RESUMO
Although efficacious vaccines have significantly reduced the morbidity and mortality of COVID-19, there remains an unmet medical need for treatment options, which monoclonal antibodies (mAbs) can potentially fill. This unmet need is exacerbated by the emergence and spread of SARS-CoV-2 variants of concern (VOCs) that have shown some resistance to vaccine responses. Here we report the isolation of five neutralizing mAbs from an Indian convalescent donor, out of which two (THSC20.HVTR04 and THSC20.HVTR26) showed potent neutralization of SARS-CoV-2 VOCs at picomolar concentrations, including the Delta variant (B.1.617.2). One of these (THSC20.HVTR26) also retained activity against the Omicron variant. These two mAbs target non-overlapping epitopes on the receptor-binding domain (RBD) of the spike protein and prevent virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Furthermore, the mAb cocktail demonstrated protection against the Delta variant at low antibody doses when passively administered in the K18 hACE2 transgenic mice model, highlighting their potential as a cocktail for prophylactic and therapeutic applications. Developing the capacity to rapidly discover and develop mAbs effective against highly transmissible pathogens like coronaviruses at a local level, especially in a low- and middle-income country (LMIC) such as India, will enable prompt responses to future pandemics as an important component of global pandemic preparedness.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Camundongos , Glicoproteína da Espícula de CoronavírusRESUMO
The emergence of new variants of SARS-CoV-2 necessitates unremitting efforts to discover novel therapeutic monoclonal antibodies (mAbs). Here, we report an extremely potent mAb named P4A2 that can neutralize all the circulating variants of concern (VOCs) with high efficiency, including the highly transmissible Omicron. The crystal structure of the P4A2 Fab:RBD complex revealed that the residues of the RBD that interact with P4A2 are a part of the ACE2-receptor-binding motif and are not mutated in any of the VOCs. The pan coronavirus pseudotyped neutralization assay confirmed that the P4A2 mAb is specific for SARS-CoV-2 and its VOCs. Passive administration of P4A2 to K18-hACE2 transgenic mice conferred protection, both prophylactically and therapeutically, against challenge with VOCs. Overall, our data shows that, the P4A2 mAb has immense therapeutic potential to neutralize the current circulating VOCs. Due to the overlap between the P4A2 epitope and ACE2 binding site on spike-RBD, P4A2 may also be highly effective against a number of future variants.
Assuntos
Enzima de Conversão de Angiotensina 2 , Anticorpos Neutralizantes , COVID-19 , SARS-CoV-2 , Animais , Humanos , Camundongos , Enzima de Conversão de Angiotensina 2/química , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , COVID-19/imunologia , COVID-19/terapia , Camundongos Transgênicos , Testes de Neutralização , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Dengue viruses (DENV) are positive-stranded RNA viruses belonging to the Flaviviridae family. DENV is the causative agent of dengue, the most rapidly spreading viral disease transmitted by mosquitoes. Each year, millions of people contract the virus through bites from infected female mosquitoes of the Aedes species. In the majority of individuals, the infection is asymptomatic, and the immune system successfully manages to control virus replication within a few days. Symptomatic individuals may present with a mild fever (Dengue fever or DF) that may or may not progress to a more critical disease termed Dengue hemorrhagic fever (DHF) or the fatal Dengue shock syndrome (DSS). In the absence of a universally accepted prophylactic vaccine or therapeutic drug, treatment is mostly restricted to supportive measures. Similar to many other viruses that induce acute illness, DENV has developed several ways to modulate host metabolism to create an environment conducive to genome replication and the dissemination of viral progeny. To search for new therapeutic options, understanding the underlying host-virus regulatory system involved in various biological processes of the viral life cycle is essential. This review aims to summarize the complex interaction between DENV and the host cellular machinery, comprising regulatory mechanisms at various molecular levels such as epigenetic modulation of the host genome, transcription of host genes, translation of viral and host mRNAs, post-transcriptional regulation of the host transcriptome, post-translational regulation of viral proteins, and pathways involved in protein degradation.
Assuntos
Vírus da Dengue , Dengue , Vírus da Dengue/fisiologia , Vírus da Dengue/patogenicidade , Vírus da Dengue/genética , Humanos , Dengue/virologia , Animais , Interações Hospedeiro-Patógeno , Replicação ViralRESUMO
OBJECTIVE AND DESIGN: The exact immunological mechanism of widespread chronic inflammatory skin disorder psoriasis has not been fully established. CD11b+Gr.1+ myeloid-derived cells are immature heterogeneous cells with T-cell suppressive property in neoplasia; however, influence of these cells on adaptive immunity is highly contextual; therefore, we dubbed these cells as myeloid-derived adjuster cells (MDAC). We studied imiquimod induced psoriasis in mouse model and evaluated for the first time the RORγt-NFAT1 axis in MDACs and the function, differentiation and interaction of these cells with T cells. MATERIALS AND METHODS: The status of T cells and MDACs; their functionality and differentiation properties, and the roles of RORγt and NFAT1 in MDACs were evaluated using flow cytometry, qRT-PCR and confocal imaging. RESULTS: We found gradual increase in T cells and MDACs and an increase in the number of IL17 -secreting MDACs and T cells in the skin of psoriatic animals. We also noted that MDAC differentiation is biased toward M1 macrophages and DCs which perpetuate inflammation. We found that psoriatic MDACs were unable to suppress T-cell proliferation or activation but seemingly helped these T cells produce more IL17. Inhibition of the RORγt/NFAT1 axis in MDACs increased the suppressive nature of MDACs, allowing these cells to suppress the activity of psoriatic T-cells. CONCLUSION: Our results indicate that altered MDAC properties in psoriatic condition sustains pathological inflammation and RORγt and NFAT1 as promising intervention target for psoriasis management.
RESUMO
Cross-reactive epitopes (CREs) are similar epitopes on viruses that are recognized or neutralized by same antibodies. The S protein of SARS-CoV-2, similar to type I fusion proteins of viruses such as HIV-1 envelope (Env) and influenza hemagglutinin, is heavily glycosylated. Viral Env glycans, though host derived, are distinctly processed and thereby recognized or accommodated during antibody responses. In recent years, highly potent and/or broadly neutralizing human monoclonal antibodies (bnAbs) that are generated in chronic HIV-1 infections have been defined. These bnAbs exhibit atypical features such as extensive somatic hypermutations, long complementary determining region (CDR) lengths, tyrosine sulfation and presence of insertions/deletions, enabling them to effectively neutralize diverse HIV-1 viruses despite extensive variations within the core epitopes they recognize. As some of the HIV-1 bnAbs have evolved to recognize the dense viral glycans and cross-reactive epitopes (CREs), we assessed if these bnAbs cross-react with SARS-CoV-2. Several HIV-1 bnAbs showed cross-reactivity with SARS-CoV-2 while one HIV-1 CD4 binding site bnAb, N6, neutralized SARS-CoV-2. Furthermore, neutralizing plasma antibodies of chronically HIV-1 infected children showed cross neutralizing activity against SARS-CoV-2 pseudoviruses. Collectively, our observations suggest that human monoclonal antibodies tolerating extensive epitope variability can be leveraged to neutralize pathogens with related antigenic profile.
Assuntos
Anticorpos Amplamente Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , SARS-CoV-2/imunologia , Anticorpos Monoclonais/imunologia , COVID-19/imunologia , Reações Cruzadas/imunologia , Humanos , Plasma/imunologiaRESUMO
We showed solvent- and concentration-triggered chiral tuning of the fibrous assemblies of two novel glycoconjugates Z-P(Gly)-Glu and Z-F(4-N)-Glu made by chemical attachment of Cbz-protected [short as Z)] non-proteinogenic amino acids L-phenylglycine [short as P(Gly)] and 4-Nitro-L-phenylalanine [short as F(4-N)] with D-glucosamine [short as Glu]. Both biomimetic gelators can form self-healing and shape-persistent gels with a very low critical gelator concentration in water as well as in various organic solvents, indicating they are ambidextrous supergelators. Detailed spectroscopic studies suggested ß-sheet secondary structure formation during anisotropic self-aggregation of the gelators which resulted in the formation of hierarchical left-handed helical fibers in acetone with an interlayer spacing of 2.4â nm. After the physical characterization of the gels, serum protein interaction with the gelators was assessed, indicating they may be ideal for biomedical applications. Further, both gelators are benign, non-immunogenic, non-allergenic, and non-toxic in nature, which was confirmed by performing the blood parameters and liver function tests on Wister rats. Streptomycin-loaded hydrogels showed efficacious antibacterial activity inâ vitro and inâ vivo as well. Finally, cell attachment and biocompatibility of the hydrogels were demonstrated which opens a newer avenue for promising biomedical and therapeutic applications.
Assuntos
Aminoácidos , Estreptomicina , Ratos , Animais , Aminoácidos/química , Solventes/química , Ratos Wistar , Hidrogéis/químicaRESUMO
DENV infection poses a major health concern globally and the pathophysiology relies heavily on host-cellular machinery. Although virus replication relies heavily on the host, the mechanistic details of DENV-host interaction is not fully characterized yet. Here, we are focusing on characterizing the mechanistic basis of virus-induced stress on the host cell. Specifically, we aim to characterize the role of the stress modulator ribonuclease Angiogenin during DENV infection. Our results suggested that the levels of Angiogenin are up-regulated in DENV-infected cells and the levels increase proportionately with DENV replication. Our efforts to knockdown Angiogenin using siRNA were unsuccessful in DENV-infected cells but not in mock-infected control. To further investigate the modulation between DENV replication and Angiogenin, we treated Huh7 cells with Ivermectin prior to DENV infection. Our results suggest a significant reduction in DENV replication specifically at the later stages as a consequence of Ivermectin treatment. Interestingly, Angiogenin levels were also found to be decreased proportionately. Our results suggest that Angiogenin modulation during DENV infection is important for DENV replication and pathogenesis.
Assuntos
Dengue , Ivermectina , Humanos , Ivermectina/farmacologia , Ribonuclease Pancreático/genética , Replicação ViralRESUMO
A series of thiazole linked Oxindole-5-Sulfonamide (OSA) derivatives were designed as inhibitors of RNA-dependent RNA polymerase (RdRp) activity of Dengue virus. These were synthesized and then evaluated for their efficacy in ex-vivo virus replication assay using human cell lines. Among 20 primary compounds in the series, OSA-15 was identified as a hit. A series of analogues were synthesized by replacing the difluoro benzyl group of OSA-15 with different substituted benzyl groups. The efficacy of OSA-15derivatives was less than that of the parent compound, except OSA-15-17, which has shown improved efficacy than OSA-15. The further optimization was carried out by adding dimethyl (DM) groups to both the sulfonamide and oxindole NH's to produce OSA-15-DM and OSA-15-17-DM. These two compounds were showing no detectable cytotoxicity and the latter was more efficacious. Further, both these compounds were tested for inhibition in all the serotypes of the Dengue virus using an ex-vivo assay. The EC50 of OSA-15-17-DM was observed in a low micromolar range between 2.5 and 5.0 µg/ml. Computation docking and molecular dynamics simulation studies confirmed the binding of identified hits to DENV RdRp. OSA15-17-DM blocks the RNA entrance and elongation site for their biological activity with high binding affinity. Overall, the identified oxindole derivatives are novel compounds that can inhibit Dengue replication, working as non-nucleoside inhibitors (NNI) to explore as anti-viral RdRp activity.
Assuntos
Antivirais , Dengue , Oxindóis , Antivirais/química , Dengue/tratamento farmacológico , Vírus da Dengue , Simulação de Acoplamento Molecular , Oxindóis/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Sulfonamidas/farmacologiaRESUMO
Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these nucleic-acid-based modalities, especially in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the receptor-binding domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan-engineered RBD protein fragment that is expressed at a purified yield of 214 mg/l in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100 °C when lyophilized, up to 70 °C in solution and stable for over 4 weeks at 37 °C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean titer of â¼415 against replicative virus, comparing favorably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance, and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.
Assuntos
Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Antivirais/biossíntese , Vacinas contra COVID-19/biossíntese , COVID-19/prevenção & controle , Receptores Virais/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/genética , Animais , Anticorpos Neutralizantes/biossíntese , Sítios de Ligação , COVID-19/imunologia , COVID-19/virologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Cobaias , Células HEK293 , Temperatura Alta , Humanos , Imunogenicidade da Vacina , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Receptores Virais/química , Receptores Virais/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Vacinação , Potência de VacinaRESUMO
Abnormal coagulation dynamics, including disseminated intravascular coagulopathy, pulmonary embolism, venous thromboembolism and risk of thrombosis are often associated with the severity of COVID-19. However, very little is known about the contribution of platelets in above pathogenesis. In order to decipher the pathophysiology of thrombophilia in COVID-19, we recruited severely ill patients from ICU, based on the above symptoms and higher D-dimer levels, and compared these parameters with their asymptomatic counterparts. Elevated levels of platelet-derived microparticles and platelet-leukocyte aggregates suggested the hyperactivation of platelets in ICU patients. Strikingly, platelet transcriptome analysis showed a greater association of IL-6 and TNF signalling pathways in ICU patients along with higher plasma levels of IL-6 and TNFα. In addition, upregulation of pathways like blood coagulation and hemostasis, as well as inflammation coexisted in platelets of these patients. Further, the increment of necrotic pathway and ROS-metabolic processes in platelets was suggestive of its procoagulant phenotype in ICU patients. This study suggests that higher plasma IL-6 and TNFα may trigger platelet activation and coagulation, and in turn aggravate thrombosis and hypercoagulation in severe COVID-19 patients. Therefore, the elevated IL-6 and TNFα, may serve as potential risk factors for platelet activation and thrombophilia in these patients.
Assuntos
COVID-19 , Micropartículas Derivadas de Células , Trombofilia , Plaquetas/metabolismo , COVID-19/complicações , Micropartículas Derivadas de Células/metabolismo , Citocinas/metabolismo , Humanos , SARS-CoV-2 , Trombofilia/complicações , Regulação para CimaRESUMO
This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the authors. This study provided an explanation for why loss of FoxP3 in inducible regulatory T cells results in reduced expression of interleukin (IL)-10 despite the absence of FoxP3 binding sites in the IL-10 promoter. STAT3 binding sites do exist in the promoter, and evidence for a direct molecular interaction between FoxP3 and STAT3 proteins was provided as an explanation of the effect of loss of FoxP3. As supporting evidence, we reported modeling of a structural interaction between these two transcription factors in Figure 4D. As the N-terminal region of FoxP3, which consists of the Exon-2 region, had not been solved at structural resolution, we mistakenly used what we deduced to be a FoxP3 related transcription factor, NFAT, in the modeling. The model depicted in Figure 4D therefore did not represent a putative interaction between FoxP3 and STAT3 as labeled, but rather a putative interaction between NFAT and STAT3. Given the incorrect labeling of Figure 4D, the lack of documentation in the paper describing exactly how the modeling was performed, the lack of evidence shown in the paper for the choice of NFAT as the modeling partner, and the limited supporting evidence for a cooperative interaction between FoxP3 and STAT3, the editors have concluded with the corresponding author that the appropriate course of action is to retract the paper. We apologize for any confusion and inconvenience caused to readers.
Assuntos
Neoplasias da Mama/fisiopatologia , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição STAT3/metabolismo , Linfócitos T Reguladores/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Modelos Moleculares , Fatores de TranscriçãoRESUMO
Apart from the canonical fingers, palm and thumb domains, the RNA dependent RNA polymerases (RdRp) from the viral order Nidovirales possess two additional domains. Of these, the function of the Nidovirus RdRp associated nucleotidyl transferase domain (NiRAN) remains unanswered. The elucidation of the 3D structure of RdRp from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), provided the first ever insights into the domain organisation and possible functional characteristics of the NiRAN domain. Using in silico tools, we predict that the NiRAN domain assumes a kinase or phosphotransferase like fold and binds nucleoside triphosphates at its proposed active site. Additionally, using molecular docking we have predicted the binding of three widely used kinase inhibitors and five well characterized anti-microbial compounds at the NiRAN domain active site along with their drug-likeliness. For the first time ever, using basic biochemical tools, this study shows the presence of a kinase like activity exhibited by the SARS-CoV-2 RdRp. Interestingly, a well-known kinase inhibitor- Sorafenib showed a significant inhibition and dampened viral load in SARS-CoV-2 infected cells. In line with the current global COVID-19 pandemic urgency and the emergence of newer strains with significantly higher infectivity, this study provides a new anti-SARS-CoV-2 drug target and potential lead compounds for drug repurposing against SARS-CoV-2.
Assuntos
Antivirais/farmacologia , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , Domínios Proteicos , SARS-CoV-2/efeitos dos fármacos , Domínio Catalítico , Simulação por Computador , RNA-Polimerase RNA-Dependente de Coronavírus/química , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , HumanosRESUMO
Peroxidases are a heterogeneous family of enzymes that have diverse biological functions. Ascorbate peroxidase is a redox enzyme that is reduced by trypanothione, which plays a central role in the redox defense system of Leishmania In view of developing new and novel therapeutics, we performed in silico studies in order to search for a ligand library and identify new drug candidates and their physiological roles against promastigotes and intracellular amastigotes of Leishmania donovani Our results demonstrated that the selected inhibitor ZINC96021026 has significant antileishmanial effect and effectively killed both free and intracellular forms of the parasite. ZINC96021026 was found to be identical to ML-240, a selective inhibitor of valosin-containing protein (VCP), or p97, a member of the AAA-ATPase protein family which was derived from the scaffold of N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ), targeting the D2-ATPase domain of the enzyme. ZINC96021026 (ML-240) thus has a broad range of cellular functions, thought to be derived from its ability to unfold proteins or disassemble protein complexes, besides inhibiting the ascorbate peroxidase activity. ML-240 may inhibit the parasite's ascorbate peroxidase, leading to extensive apoptosis and inducing generation of reactive oxygen species. Taken together, our results demonstrated that ML-240 could be an attractive therapeutic option for treatment against leishmaniasis.
Assuntos
Antiprotozoários , Ascorbato Peroxidases/antagonistas & inibidores , Leishmania donovani , Antiprotozoários/farmacologia , Simulação por Computador , Leishmania donovani/efeitos dos fármacosRESUMO
Diphenyleneiodonium (DPI) and N-acetyl-l-cysteine (NAC), two widely used anti-oxidants, were employed to evaluate the role of oxidative stress in Japanese encephalitis virus (JEV) induced autophagy, stress responses and replication. DPI and NAC exerted opposite effects on ROS levels in JEV infected mouse neuronal cells (Neuro2a), mouse embryonic fibroblasts (MEFs) and human epithelial cells (HeLa). While NAC effectively quenched ROS, DPI enhanced ROS levels, suggesting that DPI induces oxidative stress in JEV infected cells. DPI treatment of JEV infected Neuro2a cells further blocked autophagy induction and activation of all three arms of the ER stress pathway, and, inhibited virus particle release. Autophagy induction in JEV infection has been previously shown to be linked to the activation of XBP1 and ATF6 ER stress sensors. Our data suggests that DPI mediated block of autophagy is a result of inhibition of ER stress responses and is not associated with an anti-oxidative effect. Since DPI has a wide inhibitory potential for all Flavin dependent enzymes, it is likely that the signalling pathways for ER stress and autophagy during JEV infection are modulated by DPI sensitive enzymes.
Assuntos
Vírus da Encefalite Japonesa (Espécie)/efeitos dos fármacos , Oniocompostos/farmacologia , Acetilcisteína/farmacologia , Animais , Antioxidantes/farmacologia , Autofagia/efeitos dos fármacos , Células Cultivadas , Vírus da Encefalite Japonesa (Espécie)/patogenicidade , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células HeLa , Humanos , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Suínos , Replicação Viral/efeitos dos fármacosRESUMO
Endoplasmic reticulum (ER) stress and autophagy are key cellular responses to RNA virus infection. Recent studies have shown that Japanese encephalitis virus (JEV)-induced autophagy negatively influences virus replication in mouse neuronal cells and embryonic fibroblasts, and delays virus-induced cell death. Here, we evaluated the role of ER stress pathways in inducing autophagy during JEV infection. We observed that JEV infection of neuronal cells led to activation of all three sensors of ER stress mediated by eIF2α/PERK, IRE1/XBP1 and ATF6. The kinetics of autophagy induction as monitored by levels of SQSTM1 and LC3-II paralleled activation of ER stress. Inhibition of the eIF2α/PERK pathway by siRNA-mediated depletion of proteins and by the PERK inhibitor had no effect on autophagy and JEV replication. However, depletion of XBP1 and ATF6, alone or in combination, prevented autophagy induction and significantly enhanced JEV-induced cell death. JEV-infected cells depleted of XBP1 or ATF6 showed reduced transcription of ER chaperones, ERAD components and autophagy genes, resulting in reduced protein levels of the crucial autophagy effectors ATG3 and BECLIN-1. Conversely, pharmacological induction of ER stress in JEV-infected cells further enhanced autophagy and reduced virus titres. Our study thus demonstrates that a crucial link exists between the ER stress pathways and autophagy in virus-infected cells, and that these processes are highly regulated during virus infection.
Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Autofagia , Vírus da Encefalite Japonesa (Espécie)/imunologia , Interações Hospedeiro-Patógeno , Neurônios/fisiologia , Neurônios/virologia , Proteína 1 de Ligação a X-Box/metabolismo , Animais , Linhagem Celular , Camundongos , Replicação ViralRESUMO
Japanese encephalitis virus (JEV) infection-induced encephalitis causes extensive death or long-term neurological damage, especially among children, in south and south-east Asia. Infection of mammalian cells has shown induction of an unfolded protein response (UPR), presumably leading to programmed cell death or apoptosis of the host cells. UPR, a cellular response to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen, is initiated by three ER-lumen-resident sensors (PERK, IRE1 and ATF6), and involves transcriptional and translational regulation of the expression of several genes. The sensor IRE1 possesses an intrinsic RNase activity, activated through homo-dimerization and autophosphorylation during UPR. Activated IRE1 performs cytoplasmic cleavage of Xbp1u transcripts, thus facilitating synthesis of XBP1S transcription factor, in addition to cleavage of a cohort of cellular transcripts, the later initiating the regulated IRE1-dependent decay (RIDD) pathway. In this study, we report the initiation of the RIDD pathway in JEV-infected mouse neuroblastoma cells (Neuro2a) and its effect on viral infection. Activation of the RIDD pathway led to degradation of known mouse cell target transcripts without showing any effect on JEV RNA despite the fact that both when biochemically purified showed significant enrichment in ER membrane-enriched fractions. Additionally, inhibition of the IRE1 RNase activity by STF083010, a specific drug, diminished viral protein levels and reduced the titre of the virus produced from infected Neuro2a cells. The results present evidence for the first report of a beneficial effect of RIDD activation on the viral life cycle.
Assuntos
Vírus da Encefalite Japonesa (Espécie)/fisiologia , Encefalite Japonesa/enzimologia , Resposta a Proteínas não Dobradas , Replicação Viral , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Vírus da Encefalite Japonesa (Espécie)/genética , Encefalite Japonesa/genética , Encefalite Japonesa/metabolismo , Encefalite Japonesa/virologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-BoxRESUMO
BACKGROUND: Increasingly, the role of chronic inflammation and its mediators in tumor generation and progression is gaining importance in the field of cancer research. In this context, candidature of non steroidal anti-inflammatory drugs (NSAIDs) as potential anti-tumor therapeutic agent is being evaluated globally. In the present study we have evaluated the anti-cancer effect of a series of newly synthesized naproxen derivatives on human breast cancer cell lines. METHODS: MCF-7 (poorly invasive) and MDA-MB-231 (highly invasive) cells were treated with different concentrations of naproxen sodium and its derivatives in vitro, and the underlying mechanism of action was monitored by employing studies related to induction of apoptosis, activation of caspases, cell-cycle progression, synthesis of PGE2 and cellular migration. RESULTS: After a preliminary screening using MCF-7 and MDA-MB-231 cells, it was evident that naproxen derivative 4 has a better killing property compared to its parent compound naproxen sodium (NS). On further investigation, it was apparent that the observed growth inhibitory activity on MDA-MB-231 cells after treatment with 4, was not due to cell cycle arrest but due to an early induction of apoptosis and subsequent induction of caspases 3 and 9. Derivative 4 could also inhibit COX activity in MDA-MB-231 cells as evidenced by reduction in prostaglandin E2 secretion. Moreover, 4 was capable of delaying the overall migration rate of MDA-MB-231 cells in vitro. CONCLUSION: In this study we report that a naproxen-derivative (4) has powerful anti-inflammatory and anti-tumor properties as it induces appreciable amount of apoptosis in breast cancer cell line, and can also delay migration of cancer cells (MDA-MB-231) which would in turn delay cancer cell invasion and formation of secondary tumours in primary breast cancer patients. Thus, we propose that 4 is worthy of further investigation due to its potential as a therapeutic agent in anti-tumor treatment regimen.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Neoplasias da Mama/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Naproxeno/análogos & derivados , Naproxeno/farmacologia , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , CamundongosRESUMO
The ionization state of amino acids on the outer surface of a virus regulates its physicochemical properties toward the sorbent surface. Serologically different strains of the dengue virus (DENV) show different extents of infectivity depending upon their interactions with a receptor on the host cell. To understand the structural dependence of E-protein protonation over its sequence dependence, we have followed E-protein titration kinetics both experimentally and theoretically for two differentially infected dengue serotypes, namely, DENV-2 and DENV-4. We have performed E-protein protonation titration-induced single-particle chemical force spectroscopy using an atomic force microscope (AFM) to measure the surface chemistry of DENV in physiological aqueous solutions not only to understand the charge distribution dynamics on the virus surface but also to estimate the isoelectric point (pI) accurately for infectious dengue viruses. Cryo-EM structure-based theoretical pI calculations of the DENV-2 surface protein were shown to be consistent with the evaluated pI value from force spectroscopy measurements. We also highlighted here the role of the microenvironment around the titrable residues (in the 3D-folded structure of the protein) in altering the pKa. This is a comprehensive study to understand how the cumulative charge distribution on the outer surface of a specific serotype of DENV regulates a prominent role of infectivity over minute changes at the genetic level.
Assuntos
Vírus da Dengue , Dengue , Humanos , Dengue/metabolismoRESUMO
Myeloid-derived suppressor cells are heterogenous immature myeloid lineage cells that can differentiate into neutrophils, monocytes, and dendritic cells as well. These cells have been characterized to have potent immunosuppressive capacity in neoplasia and a neoplastic chronic inflammatory microenvironment. Increased accumulation of myeloid-derived suppressor cells was reported with poor clinical outcomes in patients. They support neoplastic progression by abrogating antitumor immunity through inhibition of lymphocyte functions and directly by facilitating tumor development. Yet the shifting genetic signatures of this myeloid lineage cell toward immunosuppressive functionality in progressive tumor development remain elusive. We have attempted to identify the gene expression profile using lineage-specific markers of these unique myeloid lineage cells in a tumor microenvironment and bone marrow using a liquid transplantable mice tumor model to trace the changing influence of the tumor microenvironment on myeloid-derived suppressor cells. We analyzed the phenotype, functional shift, suppressive activity, differentiation status, and microarray-based gene expression profile of CD11b+Gr1+ lineage-specific cells isolated from the tumor microenvironment and bone marrow of 4 stages of tumor-bearing mice and compared them with control counterparts. Our analysis of differentially expressed genes of myeloid-derived suppressor cells isolated from bone marrow and the tumor microenvironment reveals unique gene expression patterns in the bone marrow and tumor microenvironment-derived myeloid-derived suppressor cells. It also suggests T-cell suppressive activity of myeloid-derived suppressor cells progressively increases toward the mid-to-late phase of the tumor and a significant differentiation bias of tumor site myeloid-derived suppressor cells toward macrophages, even in the presence of differentiating agents, indicating potential molecular characteristics of myeloid-derived suppressor cells in different stages of the tumor that can emerge as an intervention target.