Publications

While having already killed more than 7 million of people worldwide in 4 years, SARS-CoV-2, the etiological agent of COVID-19, is still circulating and evolving. Understanding the pathogenesis of the virus is of capital importance. It was shown that in vitro and in vivo infection with SARS-CoV-2 can lead to cell cycle arrest but the effect of the cell cycle arrest on the virus infection and the associated mechanisms are still unclear. By stopping cells in the G1 phase as well as targeting several pathways involved using inhibitors and small interfering RNAs, we were able to determine that the cell cycle arrest in the late G1 is beneficial for SARS-CoV-2 replication. This cell cycle arrest is independent of p53 but is dependent on the CDC25A-CDK2/cyclin E pathway. These data give a new understanding in SARS-CoV-2 pathogenesis and highlight some possible targets for the development of novel therapeutic approaches.

JTD Keywords: Cell lung-cancer,exchanger nhe,g1 phase,proliferation,inhibitio, Covid-19,coronavirus,pathogenicity,replication,cdk2,cyclin e,cdc25a,treatment

Human pluripotent stem cell -derived kidney organoids offer unprecedented opportunities for studying polycystic kidney disease (PKD), which still has no effective cure. Here, we developed both in vitro and in vivo organoid models of PKD that manifested tubular injury and aberrant upregulation of renin-angiotensin aldosterone system. Single -cell analysis revealed that a myriad of metabolic changes occurred during cystogenesis, including defective autophagy. Experimental activation of autophagy via ATG5 overexpression or primary cilia ablation significantly inhibited cystogenesis in PKD kidney organoids. Employing the organoid xenograft model of PKD, which spontaneously developed tubular cysts, we demonstrate that minoxidil, a potent autophagy activator and an FDA -approved drug, effectively attenuated cyst formation in vivo. This in vivo organoid model of PKD will enhance our capability to discover novel disease mechanisms and validate candidate drugs for clinical translation.

JTD Keywords: Adenylate kinase, Adult, Animal cell, Animal experiment, Animal model, Animal tissue, Article, Autophagosome, Autophagy, Autophagy (cellular), Autosomal-dominant, Calcium homeostasis, Cilia, Cilium, Cohort analysis, Controlled study, Cyclic amp, Disease, Dominant polycystic kidney, Enzyme linked immunosorbent assay, Epithelium, Exon, Expression, Female, Food and drug administration, Framework, Generation, Growth, Hepatitis a virus cellular receptor 1, Human, Human cell, Humans, Immunohistochemistry, In vitro study, In vivo study, Kidney, Kidney organoid, Kidney polycystic disease, Male, Minoxidil, Mouse, Mutations, Nonhuman, Organoid, Organoids, Platelet derived growth factor beta receptor, Pluripotent stem-cells, Polycystic kidney diseases, Protein kinase lkb1, Renin, Sequestosome 1, Single cell analysis, Single cell rna seq, Small nuclear rna, Tunel assay, Upregulation, Western blotting, Whole exome sequencing

Naso- and oropharyngeal swabs are the Center for Disease Control and Prevention (CDC) -recommended disease sampling methods for respiratory viruses. The short swabbing time for sampling by these methods may lead to variability in test results. Further, these methods are mildly invasive and can cause discomfort, tearing or gag reflexes in tested individuals. If longer sampling time is coupled with lesser patient discomfort, test reliability and patient compliance can be improved. Towards this end, we developed cationic biopolymer membranes for the electrostatic capturing of viruses in the oral cavity. Here, chemically (EDC-NHS) crosslinked uncharged chitosan (CS) nanofiber membranes were conferred either with negative surface charge by anionic poly-aspartic acid (pAsp) coating or positive charge by cationic poly-L-lysine (PLL). Consistent with our preliminary findings of dynamic light scattering (DLS) size measurements showing large agglomerates of anionic virus-like particles (VLPs) and cationic PLL in solution, a 75% increase in VLP adsorption by PLL coated CS membranes was recorded by enzyme linked immunosorbent assay (ELISA), in comparison to untreated controls. It is envisaged that the electrostatic concentration of respiratory viruses on cationic membranes can be superior alternatives to traditional swabbing in the oral cavity.

JTD Keywords: Cationic biopolymer membranes, Disease sampling, Dynamic light scattering (dls), Electrostatic capture of viruses, Enzyme linked immunosorbent assay (elisa), Magnetic beads, Virus -like particles (vlps)

It is not well understood why diabetic individuals are more prone to develop severe COVID-19. To this, we here established a human kidney organoid model promoting early hallmarks of diabetic kidney disease development. Upon SARS-CoV-2 infection, diabetic-like kidney organoids exhibited higher viral loads compared with their control counterparts. Genetic deletion of the angiotensin-converting enzyme 2 (ACE2) in kidney organoids under control or diabetic-like conditions prevented viral detection. Moreover, cells isolated from kidney biopsies from diabetic patients exhibited altered mitochondrial respiration and enhanced glycolysis, resulting in higher SARS-CoV-2 infections compared with non-diabetic cells. Conversely, the exposure of patient cells to dichloroacetate (DCA), an inhibitor of aerobic glycolysis, resulted in reduced SARS-CoV-2 infections. Our results provide insights into the identification of diabetic-induced metabolic programming in the kidney as a critical event increasing SARS-CoV-2 infection susceptibility, opening the door to the identification of new interventions in COVID-19 pathogenesis targeting energy metabolism.Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.

JTD Keywords: complications, coronavirus, cultured-cells, disease, distal tubule, mouse, protein, reveals, spike, Ace2, Angiotensin-converting enzyme 2, Angiotensin-converting enzyme-2, Covid-19, Diabetes 2, Human kidney organoids, Sars-cov-2

The influence of the properties of different solid substrates on the tethering of two antibodies, IgG1-CR3022 and IgG1-S309, which were specifically engineered for the detection of SARS-CoV-2, has been examined at the molecular level using conventional and accelerated Molecular Dynamics (cMD and aMD, respectively). Two surfaces with very different properties and widely used in immunosensors for diagnosis, amorphous silica and the most stable facet of the face-centered cubic gold structure, have been considered. The effects of such surfaces on the structure and orientation of the immobilized antibodies have been determined by quantifying the tilt and hinge angles that describe the orientation and shape of the antibody, respectively, and the dihedrals that measure the relative position of the antibody arms with respect to the surface. Results show that the interactions with amorphous silica, which are mainly electrostatic due to the charged nature of the surface, help to preserve the orientation and structure of the antibodies, especially of the IgG1-CR3022, indicating that the primary sequence of those antibodies also plays some role. Instead, short-range van der Waals interactions with the inert gold surface cause a higher degree tilting and fraying of the antibodies with respect to amorphous silica. The interactions between the antibodies and the surface also affect the correlation among the different angles and dihedrals, which increases with their strength. Overall, results explain why amorphous silica substrates are frequently used to immobilize antibodies in immunosensors. © 2022 The Authors

JTD Keywords: amorphous silica, antibody immobilization, enzyme, gol d, gold, immobilization, immunosensor, molecu l a r dynamics, molecular dynamics, protein adsorption, sars-cov-2 immunosensor, simulations, spike protein, surface interactions, target, vaccine, Amorphous silica, Antibodies, Antibody engineering, Antibody immobilization, Antibody structure, Article, Chemical detection, Computer model, Controlled study, Dihedral angle, Gold, In-silico, Molecular dynamics, Molecular levels, Molecular-dynamics, Nonhuman, Property, Sars, Sars-cov-2 immunosensor, Severe acute respiratory syndrome coronavirus 2, Silica, Silico studies, Silicon dioxide, Solid substrates, Structure analysis, Substrate chemistry, Substrates, Van der waals forces, Virus detection

Visual impairments are a critical medical hurdle to be addressed in modern society. Müller glia (MG) have regenerative potential in the retina in lower vertebrates, but not in mammals. However, in mice, in vivo cell fusion between MG and adult stem cells forms hybrids that can partially regenerate ablated neurons.We used organotypic cultures of human retina and preparations of dissociated cells to test the hypothesis that cell fusion between human MG and adult stem cells can induce neuronal regeneration in human systems. Moreover, we established a microinjection system for transplanting human retinal organoids to demonstrate hybrid differentiation.We first found that cell fusion occurs between MG and adult stem cells, in organotypic cultures of human retina as well as in cell cultures. Next, we showed that the resulting hybrids can differentiate and acquire a proto-neural electrophysiology profile when the Wnt/beta-catenin pathway is activated in the adult stem cells prior fusion. Finally, we demonstrated the engraftment and differentiation of these hybrids into human retinal organoids.We show fusion between human MG and adult stem cells, and demonstrate that the resulting hybrid cells can differentiate towards neural fate in human model systems. Our results suggest that cell fusion-mediated therapy is a potential regenerative approach for treating human retinal dystrophies.This work was supported by La Caixa Health (HR17-00231), Velux Stiftung (976a) and the Ministerio de Ciencia e Innovación, (BFU2017-86760-P) (AEI/FEDER, UE), AGAUR (2017 SGR 689, 2017 SGR 926).Published by Elsevier B.V.

JTD Keywords: cell fusion, expression, fusion, ganglion-cells, in-vitro, mouse, müller glia, neural differentiation, organoids, regeneration, retina regeneration, stem cells, stromal cells, transplantation, 4',6 diamidino 2 phenylindole, 5' nucleotidase, Agarose, Alcohol, Arpe-19 cell line, Article, Beta catenin, Beta tubulin, Bone-marrow-cells, Bromophenol blue, Buffer, Calcium cell level, Calcium phosphate, Calretinin, Canonical wnt signaling, Cd34 antigen, Cell culture, Cell fusion, Cell viability, Coculture, Complementary dna, Confocal microscopy, Cornea transplantation, Cryopreservation, Cryoprotection, Crystal structure, Current clamp technique, Dimethyl sulfoxide, Dodecyl sulfate sodium, Edetic acid, Electrophysiology, Endoglin, Fetal bovine serum, Fibroblast growth factor 2, Flow cytometry, Fluorescence activated cell sorting, Fluorescence intensity, Glyceraldehyde 3 phosphate dehydrogenase, Glycerol, Glycine, Hoe 33342, Immunofluorescence, Immunohistochemistry, Incubation time, Interleukin 1beta, Lentivirus vector, Matrigel, Mercaptoethanol, Microinjection, Mueller cell, Müller glia, N methyl dextro aspartic acid, Nerve cell differentiation, Neural differentiation, Nitrogen, Nonhuman, Organoids, Paraffin, Paraffin embedding, Paraformaldehyde, Patch clamp technique, Penicillin derivative, Phenolsulfonphthalein, Phenotype, Phosphate buffered saline, Phosphoprotein phosphatase inhibitor, Polyacrylamide gel electrophoresis, Potassium chloride, Povidone iodine, Promoter region, Proteinase inhibitor, Real time polymerase chain reaction, Receptor type tyrosine protein phosphatase c, Restriction endonuclease, Retina, Retina dystrophy, Retina regeneration, Retinol, Rhodopsin, Rna extraction, Stem cell, Stem cells, Subcutaneous fat, Tunel assay, Visual impairment, Western blotting

The use of broadly neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) has been shown to be a promising therapeutic modality in the prevention of HIV infection. Understanding the b12-gp120 binding mechanism under physiological conditions may assist the development of more broadly effective antibodies. In this work, the main conformations and interactions between the receptor-binding domain (RBD) of spike glycoprotein gp120 of HIV-1 and the IgG1-b12 mAb are studied. Accelerated molecular dynamics (aMD) and ab initio hybrid molecular dynamics have been combined to determine the most persistent interactions between the most populated conformations of the antibody-antigen complex under physiological conditions. The results show the most persistent receptor-binding mapping in the conformations of the antibody-antigen interface in solution. The binding-free-energy decomposition reveals a small enhancement in the contribution played by the CDR-H3 region to the b12-gp120 interface compared to the crystal structure.

JTD Keywords: antibody, complex, functionals, gp120 envelope glycoprotein, hiv, immunodeficiency-virus, noncovalent interactions, simulations, software integration, Ab initio, Accelerated molecular dynamics, Accelerated molecular-dynamics, Antibodies, Antigens, Binding energy, Binding mechanisms, Computational studies, Crystal structure, Diseases, Free energy, Hiv infection, Human immunodeficiency virus, Molecular dynamics, Neutralizing antibodies, Physiological condition, Physiology, Receptor-binding domains, Therapeutic modality, Viruses

Influenza recombinant proteins and virus-like particles (VLPs) play an important role in vaccine development (e.g., CadiFluS). However, their production from mammalian cells suffers from low yields and lack of control of the final VLPs. To improve these issues, characterization techniques able to visualize and quantify the different steps of the process are needed. Fluorescence microscopy represents a powerful tool able to image multiple protein targets; however, its limited resolution hinders the study of viral constructs. Here, we propose the use of super-resolution microscopy and in particular of DNA-point accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy as a characterization method for recombinant viral proteins on both cells and VLPs. We were able to quantify the amount of the three main influenza proteins (hemagglutinin (HA), neuraminidase (NA), and ion channel matrix protein 2 (M2)) per cell and per VLP with nanometer resolution and single-molecule sensitivity, proving that DNA-PAINT is a powerful technique to characterize recombinant viral constructs.

JTD Keywords: dna-paint, hemagglutinin, influenza, neuraminidase, paint, recombinant proteins, single-molecule localization microscopy, single-particle analysis, virus-like particles, Dna-paint, Hemagglutinin, Influenza, Neuraminidase, Paint, Recombinant proteins, Single particle analysis, Single-molecule localization microscopy, Single-particle analysis, Super-resolution microscopy, Superresolution microscopy, Virus-like particles

Despite tremendous progress in the understanding of COVID-19, mechanistic insight into immunological, disease-driving factors remains limited. We generated maVie16, a mouse-adapted SARS-CoV-2, by serial passaging of a human isolate. In silico modeling revealed how only three Spike mutations of maVie16 enhanced interaction with murine ACE2. maVie16 induced profound pathology in BALB/c and C57BL/6 mice, and the resulting mouse COVID-19 (mCOVID-19) replicated critical aspects of human disease, including early lymphopenia, pulmonary immune cell infiltration, pneumonia, and specific adaptive immunity. Inhibition of the proinflammatory cyto-kines IFN? and TNF substantially reduced immunopathology. Importantly, genetic ACE2-deficiency completely prevented mCOVID-19 development. Finally, inhalation therapy with recombinant ACE2 fully protected mice from mCOVID-19, revealing a novel and efficient treatment. Thus, we here present maVie16 as a new tool to model COVID-19 for the discovery of new therapies and show that disease severity is determined by cytokine-driven immunopathology and critically dependent on ACE2 in vivo. © Gawish et al.

JTD Keywords: covid-19 mouse model, covid-19 therapy, cytokine storm, immunology, inflammation, mavie16, mouse, mouse-adapted sars-cov-2, program, recombinant soluble ace2, tmprss2, Adaptive immunity, Angiotensin converting enzyme 2, Angiotensin-converting enzyme 2, Animal, Animal cell, Animal experiment, Animal model, Animal tissue, Animals, Apoptosis, Article, Bagg albino mouse, Breathing rate, Bronchoalveolar lavage fluid, C57bl mouse, Cell composition, Cell infiltration, Controlled study, Coronavirus disease 2019, Coronavirus spike glycoprotein, Covid-19, Cytokeratin 18, Cytokine production, Dipeptidyl carboxypeptidase, Disease model, Disease models, animal, Disease severity, Drosophila-melanogaster, Enzyme linked immunosorbent assay, Expression vector, Flow cytometry, Gamma interferon, Gene editing, Gene expression, Gene mutation, Genetic engineering, Genetics, Glycosylation, High mobility group b1 protein, Histology, Histopathology, Immune response, Immunocompetent cell, Immunology, Immunopathology, Interferon-gamma, Interleukin 2, Metabolism, Mice, inbred balb c, Mice, inbred c57bl, Mouse-adapted sars-cov-2, Myeloperoxidase, Neuropilin 1, Nonhuman, Nucleocapsid protein, Pathogenicity, Peptidyl-dipeptidase a, Pyroptosis, Recombinant soluble ace2, Renin angiotensin aldosterone system, Rna extraction, Rna isolation, Sars-cov-2, Severe acute respiratory syndrome coronavirus 2, Spike glycoprotein, coronavirus, T lymphocyte activation, Trabecular meshwork, Tumor necrosis factor, Virology, Virus load, Virus replication, Virus transmission, Virus virulence

The epidemic spread of many viral infections is mediated by the environmental conditions and influenced by the ambient humidity. Single virus particles have been mainly visualized by atomic force microscopy (AFM) in liquid conditions, where the effect of the relative humidity on virus topography and surface cannot be systematically assessed. In this work, we employed multi-frequency AFM, simultaneously with standard topography imaging, to study the nanoscale wetting of individual Tobacco Mosaic virions (TMV) from ambient relative humidity to water condensation (RH > 100%). We recorded amplitude and phase vs. distance curves (APD curves) on top of single virions at various RH and converted them into force vs. distance curves. The high sensitivity of multifrequency AFM to visualize condensed water and sub-micrometer droplets, filling gaps between individual TMV particles at RH > 100%, is demonstrated. Dynamic force spectroscopy allows detecting a thin water layer of thickness ⁓1 nm, adsorbed on the outer surface of single TMV particles at RH < 60%.

JTD Keywords: amplitude-modulation am-afm, atomic-force microscopy, capillary, force reconstruction, multifrequency afm, nanoscale wetting, persistence, reconstruction, relative-humidity, surfaces, tobacco mosaic virus (tmv), tobamovirus, transmission, water, Amplitude-modulation am-afm, Force reconstruction, Multifrequency afm, Nanoscale wetting, Tobacco mosaic virus (tmv), Tobacco mosaic virus (tmv), nanoscale wetting, Tobacco-mosaic-virus

Although coronaviruses (CoVs) have long been predicted to cause zoonotic diseases and pandemics with high probability, the lack of effective anti-pan-CoVs drugs rapidly usable against the emerging SARS-CoV-2 actually prevented a promptly therapeutic intervention for COVID-19. Development of host-targeting antivirals could be an alternative strategy for the control of emerging CoVs infections, as they could be quickly repositioned from one pandemic event to another. To contribute to these pandemic preparedness efforts, here we report on the broad-spectrum CoVs antiviral activity of MEDS433, a new inhibitor of the human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of the de novo pyrimidine biosynthesis pathway. MEDS433 in-hibited the in vitro replication of hCoV-OC43 and hCoV-229E, as well as of SARS-CoV-2, at low nanomolar range. Notably, the anti-SARS-CoV-2 activity of MEDS433 against SARS-CoV-2 was also observed in kidney organoids generated from human embryonic stem cells. Then, the antiviral activity of MEDS433 was reversed by the addition of exogenous uridine or the product of hDHODH, the orotate, thus confirming hDHODH as the specific target of MEDS433 in hCoVs-infected cells. Taken together, these findings suggest MEDS433 as a potential candidate to develop novel drugs for COVID-19, as well as broad-spectrum antiviral agents exploitable for future CoVs threats.

JTD Keywords: antiviral activity, biosynthesis, broad-spectrum antiviral, combination treatment, coronavirus, dipyridamole, hdhodh inhibitor, organoids, pyrimidine, pyrimidine biosynthesis, sars-cov-2, target, virus-infection, Antiviral activ-ity, Broad-spectrum antiviral, Combination treatment, Coronavirus, Gene-expression, Hdhodh inhibitor, Organoids, Pyrimidine biosynthesis, Sars-cov-2

In the last year the COVID19 pandemic clearly illustrated the potential threat that viruses pose to our society. The characterization of viral structures and the identification of key proteins involved in each step of the cycle of infection are crucial to develop treatments. However, the small size of viruses, invisible under conventional fluorescence microscopy, make it difficult to study the organization of protein clusters within the viral particle. The applications of super-resolution microscopy have skyrocketed in the last years, converting this group into one of the leading techniques to characterize viruses and study the viral infection in cells, breaking the diffraction limit by achieving resolutions up to 10 nm using conventional probes such as fluorescent dyes and proteins. There are several super-resolution methods available and the selection of the right one it is crucial to study in detail all the steps involved in the viral infection, quantifying and creating models of infection for relevant viruses such as HIV-1, Influenza, herpesvirus or SARS-CoV-1. Here we review the use of super-resolution microscopy (SRM) to study all steps involved in the viral infection and antiviral design. In light of the threat of new viruses, these studies could inspire future assays to unveil the viral mechanism of emerging viruses and further develop successful antivirals against them.

JTD Keywords: antivirals, characterization, imaging, super-resolution, virus, Antivirals, Characterization, Imaging, Super-resolution, Virus

Rapid spread of SARS-CoV-2 virus have boosted the need of knowledge about inactivation mechanisms to minimize the impact of COVID-19 pandemic. Recent studies have shown that SARS-CoV-2 virus can be disabled by heating, the exposure time for total inactivation depending on the reached temperature (e.g. more than 45 min at 329 K or less than 5 min at 373 K. In spite of recent crystallographic structures, little is known about the molecular changes induced by the temperature. Here, we unravel the molecular basis of the effect of the temperature over the SARS-CoV-2 spike glycoprotein, which is a homotrimer with three identical monomers, by executing atomistic molecular dynamics (MD) simulations at 298, 310, 324, 338, 358 and 373 K. Furthermore, both the closed down and open up conformational states, which affect the accessibility of receptor binding domain, have been considered. Our results suggest that the spike homotrimer undergoes drastic changes in the topology of the hydrogen bonding interactions and important changes on the secondary structure of the receptor binding domain (RBD), while electrostatic interactions (i.e. salt bridges) are mainly preserved. The proposed inactivation mechanism has important implications for engineering new approaches to fight the SARS-CoV-2 coronavirus, as for example, cleaving or reorganizing the hydrogen bonds through chaotropic agents or nanoparticles with local surface resonant plasmon effect.

JTD Keywords: atomistic simulations, coronaviruses, denaturation, homotrimeric protein, inactivation, proteins, receptor binding domain, salt bridges, simulation, thermal inactivation, virus spike, Atomistic simulations, Homotrimeric protein, Receptor binding domain, Secondary-structure, Thermal inactivation, Virus spike

Engineered immunoglobulin-G molecules (IgGs) are of wide interest for the development of detection elements in protein-based biosensors with clinical applications. The strategy usually employed for the de novo design of such engineered IgGs consists on merging fragments of the three-dimensional structure of a native IgG, which is immobilized on the biosensor surface, and of an antibody with an exquisite target specificity and affinity. In this work conventional and accelerated classical molecular dynamics (cMD and aMD, respectively) simulations have been used to propose two IgG-like antibodies for COVID-19 detection. More specifically, the crystal structure of the IgG1 B12 antibody, which inactivates the human immunodeficiency virus-1, has been merged with the structure of the antibody CR3022 Fab tightly bounded to SARS-CoV-2 receptor-binding domain (RBD) and the structure of the 5309 antibody Fab fragment complexed with SARS-CoV-2 RBD. The two constructed antibodies, named IgG1-CR3022 and IgG1-S309, respectively, have been immobilized on a stable gold surface through a linker. Analyses of the influence of both the merging strategy and the substrate on the stability of the two constructs indicate that the IgG1-S309 antibody better preserves the neutralizing structure than the IgG1-CR3022 one. Overall, results indicate that the IgG1-S309 is appropriated for the generation of antibody based sensors for COVID-19 diagnosis. (C) 2021 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.

JTD Keywords: cr3022, igg1, molecular engineering, s309, Antibodies, Antibody engineering, Biosensors, Chemical detection, Clinical application, Cov, Cr3022, Crystal structure, Design, Diseases, Gold nanoparticles, Igg1, Igg1 antibody, Immobilization, Immunoglobulin g, Immunosensor, In-silico, Merging, Molecular dynamics, Molecular engineering, Orientation, Protein-based biosensors, Receptor-binding domains, S309, Sars, Sensor, Spike protein, Target, Vaccine, Viruses

We have previously provided the first genetic evidence that angiotensin converting enzyme 2 (ACE2) is the critical receptor for severe acute respiratory syndrome coronavirus (SARS-CoV), and ACE2 protects the lung from injury, providing a molecular explanation for the severe lung failure and death due to SARS-CoV infections. ACE2 has now also been identified as a key receptor for SARS-CoV-2 infections, and it has been proposed that inhibiting this interaction might be used in treating patients with COVID-19. However, it is not known whether human recombinant soluble ACE2 (hrsACE2) blocks growth of SARS-CoV-2. Here, we show that clinical grade hrsACE2 reduced SARS-CoV-2 recovery from Vero cells by a factor of 1,000-5,000. An equivalent mouse rsACE2 had no effect. We also show that SARS-CoV-2 can directly infect engineered human blood vessel organoids and human kidney organoids, which can be inhibited by hrsACE2. These data demonstrate that hrsACE2 can significantly block early stages of SARS-CoV-2 infections.

JTD Keywords: COVID-19, Angiotensin converting enzyme 2, Blood vessels, Human organoids, Kidney, Severe acute respiratory syndrome coronavirus, Spike glycoproteins, Treatment

Oncolytic adenoviruses are used as agents for the treatment of cancer. However, their potential is limited due to the high seroprevalence of anti-adenovirus neutralizing antibodies (nAbs) within the population and the rapid liver sequestration when systemically administered. To overcome these challenges, we explored using nanoparticle formulation to boost the efficacy of systemic oncolytic adenovirus administration. Methods: Adenovirus were conjugated with PEGylated oligopeptide-modified poly(β-amino ester)s (OM-pBAEs). The resulting coated viral formulation was characterized in terms of surface charge, size, aggregation state and morphology and tested for anti-adenovirus nAbs evasion and activity in cancer cells. In vivo pharmacokinetics, biodistribution, tumor targeting, and immunogenicity studies were performed. The antitumor efficacy of the oncolytic adenovirus AdNuPARmE1A coated with OM-pBAEs (SAG101) in the presence of nAbs was evaluated in pancreatic ductal adenocarcinoma (PDAC) mouse models. Toxicity of the coated formulation was analyzed in vivo in immunocompetent mice. Results: OM-pBAEs conjugated to adenovirus and generated discrete nanoparticles with a neutral charge and an optimal size. The polymeric coating with the reporter AdGFPLuc (CPEG) showed enhanced transduction and evasion of antibody neutralization in vitro. Moreover, systemic intravenous administration of the formulation showed improved blood circulation and reduced liver sequestration, substantially avoiding activation of nAb production. OM-pBAEs coating of the oncolytic adenovirus AdNuPARmE1A (SAG101) improved its oncolytic activity in vitro and enhanced antitumor efficacy in PDAC mouse models. The coated formulation protected virions from neutralization by nAbs, as antitumor efficacy was preserved in their presence but was completely lost in mice that received the non-formulated AdNuPARmE1A. Finally, coated-AdNuPARmE1A showed reduced toxicity when high doses of the formulation were administered. Conclusions: The developed technology represents a promising improvement for future clinical cancer therapy using oncolytic adenoviruses.

JTD Keywords: Oncolytic adenovirus, Pancreatic cancer, Poly(β-amino ester)s, Polymer-coated viral vectors, Systemic delivery

In marine ecosystems, the algae community is abundant and plays an important role in the food web. On the other hand, algal viruses have different advantages and disadvantages depending on the properties, such as applications of algal viruses in the advancement of molecular biology and for enhancement of biofuel production. Many environmental factors affecting growth and development of algae and virus such as temperature, salinity, ultraviolet radiation, photosynthetic active radiation, nutrients, inorganic particles, organic particles, CO2 concentration, and pH.

JTD Keywords: Algal viruses, Biofuel production, Nutrients

Here we describe a label-free electrochemical DNA sensor based on poly(3,4-ethylenedioxythiophene)-modified (PEDOT-modified) electrodes. An acetylene-terminated DNA probe, complementary to a specific "Hepatitis C" virus sequence, was immobilized onto azido-derivatized conducting PEDOT electrodes using "click" chemistry. DNA hybridization was then detected by differential pulse voltammetry, evaluating the changes in the electrochemical properties of the polymer produced by the recognition event. A limit of detection of 0.13. nM was achieved using this highly selective PEDOT-based genosensor, without the need for labeling techniques or microelectrode fabrication processes. These results are promising for the development of label-free and reagentless DNA hybridization sensors based on conducting polymeric substrates. Biosensors can be easily prepared using any DNA sequence containing an alkyne moiety. The data presented here reveal the potential of this DNA sensor for diagnostic applications in the screening of diseases, such as "Hepatitis C", and genetic mutations.

JTD Keywords: Azido-EDOT, Click chemistry, Differential pulse voltammetry, DNA biosensor, Electrochemistry, Hepatitis C virus

The electric polarizability of DNA, represented by the dielectric constant, is a key intrinsic property that modulates DNA interaction with effector proteins. Surprisingly, it has so far remained unknown owing to the lack of experimental tools able to access it. Here, we experimentally resolved it by detecting the ultraweak polarization forces of DNA inside single T7 bacteriophages particles using electrostatic force microscopy. In contrast to the common assumption of low-polarizable behavior like proteins (εr ~ 2–4), we found that the DNA dielectric constant is ~ 8, considerably higher than the value of ~ 3 found for capsid proteins. State-of-the-art molecular dynamic simulations confirm the experimental findings, which result in sensibly decreased DNA interaction free energy than normally predicted by Poisson–Boltzmann methods. Our findings reveal a property at the basis of DNA structure and functions that is needed for realistic theoretical descriptions, and illustrate the synergetic power of scanning probe microscopy and theoretical computation techniques.

JTD Keywords: Atomic force microscopy, Atomistic simulations, DNA packaging, DNA-ligand binding, Poisson-Boltzmann equation, capsid protein, DNA, double stranded DNA, amino acid composition, article, atomic force microscopy, bacteriophage, bacteriophage T7, dielectric constant, dipole, DNA binding, DNA packaging, DNA structure, electron microscopy, ligand binding, nonhuman, polarization, priority journal, protein analysis, protein DNA interaction, scanning probe microscopy, static electricity, virion, virus capsid, virus particle, atomic force microscopy, atomistic simulations, DNA packaging, DNA-ligand binding, Poisson-Boltzmann equation, Bacteriophage T7, Capsid, Cations, Dielectric Spectroscopy, DNA, DNA, Viral, DNA-Binding Proteins, Electrochemical Techniques, Ligands, Microscopy, Atomic Force, Models, Chemical, Nuclear Proteins

Five peptide sequences corresponding to the E1 protein of GBV-C [NCCAPEDIGFCLEGGCLV (P7), APEDIGFCLEGGCLVALG (P8), FCLEGGCLVALGCTICTD (P10), QAGLAVRPGKSAAQLVGE (P18), and AQLVGELGSLYGPLSVSA (P22)] were synthesized because they were capable of interfering with the HIV-1 fusion peptide (HIV-1 FP)-vesicle interaction. In this work the interaction of these peptides with the HIV-1 FP, as well as with membrane models, was analyzed to corroborate their inhibition ability and to understand if the interaction with the fusion peptide takes place in solution or at the membrane level. Several studies were carried out on aggregation and membrane fusion, surface Plasmon resonance, and conformational analysis by circular dichroism. Moreover, in vitro toxicity assays, including cytotoxicity studies in 3T3 fibroblasts and hemolysis assays in human red blood cells, were performed to evaluate if these peptides could be potentially used in anti-HIV-1 therapy. Results show that P10 is not capable of inhibiting membrane fusion caused by HIV-1 and it aggregates liposomes and fuses membranes, thus we decided to discard it for futures studies. P18 and P22 do not inhibit membrane fusion, but they inhibit the ability of HIV-1 FP to form pores in bilayers, thus we have not discarded them yet. P7 and P8 were selected as the best candidates for future studies because they are capable of inhibiting membrane fusion and the interaction of HIV-1 FP with bilayers. Therefore, these peptides could be potentially used in future anti-HIV-1 research. Part of the gang: Liposomes are deposited on a surface plasmon resonance chip (see AFM image of the chip) to observe the interaction of peptides corresponding to the E1 envelop protein of the hepatitis G virus with membranes to show how they reduce the interaction of the HIV-1 fusion peptide.

JTD Keywords: HIV-1 fusion protein, Liposomes, Membranes, Peptides, Viruses