected in endothelial cells in vitro, using quantitative real-time polymerase chain reaction and immunoblotting. These organized temporally downstream BMP-related activities, including expression of stalk-cell markers and cell proliferation, consistent with an integral role of BMP9 in vessel maturation. In vivo, the inhibitors were located in distinct zones in relation to the front of the expanding retinal network, as determined by immunofluorescence. Time-dependent changes of the CV2 location in the retina and the existence of an endothelial population with signs of oscillatory MGP expression in developing vasculature supported the in vitro findings. Loss of MGP or its BMP4-binding capacity disrupted the retinal vasculature, resulting in poorly formed networks, especially in the venous drainage areas, and arteriovenous malformations as determined by increased cell coverage and functional testing. Conclusions: Our results suggest a previously unknown mechanism of temporal orchestration of BMP4 and BMP9 activities that utilize the tandem actions of the extracellular antagonists MGP and CV2. Disruption of this mechanism may contribute to vascular malformations and disease.

Proteases are key components of the hydrolytic enzyme arsenal employed by fungal pathogens to invade their host plants. The recent advances in -omics era have facilitated identification of functional proteases involved in plant-fungus interactions. By comparison of the publically available sequences of fungal genomes we found that the number of protease genes present in the genome of Cladosporium fulvum, a biotrophic tomato pathogen, is comparable with that of hemibiotrophs. To identify host plant inducible protease genes and their products, we performed transcriptome and proteome analyses of C. fulvumin vitro and in planta by means of RNA-Seq/RT-qPCR and mass spectrometry. Transcriptome data showed that 14 out of the 59 predicted proteases are expressed during in vitro and in planta growth of C. fulvum, of which nine belong to serine proteases S8 and S10 and the rest belong to metallo- and aspartic proteases. Mass spectrometry confirmed the presence of six proteases at proteome level during plant infection. Expression of limited number of proteases by C. fulvum might sustain biotrophic growth and benefits its stealth pathogenesis.

Background Neuroleptic drug-induced parkinsonism (NIP) is a leading cause of parkinsonism, particularly in aging. Based on abnormal dopamine transporter scan results, individuals displaying chronic NIP are often diagnosed with Lewy-body Parkinson's disease (PD), but this assumption needs further substantiation. Objective To quantitate the profile of striatal dopaminergic nerve terminal density in NIP relative to PD. Methods We used the positron emission tomography ligand [11 C](+)-dihydrotetrabenazine targeting vesicular monoamine transporter type 2 (VMAT2) binding sites and collected various clinical parameters (motor ratings, olfaction, polysomnography to document rapid eye movement sleep muscle activity, quantitative sensory testing for pain thresholds) possibly predicting binding results in patients older than age 50 living with schizophrenia spectrum disorders under long-term stable antipsychotic drug treatment, with (N = 11) or without (N = 11) chart documention of chronic NIP, and compared them to healthy volunteers (N = 11) and others medicated for PD (N = 12). Results Striatal VMAT2 binding was dichotomous in the NIP group between those with spared (N = 5) or low (N = 6) PD-like values. Striatal binding reduction in the low VMAT2-NIP group was asymmetric without the gradient of maximal involvement in the posterior putamen typical of PD. Anosmia was the only nonmotor parameter measured matching the abnormal striatal VMAT2 binding status. Conclusion These preliminary observations suggest that striatal VMAT2 binding is abnormal in a fraction of chronic NIP cases and differs in spatial distribution from PD. The possibility of a drug-induced axonopathy and resultant synaptopathy, as well as the evolution of the binding deficit, warrant further longitudinal studies in a large cohort. © 2020 International Parkinson and Movement Disorder Society.

Here we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1. EstN7 is a dimer with a classical α/β hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. The conformation of the functionally important cap region is significantly different to EstN7's closest relatives, forming a bridge-like structure with reduced helical content providing greater access to the active site through more than one substrate access tunnel. However, dynamics do not appear to play a major role in cold adaption. Molecular dynamics at different temperatures, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are important for of EstN7's cold adaptation rather than changes in dynamics.

International audience; Since December 2019, coronavirus disease 2019 (COVID-19), caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has changed our lives. Elderly, and those with comorbidities represent the vast majority of patients hospitalized with severe COVID-19 symptoms, including acute respiratory disease syndrome, and cardiac dysfunction. Despite a huge effort of the scientific community, improved treatment modalities limiting the severity and mortality of hospitalized COVID-19 patients, are still required. Here, we compare the effectiveness of virus- and patients-centred strategies to reduce COVID-19 mortality. We also discuss the therapeutic options that might further reduce death rates associated with the disease in the future. Unexpectedly, extensive review of the literature suggests that SARS-CoV-2 viral load seems to be associated neither with the severity of symptoms nor with mortality of hospitalized patients with COVID-19. This may explain why, so far, virus-centred strategies using antivirals aiming to inhibit the viral replicative machinery, have failed to reduce COVID-19 mortality in patients with respiratory failure. By contrast, anti-inflammatory treatments without antiviral capacities but centred on patients, such as dexamethasone or Tocilizumab®, reduce COVID-19 mortality. Finally, since the spike protein of SARS-CoV-2 binds to Angiotensin Converting Enzyme 2 (ACE2) and inhibits its function, we explore the different treatment options focussing on rebalancing the Renin-Angiotensin System (RAS). This new therapeutic strategy could hopefully further reduce the severity of respiratory failure and limit COVID-19 mortality in elderly patients.

Description, FGF and Wnt signaling controls the phase transition of the ocular fate., The developing vertebrate eye cup is partitioned into the neural retina (NR), the retinal pigmented epithelium (RPE), and the ciliary margin (CM). By single-cell analysis, we showed that fibroblast growth factor (FGF) signaling regulates the CM in its stem cell–like property of self-renewal, differentiation, and survival, which is balanced by an evolutionarily conserved Wnt signaling gradient. FGF promotes Wnt signaling in the CM by stabilizing β-catenin in a GSK3β-independent manner. While Wnt signaling converts the NR to either the CM or the RPE depending on FGF signaling, FGF transforms the RPE to the NR or CM dependent on Wnt activity. The default fate of the eye cup is the NR, but synergistic FGF and Wnt signaling promotes CM formation both in vivo and in human retinal organoid. Our study reveals that the vertebrate eye develops through phase transition determined by a combinatorial code of FGF and Wnt signaling.

In 1995 Benington and Heller formulated an energy hypothesis of sleep centered on a key role of glycogen. It was postulated that a major function of sleep is to replenish glycogen stores in the brain that have been depleted during wakefulness which is associated to an increased energy demand. Astrocytic glycogen depletion participates to an increase of extracellular adenosine release which influences sleep homeostasis. Here, we will review some evidence obtained by studies addressing the question of a key role played by glycogen metabolism in sleep regulation as proposed by this hypothesis or by an alternative hypothesis named "glycogenetic" hypothesis as well as the importance of the confounding effect of glucocortico < ds. Even though actual collected data argue in favor of a role of sleep in brain energy balance-homeostasis, they do not support a critical and direct involvement of glycogen metabolism on sleep regulation. For instance, glycogen levels during the sleep-wake cycle are driven by different physiological signals and therefore appear more as a marker-integrator of brain energy status than a direct regulator of sleep homeostasis. In support of this we provide evidence that blockade of glycogen mobilization does not induce more sleep episodes during the active period while locomotor activity is reduced. These observations do not invalidate the energy hypothesis of sleep but indicate that underlying cellular mechanisms are more complex than postulated by Benington and Heller.

Oncolytic virotherapies (OV) hold immense clinical potential. OV based on human adenoviruses (HAdV) derived from HAdV with naturally low rates of pre- existing immunity will be beneficial for future clinical translation. We generated a low- seroprevalence HAdV-D10 serotype vector incorporating an αvβ6 integrin-selective peptide, A20, to target αvβ6-positive tumor cell types. HAdV-D10 has limited natural tropism. Structural and biological studies of HAdV-D10 knob protein highlighted low-affinity engagement with native adenoviral receptors CAR and sialic acid. HAdV-D10 fails to engage blood coagulation factor X, potentially eliminating “off-target” hepatic sequestration in vivo. We engineered an A20 peptide that selectively binds αvβ6 integrin into the DG loop of HAdV-D10 fiber knob. Assays in αvβ6+ cancer cell lines demonstrated significantly increased transduction mediated by αvβ6-targeted variants compared with controls, confirmed microscopically. HAdV-D10.A20 resisted neutralization by neutralizing HAdV-C5 sera. Systemic delivery of HAdV-D10.A20 resulted in significantly increased GFP expression in BT20 tumors. Replication-competent HAdV-D10.A20 demonstrated αvβ6 integrin-selective cell killing in vitro and in vivo. HAdV-D10 possesses characteristics of a promising virotherapy, combining low seroprevalence, weak receptor interactions, and reduced off-target uptake. Incorporation of an αvβ6 integrin-selective peptide resulted in HAdV-D10.A20, with significant potential for clinical translation.

Our group previously demonstrated that sarcoma cell lines were insensitive to epidermal growth factor receptor (EGFR) inhibitor gefitinib monotherapy. PENAO, an anti-tumour metabolic compound created in our laboratory, is currently in clinical trials. Considering the positive regulation of tumour energy production by both the EGFR signalling and tumour metabolism pathways, this study aimed to investigate the effect and mechanisms of combination therapy using gefitinib and PENAO in sarcoma cell lines in vitro and in vivo. PENAO monotherapy reduced proliferation in 12 sarcoma cell lines. Combining gefitinib and PENAO resulted in synergistic inhibition in both a time- and dose-dependent manner in 3 sarcoma cell lines with less prominent monotherapy effects. Combined treatment significantly enhanced cell death and perturbed mitochondrial function. In vivo combination therapy with PENAO and gefitinib was non-toxic to mice and significantly delayed tumour growth and prolonged survival. At 20 days after treatment, tumours from the combination treated mice were significantly smaller than those from untreated and single drug treated mice. The survival curves also showed significant difference across and between groups. The combination of PENAO and gefitinib in vitro and in vivo, shows promise as a treatment pathway in this poor outcome tumour.

Enterobacteriaceae are among the first colonizers of neonate intestine. Members of this family, such as Escherichia and Klebsiella, are considered pathobionts and as such are capable of inducing local and systemic disease under specific colonization circumstances. Interplay between developing microbiota and pathogenic function of pathobionts are poorly understood. In this study, we investigate the functional interaction between various colonization patterns on an early colonizer, K. pneumoniae. K. pneumoniae 51-5 was isolated from stool of a healthy, premature infant, and found to contain the genotoxin island pks associated with development of colorectal cancer. Using intestinal epithelial cells, macrophages, and primary splenocytes, we demonstrate K. pneumoniae 51-5 upregulates expression of proinflammatory genes in vitro. Gnotobiotic experiments in Il10−/− mice demonstrate the neonate isolate induces intestinal inflammation in vivo, with increased expression of proinflammatory genes. Regulation of microbiota assembly revealed K. pneumoniae 51-5 accelerates onset of inflammation in Il10−/− mice, most significantly when microbiota is naturally acquired. Furthermore, K. pneumoniae 51-5 induces DNA damage and cell cycle arrest. Interestingly, K. pneumoniae 51-5 induced tumors in ApcMin/+; Il10−/− mice was not significantly affected by absence of colibactin activating enzyme, ClbP. These findings demonstrate pathogenicity of infant K. pneumoniae isolate is sensitive to microbial colonization status.

Adenovirus derived vectors, based on chimpanzee adenovirus Y25 (ChAdOx1) and human adenovirus type 26 are proving critical in combatting the 2019 SARS-CoV-2 pandemic. Following emergency use authorisation, scale up in vaccine administration has inevitably revealed vaccine related adverse effects; too rare to observe even in large Phase-III clinical trials. These include vaccine-induced thrombotic thrombocytopenia (VITT), an ultra-rare adverse event in which patients develop life-threatening blood clots 5-24 days following vaccination.To investigate vector-host interactions of ChAdOx1 underpinning VITT we solved the structure of the ChAdOx1 capsid by CryoEM, and the structure of the primary receptor tropism determining fiber-knob protein by crystallography. These structural insights have enabled us to unravel key protein interactions involved in ChAdOx1 cell entry and a possible means by which it may generate misplaced immunity to platelet factor 4 (PF4), a protein involved in coagulation.We use in vitro cell binding assays to show that the fiber-knob protein uses coxsackie and adenovirus receptor (CAR) as a high affinity binding partner, while it does not form a stable interface with CD46. Computational simulations identified a putative mechanism by which the ChAdOx1 capsid interacts with PF4 by binding in the spaces between hexon proteins, with downstream implications for the causes of VITT.SummaryWe present the structure of the ChAdOx1 viral vector, derived from chimpanzee adenovirus Y25 at 4.2Å resolution1. ChAdOx1 is in global use in the AstraZeneca vaccine, ChAdOx1 nCoV-19/AZD-1222, to combat the SARS-CoV-2 coronavirus pandemic. Recently observed, rare, adverse events make detailed mechanistic understanding of this vector key to informing proper treatment of affected patients and the development of safer viral vectors.Here, we determine a primary mechanism ChAdOx1 uses to attach to cells is coxsackie and adenovirus receptor (CAR), a protein which is identical in humans and chimpanzees. We demonstrate the vector does not form a stable CD46 interaction, a common species B adenovirus receptor, via its primary attachment protein.Further, we reveal the surface of the ChAdOx1 viral capsid has a strong electronegative potential. Molecular simulations suggest this charge, together with shape complementarity, are a mechanism by which an oppositely charged protein, platelet factor 4 (PF4) may bind the vector surface. PF4 is a key protein involved in the formation of blood clots2, and the target of auto-antibodies in heparin-induced immune thrombotic thrombocytopenia (HITT)3, an adverse reaction to heparin therapy which presents similarly to vaccine-induced immune thrombotic thrombocytopenia (VITT), a rare complication of ChAdOx1 nCoV-19 vaccination4–6. We propose a mechanism in which the ChAdOx1-PF4 complex may stimulate the production of antibodies against PF4, leading to delayed blood clot formation, as observed in VITT.

Triple-negative breast cancers (TNBCs) are very heterogenous, molecularly diverse, and are characterized by a high propensity to relapse or metastasize. Clinically, TNBC remains a diagnosis of exclusion by the lack of hormone receptors (Estrogen Receptor (ER) and Progesterone Receptor (PR)) as well as the absence of overexpression and/or amplification of HER2. DNA methylation plays an important role in breast cancer carcinogenesis and TNBCs have a distinct DNA methylation profile characterized by marked hypomethylation and lower gains of methylations compared to all other subtypes. DNA methylation is regulated by the balance of DNA methylases (DNMTs) and DNA demethylases (TETs). Here, we review the roles of TETs as context-dependent tumor-suppressor genes and/or oncogenes in solid tumors, and we discuss the current understandings of the oncogenic role of TET1 and its therapeutic implications in TNBCs.

Background DNA methylation alterations have similar patterns in normal aging tissue and in cancer. In this study, we investigated breast tissue-specific age-related DNA methylation alterations and used those methylation sites to identify individuals with outlier phenotypes. Outlier phenotype is identified by unsupervised anomaly detection algorithms and is defined by individuals who have normal tissue age-dependent DNA methylation levels that vary dramatically from the population mean. Methods We generated whole-genome DNA methylation profiles (GSE160233) on purified epithelial cells and used publicly available Infinium HumanMethylation 450K array datasets (TCGA, GSE88883, GSE69914, GSE101961, and GSE74214) for discovery and validation. Results We found that hypermethylation in normal breast tissue is the best predictor of hypermethylation in cancer. Using unsupervised anomaly detection approaches, we found that about 10% of the individuals (39/427) were outliers for DNA methylation from 6 DNA methylation datasets. We also found that there were significantly more outlier samples in normal-adjacent to cancer (24/139, 17.3%) than in normal samples (15/228, 5.2%). Additionally, we found significant differences between the predicted ages based on DNA methylation and the chronological ages among outliers and not-outliers. Additionally, we found that accelerated outliers (older predicted age) were more frequent in normal-adjacent to cancer (14/17, 82%) compared to normal samples from individuals without cancer (3/17, 18%). Furthermore, in matched samples, we found that the epigenome of the outliers in the pre-malignant tissue was as severely altered as in cancer. Conclusions A subset of patients with breast cancer has severely altered epigenomes which are characterized by accelerated aging in their normal-appearing tissue. In the future, these DNA methylation sites should be studied further such as in cell-free DNA to determine their potential use as biomarkers for early detection of malignant transformation and preventive intervention in breast cancer.

The Hbl toxin is a three-component haemolytic complex produced by Bacillus cereus sensu lato strains and implicated as a cause of diarrhoea in B. cereus food poisoning. While the structure of the HblB component of this toxin is known, the structures of the other components are unresolved. Here, we describe the expression of the recombinant HblL1 component and the elucidation of its structure to 1.36 Å. Like HblB, it is a member of the alpha-helical pore-forming toxin family. In comparison to other members of this group, it has an extended hydrophobic beta tongue region that may be involved in pore formation. Molecular docking was used to predict possible interactions between HblL1 and HblB, and suggests a head to tail dimer might form, burying the HblL1 beta tongue region.

Cold-active esterases hold great potential for undertaking useful biotransformations at low temperatures. Here, we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1, which has an apparent melting temperature of 26{degrees}C. EstN7 is a dimer with a classical /{beta} hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. However, dynamics do not appear to play a major role in cold adaption. Comparison of B-factors with the closest related mesophilic and thermophilic esterases suggests there is little difference in dynamics with the catalytically important N-terminal cap comprising the main dynamic element. Molecular dynamics, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. The conformation of the cap region is significantly different to EstN7s closest relatives, forming a bridge-like structure with reduced helical content providing more than one access tunnel through to the active site. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are the main drivers of EstN7s cold adaptation rather than changes in dynamics.

Adenoviruses are powerful tools experimentally and clinically. To maximize efficacy, the development of serotypes with low preexisting levels of immunity in the population is desirable., The human adenovirus (HAdV) phylogenetic tree is diverse, divided across seven species and comprising over 100 individual types. Species D HAdV are rarely isolated with low rates of preexisting immunity, making them appealing for therapeutic applications. Several species D vectors have been developed as vaccines against infectious diseases, where they induce robust immunity in preclinical models and early phase clinical trials. However, many aspects of the basic virology of species D HAdV, including their basic receptor usage and means of cell entry, remain understudied. Here, we investigated HAdV-D49, which previously has been studied for vaccine and vascular gene transfer applications. We generated a pseudotyped HAdV-C5 presenting the HAdV-D49 fiber knob protein (HAdV-C5/D49K). This pseudotyped vector was efficient at infecting cells devoid of all known HAdV receptors, indicating HAdV-D49 uses an unidentified cellular receptor. Conversely, a pseudotyped vector presenting the fiber knob protein of the closely related HAdV-D30 (HAdV-C5/D30K), differing in four amino acids from HAdV-D49, failed to demonstrate the same tropism. These four amino acid changes resulted in a change in isoelectric point of the knob protein, with HAdV-D49K possessing a basic apical region compared to a more acidic region in HAdV-D30K. Structurally and biologically we demonstrate that HAdV-D49 knob protein is unable to engage CD46, while potential interaction with coxsackievirus and adenovirus receptor (CAR) is extremely limited by extension of the DG loop. HAdV-C5/49K efficiently transduced cancer cell lines of pancreatic, breast, lung, esophageal, and ovarian origin, indicating it may have potential for oncolytic virotherapy applications, especially for difficult to transduce tumor types. IMPORTANCE Adenoviruses are powerful tools experimentally and clinically. To maximize efficacy, the development of serotypes with low preexisting levels of immunity in the population is desirable. Consequently, attention has focused on those derived from species D, which have proven robust vaccine platforms. This widespread usage is despite limited knowledge in their basic biology and cellular tropism. We investigated the tropism of HAdV-D49, demonstrating that it uses a novel cell entry mechanism that bypasses all known HAdV receptors. We demonstrate, biologically, that a pseudotyped HAdV-C5/D49K vector efficiently transduces a wide range of cell lines, including those presenting no known adenovirus receptor. Structural investigation suggests that this broad tropism is the result of a highly basic electrostatic surface potential, since a homologous pseudotyped vector with a more acidic surface potential, HAdV-C5/D30K, does not display a similar pantropism. Therefore, HAdV-C5/D49K may form a powerful vector for therapeutic applications capable of infecting difficult to transduce cells.

Astrocytes connect the vasculature to neurons and mediate the supply of nutrients and biochemicals. They also remove metabolites from the neurons and extracellular environment. They are involved in a growing number of physiological and pathophysiological processes. Understanding the biophysical, physiological, and molecular interactions in this neuro-glia-vascular ensemble (NGV) and how they support brain function is severely restricted by the lack of detailed cytoarchitecture. To address this problem, we used data from multiple sources to create a data-driven digital reconstruction of the NGV at micrometer anatomical resolution. We reconstructed 0.2 mm3 of rat somatosensory cortical tissue with approximately 16000 morphologically detailed neurons, its microvasculature, and approximately 2500 morphologically detailed protoplasmic astrocytes. The consistency of the reconstruction with a wide array of experimental measurements allows novel predictions of the numbers and locations of astrocytes and astrocytic processes that support different types of neurons. This allows anatomical reconstruction of the spatial microdomains of astrocytes and their overlapping regions. The number and locations of end-feet connecting each astrocyte to the vasculature can be determined as well as the extent to which they cover the microvasculature. The structural analysis of the NGV circuit showed that astrocytic shape and numbers are constrained by vasculature’s spatial occupancy and their functional role to form NGV connections. The digital reconstruction of the NGV is a resource that will enable a better understanding of the anatomical principles and geometric constraints which govern how astrocytes support brain function.Table of contentsMain pointsThe Blue Brain Project digitally reconstructs a part of neocortical Neuro-Glia-Vascular organizationInterdependencies and topological methods allow dense in silico reconstruction from sparse experimental dataThe polarized role of protoplasmic astrocytes constrains their shapes and numbersTable of contents image

BackgroundGenetics and biology may influence the age of onset of anorexia nervosa (AN). The aims of this study were to determine whether common genetic variation contributes to age of onset of AN and to investigate the genetic associations between age of onset of AN and age at menarche.MethodsA secondary analysis of the Psychiatric Genomics Consortium genome-wide association study (GWAS) of AN was performed, which included 9335 cases and 31,981 screened controls, all from European ancestries. We conducted GWASs of age of onset, early-onset AN (ResultsTwo loci were genome-wide significant in the typical-onset AN GWAS. Heritability estimates (single nucleotide polymorphism–h2) were 0.01–0.04 for age of onset, 0.16–0.25 for early-onset AN, and 0.17–0.25 for typical-onset AN. Early- and typical-onset AN showed distinct genetic correlation patterns with putative risk factors for AN. Specifically, early-onset AN was significantly genetically correlated with younger age at menarche, and typical-onset AN was significantly negatively genetically correlated with anthropometric traits. Genetic risk scores for age of onset and early-onset AN estimated from independent GWASs significantly predicted age of onset. Mendelian randomization analysis suggested a causal link between younger age at menarche and early-onset AN.ConclusionsOur results provide evidence consistent with a common variant genetic basis for age of onset and implicate biological pathways regulating menarche and reproduction. Background: Genetics and biology may influence the age of onset of anorexia nervosa (AN). The aims of this study were to determine whether common genetic variation contributes to age of onset of AN and to investigate the genetic associations between age of onset of AN and age at menarche. Methods: A secondary analysis of the Psychiatric Genomics Consortium genome-wide association study (GWAS) of AN was performed, which included 9335 cases and 31,981 screened controls, all from European ancestries. We conducted GWASs of age of onset, early-onset AN (2) were 0.01–0.04 for age of onset, 0.16–0.25 for early-onset AN, and 0.17–0.25 for typical-onset AN. Early- and typical-onset AN showed distinct genetic correlation patterns with putative risk factors for AN. Specifically, early-onset AN was significantly genetically correlated with younger age at menarche, and typical-onset AN was significantly negatively genetically correlated with anthropometric traits. Genetic risk scores for age of onset and early-onset AN estimated from independent GWASs significantly predicted age of onset. Mendelian randomization analysis suggested a causal link between younger age at menarche and early-onset AN. Conclusions: Our results provide evidence consistent with a common variant genetic basis for age of onset and implicate biological pathways regulating menarche and reproduction.

The gut microbiota modulates brain physiology, development, and behavior and has been implicated as a key regulator in several central nervous system disorders. Its effect on the metabolic coupling between neurons and astrocytes has not been studied to date, even though this is an important component of brain energy metabolism and physiology and it is perturbed in neurodegenerative and cognitive disorders. In this study, we have investigated the mRNA expression of 6 genes encoding proteins implicated in the astrocyte-neuron lactate shuttle (Atp1a2, Ldha, Ldhb, Mct1, Gys1, Pfkfb3), in relation to different gut microbiota manipulations, in the mouse brain hippocampus, a region with critical functions in cognition and behavior. We have discovered that Atp1a2 and Pfkfb3, encoding the ATPase, Na+/K+ transporting, alpha 2 subunit, respectively and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, two genes predominantly expressed in astrocytes, were upregulated in the hippocampus after microbial colonization of germ-free mice for 24 h, compared with conventionally raised mice. Pfkfb3 was also upregulated in germ-free mice compared with conventionally raised mice, while an increase in Atp1a2 expression in germ-free mice was confirmed only at the protein level by Western blot. In a separate cohort of mice, Atp1a2 and Pfkfb3 mRNA expression was upregulated in the hippocampus following 6-week dietary supplementation with prebiotics (fructoand galactooligosaccharides) in an animal model of chronic psychosocial stress. To our knowledge, these findings are the first to report an influence of the gut microbiota and prebiotics on mRNA expression of genes implicated in the metabolic coupling between neurons and astrocytes. (c) 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

The CD8 T cell response to the HLA-A2-restricted epitope LLWNGPMAV (LLW) of the non-structural protein 4b of Yellow Fever Virus (YFV) is remarkably immunodominant, highly prevalent and powerful in YFV-vaccinated humans. Here we used a combinatorial peptide library screening in the context of an A2/LLW-specific CD8 T cell clone to identify a superagonist that features a methionine to isoleucine substitution at position 7. Based on in silico modeling, the functional enhancement of this LLW-7I mutation was associated with alterations in the structural dynamics of the peptide in the major histocompatibility complex (pMHC) binding with the T cell receptor (TCR). While the TCR off-rate of LLW-7I pMHC is comparable to the wild type peptide, the rigidity of the 7I peptide seems to confer less entropy loss upon TCR binding. This LLW-7I superagonist is an example of improved functionality in human CD8 T cells associated with optimized ligand rigidity for TCR binding and not with changes in TCR:pMHC off-rate kinetics.

The SARS-CoV-2 virus is responsible for the coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The Spike glycoproteins of SARS-CoV-2 mediates viral entry and is the main target for neutralizing antibodies. Understanding the antibody response directed against SARS-CoV-2 is crucial for the development of vaccine, therapeutic and public health interventions. Here we perform a cross-sectional study on 106 different SARS-CoV-2-infected individuals to evaluate humoral responses against SARS-CoV-2 Spike. The vast majority of infected individuals elicits anti-Spike antibodies within 2 weeks after the onset of symptoms. The levels of receptor-binding domain (RBD)-specific IgG persist overtime, while the levels of anti-RBD IgM decrease after symptoms resolution. While most of individuals develop neutralizing antibodies within two weeks of infection, the level of neutralizing activity is significantly decreased over time. Our results highlight the importance of studying the persistence of neutralizing activity upon natural SARS-CoV-2 infection., Graphical Abstract, Highlights • Antibodies against SARS-CoV-2 Spike correlate with COVID-19 disease severity • RBD-specific IgM and IgA decline more rapidly than IgG • SARS-CoV-2 neutralizing antibodies are elicited within two weeks of infection • Neutralizing antibodies decline significantly after resolution of the infection

DNA methylation is an epigenetic mark that is altered in cancer and aging tissues. The effects of extrinsic factors on DNA methylation remain incompletely understood. Microbial dysbiosis is a hallmark of colorectal cancer, and infections have been linked to aberrant DNA methylation in cancers of the GI tract. To determine the microbiota’s impact on DNA methylation, we studied the methylomes of colorectal mucosa in germ-free (no microbiota) and specific-pathogen-free (controlled microbiota) mice, as well as in Il-10 KO mice (Il10−/−) which are prone to inflammation and tumorigenesis in the presence of microbiota. The presence of microbiota was associated with changes in 5% of the methylome and Il10−/− mice showed alterations in 4.1% of the methylome. These changes were slightly more often hypo than hypermethylation and affected preferentially CpG sites located in gene bodies and intergenic regions. Mice with both Il-10 KO and microbiota showed much more pronounced alterations, affecting 18% of the methylome. When looking specifically at CpG island methylation alterations, a hallmark of aging and cancer, 0.4% were changed by the microbiota, 0.4% were changed by Il10−/−, while 4% were changed by both simultaneously. These effects are comparable to what is typically seen when comparing colon cancer to normal. We next compared these methylation changes to those seen in aging, and after exposure to the colon carcinogen Azoxymethane (AOM). Aging was associated with alterations in 18% of the methylome, and aging changes were accelerated in the Il10−/− /SPF mice. By contrast, AOM induced profound hypomethylation that was distinct from the effects of aging or of the microbiota. CpG sites modified by the microbiota were over-represented among DNA methylation changes in colorectal cancer. Thus, the microbiota affects the DNA methylome of colorectal mucosa in patterns reminiscent of what is observed in aging and in colorectal cancer.

In order for sustainable nanomaterials such as cellulose nanocrystals (CNCs) to be utilized in industrial applications, a large-scale production capacity for CNCs must exist. Currently the only CNCs available commercially in kilogram scale are obtained from wood pulp (W-CNCs). Scaling the production capacity of W-CNCs isolation has led to their use in broader applications and captured the interest of researchers, industries and governments alike. Another source of CNCs with potential for commercial scale production are tunicates, a species of marine animal. Tunicate derived CNCs (T-CNCs) are a high aspect ratio CNC, which can complement commercially available W-CNCs in the growing global CNC market. Herein we report the isolation and characterization of T-CNCs from the tunicate Styela clava, an invasive species currently causing significant harm to local aquaculture communities. The reported procedure utilizes scalable CNC processing techniques and is based on our experiences from laboratory scale T-CNC isolation and pilot scale W-CNC isolation. To our best knowledge, this study represents the largest scale where T-CNCs have been isolated from any tunicate species, under any reaction conditions. Demonstrating a significant step towards commercial scale isolation of T-CNCs, and offering a potential solution to the numerous challenges which invasive tunicates pose to global aquaculture communities.

The evolution of DNA methylation at genome level and methylation intra-tumor heterogeneity (ITH) during early lung carcinogenesis has not been systematically studied. We performed multiregional reduced representation bisulfite sequencing (RRBS) of 62 resected lung nodules from 39 patients including atypical adenomatous hyperplasia (AAH, n=14), adenocarcinoma in situ (AIS, n=15), minimally invasive adenocarcinoma (MIA, n=22), and invasive adenocarcinoma (ADC, n=11). We observed significantly higher level of methylation ITH in later-stage lesions and gradual increase in both hyper- and hypomethylation compared to matched normal lung tissues over the course of neoplastic progression. The phyloepigenetic patterns inferred from methylation aberrations resembled those based on somatic mutations suggesting parallel methylation and genetic evolution during the early lung carcinogenesis. De-convolution of transcriptomic profiles from a previously published cohort and RBBS data from the current cohort demonstrated higher ratios of T regulatory cells (Tregs) versus CD8+ T cells in later-stage diseases implying progressive immunosuppression with neoplastic progression. Furthermore, increased global hypomethylation was associated with higher mutation burden, higher copy number aberration burden, higher allelic imbalance burden as well as higher Treg/CD8 ratio highlighting the potential impact of methylation states on chromosomal instability, mutagenesis and the tumor immune microenvironment.

The human adenovirus (HAdV) phylogenetic tree is diverse, divided across seven species and comprising over 100 individual types. Species D HAdV are rarely isolated with low rates of pre-existing immunity, making them appealing for therapeutic applications. Several species D vectors have been developed as vaccines against infectious diseases where they induce robust immunity in pre-clinical models and early phase clinical trials. However, many aspects of the basic virology of species D HAdV, including their basic receptor usage and means of cell entry, remain understudied.Here, we investigated HAdV-D49, which previously has been studied for vaccine and vascular gene transfer applications. We generated a pseudotyped HAdV-C5 presenting the HAdV-D49 fiber knob protein (HAdV-C5/D49K). This pseudotyped vector was efficient at infecting cells devoid of all known HAdV receptors, indicating HAdV-D49 uses an unidentified cellular receptor. Conversely, a pseudotyped vector presenting the fiber knob protein of the closely related HAdV-D30 (HAdV-C5/D30K), differing in four amino acids to HAdV-D49, failed to demonstrate the same tropism. These four amino acid changes resulted in a change in isoelectric point of the knob protein, with HAdV-D49K possessing a basic apical region compared to a more acidic region in HAdV-D30K. Structurally and biologically we demonstrate that HAdV-D49 knob protein is unable to engage CD46, whilst potential interaction with CAR is extremely limited by extension of the DG loop. HAdV-C5/49K efficiently transduced cancer cell lines of pancreatic, breast, lung, oesophageal and ovarian origin, indicating it may have potential for oncolytic virotherapy applications, especially for difficult to transduce tumour types.ImportanceAdenoviruses are powerful tools experimentally and clinically. To maximise efficacy, the development of serotypes with low pre-existing levels of immunity in the population is desirable. Consequently, attention has focussed on those derived from species D, which have proven robust vaccine platforms. This widespread usage is despite limited knowledge in their basic biology and cellular tropism.We investigated the tropism of HAdV-D49, demonstrating it uses a novel cell entry mechanism that bypasses all known HAdV receptors. We demonstrate, biologically, that a pseudotyped HAdV-C5/D49K vector efficiently transduces a wide range of cell lines, including those presenting no known adenovirus receptor. Structural investigation suggests that this broad tropism is the result of a highly basic electrostatic surface potential, since a homologous pseudotyped vector with a more acidic surface potential, HAdV-C5/D30K, does not display a similar pan-tropism. Therefore, HAdV-C5/D49K may form a powerful vector for therapeutic applications capable of infecting difficult to transduce cells.

Summary: T cell recognition of peptides presented by human leukocyte antigens (HLAs) is mediated by the highly variable T cell receptor (TCR). Despite this built-in TCR variability, individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD8+ T cells. Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA. However, few examples exist for CD4+ T cells. Here, we investigate CD4+ T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity. We identify five internal epitopes that are commonly recognized by CD4+ T cells in five HLA-DR1+ subjects and show conservation across viral strains and zoonotic reservoirs. TCR repertoire analysis demonstrates several shared gene usage biases underpinned by complementary biochemical features evident in a structural comparison. These epitopes are attractive targets for vaccination and other T cell therapies.

Introduction International travellers contribute to the rapid spread of Zika virus (ZIKV) and its sentinel identification globally. We describe ZIKV infections among international travellers seen at GeoSentinel sites with a focus on ZIKV acquired in the Americas and the Caribbean, describe countries of exposure and traveller characteristics, and assess ZIKV diagnostic testing by site. Methods Records with an international travel-related diagnosis of confirmed or probable ZIKV from January 2012 through December 2019 reported to GeoSentinel with a recorded illness onset date were included to show reported cases over time. Records from March 2016 through December 2019 with an exposure region of the Americas or the Caribbean were included in the descriptive analysis. A survey was conducted to assess the availability, accessibility and utilization of ZIKV diagnostic tests at GeoSentinel sites. Results GeoSentinel sites reported 525 ZIKV cases from 2012 through 2019. Between 2012 and 2014, eight cases were reported, and all were acquired in Asia or Oceania. After 2014, most cases were acquired in the Americas or the Caribbean, a large decline in ZIKV cases occurred in 2018–19. Between March 2016 and December 2019, 423 patients acquired ZIKV in the Americas or the Caribbean, peak reporting to these regions occurred in 2016 [330 cases (78%)]. The median age was 36 years (range: 3–92); 63% were female. The most frequent region of exposure was the Caribbean (60%). Thirteen travellers were pregnant during or after travel; one had a sexually acquired ZIKV infection. There was one case of fetal anomaly and two travellers with Guillain-Barré syndrome. GeoSentinel sites reported various challenges to diagnose ZIKV effectively. Conclusion ZIKV should remain a consideration for travellers returning from areas with risk of ZIKV transmission. Travellers should discuss their travel plans with their healthcare providers to ensure ZIKV prevention measures are taken.

The viruses that penetrate the central nervous system (CNS) possess neuroinvasive properties and are usually also neurotropic, meaning that they infect neural cells (neurons and glial cells) and by doing so may also become neurovirulent as they can participate in the development of neurological diseases. In the long list of viruses that are neuroinvasive, neurotropic, and potentially neurovirulent, the coronaviruses, from the order Nidovirales, are prevalent and have been occasionally associated with neuro-degenerative diseases of the CNS including multiple sclerosis, Parkinson’s disease, and even Alzheimer’s disease. Coronaviruses are classified in three different genera, namely a-, fi-, and 7-coronaviruses and are ubiquitous mainly respiratory and enteric pathogens, with neurotropic and neuroinvasive properties in various hosts including humans, cats, pigs, and rodents. Human coronaviruses are recognized respiratory pathogens, however, infectious particles, antigens or RNA, were detected in tissues other than the respiratory tract, including the CNS.

Cyclin-dependent kinase 9 (CDK9) plays a key role in transcription elongation, and more recently it was also identified as the molecular target of a series of diaminothiazole compounds that reverse epigenetic silencing in a phenotypic assay. To better understand the structural basis underlying these compounds’ activity and selectivity, we developed a comparative modeling approach that we describe herein. Briefly, this approach draws upon the strong structural conservation across the active conformation of all protein kinases, and their shared pattern of interactions with Type I inhibitors. Because of this, we hypothesized that the large collection of inhibitor/kinase structures available in the Protein Data Bank (PDB) would enable accurate modeling of this diaminothiazole series in complex with each CDK family member. We apply this new comparative modeling pipeline to build each of these structural models, and then demonstrate that these models provide retrospective rationale for the structure-activity relationships that ultimately guided optimization to the lead diaminothiazole compound MC180295 (14e). We then solved the crystal structure of the 14e/CDK9 complex, and found the resulting structure to be in excellent agreement with our corresponding comparative model. Finally, inspired by these models, we demonstrate how structural changes to 14e can be used to rationally tune this compound’s selectivity profile. With the emergence of CDK9 as a promising target for therapeutic intervention in cancer, we anticipate that comparative modeling can provide a valuable tool to guide optimization of potency and selectivity of new inhibitors targeting this kinase.

Food fortification with folic acid and increased use of vitamin supplements have raised concerns about high folic acid intake. We previously showed that high folic acid intake was associated with hepatic degeneration, decreased levels of methylenetetrahydrofolate reductase (MTHFR), lower methylation potential, and perturbations of lipid metabolism. MTHFR synthesizes the folate derivative for methylation reactions. In this study, we assessed the possibility that high folic acid diets, fed to wild-type and Mthfr+/- mice, could alter DNA methylation and/or deregulate hepatic cholesterol homeostasis. Digital restriction enzyme analysis of methylation in liver revealed DNA hypomethylation of a CpG in the lipolysis-stimulated lipoprotein receptor (Lsr) gene, which is involved in hepatic uptake of cholesterol. Pyrosequencing confirmed this methylation change and identified hypomethylation of several neighboring CpG dinucleotides. Lsr expression was increased and correlated negatively with DNA methylation and plasma cholesterol. A putative binding site for E2F1 was identified. ChIP-qPCR confirmed reduced E2F1 binding when methylation at this site was altered, suggesting that it could be involved in increasing Lsr expression. Expression of genes in cholesterol synthesis, transport or turnover (Abcg5, Abcg8, Abcc2, Cyp46a1, and Hmgcs1) was perturbed by high folic acid intake. We also observed increased hepatic cholesterol and increased expression of genes such as Sirt1, which might be involved in a rescue response to restore cholesterol homeostasis. Our work suggests that high folic acid consumption disturbs cholesterol homeostasis in liver. This finding may have particular relevance for MTHFR-deficient individuals, who represent ~10% of many populations.

Tumor-associated peptide-human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface-expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents.

20-Hydroxyecdysone (20E) is a steroid hormone that plays a key role in insect development through nuclear ecdysone receptors (EcRs) and at least one membrane GPCR receptor (DopEcR) and displays numerous pharmacological effects in mammals. However, its mechanism of action is still debated, involving either an unidentified GPCR or the estrogen ERβ receptor. The goal of our study was to better understand 20E mechanism of action.A mouse myoblast cell line (C2C12) and the gene expression of myostatin (a negative regulator of muscle growth) was used as a reporter system of anabolic activity. Experiments using protein-bound 20E established the involvement of a membrane receptor. 20E-like effects were also observed with Angiotensin-(1-7), the endogenous ligand of Mas. Additionally, the effect on myostatin gene expression was abolished by Mas receptor knock-down using small interfering RNA (siRNA) or pharmacological inhibitors.17β-Estradiol (E2) also inhibited myostatin gene expression, but protein-bound E2 was inactive, and E2 activity was not abolished by angiotensin-(1-7) antagonists. A mechanism involving cooperation between Mas receptor and a membrane-bound palmitoylated estrogen receptor is proposed.The possibility to activate the Mas receptor with a safe steroid molecule is consistent with the pleiotropic pharmacological effects of ecdysteroids in mammals and indeed this mechanism may explain the close similarity between angiotensin-(1-7) and 20E effects. Our findings open a lot of possible therapeutic developments by stimulating the protective arm of the renin-angiotensin-aldosterone system (RAAS) with 20E.

Promoter DNA methylation is a well-established mechanism of transcription repression, but its global correlation with gene expression is surprisingly weak. This weak correlation is due in part to the failure of current methylation quantification methods to consider the heterogeneity among sequenced bulk cells. Here, we introduce Cell Heterogeneity–Adjusted cLonal Methylation (CHALM) as a novel methylation quantification method. CHALM better explains the functional consequences of DNA methylation, such as its correlations with gene expression and H3K4me3. When applied to different methylation datasets, the CHALM method has uniquely detected differentially methylated genes that exhibit distinct biological functions supporting underlying mechanisms.

Calpain is a Ca2+-activated, heterodimeric cysteine protease consisting of a large catalytic subunit and a small regulatory subunit. Dysregulation of this enzyme is involved in a range of pathological conditions such as cancer, Alzheimer's disease and rheumatoid arthritis, and thus calpain I is a drug target with potential therapeutic applications. Difficulty in the production of this enzyme has hindered structural and functional investigations in the past, although heterodimeric calpain I can be generated by Escherichia coli expression in low yield. Here, an unexpected structure discovered during crystallization trials of heterodimeric calpain I (CAPN1C115S + CAPNS1ΔGR) is reported. A novel co-crystal structure of the PEF(S) domain from the dissociated regulatory small subunit of calpain I and the RNA-binding chaperone Hfq, which was likely to be overproduced as a stress response to the recombinant expression conditions, was obtained, providing unexpected insight in the chaperone function of Hfq.

Purpose: Virotherapies are maturing in the clinical setting. Adenoviruses (Ad) are excellent vectors for the manipulability and tolerance of transgenes. Poor tumor selectivity, off-target sequestration, and immune inactivation hamper clinical efficacy. We sought to completely redesign Ad5 into a refined, tumor-selective virotherapy targeted to αvβ6 integrin, which is expressed in a range of aggressively transformed epithelial cancers but nondetectable in healthy tissues. Experimental Design: Ad5NULL-A20 harbors mutations in each major capsid protein to preclude uptake via all native pathways. Tumor-tropism via αvβ6 targeting was achieved by genetic insertion of A20 peptide (NAVPNLRGDLQVLAQKVART) within the fiber knob protein. The vector's selectivity in vitro and in vivo was assessed. Results: The tropism-ablating triple mutation completely blocked all native cell entry pathways of Ad5NULL-A20 via coxsackie and adenovirus receptor (CAR), αvβ3/5 integrins, and coagulation factor 10 (FX). Ad5NULL-A20 efficiently and selectively transduced αvβ6+ cell lines and primary clinical ascites-derived EOC ex vivo, including in the presence of preexisting anti-Ad5 immunity. In vivo biodistribution of Ad5NULL-A20 following systemic delivery in non–tumor-bearing mice was significantly reduced in all off-target organs, including a remarkable 107-fold reduced genome accumulation in the liver compared with Ad5. Tumor uptake, transgene expression, and efficacy were confirmed in a peritoneal SKOV3 xenograft model of human EOC, where oncolytic Ad5NULL-A20–treated animals demonstrated significantly improved survival compared with those treated with oncolytic Ad5. Conclusions: Oncolytic Ad5NULL-A20 virotherapies represent an excellent vector for local and systemic targeting of αvβ6-overexpressing cancers and exciting platforms for tumor-selective overexpression of therapeutic anticancer modalities, including immune checkpoint inhibitors. Clin Cancer Res; 24(17); 4215–24. ©2018 AACR.

Acute myeloid leukemia (AML) often harbors mutations in epigenetic regulators, and also has frequent DNA hypermethylation, including the presence of CpG island methylator phenotypes (CIMPs). Although global hypomethylation is well known in cancer, the question of whether distinct demethylator phenotypes (DMPs) exist remains unanswered. Using Illumina 450k arrays for 194 patients from The Cancer Genome Atlas, we identified two distinct DMPs by hierarchical clustering: DMP.1 and DMP.2. DMP.1 cases harbored mutations in NPM1 (94%), FLT3 (71%) and DNMT3A (61%). Surprisingly, only 40% of patients with DNMT3A mutations were DMP.1, which has implications for mechanisms of transformation by this mutation. In contrast, DMP.2 AML was comprised of patients with t(8;21), inv(16) or t(15;17), suggesting common methylation defects connect these disparate rearrangements. RNA-seq revealed upregulated genes functioning in immune response (DMP.1) and development (DMP.2). We confirmed these findings by integrating independent 450k data sets (236 additional cases), and found prognostic effects by DMP status, independent of age and cytogenetics. The existence of DMPs has implications for AML pathogenesis and may augment existing tools in risk stratification.

The Dothideomycete Pseudocercospora fijiensis, previously Mycosphaerella fijiensis, is the causal agent of black Sigatoka, one of the most destructive diseases of bananas and plantains. Disease management depends on fungicide applications, with a major contribution from sterol demethylation-inhibitors (DMIs). The continued use of DMIs places considerable selection pressure on natural P. fijiensis populations, enabling the selection of novel genotypes with reduced sensitivity. The hitherto explanatory mechanism for this reduced sensitivity was the presence of non-synonymous point mutations in the target gene Pfcyp51, encoding the sterol 14α-demethylase enzyme. Here, we demonstrate a second mechanism involved in DMI sensitivity of P. fijiensis. We identified a 19-bp element in the wild-type (wt) Pfcyp51 promoter that concatenates in strains with reduced DMI sensitivity. A polymerase chain reaction (PCR) assay identified up to six Pfcyp51 promoter repeats in four field populations of P. fijiensis in Costa Rica. We used transformation experiments to swap the wt promoter of a sensitive field isolate with a promoter from a strain with reduced DMI sensitivity that comprised multiple insertions. Comparative in vivo phenotyping showed a functional and proportional up-regulation of Pfcyp51, which consequently decreased DMI sensitivity. Our data demonstrate that point mutations in the Pfcyp51 coding domain, as well as promoter inserts, contribute to the reduced DMI sensitivity of P. fijiensis. These results provide new insights into the importance of the appropriate use of DMIs and the need for the discovery of new molecules for black Sigatoka management.

During the last three decades, important progress in bone cell biology and in human and mouse genetics led to major advances in our understanding of the life and functions of cells of the osteoblast lineage. Previously unrecognized sources of osteogenic cells have been identified. Novel cellular and molecular mechanisms controlling osteoblast differentiation and senescence have been determined. New mechanisms of communications between osteogenic cells, osteocytes, osteoclasts, and chondrocytes, as well as novel links between osteogenic cells and blood vessels have been identified. Additionally, cells of the osteoblast lineage were shown to be important components of the hematopoietic niche and to be implicated in hematologic dysfunctions and malignancy. Lastly, unexpected interactions were found between osteogenic cells and several soft tissues, including the central nervous system, gut, muscle, fat, and testis through the release of paracrine factors, making osteogenic cells multifunctional regulatory cells, in addition to their bone-making function. These discoveries considerably enlarged our vision of the life and functions of osteogenic cells, which may lead to the development of novel therapeutics with immediate applications in bone disorders. © 2017 American Society for Bone and Mineral Research.

More and more evidence shows how brain energy metabolism is the linkage between physiological and morphological synaptic plasticity and memory consolidation. Different types of memory are associated with differential inputs, each with specific inputs that are upstream diverse molecular cascades depending on the receptor activity. No matter how heterogeneous the response is, energy availability represents the lowest common denominator since all these mechanisms are energy consuming and the brain networks adapt their performance accordingly. Astrocytes exert a primary role in this sense by acting as an energy buffer; glycogen granules, a mechanism to store glucose, are redistributed at glance and conveyed to neurons via the Astrocyte–Neuron Lactate Shuttle (ANLS). Here, we review how different types of memory relate to the mechanisms of energy delivery in the brain.

Accumulating evidence indicates that motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is a non-cell-autonomous process and that impaired glutamate clearance by astrocytes, leading to excitotoxicity, could participate in progression of the disease. In astrocytes derived from an animal model of ALS (hSOD1G93A rats), activation of type 5 metabotropic glutamate receptor (mGluR5) fails to increase glutamate uptake, impeding a putative dynamic neuroprotective mechanism involving astrocytes. Using astrocyte cultures from hSOD1G93A rats, we have demonstrated that the typical Ca2+ oscillations associated with mGluR5 activation were reduced, and that the majority of cells responded with a sustained elevation of intracellular Ca2+ concentration. Since the expression of protein kinase C epsilon isoform (PKCɛ) has been found to be considerably reduced in astrocytes from hSOD1G93A rats, the consequences of manipulating its activity and expression on mGluR5 signaling and on the regulation of glutamate uptake have been examined. Increasing PKCɛ expression was found to restore Ca2+ oscillations induced by mGluR5 activation in hSOD1G93A -expressing astrocytes. This was also associated with an increase in glutamate uptake capacity in response to mGluR5 activation. Conversely, reducing PKCɛ expression in astrocytes from wild-type animals with specific PKCɛ-shRNAs was found to alter the mGluR5 associated oscillatory signaling profile, and consistently reduced the regulation of the glutamate uptake-mediated by mGluR5 activation. These results suggest that PKCɛ is required to generate Ca2+ oscillations following mGluR5 activation, which support the regulation of astrocytic glutamate uptake. Reduced expression of astrocytic PKCɛ could impair this neuroprotective process and participate in the progression of ALS.

Epigenetic alterations such as DNA methylation defects and aberrant covalent histone modifications occur within all cancers and are selected for throughout the natural history of tumor formation, with changes being detectable in early onset, progression, and ultimately recurrence and metastasis. The ascertainment and use of these marks to identify at-risk patient populations, refine diagnostic criteria, and provide prognostic and predictive factors to guide treatment decisions are of growing clinical relevance. Furthermore, the targetable nature of epigenetic modifications provides a unique opportunity to alter treatment paradigms and provide new therapeutic options for patients whose malignancies possess these aberrant epigenetic modifications, paving the way for new and personalized medicine. DNA methylation has proven to be of significant clinical utility for its stability and relative ease of testing. The intent of this review is to elaborate upon well-supported examples of epigenetic precision medicine and how the field is moving forward, primarily in the context of aberrant DNA methylation.

In mammals, caloric restriction consistently results in extended lifespan. Epigenetic information encoded by DNA methylation is tightly regulated, but shows a striking drift associated with age that includes both gains and losses of DNA methylation at various sites. Here, we report that epigenetic drift is conserved across species and the rate of drift correlates with lifespan when comparing mice, rhesus monkeys, and humans. Twenty-two to 30-year-old rhesus monkeys exposed to 30% caloric restriction since 7–14 years of age showed attenuation of age-related methylation drift compared to ad libitum-fed controls such that their blood methylation age appeared 7 years younger than their chronologic age. Even more pronounced effects were seen in 2.7–3.2-year-old mice exposed to 40% caloric restriction starting at 0.3 years of age. The effects of caloric restriction on DNA methylation were detectable across different tissues and correlated with gene expression. We propose that epigenetic drift is a determinant of lifespan in mammals., Caloric restriction has been shown to increase lifespan in mammals. Here, the authors provide evidence that age-related methylation drift correlates with lifespan and that caloric restriction in mice and rhesus monkeys results in attenuation of age-related methylation drift.

J. Yao et al. demonstrate that loss of MGP, a BMP inhibitor, causes abnormal hepatic differentiation in lungs. They find that interactions between endothelium and epithelium separate pulmonary from hepatic differentiation during development. Lack of MGP triggers hepatic differentiation in the pulmonary epithelium, as regulated by the endothelium., The vascular endothelium is critical for induction of appropriate lineage differentiation in organogenesis. In this study, we report that dysfunctional pulmonary endothelium, resulting from the loss of matrix Gla protein (MGP), causes ectopic hepatic differentiation in the pulmonary epithelium. We demonstrate uncontrolled induction of the hepatic growth factor (HGF) caused by dysregulated cross talk between pulmonary endothelium and epithelium in Mgp-null lungs. Elevated HGF induced hepatocyte nuclear factor 4 α (Hnf4a), which competed with NK2 homeobox 1 (Nkx2.1) for binding to forkhead box A2 (Foxa2) to drive hepatic differentiation in Mgp-null airway progenitor cells. Limiting endothelial HGF reduced Hnf4a, abolished interference of Hnf4a with Foxa2, and reduced hepatic differentiation in Mgp-null lungs. Together, our results suggest that endothelial–epithelial interactions, maintained by MGP, are essential in pulmonary cell differentiation.

Bone morphogenetic protein (BMP) 10, a cardiac-restricted BMP family member, is essential in cardiomyogenesis, especially during trabeculation. Crossveinless-2 [CV2, also known as BMP endothelial cell precursor derived regulator (BMPER) ] is a BMP-binding protein that modulates the activity of several BMPs. The objective of this study was to examine the combined effects of BMP10 and CV2 on cardiomyocyte differentiation using mouse dedifferentiated fat (mDFAT) cells, which spontaneously differentiate into cardiomyocyte-like cells, as a model. Our results revealed that CV2 binds directly to BMP10, as determined by co-immunoprecipitation, and inhibits BMP10 from initiating SMAD signaling, as determined by luciferase reporter gene assays. BMP10 treatment induced mDFAT cell proliferation, whereas CV2 modulated the BMP10-induced proliferation. Differentiation of cardiomyocyte-like cells proceeded in a reproducible fashion in mDFAT cells, starting with small round Nkx2.5-positive progenitor cells that progressively formed myotubes of increasing length that assembled into beating colonies and stained strongly for Troponin I and sarcomeric alpha-actinin. BMP10 enhanced proliferation of the small progenitor cells, thereby securing sufficient numbers to support formation of myotubes. CV2, on the other hand, enhanced formation and maturation of large myotubes and myotube-colonies and was expressed by endothelial-like cells in the mDFAT cultures. Thus BMP10 and CV2 have important roles in coordinating cardiomyogenesis in progenitor cells.