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Infection with SARS-CoV-2 sets off a molecular arms race between virus replication and host cell defense. In this issue of Cell, Flynn, Belk, et al. integrate an advanced large-scale RNA-centered approach with custom CRISPR screens to functionally characterize the interactome of the SARS-CoV-2 RNA genome during infection.
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COVID-19 , SARS-CoV-2 , Amigos , Interações Hospedeiro-Patógeno/genética , Humanos , RNA Viral/genéticaRESUMO
The 2021 Annual Joint Meeting (AJM) and Young Investigators' Forum of the Canadian Society for Clinical Investigation / Société Canadienne de Recherches Clinique (CSCI/SCRC) and Clinician Investigator Trainee Association of Canada/Association des Cliniciens-Chercheurs en Formation du Canada (CITAC/ACCFC) was hosted virtually on November 14-16th, 2021. The theme of the AJM was "Communication, Collaboration, and Tools for the Next Generation of Clinician Scientists", and emphasized lectures, panels and interactive workshops designed to provide knowledge and skills for professional development of clinician investigator trainees. The opening remarks were given by Nicola Jones (President of CSCI/SCRC) and Adam Pietrobon (Past President of CITAC/ACCFC). The keynote speaker was Dr. Timothy Caulfield, who delivered the presentation titled "Communication in the Era of Misinformation". Dr. Michael Hill (University of Calgary) received the CSCI Distinguished Scientist Award and Dr. Philippe Campeau (Université de Montréal) received the CSCI Joe Doupe Young Investigator Award. Each of the scientists delivered award winning talks during the symposium titled "All the King's Horses and All the King's Men" and "Understanding Growth Plate Disorders to Better Treat Them", respectively. The three interactive workshops included "Data Visualization", "Science Communication on Social Media" and "Mentorship in Action". The two panels were "CIHR Engagement: Challenges and Opportunities in the Clinician Investigator Career Path" and "Early Career Investigator Panel". The AJM also included presentations from clinician investigator trainees from across the country. Over 60 abstracts were showcased at this year's meeting, most of which are summarized in this review. Six outstanding abstracts were selected for oral presentations during the President's Forum.
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Pesquisa Biomédica , Médicos , Canadá , Humanos , Mentores , PesquisadoresRESUMO
Fungi inhabit extraordinarily diverse ecological niches, including the human body. Invasive fungal infections have a devastating impact on human health worldwide, killing â¼1.5 million individuals annually. The majority of these deaths are attributable to species of Candida, Cryptococcus, and Aspergillus Treating fungal infections is challenging, in part due to the emergence of resistance to our limited arsenal of antifungal agents, necessitating the development of novel therapeutic options. Whereas conventional antifungal strategies target proteins or cellular components essential for fungal growth, an attractive alternative strategy involves targeting proteins that regulate fungal virulence or antifungal drug resistance, such as regulators of fungal stress responses. Stress response networks enable fungi to adapt, grow, and cause disease in humans and include regulators that are highly conserved across eukaryotes as well as those that are fungal-specific. This review highlights recent developments in elucidating crystal structures of fungal stress response regulators and emphasizes how this knowledge can guide the design of fungal-selective inhibitors. We focus on the progress that has been made with highly conserved regulators, including the molecular chaperone Hsp90, the protein phosphatase calcineurin, and the small GTPase Ras1, as well as with divergent stress response regulators, including the cell wall kinase Yck2 and trehalose synthases. Exploring structures of these important fungal stress regulators will accelerate the design of selective antifungals that can be deployed to combat life-threatening fungal diseases.
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Antifúngicos/química , Candida/metabolismo , Proteínas Fúngicas/metabolismo , Antifúngicos/metabolismo , Sítios de Ligação , Calcineurina/química , Calcineurina/metabolismo , Desenho de Fármacos , Proteínas Fúngicas/química , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Simulação de Dinâmica Molecular , Micoses/microbiologia , Micoses/patologiaRESUMO
The 2020 Annual General Meeting (AGM) and Young Investigators' Forum of the Canadian Society for Clinical Investigation / Société Canadienne de Recherches Clinique (CSCI/SCRC) and Clinician Investigator Trainee Association of Canada/Association des Cliniciens-Chercheurs en Formation du Canada (CITAC/ACCFC) was the first meeting to be hosted virtually. The theme was "Navigating Uncertainty, Embracing Change and Empowering the Next Generation of Clinician-Scientists", and the meeting featured lectures and workshops that were designed to provide knowledge and skills for professional development of clinician investigator trainees. The opening remarks were given by Jason Berman (President of CSCI/SCRC), Tina Marvasti (President of CITAC/ACCFC) and Nicola Jones (University of Toronto Clinician Investigator Program Symposium Chair). Dr. Michael Strong, President of the Canadian Institutes of Health Research, delivered the keynote presentation titled "CIHR's COVID-19 Response and Strategic Planning". Dr. John Bell (University of Ottawa) received the CSCI Distinguished Scientist Award, Dr. Stanley Nattel (Université de Montréal) received the CSCI-RCPSC Henry Friesen Award (RCPSC; Royal College of Physicians and Surgeons of Canada) and Dr. Meghan Azad (University of Manitoba) received the CSCI Joe Doupe Young Investigator Award. Each scientist delivered talks on their award-winning research. The interactive workshops were "Developing Strategies to Maintain Wellness", "Understanding the Hidden Curriculum: Power and Privilege in Science and Medicine", "Hiring a Clinician Scientist Trainee: What Leaders Are Looking For" and "COVID-19: A Case Study for Pivoting Your Research". The AGM included presentations from clinician investigator trainees nationwide. Over 70 abstracts were showcased, most are summarized in this review, and six were selected for oral presentations.
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Pesquisa Biomédica , Pesquisadores , Canadá , Congressos como Assunto , HumanosRESUMO
Individuals infected with the SARS-CoV-2 virus present with a wide variety of symptoms ranging from asymptomatic to severe and even lethal outcomes. Past research has revealed a genetic haplotype on chromosome 3 that entered the human population via introgression from Neanderthals as the strongest genetic risk factor for the severe response to COVID-19. However, the specific variants along this introgressed haplotype that contribute to this risk and the biological mechanisms that are involved remain unclear. Here, we assess the variants present on the risk haplotype for their likelihood of driving the genetic predisposition to severe COVID-19 outcomes. We do this by first exploring their impact on the regulation of genes involved in COVID-19 infection using a variety of population genetics and functional genomics tools. We then perform a locus-specific massively parallel reporter assay to individually assess the regulatory potential of each allele on the haplotype in a multipotent immune-related cell line. We ultimately reduce the set of over 600 linked genetic variants to identify four introgressed alleles that are strong functional candidates for driving the association between this locus and severe COVID-19. Using reporter assays in the presence/absence of SARS-CoV-2, we find evidence that these variants respond to viral infection. These variants likely drive the locus' impact on severity by modulating the regulation of two critical chemokine receptor genes: CCR1 and CCR5. These alleles are ideal targets for future functional investigations into the interaction between host genomics and COVID-19 outcomes.
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COVID-19 , Homem de Neandertal , Viroses , Humanos , Animais , COVID-19/genética , Homem de Neandertal/genética , SARS-CoV-2/genética , Genética PopulacionalRESUMO
Exo-enzymatic glycan labeling strategies have emerged as versatile tools for efficient and selective installation of terminal glyco-motifs onto live cell surfaces. Through employing specific enzymes and nucleotide-sugar probes, cells can be equipped with defined glyco-epitopes for modulating cell function or selective visualization and enrichment of glycoconjugates. Here, we identifyCampylobacter jejunisialyltransferase Cst-II I53S as a tool for cell surface glycan modification, expanding the exo-enzymatic labeling toolkit to include installation of α2,8-disialyl epitopes. Labeling with Cst-II was achieved with biotin- and azide-tagged CMP-Neu5Ac derivatives on a model glycoprotein and native sialylated cell surface glycans across a panel of cell lines. The introduction of modified Neu5Ac derivatives onto cells by Cst-II was also retained on the surface for 6 h. By examining the specificity of Cst-II on cell surfaces, it was revealed that the α2,8-sialyltransferase primarily labeled N-glycans, with O-glycans labeled to a lesser extent, and there was an apparent preference for α2,3-linked sialosides on cells. This approach thus broadens the scope of tools for selective exo-enzymatic labeling of native sialylated glycans and is highly amenable for the construction of cell-based arrays.
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Polissacarídeos , Sialiltransferases , Sialiltransferases/metabolismo , Membrana Celular/metabolismo , Polissacarídeos/metabolismo , Glicoconjugados , EpitoposRESUMO
Background: Aframomum sp. is a genus of plants in the Zingiberaceae family. It includes several species, some of which are used in cosmetics for their various properties, making them useful in skincare products, particularly for anti-aging, moisturizing, and brightening the skin. However, to date, there is no experimental evidence on its natural extracts obtained or modified using microorganisms (bio-fermentation) as an anti-aging agent. Objective: The present study aimed to evaluate the antiaging effect of a Bio-fermented Aframomum angustifolium (BAA) extract on 3D bioprinted skin equivalent. Methods: The consortium of microorganisms contained Komagataeibacter, Gluconobacter, Acetobacter, Saccharomyces, Torulaspora, Brettanomyces, Hanseniaspora, Leuconostoc, Lactobacillus, Schizosaccharomyces. It was developed on a media containing water, sugar, and infused black tea leaves. The seeds of Aframomum angustifolium previously grounded were mixed with the culture medium, and the ferments in growth; this fermentation step lasted 10 days. Then, the medium was collected and filtered (0.22 µm) to obtain the BAA extract. To enhance our comprehension of the impact of BAA extract on skin aging, we developed skin equivalents using bio-printing methods with the presence or absence of keratinocyte stem cells (KSC). These skin equivalents were derived from keratinocytes obtained from both a middle-aged donor, with and without KSC. Moreover, we examined the effects of treating the KSC-depleted skin equivalents with Bio-fermented Aframomum angustifolium (BAA) extract for 5 days. Skin equivalents containing KSC-depleted keratinocytes exhibited histological characteristics typical of aged skin and were compared to skin equivalents derived from young donors. Results: The BAA extract contained specific organic acids such as lactic, gluconic, succinic acid and polyphenols. KSC-depleted skin equivalents that were treated with BAA extract exhibited higher specular reflection, indicating better hydration of the stratum corneum, higher mitotic activity in the epidermis basal layer, improved dermal-epidermal connectivity, and increased rigidity of the dermal-epidermal junction compared to non-treated KSC-depleted equivalents. BAA extract treatments also resulted in changes at the dermis level, with an increase in total collagen and a decrease in global laxity, suggesting that this extract could help maintain youthful-looking skin. Conclusion: In summary, our findings indicated that BAA extract treatments have pleiotropic beneficial effects on skin equivalents and that the bio-fermentation provides new biological activities to this plant.
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The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has emphasized the serious threat to human health posed by emerging coronaviruses. Effective broadly-acting antiviral countermeasures are urgently needed to prepare for future emerging CoVs, as vaccine development is not compatible with a rapid response to a newly emerging virus. The green tea catechin, epigallocatechin gallate (EGCG), has broad-spectrum antiviral activity, although its mechanisms against coronavirus (CoV) infection have remained unclear. Here, we show that EGCG prevents human and murine CoV infection and blocks the entry of lentiviral particles pseudotyped with spike proteins from bat or highly pathogenic CoVs, including SARS-CoV-2 variants of concern, in lung epithelial cells. Mechanistically, EGCG treatment reduces CoV attachment to target cell surfaces by interfering with attachment to cell-surface glycans. Heparan sulfate proteoglycans are a required attachment factor for SARS-CoV-2 and are shown here to be important in endemic HCoV-OC43 infection. We show that EGCG can compete with heparin, a heparan sulfate analog, for virion binding. Our results highlight heparan sulfate as a conserved cell attachment factor for CoVs, and demonstrate the potential for the development of pan-coronavirus attachment inhibitors, which may be useful to protect against future emerging CoVs.
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Tratamento Farmacológico da COVID-19 , Catequina , Animais , Antivirais/farmacologia , Catequina/análogos & derivados , Catequina/farmacologia , Heparitina Sulfato , Humanos , Camundongos , Pandemias , SARS-CoV-2 , CháRESUMO
Recognition of viral infection by pattern recognition receptors is paramount for a successful immune response to viral infection. However, an unbalanced proinflammatory response can be detrimental to the host. Recently, multiple studies have identified that the SARS-CoV-2 spike protein activates Toll-like receptor 4 (TLR4), resulting in the induction of proinflammatory cytokine expression. Activation of TLR4 by viral glycoproteins has also been observed in the context of other viral infection models, including respiratory syncytial virus (RSV), dengue virus (DENV) and Ebola virus (EBOV). However, the mechanisms involved in virus-TLR4 interactions have remained unclear. Here, we review viral glycoproteins that act as pathogen-associated molecular patterns to induce an immune response via TLR4. We explore the current understanding of the mechanisms underlying how viral glycoproteins are recognized by TLR4 and discuss the contribution of TLR4 activation to viral pathogenesis. We identify contentious findings and research gaps that highlight the importance of understanding viral glycoprotein-mediated TLR4 activation for potential therapeutic approaches.
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In vitro evolution and whole genome analysis were used to comprehensively identify the genetic determinants of chemical resistance in Saccharomyces cerevisiae. Sequence analysis identified many genes contributing to the resistance phenotype as well as numerous amino acids in potential targets that may play a role in compound binding. Our work shows that compound-target pairs can be conserved across multiple species. The set of 25 most frequently mutated genes was enriched for transcription factors, and for almost 25 percent of the compounds, resistance was mediated by one of 100 independently derived, gain-of-function SNVs found in a 170 amino acid domain in the two Zn2C6 transcription factors YRR1 and YRM1 (p < 1 × 10-100). This remarkable enrichment for transcription factors as drug resistance genes highlights their important role in the evolution of antifungal xenobiotic resistance and underscores the challenge to develop antifungal treatments that maintain potency.
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Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Xenobióticos/metabolismo , Xenobióticos/farmacologiaRESUMO
Accurate population surveillance of SARS-CoV-2 infection has been hampered by limited testing and inadequate serological assays. In a recent issue of Med, Hippich et al.1 describe a two-step antibody test with 100% specificity, revealing higher-than-reported SARS-CoV-2 exposure rates in children.
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Anticorpos Antivirais/sangue , COVID-19/diagnóstico , SARS-CoV-2/imunologia , Doenças Assintomáticas , COVID-19/epidemiologia , COVID-19/virologia , Teste Sorológico para COVID-19 , Criança , Diabetes Mellitus Tipo 1/patologia , Feminino , Humanos , Masculino , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificaçãoRESUMO
Chronic hepatitis C virus (HCV) infections continue to be a major contributor to liver disease worldwide. HCV treatment has become highly effective, yet there are still no vaccines or prophylactic strategies available to prevent infection and allow effective management of the global HCV burden. Glycan-dependent interactions are crucial to many aspects of the highly complex HCV entry process, and also modulate immune evasion. This review provides an overview of the roles of viral and cellular glycans in HCV infection and highlights glycan-focused advances in the development of entry inhibitors and vaccines to effectively prevent HCV infection.
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Fungi infect over a billion people worldwide and contribute substantially to human morbidity and mortality despite all available therapies. New antifungal drugs are urgently needed. Decades of study have revealed numerous protein targets of potential therapeutic interest for which potent, fungal-selective ligands remain to be discovered and developed. To measure the binding of diverse small molecule ligands to their larger protein targets, fluorescence polarization (FP) can provide a robust, inexpensive approach. The protocols in this article provide detailed guidance for developing FP-based assays capable of measuring binding affinity in whole cell lysates without the need for purification of the target protein. Applications include screening of libraries to identify novel ligands and the definition of structure-activity relationships to aid development of compounds with improved target affinity and fungal selectivity. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Use of saturation binding curves to optimize tracer and lysate protein concentrations Basic Protocol 2: Establishment of competition binding experiments Support Protocol 1: Preparation of fungal cell lysates Support Protocol 2: Preparation of human HepG2 cell lysate.
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Fungos , Proteínas , Ligação Competitiva , Polarização de Fluorescência , Humanos , LigantesRESUMO
The essential eukaryotic chaperone Hsp90 regulates the form and function of diverse client proteins, many of which govern thermotolerance, virulence, and drug resistance in fungal species. However, use of Hsp90 inhibitors as antifungal therapeutics has been precluded by human host toxicities and suppression of immune responses. We recently described resorcylate aminopyrazoles (RAPs) as the first class of Hsp90 inhibitors capable of discriminating between fungal (Cryptococcus neoformans, Candida albicans) and human isoforms of Hsp90 in biochemical assays. Here, we report an iterative structure-property optimization toward RAPs capable of inhibiting C. neoformans growth in culture. In addition, we report the first X-ray crystal structures of C. neoformans Hsp90 nucleotide binding domain (NBD), as the apoprotein and in complexes with the non-species-selective Hsp90 inhibitor NVP-AUY922 and three RAPs revealing unique ligand-induced conformational rearrangements, which reaffirm the hypothesis that intrinsic differences in protein flexibility can confer selective inhibition of fungal versus human Hsp90 isoforms.
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Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Fungos/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Pirazóis/farmacologia , Animais , Antifúngicos/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Microssomos Hepáticos/metabolismo , Ligação Proteica , Pirazóis/química , Especificidade da Espécie , Relação Estrutura-AtividadeRESUMO
The molecular chaperone Hsp90, essential in all eukaryotes, plays a multifaceted role in promoting survival, virulence, and drug resistance across diverse pathogenic fungal species. The chaperone is also critically important, however, to the pathogen's human host, preventing the use of known clinical Hsp90 inhibitors in antifungal applications due to concomitant host toxicity issues. With the goal of developing Hsp90 inhibitors with acceptable therapeutic indices for the treatment of invasive fungal infections, we initiated a program to design and synthesize potent inhibitors with selective activity against fungal Hsp90 isoforms over their human counterparts. Building on our previously reported derivatization of resorcylate natural products to produce fungal-selective compounds, we have developed a series of synthetic aminopyrazole-substituted resorcylate amides with broad, potent, and fungal-selective Hsp90 inhibitory activity. Herein we describe the synthesis of this series, as well as biochemical structure-activity relationships driving selectivity for the Hsp90 isoforms expressed by Cryptococcus neoformans and Candida albicans, two pathogenic fungi of major clinical importance.
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Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Proteínas Fúngicas/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Pirazóis/farmacologia , Aminação , Animais , Antifúngicos/síntese química , Antifúngicos/química , Candida albicans/metabolismo , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Cryptococcus neoformans/metabolismo , Desenho de Fármacos , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos , Pirazóis/síntese química , Pirazóis/química , Relação Estrutura-AtividadeRESUMO
New strategies are needed to counter the escalating threat posed by drug-resistant fungi. The molecular chaperone Hsp90 affords a promising target because it supports survival, virulence and drug-resistance across diverse pathogens. Inhibitors of human Hsp90 under development as anticancer therapeutics, however, exert host toxicities that preclude their use as antifungals. Seeking a route to species-selectivity, we investigate the nucleotide-binding domain (NBD) of Hsp90 from the most common human fungal pathogen, Candida albicans. Here we report structures for this NBD alone, in complex with ADP or in complex with known Hsp90 inhibitors. Encouraged by the conformational flexibility revealed by these structures, we synthesize an inhibitor with >25-fold binding-selectivity for fungal Hsp90 NBD. Comparing co-crystals occupied by this probe vs. anticancer Hsp90 inhibitors revealed major, previously unreported conformational rearrangements. These insights and our probe's species-selectivity in culture support the feasibility of targeting Hsp90 as a promising antifungal strategy.